Regimen-specific effects of RNA-modified chimeric antigen receptor T cells in mice with advanced leukemia

Abstract

Cytotoxic T lymphocytes modified with chimeric antigen receptors (CARs) for adoptive immunotherapy of hematologic malignancies have demonstrated activity in early phase clinical trials. While T cells bearing stably expressed CARs are efficacious and have potential long-term persistence, temporary expression of a CAR via RNA electroporation is also potentially efficacious in preclinical models. Temporary CAR expression using RNA presents a method of testing CARs clinically with additional safety where there may be concerns about possible chronic "on-target, off-tumor" toxic effects, as the degradation of RNA ensures complete removal of the CAR over time without relying on suicide induction systems. CD19-directed RNA CAR T cells were tested in vivo for efficacy and comparison to lentiviral vector (LV)-generated stable CAR T cells. We tested the hypothesis that multiple infusions of RNA CAR T cells preceded by lymphodepleting chemotherapy could mediate improved survival and sustained antitumor responses in a robust leukemia xenograft model. The saturation strategy using rationally designed multiple infusions of RNA CARs based on multiple model iterations approached the efficacy of a stable LV expression method. Two-color imaging revealed that relapse was a locoregional phenomenon in both the temporary and the stable expression models. In marked contrast to stably expressed CARs with retroviral or LV technology, the efficacy of RNA CARs appears independent of the costimulatory signaling endodomains likely because they more influence proliferation and persistence rather than short-term efficacy. The efficacy of the RNA CAR infusions may approach that of stably expressed CARs, offer theoretically safer initial clinical testing in addition to suicide systems, and allow for rapid and effective iterative preclinical modeling for the testing of new targets.

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.

FIG. 2.

Interval lymphodepletion produces a sustained antitumor effect with multiple infusions of mRNA CAR T cells. (A) CTX plus mRNA CAR T cells produce a more sustained disease control than that without CTX. NSG mice bearing Nalm-6 CBG were treated with 10⁷ mRNA CAR T cells on days 7, 14, and 28 with 60 mg/kg CTX IP 24 hr before injections 2 and 3. While disease responded to the first injection of anti-CD19 CAR T cells, only with CTX did subsequent infusions have any impact on disease progression. About 10⁷ stably transduced CAR T cells (via lentiviral vector) produce a sustained disease response as well, and this is not lost with CTX treatment. mRNA CAR T cells directed against mesothelin with CTX do not affect disease progression. (B) The reduction in disease burden correlates with increased median survival (n=5 per arm).

FIG. 3.

Disease progression in animals treated with CTX and mRNA CAR T cells. Animals are from experiment described in Fig. 2. Mesothelin-directed T cells and cells administered without interval CTX show progression after the first injection. Repeat T-cell infusions with CTX show sustained disease response, with subsequent infusions able to impact relapsing disease (third row, change between days 21 and 29). Once infusions are stopped the disease does progress, with the exception of those mice treated with stably expressed 19–41BB–z. In the lentiviral vector 19–41BB–z animals, which received CTX on day 27, disease is eliminated quickly and no harbor sites are seen but no reduction in later xenogeneic GVHD is seen either. GVHD, graft-versus-host disease.

FIG. 4.

(A) There is little difference among RNA CAR constructs in survival, though CTX is required for multiple infusions to have efficacy. NSG mice (n=5 per experiment, repeated twice) were given 10⁶ Nalm-6 CBG on day 0 followed by the 3 infusion schedule of 10⁷ mRNA CAR T cells or a single infusion of 10⁷ stably transduced CAR T cells. All anti-CD19 CARs have statistically superior improved median overall survival (p<0.0001) to saline alone, CTX alone, a mesothelin CAR, and the 19–BBz CAR with no interval CTX. There was no significant difference between 19–z, 19–28z, 19–BBz, or 19–28BBz in survival. (B) In this experiment, NSG mice received 10⁶ of a primary pediatric high-risk leukemia modified to express the CBG luciferase. Constructs compared include CARs with unmodified sequence cassettes within the CAR or codon optimized versions to reduce internal open reading frames. While the 19–BBz wild type produced the most rapid initial disease response by bioluminescence, the effect was not sustained at the third infusion possibly because too much time had elapsed allowing leukemia to progress. (C) Survival was followed for 126 days, and no statistically significant differences in median overall survival could be appreciated between mRNA constructs (p=0.22). Stably modified CARs were superior in overall survival, with five mice both clearing leukemia and not developing lethal xenogenic GVHD. (D) Day 100 image of mice from the lentiviral CAR groups, demonstrating the jaw/tooth harbor site and the appearance of locoregional relapse.

FIG. 5.

Dose splitting impacts efficacy of mRNA CAR T cells. (A) This is a composite survival of 4 independent experiments (with 3 different donors, n=20) using 10⁶ Nalm-6 on day 0 and either a single dose 2.5×10⁷ on day 7, or split infusions on days 7, 14, and 28 with interval CTX at 60 mg/kg IP×1 24 hr before CTL doses 2 and 3. Survival is significantly improved by the 20–5–5 schedule despite the total dose of T cells being equivalent (p=0.00103). The only long-term survivors are observed with this dose schedule. (B) Compressed T-cell infusion schedule further improves efficacy. This composite of 2 experiments with 2 donors demonstrates the significant effect of a compressed infusion schedule of 20–5–5 on days 7–14–21 (n=15, p<0.0001). The compressed infusion and CTX schedule did result in some meso–41BBz-treated mice surviving beyond CTX-only controls, perhaps reflecting a slight allogeneic effect of this donor against Nalm-6 (see Supplementary Fig. S2 for further investigation). The difference approached significance for control versus meso–41BBz (p=0.09), while the comparison of control versus anti-CD19-targeted RNA CAR T cells from the same donor was highly significant (p<0.001).