CAR-Modified Th1/Tc1-Polarized T-Rapa Cells Dissociate Inflammatory Cytokine Secretion from Anti-Tumor Cytotoxicity

Introduction: Despite some striking clinical success thus far, chimeric antigen receptor (CAR) engineered cells have the potential to cause severe side effects. Neurotoxicity and cytokine release syndrome (CRS), the latter characterized by increased levels of cytokines such as IL-6, IFN-γ, and MCP-1, are common adverse events associated with CAR therapy. Lymphodepleting preconditioning regimens are associated with improved clinical responses to CAR therapy, yet lymphodepletion has also been identified as a risk factor for CRS. Understanding and management of these toxicities has improved significantly, however these conditions are challenging to treat and can be life-threatening. The ability to limit or prevent initiation of CRS would greatly improve the safety of CAR therapy.

Previous clinical trials have shown that T-Rapa cells (patient T cells that have been grown exvivo in rapamycin) can be successfully infused back into autologous recipients after a low-dose conditioning regimen. After infusion, these T-Rapa cells have potent effector functions and demonstrate long-term persistence. Here we determine that T-Rapa cells, engineered by lentivirus-mediated gene transfer to express an anti-CD19 CAR, are just as effective at killing tumor cells as similarly-engineered pan T cells but produce dramatically less IFN-γ, for example.

Methods: Human CD3+ cells were treated with rapamycin in the presence of IFN-α and IL-2 to produce T-Rapa cells with a Th1/Tc1 phenotype. An anti-CD19-41BB-CD3ζ CAR construct was subcloned into a lentiviral vector backbone containing an IRES-eGFP element. Vector was prepared and used to transduce T or T-Rapa cells. Transgene expression was assessed by FACS for eGFP and Protein L staining for the CAR. CAR-T cells were then expanded using CD3/CD28 beads in the presence of IL-2. The expanded cells were used in assays including FACS assessment of T cell phenotype, co-culture assays, and ⁵¹Cr release assays in comparison with non-rapamycin treated CAR T cells and non-transduced controls.

Results: Following transduction and expansion, similar eGFP and CAR expression levels were found in T and T-Rapa cells transduced at the same MOI. CAR-T and CAR-T-Rapa cells developed from multiple independent T cell donors exhibited similar phenotypes at days 5 and 14 post-thaw, as determined by analyses of T-cell subset and exhaustion markers including CD45RO, CD127, CCR7, CD95, CD25, CXCR3, CTLA-4, PD-1, LAG-3, and TIM-3. Both CAR-T and CAR-T-Rapa cells exhibited comparable levels of cytotoxicity against CD19+ Raji, SUP-B15 and RS4;11 cancer cell lines after coculture for 4 hours in a ⁵¹Cr release assay. Further, both T and T-Rapa CAR cells produced similar amounts of IL-2 following a 24-hour coculture with CD19+ Raji, SUP-B15 and RS4;11 cancer cell lines, as measured by ELISA. Interestingly, CAR-T-Rapa cells produced significantly less IFN- γ that CAR-T cells after 24 hours of coculture with CD19+ tumor cells. This observation was consistent for CAR-T and CAR-T-Rapa cells assessed at both day 5 and day 14 post-thaw.

Conclusions: T-Rapa cells can be successfully transduced with a CAR vector, and show comparable T cell subset, exhaustion phenotype, and cytotoxicity to CAR-T cells that have not been treated with rapamycin. In spite of these similarities, when challenged with CD19+ tumor cells, CAR-T-Rapa cells produced less IFN-γ than CAR-T cells. Decreased production of IFN- γ may reduce the risk and severity of CRS, improving the safety of CAR therapy. Additional cytokine production studies, as well as in vivo studies, are underway to further characterize T-Rapa cells as a novel CAR effector cell type.