Abstract 3233: Reduced inflammatory cytokine production by CAR-modified Th1/Tc1-polarized T-Rapa cells is not sufficient to reduce monocyte-driven production of IL-6
Authors: Oldham Robyn A. , Felizardo Tania , Zhu Nathaniel , Fowler Daniel H. , Medin Jeffrey A. 
Published: September 2020

Abstract
Introduction: Chimeric antigen receptor (CAR) engineered T cells have delivered remarkable anti-tumor effects. However, the potential for severe and life-threatening cytokine release syndrome (CRS) represents a therapeutic obstacle. CRS is in large part IL-6 mediated, as indicated by the recent FDA approval of tocilizumab after CAR-T therapy. However, because CRS remains a significant clinical toxicity, additional approaches are necessary.
Previous experimental data has shown that T-Rapa cells (T cells grown ex vivo in rapamycin) secrete reduced levels of some cytokines, and express a central memory phenotype that is associated with increased in vivo persistence. In the clinical context, allogeneic T-Rapa cells of a Th2 phenotype were associated with a low rate of GVHD, which is, in part, a cytokine-mediated syndrome. As such, T-Rapa cells represent a novel CAR effector cell type (CAR-T-Rapa). We hypothesized that Th1/Tc1 polarized CAR-T-Rapa cells would mediate efficient cytotoxicity, secrete reduced inflammatory T cell cytokines, and thereby result in a reduced propensity for IL-6 secretion, which is predominantly a monocyte-derived cytokine.
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, and were transduced with a CD19-41BB-CD3ζ CAR. T cells were expanded and 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. ELISA and Luminex were used for assessment of cytokine levels in cell culture supernatants.
Results: CAR-T and CAR-T-Rapa cells exhibited equal levels of cytotoxicity against CD19+ tumor cell lines. Both CAR-T and CAR-T-Rapa cells produced comparable amounts of IL-2 and undetectable IL-6 following co-culture with CD19+ tumor cell lines. However, CAR-T-Rapa cells produced significantly less IFN- γ, IL-4, TNF-α, and GM-CSF than CAR-T cells. When T cells were co-cultured with both tumor cells and monocytes, a high level of IL-6 secretion was detected in both CAR-T and CAR-T-Rapa conditions.
Conclusions: CAR-T-Rapa cells are effective killers, while producing considerably less inflammatory cytokines in comparison to CAR-T cells. However, in the monocyte-replete condition, CAR-T-Rapa cells did not result in reduced IL-6 secretion, thereby indicating that controlling T cell inflammatory cytokine levels may not be sufficient to mitigate CRS.
Citation Format: Robyn A. Oldham, Tania Felizardo, Nathaniel Zhu, Daniel H. Fowler, Jeffrey A. Medin. Reduced inflammatory cytokine production by CAR-modified Th1/Tc1-polarized T-Rapa cells is not sufficient to reduce monocyte-driven production of IL-6 [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3233.