Background CAR T cell therapy has been successful for targeting blood cancers, but treatment of solid cancers has been limited due to the heterogenous nature of tumour-associated antigen expression on solid cancers, and the suppressive tumour microenvironment.1 Another major obstacle to CAR T cell therapy is activation-induced cell death (AICD) of the CAR T cells.2 In this study, we expressed the anti-apoptotic cellular FLICE-like inhibitory protein (c-FLIP short; c-FLIPs) together with the CAR construct to enhance CAR T cell persistence.3
Materials and Methods The anti-Her2 FRP5 CAR T construct with P2A-linked cFLIPs or cFLIPp43 was cloned into the Sleeping Beauty (SB) transposon vector (pSBtet-GP) or lentiviral vector, under the control of either a tet-on or a constitutive promoter. Construct expression was validated by qPCR and immunoblot analysis. CAR T cells were generated by SB transposition or lentiviral transduction of CD3/CD28 stimulated primary human T cells that were subsequently maintained with IL-2. Mitochondrial function and apoptosis were determined by resazurin assay and by flow cytometry using tetramethyl rhodamine (TMRE).
Results Overexpression of cFLIP (cFLIPp43 and cFLIPs) in pSBtet-GP demonstrated protection in both Jurkat T cell line and primary human T cells. pSBtet-GP was modified to overexpress cFLIPs and cFLIPp43 under tet-on promoter, with the anti-her2 CAR, GFP and rtTA under constitutive promoter. Transfer of the inducible cassette from the SB transposon to a lentiviral system resulted in a significant loss of tightness. Doxycycline treated CAR T cells showed only ~13-fold overexpression of cFLIPs or cFLIPp43 compared to untreated cells, and doxycycline significantly inhibited (approximately 30%) primary CAR T cell expansion. In contrast, constitutive expression of CAR-cFLIPs or cFLIPp43 construct gave a >3 × 105-fold cFLIP overexpression, as compared to CAR-only control. While the transduction efficiency of CAR-only was around 70–80% control in primary T cells, this dropped to 20–25% when using the more genetically complex tet-on system.
Conclusions cFLIP protects T cells from Fas-induced apoptosis. The tet-on system demonstrates several drawbacks in the lentiviral system, including toxicity of the inducer drug (and/or squelching effects resulting in lowered viability), loss of responsiveness and lowered transduction frequencies. Therefore, a constitutive promoter system is preferred in lentiviral systems for the control of genes of interest within CAR T cells, while the SB transposon system may be preferred for tet-on control within CAR T cells.
Pitt JM, Marabelle A, Eggermont A, Soria JC, Kroemer G, and Zitvogel L. Targeting the tumor microenvironment: removing obstruction to anticancer immune responses and immunotherapy. Ann Oncol 2016;27:1482–1492.
Yeku O, Li X, Brentjens RJ. Adoptive T-Cell Therapy for Solid Tumors. Am Soc Clin Oncol Educ Book 2017;37:193–204.
Dohrman A, Kataoka T, Cuenin S, Russell JQ, Tschopp J, and Budd RC. Cellular FLIP (long form) regulates CD8+ T cell activation through caspase-8-dependent NF-kappa B activation. J Immunol 2005; 174:5270–5278.
Disclosure Information G.M.Y. Tan: None. S.M.A.R. Hosseini: None. A. Poudel: None. A.D. Mclellan: None.
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