The role of anti-apoptotic c-FLIP in CAR-T cells targeting solid tumours
Chimeric antigen receptor (CAR) T-cell therapy has recently emerged as a promising treatment for targeting haematological malignancies, however, this treatment of solid tumours is limited. CAR T cell treatment of solid tumours has resulted in poor performance of adoptively transferred T cells, potentially due to Fas ligand (FasL)-induced apoptosis. Studies show that cells within of the tumour microenvironment such as tumour cells, epithelial cells and myeloid-derived suppressor cells express high levels of FasL that trigger apoptosis in CAR T cells. Tonic signalling of CAR T cells also induces the secretion of FasL, which initiates activation-induced cell death, thus resulting in only short-term killing by CAR T cells. In this study, the anti-apoptotic cellular FLICE-like inhibitory protein (c-FLIP) was co-expressed with the anti-Her2 FRP5 CAR construct to enhance the anti-tumour response to solid tumours. Two isoforms of c-FLIP (c-FLIPp43 and c-FLIPs) were cloned into Sleeping-Beauty (SB) transposon vector (pSBtet-GP) to validate the anti-apoptotic activities in the Jurkat T cell line and human primary T cells. Mitochondrial function and apoptosis were determined by resazurin assay and by flow cytometry using tetramethyl rhodamine ethyl ester (TMRE). A series of expression cassettes were developed using the SB transposon system or lentiviral-based system, these were under the control of either Tet-On or a constitutive promoter to express c-FLIP gene together with the anti-Her2 CAR construct. CAR T cells were generated by lentiviral transduction of CD3 / CD28 stimulated primary human T cells that were subsequently maintained with IL-2. The potential of c-FLIP expression in the function of CAR T cells was evaluated through in vitro functional assays and an in vivo tumour-bearing xenograft model. Overexpression of cFLIP with pSBtet-GP protected both the Jurkat T cell line and primary human T cells. pSBtet-GP was modified to overexpress cFLIP under the Tet-On promoter, while the anti-Her2 CAR, green fluorescent protein (GFP) and reverse transactivator (rtTA) were under a constitutive promoter. The developed SB-based Tet-On cassette displayed high gene induction with low basal activity. However, the transfer of the Tet-On cassette from the SB system to lentiviral system resulted in a significant loss of tightness and gene induction. Doxycycline-treated c-FLIP Her2-CAR T cells showed only a minimal fold induction of c-FLIP as compared to Her2-CAR control only. In contrast, constitutive expression of c-FLIPp43 Her2-CAR T cells gave a significant enhanced c-FLIP expression, as compared to the Her2-CAR control only. Functional analyses revealed that the overexpression of c-FLIP in Her2-CAR T cells enhanced CAR T cell survival against AICD, produced higher concentration of IL-2 cytokine upon antigen stimulation and exhibited higher frequency of CXCR3+ expression in memory T subpopulations (TCM and TEM). Interestingly, the expression of c-FLIP in Her2-CAR T cells significantly lowered the secretion of interferon-γ, perforin and granzyme activities upon antigen stimulation without affecting CAR T cell cytotoxicity function. However, the overexpression of c-FLIP failed to protect CAR T cells from AICD in vivo, with a reducing overall survival rate of tumour-bearing mice observed. Both c-FLIP isoforms (c-FLIPp43 and c-FLIPs) protected T cells from FasL-induced apoptosis. The findings in this study demonstrate that c-FLIP expression protects CAR T cells from FasL-induced apoptosis but reduces the anti-tumour immunity of CAR T cells through the inhibition of the perforin / granzyme pathway. The findings of this study provide a new understanding on the effects of controlling extrinsic apoptosis pathway suppression in CAR T cells. Additionally, this study also provides insights on the possible roles of c-FLIP in modulating T cell cytolytic pathways.
Advisor: McLellan, Alexander
Degree Name: Doctor of Philosophy
Degree Discipline: Microbiology and Immunology
Publisher: University of Otago
Keywords: CAR-T; cancer; immunotherapy; anti-apoptotic
Research Type: Thesis