Background CAR T cell therapy for solid tumours has achieved limited success compared to its application to B cell malignancies. One reason for this failure is the low differentiation rate to memory subsets and low persistence of CAR T cells due to activation-induced cell death (AICD) in lymphoid tissue and the tumour microenvironment. In this study, we have expressed the MCL1 gene within CAR T cells to overcome losses by AICD in adoptively transferred T cells. The MCL1 gene expresses two isoforms; the long isoform localises to the outer membrane of mitochondria and inhibits the CD95 signalling death pathway, while the short isoform localises to the inner membrane of mitochondria to enhance mitochondrial oxidation, phosphorylation and fusion. In addition, we have also utilized a microRNA (miR) 429 to promote memory T cell formation through the suppression of genes such as T-cell-restricted intracellular antigen-1 (TIA-1), T cell activation inhibitor, mitochondrial (TCAIM) and mitochondrial fission factor (MFF).
Materials and Methods Overexpression of MCL1 was confirmed at both mRNA and protein level by real time RT-PCR (qPCR) and western blot. Similarly, overexpression of miR-429 was measured by qPCR and specific binding of miR-429 to the 3′ UTR of target genes was confirmed by luciferase reporter assay. Mitochondrial depolarization and cell viability were assessed by TMRE mitochondrial membrane potential assay (flow-cytometry) and resazurin assay. The effect of MCL1 or miR429 overexpression on HER2-CAR T cells was determined by flow cytometry. Soluble leucine-zipper CD95L (https://www.addgene.org/104349/) was expressed and purified from Expi293 cells.
Results Overexpression of MCL1 in both Jurkat T cells and primary human T cells protected cells against mitochondria depolarization as well as the loss of cell viability in response to CD95L-triggering. Expression of miR429 downregulated TIA1, TCAIM and MFF. A HER2-CAR construct with either MCL1 or miR429 in a lentiviral system was successfully designed and transduced into primary T cells. Mitochondria in transduced T demonstrated enlarged and fusion morphology - a classic feature of memory T cells.
Conclusions Overexpressing MCL1 or miR429 significantly improves mitochondrial function in T cells. This approach will be used to increase persistence of adoptively transferred CAR T cells.
Disclosure Information A. Hosseini Rad: None. G. Min Yi Tan: None. A. Poudel: None. A. McLellan: None.
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