Strategies to enhance CAR T cell function in solid tumours utilising coding and non-coding genes
Cancer immunotherapy has emerged as an effective treatment to provide benefits to cancer patients. One of the main treatment modalities of cancer immunotherapy is the adoptive transfer of T cells with genetically engineered receptors known as chimeric antigen receptor (CAR), which has recently emerged as a promising approach for haematological cancers. Although some success has been achieved in treating leukaemia, treatment of solid tumours by this approach is limited mainly due to poor infiltration of cancer targeting lymphocytes and their inhibition by immunosuppressive tumour microenvironment (TME) created by hypoxia, low pH, and immunosuppressive metabolites within solid tumours. In this thesis, we attempted to exploit immunosuppressive metabolites (e.g. adenosine and low pH) in solid TME to reduce their suppressive effect on T cells by designing chimeric receptors with CXCR3 signalling domain fused to the extracellular metabolite sensing receptor. Functionality of these chimeric receptors were assessed using calcium flux assay, ERK phosphorylation assay and migration assays. While the assays were effective at assessing signal transduction via non-modified CXCR3 in response to the cognate ligand, CXCL11, signalling via chimeric receptors was not detected in the presence of respective stimuli. Immunosuppressive molecules in TME also activate protein kinase A (PKA) in T cells which results in the localization of the PKA regulatory subunit 1A (PRKAR1A) to the immune synapse inhibiting several central proteins involved in the T-cell signalling cascade and leading to T cell inactivation. While research have targeted PRKAR1A using peptides to supress its activity, we used a different approach by overexpressing microRNAs (miRNAs) to downregulate PRKAR1A expression. Overexpression of either miR96/183 or miR155 significantly downregulated PRKAR1A expression in HEK293 cells at both mRNA and protein level. We further validated PRKAR1A 3' UTR as a direct target of both miRNAs using luciferase assay. Additionally, miR96/183 and miR155 were found to target other inhibitory proteins of TCR signalling such as TET2, FOXO3 and PTPN2 which might have additional advantage to enhance T cell activation. Consistently, overexpression of miRNA96/183 enhanced IL-2 production while overexpression of miR155 enhanced both IL-2 production and CD69 expression on Jurkat T cells following anti-CD3/CD28 stimulation compared to controls with normal expression of respective microRNAs. We revisited the tetracycline-inducible sleeping beauty system to expand miRNA overexpression approach in CAR T cell therapy and generated a vector to induce miRNA/coding gene overexpression in the presence of tetracycline while constitutively expressing second generation HER2 CAR. To study the effect of miRNA overexpression in HER2 CAR in primary human T cells, a third-generation lentiviral system was also optimised by exploring both single and dual promoter systems to co-express non-coding (miRNA) and coding genes (HER2-CAR and GFP) from the same vector. Although cytokine production (IL-2 and IFN-γ) and CD69 expression were similar between HER2 CAR±miRNA transduced T cells when co-cultured with HER2 positive MCF-7 cells, the profile of phenotypic marker expression was different. Overall, this thesis demonstrates that the miRNA overexpression in T cells could be a useful approach to combat intrinsic inhibitory molecules. The modified sleeping beauty vector and the lentiviral vectors developed in this study can be utilized to combine CAR T cell therapy with other approaches to enhance the efficacy of the treatment in cancer immunotherapy. Therefore, this thesis provides useful resource for the development of various strategies in future to enhance the efficacy of anti-cancer T cells or CAR T cells in solid tumour.
Advisor: McLellan, Alexander
Degree Name: Doctor of Philosophy
Degree Discipline: Department of Microbiology and Immunology
Publisher: University of Otago
Keywords: Solid tumour; Protein Kinase A; miRNA; HER2 CAR T cells
Research Type: Thesis