Sir Peter MacCallum Department of Oncology - Theses
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ItemInvestigating the role of Mucosal-Associated Invariant T (MAIT) cells in cancer( 2021)The success of immunotherapy in patients has highlighted the importance of the anti-tumour role of the immune system. The function of conventional T cells within the tumour microenvironment (TME) have been intensively studied, while the role of mucosal-associated invariant T (MAIT) cells is yet to be determined. MAIT cells are abundant in humans and enriched in mucosal tissues, such as the colon and lung, and have been found within primary and metastatic tumours. Upon activation, MAIT cells exert rapid effector functions and can secrete both the anti-tumour cytokines (IFNg and TNF) and pro-tumour cytokines (IL-17 and IL-22). MAIT cells also produce granzyme B and perforin, suggesting they are capable of killing target cells. Although direct evidence of MAIT cells precise function in cancer is limited, some studies show that increased numbers of MAIT cells within tumours are correlated with a good prognosis, whilst other studies have indicated MAIT cells are associated with a poorer prognosis. These divergent results have made it difficult to interpret whether MAIT cells have an anti-tumour or pro-tumour role. Therefore, this thesis investigated the role of MAIT cells in cancer and the potential for MAIT cells to be exploited for adoptive cellular therapy. The first results chapter of this thesis explores the anti-tumour role of MAIT cells in both murine and ex vivo human models. It was observed that at steady-state, MAIT cells negatively regulate NK cell maturation and anti-tumour activity. Conversely, activating MAIT cells through either pulsing of tumour targets or intranasal administration of free MAIT cell antigen, led to robust protection against the development of lung metastases. Upon further investigation, it was discovered that activated MAIT cells enhance NK cell maturation and anti-tumour activity in an IFNg-dependent manner. This chapter proposes the existence of a MAIT-NK cell axis that can control NK cell mediated anti-tumour efficacy. The second results chapter aims to further improve the anti-tumour efficacy of activated MAIT cells, by combining this therapy with additional immunotherapies. The additional immunotherapies tested in combination with MAIT cell activation were selected on the basis of their ability to activate MAIT cells and/or NK cells. Notably, additional therapies that increased both MAIT cell and NK cell activity were most promising. This chapter also found that intravenous administration of MAIT cell antigen led to systemic expansion of MAIT cells and an increase in MAIT cells within the tumour tissue, broadening the application of activating MAIT cells in the clinic. The third results chapter aims to investigate the potential of MAIT cells in the context of Chimeric antigen receptor (CAR) T cell therapy. CAR T cell therapy is currently ineffective in solid tumours, due to the immunosuppressive TME, antigen heterogeneity, poor trafficking to solid tumours and decreased persistence. Furthermore, this therapy requires autologous generation of CAR cells in order to avoid graft versus host disease (GVHD). Excitingly, MAIT cells represent an allogeneic source of CAR cells as they are not restricted to conventional MHC. Chapter 5 demonstrates that MAIT cells are able to be efficiently transduced with CAR and upon target recognition CAR MAIT cells produce cytokines and directly kill tumour cells. Collectively, this data illustrates the potential anti-tumour activity of MAIT cells through a MAIT-NK cell axis. Furthermore, this thesis demonstrates the potential for MAIT cells to be used in adoptive cellular therapy, namely as CAR MAIT cells.
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ItemUnderstanding the role of adenosine receptor signalling in chimeric antigen receptor (CAR) T cell therapy in solid cancer( 2021)Chimeric antigen receptor (CAR) T cell therapies have been highly effective and clinically approved for treating haematological malignancies, however trials in solid cancers have shown limited efficacy, likely due in part to the increased complexity of the immunosuppressive tumour microenvironment (TME) in solid cancers. CAR T cells are inhibited by immunosuppressive proteins, cytokines or physical barriers deployed by the tumour to evade and avoid destruction by anti-tumour immunity. One such process involves the accumulation of extracellular adenosine (eADO), in the TME which has potent immunosuppressive effects on T cells and other immune cells. eADO has four known G protein coupled receptors, the A1R, A2AR, A2BR and A3R, of which the A2AR is primarily responsible for suppressing T cell function. Our previous studies highlighted a major impediment to pharmacological blockade of the A2AR which was predicted to be hindered by poor solubility and suboptimal in vivo pharmacokinetic profile [1]. This became apparent when comparing the effectiveness with genetic deletion of A2AR in CAR T cells to pharmacological blockade, in which the CAR T cells generated from A2AR-/- mice elicited comparatively greater efficacy in vivo when combined with anti-PD-1 blockade [1, 2]. This thesis therefore investigated multiple gene editing strategies to modulate adenosine receptor signalling, firstly by overexpressing the alternative signalling A1R or A3R in human or mouse CAR T cells. A1R or A3R have been shown to act by the opposing downstream signalling pathway to A2AR, and thus it is hypothesised that A1R or A3R overexpression can reverse suppression and supercharge CAR T cells in the presence of eADO. Interestingly, A1R or A3R overexpression did not confer protection to suppression by eADO in both mouse and human models, but A1R expression instead enhanced effector and terminal differentiation, activation, and baseline cytokine production of CAR T cells. This however translated to higher expression of exhaustion markers, loss of memory associated gene expression and reduced stem-like memory fraction in the CAR T cell product, ultimately leading to reduced persistence in vivo, and limiting the therapeutic efficacy of this approach. Alternatively, a previous publication from our lab briefly examined short-hairpin RNA (shRNA) mediated silencing of A2AR expression [1]. While shRNA-mediated silencing of the A2AR was able to partially reverse suppression by eADO, much like A1R expression, it also led to effector differentiation, activation, and increased baseline cytokine production. Importantly, while shRNA-mediated silencing of the A2AR also resulted in reduced persistence in vivo, it was able to mediate modest anti-tumour efficacy leading to reduced tumour growth and increased mouse survival. Both overexpression and knockdown approaches are limited by sub-optimal persistence in vivo which limited their overall therapeutic efficacies. Yet these results contradicted our prior observations of CAR T cells derived from A2AR-/- mice and from studies in the Lymphocytic choriomeningitis virus (LCMV) setting, whereby A2AR deletion was linked to increased T cell numbers [1, 3]. Therefore, the final gene-editing approach examined in this thesis utilised CRISPR/Cas9 protocols to achieve full deletion of the A2AR in CAR T cells. CRISPR/Cas9 methodologies are currently being used in clinical trials and therefore deleting the A2AR in CAR T cells using this approach is highly novel and clinically translatable. To reasons unknown, CRISPR/Cas9 mediated deletion of A2AR had minimal effects on CAR T cell memory phenotypes and no adverse effects on engraftment or persistence in vivo. Furthermore, CRISPR/Cas9-mediated deletion of A2AR in CAR T cells led to enhanced therapeutic efficacy in both mouse and human models, thus representing a potent approach to targeting the A2AR. In conclusion, future studies comparing full A2AR deletion to A2AR silencing/ pharmacological blockade or A1R overexpression may be of interest to fully elucidate the mechanisms of adenosine receptor signalling on T cell persistence and memory.