Sir Peter MacCallum Department of Oncology - Theses
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ItemCRISPR/Cas9 Engineering of Next Generation Armoured CAR T Cells( 2023-08)Chimeric antigen receptor (CAR) T cell therapy has achieved remarkable successes in the treatment of certain B cell haematological malignancies; however, its efficacy in the solid tumour setting remains limited. This is attributed to a number of factors, including immunosuppression in the tumour microenvironment (TME), T cell exhaustion, tumour antigen heterogeneity and limited trafficking of CAR T cells into the tumour. A promising strategy to overcome these limitations is by engineering, or “armouring”, CAR T cells to express factors that have the potential to enhance therapeutic efficacy, such as proinflammatory cytokines, chemokines, transcription factors, cell surface molecules and synthetic constructs. This strategy has been assessed in the context of various forms of adoptive cellular therapy (ACT), including tumour-infiltrating lymphocyte (TIL) therapy, as well as transgenic TCR and CAR T cell therapy. Preclinical studies exploring a vast range of armouring genes have demonstrated promising results, with armoured T cell products mediating enhanced anti-tumour efficacy through the capacity to modulate the TME, engage host anti-immune responses and directly enhance T cell function. However, a key limitation of this approach is the potential toxicities caused by peripheral expression of the armouring gene by the engineered T cells, highlighted by the life-threatening side effects observed in an early clinical trial assessing this approach with IL-12-engineered TILs. While strategies have been developed to limit transgene expression to the tumour, such as the synthetic NFAT promoter system, none thus far have successfully demonstrated the ability to prevent clinical toxicities. The advent of CRISPR/Cas9 gene editing, including recent advancements in the engineering of primary T cells, has introduced unique opportunities to enhance the design of armoured T cells. In particular, CRISPR-mediated homology-directed repair (CRISPR-HDR) enables the precise modification of specified genomic sites, including the introduction of a complementary DNA (cDNA) sequence into a target gene locus. Hence, in the context of armoured T cells, CRISPR-HDR can be used to insert armouring genes into a specific endogenous site, creating the possibility to leverage endogenous gene regulatory mechanisms to drive transgene expression. By targeting the armouring gene into the site of an endogenous gene that is exclusively upregulated in intratumoural CAR T cells, this should lead to tumour-restricted expression of the armouring gene. Additionally, integration of the armouring gene can concurrently disrupt expression of the endogenous target gene. Thus, by selecting a target gene that is not only specifically expressed in CAR T cells within the tumour, but also regulates inhibitory T cell functions, this would enable knock-in of a transgene that can enhance therapeutic efficacy while simultaneously knocking out an endogenous inhibitory gene. Therefore, this study assessed the overarching hypothesis that that engineering armoured T cells in accordance with this “knock-in, knock-out” approach would lead to enhanced anti-tumour efficacy while maintaining a favourable safety profile. To explore this hypothesis, a CRISPR-HDR protocol was first developed to engineer primary murine and human transgenic TCR and CAR T cells at high editing efficiencies. This protocol was subsequently applied to generate armoured transgenic TCR and CAR T cells expressing various proinflammatory cytokines under the control of endogenous tumour-specific promoters, and assessed for therapeutic efficacy, mechanism and safety in syngeneic murine models and human xenograft models of ACT. T cells engineered to express TNF, IFN-gamma, IL-2 and IL-12 in a tumour-restricted manner mediated significantly enhanced anti-tumour efficacy; however, their therapeutic effect was largely dependent on the endogenous promoter used. The PD-1 promoter was found to support potent transgene expression in the tumour, which was required for the therapeutic efficacy of TNF, IFN-gamma and IL-2. However, its low level of peripheral expression led to toxicities when used to drive IL-12 expression. Instead, the NR4A2 promoter, which supported highly tumour-restricted transgene expression, was capable of regulating IL-12 production with minimal toxicities while mediating a robust therapeutic response. Hence, these data demonstrate that this CRISPR-HDR strategy is highly customisable, enabling the use of different endogenous promoters to achieve the optimal expression pattern for specific transgenes. In summary, the current study details the development of a novel CRISPR-HDR approach for engineering next generation armoured T cells for ACT, and presents promising preclinical data demonstrating the efficacy, safety, feasibility and translational potential of this approach.
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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.