Show simple item record

dc.contributor.authorSek, Kevin Chen Ming
dc.description© 2021 Kevin Chen Ming Sek
dc.description.abstractChimeric 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.
dc.rightsTerms and Conditions: Copyright in works deposited in Minerva Access is retained by the copyright owner. The work may not be altered without permission from the copyright owner. Readers may only download, print and save electronic copies of whole works for their own personal non-commercial use. Any use that exceeds these limits requires permission from the copyright owner. Attribution is essential when quoting or paraphrasing from these works.
dc.subjectAdenosine A2 receptor
dc.subjectAdenosine A1 receptor
dc.subjectAdenosine A3 receptor
dc.subjectChimeric Antigen Receptor
dc.subjectGene editing
dc.subjectCell engineering
dc.subjectT lymphocytes
dc.subjectCheckpoint blockade
dc.subjectGene Knockdown
dc.subjectGene Knockout
dc.subjectAdenosine agonists
dc.subjectAdenosine antagonists
dc.titleUnderstanding the role of adenosine receptor signalling in chimeric antigen receptor (CAR) T cell therapy in solid cancer
dc.typePhD thesis
melbourne.affiliation.departmentSir Peter MacCallum Department of Oncology
melbourne.affiliation.facultyMedicine, Dentistry & Health Sciences
melbourne.thesis.supervisornamePaul Beavis
melbourne.contributor.authorSek, Kevin Chen Ming
melbourne.thesis.supervisorothernameLev Kats
melbourne.thesis.supervisorothernamePhillip Darcy
melbourne.tes.fieldofresearch1320409 Tumour immunology
melbourne.tes.fieldofresearch2320402 Applied immunology (incl. antibody engineering, xenotransplantation and t-cell therapies)
melbourne.accessrights This item is embargoed and will be available on 2023-09-03. This item is currently available to University of Melbourne staff and students only, login required.

Files in this item


There are no files associated with this item.

This item appears in the following Collection(s)

Show simple item record