Sir Peter MacCallum Department of Oncology - Theses

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    Avenues to enhance chimeric antigen receptor (CAR) T cell stemness and therapeutic efficacy against solid tumours
    Chan, Jack Daniel ( 2023-02)
    Chimeric antigen receptor (CAR) T cell therapy has demonstrated remarkable activity in B cell malignancies leading to multiple Food and Drug Administration (FDA) approvals. However, in solid tumours, CAR T cells have only yielded limited efficacy. This is thought to be due to several factors including poor persistence of CAR T cells and terminal differentiation. In the present thesis, two approaches for improving the persistence and efficacy of CAR T cells against solid tumours were explored. The first strategy involved overexpressing T cell memory-associated transcriptional regulators in CAR T cells. Clinical data has shown that CAR T cell persistence and therapeutic outcomes are associated with the adoptive transfer of less differentiated, “stem-like” CAR T cells that assume a similar phenotype to circulating memory T cell subsets. I hypothesised that the overexpression of the transcriptional regulators associated with memory T cells would reduce CAR T cell differentiation, thereby leading to their improved persistence and enhanced anti-tumour efficacy. FOXO1 was identified as a primary candidate, and the impact of overexpressing a constitutively active form of Foxo1 (Foxo1-ADA) in HER2 directed murine CAR T cells and wild type FOXO1 in human Lewis Y directed CAR T cells was evaluated. Foxo1-ADA overexpression improved the therapeutic efficacy of CAR T cells in breast and colon carcinoma tumour models. Additionally, Foxo1-ADA overexpression enhanced CAR T cell polyfunctionality and mitochondrial health in vivo. I found that the overexpression of wild type FOXO1 similarly improved the stem-like characteristics of human CAR T cells, enhancing overall respiratory capacity and therapeutic responses in a human ovarian cancer model. Our data indicates that reinforcement of a stem-like program through FOXO1 overexpression significantly enhances CAR T cell responses against solid tumours. The second approach involved the use of a dual-specific CAR T cell that expressed two distinct CARs against a solid tumour and B cell antigen. The success of CD19 directed CAR T cells may be attributed to engaging antigens outside of a solid tumour microenvironment (TME). Therefore, this approach aimed to co-opt these benefits in the solid tumour setting by co-transducing a unique T cell designated as a ‘dual CAR T cell’ with two CARs having specificity for the human HER2 and murine CD19 antigens. I hypothesised that by first engaging the CD19 antigen in sites away from the immunosuppressive TME, dual CAR T cells would be primed to better mount responses against solid tumours. Dual CAR T cells demonstrated higher polyfunctionality in vitro against HER2 expressing tumour cells when primed through the CD19 directed CAR and mediated enhanced therapeutic efficacy against orthotopic breast tumours in vivo. Notably, dual CAR T cells demonstrated improved persistence with a similar differentiation phenotype relative to control T cells. These results indicate that maintenance of a less differentiated CAR T cell phenotype and engagement of target antigens outside of the TME are instrumental for maintaining CAR T cell persistence. These investigations contribute to the growing body of work that aims to enhance CAR T cell stemness. As these studies have been conducted in both syngeneic and human CAR T cell models, I believe that this work carries high translational potential.
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    Novel approaches to harness the anti-tumour activity of natural killer cells
    Freeman, Andrew John ( 2022)
    Despite revolutionary advances in cancer treatment with immunotherapy, durable clinical benefit is limited to a subset of patients. Current forms of immunotherapy, including checkpoint inhibition and chimeric antigen receptor T cells, are primarily restricted to targeting cytotoxic CD8+ T cells of the adaptive immune system. Natural killer (NK) cells are the cytotoxic effector cells of the innate immune response and are emerging as a promising and alternative target for cancer immunotherapy, however, comprehensive functional genetic studies examining anti-tumour NK cell activity are limited. Using genome-scale clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR associated protein 9 screening technology, we sought to comprehensively identify and validate tumour genes that potently influence sensitivity to primary NK cells. We first demonstrate that B16-F10 mouse melanoma cells lacking genes encoding proteins involved in interferon-gamma (IFN-gamma) signaling and antigen processing/presentation are highly sensitive to killing by NK cells, a process dependent on the absence of major histocompatibility complex class I expression. As checkpoint inhibition-resistant melanoma patients present with loss-of-function mutations within these pathways, our findings highlight intratumoural NK cells as a potent strategy to limit and/or overcome resistance to CD8+ T cell attack during conventional checkpoint inhibition immunotherapy. We additionally identify Rnf31 as a novel negative regulator of tumour cell sensitivity to NK cell killing. Rnf31 encodes for HOIP, which has an established role in driving tumour necrosis factor (TNF)-mediated gene induction and inhibition of TNF-induced cell death in certain cell types. HOIP-deficient melanoma cells not only exhibited increased sensitivity to NK cells, but also to antigen-specific CD8+ T cells. We surprisingly demonstrate that HOIP protects tumour cells from apoptosis induced by combined IFN-gamma and TNF, rather than TNF alone. We provide valuable mechanistic insight into the transcription-dependent form of apoptosis induced by combined IFN-gamma and TNF limited by HOIP, pharmacological validation using a HOIP inhibitor, and in vivo validation using HOIP-deficient B16-F10 ovalbumin-expressing tumours that exhibit enhanced CD8+ T cell-mediated control. Inhibition of tumour HOIP activity may therefore enhance NK and CD8+ T cell anti-tumour responses through unlocking the apoptotic potential of combined IFN-gamma and TNF secreted by these immune cells, representing a potential new combinatorial target for current and emerging immunotherapies. Collectively, we impartially identify tumour-specific genes that powerfully modulate tumour cell sensitivity to primary NK cells. We provide validation of established yet clinically relevant tumour genes associated with evasion from CD8+ T cells that mediate resistance to checkpoint inhibition immunotherapy, and additionally validate Rnf31 as a novel tumour regulator of not only sensitivity to NK cells, but also CD8+ T cells. We lastly present appropriate methodology and molecular tools that may facilitate analogous screening within NK cell lines to identify targetable regulators of tumour cell-induced IFN-gamma production by NK cells. Taken together, we advocate that NK cells may play an important role in combating resistance to checkpoint inhibition therapy in melanoma and identify HOIP as a potential combinatorial tumour target that may enhance future NK cell-based immunotherapy endeavours through secreted IFN-gamma and TNF, which may form the basis of a future immunotherapeutic strategy.
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    Investigating the role of Mucosal-Associated Invariant T (MAIT) cells in cancer
    Petley, Emma Victoria ( 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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    Investigating mechanisms for Elotuzumab and Lenalidomide therapy in Multiple Myeloma
    Richardson, Kelden James ( 2021)
    Multiple myeloma (MM) is a haematological malignancy of plasma cells with disease present in the bone marrow. Despite the success of new therapies in extending life expectancy, MM is still considered incurable and requires further investigation into new treatments. In the context of refractory/relapsed MM (RRMM), the combination of the two immunotherapies Elotuzumab (Elo, anti-SLAMF7 mAb) and Lenalidomide (Len, IMiD) has resulted in an 82% objective response rate and 16% complete remission in patients. This success led to FDA approval of Elo plus Len treatment for patients with RRMM and has warranted further investigations into the exact mechanism of action. Elo targets SLAMF7 antigen which is highly expressed on MM tumour cells and activates NK cells for antibody dependent cell-mediated cytotoxicity (ADCC) resulting in MM tumour cell death. Len is an immunomodulatory agent commonly used to treat a wide range of haematological cancers. Len has been shown to activate various cells of the immune system as well as directly suppress tumour progression. This thesis aims to identify how the mechanisms of Len intersect with, and enhance, Elo activated NK cell control of tumours. The work from this thesis revealed that to induce ADCC, Elo required both expression of SLAMF7 on MM target cells and NK cell CD16 engagement. Elo-activated NK cells had increased expression of CD107a and CD69, as well as loss of CD16 expression which was a result of ADAM17 induced cleavage. Elo activation of NK cells also secreted increased levels of cytokines and chemokines associated with recruitment of effector immune cells. In both in-vitro and in-vivo studies, Len significantly enhanced Elo-induced healthy donor NK cell killing of MM tumour cells. In contrast, only 4/12 RRMM patient (responders) NK cells induced increased MM cell killing in the context of Elo plus Len. Important for this increased cytotoxicity was the expression of CD54 on NK cells that allowed differentiation between these responder and non-responder patients. CD54 was also more highly expressed on NK cells activated by Elo plus Len compared to Elo activation alone. However, further phenotypic studies and RNA sequencing did not reveal any further mechanisms in NK cells by which Len enhances Elo activation. Further studies revealed that MM tumour (myeloma) cells specifically upregulated CD54 and CD11a expression in response to Elo plus Len activation of NK cells and this did not occur with Elo treatment alone. This increase in CD54/CD11a expression on myeloma cells was also dependent on the presence of CD14+ monocytes. RNA sequencing also revealed enrichment of cytokine and chemokine signalling/secretion pathways in MM cells specifically in the context of Elo and Len activated NK cells and monocytes. In conclusion, these data indicate that a complex network of direct and indirect mechanisms between NK cells, monocytes and myeloma cells contribute to the Elo plus Len treatment induced response in RRMM patients. Elo alone activates NK cells and monocytes, whereas the Len effect is largely on myeloma cells. Collectively the two treatments led to robust myeloma antibody dependent cellular cytotoxicity (ADCC). There was a strong correlation between increased myeloma cell CD54/CD11a expression and myeloma cell ADCC suggesting that stabilisation of the immune synapse with strong adhesion may be a key factor for this improved myeloma cell killing. Finally, this study revealed that upregulation of MM cell CD54 expression on MM cells may be a useful predictive biomarker to stratify RRMM patients for Elo plus Len therapy.
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    Understanding the role of adenosine receptor signalling in chimeric antigen receptor (CAR) T cell therapy in solid cancer
    Sek, Kevin Chen Ming ( 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.
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    Novel biomarkers for melanoma immunotherapy
    Wong, Ngai Man Annie ( 2020)
    Traditionally, metastatic melanoma had a dismal prognosis, but the recent advent of immune checkpoint inhibitors (ICI) has extended survival from months to years for some patients. There is an urgent need to identify prognostic and predictive biomarkers for melanoma patients treated with ICI, given that only a minority of patients respond, coupled with the potential treatment related toxicities. This thesis aimed to investigate clinical factors, functional PET imaging and tumour immune profiling as candidate biomarkers for ICI in patients with melanoma. Firstly, Chapter 3 focused on baseline performance status as a biomarker for outcome following anti-PD-1. The hypothesis was that unlike cytotoxic chemotherapy, baseline performance status was not correlated with outcome following ICI, owing to its distinct mechanism of action. However, in the cohort of 91 patients treated with anti-PD-1 at Peter MacCallum Cancer Centre, poor performance status was correlated with poor survival and low response rate to anti-PD-1. Furthermore, patients with poor performance status were more likely to be hospitalised and more likely to die in hospital. Patient characteristics and blood parameters were further examined in Chapter 4, but specific to a cohort of patients with melanoma brain metastases. Melanoma commonly metastasise to the central nervous system and this is associated with extremely poor survival. Recently, combination ICI has resulted in intracranial responses and durable survival. Most of the existing literature in biomarkers in melanoma brain metastases also predates the introduction of ICI, therefore investigation of biomarkers in patients with melanoma brain metastases treated with ICI is needed. A post-hoc analysis of patients with melanoma brain metastases as part of the phase II Anti-PD1 Brain Collaboration study was performed to identify possible predictors of clinical outcome or toxicity. In this study, patients were randomised to receive either nivolumab monotherapy or nivolumab in combination with ipilimumab. High C-reactive protein, a marker of systemic inflammation, was correlated with poor survival. Treatment with combination ICI, hypernatraemia and increased body mass index were associated with higher likelihood of severe toxicity at 120 days, whereas CRP was not associated with higher toxicity. The thesis then went on to examine the role of FDG PET functional imaging as a source of biomarkers for outcome following ICI in Chapter 5. Baseline pre-treatment tumoural FDG-PET avidity (measured by SUVmax or metabolic tumour volume) as well as FDG-avidity in the immune system (measured by spleen to liver ratio) were assessed in relation to survival outcomes. Interestingly, tumoural PET avidity was not correlated with survival, whereas high spleen to liver ratio was correlated with poor survival after ipilimumab. This was subsequently validated in a combined cohort of patients from two separate European centres. High spleen to liver ratio was correlated with low albumin in a multivariate analysis, thus suggesting a possible association with systemic inflammation. Early on-treatment PET (EOT PET) were assessed in a small subset of 16 patients, and several challenges were identified that may limit the use of FDG PET in this early juncture as a biomarker for outcome after ICI. In-depth characterisation of tumoural immune landscape is crucial to improving the understanding of melanoma immuno-biology, with potential implications for biomarker development. Chapter 6 aimed to compare the immune profile of UV related skin cancers (melanoma, cutaneous squamous cell carcinoma and Merkel cell carcinoma) using orthogonal methods of bulk RNA-sequencing and multi-spectral immunohistochemistry. The three skin cancers showed distinct immune landscapes, with melanoma having a significantly higher intratumoural T cell infiltrate compared to Merkel cell carcinoma, whereas PD-L1 density was highly variable across three skin cancers. Transcriptomic analyses of melanoma samples with high PD-L1 density were associated with upregulation of genes related to leucocyte proliferation, migration and adaptive immune responses, in contrast to MCC samples with high PD-L1 density, where such a signature was not observed. Lastly, an in-depth case study of six patients highlighted how multi-factorial biomarkers such as clinical factors, functional PET imaging, baseline blood parameters, and multi-spectral immunohistochemistry can be applied together. In conclusion, this thesis evaluated multi-factorial biomarkers including clinical, functional imaging and tumoural immune profiling biomarkers. These studies add to the evolving literature on biomarkers associated with ICI treatment. It is envisaged that with time, these complementary methods of understanding the patient and tumoural immune environment can aid rational selection of immune based therapies for patients with advanced melanoma.
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    Examining the effects of BRAF, MEK and CDK4/6 inhibition on anti-tumor immunity in BRAFV600 melanoma
    Lelliott, Emily Jane ( 2020)
    The recent advent of targeted and immune-based therapies has revolutionized the treatment of melanoma, and transformed outcomes for patients with metastatic disease. However, the mechanisms underpinning the clinical efficacy of these approaches are still being elucidated. The majority of patients develop resistance to the current standard-of-care targeted therapy, dual BRAF and MEK inhibition (BRAFi+MEKi), prompting evaluation of a new combination incorporating a CDK4/6 inhibitor. Based on promising preclinical data, combined BRAF, MEK and CDK4/6 inhibition (triple therapy) has recently entered clinical trials for the treatment of BRAFV600 melanoma. Interestingly, while BRAFi+MEKi therapy was initially developed on the basis of potent tumor-intrinsic effects, it was later discovered to have significant immune-potentiating activity. Recent studies have also identified immune-related impacts of CDK4/6 inhibition, though these are less well defined and appear to be both immune-potentiating and immune-inhibitory. BRAFV600 melanoma patients are also eligible for immunotherapies, and hence the immunomodulatory activity of these targeted inhibitors makes first-line treatment decisions complex. The aim of this thesis was to examine the immunomodulatory effects of BRAF, MEK and CDK4/6 inhibition, with an ultimate goal of providing critical information to aid in the clinical management of BRAFV600 melanoma patients. Examining mechanisms of the immunomodulatory effects of targeted therapies requires preclinical mouse models of melanoma that are both immunogenic, and harbor the oncogenic drivers targeted by the therapies being evaluated. To address this, we developed a novel immunogenic BrafV600ECdkn2a-/-Pten-/- melanoma mouse model, called YOVAL1.1. YOVAL1.1 tumors are transplantable in immunocompetent mice and amenable to standard-of-care melanoma therapies, including BRAFi+MEKi and immune checkpoint blockade. This, coupled with the Cdkn2a status, which infers some sensitivity to CDK4/6 inhibitors, makes this an ideal preclinical model to evaluate the immunomodulatory effects of the triple therapy. Using this model, we demonstrated that triple therapy promotes durable tumor control through tumor-intrinsic mechanisms, while promoting immunogenic cell death and T cell infiltration. However, despite this, tumors treated with triple therapy were unresponsive to immune checkpoint blockade. Flow cytometric and single cell RNA-seq analyses of tumor infiltrating immune populations revealed that triple therapy markedly depleted pro-inflammatory macrophages and cross priming CD103+ dendritic cells, the absence of which correlated with poor overall survival and clinical responses to immune checkpoint blockade in melanoma patients. Indeed, immune populations isolated from tumors of mice treated with triple therapy failed to stimulate T cell responses ex vivo. Hence, while combined BRAF, MEK and CDK4/6 inhibition demonstrated favorable tumor-intrinsic activity, these data suggest that collateral effects on tumor-infiltrating myeloid populations may impact on anti-tumor immunity. Several recent studies have reported immune-potentiating effects of CDK4/6 inhibition, and subsequent synergy with immune checkpoint blockade. However, T cells are the primary target of these immunotherapies, and an understanding of the direct effects of CDK4/6 inhibition on this cellular subset was lacking. In this thesis, using integrated epigenomic, transcriptomic and single cell CITE-seq analyses, we identified a novel role for CDK4/6 in regulating T cell fate. Specifically, we demonstrated that CDK4/6 inhibition promoted the phenotypic and functional acquisition of T cell memory. Genome-wide CRISPR/Cas9 screening and phospho-proteomics revealed that memory formation in response to CDK4/6 inhibition was cell intrinsic and required RB. Pre-conditioning human CAR T cells with a CDK4/6 inhibitor enhanced their persistence and tumor control, and clinical treatment with a CDK4/6 inhibitor promoted expansion of memory T cells in a melanoma patient, priming a response to immune checkpoint blockade. Collectively these findings highlight the multi-faceted immunomodulatory activity of BRAF, MEK and CDK4/6 inhibition. The addition of a CDK4/6 inhibitor to dual BRAFi+MEKi led to the depletion of intratumoral myeloid subsets that may be critical for supporting a therapeutically beneficial T cell response. In contrast, as an individual therapy, CDK4/6 inhibition promoted effector and memory T cell activity, suggesting that, with optimal scheduling to prevent myeloid depletion, CDK4/6 inhibitors may be used to enhance and prolong BRAFi/MEKi-induced anti-tumor T cell immunity. Defining the mechanisms that underpin the clinical efficacy of these available therapies is a critical step forward in optimising novel combination and scheduling approaches to combat melanoma and improve patient outcomes.
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    Profiling the immune and genomic landscape of anal squamous cell carcinoma and establishing preclinical models to explore new therapeutic options
    Guerra, Glen Robert ( 2019)
    Anal SCC is a rare disease that has increased significantly in both incidence and mortality over the last fourty years. Definitive chemoradiotherapy is the primary modality of treatment, offering a 5-year overall survival rate of 65%. For patients with locally persistent or recurrent disease, salvage surgery is an option with a 5-year overall survival of 50%. However, for those patients with un-resectable locoregional or metastatic disease, there are limited treatment options, and patients face a dismal outcome. Progress in identifying new treatment options for patients with anal cancer has been hampered by a deficiency in understanding the underpinnings of the disease and a lack of appropriate preclinical models. This thesis has focussed on addressing both of these deficiencies in addition to assessing the success of salvage surgery at a quaternary centre in Australia. Firstly, an attempt has been made to further our understanding of the biology of Anal SCC. This was undertaken by exploring the immune and genomic landscape of ASCC, to identify potential prognostic and therapeutic biomarkers. This has provided insight into the prognostic power of assessing the CD8+ immune infiltrate in Anal SCC. It has also identified PI3K aberrations as a frequent genomic event that may serve as a future therapeutic target. Secondly, it has led to the establishment of both human and mouse preclinical models of this disease. This includes the world’s first panel of human anal SCC cells lines and a syngeneic mouse model. Both of these pre-clinical models have been validated and characterised, with features closely resembling the human disease. These models can now act as a platform to further explore and facilitate investigation into potential new therapeutic options in this disease.
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    Study of the immunomodulatory effects of radiation therapy in solid cancers
    Sia, Joseph Ikin ( 2020)
    Radiation therapy (RT) has evolved over more than a century into a well-established, highly sophisticated and major cancer treatment modality today. A paradigm shift that has occurred within the last two decades is the growing understanding that RT can induce host immune responses that contribute to tumour control, beyond direct radiation-induced cytotoxicity. Contemporaneously, the advent of modern cancer immunotherapy such as immune checkpoint inhibitors has revolutionised the field of oncology and highlighted the potential of harnessing the immune system to suppress and eradicate tumours. Inevitably, resistance to cancer immunotherapy has also brought into focus immunological barriers that preclude cancer immunity. In this context, an increasing body of pre-clinical and clinical studies substantiate the use of RT as a unique candidate to complement cancer immunotherapy in non-overlapping mechanisms to overcome such barriers. However, instruction on the optimal integration of RT and cancer immunotherapy is scarce. For the radiation oncologist, an outstanding gap in knowledge is how radiation dose-fractionation influences the immunomodulatory effects of RT and its synergy with cancer immunotherapy. In this PhD project, mouse models of solid cancer were used to systematically interrogate this question by employing a series of rationally selected radiation dose-fractionation regimens to dissect the immunological impact of dose per fraction (DPF) from that of total dose, as represented by biological effective dose (BED). In orthotopic AT3-OVA mammary carcinomas, radiation-induced CD8+ T cell responses were found to be regulated by radiation DPF, rather than BED. By contrast, radiation-induced natural killer (NK) cell responses in the same tumours were independent of radiation DPF but required a sufficient BED. Mechanistic investigations examining the cellular and transcriptional changes in AT3-OVA tumours evoked by radiation demonstrated that the differential regulation of anti-tumour immune responses by radiation DPF and BED was not primarily dictated by differences in tumour cell-intrinsic immunogenicity, but rather by the effector and suppressor dynamics in the tumour immune microenvironment, of which regulatory T cells played a central role. Furthermore, cross-examination of subcutaneously implanted MC38 colon carcinomas and publicly available transcriptomic data of human cancers pre- and post-RT suggested that radiation-induced immune responses are also significantly shaped by the tumour type. Lastly, the impact of radiation dose-fractionation on the anti-tumour activity of immune checkpoint inhibitors targeting the adaptive and innate immune arms was examined in AT3-OVA tumours, confirming the corollary that RT and immune checkpoint inhibitors do not universally synergise, but require selection of radiation regimens and checkpoint targets that are predicated on biological rationale. Overall, this PhD project represents a comprehensive side-by-side pre-clinical study of the effects of radiation dose-fractionation on host anti-tumour immune responses. Results presented herein contribute towards a clearer understanding of this complex and clinically urgent question. More broadly, insights from this project will help guide the refashioning of RT into an exciting key adjunct in the immuno-oncology era.
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    Dual-specific Chimeric Antigen Receptor T Cells and an Indirect Vaccine against Pancreatic Cancer
    Ali, Aisheh Ibrahim ( 2020)
    Pancreatic cancer is one of the most aggressive malignancies with an overall 5-year survival rate of <7%. Pancreatic cancer is highly resistant to radiotherapy and chemotherapy, and surgery is not feasible in most patients. In this thesis, I developed a new form of treatment for pancreatic cancer, based on immunotherapy. Adoptive cell transfer (ACT) is a promising form of cancer immunotherapy, which involves the isolation and reinfusion of tumour specific T lymphocytes into patients. While ACT can eliminate substantial burdens of some leukaemia, the ultimate challenge remains the eradication of large solid tumours and metastases for most cancers, including pancreatic cancer. In this thesis, an enhanced ACT treatment strategy for pancreatic cancer was developed, which was termed ‘ACTIV: Adoptive Cell Transfer Incorporating Vaccination’. This treatment included dual-specific T cells that expressed a chimeric antigen receptor (CAR) specific for the tumour antigen Her2, and a TCR specific for the melanocyte protein (pMEL, gp100). These dual specific T cells were termed ‘CARaMEL T cells’. CARaMEL T cells were administered together with an injection of a recombinant vaccinia virus vaccine expressing gp100 (VV-gp100). We hypothesized that adoptively transferred CARaMEL T cells would proliferate mediated by their gp100 TCR, in response to the VV-gp100 vaccine, and kill Her2+ tumours through their anti-Her2 CAR. Functional assays performed in vitro indicated that murine CARaMEL T cells mediated antigen-specific cytokine secretion and killing abilities against pancreatic cancer cells, and demonstrated potent proliferative ability in response to gp100 antigen, confirming our hypothesis. In addition, I found that ACTIV therapy inhibited tumour growth and prolonged the survival of mice bearing Her2+ subcutaneous murine pancreatic tumour. However, tumours usually relapsed after ACTIV therapy administration. Therefore, I directed my study to augment the anti-tumour activity of ACTIV therapy by the administration of either a histone deacetylase inhibitor (Panobinostat) or an immune agonist monoclonal antibody specific for CD40. Panobinostat significantly suppressed the growth of pancreatic cancer cells in vitro through apoptosis and cell cycle arrest. Also, Panobinostat significantly increased the growth suppression of pancreatic cancer cells mediated by CARaMEL T cells. In addition, I found that the combination of ACTIV therapy and Panobinostat significantly reduced the tumour growth and prolonged the survival of mice bearing Her2+ subcutaneous murine pancreatic tumours. In addition, administration of an agonist CD40 monoclonal antibody with ACTIV therapy significantly reduced the tumour growth and prolonged survival of mice bearing subcutaneous Her2+ pancreatic tumours through a T-cell-dependent immune mechanism. Finally, I explored the clinical translational potential for ACTIV therapy through the generation of human CARaMEL T cells expressing both a Her2-specific CAR and a gp100-TCR. In vitro functional assays indicated that human CARaMEL T cells mediated powerful and antigen-specific killing and cytokine secretion against Her2, together with a strong proliferative ability in response to gp100 antigen. In addition, I found that the administration of both human CARaMEL T cells and an adenovirus vaccine expressing gp100 led to potent anti-tumour activity against subcutaneous human Her2+ pancreatic tumours in immunodeficient mice.