Fumagillin is the natural product isolated from fungus Aspergillus fumigatus, and is recognized as a potent anti-angiogenic compound. Substantial investigation has been focused on the anti-tumor activities of fumagillin and its analogues, some of which have been adopted in pre-clinical and clinical studies. However, investigation on the immunomodulating activities of this class of compounds is limited and results have been controversial. As there is intense interest in elucidating the interrelation between immune modulation and tumor development, novel immunopharmacological properties of chemotherapeutic agents have recently been explored for their therapeutic potentials in clinical applications. As a combination to both these research topics, fumagillin and its synthetic analogues were firstly investigated on different types of immune cells, such as T lymphocytes, dendritic cells and macrophages. F23, a fumagillin analogue with potent immunological activities, was further examined in three different murine cancer models, EL4 lymphoma, CT26 colon carcinoma and 4T1 mammary carcinoma, and their anti-tumor activities and intrinsic immunomodulatory effects were explored. Fumagillin and its analogues exert diversified functions in different types of immune cells. For example, they showed inhibitory effects on cell proliferation and cytokine production of T lymphocytes upon polyclonal stimulation, stimulatory effects on dendritic cells by inducing a highly-matured population, which contributed to induction of syngeneic and allogeneic lymphocyte proliferation and a preference to Th1 polarization, and multiple effects on macrophages based on phenotypic and cytokine analyses. Studies in murine cancer models showed that the fumagillin analogue F23 caused substantial inhibition of tumor development in three cancer models to different extents, with pronounced inhibitory effects on the expansion and functions of myeloid-derived suppressor cells (MDSCs), the signature cell population responsible for tumor progression and refractoriness to chemotherapeutic and immunotherapeutic agents, thereby suggesting the novel immunopharmacological properties of fumagillin and its analogues contributed to tumor suppression. / published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy

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Oncolytic viruses are characterised by their ability to selectively infect and kill tumour cells. Recently it has emerged that they can exert an additional anticancer mechanism stimulating adaptive immune-mediated cancer cell killing. Enadenotucirev (EnAd, formerly known as ColoAd1), is a chimeric Ad11p/Ad3 virus group B oncolytic adenovirus that binds CD46 and is under development for the systemic treatment of metastatic carcinomas. The central aim of this thesis was to to assess whether EnAd provides an adjuvant effect on tumour-associated antigen presenting cells (APCs) that could drive T_H1 polarisation for an effective anti-tumour immune response. This thesis describes the potent oncolytic properties, fast replication and high numbers of virus progeny production by EnAd in cancer cells. Recombinant EnAd variants were engineered to investigate the roles of the mutant regions in the genome of EnAd, and how these influence the modified phenotype. A chemical drug panel was used to identify pathways and cellular factors involved in cellular production of EnAd, finding that several mTOR inhibitors and microtubule inhibitors could improve virus replication. An in vitro system using partially matured human monocyte-derived dendritic cells (DCs), which displayed a similar phenotype to tumour-infiltrating DCs, was used to explore the effect of EnAd on APC responses. EnAd induced a strong adjuvant effect on these cells by up-regulating surface markers and secretion of pro-inflammatory factors. Further mechanistic experiments, alongside a CAR-binding group C adenovirus 5, indicated these adjuvant effects were virus particle-mediated and dependent on CD46 binding. To understand the functional implications downstream of these interactions, T cell activation and phenotype was assessed using a mixed lymphocyte reaction approach. The data indicated EnAd was a good candidate compared to other adenoviruses, that may steer the response of activated T-cells towards a T_H1 phenotype, for an effective immune response. In conclusion, the potent oncolytic properties of EnAd virus may provide an adjuvant effect on tumour-associated APCs, helping to harness an adaptive immune response.

The most exciting area in current cancer research is immuno-oncology, which aims to develop immunotherapy that activates the human immune system to attack cancers. However, we still lack broadly effective drugs and drug targets for this promising new cancer treatment modality. In an attempt to seek new immuno-oncology drugs that particularly target the antitumor innate immunity, our lab had previously screened traditional Chinese herbal medicine and found that water extract from a medicinal plant, Alocasia Cucullata (AC), has strong anticancer activity in mouse solid tumor models and acts partly by promoting antitumor, proinflammatory macrophages. However, the active components responsible for this exciting immuno-oncology activity and the corresponding immune targets are unknown. Therefore, the aim of my PhD study is to develop chemical biology strategies to isolate and purify the active components of AC from the crude water extract and identify the corresponding cellular targets and mechanisms. Results from my study identified two separable activities and active components, one smaller than 3K and the other larger than 100K, which work synergistically to simulate antitumor macrophages. Further analysis revealed the >100K active component is a large polysaccharide that binds to multiple Toll-like Receptors (TLRs) critical for activating proinflammatory M1-type macrophages. Identity of the Nonetheless, I was able to clean up this fraction by 50 fold and perform RNAseq to examine the innate immune targets of this intriguing drug lead and found it acts to differentiate monocytes to macrophages. Overall my PhD thesis has explored new chemical biology strategies to purify and characterize active components from traditional Chinese medicine towards new drug development and developed a variety of cell-based immune activity assays for identifying and characterizing novel innate immune drug targets and mechanisms

Background: High-grade serous carcinoma (HGSC) can exhibit high intratumoral heterogeneity (ITH). Despite a strong association between tumor-infiltrating lymphocytes (TIL) and survival in HGSC, ITH may have profound impacts on the anti-tumor T cell response. Yet, it is unknown how anti-tumor T cell responses contend with ITH over time in HGSC. Previous studies in melanoma and HGSC both showed tumor-reactive T cell clones emerge over time with their cognate tumor-antigens. Therefore, I hypothesized patients would share a common mechanism of T cell evolution to respond to ITH in HGSC. If so, I expect to see similar patterns of tumor recognition between primary and recurrent disease. Methods: Tumor-associated lymphocytes (TAL) were expanded from primary and recurrent ascites samples using high-dose IL-2 and a rapid-expansion protocol (REP). Following expansion, TAL were assessed for recognition of autologous tumor by IFN-γ ELISPOT and flow cytometry for CD137. CD137+ tumor-reactive TAL were FACS-purified and the tumor-reactive T cell repertoire was profiled by deep sequencing of TCRβ chains (TCRseq). Tumor-reactive TCR clonotypes were compared between primary and recurrent disease to elucidate differences in tumor-reactive populations over time in HGSC. Results: Patient TAL recognized tumor in two out of three cases. In patient IROC 060, the tumor became more immunogenic between primary and recurrent disease, which may reflect expression of new antigens and/or loss of an immunosuppressive phenotype. In patient IROC 106, the tumor remained immunogenic between primary and recurrent disease, which may reflect maintenance of stable antigen expression and an immune-sensitive phenotype. Patient IROC 034 did not exhibit any tumor-reactivity, suggesting tumor-reactivity is not ubiquitous in HGSC. FACS-purification of CD137+ T cells followed by TCRseq was successfully performed on T cell populations of both high- and low-abundance, suggesting TCRseq can be performed on populations containing very few T cells. TCRseq results that profiled the clonal repertoire of tumor-reactive TAL from primary and recurrent disease in two patients, IROC 060 and IROC 106, showed both patients had evidence of T cell loss and T cell emergence between primary and recurrent disease. Further, IROC 106 had evidence of T cell clones that were maintained between primary and recurrent disease. Conclusions: Anti-tumor T cell responses from ascites are both diverse between patients and dynamic within a patient, suggesting various mechanisms of T cell evolution to contend with ITH in HGSC. I developed a pipeline for the identification of tumor-reactive TCR sequences without the need for a priori knowledge of specific antigens. Additionally, this pipeline is feasible for very low-abundance samples, such as tumor-reactive T cells. Significance: This study provides early insights into how TAL contend with ITH in HGSC. Ultimately, these results will inform the design of adoptive T cell therapy for recurrent HGSC. / Graduate

Recent developments in the expansion and manipulation of primary NK cells has allowed this source of effective anti-tumour cells to be exploited for cell-based cancer immunotherapies. While ex vivo expanded primary NK cells are highly effective in the treatment of haematological malignancies, their efficacy against the solid tumour has been limited due to the presence of immune-regulatory factors in the tumour microenvironment. These factors can abrogate NK cell function by down regulating the expression of NK activating receptors, thus preventing these highly cytotoxic effector cells from activating in response to tumour challenge. Our work explores whether the expression of a tumour specific chimeric antigen receptor (CAR) on ex vivo expanded primary NK cells would allow the lost activatory signalling to be recouped, and regain their efficacy against the solid tumour. Unfortunately, the use of primary NK cells as effectors in CAR based cell immunotherapies has been hampered by the technical limitations of producing large numbers of CAR positive primary NK cells. This has led many researchers to utilise the NK-92 cell line instead of primary cells. We demonstrate that ex vivo expanded primary CAR NK cells can be produced efficiently and demonstrate higher anti-tumour functionality than CAR NK-92. Finally, due to the intricacies of NK cell biology, they are able to effectively discriminate between healthy and malignant targets thus preventing their cytotoxic function from being directed towards the incorrect target. This could be a key advantage in the use of primary NK cells over T cells as effectors of CAR as the off-tumour/on-target adverse effects seen with CAR T cells has severely hampered this clinical strategy. We have shown that CAR T cells but not CAR NK cells are reactive towards phenotypically non-malignant, clinically relevant, healthy cells expressing the CAR target. / Thesis / Master of Science (MSc)

The CD40 ligand (CD40L) molecule is central to innate and adaptive immunity. CD40L expression is very tightly regulated whereas its CD40 receptor is constitutively expressed by many different cell types. CD40L is expressed transiently on helper T cells (Th) only after activation by specific immune recognition molecules carried by professional antigen presenting cells, in particular, dendritic cells (DC). CD40L subsequently binds to CD40 on DC to enable full Th activation. CD40 ligated DC produce interleukin-12 (IL-12) and contribute both to the development of IFNγ-secreting natural killer cells, a vital component of innate immunity, and of IFNγ-secreting type 1 Th (Th1) cells. CD40 ligated DC also contribute to the development of IL-4- and IL-10-secreting Th2 cells. CD40L on Th cells also binds CD40 on macrophages to enhance their cytotoxic functions. CD40L-expressing Th cells provide the ‘help’ pivotally required to activate other components of adaptive immunity responsible both for clearing invading pathogens and generating the memory cells required to prevent re-infection. Th-supplied CD40L binds (i) B cell CD40 to switch production of antibodies to more potent effector molecules that have higher avidity for antigen, and (ii) DC CD40 to prime then expand antigen-specific cytotoxic T lymphocytes (CTL). Activated NK cells and CTL are required both to eradicate malignant cells and cells infected with viruses or other intracellular pathogens. Genetic CD40L deficiency causes the very rare HyperIgM Syndrome Type 1 (HIGM1), which is realistically modelled by genetically engineered CD40L-deficient mice. Neither CD40L-deficient patients nor mice make effective antibodies or mount cellular immune responses that would defend them against intracellular pathogens such as parasites. Consequently, the only potentially curative therapy is allogeneic stem cell transplantation or CD40L gene replacement. Here, we used a retroviral vector, which constitutively expressed CD40L, to genetically modify CD40L-deficient bone marrow cells, which were used to reconstitute partially the immunity of CD40L-deficient mice. The crucial importance of tight regulation of CD40L expression was revealed when these mice later developed lethal thymic T cell malignancy. Growing tumours escape immune vigilance by genetic alterations that reduce their sensitivity to IFNγ. Using murine tumour models, we incorporated transgenic CD40L expression in therapeutic tumour vaccines to show that CD40L gene transfer augmented the immunogenicity of the host’s tumour thus reducing its tumorigenicity. We translated this finding clinically to safety and immunogenicity testing of a transgenic CD40L- and IL-2- expressing leukaemia vaccine. Finally, the common viral respiratory pathogen, respiratory syncytial virus (RSV) mainly infects young infants and the elderly to cause potentially lethal pneumonia. Both groups have reduced cellular and humoral immunity, which predisposes them to re-infection with RSV. Using a murine model, we showed first that simultaneous adenoviral expression of CD40L augmented primary RSV-specific Th1 responses that were associated with accelerated pulmonary viral clearance. Second, we showed that expression of CD40L in RSV-F and RSV-G subunit DNA vaccines elevated antibody and cellular immune responses to RSV challenge four and eight months after the initial immunisation. These results demonstrate the potent ability of CD40L gene transfer to solve the absolute immune deficiency caused by genetic lesions of CD40L. However, physiological regulation of the transgene is required to prevent serious adverse consequences. In contrast, no adverse effects were observed after transgenic CD40L expression was used to overcome relative immune deficiencies imposed by malignancy and RSV infection. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1298200 / Thesis (Ph.D.) - University of Adelaide, School of Medicine, 2007