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Tumour associated macrophages (TAMs) are implicated in cancer progression but can also exert anti-tumour activities. In this thesis, a strategy to redirect endogenous T cell cytotoxicity towards cancer supporting (M2-like) TAMs is explored. A panel of novel bi- and tri-valent T cell engagers (BiTEs/TriTEs) was generated, recognising CD3ε on T cells and M2-like macrophage markers CD206 or folate receptor β (FRβ). Primary human T cells activated by the BiTEs/TriTEs preferentially killed M2- over M1-polarised autologous macrophages, with EC50 values in the nanomolar range. This work represents the first to demonstrate the feasibility of redirecting T cells to kill macrophages. To avoid on-target off-tumour toxicities, we propose localising BiTE/TriTE expression to the tumour with an engineered oncolytic virus. We confirmed the feasibility of this approach by modifying an oncolytic adenovirus in early-phase clinical trials for solid cancers, enadenotucirev (EnAd), to express the BiTEs. Critically, T cell engager-armed EnAd retained its oncolytic and replicative capacities, whilst mediating BiTE expression from infected cancer cells. In immunosuppressive human malignant ascites samples, free and EnAd-encoded FRβ-targeting BiTEs (but not CD206-targeting T cell engagers) triggered endogenous T cell activation and IFN-γ production, leading to increased T cell numbers and a reduction in the number of CD11b+CD64+ ascites macrophages. Strikingly, surviving macrophages exhibited a general increase in M1-like marker expression, suggestive of a repolarisation of the microenvironment towards a more immune-responsive state. Taken together, this approach to deplete cancer-promoting TAMs in the context of the immune-stimulatory effects of BiTEs and oncolytic viruses offers a powerful new strategy for removing barriers to anti-tumour immunity in patients with cancer.

Many oncolytic adenoviruses are in clinical development for the treatment of solid tumours and Enadenotucirev (EnAd), a chimeric group B adenovirus, is one leading candidate. Little is known about its activity in hypoxic regions, a feature present in virtually all solid carcinomas and well known to confer resistance to traditional therapies. Hypoxia may account for the long term persistence of adenovirus in tumours and incomplete tumour coverage, as intratumoral and even intravenous virus delivery may expose viruses to hypoxia due to perfusion changes in the tumour vasculature. We set out to define how hypoxia influences EnAd's oncolytic activity through an in-depth analysis of the viral infection in cancer cells, its dissemination to bystander cells in hypoxic conditions in vitro, and characterisation of virus dissemination in animal tumour models. Work in this thesis showed that hypoxia in vitro actually boosts expression of virus early and late gene products leading to increased production of infectious virus (2 to 12-fold). It also increases expression of virus-encoded biotherapeutic agents. The mechanism appears to involve hypoxia specific over-expression of the immediate-early gene E1A, and we provide evidence that a hypoxia responsive element within the promoter of this key viral gene could be induced in a HIF 2α dependent fashion. Nevertheless, in conditions involving cell-to-cell spread in vitro, this ability of hypoxic cancer cells to produce greater number of virus particles is masked, due to poor paracellular spread reflecting the presence, or absence, of a factor smaller than 5 kilodaltons, yet to be characterised. We also assessed the impact of hypoxia in vivo by combining EnAd with atovaquone, a mitochondrial Complex-III inhibitor that reduces the cellular oxygen consumption rate, thereby alleviating hypoxia in tumours. Oral atovaquone treatment, which does not increase virus production in vitro, led to an unprecedented increase of tumoural viral spread while significantly reducing the hypoxic fraction, suggesting that hypoxia could be a key therapeutic barrier. In conclusion, hypoxia boosts the oncolytic activity of adenoviruses, but can inhibit intercellular spread. The overall impact of hypoxia upon the success of oncolytic adenovirus therapy will likely rely on the balance of improved expression of virus-encoded therapeutics, anti-tumour effects of pro inflammatory (antiviral) signalling, and limited viral spread.

Epithermal Growth Factor Receptor (EGFR) is overexpressed in up to 90% of head and neck squamous cell carcinoma (HNSCC). Cetuximab is the first and the only anti-EGFR monoclonal antibody which received approval from FDA for the treatment of HNSCC. However, most patients either do not respond to cetuximab or develop acquired resistance. The aim of my D.Phil. study was to investigate the role of ADAM10 and 17 in resistance mechanisms of cetuximab in HNSCC. Chronic exposure to cetuximab led to an activation of HER receptors and downstream signalling pathways in HNSCC cell lines. Higher levels of ADAM10 and 17 and their substrates, BTC and NRG-1 were found in cetuximab resistant cells compared to their parental cells, suggesting the involvement of ADAM-mediated ligands’ release in reactivation of HER receptors. Inhibition or knockdown of ADAM10 and 17 enhanced cetuximab response and reversed cetuximab resistance in HNSCC cells. In addition, results from this study showed that the combination of cetuximab with EGFR-TKIs had greater effect in parental cells and reversed cetuximab resistance in HNSCC cells. Upregulation of ADAM10 and 17 also was observed in HNSCC TMAs compared to normal head and neck TMAs. High nuclear ADAM10 and cytoplasmic ADAM17 expression levels were associated with shorter DFS. By evaluating tumour excision samples from HNSCC patients who underwent a cetuximab window study high ADAM10 and 17 expression levels were found to be associated with poor response to cetuximab. In conclusion cetuximab-induced ADAM-mediated ligands’ release is a potential mechanism of resistance to cetuximab in HNSCC. Thus, targeting ADAM10/17 or subsequent HER activations may represent an important strategy in overcoming resistance to cetuximab in HNSCC. Results from this study also suggest the implication of ADAM10 and 17 as potential prognostic and predictive biomarkers in HNSCC although further validation is required.

Despite expressing antigens that can induce immune surveillance and immune eradication, tumours demonstrate the capacity to evade anti-tumour immunity. Recently, this has been attributed to the ability of tumours to induce a local immunosuppressed micro-environment, which is a major obstacle to successful natural and vaccine induced anti-tumour immunity. Soluble factors such as transforming growth factor beta (TGFβ), and interleukin 10 (IL-10), released by cancer and stromal cells, are thought to play a significant role in this local immunosuppression. In order to assess the influence of antagonising these soluble factors locally on tumour biology and tumour immunity, a murine CT26 colorectal carcinoma model that can express cytokine antagonists under Doxycycline (Dox) control was engineered. Two stable CT26 cell lines expressing Dox-inducible soluble extracellular domain of TGFβ receptor II (STGFβRII) or soluble extracellular domain of IL-10 receptor (SIL-10R), were established. Expression of STGFβRII in vitro and in vivo was only evident after Dox treatment. When Dox was administered directly following subcutaneous (s.c.) inoculation of STGFβRII-expressing CT26 cells into Balb/c mice, tumour growth was significantly suppressed. Interestingly, inducing STGFβRII in well-established tumours showed less suppression of tumour growth. To assess the effect of expressing STGFβRII on tumour immunity the RNA expression of 22 common cytokines was measured in the tumours of mice receiving Dox and control mice. The levels of some of these cytokines were modulated by STGFβRII expression (e.g TGFβ,Tnfsf9, IL-2). We also tested for any additive effect between expressing STGFβRII, and the administration of anti-CTLA-4 antibody on tumour growth, and tumour immunity. The model described here could help address various limitations seen previously in studies of TGFβ blockade. It provides means of effective local antagonism, and it addresses immunological endpoints which have been limited in previous studies. Because of its tight regulation, the model also allows identification of the best timing of TGFβ blockade alone or in combination with other immunotherapeutics.

Over one in five cancer patients will develop brain metastases and prognosis remains poor. Effective chemotherapeutics for primary systemic tumours have limited access to brain metastases owing to the blood-brain barrier (BBB). The aim of this study was to develop a strategy for specifically permeabilising the BBB at sites of cerebral metastases. Tumour necrosis factor was injected intravenously into mouse models of haematogenously induced brain metastasis. BBB permeability was assessed through histology and in vivo MRI and SPECT. Tumour burden and neuroinflammation were assessed after injection of TNF with Caelyx or a novel therapeutic. Mechanism of permeabilisation was investigated through histology and receptor-specific agonist antibodies. Administration of TNF dose-dependently permeabilised the BBB to exogenous tracers selectively at sites of brain metastasis, with peak effect after six hours. Metastasis-specific uptake of radiolabelled trastuzumab was also demonstrated following systemic cytokine administration. Administration of liposomal doxorubicin formulations in conjunction with TNF reduced tumour burden and mean metastasis size. Localised expression of TNFR1 was evident on the vascular endothelium associated with brain metastases. Human brain metastases displayed a similar TNF receptor profile compared to the mouse model. These findings describe a new approach to selectively permeabilise the BBB at sites of brain metastases, thereby enabling detection of currently invisible micrometastases and facilitating tumour-specific access of chemotherapeutic agents. We hypothesize that this permeabilisation works primarily though TNFR1 activation and, owing to the similar TNFR1 expression profiles in mouse models and human condition, the strategy has the potential for clinical translation.

Systemic administration of adenovirus type 5 (Ad5) vectors for the treatment of cancer is limited by poor circulation kinetics and inefficient uptake from the bloodstream into tumours. This study reports a novel method for linkage of highly-PEGylated gold nanoparticles (AuPEG) to Ad5 by a single reduction cleavable bond. The resulting ‘dandelion’ structure provides very effective steric shielding with only minimal and reversible modification of the Ad5 capsid. This ablates in vitro cell infection, improves protection against the binding of antibodies, and enhances in vivo circulation kinetics. Focused ultrasound is a promising technology for the non-invasive, targeted treatment of cancer. In the context of drug delivery, cavitational energy generated upon exposure of ultrasound contrast agents to focused ultrasound can be used as a powerful stimulus to move therapeutics over distances of hundreds of microns away from blood vessels. In addition to providing a platform for effective stealthing, conjugation of AuPEG to Ad5 also increases the effective density of Ad5. This increase in density imparts a second major advantage on the strategy, observed for the first time in the present study: denser particles are transported significantly farther by cavitation-induced microstreaming than identically-sized particles of lower density. Specifically, in in vitro tests using a tumour-mimicking flow-channel phantom model and in in vivo experiments using tumour bearing mice, Ad5–AuPEG was delivered farther from vessels in response to ultrasound induced cavitation than either naked Ad5 or polymer-coated Ad5. The enhancements in stealthing and improvements in response to ultrasound provided by this strategy enabled up to 12% (S.D. 0.97) of the injected dose to be deposited in the tumour, compared to just 0.12% (S.D. 0.05) for Ad5 without ultrasound (p < 0.001). Consequently, in a survival study, mice treated with Ad5–AuPEG with focussed ultrasound had the slowest tumour growth and longest survival rate when compared to mice treated with Ad5 alone, Ad5–AuPEG alone, or Ad5 with focussed ultrasound. These results provide compelling evidence that the combination of focussed ultrasound with density-augmented stealthed Ad5 results in improved delivery to tumours and therapeutic efficacy. This combination of ultrasound with particle modification for optimal cavitation-mediated delivery has the potential to be applied to a broad range of anti-cancer nano-medicines and therapeutics to augment their bio-availability for improved cancer treatment.

Background: The cardiomyocyte cell membrane is composed of the surface membrane and a complex system of membrane invaginations, the t-tubules. In keeping with its role in synchronisation of electromechanical coupling, the t-tubular system is distinguished from the cell membrane by its ion channel composition. Variability of the architecture of the t-tubular system might modulate electrophysiological function at the cellular level. Light and electron microscopy-based methods were established to generate three-dimensional models of t-tubular structure. Effects of t-tubular variability on electrophysiological function were investigated using a pseudo-two dimensional model of the ventricular cardiomyocyte. Methods: Confocal microscopy and electron tomography were used to generate three-dimensional models. Based on the distribution of the t- tubular membrane, electrophysiological parameters were calculated using a mathematical model. Results and Discussion: Three-dimensional models of t-tubular structure could be generated using different staining protocols for confocal microscopy and different fixation protocols for electron tomography. Advantages and applications of the different methods were discussed. The t-tubular fraction varied considerably among different cardiomyocytes. In the model this gave rise into changes in ionic currents; however variation of t-tubular density lead to heterogeous changes in repolarisation only after simulated inhibition of repolarisation-related membrane currents. Conclusions: Electron tomography allows generation of structural models at resolution of a few nanometres, however optimisation of fixation protocols is a critical step. Larger volumes can be acquired in living cells with confocal microscopy; however, a large fraction of the t-tubular system is sub-resolution. The variability of the t-tubular system may contribute to electrophysiological heterogeneity in patho-physiologically challenged states, and it forms an interesting target for functional modelling based on three-dimensional models of t-tubular structure.