Immunotherapy for the treatment of poor prognosis cancers

Cancer is amongst the leading causes of death worldwide and the number one cause in the developed world. Every year there are close to 10 million cancer related deaths and this corresponds to hundreds of millions of euro in health care costs and lost productivity, placing a substantial drain on the economy. The efficacy of traditional treatment modalities for cancer therapy, such as surgery, radiotherapy and chemotherapy has plateaued, and while they are undoubtedly effective at prolonging patient lifespan, there is a high rate of adverse side effects and fatal reoccurrence. Currently, there is a huge amount of interest in the areas of cancer immunosurveillance and cancer immuno-editing, which explain some of the complex interactions between the host immune system and cancer. If left unchecked, cancerous malignancies have the ability to generate an immunosuppressive microenvironment, effectively shielding themselves from elimination and promoting tumour growth and progression. To overcome this, the potential of the immune system must be harnessed and the work undertaken in this thesis sought to contribute to this goal. Focus was placed on using novel therapies, combining tumour ablation with immune-modulating antibodies to maximise tumour elimination in an immune dependent manner, to overcome immunosuppression and promote immune activation. Chapter 2 focuses on the use of ECT as a method of tumour ablation and its effects on the immune system. ECT proved to be effective at inhibiting the tumour growth both in vitro and in vivo, and conferred significant survival advantages in both small and large animal models. More importantly, ECT proved to cause tumour death in an immune dependent manner, displaying the hallmarks of Immunogenic Cell Death, increases in immune cell infiltration and generating tumour-specific immune responses. Chapter 3 focuses on combining ECT with immune checkpoint blockade inhibitors; anti- CTLA-4 and anti-PD-1. Both combinations proved to be effective at inhibiting both primary and distal tumour growth, indicating the generation of tumour specific immune responses and prolonged animal survival. In addition, the treatments caused increases in the levels of certain intra-tumoural immune cell subsets and modulated the cytokine profile of treated animals in a way that was favourable overall. Chapter 4 focuses on the combining ECT with an anti-iCOS agonist antibody, capable of causing immune co-stimulation. This novel combinational therapy proved to be the most effective by far, with a high cure rate achieved across a number of different in vivo tumour models. Total regression was seen in both primary and distal tumours, as well as spontaneous metastases, with the tumour specific immune response generated conferring total protection to animals on tumour rechallenge. Overall the data presented here adds further insight into the area of cancer immunotherapy with some of the novel combinational therapies demonstrating substantial clinic potential.

Gene delivery for the treatment of prostate cancer

Prostate cancer is one of the most common cancers diagnosed in men. Whilst treatments for early-stage disease are largely effective, current therapies for metastatic prostate cancer, particularly for bone metastasis, offer only a few months increased lifespan at best. Hence new treatments are urgently required. Small interfering RNA (siRNA) has been investigated for the treatment of prostate cancer where it can ‘silence’ specific cancer-related genes. However the clinical application of siRNA-based gene therapy is limited due to the absence of an optimised gene delivery vector. The optimisation of such gene delivery vectors is routinely undertaken in vitro using 2D cell culture on plastic dishes which does not accurately simulate the in vivo bone cancer metastasis microenvironment. The goal of this thesis was to assess the potential of two different targeted delivery vectors (gold or modified β-cyclodextrin derivatives) to facilitate siRNA receptor-mediated uptake into prostate cancer cells. Furthermore, this project aimed to develop a more physiologically relevant 3D in vitro cell culture model, to mimic prostate cancer bone metastasis, which is suitable for evaluating the delivery of nanoparticulate gene therapeutics. In the first instance, cationic derivatives of gold and β-cyclodextrin were synthesized to complex anionic siRNA. The delivery vectors were targeted to prostate cancer cells using the anisamide ligand which has high affinity for the sigma receptor that is overexpressed by prostate cancer cells. The gold nanoparticle demonstrated high levels of uptake into prostate cancer PC3 cells and efficient gene silencing when transfection was performed in serum-free media. However, due to the absence of a poly(ethylene glycol) (PEG) stabilising group, the formulation was unsuitable for use in serum-containing conditions. Conversely, the modified β-cyclodextrin formulation demonstrated enhanced stability in the presence of serum due to the inclusion of a PEG chain onto which the anisamide ligand was conjugated. However, the maximum level of gene silencing efficacy from three different prostate cancer cell lines (DU145, VCaP and PC3 cells) was 30 %, suggesting that further optimisation of the formulation would be required prior to application in vivo. In order to develop a more physiologically-relevant in vitro model of prostate cancer bone metastasis, prostate cancer cells (PC3 and LNCaP cells) were cultured in 3D on collagenbased scaffolds engineered to mimic the bone microenvironment. While the model was suitable for assessing nanoparticle-mediated gene knockdown, prostate cancer cells demonstrated a phenotype with lower invasive potential when grown on the scaffolds relative to standard 2D cell culture. Hence, prostate cancer cells (PC3 and LNCaP cells) were subsequently co-cultured with bone osteoblast cells (hFOB 1.19 cells) to enhance the physiological relevance of the model. Co-cultures secreted elevated levels of the MMP9 enzyme, a marker of prostate cancer metastasis, relative to prostate cancer cell monocultures (2D and 3D) indicating enhanced physiological relevance of the model. Furthermore, the coculture model proved suitable for investigating nanoparticle-mediated gene silencing. In conclusion, the work outlined in this thesis identified two different sigma receptor-targeted gene delivery vectors with potential for the treatment of prostate cancer. In addition, a more physiologically relevant model of prostate cancer bone metastasis was developed with the capacity to help optimise gene delivery vectors for the treatment of prostate cancer.

Treatment trials for neonatal seizures: the effect of design on sample size

Neonatal seizures are common in the neonatal intensive care unit. Clinicians treat these seizures with several anti-epileptic drugs (AEDs) to reduce seizures in a neonate. Current AEDs exhibit sub-optimal efficacy and several randomized control trials (RCT) of novel AEDs are planned. The aim of this study was to measure the influence of trial design on the required sample size of a RCT. We used seizure time courses from 41 term neonates with hypoxic ischaemic encephalopathy to build seizure treatment trial simulations. We used five outcome measures, three AED protocols, eight treatment delays from seizure onset (Td) and four levels of trial AED efficacy to simulate different RCTs. We performed power calculations for each RCT design and analysed the resultant sample size. We also assessed the rate of false positives, or placebo effect, in typical uncontrolled studies. We found that the false positive rate ranged from 5 to 85% of patients depending on RCT design. For controlled trials, the choice of outcome measure had the largest effect on sample size with median differences of 30.7 fold (IQR: 13.7–40.0) across a range of AED protocols, Td and trial AED efficacy (p<0.001). RCTs that compared the trial AED with positive controls required sample sizes with a median fold increase of 3.2 (IQR: 1.9–11.9; p<0.001). Delays in AED administration from seizure onset also increased the required sample size 2.1 fold (IQR: 1.7–2.9; p<0.001). Subgroup analysis showed that RCTs in neonates treated with hypothermia required a median fold increase in sample size of 2.6 (IQR: 2.4–3.0) compared to trials in normothermic neonates (p<0.001). These results show that RCT design has a profound influence on the required sample size. Trials that use a control group, appropriate outcome measure, and control for differences in Td between groups in analysis will be valid and minimise sample size.