Abstract
Melanoma is the most serious form of skin cancer, with New Zealand having the world’s highest incidence rates. It is characterised by an aggressive clinical behaviour, associated with metastasis to distant organs and poor responses to chemo- and radiotherapy. Immunotherapeutic strategies, such as cancer vaccines, designed to inhibit melanoma cell proliferation by inducing cell death offer a promising and less toxic treatment option as they have the potential to stimulate immunity to tumour-associated antigens while sparing healthy cells. However, cancer-induced immune-suppression inhibits the generation of anti-tumour immune responses and needs to be overcome in order for the vaccine to be successful. The aim of the work described in this thesis was to investigate the ability of experimental particulate delivery systems to deliver a tumour-associated antigen, a vaccine adjuvant and a novel immune-modulator to stimulate anti-tumour adaptive immune responses.
The immune-stimulatory and physico-chemical properties of two experimental (chitosan nanoparticles (CNP), cubosomes) and two commercially available (alum, Incomplete Freund’s adjuvant (IFA)) particulate formulations were assessed in vitro and in vivo since it has been suggested that particles enhance immune responses to tumour-associated antigens by acting as danger signals and thus stimulating antigen presenting cells. CNP, IFA and alum stimulated the up-regulation of cell surface markers on dendritic cells but only the positively charged CNP and alum triggered the activation of the NACHT leucine-rich repeat protein 3 (NLRP3) inflammasome, an intracellular multi-protein complex, crucial for the processing and secretion of the pro-inflammatory cytokine interleukin 1 beta (IL-1β).
A tumour-associated self-antigen (long TRP2-peptide), consisting of amino acid sequences known to stimulate CD4+ and CD8+ T-cells responses, was then synthesised and investigated for its ability to stimulate adaptive immune responses when given with the particulate formulations. Mice were immunised subcutaneously twice with the particles (CNP, alum, IFA) containing the long TRP2-peptide or the two corresponding short peptides and a vaccine adjuvant (S-[2, 3-bis (palmitoyloxy)propyl] cysteine (Pam2Cys), Interferon-α). Cytotoxic T-cell responses were greater in mice immunised with the long TRP2-peptide as compared to the two short peptides; however, the combination of the particulate formulations and adjuvants did not result in a significant improvement of adaptive immune responses. In contrast, the formulation of the long TRP2-peptide into cationic liposomes supplemented with α-galactosylceramide (α-GalCer), known to stimulate adaptive immune responses when administered intravenously, induced strong cytotoxic T-cell responses.
Consequently, the ability of this vaccine composition to inhibit tumour growth was investigated in mice with subcutaneous tumours, induced by the injection of B16F10luc2 melanoma cells six days prior to the vaccination. To further enhance the anti-tumour efficacy of the vaccine the effect of the novel dual cyclooxygenase-2/5-lipoxygenase (COX-2/5-LO) inhibitor, licofelone, alone and in combination with the vaccine was studied. COX-2 inhibitors have been demonstrated to enhance therapeutic cancer vaccines and the dual inhibitor licofelone was hypothesised to show superior ability to inhibit tumour growth when given in combination with a vaccine. Mice immunised with the vaccine alone demonstrated a prolonged survival and this was further enhanced when licofelone was added to the vaccine. Phenotypic analysis of immune cell populations after immunisation revealed that the levels of immune-suppressive myeloid-derived suppressor cells (MDSCs) were decreased, perhaps explaining the improved survival rates of mice immunised with the vaccine containing licofelone. Although the mechanism of action of licofelone needs further investigation, the combined treatment of licofelone, the long TRP2-peptide and α-GalCer may present a potential approach for the induction of anti-cancer immunity in melanoma patients.
In conclusion, this thesis supports the use of the long TRP2-peptide, the adjuvant α-GalCer and the COX-2/5-LO inhibitor licofelone prepared in cationic liposomes or saline as a promising vaccine candidate for the treatment of melanoma. The composition induced strong CD8+ T-cell responses in healthy mice and prevented tumour outgrowth in approximately half of tumour-bearing mice.