Ovarian cancer, with the highest mortality rate amongst gynaecological malignancies in Australia, is the eighth most common cancer and the fifth cause of cancer-related deaths in women. Currently, five-year survival for women diagnosed with ovarian cancer is only 40 % and despite many patients experiencing remission, approximately 80 % of them will relapse due to residual micrometastasis. The limited impact of standard therapies on the prognosis for recurrent chemotherapy-resistant disease and the need to identify less toxic alternatives has motivated the development of strategies to combat the aggressive and life-threatening burden of ovarian cancer. A novel therapy against cancer utilises dendritic cells (DC), potent antigen presenting cells, to deliver tumour antigens to the immune system for the stimulation of cytotoxic T-lymphocyte (CTL) responses. DC immunotherapy has been used for the treatment of patients with ovarian cancer; however, clinical responses after the injection of antigen-loaded DC have been disappointing. Therefore, the identification of additional tumour associated antigens (TAA) is required. A TAA highly expressed in ovarian cancer cells, CA125, is a candidate target for DC-based immunotherapy. Initially, CTL responses to CA125 were studied in the context of HLA-A*0201. CD8+ T-cell responses specific for CA125 peptides (with high affinity for the MHC class I) were generated from cultures initiated with peptide-loaded monocyte-derived DC (Mo-DC). To expand the evaluation of T-cell recognition of CA125 to non-HLA-A*0201 individuals, messenger RNA (mRNA) was investigated as an antigen-loading vehicle. RNA encodes for the repertoire of epitopes presented by the TAA, potentially inducing immune responses in the context of multiple MHC class I and II molecules to known/unknown antigens. One focus of this study was to investigate a novel mRNA transfection system utilising mannan for the delivery of mRNA into DC. Initially the immunomodulating effect of mannan was examined in terms of DC activation. Mannan induced the phenotypic and functional maturation of immature Mo-DC in vitro. Next, the ability of oxidised mannan (OxM) linked to mRNA was investigated for its capacity to deliver enhanced green fluorescent protein (EGFP) mRNA into DC. We observed high transfection efficiencies in the murine and in human DC systems using low mRNA concentrations, in the absence of significant cell viability impairment. Interestingly, upon mRNA delivery via the OxM-PEI complex, DC maturation was induced to considerably higher levels as compared with that achieved with electroporation and non-transfected controls, this was measured by phenotype (CD83) and IL-12 secretion. Within this study, OxM-PEI did not deliver TAA encoding mRNA into DC for the stimulation of CTL. In summary, mannan is a novel strategy to deliver mRNA and a strong maturation signal simultaneously to human Mo-DC. The functional capacity of this system requires further investigation before it can be considered for clinical use. Electroporation has evolved as a superior method for mRNA delivery into DC as reported in the literature. Therefore, a comprehensive study was performed encompassing the critical issues associated with transfection efficiency, in order to standardise an electroporation protocol for use in DC immunotherapy schedules. EGFP was used as a model antigen to optimise mRNA uptake by Mo-DC by monitoring the expression of the reporter gene by FACS analysis. Influenza matrix protein 1 mRNA was, then, utilised as a model antigen for MHC class I restricted antigen presentation, for confirmation of the optimised loading parameters. The efficiency of this delivery system was assessed using CA125 mRNA in stimulating antigen-specific T-cell responses in PBMC of healthy individuals. CD4+ and CD8+ antigen-specific T-cell responses were generated recognising CA125 mRNA loaded Mo-DC and also ovarian cancer cell lines endogenously expressing CA125. This study has identified CA125 specific T-cell responses in healthy donors, allowing further investigation into the potential for its use as a candidate TAA in ovarian cancer immunotherapy. Furthermore, the use of Mo-DC transfected with mRNA encoding TAA is a promising strategy for the delivery of TAA in the generation of antigen-specific T-cell responses. In summary, the results gained from this PhD thesis should be taken into consideration when designing future DC immunotherapy strategies to combat one of the leading causes of cancer mortality in women, ovarian cancer.
| Identifer | oai:union.ndltd.org:ADTP/265487 |
| Date | January 2007 |
| Creators | Maxwell, Tammy Joy |
| Publisher | Queensland University of Technology |
| Source Sets | Australiasian Digital Theses Program |
| Detected Language | English |
| Rights | Copyright Tammy Joy Maxwell |
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