Identification of candidate tumour antigen peptides with immunogenic potential for use in cancer immunotherapy
It is now understood that CD4+ T cells play a central role in antitumour immunity; they are important in the priming of CTL and can augment cytotoxicity; they are also responsible for enhancing antitumour responses via indirect mechanisms such as recruitment of accessory cells to the tumour site and they are crucial in the formation of memory T cells. CD4+ T cells recognise antigen in the context of peptide presented on MHC class II molecules, therefore recent research has focused on the identification of these peptides in order to formulate more effective immunotherapeutic strategies. As such, the aim of this study was to identify novel MHC class II HLA-DR1 and HLA-DR4 restricted immunogenic peptides derived from the MART-1 and Tyrosinase tumour antigens. A computer algorithm (SYFPEITHI; available on the World Wide Web) was used to predict immunogenic peptides from the two tumour antigens; these peptides were then used to immunise HLA-DR1 and HLA-DR4 transgenic mice in order to assess their immunogenicity. At the same time efforts were made to optimise the screening process by fully characterising the BM-DC used in proliferation assays and employing antioxidants in conjunction with T cell culture. A second aspect of the project utilised p53 peptides in order to investigate the effects of protein specific T cell help on the generation of effector and memory CTL. Efforts were made to maximise the efficiency of the MHC class II peptide screening method by optimising expression of co-stimulatory molecules and cytokine production by BM-DC. Testing of BM-DC derived from FVB/N-DR1, C57bl/6-DR4 and C57bl/6 HHD II HLA-A2 transgenic mice revealed that differing maturation protocols were required to generate BM-DC with the optimal T cell stimulatory capacity, depending upon the strain of transgenic mice employed. The screening process was further optimised by the use of Vitamin E in conjunction with T cell culture; this antioxidant was found to increase peptide specific proliferative responses against the immunised peptide. Using the optimised screening protocols, immunisation of transgenic mice with predicted epitopes led to the discovery of the novel HLA-DR1 restricted MART-129-43 and the HLA-DR1/DR4 restricted Tyrosinase147-161 peptides. Further experiments also indicated that the Tyrosinase protein was processed by murine dendritic cells to produce the Tyrosinase147-161 peptide. This study demonstrated that HLA-DR restricted responses to novel peptide can be obtained in HLA-DR1 and HLA-DR4 transgenic mice.