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Title page, table of contents and abstract only. The complete thesis in print form is available from the University of Adelaide Library. / The last decade has witnessed a huge expansion in the field of Tumour Immunology. A large number of tumour antigens recognized by T lymphocytes have been identified and new techniques have enabled the direct ex vivo analysis of epitope-specific cytotoxic T lymphocytes (CTL). These developments provide the tools with which spontaneous tumour antigen-specific CTL responses can be characterized in detail, have facilitated the development of antigen-specific tumour immunotherapeutic strategies, and have heralded a new era of immunomonitoring in clinical trials. In this work, ex vivo phenotypic and functional analyses of CTL specific for an HLA-A*0201-restricted epitope encoded by the mel an-A tumour differentiation antigen (melan-A₂₆-₃₅) were performed on samples from melanoma patients and normal healthy donors. Ex vivo tetramer analysis revealed circulating melan-A₂₆-₃₅-specific T lymphocytes in 50% of both patients and normal donors. Phenotypic analysis of tetramer+ cells was correlated with ex vivo assays of CTL function. In the normal donors and approximately 50% of patients, the melan-A₂₆-₃₅-specific cells were always phenotypically and functionally naIve. However, in the remaining patients, a proportion of melan-A₂₆-₃₅-specific cells were phenotypically antigen-experienced, and functional responses to peptide could readily be detected ex vivo. The observation in patients, that tumour antigen-specific CTL responses with effector function are "too little, too late", provided the basis for a clinical trial of recombinant plasmid DNA and Modified Vaccinia Ankara in patients with surgically-treated melanoma and a high risk of disease recurrence. Both vaccines were engineered to encode the same polyepitope string of seven HLA-A2-and HLA-Alrestricted tumour antigen epitopes, including the high affinity melan-A₂₆-₃₅ analogue. Immunomonitoring, which included detailed kinetic analyses of tetramer+ cells, demonstrated that MV A was capable of generating an expansion of melan-A₂₆-₃₅-specific CTL with effector function in approximately 50% of patients. However, no responses to the other tumour antigen epitopes were seen. A detailed analysis of CTL responses specific for recently-identified vaccinia-encoded epitopes (including a new epitope identified as part of this work) demonstrated that CTL responses specific for the viral vector were dominant over those for the recombinant epitopes. This finding has important implications for the future design of recombinant viral vaccmes. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1152149 / Thesis (Ph.D.) -- University of Adelaide, Dept. of Medicine, 2005

Title page, table of contents and abstract only. The complete thesis in print form is available from the University of Adelaide Library. / The last decade has witnessed a huge expansion in the field of Tumour Immunology. A large number of tumour antigens recognized by T lymphocytes have been identified and new techniques have enabled the direct ex vivo analysis of epitope-specific cytotoxic T lymphocytes (CTL). These developments provide the tools with which spontaneous tumour antigen-specific CTL responses can be characterized in detail, have facilitated the development of antigen-specific tumour immunotherapeutic strategies, and have heralded a new era of immunomonitoring in clinical trials. In this work, ex vivo phenotypic and functional analyses of CTL specific for an HLA-A*0201-restricted epitope encoded by the mel an-A tumour differentiation antigen (melan-A₂₆-₃₅) were performed on samples from melanoma patients and normal healthy donors. Ex vivo tetramer analysis revealed circulating melan-A₂₆-₃₅-specific T lymphocytes in 50% of both patients and normal donors. Phenotypic analysis of tetramer+ cells was correlated with ex vivo assays of CTL function. In the normal donors and approximately 50% of patients, the melan-A₂₆-₃₅-specific cells were always phenotypically and functionally naIve. However, in the remaining patients, a proportion of melan-A₂₆-₃₅-specific cells were phenotypically antigen-experienced, and functional responses to peptide could readily be detected ex vivo. The observation in patients, that tumour antigen-specific CTL responses with effector function are "too little, too late", provided the basis for a clinical trial of recombinant plasmid DNA and Modified Vaccinia Ankara in patients with surgically-treated melanoma and a high risk of disease recurrence. Both vaccines were engineered to encode the same polyepitope string of seven HLA-A2-and HLA-Alrestricted tumour antigen epitopes, including the high affinity melan-A₂₆-₃₅ analogue. Immunomonitoring, which included detailed kinetic analyses of tetramer+ cells, demonstrated that MV A was capable of generating an expansion of melan-A₂₆-₃₅-specific CTL with effector function in approximately 50% of patients. However, no responses to the other tumour antigen epitopes were seen. A detailed analysis of CTL responses specific for recently-identified vaccinia-encoded epitopes (including a new epitope identified as part of this work) demonstrated that CTL responses specific for the viral vector were dominant over those for the recombinant epitopes. This finding has important implications for the future design of recombinant viral vaccmes. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1152149 / Thesis (Ph.D.) -- University of Adelaide, Dept. of Medicine, 2005

Title page, table of contents and abstract only. The complete thesis in print form is available from the University of Adelaide Library. / The last decade has witnessed a huge expansion in the field of Tumour Immunology. A large number of tumour antigens recognized by T lymphocytes have been identified and new techniques have enabled the direct ex vivo analysis of epitope-specific cytotoxic T lymphocytes (CTL). These developments provide the tools with which spontaneous tumour antigen-specific CTL responses can be characterized in detail, have facilitated the development of antigen-specific tumour immunotherapeutic strategies, and have heralded a new era of immunomonitoring in clinical trials. In this work, ex vivo phenotypic and functional analyses of CTL specific for an HLA-A*0201-restricted epitope encoded by the mel an-A tumour differentiation antigen (melan-A₂₆-₃₅) were performed on samples from melanoma patients and normal healthy donors. Ex vivo tetramer analysis revealed circulating melan-A₂₆-₃₅-specific T lymphocytes in 50% of both patients and normal donors. Phenotypic analysis of tetramer+ cells was correlated with ex vivo assays of CTL function. In the normal donors and approximately 50% of patients, the melan-A₂₆-₃₅-specific cells were always phenotypically and functionally naIve. However, in the remaining patients, a proportion of melan-A₂₆-₃₅-specific cells were phenotypically antigen-experienced, and functional responses to peptide could readily be detected ex vivo. The observation in patients, that tumour antigen-specific CTL responses with effector function are "too little, too late", provided the basis for a clinical trial of recombinant plasmid DNA and Modified Vaccinia Ankara in patients with surgically-treated melanoma and a high risk of disease recurrence. Both vaccines were engineered to encode the same polyepitope string of seven HLA-A2-and HLA-Alrestricted tumour antigen epitopes, including the high affinity melan-A₂₆-₃₅ analogue. Immunomonitoring, which included detailed kinetic analyses of tetramer+ cells, demonstrated that MV A was capable of generating an expansion of melan-A₂₆-₃₅-specific CTL with effector function in approximately 50% of patients. However, no responses to the other tumour antigen epitopes were seen. A detailed analysis of CTL responses specific for recently-identified vaccinia-encoded epitopes (including a new epitope identified as part of this work) demonstrated that CTL responses specific for the viral vector were dominant over those for the recombinant epitopes. This finding has important implications for the future design of recombinant viral vaccmes. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1152149 / Thesis (Ph.D.) -- University of Adelaide, Dept. of Medicine, 2005

The immune system, which defends the body against a wide array of threats, is gaining a growing role in the fight against cancer. For an immunotherapy to be successful, it needs to overcome intrinsically weak tumour-specific immune responses. There are two broad approaches to achieving this goal: targeting the various arms of the immune system or targeting the cancer and its microenvironment. The experiments discussed in this thesis adopt the second approach. Tumours were transduced with a combination of costimulatory molecules: CD48, CD54, CD70 & CD86, the chemokine CX3CL1 and the cytokines: IFNγ, GM-CSF and IL-12. Transduction of costimulatory molecules enhances priming in-vitro and cause tumour rejection and delayed tumour growth in-vivo. This effect is demonstrated with single costimulatory molecules but is more pronounced when multiple costimulatory molecules are transduced. Addition of the cytokines and chemokine enhanced tumour rejection, and also resulted in partial rejection of contralateral parental tumours. Attempts to enhance anti-tumour memory by fusing IL-2 and IL-15 to their respective receptors are also discussed. Work in a human/mouse chimeric PD-1 mouse model shows that transduction of multiple costimulatory molecules is able to overcome intrinsic anti-PD-1 resistance. Radiation is known to result in upregulation of several costimulatory molecules within tumours or their infiltrating dendritic cells. The experiments presented here suggest that radiation therapy may be useful in overcoming anti-PD-1 therapy resistance. In human trials, approximately three quarters of cancers fail to respond to anti-PD-1 therapies. Understanding and potentially overcoming anti-PD-1 therapy resistance is therefore of great interest.

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>Magister Scientiae - MSc / Retinoblastoma Binding Protein 6 (RBBP6) is a 200 KDa multi-domain protein that has been shown to play a role in mRNA processing, cell cycle arrest and apoptosis. RBBP6 interacts with tumour suppressor proteins such as p53 and pRb and has been shown cooperate with Murine Double Minute 2 (MDM2) protein in catalyzing ubiquitination and suppression of p53. Unpublished data from our laboratory has suggested that RBBP6 and MDM2 interact with each other through their RING finger domains. RBBP6 has also been shown to have its own E3 ubiquitin ligase activity, catalyzing ubiquitination of Y-Box Binding Protein 1 (YB-1) in vitro and in vivo. YB- 1 is a multifunctional oncogenic protein that is generally associated with poor prognosis in cancer, tumourigenesis, metastasis and chemotherapeutic resistance. Unpublished data from our laboratory shows that RBBP6 catalyzes poly-ubiquitination of YB-1, using Ubiquitin-conjugating enzyme H1 (UbcH1) as E2 ubiquitin conjugating enzyme. We have furthermore shown that the zinc knuckle of RBBP6 interacts specifically with the Ubiquitin-associated domain (UBA) domain of UbcH1.