Harries, Mark; (2000) Enhancing anti-melanoma immune responses. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Gene therapy strategies, designed to enhance an anti-tumour immune response in patients with melanoma, can be broadly classified into approaches that aim to modify tumour cells and those that aim to transduce cells of the immune system. This thesis describes work carried out on aspects of both approaches. Melanoma cells transduced to secrete interleukin-12 (IL-12), either alone, or in combination with other proteins, can potentially enhance anti-tumour immune responses. Retroviral vectors were constructed to express this hetero-dimeric cytokine in which an internal ribosome entry site (IRES) initiates translation of the p40 subunit. Vectors containing an IRES from either polio virus (PV), encephalomyocarditis virus (EMCV), foot and mouth disease virus (FMDV) or murine leukaemia virus (MLV) were compared with a vector expressing human IL-12 as a single protein (Flexi-12; in which the two IL-12 subunits are linked by a peptide). All vectors produced high titre virus and directed synthesis of IL-12 in target cells. The bicistronic vectors containing the IRES from EMCV and PV were the most effective, producing similar levels of IL-12 to that obtained with Flexi-12. Target cell specificity of IRES function was also observed. In addition, a new IRES was identified in human herpes virus-8 (HHV-8). This IRES facilitates translation of the viral FLICE inhibitory protein (v-FLIP) from a bicistronic message that also encodes a viral D-type cyclin (v-cyclin). The IRES was found to extend upstream from the initiation codon of v-FLIP into the coding region of v-cyclin and is the first description of an IRES in a DNA virus. In a bicistronic IL-12 vector, translation of the p40 subunit from the v-FLIP IRES was more efficient than from the EMCV IRES. Engineering dendritic cells to present melanoma antigens can also potentially enhance anti-melanoma immune responses. Retroviral vectors were constructed to express melanoma antigens (MAGE-3, Melan-A and NY-ESO-1) and modified constructs targeted for ubiquitination and proteosomal degradation. HLA-A2+ EBV transformed B cell lines expressing the modified proteins were used as targets for lysis by melanoma antigen specific, cytotoxic T lymphocyte (CTL) clones. HLA-A2 restricted CTL clones were able to lyse cells expressing unmodified Melan-A and NY-ESO-1 but not MAGE-3. However, there was no enhancement of CTL lysis seen with cells expressing the modified constructs.

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