Hardwick, Nicola Rina; (2000) Mechanisms of anti-tumour immune stimulation following GDEPT. Masters thesis (M.Phil), UCL (University College London).

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Abstract

Studies undertaken in this and other laboratories have shown the HSV-tk/GCV system to be effective in the local control of experimental tumours, and a potent 'bystander effect' both in vitro and in vivo. In addition, the development of systemic anti-tumour immunity in treated animals has been widely reported. Previous work in this laboratory has shown that the systemic protection generated in mice after eradication of CMT93 tumours with HSV-tk/GCV was consistently lower than in mice cured of B16 tumours. Alternative prodrug systems were therefore investigated in the CMT93 model with the aim of improving the levels of post treatment anti tumour immunity. CMT93 derived cell lines expressing HSV-tk, CDA and ntr were first compared in vitro. A potent bystander effect was seen in all three cell lines, with a strong density dependence being seen in HSV-tk and ntr expressing cells which was not apparent in CDA expressing cells. Local control of tumours derived from CMT93-ntr, CMT93-tk and CMT93-CDA cells was possible when the relevant prodrugs were administered for 7 days. Expression of HSV-tk in CMT93 cells did not alter their tumourigenicity. Conversely, expression of CDA often caused a decrease in tumourigenicity which resulted in complete tumour regression and the development of protective immunity. This protection was greatly reduced when CDA expressing tumours were eliminated with prodrug rather than being left to regress spontaneously. This implies that leaving the immunogenic CDA protein in place longer provided a more effective immunising signal. Expression of ntr in CMT93 caused a dramatic reduction in tumourigenicity, but did not cause a corresponding increase in immunogenicity. An adenoviral vector expressing both HSV-tk and CDA was constructed, with the aim of using both prodrug systems together. In vitro experiments indicated that there was a synergistic interaction between the two prodrug systems. The development of post GDEPT anti-tumour immunity was further investigated with regard to heat shock protein induction. After in vitro prodrug treatment, the levels of surface Hsp70.1 were found to increase on B16-tk cells, but on none of the CMT93 lines. Over-expression of Hsp70.1 increased the immunogencity of both CMT93 and B16 cells. Further investigation suggested that this effect was T cell mediated, and in vitro experiments with bone marrow derived cells support the hypothesis that the increase in heat shock protein may play a role in chaperoning tumour antigens in to APCs and hence promote cross priming.

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