INTERFAZ CON SENSOR INDUCTIVO

Since human tumours are likely to exhibit great antigenic individuality, the idea of designing general antigen based vaccination strategies is likely to be impractical. Heat shock protein based vaccines may prove to be an effective alternative, since they have been clearly demonstrated to ellicit tumour specific immunity. In theory, hsp preparations from surgically resected tumours could be used to vaccinate patients, without the need for characterisation of the antigenic epitopes present in the tumour (Srivastava, 1994).

Presumably, such vaccines would ellict a more potent effect than whole cell vaccines or peptides alone due to the ‘adjuvant-like’ effect of hsps whereby peptides are directed into the MHC I pathway.

Hsps have been used to successfully vaccinate against rat hepatoma, murine sarcoma (meth A), murine thymoma, B 16 melanoma and mouse lung carcinoma amongst others (Li, 1997) suggesting that hsp vaccination might have widespread application. In addition, purification of hsps from tumour tissue is well-documented. An alternative would be to use standardised prepartions of hsps (since they are well-conserved between tumour types and different species) and to reconstitute these with tumour antigens eluted from the MHC molecules of patients tumours. Blachere e.a. have shown that hsp-peptide complexes reconstituted in vitro are taken up and presented by macrophages in the same way as hsp-peptide complexes generated in vivo. (Blachere, 1997)

In addition,there is evidence to suggest that gp96 associated peptides are not limited to the MHC class I ligandof the cell from which the gp96 were isolated . This was shown by Arnold et.al. who found that b-gal specific CTLS could be generated in mice with a different MHC to that of the cell from which the gp96 was isolated. (Arnold, 1995). This

is confirmed by the work of Nieland e.a. who showed the presence of VSV peptides in gp96 purified from VSV infected cells, as well as demonstrating the ability of gp96 to prime VSV-specific cytotoxic T cells. (Nieland, 1996). They found that the immunogenic peptide associated with gp96 in VSV infected cells regardless of the MHC haplotype of the cell (Nieland, 1996). A clinical application of this could be the immunisation of patients with gp96 preparations isolated fom tumour cells expressing common antigens, or cells infected with viruses known to induce specific CTL responses (Arnold, 1995). The possibility of inducing a pathological autoimmune response with hsp preparations is a danger that should be considered, but Srivastava e.a report no signs of toxicity in several hundred mice immunised with gp96 derived from a wide variety of cell lines. In addition, a recent phase I clinical trial in which patients were immunised with autologous cancer- derived gp96 did not show any signs of autoimmunity (Tamura, 1997)

Aims of the work presented in this thesis:

The HSV-tk/GCV system has been shown to be effective in local control of experimental tumours by this laboratory and many others (see introduction).

Interestingly, this control was reported by some to more effective in immunocompetant mice (Vile, 1994) (Gagandeep, 1996) (Ramesh, 1996). In addition, control of distant métastasés was reported, this being unlikely to be due to a B.E. which requires cell-cell contact. Therefore the involvement of an immune response was suggested (Vile, 1994). Most significantly it has also been shown that after killing tumours with the HSV- tk/GCV system, protection to rechallenge with parental cells can be generated (Vile,

1994) (Barba, 1994) (Caruso, 1993). Many studies indicated that expression of HSV-tk did not intrinsically increase the immunogenicity of the tumour cells and so the presence of the foreign protein could not explain the development of anti-tumour immunity (Vile,

1994).

Further investigation of HSV-tk/GCV killing in this laboratory suggested that it was the process of killing that was important in development of anti-tumour immunity, perhaps because this results in rapid release of tumour antigens which are previously unavailable to the immune system. Histological examination of dying B 16 tumours treated with HSV-tk/GCV revealed the presence of a mononuclear cell infiltrate. This suggested that destruction of the tumour infrastmcture increased the ability of

lymphocytes to penetrate these tumours, and thus prime an immune response. However, when this phenomenon was investigated in a different cell line CMT93, inferior protection was generated despite the chosen cell line being MHCI positive. Further investigation suggested that the type of cell death is also important.

Comparison of the B 16 and CMT93 models showed that better protection was generated by B 16-tk tumours which died primarily by necrosis, than CMT93-tk tumours which died primarily by apoptosis (Melcher, 1998). In addition, RT-PCR analysis of the dying B 16-tk tumours revealed a profile of cytokines at the tumour site that is suggestive of a Thl immune response (Vile, 1997), a finding also reported by others in a murine fibrosarcoma model (Ramesh, 1996). Therefore it could be that necrotic cell death, and the accompanying inflammation may provide the necessary danger signal to break tolerance to tumour antigens, and help recruit immune effector cells to the area . These infiltrating cells then release cytokines, creating an

immunostimulatory environment in an otherwise non-immunogenic tumour. If tumour antigens are taken up by professional antigen presenting cells such as dendritic cells, this could result in cross-priming of T cells and development of a

CTL response. Consistent with this theory is the finding that up regulation of B7 and ICAM, occurred on a macrophage cell line after exposure to HSV-tk/GCV treated tumour lysate/supematant (Ramesh, 1996).

The inferior protection consistently seen in the CMT93 model, could therefore be due to the fact that the cells die primarily by apoptosis which is beheved to be

immunologically silent. However this protection could be enhanced by co-expressing GM-CSF with HSV-tk in the CMT93 tumours. By providing cytokines at the site of the dying tumour, a more immunostimulatory environment is therefore created even within the context of apoptotic cell death (Castleden, 1997).

The HSV-tk gene has been found to be of variable immunogenicity, while work with the CDA gene has consistently demonstrated an anti-CDA immune response in mice. When immunocompetant mice were immunised with normal, syngeneic cells that expressed CDA, subsequent protection to unrelated CDA+ tumour cells was generated (Mullen , 1994(ii)). As with the HSV-tk system, effective local control of tumours was also possible (Huber et al, 1993), with a potent bystander effect being seen and protective immunity to rechallenge developing after in vivo killing (Mullen, 1994(ii)). Therefore, the CDA/5FC system might provide a way of improving the immunostimulation seen after GDEPT in the CMT93 model.

The aims of my project were therefore:

i) To compare the CDA/5FC system to the HSV-tk system in the CMT93 model with regard to local control of primary tumours and protection to rechallenge.

ii) To investigate the possibility of using both the HSV-tk/GCV and CDA/5FC systems together in order to exploit the different features of each.

M l Standard solutions and media

All solutions were prepared using Milli-Q water and were autoclaved before use. A list of stock solutions used in this work can be found in Appendix 1. All chemicals were supplied by the Sigma-Aldrich Company Ltd., Poole, Dorset, UK., unless otherwise stated.