Variables a ser incorporadas en el análisis

DC+MUC264-272 DC+MUC13-21 DC+MUC460-468 PBS 148

Immunisation with peptides and as well as with W-MUC1 induced significant protection of the mice against tumour growth compared to mice immunised with IFA alone (p=0.0008, p=0.005 and p=0.02 respectively). Peptides and MUC^®®*^®®, which failed to induce peptide specific CTL responses (Figure 6.1), were also not able to protect mice against tumour challenge. Mice immunised with these peptides did not survive significantly better than mice that had received IFA alone (p=0.536 and p=0.926 respectively) (Figure 6.4A) and tumour growth was recorded by measuring three dimensions. No changes in tumour status were recorded after the last day shown.

Figure 6.4: MUC1 peptide vaccination protects A2/K^ mice against tumour challenge.

A: Groups o f 8 A2/K^ mice were immunised s.c. on day - 2 8 and -1 4 with lOOpg o f M U C l peptide in IFA, IFA alone o r with 10^ pfu rec. VV-MUC1 i.p. B: Alternatively, mice were im m unised i.v. with 5x10^ DC p e r mouse loaded with 20pg/m l o f M U C l peptide, o r with PBS alone i.v. 14 days after the last immunisation, on day 0 all mice were challenged s.c with 10^ B16-MUC1-A2/K^

Vaccination with peptides emulsified in I FA has in certain cases been shown to induce epitope specific tolerance (Nieland et al., 1999; Toes et al., 1996). On the other hand, presentation of these peptides in an appropriate costimulatory context, for instance by loading of the peptides on dendritic cells, was shown to prevent tolerance induction and instead result in CTL-priming (Toes et al., 1997). To exclude the possibility that we might overlook the immunogenic potential of one or more of the MUC1-derived peptides under investigation, in particular peptides and A2/K^ mice were immunised, prior to tumour challenge, with 5x10^ peptide loaded spleen derived DC (Figure 6.4B). This experiment confirmed that peptides and induced significant protection against tumour challenge (p=0.006, p=0.002 and p=0.001 respectively). Peptides and , even when loaded on DC, failed to result in significant protection of the mice. (p=0.122 and p=0.172 respectively) (Figure 6.4B).

6.3 Discussion:

The human polymorphic epithelial mucin MUC1 is over-expressed on a number of epithelial and haematological malignancies and therefore is a potential target for T- cell meditated immunity. We have identified three non-VNTR MUC1-derived HLA- A*0201 restricted CTL epitopes. In contrast to most previously reported MUC1- derived CTL-epitopes (Apostolopoulos et al., 1997; Jerome et al., 1991; Noto et al., 1997), the epitopes identified in this study comply to the HLA-A*0201 motif, show strong binding to this molecule and map outside the TR region of MUC1. In addition, the data demonstrates that vaccination of A2/K^ mice with peptides

and elicits peptide-specific CTL immunity capable of protecting mice against a challenge with MUC1-expressing tumour cells. The latter experiment suggests that peptides and IVIUC^^"^^^ are not merely immunogenic, but that they may represent naturally processed CTL epitopes. Moreover, the tumour-challenge experiments in A2/K^ mice indicate that these peptides can indeed serve as targets in a CTL-mediated immune response against MUC-1 over-expressing, HLA-A*0201-positive tumours in vivo.

In a recent report by Brossart and coworkers (Brossart et al., 1999) two other MUC1-derived HLA-A*0201-restricted CTL epitopes have been described: peptides MUC^^'^° and (see Table 6.1 for sequences). Like the epitopes identified in this thesis, these map outside the VNTR sequence and are in compliance with the binding motif for HLA-A*0201. CTL raised against these peptides recognise MUC1-expressing tumour cells, indicating that also these peptides represent naturally processed epitopes (Brossart et al., 1999). The fact that independent attempts to identify non-VNTR MUC1-derived HLA-A*0201- restricted T cell epitopes resulted in the identification of complementary sets of epitopes can best be explained by differences in experimental approach. Brossart and coworkers have screened the MUC1 sequence for peptides matching the motif for HLA-A*0201-binding, after which they have selected two peptides for induction of CTL responses without prior analysis of their capacity to bind to HLA-A*0201. Although this publication does not provide an elaborate description of the basis on 151

which these two peptides were selected for further studies, it is conceivable that these peptides were selected because of their high score in the HLA-A*0201 motif as made available by Rammensee et al. (Rammensee et al., 1993) http://www.uni- tuebingen.de/uni/kxi/immunol.html. Accordingly, peptides and

were found to score high in computer scoring available in the Lab (DAmaro et al., 1995). These two peptides were in fact among the set of 90 MUCI-derived peptides tested for HLA-A*02Q1 binding. Although these peptides indeed showed weak binding in our assays, the binding data did not encourage further experiments concerning immunogenicity of these peptides. Instead, the experiments presented here led to the identification of three other MUC-derived, HLA-A*0201-restricted epitopes: and IVIUC^®^'^^^.

In conclusion, three independent and complementary research projects aimed at the identification of HLA-A*0201-restricted MUC1 epitopes have resulted in the identification of six distinct immunogenic peptides, one derived from the VNTR sequence (Apostolopoulos et al., 1997) and five derived from the non-VNTR areas of the MUC1 protein ((Brossart et al., 1999), this chapter). Analysis of T cell immunity against these peptides in cancer patients with MUC1-positive tumours will be required to show which of these peptides constitute targets of the natural and/or vaccine-induced CTL response against MUC1.