Definition of MHC-restricted CTL epitopes from non-variable number of tandem repeat sequence of MUC1

Mucin 1 (MUC1) is expressed ubiquitously on breast cancer cells and is a po tential target for the generation of cytotoxic T cells for vaccination agai nst breast cancer. Thus far studies of the immunogenicity of MUC1 have used peptides from the variable number of tandem repeat (VNTR); mice so immunis ed can generate strong cellular and antibody responses to the VNTR of human MUC1. We now demonstrate that significant CTL and CTLp can be induced to o ther regions of MUC1. Using the whole native MUC1 molecule, the human milk fat globule membrane antigen (HMFG) linked to mannan, cytotoxic T cell prec ursors (CTLp) can be generated in BALB/c, C57BL/6, transgenic HLA-A*0201/K- b and double transgenic HLA-A*0201/ K-b x human MUC1 (A2 K(b)MUC1) mice. By immunising with HMFG and testing selectively on (a) extracellular (non-VNT R); (b) VNTR and (c) intracellular peptides, it was shown that all three re gions generated effective CTL. Further, the CTL responses to non-VNTR pepti des were as strong as those generated to the VNTR. Epitope prediction algor ithms were not particularly helpful to describe CTL epitopes: overlapping p eptides had to be synthesised and tested to find the epitopes, Thus, for CT L generation, the whole HMFG molecule is a powerful immunogen when linked t o mannan, especially as multiple peptide epitopes for presentation by many Class I molecules are contained within the one molecule. Furthermore, Class I restricted MUC1 CTL were generated in double transgenic A2 K(b)MUC1 mice by immunising with mannan-native mucin (HMFG), suggesting that tolerance t o MUC1 can be overcome with mannan-HMFG. (C) 2000 Elsevier Science Ltd. All rights reserved.