Selective amino acid substitutions of a subdominant Epstein-Barr virus LMP2-derived epitope increase HLA/peptide complex stability and immunogenicity: implications for immunotherapy of Epstein-Barr virus-associated malignancies

The latent membrane protein 2 is an immunogenic antigen expressed in Epstei n-Barr virus (EBV)-associated tumors and consequently it may represent a ta rget for specific cytotoxic T lymphocyte (CTL)-based immunotherapies. Howev er, the efficacy of such a therapy is limited by the poor immunogenicity of the protein that induces weak CTL responses directed to the CLGGLLTMV (CLG ) epitope only in the minority of EBV-seropositive donors. We have now demo nstrated that selective peptide stimulation of peripheral blood lymphocytes induced CLG-specific CTL in all donors, suggesting that this epitope can b e a suitable target for specific immunotherapies. We found that the CLG pep tide has a low affinity for HLA-A*0201 and does not produce stable complexe s, both factors that are likely to determine the strength of CTL responses to this epitope. Therefore, we synthesized and tested CLG analogues carryin g single or combined amino acid substitutions to increase HLA/peptide stabi lity. Among the analogues tested we identified two peptides which, compared to the natural epitope, showed higher affinity for HLA-A*0201 molecules, a nd produced stable complexes. These peptides demonstrated a potent, specifi c stimulatory capacity and could be used for selective CTL-based therapies.