| With the development of molecular immunology and molecular biology of tumor antigens, tumor vaccine therapy becomes the leading area in tumor treatment research. In previous investigations, it has been proved that specific cell immunity mediated by cytotoxic T lymphocyte (CTL) is the main anti-tumor mechanism. Immune recognition studies of tumor antigen indicated that tumor-specific CTL could specifically recognize the antigen located on the surface of tumor cell. Tumor antigen can be processed and presented by antigen presenting cells (APC) in the form of MHC-peptide complex which can be recognized by CTL. Then, CTL differentiates into the effector CTL with the activity of killing tumor cells. Therefore, it is the key part to seek for CTL epitopes and vaccine forms to trigger the CTL response effectively.A rational approach to the development of vaccine for the treatment of patients with cancer depends on the identification of tumor antigen and their CTL epitopes that are essential element to design vaccine molecules. MAGE-2 is a tumor antigen identified by Boon in 1989.MAGE-2 expresses in a series of human tumor tissues but not normal tissues except for testis and placenta. Till now, two MAGE-2-derived HLA-A2-restricted peptides were shown to be capable of eliciting a CTL response in transgenic mice, although no direct evidence showed that these CTL could recognize HLA-A2 human tumor cell line. Thus, in this paper, we used the supermotif method combined with thequantitative motif plot to predict CTL epitopes of MAGE-2. As a results, nine HLA-A2-restricted CTL epitopes i.e. MAGE-248-56 (VTLGEVPAA), MAGE-2108-116 (AISRKMVEL), MAGE-2112-120 (KMVELVHFL), MAGE-2 153-161 (KASEYLQLV), MAGE-2171-179 (PISHLYILV), MAGE-2176-184 (YILVTCLGL), MAGE-2220-228 (KIWEELSML), MAGE-2237-245 (SVFAHPRKL), MAGE-2271-279 (FLWGPRALI) were predicted. Four epitopes, MAGE-2112-120, MAGE-2171-179, MAGE-2220-228, MAGE-2271-279., which have higher predictive value than the remains were selected for molecular simulation studies. In molecular simulation those four candidate epitopes were proved to bind HLA-A2 with a distance between the two major anchor residues from 15 ? to 20 ?. Each candidate epitope had similar non-bond energy and hydrogen bond number, which were in accordance with the requirement of HLA-A2-restricted CTL epitope. Among those four predicted CTL epitopes, MAGE-2112-120 had been proved as the HLA-A2-restricted epitope by Dr Visseren in 1997.In further study, all the four predicted CTL epitopes were synthesized by using Fmoc chemistry with the peptide synthesizer ABI431A. The resin-peptide compound was cleavaged by TFA. The synthesized epitope peptides were purified by HPLC on Acta explorer. The purity of synthesized peptides were over 90% by reversed phase HPLC method. The molecular weight of peptides was validated by mass-spectrum.To confrim those predicted epitopes, the standard 51Cr release assay was carried out. Synthetic peptide-stimulated human peripheral blood mononuclear cells (PBMCs) of HLA-A2+ healthy donors were employed as effector cells. The target cell was human melanoma cell line LB373-MEL expressing MAGE-2 and HLA-A2 protein. When the ratio of effector to target was 100 to 1, MAGE-2112-120 stimulated PBMC could lyse 71.2% of target cells, and 74.4% for MAGE-2220-228, 68.2% for MAGE-2271-199. However, no obvious lysis was found for MAGE-2171-179. MAGE-2112-120, MAGE-2220-228 andMAGE-2271-279, were proved by the peptide binding assay with T2 cells by FACScan method. The results showed that the florescence index was 0.79 for MAGE-2112-120, 0.61 for MAGE-2220-228, 0.24 for MAGE-2271-279. These results showed that candidate peptide MAGE-2220-228 and MAGE-2271-279 were novel identified CTL epitopes and MAGE-2220-228 could bind HLA-A2 with higher affinity.To improve linear peptide immunogenicity, we performed the molecular engineering experiment of the CTL epitope peptide MAGE-2220-228. Multiple antigen peptide (MAP), taking lysine with small molecular weight and weak immunogenicity as...
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