Identification And Modification Of An HLA-A~*0201-restricted Cytotoxic T Lymphocyte Epitope From Tumor Antigen PL2L60

Biological therapeutics has become more and more important in tumor therapy. The development of tumor vaccines, one of the hottest approaches to tumor therapeutics, made a lot of progress in recent years. In contrast to conventional vaccines, tumor vaccines are always used for treatment, not for prevention.The most significant advantage of using tumor vaccines is the induction of systemic specific anti-tumor immunity and the development of immune memory, which can be used both to monitor tumor recurrence and to generate long-lasting anti-tumor effects. Tumor vaccines can be used for effective treatment and prevention of recurrence and metastasia. The main areas for future tumor vaccine research could be in:searching for new targets, employing new targeting strategies, generating new methods for delivery and looking for new carriers for therapeutic vaccines.PurposeThe purpose of this study was to screening and identification of tumor cells in high expression of cancer-testis antigen PL2L60of HLA-A2restrictive CTL candidate epitope and modified the advantage epitope,1CF can enhance binding affinity and stability and immunogenicity of epitope.MethodsThe prediction of HLA-A*0201epitopes in PL2L60protein was carried out by using computer-based T cell epitope prediction programs including NetCTL1.2, BIMAS, SYFPEITHI and IEDB. Based on the prediction results of the three programs, native peptides and their analogues with higher scores towards HLA-A*0201allele were selected for further study. The analogues were designed by altering the native peptides with tyrosine at positionl （1Y） and tyrosine analogues. Native peptides and all substituted peptides were synthesized by standard solid-phase methods, purified by HPLC, and confirmed by mass spectrometry （ESI-MS）. Sequentially their binding affinity and stability to HLA-A*0201molecules were evaluated by T2cells binding and stable assay. RT-PCR and Western blot was used to analyze the expression of PL2L60cancer cell lines. Peptides were selected to investigate their ability to induce T cell response by using ELISPOT, intracellular cytokine staining and cytotoxicity assay in vitro and in vivo. Through the enzyme degradation stability analysis to compare wild type peptide and substituted peptides in human serum peptide the degradation rate.Results1. Based on the four prediction programs, nine native peptides P42（MLLKGEIL）, p49（LLLPELSFM）, p56（FMTGIPEKM）, p274（ILLQINCKL）, P281（KLGGELWGV）, p317（FVASINLTL）, p400（YQPKMVVFV）, p418（YLAAPQNFV）, p522（QLCENLFFL） were selected. These nine peptides were synthesized by standard solid Fmoc method and their molecular weights were verified by MS.2. Among the nine candidate peptides, the native peptide P281showed potent binding affinity to HLA-A*0201（FI values were2.42）. The DC50of the peptide was longer than4h. So we choosed this peptide to modified. These eight analogues showed potent binding affinity.3. The experiment of P281and analogues in vivo and vitro.â‘ ELISPOT and intracellular cytokine staining assay were employed to test their ability to induced CTL response in vitro. The CTLs induced by P281₁y、P281₁Nal、P281₁CF and P281₁F could showed more potent activity which could induce more amounts of IFN-γ in PBMCs from HLA-A*02+healthy donors.â‘¡Cytotoxic activity in vivo and vitro, P281₁CF and P281₁F were able to elicit specific CTLs which could lyse target cells.â‘¢Cross-recognition experiment show elicit specific CTLs of P281₁CF and P281₁F can recognize T2cells loaded with peptide P281and T2cells loaded with their variants.â‘£ELISPOT and ELISA assay were employed to test their ability to induced CTL response in vivo. The CTLs induced by P281₁CF and P281₁F could showed more potent activity which could induce more amounts of IFN-y in transgenic mice.（5）The mentioned modification strategy1y、1Nal、1CF and1F also were used to recognized advantage epitope P271and P154.4. Enzyme degradation stability analysis compared the degradation rate of native peptide and analogues in human serum peptide. The results show that P281₁CF more stable than other peptide, show high resistance against proteolytic degradation.Conclusions1. The native peptide P281and their analogues show potent binding affinity and stability to HLA-A*0201molecule. Some analogues showed stronger affinity and stability than the native peptide.2. ELISPOT and intracellular cytokine staining assay showed the P281₁CF can induced specific CTLs and more amounts of IFN-γ in PBMCs from HLA-A2+healthy donors.3. The results of ELISPOT、ELISA and LDH release assays in vivo by using HLA-A2.1/Kb transgenic mice suggested that P281₁CF could be naturally processed and presented, and induce potent specific CTL response in vivo.4. Enzyme degradation stability analysis showed that P281₁CF more stable than other peptide, show high resistance against proteolytic degradation.5. The mentioned modification strategy1y、1Nal、1CF and1F not only can used P281but also can used the other epitope. The efficacy is more stable modified by1CF.6. In conclusion, our results indicated that P281could serve as a good candidate to develop peptide vaccines against PL2L60-positive cancer; P281₁CF can enhance binding affinity and stability and immunogenicity of epitope.