Prediction, screening, and identification of HLA-A2.1 -restricted CTL epitopes derived from Ewing's sarcoma EWS-FLH fusion protein

ZHAO Jinhua; HUANG Lu; CHEN Wenzhao; CAO Kai; AN Hong; YUAN Keng; SHU Yong; HUANG Shanhu; LIAO Xiang; ZHANG Zhanmin; DENG Gaorong

Abstract：EWS-FLI1 fusion protein expressed in Ewing's sarcoma (ES) is a new therapeutical target for ES treatment.The aim of this study is to predict and screen the HLA-A2.1-restricted CTL epitopes of Ewing's sarcoma EWS-FLI1 fusion protein, and to primarily I-dentify the predicted HLA-A2.1 -restricted CTL epitope candidates.The HLA-A2.1-restricted CTL epitopes of EWS-FLI1 fusion protein were synthetically predicted by BIMAS, SYFPEITHI, Predep and 1EDB methods, and then screened by the polynomial and quantitative motif method combined with supermotif prediction method as well.Docking of the screened peptides and HLA-A2.1 was observed by molecular dynamics simulation, while all peptides were synthesized with standard Fmoc strategy.After being stimulated by peptides, the CTL granulysin release test and the test of killing effects of CTL on target cells were performed to validate the immunological effects of screened epitopes.Eight CTL epitopes were predicted, among which 4 peptides were the candidates to be synthesized.The candidates docking with HLA-A2.1 were primarily validated by molecular dynamics simulation.All synthesized peptides were detected beyond 98 % in purity by reverse phase high performance liquid chromatography (RP-HPLC) and the values of molecular weight of them were conformed to match their theoretical values by mass spectrometry (MS).The granulysin release test and killing effects of CTL on target cells confirmed that all screened peptides were able to induce the cytotoxic effect of T lymphocytes.Moreover, peptide QIQLWQFLL(EWS-FLI1 304) was the most effective epitope of EWS-FLI1.All the results demonstrates that our work efficiently predict, screened, and identified the peptides of EWS-FLI1.