Identification of human leukemia antigen A*0201restricted epitopes derived from epidermal growth factor pathway substrate number 8

Tcellmediated immunotherapy of hematological malignancies requires selection of targeted tumorassociated antigens and Tcell epitopes contained in these tumor proteins. Epidermal growth factor receptor pathway substrate 8 (EPS8), whose function is pivotal for tumor proliferation, progression and metastasis, has been found to be overexpressed in most human tumor types, while its expression in normal tissue is low. The aim of the present study was to identify human leukemia antigen (HLA)A*0201restricted epitopes of EPS8 by using a reverse immunology approach. To achieve this, computer algorithms were used to predict HLAA*0201 molecular binding, proteasome cleavage patterns as well as translocation of transporters associated with antigen processing. Candidate peptides were experimentally validated by T2 binding affinity assay and brefeldinA decay assay. The functional avidity of peptidespecific cytotoxic T lymphocytes (CTLs) induced from peripheral blood mononuclear cells of healthy volunteers were evaluated by using an enzymelinked immunosorbent spot assay and a cytotoxicity assay. Four peptides, designated as P455, P92, P276 and P360, had high affinity and stability of binding towards the HLAA*0201 molecule, and specific CTLs induced by them significantly responded to the corresponding peptides and secreted IFNγ. At the same time, the CTLs were able to specifically lyse EPS8expressing cell lines in an HLAA*0201restricted manner. The present study demonstrated that P455, P92, P276 and P360 were CTL epitopes of EPS8, and were able to be used for epitopedefined adoptive Tcell transfer and multiepitopebased vaccine design.