Identification Of HLA-A2-Restricted CTLs Epitopes From A Cancer/Testis Antigen KM-HN-1

The therapeutic options for treatment of tumor patients are limited to 3 fundamental modalities:(a) surgical resection,(b) chemotherapy and(c) radiation therapy.However, despite the curative effects,they have some inevitable drawbacks and often fail to eradicate the tumors.The development of progressive tumor is thought to result from the successful escape of tumor cells from cancer immunosurveillance¹.Therefore alerting the immune system to the escaping tumor cells is thought to be a promising alternative therapeutic approach against cancer.Cytotoxic Tlymphocytes(CTLs) are thought to play an important role in the eradication of tumors by self immune system,because in vitro analysis revealed that they can effectively lyse autologous tumors in a peptide-MHC(pMHC) restricted manner².Therefore antigenic peptide based immunotherapeutics by immunizing patients with peptides or with peptides pulsed autologous antigen-presenting cells(APCs) tends to be an attractive cancer-specific immunotherapeutic approach.For the peptide based immunotherapy,an ideal tumor antigen as the target is a prerequisite for the successful therapy.The cancer-testis antigen(CT antigen) family has been an attractive target for peptide-based immunotherapy due to their exclusive expression pattern (tumor,testis & placenta)^3,4 and high immunogenicity^5,6.Clinical trails suggested that CT antigens are good targets for peptide based immunotherapy^7,8.The KM-HM-1 antigen is a novel CT antigen identified in 2004⁹.It is found to be highly expressed in a variety of cancer tissues and cancer cell lines but not in normal tissues except testis.More importantly,it can induce coordinated humoral and cellular immune responses in cancer patients,which reveals its high immunogenicity.Therefore,we consider it a good target for tumor immunotherapy.HLA-A*0201 is the most frequent HLA-A allele in the Chinese population with an estimated frequency of＞50%.In this study,we used synthetic KM-HN-1 peptides consisting HLA-A*0201 binding motifs to identify HLA-A*0201 restricted CTLs epitopes from CT antigen KM-HN-1.The HLA-A*0201 restricted CTLs epitopes were identified following a four-step procedure:(a) in silico epitope prediction of antigen KM-HN-1(b) peptide HLA-A*0201 binding and pMHC complex stability assay(c) in vitro generation of the predicted epitope-specific CTLs from both healthy and cancer patients(d) Testing of the induced CTLs toward T2 cells pulsed with peptides or toward various tumor cells expressing both antigen KM-HN-1 and HLA-A*0201.As a result,we identified HLA-A*0201-binding peptides KM-HN-1_203-211(FLPTAPPNV) and KM-HN-1_321-329(KLLPFRETV) which are naturally processed and presented T-cell epitopes capable of inducing KM-HN-1 specific CTLs in both healthy donors and cancer patients in vitro.Peptide KM-HN-1_203-211(FLPTAPPNV) is of higher immunogenicity compared with peptide KM-HN-1_321-329(KLLPFRETV),it may be the dominant epitope of CT antigen KM-HN-1.These identified epitopes would be useful in further evaluating the clinical utility of peptide-based,cancer-specific immunotherapy against various cancers.