| Recently, cancer has been the first leading cause of death. In this serioussituation, the prevention and treatment of cancer is so urgent. With the developmentof biotechnology and medicine, cancer immunotherapy has been gradually applied inclinical cancer cases. The cancer vaccine is one of the most promising approaches totumor immunotherapy, and now most vaccines are usually used for treatment.Furthermore, the peptide vaccine has drawn an increasing amount of attention withits high specificity, simple synthesis and non-carcinogenic potential.PurposeTo identify short peptides (SP) derived from a novel ancer/testis antigen (CTA),αN-catenin (CTNNA2), which can induce CTLs. Besides, to enhance the bindingaffinity and stability of epitopes, altering the Serine at position4of two nativepeptides with the phosphorylated Serine was performed.MethodsFirstly, the prediction of HLA-A*0201restricted CTL epitopes derived fromCTNNA2protein was conducted by using recently developed computer algorithmssuch as NetCTL1.2, BIMAS and SYFPEITHI. Meanwhile, two phosphopeptideswere designed. Secondly, native peptides and phosphopeptides were synthesized bythe standard Fmoc chemistry and purified by RP-HLPC. The sequences of peptideswere determined by mass spectrometry. Sequentially, their binding affinity toHLA-A*0201molecules and pMHC complexes stability were measured using the T2cell line. The expression of CTNNA2mRNA in tumor cell lines was investigated byRT-PCR. Finally, CTNNA2-specific human CTLs were induced from PBMCs ofthree HLA-A2+healthy donors, after which the frequency and immunogenicity ofCTLs were detected with cytokine flow cytometry (CFC), ELISPOT and cytotoxicitysssay. Results1. On the basis of an integrated analysis of the three prediction algorithms.P129(ALLSAVTRL), P138(LILADMADV), P148(RLLSHLKIV), P279(ALNEFDNKI), P345(LLSEYMNNT), P495(VLTEAVDDI), P569(KVLEATKLL), P581(VMPRFAEQV), P721(IMMEMTDFT), P779(LMNAVVLTV) and P784(VLTVKASYV) were selected. All peptides weresynthesized with the solid Fmoc method. The purity (>95%) and the sequences ofpeptides were confirmed by RP-HLPC and MS, respectively.2. We measured and confirmed the binding affinity of the eleven peptides bythe peptide-T2binding assay. Actually, native peptides P148, P581, P129, P138,P345and P721could potentially bind to HLA-A*0201molecules (FI values were1.81,1.51,1.46,1.31,1.30and1.21, respectively). The DC50of peptides exceptP129were longer than4h. Two phosphopeptides also showed high binding affinityand long half-life.3. Intracellular cytokine staining and ELISPOT assays were used to test theability of these CTNNA2deriving epitopes to induce CTL responses in vitro. P129,P138, P148, P581and P721could evidently stimulate stronger CTL responses,which produced large numbers of IFN-γ-secreting cells. While P345did not producean efficient result like this. Noticeably, the results of P138, P581and P721werehighly significant in both of the ELISPOT assay and cytotoxic assay. In particular,CTLs induced by these peptides, could effectively lyze peptide-loaded T2cells andCTNNA2+/HLA-A2+cancer cell lines such as MCF-7and MDA-MB-231. Incontrast, phosphopeptides were not so capable of stimulating cytotoxic activities.Besides, intracellular cytokine staining assay results showed that specific CTLsproduced by loading P129-4(pSer)and P148-4(pSer)could secrete large amounts ofIFN-γ.Conclusions1. Native peptides P138, P148, P345, P581and P721derived from CTNNA2and two phosphopeptides showed potent binding affinity to HLA-A*0201moleculesand strong pMHC stability. According to the assays, the phosphopeptides displayedsuperior binding affinity and stability than the native counterparts.2. All peptide-specific cytotoxic T lymphocytes were induced from the PBMCs of different HLA-A2+healthy donors stimulated by using CTNNA2peptides(P129, P138, P148, P581and P721, respectively). And we detected greater numbersof peptide-specific T cells secreted IFN-γ by ICS and ELISPOT assays.3. LDH assay showed that CD8+CTLs stimulated by P138, P581and P721could significantly lyse cancer cell lines like MCF-7and MDA-MB-231. Wehypothesized that the cytotoxic activity was observed in HLA-restricted andCTNNA2-specific manners.4. With the illustration of CFSE, cytotoxic capability of P138, P129, P581andP721appeared stronger than their counterparts of the other candidate peptides.5. In conclusion, the results of our study demonstrated that candidate peptidesP129, P138, P148, P581and P721derived from CTNNA2could serve as HLA-A2restricted CTL epitopes. The effort of pSer altering of native candidate peptides wasproved effective to enhance the binding affinity to HLA-A2molecules and thepMHC stability. |
