靶向NSCLC EGFR exon20插入突变的新抗原疫苗筛选和功能研究

Screening and immune activity detection of neoantigen vaccine targeting epidermal growth factor receptor mutation of non-small cell lung cancer

目的:筛选具有免疫源性HLA-A*02限制性表皮生长因子受体(epidermal growth factor receptor,EGFR)exon20插入突变编码的优势表位肽,为携带EGFR exon20插入突变的非小细胞肺癌(non-small cell lung cancer,NSCLC)提供新的免疫治疗手段。方法:通过IEDB、NetMHC 4.0和SYFPEITHI软件,筛选EGFR exon20插入突变编码的HLA-A*02限制性表位,针对细胞毒性T淋巴细胞表位集中区域设计多肽疫苗,通过体外实验验证其免疫活性。结果:V769_D770insASV为EGFR exon20插入突变最高频突变位点(19.35%),经软件预测其编码的多肽YVMASVASV与HLA-A*02具有较强的结合力。围绕核心序列设计两条优势表位多肽E-ASV-10和E-ASV-19,体外可诱导HLA-A~*02限制性T细胞的扩增和活化,上调4-1BB^+CD25^+细胞比例,促进CD4^+、CD8^+T细胞IFN-γ的合成和释放。多肽E-ASV-10可刺激C57BL/6小鼠的T细胞分泌IFN-γ,对携带V769_D770insASV的小鼠NSCLC细胞LLCasv具有杀伤活性。结论:E-ASV-10和E-ASV-19多肽不仅可以在体外诱导人HLA-A*02限制性T细胞的扩增和活化,而且在C57BL/6小鼠中诱导特异性靶向携带V769_D770insASV突变的小鼠肺腺癌(lewis lung cancer,LLC)的杀伤性T细胞,提示其作为一种新的肿瘤疫苗或可用于治疗携带相应EGFR exon20插入突变的NSCLC,具有潜在临床应用前景。

Objective:This study aims to screen the optional epitope peptides of HLA-A*02 restricted human epidermal growth factor receptor(EGFR)exon20 insertion mutations in order to provide a novel immunotherapeutic strategy for non-small cell lung cancer(NSCLC).Methods:HLA-A*02 restricted epitopes derived from EGFR exon20 insertion mutation were predicted by IEDB,NetMHC4.0 and SYFPEITHI.The cytotoxic T lymphocyte(CTL)epitope-concentrated area was analyzed,and the appropriate length of polypeptides were filtered.Both cell experiments and mice models were implemented to verify the immunogenicity and antitumor efficiency of the candidate polypeptides.Results:V769_D770insASV(19.35%)mutation is a high-frequency EGFR exon20 insertion in NSCLC,which encoded YVMASVASV polypeptide with higher MHC class I binding score than other insertions.E-ASV-10 and E-ASV-19,both possessing the core sequence of YVMASVASV polypeptide,were capable to induce the expansion and activation of HLA-A*02 restricted T cells in vitro,displaying increase in the proportion of 4-1BB+CD25^+population and the production and release of IFN-γin CD4^+and CD8^+T cells.E-ASV-10 stimulated the amplification of cytotoxic T cells against the LLC cells carrying th eEGFR exon20 V769_D770insASVmutation(LLCasv)in C57BL/6 mice.Conclusions:E-ASV-10 and E-ASV-19 could induce the expansion and activation of human HLA-A*02 restricted T cells in vitro,and stimulate specific cytotoxic CTL response against LLCasv in C57BL/6 mice,which might become an promosing immunotherapeutic approach for NSCLC patients carrying EGFR exon20 V769_D770insASV mutation.