非小细胞肺癌新抗原共享肽库的构建及功能验证  

Construction and functional verification of neoantigen shared peptide library for non-small cell lung cancer

作　　者：徐铭远 陈仿军[1] 苏舒[1] 吕昕[1] 王立峰[1] XU Mingyuan;CHEN Fangjun;SU Shu;LYU Xin;WANG Lifeng(Comprehensive Cancer Centre of Nanjing Drum Tower Hospital,Affiliated Hospital of Nanjing University Medical School,Nanjing,Jiangsu 210008,China)

机构地区：[1]南京大学医学院附属鼓楼医院肿瘤中心,江苏南京210008

出　　处：《中华肿瘤防治杂志》2023年第19期1154-1160,1167,共8页Chinese Journal of Cancer Prevention and Treatment

基　　金：江苏省卫生计生委医学科研课题(H2018111)。

摘　　要：目的 构建基于非小细胞肺癌(NSCLC)热点突变的共享新抗原肽库,体外验证其免疫原性,从而建立针对肺癌患者快速、高效且可行的个体化新抗原鉴定及应用新策略。方法 收集2020-01-01-2021-08-31南京大学医学院附属鼓楼医院肿瘤中心收治的12例晚期NSCLC患者癌组织、血清循环肿瘤DNA(ctDNA)和血液样本。针对中国人群常见的人类白细胞抗原(HLA)分型(HLA-A*0201/0203/0206、HLA-A*2402和HLA-A*1101),使用T细胞表位预测工具NetMHCpan 4.0,优选出高亲和力表位肽并构建新抗原肽库。通过二代测序鉴定肿瘤特异性突变,聚合酶链式反应(PCR)-测序分型方法(SBT)技术鉴定HLA精确分型,设置试验组(加新抗原肽)、阳性对照组(加植物血凝素)和阴性对照组(不加肽),通过酶联免疫斑点试验(ELISPOT)和流式细胞术检测活化T细胞标志物IFN-γ及CD137的表达,评估其免疫原性,鉴定具有免疫原性的新抗原表位肽。结果 选择表皮生长因子受体(EGFR)、鼠类肉瘤病毒癌基因(KRAS)和TP53 3个驱动基因中的8个热点突变,针对5个中国人群常见HLA,设计和构建高亲和力新抗原肽14条。在12例患者中进行新抗原共享肽库表位肽的免疫原性验证,共验证新抗原肽10条,其中11例(91.60%)患者对新抗原肽有反应性,90.00%(9/10)的新抗原肽对相匹配的患者有反应,共计9条新抗原肽被证实存在免疫原性。在1例患者中,尽管其相匹配的新抗原肽(TP53-2)多轮刺激后能观察到IFN-γ表达及CD137阳性T细胞增加的趋势,但与阴性对照组相比差异无统计学意义,P>0.05。结论 本研究成功构建基于中国人群NSCLC热点突变的共享新抗原肽库,并验证了其免疫原性。为存在驱动热点突变的肺癌人群提供了一个快速鉴定和获取新抗原的新思路,可加速个体化新抗原为基础的免疫治疗的临床应用。Objective To establish a rapid,efficient,and feasible personalized new antigen identification and application strategy for patients with lung cancer by constructing a shared-neoantigen peptides library based on hot mutations in non-small cell lung cancer(NSCLC)and further testing its'immunogenicity in vitro analysis.Methods Cancer tissue,serum circulating tumor DNA(ctDNA),and blood samples were collected from 12 patients with advanced NSCLC who were ad-mitted to the Cancer Center of Nanjing Drum Tower Hospital Affiliated to Nanjing University Medical School from January 1,2020 to August 31,2021.High-affinity epitope peptides of common human leukocyte antigen(HLA)types(HLA-A*0201/0203/0206,HLA-A*2402,HLA-A*1101)in the Chinese population were screened by going through dominant gene mu-tations in NSCLC combining with T cells epitope prediction tool NetMHCpan 4.O.Tumor specific mutations were identi-fied by second generation sequencing,and HLA precise typing was identified by Polymerase chain reaction sequencing typing(SBT).The test group was set up with the neoantigenic peptide,the positive control group with phytohaemaggluti-nin(PHA),and the negative control group without the peptide.Following multi-rounds in vitro stimulations,the expres-sion levels of IFN-and CD137 in activating T cells were assessed by ELISPOT and flow cytometry separately.The im-munogenicity was evaluated and a new epitope peptide with immunogenicity was identified.Results Fourteen high-affinity neoantigen peptides were devised and synthesized for five widespread HLA groups among Chinese populations,against,eight hotspot mutations within three driver genes,including EGFR,TP53 and KRAS.Immunogenicity validation of neoantigenic shared peptide library epitope peptides was performed in 12 patients,and 10 neoantigenic peptides were validated,of which 11(91.60%)patients showed reactivity to the neoantigenic peptides and 90.00%(9/10)of the neoantigenic peptides responded to matched patients,for a total of 9 neoantigenic peptides with confirmed immunog

关 键 词：非小细胞肺癌 热点突变 新抗原肽 免疫治疗 免疫原性 

分 类 号：R734.2[医药卫生—肿瘤]