基于体细胞突变数据库筛选免疫原性结直肠癌高频新抗原的方法

A method of screening highly common neoantigens with immunogenicity in colorectal cancer based on public somatic mutation library

结直肠癌是世界高发和高致死率的恶性肿瘤。靶向新抗原的免疫治疗已被证实可以诱导癌症患者肿瘤持续消退,但这些特异性新抗原,仅适用于个体精准治疗。随着大量的高频肿瘤基因突变被发现,这些与突变相关的高频新抗原可覆盖更多人群,具有较强的临床意义。然而目前结直肠癌中是否也存在高频新抗原仍不清楚。本研究利用来源于321个结直肠癌患者的体细胞突变数据库,联合1种标准过滤和7种预测算法,筛选并获得了25个基于中国人高频分型HLA-A^*1101限制性的高频新抗原,它们均具有高亲和力(IC50<50 nmol/L)和高呈递分值(>0.90);其中,除了阳性对照多肽KRAS_G12V8-16外,11个高频新抗原能够在体外诱导细胞毒性T淋巴细胞(cytotoxic T lymphocyte,CTL)分泌γ干扰素(interferon gamma,IFN-γ),证实具有免疫原性。选取免疫原性最强的新抗原C1orf170_S418G413-421及阳性对照多肽KRAS_G12V8-16体外刺激T细胞,利用流式细胞分选及单细胞转录组测序技术,获得其特异性CTL的免疫组库信息,所构建的TCR-T(T-cell receptor engineered T cell)能够识别新抗原并分泌细胞因子。以上结果表明,本研究开发了一种利用体细胞数据库预测并体外筛选验证具有免疫原性高频新抗原的方法,为结直肠癌及其他癌种的多肽、DC(dendritic cells)疫苗、TCR-like抗体、TCR-T等免疫治疗提供了重要的多肽靶点和TCR信息,具有实际的临床应用价值。

Colorectal cancer(CRC)is a malignant cancer with high incidence and mortality in the world.Immunotherapy targeting neoantigens can induce durable tumor regression in cancer patients,but is almost limited to personalized precision therapy,due to the individual differences of unique neoantigens.With the discovery of many common oncogenic mutations,and such mutation-associated neoantigens could cover more patients,and hence are valuable in clinical field.However,whether the common neoantigens can be identified in CRC is unknown.Combining the somatic mutations data from 321 CRC patients with a filter standard and 7 predicted algorithms,we screened and obtained 25 HLA-A^*1101-restricted common neoantigens with a high binding affinity(IC50<50 nmol/L)and presentation score(>0.90).Besides the positive epitope KRAS_G12V8-16,11 out of 25 common neoantigens specifically induced in vitro prestimulated cytotoxic lymphocyte(CTL)to secrete interferon gamma(IFN-γ).Moreover,combining cell-sorting technology and single-cell RNA sequencing,the immune repertoire profiles of C1orf170_S418G413-421 and KRAS_G12V8-16-specific CTL were analyzed and validated.Their related T-cell receptor engineered T cell(TCR-T)cells could also recognize the neoantigens and secrete IFN-γ.Hence,we have established a method to screen for common neoantigens with immunogenicity in CRC based on the public somatic mutation library.It can provide essential peptide and TCR information for immunotherapies,such as peptides,dendritic cells(DC)vaccines,TCR-like antibodies,TCR-T,etc.,for the CRC and other cancers,which has practical application value in the clinics.