基于PIK3CA热点突变的宫颈癌新型靶向性治疗疫苗的设计及评价

Recent understandings from data of cancer genome sequencing and mechanical analysis of checkpoint blocking therapy made the researchers in vaccinology realized that mutations-derived neoantignes are better than those used previously, the formers of which can induce strong immune response that can kill tumor cells. However, they can not employed as targets for vaccine design due to their extremely low frequency in patient populations. Genes harbor hotspot mutations, which both are with high immunogenicity and with enough higher frequency in patient populations, are extremely rare. We deeply diged the sequencing data from more than 130 cancer types and found a gene, PIK3CA, with hotspot mutations in cervical cancer. Exome sequencing from 102 cervical cancer samples confirmed that the mutation rate in Chinese people was 16.7%, with E542K, E545K and H1047R as hotspot mutations. We further identified five T-cell epitopes by immunological bioinformatics and immunogenicity screening assays. In this program, we would format a vaccine with these identified epitopes, and evaluate their efficiency in tumor killing with transgenic mice with humanized MHC complex. The frequency and phenotype of mutation specific T cells in patients with PIK3CA mutation would be detected by pentamers. The project is expected to complete the preclinical assays of a novel therapeutic targeted vaccine of cervical cancer.

肿瘤基因组测序数据及免疫检查点阻断治疗的临床突破及机制分析，使疫苗学界认识到，突变产生的新抗原(neoantigen)明显优于既往抗原，能诱导强烈的杀伤肿瘤的免疫应答。但单个突变在群体中极低的频率使其不能用来作为疫苗靶标。热点(hotspot)突变兼具免疫原性强和群体频率高两个优点，但数量极其稀少。我们前期对多达130余种肿瘤的测序数据的挖掘分析，找到一个在宫颈癌中具有热点突变的基因PIK3CA，进一步对102例宫颈癌标本测序发现，PIK3CA在中国人群中的突变率为16.7%，其热点突变为E542K、E545K及H1047R。随后的免疫信息学预测及免疫原性筛选鉴定了5个T细胞表位。本项目拟以此表位为抗原制备多肽疫苗，借助人源化MHC转基因小鼠评价疫苗的肿瘤杀伤效应。并用五聚体技术检测临床病人血液中表位特异性T细胞频率及表型。本项目有望完成一个宫颈癌新型靶向性疫苗候选药物的临床前实验。

项目背景.宫颈癌的治疗目前仍以手术结合放化疗为主，对于中晚期患者尚无更有效的治疗手段。基于肿瘤相关/特异性抗原肽的肿瘤治疗性肽疫苗具有个体化、激发宿主主动免疫、靶向性强、显著增强免疫检测点治疗效果和毒副作用低等多个优势。本研究旨在建立从宫颈原位癌组织中直接提取、鉴定和筛选MHC-Ⅰ类分子相关抗原肽的方法和策略，为宫颈癌治疗性多肽疫苗研究提供新的思路和方法。.主要研究内容和重要结果.1. 基于定制肽数据库的MIP高通量质谱鉴定和特征分析.基于Universal Protein和Human isoform protein两个数据库建立了定制肽库。样本库中符合条件的21对宫颈癌和正常宫颈组织，经过IAC分离纯化获得MIP样品。再经过LC-MS/MS检测、定制肽库匹配、netMHCpan4.0预测，最终从每个样品中鉴定出含肽数量不等（3470-24392）的MIP数据集。.2. 宫颈癌相关抗原肽的筛选.根据MIP来源蛋白的组织来源、与肿瘤相关性分析、被检测出的频次等步骤筛选出宫颈癌组织特异性MIP来源蛋白GPX1、MYO9B、MCM4、PNKP、CKAP5、LAMB1和EIF3D。.3. 宫颈癌相关抗原肽免疫原性的验证.来源于上述7个蛋白的12条候选肽，采用健康捐献者和宫颈癌患者的PBMC，通过间接法和直接法证实候选抗原肽刺激PBMC分泌IFN-γ升高。.关键数据及其科学意义.1. 创新性建立了限制长度的定制肽数据库，成功鉴定出宫颈癌和正常宫颈组织来源的MIP。.2. 成功建立了基于分析MIP来源蛋白筛选自体蛋白来源的宫颈癌MHC-Ⅰ类分子相关抗原肽的策略，并筛选出系列候选宫颈癌相关自体蛋白抗原。.3. 体外实验验证了筛选到的系列宫颈癌MHC-Ⅰ类分子相关抗原肽的免疫原性，为将来宫颈癌治疗性肽疫苗研制提供了新靶标。