基于不同CART细胞亚群靶向HER2阳性胃癌的抗肿瘤疗效和机制研究

The role of chimeric antigen receptor T cells (CART) in the treatment of solid tumors is increasingly important. CART cells targeting HER2 positive gastric cancer have been proved initially effective in the basic research, but the disease control time is not satisfied. Each T cell population can be divided into initial T cells (TN), memory stem cell-like T cells (TSCM), central memory cells (TCM), effector memory T cells (TEM) and effector T cells (TEFF) based on the different response to antigen. Functions and life span of different subsets are not similar. Hence, optimization and quantify the components of reinfusion CART cells are important measures to improve the anti-tumor efficacy and maintain long-lasting immune response. Previously, we have successfully isolated different T cell subsets and modified into CART cells accordingly. The antitumor ability and phenotypic changes of each CART cell subpopulation have been analyzed in vitro. The combination of CD4+ TN and CD8+ TCM group showed the best anti-tumor effect in the lymphoma model. Therefore, we hypothesized that for gastric cancer, we can improve the curative effect by optimizing the components of CART cells targeting HER2, of which CD4+ TN and CD8+ TCM group may be the most effective combination. The project uses gastric cancer as a solid tumor representative and aims to improve anti-solid tumor efficiency of CART cell and prolong disease-free survival though the optimization of CART cell components. Ultimately, this project can facilitate the accurate treatment of CART cells in solid tumor and offer new strategy for the good clinical management of CART cells.

嵌合抗原受体T细胞(CART)在实体瘤综合治疗的地位越来越重要。靶向HER2阳性胃癌CART细胞治疗已初有成效，但疾病控制时间短。T细胞按对抗原应答不同分为初始T（TN）、记忆性干细胞样T(TSCM)、中央记忆型T(TCM)、效应记忆型T(TEM)和效应T细胞(TEFF)，不同亚群功能和寿命不同，故优化和量化回输的CART细胞亚群是提高抗肿瘤疗效和持久免疫应答的关键。我们前期已成功分离不同T细胞亚群并修饰成CART细胞，分析各CART细胞亚群活性及表型，在淋巴瘤模型中证实CD4+TN联合CD8+TCM抗肿瘤疗效最优。因此我们推测在胃癌中可通过优化靶向HER2的CART细胞成分来提高疗效，其中CD4+TN联合CD8+TCM可能为最优组合。本项目以胃癌为实体瘤切入点，通过优化CART细胞成分最终提高抗实体瘤有效率、延长无病生存期，为实现CART细胞实体瘤精准治疗和临床规范化应用提供新策略。

本项目计划以HER2高表达的人胃癌细胞为研究模型，围绕着不同CART细胞亚群及相应组合在实体瘤中体内体外的抗肿瘤疗效的优化这一核心内容展开研究，即通过充分优化和量化T细胞亚群的组成及比例，来提高靶向HER2的CART细胞的在胃癌的抗肿瘤效应，基于我们前期假设：CD4+TN联合CD8+TCM组可能会成为最有效的抗肿瘤组合，最终成为提高CART抗实体瘤疗效的一个突破口。课题组按计划完成了筛选高表达HER2的胃癌细胞、建立HER2-CAR病毒包装体系、感染T细胞，初步探索靶向HER2的CART细胞的抗肿瘤活性，整个研究最重要的工作是优化TN、TCM、TEM、TM等T细胞亚群的分选策略，发现CD8+T M较CD8+T CM更易分选、扩增更快，因此我们采用CD8+T M代替CD8+T CM完成后期实验；我们成果将各个T细胞亚群感染病毒后建立CART细胞，通过体外比较各个CART细胞亚群或者组合的抗肿瘤效应，发现CD8+ CAR-TM 联合CD4+ CAR-TN的组合在体外的抗肿瘤效果较好且分泌的细胞因子也较多；也初步探索了体内各个CAR-T亚组抗肿瘤疗效，完成本项目研究计划。此外，我们构建了表达荧光素酶的胃癌细胞系和筛选最佳动物模型，计划再次验证CD8+ CAR-TM 联合CD4+ CAR-TN组合在体内的抗肿瘤效果，同时后期继续收集HER2阳性的胃癌患者的标本以进行体外杀伤实验的验证。在这个项目的支持下，考虑到癌细胞和癌相关成纤维细胞产生的细胞外基质和胶原纤维阻碍了免疫效应细胞向肿瘤的归巢，CAR-T细胞难以进入到肿瘤组织，我们在目前HER2-CAT结构的基础上再联合基质金属蛋白酶-8，以增加CAR-T细胞在肿瘤组织的浸润，促进其抗肿瘤疗效及抗肿瘤的持久性。到目前为止，我们在国外杂志上发表已标注该基金的 SCI 论文3篇（其中第一标注SCI论文2篇）。期望通过本研究为实现CART细胞在实体瘤的精准治疗和临床规范化应用提供新的策略。