CD19 CAR-T是常规治疗失败的DLBCL治疗方法之一，但CD19阴性(CD19-)复发是当前的重大难题。本项目组具备多链结构CAR-T技术(Cancer Immunol Res 2015)。前期研究筛选出多个(包括CD30)介导DLBCLCD19-复发的关键分子，发现CD30单抗抑制CD19-肿瘤生长。据此提出假设：CD30是介导DLBCLCD19-复发的关键分子，靶向CD30/CD19是解决CD19-复发的有效策略。同时，多链CAR的激活机制以及比单链CAR优越的肿瘤杀伤机制国际上几无报道。本项目拟验证DLBCLCD19-复发关键分子，筛选优化CD30scFv，体外构建高活性多链嵌合CD30/CD19 CAR-T，阐明多链CAR激活机制以及探讨其优越的肿瘤杀伤的机制；动物实验验证CD30/CD19 CAR-T对CD19-复发的疗效，临床验证靶向CD30/CD19克服CD19-复发。

CD19 CAR-T is one of the effective therapeutic strategies for relapsed-refractory diffuse large B-cell Lymphoma (r/r DLBCL) which is failed to response to traditional therapies. However, CD19 negative relapse is the main obstacle faced in applying CD19 CAR-T in DLBCL. We have previously established a multichain chimeric antigen receptor T-cell technology (Cancer Immunol Res 2015). Our preliminary studies have identified multiple targets (including CD30) which might be critical for the development of CD19 negative relapse. We also find that CD30 monoclonal antibody suppress the proliferation of CD19 negative lymphoma. Thus, we speculate CD30 might be critical for the development of CD19 negative relapse, and targeting CD30 could be an effective strategy to overcome CD19 negative relapse. According to our understanding, the activation mechanism of multi-chain CAR-T have not been reported; and the mechanism of muti-chain CAR-T for having potent tumor-killing ability than single-chain CAR-T also have not been reported. In this project, we are going to confirm the previously identified molecules which could be critical for CD19 negative relapse. We will screen and optimize the CD30 scFv. Moreover, we will construct high efficiency CAR-T cells by using the optimized CD30 scFv and multichain chimeric antigen receptor T-cell technology. We will further elucidate the mechanism of the activation of multi-chain CAR-T cells, and the mechanism of muti-chain CAR-T for having potent tumor-killing ability than single-chain CAR-T. Finally, we will conduct clinical observation to verify whether targeting both of CD30 and CD19 could be an effective way to overcome CD19 negative relapse. The finding of this project will provide theoretical basis for overcoming CD19 negative relapse.