尽管CD19 CAR-T细胞疗法取得了巨大成功，但其临床疗效受到高复发率的极大阻碍。本研究设计并比较了四种CD19/CD22双特异性CAR-T细胞的结构，不同的连接剂和抗体序列顺序。我们检测了这四种CAR-T细胞在体外的细胞毒性、细胞因子分泌水平、可持续杀伤能力、分化和疲劳。通过NSG小鼠评估了最佳的Bis-C CAR-T细胞的疗效。使用(EAAAK)3作为连接剂的两种CD19/CD22双特异性CAR-T细胞的结构具有更显著的细胞毒性和细胞因子分泌水平。在连续杀伤过程中，Bis-C CAR-T细胞表现出更好的持续杀伤能力、记忆表型分化和疲劳。在模拟CD19阴性复发的体内实验中，与CD19 CAR-T相比，Bis-C CAR-T能更好地控制CD19低表达或不表达组的小鼠肿瘤进展。本研究生成了一种新的双特异性CAR-T细胞，可以同时针对CD19或CD22阳性肿瘤细胞，为解决B细胞肿瘤中单一靶向CAR-T疗法的局限（反应有限或复发）提供了一种新策略。© 2023 The Authors. 由John Wiley & Sons Ltd发表的《欧洲血液学杂志》。

Despite the great success of CD19 CAR-T cell therapy, its clinical efficacy has been greatly hampered by the high relapse rate. In this study, we designed and compared four structures of CD19/CD22 bispecific CAR-T cells with different linkers and different orders of the antibody sequences.We detected the cytotoxicity, cytokine secretion levels, sustainable killing ability, differentiation, exhaustion of these four CAR-T cells in vitro. The optimal Bis-C CAR-T cells were evaluated the efficacy using NSG mice.The two structures of CD19/CD22 bispecific CAR-T cells using (EAAAK)3 as linker had more significant cytotoxicity and cytokine secretion levels. In the process of continuous killing, Bis-C CAR-T cells showed better sustained killing ability, memory phenotype differentiation, and exhaustion. In the in vivo experiment mimicking CD19-negative relapse, Bis-C CAR-T was more able to control the tumor progression of mice in the CD19 low expression or no expression groups than CD19 CAR-T.This study has generated a novel bispecific CAR-T cell that can simultaneously target CD19 or CD22 positive tumor cells, providing a new strategy to address the limitations of single-targeted CAR-T therapy in B-cell tumors (limited response or relapse).© 2023 The Authors. European Journal of Haematology published by John Wiley & Sons Ltd.