CD19/CD22双靶点CART治疗通过减少靶点导致的免疫逃逸来提高临床疗效，但仍有一半病人复发而机制不明。我们前期研究发现，约30%的复发病人CART细胞功能正常，但肿瘤细胞表面抗原密度降低并伴随抗原膜定位功能相关的CD19移码突变和CD22结构变异，与单靶点治疗复发获得的CD19变异谱显著不同。我们提出双靶点治疗特异的基因组变异引起抗原密度降低从而显著削弱CART细胞杀伤功能的假说。将在大样本复发人群全面刻画双靶点及复合物的基因组变异谱，验证前期发现的移码突变等新变异在复发人群的重现性，解析密度降低的遗传基础。进而功能实验直接验证这些双靶点治疗特异的CD19和CD22变异能直接导致双靶点抗原密度降低，并最终确立双靶点密度降低对CART细胞杀瘤功能的削弱作用。将揭示双靶点CART治疗复发特异关键机制，为优化该疗法和设计科学治疗方案提供认识，为临床预后评估和复发监控提供易检测的分子标记。

Chimeric Antigen Receptor-modified T (CART) cell immunotherapy showed promising efficacy in B-cell malignancies. Dual specificity CD19 and CD22 CART cell immunotherapy improves clinical outcome by reducing immune escape caused by target loss in the last two years. However, about half of patients still relapse and dual targets remain expressed. Our preliminary data show that approximately 30% of patients relapsed with normal function of CART cells, but carried novel frameshift mutations in CD19 and structural variants in CD22 that are associated with antigen surface localization, and accompanied by reduced dual-target antigen density at relapse. This genomic profile is markedly different from that acquired in patients relapsed after single-target CART immunotherapy. Based on these data, we proposes a hypothesis that dual-target CART cell immunotherapy-specific genomic alterations lead to a decrease in dual-target antigen density and significantly impair CART cell killing function under dual-target CART immunotherapy. We will comprehensively analyze genomic mutations and transcriptomic aberrations of CD19, CD22, and genes in their complexes using a large relapsed patient cohort. We will verify the reoccurrence of these novel mutations including frameshift and structural variations that were identified in our previous study in relapsed populations, and explore the genetic bases of antigen density decrease of these two targets. Furthermore, we will verify whether genomic alterations of CD19 and CD22 specific in dual-target CART cell immunotherapy directly cause a decrease in density of these two antigens using functional experiments. Finally, we will examine whether the antigen density reduction of dual targets attenuates the cytotoxicity and efficacy of CART cells under dual-target CART immunotherapy using in vitro and in vivo functional experiments. This study will reveal the key genetic mechanisms of dual-target CART immunotherapy-associated relapse, and provide insights for optimizing this CART immunotherapy technique and designing a more scientific therapeutic regimen. Moreover, this study will offer easily measurable biomarkers for clinical outcome and relapse monitoring.