以CD19为靶点的CAR T治疗在B-ALL及NHL等恶性血液肿瘤中取得突出临床疗效，但肿瘤细胞靶向抗原缺失/突变或CAR T细胞“耗竭”会导致治疗抵抗与复发。我们已有临床前数据显示，分别针对B-ALL/NHL两种血液肿瘤，双靶点CD19-CD22/CD19-CD20-CAR T较单靶点T抗瘤活性强，提示双靶点CAR T具有降低肿瘤抵抗发生的潜能，以此为基础，本项目将从细胞培养/实验动物/临床试验层面研究：1）与单/双靶点CAR T相互作用过程中肿瘤细胞的抵抗性，单细胞水平鉴定抵抗克隆的表型、基因组与转录组特征，通过全基因组CRISPR /Cas9文库筛选，识别介导抵抗的关键细胞信号途径及分子，并进行回复验证；2）监测抗瘤过程CAR T的耗竭状态，阐明去耗竭是否可有效逆转肿瘤对CAR T治疗的抵抗。本项目研究将为优越的CAR T临床治疗策略设计与肿瘤去抵抗联合方案的制定奠定基础。

Chimeric antigen receptor-engineered T(CAR-T) cells have yielded unprecedented efficacy in B cell malignancies, most remarkably in anti-CD19 CAR-T cells for B cell acute lymphoblastic leukemia with up to a 90% complete remission rate. However, more than 50% of responders have occurred resistance to this novel immunotherapy and suffered relapses 6 months post CAR-T cell infusion. Two modes of disease recurrence have been seen: CD19 positive and CD19 negative. CD19 positive relapse is attributed to the rapid disappearance of CAR-T cells or decreased function of those T cells (known as T cell exhaustion, an acquired state of T cell dysfunction – is a hallmark of cancer), whereas CD19 negative relapse may be an example of tumor editing such as target antigen loss, mutation or leukemic lineage switch due to the selective pressure exerted by CAR-T cell therapy, highlighting the shortcoming of single-target CAR-T cell therapy. To prevent and treatment of these relapses, our group designed a CD19/CD22 bispecific CAR for B-ALL, and enhanced anti-tumor activity was demonstrated by the CD19/CD22 bispecific CAR compared to the unispecific CARs targeting either CD19 or CD22 in experimental studies. Consistent outcomes have also been observed in the preclinical researches focusing on CD19/CD20 bispecific CAR for B cell non-Hodgkin lymphoma. These findings provide the preliminary evidence that generating T cells capable of recognizing multiple antigens may be an effective alternative to address the challenge of resistance and relapse after CAR-T cell therapy. On this basis, our group will further investigate these bispecific CARs for hematological malignancies in vitro and in vivo including animal models and humans to determine the underlying mechanism of tumor editing especially in the setting of bispecific CARs and development of strategy for reversing T cell exhaustion: 1) explore characteristics of genomics and transcriptomics of the evolutionary tumor clone contributing the adaptive resistance to the unispecific or bispecific CARs at single cell level, and through CRISPR /Cas9 to screen the whole genome library to investigate potential signal transduction pathways that meditate those adaptive resistance and identify key molecules which are critical for the signal transduction pathways, then carry out the functional complementation analysis to confirm the function of those molecules.2) Dynamic monitor the status of CAR-T cell exhaustion by testing the phenotype of CAR-T cells , to illustrate that whether reversing T cell exhaustion will be beneficial to address the resistance to CAR-T cell therapy. Overall, these abovementioned work will pave the road to optimize CAR-T cell therapy, and will also provide the foundation for the development of combination therapy in an effort to reverse resistance to CAR-T cells.