嵌合抗原受体T细胞（CAR-T cells）已在血液瘤治疗上取得巨大进展，但对实体瘤治疗并没有很好的疗效, 一个重要原因是实体瘤的肿瘤微环境抑制了CAR-T细胞的功能， T细胞表面的抑制性受体与肿瘤细胞表面的抑制性配体结合启动抑制信号通路，引起T细胞功能耗竭。如何阻止CAR-T细胞在肿瘤微环境中的耗竭，是一个重要的科学问题。本项目将从卵巢癌临床样本中建立原代肿瘤细胞系，建立诱发CAR-T细胞耗竭的体外和体内肿瘤模型。基于实验室已有的CAR-T基因编辑平台，我们将用CRISPR-Cas9技术敲除CAR-T细胞的主要免疫检查点基因，验证单一敲除不同基因或敲除不同组合基因是否能使CAR-T细胞抵御实体瘤微环境的免疫抑制，获得肿瘤杀伤效果最佳的基因编辑CAR-T细胞，最后使用PDX小鼠模型验证其治疗实体肿瘤的功能。这项研究将为应用CAR-T细胞治疗实体瘤提供新的策略。

Chimeric antigen receptor (CAR) T cell therapy has been applied successfully in treating patients with advanced refractory B cell malignancy. However, many challenges remain in extending its application toward the treatment of solid tumors. The immunosuppressive nature of the tumor microenvironment (TME) is considered as one of the key factors limiting CAR-T efficacy. The TME expresses various ligands that activate immune checkpoint receptors on T cell surface, leading to T cell exhaustion. It is highly desirable to establish a method that renders CAR-T cells non-responsive to the TME immune suppression. In this study, we will first derive mesothelin positive tumor cell lines and stromal cell lines from primary ovarian tumor samples and establish tumor models in vitro and in vivo. We will use anti-mesothelin CAR-T cells to treat different tumor lines and identify the best model that induces CAR-T cell exhaustion. Using CRISPR-Cas9, we will knockout multiple immune check point genes independently or in different combinations in CAR-T cells, and compare their anti-tumor efficacy with non-edited CAR-T cells, using the optimized tumor model. Finally, we will test gene edited CAR-T cells using PDX models. This study will facilitate the development of more potent CAR-T therapy for treating solid tumors.