PD-1免疫检查点缺失的CAR-T细胞的抗肿瘤作用及机制研究

Chimeric antigen receptor-modified T cells (CAR-T) have been endowed with specificity to tumor cells by which they have the tumor-killing ability. Cancer immunotherapy based on CAR-T cells are applied to conquer leukemia and other hematologic malignancies and landmarks of breakthrough has been made, but not for solid tumors. Programmed cell death protein 1 (PD-1) is a key immune check point on effector T cells. Many solid tumors express the ligands of PD-1 and inhibit the anti-tumor capacity of CAR-T cells after the ligation of PD-1 with its ligands. In our previous study, by exploitation of lentiviral platform and RNA interference technology, we produced CAR-T cells with tumor specificity and simultaneously without the expression of PD-1. CAR-T cells lack PD-1 exhibited enhanced cytotoxicity to tumor cells, elevated tumor infiltration and growth and metastatic inhibition, compared to that with PD-1 expression. Then we hypothesize that CAR-T cell lack of PD-1 have enhanced tumor-killing efficacy for solid tumors. In this grant, we will further study the tumor-killing kinetics and mechanism of CAR-T lack PD-1 on the basis of our previous results. Issues like bio-distribution, survival time and safety of the CAR-T will be evaluated. we are aim to produce CAR-T cells beyond the inhibition of immune check point and enhance the efficacy of cancer immunotherapy based on CAR-T cells for the treatment of solid tumors.

嵌合型抗原受体修饰的T细胞（CAR-T）被赋予特异的肿瘤靶向识别和杀伤能力。基于CAR-T细胞的肿瘤免疫治疗对白血病等血液肿瘤疗效显著，但对实体肿瘤的疗效不理想。程序性死亡受体-1（PD-1）是效应T细胞的一个关键免疫检查点，多种实体肿瘤细胞均表达PD-1的配体并与之结合进而抑制CAR-T细胞的肿瘤杀伤活性。在前期研究中，我们利用慢病毒载体平台和RNA沉默技术，制备了具有肿瘤特异性同时缺乏PD-1分子的CAR-T细胞，发现缺少PD-1的CAR-T细胞具有更强的细胞杀伤活性及肿瘤浸润能力并明显抑制肿瘤的生长和转移。因此我们推测：PD-1的缺失能够增强CAR-T细胞对实体肿瘤疗效。在本项目中，我们拟在此基础上，进一步研究缺少PD-1的CAR-T细胞的抗肿瘤活性及机制，检测其在瘤内的分布与存活时间，评估其毒副作用，以期获得一种不受免疫检查点调控的效应T细胞，提高CAR-T细胞对实体瘤的疗效。

程序性死亡受体-1(PD-1)是效应T细胞的一个关键免疫检查点，多种实体肿瘤细胞均表达PD-1的配体并与之结合进而抑制CAR-T细胞的肿瘤杀伤活性。嵌合型抗原受体修饰的T细胞(CAR-T)对白血病等血液肿瘤疗效显著，但对实体肿瘤的疗效不理想。我们在前期研究中发现，缺乏PD-1分子的CAR-T细胞，发现缺少PD-1的CAR-T细胞具有更强的细胞杀伤活性及肿瘤浸润能力并明显抑制肿瘤的生长和转移。本项目中，我们在此基础上，进一步研究缺少PD-1的CAR-T细胞的抗肿瘤活性及机制，检测其在瘤内的分布与存活时间，评估其毒副作用，以期获得一种不受免疫检查点调控的效应T细胞，提高CAR-T细胞对实体瘤的疗效。在本项目的资助下，目前我们证实缺失PD-1表达的CAR-T细胞，CAR-T细胞具有识别VEGFR-1或EpCAM的肿瘤靶向杀伤的特异性；我们也对比了PD-1缺失的CAR-T细胞对PDL-1阳性和阴性实体肿瘤细胞的杀伤活性差异，证实了PD-1的缺失能够逆转PD-1/PD-L1信号通路对CAR-T细胞杀伤活性的抑制；我们比对正常表达PD-1的CAR-T细胞和缺失PD-1的CAR-T细胞在对靶细胞杀伤时相应胞内信号通路的差异，同时富集了相应的差异信号通路，获得PD-1影响CAR-T细胞功能的分子机制基础；我们检测并证实了PD-1缺失的CAR-T细胞在体内的抗实体肿瘤作用，证实PD-1缺失能够增强CAR-T细胞在体内抗实体肿瘤；同时我们也检测了PD-1缺失的CAR-T细胞在体内的生物学分布，各主要脏器的损伤情况并检测血液学生化指标，证实了PD-1缺失的CAR-T细胞在小鼠体内应用的安全性。总之，本项目制备了一种不受PD-1免疫检查点抑制的肿瘤特异性效应T细胞并证实了其抗实体肿瘤的作用并阐明了其潜在的机制，为推进CAR-T细胞抗实体肿瘤的基础研究及临床转化提供了实验依据。