靶向Her3的人同种异体CAR-T的建立及其对肿瘤的杀伤作用

In recent years, radiotherapy or chemotherapy combined with chimeric antigen receptor T-cells (CAR-T) has become a new strategy for cancer therapy, however, deficiencies exist in the preparation of CAR-T by using autologous T cells. As a result of radiotherapy or chemotherapy, autologous T cells isolated from patients are often insufficient or dysfunction, resulting in the preparation of CAR-T not only long cycle, time-consuming, but also the quality and quantity of cells are difficult to be guaranteed. In this project, healthy human allogeneic T cells are to be used to establish universal CAR-T, and the killing effects of CAR-T on tumors and the elimination of autoimmunity will be tested. Firstly in order to get the universal allogeneic donor-derived cells for CAR-T construct, the HLA-A and HLA-B locus genes in T cells would be knocked out by the CRISPR/Cas9 technique; Then the human allogeneic CAR-T targeting the human epidermal growth factor receptor 3 (Her3) will be established; Lastly, Her3-positive cells from breast cancer, non-small cell lung cancer, colorectal cancer and esophageal cancer cell lines will be used as targeted cells. The killing capacity of allogeneic CAR-T on the Her3-positive cell lines will be evaluated both in vivo and in vitro, and the immune rejection effects of CAR-T will be judged both in the humanized hematopoietic immune system mice and Balb/C mice. The results in this project will provide a new method for immunotherapy of Her3-positive solid tumors, which has important theoretical and practical significance.

近年来放疗或化疗联合CAR-T应用已成为肿瘤治疗的新策略，但利用患者自体T细胞制备CAR-T的方法存在不足。由于经过放疗或化疗，从患者自体分离的T细胞往往数量不足或功能障碍，导致CAR-T制备不仅周期长、费时费力，而且细胞质量和数量不易保证。本项目拟采用人同种异体T细胞建立通用型CAR-T，观察其对肿瘤的杀伤作用和自身免疫原性的消除。项目首先利用CRISPR/Cas9技术敲除健康人T细胞HLA-A和HLA-B座位基因，获得通用型同种异体供体T细胞；然后以Her3为靶标建立人同种异体CAR-T；最后以Her3阳性的乳腺癌、非小细胞肺癌和结肠直肠癌细胞作为靶细胞，在细胞和动物水平检测CAR-T对靶细胞的杀伤作用，并利用人源化造血免疫系统小鼠和Balb/C小鼠对同种异体CAR-T的免疫排斥反应进行评价。本项目将为Her3阳性实体肿瘤的免疫治疗提供新的方法，具有重要的理论和实际意义。

嵌合抗原受体T细胞（Chimeric antigen receptor T cells, CAR-T）疗法是当前肿瘤免疫治疗领域最具有前景的治疗方法之一，该疗法在血液病肿瘤方面取得了令人鼓舞的研究成果。对于实体肿瘤的CAR-T疗法，由于缺乏实体肿瘤特异性靶点、肿瘤微环境的免疫抑制、归巢浸润至肿瘤部位和细胞扩增不足等问题，实体肿瘤的疗效仍有待提高。关于CAR-T细胞的制备，目前多采用患者自体T细胞。然而临床应该中最大的问题是由于前期的放疗或化疗，患者自体T细胞往往数量不足或功能障碍，且体外制备周期长、费时费力，不适合大规模的临床使用。本研究利用同种异体健康人T细胞，制备靶向人表皮生长因子受体2（HER2）的通用型 CAR-T细胞，并检测其对HER2阳性肿瘤细胞的杀伤能力和自身免疫原性的消除。HER2在非小细胞肺癌、乳腺癌、结直肠癌、胃癌等多种实体肿瘤细胞中均有过表达，是经典的实体肿瘤治疗靶点，针对HER2的靶向治疗药物已成功应用于临床治疗。本研究首先利用靶向HER2的单链抗体构建了表达靶向HER2嵌合抗原受体（CAR）的慢病毒载体，该载体与慢病毒包装载体共转染至293-T细胞，包装慢病毒，将慢病毒转导至健康人T细胞制备了靶向HER2的CAR-T。随后利用CRISRP/Cas9技术敲除了CAR-T细胞的T细胞受体(TCR)和人类白细胞抗原I (HLA-I)，制备靶向HER2的通用型CAR-T细胞（Universal CAR-T, U CAR-T），CCK-8试验表明制备的U CAR-T在体外可以杀伤HER2阳性的非小细胞肺癌细胞系PC9。利用NSG小鼠建立了非小细胞肺癌的荷瘤小鼠模型，U CAR-T细胞可以显著抑制肿瘤细胞的生长。混合淋巴细胞试验表明U CAR-T细胞能够抵抗同种异体的免疫排斥。总之本研究为HER2阳性肿瘤患者的免疫治疗提供了新的治疗方法，同时为实现应用通用型同种异体CAR-T治疗实体肿瘤奠定了基础。

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