近来，以T细胞为基础的免疫治疗虽取得了显著的成功，但在实体瘤的治疗中仍面临着严峻的挑战。申请人团队前期成功建立了CRISPR-Cas9介导的T细胞PD-1分子敲除技术，并在体内外证实肿瘤穿透肽iRGD能够显著促进T细胞进入肿瘤实质。基于此，我们认为通过选择特异性的抗原靶点，增加T细胞的肿瘤靶向穿透性，同时敲除T细胞的PD-1分子，可克服肿瘤免疫耐受，提高T细胞的抗肿瘤效果。本项目拟以癌睾抗原NY-ESO-1特异性表达的实体瘤为研究对象，构建NY-ESO-1TCR工程化的T细胞，通过CRISPR-Cas9技术敲除TCR-T的PD-1分子，以逆转TCR-T细胞的免疫耐受；并将iRGD肽嵌插到T细胞膜上，赋予TCR-T细胞靶向穿透肿瘤组织的能力；通过体内外模型研究改造后的T细胞在实体瘤中的抗肿瘤作用和机理。预期结果有望建立一种新型的T细胞治疗模式，为其潜在的临床应用提供理论和实验依据。

Recently, T cells-based immunotherapy has achieved great success. However, it still faces severe challenges in the treatment of solid tumors. In our previous study we have successfully established CRISPR-Cas9 mediated PD-1 knock-out technology in human T cells. Also we confirmed that tumor penetrating peptide iRGD can significantly promote T cells to penetrate into the tumor tissue deeply and extensively both in vivo and in vitro. Based on this, we speculate that by selecting specific antigen targets, increasing the penetrability of T cells, and knocking the PD-1 molecules out of T cells, we will overcome the tumor immune-tolerance and enhance the anti-tumor effect of T cells. This project will intend to use cancer testis antigen NY-ESO-1 positive solid tumors as the study object. NY-ESO-1 TCR engineered T cells will be constructed and PD-1 will be disrupted by CRISPR-Cas9 system to reverse acquired immune tolerance. Besides, the iRGD peptide will be inserted into the membrane of T cells, endowing TCR-T cells with penetrating power. Anti-tumor effect and mechanism will be studied both in vitro and in vivo. This project is expected to establish a new technology platform of T cell therapy for clinical applications.