iRGD修饰联合PD-1基因敲除增强NY-ESO-1 TCR-T 抗肿瘤的研究

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.

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。

肢端型黑色素瘤是我国最常见的恶性黑色素瘤亚型，其肿瘤突变负荷较低，抑制性肿瘤免疫微环境特征显著。应用PD-1单抗联合NY-ESO-1 TCR-T细胞疗法治疗肢端型黑色素瘤具有极大潜力，但是，TCR-T 细胞疗法用于临床仍会面临诸多困境。为克服以上困境，本研究相继开展实验发现：（1）我国肢端型恶性黑色素瘤患者NY-ESO-1抗原表达阳性率最高，但强阳性率偏低，同时NY-ESO-1抗原表达阳性患者肿瘤免疫微环境中T淋巴细胞浸润显著，提示肿瘤细胞的NY-ESO-1抗原表达联合过继细胞疗法治疗恶性黑色素瘤具有临床可行性。（2）NY-ESO-1 TCR-T和PD-1敲除的T淋巴细胞可维持正常细胞活性和免疫效应功能； PD-1-KO-NY-ESO-1 TCR-T淋巴细胞体外免疫效应强于NY-ESO-1 TCR-T和PD-1敲除的T淋巴细胞，但是细胞增殖活性略有下降；iRGD嵌插的T 淋巴细胞在体外3D 肿瘤细胞球及体内腹腔播散肿瘤模型中可提高T 淋巴细胞的肿瘤靶向穿透能力及抗肿瘤作用。（3）通过瘤内注射抗原肽可将抗原肽负载到肿瘤细胞表面的MHC分子上，增强特异性CTL的靶向识别与杀伤；联合PD-1单抗可阻断免疫微环境中PD-1/PD-L1等负性分子的抑制效应，进而显著抑制了肿瘤生长速率。（4）将肿瘤靶向纳米脂质体（iRGD-NO-NP）递送至肿瘤微环境，可实现主动肿瘤靶向，通过NO释放，改善肿瘤血管灌注，促进肿瘤微环境中肿瘤相关巨噬细胞（TAM）由M2 型转化为M1型，使肿瘤转化为利于免疫治疗的“热”肿瘤。综上所述，本研究通过制备NY-ESO-1 TCR-T淋巴细胞，予以iRGD嵌插修饰，并与PD-1单抗、相应抗原肽及NO负载纳米脂质体等多种疗法相联合，成功为恶性黑色素瘤提供了一种个体化与靶向精准免疫治疗的新模式并明确其临床应用潜力。