免疫检查点分子Tim-3沉默的CAR-T细胞的抗肿瘤作用及机制研究

Chimeric antigen receptors engineered T cells (CAR-T) have been modified with a recombinant receptor molecule recognize cell surface antigens directly and are independent of major histocompatibility complex (MHC) restrictions. CAR-T cell therapy has made significant achievements in hematological malignancies, but the efficacy is not ideal for solid tumors. In patients with malignancies, immune check point Tim-3 was expressed upon T cells, which could lead to promote peripheral immune tolerance directly or indirectly and inhibit the anti-tumor immunity. Our previous study found that 8% of the cultured CAR-T cells expressed Tim-3, and after co-culture with tumor cells, the rate further rise to about 30%. Therefore, we hypothesized that Tim-3 is the key immune check point for limiting CAR-T therapy, and CAR-T cells lack Tim-3 could enhance the tumor-killing efficacy for the treatment of solid tumors. In this project, we aim to create Tim-3 silenced CAR-T cells, reveal its anti-tumor function and mechanism, and evaluate its safety, in order to 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）技术将识别肿瘤抗原能力和T细胞介导的杀伤功能结合于一体，具有肿瘤靶向性、MHC非限制性等优点。CAR-T细胞治疗已经在血液系统肿瘤中取得了显著成绩，而其对实体肿瘤疗效仍不理想。在肿瘤病人中，免疫检查点分子T细胞免疫球蛋白黏液素3（Tim-3）常表达于T细胞表面，可直接或间接地促进外周免疫耐受，抑制机体抗肿瘤免疫。我们的前期研究发现，CAR-T细胞体外培养后Tim-3表达升高至约8%，而与肿瘤细胞共培养后，Tim-3进一步升高至约30%。因此，我们推测Tim-3是限制CAR-T 疗法对实体瘤疗效的关键免疫检查点，Tim-3的缺失能够增强CAR-T细胞对实体肿瘤的疗效。在本项目中，我们拟制备一种免疫检查点分子Tim-3沉默的CAR-T 细胞，研究其抗实体肿瘤活性，阐明其抗肿瘤作用机制，并对其安全性进行评估，以期提高CAR-T 细胞治疗实体肿瘤的疗效。

嵌合型抗原受体修饰的T细胞（CAR-T）技术将识别肿瘤抗原能力和T细胞介导的杀伤功能结合于一体，具有肿瘤靶向性、MHC非限制性等优点。CAR-T细胞治疗已经在血液系统肿瘤中取得了显著成绩，而其对实体肿瘤疗效仍不理想。在肿瘤病人中，免疫检查点分子T细胞免疫球蛋白黏液素3（Tim-3）常表达于T细胞表面，可直接或间接地促进外周免疫耐受，抑制机体抗肿瘤免疫。我们的前期研究发现，CAR-T细胞体外培养后Tim-3表达升高。因此，我们推测Tim-3是限制CAR-T疗法对实体瘤疗效的关键免疫检查点，Tim-3的缺失能够增强CAR-T细胞对实体肿瘤的疗效。在本项目中，我们筛选出干扰效果满意的Tim-3-shRNA序列，成功构建Tim-3沉默的第三代嵌合抗原受体基因，并完成了其对外周血T淋巴细胞的修饰使其在T细胞内稳定的表达。Tim-3沉默的CAR-T细胞在体外大量扩增培养后，约60%的淋巴细胞为CD8+T细胞，并大部分(约80%)为中心记忆T细胞(CD45RO+CD62L+)，表明Tim-3沉默后其表型不受影响。与没有沉默的CAR-T细胞相比，Tim-3沉默的CAR-T细胞体外杀伤能力更强，并大量释放以IFN-γ和TNF-α为主的Th1细胞因子。同时，Tim-3沉默的CAR-T细胞在体内也能够显著地抑制肿瘤的形成与生长。因此，本项目制备了一种不受Tim-3免疫检查点调控的肿瘤特异性效应T细胞，在进入实体瘤内部后，其免疫活性将大大提高并可以显著提高对实体瘤的抗肿瘤活性，有望突破CAR-T细胞对实体瘤治疗的限制瓶颈。