National Translational Science Center for Molecular Medicine, Xi'an, 710032, China.
Fourth Military Medical University, Xi'an, 710032, China.
Front Med. 2019 Feb;13(1):57-68. doi: 10.1007/s11684-019-0683-y. Epub 2019 Feb 5.
Lung cancer is the most common incident cancer and the leading cause of cancer death. In recent years, the development of tumor immunotherapy especially chimeric antigen receptor T (CAR-T) cell has shown a promising future. Epidermal growth factor receptor variant III (EGFRvIII) is a tumor-specific mutation expressed in various types of tumors and has been detected in non-small cell lung cancer with a mutation rate of 10%. Thus, EGFRvIII is a potential antigen for targeted lung cancer therapy. In this study, CAR vectors were constructed and transfected into virus-packaging cells. Then, activated T cells were infected with retrovirus harvested from stable virus-producing single clone cell lines. CAR expression on the surfaces of the T cells was detected by flow cytometry and Western blot. The function of CAR-T targeting EGFRvIII was then evaluated. The EGFRvIII-CAR vector was successfully constructed and confirmed by DNA sequencing. A stable virus-producing cell line was produced from a single clone by limited dilution. The culture conditions for the cell line, including cell density, temperature, and culture medium were optimized. After infection with retrovirus, CAR was expressed on more than 90% of the T cells. The proliferation of CAR-T cells were induced by cytokine and specific antigen in vitro. More importantly, EGFRvIII-CART specifically and efficiently recognized and killed A549-EGFRvIII cells with an effector/target ratio of 10:1 by expressing and releasing cytokines, including perforin, granzyme B, IFN-γ, and TNF-α. The in vivo study indicated that the metastasis of A549-EGFRvIII cells in mice were inhibited by EGFRvIII-CART cells, and the survival of the mice was significantly prolonged with no serious side effects. EGFRvIII-CART showed significantly efficient antitumor activity against lung cancer cells expressing EGFRvIII in vivo and in vitro. Therefore, CAR-T targeting EGFRvIII is a potential therapeutic strategy in preventing recurrence and metastasis of lung cancer after surgery.
肺癌是最常见的癌症，也是癌症死亡的主要原因。近年来，肿瘤免疫治疗的发展，尤其是嵌合抗原受体 T 细胞（CAR-T）的发展，展现了广阔的前景。表皮生长因子受体变异 III（EGFRvIII）是一种在多种肿瘤中表达的肿瘤特异性突变，在非小细胞肺癌中的突变率为 10%。因此，EGFRvIII 是一种潜在的针对肺癌的治疗靶点。本研究构建了 CAR 载体并转染到病毒包装细胞中，然后用收获自稳定病毒产生单克隆细胞系的逆转录病毒感染激活的 T 细胞。通过流式细胞术和 Western blot 检测 T 细胞表面 CAR 的表达。然后评估了靶向 EGFRvIII 的 CAR-T 的功能。成功构建了 EGFRvIII-CAR 载体，并通过 DNA 测序进行了验证。通过有限稀释从单个克隆中产生了稳定的病毒产生细胞系。优化了细胞系的培养条件，包括细胞密度、温度和培养基。感染逆转录病毒后，CAR 在超过 90%的 T 细胞上表达。细胞因子和特异性抗原在体外诱导 CAR-T 细胞的增殖。更重要的是，EGFRvIII-CART 通过表达和释放细胞因子，包括穿孔素、颗粒酶 B、IFN-γ 和 TNF-α，以效应细胞/靶细胞比为 10:1 特异性和有效地识别和杀伤 A549-EGFRvIII 细胞。体内研究表明，EGFRvIII-CART 细胞抑制了 A549-EGFRvIII 细胞在小鼠体内的转移，并且小鼠的存活率显著延长，没有严重的副作用。EGFRvIII-CART 在体内和体外对表达 EGFRvIII 的肺癌细胞具有显著的抗肿瘤活性。因此，针对 EGFRvIII 的 CAR-T 是预防肺癌手术后复发和转移的一种潜在治疗策略。
