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 Weste 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-y,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.