Abstract
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.
Highlights
The incidence of cancer in China has increased rapidly since the 21st century [1,2]
The cytolytic activities of the chimeric antigen receptor T (CAR-T) cells or Peripheral blood mononuclear cells (PBMCs) were measured by lactate dehydrogenase (LDH) release assay
The amount of cytokine released by the effect cells was measured through enzyme-linked immunosorbent assay (ELISA)
Summary
The incidence of cancer in China has increased rapidly since the 21st century [1,2]. In 2015, approximately 4 292 000 of cancer cases was reported in China, with mortality reaching up to 2 814 000 and five-year-overall survival rate of 36.9%. Lung cancer is the most common incident cancer and the leading cause of cancer death [3]. Despite the continuous progress in traditional treatments, including surgery, chemotherapy, and radiotherapy, the prognosis of lung cancer, especially for advanced stage, is far from satisfactory [4]. The metastasis in advanced stage remains a tricky problem in medical practice [5]. The immune system plays an important role in inhibiting
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