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Frontiers of Medicine

ISSN 2095-0217

ISSN 2095-0225(Online)

CN 11-5983/R

Postal Subscription Code 80-967

2018 Impact Factor: 1.847

Front. Med.    2019, Vol. 13 Issue (1) : 57-68    https://doi.org/10.1007/s11684-019-0683-y
RESEARCH ARTICLE
Chimeric antigen receptor T cell targeting EGFRvIII for metastatic lung cancer therapy
Zhao Zhang1,2, Jun Jiang3, Xiaodong Wu1,2, Mengyao Zhang4, Dan Luo1,2, Renyu Zhang1,2, Shiyou Li3(), Youwen He5(), Huijie Bian1,2(), Zhinan Chen1,2()
1. National Translational Science Center for Molecular Medicine, Xi’an 710032, China
2. Department of Cell Biology, Fourth Military Medical University, Xi’an 710032, China
3. Beijing Institute of Genomics, Chinese Academy of Science, Beijing 100101, China
4. Beijing Institute of Biotechnology, Academy of Military Medical Sciences, Beijing 100142, China
5. Department of Immunology, Duke University Medical Center, Durham, NC 27710, USA
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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-g, 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.

Keywords chimeric antigen receptor T cells      epidermal growth factor receptor      lung cancer      immunotherapy      tumor immunology     
Corresponding Authors: Shiyou Li,Youwen He,Huijie Bian,Zhinan Chen   
Just Accepted Date: 29 December 2018   Online First Date: 18 February 2019    Issue Date: 12 March 2019
 Cite this article:   
Zhao Zhang,Jun Jiang,Xiaodong Wu, et al. Chimeric antigen receptor T cell targeting EGFRvIII for metastatic lung cancer therapy[J]. Front. Med., 2019, 13(1): 57-68.
 URL:  
http://academic.hep.com.cn/fmd/EN/10.1007/s11684-019-0683-y
http://academic.hep.com.cn/fmd/EN/Y2019/V13/I1/57
Fig.1  Expression of chimeric antigen receptor (CAR) of cell membrane. EGFRvIII-CAR was constructed by the extracellular domain of single chain antibody against EGFRvIII, and the intracellular domain consisting of CD28, 4-1BB, and CD3z. MOCK-CAR only contains the transmembrane and intracellular domains.
Fig.2  Establishment of retrovirus-producing cell line and optimization of culture conditions. (A) CAR expression plasmid was transfected into Phoenix Eco cells by lipotamine 2000 (scale bar= 50 μm). (B) PT67 cells were infected by the supernatant of the Phoenix cells (scale bar= 50 μm). (C - E) Virus titer of the producing cell line harvested from RV-E70 cultured in different conditions. (F) Virus titers of RV-E70 and MOCK-C291 virus producing cell line after cultured and passaged for 10 generations. *P<0.05, **P <0.01.
Fig.3  Isolation and infection of PBMCs. (A) Ratio of CD3+CD4+ and CD3+CD8+ T cells in PBMCs before (above) and after (below) cultured in vitro. (B) Expression of CD25 and CD69 before (above) and after (below) T cells were activated by OKT-3 in vitro. (C) Expression of EGFRvIII scFv on uninfected PBMCs (above) and infected PBMCs were detected by FACS analysis. (D) Western blot analysis indicated both wild type CD3ζand chimeric antigen receptor CD3ζ were expressed in CAR-T cells.
Fig.4  Proliferation of PBMCs, MOCK-CART and EGFRvIII-CART in vitro. (A) Cytokine dependent cell proliferation. (B) Antigen dependent cell proliferation. n = 8, **P<0.01.
Fig.5  Cytotoxicity of PBMCs, MOCK-CART and EGFRvIII-CART in vitro. (A) Percentage of target cell lysis at different E:T ratios cocultured with tumor cells in vitro detected by LDH release assay. n = 8, **P<0.01 vs. other groups, ##P<0.01 vs. A549-blank and A549-EGFRvIII-blank groups. (B) Cytotoxicity of PBMCs, MOCK-CART and EGFRvIII-CART analyzed by RTCA system at the E:T ratio of 10:1. n = 3.
Fig.6  Expression and release of cytokines of effector cells cocultured with target cells in vitro. (A) Representative results of expression of IFN-γ, TNF-α, granzyme B and perforin in PBMCs, MOCK-CART and EGFRvIII-CART detected by intracellular staining. (B) Cytokines released from effector cells in the supernatant analyzed by ELISA. n = 6, **P <0.01.
Fig.7  Antitumor response of EGFRvIII-CART cells in vivo. (A) NPG mice were randomized into four groups and received i.v. injections of A549 tumor cells on day 0. Effector cells were injected for 3 times on day 7, 9, and 11 after tumor cell injection. PBMCs were collected once a week, and mice were observed for 90 days. (B) Lung tissue samples of mice in different groups. Normal lung tissue was pink and smooth and had no lesions, and tumor tissues appeared dark red and had granular nodules. (C) Weight of lung tissue in each group. n = 8, **P<0.01. (D) Survival curve of mice in each group. (E) Percentage of human CD3+ T cells in peripheral blood of mice in each groups analyzed by FACS. n = 8.
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