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CRISPR-Cas9 mediated LAG-3 disruption in CAR-T cells

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Abstract

T cells engineered with chimeric antigen receptor (CAR) have been successfully applied to treat advanced refractory B cell malignancy. However, many challenges remain in extending its application toward the treatment of solid tumors. The immunosuppressive nature of tumor microenvironment is considered one of the key factors limiting CAR-T efficacy. One negative regulator of Tcell activity is lymphocyte activation gene-3 (LAG-3). We successfully generated LAG-3 knockout Tand CAR-T cells with high efficiency using CRISPR-Cas9 mediated gene editing and found that the viability and immune phenotype were not dramatically changed during in vitro culture. LAG-3 knockout CAR-T cells displayed robust antigen-specific antitumor activity in cell culture and in murine xenograft model, which is comparable to standard CAR-T cells. Our study demonstrates an efficient approach to silence immune checkpoint in CAR-T cells via gene editing.

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Acknowledgements

We would like to thank Junning Wei and Yi Yang (Beijing Cord Blood Bank) for their help in preparing the cord blood samples. This work was supported by the National Natural Science Foundation of China (No. 31471215), the Strategic Priority Research Program of the Chinese Academy of Sciences (No. XDA01010409), and the National High Technology Research and Development Program of China (863 Program, No. 2015AA020307). Haoyi Wang is supported by the “Young Thousand Talent Project.”

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Authors and Affiliations

  1. Department of Hematology, Xuanwu Hospital, Capital Medical University, Beijing, 100053, China

    Yongping Zhang & Changqing Xia

  2. State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, 100190, China

    Xingying Zhang, Chen Cheng, Wei Mu, Xiaojuan Liu, Na Li, Xiang Liu & Haoyi Wang

  3. University of Chinese Academy of Sciences, Beijing, 100049, China

    Xingying Zhang, Wei Mu & Haoyi Wang

  4. Graduate School, University of Science and Technology of China, Hefei, 230026, China

    Chen Cheng

  5. Department of Pathology, Immunology and Laboratory Medicine, University of Florida, Florida, FL, 32611, USA

    Changqing Xia

  6. Beijing Cord Blood Bank, Beijing, 100176, China

    Xiaofei Wei

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  1. Yongping Zhang
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Correspondence to Changqing Xia or Haoyi Wang.

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Zhang, Y., Zhang, X., Cheng, C. et al. CRISPR-Cas9 mediated LAG-3 disruption in CAR-T cells. Front. Med. 11, 554–562 (2017). https://doi.org/10.1007/s11684-017-0543-6

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  • DOI: https://doi.org/10.1007/s11684-017-0543-6

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