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Background <p>Colorectal cancer (CRC) is a highly prevalent malignancy of the digestive system worldwide, necessitating the development of safe and effective therapeutic strategies. While chimeric antigen receptor T-cell (CAR-T) therapy has achieved remarkable success in hematologic malignancies, its application in solid tumors remains limited. This study aimed to explore CAR-based technology for treating solid tumors, focusing on CRC.</p> Methods <p>A CAR gene sequence targeting EpCAM for NK cells was designed and synthesized. The CAR fragment, along with co-expressed IL-21 and CCL19 genes, was cloned into the pCHD-CMV-MCS-EF1-copGFP-T2A-puro lentiviral vector. Successful construction was confirmed by restriction enzyme digestion, PCR, and sequencing. The vector was packaged using a lentiviral three-plasmid system, and viral particles were concentrated via PEG-8000 centrifugation. Transgenic NK-92 cells (21X19EpCAM-CAR-NK) were generated by infecting NK cells with concentrated lentivirus.</p> Results <p>The pCHD-21X19EpCAM-CAR recombinant lentiviral vector was successfully constructed and packaged, yielding viral titers of 5 × 10<sup>5</sup> TU/mL. Transgenic NK-92 cells expressing the CAR construct were obtained, with a 1940.17 ± 402.22-fold expansion of NK cells from peripheral blood mononuclear cells (PBMCs).</p> Conclusions <p>This study successfully establishes a robust platform for CAR-NK cell therapy against colorectal cancer through the construction of the pCHD-21X19EpCAM-CAR vector and its application in primary human NK cells. Our work introduces significant novelty by developing the first lentiviral vector that co-expresses the immunomodulators IL-21 and CCL19 with an EpCAM-targeting CAR. This unique design contributed to an unprecedented 1940-fold expansion of NK cells from PBMCs, significantly surpassing existing methods. Together, these achievements provide a compelling preclinical foundation for a new therapeutic strategy aimed at overcoming the immunosuppressive tumor microenvironment in CRC.</p>

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Construction of the pCHD-21X19EpCAM-CAR lentiviral vector and generation of CAR-engineered natural killer cells from human PBMCs

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年4月6日

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  • Bingping Wang,
  • Jinkai Duan,
  • Hulin Ma,
  • Meng Ren,
  • Liangquan Chen,
  • Rina Su,
  • Hao Zhang,
  • Shuang Zhang,
  • Yanwei Gao

摘要

Background

Colorectal cancer (CRC) is a highly prevalent malignancy of the digestive system worldwide, necessitating the development of safe and effective therapeutic strategies. While chimeric antigen receptor T-cell (CAR-T) therapy has achieved remarkable success in hematologic malignancies, its application in solid tumors remains limited. This study aimed to explore CAR-based technology for treating solid tumors, focusing on CRC.

Methods

A CAR gene sequence targeting EpCAM for NK cells was designed and synthesized. The CAR fragment, along with co-expressed IL-21 and CCL19 genes, was cloned into the pCHD-CMV-MCS-EF1-copGFP-T2A-puro lentiviral vector. Successful construction was confirmed by restriction enzyme digestion, PCR, and sequencing. The vector was packaged using a lentiviral three-plasmid system, and viral particles were concentrated via PEG-8000 centrifugation. Transgenic NK-92 cells (21X19EpCAM-CAR-NK) were generated by infecting NK cells with concentrated lentivirus.

Results

The pCHD-21X19EpCAM-CAR recombinant lentiviral vector was successfully constructed and packaged, yielding viral titers of 5 × 105 TU/mL. Transgenic NK-92 cells expressing the CAR construct were obtained, with a 1940.17 ± 402.22-fold expansion of NK cells from peripheral blood mononuclear cells (PBMCs).

Conclusions

This study successfully establishes a robust platform for CAR-NK cell therapy against colorectal cancer through the construction of the pCHD-21X19EpCAM-CAR vector and its application in primary human NK cells. Our work introduces significant novelty by developing the first lentiviral vector that co-expresses the immunomodulators IL-21 and CCL19 with an EpCAM-targeting CAR. This unique design contributed to an unprecedented 1940-fold expansion of NK cells from PBMCs, significantly surpassing existing methods. Together, these achievements provide a compelling preclinical foundation for a new therapeutic strategy aimed at overcoming the immunosuppressive tumor microenvironment in CRC.