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

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. 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. 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). 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.