Expression of chimeric antigen receptors in natural killer cells with a regulatory-compliant non-viral method

Cancer Gene Ther. 2010 Mar;17(3):147-54. doi: 10.1038/cgt.2009.61. Epub 2009 Sep 11.


Natural killer (NK) cells hold promise for cancer therapy. NK cytotoxicity can be enhanced by expression of chimeric antigen receptors that re-direct specificity toward target cells by engaging cell surface molecules expressed on target cells. We developed a regulatory-compliant, scalable non-viral approach to engineer NK cells to be target-specific based on transfection of mRNA encoding chimeric receptors. Transfection of eGFP mRNA into ex vivo expanded NK cells (N=5) or purified unstimulated NK cells from peripheral blood (N=4) resulted in good cell viability with eGFP expression in 85+/-6% and 86+/-4%, 24 h after transfection, respectively. An mRNA encoding a receptor directed against CD19 (anti-CD19-BB-z) was also transfected into NK cells efficiently. Ex vivo expanded and purified unstimulated NK cells expressing anti-CD19-BB-z exhibited enhanced cytotoxicity against CD19(+) target cells resulting in > or =80% lysis of acute lymphoblastic leukemia and B-lineage chronic lymphocytic leukemia cells at effector target ratios lower than 10:1. The target-specific cytotoxicity for anti-CD19-BB-z mRNA-transfected NK cells was observed as early as 3 h after transfection and persisted for up to 3 days. The method described here should facilitate the clinical development of NK-based antigen-targeted immunotherapy for cancer.

MeSH terms

  • Antigens, CD19 / metabolism*
  • Cell Line
  • Cell Line, Tumor
  • Cell Survival / genetics
  • Cell Survival / physiology
  • Cells, Cultured
  • Electroporation
  • Green Fluorescent Proteins / genetics
  • Green Fluorescent Proteins / metabolism
  • Humans
  • Immunophenotyping
  • Killer Cells, Natural / cytology
  • Killer Cells, Natural / metabolism*
  • Receptors, Antigen / genetics
  • Receptors, Antigen / metabolism*
  • Transfection


  • Antigens, CD19
  • Receptors, Antigen
  • Green Fluorescent Proteins