Intrinsic Functional Potential of NK-Cell Subsets Constrains Retargeting Driven by Chimeric Antigen Receptors

Cancer Immunol Res. 2018 Apr;6(4):467-480. doi: 10.1158/2326-6066.CIR-17-0207. Epub 2018 Feb 19.


Natural killer (NK) cells hold potential as a source of allogeneic cytotoxic effector cells for chimeric antigen receptor (CAR)-mediated therapies. Here, we explored the feasibility of transfecting CAR-encoding mRNA into primary NK cells and investigated how the intrinsic potential of discrete NK-cell subsets affects retargeting efficiency. After screening five second- and third-generation anti-CD19 CAR constructs with different signaling domains and spacer regions, a third-generation CAR with the CH2-domain removed was selected based on its expression and functional profiles. Kinetics experiments revealed that CAR expression was optimal after 3 days of IL15 stimulation prior to transfection, consistently achieving over 80% expression. CAR-engineered NK cells acquired increased degranulation toward CD19+ targets, and maintained their intrinsic degranulation response toward CD19- K562 cells. The response of redirected NK-cell subsets against CD19+ targets was dependent on their intrinsic thresholds for activation determined through both differentiation and education by killer cell immunoglobulin-like receptors (KIR) and/or CD94/NKG2A binding to self HLA class I and HLA-E, respectively. Redirected primary NK cells were insensitive to inhibition through NKG2A/HLA-E interactions but remained sensitive to inhibition through KIR depending on the amount of HLA class I expressed on target cells. Adaptive NK cells, expressing NKG2C, CD57, and self-HLA-specific KIR(s), displayed superior ability to kill CD19+, HLA low, or mismatched tumor cells. These findings support the feasibility of primary allogeneic NK cells for CAR engineering and highlight a need to consider NK-cell diversity when optimizing efficacy of cancer immunotherapies based on CAR-expressing NK cells. Cancer Immunol Res; 6(4); 467-80. ©2018 AACR.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antigens, Neoplasm / genetics
  • Antigens, Neoplasm / immunology
  • Cell Line, Tumor
  • Cytotoxicity, Immunologic
  • Electroporation
  • Gene Expression
  • HLA Antigens / genetics
  • HLA Antigens / immunology
  • Humans
  • Killer Cells, Natural / immunology*
  • Killer Cells, Natural / metabolism*
  • Lymphocyte Activation / immunology
  • Lymphocyte Subsets / immunology*
  • Lymphocyte Subsets / metabolism*
  • Mice
  • NK Cell Lectin-Like Receptor Subfamily C / antagonists & inhibitors
  • Receptors, Antigen, T-Cell / genetics
  • Receptors, Antigen, T-Cell / metabolism*
  • Receptors, Chimeric Antigen / genetics
  • Receptors, Chimeric Antigen / metabolism*
  • Receptors, KIR / antagonists & inhibitors


  • Antigens, Neoplasm
  • HLA Antigens
  • NK Cell Lectin-Like Receptor Subfamily C
  • Receptors, Antigen, T-Cell
  • Receptors, Chimeric Antigen
  • Receptors, KIR