Clinical grade manufacturing of genetically modified, CAR-expressing NK-92 cells for the treatment of ErbB2-positive malignancies

Cancer Immunol Immunother. 2018 Jan;67(1):25-38. doi: 10.1007/s00262-017-2055-2. Epub 2017 Sep 6.


Background: The NK-92/5.28.z cell line (also referred to as HER2.taNK) represents a stable, lentiviral-transduced clone of ErbB2 (HER2)-specific, second-generation CAR-expressing derivative of clinically applicable NK-92 cells. This study addresses manufacturing-related issues and aimed to develop a GMP-compliant protocol for the generation of NK-92/5.28.z therapeutic doses starting from a well-characterized GMP-compliant master cell bank.

Materials and methods: Commercially available GMP-grade culture media and supplements (fresh frozen plasma, platelet lysate) were evaluated for their ability to support expansion of NK-92/5.28.z. Irradiation sensitivity and cytokine release were also investigated.

Results: NK-92/5.28.z cells can be grown to clinically applicable cell doses of 5 × 108 cells/L in a 5-day batch culture without loss of viability and potency. X-Vivo 10 containing recombinant transferrin supplemented with 5% FFP and 500 IU/mL IL-2 in VueLife 750-C1 bags showed the best results. Platelet lysate was less suited to support NK-92/5.28.z proliferation. Irradiation with 10 Gy completely abrogated NK-92/5.28.z proliferation and preserved viability and potency for at least 24 h. NK-92/5.28.z showed higher baseline cytokine release compared to NK-92, which was significantly increased upon encountering ErbB2(+) targets [GZMB (twofold), IFN-γ (fourfold), IL-8 (24-fold) and IL-10 (fivefold)]. IL-6 was not released by NK cells, but was observed in some stimulated targets. Irradiation resulted in upregulation of IL-8 and downregulation of sFasL, while other cytokines were not impacted.

Conclusion: Our concept suggests NK-92/5.28.z maintenance culture from which therapeutic doses up to 5 × 109 cells can be expanded in 10 L within 5 days. This established process is feasible to analyze NK-92/5.28.z in phase I/II trials.

Keywords: CAR; Cancer immunotherapy; Glioblastoma; HER2; NK-92; Natural killer cells.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Blood Platelets / physiology
  • Cell Culture Techniques*
  • Cell Line, Tumor
  • Cell Proliferation
  • Clinical Trials as Topic
  • Culture Media
  • Cytokines / metabolism
  • Cytotoxicity, Immunologic
  • Genetic Therapy
  • Humans
  • Immunotherapy, Adoptive / methods*
  • Killer Cells, Natural / immunology*
  • Killer Cells, Natural / transplantation
  • Neoplasms / immunology
  • Neoplasms / therapy*
  • Radiation Tolerance
  • Receptor, ErbB-2 / metabolism*


  • Culture Media
  • Cytokines
  • ERBB2 protein, human
  • Receptor, ErbB-2