title: Fully automated expansion and activation of clinical-grade natural killer cells for adoptive immunotherapy.
abstract: OBJECTIVE
  Ex vivo expansion of natural killer (NK) cells is a strategy to produce large numbers of these effector cells for immunotherapy. However, the transfer of bench-top expansion protocols to clinically applicable methods is challenging for NK cell-based therapy because of regulatory aspects and scale-up issues. Therefore, we developed an automated, large-scale NK cell expansion process.
  
  
  METHODS
  Enriched NK cells were expanded with interleukin-2 and irradiated clinical-grade Epstein-Barr virus-transformed lymphoblastoid feeder cells with the use of an automated system in comparison to manual expansion, and the cells were investigated for their functionality, phenotype and gene expression.
  
  
  RESULTS
  Automated expansion resulted in a mean 850-fold expansion of NK cells by day 14, yielding 1.3 (± 0.9) × 10(9) activated NK cells. Automatically and manually produced NK cells were comparable in target cell lysis, degranulation and production of interferon-γ and tumor necrosis factor-α and had similar high levels of antibody-dependent cellular cytotoxicity against rituximab-treated leukemic cells. NK cells after automated or manual expansion showed similar gene expression and marker profiles. However, expanded NK cells differed significantly from primary NK cells including upregulation of the functional relevant molecules TRAIL and FasL and NK cell-activating receptors NKp30, NKG2D and DNAM-1. Neither automatically nor manually expanded NK cells showed reduced telomere length indicative of a conserved proliferative potential.
  
  
  CONCLUSIONS
  We established an automated method to expand high numbers of clinical-grade NK cells with properties similar to their manually produced counterparts. This automated process represents a highly efficient tool to standardize NK cell processing for therapeutic applications.
authors.affiliation: Miltenyi Biotec GmbH, Bergisch Gladbach, Germany; Innate Immunity Group, German Cancer Research Center, Heidelberg, Germany. Electronic address: markusgr@miltenyibiotec.de.
authors.forename: Markus
authors.initials: M
authors.lastname: Granzin
authors.affiliation: Miltenyi Biotec GmbH, Bergisch Gladbach, Germany.
authors.forename: Stephanie
authors.initials: S
authors.lastname: Soltenborn
authors.affiliation: Miltenyi Biotec GmbH, Bergisch Gladbach, Germany.
authors.forename: Sabine
authors.initials: S
authors.lastname: Müller
authors.affiliation: Miltenyi Biotec GmbH, Bergisch Gladbach, Germany.
authors.forename: Jutta
authors.initials: J
authors.lastname: Kollet
authors.affiliation: National Heart, Lung and Blood Institute, Hematology Branch, Bethesda, Maryland, USA.
authors.forename: Maria
authors.initials: M
authors.lastname: Berg
authors.affiliation: Innate Immunity Group, German Cancer Research Center, Heidelberg, Germany.
authors.forename: Adelheid
authors.initials: A
authors.lastname: Cerwenka
authors.affiliation: National Heart, Lung and Blood Institute, Hematology Branch, Bethesda, Maryland, USA.
authors.forename: Richard W
authors.initials: RW
authors.lastname: Childs
authors.affiliation: Miltenyi Biotec GmbH, Bergisch Gladbach, Germany. Electronic address: volker@miltenyibiotec.de.
authors.forename: Volker
authors.initials: V
authors.lastname: Huppert
mesh_terms: D000920:Antibody-Dependent Cell Cytotoxicity / Q000276:immunology; D001331:Automation; D015415:Biomarkers / Q000378:metabolism; D049109:Cell Proliferation; D002478:Cells, Cultured; D005434:Flow Cytometry; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D006801:Humans; D016219:Immunotherapy, Adoptive / Q000379:methods*; D020014:K562 Cells; D007694:Killer Cells, Natural / Q000166:cytology* / Q000276:immunology*; D007700:Kinetics; D008213:Lymphocyte Activation / Q000276:immunology*; D059505:Telomere Homeostasis
chemical_list: D015415:Biomarkers
keywords: CliniMACS Prodigy; NK cell expansion; NK cell therapy; automation; immunotherapy; natural killer cells