Good manufacturing practice-compliant cell sorting and large-scale expansion of single KIR-positive alloreactive human natural killer cells for multiple infusions to leukemia patients

Cytotherapy. 2010 Oct;12(6):750-63. doi: 10.3109/14653241003786155.


Background aims: Alloreactive natural killer (NK) cells are potent effectors of innate anti-tumor defense. The introduction of NK cell-based immunotherapy to current treatment options in acute myeloid leukemia (AML) requires NK cell products with high anti-leukemic efficacy optimized for clinical use.

Methods: We describe a good manufacturing practice (GMP)-compliant protocol of large-scale ex vivo expansion of alloreactive NK cells suitable for multiple donor lymphocyte infusions (NK-DLI) in AML. CliniMACS-purified NK cells were cultured in closed air-permeable culture bags with certified culture medium and components approved for human use [human serum, interleukin (IL)-2, IL-15 and anti-CD3 antibody] and with autologous irradiated feeder cells.

Results: NK cells (6.0 ± 1.2 x 10(8)) were purified from leukaphereses (8.1 ± 0.8 L) of six healthy donors and cultured under GMP conditions. NK cell numbers increased 117.0 ± 20.0-fold in 19 days. To reduce the culture volume associated with expansion of bulk NK cells and to expand selectively the alloreactive NK cell subsets, GMP-certified cell sorting was introduced to obtain cells with single killer immunoglobulin-like receptor (KIR) specificities. The subsequent GMP-compliant expansion of single KIR+ cells was 268.3 ± 66.8-fold, with a contaminating T-cell content of only 0.006 ± 0.002%. The single KIR-expressing NK cells were cytotoxic against HLA-mismatched primary AML blasts in vitro and effectively reduced tumor cell load in vivo in NOD/SCID mice transplanted with human AML.

Conclusions: The approach to generating large numbers of GMP-grade alloreactive NK cells described here provides the basis for clinical efficacy trials of NK-DLI to complement and advance therapeutic strategies against human AML.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antineoplastic Protocols
  • Cell Culture Techniques / methods*
  • Cell Line, Tumor
  • Cell Proliferation*
  • Cytotoxicity, Immunologic
  • Guideline Adherence
  • Humans
  • Immunotherapy, Adoptive*
  • Infusions, Intravenous
  • Isoantigens / immunology
  • Killer Cells, Natural / immunology
  • Killer Cells, Natural / metabolism*
  • Killer Cells, Natural / pathology
  • Killer Cells, Natural / transplantation
  • Leukemia, Myeloid, Acute / immunology
  • Leukemia, Myeloid, Acute / pathology
  • Leukemia, Myeloid, Acute / therapy*
  • Mice
  • Mice, SCID
  • Neoplasm Transplantation
  • Receptors, KIR / metabolism
  • Tumor Burden


  • Isoantigens
  • Receptors, KIR