Abstract
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 × 108) 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.
Acknowledgments
The authors would like to thank S. Sendelov for technical help, V. Jäggin for cell sorting, M. Dessing for performing GMP cell sorting at the BD Europe Technology Center, Allschwil, Switzerland, and H. Himmelreich for critically reading the manuscript. The authors would like to thank C. Arber and the Hematology Diagnostic Team, University Hospital Basel, for their support and for performing HLA typing.
This work was supported by grants from the Oncosuisse OCS-02175-02-2008, Stiftung für Hämatologische Forschung, Novartis Stiftung 06C84, Stiftung zur Krebsbekämpfung 226, Freiwillige Akademische Gesellschaft and Krebsliga beider Basel 04/2008.
Disclosure of interest: The authors declare no potential conflict of interest.
Supplementary material available online