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.
OBJECTIVE
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.
Siegler
Uwe
U
Experimental Hematology, Department of Biomedicine, University Hospital Basel, Basel, Switzerland.
Meyer-Monard
Sandrine
S
Jörger
Simon
S
Stern
Martin
M
Tichelli
André
A
Gratwohl
Alois
A
Wodnar-Filipowicz
Aleksandra
A
Kalberer
Christian P
CP
D000818:Animals; D024221:Antineoplastic Protocols; D018929:Cell Culture Techniques / Q000379:methods*; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D003602:Cytotoxicity, Immunologic; D019983:Guideline Adherence; D006801:Humans; D016219:Immunotherapy, Adoptive; D007262:Infusions, Intravenous; D007519:Isoantigens / Q000276:immunology; D007694:Killer Cells, Natural / Q000276:immunology / Q000378:metabolism* / Q000473:pathology / Q000637:transplantation; D015470:Leukemia, Myeloid, Acute / Q000276:immunology / Q000473:pathology / Q000628:therapy*; D051379:Mice; D016513:Mice, SCID; D009368:Neoplasm Transplantation; D054340:Receptors, KIR / Q000378:metabolism; D047368:Tumor Burden
D007519:Isoantigens; D054340:Receptors, KIR