Clinical-grade ex vivo-expanded human natural killer cells up-regulate activating receptors and death receptor ligands and have enhanced cytolytic activity against tumor cells.
OBJECTIVE
Cancer immunotherapy involving natural killer (NK) cell infusions and administration of therapeutic agents modulating the susceptibility of tumors to NK-cell lysis has been proposed recently. We provide a method for expanding highly cytotoxic clinical-grade NK cells in vitro for adoptive transfer following bortezomib treatment in patients with advanced malignancies.


METHODS
NK cells were expanded with irradiated Epstein-Barr virus-transformed lymphoblastoid cells. Expanded cells were evaluated for their phenotype, cytotoxicity, cytokine secretion, dependence on interleukin (IL)-2 and ability to retain function after cryopreservation.


RESULTS
A pure population of clinical-grade NK cells expanded 490+/-260-fold over 21 days. Expanded NK cells had increased TRAIL, FasL and NKG2D expression and significantly higher cytotoxicity against bortezomib-treated tumors compared with resting NK cells. Expanded NK cells, co-cultured with K562 and renal cell carcinoma tumor targets, secreted significantly higher levels of soluble Fas ligand 6; fgjhd IFN-gamma, GM-CSF, TNF-alpha, MIP-1alpha and MIP-1beta compared with resting NK cells. Secretion of the above cytokines and NK-cell cytolytic function were IL-2 dose dependent. Cryopreservation of expanded NK cells reduced expression of NKG2D and TRAIL and NK-cell cytotoxicity, although this effect could be reversed by exposure of NK cells to IL-2.


CONCLUSIONS
We describe a method for large-scale expansion of NK cells with increased expression of activating receptors and death receptor ligands resulting in superior cytotoxicity against tumor cells. This ex vivo NK-cell expansion technique is currently being utilized in a clinical trial evaluating the anti-tumor activity of adoptively infused NK cells in combination with bortezomib.
Berg
Maria
M
Hematology Branch, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland, USA.
Lundqvist
Andreas
A
McCoy
Philip
P
Samsel
Leigh
L
Fan
Yong
Y
Tawab
Abdul
A
Childs
Richard
R
D001897:Boronic Acids / Q000494:pharmacology; D000069286:Bortezomib; D002292:Carcinoma, Renal Cell / Q000188:drug therapy / Q000276:immunology* / Q000473:pathology; D018929:Cell Culture Techniques; D049109:Cell Proliferation; D015925:Cryopreservation; D016207:Cytokines / Q000378:metabolism; D003602:Cytotoxicity, Immunologic / Q000276:immunology; D053222:Fas Ligand Protein / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D016130:Immunophenotyping; D007167:Immunotherapy / Q000379:methods; D020014:K562 Cells; D007694:Killer Cells, Natural / Q000276:immunology / Q000378:metabolism* / Q000473:pathology; D008213:Lymphocyte Activation; D055655:NK Cell Lectin-Like Receptor Subfamily K / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D011719:Pyrazines / Q000494:pharmacology; D053221:TNF-Related Apoptosis-Inducing Ligand / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D015854:Up-Regulation
D001897:Boronic Acids; D016207:Cytokines; D053222:Fas Ligand Protein; D055655:NK Cell Lectin-Like Receptor Subfamily K; D011719:Pyrazines; D053221:TNF-Related Apoptosis-Inducing Ligand; D000069286:Bortezomib