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

Hematology Branch, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland, USA.
Cytotherapy (Impact Factor: 3.29). 04/2009; 11(3):341-55. DOI: 10.1080/14653240902807034
Source: PubMed


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.
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.
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.
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.

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Available from: J. Philip Mccoy, Sep 03, 2014
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    • "Various methods for large-scale and clinical-grade ex vivo NK-cell expansion have been reported with this aim (83–92). Due to the advantage of aseptic conditions in a closed system, peripheral blood mononuclear cells (PBMCs) collected by leukapheresis are frequently used as source for goods manufacturing practice (GMP)-compliant expansion of NK cells (84, 85, 87). "
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    ABSTRACT: Natural killer (NK) cells play an essential role in the fight against tumor development. Over the last years, the progress made in the NK-cell biology field and in deciphering how NK-cell function is regulated, is driving efforts to utilize NK-cell-based immunotherapy as a promising approach for the treatment of malignant diseases. Therapies involving NK cells may be accomplished by activating and expanding endogenous NK cells by means of cytokine treatment or by transferring exogenous cells by adoptive cell therapy and/or by hematopoietic stem cell transplantation. NK cells that are suitable for adoptive cell therapy can be derived from different sources, including ex vivo expansion of autologous NK cells, unstimulated or expanded allogeneic NK cells from peripheral blood, derived from CD34+ hematopoietic progenitors from peripheral blood and umbilical cord blood, and NK-cell lines. Besides, genetically modified NK cells expressing chimeric antigen receptors or cytokines genes may also have a relevant future as therapeutic tools. Recently, it has been described the derivation of large numbers of functional and mature NK cells from pluripotent stem cells, both embryonic stem cells and induced pluripotent stem cells, which adds another tool to the expanding NK-cell-based cancer immunotherapy arsenal.
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    • "Various feeder cell-based systems have been used for NK cell expansion from peripheral blood mononuclear cells (PBMC), including third-party Epstein–Barr virus transformed lymphoblastoid B cell lines (EBV–BLCL), genetically modified K562 cells, or irradiated autologous cells (21–24). Ex vivo expansion of bulk peripheral NK cells using third-party EBV–BLCL feeders approximately yields a 180-fold NK cell expansion after 2 weeks of culture (22). Another expansion technique, yielding clinical valuable amounts of NK cells, is based upon K562 cell feeder double-transduced with IL-15 and 4-1BB (CD137) co-stimulatory ligand (K562–mb15–41BBL) (23). "
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    ABSTRACT: Blurring the boundary between innate and adaptive immune system, natural killer (NK) cells are widely recognized as potent anti-leukemia mediators. Alloreactive donor NK cells have been shown to improve the outcome of allogeneic stem-cell transplantation for leukemia. In addition, in vivo transfer of NK cells may soon reveal an important therapeutic tool for leukemia, if tolerance to NK-mediated anti-leukemia effects is overcome. This will require, at a minimum, the ex vivo generation of a clinically safe NK cell product containing adequate numbers of NK cells with robust anti-leukemia potential. Ideally, ex vivo generated NK cells should also have similar anti-leukemia potential in different patients, and be easy to obtain for convenient clinical scale-up. Moreover, optimal clinical protocols for NK therapy in leukemia and other cancers are still lacking. These and other issues are being currently addressed by multiple research groups. This review will first describe current laboratory NK cell expansion and differentiation techniques by separately addressing different NK cell sources. Subsequently, it will address the mechanisms known to be responsible for NK cell alloreactivity, as well as their clinical impact in the hematopoietic stem cells transplantation setting. Finally, it will briefly provide insight on past NK-based clinical trials.
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    • "However, the clinical responses of these studies have been unsatisfactory, and thus further efforts will be needed. Development of ex vivo expansion and activation of NK cells has been extensively assessed by groups at St. Jude Children's Research Hospital (Leung et al., 2005), the National Institutes of Health (Berg et al., 2009), Singapore (Suck et al., 2011), and the Karolinska Institute (Sutlu et al., 2010). These methods have generally been based on CD56-positive selection with or without CD3-depletion/CD3-suppression followed by IL-2 and/or IL-15 stimulation, and in some case virus-infected feeder cells have been used to obtain highly activated NK cells. "
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    ABSTRACT: Adoptive immunotherapy using natural killer (NK) cells has been a promising treatment for intractable malignancies; however, there remain a number of difficulties with respect to the shortage and limited anticancer potency of the effector cells. We here established a simple feeder-free method to generate purified (>90%) and highly activated NK cells from human peripheral blood-derived mononuclear cells (PBMCs). Among the several parameters, we found that simply 1) CD3-depletion, 2) high dose IL-2, and 3) use of a specific culture medium were sufficient to obtain highly purified, expanded (~200-fold) and activated CD3-/CD56+ NK cells from PBMCs that we named zenithal-NK cells (Z-NK). Almost all Z-NK cells expressed the lymphocyte-activated marker CD69, and showed dramatically high expression of activation receptors (i.e., NKG2D), interferon-γ, perforin and granzyme B. Importantly, only 2 hours of reaction at an effector/target ratio of 1:1 was sufficient to kill almost all K562 cells, and the antitumor activity was also replicated in tumor-bearing mice in vivo. Cytolysis was specific for various tumor cells, but not for normal cells, irrespective of MHC class I expression. These findings strongly indicate that Z-NK cells are purified, expanded, and near-fully activated human NK cells and warrant further investigation in a clinical setting.
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