Elsevier

Cytotherapy

Volume 11, Issue 3, 2009, Pages 341-355
Cytotherapy

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

https://doi.org/10.1080/14653240902807034Get rights and content

Background aims

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-γ, GM-CSF, TNF-α, MIP-1α and MIP-1β 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.

Introduction

Natural killer (NK) cells are innate immune lymphocytes that are identified by the expression of CD56 surface antigen (Ag) and lack of CD3 [1,2]. NK cells have the ability to kill target cells directly through the release of granules containing perforin and serine proteases (granzymes) and/or by surface-expressed ligands that engage and activate death receptors expressed on target cells. They can also mediate antibody (Ab)-dependent cellular cytotoxicity (ADCC) via the membrane receptors FcγRIII (CD16) [3]. Unlike T cells, NK cells do not require the presence of a specific tumor Ag to kill cancer cells, rather their recognition of targets is regulated through a balance of activating and inhibitory signals. Even in the presence of an activating ligand, inhibitory ligands can initiate overriding signals that culminate in a net suppression of NK-cell function. The inactivation of NK cells by self-HLA molecules is a potential mechanism by which malignant cells evade host NK-cell mediated immunity [4,5].

Recently, we and others observed that the proteasome inhibitor bortezomib (Velcade, PS-341) sensitized malignant cells to tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL)-dependent NK-cell lysis 6., 7., 8.. This effect appeared to overcome killer cell immunogloblin – like receptors (KIR)-mediated suppression of NK-cell function, enhancing autologous NK-cell cytotoxicity against patient tumor cells in vitro. Based on this finding, we pursued a method for large-scale expansion of clinical-grade NK cells to evaluate the anti-cancer effects of autologous adoptively infused NK cells following bortezomib treatment in patients with cancer.

Only a few trials investigating adoptive NK-cell infusions in humans with cancer have been conducted to date (reviewed in [9,10]). Because NK cells represent only a minor fraction of human lymphocytes, the small number of NK cells isolated following a typical leukapheresis procedure has precluded phase I trials evaluating NK-cell dose-dependent tumor cytotoxicity in humans with cancer.

Several methods for expansion and activation of NK cells in vitro have been investigated, including overnight and long-term culture with cytokines [11,12] and the use of peripheral blood mononuclear cells (PBMC) [13], K562 cells [14] and Epstein–Barr virus-transformed lymphoblastoid cell lines (EBV-LCL) as feeder cells [15, 16]. We have previously developed [17] and now optimized an improved method for large-scale expansion of human NK cells in bags using irradiated EBV-LCL feeder cells and interleukin (IL)-2. The EBV-LCL cell line, used in our studies, has been proven previously [18] to be safe for use in clinical trials; cells have met release test criteria for the presence of viral contaminants and infectious EBV. We explored the phenotype, cytotoxic potential against tumor cells and cytokine secretion of these expanded NK cells compared to freshly isolated cells. We also investigated the effects of IL-2 withdrawal on phenotype and function of expanded cells and, finally, the effects of cryopreservation and thawing.

We show that NK-cell phenotype and function are modulated following in vitro expansion. As a consequence of these changes, NK-cell cytolytic activity against bortezomib-treated tumors is significantly higher with expanded compared with fresh NK cells.

Section snippets

Cell isolation, culture and cryopreservation

Human NK cells were isolated from PBMC obtained from multiple different healthy volunteers and one patient with metastatic sarcoma. Depletion of CD3+ T cells and a subsequent positive selection of CD56+ cells were performed on a CliniMACS system (Miltenyi Biotec Inc., Auburn, CA, USA). The cells were analyzed immediately after purification for phenotypic markers and cytotoxicity and were then either expanded or cryopreserved for future analysis. For NK expansions the following parameters were

Expansion kinetics of NK cells

Previously, small-scale laboratory-based experiments have shown that NK-cell lines can be expanded in vitro using a variety of different methods [16,17]. We sought to optimize the conditions for large-scale NK-cell expansions using GMP conditions for NK-cell-based clinical trials in humans with cancer.

When allogeneic PBMC were used as feeder cells, NK cells were most efficiently expanded by 25 Gy-irradiated feeder cells added to cultures at a 20:1 ratio in culture medium containing 500 IU/mL

Discussion

Although there has been increased interest in exploring the anti-tumor effects of adoptively infused NK cells in cancer patients, the small number of cells isolated following a typical apheresis procedure has precluded trials assessing a relationship between NK-cell dose and tumor response. We present a functionally closed in vitro system using irradiated EBV-LCL feeder cells resulting in large-scale expansion of highly cytotoxic clinical-grade NK cells.

In contrast to NK-cell expansion

Acknowledgements

This research was supported by the intramural research program of NIH, National Heart, Lung, and Blood Institute, Hematology Branch. We wish to acknowledge ACKC (Action to Cure Kidney Cancer) and The Dean R. O'Neill Memorial Fellowship for generous contributions supporting this research. The authors would also like to thank Dr E. J. Read, Dr David Stroncek, Dr Hanh Khuu, Vicki Fellows and Virginia David-Ocampo from the Department of Transfusion Medicine in NIH for their valuable contribution to

References (27)

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