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
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)
- et al.
Biology and clinical relevance of human natural killer cells
Blood
(1990) Biology of natural killer cells
Adv Immunol
(1989)- et al.
Natural killer cell receptors: new biology and insights into the graft-versus-leukemia effect
Blood
(2002) - et al.
The proteasome inhibitor PS-341 sensitizes neoplastic cells to TRAIL-mediated apoptosis by reducing levels of c-FLIP
Blood
(2003) - et al.
Natural-killer-cell-based treatment in haematopoietic stem-cell transplantation
Best Pract Res Clin Haematol
(2006) - et al.
A new method for in vitro expansion of cytotoxic human CD3−CD56+ natural killer cells
Hum Immunol
(2001) - et al.
Genetic modification of primary natural killer cells overcomes inhibitory signals and induces specific killing of leukemic cells
Blood
(2005) - et al.
Increased proliferation, lytic activity, and purity of human natural killer cells cocultured with mitogen-activated feeder cells
Cell Immunol
(1991) - et al.
Enhanced cytotoxicity of allogeneic NK cells with killer immunoglobulin-like receptor ligand incompatibility against melanoma and renal cell carcinoma cells
Blood
(2004) - et al.
Manufacturing of gene-modified cytotoxic T lymphocytes for autologous cellular therapy for lymphoma
Cytotherapy
(2006)
Requirement of homotypic NK-cell interactions through 2B4 (CD244)/CD48 in the generation of NK effector functions
Blood
NK cell and DC interactions
Trends Immunol
Differential effects of donor T-cell cytokines on outcome with continuous bortezomib administration after allogeneic bone marrow transplantation
Blood
Cited by (244)
Attack of the clones: An NK cell origins story
2023, Molecular Therapy OncolyticsExpanded NK cells used for adoptive cell therapy maintain diverse clonality and contain long-lived memory-like NK cell populations
2023, Molecular Therapy OncolyticsAllogeneic natural killer cell therapy
2023, BloodHarnessing natural killer cells for the treatment of multiple myeloma
2022, Seminars in Oncology