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 for a relationship between NK cell dose and tumor response. Here we present a functionally closed in vitro system using irradiated EBV-LCL feeder cells resulting in the large scale expansion of highly cytotoxic clinical grade NK cells.
In contrast to NK cell expansion protocols that require culturing in plastic flasks and multiple rounds of stimulation with feeder cells, the expansion technique presented here utilized sterile bags, required only a single round of stimulation with irradiated EBV-LCL feeder cells, and achieved substantial NK expansions in the range of 250-850 fold over a 2-3 week interval. With a starting population of 200 million immunomagnetic bead-purified CD3-
NK cells isolated after a typical 15 liter apheresis, this expansion technique would achieve a final NK-cell product in the range of 3 × 1010
cells, a number that would seem sufficient for phase I studies. To address the safety of using EBV-LCL cells for NK-cell expansion, 3 expanded NK-cell products were tested by in situ
hybridization for EBV-encoded early small RNAs (EBER) and were all found to be negative. The TM-LCL feeder cell line used here to expand NK cells was previously used by others to expand T-cell lines in vitro
utilizing GMP-compliant components (18
). To avoid expanding T cells which proliferate rapidly under these culture conditions (data not shown), a 2 step CD3+
T cell depletion followed by a CD56+
selection was used to enrich for an NK- cell population that typically had <0.5% T-cell contamination.
The most efficient large scale NK-cell expansions were achieved when cells were cultured in Baxter “Lifecell” bags. In contrast, cultures generated in Teflon-coated bags resulted in relatively limited NK-cell expansions (data not shown). The viability and expansion rates of NK cells were at their greatest 9 to 15 days following the initiation of cell cultures and declined after 21 days. Several attempts to re-expand NK cells with EBV-LCL feeder cells after cells had been cultured for ≥14 days were mostly unsuccessful. Regardless of culture vessels used for expansions of NK cells, the phenotype and lytic activity of the expanded cells were similar.
Although NK cells can be activated by IL-2, IL-2 alone fails to expand NK cells in vitro. In contrast, NK cells stimulated with EBV-LCL feeders expanded dramatically, had an activated phenotype, and as a consequence of upregulated expression of NKG2C, NKG2D, FasL, TRAIL and granzymes A and B, were significantly more cytotoxic against tumor cells compared to fresh NK cells. We also observed that expression of CD244 (2B4) and CD48 was augmented in expanded compared to resting NK cells. The function of CD48 and CD244 on expanded human NK cells is not entirely understood. Although one study reported increased expression of CD244 could have an inhibitory effect on the function of NK cells (20
), murine data have shown that homotypic interactions between these molecules prevent fratricide and enhance NK cell expansion and cytolytic activity (21
Compared to non-expanded NK cells, expanded NK cells secreted either spontaneously or following co-culture with tumor targets (K562 and RCC cells) higher levels of IFNγ, IL-2, FasL and TRAIL. In contrast, non-expanded NK cells secreted higher levels of IL-1ra which was not produced by expanded cells. An unexpected and previously unobserved finding was that TNFa secretion increased when NK cells were co-cultured with bortezomib-treated RCC cells. The biologic significance of this finding is unknown, although TNFa can be directly cytotoxic to tumor cells and can have a positive immunoregulatory function, inducing dendritic cell maturation, activation, and Ag cross-presentation resulting in augmented T-cell cytokine secretion (22
). Contrary to previous reports, only very low levels of IL-10 were detected in expanded NK cells cultured in IL-2. In contrast to when IL-12 is combined with IL-2, IL-2 alone is a weak stimulator of IL-10 secretion. IL-10 has been shown to have anti-inflammatory effects, inhibiting macrophage and DC activation and maturation and secretion of multiple pro-inflammatory cytokines (24
). Therefore, lack of IL-10 secretion would seem desirable when expanded NK cells are used in the context of tumor immunotherapy.
The net effect of changes in NK cell phenotype and cytokine secretion resulted in expanded NK cells having markedly higher levels of cytotoxicity against tumor cells compared to non-expanded cells. Although, it is likely that both an increase in expression of activating receptors and molecules that induce tumor apoptosis (i.e. TRAIL, FasL, granzyme B, etc.) in expanded NK cells contributed to their enhanced cytotoxicity, blocking experiments to define the exact contribution of individual pathways to augmented NK-cell cytolytic function were not performed in this analysis.
Previously, we and others have shown that the proteosome inhibitor bortezomib enhances TRAIL-mediated cytotoxicity against tumor cells in vitro (26
) and in vivo (6
). In experiments conducted in this study, we observed that lysis of bortezomib-treated RCC tumors was dramatically higher with expanded compared to resting NK cells, providing strong evidence that increased surface expression of TRAIL on expanded NK cells substantially augmented their tumor lysis at least in part via TRAIL apoptotic pathways. In contrast, it is unlikely that changes in NK cell inhibitory receptors played any role in augmenting NK-cell cytotoxicity, as CD158b/KIR2DL2/3, KIR3DL1 and NKG2A expression remained unchanged or increased slightly following NK cell expansion.
The changes in phenotype and maintenance of cytotoxicity against tumor cells by expanded NK cells were dependent on IL-2. Withdrawal of IL-2 from expanded NK cell populations rapidly resulted in substantial reductions in NK-cell killing of tumor cells. Whether the exogenous administration of IL-2 would be required to maintain high levels of NKG2D, TRAIL, and tumor cytotoxicity in vivo of adoptively-infused expanded NK cells is currently being investigated in an animal model.
The ability to cryopreserve and subsequently thaw NK cells while maintaining their cytolytic activity could logistically facilitate clinical trials evaluating multiple rounds of adoptive NK cell infusions. Although expanded NK cells that were frozen then subsequently thawed maintained high viability, their cytolytic capacity was substantially lower than that of expanded NK cells that had never undergone cryopreservation. Thawed NK cells had lower surface expression of TRAIL and NKG2D and were more likely to contain populations that were dim or negative for CD16. These findings suggest thawed adoptively-infused NK cells might have reduced cytotoxic potential compared to expanded NK cells that are maintained fresh in culture. Importantly, the cytotoxicity of expanded NK cells that were frozen then thawed could be rescued by culturing in IL-2-containing medium for 16 hours, although the overall viability of these populations was lower than that of non-thawed cells.
In conclusion, we show a method for the large scale production of in vitro-expanded NK cells using irradiated EBV-LCL feeder cells and a functionally closed “bag-based” culture system. In vitro-expanded NK cells had altered cytokine secretion profiles, were phenotypically distinct from non-expanded NK cells and were significantly more cytotoxic to tumor cells.
Expanded cells had increased surface expression of the NKG2D and TRAIL and greatly enhanced TRAIL-mediated cytotoxicity against bortezomib-treated tumors compared to non-expanded NK cells. Based on these findings, a phase I trial has recently been initiated in patients with advanced metastatic tumors and hematological malignancies to investigate the safety and anti-tumor effects of escalating doses of adoptively-infused ex vivo-expanded autologous NK cells. NK-cell doses in this trial will range from 5 × 106 to 108 NK cells/kg and will be given every 3 weeks following treatment with bortezomib and concomitant with IL-2 administration.