Interleukin-2 Gene Transduction in Human Natural Killer Cells Augments Their Survival and Anti-Leukemic Capacity (2008)

Abstract Novel therapies are required to circumvent the resistance of leukemic cells to chemotherapy and improve cure rates. Infusions of natural killer (NK) cells might enhance the anti-leukemic effects of chemotherapy and hematopoietic stem cell transplantation. Indeed, haploidentical NK cells were shown to induce remission in patients with high-risk acute myeloid leukemia (AML; Miller at al. Blood105: 3051, 2005). Current NK-cell therapy protocols include administration of interleukin (IL)-2 to sustain the survival and activation of the infused NK cells in vivo. In this study, we tested whether enforcing the expression of the IL-2 gene in NK cells would abrogate their requirement for exogenous IL-2, prolong their survival and augment their anti-leukemic capacity., We previously reported a method that induces NK cells to proliferate and reliably allows their genetic modification (Imai et al, Blood106: 376, 2005). We used this method to expand primary NK cell from healthy individuals for 7 days. After depletion of residual T cells, NK cells were transduced with a retroviral vector containing human IL-2 cDNA and GFP. Fourteen days after transduction, more than 99% of cells were CD3− CD56+ NK cells; median transduction efficiency, assessed by GFP positivity, was 70.0% (40.4%–91.4%, n=9). NK cells expressing IL-2 cDNA (NK-gIL2) showed intracellular expression of IL-2 and secreted IL-2 (1.3 ± 0.5 U/106 cells/24 hours). Karyotyping of NK-gIL2 cells propagated for more than 6 weeks revealed no chromosomal aberration. The cytotoxicity of NK-gIL2 cells against the AML cell line K562 was similar to that of NK cells transduced with a vector containing GFP only when the cytotoxicity assay was performed with 4-hour coculture. However, NK-gIL2 exerted a much more potent cytotoxicity if the cultures were prolonged to 7 days. For example, residual leukemia was 58.4% ± 1.3% versus 97.3% ± 2.1% in control culture at a 0.5:1 E:T ratio. The superiority of NK-gIL2 was particularly evident in experiments where exogenous IL-2 was withdrawn for 72 hours before testing. In 4-hour cytotoxicity assays performed this way, cytotoxicity against K562 cells, as well as the T-ALL cell line MOLT4 and the Burkitt lymphoma cell line Daudi was significantly higher than that exerted by control NK cells (e.g., K562 killing: 82.9% ± 1.1% versus 30.9% ± 4.1% at a 1:1 ratio). NK-gIL2 showed significantly lower apoptotic rates than control NK cells after 72 hours of culture without exogenous IL-2 by Annexin V/7AAD staining. Survival after 7 days of culture in the absence of exogenous cytokines was also much higher in NK-gIL2 than NK cells transduced with GFP only. Cell death of NK cells may occur after killing of K562. We found that this was significantly lower in NK-gIL2 than in control NK cells, thus explaining the more potent anti-leukemic activity of NK-gIL2 in long-term culture. The addition of more than 100 U/ml IL-2 was needed to suppress cell death in control NK cells to a degree similar to that measured for NK-gIL2 cells in cultures without exogenous IL-2, suggesting that secretion of low amounts of IL-2 in an autocrine/paracrine fashion could sustain NK cell survival better than exogenous IL-2. We also found that the expression of the natural cytotoxicity receptor p30 and p44 was higher in NK-gIL2 cells than in control NK cells after 72-hour of IL-2 depletion, in line with their higher cytotoxic activities after IL-2 withdrawal. Our results indicate that endogenous IL-2 production can replace the requirement for exogenous IL-2 in NK cells and significantly improve their survival and cytotoxicity. An in vivo infusion of 1010 NK-gIL2 cells would result in the secretion of 1 × 104 U IL-2, an amount much lower than that administered in most immunotherapy protocols (1–2 × 106 U/sqm/day). These data suggest that infusion of NK-gIL2 could be more effective than that of unmodified NK cells while minimizing adverse effects of IL-2 administration.