Interleukin-2 Gene Transduction in Human Natural Killer Cells Augments Their Survival and Anti-Leukemic Capacity
Authors: Imai Chihaya , Takachi Takayuki , Iwabuchi Haruko , Imamura Masaru , Nemoto Tae , Campana Dario , Uchiyama Makoto 
Published: April 2020

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 The superiority of NK-gIL2 was particularly evident in experiments where exogenous IL-2 was withdrawn for 72 hours before testing.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.