HIGHGLY EFFICIENT ACTIVATION AND EXPANSION OF NATURAL KILLER CELLS FOR CLINICAL USE IN CANCER IMMUNOTHERAPY

Abstract

Natural killer (NK) cells can detect and kill tumor cells and infusion of NK cells to cancer patients may be a promising option to treat cancer. In this context, ex vivo expansion is used to produce large quantities of activated NK cells, because sufficient numbers of these effector cells are essential for successful NK cell based adoptive cancer immunotherapy. The development of efficient NK cell expansion protocols and the transfer of these protocols to clinically applicable methods represent a major challenge. To overcome this issue, the aim of my project was to develop a clinically applicable method that yields large numbers of highly functional NK cells. First, a fully automated technical process was developed to activate and expand NK cells with (interleukin) IL-2 and irradiated clinical-grade feeder cells (EBV-LCL). In comparison to the manual procedure, the automated process yielded similar NK cells in terms of cell numbers, surface marker profile, gene expression and in vitro effector functions. Upon expansion, NK cells up-regulated functional surface molecules, such as TRAIL, FasL, NKG2D and DNAM-1, they increased the production of interferon (IFN)-g and tumor necrosis factor (TNF)-a and they became more cytotoxic against tumor cell lines. Next, because in the used protocol NK cell expansion was restricted to a period of 2-4 weeks, a more efficient protocol for long-term expansion was developed. Manual NK cell expansion with EBV-LCL and IL-2 induced a 22– fold mean NK cell expansion after one week that was significantly increased to 53–fold by addition of IL-21. Furthermore, repeated stimulation with irradiated EBV-LCL and IL-2 and addition of IL-21 at the initiation of the culture allowed sustained NK cell proliferation with 1011–fold NK cell expansion after six weeks, which is an unprecedented high expansion rate not achieved by any other method so far. Most importantly, adoptive transfer of NK cells expanded with this optimized protocol led to significant inhibition of tumor growth in a melanoma xenograft mouse model, proofing the therapeutic efficacy of the ex vivo generated NK cells. This anti-tumor efficacy was superior over that from conventionally IL-2 activated NK cells, demonstrating that the improved NK cell expansion method enhanced not only the quantity but also the therapeutic quality of NK cells. In conclusion, the outcome of this project is a fully automated process for ex vivo production of NK cells and an optimized protocol for NK cell expansion with unparalleled efficacy. The expanded NK cells possess potent anti-tumor features and showed therapeutic efficacy in a preclinical melanoma xenograft model. Thereby, the project serves clinical needs and makes it possible to generate high cell doses of functional NK cells for the use in cancer immunotherapy.