Preparation of highly activated natural killer cells for advanced lung cancer therapy

Abstract

Background: Natural killer (NK) cells can be used as an adoptive immunotherapy to treat cancer patients. Purpose: In this study, we evaluated the efficacy of highly activated NK (HANK) cell immunotherapy in patients with advanced lung cancer. Patients and methods: Between March 2016 and September 2017, we enrolled 13 patients who met the enrollment criteria. Donor peripheral blood monocytes were isolated from patients and the NK cells were expanded. After 12 days of culture, the cells were collected and infused intravenously on days 13 to 15. The enrolled patients received at least one course including three times of infusions. The lymphocyte subsets, cytokine production, and the expression of carcinoembryonic antigen (CEA) and thymidine kinase 1 (TK1) were measured before treatment and after the last infusion. Results: No side effects were observed. After a three-month follow-up, the percentage of patients who achieved stable disease and progressive disease was 84.6% and 15.4%. Moreover, the level of IFN-γ was significantly higher after treatment and the level of CEA decreased substantially. The overall immune function of the patients who received the NK cell therapy remained stable. Conclusion: This is the first study to describe the efficacy of NK cell therapy of patients with advanced lung cancer. These clinical observations demonstrated that NK cell is safe and efficient for advanced lung cancer therapy.

Keywords: adoptive immunotherapy; highly activated natural killer cells; immune function; lung cancer.

Figure 1

NK expansion process. PBMCs were obtained from peripheral blood (approximately 50 mL) of each cancer patient. The plasma was prepared, and the lymphocytes were separated using human lymphocyte separation solution. The lymphocytes were resuspended in NK cell culture medium and cultured. For the first week, the cells were cultured in a T175 culture flask. From day 8, the cells were cultured in a 2-L cell culture bag. The first sterility test was performed on day 8, while the quality control (cell viability ≥80%, NK cell purity ≥80%, endotoxin ≤1 EU/mL, and bacteria, fungi, and mycoplasma culture-negative) and sterility tests were performed on day 12. On day 13, approximately 3–5×10⁹ HANK cells were harvested into a blood transfusion bags each day for infusion at a concentration of approximately 2×10⁷/mL. Abbreviations: NK, natural killer; PBMC, peripheral blood mononuclear cell; HANK, highly activated natural killer.

Figure 2

The CD3^–CD56⁺ NK cell proportion before and after in vitro expansion. (A) The proportion of NK cells before the expansion. (B) The proportion of NK cells after expansion on day 12. Abbreviation: NK, natural killer.

Figure 3

Effects of ex vivo expansion on expression of NK cells. NK cells were stained with conjugated antibodies like PE or APC, etc., against activating receptors such as NKG2D, NKp30, NKp44, and NKp46 or an inhibitory receptor such as NKG2A. Flow cytometry was performed after incubation. The presented data show the results from one donor. The error bars represent SDs, according equal weight to all three assays. Abbreviations: NK, natural killer; NKG2, NK group 2.