Expanded and Activated Natural Killer Cells for Immunotherapy of Hepatocellular Carcinoma

Abstract

Viral infection of the liver is a major risk factor for hepatocellular carcinoma (HCC). Natural killer (NK) cells recognize virally infected and oncogenically transformed cells, suggesting a therapeutic role for NK-cell infusions in HCC. Using the K562-mb15-41BBL cell line as a stimulus, we obtained large numbers of activated NK cells from the peripheral blood of healthy donors. Expanded NK cells exerted remarkably high cytotoxicity against HCC cell lines, which was generally much higher than that of unstimulated or IL2-activated NK cells. In immunodeficient NOD/scid IL2RGnull mice engrafted with Hep3B, treatment with expanded NK cells markedly reduced tumor growth and improved overall survival. HCC cells exposed for 48 hours to 5 mmol/L of sorafenib, a kinase inhibitor currently used for HCC treatment, remained highly sensitive to expanded NK cells. HCC cell reductions of 39.2% to 53.8% caused by sorafenib in three cell lines further increased to 80.5% to 87.6% after 4 hours of culture with NK cells at a 1:1 effector-to-target ratio. NK-cell cytotoxicity persisted even in the presence of sorafenib. We found that NKG2D, an NK-cell-activating receptor, was an important mediator of anti-HCC activity. We therefore enhanced its signaling capacity with a chimeric NKG2D-CD3z-DAP10 receptor. This considerably increased the anti-HCC cytotoxicity of expanded NK cells in vitro and in immunodeficient mice. The NK expansion and activation method applied in this study has been adapted to clinical-grade conditions. Hence, these results warrant clinical testing of expanded NK-cell infusions in patients with HCC, possibly after genetic modification with NKG2D-CD3z-DAP10.

Novel Strategy to Expand Super-Charged NK Cells with Significant Potential to Lyse and Differentiate Cancer Stem Cells: Differences in NK Expansion and Function between Healthy and Cancer Patients

“…It remains to be determined if such signals by engineered K562s (Table S1 in Supplementary Material) are inferior to those delivered by the OCs since the rate of expansion of NK cells by engineered K562 cells is lower by a magnitude of 100-fold from OC-expanded NK cells (Table S1 and Figures S3A,B in Supplementary Material). In addition, continuous stimulation by the engineered K562s is required to maintain NK expansion whereas only one stimulation with OCs is sufficient to expand super-charged NK cells for over a month (51). Moreover, OC-expanded NK cells, unlike primary NK cells, withstand freezing temperatures quite well and retain their super-charged characteristics and expansion rates with no loss of viability or function (Figure S5 in Supplementary Material).…”

The Rise of Allogeneic Natural Killer Cells As a Platform for Cancer Immunotherapy: Recent Innovations and Future Developments

“…Besides specific targeting of tumor antigens and strategies to promote ADCC, Nkarta therapeutics developed NKG2D CARs (NKG2D-CD3ζ-DAP10) using NK-92 cells and PBNK cells, which exhibited enhanced cytotoxicity against osteosarcoma and hepatocellular carcinoma when compared to activated and expanded PBNK cells (100, 101). mRNA-based genetic engineering has been used to enhance migration of NK cells to tumors.…”

Natural Killer Cell Expansion with Autologous Feeder Layer and Anti-CD3 Antibody for Immune Cell Therapy of Hepatocellular Carcinoma

“…The cytotoxic effect of NK-92MI cell line (Shi et al, 2016) and also expanded NK cells using irradiated HFWT cells (Peng et al, 2004) or genetically modified K562 feeder cells (Kamiya et al, 2016) against target HCC cells were reported in previous studies. Even though these methods are very efficient, but as they were cancer cell-based approaches, therefore, they can be dangerous if any viable tumoric cells have been mixed with the expanded NK cells.…”

“…One of the problem solving approaches is transplantation of NK cells to HCC patients, but the amount of NK cells derived from various sources is less than clinical use. Therefore, many techniques have been proposed for activation and expansion of NK cells (Cho and Campana, 2009;Ahn et al, 2013;Kamiya et al, 2016;Lee et al, 2017).…”