The naturally occurring oncolytic virus, reovirus, exhibits cytotoxic effects on cancer cells with an activated RAS-signaling pathway. In addition to their direct cytotoxic effects they also activate the innate and adaptive immune responses to facilitate tumor clearance. Reovirus is safe, and currently is in clinical testing for the treatment of multiple clinical cancer histologies. NK cells are innate immune effectors that mediate antibody dependent cellular cytotoxicity (ADCC) against tumor cells. Here we investigated the direct effect of reovirus on NK cell mediated ADCC against two EGFR (Epidermal Growth Factor) positive colorectal cancer cell lines: DLD-1 (KRAS mut) and Caco-2 (KRAS WT). NK cells isolated from human PBMCs were cultured with 1pfu of reovirus for 12 hrs. These reovirus treated NK cells were co-cultured with DLD-1 cells or Caco2 cells (E:T-1:1) coated with increasing concentrations anti-EGFR antibody cetuximab. ADCC was measured after 4hrs using a lactate dehydrogenase (LDH) based cytotoxicity assay. We observed that the reovirus treated NK cells (Reo-NK cells) exhibited a ~16-fold increase in cytotoxicity against DLD-1 (16.3% ±1.5, n=3) compared to untreated NK cells (NK cells), even in the absence of any cetuximab antibody. In the presence of cetuximab antibody, NK cells showed a dose dependent increase in ADCC, with maximum ADCC, observed at 0.1 µg/ml of cetuximab (DLD-NK: 33.4%± 7.1, Caco2-NK: 5.7%± 0.071, n=3). Interestingly, Reo-NK cells showed maximum ADCC even at 0.01 µg /ml of cetuximab (DLD1-Reo-NK: 39.1±7.4, DLD1-NK: 26.7±2.4%; Caco2-Reo-NK: 9.2±0.4, Caco2-NK: 4.0±0.8, n=3). To further investigate the factors contributing to the increased cytotoxic potential of Reo-NK cells we performed flow cytometry analysis to determine the expression of activation and degranulation markers on NK cells. We observed that in presence of tumor cells Reo-NK cells exhibited a 2-fold increased expression of activation marker CD69 when compared to untreated NK cells (Reo-NK:70.4%, NK:35.2%). A similar increase was also observed when Reo-NK cells were co-cultured with target cells coated with 0.01 µg /ml of cetuximab (Reo-NK: 85.1%; NK: 72.7%). Expression of the degranulation marker CD107a is increased on NK cells during ADCC. There was a ~3-fold increase in expression of CD107a on Reo-NK cells when compared to NK cells (Reo-NK: 14.6%; NK: 4.45%). To further investigate if perforin-based cytotoxicity was responsible for the increased ADCC by Reo-NK cells, we treated Reo-NK and NK cells with concanamycin A (CMA), an inhibitor of perforin. We found that CMA treatment reduced Reo-NK cell mediated ADCC by 1.5 fold (CMA treated-28.2%, CMA untreated 18.9%) indicating a perforin-mediated cytotoxicity contributes to the increased cytotoxicity of Reo-NK cells. Thus, in this study our results demonstrated that human NK cells when treated with reovirus show increases in activation, degranulation and cytotoxicity when compared to untreated NK cells. Importantly, reovirus treated NK cells lowered the threshold of cetuximab required to achieve maximum ADCC. We propose that reovirus activated NK cells are a potential candidate for cell based immunotherapy in combination with FDA approved tumor targeting antibodies. Further studies are ongoing to investigate the underlying mechanisms that contribute to the increase in cytotoxicity by NK cells treated with reovirus.
Citation Format: Xing Zhao, Narendiran Rajasekaran, Cariad Chester, Atsushi Yonezawa, Suparna Dutt, Matt Coffey, Holbrook Kohrt. Reovirus treated NK cells exhibit enhanced cetuximab mediated antibody- dependent cellular cytotoxicity against colorectal cancer cell lines. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B082.
- ©2016 American Association for Cancer Research.