Natural killer (NK) lymphocytes exhibit potent responses against transformed and pathogen-infected cells. NK cells can act rapidly as they monitor the bloodstream and tissues for tumorigenic and infected cells that lack “self” markers of MHC class I. Upon recognition of a foreign invader or tumor cell, NK cells release cytotoxic proteins such as interferon-gamma and granzyme B. Because of their ability to recognize stressed cells lacking “self” markers, human NK cells have shown promise as therapeutics in clinical trials to reduce disease burden in patients with therapy-resistant or advanced-stage blood cancers. With more than one hundred NK cell- and induced NK cell-based clinical trials ongoing for blood cancers and refractory solid tumors, there is an ever-increasing need for NK cell therapies to treat cancer and immune diseases. We sought to develop novel, clinically relevant, and robust methods for human NK cell expansion. In addition, to simplify the processing of cells for characterization, we developed a flow cytometry based killing assay to monitor NK activity. Therefore, within the same experiment, we can analyze NK cell phenotype profiles with specific antibody panels while also assessing NK cell killing activity. Using optimized serum- and xeno-free conditions with minimal cell manipulations, we have expanded highly purified NK cells up to 500-fold in 14 days from human peripheral blood mononuclear cells (PBMCs) using optimized cytokines and Cloudz CD2/NKp46 microspheres. To characterize the resulting NK cell population, we used a unique antibody panel to phenotype the NK cells by flow cytometry. Within the same experiment, NK killing assays against K562 cells can be performed using a flow cytometry-based killing assay, which has the advantage of using one instrument for all analyses, shortened processing times, and reproducibility. Using this workflow, we can identify different culture conditions that adversely affect NK activity. Together, this data presents a comprehensive and clinically relevant workflow for the expansion of highly purified NK cells and assessing their killing potential. Furthermore, this workflow can be used for screening the activity of chimeric antigen receptor (CAR) expressing immune cells against specific cancer antigens.

Citation Format: Jody Bonnevier, Christine Goetz, Li Peng, Jamie Van Etten, Bora Faulkner, Christopher Hammerbeck, Ariel Miller, Gabriella Perell, Christopher Johnson, Joseph Lomakin, David Hermanson, Kevin Flynn. Assessment of natural killer (NK) cell activity for immunotherapy using a novel flow cytometry-based killing assay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB141.