Abstract 3251: Assessment of NK cell-mediated killing and phenotypic analysis using advanced flow cytometry and an optimized multiplexed assay

In this study, a novel multiplexed assay was developed that simultaneously measures NK cell phenotype and function in relation to tumor target killing. Natural Killer (NK) cells are an essential part of the innate immune system, interface with the adaptive immune system, and play a critical role in tumor immune surveillance and anti-tumor responses. Although chimeric antigen receptor (CAR)-modified T cells have proven to be highly effective as an anti-cancer therapeutic, there are potential problems associated with their use, particularly the risk of inducing graft-versus-host disease (GVHD) resulting in aberrant cytokine release. In contrast to T cells, NK cells are potent cytotoxic effector cells that do not require MHC restriction and have a low risk of inducing GVHD. Recent advances in the development of CAR-modified NK cells have shown great promise for clinical utilization in cancer immunotherapy with capability for both direct tumor killing as well as for use in combination therapy with monoclonal antibodies to boost NK cell antibody-dependent cell-mediated cytotoxicity (ADCC). A critical process in the development of new NK cell-mediated therapeutics is the ability to expediently assay, screen, and analyze data for NK cell activation and tumor killing. To address this need, a novel multiplexed assay was developed that measures tumor target cell killing, target-dependent expression of activation markers (CD69 and CD25), and the quantitation of secreted granzyme B, IFNγ, and TNFα in a single well of a microtiter plate. To validate the assay, a direct NK cell-tumor cell killing model was used. Purified human NK cells were mixed with target cells at different ratios. Tumor cell killing, NK cell activation status, and quantification of secreted proteins were measured at 4 and 24 hours after co-culture. Assay data acquisition was performed on Intellicyt iQue3® platform for advanced flow cytometry. Data analysis and visualization was performed using the integrated ForeCyt® software package. Tumor cell killing along with granzyme B release, was evident by 4 hours following co-culture, and both were increased with the increase in effector to target ratio (E:T). Killing was further enhanced when NK cells were stimulated with IL-2 and IL-15. As expected, an increase in CD69 and CD25 expression was also observed following cytokine stimulation with further increase upon co-culture of NK cells with tumor cell targets, along with increased levels of secreted IFNγ