Characterization of Natural Killer Cell Activation and Functionality for Cell Therapy Applications

Abstract

Natural killer (NK) cells are a promising emerging allogeneic cell therapy due to their cytotoxic effector and cytokine producing functions and lack of induction of Graft-vs-Host Disease. For allogeneic clinical applications, NK cells isolated from a single donor must be expanded through an efficient large-scale biomanufacturing process to treat many patients and produce an economical off-the-shelf therapy. Critically, the cultured NK cells must maintain functionality post-expansion to be an effective cellular therapy. Through multiple rounds of activation with K562 artificial antigen-presenting cells (aAPCs), the quantity of NK cells can expand by several orders of magnitude. However, the changes that chronic stimulation might induce in cell cycle status, metabolism, and ex vivo functionality of NK cells are not well understood. Even less is known regarding how changes in these cell characteristics may influence their in vivo functionality. In this work, we conducted a systematic evaluation of the activation and expansion process of NK cells through transcriptome analysis, dynamics of chromatin accessibility, metabolic characterization, and phenotypic analysis of exhaustion and senescence over time. By understanding the transcriptional and epigenetic signature of the K562-activated NK cells, we have identified potential genes and transcription factors that may regulate K562 activation in order to develop a bioprocess that phases out these feeder cells from the culture process. Furthermore, rigorous characterization of NK cell growth and receptor expression during expansion revealed that prolonged stimulation results in an immature, exhausted, cytokine-producing phenotype over time. Changes in NK growth kinetics corresponds to shifts in NK cytotoxicity in select target cancer cell lines, indicating proliferative potential may be an indicator of a good donor for clinical use. Collectively, understanding the effects of activation and consequent proliferation of NK cells would remove a major roadblock in the biomanufacturing of NK cells, thus laying the groundwork for their potential use as an off-the-shelf allogeneic cellular therapy. Insights into mechanisms underlying activation and expansion provide a path to develop strategies to eliminate K562 aAPCs altogether, which would be desirable from a regulatory standpoint and further ease the transition from benchtop to biomanufacturing as well as improve efficacy in the clinic.