Natural killer (NK) cells, which have both cytotoxic and cytolytic effector functions to kill cancer cells, are an ideal allogeneic therapy as they do not induce Graft-vs-Host disease and can be expanded by several orders of magnitude through multiple rounds of activation with K562 artificial antigen presenting cells. However, the changes that extended stimulation of NK cells might incur in cell cycle status, metabolism, and functionality necessary for production of sufficient cell quantities are not well understood and thus were the focus of this study. We observed that repeated activation allowed for 10<sup>6</sup>-10<sup>8</sup> fold expansion, although a reduced growth rate was observed from approximately day 21 onwards, potentially signifying the presence of senescent cells in later stages of culture. Flow cytometry was used to further evaluate expression of various markers for exhaustion while functional cell cytotoxicity assays were used to determine the presence of important secreted factors for NK cell functionality. IncuCyte assays measured the rate of killing during the different growth phases. Together, this allowed us to explore the relationship between NK proliferative capacity and cytotoxic ability. RNA-sequencing analysis was used to explore activation-associated gene expression dynamics, while ATAC-sequencing was utilized to unveil the dynamic nature of epigenetic/transcriptional modifications during NK cell activation. Activation was found to be accompanied by an increase in oxygen consumption and upregulation of important metabolic pathways, demonstrating that metabolism is an important indicator of activation. Extensive characterization of prolonged K562 stimulation through transcriptome analysis, dynamics of chromatin accessibility, metabolic characterization, and phenotypic analysis of exhaustion and senescence allows us to evaluate the activation and expansion process of NK cells over continuous long-term culture. Insights into mechanisms underlying activation and expansion will provide a path to develop strategies for large-scale biomanufacturing while maintaining functionality, thus laying the groundwork as a potential off-the-shelf allogeneic cellular therapy.

Characterization of the effect of extended K562 stimulation on natrual killer cell functionality for emergent off-the-shelf allogeneic therapy applications

Macromolecular Chemistry: The Second Century

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ACS Spring 2020 National Meeting & Expo

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.