Adoptive cell therapy represents a promising approach in cancer treatment that involvesthe administration of ex vivo activated, expanded and genetically modified allogeneic or autologous immune cells. Natural killer cells have emerged as versatile and safe candidatesfor this therapy due to their innate ability to target and eliminate abnormal cells and modulate immune responses. Our study focuses on the NK-92 cell line, which reflects the characteristics of highly activated blood-derived NK cells that exhibit high cytotoxicity.This method offers a cost-effective alternative to allogeneic primary NK cells by reducing donor-to-donor variability and eliminating the need for expensive cell isolation and purification techniques, enabling their use as an off-the-shelf therapeutic agent.Despite the potential of NK cell-based therapies, expansion processes remain poorly understood and lack standardization. Rapid adaptations in immune cell metabolism further complicate the predictability of lymphocyte expansion. We aim to employ Qualityby Design principles to ensure consistent qualitative and quantitative characteristics of therapeutic NK cells. By elucidating the interaction between quality attributes and process parameters, we aim to ensure the consistent quality of the living product. To overcome the challenges that arise from the slow growth kinetics of NK-92 cells, our study employed a pseudo-static cultivation method for daily phenotypic characterization,assessment of cytokine production, and comparative cytotoxicity evaluation. Furthermore,we aimed to gain a deeper understanding of the basic metabolic requirements of the NK-92 expansion process by monitoring nutrient uptake and metabolite release.Our investigation uncovered a metabolic fingerprint of NK-92 cells, characterized by heavyutilization of L-serine, L-arginine, L-asparagine, and L-glutamine. Moreover, we observedthe development of a dysfunctional phenotype characterized by reduced metabolic activity,cytotoxicity, impaired degranulation, and downregulation of activating receptors. We hypothesized that this is associated with shear stress induced during cell handling orlinked to nutrient depletion and the accumulation of inhibitory metabolites. Furthermore,our research has demonstrated that flow cytometry is the optimal method to evaluate cellular cytotoxicity because of its superior sensitivity, applicability, and the information density of the obtained data. In conclusion, the insights gained from this project provide a valuable basis for future experiments that will allow us to understand and improve the manufacturing process of therapeutic NK cells.
