Optimization of in vitro Human Natural Killer Cell Expansion to Enhance Standardization and Translational Application in Cancer Immunotherapy

While cancer research faces numerous challenges, inter- and intratumoral heterogeneity most directly constrains the development of antigen-specific therapies. Natural Killer (NK) cells, as essential components of the innate immune system, offer an antigen-independent modality by recognizing and eliminating tumor-associated stress signals. Their rapid activation and potent cytotoxic effector functions are mediated by activating receptors and complemented by antibody-dependent cellular cytotoxicity via CD16a. Crucially, inhibitory receptors ensure tolerance toward healthy cells, collectively positioning them as compelling candidates for cellular immunotherapies. However, the translational application of NK cells necessitates precise control and optimization of ex vivo expansion to secure both quantitative yield and functional quality. These challenges are addressed in this thesis by optimizing in vitro NK cell cultivation parameters, investigating donor variability, and introducing a streamlined, flow cytometry-based killing assay for functional evaluation. Investigation of different media and serum supplements under interleukin (IL) 2 stimulation identified NK MACS® medium supplemented with human AB serum as optimal for NK cell expansion while limiting T cell contamination. Pre-enrichment by negative selection using the RosetteSep™ human NK cell enrichment cocktail increased final cell yield at high purity compared to cultures initiated from peripheral blood mononuclear cells, despite lower relative fold-changes. In the G-Rex® 24-well format, seeding density emerged as a key driver of final cell yield and surface marker expression, as higher seeding densities resulted in higher NK cell numbers and increased expression of the activating receptors CD16a, NKp46, and NKG2D. Donor-dependent heterogeneity persisted despite harmonized culture conditions. Some donors displayed significantly increased proliferation and elevated activating receptor expression, whereas others showed reduced expansion and atypical trajectories of markers such as NKp46 or ICAM-1. To explore genetic contributors, long-read sequencing targeted selected loci encoding the phenotypically investigated activating NK cell markers and IL 2 receptor subunits. Although no consistent genotype-phenotype correlation emerged across donors, clinically relevant single-nucleotide polymorphisms were detected, and one donor carried a distinct haplotype at an investigated locus. In the final part of this thesis, a ready-to-thaw, flow cytometry-based killing assay concept for the assessment of NK cell cytotoxicity is introduced. The concept relies on cryopreservation of pre-stained target cells that, after thawing, can be used directly without washing, enabling the decoupling of target cell preparation from the actual assay workflow. Moreover, cryopreservation in small aliquots facilitates freezing, co-incubation, and acquisition all in the same tube, thereby significantly streamlining the assay workflow, enhancing operational efficiency, and improving reproducibility. Collectively, the results provide a solid foundation for standardized NK cell in vitro expansion, including implications for further research on donor variability, and for a streamlined assay for cytotoxicity assessment, thereby supporting the translational application in immunotherapy.