358 Efficient expansion of clinical-grade nature killer cells using the G-rex bioreactor for adoptive immunotherapy trials

Background Natural killer (NK) cell-based immunotherapy offers a promising strategy for treating a wide range of hematologic and solid malignancies. However, the current challenge is to establish a robust, scalable, and Good Manufacturer Product (GMP)-compliant expansion system to achieve sufficient functional NK cells that meet the clinical needs. We report the optimization of a xeno-free, feeder-free bioreactor system for the large-scale expansion of GMP-grade NK cells.Methods Clinical-grade NK cells were isolated from commercially available peripheral blood mononuclear cell apheresis (PBA) products using the LP3/56 program on the CliniMACS Prodigy platform to enrich CD3 –CD56+ cells. Expansion was conducted in the G-Rex bioreactor system across multiple formats (2 cm2, 5 cm2-G-Rex6 well plates, and G-Rex50M systems with a 500 mL working volume). Cells were cultured in Excellerate™ Human NK xeno-free medium supplemented with defined cytokine combinations (IL-2, IL-15, IL-18, and IL-21). Cultures were maintained for 10–14 days without media exchange or cell splitting. NK cell identity and purity was confirmed using the BD 4-Color TBNK flow cytometry kit.Results NK cell enrichment on the CliniMACS Prodigy resulted in 94% pure starting population. With an initial seeding density of 5 × 10 5 to 1 × 106 cells/cm2, the system consistently achieved a final density of approximately 2 × 107 cells/cm2 across all formats. Importantly, endpoint viability remained ≥90%, demonstrating the G-Rex system’s capacity to support high-density cell growth through enhanced gas exchange and nutrient. Across all platforms, NK cells (CD3–CD16+CD56+) constituted >96% of expanded cells were with minimal contamination from residual T cells (<1%), including negligible levels of CD4+ and CD8+ subsets. The NK cell population maintained strong expression of CD56 and CD16, consistent with a mature and cytotoxic phenotype suitable for therapeutic use. Preliminary cytokine screening revealed that specific combinations significantly enhanced expansion efficiency while preserving NK cell identity and function.Conclusions We have developed a highly efficient, scalable, xeno- and feeder-free bioreactor platform for the expansion of clinical-grade NK cells from PBA products. This system eliminates the need for feeder cells and splitting, thereby reducing contamination risk, labor intensity, and cost, while remaining GMP-compliant. The incorporation of defined cytokine cocktails and lack of feeder cells further enhances batch consistency and reproducibility. This strategy supports the scalable production of feeder-free off-the-shelf NK cell therapies for current and future clinical trials.Acknowledgements This research was funded by Vera Bradley Foundation and Brown Center for Immunotherapy, Indiana University.