Improved Expansion of Peripheral Blood and iPSC-Derived Natural Killer Cells for Clinical Applications

NK cells are innate immune cells that are able to recognize and kill tumor cells without prior sensitization. Their potent cytotoxic activity makes them an attractive option for cell-based cancer immunotherapy. Clinical application of NK cells requires large-scale expansion and activation, which is typically achieved using engineered feeder cells. Current methods typically utilize K562 cells expressing 41BBL and membrane-bound (mb) IL-21 that have been widely used in both research and clinical settings. However, there is still a need for alternative sources that can enhance NK cell expansion more effectively.

In this study, we investigated four novel genetically modified feeder cell lines, also commonly called artificial antigen presenting cells (aAPCs) to enhance NK cell activation and expansion. Specifically, K562 and 721.221 cells were engineered to express either OX40L or 41BBL co-stimulatory molecules along with mbIL18 and IL21, as well as B7H6, which stimulates NKp30 on NK cells. Therefore, we developed four different new aAPCs: 1) K562-OX40L-mbIL18/21-B7H6, 2) K562-41BBL-mbIL18/21-B7H6, 3) 721.221-OX40L-mbIL18/21-B7H6 and 4) 721.221-41BBL-mbIL18/21-B7H6. We compared these new feeder cells to “standard” K562-41BBL-mbIL21 cell for ability to expand NK cells from whole PBMCs, sorted CD56⁺ peripheral blood (PB) NK cells, and iPSC-derived NK cells. Cultures were stimulated with irradiated aAPCs co-cultured with NK cells at 1:1 ratio.

Our studies demonstrate that the novel 721.221 feeder cells expressing either 41BBL or OX40L with B7H6-mbIL-18/21 significantly enhanced the expansion of CD56⁺ sorted PB-NK cells and iPSC-NK cells compared to standard aAPCs. Specifically, iPSC-derived NK cells expanded to over 10⁹ cells starting from 6×10⁶ cells after 6 weeks without any significant reduction in viability when stimulated with our novel engineered aAPCs. Among these novel engineered aAPCs, 721-41BBL-B7H6-mbIL18/21 induced the expansion of iPSC-NK cells to >4×10⁹ cells, 721-OX40L-B7H6-mbIL18/21 induced their expansion to >3.5×10⁹ cells, and K562-41BBL-B7H6-mbIL18/21 co-culture with iPSC-NK cells induced the expansion to >3×10⁹ cells over 6-week culture period. Similar expansion trend was observed with the expansion PB-NK cells using these novel aAPCs In all studies, the 721.221 engineered aAPCs demonstrated superior NK cell expansion compared to similar K562-engineered aAPCs. Notably, the iPSC-derived NK cells exhibited significantly improved expansion compared to PB-NK cells using each of the new aAPCs.

We next tested the functional ability of expanded PB-NK cells and iPSC-NK cells to mediate anti-tumor activity against various tumor cell lines, including K562 cells (erythroleukemia), CAL27 (head and neck squamous cell carcinoma), A1847 (ovarian cancer), and SKOV3 (ovarian cancer) tumor cell lines similar to standard aAPCs expanded NK cells. A standard flow cytometry-based cytotoxicity assay was used to demonstrate a high level of anti-tumor activity by the new aAPC-expanded NK cells. For example, the 721-41BBL-B7H6-mbIL-18/21 activated and expanded iPSC NK cells demonstrated significant cytotoxic activity, inducing over 85% killing of K562 cells, 77% killing of CAL27 cells, 75% killing of A1847 cells, and 68% killing of SKOV3 cells. In all cases, the anti-tumor activity mediated by NK cells expanded with new aAPCs was as good or better than those expanded using the standard aAPCs.

Our findings demonstrate that 721.221 cells engineered to express 41BBL or OX40L, combined with mbIL18/21 and B7H6 significantly enhance the expansion of PB-NK cells and iPSC-NK cells and provide an important and superior alternative to traditional K562-based aAPCs. This advancement is useful to enhance the efficacy and scalability of NK cell-based therapies.