365 Oxygen levels found in bone marrow (5%) provide an optimal niche for the differentiation of induced-pluripotent stem cell-derived NK cells for the treatment of AML

Background

Acute myeloid leukemia (AML) incidence increases with age. Five year survival for those over 65 is less than 11%, highlighting the need for safe interventions to improve outcomes. Adoptive natural killer (NK) cell products have achieved success as a ‘bridge to transplant’ in refractory leukemia and lymphoma, inducing remission to a point where patients are eligible for stem cell transplantation. Multiplexed-engineered induced pluripotent stem cells (iPSCs) are a reproducible source of highly functional NK cells (iNK) for on-demand treatment and broad patient access. Clinical trials are currently testing iNK cells with therapeutic antibodies for the treatment of leukemia and lymphoma (NCT04023071, NCT04614636, NCT04714372). We have developed a protocol for the production of highly functional iNK cells, engineered for greater anti-tumor effect. However, we hypothesized that performing NK cell lineage commitment under physiological oxygen conditions found in bone marrow (5%) would create a niche that could support the generation of a more functional cell product. Methods iPSCs are matured into CD34+ precursors, then differentiated into iNK cells that are subsequently expanded to clinically-relevant quantities (figure 1A). We have previously published on iNK cells that consist of three unique edits: high-affinity non-cleavable CD16, membrane bound IL-15 and knockout of CD38. Using these cells, we performed stage specific differentiation from CD34+ precursors in 5% oxygen (‘physoxic iNK’) or conventional 20% oxygen, with subsequent expansion in 20% oxygen. iNK cells were compared for their phenotype (CyTOF), proliferation (flow cytometry), cytotoxicity (live cell imaging), metabolic stability (reactive oxygen species staining by flow cytometry) and ability to control tumor (xenograft mouse model).

Results

CyTOF analysis revealed a more naive phenotype in physiological oxygen conditions that persisted after expansion. However, these cells were equally capable of natural cytotoxicity and antibody-dependent cellular cytotoxicity. In a xenograft model of AML (NSG mice with HL60-GFP/luciferase; figure 1B) there was greater persistence of physoxic iNK cells in blood and bone marrow (figure 1C), correlating with greater tumor control within the bone marrow (figure 1D) and across the whole animal (figure 1E). When exposed to oxidative stress, physoxic iNK cells were more resilient, with lower reactive oxygen species detected in their mitochondria, suggesting greater tumor control arose from greater persistence within the animal, rather than better cytotoxicity.

Conclusions

These data suggest that manufacturing therapeutic NK cells in a physiological environment at a unique stage of lineage commitment can generate resilient cells with a greater durability for anti-tumor activity.

Acknowledgements

This project was supported by grants from the Department of Defense (CA200922) and National Institutes of Health (R35 CA197292, P01 CA111412). Parts of the figure were drawn using images from Servier Medical Art and from Biorender.

Ethics Approval

The University of Minnesota human research protection program determined this was not human research (institutional review board ID: STUDY00013106)