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Abstract
Background Cancer-targeting natural killer (NK) cells show substantial promise in allogeneic immunotherapy settings due to their desirable cytotoxicity and safety profiles. However, NKs from adult peripheral blood or umbilical cord blood units (CBUs) are restricted by critical factors: insufficient cell yield, unpredictable efficacy due to donor variability, NK resistance to genetic manipulation, and reliance on activation to expand NKs, which may induce exhaustion.
Methods To overcome these challenges, we have pursued a strategy enabled by IBR403, our proprietary small molecule that enables large-scale expansion of cord blood hematopoietic stem cells (HSCs) and differentiation of clinically relevant quantities of NK cells. HSC expansion allows CBU segments (figure 1) to be used for low-cost, high-throughput screening; this enables intentional leveraging of donor variability to improve allogeneic efficacy. We developed optimized NK differentiation protocols that enhance expansion, purity, and cytotoxicity of NK cells derived from IBR403-expanded HSCs from a variety of cord blood donors.
Results To validate our process, we expanded HSCs from CBU segments for 28 days using IBR403. Extrapolating from this expansion, a full CBU could produce thousands of doses of NKs without requiring feeder cells. Using our optimized NK differentiation protocol, segment-derived HSCs can yield sufficient numbers of NKs to carry out phenotyping, degranulation, intracellular cytokine staining, and cytotoxicity assays against ten target cell lines. Furthermore, we can isolate and expand NKs from the same segment and functionally assay these in parallel with their matched HSC-derived NKs. Our protocol generated a 15-fold higher CD56 yield and a 900% increase in cytotoxicity against HCT116 when compared to commercial reagents (figure 2).
Conclusions Our HSC expansion molecule allows us to investigate and link intrinsic functional variability within CBUs to donor genotype. This process enables us to screen for optimal donors using only CBU segments, while preserving the full CBU for clinical manufacturing. Furthermore, our ability to isolate and expand cord blood HSCs and NKs from the same CBU segment enables us to validate functional differences observed between HSC-derived NKs from different donors and verify their overall functional relatedness to cord blood NKs. Overall, our HSC expansion, NK differentiation, and assay pipeline improves donor selection for cancer patients and transforms cord blood into a versatile and scalable asset for cellular therapies.
Diagram of a CBU. Each section is heat sealed to separate segments from the full unit.
Comparison of optimized differentiation methods to commercial reagents. Modifications of the protocol resulted in improvements in CD56 yield (A) and target cell cytotoxicity (B).
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