Advancing acute myeloid leukemia immunotherapy: transcriptomic profiling-guided donor selection combined with an innovative natural killer cell expansion protocol

Acute myeloid leukemia is an aggressive malignancy with limited treatment options and high relapse rates. Cellular immunotherapy using natural killer (NK) cells offers a promising approach to improve treatment outcomes by employing their innate cytotoxic potential. However, the results of clinical studies show inconsistent efficacy, which could be explained by various aspects, including variable donor-host combinations or differences in cryopreservation steps affecting the viability and cytotoxicity of the final immunotherapeutic product. Here we introduce a novel strategy involving an intermediate cryopreserved product prepared on day 7 of NK-cells expansion, followed by an additional 10-12 days of culture. This approach allowed substantial recovery of NK-cell function after recultivation. Furthermore, since NK cells from different donors display considerable functional and proliferation variability, we performed detailed functional analyses of multiple donors to identify functional and molecular signatures linked to cytotoxicity and expansion potential, combining cytotoxicity assays, immunophenotyping and global transcriptomic profiling. This revealed a distinct gene expression signature distinguishing NK cell donors with superior cytotoxicity and expansion potential, linked to activation and cellular stress responses. Our findings demonstrate that our current Good Manufacturing Practice-compliant expansion method with intermediate cryopreservation supports improved therapeutic timing and preserves NK-cell quality. In parallel, the proposed donor selection approach may enhance manufacturing efficiency by identifying donors whose NK cells have greater cytotoxic and expansion potential.