Bulk and single-cell transcriptomics identify gene signatures of stem cell-derived NK cell donors with superior anti-tumor activity [scRNA-seq]

Allogeneic natural killer (NK) cell therapies are a valuable treatment option for cancer, given their remarkable safety and favorable efficacy profile. Although the use of allogeneic donors allows for off-the-shelf and timely patient treatment, intrinsic interindividual differences put clinical efficacy at risk. The identification of donors with superior anti-tumor activity is essential to ensure the success of adoptive NK cell therapies. Here, we investigated the heterogeneity of 10 umbilical cord blood stem cell-derived NK cell batches. First, we evaluated the donors' cytotoxic potential against tumor cell lines from solid and hematological cancer indications, to distinguish a group of superior, “excellent” killers (4/10), compared to “good” killers (6/10). Next, bulk and single-cell RNA-Sequencing, performed at different stages of NK differentiation, revealed distinct transcriptomic features of the two groups. Excellent donors showed an enrichment in cytotoxicity pathways and a depletion of myeloid traits, linked to the presence of a larger population of effector-like NK cells early on during differentiation. Consequently, we defined a multi-factorial gene expression signature able to predict the donors' anti-tumor potential. Our study contributes to the identification of key traits of superior NK cell batches, supporting the development of efficacious NK therapeutics and the achievement of durable anti-tumor responses Overall design: CD34+ cells from 10 UCB donors were seeded in 6-wells tissue culture treated plates (Corning Incorporated) for expansion culture in Glycostem Basal Growth Medium (GBGM®; Fertipro) supplemented with human serum (Sanquin), thrombopoietin (TPO), interleukin (IL)-7, FMS-like tyrosine kinase 3 ligand (Flt-3L), granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-6, stem cell factor (SCF) (all from Cellgenix) and Neupogen (G-CSF; Amgen) as previously described. After 9 days, TPO was replaced by IL-15 (Cellgenix) and after 14 days of NK progenitor expansion, Flt-3L was replaced by Proleukin (IL-2; Novartis) for the NK cell differentiation phase. Cells were cultured until day 28 and cryopreserved. Upon thawing, cells were directly used for assays, or cultured for an additional 7 days in differentiation medium before use. 8 donors were selected to sequence at day28 directly after thawing.