Single-Cell Transcriptional Landscape of W-NK1, an Off-the-Shelf Natural Killer Cell Therapy
Authors: Mahajan Nitin , Arthur Laura , Vadakekolathu Jayakumar , Muth John , Davidson-Moncada Jan K , Rutella Sergio 
Published: December 2024

Background
Natural killer (NK) cells play a critical role in tumor eradication. W-NK1 is a non-edited, cryopreserved off-the-shelf NK cell therapy product with a ‘memory-like’ phenotype that addresses challenges faced by adoptive cell therapies, such as poor resilience to adverse and immunosuppressive tumor microenvironments (TME). W-NK1 not only overcomes nutrient deprivation, inhibitory ligands, and reduced chemokine expression in the TME, but also recruits and activates dendritic and T cells through cytokine and chemokine production, thereby enhancing the overall immune response. This study aims to elucidate the molecular underpinnings of W-NK1's activity as well as its cellular heterogeneity using bulk and single-cell RNA-sequencing (sc-RNA-seq) approaches.
Methods
W-NK1 were manufactured from healthy donor peripheral blood NK cells (herein referred to as conventional NK, cNK) which are activated, expanded, and cryopreserved to create an off-the-shelf memory NK-cell product. We then performed bulk RNA sequencing (RNA-seq) and scRNA-seq on both cNK (before cytokine priming) and W-NK1. Utilizing transfer learning, we compared W-NK1's single-cell transcriptomes to recently published reference maps encompassing NK cells from healthy donors (GSE197037 and https://zenodo.org/doi/10.5281/zenodo.8434223).
Results
We identified over 7,000 differentially expressed genes (DEGs) at a false discovery rate (FDR) &lt;0.01. Upregulated genes in W-NK1 included IL2RA, TPX, and MKI67, indicating an activated and proliferative state post-cytokine expansion. Additionally, genes involved in NK-cell regulation, such as CSF2 encoding GM-CSF, and BIRC5 encoding survivin, were also highly expressed. We observed reduced intra-donor variance in cluster proportions in W-NK1 compared to cNK cells, with tighter clustering in principal component analysis suggesting low batch-to-batch heterogeneity of the cell therapy product. Unbiased clustering and UMAP visualization of sc-RNA-seq data from more than 49,000 cells across four donors revealed eight cellular states (c0-c7), most of which were significantly differentially abundant in W-NK1 compared to cNK cells, as supported by linear modeling of sc-RNA-seq data. W-NK1's cytolytic capacity was supported by the high expression of established (perforin and GZMB) as well as novel cytotoxicity genes (FGFBP2, SPON2). Pseudotime inference illustrated a progression of maturation from cytotoxic W-NK1 in c0 to ‘hyper-metabolic’ and proliferative states in c7, driven by cytokine priming. Transfer learning highlighted unique transcriptional features of W-NK1, including high expression of NKG2A and low relatedness with adaptive NK states characterized by human cytomegalovirus-induced inflammatory memory. Pathway analysis indicated significant enrichment in glycolysis, amino acid and fatty acid metabolism, DNA replication, and cell cycle-related gene programs in W-NK1 compared to cNK cells.
Conclusion
Our multi-omic analysis highlights potential advantages of W-NK1 in overcoming a hostile and nutrient-deprived TME, such as enhanced proliferation and metabolic fitness, and augmenting immune responses. These findings position W-NK1 as a promising candidate for cancer immunotherapy.