An integrated multi-omics investigation of W-NK1, a cytokine-primed non-engineered natural killer cell therapy product

BackgroundNatural killer (NK) cells originate from bone marrow precursors and mediate effective anti-tumor responses. Clinical trials of cytokine-primed memory-like (ML) NK cells in acute myeloid leukemia (AML) have demonstrated activity without major toxicity, including graft-versus-host disease or cytokine release syndrome. However, broad application of non-expanded, non-engineered ML NK cells has been hindered by limited availability of NK cells from a single donor, thereby precluding aggressive dose escalation and repeat dosing. W-NK1 is derived from human peripheral blood mononuclear cells undergoing ML reprogramming with a proprietary heteromeric fusion protein complex including IL-12, IL-15 and IL-18. MethodsWe conducted a multi-omics characterization of W-NK1 by interrogating its transcriptomic, proteomic and metabolic profile. Using functional assays, we assessed W-NK1s cytotoxicity under adverse culture conditions, as well as W-NK1s trafficking and killing abilities in immunodeficient mice engrafted with THP-1 AML. Finally, we evaluated W-NK1s phenotype and in vivo expansion kinetics in one patient with AML enrolled in study NCT05470140. ResultsW-NK1 displayed an activated, hyper-metabolic, and proliferative state differing from unstimulated conventional NK cells (cNK) from healthy donors. When compared to external single-cell NK datasets, W-NK1 was largely annotated as NKG2A+ and showed low relatedness with adaptive NK states characterized by HCMV-induced inflammatory memory. W-NK1 outperformed cNK cells in terms of in vitro killing of a broad panel of AML cell lines, with no appreciable cytotoxicity against normal cell lines. The expression of nutrient transporters was higher in W-NK1 compared to cNK cells and was retained even in adverse culture conditions designed to mimic an immunosuppressive tumor microenvironment. In mice engrafted with THP-1 AML, W-NK1 trafficked and efficiently homed to the bone marrow, where it mediated better tumor control than cNK cells. W-NK1 expanded, underwent phenotypic changes and persisted with effective elimination of circulating AML blasts through day 14 after infusion in one patient treated on clinical trial NCT05470140. Immunofluorescence staining of BM sections collected on day 28 showed increased expression of both CD56 and CD3 compared to a pre-treatment biopsy. ConclusionsOur study offers a comprehensive characterization of W-NK1 as an effective cell therapy product for AML and solid tumor malignancies. What is already known on this topicNatural killer (NK) cells have been shown to be safe and effective for treating certain human malignancies. Nonetheless, limitations for adoptive cell therapy exist which include trafficking / homing to tumor tissues as well as metabolic resilience in an adverse microenvironment. What this study addsW-NK1 is distinct transcriptionally and functionally from conventional NK cells with improved anti-tumor effector functions and metabolic adaptation in hostile culture conditions. Moreover, W-NK1 was readily detectable post-infusion in a patient with refractory acute myeloid leukemia. How this study might affect research, practice or policyOur in vitro and in vivo findings indicate that W-NK1 is an effective NK-cell therapy product and augur positively for patients being treated in phase I immunotherapy clinical trials.