Enhancing Non-Small Cell Lung Cancer Treatment Utilizing Natural Killer Cell-Derived Extracellular Vesicles (nkevs) As a Novel Adoptive Cellular Therapeutic.

e20079 Background: An established mechanism contributing to immune checkpoint inhibitor (ICI) therapy failure is tumor evasion of T-cell responses via downregulation of human leukocyte antigen (HLA). Conversely, the effector function of Natural Killer (NK) cells is enhanced in the absence of HLA expression, making NK-based cellular therapies an attractive option for ICI resistant tumors. NK-derived extracellular vesicles (NKEVs) have the potential to become a personalized adoptive cellular therapeutic that may overcome challenges associated with traditional NK cell-based therapies failing to deliver adequate numbers of cells to the tumor. We sought to investigate the preclinical efficacy of an approach using selective isolation of NK cells and harvesting of NKEVs in NSCLC. Methods: From 10 NSCLC patients, tumor tissue was collected, and blood mononuclear cells (PBMCs) were retrieved pre- and -post surgery. Single-cell RNAseq (scRNAseq) was used to examine the cellular landscape in PBMCs and tumor tissue. NK cells were isolated from PBMCs, expanded in vitro, and NK-derived EVs (NKEV) were collected and the EV RNA and protein cargo was characterized using proteomics and transcriptomics. The functional capabilities of patient derived NKEVs were assayed with patient derived organoids. Results: scRNAseq highlighted significant cellular heterogeneity, particularly in the cancer cell population. Within the tumor, infiltrating immune cells were lymphoid and myeloid in origin. Over 200,000 PBMCs were analyzed with scRNAseq, revealing differences associated with cancer subtype, tumor mutation burden and experimental time point. Exposure to patient NKEV treatment resulted in a 40-45% decrease in organoid viability, significantly lowering the cisplatin dose required to elicit a cytotoxic response. NKEVs derived from healthy controls were less efficient. In Nivolumab-treated PBMC organoid co-culture experiments, NKEV addition increased the proportion of infiltrating CD8+ T cells and CD56+ NK cells compared to PD-1 inhibitors alone, particularly from the pre-tumor resection PBMCs. Conclusions: This work demonstrated that NKEVs can be successfully harvested from patient derived, expanded NK cells, and highlighted their anti-tumor properties in combination with standard-of-care therapies. Additional in-depth immune analysis will be presented. [Table: see text]