Abstract B062: Ascites-derived natural killer (NK) cells exhibit chronic overstimulation and metabolic dysfunction in patients with advanced epithelial ovarian cancer

Abstract Background: Immune checkpoint inhibitors show minimal activity in patients (pts) with epithelial ovarian cancer (EOC), in part due to an immunosuppressive tumor microenvironment (TME). We are currently investigating the safety and preliminary efficacy of an autologous intraperitoneal cytokine-induced memory-like NK cell therapy in pts with recurrent EOC (NCT06321484). We aimed to define NK cell dysregulation in EOC ascites to inform potential mechanisms of resistance to NK cell therapies in the TME. Methods: Ascites and matched peripheral blood (PB) were collected from 4 pts with advanced EOC. O-link target 48 was performed on paired ascites and serum from 3 pts; Luminex was performed on ascites from 4 additional EOC pts. 10x Single cell RNA sequencing (scRNAseq) was performed on cells isolated from paired ascites and PB from 3 pts; downstream analyses were conducted using Seurat, ggplot2 and EnrichR using Wilcoxon rank sum test for significance. Results: Consistent with prior studies, ascites-derived NK (A-NK) cells exhibited reduced cytotoxic capacity compared with matched PB- derived NK (PB-NK) cells. PB-NK cells showed activation (IFN-g release) and degranulation (CD107a+) in response to cytokine stimulation and target cell exposure, respectively; however matched A-NK cells did not respond to target stimulation. Multiplex immunoassays performed to identify ascites factors contributing to NK cell dysfunction showed enrichment of multiple immunomodulatory secreted factors including CXCL10, IL-6, TGF-b, VEGF-A, CXCL9, CCL2, and CCL8. In line with the functional data, scRNAseq analysis revealed lower expression of cytotoxicity-related genes (PRF1, GZMB, GZMA, GNLY, NKG7) in A-NK compared to PB-NK cells (p<0.0001, n=3 paired samples). Additionally, A-NK showed significant enrichment in type I interferon (IFN) signaling (p≤0.0001), suggesting chronic overstimulation. Flow cytometry revealed elevated CD69 (p<0.05) and NKG2A on A-NK, hallmarks of chronic overstimulation. In a module score analysis using genes for glycolysis and oxidative phosphorylation (e.g. CAPN5, STC2, VEGFA, HK2, CTH, GNE, GLRX, VLDR and CYC1, GRPEL1, TIMM17A), A-NK showed significantly lower expression than PB-NK (p<0.0001). To address this immune dysfunction, we engineered an IL-12-secreting CAR-NK cells targeting mesothelin that maintain high cytotoxic activity in the presence of EOC ascites. Notably, IL-12 CAR NK cells highly express the genes within our metabolic modular score, suggesting increased metabolic fitness. Conclusions: Ascites-derived NK cells demonstrate marked immune and metabolic dysfunction, and we hypothesize that IL12-engineering may overcome ascites-induced dysfunction via metabolic reprogramming. Ongoing efforts are focused on RNA and ATAC sequencing analyses of ascites-exposed IL-12-secreting CAR NK to elucidate epigenetic changes that mediate their resistance to the TME. Citation Format: Grace C. Birch, Mubin Tarannum, Maily Nguyen, Khanlinh Dinh, Mila Stanojevic, Ursula A. Matulonis, Rizwan Romee, Rebecca L. Porter. Ascites-derived natural killer (NK) cells exhibit chronic overstimulation and metabolic dysfunction in patients with advanced epithelial ovarian cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Ovarian Cancer Research; 2025 Sep 19-21; Denver, CO. Philadelphia (PA): AACR; Cancer Res 2025;85(18_Suppl):Abstract nr B062.