Abstract 3181: A novel memory-like NK cell CAR targeting the proximal mesothelin domain shows promising pre-clinical activity in ovarian cancer using cell lines, patient-derived organoid tumors, and xenograft mice
Authors: Tarannum Mubin , Dinh Khanhlinh , Vergara Juliana , Ay Oyku , Hill Sarah , Romee Rizwan 
Published: April 2023

Abstract
Epithelial ovarian cancer (OVC) is the leading cause of death among gynecological malignancies. Natural killer (NK) cells are an attractive platform for immunotherapy owing to their superior safety, multiple mechanisms of cytotoxicity, and reduced risk of alloreactivity. Recently, paradigm-shifting studies have shown that brief activation of NK cells with inflammatory cytokines induces differentiation into cytokine induced memory-like (CIML) feature. CIML NK cells have demonstrated potent activity in preclinical and in recent clinical trial settings, suggesting CIML NK cells as a novel platform for genetically modified chimeric antigen receptor (CAR) cells for OVC therapy. Based on patient samples and patient-derived organoids (PDOs), we confirmed mesothelin (MSLN) as an attractive candidate for NK cells. Here, we designed and tested aMSLN CAR CIML NK cells for their functionality against OVC cell lines, PDOs, and xenograft mice. We designed the aMSLN CAR using the ScFv sequence against the proximal domain of MSLN with 4-1BB and CD3z as co-stimulatory domains. Primary NK cells were purified from peripheral blood, activated using IL-12, IL-18, and IL-15 resulting in CIML NK cells. The CAR gene was transduced into CIML NK cells via our optimized BaEV lentiviral system to achieve high transduction efficiency of 40-60%. The direct cytotoxic effect of NK cells was tested against OVC cell lines where the cells were co-cultured with NK cells for 6 hours and analyzed using apoptotic markers. Compared to CIML NK cells, aMSLN CAR CIML NK cells showed enhanced cytotoxicity against OVCAR3 (47.9% vs 14.8%), SKOV3 (29.3% vs 8.0%) and OVCAR8 (60.7% vs 39.8%) cells at a 10:1 ratio. In addition, the aMSLN CAR CIML NK cells exhibit long-term cytotoxicity when evaluated over a period of 3 days; killing efficiency of 71.7% vs 37.6% and 54.6% vs 32.8% for aMSLN CAR CIML NK cells compared to CIML NK cells in OVCAR8 cells and SKOV3 cells, respectively. To test the therapeutic potential of NK cells in a relevant in vitro model, we used PDOs. After 18-hour co-culture, the cytotoxicity was determined by evaluating apoptotic EpCAM+ cancer cells amongst the organoid cell population. Results showed that aMSLN CAR CIML NK cells caused increased apoptotic cells compared to CIML NK cells; 58.2% vs 11.0%, 4.5% vs 61.0%, and 10.5% vs 32.0% in three different PDOs. Finally, we tested the in vivo therapeutic efficacy of aMSLN CAR CIML NK cells against intraperitoneal OVCAR8-mcLuc model wherein we observed significant tumor control, with 80.86% tumor growth inhibition as measured by BLI after 2 doses of 5 million/animal NK cells. We are currently evaluating the functionality of aMSLN CAR CIML NK cells after their exposure to ascites from OVC patients as well as developing methods to enhance their function in highly immunosuppressive tumor microenvironment.
Citation Format: Mubin Tarannum, Khanhlinh Dinh, Juliana Vergara, Oyku Ay, Sarah Hill, Rizwan Romee. A novel memory-like NK cell CAR targeting the proximal mesothelin domain shows promising pre-clinical activity in ovarian cancer using cell lines, patient-derived organoid tumors, and xenograft mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3181.