Abstract 568: A novel memory-like NK cell CAR targeting proximal mesothelin domain shows promising preclinical activity in ovarian cancer using cell lines and patient derived organoids

Abstract

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 target killing, 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) NK cells. CIML NK cells have demonstrated potent anti-leukemia activity in preclinical settings and in recent clinical trials and therefore represent a novel platform for genetically modified chimeric antigen receptor (CAR) cells for OVC therapy. Mesothelin (MSLN) is expressed in around 82% of OVCs and its expression is corelated with chemoresistance, increased metastasis, and poor overall survival in patients. We hypothesize arming CIML NK cells with a CAR targeting the proximal mesothelin domain will enhance their antitumor responses. We designed and tested anti-MSLN CAR CIML NK cells for their anti-cancer functionality against OVC cell lines and patient-derived organoids (PDOs). We first validated the mesothelin expression in 13 OVC patient samples which, based on pathological scoring showed high mesothelin in 8, moderate in 3, and low in 2 patient samples. PDOs also showed high expression of mesothelin, confirming it as an attractive candidate for CAR cells. We designed the aMSLN-CAR using an ScFv sequence against the proximal domain of the protein with 4-1BB and CD3ζ as co-stimulatory domains. Primary NK cells were purified from peripheral blood, activated using IL-12, IL-18, and IL-15 to afford CIML NK cells. The CAR gene was transduced into CIML NK cells via our optimized baboon lentiviral system to achieve a high transduction efficiency of 40-60%. The direct cytotoxic effect of these NK cells was tested against OVC cell lines. OVC cell lines were co-cultured with NK cells at various effector:target (E:T) ratios 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%) at 10:1 ratio. To test the therapeutic potential of CAR CIML NK cells in vitro, three different PDOs were used. Cytotoxicity was determined by evaluating apoptotic EpCAM+ cancer cells amongst the organoid cell population after 18-hour co-culture with NK cells. The aMSLN-CAR CIML NK cells were shown to cause increased apoptosis in cells from PDOs compared to CIML NK cells; 58.2% vs 11.0%, 4.5% vs 61.0%, and 10.5% vs 32.0%. We are currently evaluating the in vivo functionality of aMSLN-CAR CIML NK cells in a xenograft mouse model using OVC cell lines and PDOs. The successful application of CAR CIML-NK cells will pave way for the use of NK cell platforms for other solid tumors. Citation Format: Mubin Tarannum, Juliana A. Vergara, Yasmin Abdulhamid, Khanhlinh Dinh, Katherine N. Lynch, Sarah Hill, Rizwan Romee. A novel memory-like NK cell CAR targeting proximal mesothelin domain shows promising preclinical activity in ovarian cancer using cell lines and patient derived organoids [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 568.