Circumventing IFN-γ induced resistance in ovarian cancer with a double-hit: NKG2A knockout of PRAME-TCR expressing NK cells

The success of NK cell therapies against solid tumors remains limited, possibly due to tumor resistance mechanisms associated with the upregulation of inhibitory ligands. Previous studies have demonstrated that expanded NK cells can lyse ovarian cancer cells and produce IFN-γ. However, secretion of IFN-γ within the tumor microenvironment, leads to the upregulation of both classical HLA class I and the non-classical HLA-E on bystander tumor cells, thereby contributing to resistance against NK cell-mediated cytotoxicity. To overcome this IFN-γ induced resistance, we developed NKG2A-knockout NK: TCR cells targeting PRAME (NK: PRAME NKG2A KO ), a tumor-associated antigen expressed in ovarian cancer. Primary NK cells were isolated from PBMCs, stimulated with cytokines, and genetically modified using CRISPR-Cas9 to knockout the KLRC1 gene, which encodes NKG2A. After stimulation, the NK cells were further engineered to express the PRAME-specific TCR. NK: PRAME NKG2A KO were compared with control NK: PRAME NKG2A WT and NK: MOCK NKG2A KO cells for effector function against PRAME-positive ovarian cancer cell lines and primary ovarian cancer cells. To mimic the pro-inflammatory tumor environment, ovarian cancer cell lines were pre-treated with IFN-γ. First we observed that NK: PRAME cells without a KLRC1 knockout were not effective to lyse IFN-γ treated ovarian cancer cells, irrespective of upregulated HLA class I expression. To overcome HLA-E mediated inhibition, the CRISPR-Cas9 induced KLRC1 knockout was successfully achieved without negatively impacting NK: TCR cell engineering, expansion and further alterations in phenotype. As a result, the NK: PRAME NKG2A KO cells exhibited increased cytotoxicity against these IFN-γ treated tumor cells. This dual-targeting strategy offers a unique advantage by enabling the targeting of both HLA-positive and HLA-negative tumor cells, promoting a pro-inflammatory environment and enhancing the efficacy of TCR-based immunotherapy for ovarian cancer and other solid tumors.