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

Journal of Ovarian Research https://doi.org/10.1186/s13048-026-02156-0 Article in Press Circumventing IFN-γ induced resistance in ovarian cancer with a double-hit: NKG2A knockout of PRAME-TCR expressing NK cells Els P. Hees, Oliva W. Rensing, Renate S. Hagedoorn, Cilia R. Pothast, Anne K. Wouters, Rosa A. Amerongen, J. H. Frederik Falkenburg, Rosa Groot & Mirjam H.M. Heemskerk Received: 23 February 2026 Accepted: 29 May 2026 Cite this article as: Hees E.P., Rensing O.W., Hagedoorn R.S. et al. Circumventing IFN-γ induced resistance in ovarian cancer with a double-hit: NKG2A knockout of PRAME-TCR expressing NK cells. J Ovarian Res (2026). https://doi.org/10.1186/ s13048-026-02156-0 A S S We are providing an unedited version of this manuscript to give early access to its findings. Before final publication, the manuscript will undergo further editing. Please note there may be errors present which affect the content, and all legal disclaimers apply. E R P If this paper is publishing under a Transparent Peer Review model then Peer Review reports will publish with the final article. I T R E L C IN © The Author(s) 2026. Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/. ARTICLE IN PRESS Circumventing IFN-γ induced resistance in ovarian cancer with a double-hit: NKG2A knockout of PRAME-TCR expressing NK cells Els P. van Hees1, Oliva W. Rensing1, Renate S. Hagedoorn1, Cilia R. Pothast1, Anne K. Wouters1, Rosa A. van Amerongen1, J.H. Frederik Falkenburg1, Rosa de Groot1, Mirjam H.M. Heemskerk1 1 Leiden University Medical Center (LUMC), Leiden, Netherlands, Department of Hematology Corresponding author: Mirjam H.M. Heemskerk () S S E R P Keywords: Cancer, NK cell therapy, TCR, Interferon-γ, Ovarian Abstract E L C IN Background: The success of NK cell therapies against solid tumors remains limited, I T AR 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 nonclassical 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:PRAMENKG2A KO), a tumorassociated antigen expressed in ovarian cancer. Methods: Primary NK cells were isolated from PBMCs, stimulated with cytokines, and genetically modified using CRISPR-Cas9 to knockout the KLRC1 gene, which encodes ARTICLE IN PRESS NKG2A. After stimulation, the NK cells were further engineered to express the PRAMEspecific TCR. NK:PRAMENKG2A KO were compared with control NK:PRAMENKG2A WT and NK:MOCKNKG2A 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. Results: 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 S S E R P engineering, expansion and further alterations in phenotype. As a result, the NK:PRAMENKG2A KO cells exhibited increased cytotoxicity against these IFN-γ treated tumor cells. E L C IN I T AR Conclusions: This dual-targeting strategy offers a unique advantage by enabling the targeting of both HLA-positive and HLA-negative tumor cells, promoting a proinflammatory environment and enhancing the efficacy of TCR-based immunotherapy for ovarian cancer and other solid tumors. Background Over the last decade, adoptive cell therapy (ACT) has shown significant clinical potential, especially since the rise of chimeric antigen receptor (CAR-T) and T cell receptor (TCR-T) therapy (1, 2). Nevertheless, there are still limitations associated with these therapies. Besides adverse events, such as cytokine release syndrome (CRS) and neurotoxicity, therapeutic resistance can arise, most commonly through ARTICLE IN PRESS downregulation of the target antigen on tumor cells (3-5). For TCR-T therapies, this escape mechanism can involve reduced expression of both the target protein and the presenting HLA molecule (5). To overcome HLA-dependent immune escape, natural killer (NK) cells could be used as an alternative source for TCR therapy, since NK cells are capable of recognizing and eliminating tumor cells with minimal HLA expression (610). By introducing tumor-specific TCRs into NK cells (NK:TCR), a dual-targeting approach can be achieved: TCR-medicated recognition of intracellular tumor-associated antigens presented on HLA molecules, combined with NK-mediated recognition of HLAdeficient tumor cells(11, 12). S S E R P NK:TCR therapy could also be an interesting strategy for the treatment of solid tumors, such as ovarian cancer, as they often exhibit heterogenous HLA expression, which IN makes them not always suitable for TCR-T therapy (13). Intrinsically, NK cells have E L C shown promising results in vitro for treatment of ovarian cancer. However, their clinical I T AR efficacy remains limited (14-16). One possible reason for this could be the interferongamma (IFN-γ) production of NK cells upon tumor interaction, inducing HLA class I and HLA-E expression on tumor cells, which could eventually provoke resistance to NKmediated cytotoxicity (13, 17-20). However, this upregulation of HLA class I offers possibilities for NK:TCR therapies, since ovarian cancer cells express a variety of intracellular tumor-associated antigens recognized by tumor-specific TCRs (21, 22). Over the years, we have identified several high-affinity TCRs specific to various antigens expressed in ovarian cancer and present (...truncated)