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Circumventing IFN-γ induced resistance in ovarian cancer with a double-hit: NKG2A knockout of PRAME-TCR expressing NK
Els P van Hees1, Oliva W Rensing1, Renate S Hagedoorn1
1Department of Hematology, Leiden University Medical Center (LUMC), Leiden, Netherlands.
Background:
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: PRAMENKG2A KO), a tumor-associated 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 NKG2A. After stimulation, the NK cells were further engineered to express the PRAME-specific 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 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.
Conclusions:
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.
Insights
Engineered Natural Killer (NK) cells targeting PRAME with NKG2A knockout overcome IFN-γ-induced resistance in ovarian cancer. This dual-targeting strategy enhances NK cell cytotoxicity against both HLA-positive and HLA-negative tumor cells.
Area of Science:
- Immunotherapy
- Oncology
- Cellular Biology
Background:
- Natural Killer (NK) cell therapy efficacy against solid tumors is limited by tumor resistance mechanisms, including upregulation of inhibitory ligands.
- Interferon-gamma (IFN-γ) secreted in the tumor microenvironment can increase HLA class I and HLA-E expression on tumor cells, promoting resistance to NK cell-mediated cytotoxicity.
- A strategy was developed to overcome IFN-γ-induced resistance by creating NKG2A-knockout NK: TCR cells targeting PRAME, a tumor-associated antigen in ovarian cancer.
Purpose of the Study:
- To develop and evaluate NKG2A-knockout NK: TCR cells targeting PRAME (NK: PRAMENKG2A KO) for enhanced efficacy against ovarian cancer.
- To assess the ability of these engineered NK cells to overcome IFN-γ-induced resistance mediated by HLA-E.
- To compare the effector function of NK: PRAMENKG2A KO cells with control NK cells in a simulated pro-inflammatory tumor environment.
Main Methods:
- Primary NK cells were isolated, stimulated, and genetically modified using CRISPR-Cas9 to knockout the KLRC1 gene (encoding NKG2A).
- NK cells were further engineered to express a PRAME-specific T-cell receptor (TCR).
- Effector functions of NK: PRAMENKG2A KO cells were compared to control NK: PRAMENKG2A WT and NK: MOCKNKG2A KO cells against ovarian cancer cell lines and primary cells pre-treated with IFN-γ.
Main Results:
- NK: PRAME cells without NKG2A knockout showed limited efficacy against IFN-γ-treated ovarian cancer cells, despite upregulated HLA class I.
- Successful CRISPR-Cas9 mediated KLRC1 knockout was achieved without compromising NK: TCR cell engineering, expansion, or phenotype.
- NK: PRAMENKG2A KO cells demonstrated significantly increased cytotoxicity against IFN-γ-treated ovarian cancer cells, overcoming HLA-E-mediated inhibition.
Conclusions:
- The dual-targeting strategy using NKG2A-knockout NK: TCR cells effectively targets both HLA-positive and HLA-negative tumor cells.
- This approach promotes a pro-inflammatory tumor microenvironment, enhancing the efficacy of TCR-based immunotherapy.
- The developed NK: PRAMENKG2A KO cells represent a promising advancement for ovarian cancer and other solid tumor treatments.
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