Glypican-3-Specific CAR NK Cells Co-Secreting IL-15 and IFN-α Have Increased Anti-Tumor Function Versus Hepatocellular Carcinoma In Vitro.

Chimeric antigen receptor (CAR)-modified natural killer (NK) cells represent a promising immunotherapeutic approach for the treatment of oncological malignancies such as hepatocellular carcinoma (HCC). In this work, we have engineered primary human NK cells, re-directing them so they can specifically recognize Glypican-3 (GPC3), an immunotherapeutic target for HCC. In previous studies, we have demonstrated that IFN-α significantly enhances NK cells' anti-tumor and anti-viral cytotoxicity. Fourth-generation self-inactivating lentiviral vectors were used to deliver a transgenic expression of IFN-α or its co-expression with IL-15 (which induces NK cells expansion, survival, and function), aiming to enhance CAR-GPC3 NK cells' anti-tumor response against HCC. We optimized a protocol for efficient transduction of primary NK cells, demonstrating that CAR expression is maintained at high levels over time. Exposure of HCC ectopically expressing GPC3+ to CAR-GPC3-IL15 and CAR-GPC3-IL15-IFNα NK cells demonstrated significant in vitro cytotoxicity and cytokine production, dependent on GPC3 expression. To prevent undesired side effects of CAR-NK cell immunotherapy, co-delivery with a suicide gene is advised as a safety measure. Thus, a truncated epidermal growth factor receptor (tEGFR) was co-delivered with the anti-GPC3 CAR, which efficiently promoted the suicide of the CAR-NK used in this work. Our study demonstrates the efficacy of re-directed CAR-GPC3 primary NK cells, encouraging further preclinical and clinical translation studies and strengthening the potential of these cells as a novel treatment option for patients with HCC.