Fourth generation Glypican-3–specific CAR-NK cells co-secreting IL-15 and IFN-α have increased anti-tumor function versus hepatocellular carcinoma

Abstract

Abstract Hepatocellular carcinoma (HCC) ranks as the third most frequent cause of cancer-related mortality, indicating the urgent need for effective therapies. Glypican-3 (GPC3) is up-regulated in HCC and not expressed in normal liver cells, representing a strategic target. Chimeric antigen receptor (CAR)-modified natural killer (NK) cells are a promising immunotherapeutic approach for cancer treatment. However, their potential applications have not been fully explored in HCC. We have patented a protocol of purification and expansion of clinically relevant numbers of NK cells starting from the liver perfusate from deceased donors. Further, we have demonstrated that activating NK cells with interferon-alpha (IFN-α) potentiates the immune response in vitro and in vivo both in a murine model of hepatocarcinoma and of viral hepatitis. In this study, we engineered primary CAR-NK cells to acquire specificity for GPC3. The construct consists of the intracellular signaling region CD28, 4–1BB, CD3ζ and CD8α hinge domain and of the truncated form of human epidermal growth factor receptor (tEGFR) as suicide gene. The fourth-generation construct is designed to secrete the transgenic cytokine IL-15, essential for NK cells function and survival. A second plasmid was also generated differing for the additional secretion of IFN-α. Here we show that we could achieve a transduction efficiency of above 50%. Exposure of GPC3+ HCC cells to CAR-NK cells resulted in enhanced specific cytotoxicity and cytokine production such al IL-2 and IFN-γ; moreover IFN-α secretion significantly increased anti-GPC3 function. Our data encourage preclinical proof-of-concept studies to clinically translate GPC3-specific NK cells as a treatment option for HCC patients. This work was supported by Fondazione Ri.MED (Fondo Generale, ID 10019) and ISMETT (ATMP-P A105412 project, ID I00000219).