USP22 inhibition potentiates GPC3 chimeric antigen receptor macrophages efficacy in hepatocellular carcinoma by downregulating tumor CD24 expression.

Although immunotherapy-based regimens have improved overall survival for some patients with advanced hepatocellular carcinoma (HCC), many patients ultimately experience disease progression. Macrophages are abundant in the HCC microenvironment and represent promising therapeutic effectors, yet the potential of combining chimeric antigen receptor macrophages (CAR-M) with macrophage checkpoint targeting remains underexplored. Given that glypican-3 (GPC3), a cell-surface heparan sulfate proteoglycan, is highly expressed in HCC, we developed a GPC3-targeted CAR-M system and evaluated its efficacy alone and in combination with USP22 inhibition. USP22, a deubiquitinating enzyme previously linked to tumor CD24 expression, was selected as a combinatorial target to potentially relieve CD24-associated phagocytic suppression. In vitro assays demonstrated that GPC3 CAR-M cells exhibited specific binding, phagocytosis, and potent cytotoxicity against multiple GPC3-positive HCC cell lines. RNA sequencing and flow cytometry revealed a statistically significant positive correlation between USP22 and the macrophage checkpoint CD24. The addition of the USP22 inhibitor significantly enhanced the tumor-killing capacity of CAR-M in a dose-dependent manner and was associated with reduced CD24 expression on tumor cells. This combinatory strategy robustly suppressed tumor growth in both a murine peritoneal dissemination model and a patient-derived xenograft (PDX) model of HCC. Importantly, the enhanced efficacy was consistently observed across CAR-M cells derived from THP-1, human monocyte-derived macrophages (hMDMs), and human pluripotent stem cells (hPSCs), underscoring its broad applicability. Our findings provide proof-of-concept preclinical evidence supporting further evaluation of combining GPC3 CAR-M therapy with USP22 inhibition as a potential immunotherapeutic strategy for HCC.