Targeted delivery of IFN-α-anti-GPC3 fusion protein via mRNA-LNP platform elicits potent anti-tumor immunity in hepatocellular carcinoma.

This study aimed to utilize the mRNA-lipid nanoparticle (mRNA-LNP) platform to achieve in situ hepatic expression of an interferon-α (IFN-α)/anti-glypican-3 (anti-GPC3) fusion protein (GPA01), enhancing IFN-α targeting and antitumor activity to provide a precision therapy strategy for GPC3-positive hepatocellular carcinoma (HCC). mRNA encoding a GPC-3/IFN-α bispecific fusion protein was designed and synthesized, encapsulated in lipid nanoparticles, and transfected into HCC cell lines (HepG2) for in vitro characterization of protein expression, binding activity, and gene induction. Orthotopic HCC models (HepG2-luc) and subcutaneous tumor model (Hepa 1-6/hGPC3-hi) were established in mice to evaluate tumor growth, survival, and immune cell infiltration following treatment with mRNA-LNP or control agents. Safety was assessed in human IFNAR transgenic mice. In vitro experiments demonstrated successful transfection and bioactive fusion protein expression by mRNA-LNP, with transfected supernatants showing specific GPC3 binding and interferon-stimulated gene (ISG) induction. In vivo studies revealed that GPC-3/IFN-α mRNA-LNP significantly inhibited tumor growth, prolonged median survival, and increased intratumoral CD8⁺ T cell and NK cell infiltration compared to controls, with favorable safety profiles. Combination therapy with PD-1 antibody (PD-1 Ab) exerted synergistic antitumor effects, primarily dependent on CD8⁺ T cell infiltration. Safety evaluations in human IFNAR transgenic mice showed good tolerability at single doses of 1-10 mpk, with transient changes in select biomarkers. Repeated dosing (6 or 10 mpk) identified a maximum tolerated dose (MTD) of 6 mpk, at least 40-fold higher than the minimal effective dose (MED, 0.15 mpk). mRNA-LNP-mediated delivery of IFN-α-anti-GPC3 fusion protein achieves targeted in situ hepatic expression, significantly enhancing antitumor activity with a broad therapeutic window. This strategy offers a novel approach for precision immunotherapy in HCC, holding substantial potential for clinical translation.