IL-2(N88D)/CD25 Fusion Protein Enhances Expansion of Antigen-Specific CD8⁺ T Cells to Suppress Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) remains a major cause of cancer-related death worldwide, with limited efficacy of current immunotherapies due to an immunosuppressive tumor microenvironment and dysfunctional CD8⁺ tumor-infiltrating lymphocytes (TILs). Among these, PD-1⁺CD25⁺ CD8⁺ T cells represent a tumor-reactive subset linked to IL-2 responsiveness and favorable responses to PD-1 blockade. In this study, we integrated single-cell and spatial transcriptomics with clinical immunotherapy cohort analyses to characterize the phenotype and function of PD-1⁺CD25⁺ CD8⁺ TILs in human HCC. To therapeutically exploit this subset, we engineered IL-2(N88D)/CD25 fusion proteins with trimeric receptor bias, selectively promoting the expansion of PD-1⁺CD25⁺ CD8⁺ T cells. These constructs were tested in multiple murine HCC models, including orthotopic and hydrodynamic tail vein injection systems. PD-1⁺CD25⁺ CD8⁺ TILs were enriched in HCC tumors, particularly in immunotherapy responders, and exhibited enhanced tumor reactivity and neoantigen specificity. Treatment with IL-2/CD25 led to increased CD8⁺ T cell infiltration and delayed tumor progression with minimal toxicity, while the IL-2(N88D)/CD25 variant further boosted infiltration, activation, and survival outcomes. These findings highlight the therapeutic potential of selectively activating antigen-specific CD8⁺ T cells via IL-2(N88D)/CD25 fusion proteins, offering a promising strategy for improving HCC immunotherapy. An IL-2(N88D)/CD25 fusion protein selectively expands antigen-specific CD8⁺ T cells, enhances antitumor immunity, and suppresses hepatocellular carcinoma while minimizing regulatory T cell expansion.