A high-affinity anti-ITGB5 nanobody for hepatocellular carcinoma: Antitumor efficacy and tumor microenvironment reprogramming.

Although immune checkpoint blockade has improved systemic therapy for hepatocellular carcinoma (HCC), durable benefit is limited by primary resistance, poor tumor penetration of antibodies, and an immunosuppressive tumor microenvironment. We identified integrin β5 (ITGB5) as an oncogenic and immune-associated target in HCC and developed an ITGB5-directed nanobody. Integrative analyses of TCGA/ICGC/TIMER2.0/Human Protein Atlas datasets, tissue microarrays, and functional assays showed that ITGB5 is upregulated in HCC and correlates with poor prognosis, higher tumor grade, increased immunosuppressive macrophage infiltration, reduced CD8+ T-cell abundance, and predicted resistance to sorafenib, oxaliplatin, and axitinib. Gain- and loss-of-function studies demonstrated that ITGB5 promotes proliferation, clonogenicity, migration/invasion, cell-cycle progression, and tumor growth while suppressing apoptosis, with transcriptomic signatures implicating extracellular matrix-receptor interaction, cytokine signaling, TNF/NF-κB, MAPK, and TGF-β pathways. From a yeast-display library we isolated AntiITGB5-Nb#2, a high-affinity nanobody that recognizes human and murine ITGB5, validated by flow cytometry, immunofluorescence, molecular docking, and surface plasmon resonance. AntiITGB5-Nb#2 inhibited growth and motility of ITGB5-high HCC cells and suppressed tumor progression in subcutaneous and orthotopic models, including an immunocompetent murine HCC model, without overt toxicity. Single-cell RNA sequencing revealed tumor microenvironment remodeling characterized by enhanced cytotoxic immune infiltration and reduced immunosuppressive signaling. These results establish ITGB5 as a prognostic, therapeutically actionable target in HCC and support AntiITGB5-Nb#2 as a nanobody-based strategy to complement current therapies.