Engineering tumor-specific interferon-alpha-Nivolumab pro-immunocytokine for enhanced melanoma immunotherapy.

Metastatic melanoma is an aggressive skin cancer with poor prognosis and limited treatment options at advanced stages. While immune checkpoint blockades (ICBs), such as the anti-PD-1 antibody Nivolumab, have improved survival-raising the 1-year rate to 72.9%-durable responses remain limited due to immune evasion, often associated with MHC I downregulation. To address this, we engineered interferon-alpha (IFNα)-Nivolumab, a tumor-selective pro-immunocytokine that combines IFNα signaling with PD-1 blockade. IFNα-Nivolumab remains stable and is activated by tumor-specific proteases. In vitro, its PD-1 binding was reduced by 95.8% before activation and fully restored following MMP-2/9 treatment. In a humanized ASID mouse melanoma model, IFNα-Nivolumab significantly reduced tumor volume compared to Nivolumab or combined IFNα and Nivolumab. Immunohistochemistry showed increased CD4+/CD8+ T cell infiltration and enhanced MHC I expression. Elevated IFNγ, but not TNFα, levels indicated strong cytotoxic immune responses with limited inflammation. By selectively enhancing immune activity within the tumor microenvironment while minimizing systemic toxicity, IFNα-Nivolumab upregulates MHC I expression and enhances therapeutic efficacy compared with Nivolumab alone or the combination of Nivolumab and IFNα. This strategy offers improved therapeutic efficacy and a favorable safety profile, representing a promising advance in overcoming resistance to ICBs in melanoma.