Non-α-biased IL-2 enhances both intratumoral and subcutaneous CpG/α-OX40 therapy, unleashing systemic antitumor immunity in mice

Background The CpG/α-OX40 in situ vaccine strategy has shown efficacy in immune-hot tumors but remains ineffective in immune-cold tumors possibly due to immunosuppressive microenvironments characterized by myeloid-derived suppressor cells (MDSCs) infiltration and T cell exclusion. This study aims to investigate the antitumor effect of non-α-biased IL-2 combined with CpG/α-OX40 in mice. Methods Using 4T1 breast cancer and CT26 colon cancer models, we evaluated the antitumor effects of combining CpG/α-OX40 with a non-α-biased IL-2 variant via both intratumoral and subcutaneous routes. Tumor growth, lung metastasis, immune profile, and in vitro function analysis were assessed through flow cytometry, transcriptomic analysis, and T cell suppression assays. Results The combination therapy significantly inhibited primary and distant tumor growth and reduced lung metastasis in 4T1 models. Subcutaneous administration induced complete tumor regression in 55% (5/9) of CT26-tumor-bearing mice and conferred durable tumor-specific memory. Mechanistically, the treatment enhanced CD8 + T cell activation, metabolic reprogramming, and IFN-γ production, while suppressing MDSC expansion and immunosuppressive function. Conclusions These findings demonstrate that non-α-biased IL-2 synergizes with CpG/α-OX40 to overcome microenvironmental immunosuppression and achieve systemic antitumor immunity via a subcutaneous route, offering a translatable combinatorial strategy for immune-cold tumors.