Severe acidity in human tumors for pH-activatable cytokine therapy

Introduction: Precise tumor targeting is essential for improving the therapeutic index of cancer immunotherapies. Systemic cytokine therapies, such as interleukin-2 (IL-2), suffer from narrow therapeutic windows due to dose-limiting toxicities. While receptor engineering and prodrug approaches have been widely investigated, their application is often limited to tumor subtypes that express the corresponding targets. In contrast, tumor acidity is a universal hallmark of the microenvironment, driven by elevated glycolysis, and offers a broadly applicable opportunity for tumor-specific therapy. However, the spatial heterogeneity of tumor acidity remains poorly understood, and its therapeutic application has not been fully demonstrated. Methods: To systematically evaluate the heterogeneity of tumor acidity and its potential for targeted therapy, we employed ultra-pH-sensitive (UPS) nanoparticles that exhibit sharp ON/OFF transitions at defined pH thresholds. In 3D tumor cultures, we embedded UPS nanoprobes within extracellular matrix gels and assessed the spatial distribution of acidity at single-cell resolution. In human cancer patients, we evaluated the tumor-imaging properties of this acidity-targeting strategy and further investigated acidity patterns by analyzing UPS nanoparticle distribution in tumor tissues following intravenous injection. Building on these findings, we developed a formulation strategy to encapsulate IL-2-Fc into UPS micelles, enabling selective cytokine release in acidic microenvironments. This platform aims to provide a comprehensive understanding of spatial acidity and to test the therapeutic index of pH-activatable cytokine delivery. Results: Through this work, we identified a severe acidity phenotype in cancer, where extracellular pH drops below 5.3 due to spatially polarized lactate export. Targeting acidity allows demarcation of tumor from surrounding tissue in intraoperative imaging, even when tumors are as small as 2 mm. Spatial transcriptomic analysis of human head and neck tumors after UPS nanoprobe injection revealed that these severely acidic regions co-localize with immune-infiltrated stromal zones. These regions serve as critical entry points for acid-targeting nanosystems, linking tumor metabolism, immune cell infiltration, and immune suppression in both preclinical models and human tissues. The UPS/IL-2-Fc formulation selectively releases IL-2-Fc within these regions and demonstrates potent antitumor efficacy with markedly reduced systemic toxicity, expanding the therapeutic window by over 10-fold compared to free IL-2-Fc in preclinical models. Conclusion: This study identifies severe acidity as a spatially heterogeneous and translatable hallmark of the tumor microenvironment, enabling tumor-targeted imaging and immunotherapy.