Collagen-binding IL-12 remodels immunosuppresive microenvironment and improves survival in recurrent glioblastoma

Recurrent GBM (rGBM) is the most aggressive malignant brain tumors, with a median survival of 6 months. rGBM establishes a severe immunosuppressive tumor microenvironment (TME) with increased Tregs and impaired CD8 T cell recruitment, rendering immunotherapies effective in other cancers ineffective. While IL-12 is a potent immunostimulatory cytokine capable of remodeling this immunosuppressive TME, systemic delivery causes severe toxicity, hindering clinical application. Therefore, technology to selectively deliver IL-12 to rGBM tumors is critically needed. rGBM acquires treatment resistance through construction of a unique extracellular matrix (ECM); therefore, we conceived of utilizing this as a reservoir to accumulate IL-12. Through screening, we focused on COL I & III, minimally expressed in normal brain but highly upregulated after radiotherapy and in rGBM. We created CBD-IL-12 by fusing IL-12 with the collagen-binding domain (CBD) of von Willebrand factor (vWF), leveraging vWF's selective binding to COL I & III at sites of vascular disruption, inflammation, and tumors while sparing normal organs. Biodistribution assays in a mouse rGBM model demonstrated selective CBD-IL-12 accumulation in rGBM tumors without accumulation in other COL I & III-high organs. Toxicity studies in mice and dogs showed CBD-IL-12 significantly reduced peripheral IFN-gamma levels compared to IL-12 without hepatic or pancreatic dysfunction. Spectral flow cytometry and single-cell RNA-seq revealed that CBD-IL-12 increased effector memory CD8 T cells, M1-like macrophages, and DCs while reducing Tregs, exhausted T cells, M2-like macrophages, and MDSCs, dramatically reversing immunosuppression. CBD-IL-12 (25μg i.v., twice) prolonged survival by 260% with a 13% CR rate in rGBM mice models. Combining CBD-IL-12 with radiotherapy achieved 63% CR. Notably, in our rGBM model where radiotherapy alone provided no survival benefit, combination with CBD-IL-12 achieved dramatic tumor suppression, likely through radiation-induced COL I & III upregulation and enhanced macrophage/microglia recruitment. Long-term survivors demonstrated durable immune memory by rechallenge testing. For clinical translation, we engineered humanized CBD-IL-12 and validated its efficacy in 3D human GBM slice cultures, demonstrating increased CD8 T cells and reduced Tregs. Collagen-binding IL-12 overcomed rGBM immunosuppression with potent antitumor effects, representing a promising novel immunotherapeutic approach.