DDEL-21. Tumor-collagen binding IL-12 Immunotherapy for recurrent Glioblastoma

Abstract BACKGROUND Recurrent Glioblastoma (rGBM) remains a major therapeutic challenge due to its immunosuppressive microenvironment and poor response to current immunotherapies. Interleukin-12 (IL-12) is a potent anti-tumor cytokine, but its systemic use is limited by immune-related toxicity in normal tissues. Thus, strategies for tumor-selective IL-12 delivery are needed. We aimed to identify anchors for selective IL-12 delivery to tumors and focused on Collagen type I and III (COLI&III), which are minimal in normal brain but highly upregulated during glioblastoma recurrence following radiation and chemotherapy. METHODS We engineered CBD-IL-12 by fusing the collagen-binding domain (CBD) of von Willebrand Factor (vWF) to IL-12. vWF binds selectively to extravascular collagen at sites of vascular injury, inflammation, or tumor, but not to normal tissues, offering a mechanism for tumor-specific delivery. We investigated the tumor-selective accumulation, immunosuppressive microenvironment remodeling, and antitumor effects of CBD-IL-12 using clinically relevant syngeneic rGBM mouse models established from post-radiation tumor cells, along with patient-derived 3D tumor slice cultures. RESULTS Using a fluorescently labeled biodistribution assay, CBD-IL-12 selectively accumulated in rGBM tumors but not in normal brain or COLI/III-expressing organs, and markedly reduced the off-target side effects. Single-cell RNA sequencing revealed that CBD-IL-12 reduced regulatory T and exhausted T cells, while increased CD8+ T cells, anti-tumor macrophages and dendritic cells. In a syngeneic rGBM model, CBD-IL-12 (25µg, i.v.) monotherapy extended survival by up to 260%, with a 13% complete response rate. Combined treatment with radiation and CBD-IL-12 improved the complete response rate to 62.5%. CBD-IL-12 showed no effect in rGBM tumors with COLI/III-knockout, demonstrating that its efficacy depends on COLI&III-expression. We have developed a humanized CBD-IL-12 for clinical application, which effectively remodeled the immunosuppressive microenvironment in patient-derived 3D tumor slice cultures. CONCLUSIONS Collagen-binding IL-12 selectively targets rGBM and effectively reprograms its immunosuppressive microenvironment, with the potential to revolutionize cytokine-based treatment for rGBM.