Abstract We have developed an antibody-enzyme fusion protein designed to overcome cancer treatment limitations due to immunosuppressive tumor microenvironments (TMEs) through a unique three-pronged mechanism of action. Our approach consists of a tumor-targeting nanobody fused to a xanthine oxidase (XO) enzyme domain modified to maximize superoxide production activity. The mechanism of action consists of: 1) the tumor-targeting nanobody domain binds to target-expressing cells, inhibiting growth with selectivity; 2) the XO enzyme domain produces extracellular superoxide, triggering cell death of the target and surrounding cells; and 3) the dying cells undergo immunogenic cell death (ICD), releasing damage-associated molecular patterns (DAMPs) into the TME to recruit immune cells. This plug-and-play approach delivers therapeutic enzymes specifically to the tumor microenvironment, bypassing conventional limitations and establishing a novel platform for oncology treatment. In this study, we investigated the feasibility of our approach in models of glioblastoma (GBM) using an EGFR-targeted fusion protein. A key molecular feature of GBM is EGFR amplification and mutation, however current EGFR-targeted therapies like cetuximab or kinase inhibitors have limited efficacy due to heterogeneous tumors and adaptive resistance. Due to an immunosuppressive TME, GBM tumors are frequently resistant to immunotherapies such as checkpoint blockade and CAR-T therapy. Approaches designed to induce an inflammatory response show promise in GBM, such as oncolytic viruses that trigger ICD. To validate the target binding and enzyme activity of our lead molecule, VISK-103, we have performed enzyme activity assays, ELISA binding assays, and flow cytometric analyses. To demonstrate proof of concept, we have measured efficacy both in vitro and in vivo, using cell viability assays and a mouse GBM xenograft model. In addition, we have employed several in vitro techniques and have demonstrated production of ICD markers (e. g. eATP, HMGB1) and measured immune activation via co-culture experiments. Our results indicate that VISK-103 treatment can bind to EGFR-positive GBM cells with a KD in the nanomolar range. VISK-103 treatment induces cell death in EGFR-positive cells with a sub uM IC50 in vitro for GBM cell lines. In a xenograft model of GBM, VISK-103 treatment led to a ∼50% reduction in tumor burden and prolonged animal survival. Additionally, VISK-103 treatment causes release of DAMPs and activation of dendritic cells in co-culture models with an over 3-fold increase in IL-1b release using VISK-103-treated cells compared to a chemotherapy control. Taken together, we are developing a first-in-class, plug-and-play platform using an antibody-enzyme scaffold that couples cell-selective killing with immune system engagement to provide a multi-pronged approach to GBM treatment. Citation Format: Camille H. Cushman, Bushra Dabbagh, Karistan Swan, Aakanksha Singh Parihar, Daniel T. Dransfield, Eric Fossel. Targeting glioblastoma using an antibody-enzyme fusion protein with a multi-pronged mechanism of action abstract. In: Proceedings of the AACR Special Conference in Cancer Research: Brain Cancer; 2026 Mar 23-25; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2026;86 (6Suppl): Abstract nr A019.
Cushman et al. (Mon,) studied this question.