Treatment of glioblastoma (GBM) represents an unmet need in medicine. We have been pursuing a therapeutic approach of delivering potent targeted and specific cytotoxins using convection-enhanced delivery (CED). We and others found that patients with GBM over-express interleukin 13 receptor alpha 2 (IL-13RA2), EphA2, EphA3 and EphB2 receptors. These receptors are present in various pathophysiological compartments of GBM and all four are expressed in tumor cells of the core of tumor and in locally-infiltrating tumor cells, while EphA2 is also found in tumor neovasculature. Further, IL-13RA2, EphA2, and EphA3 are associated with, and play crucial roles in, the pathobiology of glioma stem-like cells (GSC). Finally, the EphA3 receptor can be readily detected in M2 tumor-associated macrophages (TMA). Thus, collectively, IL-13RA2, EphA2, EphA3 and EphB2 are over-expressed in principal GBM compartments shown to be involved in tumor progression and/or resistance to therapies. One of the Eph receptor ligands, ephrinA5 (eA5), binds EphA2, EphA3 and EphB2 receptors. In the current project, we will pursue the novel idea of targeting all four receptors with one pharmaceutical compound delivered using monitored and effective CED. We have already engineered an agent based on eA5 and IL-13 mutants targeting all four receptors using an IgG1 scaffold and conjugated it to a modified bacterial toxin to form QUAD 3.0-PE38QQR. The conjugate is safe and effective in GBM tumors. We will continue this exciting line of research through three Specific Aims. In Specific Aim 1, we will evaluate QUAD 3.0-PE38QQR distribution, safety and anti-tumor activity in treating canine high-grade gliomas, which represents the closest model of human disease. In Specific Aim 2, we will develop QUAD 3.0-PE38QQR for first-in-human Phase I clinical trial in patients with recurrent GBM. We will make QUAD 3.0-PE38QQR under Good Manufacturing Practices (GMP) conditions. The QUAD-CTX will undergo pre-clinical animal studies, based on pre-IND discussions with the FDA. Studies will be perfromed under Good Laboratory Practices (GLP) conditions in order to obtain an Investigational New Drug (IND). In the third Specific Aim, we will perform Phase I clinical trial with QUAD 3.0-PE38QQR in patients with recurrent GBM. The focus will be on obtaining optimal volume of distribution of the CED-administered drug, its safety, initial efficacy and evidence of inducing immune responses. Thus, with one therapeutic agent and improved delivery system, we will be eliminating tumor cells and abnormal cells of the tumor microenvironment promoting its growth. This approach also addresses crucial issues of inter- and intra-tumoral heterogeneity and is also expected to evoke an in situ vaccination or so called “tumor inflaming” effect. We envision that this all-out assault, termed by us “molecular resection”, will result in a more effective management of GBM.