PROJECT SUMMARY/ABSTRACT Glioblastoma (GBM) is the most common and aggressive primary brain tumor in adults. Current therapies remain unsuccessful in improving overall survival; thus, the identification of novel therapies for GBM is critical. We have pioneered a novel, targeted immunotoxin (IT)-based cytotoxic therapy, D2C7-IT, that targets epidermal growth factor receptor (EGFR) and mutant EGFR variant III (EGFRvIII), established driver oncogenes of GBM. In preclinical studies, D2C7-IT targets and kills a substantial number of tumor cells and prolongs survival but is unable to generate cures in all treated animals because of the presence of a highly immunosuppressive GBM microenvironment. The majority of the immune cells in the GBM microenvironment are tumor-associated macrophages (TAMs), which promote tumor cell growth and inhibit antitumor T cell responses. Therefore, eliminating TAM-mediated immunosuppression is anticipated to enhance D2C7-IT-induced antitumor immune responses. CD40 is an immune co-stimulatory molecule whose activation is known to re-educate TAMs, and also induce T cell responses. Thus, the central hypothesis driving the present proposal is that overcoming TAM immunosuppression and tumor-promoting activities via CD40 co-stimulation will improve the efficacy of the cytotoxic D2C7-IT therapy. Accordingly, our preliminary studies have demonstrated that (1) in a mouse glioma model, D2C7-IT+αCD40 functions synergistically to prolong survival and generate significant cures, (2) brain resident microglia is the principal antigen-presenting cells (APCs) activated by the combination therapy, and (3) αCD40 treatment engages CD8+ effector T cells that are antitumorigenic only when combined with cytotoxic D2C7-IT. Our results strongly imply that αCD40 alters either the development or activity of TAMs in GBM and activates microglia/T cells. Demonstrating the antitumor efficacy of the D2C7-IT+αCD40 therapy in relevant brain tumor models and gaining insights into their mechanism of action will greatly aid in the clinical translation of D2C7-IT+αCD40 therapy. Therefore, we propose to pursue three Specific Aims to characterize D2C7-IT+αCD40 antitumor efficacy, TAM re-education, and microglia/T cell activation mechanisms: Aim 1: Evaluate whether αCD40 overcomes TAM immunosuppression and enhances D2C7-IT efficacy in two preclinical immunocompetent glioma models. Aim 2: Define whether microglial CD40/MHCII molecules are the mediators of D2C7-IT+αCD40 antitumor immune response. Aim 3: Determine whether D2C7-IT+αCD40 therapy stimulates CD8+ effector T cell response capable of eliminating antigen-positive as well as antigen-negative tumors. The proposed research is significant because it will result in the development of a therapeutic strategy for simultaneous tumor cell killing, reversal of TAM immunosuppression, activation of microglia and T cells, and ultimately could be translated and tested in the clinic.