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+aCD40 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) aCD40 treatment engages CD8+ effector T cells that are antitumorigenic only when combined
with cytotoxic D2C7-IT. Our results strongly imply that aCD40 alters either the development or activity of TAMs
in GBM and activates microglia/T cells. Demonstrating the antitumor efficacy of the D2C7-IT+aCD40 therapy in
relevant brain tumor models and gaining insights into their mechanism of action will greatly aid in the clinical
translation of D2C7-IT+aCD40 therapy. Therefore, we propose to pursue three Specific Aims to characterize
D2C7-IT+aCD40 antitumor efficacy, TAM re-education, and microglia/T cell activation mechanisms: Aim 1:
Evaluate whether aCD40 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+aCD40 antitumor immune response. Aim 3: Determine whether D2C7-IT+aCD40 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.