Recruitment of Immune Effector Cells against Glioblastoma-Multiforme by a MHC-Chlorotoxin Chimeric Protein

Abstract

Glioblastoma Multiforme is the most common malignant primary brain tumor, having a mean overall survival <2 years. The lack of an efficient immune response against the tumor have been attributed to its immunosuppressive capabilities and an immunosuppressing local environment. Aim: We set out to design a chimeric molecule that recognizes and binds tissue inducible metalloproteinase known to be induced in GBM cells (MMP-2) on one end. Its other end, the effector domain, mobilizes and recruits cytotoxic T-cells to mount an effective anti-tumor reaction. Methods: The targeting moiety is the small 36-amino acids Chlorotoxin, derived from the venom of the Israeli Yellow scorpion. The effector end is a single chain HLA-A2 (Human leukocyte antigen subtype A2) covalently bound to phosphoprotein-65 derived from the cytomegalovirus, to which most of the human population has developed a specific immune response. Results: The molecular construct was cloned and expressed in E.coli. The protein product was isolated, purified, and then folded in vitro. Various activity assays employed demonstrated retained activity of each domain, including flow-cytometry, intracellular staining, fluorescence immunohistochemistry, radiolabeled toxicity assays etc. Initial in-vivo studies show great promise. Conclusions: We present a proof of concept study for a new immunotherapy approach to battle GBM. A molecular construct which contains a non-antibody compact and highly specific targeting domain, combined with the ability to recruit anti-CMV T-cell lymphocyte population. The recruitment of potent memory CTL’s to the tumor’s milieu may prove resistant to the previously described local immunosuppressive environment brought about by the tumor.