Expression optimization and functional testing of a scorpion toxin/antibody fragment fusion protein, ACDClx, for the immunotherapeutic treatment of glioblastoma

Abstract

The prognosis for patients with glioblastoma (GBM), a deadly form of brain tumor with a median survival of <15 months, has not substantially improved since the 1970s. To overcome the myriad of treatment obstacles for GBM, we sought to design a new therapeutic capable of selectively enhancing T cell recognition and killing of all GBM cells while preserving healthy tissue. For this purpose, we have designed, expressed, and performed initial testing of the novel bispecific fusion protein, anti-CD3/chlorotoxin (ACDClx). ACDClx is a fusion of anti-CD3 heavy and light variable regions with a non-toxic, GBM-selective scorpion-derived peptide, chlorotoxin, which has been shown to bind to 100% of GBM cells with no selectivity for any healthy tissue types, including brain. ACDClx was expressed in leaves of N. benthamiana, purified, and tested for the capacity to selectively activate T cells. Initial experiments showed that ACDClx activates T cells, measured via calcium flux and CD69 expression, only if T cells, GBM cells, and ACDClx were all present. Mock purified protein in the presence GBM cells and T cells did not result in T cell activation, nor did ACDClx alone. Before further testing in vitro and in vivo, we sought to increase the production of ACDClx, the expression of which is significantly hampered due to its six disulfide bonds. To this end, we have produced 7 new constructs (5 for N. benthamiana, 2 for E. coli), 5 of which include a unique fusion partner for ACDClx to enhance solubility. Ongoing work includes evaluation of the most efficient construct, followed by evaluation in an immunocompetent tumor model of GBM.