Fusion protein immunotherapies such as the bispecific T cell engager (BiTE) have displayed promising potential as cancer treatments capable of engaging the immune system against tumor cells. It has been shown that chlorotoxin, a 36-amino peptide found in the venom of the deathstalker scorpion (Leiurus quinquestriatus), binds specifically to glioblastoma (GBM) cells without binding healthy tissue, making it an ideal GBM cell binding moiety for a BiTE-like molecule. However, chlorotoxin’s four disulfide bonds pose a folding challenge outside of its natural context and impede production of the recombinant protein in various expression systems, including those relying on bacteria and plants. To overcome this difficulty, we have engineered a truncated chlorotoxin variant (Cltx∆15) that contains just two of the original eight cystine residues, thereby capable of forming only a single disulfide bond while maintaining its ability to bind GBM cells. We further created a BiTE (ACDClx∆15) which tethers Cltx∆15 to a single chain ⍺-CD3 antibody in order to bring T cells into contact with GBM cells. The gene for ACDClx∆15 was cloned into a pET-11a vector for expression in Escherichia coli and isolated from inclusion bodies before purification via affinity chromatography. Immunoblot analyses confirmed that ACDClx∆15 can be expressed in E. coli and purified with high yield and purity; moreover, flow cytometry indicated that ACDClx∆15 is capable of binding GBM cells. These data warrant further investigation into the ability of ACDClx∆15 to activate T cells against GBM cells.

Engaging the immune system to attack neoplastic glial cells in the brain may be a promising approach to eliminate glioblastoma (GBM), a deadly form of primary brain cancer with low median survival. A bispecific fusion protein, anti-CD3/chlorotoxin (ACDClx), has been developed to engage cytotoxic T cells for destruction against GBM with little to no expected toxicity to surrounding healthy tissue. Previously, ACDClx has been demonstrated to induce calcium flux in T cells, indicating activation when cultured with GBM cells in vitro. Here, ACDClx fails to demonstrate successful binding to the CD3 domain of the T-cell receptor on CD4 T cells in vitro and fails to bind GBM cells despite demonstrated binding of chlorotoxin to the same cell line. This data warrants further investigation into the binding characteristics of ACDClx to target cells.