Brain Tumor Research Laboratory
Walter A. Hall, MD, Professor
Anti-Angiogenic Targeted Toxins against Malignant Gliomas
The prognosis for patients with glioblastoma multiforme (GBM) treated with conventional therapies remains poor. Glioblastoma multiforme is the most common brain tumor in adults and represents 40% of all brain tumors seen annually. One attractive alternative treatment is recombinant targeted toxin (TT) therapy. Targeted toxins are hybrid molecules containing a potent catalytic toxin and a carrier ligand that selectively recognizes the target tumor. After entry into the cancer cell, the TT will kill the cell by blocking protein synthesis (Figure 1). Although TT therapy has been around for a number of years, its use has been limited by organ toxicity when administered systemically.
Injection of the TT directly into the brain tumor will circumvent the problem of systemic toxicity. Brain cancer therapy affords the advantage of local intracranial delivery and clinical responses for this disease have far exceeded those for other cancers with reported response rates in excess of 50%. These encouraging results are mainly because localized delivery can concentrate therapy at the site of tumor diminishing the risk to nontarget organs. Another major advantage of using TT is their effectiveness against both dividing and non-dividing cancer cells in contrast to radiation therapy and conventional chemotherapy that are directed against dividing cells.
Past Clinical TrialsA group at the National Institutes of Health evaluated the toxicity of a TT called TransMID consisting of transferrin as the ligand linked to an altered form of diphtheria toxin (DT). A prospective trial with TransMID was conducted in 18 patients. To distribute TransMID in the tumor and into areas of the brain infiltrated with tumor, they used high flow microinfusion to establish interstitial convection and enhance distribution. Following a positive biopsy, one to three infusion catheters were placed in the tumor. Intratumoral infusions were well tolerated and therapeutic responses were observed. At least a 50% decrease in tumor volume occurred in nine of 15 evaluable patients.
Several other targeted toxins have been developed and tested in clinical trials. These include mutated forms of Pseudomonas exotoxin linked to proteins that targeted the interleukin-13 receptor, the interleukin-4 receptor, and the epidermal growth factor receptor. Only TransMID contains an altered DT. The results of these trials are presently being analyzed. Currently, there are no open clinical trials for TT therapy.
Future Clinical Trials
We have developed a TT against GBM for future use in early phase clinical trials. A recombinant hybrid fusion protein DTAT (Figure 2) was created using a mutated 390-amino acid portion of DT, a linker, and the non-internalizing 135-amino acid terminal (AT) fragment of human urokinase-type plasminogen activator (uPA). DTAT has the advantage over other fusion proteins of targeting the uPA receptor on malignant glioma cells and the endothelial cells of the neovasculature that supplies the tumor. The toxicity, specificity and efficacy of DTAT were evaluated in an intracranial model of human GBM.
The maximum tolerated dose (MTD) of DTAT was determined for three consecutive injections administered every-other-day and by convection enhanced delivery. Intratumoral injection of 0.15 mg every other day for 3 doses was performed in a murine intracranial U87MG GBM model. Convection enhanced delivery of DTAT in nude mice with established intracerebral U87MG brain tumors resulted in significant reductions in tumor volume and prolongation of survival in treated animals compared to controls (P < 0.0001). MR imaging was performed to evaluate the brain for neurotoxicity and the tumor for a therapeutic response. The total MTD for DTAT was 0.45 mg and the total MTD using CED was 5 mg. Toxicity was manifest as weight loss and cerebral infarction.
Experiments in uPA knockout mice revealed that toxicity was not receptor related. MR imaging demonstrated a marked decrease in tumor burden and no evidence of intracranial hemorrhage or vascular leak syndrome. DTAT is an active agent against intracranial GBM without demonstrating evidence of hemorrhage on MR imaging or neurotoxicity on histological examination. Toxicity studies are being conducted in a porcine model. These results support the further development of DTAT for future clinical trials in patients with GBM.
Figure 1. Mechanism of action for targeted toxins. After entry into the cytosol, they are transported in vesicles to the Golgi apparatus where the toxin is cleaved. The toxin will inhibit protein synthesis by preventing the addition of amino acids. Blocking protein synthesis kills the cell.
Figure 2. DTAT. Schematic of the plasmid for inserting the gene sequences into bacteria in order to produce the anti-angiogenic targeted toxin, DTAT.