Interstitial diphtheria toxin-epidermal growth factor fusion protein therapy produces regressions of subcutaneous human glioblastoma multiforme tumors in athymic nude mice.

PURPOSE: The novel fusion protein, DAB389EGF, composed of the catalytic and translocation domains of diphtheria toxin (DAB389) fused with a His-Ala linker to human epidermal growth factor (EGF) was tested for antiglioma efficacy in an in vivo model of human glioma. EXPERIMENTAL DESIGN: Female athymic nude mice (ages 4-6 weeks) were inoculated s.c. with 10 million U87MG human glioma cells in the right flank. When tumor volumes reached approximately 100 mm3 (approximately 6-8 days), i.t. injections of saline, DAB389IL2, or DAB389EGF 1, 3, 5 or 10 microg in 50 microL were given every other day for three to six doses. Animals were monitored twice daily and tumor measurements were made by calipers. RESULTS: The maximal tolerated dose (MTD) of DAB389EGF was 3 microg every other day. Above the MTD, animals experienced loss of activity, reduced oral intake, and dehydration. Blood chemistries confirmed elevated blood urea nitrogen, creatinine, aspartate transaminase, and alanine transaminase. Histopathology revealed renal tubular necrosis. At the MTD, tumor regression was seen in all animals. Relapses occurred in 4 of 16 (25%) of animals after 1 month. These tumors contained EGF receptor, were sensitive in vitro to DAB389EGF, and responded to a second course of i.t. DAB389EGF. CONCLUSIONS: DAB389EGF fusion protein shows in vivo antiglioma efficacy in a s.c. tumor model and warrants further preclinical testing in an i.c. tumor model for eventual treatment of patients with recurrent or refractory EGF receptor-positive glioblastoma multiforme.

Safety evaluation of DT388IL3, a diphtheria toxin/interleukin 3 fusion protein, in the cynomolgus monkey.

We developed a fusion toxin, DT388IL3, consisting of the catalytic and translocation domains of diphtheria toxin (DT388) linked to interleukin 3 (IL3) for the treatment of patients with acute myeloid leukemia (AML). Our goal in this study was to estimate a range for the maximum tolerated dose (MTD) and to evaluate the dose-limiting toxicity (DLT) of DT388IL3 in cynomolgus monkeys (Macaca fasicularis), which possess cross-reactive IL3 receptors. In our previous study, we administered up to six infusions of DT388IL3 at 40, 60, or 100 microg/kg every other day to three pairs (one male monkey and one female monkey) of young adult monkeys. In five of six monkeys, results showed a dose-dependent increase in malaise and anorexia but no consistent abnormalities in serum chemistries or blood counts. There was no evidence of organ damage by blood tests or histopathology. However, the female treated at 100 microg/kg, died of moderate to severe vasculitis of multiple tissues. Based on these findings, this study repeated the 100 microg/kg group and added a group that received 150 microg/kg in an effort to confirm a dose response. Two female monkeys were treated with up to six infusions of DT388IL3 at 100 microg/kg or 150 microg/kg every other day. One additional female monkey was treated as a negative control. Monkeys in the 100 microg/kg group showed moderate malaise and anorexia, but no consistent abnormalities in blood counts or serum chemistries. Moderate elevations of liver enzymes were noted in the 150 microg/kg group in addition to severe malaise and anorexia. No significant findings were revealed at gross necropsy. The histopathological findings revealed regenerative myeloid hyperplasia and hepatic degeneration and regeneration in the 150 microg/kg group. Similar lesions of less severity were detected in the 100 microg/kg group. DT388IL3 plasma half-life was approximately 20 min with a peak concentration of approximately 2 microg/ml (30,000 pM). The IC50 for AML blasts in vitro was 6 pM. Collectively, our results suggest that DT388IL3 can be tolerated at doses up to 100 microg/kg in a nonhuman primate, which is higher than previously reported for other AML directed diphtheria toxin fusion proteins, and should in principle allow for dose escalation with reduced toxic side effects. Based on these findings a phase I clinical trial has recently been initiated with DT388IL3 for the treatment of AML.

Toxicology and pharmacokinetics of DT388IL3, a fusion toxin consisting of a truncated diphtheria toxin (DT388) linked to human interleukin 3 (IL3), in cynomolgus monkeys.

The fusion toxin DT388IL3 composed of the catalytic and translocation domains of diphtheria toxin (DT388) linked to interleukin-3 (IL3) was administered to 6 cynomolgus monkeys which possessed cross-reactive IL3 receptors. Groups of 2 animals (1 male and 1 female) received up to 6 every other day slow intravenous infusions of 40, 60, or 100 microg/kg DT388IL3. Monkeys given 40 or 60 microg/kg showed mild or moderate transient malaise and anorexia, respectively, without evidence of organ damage by blood tests or histopathology. Animals treated at 100 microg/kg showed severe malaise and anorexia. The female monkey had moderate to severe vasculitis in multiple tissues. Necropsies were performed on the 40 microg/kg monkeys on day 14 and the 100 microg/kg monkeys on days 6 and 7. DT388IL3 plasma half-life was approximately 30 min with a peak concentration of 0.45 microg/ml or 10,000 pM (IC50 for AML blasts treated in vitro was 6 pM). Immune responses were minimal in 4 animals tested at 12 days and 2 animals tested at 30 days post treatment with anti-DT388IL3 levels < 1 microg/ml. Bone marrow aspirates were obtained on all animals at day 19 or at necropsy and revealed myeloid suppression in the females and myeloid hyperplasia in the males irrespective of dose groups. The maximal tolerated dose of 60 microg/kg for 6 doses is markedly higher than other recombinant diphtheria toxins and provides a dose level sufficient for anti-leukemic activity in vitro and in rodent models. Thus, we propose this agent is a promising drug for AML patients.

Expression and purification of the recombinant diphtheria fusion toxin DT388IL3 for phase I clinical trials.

A genetically engineered fusion toxin targeted to acute myeloid leukemia (AML) blasts was designed with the first 388 amino acid residues of diphtheria toxin with an H-M linker fused to human interleukin-3. The cDNA was subcloned in the pRK bacterial expression plasmid and used to transform BLR (DE3) Escherichia coli. A single transformed colony was grown in Superbroth with ampicillin; bacteria were centrifuged at an OD(650) of 1.3; master cell bank aliquots of bacteria in 30% glycerol/Superbroth were frozen and stored at -80 degrees C. Master cell bank bacteria were diluted 1500-fold into Superbroth and recombinant protein was induced with 1 mM IPTG at an OD(650) of 0.6. After two additional hours of fermentation, inclusion bodies were isolated, washed, and denatured in guanidine hydrochloride and dithioerythritol. Recombinant protein was refolded by diluted 100-fold in cold buffer with arginine and oxidized glutathione. After dialysis, purified protein was obtained after anion-exchange, size exclusion on FPLC, and polymyxin B affinity chromatography. The final material was filter sterilized, aseptically vialed, and stored at -80 degrees C. Seventy-five 3-L bacterial culture preparations were made and pooled for the AT-1 batch (568 mL) and twenty-four 3-L bacterial culture preparations were made and pooled for the AT-2 batch (169 mL). The final product was characterized by Coomassie Plus protein assay, Coomassie-stained SDS-PAGE, limulus amebocyte lysate endotoxin assay, human AML TF/H-ras cell cytotoxicity assay, sterility, tandem mass spectroscopy, IL3 receptor binding affinity, ADP ribosylation activity, inhibition of normal human CFU-GM, disulfide bond analysis, immunoblots, peptide mapping, stability, HPLC TSK3000, N-terminal sequencing, E. coli DNA contamination, C57BL/6 mouse toxicity, cynomolgus monkey toxicity, and immunohistochemistry. Yields were 25.7+/-5.6 mg/L bacterial culture of denatured fusion toxin. After refolding and chromatography, final yields were 20+/-11% or 5 mg/L. Vialed product was sterile. Batches were in 0.25 M sodium chloride/5 mM Tris, pH 8, and had protein concentrations of 1.8-1.9 mg/mL. Purity by SDS-PAGE was 99+/-1%. Aggregates by HPLC were <1 %. Potency revealed a 48 h IC(50) of 6-8 pM on TF/H-ras cells. Endotoxin levels were 1 eu/mg. The remaining chemical and biologic assays confirmed the purity, composition, and functional activities of the molecule. The LD(10) in mice was 250 microg/kg/day every other day for six doses. The MTD in monkeys was 60 microg/kg/day every other day for six doses. Drug did not react with tested frozen human tissue sections by immunohistochemistry. There was no evidence of loss of solubility, proteolysis aggregation, or loss of potency over 6 months at -80 and -20 degrees C. Further, the drug was stable at 4 and 25 degrees C in the plastic syringe and administration tubing for 24 h and at 37 degrees C in human serum for 24 h. The synthesis of this protein drug should be useful for production for clinical phase I/II clinical trials and may be suitable for other diphtheria fusion toxins indicated for clinical development.

Diphtheria toxin-murine granulocyte-macrophage colony-stimulating factor-induced hepatotoxicity is mediated by Kupffer cells.

DT388GMCSF, a fusion toxin composed of the NH2-terminal region of diphtheria toxin (DT) fused to human granulocyte-macrophage colony-stimulating factor (GMCSF) has shown efficacy in the treatment of acute myeloid leukemia. However, the primary dose-limiting side effect is liver toxicity. We have reproduced liver toxicity in rats using the rodent cell-tropic DT-murine GMCSF (DT390mGMCSF). Serum aspartate aminotransferase and alanine aminotransferase were elevated 15- and 4-fold, respectively, in DT390mGMCSF-treated rats relative to controls. Histologic analysis revealed hepatocyte swelling; however, this did not lead to hepatic necrosis or overt histopathologic changes in the liver. Immunohistochemical staining showed apoptotic cells in the sinusoids, and depletion of cells expressing the monocyte/macrophage markers, ED1 and ED2, indicating that Kupffer cells (KC) are targets of DT390mGMCSF. In contrast, sinusoidal endothelial cells seemed intact. In vitro, DT390mGMCSF was directly cytotoxic to primary KC but not hepatocytes. Two related fusion toxins, DT388GMCSF, which targets the human GMCSF receptor, and DT390mIL-3, which targets the rodent IL-3 receptor, induced a less than 2-fold elevation in serum transaminases and did not deplete KC in vivo. In addition, DTU2mGMCSF, a modified form of DT390mGMCSF with enhanced tumor cell specificity, was not hepatotoxic and was significantly less toxic to KC in vivo and in vitro. These results show that DT390mGMCSF causes liver toxicity by targeting KC, and establish a model for studying how this leads to hepatocyte injury. Furthermore, alternative fusion toxins with potentially reduced hepatotoxicity are presented.

Diphtheria toxin-epidermal growth factor fusion protein and Pseudomonas exotoxin-interleukin 13 fusion protein exert synergistic toxicity against human glioblastoma multiforme cells.

The cytotoxicity of combinations of a diphtheria toxin-human epidermal growth factor fusion protein (DAB(389)EGF) and a Pseudomonas exotoxin-human interleukin 13 fusion protein (IL13PE38QQR) was tested against 14 human glioma cell lines. After cells were cultured for 48 h with various concentrations of the fusion proteins, the percentage reductions in thymidine incorporation were determined. Seven of fourteen cell lines were highly sensitive to DAB(389)EGF alone, and six cell lines were highly sensitive to IL13PE38QQR alone with IC(90)'s < 100 pM. When combined, synergistic cell killing was observed for seven of the cell lines based upon concave isobolograms and combination indices (CI's) of 0.2 to 0.7. Supraadditive cytotoxicity was confirmed by measurements of induction of apoptosis. Receptor expression was assessed by flow cytometry and confocal microscopy. Marked heterogeneity of expression of EGFR and IL13Ralpha2 was seen on all the glioma cell lines. This heterogeneity may contribute to incomplete cell killing with the individual fusion proteins and synergistic cell kill with the combination. These results suggest that both fusion proteins may yield antitumor effects in patients with recurrent gliomas and that combination fusion protein intracranial therapy of malignant gliomas may yield an improved therapeutic index.

A diphtheria toxin-epidermal growth factor fusion protein is cytotoxic to human glioblastoma multiforme cells.

The cytotoxicity of a diphtheria toxin-human epidermal growth factor fusion protein (DAB(389)EGF) was tested against 14 human glioma cell lines. After cells were cultured for 48 h with various concentrations of DAB(389)EGF, the percentage reduction in thymidine incorporation was determined. For 13 of 14 cell lines, potent cytotoxicity was observed, with IC(50)s of 0.4-50 pM. The epidermal growth factor receptor (EGFR) density of these cell lines was determined by immunofluorescence microscopy, flow cytometry, and radioligand binding. These assays correlated well with each other and demonstrated EGFR levels of 15,000-230,000/cell for 13 of 14 cell lines. The cell line U138MG, which lacked EGFR, was the only cell line insensitive to DAB(389)EGF. Linear regression analysis showed a good correlation between EGFR density and DAB(389)EGF sensitivity (P < 0.001) and between results of flow cytometry and radiolabeled binding assays of EGFR density (P = 0.01). DAB(389)EGF may have potential for intracranial therapy of EGFR-positive glioblastomas.

DAB389EGF fusion protein therapy of refractory glioblastoma multiforme.

Primary brain tumors including anaplastic astrocytomas and glioblastoma multiforme are difficult to treat because of their locally invasive nature and chemoradioresistance. Novel therapies are needed. One class of therapeutics is fusion proteins consisting of peptide toxins fused to brain tumor selective ligands. DAB389EGF is a fusion protein composed of the catalytic and translocation domains of diphtheria toxin fused via a His-Ala linker to human epidermal growth factor (EGF). DAB389EGF is selectively toxic to EGF receptor (EGFR) overexpressing cells. Close to half of all high-grade primary brain tumors have EGFR gene amplification and EGFR overexpression. With the use of convection-enhanced delivery (CED), DAB389EGF may be delivered locally at high concentrations to the brain tumor. CED would avoid many of the pharmacologic and toxicologic barriers which have limited effective use of this agent including rapid clearance from the circulation, high anti-diphtheria toxin antibody titers in the blood and toxicities to the liver and kidney. Both cell lines and animal models are available to assess the potential of this agent for brain tumor therapy. Since significant amounts of clinical grade DAB389EGF are available, some careful additional preclinical efficacy work should lead to testing of this agent in patients within the next few years.