Evaluation of a bispecific biological drug designed to simultaneously target glioblastoma and its neovasculature in the brain

Laboratory investigation

Restricted access

Object

The authors of this study aimed to genetically design a bispecific targeted toxin that would simultaneously target overexpressed markers on glioma as well as the tumor vasculature, to mutate certain amino acids to reduce the immunogenicity of this new drug, and to determine whether the drug was able to effectively reduce aggressive human brain tumors in a rat xenograft model via a novel hollow fiber (HF) catheter delivery system.

Methods

A new bispecific ligand-directed toxin (BLT) was created in which 2 human cytokines—epidermal growth factor ([EGF], targeting overexpressed EGF receptor) and amino acid terminal fragment ([ATF], targeting urokinase plasminogen activator receptor)—were cloned onto the same single-chain molecule with truncated Pseudomonas exotoxin with a terminal lysyl-aspartyl-glutamyl-leucine (KDEL) sequence. Site-specific mutagenesis was used to mutate amino acids in 7 key epitopic toxin regions that dictate the B cell generation of neutralizing antitoxin antibodies to deimmunize the drug, now called “EGFATFKDEL 7mut.” Bioassays were used to determine whether mutation reduced the drug's potency, and enzyme-linked immunosorbent assay studies were performed to determine whether antitoxin antibodies were decreased. Aggressive brain tumors were intracranially established in nude rats by using human U87 glioma genetically marked with a firefly luciferase reporter gene (U87-luc), and the rats were stereotactically treated with 2 intracranial injections of deimmunized EGFATFKDEL via convection-enhanced delivery (CED). Drug was administered through a novel HF catheter to reduce drug backflow upon delivery.

Results

In vitro, EGFATFKDEL 7mut selectively killed the human glioblastoma cell line U87-luc as well as cultured human endothelial cells in the form of the human umbilical vein endothelial cells. Deimmunization did not reduce drug activity. In vivo, when rats with brain tumors were intracranially treated with drug via CED and a novel HF catheter to reduce backflow, there were significant tumor reductions in 2 experiments (p < 0.01). Some rats survived with a tumor-free status until 130 days post–tumor inoculation. An irrelevant BLT control did not protect establishing specificity. The maximal tolerated dose of EGFATFKDEL 7mut was established at 2 μg/injection or 8.0 μg/kg, and data indicated that this dose was nontoxic. Antitoxin antibodies were reduced by at least 90%.

Conclusions

First, data indicated that the BLT framework is effective for simultaneously targeting glioma and its neovasculature. Second, in the rodent CED studies, newly developed HF catheters that limit backflow are effective for drug delivery. Third, by mutating critical amino acids, the authors reduced the threat of the interference of neutralizing antibodies that are generated against the drug. The authors' experiments addressed some of the most urgent limitations in the targeted toxin field.

Abbreviations used in this paper: ALT = alanine transaminase; ATF = amino acid terminal fragment; BLT = bispecific liganddirected toxin; CED = convection-enhanced delivery; EGF = epidermal growth factor; EGFR = EGF receptor; ELISA = enzyme-linked immunosorbent assay; HF = hollow fiber; HUVEC = human umbilical vein endothelial cell; IC50 = half maximal inhibitory concentration; KDEL = lysyl-aspartyl-glutamyl-leucine; MTD = maximal tolerated dose; PE = Pseudomonas exotoxin; PE38 = truncated PE; uPA = urokinase plasminogen activator; uPAR = uPA receptor.

Article Information

Address correspondence to: Daniel A. Vallera, Ph.D., University of Minnesota Cancer Center, MMC: 367, Minneapolis, Minnesota 55455. email: valle001@umn.edu.

Please include this information when citing this paper: published online February 4, 2011; DOI: 10.3171/2010.11.JNS101214.

© AANS, except where prohibited by US copyright law.

Headings

Figures

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    A map illustrating the construction of EGFATFKDEL 7mut. The EGFATFKDEL 7mut gene consisted of DNA fragments encoding an NcoI restriction site, an ATG initiation codon, and then human EGF. The EGF was followed downstream by a fragment encoding 135-ATF followed by the 7–amino acid linker EASGGPE and then the first 362 amino acids of the PE38 molecule. Finally, the final 5 amino acids of PE38 (REDLK) were replaced with KDEL as an endoplasmic reticulum retention sequence and then a NotII restriction site at the 3′ end of the construct. The resultant gene was spliced into the pET28c bacteria expression vector under control of an isopropyl βD-thiogalactopyranoside–inducible T7 promoter. A vector map is also included in the lower panel, illustrating important restriction sites and promoter locations.

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    Graphs showing the selective activity of EGFATFKDEL. A: Experiment comparing the effects of nonmutated parental EGFATFKDEL and to mutated EGFATFKDEL 7mut showing that mutation did not compromise drug activity. Activity was determined by analyzing 3H-leucine uptake after a 72-hour incubation with targeted toxins. The CD3CD3KDEL was an irrelevant control recognizing CD3+ T cells. Each data point represents an average of triplicate measures ± SD. B: Experiment showing the effect of EGFATFKDEL 7mut against U118 glioblastoma cells to illustrate the effect of the drug against a second glioblastoma cell line. C: Experiment showing the effect of EGFATFKDEL 7mut against HUVEC, illustrating the drug targeting the endothelial cells. The bispecific drugs, mutated EGFATFKDEL 7mut and parental EGFATFKDEL are compared with monospecific EGFKDEL, ATFKDEL, and a mixture of monospecific EGFKDEL and ATFKDEL. Cells were tested in a 3H-thymidine uptake assay to determine their ability to target endothelial cells. D: A blocking assay showing that the EGF and ATF ligands mediated the selective activity of the EGFATFKDEL 7mut molecule. The U87 cells were incubated with 1 nM of EGFATFKDEL 7mut and 100 nM of α-uPA antibody, EGF, α-PE, or a mixture of α-uPA and EGF. The nonspecific α-Ly5.2 antibody was included as a negative control.

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    A: Kaplan-Meier survival curve for animals in Experiment 2, indicating the long-term survival of EGFATFKDEL-treated rats. B: Plot of tumor growth (bioluminescence) of animals studied in Experiment 2, indicating that negative control 2219KDEL 7mut had no effect on tumor growth. Total bioluminescence over time was plotted for both the 2219KDEL 7mut group and the untreated group. Graph shows that tumor grew at a similar rate in both groups, indicating that the anti–brain tumor effect of EGFATFKDEL 7mut was selective.

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    Graphs showing that EGFATFKDEL 7mut did not affect kidney and liver enzymes. In a separate experiment, normal rats (5 animals) were given 2 μg EGFATFKDEL 7mut intracranially on the same 2-injection schedule. To study systemic toxicity, 14 days after the final injection rats were bled and ALT levels and blood urea nitrogen (BUN) levels were determined. A group of 5 control rats were not treated but were studied in an identical manner. Alanine transferase (A) and BUN (B) levels, represented on the y axis, were determined. Data are shown as the means ± SDs.

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    Graph illustrating that EGFATFKDEL 7mut was nonimmunogenic and did not induce serum antitoxin antibody. The comparative ability of EGFATFKDEL and EGFATFKDEL 7mut to induce the production of antitoxin (PE38KDEL) antibodies in immunocompetent mice was determined by measuring anti–PE38(KDEL) serum IgG levels in samples from mice immunized weekly with 0.25 μg of parental EGFATFKDEL or deimmunized EGFATFKDEL 7mut (5 animals per group). Animals were bled on Days 75 and 90. The Day 75 serum was taken after 11 immunizations, and the Day 90 measurement after 13 immunizations. Serum IgG antitoxin levels were made using an indirect ELISA, and quantification of antibodies was determined using a standard curve generated with M40-1 anti–PE(KDEL) antibody. Data comparisons between the EGFATFKDEL-immunized and the deimmunized EGFATFKDEL 7mut groups were significant according to the Student t-test (p < 0.05). The bars showing serum antitoxin levels in EGFATFKDEL 7mut immunized mice were too low to register on the graph.

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    In vivo intracranial tumor studies showing that EGFATFKDEL 7mut had a potent antiglioma affect. Athymic nude rats were given 4 × 105 U87-luc cells intracranially on Day 0. In Experiment 1 on Days 7 and 14, a group of rats was either left untreated or treated with 2 μg EGFATFKDEL 7mut delivered via CED using the HF catheter. Drug was delivered at the same coordinates as tumor. Each animal was subjected to weekly imaging to monitor tumor growth. The physical appearance and behavior bias of animals were monitored daily. In Experiment 2, animals were either left untreated or treated with 2 μg EGFATFKDEL 7mut on Days 4 and 11. Bioluminescence is shown on each picture as a function of photons/s/cm2/sr. The intensity of the signal is illustrated by the color bar, with red representing the highest signal intensity. Red crosses indicate death of the animal. Photomicrographs showing striatal sections from 2 rats, R15 and R17, taken on Day 130 and verifying that the rats were tumor-free and that the tissue looks normal. The needle track is visible on the section from R15. ND = no data at that time point. Note that hair patterns vary from week to week because nude rats elicit abortive hair growth.

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