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A Diphtheria Toxin-Epidermal Growth Factor Fusion Protein Is Cytotoxic to Human Glioblastoma Multiforme Cells¹

Department of Cancer Biology and Pathology, Wake Forest University School of Medicine, Winston-Salem, North Carolina 27157

	ABSTRACT

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The cytotoxicity of a diphtheria toxin-human epidermal growth factor fusion protein (DAB₃₈₉EGF) was tested against 14 human glioma cell lines. After cells were cultured for 48 h with various concentrations of DAB₃₈₉EGF, the percentage reduction in thymidine incorporation was determined. For 13 of 14 cell lines, potent cytotoxicity was observed, with IC₅₀s 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 DAB₃₈₉EGF. Linear regression analysis showed a good correlation between EGFR density and DAB₃₈₉EGF sensitivity (P < 0.001) and between results of flow cytometry and radiolabeled binding assays of EGFR density (P = 0.01). DAB₃₈₉EGF may have potential for intracranial therapy of EGFR-positive glioblastomas.

	INTRODUCTION

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There are 20,000 cases of primary brain tumors per year in the United States (1) . More than 80% of these tumors are high-grade infiltrative astrocytoma/GBM,³ which arise from malignant transformation of astrocytes (2) . GBMs are the third leading cause of cancer-related death in adolescents and adults in the age range 15–34 years. The median survival for GBM patients is 1 year for newly diagnosed patients and 6 months for patients with recurrent disease (3) . Fewer than 20% of GBM patients are alive at 2 years. Thus, this disease is associated with a very poor prognosis. Chemotherapy- and radiotherapy-resistant tumor cells and local tumor growth are the main causes of death, with 90% of recurrences at or adjacent to the site of origin of the disease (4 , 5) . Novel agents with different mechanisms of action are needed for these brain tumors.

Fusion proteins targeting cell surface receptors selectively overexpressed on GBMs represent one such different approach. The brain tumor-selective ligand directs the protein to the glioma cell surface; the peptide toxin is then internalized into the cell, translocates to the cytosol, and catalytically inactivates protein synthesis, leading to cell death. Several fusion proteins have been synthesized for brain tumor therapy that are reactive with high-affinity IL13 receptors, Tf receptors, urokinase receptors, and IL4 receptors (6, 7, 8, 9, 10) . CED was used to introduce these agents into the brain tumor interstitium. This method creates a bulk flow that supplements diffusion and achieves drug concentrations orders of magnitude higher than concentrations achieved by systemic administration for large areas of the brain parenchyma (6) . Clinical remissions lasting years were observed in many patients (6 , 8) . Because of the toxicities with several of the agents (e.g., Tf-CRM107 and IL4[38–37]-PE38KDEL) and the lack of binding to all brain tumors with others (e.g., IL13PE38QQR), there is a need for additional brain tumor fusion proteins.

An attractive target is the EGFR. Glioma cells often express 100,000 EGFR molecules/cell, whereas normal brain tissues (glial, neuronal, and endothelial cell lineages) display only a few thousand EGFR molecules/cell (11) . Half of the glioblastomas show amplification of the EGFR gene (up to 60-fold) and overexpression of EGFR mRNA and protein (12) . Increased EGFR expression is correlated with a poorer prognosis in brain tumor patients (13) . Furthermore, EGFR overexpression may be critical for maintenance of the malignant phenotype because glioma cells possess an EGFR/tumor growth factor- autocrine loop (14) . This autocrine loop produces constitutive stimulation of pathways important for cell survival, spreading, and proliferation. Because the EGFR binds EGF with high affinity and the EGF-EGFR complex internalizes efficiently by receptor-mediated endocytosis, the EGFR is an excellent target for fusion proteins with toxins such as ricin, PE, and DT. On the basis of these observations, we chose to investigate the antiglioma activity of a DT-EGF fusion molecule, DAB₃₈₉EGF.

DNA encoding a methionine, the catalytic and translocation domains of DT (amino acid residues 1–387), a His-Ala linker, and amino acids 1–53 of EGF was inserted into a bacterial expression plasmid under the control of a trc promoter and used to transform Escherichia coli (15) . After induction with isopropyl ß-D-thiogalactopyranoside, inclusion bodies were washed, denatured, and renatured. Recombinant protein was purified by anion-exchange chromatography and filter sterilization. DAB₃₈₉EGF had a molecular weight of 48,522, reacted with antibodies to DT and EGF on immunoblots, and was cytotoxic at pM concentrations to the EGFR-positive A431 human vulvar carcinoma cell line. Cell killing was dependent on endosome acidification.

The fusion protein had an affinity for the EGFR that was 15–30-fold lower than for EGF itself. Rats administered DAB₃₈₉EGF by daily bolus i.v. infusions for 10–14 days had a MTD of 30 µg/kg/day and a DLT of renal and hepatic injury (16) . The circulating half-life was <1 min with peak levels of 20 ng/ml at doses of 40 µg/kg. [³⁵S]methionine-labeled DAB₃₈₉EGF administered i.v. to rats distributed rapidly to the liver (61% of the injected dose) and kidney (6% of injected dose). Antibody formation to DT occurred within 1 month in all animals.

DAB₃₈₉EGF was slightly more toxic to cynomolgus monkeys after i.v. infusions with an MTD of 20 µg/kg/day for 10 days. The DLT was also liver and kidney damage. Nude mice inoculated s.c. with A549 human EGFR-positive lung adenocarcinoma cells and treated starting 24 h later with DAB₃₈₉EGF (25 µg/kg/day for 10 days) had a 50% tumor growth inhibition by day 17 with complete tumor regressions in 2 of 10 treated mice. Three Phase I/II clinical studies were conducted with five to nine 30-min i.v. infusions of DAB₃₈₉EGF over 1–3 weeks in 72 patients with EGFR-positive metastatic carcinomas (17) . Peak DAB₃₈₉EGF serum levels were 0–50 ng/ml. One-fourth of the patients had high pretreatment anti-DT antibody titers; all patients had high anti-DT antibody titers 1 month posttreatment. The MTD was 6 µg/kg/day, and the DLT was renal and liver injury. Only one 6-month partial remission in a patient with non-small cell lung carcinoma was observed.

Many of the properties of DAB₃₈₉EGF that compromised clinical activity with systemic administration should be beneficial for brain tumor CED therapy. The short half-life, circulating anti-DT antibodies, and clearance by the liver and kidneys should not influence local delivery in the brain. Normal tissue toxicities should be reduced because high concentrations of fusion protein will be present only in the brain and brain tumor. With the known clinical experience with DAB₃₈₉EGF in patients, we wanted to extend the work to a brain tumor application. In this report, we describe the sensitivity of a battery of human glioma cell lines to DAB₃₈₉EGF and correlate the sensitivity with EGFR expression. The results establish a rationale for CED DAB₃₈₉EGF therapy of recurrent EGFR-positive gliomas.

	MATERIALS AND METHODS

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DAB₃₈₉EGF.
DAB₃₈₉EGF was synthesized and partially purified as described previously (16) . Vials contained 500 µg of sterile-filtered, lyophilized DAB₃₈₉EGF in PBS (pH 7.2) containing 1% mannitol and 50 µM EDTA (lot no. 3E13EB2; Ligand Pharmaceuticals). Vials were stored at -80°C. Fusion protein was prepared for experiments by adding 2 ml of sterile water and gently stirring. Dissolved fusion protein was stored at 4°C.

Cell Lines.
Cell lines A431, U373MG, and U138MG were obtained from the Wake Forest University Tissue Culture Core Laboratory. Cell lines A172, DBTRG05MG, T98G, U87MG, and U138MG were purchased from the American Type Culture Collection (Rockville, MD). Cell lines LN405, GAMG, DKMG, GMS10, 42MGBA, SNB19, and 8MGBA were purchased from the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (Braunschweig, Germany). A431 cells were grown in DMEM containing 10% fetal bovine serum, penicillin/streptomycin, and L-glutamine. U373MG and U138MG cells were grown in Eagle’s medium supplemented with penicillin/streptomycin, L-glutamine, nonessential amino acids, 0.1 g/l sodium pyruvate, and 10% fetal bovine serum. A172 and U118MG cells were grown in Dulbecco’s medium containing 1.5 g/l sodium bicarbonate, penicillin/streptomycin, L-glutamine, and 10% fetal bovine serum. LN405 and SNB19 cells were expanded in Dulbecco’s medium containing 10% fetal bovine serum. GAMG cells were grown in Dulbecco’s medium containing 10% fetal bovine serum, L-glutamine, and nonessential amino acids. DKMG cells were grown in RPMI 1640 supplemented with 10% fetal bovine serum and L-glutamine. GMS10 cells were grown in Dulbecco’s medium containing 10% fetal bovine serum and L-glutamine. DBTRG05MG cells were grown in RPMI 1640 supplemented with penicillin/streptomycin, L-glutamine, 0.1 g/l sodium pyruvate, 10% fetal bovine serum, 4.5 g/l glucose, 1.5 g/l sodium bicarbonate, sodium pyruvate, 1 mg/ml AMP, 10 mg/l adenine, 1 mg/l thymidine, 50 mg/l L-isoleucine, 100 mg/l L-cystine, 5.95 g/l HEPES, 50 mg/l L-proline, and 15 mg/l hypoxanthine. T98G and U87MG cells were grown in Eagle’s medium containing sodium pyruvate, 10% fetal bovine serum, nonessential amino acids, L-glutamine, penicillin/streptomycin, and 1.5 g/l sodium bicarbonate. 42MGBA cells were grown in 50% RPMI 1640/50% minimal essential medium with Earle’s salts plus 20% fetal bovine serum. 8MGBA cells were grown in minimal essential medium with Earle’s salts plus 20% fetal bovine serum. Cultures were incubated at 37°C in a humidified atmosphere containing 5% CO₂ in air.

Cell Line Sensitivity to DAB₃₈₉EGF.
Aliquots of 5 x 10³ cells were incubated in triplicate in 100 µl of medium (same as that used to grow the cells) in Costar 96-well flat-bottomed plates. After 24 h, 50 µl of DAB₃₈₉EGF in medium were added to each column to yield concentrations ranging from 0 to 10,000 pM, and the cells were incubated at 37°C in 5% CO₂ for another 48 h. After the incubation, 1 µCi of [³H]thymidine or [³H]leucine (NEN DuPont, Boston, MA) in 50 µl of medium was added to each well, and incubation was continued for an additional 16 h at 37°C in 5% CO₂. Plates were frozen at -80°C and thawed, cell lysates were harvested using a Skatron Cell Harvestor (Skatron Instruments, Lier, Norway) on to glass fiber mats, and the cpm of incorporated radiolabel were counted using an LKB liquid scintillation counter gated for ³H. The IC₅₀ was defined as the concentration of toxin that inhibited thymidine or leucine incorporation by 50% compared with control wells. The percentage of maximum [³H]thymidine or [³H]leucine incorporation was plotted versus the log of the toxin concentration, and nonlinear regression with a variable-slope sigmoidal dose-response curve was generated along with IC₅₀ with use of GraphPad Prism software (GraphPad Software, San Diego, CA). All assays were performed in triplicate.

Detection of EGFR by Immunofluorescence Microscopy.
Aliquots of 100,000 cells for each of the cell lines were grown overnight attached to the surface of 35-mm Costar tissue culture dishes in 1 ml of medium. Cells were washed twice with cold PBS, blocked with 1% BSA in PBS, incubated for 60 min on ice with either 10 µg/ml murine monoclonal antibody to the extracellular domain of the EGFR (31G7; Zymed Laboratories, South San Francisco, CA) or 10 µg/ml murine monoclonal antibody isotype control (08-6599; Zymed Laboratories), washed three times with PBS, incubated for 60 min on ice with 25 µg/ml affinity-purified goat antimouse immunoglobulin conjugated to rhodamine in PBS/BSA (115-025-062; Jackson ImmunoResearch, West Grove, PA), washed three times with PBS, fixed with 3.7% formaldehyde in PBS, mounted under a no. 1 coverslip in glycerol-PBS (90:10), and examined under a Zeiss Axioplan epifluorescence microscope. Photomicrographs were obtained using a Zeiss Axiocam charge-coupled device camera with a fixed magnification, exposure, and gain setting for all cell lines.

Flow Cytometry Measurements of EGFR Density.
Two aliquots, each containing 1 x10⁶, for each of the cell lines were pelleted at 600 x g and resuspended in 80 µl of PBS/BSA. To one of each aliquot of cells, we added 20 µl of R-phycoerythrin-conjugated mouse anti-EGFR antibody (EGFR.1; BD Biosciences, Mountain View, CA) or phycoerythrin-conjugated mouse IgG2b isotype control antibody (27-35; BD Biosciences). The cells were incubated at 4°C for 30 min, washed with PBS/BSA, and resuspended in 1 ml of PBS/BSA. We added 250 µl of 3.7% formaldehyde to each tube, and the cells were assayed on an EPICS-XL flow cytometry (Coulter, Hialeah, FL) with filters set for phycoerythrin fluorescence detection. QuantiBrite beads (BD Biosciences) were used to determine the ratio of phycoerythrin fluorescence channel/number of phycoerythrin molecules. The phycoerythrin conjugate used had one molecule of phycoerythrin per antibody. EGFR expression was calculated assuming single antibody molecules bound per EGFR (also referred to as ABC).

Radiolabeled EGF Binding.
We cultured 100,000-cell aliquots in medium in wells of Costar 24-well flat-bottomed plates overnight at 37°C in 5% CO₂. Wells were then washed three times with 200 µl of PBS, incubated 30 min at 37°C in 5% CO₂ in 200 µl of binding buffer [RPMI 1640 plus 0.2% sodium azide plus 2.5% BSA plus 20 mM HEPES (pH 7.4)] with various concentrations (1–2000 pM) of ¹²⁵I-labeled EGF (195 mCi/mg; ICN, Irvine, CA) with or without unlabeled (400 nM) human EGF (236-EG; R&D Systems, Minneapolis, MN). Wells were then incubated for 1 h at 37°C in 5% CO₂; medium was then collected from the wells, transferred to glass tubes, and counted. Wells were then washed three times with PBS and incubated 30 min with 300 µl of 1 M sodium hydroxide. Well contents and two 300-µl water washes were transferred to glass tubes and counted. Counts were measured in a Packard Auto-Gamma 5650 gamma counter gated for ¹²⁵I with 50% counting efficiency. Background cpm were calculated by linear extrapolation from incubation with excess unlabeled EGF. Specific binding saturation curves were made using GraphPad Prism (Graph Pad Software). Nonlinear regression analysis was used to calculate the K_d and B_max of the EGFR.

	RESULTS

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Sensitivity of Cultured Cells to DAB₃₈₉EGF.
The A431 epidermoid carcinoma cell line was sensitive to DAB₃₈₉EGF. The mean (±SD) IC₅₀ for proliferation inhibition (thymidine incorporation) after 48 h of incubation was 0.9 ± 0.7 pM. Similar sensitivity was observed with 13 of 14 glioma cell lines (see Table 1 ). The cytotoxicity assays yielded reproducible IC₅₀s with ranges <30% from the mean (Fig. 1) . The potency was high for the 13 sensitive cell lines, ranging from 0.4 to 50 pM. The cell line with low or absent EGFR (U138MG) showed no sensitivity to DAB₃₈₉EGF. Protein synthesis inhibition by DAB₃₈₉EGF was measured in U373MG and U87MG cells and yielded IC₅₀s within 50% of the proliferation inhibition IC₅₀s. The protein synthesis inhibition IC₅₀s were 1.2 pM and 2.1 pM, and the proliferation inhibition IC₅₀s were 0.8 pM and 3.1 pM, respectively.

View larger version (17K): [in this window] [in a new window] [Download PPT slide]   Fig. 1. Cytotoxicity of DAB389EGF (lot 3E13EB2) toward glioma cells. Five thousand cells were incubated 48 h with the indicated concentrations of fusion protein and then pulsed for 16 h with 1 µCi of [3H]thymidine. Cells were harvested to glass fiber mats with a cell harvester and counted on a Betaplate scintillation counter. , U373MG; , U138MG. Bars, SD. IC50s were 0.4 pM and >150 pM, respectively.

View larger version (117K): [in this window] [in a new window] [Download PPT slide]   Fig. 2. Fluorescent antibody staining of glioma cells. One hundred thousand cells were grown attached to the surface of tissue culture dishes, washed, blocked with BSA, incubated with murine monoclonal antibody to EGFR or a isotype control, rewashed, reacted with affinity-purified goat antimouse immunoglobulin conjugated to rhodamine, washed, fixed, mounted, and examined under a Zeiss Axioplan epifluorescence microscope. Photomicrographs were obtained with a fixed shutter setting for all cell lines. A, A431; B, U373MG; C, U87MG; D, 42MGBA; E, SNB19; F, 8MGBA; G, GAMG; H, LN405; I, T98G; J, DKMG; K, GMS10; L, A172; M, U118MG; N, DBTRG05MG; O, U138MG.

Radiolabeled EGF Binding.
We observed a single class of receptors with a K_d of 1 nM. Similar to the results with flow cytometry, the B_max varied from 15,000 to 230,000 (Table 1) .

Correlation of DAB₃₈₉EGF IC50, Radiolabeled EGF Binding, and EGFR Flow Cytometry.
There was a strong negative correlation between EGFR determined by radiolabeled binding and the DAB₃₈₉EGF thymidine incorporation inhibition IC₅₀ based on linear regression (Fig. 3A ; P < 0.001). Confirming the results of the EGFR estimation, the EGFR density measured by flow cytometry and radiolabeled binding showed a positive correlation by linear regression (P = 0.01; Fig. 3B ).

View larger version (16K): [in this window] [in a new window] [Download PPT slide]   Fig. 3. A, scatterplot of DAB389EGF IC50 and EGFR Bmax. Linear regression yielded P < 0.001. B, scatterplot of EGFR derived from radioligand binding (Bmax) and flow cytometry (ABC). Linear regression yielded P = 0.01.

	DISCUSSION

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The binding affinity of radiolabeled EGF for the EGFR on the glioblastoma cell lines (K_d = 1 nM) and the receptor density/cell (B_max = 20,000–200,000) matched the results of previous studies of EGFR on glioblastoma cells (19) . The high level of EGFR may occur as part of the selection pressure for establishment of glioblastoma cell lines. However, as described above, EGFR overexpression is common in high-grade gliomas, associated with a worse prognosis, and may be part of the oncogenic process leading to tumor cell proliferation (12, 13, 14) .

EGFR density correlated with DAB₃₈₉EGF sensitivity, although because most glioma cell lines overexpressed EGFR, there were limited data with low to intermediate EGFR densities. The correlation was expected because DAB₃₈₉EGF must bind EGFR before cell intoxication (15) . Although some correlation may be expected, it was important to test the hypothesis for several reasons. First, receptor number has not always correlated with fusion protein sensitivity. For complex heterodimer or heterotrimeric receptors, such as the IL2 receptor, the numbers of low-affinity receptors did not correlate with fusion protein sensitivity (16) . In other cases, such as the granulocyte-macrophage colony-stimulating factor receptor, only low receptor numbers may be needed for DT fusion protein intoxication, and additional cell surface receptors did not significantly modify sensitivity (16) . Second, EGFR mutations are frequent in gliomas and may be associated with altered ligand binding or internalization (20) . Thus, we needed to obtain some information on the correlation of DAB₃₈₉EGF sensitivity and EGFR density even for a set of glioma cell lines. Although not necessarily reflecting the behavior of patient glioma cells in vivo, our observation of a correlation between sensitivity and receptor density among cell lines suggests that some measurements of EGFR may be useful in the design of clinical trials with DAB₃₈₉EGF. Patients with high-EGFR-expressing brain tumors may be more likely to respond.

This study provides some preclinical rationale for the further development of DAB₃₈₉EGF for therapy of refractory gliomas. The availability of clinical grade reagent and the extensive previous preclinical and clinical pharmacology and toxicology information with this drug should lead to its more rapid development (16) . Although several fusion proteins are being tested for interstitial therapy of gliomas at present, the therapeutic window has been reduced because of tumor cell heterogeneity and reactivity with normal brain tissues. Thus, there remains a strong need for novel agents that are active and safe in patients with this disease.

	FOOTNOTES

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by NIH Grants R01CA76178 (to A. E. F.), R01CA90263 (to A. E. F.), R21CA90550 (to A. E. F.), and T32RR007009 (to K. A. C.).

² To whom requests for reprints should be addressed, at Hanes 4046, Wake Forest University School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27157. Phone: (336) 716-3313; Fax: (336) 716-0255; E-mail: afrankel{at}wfubmc.edu

³ The abbreviations used are: GBM, glioblastoma multiforme; IL, interleukin; Tf, transferrin; CED, convection-enhanced delivery; EGFR, epidermal growth factor receptor; PE, Pseudomonas exotoxin; DT, diphtheria toxin; MTD, maximum tolerated dose; DLT, dose-limiting toxicity; ABC, antibody binding capacity; K_d, affinity constant; B_max, maximum number of binding sites.

Received 11/20/02. Accepted 2/19/03.

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