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Clinical Cancer Research 13, 6486, November 1, 2007. doi: 10.1158/1078-0432.CCR-07-0938
© 2007 American Association for Cancer Research
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Cancer Therapy: Preclinical

A Bispecific Recombinant Cytotoxin (DTEGF13) Targeting Human Interleukin-13 and Epidermal Growth Factor Receptors in a Mouse Xenograft Model of Prostate Cancer

Brad J. Stish1, Hua Chen1, Yanqun Shu1, Angela Panoskaltsis-Mortari2 and Daniel A. Vallera1

Authors' Affiliations: Departments of 1 Therapeutic Radiology-Radiation Oncology and 2 Pediatrics, Section on Molecular Cancer Therapeutics, University of Minnesota Cancer Center, Minneapolis, Minnesota

Requests for reprints: Daniel A. Vallera, University of Minnesota Cancer Center, MMC 367, Minneapolis, MN 55455. Phone: 612-626-6664; Fax: 612-624-3913; E-mail: valle001{at}umn.edu.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Purpose: Overexpressed cytokine receptors are considered valid targets for new biologicals targeting prostate cancer. However, current reagents are limited in efficacy. Our goal was to determine the advantages of simultaneously targeting two established targets, epidermal growth factor receptor and interleukin-13 (IL-13) receptor, with a new bispecific cytotoxin in which both EGF and IL-13 cytokines were cloned onto the same single-chain molecule with truncated diphtheria toxin (DT390).

Experimental Design: In vitro experiments measured the potency of bispecific DTEGF13 and compared its activity to its monospecific counterparts, DTEGF and DTIL13. We determined whether the presence of both cytokine ligands on the same molecule was responsible for its superior activity. In vivo, DTEGF13 was given i.t. to athymic nude mice with established PC-3 human prostate cancer tumor xenografts on their flanks.

Results: In vitro, DTEGF13 was more potent than the monospecific cytotoxins against human prostate cancer lines. Enhanced activity was related to the presence of both cytokines on the same single-chain molecule and was not attributed to enhanced binding capacity. Killing was receptor specific. Cytotoxicity could be blocked with anti-EGF and anti–IL-13 antibodies. In vivo, DTEGF13, but not monospecific DTEGF or DTIL13, significantly inhibited the growth of established PC-3 tumors in nude mice (P < 0.0001).

Conclusions: These data show for the first time that simultaneous targeting of cytokine receptors with two ligands on the same molecule has pronounced anticancer advantages. In an animal model in which human DTEGF13 is cross-reactive with mouse, DTEGF13 was highly effective in checking aggressive prostate tumor progression and was reasonably tolerated.

Prostate cancer is the second leading cause of death among men in America (1) and androgen deprivation therapy is the most common and a successful form of treatment (2). However, metastatic hormone-refractory prostate cancer often develops and is the main cause of death among patients (3). Novel therapies are needed that can selectively target and destroy androgen-independent tumors. Previous research has shown that cytotoxins show potential for the treatment of metastatic hormone-refractory prostate cancer (4). Cytotoxins represent a broad class of therapeutic agents that are created by linking a highly specific cytokine to a catalytic toxin molecule.

Epidermal growth factor (EGF) is the main ligand of the EGF receptor (EGFR), a transmembrane signaling protein from the erbB family (5). Studies have revealed a link between EGFR signaling pathways and malignancy (6). Clinically, there is a strong correlation between EGFR expression levels and disease progression of hormone-refractory prostate cancer (7). A number of therapies targeting EGFR, including monoclonal antibodies (8), small-molecule kinase inhibitors (9), and immunotoxins (10), have shown promising but inconsistent results.

Interleukin-13 (IL-13; refs. 11, 12), secreted by activated type 2 T cells and mast cells (13), is a pleiotropic lymphokine regulating inflammatory and immune responses. It modulates human monocyte and B-cell functions but not T-cell function (14). IL-13 receptors (IL-13R) are found to be overexpressed in solid tumor cells including glioblastoma (15–19), renal cell carcinoma (20), AIDS Kaposi's sarcoma (21), and cancers of the prostate (22), ovary (23), and head and neck (24). IL-13 has proved a useful ligand for therapy because, although it is overexpressed on tumors, the only normal cells targeted are B cells and monocytes. It seems that the IL-13R functions as a tumor-specific, high-affinity target, and incorporating IL-13 into a cytotoxin may be a beneficial strategy.

Diphtheria toxin (DT) is an ideal molecule for cytotoxin construction due to its irreversible catalytic activity, and research showed that a single molecule delivered to the cytosol is sufficient to bring about cell killing (25). The truncated form of DT used in this study (DT390) was selected due to previous research describing a series of internal frame deletion mutations that established amino acid 389 as the best location for genetic fusion of DT to targeting ligands. DT390 contains the A fragment of native DT that catalyzes ADP ribosylation of elongation factor 2, leading to irreversible inhibition of protein synthesis and cell death (26, 27).

Investigators have shown that mixtures of cytotoxins can be more effective than individual cytotoxins. For example, Frankel et al. (28) showed that a mixture of EGF and IL-13 cytotoxins was more effective than individual cytotoxins. In this report, we describe for the first time the advantages of DTEGF13, a bispecific cytotoxin created by linking the separate EGF and IL-13 ligands to DT390. DTEGF13 showed increased activity toward the PC-3 and DU-145 human prostate cancer cell lines when compared with monospecific cytotoxin targeting EGFR or IL-13R individually. Furthermore, DTEGF13 was more potent than mixtures of individual cytotoxins. In vivo, DTEGF13 was able to more effectively inhibit the growth of PC-3 flank tumors in a nude mouse model. This research shows for the first time that a single-chain bispecific cytotoxin simultaneously targeting EGFR and IL-13R offers a significantly greater anticancer effect over targeting either receptor alone.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Construction of DTEGF13
Synthesis and assembly of hybrid genes encoding the single-chain DTEGF13 was accomplished using DNA shuffling and DNA cloning techniques. The fully assembled fusion gene (from 5' end to 3' end) consisted of an Nco1 restriction site, an ATG initiation codon, the first 389 amino acids of the DT molecule (DT390), the seven-amino-acid EASGGPE linker, the genes for human EGF and IL-13 linked by a 20-amino-acid segment of human muscle aldolase (hma), and a XhoI restriction site. The resultant 1,755-bp NcoI/XhoI fragment gene was spliced into the pET21d expression vector under control of an isopropyl-ß-D-thiogalactopyranoside–inducible T7 promoter (Fig. 1 ). DNA sequencing analysis (Biomedical Genomics Center, University of Minnesota) was used to verify that the gene was correct in sequence and had been cloned in frame. Genes for monospecific cytotoxins splicing DT390 into human EGF (DTEGF) and human IL-13 (DTIL13) were created using the same techniques.


Figure 1
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  		Fig. 1. Construction and purification of DTEGF13. Top, the gene fragment encoding the single-chain bispecific cytotoxin DTEGF13 was created using overlap extension PCR. This construct consisted of (from 5' to 3') a truncated diphtheria toxin molecule (DT390), a seven-amino-acid (EASPPGE) linker, human EGF, a flexible 20-amino-acid segment of human muscle aldolase (hma), and IL-13. Using the NcoI/XhoI restriction sites, the sequence for DTEGF13 was cloned in the pET21d bacterial expression vector. Bottom, size and purity of recombinant proteins used in this study were confirmed following SDS-PAGE and Coomassie blue staining. Lane 1, molecular weight standards; lane 2, EGF13; lane 3, DTIL13; lane 4, DTEGF; lane 5, DTEGF13.

 
An additional bivalent fusion protein containing the DT390 fragment was created as specificity controls. DT2222 was produced by joining two repeating sFvs specific for human anti-CD22 to DT390. CD22 is a human B lymphocyte–specific glycoprotein that shows increased expression in the majority of B-cell leukemias and lymphomas (29).

Inclusion body isolation
Plasmids were transformed into Escherichia coli strain BL21(DE3) (Novagen). After overnight culture, bacteria were grown in 800-mL Luria broth containing 50 mg/mL carbenicillin in a 2-L flask at 37°C with shaking. Gene expression was induced when culture media reached an A600 of 0.65 with the addition of 8 mL of 100 mmol/L isopropyl-b-D-thiogalactopyranoside (FisherBiotech). Two hours after induction, bacteria were harvested by centrifugation. Cell pellets were suspended and homogenized in a buffer solution (50 mmol/L Tris, 50 mmol/L NaCl, and 5 mmol/L EDTA pH 8.0) using a polytron homogenizer. After sonication and centrifugation, the pellets were extracted with 0.3% sodium deoxycholate, 5% Triton X-100, 10% glycerin, 50 mmol/L Tris, 50 mmol/L NaCl, 5 mmol/L EDTA (pH 8.0), and then washed.

Refolding and purification
Inclusion bodies were dissolved at 20:1 (mg wet weight/mL) in solubilization buffer, 7 mol/L guanidine hydrochloride, 50 mmol/L Tris, 50 mmol/L NaCl, 5 mmol/L EDTA, and 50 mmol/L DTT, pH 8.0. After 1-h incubation at 37°C, pellets were removed by centrifugation. The supernatant was diluted 20-fold with refolding buffer and incubated at 4°C for 2 days. Refolding buffer consisted of 50 mmol/L Tris-HCl, 50 mmol/L NaCl, 0.8 mmol/L L-arginine, 20% glycerin, 5 mmol/L EDTA, and 1 mmol/L oxidized glutathione, pH 8.0. The denaturant was removed following 10-fold dialysis against 20 mmol/L Tris-HCl, pH 9.0. Refolded proteins were purified by fast protein liquid chromatography ion-exchange chromatography (Q sepharose Fast Flow, Sigma) using a continuous gradient from 0.2 to 0.5 mol/L NaCl in 20 mmol/L Tris-HCl (pH 9.0) over 4 column volumes.

Cell culture
The human prostate cancer cell lines DU-145 (30) and PC-3 (31), the colorectal cancer cell line HT-29 (32), and the Burkitt's Lymphoma cell line Daudi (33) were obtained from American Type Culture Collection. Cells were maintained in RPMI 1640 (Cambrex) supplemented with 10% fetal bovine serum, 2 mmol/L L-glutamine, 100 units/mL penicillin, and 100 µg/mL streptomycin. All carcinoma cells were grown as monolayers and Daudi cells in suspension using culture flasks. Cell cultures were incubated in a humidified 37°C atmosphere containing 5% CO2. When adherent cells were 80% to 90% confluent, they were passaged using trypsin-EDTA for detachment. Only cells with viability >95%, as determined by trypan blue exclusion, were used for experiments.

Bioassays to measure in vitro cytotoxin activity. To determine the effect of DTEGF13 on DU-145, HT-29, and Daudi cells, proliferation assays measuring [3H]thymidine incorporation were used (34). Cells (104 per well) were plated out in a 96-well flat-bottomed plate and incubated overnight at 37°C with 5% CO2 to allow cells to adhere. Immunotoxins in varying concentrations were added to wells in triplicate. Incubation at 37°C and 5% CO2 continued for 72 h. [methyl-3H]Thymidine (GE Healthcare, United Kingdom) was added (1 µCi/well) for the final 8 h of incubation. Plates were frozen to detach cells and cells were then harvested onto a glass fiber filter, washed, dried, and counted using standard scintillation methods. Protein synthesis inhibition of PC-3 cells exposed to cytotoxin was analyzed by measuring [3H]leucine incorporation. Assays measuring [3H]leucine uptake only differed from [3H]thymdine assays in that they were done in leucine-free media and incubation with labeled leucine lasted for 24 h. Data from proliferation assays are reported as percentage of control counts.

Blocking studies were conducted to test the specificity of DTEGF13. Briefly, anti-EGF or anti–IL-13 (R&D Systems) was added to media containing 0.1 nmol/L DTEGF13 at a final concentration of 50 µg/mL. Resulting mixtures were added to wells containing PC-3 cells and proliferation was measured by [3H]leucine uptake. The mouse leukocyte-specific antibody Ly5.2 was included as a negative control (35).

Binding and internalization of radiolabeled DTEGF13. To measure the binding and internalization efficiency of DTEGF13, an aliquot of the protein was labeled with 111In (36). Briefly, the MX-DTPA 1B4M chelating agent was conjugated to proteins at a 2.5:1 molar ratio using a conjugation buffer consisting of 5 mmol/L sodium bicarbonate, 15 mmol/L sodium chloride, and 0.5 mmol/L EDTA at pH 9.2. Approximately 250 µg of 1B4M-chelated DTEGF13 were labeled with 20 µCi of 111In with a labeling efficiency of >90%. PC-3 cells (3 x 105 per tube) were then suspended in 100 µL of RPMI 1640 and placed at 4°C for 30 min. One hundred microliters of 600 nmol/L 111In-labeled DTEGF13 in ice-cold RPMI 1640 were then added to each tube and then cells were incubated for 30 min at 4°C to prevent internalization. After two washes with cold PBS, cells were resuspended and placed at 37°C for specified incubation time period. Two samples were saved to calculate initially bound protein. After incubation, cells were pelleted and medium was aspirated from each tube. Cells were washed twice with 500-µL PBS. All incubation media and PBS from washes were pooled for each tube and saved as unbound fraction. PC-3 cells were then washed twice with RPMI 1640 (pH 3.0) to release bound protein and medium was saved as bound fraction. Cell pellets were also saved, and associated radioactivity was counted as internalized protein. Radioactivity of all tubes was counted with a gamma counter (Perkin-Elmer). Data are calculated as percentage of initially bound activity present in each fraction.

To quantitatively compare the binding of each cytotoxin to target cells, we used a FACS-based saturation binding assay. Briefly, all cytotoxins were FITC labeled at a 1:24 ratio (protein/FITC) for 3 h in a 50 mmol/L borate buffer solution (50 mmol/L boric acid, pH 9.0). Labeled protein was separated from unbound FITC using NAP-5 size exclusion column (GE Healthcare). FITC-labeled immunotoxins were incubated with 106 PC-3 cells in a 100-µL volume of buffer (PBS + 2%FBS) for 45 min. Following three washes with 500 µL of buffer, cells were analyzed with FACSCalibur. To determine Kd and Bmax mean fluorescence intensity was plotted versus immunotoxin concentration and analyzed with Prism 4 software (GraphPad Software).

In vivo efficacy studies. Male nu/nu mice were purchased from the National Cancer Institute, Frederick Cancer Research and Development Center, Animal Production Area and housed in an Association for Assessment and Accreditation of Laboratory Animal Care–accredited specific pathogen-free facility under the care of the Department of Research Animal Resources, University of Minnesota. Animal research protocols were approved by the University of Minnesota Institutional Animal Care and Use Committee. All animals were housed in microisolator cages to minimize the potential of contaminating virus transmission.

For flank tumor studies, mice were injected in the left flank with 4 x 106 (experiment 1) or 6 x 106 (experiment 2) PC-3 cells suspended in 100 µL of a 1:1 RPMI 1640/Matrigel mixture. Once palpable tumors had formed (day 18), mice were divided into groups and treated with multiple injections of DTEGF13. All cytotoxins were administered by i.t. injection using 3/10 mL syringes with 29-gauge needles. All treatments were given in a 100-µL volume of sterile PBS. Tumor size was measured using a digital caliper, and volume was determined as a product of length, width, and height.

Statistical analyses. All statistical analyses of in vivo data were done using Prism 4 (GraphPad, Inc.). Groupwise comparisons of mean data were made by Student's t test. P < 0.05 was considered significant.


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Construction and purification of DTEGF13. After NcoI/XhoI digestion, the DNA fragment encoding the bispecific cytotoxin DTEGF13 was cloned into the pET21d expression vector under control of an isopropyl-ß-D-thiogalactopyranoside–inducible T7 promoter (Fig. 1). Constructs containing the monospecific cytotoxin genes (DTEGF and DTIL13) were also synthesized. DNA sequencing analysis was done by the University of Minnesota Microchemical Facilities (University of Minnesota, Minneapolis, MN). After refolding and purification, DTEGF was of the appropriate molecular weight of 63.6 kDa. All cytotoxins were >95% pure when analyzed by SDS-PAGE (Fig. 1).

The ability of DTEGF13 to specifically kill prostate cancer cells. To determine the ability of DTEGF13 to kill EGFR-expressing and IL-13R–expressing carcinoma cells, it was tested against the EGFR+ and IL-13R+ prostate cancer cell line PC-3. Figure 2A shows that monospecific DTIL13 was able to kill PC-3 cells, with an IC50 of 0.038 nmol/L. Monospecific DTEGF was far less effective, inhibiting only 20% of protein synthesis at 100 nmol/L. However, the bispecific cytotoxin DTEGF13 showed an IC50 of 0.042 pmol/L, representing a 905-fold increase in activity as compared with DTIL13 and at least a 7-log increase in activity as compared with DTEGF. In Fig. 2B, DTEGF13 and the monospecific cytotoxin showed minimal activity against the EGF13R–, IL-13R– cell line Daudi.


Figure 2
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  		Fig. 2. Specific cytotoxicity of DTEGF13. A, the ability of DTEGF13 to specifically kill EGFR- and IL-13R–expressing target cancer cells was tested using a protein synthesis inhibition assay. The effects of DTEGF13, DTIL13, and DTEGF were tested by incubating the cytotoxins with PC-3 prostate cancer cells. Protein synthesis of PC-3 cells was measured by [3H]leucine incorporation after 72 h. B, the specific cytotoxicity of DTEGF13 was assayed by measuring its effect on the proliferation of the EGFR– and IL-13R– Daudi lymphoma cell line. [3H]Thymidine uptake was used to measure cell proliferation. Points, mean of triplicate samples; bars, SD. IC50 is the concentration of cytotoxin that inhibits 50% of the percent control activity. C, to test the importance of both EGF and IL-13 in the activity of DTEGF13, blocking assays were conducted in which antibodies were mixed with cytotoxin and then added to cells. Proliferation assays were then conducted. About 50 µg/mL of antibody ({alpha}-EGF, {alpha}-IL-13, or {alpha}-Ly5.2, a negative control antibody recognizing murine CD45) were used to block 0.1 nmol/L DTEGF13 activity. Protein synthesis of PC-3 cells was measured by [3H]leucine incorporation. D, the proliferation of DU-145 human prostate cancer cells incubated with DTEGF13, DTEGF, DTIL13, or DT2222 (negative control) was measured by [3H]thymidine incorporation 72 h after cytotoxin exposure.

 
We also synthesized the EGF13 protein devoid of DT390 with a purity of >95% (Fig. 1, lane 2). When as much as 1,000 nmol/L was added to PC-3 cells, it was not inhibitory, indicating that the EGF13 moiety by itself had no anticancer activity (data not shown).

The ability of anti-EGF and anti–IL-13 antibodies to block the killing of DTEGF13. To confirm that the EGF and IL-13 ligands were both active in DTEGF13, a blocking experiment was done. In Fig. 2C, 50 µg/mL of anti-EGF or anti–IL-13 antibodies were used to block the killing of PC-3 cells by DTEGF13. When added to 0.1 nmol/L DTEGF13, both antibodies were capable of blocking ~70% to 80% of the cytotoxic effect, but neither of the antibodies completely blocked cell killing, likely because when only one ligand was blocked, the other ligand remained active. Blocking with both antibodies in a separate assay resulted in 100% blocking (data not shown). The addition of antimouse Ly5.2 monoclonal antibody had no blocking effect. Together, these findings indicated that both ligands were active on the DTEGF13 molecule.

The ability of DTEGF13 to kill the prostate cell line DU-145. DTEGF13 was tested against a second prostate cancer cell line, DU-145. Figure 2D shows that against DU-145 cells, monospecific DTIL13 was mostly ineffective, with an IC50 of >100 nmol/L. Monospecific DTEGF was more effective with an IC50 of 0.018 nmol/L. However, note that DTEGF was incapable of entirely inhibiting DU-145 cell proliferation, even at 100 nmol/L. In contrast, DTEGF13 (IC50 of 0.0021 nmol/L) entirely inhibited the DU-145 response at concentrations as low as 0.1 nmol/L. DT2222, a negative control immunotoxin, was minimally inhibitory and did not reach an IC50. Similar findings were observed with the LNCaP-derived C4-2 prostate cancer cell line (data not shown). Together, these cell line data showed that combining an EGF ligand and an IL-13 ligand on a single-chain molecule increased the potency against a number of different prostate cancer lines compared with either monospecific cytotoxin.

Increased activity of DTEGF13 is due to the presence of EGF and IL-13 ligands on a single molecule. DTEGF13 was highly active against the HT-29 human colorectal cancer cell line (Fig. 3 ). To determine if the increased activity of bispecific cytotoxin was due to the presence of the two different ligands on the same single-chain molecule, proliferation assays were done comparing HT-29 cells treated with DTEGF13 to cells treated with a mixture of equimolar concentrations of monomeric DTEGF and DTIL13. This mixture of monospecific cytotoxins provides a number of binding molecules equivalent to the binding molecules on single-chain DTEGF13. Figure 3 shows a representative experiment in which the mixture of DTEGF and DTIL13 showed the same activity as DTEGF alone. The DTEGF13 molecule had an IC50 of 0.0015 nmol/L, which was 307-fold more potent than the IC50 of the DTEGF and DTIL13 mixture. These data show that increased activity observed with DTEGF13 is mostly due to the presence of the two different ligands on a single-chain molecule.


Figure 3
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  		Fig. 3. Superior activity of DTEGF13 is due to the presence of both ligands on a single-chain molecule. The proliferation of HT-29 cells incubated with bispecific DTEGF13 was compared with those of cells incubated with DTEGF and DTIL13 and cells exposed to equimolar mixtures of monospecific DTEGF and DTIL13. Proliferation was tested by measuring [3H]thymidine uptake 72 h after cytotoxin exposure. Points, mean of triplicate samples; bars, SD.

 
Binding and internalization of DTEGF13. To measure the binding and internalization of DTEGF13, the cytotoxin was radiolabeled with 111In and incubated with PC-3 cells for varying time periods. After incubation, the bound fraction of each protein was preferentially released, and all remaining radioactivity associated with the cells was considered to be the result of internalized cytotoxin. Figure 4A shows the bound and internalized fractions over a 1-h period. As expected over the first hour, binding decreased as the cytotoxin moved into the cells and internalization increased. Comparative binding studies were done using radiolabeled DTEGF, DTIL13, and DTEGF13 and they did not show that DTEGF13 enhanced the binding compared with the monospecific agents. Thus, a flow cytometry–based saturation binding assay was used to compare binding of DTEGF, DTIL13, and DTEGF13 to PC-3 cells (Fig. 4B). Using this assay, the binding affinity was not greater for DTEGF13 compared with the monospecific agents. The binding affinities (Kd) for DTEGF13, DTIL13, and DTEGF were 235, 276, and 103 nmol/L, respectively. Interestingly, the maximum number of binding sites (93.3) of DTEGF13 was higher than that of DTEGF (78.4) or DTIL13 (61.8), indicating that DTEGF13 reacted with the greatest number of PC-3 cell-surface receptors and confirming that DTEGF13 had broader reactivity. Studies with radiolabeled cytotoxin indicated that DTEGF13 was not internalized any better than the monspecific agents.


Figure 4
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  		Fig. 4. Binding of cytotoxins. A, PC-3 cells were incubated with 111In-labeled DTEGF13 to measure the binding and internalization over time. Points, mean percentage of initially bound protein in each fraction; bars, SD. B, binding affinity of DTEGF13, DTEGF, and DTIL13 was determined by fluorescence-activated cell sorting analysis of PC-3 cells incubated with increasing concentrations of each cytotoxin that had been labeled with FITC. Kd and maximum number of binding sites values were determined using Prism 4 software and represent binding affinity and capacity, respectively.

 
Efficacy DTEGF13 in an intratumoral nude mouse flank tumor model. To test the ability of DTEGF13 to inhibit tumor growth in vivo, PC-3 cells were injected into the flanks of nude mice in two separate experiments. Once the tumors were established and palpable, mice were treated with multiple i.t. injections. DTEGF13 was studied in a mouse model because human EGF and IL-13 (of DTEGF13) react with mouse EGFR and IL-13R, respectively (37, 38). Figure 5A shows mean tumor volume data from the first experiment in which groups of mice (n = 4-5 per group) were given injections (2.5 µg/injection) of DTEGF13, DTEGF, or DTIL13 on days 1, 3, 5, 8, 10, 12, 23, and 25. Control mice in this experiment received no treatment. The multiple injections of DTEGF13 were effective at preventing tumor growth compared with the untreated controls until injections were halted on day 25. On day 29, in DTEGF13-treated mice, tumors had reoccurred in two of four mice and were undetectable in two of four mice. DTEGF- and DTIL13-treated tumors grew at a similar rate as the untreated control tumors and thus had no significant inhibitory affect.


Figure 5
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  		Fig. 5. The effect of treatment of established PC-3 flank tumors with DTEGF13. In experiment 1 (Exp. 1), nude mice bearing PC-3 flank tumors were treated i.t. with DTEGF13, DTIL13, or DTEGF (n = 4-5/treatment group). Mean tumor volumes (A) and mean weights (B) of mice are shown in each treatment group. The arrows along the abscissa indicate days of injection. C, a separate experiment 2 (Exp. 2) shows the effect of DTEGF13 on individual flank tumors. Tumors were treated with five i.t. injections of 2.5 µg of either DTEGF13 or DT2222. The growth of individual tumors is plotted over time.

 
Weight loss is frequently used in cytotoxin and immunotoxin studies as an indication of toxicity. Figure 5B shows that weight loss resulting from the DTEGF13 treatment at the termination of the experiment after the multiple doses did not exceed 10% of the pretreatment weight. Thus, this dosage of DTEGF13 was tolerated.

In a second experiment, established PC-3 tumors were treated every other day between days 1 and 10 (total of five injections) with i.t. injections of DTEGF13 or negative control immunotoxin DT2222 (Fig. 5C). Tumor volumes are shown for individual treated mice. Tumors treated with DT2222 continued to escalate in size despite treatment. In contrast, treatment with DTEGF13 in five of five mice inhibited tumor growth and kept the tumor growth in check even on day 57, despite the fact that treatments were stopped on day 10. Differences between DT2222-treated mice and DTEGF13-treated mice were significant (P < 0.0001) as calculated on the final day of the study. There was no significant weight loss in these mice due to treatment (not shown).

In an independent study, the tumors of two mice receiving multiple DTEGF13 treatments were photographed at various times after treatment (Fig. 6 ). Animals received five injections into their tumors (which were ~0.2 cm3) over a 10-day interval. The tumor on mouse 1 regressed more slowly, showing slight signs of ulceration as early as day 2. By day 28, it shrunk ~80% of its original tumor size. By day 47, it was entirely undetectable. The tumor on mouse 2 shrunk more quickly. Tumor size was reduced 100% by day 10. This tumor reoccurred 20 days later.


Figure 6
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  		Fig. 6. Visual regression of PC-3 flank tumors treated with DTEGF13. Two male nude mice bearing PC-3 flank tumors were each treated with i.t. injections of DTEGF13. The progressive effect on tumor volume over time is shown in photographs. Mouse 1 was injected a total of nine times between days 0 and 25, whereas mouse 2 was injected five times every other day between days 0 and 10.

 
Together, these studies show that in a model in which the human EGF and IL-13 of DTEGF13 are cross-reactive with mouse EGF and IL-13, DTEGF13 is a highly effective antitumor agent. The agent is highly selective in its action against prostate cancer and that both EGF and IL-13 moieties positioned on the same molecule are necessary for its superior antitumor effect.


   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
The original contribution of this study is the observation that when IL-13 and EGF are present on the same molecule with truncated diphtheria toxin, they make up a new anticarcinoma agent with greater activity than cytotoxin made with either cytokine alone. This new anticancer agent was highly effective against PC-3 prostate cancer cells in vitro, displaying an IC50 of 0.042 pmol/L, which represents a 905-fold increase in activity over DTIL13 and a 7-log increase in activity over DTEGF. Additionally, it had striking effects against prostate cancer tumors in vivo. Despite the aggressive nature of the PC-3 prostate flank tumor in xenografted nude mice, all of the tumors responded and some did not reoccur after treatment. The fact that multiple injections of DTEGF13 were tolerated in animals was important because DTEGF13 was studied in a mouse model in which human EGF and IL-13 are cross-reactive with mouse EGFR and IL-13R, respectively.

Interestingly, the combined effect of the monomeric DTEGF and DTIL13 cytotoxins was not greater than their individual effects. If the enhanced activity of bispecific DTEGF13 could be explained by enhanced binding capacity alone, then we would expect greater activity by the combined monomeric agents and this was not the case in our studies. Binding affinity studies suggested that DTEGF13 did not have superior affinity compared with the monomeric agents, but did bind to more surface receptors. Also remarkable was the fact that DTIL13 was not very potent against PC-3, DU-145, and HT-29 target cells. This same immunotoxin kills U373 MG glioblastoma cells in picomolar concentrations (39). Still, when the IL-13R is targeted by IL-13 on the same molecule as EGF, the presence of both cytokines on the same molecule markedly enhanced anticancer activity thereby indicating a relationship between the EGFR and IL-13R pathways that may somehow relate to the simultaneous triggering of both pathways. In a recent report, Zhen et al. (40) studied the interrelationships between the IL-13R and EGFR pathways in a primary normal human bronchial epithelial cell culture system. Microarray analysis revealed that the two pathways seem to have independent effects on transcript expression. However, there were a few common effects such as the decreased expression of FOXA2 during signaling. Perhaps the superiority of DTEGF13 somehow relates to these shared elements. We synthesized and tested a hybrid molecule consisting of the EGF and IL-13 cytokines devoid of toxin. This EGF13 molecule did not inhibit proliferation of carcinomas, indicating that signaling interference alone does not explain the result and that internalization of the toxin is requisite for the anticancer effect.

An important aspect of this study is the superior effect of simultaneously targeting two markers, EGFR and IL-13R, which are very well-established therapeutic markers for carcinoma and glioblastoma. Drugs targeting each of these receptors are currently in clinical trials, making a combination molecule that has even greater activity highly attractive. One key issue will be to measure the therapeutic index of DTEGF13 and determine if it is more or less toxic to nontarget organs than the monomeric agents. Encouragingly, the animals in these studies did tolerate multiple i.t. injections despite the fact that DTEGF13 does bind mouse EGFR and IL-13R.

The goal of our flank tumor model was to determine if DTEGF13 had any effect in a localized in vivo setting, and we were able to accomplish this goal. However, the flank tumor model is inappropriate as a model for clinical prostate cancer and for addressing the issue of whether DTEGF13 will be effective for systemic therapy. Preliminary studies have indicated that DTEGF13 will have systemic activity, but a detailed study is required to determine the optimal dosage, route of administration, dose schedule, and whether it should be delivered by pump or injection. In the event that DTEGF13 is not effective systemically, a powerful advantage of recombinant cytotoxins is their ability to undergo genetic modification. Alterations have been described that enhance affinity and pharmacokinetics and reduce toxicity and immunogenicity (41). Such alteration is currently under study and may benefit DTEGF13.

Findings with other bispecific cytotoxins and immunotoxins have been reported. Investigators reported that the antiglioblastoma bispecific cytotoxin DTAT13 simultaneously targeted the cytokine receptors urokinase-type plasminogen activator receptor and IL-13R with the same DT390 cassette used in DTEGF13 (42). This bispecific molecule was made by genetically attaching human IL-13 to the human urokinase ATF-DT390 gene. Rendering this molecule bispecific did not enhance (or hinder) the activity of the molecule against human U373 MG glioblastoma cells. However, it did show a broader spectrum of reactivity to glioblastoma cell lines and unique pharmacokinetics. Another bispecific immunotoxin simultaneously targeted EpCAM and erbB2, resulting in a hybrid that again had significantly greater activity against the two monomeric immunotoxins. Unlike DTEGF13, internalization studies of DTEpCAM/anti-erbB2 hybrid revealed a higher level of internalization with the bispecific immunotoxin rather than the monomeric immunotoxin (43). A bispecific immunotoxin was reported that selectively and simultaneously targeted human CD22 and CD19 on B cells and was effective in the therapy of systemic B-cell malignancy in a scid/hu mouse model (12). As in the case of DTEGF13 for human carcinoma, the BIT DT2219 for human B-cell leukemia was much more effective than the corresponding monospecific immunotoxin. Together, these studies imply that the activity of each of these hybrid molecules may be unique and the mechanism of action may be very complex and not simply be related to binding.

Ways of increasing limited IL-13 cytotoxin activity have been under investigation. For example, investigators reported that human adrenomedullin has the ability to augment IL-13R{alpha}2 expression and thus sensitize tumors to IL-13 immunotoxin (44). Although the therapeutic use of secondary sensitizing agents may prove very useful, this approach is still clinically unproven. On the other hand, the addition of an enhancing moiety such as EGF on the same molecule will not require separate studies for Food and Drug Administration approval. In addition, it is still possible that sensitizing substances like adrenomedullin, which up-regulates receptors, will still be highly advantageous in promoting the activity of DTEGF13.

The mouse experiments described in this article indicated that DTEGF13 was tolerated in terms of toxicity and selective in its efficacy because a control immunotoxin made with the same DT390 cassette did not inhibit tumor growth. Still, the effectiveness of the bispecific immunotoxin will need to be determined in a systemic model and also against larger tumors. More clinically relevant mouse models looking at orthotopic growth of human tumors in the murine prostate as well as bone metastases models have been described and will be useful for future studies (45). Additional studies to optimize dose and dose schedule and determine the maximum tolerated dose and extent of the therapeutic window will be done.

In conclusion, DTEGF13 represents a powerful new anti–prostate cancer agent that is effective against colon carcinoma as well. Its construction is based on molecules that react with popular and established cancer targets, IL-13R and EGFR. In vitro studies show conclusive proof that the presence of both ligands on the same molecule is responsible for its superior activity. Animal studies in a model in which human DTEGF13 is cross-reactive with mouse indicates that it is highly effective in checking aggressive prostate tumor progression and is reasonably tolerated.


   Footnotes
 
Grant support: U.S. Public Health Service grants RO1-CA36725 and RO1-82154 awarded by the National Cancer Institute, National Institute of Allergy and Infectious Diseases, Department of Health and Human Services and the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research.

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.

Received 4/23/07; revised 7/12/07; accepted 7/26/07.


   References
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 Abstract
 Materials and Methods
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 Discussion
 References
 

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