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Pancreas
Genetically designing a more potent antipancreatic cancer agent by simultaneously co-targeting human IL13 and EGF receptors in a mouse xenograft model
  1. D A Vallera1,
  2. B J Stish1,
  3. Y Shu1,
  4. H Chen1,
  5. A Saluja2,
  6. D J Buchsbaum3,
  7. S M Vickers2
  1. 1 University of Minnesota Cancer Center, Section on Molecular Cancer Therapeutics, Department of Therapeutic Radiology-Radiation Oncology, Minneapolis, MN, USA
  2. 2 Department of Surgery, Minneapolis, MN, USA
  3. 3 Department of Radiation Oncology, University of Alabama at Birmingham, Birmingham, AL, USA
  1. Dr D A Vallera, University of Minnesota Cancer Center, MMC: 367, Minneapolis, MN 55455, USA; valle001{at}umn.edu

Abstract

Objective: Investigators are currently interested in the epidermal growth factor receptor (EGFR) and interleukin 13 receptor (IL13R) as potential targets in the development of new biologicals for pancreatic cancer. Attempts to develop successful agents have met with difficulty. The novel approach used here was to target these receptors simultaneously with EGF and IL13 cloned on the same bispecific single-chain molecule with truncated diphtheria toxin (DT390) to determine if co-targeting with DTEGF13 had any advantages.

Design: Proliferation experiments were performed to measure the potency and selectivity of bispecific DTEGF13 and its monospecific counterparts against pancreatic cancer cell lines PANC-1 and MiaPaCa-2 in vitro. DTEGF13 was then administered intratumourally to nude mice with MiaPaCa-2 flank tumours to measure efficacy and toxicity (weight loss).

Results: In vitro, bispecific DTEGF13 was 2800-fold more toxic than monospecific DTEGF or DTIL13 against PANC-1. A similar enhancement was observed in vitro when MiaPaCa-2 pancreatic cancer cells or H2981-T3 lung adenocarcinoma cells were studied. DTEGF13 activity was blockable with recombinant EGF13. DTEGF13 was potent (IC50 = 0.00017 nM) against MiaPaCa-2, receptor specific and significantly inhibited MiaPaCa-2 tumours in nude mice (p<0.008).

Conclusions: In vitro studies show that the presence of both ligands on the same bispecific molecule is responsible for the superior activity of DTEGF13. Intratumoural administration showed that DTEGF13 was highly effective in checking aggressive tumour progression in mice. Lack of weight loss in these mice indicated that the drug was tolerated and a therapeutic index exists in an “on target” model in which DTEGF13 is cross-reactive with native mouse receptors.

https://doi.org/10.1136/gut.2007.137802

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    Pancreatic adenocarcinoma is one of the most lethal cancers. Only 10–15% of patients’ tumours are resectable at diagnosis because of aggressive growth.1 More than 32 000 patients each year die of this cancer in the USA alone mostly due to its highly aggressive nature and rapid metastasis.2 Pancreatic cancer is most commonly treated with the nucleoside analogue gemcitabine, but the median survival time is <6 months.3 ,4 Studies have shown that pancreatic tumours overexpress epidermal growth factor receptor (EGFR),5 and others have shown that fusion toxins targeting the EGFR show promise for pancreatic cancer therapy.6–9 Also, interleukin 13 receptors (IL13Rs) are expressed on pancreatic tumours. For example, investigators showed that six of six pancreatic cancer cell lines examined expressed IL13Rα1 and IL4Rα, one cell line expressed IL13Rα2, and five pancreatic cancer cell lines expressed γc.10 IL13R-directed fusion toxins also have successfully been shown to kill pancreatic tumour cells.10 ,11

    EGF is the main ligand of the EGFR, a transmembrane signalling protein from the erbB family.12 Studies have revealed a link between EGFR signalling pathways and malignancy.13 IL1314 ,15 secreted by activated type 2 T cells and mast cells,16 is a pleiotropic lymphokine regulating inflammatory and immune responses. It modulates human monocyte and B cell functions but not those of T cells.17 IL13Rs are found to be overexpressed on solid tumour cells including glioblastoma,18–21 renal cell carcinoma,22 AIDS Kaposi’s sarcoma23 and cancers of the prostate,24 ovary,25 and head and neck.26 IL13 has proven a useful ligand for therapy because together with IL13R being overexperessed on tumours, its expression on normal cells is limited to B cells and monocytes. It appears that IL13R may function as a tumour-specific, high affinity target, and incorporating IL13 into a cytotoxin (CT) may be a beneficial strategy.

    In this study, we created a novel single-chain recombinant bispecific CT by linking a fragment of diphtheria toxin (DT) to human EGF and IL13, forming the fusion protein DTEGF13. DT is used for CT construction because a single molecule delivered to the cytosol is sufficient to bring about cell killing.27 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.28 DT390 contains the A fragment of native DT that catalyses ADP ribosylation of elongation factor 2 (EF-2) leading to irreversible inhibition of protein synthesis and cell death.29 ,30

    This study set out to address the hypothesis that including two well-established targeting ligands on the same single-chain molecule would have distinct targeting advantages over the same monospecific targeting agents. Thus, a new bispecific CT, DTEGF13, was bioengineered and compared with the monospecific CTs DTEGF and DTIL13. The agents were tested against several aggressive human pancreatic tumour cell lines in vitro and against MiaPaCa-2 cells in vivo, which are readily xenografted into athymic nude mice.

    MATERIALS AND METHODS

    DTEGF13 construction

    DNA shuffling and PCR assembly techniques were used to assemble the genes encoding the single-chain bispecific CT DTEGF13. From the 5′ end to the 3′ end, the assembled gene consisted of an NcoI 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 IL13 linked by a 20 amino acid segment of human muscle aldolase (hma), and an XhoI restriction site (fig 1A). The primers used for the final assembly of DTEGF13 are shown in table 1. The final 1755 bp NcoI/XhoI target gene was spliced into the pET21d expression vector under control of an isopropyl-β-d-thiogalactopyranoside (IPTG)-inducible T7 promoter. The gene was correct in sequence and has been cloned (Biomedical Genomics Center, University of Minnesota). The monospecific agents DTEGF and DTIL13 were created using the same techniques.

    Figure 1
    Figure 1 Construction and purification of DTEGF13. (A) The gene fragment encoding the single-chain bispecific immunotoxin DTEGF13 was created using overlap extension PCR. This construct consisted of: (from the 5′ to the 3′ end) a truncated diphtheria toxin molecule (DT390), a seven amino acid (EASPPGE) linker, human epidermal growth factor (EGF), a flexible 20 amino acid segment of human muscle aldolase (hma), and interleukin 13 (IL13). Using the NcoI/XhoI restriction sites, the sequence of DTEGF13 was cloned in the pET21d bacterial expression vector. (B) Sodium dodecyl sulphate-polyacrylamide gel electrophoresis was used to analyse the expression and purification of DTEGF13. Lane 1, inclusion bodies isolated from Escherichia coli following isopropyl-β-d-thiogalactopyranoside (IPTG) induction of DTEGF13 expression. Lane 2, molecular weight standards. Lane 3, DTEGF13 (63.6 kDa) protein following in vitro refolding and purification.
    View this table:
    Table 1 Sequence of oligonucleotides used to assemble DTEGF13

    A pair of bivalent immunotoxins targeting haematological malignancies were produced and used as specificity controls in this study. DT2222, a bivalent fusion toxin control containing the same DT390 cassette, was produced by joining two repeating scFvs specific for human CD22 to DT390. CD22 is human B lymphocyte-specific glycoprotein that is expressed in the majority of B cell leukaemias and lymphomas.31 Bic3 is a T cell-specific immunotoxin consisting of two consecutive scFvs recognizing the human CD3∊ linked to DT390.32

    Isolation of inclusion bodies, refolding and purification

    These procedures were previously described.32 Plasmids were transformed into Escherichia coli strain BL21(DE3) (Novagen, Madison, Wisconsin, USA). Following overnight culture, bacteria were grown in Luria broth. Gene expression was induced with the addition of IPTG (FischerBiotech, Fair Lawn, New Jersey, USA). Two hours after induction, bacteria were harvested by centrifugation. Cell pellets were suspended and homogenised. Following sonication and centrifugation, the pellets were extracted and washed. Inclusion bodies were dissolved and the protein refolded. Refolded proteins were purified by fast protein liquid chromatography ion exchange chromatography (Q Sepharose Fast Flow, Sigma, St Louis, Missouri, USA) using a continuous gradient.

    Tissue culture

    The human pancreatic cell lines MiaPaCa-2 and PANC-1, and the Burkitt’s lymphoma cell line Daudi33 were obtained from the American Type Culture Collection (ATCC, Rockville, Maryland, USA). The H2981-T3 human lung adenocarcinoma line has been described previously.34 Cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM; MiaPaCa-2 and PANC-1) or RPMI-1640 medium (H2981-T3 and Daudi) (Cambrex, East Rutherford, New Jersey, USA) supplemented with 10% fetal bovine serum, 2 mmol/l l-glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin. All carcinoma cells were grown as monolayers, and Daudi cells were grown in suspension using culture flasks. Cell cultures were incubated in a humidified 37°C atmosphere containing 5% CO2. When adherent cells were 80–90% confluent they were passaged using trypsin-EDTA for detachment. Only cells with viability >95%, as determined by trypan blue exclusion, were used for experiments.

    Measuring cell kill by proliferation inhibition

    To determine the effect of DTEGF13 on pancreatic cancer cells, proliferation assays measuring [3H]thymidine incorporation were performed.32 Cells (104/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. CTs 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, Little Chalfont, Buckinghamshire, UK) was added (1 μCi per well) for the final 8 h of incubation. Plates were frozen to detach cells and cells were then harvested onto a glass fibre filter, washed, dried and counted using standard scintillation methods. Data from proliferation assays are reported as the percentage of control counts. For blocking studies, increasing concentrations of EGF13 were mixed with 1 nM DTEGF13, and subsequent mixtures were added to wells containing MiaPaCa-2 cells. All other aspects of blocking assays were identical to the procedure listed above for proliferation assays.

    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 minimise the potential of contaminating virus transmission. Three different flank tumour studies were performed for this study. In each of the experiments, tumour volume was determined as a product of W×L×H as measured by calipers. Animal weights were also monitored as an indication of treatment-related toxicity. Mice with flank tumours >2.0 cm3 were euthanised in accordance with University of Minnesota Research Animal Resources guidelines.

    Experiment 1

    Flank tumours were initiated by injecting 1×107 MiaPaCa-2 cells in DMEM into the left flank of nude mice (n = 20). On day 22, mice with tumours exceeding 50 mm3 in volume were randomised into DTEGF13 treatment or no treatment groups. Treatment mice received 2.5 μg of DTEGF13 in 100 μl of phosphate-buffered saline (PBS) injected intratumourally. A total of 10 injections were given on days 22, 24, 27, 29, 31, 36, 38, 42, 45 and 48 (fig 5B).

    Figure 5
    Figure 5 Effect of intratumoural injection of DTEGF13 on MiaPaCa-2 flank tumours: experiment 1. MiaPaCa-2 flank tumours were established by injecting 1×107 cells into the left flank of male nude mice. Once palpable tumours were established (day 22) mice were divided into two groups, (A) no treatment or (B) DTEGF13 treatment. Mice in the DTEGF13 group received 2.5 μg of DTEGF13 injected intratumourally as often as indicated by the arrows on the graph.

    Experiment 2

    One day prior to cell injection, male nude mice were irradiated with 3 Gy using an x ray irradiator. Flank tumours were established by injecting 1×107 MiaPaCa-2 cells in a 1:1 mixture of DMEM and Matrigel (BD Biosciences, San Jose, California, USA). When tumours reached approximately 50 mm3 (day 18), mice were divided into groups (n = 5/group) and treatment was initiated. Four injections of 2.5 μg of DTEGF13, DTEGF or DTIL13 were given intratumourally every other day on days 18, 20, 22 and 24. Mice in the control group received intratumoural injections of 100 μl of PBS.

    Experiment 3

    For the final flank tumour study, nude mice were injected with 1×107 MiaPaCa-2 cells in a 1:1 mixture of DMEM and Matrigel. On day 15 when flank tumours were approximately 75 mm3, mice were divided into treatment groups (n = 6/group). Treatment mice received a total of six intratumoural injections of 2.5 μg of either DTEGF13 or the B cell-targeting immunotoxin DT2222. Injections were given on days 15, 17, 19, 23, 25 and 28.

    Statistical analyses

    All statistical analysis was performed using Prism 4 (Graphpad Software, San Diego, California, USA). Groupwise comparisons of single data points were made by Student t test. p Values <0.05 were considered significant.

    RESULTS

    Purification of DTEGF13

    Following refolding and purification, batches of DTEGF13 were analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) (fig 1B) and size exclusion chromatography. Both analyses revealed that DTEGF13 was the expected molecular weight (63.6 kDa) and purity was ⩾95%. Final yields of purified protein were 5–10 mg/l of culture.

    The ability of DTEGF13 to inhibit proliferation of pancreatic tumour cells

    To determine the ability of DTEGF13 to kill EGFR-expressing and IL13R-expressing pancreatic cancer cells, it was tested against the EGFR+ and IL13R+ PANC-1 tumour cell line (fig 2). Bispecific DTEGF13 showed an IC50 (concentration that inhibits 50% of cell proliferation) of 0.035 nM, indicating that it was at least 2800-fold more toxic than either DTEGF or DTIL13 which did not reach an IC50. Bic3, a T cell-targeting immunotoxin containing the DT390 molecule, was included as a specificity control. DTEGF13 also showed potent cytotoxicity against the SW-1990 and ASPC-1 pancreatic cancer cell lines, with an IC50 of 0.00013 and 0.052 nM, respectively (not shown). Killing in proliferation assays was verified by trypan blue staining (not shown).

    Figure 2
    Figure 2 Potent cytotoxicty of the bispecific cytotoxin DTEGF13. The in vitro activity of bispecific DTEGF13 and monospecific DTEGF and DTIL13 was determined by measuring [3H]thymidine incorporation into PANC-1 cells following a 72 h incubation with varying concentrations of cytotoxin. Bic3, a T cell-targeting immunotoxin containing DT390, was included as a negative control. Data are expressed as a percentage of [3H]thymidine relative to control cells incubated in medium alone (control counts = 30 290 (5091)). Points represent the mean of triplicate measures (SD). IC50 indicates the concentration of cytotoxin that inhibits 50% of cell proliferation relative to untreated cells.

    Increased activity of DTEGF13 is due the presence of EGF and IL13 ligands on a single molecule

    Proliferation assays were conducted in order to determine if the increased activity of DTEGF13 was a result of the increased number of binding molecules present on a bispecific CT. Figure 3 shows the data comparing the activity of DTEGF13 with that of the monospecific DTEGF and DTIL13, as well as a combination of both monospecific CTs against MiaPaCa-2 pancreatic (fig 3A) and H2981-T3 lung (fig 3B) cancer cells. A mixture of DTEGF and DTIL13 results in an identical number of ligands to those present in the same concentration of DTEGF13. Against MiaPaCa-2 cells the monospecific DTEGF was able to kill with an IC50 of 0.007 nM. Monospecific DTIL13 was less effective, with an IC50 of 30 nM. However, DTEGF13 showed an IC50 of 0.00017 nM, representing a 41-fold increase in activity as compared with DTEGF and a 176 000-fold increase in activity as compared with DTIL13. Interestingly, a mixture of DTEGF and DTIL13 showed no increased activity over DTEGF alone. DTEGF13 also demonstrated broad reactivity with a number of EGFR+ and IL13R+ carcinoma cell lines, including the human lung cancer cell line H2981-T3 (fig 3B). As in the case of MiaPaCa-2, DTEGF13 was the most effective, with an IC50 of 0.002 nM. DTEGF showed an IC50 of 0.25 nM and DTIL13 showed an IC50 of 100 nM, an increase of 125-fold and 50 000-fold, respectively. Once again, a combination of DTEGF and DTIL13 showed no advantage in cytotoxicity over either monospecific CT. These data demonstrate that the superior activity of DTEGF13 is due to the presence of both ligands on a single-chain molecule.

    Figure 3
    Figure 3 Enhanced cytotoxicity of bispecific DTEGF13 is dependent on linking both epidermal growth factor (EGF) and interleukin (IL) 13 on a single-chain molecule. The antiproliferative effect of DTEGF13, DTEGF, DTIL13 and a mixture of DTEGF/DTIL13 on (A) MiaPaCa-2 (pancreatic) and (B) H2981-T3 (lung) cancer cells was tested by measuring [3H]thymidine uptake 72 h following cytotoxin exposure. Points on each graph represent the mean of triplicate samples (SD). Control counts  =  (A) 72 048 (9826), (B) 64 876 (11 866).

    Specificity of DTEGF13 cytotoxicity

    In order to test the specificity of DTEGF13 activity, two different assays were conducted. Figure 4A shows that neither DTEGF13 nor the monospecific DTIL13 or DTEGF CT had any effect on the proliferation of EGFR– and IL13R– Daudi lymphoma cells. In fig 4B we demonstrate that the activity of 1 nM DTEGF13 can be blocked by adding saturating concentrations of EGF13 to cultures of MiaPaCa-2 cells. EGF13 is a recombinant molecule identical to DTEGF13 except that it lacks the DT390 fragment. A 10-fold excess of EGF13 was able to inhibit >80% of DTEGF13-mediated cell killing. Higher concentrations of EGF13 fully abrogated CT activity. The specificity of blocking was confirmed by the fact that even a 1000-fold excess of 2219EA, a B cell-targeting bispecific protein, was unable to affect the activity of DTEGF13. In other studies, we showed that anti-EGFR and anti-IL13 antibodies blocked DTEGF13 activity.35 ,36 Together, these data and the Bic3 data in fig 2 demonstrate the highly specific nature of DTEGF13 cytotoxicity.

    Figure 4
    Figure 4 Specificity of DTEGF13. (A) Epidermal growth factor receptor negative (EGFR–) and interleukin 13 receptor negative (IL13R–) Daudi cells were incubated with DTEGF13, DTEGF and DTIL13. (B) DTEGF13-mediated cytotoxicity was inhibited by adding increasing concentrations of bispecific EGF13, which lacks a DT390 moiety, to cultures of MiaPaCa-2 cells incubated with 1 nM DTEGF13. 2219EA, a bispecific B cell-targeting molecule, was used as an irrelevant negative control. In both experiments, cell proliferation was determined by measuring [3H]thymidine incorporation following 72 h incubation time. Data are expressed as the percentage of cell-associated [3H]thymidine at each concentration relative to control cells. Points represent the means (SD).

    Efficacy of intratumoural DTEGF13 in a MiaPaCa-2 nude mouse flank tumour model

    To test the ability of DTEGF13 to inhibit pancreatic tumour growth in vivo, human MiaPaCa-2 cells were xenografted into the flank of nude mice. Once the tumours were established and palpable, mice were treated with multiple intratumoural injections. For experiment 1 shown in fig 5, 1×107 tumour cells suspended in DMEM were injected into mice that had not been subjected to total body irradiation (TBI). This method yielded a poor tumour establishment rate (<40% of injected animals). Three of the animals that did form tumours were given an aggressive course of 10 injections of DTEGF13 over the course of 3 weeks, as established from earlier pilot experiments. In this experiment, control tumour growth was slower than desired in most animals (fig 5A). However, fig 5B shows that the course of DTEGF13 treatment appeared to keep tumour growth in check over the duration of the short study.

    In experiment 2 (fig 6), animals were given 3 Gy TBI 1 day prior to the injection of 1×107 MiaPaCa-2 cells. Cells were injected in a 1:1 mixture of DMEM and Matrigel in order to promote better tumour growth. The combination of TBI and Matrigel increased the tumour take rate to >95%. When treatment was initiated (day 18), mice in the treatment groups received four intratumoural injections of 2.5 μg of either DTEGF13, DTEGF or DTIL13 given every other day. Control mice received intratumoural injections of PBS on the same schedule. Figure 6A shows that the highest degree of antitumour efficacy was achieved with DTEGF13 administration. Figure 6B shows the tumour volumes of the individual animals in the DTEGF13 treatment group. Each of the animals showed a noticeable decrease in tumour volume, with one tumour completely regressing. However, fig 6C shows that treatment-related toxicity was heightened by the TBI given to the animals in this experiment. Weight loss and mortality (3/5 animals) occurred despite following a previously well-tolerated treatment regimen. Others have reported that immunotoxin toxicity can be exacerbated by irradiation.33

    Figure 6
    Figure 6 Effect of intratumoural injection of DTEGF13 on MiaPaCa-2 flank tumours: experiment 2. Prior to injection of tumour cells, male nude mice were irradiated with 3 Gy using an x ray irradiator. Flank tumours were then established by injecting 1×107 MiaPaCa-2 cells in a 1:1 mixture of Dulbecco’s modified Eagle’s medium (DMEM):Matrigel. When tumours reached approximately 50 mm3, mice were divided into groups and treated with intratumoural injections of 2.5 μg of DTEGF13, DTIL13, DTEGF or phosphate-buffered saline. Four injections were given every other day as indicated by the arrows on the graph. (A) Average tumour volume of animals in each treatment group. (B) Individual tumour volumes and (C) animal weights of individual mice in the DTEGF13 treatment group.

    For experiment 3 (fig 7), unirradiated mice were injected subcutaneously in the left flank with 1×107 MiaPaCa-2 cells suspended in 100 μl of a 1:1 mixture of DMEM and Matrigel. This method facilitated 100% tumour establishment without introducing the unwanted side effects related to TBI. Figure 7A shows a significant antitumour effect of DTEGF13 compared with the tumour progression observed in groups of mice that were untreated or were treated with the negative control DT2222. Tumour growth was contained, but relapses did occur following cessation of DTEGF13 treatment. A course of six intratumoural injections of 2.5 μg of DTEGF13 was tolerated with no significant toxicity as evidenced by animal weight (fig 7B).

    Figure 7
    Figure 7 Effect of intratumoural administration of DTEGF13 on MiaPaCa-2 flank tumours: experiment 3. (A) A final xenograft model of pancreatic cancer was established by injecting male nude mice (no prior irradiation) with 1×107 MiaPaCa-2 cells in a 1:1 mixture of Dulbecco’s modified Eagle’s medium (DMEM):Matrigel. Mice were randomised into three groups (n = 6/group) on day 15 when the average tumour volume was approximately 75 mm3. Treated animals were injected intratumourally with 2.5 μg of either DTEGF13 or the B cell lymphoma-targeting DT2222. A total of six injections were given over the course of 2 weeks as indicated by the arrows on the graph. (B) Weights of animals in the DTEGF13 treatment group.

    Together, these studies show that in a model in which the human EGF and IL13 of DTEGF13 are cross-reactive with mouse EGFR and IL13R, DTEGF13 is a highly effective antitumour agent. The agent is highly selective in its action against pancreatic cancer, and both EGF and IL13 moieties positioned on the same molecule are necessary for its superior antitumour effect.

    DISCUSSION

    The original contribution of this study is that IL13 and EGF were cloned into the same single-chain molecule with truncated DT. The new recombinant hybrid antipancreatic cancer agent had far greater activity than CTs made with either cytokine alone. This new DTEGF13 was potent and highly effective against MiaPaCa-2 tumour cells in vitro, displaying an IC50 of 0.00017 nM, representing a 41-fold increase in activity over DTEGF and a 176 000-fold increase in activity over DTIL13. Studies with mixtures of monomeric CT showed that the enhanced activity of DTEGF13 was dependent on having both ligands on the same single-chain molecule. Additionally, DTEGF13 had striking effects against pancreatic tumours in vivo. Despite the aggressive nature of the Mia-PaCa-2 flank tumour in xenografted nude mice, all of the tumours responded, including one tumour that did not reoccur after treatment. Another important aspect of this study is that EGF and IL13 are cross-reactive with mouse EGFR and IL13R in this mouse model (constituting an “on target” model). Multiple injections of DTEGF13 were tolerated. Thus, a therapeutic index clearly exists.

    Others have investigated the potential of using IL13R- and EGFR-targeted toxins for therapy of this aggressive pancreatic cancer. For example, investigators have enhanced IL13Rα2 gene expression in pancreatic cancer cells and showed that these IL13R-expressing tumours had susceptibility to IL13 CT.11 Interestingly, these investigators reported a dominant infiltration of cells including macrophages and natural killer cells in the regressing tumours. Since macrophages were found to produce nitric oxide, IL13Rα2-targeted cancer therapy involved not only a direct tumour cell killing by IL13 CT but also activation of the innate immune response at the tumour site. It will be important to determine whether DTEGF13 is activating the immune response. This study also points out that IL13 CTs are not effective against all pancreatic tumours, probably because they differ in the amount of surface IL13R expressed. Since DTEGF13 appears to require limited IL13R expression to enhance tumour kill, it is an attractive antipancreatic cancer agent.

    For EGF, studies show that a recombinant immunotoxin made by genetically fusing the anti-EGFR single-chain variable fragment to truncated Pseudomonas aeroginosa exotoxin A showed specific binding to and toxicity against the EGFR+ metastatic pancreatic carcinoma cell line L3.6pl, but not to control cell systems.6 ,7 Both single and multiple injection treatment protocols resulted in a significant reduction in the average number of lung metastases in tumour-bearing animals, indicating that targeting the EGFR with CT is an effective strategy against disseminated human pancreatic carcinoma cells. Together, these studies suggest that targeting EGFR and IL13R simultaneously may have clear advantages.

    In the in vivo experiments performed in this study, experiment 1 showed that DTEGF13 was effective, but control tumour growth was slow. In experiment 2, DTEGF13 was effective again, but toxicity was enhanced because TBI was given to enhance tumour growth. Others have shown in human lymphoma models that irradiation can enhance immunotoxin-related side effects.37 ,38 This appears also to be true of DTEGF13. Others are exploring the efficacy of targeting EGFR with radiolabelled antibodies.39 ,40 Combining this form of therapy with our DTEGF13 CT therapy would be inadvisable without thorough investigation. However, other forms of combined therapy may be very effective. For example, phase 1 studies are currently examining combining anti-EGFR antibody therapy with gemcitabine chemotherapy.41 Gemcitabine is the most successful form of chemotherapy for pancreatic cancer. Because CTs have a mechanism of killing that is entirely different from the mechanism of killing of chemotherapy, many studies have shown that chemotherapy can be successfully combined with immunotoxin or CT therapy.42–44 Thus, the probability is high that the effectiveness of DTEGF13 therapy can be heightened by combining it with chemotherapy. In fact, investigators have combined IL4 CT with gemcitabine, heightening antipancreatic cancer efficacy.45 In fact, our preliminary studies with MiaPaca-2 show that adding 50 nM gemcitabine reduces the IC50 of DTEGF13 >3 logs in vitro. In experiment 3, we successfully enhanced tumour growth by injecting cells in Matrigel, and DTEGF13 was highly effective at inhibiting flank tumour growth.

    The combined effect of the monomeric DTEGF and DTIL13 CT was not greater than the individual effects of the monomeric CT, but when IL13R and EGFR were targeted simultaneously by linking both cytokines on the same molecule, the anticancer effect was enhanced. The reason could potentially relate to enhanced receptor numbers, enhanced internalisation or differences in intracellular, subcellular compartmentalisation. Studies with prostate cancer cells show that enhanced activity of DTEGF13 may not be solely attributed to binding.46 Past studies show that immunotoxin activity relates to compartmentalisation within the cell. Studies of the inter-relationships of the IL13R and EGFR pathways in a primary normal human bronchial epithelial cell culture system via microarray analysis indicate that the two pathways have independent effects.40 The enhanced activity of DTEGF13 could relate to improved internalisation whereby the toxin is delivered more efficiently to the cytosol, the main target organelle of catalytic toxins. The ligands themselves are not contributing to the cytotoxicity, since we synthesised EGF13 devoid of toxin and this molecule did not inhibit proliferation of cells that were killed by DTEGF13.

    As discussed, toxicity issues will need to be addressed more thoroughly. Mice tolerated DTEGF13 very well at the dose of 2.5 μg/injection (100 μg/kg). However, tolerability was dependent on spacing the doses. Intratumoural injection is not an effective means of delivery since areas of the tumour are often not injected and relapses occur in these neglected tumour areas. Moreover, injection precipitously spikes the dosage, creating steep peaks and rapid troughs. Studies by others indicate that pump delivery may be superior, providing constant dose delivery over a far longer period of time.

    Other bispecific fusion toxins have been reported. In some reports, inclusion of a second ligand enhances activity,47 ,48 but not in others.49 Currently, no method exists which predicts whether a given bispecific will be successful.

    In conclusion, DTEGF13 represents a powerful new antipancreatic cancer agent. Its construction is based on molecules that react with well-studied and established cancer targets, IL13R 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 the native mouse receptors indicate that it is highly effective in checking aggressive tumour progression and reasonably tolerated. DTEGF13 may be a useful alternative therapy for pancreatic cancer and perhaps other carcinomas.35 ,36

    Acknowledgments

    This work was supported in part by the US Public Health Service Grants RO1-CA36725, RO1-CA082154 and P20 CA101955 awarded by the NCI and the NIAID, DHHS.

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    Footnotes

    • Competing interests: None.