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Keywords:

  • IL-13 cytotoxin;
  • IL-13Rα2 chain;
  • adenoviral vector;
  • molecular targeting

Abstract

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Previous studies demonstrated that IL-13Rα2 chain–overexpressing cancer cells were highly sensitive to IL-13 cytotoxin (IL13-PE38QQR) and could be targeted by cytotoxin treatment. However, the majority of human tumors do not express high levels of IL-13Rα2 chain. To expand the IL-13 cytotoxin–mediated cancer targeting therapy, we combined cytotoxin treatment with gene transfer of IL-13Rα2 chain. We constructed a recombinant adenoviral vector carrying the human IL-13Rα2 gene (Ad-IL-13Rα2), which expresses high levels of IL-13Rα2 chain on infected cells. Human cancer cell lines A549 and HOS, which originally show no IL-13Rα2 expression and little sensitivity to IL-13 cytotoxin, were effectively converted to become sensitive to this cytotoxin after Ad-IL-13Rα2 infection. The CC50 of IL-13 cytotoxin for Ad-IL-13Rα2-infected A549 cells was <10 ng/ml, whereas the CC50 for uninfected or control vector-infected cells was >500 ng/ml. We also examined the antitumor activity of IL-13 cytotoxin in an established xenograft model of cytotoxin-resistant human lung tumor. Only a single i.t. injection of Ad-IL-13Rα2 markedly enhanced the sensitivity of established tumors to IL-13 cytotoxin treatment; furthermore, this antitumor effect was significantly sustained for more than 1 month after the last treatment with IL-13 cytotoxin. Taken together, these results suggest the combination of adenoviral vector–mediated IL-13Rα2 gene transfer and IL-13 cytotoxin administration can be an effective targeting approach for several types of IL-13 cytotoxin–resistant cancers which show no or little expression of IL-13Rα2 chain. © 2005 Wiley-Liss, Inc.

Targeted anticancer therapies have become increasingly successful in the treatment of various cancers. To limit nonspecific toxicity and to improve the efficiency of cancer therapy, tumor markers, which are generally overexpressed on the surface of tumor cells, can be selectively targeted. Previous studies of our and other groups have shown that IL-13Rα2 chain is one of the tumor-associated markers1, 2 that is overexpressed on several types of human cancer cell, such as RCC,3, 4, 5 glioma,4, 5, 6, 7, 8, 9, 10 AIDS-associated Kaposi's sarcoma,11, 12 head-and-neck cancer13, 14 and some ovarian cancers.4, 15 To establish a novel targeting therapy for such cancers, we had tried to target IL-13Rα2 chain by developing a potential therapeutic agent, IL-13 cytotoxin (IL13-PE38QQR),16 which is a recombinant fusion protein composed of IL-13 and a modified form of PE. Previous studies have proved that IL-13 cytotoxin is highly selective and potent in killing cancer cells that overexpress IL-13Rα2 chain.3, 5, 6, 7, 8, 9, 10, 11, 12, 14, 16, 17 Preclinical studies regarding the safety and toxicity of IL-13 cytotoxin have been performed in mice, rats and monkeys; and all animals tolerated this therapy well, with minimal toxicity to vital organs.18 Based on these results, 3 phase I/II clinical trials of this cytotoxin in adults with malignant glioma have been initiated, and all are currently ongoing in the United States.18 However, the majority of human tumors do not express high levels of IL-13Rα2 chain;4, 14 thus, such tumor cells were practically resistant to this cytotoxin.14

IL-13 is a pleiotropic and Th2-dominated immune regulatory cytokine,19, 20 which plays a central role in allergic asthma21, 22, 23 and mediates diverse responses during helminth infection.24, 25 It is critical for tumor immunosurveillance26, 27 and modulates apoptosis or tumor cell growth.28, 29 Two different components of the IL-13 receptor have been identified, which are known as IL-13Rα1 chain30, 31, 32 and IL-13Rα2 chain.33, 34 IL-13Rα1 chain alone binds IL-13 with low affinity. Therefore, IL-13Rα1 chain requires the recruitment of IL-4Rα chain30, 31, 32, 35 to form a high-affinity heterodimer receptor complex, which mediates the IL-13 signal-transduction pathway. IL-13Rα2 chain by itself can bind IL-13 with higher affinity, but it does not mediate IL-13 signaling35, 36 because it has a very short cytoplasmic domain that lacks the conserved signaling motifs. In particular, IL-13Rα2 chain has a unique property of ligand internalization after binding,36, 37 which has also been identified as a soluble protein in mouse serum and urine;38 thus, it can be a specific inhibitor of IL-13 signaling or a decoy receptor for IL-13.39, 40, 41 Moreover, IL-13Rα2 chain may inhibit IL-13-mediated biologic functions in tumor growth and recurrence.26, 27, 42, 43

To utilize the attractive properties of IL-13Rα2 chain in IL-13 cytotoxin–mediated cancer therapy, we previously tried to combine the cytotoxin treatment with gene transfer of IL-13Rα2 chain.13, 14, 44, 45, 46 In the present study, we generated a replication-defective adenovirus, Ad-IL-13Rα2, which can express high levels of IL-13Rα2 chain in infected cells. We confirmed that adenovirus-mediated gene transfer of IL-13Rα2 chain significantly enhanced the in vitro cytotoxic effect of IL-13 cytotoxin in cytotoxin-resistant cancer cells and the in vivo antitumor activity of the cytotoxin in an established human tumor model which is resistant to the cytotoxin. Our results demonstrate that combination therapy of adenoviral gene transfer of IL-13Rα2 chain and IL-13 cytotoxin treatment can offer a potent targeting approach to cancer therapy.

Material and methods

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Recombinant cytokines and immunotoxin

Recombinant human IL-4 and IL-13 were purchased from Peprotech (London, UK). Recombinant IL-13 cytotoxin (IL13-PE38QQR) was produced and purified as previously described.14

Cell lines

A549 (human lung cancer), PM-RCC (renal cell carcinoma) and 293 (human embryonic kidney) cell lines were cultured in DMEM (Sigma, St. Louis, MO) containing 10% FBS, 100 units/ml penicillin and 100 μg/ml streptomycin (Nacalai Tesque, Kyoto, Japan). Human osteosarcoma cell lines HOS and MG-63 were cultured in Eagle's minimum essential medium (Sigma) containing 10% FBS, 100 units/ml penicillin and 100 μg/ml streptomycin.

Construction of replication-defective ΔE1ΔE3 adenoviral vector

A replication-defective ΔE1ΔE3 adenoviral vector expressing the IL-13Rα2 chain gene under CMV promoter, which was termed Ad-IL-13Rα2, was constructed as described previously.47 The human IL-13Rα2 chain cDNA fragment was inserted into the NotI site of shuttle plasmid pCMV-SV2+,48 which contains a portion of the adenovirus type 5 (Ad5) genome and the CMV early promoter/enhancer. The shuttle plasmid carrying the expression cassette of IL-13Rα2 chain instead of the Ad5 E1 region and adenoviral genomic plasmid pJM17 (Microbix Biosystems, Toronto, Canada) containing the Ad5 genome with E1 and E3 deletions were cotransfected into 293 cells using the Calcium-Phosphate Transfection System (Life Technologies, Grand Island, NY). By homologous recombination between the above 2 plasmids in 293 cells, cytopathic effects were observed within 2 weeks after transfection, after which cells were harvested. Viruses were released from cells by freeze-thawing using liquid N2. Another replication-defective adenoviral vector expressing the β-galactosidase gene, named Ad-LacZ, was constructed in the same way and utilized as an adenoviral infection control. Both adenoviral vectors were purified from a single plaque clone, propagated in 293 cells, and concentrated by the cesium chloride-density gradient centrifugation method followed by dialysis. Stocks of recombinant viruses were prepared and stored at –80°C until use. The titer of recombinant virus stocks was checked on 293 cells by a plaque-forming assay.

Conditions of recombinant adenoviral vector infection

Cells were plated in 6-well (1.0 × 105/well) or 24-well (2.0 × 104/well) culture plates and incubated at 37°C in a humidified 5% CO2 incubator for 24 hr. Cells were infected with purified adenoviral vector at several concentrations in 800 μl (6-well plate) or 200 μl (24-well plate) of serum-free medium at 37°C in a humidified 5% CO2 incubator for 2 hr. Then, fresh complete medium was added to each well.

RT-PCR analysis

Total RNA of cells was extracted using Trizol reagent (Life Technologies). Each 2.5 μg of the RNA samples was used for an RT reaction. The reaction was performed at 37°C for 50 min in a final volume of 20 μl of reaction mixture containing 50 mM TRIS-HCl (pH 8.3), 3 mM MgCl2, 75 mM KCl, 0.5 mM of each dNTP, 40 units of RNase inhibitor (Nacalai Tesque), 500 ng of oligo-d(T)20 primer and 200 units of MMLV reverse transcriptase (Life Technologies). Each 1 μl aliquot of the cDNA samples was amplified in a final volume of 50 μl of PCR mixture containing 10 mM TRIS-HCl (pH 8.8), 2 mM MgCl2, 50 mM KCl, 0.2 mM of each dNTP, 2.5 units of SuperTaq DNA polymerase (Ambion, Austin, TX) and 0.2 μM each of specific primers for human IL-13Rα2 chain cDNA (5′-AATGGCTTTCGTTTGCTTGG-3′, 5′-ACGCAATCCATATCCTGAAC-3′, in Fig. 1; 5′-ATGGCTTTCGTTTGCTTGG-3′, 5′-CATGTATCACAGAAAAATT-3′ in Fig. 2), human IL-13Rα1 chain cDNA (5′-TATTTTAGTCATTTTGGCGACAAAC-3′, 5′-GTTATGTAGAGTGTGGAATTGCGCT-3′) or human IL-4Rα chain cDNA (5′-GACCTGGAGCAACCCGTATC-3′, 5′-CATAGCACAACAGGCAGACG-3′). An internal control experiment in RT-PCR analysis was performed using a human β-actin primer pair (R&D Systems, Minneapolis, MN) or GAPDH-specific primers (5′-ACCACAGTCCATGCCATCAC-3′, 5′-TCCACCACCCTGTTGCTGTA-3′). PCR was carried out for 30 cycles at 95°C for 30 sec, 55°C for 30 sec and 72°C for 60 sec. PCR product was run in a 1% agarose gel. The intensities of each amplified IL-13Rα2 cDNA band were analyzed by Scion Image software (version 4.02; Scion, Frederick, MD) and exhibited as values relative to internal control, β-actin or GAPDH.

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Figure 1. Correlation between IL-13Rα2 chain expression and sensitivity to IL-13 cytotoxin in cancer cell lines. (a) Total RNA extracted from A549, HOS, MG-63 and PM-RCC cells was assessed for mRNA expression of IL-13 receptor components (IL-13Rα1, IL-13Rα2 and IL-4Rα) by RT-PCR. The intensity of each IL-13Rα2 band was calculated with Scion Image software and exhibited as a value relative to internal control, β-actin. (b) Human cancer cell lines (A549, HOS, MG-63 and PM-RCC) were seeded at 2.0 × 104 cells/well in 24-well plates and incubated with or without IL13-PE38QQR (100 ng/ml) for 72 hr. Living cells were counted by trypan blue dye exclusion. Viable cells (% of control) are exhibited as a value relative to each nontreated control. Data are means ± SEM from triplicate determinations of 2 representative experiments. Open bars, without treatment; closed bars, treated with IL13-PE38QQR. *p < 0.05.

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Figure 2. Expression of IL-13Rα2 chain in cancer cells infected with Ad-IL-13Rα2 vector. (a) A549 and HOS cells in 24-well plates (2.0 × 104 cells/well) were infected with Ad-IL-13Rα2 at MOI 1 and 10 for 2 hr. Total RNA was extracted from cells at the indicated time points (24, 48 and 72 hr after infection), and RT-PCR was performed to assess for expression of IL-13Rα2 chain mRNA. Mock, uninfected control. (b) A549 and HOS in 6-well plates (1.0 × 105 cells/well) were infected with Ad-IL-13Rα2 at MOI 1, 10 and 100 for 2 hr. Cells were collected 48 hr after infection and stained with FITC-conjugated antihuman IL-13Rα2 chain or FITC-conjugated IgG1 isotype-matched control or not stained. FITC-labeled cells were analyzed with a flow cytometer.

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Flow cytometry

A549 and HOS cells were infected with Ad-IL-13Rα2 at MOI 1–100 and, after 48 hr, harvested and incubated with FITC-conjugated mouse MAb against human IL-13Rα2 chain (1:100 dilution; Diaclone, Besançon, France) or FITC-conjugated mouse IgG1 isotype-matched control antibody (1:100 dilution; Dako Cytomation, Glostrup, Denmark) in the dark on ice for 2 hr. FITC-labeled cells were analyzed by a Coulter EPICS-XL flow cytometer (System II software; Beckman Coulter, Miami, FL).

Evaluation of sensitivity to IL-13 cytotoxin on cancer cell lines

Cancer cell lines (A549, HOS, MG-63 and PM-RCC) were treated with IL13-PE38QQR (final concentration 100 ng/ml) at 37°C in a humidified 5% CO2 incubator for 72 hr, washed and harvested. The cytotoxic effect of IL13-PE38QQR was evaluated by counting viable cells using trypan blue dye exclusion and exhibited as a value relative to that of nontreated control.

Analysis of the cytotoxic effect of IL-13 cytotoxin on adenoviral vector–infected cells

A549 and HOS cells were infected with either Ad-IL-13Rα2 or Ad-LacZ at different concentrations (MOI 0–100) followed by IL13-PE38QQR (100 ng/ml) treatment for 72 hr or infected with adenoviral vectors at MOI 10 followed by treatment with different concentrations of IL13-PE38QQR (0–500 ng/ml) for 72 hr. Cell viability was determined and exhibited as a value relative to that of nontreated control.

In vitro proliferation assay

A549 cells were infected with Ad-IL-13Rα2 or Ad-LacZ at MOI 10 and treated with IL13-PE38QQR (100 ng/ml every 3 days) for 14 days. The number of living cells was assessed on the indicated days (day 3, 7, 11 and 14).

Competitive effect of IL-13 on cytotoxicity of IL-13 cytotoxin

A549 cells were infected with Ad-IL-13Rα2 or Ad-LacZ at MOI 10 followed by IL13-PE38QQR (10 ng/ml) treatment and incubated in the presence or absence of recombinant human IL-13 or IL-4 (500 ng/ml) for 72 hr.

Human lung cancer xenograft model and treatment protocol

Four-week-old nude mice, BALB/cA jcl-nu/nu (Clea, Tokyo, Japan), were housed in sterilized filter-topped cages and maintained in a pathogen-free condition. Animal care was in accordance with the guidelines of the Nagasaki University Animal Research Advisory Committee. Tumorigenic human lung cancer A549 cells (5.0 × 106/body) were injected s.c. into the dorsal surface of mice, and that day was appointed as day 0 after transplantation of tumors. Palpable tumors developed within 5–7 days. Mice bearing A549 tumors received i.t. injection of 1.0 × 109 pfu/body of Ad-IL-13Rα2 or Ad-LacZ on day 5 and i.t. injection of IL13-PE38QQR (250 μg/kg/day) or PBS (excipient) on days 7, 8, 9, 10 and 11 using a 25 μl microsyringe (Hamilton, Reno, NV) with a 26-gauge needle. Tumor size was measured by caliper every 3 days. Approximate tumor size was calculated as the product of the 2 perpendicular diameters.

Statistical analysis

Data were presented as means ± SEM or SD. Differences between 2 groups were compared using 2-tailed Student's t-test. One-way ANOVA was used to examine differences between the multiple treatment groups in competition assay for the cytotoxic effect of IL-13 cytotoxin and in the tumor size measurement experiment. When ANOVA indicated a significant difference (p < 0.05), a post hoc multiple comparison test was performed using Dunnett's 2-tailed t-test or Tukey's HSD test. Differences were considered statistically significant at p < 0.05.

Results

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

IL-13Rα2 chain expression and sensitivity to IL-13 cytotoxin in various cancer cell lines

To confirm the correlation between expression of IL-13Rα2 chain and sensitivity to IL-13 cytotoxin (IL13-PE38QQR) treatment in cells, several human cancer cell lines (A549, HOS, MG-63 and PM-RCC) were examined for mRNA expression of IL-13Rα2 chain by RT-PCR analysis and for sensitivity to IL-13 cytotoxin. As shown in Figure 1a, all of these cancer cell lines expressed mRNA of both IL-4Rα and IL-13Rα1 at equivalent levels, whereas expression of IL-13Rα2 mRNA was observed only in MG-63 and PM-RCC cells but not in A549 and HOS cells. In MG-63 and PM-RCC cells, treatment with IL-13 cytotoxin (final concentration 100 ng/ml) actually showed 70–80% of the cytotoxicity (Fig. 1b). By contrast, in both IL-13Rα2 mRNA-negative cell lines, A549 and HOS, IL-13 cytotoxin treatment showed <5% of the cytotoxicity, regardless of mRNA expression of IL-13Rα1 and IL-4Rα (Fig. 1a). In addition, we found that the other human cancer cell lines, Caco-2 (colon cancer), PANC-1 (pancreatic cancer) and MDA-MB-231 (breast cancer), expressing mRNA of IL-4Rα and IL-13Rα1 but not IL-13Rα2, were similarly resistant to IL-13 cytotoxin (data not shown). These results suggest that the cytotoxicity of IL-13 cytotoxin to these cancer cell lines is dependent on the expression of IL-13Rα2 chain and independent of expression of IL-13Rα1 chain and IL-4Rα chain.

IL-13Rα2 chain expression in cancer cells provided by adenoviral vector–mediated gene transfer

For the purpose of IL-13Rα2 chain delivery to target cancer cells, we generated a recombinant replication-defective adenoviral vector, Ad-IL-13Rα2.

To confirm the expression of IL-13Rα2 mRNA by Ad-IL-13Rα2 infection, A549 and HOS cells, which showed no expression of IL-13Rα2 chain (Fig. 1a), were infected with Ad-IL-13Rα2 at different infection-doses (MOI 1, 10), and mRNA expression was detected by RT-PCR. As shown in Figure 2a, expression of IL-13Rα2 mRNA in both A549 and HOS cells was detected within 24 hr after Ad-IL-13Rα2 infection at MOI 10 and at 48 hr after MOI 1 infection. The high level of IL-13Rα2 mRNA expression was sustained for 7 days after infection at MOI 1 and for 14 days at MOI 10 in A549 cells (data not shown).

To confirm the expression of IL-13Rα2 protein on the surface of Ad-IL-13Rα2-infected cells, we performed flow cytometry using FITC-conjugated antibody against human IL-13Rα2 chain. As shown in Figure 2b, expression of IL-13Rα2 chain was detected at the surface of both A549 and HOS cells infected with Ad-IL-13Rα2 vector in a dose-dependent manner.

Both RT-PCR and flow-cytometric data showed that expression of IL-13Rα2 chain on A549 cells was 2- to 4-fold higher than that on HOS cells (Fig. 2a, ratio of cDNA band intensity and Table I). We also evaluated the efficiency of adenoviral vector–based gene transfer in these cell lines using Ad-LacZ, which expresses the β-galactosidase gene, and observed that A549 cells were more susceptible to adenoviral infection compared to HOS cells (data not shown). Thus, the difference in adenovirus-mediated gene expression between these cell lines appears to be consistent with the susceptibility to adenoviral infection.

Table I. Mean Value of Fluorescence Intensity1 on Cell Surface After Ad-IL-13Rα2 Vector Infection
Cell line/treatmentNontreatedIsotype controlAnti-IL-13Rα2
  • 1

    Mean value represents total amount of FITC-intensity/total count of cells of each sample.

A549
 Mock0.1790.1870.208
 Ad-IL-13Rα2 MOI 10.1690.1760.243
 Ad-IL-13Rα2 MOI 100.2160.2354.03
 Ad-IL-13Rα2 MOI 1000.2560.26713.6
HOS
 Mock0.1510.1560.193
 Ad-IL-13Rα2 MOI 10.1610.1690.235
 Ad-IL-13Rα2 MOI 100.1790.1820.704
 Ad-IL-13Rα2 MOI 1000.2210.2206.62

In vitro IL-13Rα2 gene transfer by Ad-IL-13Rα2 augments sensitivity of cancer cells to IL-13 cytotoxin

To investigate an effect of Ad-IL-13Rα2 vector on the sensitivity of the target cancer cells to IL-13 cytotoxin, cytotoxin-resistant cancer cells A549 and HOS (Fig. 1b) were infected with Ad-IL-13Rα2 at different infection-doses (MOI 0–100) followed by treatment with IL-13 cytotoxin (100 ng/ml) for 72 hr and the number of viable cells determined. As shown in Figure 3a,b, both A549 and HOS cells infected with Ad-IL-13Rα2 vector at different infection-doses were effectively killed by IL-13 cytotoxin in a dose-dependent manner, whereas control cells which were uninfected or infected with Ad-LacZ vector did not respond to IL-13 cytotoxin at all. The sensitivity of Ad-IL-13Rα2-infected A549 cells to IL-13 cytotoxin was relatively higher than that of Ad-IL-13Rα2-infected HOS cells, which correlated with the difference in IL-13Rα2 chain expression between these 2 types of cancer cell after Ad-IL-13Rα2 infection (Fig. 2a,b).

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Figure 3. In vitro effect of Ad-IL-13Rα2 infection on the sensitivity to IL-13 cytotoxin. A549 (a) and HOS (b) cells in 24-well plates (2.0 × 104 cells/well) were infected with Ad-IL-13Rα2 or Ad-LacZ vector (MOI 1–100) for 2 hr and incubated with or without IL13-PE38QQR (100 ng/ml) for another 72 hr. Viable cells (% of control) are exhibited as values relative to each nontreated control. Data present means ± SEM from triplicate determinations of 2 representative experiments. Open bars, without treatment; closed bars, treated with IL13-PE38QQR. *p < 0.05 and **p < 0.01. (c) A549 and HOS in 24-well plates (2.0 × 104 cells/well) were infected with Ad-IL-13Rα2 or Ad-LacZ at MOI 10 for 2 hr. Cells were incubated with various concentrations of IL13-PE38QQR (0–500 ng/ml) for another 72 hr. Cell viability (% of control) of each toxin-treated group is exhibited as a value relative to each nontreated control. Data present means ± SEM from triplicate determinations of 2 representative experiments. (d) A549 (5.0 × 103) cells were infected with Ad-IL-13Rα2 or Ad-LacZ at MOI 10 for 2 hr and incubated with or without IL13-PE38QQR (100 ng/ml) for 14 days. Cell number was calculated at the indicated days after infection (days 3, 7, 11 and 14). Data represent means ± SEM from triplicate determinations of 2 representative experiments.

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Furthermore, we examined the dose effect of IL-13 cytotoxin (10–500 ng/ml for 72 hr) on both A549 and HOS cells infected with Ad-IL-13Rα2 at MOI 10 (Fig. 3c). When Ad-IL-13Rα2-infected A549 cells were treated with IL-13 cytotoxin at doses above 50 ng/ml, >80% of them were killed. The CC50 (Table II) of IL-13 cytotoxin for Ad-IL-13Rα2-infected A549 cells was <10 ng/ml, which was much more highly sensitive than that of PM-RCC (CC50 approx. 60 ng/ml). The viability of HOS cells infected with Ad-IL-13Rα2 was also reduced by IL-13 cytotoxin treatment in a dose-dependent manner (Fig. 3c, CC50 almost 200 ng/ml). However, both A549 and HOS cells after no infection or infection with Ad-LacZ vector were not sensitive to IL-13 cytotoxin (CC50 >500 ng/ml).

Table II. Cytotoxic Activity of IL13-PE38QQR on Cancer Cells
Cell line/treatmentCC50 (ng/ml)
PM-RCC59.1 ± 8.4
A549
 Mock> 500
 Ad-LacZ MOI 10> 500
 Ad-IL-13Rα2 MOI 107.1 ± 3.2
HOS
 Mock> 500
 Ad-LacZ MOI 10> 500
 Ad-IL-13Rα2 MOI 10208.8 ± 15.6

We also examined the long-term effect of IL-13 cytotoxin treatment on the growth of target cells infected with Ad-IL-13Rα2. A549 cells were infected with Ad-IL-13Rα2 at MOI 10 and incubated in the presence of IL-13 cytotoxin (100 ng/ml) for 14 days. As shown in Figure 3d, A549 cells without adenoviral vector infection demonstrated vigorous tumor cell proliferation regardless of treatment with IL-13 cytotoxin. Ad-IL-13Rα2-infected cells hardly proliferated for the first 7 days despite the absence of treatment with IL-13 cytotoxin; but afterward, cells grew linearly in a similar fashion to untreated/uninfected cells. A similar tendency was also observed in Ad-LacZ-infected cells. The number of Ad-IL-13Rα2-infected cells evidently decreased along with IL-13 cytotoxin treatment, and almost all cells were killed within 3–7 days after initiation of the combination treatment.

IL-13, but not IL-4, competitively inhibits cytotoxicity of IL-13 cytotoxin in cancer cells infected with Ad-IL-13Rα2

IL-13 cytotoxin, which is a derivation of IL-13, can also bind to 2 types of receptor, physiologic IL-13Rα1/IL-4Rα and decoy IL-13Rα2. Previous studies have demonstrated that the attachment of radiolabeled IL-13 to the functional heterodimer receptor can be competed by IL-4 as well as IL-13,30, 31, 32, 49 whereas its attachment to IL-13Rα2 is competed by IL-13 but not IL-4.2, 9, 10, 15, 33, 36 To confirm that the cytotoxicity of IL-13 cytotoxin was specifically mediated through IL-13Rα2 chain delivered by Ad-IL-13Rα2 vector, A549 cells were infected with Ad-IL-13Rα2 at MOI 10 followed by treatment with IL-13 cytotoxin (10 ng/ml) in the presence of a 50-fold dose of human IL-13 or IL-4. As shown in Figure 4, IL-13 and IL-4 did not affect the growth of uninfected or Ad-LacZ-infected cells, regardless of treatment with IL-13 cytotoxin. In contrast, IL-13 cytotoxin was significantly cytotoxic to only Ad-IL-13Rα2-infected cells (p < 0.05); furthermore, the cytotoxic effect was completely abolished by a 50-fold amount of IL-13 (p < 0.01) but not the same amount of IL-4. These results strongly suggest that the cytotoxic effects of IL-13 cytotoxin are selectively mediated through IL-13Rα2 chain but not the functional receptor of the IL-13Rα1/IL-4Rα heterodimer complex.

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Figure 4. Excess dose of IL-13 inhibits competitively the cytotoxicity of IL-13 cytotoxin in IL-13Rα2 chain–transferred cancer cells. A549 in 24-well plates (2.0 × 104 cells/well) were infected with Ad-IL-13Rα2 or Ad-LacZ vector at MOI 10 for 2 hr and incubated with IL13-PE38QQR (10 ng/ml) with or without a 50-fold amount (500 ng/ml) of recombinant human IL-13 or IL-4 for another 72 hr. Viable cells (% of control) are exhibited as values relative to each nontreated control. Data present means ± SEM from triplicate determinations of 2 representative experiments. White bars, nontreated group; black bars, group treated with IL13-PE38QQR in the absence of recombinant IL-13; light gray bars, group treated with IL13-PE38QQR in the presence of recombinant IL-13; dark gray bars, group treated with IL13-PE38QQR in the presence of recombinant IL-4. Symbols represent statistical significance: *p < 0.05 (Student's t-test) compared to nontreated group; #p < 0.01 (one-way ANOVA followed by Dunnett's 2-tailed t-test) compared to group treated with IL13-PE38QQR alone.

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Administration i.t. of Ad-IL-13Rα2 vector followed by IL-13 cytotoxin leads to significant regression and sustained growth inhibition of A549 tumor xenografts in nude mice

To confirm the combination effect of Ad-IL-13Rα2 infection and IL-13 cytotoxin treatment in vivo, we established a mouse xenograft model of the cytotoxin-resistant human tumor. A549 cells were s.c.-implanted in nude mice (day 0), and then the established tumors received i.t. injection of Ad-IL-13Rα2 or Ad-LacZ on day 5. Subsequently, IL-13 cytotoxin (250 μg/kg/day) or PBS was administered to the tumors on a daily basis for 5 days from day 7 through day 11. From adenoviral vector injection (day 5) to initiation of IL-13 cytotoxin treatment (day 7), no significant differences in the size of tumors between all experimental groups were observed (p > 0.1, one-way ANOVA), regardless of different adenoviral vector injections. However, as shown in Figure 5a,b, tumors treated with i.t. administration of Ad-IL-13Rα2 followed by IL-13 cytotoxin significantly showed immediate regression and sustained stationary growth (p < 0.05 vs. control groups from day 11 to day 54 after implantation) for 43 days. At the end of a 2-month course of i.t. combined treatment with Ad-IL-13Rα2 and IL-13 cytotoxin (on day 54), tumor size was approximately 70% less than that of control groups (p < 0.01). However, tumor size after Ad-LacZ injection followed by IL-13 cytotoxin treatment showed no statistical difference compared to tumors injected with Ad-LacZ followed by excipient injection (p > 0.1). Among all treated mice, noticeable histologic changes or apparent side effects were not observed in nontargeted tissues such as liver and kidneys; moreover, macroscopically detectable toxic signs including loss of body weight were not noticed either (data not shown).

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Figure 5. Antitumor effect of i.t. administration of Ad-IL-13Rα2 followed by IL-13 cytotoxin in a human lung tumor xenograft model. Male athymic nude mice were s.c.-injected with A549 cells (5.0 × 106/body) on day 0. Tumor-bearing mice received i.t. injection of 1.0 × 109 pfu of Ad-IL-13Rα2 (open and closed circles) or Ad-LacZ (open and closed squares) on day 5, followed by i.t. administration of IL13-PE38QQR (250 μg/kg, closed circles and squares) or PBS (open circles and squares) for 5 days (from day 7 to day 11 after implantation). (a) Tumor size was calculated by multiplying the length and width of the tumor. Each data point represents mean ± SD of the tumor sizes of mice (n = 4) in 4 experimental groups. Arrowhead indicates i.t. injection of adenoviral vectors. Arrows indicate i.t. administration of IL13-PE38QQR or PBS. Statistical analysis was performed at each time point. *p < 0.05 and **p < 0.01 (one-way ANOVA followed by Tukey's HSD test) compared to control groups. (b) A549 tumors of each treated group were observed at day 36 after implantation. Upper panels show Ad-LacZ-infected tumors, and lower panels show Ad-IL-13Rα2-infected tumors. Left panels show tumors treated with PBS, and right panels show tumors treated with IL13-PE38QQR after i.t. injection of adenoviral vectors.

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Discussion

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

We first demonstrated that delivery of IL-13Rα2 chain using adenoviral vector to IL-13 cytotoxin-resistant cancer cells (Fig. 2) remarkably enhanced the sensitivity of these cells to IL-13 cytotoxin in vitro (Figs. 3, 4). The cytotoxic activity of IL-13 cytotoxin to cancer cells appears to specifically require expression of IL-13Rα2 chain because IL-13Rα2-negative cells hardly showed any response to IL-13 cytotoxin treatment despite expression of both IL-13Rα1 and IL-4Rα (Fig. 1), and excess dose of IL-13, but not IL-4, significantly abolished the cytotoxic effect of IL-13 cytotoxin on Ad-IL-13Rα2-infected cancer cells (Fig. 4). Furthermore, we demonstrated that in vivo i.t. administration of Ad-IL-13Rα2 vector markedly enhanced the antitumor activity of IL-13 cytotoxin in the cytotoxin-resistant human tumor xenograft model (Fig. 5). These results are consistent with our previous findings, in which plasmid-based gene transfer of IL-13Rα2 chain showed enhanced sensitivity of tumor cells to IL-13 cytotoxin treatment.13, 14, 44, 45, 46 In the present study, only one injection of Ad-IL-13Rα2 was sufficient for IL-13 cytotoxin treatment to cause immediate and significant regression of these established tumors and sustained growth inhibition for more than 1 month after the last administration of IL-13 cytotoxin. Both in vitro and in vivo experiments demonstrated that the antitumor activity of combined treatment with Ad-IL-13Rα2 and IL-13 cytotoxin on the target cancer cells or tumors was independent of the toxic effects derived from adenoviral vector itself (Figs. 3–5).

The i.t. administration of IL-13 cytotoxin, even at high daily doses (250 μg/kg for 5 days), did not show any nonspecific toxic effects on tumors infected with the control vector (Fig. 5). Our previous studies have already confirmed that i.p. or i.v. approaches of IL-13 cytotoxin can also kill tumors expressing IL-13Rα2 chain8, 11, 13, 50, 51 without any nonspecific toxic signs;50, 51 thus, this cytotoxin should not cause nonspecific side effects or collateral damage to nontargeted normal cells. Moreover, our previous study also showed that the half-life of IL-13 cytotoxin in the blood is relatively short.11 If any serious side effect was caused by IL-13 cytotoxin, there is a possibility that it would be readily controlled by adjustment of the toxin dose even after gene transfer of IL-13Rα2 chain. Except for a certain kind of cancer cells,43 IL-13Rα2 chain by itself is not cytotoxic to targeted cancer cells, even though it is overexpressed on cells after gene transfer; this was confirmed in our data (Figs. 2–5). These findings support the contention that the high-level expression of IL-13Rα2 chain using our adenoviral vector on the surface of targeted cancer cells is not only a safe procedure but also a promising strategy for cancer targeting therapy using IL-13 cytotoxin, which itself is safe and specific.

Our previous study showed that in vivo overexpression of IL-13Rα2 chain inhibited tumorigenicity of pancreatic and breast cancer cell lines in nude mice.42 The mechanism of such antitumorigenic activity of IL-13Rα2 chain was attributed to infiltration of inflammatory cells, including neutrophils, and production of cytokines.42 In the end of our in vivo experiment, histologic analysis also showed that prominent infiltration of inflammatory cells was indeed induced in only Ad-IL-13Rα2-infected tumor tissues, whereas tumor tissues obtained from mice injected with Ad-IL-13Rα2 followed by IL-13 cytotoxin treatment were free from inflammatory cells (data not shown). Such infiltration of inflammatory cells was specifically observed in tumors administered Ad-IL-13Rα2 vector but not Ad-LacZ control vector (data not shown); thus, it appeared to be relatively a long-term and IL-13Rα2 chain–specific phenomenon, not a distinctive inflammatory response caused by adenoviral vector itself, because it had been observed nearly 2 months after administration of adenoviral vectors. In addition, a soluble form of IL-13Rα2 can prevent IL-13-mediated suppression of tumor immunosurveillance.26, 27 Therefore, i.t. introduction of IL-13Rα2 chain into established tumors may efficiently lead to activation of an antitumorigenic response and tumor immunosurveillance in tumor tissues.

To realize clinical applications of our combination approach based on in vivo injection of Ad-IL-13Rα2 vector, we need to achieve not only high efficiency but also more specific gene transfer and expression to target tumor cells because adenovirus-based vectors can infect a broad variety of mammalian cell types, not only cancer cells but also normal cells. Previously, it was reported that use of promoter and expression regulatory elements of tumor- or tissue-specific molecules, such as α-fetoprotein, carcinoembryonic antigen, human telomerase reverse transcriptase and prostate-specific antigen, successfully regulated the expression of transgenes in a tumor- or tissue-specific fashion after systemic gene transfer using adenoviral vector.52, 53, 54, 55 Therefore, these targeting techniques of adenovirus-based vectors will further improve our combination strategy.

In conclusion, our studies prove that adenoviral vector–mediated gene delivery of IL-13Rα2 chain combined with IL-13 cytotoxin treatment effectively and selectively kill targeted cancer cells in vitro and in vivo and suggest that our combined strategy offers a potent targeting approach for cancer treatment.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

We thank Dr. B.H. Joshi (Laboratory of Molecular Tumor Biology, Center for Biologics Evaluation and Research, FDA) for providing purified recombinant IL13-PE38QQR; Dr. Y. Hishikawa and Ms. M. Kawakatsu (Laboratory of Histology and Cell Biology, Nagasaki University) for histologic analysis; and Dr. Y. Niu, Ms. Y. Ma, Ms. N. Okamoto, Ms. R. Takashima, Ms. I. Fujii and Mr. H.F. Rafidinarivo (Laboratory of Molecular Biology of Infectious Agents, Nagasaki University) for technical assistance. We also thank Dr. M. Kawakami (Laboratory of Molecular Tumor Biology, Center for Biologics Evaluation and Research, FDA) for critical reading of this manuscript. This work was supported in part by a grant-in-aid from the Ministry of Education, Science, Technology, Sports and Culture of Japan (to T.M.).

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  6. Acknowledgements
  7. References
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