In vivo electroporation-mediated transfer of interleukin- 12 ... - Nature

Gene Therapy (2001) 8, 1234–1240  2001 Nature Publishing Group All rights reserved 0969-7128/01 $15.00 www.nature.com/gt

RESEARCH ARTICLE

In vivo electroporation-mediated transfer of interleukin12 and interleukin-18 genes induces significant antitumor effects against melanoma in mice T Kishida1, H Asada1, E Satoh1, S Tanaka2, M Shinya1, H Hirai1, M Iwai2, H Tahara3, J Imanishi1 and O Mazda1 1 3

Department of Microbiology and 2Third Department of Internal Medicine, Kyoto Prefectural University of Medicine, Kyoto; and Department of Surgery, Institute of Medical Science, University of Tokyo, Tokyo, Japan

Direct intratumoral transfection of cytokine genes was performed by means of the in vivo electroporation as a novel therapeutic strategy for cancer. Plasmid vectors carrying the firefly luciferase, interleukin (IL)-12 and IL-18 genes were injected into established subcutaneous B16-derived melanomas followed by electric pulsation. When plasmid vectors with Epstein–Barr virus (EBV) nuclear antigen 1 (EBNA1) gene were employed, the expression levels of the transgenes were significantly higher in comparison with those obtained with conventional plasmid vectors. In consequence of the transfection with IL-12 and IL-18 genes, serum concentrations of the cytokines were significantly elevated, while interferon (IFN)-␥ also increased in the sera of the animals.

The IL-12 gene transfection resulted in significant suppression of tumor growth, while the therapeutic effect was further improved by co-transfection with IL-12 and IL-18 genes. Repetitive co-transfection with IL-12 and IL-18 genes resulted in significant prolongation of survival of the animals. Natural killer (NK) and cytotoxic T lymphocyte (CTL) activities were markedly enhanced in the mice transfected with the cytokine genes. The present data suggest that the cytokine gene transfer can be successfully achieved by in vivo electroporation, leading to both specific and nonspecific antitumoral immune responses and significant therapeutic outcome. Gene Therapy (2001) 8, 1234–1240.

Keywords: electroporation; gene therapy; cytokine; Epstein–Barr virus-based vector; episomal vector; melanoma

Introduction Cytokine gene transfer may provide a powerful strategy for gene therapy of cancer. The effectiveness has been demonstrated in some animal model systems.1,2 A safe and efficient method is necessary for cytokine gene delivery into tumors. To reduce possible adverse effects of the cytokines, it is also required that the delivery process can be accurately controlled, gene expression being exclusively localized in tumors. Concerning these issues, a physical means, such as electroporation, may be very suitable for cytokine gene transfer. Electroporation has been widely used for transferring exogenous genes into various cells in vitro. Recently, some reports demonstrated that the electroporation is also useful for in vivo gene delivery.3 Marker genes were successfully transferred into normal tissues of animals including skeletal muscle,4,5 liver,6 skin7 and cornea.8 Tumors implanted in animals were also revealed to be good targets for in vivo electroporation; melanoma,9 hepatocellular carcinoma,10 and glioma11 were effectively transfected with marker genes. Moreover, in an earlier report, therapeutic experiments were performed

Correspondence: O Mazda, Department of Microbiology, Kyoto Prefectural University of Medicine, Kamikyo, Kyoto 602–8566, Japan Received 19 January 2001; accepted 11 May 2001

resulting in remarkable therapeutic outcome. The herpes simplex virus-type I thymidine kinase gene was transferred into tumors derived from the CT26 colon carcinoma cell line. Following systemic administration with ganciclovir, the growth of tumors was significantly inhibited, suggesting the usefulness of electroporation for in vivo gene therapy.12 As far as we know, however, there have been few reports in which a cytokine gene was delivered into tumors by means of the electroporation. Interleukin (IL)-12 plays critical roles in the induction of the cell-mediated immune responses.13 It augments proliferation and killing activity of cytotoxic T lymphocytes (CTL) as well as natural killer (NK) cells.14,15 IL-12 also facilitates T helper type 1 (Th1) response and induces interferon (IFN)-␥ production. On the other hand, IL-18 is originally identified as the IFN-␥ inducing factor.16 IL18 also augments cytotoxic activities of CTL and NK cells, and synergizes with IL-12 in the IFN-␥ production.17 Therefore, transduction of IL-12 and IL-18 genes may enhance both specific and nonspecific antitumor immune responses. Actually, IL-12 gene transfer has been demonstrated in some animal model systems.1 Osaki et al18 reported a synergistic antitumor effect of IL-18 gene transduction and recombinant IL-12 administration. However, most of these earlier studies employed retrovirus,18–21 or adenovirus vectors,22,23 while cationic liposome was also used in some studies.24 In the present study, IL-12 and IL-18 genes were trans-

Cytokine gene therapy by electroporation in vivo T Kishida et al

ferred into B16 solid tumors via electroporation, as a novel approach to gene therapy of cancer.

Results B16 tumors can be efficiently transfected in vivo by means of electroporation We examined whether B16 solid tumors can be effectively transfected in vivo by electroporation. Since authors have previously reported that markedly high transfection efficiency can be obtained in vivo by using the EBV-based plasmid vectors,25–27 pGEG.luc and pG.luc were constructed and compared; the former is an EBV-based plasmid vector, while the latter is a conventional plasmid vector (Figure 1). These plasmids were injected into B16 tumors established in syngenic mice followed by electric pulsation (day 0). The same procedure was repeated on day 2. Figure 2a shows luciferase activities in the tumors assessed on day 4, demonstrating that the transfection with pGEG.luc elicited an approximately 20-fold higher expression of marker gene than that with pG.luc (P ⬍ 0.004). Luciferase activity was not demonstrated in the control tumors that had been given the pGEG.luc injection but not a pulsation, indicating that the naked plasmid DNA per se is not effective in transducing a gene into the tumors. Histological analysis of tumors transfected with pGEG.EGFP revealed that 15–20% of tumor cells strongly expressed the marker gene 3 days after the single transfection (Figure 2b). The EGFP-positive cells existed less than 3 mm from the central electrode. Significant tumor growth inhibition by electroporationmediated transfection with IL-12 and IL-18 genes The in vivo electroporation system was applied to the cytokine gene transfer into the tumors. The IL-12 and IL18 genes were inserted into the EBV-based plasmid vectors and the resultant pGEG.mIL-12 and pGEG.mIL-18 were transfected into the B16 tumors as above (Figure 1). The pG.mIL-12 and pG.mIL-18 were also constructed and subjected to the in vivo transfection experiments. The ELISA analysis demonstrated elevation in cytokine levels in sera of the animals (Figure 3a and b). On day 4, mice

transfected with pGEG plasmids exhibited significantly higher levels of the cytokines compared with those given pG plasmids (IL-12, P ⬍ 0.01; IL-18, P ⬍ 0.01). Moreover, the EBV-based plasmids elicited more persistent expression than the conventional plasmids, as revealed by the serum cytokine levels on day 6 (IL-12, P ⬍ 0.01; IL-18, P ⬍ 0.05). IFN-␥ was also increased in the sera of the transfected mice, suggesting the biological activity in vivo of the generated IL-12 and IL-18 (Figure 3c). Similarly, a significantly larger amount of IFN-␥ was produced in case the EBV-based plasmids were used (day 4, P ⬍ 0.0001; day 6, P ⬍ 0.05). To estimate antitumor effects of the cytokine gene transfer, tumors were transfected with pGEG.mIL-12 alone or a combination of pGEG.mIL-12 and pGEG.mIL18. As a control, some tumors received pGEG.luc. Following two transfections at an interval of 2 days, tumor size was measured (Figure 4a). Tumor growth was significantly suppressed by the combination of pGEG.mIL-12 and pGEG.mIL-18, and to a lesser extent, by pGEG.mIL12 alone (pGEG.mIL-12 and pGEG.mIL-18 versus control, P ⬍ 0.0001; pGEG.mIL-12 alone versus control, P ⬍ 0.01; pGEG.mIL-12 and pGEG.mIL-18 versus pGEG.mIL-12 alone, P ⬍ 0.02). However, the genetic transfections failed to significantly prolong the longevity of the mice, as demonstrated by Kaplan–Meier analysis (Figure 4b). To obtain a more significant therapeutic effect, a more intensive protocol was conducted. Two times transfection was performed on days 0 and 2, and the treatments were repeated again on days 10 and 12. Figure 5a shows that the genetic therapy drastically suppressed the growth of tumors (P ⬍ 0.0001 compared with the control group). The Kaplan–Meier analysis demonstrates that multiple transfection with IL-12 and IL-18 genes also significantly prolonged the survival of mice (P ⬍ 0.01 compared with the control group), suggesting that the repetitive treatment is safe and effective (Figure 5b). Continuous treatments with cytokine genes did not lead to cure of the tumor. In contrast, any therapeutic effect was not observed in mice given injection with the plasmids, but not pulsation. In the above tumor-bearing animal models, a relatively

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Figure 1 Plasmids used in this study. Prom, promoter; polyA, SV40 polyA additional signal. Gene Therapy


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Figure 2 Reporter gene expression in tumors transfected by electroporation. (a) Established B16 tumors were injected with 5.5 pmol of either pGEG.luc ( ) or pG.luc ( ) followed by electric pulsation. As a control, some tumors were given the pGEG.luc injection but not a pulsation ( ). The treatments were performed on days 0 and 2. On day 4, tumors were excised and luciferase activity was measured. The luciferase activities are standardized with the amount of protein. Bars, s.d. of quadruplicate samples. (b) Tumors were transfected with 5.5 pmol of the indicated plasmids as in (a). Three days later, the tumors were frozen and cryosectioned into 10 ␮m serial slices. GFP expression was visualized with a fluorescence microscope. Bar corresponds to 200 ␮m.

large number of B16 cells were implanted, so that the tumors may not have been fully vascularized when transfected. Then we performed another experiment, in which a smaller number of B16 cells (105) were inoculated and the resultant tumors were transfected 7 and 12 days after implant. The pGEG.mIL-12 transfer drastically inhibited the growth of the tumors under this experimental condition (pGEG.mIL-12 versus pGEG.luc, P ⬍ 0.0001) (Figure 6). NK and CTL activities were enhanced by IL-12 and IL18 gene transfection To elucidate the mechanisms that mediate the antitumor effect, we assessed the NK and CTL activities in the Gene Therapy

Figure 3 Cytokine production in vivo resulted from the genetic treatment. B16 tumors received injection with 5.5 pmol of pGEG.luc ( ), a mixture of 5.5 pmol each of pG.mIL-12 and pG.mIL-18 ( ), or a mixture of 5.5 pmol each of pGEG.mIL-12 and pGEG.mIL-18 ( ), followed by electric pulsation. The treatments were performed on days 0 and 2. On days 4 and 6, mice were killed and sera were collected. As a control, sera were also obtained from normal C57/BL6 mice ( ). IL-12 (a), IL-18 (b), and IFN-␥ (c) concentrations in the sera were measured by ELISA. Bars, s.d. of triplicate samples.

tumor-bearing animals that had been transfected with pGEG.mIL-12 and pGEG.mIL-18. As shown in Figure 7a, strong cytotoxic activity against B16 cells was demonstrated in the tumor-bearing mice transfected with IL-12 and IL-18 genes, while pGEG.luc transfection did not significantly elevate the tumoricidal activity in this experimental condition. The NK activity was also remarkably augmented by the cytokine gene transfection, whereas pGEG.luc transfection failed to elevate the NK activity (Figure 7b).


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Figure 4 In vivo cytokine gene therapy for established B16 solid tumors. Tumors were injected with either 5.5 pmol of pGEG.mIL-12 ( ) (n = 8), a mixture of 5.5 pmol each of pGEG.mIL-12 and pGEG.mIL-18 ( ) (n = 8), or 5.5 pmol of pGEG.luc (왖) (n = 8), followed by electric pulsation. The treatments were performed twice on days 0 and 2 (arrowheads). The sizes of the tumors (a) and percent survival of the mice (b) are shown. Bars, s.d. *Tumor volume of the pGEG.luc-treated group is not plotted for days 14 and 16, because the survival was less than 70% on these days.

Figure 5 Repetitive cytokine gene transfection resulted in more significant therapeutic effects against B16 tumors. Tumors were injected with a mixture of 5.5 pmol each of pGEG.mIL-12 and pGEG.mIL-18 ( ) (n = 10), or 5.5 pmol of pGEG.luc ( ) (n = 10), followed by electric pulsation. As a control, other tumors were given 5.5 pmol each of pGEG.mIL12 and pGEG.mIL-18 but not pulsation (왖) (n = 10). The treatments were performed on days 0, 2, 10, and 12 (arrowheads). The sizes of tumors (a) and percent survival of mice (b) are shown. Bars, s.d.

Discussion The present study demonstrates that in vivo electroporation is quite useful in cytokine gene transfer into tumors, suggesting a novel therapeutic strategy of gene therapy of cancer. The electroporation-mediated transfer of IL-12 and IL-18 genes markedly enhanced CTL and NK activities, resulting in the significant therapeutic outcome in terms of tumor growth suppression and prolongation of survival of the animals. The present report is also the first one in which the EBV-based plasmid vector was delivered by electroporation in vivo. Compared with conventional plasmid vectors, the EBV-based plasmid vectors enabled higher expression of the luciferase, as well as cytokine genes (Figures 2a and 3). This is consistent with our previous reports showing that the EBV-based plasmid vectors are quite useful in improving transfection efficiency by a variety of nonviral delivery systems, including cationic liposomes,28,29 cationic polymers,25,26,29,30 and particle bombardment.31 The high level expression obtained by the EBV-based plasmid vectors may be ascribed to multiple functions of EBNA1, such as nuclear transfer of the plasmid,32,33 binding of plasmid to nuclear matrix,34 and transcriptional up-regulation.35–38 IL-12 plays key roles in the regulation of cellular

Figure 6 Growth suppression of tumors derived from a relatively small number of B16 cells. One hundred thousand B16 cells were inoculated into the flanks of mice. Seven days later, palpable tumors were developed, and 5.5 pmol of pGEG.mIL-12 ( ) or pGEG.luc ( ) were transduced into the tumors as in Figure 2 (day 0). The treatment was repeated on day 5. Bars, s.d.

immunity. IL-12 has been shown to be quite effective in inducing antitumoral immune responses and increasing both CTL and NK activities. IL-12 and IL-18 do not synergistically elevate cytotoxic activities of CTL and NK cells in vivo.39 The B16 cells transfected with both pGEG.mILGene Therapy


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tered to a patient, the serum concentration transiently reaches a very high level that causes the systemic adverse effects. In contrast, gene transfer may provide a relatively low level of IL-12 at the target tissue, and the level may be kept relatively constant. This could generate an antitumor effect without causing severe systemic toxicity. In vivo electroporation has been applied to chemotherapy of some malignancies. Clinical trials are being conducted against malignant melanoma, Wilms tumor and soft tissue sarcoma, so that an antitumor reagent, bleomycin, is delivered into the tumor cells. Much evidence has accumulated to demonstrate the safety of the in vivo electroporation in clinical use.46–49 Concerning the immunogenicity of the EBNA1, it is widely known that the viral protein interferes with the antigen processing and escapes from immune recognition.50,51 In the present system, repetitive treatments were effective without causing any severe adverse effect. This is consistent with our previous study in which the EBV-based plasmid vector was conjugated with a cationic polymer and repeatedly injected into some xenograft tumor models.26 Thanks to the efficacy, simplicity, and potential costeffectiveness of the system, the electroporation-mediated cytokine gene transfer may be applicable to clinical usage. Further preclinical development is now under way for the evaluation of the efficacy and safety of the system.

Materials and methods Figure 7 IL-12 and IL-18 gene transfer enhances both NK and CTL activities. Transfection with a combination of pGEG.mIL-12 and pGEG.mIL-18 ( ) (n = 3), or pGEG.luc alone ( ) (n = 3), was performed as described in the legend to Figure 4. On day 6, splenic cells were obtained from the mice, and cytotoxic activities against B16 (a) and YAC1 (b) cells were assessed as described in the Materials and methods. As another control, splenic cells of normal mice were also tested (왖) (n = 3). Bars, s.d.

12 and pGEG.mIL-18 elevated CTL and NK activities in vivo to the same extent as the pGEG.mIL12-transfected B16 did (Asada et al., unpublished observations). On the other hand, it has been demonstrated that production of IFN-␥ is essential for the antitumor effect of IL-12, while IL-18 synergizes with IL-12 in IFN-␥ production.40 In our experiments, intratumoral delivery of IL-12 and IL-18 genes induced IFN-␥ production in vivo, as indicated by a high serum level in the transfected animals (Figure 3). The therapeutic effect obtained by co-transfection with IL-12 and IL-18 genes was significantly stronger than that with IL-12 gene alone (Figure 4), suggesting the cooperation of these cytokines in our system. Systemic administration of recombinant IL-12 causes dose-dependent adverse effects in mice41 and human.42–44 IFN-␥ production brings about an acute systemic inflammatory response with marked injury to some particular organs, ie the liver, lung and intestine.45 We have intensively examined these organs from tumor-bearing mice that had been transfected with pGEG.mIL-12 and pGEG.mIL-18. No sign of inflammation or cell damage was observed in all the organs surveyed (data not shown). The discrepancy between recombinant protein therapy and gene therapy may be ascribed to the different distribution and kinetics of IFN-␥ levels provided by these treatments. In case recombinant IL-12 is adminisGene Therapy

Plasmid vectors The pGEG.luc is composed of (1) the firefly luciferase gene (derived from pGV-P; Wako Pure Chemicals, Osaka, Japan), located between CAG promoter52 and SV40 polyA additional signal; (2) EBV oriP (derived from P220.2);53 (3) EBV EBNA1 gene (derived from p220.2) under the control of another CAG promoter; (4) the ampicillin resistant gene; and (5) the replication origin for E. coli (Figure 1). pGEG.mIL-12 contains a bicistronic expression cassette with murine IL-12 p35 and p40 genes,18 while pGEG.mIL-18 contains murine IL-18 cDNA fused with the leader sequence of the human parathyroid hormone gene.18 pGEG.EGFP carries the enhanced green fluorescent protein (EGFP) gene (SalI– NotI 0.7 kb fragment derived from pEGFP.N3 (Clontech, Palo Alto, CA, USA)). pG.luc, pG.mIL-12 and pG.mIL18 were constructed from pGEG.luc, pGEG.mIL-12 and pGEG.mIL-18, respectively, by deleting both CAGEBNA1 and oriP. Plasmids were purified using Qiagen Maxiprep Endo-free kits (Qiagen, Hilden, Germany). Cells A mouse melanoma cell line, B16, and YAC-1 cells were maintained in RPMI 1640 medium (Nacalai Tesque, Kyoto, Japan) supplemented with 100 U/ml penicillin, 100 ␮g/ml streptomycin and 10% fetal bovine serum. Mice Female C57/BL6 mice were purchased from Shimizu Laboratory Suppliers Co. Ltd (Kyoto, Japan). They received humane care in compliance with the Guide to the Care and Use of Laboratory Animals. In vivo electroporation B16 cells were subcutaneously injected into the flanks of mice (females, 5–6 weeks of age). Unless otherwise


stated, 106 cells were injected, and 4 days later, tumors of an average volume of 75 mm3 developed. Plasmid DNA (5.5 pmol) was diluted in 50 ␮l of K-PBS (30 mm NaCl, 120 mm KCl, 3 mm Na2HPO4, 1.5 mm KH2PO4, 5 mm MgCl2) and injected percutaneously into the tumors by using a syringe with a 27-gauge needle. Tumors were pulsed with an electroporater, CUY21 (Tokiwa Science, Tokyo, Japan) equipped with a 0.5 cm diameter array of seven needle electrodes. In the needle array electrodes, a single center needle is encircled by six needles. Electric current is passed from the center needle to the surrounding needles, or in the opposite direction. Six square-wave pulses were delivered at a frequency of 1 s−1, with a pulse length of 100 ms and a voltage of 50 V. Three pulses were followed by other three pulses of the opposite polarity. The diameters of tumors were measured with a digital caliper and tumor volume was calculated by the formula: v = a × b2/2 mm3, where a = long diameter and b = short diameter. Luciferase assay Tumors were homogenized in 200 ␮l reporter lysis buffer (Promega, Madison, WI, USA) using a sonicater. After freezing and thawing twice, the extract was centrifuged at 14 000 g for 5 min. Luciferase activity in the supernatant was measured using a Luciferase assay kit (Promega) according to the manufacturer’s protocol. Photoemission was measured during a 10 s period using a luminometer. Protein concentration in the supernatant was measured as described.29 Histological analysis Tumors were embedded in the OCT compound and immediately frozen at −80°C. Serial 10 ␮m sections were made, and GFP expression was visualized with a fluorescence microscope with excitation at 488 nm. ELISA Sera were obtained from the carotid artery of mice. Cytokine concentration in the sera was assayed using a mouse IL-12 ELISA kit (BioSource International, Camarillo, CA, USA), a mouse IFN-␥ ELISA kit (BioSouce International) or a mouse IL-18 ELISA kit (MBL, Nagoya, Japan), according to the supplier’s instructions. Cytotoxicity assays B16 and YAC-1 cells were used as the target cells for CTL and NK cytotoxicity, respectively. The standard 51Crrelease assay was performed as described elsewhere.54 Briefly, target cells were incubated with 370 kBq Na251 CrO4 for 60 min at 37°C. After washing, the target cells were seeded in a microtiter plate at a density of 1 × 104/well. As the effector cells, splenic cells were obtained from the tumor-bearing animals on day 6 of the transfection and seeded into the microtiter plate at various effector to target ratios. After 4 h of incubation at 37°C in 5% CO2/95% humidified air, supernatants were collected and radioactivity was measured on a gamma counter. Specific 51Cr-release was calculated with the standard formula.54 Statistical evaluation For the luciferase assay, cytotoxicity assays and comparison of tumor volumes, Student’s t test was used. For the

Kaplan–Meier analyses, survival differences between groups were evaluated using Logrank test.

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Acknowledgements We would like to thank Dr Jun-ichi Miyazaki (Department of Nutrition and Physiological Chemistry, Osaka University Medical School) for kindly providing us with the CAG promoter, and Drs Kazuko Uno and Atsuko Kishi (Louis Pasteur Center for Medical Research, Kyoto) for helpful discussion. We also thank Ms Kyoko Kimura for her excellent secretarial assistance. This research was supported by a grant-in-aid for scientific research from the Ministry of Education, Science, Sports and Culture, Japan.

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