DNA immunization using constant-current electroporation ... - Nature

Cancer Gene Therapy (2008) 15, 108–114 r

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ORIGINAL ARTICLE

DNA immunization using constant-current electroporation affords long-term protection from autochthonous mammary carcinomas in cancer-prone transgenic mice C Curcio1, AS Khan2, A Amici3, M Spadaro1, E Quaglino1, F Cavallo1, G Forni1 and R Draghia-Akli2 1

Molecular Biotechnology Center, Department of Clinical and Biological Sciences, University of Turin, Turin, Italy; 2VGX Pharmaceuticals, Immune Therapeutics Division, The Woodlands, TX, USA and 3Department of Molecular, Cellular and Animal Biology, University of Camerino, Camerino, Italy A recently developed, adaptive constant-current electroporation technique was used to immunize mice with an intramuscular injection of plasmid coding for the extracellular and transmembrane domains of the product of the rat neu664V-E oncogene protein. In wild-type BALB/c mice, plasmid electroporation at lower current settings elicits higher antibody titers, a strong cytotoxic response and completely protects all mice vaccinated with 10, 25 and 50 mg of plasmid against a lethal challenge of rat neu þ carcinoma cells. BALB/c mice transgenic for the transforming rat neu664VE (ErbB-2, Her-2/neu) oncogene (BALB-neuT664VE) develop an invasive mammary gland carcinoma by 20 weeks of age. Remarkably, when transgenic BALB-neuT664VE mice were vaccinated at a 10- week interval with 50 mg of plasmid with 0.2 A electroporation, mice remained tumor free for more than a year. A single administration of plasmid associated with electroporation was enough to markedly delay carcinogenesis progression in mice with multiple microscopic invasive carcinomas, and keep about 50% of mice tumor free at one year of age. Thus, vaccination using a clinically relevant dose of plasmid encoding the extracellular and transmembrane domains of the neu oncogene delivered by electroporation prevents long-term tumor formation. These improvements in the efficacy of this cancer vaccine regimen vastly increase its chances for clinical success. Cancer Gene Therapy (2008) 15, 108–114; doi:10.1038/sj.cgt.7701106; published online 9 November 2007 Keywords: cancer vaccine; plasmid; electroporation; Her-2/neu

Introduction

Viable DNA vaccine candidates for human cancer have to overcome the same major hurdle as other plasmid-based biologics: how to increase efficacy without vastly increasing the dose. Recent technological advancements in increasing DNA vaccine uptake and expression using a constant-current electroporation (CCE) technique, could help to overcome this limitation.1–3 Many recent studies using in vivo electroporation to enhance the uptake and expression of DNA vaccines have been performed with high doses of plasmid and voltagebased methods. This technique involves delivering a predetermined voltage between the electrodes, without taking into account the resistance of the tissue to be treated. In a previous study,4 we have measured the tissue Correspondence: Dr R Draghia-Akli, VGX Pharmaceuticals, Immune Therapeutics Division, 2700 Research Forest Drive, Suite 180, The Woodlands, TX 77381, USA. E-mail: [email protected] Received 9 April 2007; revised 13 August 2007; accepted 18 August 2007; published online 9 November 2007

resistance of muscles in different species, and determined that it can vary from subject to subject, from muscle to muscle within the same animal and during the electroporation due to cellular uptake of the formulation volume. The CELLECTRAt device is able to measure the tissue resistance before, between and during the electroporation pulses and adjust the voltage to account for these individual changes. This feature allows the device to maintain a true constant-current and a square wave through the muscle tissue during electroporation, preventing heating of a tissue5 and consequently reducing tissue damage and pain, as well as contributing to the overall increase in plasmid uptake and expression.4,6 There have been numerous reviews of the advantages and disadvantages of various models of cancer vaccine, therapeutic versus prophylactic vaccines and transplantable versus autochthonous tumors7 as well as the current tools such as adjuvants that have been used to enhance the immune responses generated by the vaccines.8 Furthermore, there has been a recent surge in interest in using electroporation to enhance the delivery of plasmids or drugs with anti-tumor effects, as reviewed by Gothelf et al.9 and Heller et al.10

DNA electroporation prevents mammary carcinomas C Curcio et al

Female BALB/c mice transgenic for the transforming rat neu664VE oncogenes (BALB-neuT664VE mice) display p185neu, the protein product of rat neu oncogene, on the surface of the cells of their rudimentary mammary glands at 3 weeks of age. As a natural consequence of p185neu overexpression and signal transduction, foci of atypical hyperplasia appear first in the terminal buds and then as lateral buds sprouting from ducts and ductules in all the 10 glands. The lateral buds give rise to foci of carcinoma in situ around the 15th week, progressing to invasive lobular carcinomas by the 20th week. Carcinomas are present in all glands 10 weeks later and are palpable by the 33rd week.11 Mammary carcinomas in BALB-neuT664VE mice exhibit either a partial or complete downregulation of MHC class I expression, perhaps as a method to escape immune recognition.12 Induction of an immune response able to protect against neu-driven carcinogenesis is thus a daunting challenge.13 We have previously demonstrated prevention of tumor growth using a plasmid coding for the extracellular and transmembrane domains of the p185neu (EC-TM plasmid).14 In the present paper, we are presenting optimization studies to deliver cancer vaccines using the CCE method. We were able to induce long-term protection in BALB-neuT664VE mice using a substantially reduced dose compared to classical voltage-based electroporation of plasmid vaccines using this technique, which may be important for further clinical developments of this technology, including possible clinical trials. A single vaccination using this regimen was able to prevent the formation of multifocal, pre-neoplastic lesions in all mammary glands, and vaccinations in 10 week intervals were able to keep all BALB-neuT664VE mice free from tumors to more than one year of age.

Materials and methods

Mice Female BALB/c mice (6-weeks old) were purchased at Charles River Laboratories (Calco, Italy). BALBneuT664VE mice were bred under specific pathogen-free conditions by Charles River (Calco, Italy). BALBneuT664VE mice knocked out for Fc-gRI/III (BALBneuT664VE/FcgRI/III) were generated by crossing BALB-neuT664VE mice with BALB/c mice KO for the FcgRI/III receptor, kindly provided by Dr R Clynes (Columbia University, NY). Mammary glands were inspected at weekly intervals to note tumor appearance. Tumor masses were measured with calipers in two perpendicular diameters. Progressively growing masses of more that 1 mm mean diameter were regarded as tumors. Percentage of mice without tumor (tumor-free mice) was evaluated. Mice were treated according to the European Union guidelines. Plasmid and electroporation The pcDNA3 vector coding the extracellular and transmembrane domains of p185neu (EC-TM plasmid) was produced and used as previously described.15 Briefly,

DNA was precipitated, suspended in sterile saline at 1 mg ml1, and stored in aliquots at 20 1C for use in immunization protocols. For DNA electroporation, ECTM or pcDNA3 plasmid in 25 ml sterile water with 0.1% poly-L-glutamate were injected into the gastrocnemius muscle of anesthetized mice.4 Animals were injected with 5–50 mg of plasmid at each time specified. Two constant-current square-wave pulses of 20 ms in duration of 1 s between pulses were applied after 4 s using CELLECTRAt device and micro-electrodes (VGX Pharmaceuticals, Immune Therapeutics Division, The Woodlands, TX). Micro-electrodes are in an isosceles triangle shape with the long sides 5 mm and the short side 3 mm apart. A constant-current setting of 0.2 A was used for all cancer vaccine experiments, except for the electroporation conditions experiment in which 0.2, 0.3 and 0.4 A were used. The muscle resistance was measured to be between 800 and 1000 O, with the calculated voltage between 160 and 195 V.

Assessment of anti-p185neu antibody Sera from BALB/c mice were collected 2, 4 or 6 weeks after vaccination and diluted 1:50 in PBS-azide-bovine serum albumin (PBS-azide-BSA) (Sigma-Aldrich, St Louis, MO). The presence of anti-p185neu antibodies was determined by flow cytometry using BALB/c NIH3T3 fibroblasts, wild-type or stably co-transfected with the wild-type p185neu mouse class I H-2Kd and B7.1 genes (BALB/c NIH3T3-NKB). FITC-conjugated goat anti-mouse antibodies specific for mouse IgG Fc (DakoCytomation, Italy), were used to detect bound primary antibodies. Normal mouse serum was the negative control. The monoclonal Ab4 antibodies (Oncogene Research Products, Cambridge, MA, USA), which recognizes an extracellular domain of p185neu protein, were used as a positive control. Serial Ab4 dilutions were used to generate a standard curve to determine the concentration (mg ml1) of anti-p185neu antibodies in serum. Flow cytometry analysis was performed with CyAn ADP (DakoCytomation, Italy). Isotype determination was carried out by an indirect immunofluorescence procedure. Dilution (1:20) of sera in PBS-azide-BSA were incubated with 2 105 BALB/c NIH3T3-NKB cells for 45 min at 4 1C. After washing, the cells were incubated for 30 min with rat biotin-conjugated antibodies antimouse IgG1, IgG2a, IgG2b and IgG3 (Caltag Laboratories, Burlingame, CA), and then for 30 min with 5 ml of streptavidin–phycoertrin (DAKO) and resuspended in PBS-azide-BSA containing 1 mg ml1 propidium iodide. Samples were evaluated by CyAn ADP and data are reported as the percentage of p185neu þ cells. In vivo cytotoxicity assay In vivo cytotoxicity assay was performed as described by Ritchie et al.,16 with slight modifications. Briefly, a singlecell suspension of 107 naive Spc ml1 was labeled with two different concentrations (0.5 or 5.0 mM) of the fluorescent dye CFSE (Molecular Probes, Leiden, The Netherlands). Spc labeled with 5 mM were also pulsed with p185neu (63–71) 9-mer peptide with H-2Kd restriction element17

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for 1 h at room temperature. The two Spc populations were mixed together in equal amounts and injected intravenously into control and electroporated mice. BALB/c mice were killed 14, 28 and 42 days after vaccination, and single-cell suspensions from spleens were processed individually to evaluate the presence of highand low-CFSE cells by CyAn ADP. The specific cytolytic activity was calculated as: 100 (percentage CFSE-low cellspercentage CFSE-high cells)/percentage CFSE-low cells.18

Cell line and tumor challenge TUBO cells15 are a cloned line derived from a mammary carcinoma of a BALB-neuT664VE female. p185neu is highly expressed on the cell membrane of TUBO cells that were cultured in DMEM with 20% FBS. Mice were challenged subcutaneously in the left flank with 0.2 ml of a single suspension containing the minimal lethal dose of TUBO cells (105). The cages were coded, and the incidence and the growth of tumors were evaluated twice weekly with the investigator blinded to the treatment group. Neoplastic masses were measured with calipers in the two perpendicular diameters. At the end of this period, tumor-free mice were classed as survivors. Mice were killed for humane reasons when the tumor exceeded 10 mm in mean diameter. Data from two independently performed experiments were cumulated, as they yielded identical results.

from 0.2–0.4 A (Figure 1). Since, the most evident immune response induced by vaccination with EC-TM plasmids is the rise of anti-p185neu antibodies,15 two weeks after vaccination sera were collected and the specific antibody titers were evaluated. The titer of antip185neu antibodies elicited appears to be inversely proportional to the intensity of the electric current amperage, although the differences were not statistically significant. The lowest current setting, 0.2 A, was then used for all the other experiments. Induction of antibodies to p185neu protein was then evaluated in mice constant current electroporated with increasing doses of EC-TM plasmid. Sera from individual mice collected 2 and 4 weeks after CCE showed that all doses of EC-TM plasmid were able to induce anti-p185neu antibodies at both 2 (data not shown) and 4 weeks after vaccination. Four weeks after electroporation, sera from mice constant current electroporated with 50 mg of EC-TM plasmid showed the highest titers (Figure 2a). In addition, sera collected 2 weeks after CCE were pooled to evaluate isotype composition of anti-p185neu antibodies, showing that the 0.2 A condition strongly induces an isotype

Results

Induction of anti-p185neu response in wild-type BALB/c mice Mice were injected with 50 mg of EC-TM plasmid and administered CCE at different constant current-settings

Figure 1 The optimal current voltage for EC–TM plasmid electroporation assessed in function of the serum antibody. BALB/c mice were vaccinated with 50 mg of EC-TM plasmid and electroporated with 0.2, 0.3 or 0.4 A. Two weeks later sera were collected and the specific antibody titers were evaluated. While no statistically significant differences were evident, a trend toward an inverse correlation between the current intensity (Amperes, A) of CCE and the antibody response was detected. Each symbol indicates the average sera of an individual mouse, while the horizontal bars indicate the median value.

Cancer Gene Therapy

Figure 2 Induction of p185neu antibody response. (a) BALB/c mice were vaccinated with 5, 10, 25 and 50 mg of EC-TM plasmid and 0.2 A CCE. Anti-p185neu antibody titers in sera collected 4 weeks after vaccination. The mean titer of antibody appears to increase with the amount of plasmid injected. The antibody titer of mice vaccinated with 50 mg plasmid was significantly higher from those vaccinated with 5, 10 and 25 mg (P ¼ 0.0041, 0.0244 and 0.0424 respectively). Each symbol depicts the average sera of an individual mouse, while the horizontal bars indicate the median value. (b) Sera from individual mice were collected 2 weeks after electroporation and pooled to evaluate isotype composition of anti-p185neu antibodies. Data are reported as the percentage of p185neu þ cells stained after incubation with immune sera and specific anti-IgG subclasses FITCconjugated antibodies.


switch to IgG2a (Figure 2b), the most effective one for antitumor activity against neu þ tumors.15 It was previously shown19 that effective EC-TM vaccination of BALB/c mice is able to induce a cytotoxic response against the immunodominant p185neu (63–71) 9-mer peptide.15 To assess whether the 0.2 A CCE condition used with EC-TM plasmids was also able to induce a cytotoxic T-cell response, BALB/c mice constant current electroporated with 50 mg of empty vector or vector coding for EC-TM plasmid were analyzed in vivo for their cytotoxic activity against syngeneic spleen cells pulsed with the immunodominant (63–71) 9-mer peptide (Figure 3). A cytotoxic response was evident 14 days after CCE of EC-TM plasmid and was still evident 28 and 42 days later.

Rejection of a neu þ tumor cell challenge by wild-type BALB/c mice A dose–response experiment was set up to examine the effects of escalating doses of EC-TM plasmid constant current electroporated on the ability of BALB/c mice to reject a challenge of neu664VE þ TUBO cells (Table 1). Mice were vaccinated with 5, 10, 25 and 50 mg of EC-TM plasmid and CCE with 0.2 A. Forty-two days later, control and immunized mice were challenged with a lethal dose of TUBO cells (1 105). All mice that received CEE of empty vector developed a fast-growing tumor by day 30, whereas all those vaccinated with 10, 25 or 50 mg of EC-TM plasmid remained tumor free for 150 days after challenge (192 days after vaccination). Interestingly, two

out of eight of those constant current electroporated with 5 mg of EC-TM plasmid remained tumor free. When tumor-free mice were challenged again with TUBO cells, 206 days after the initial CCE (164 days after the first challenge), all remained tumor free 100 days after the second challenge, when the experiment was ended.

Inhibition of autochthonous carcinomas in transgenic BALB-neuT664VE mice To assess the potential of EC-TM plasmid vaccination with CCE in the control of progressive stages of neudriven mammary carcinogenesis, transgenic BALBneuT664VE mice were constant current electroporated

Table 1 Optimal DNA dose that induces rejection of TUBO cell implantation was evaluated in BALB/c mice Tumors/total mice CCE with increasing amount of:

First TUBO cell challenge TUBO cell re-challenge

pCDNA3 plasmid

EC-TM plasmid

50 mg

5 mg 10 mg 25 mg 50 mg

a

8/8 6/6b

6/8 0/2

0/8 0/8

0/8 0/8

0/8 0/8

a The experiment was performed twice and the results were cumulated. b As control, otherwise untreated BALB/c mice were challenged with the same preparation of TUBO cells.

Figure 3 Induction of a cytotoxic response to p185neu. The cytotoxic response elicited in BALB/c mice vaccinated with 50 mg EC-TM and CCE with 0.2 A was evaluated 14 (a), 28 (b) and 42 (c) days later by injecting intravenously Spc untreated or pulsed with r-p185neu (63–71) 9-mer peptide and labeled with two concentrations (0.5 or 5.0 mM) of the CFSE. The percentage of lysis is shown.

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Figure 4 Inhibition of progressive stages of mammary carcinogenesis through CCE with EC-TM plasmids. (a) Protection afforded by repeated CCE (arrows) on BALB-neuT664VE and BALB-neuT664VE/FcgRI/III KO mice started when mice display diffuse atypical hyperplasia and multifocal in situ carcinomas in all mammary glands. Seventy-day-old mice were treated using CCE every 10 weeks (arrows). EC-TM plasmid: BALB-neuT664VE mice, red line, nine mice from two independently vaccinated groups; BALB-neuT664VE/FcgRI/III KO black line, four mice. Control pcDNA3 plasmid: BALB-neuT664VE red dotted line, nine mice from two independently electroporated groups; BALB-neuT664VE/FcgRI/ III KO black dotted line, four mice. Gray lines show tumor appearance in untreated BALB-neuT664VE mice (five mice, continuous line) and in BALB-neuT664VE/FcgRI/III KO (dotted line). (b) Protection afforded by a single administration of EC-TM plasmid with CCE performed at 100-day of age (arrow) on BALB-neuT664VE mice with multiple microscopic invasive carcinomas in all mammary glands. Mice treated with EC-TM plasmid and CCE, red line, five mice; with control pcDNA3, black line, three mice, or left untreated, gray line five mice. In all these experiments, mice of the same age were randomly assigned to the concurrently treated control and experimental groups. Mice with at least one progressively growing mammary tumor 41 mm mean diameter were classified as tumor-bearing mice.

with EC-TM plasmid every 10 weeks starting when they already display diffuse mammary atypical hyperplasia and multifocal in situ carcinomas in all their mammary glands (10 weeks of age).20 All BALB-neuT664VE and BALB-neuT664VE/FcgRI/III KO mice injected with the empty vector pcDNA3 develop a tumor by day 160 (Figure 4a). Eight out of nine BALB-neuT664VE mice (89%) immunized with EC-TM were tumor free at 450 days of age. The appearance of tumors by day 170 in all BALB-neuT664VE/FcgRI/III KO mice constant current electroporated with EC-TM (4/4) (Po0.0001 versus immunized BALB-neuT664VE mice) suggests that ADCC play a major role in the protection elicited by CCE of ECTM. Data from immunized BALB-neuT664VE are from two independent experiments of four and five mice each that were cumulated as they provided homogeneous results. Lastly, to further assess the potential of EC-TM plasmid vaccination using CCE, BALB-neuT664VE mice in which the neu-driven carcinogenesis progressed for further 30 days to multiple microscopic invasive carcinomas,20 were treated with EC-TM plasmid and CCE only once. Also in these stringent conditions, CCE of EC-TM plasmid afforded a significant delay of tumor appearance (Po0.0001 versus untreated mice and mice CCE with control pc-DNA3 plasmid) (Figure 4b).

Discussion

Administration of a clinically relevant and efficacious dose is a major goal of every investigator using DNA vaccines in animal models. Electroporation has been shown to be an efficient method for enhancing the uptake

Cancer Gene Therapy

and expression of DNA vaccine candidates, enabling the use of a lower dose of vaccine with similar or higher efficacy.21 Previous studies have yielded exciting shortterm results but long-term prevention of tumor development with a minimum of intervention has remained elusive. To address these questions, we have designed and conducted the studies described herein. We have demonstrated in two independently conducted experiments that significantly lower doses of EC-TM plasmid in intramuscular injection followed by low-current intensity CCE, can be used to achieve long-term protection from breast carcinoma in both tumor implantation and transgenic mice models. This combination of low plasmid doses and mild electroporation conditions is paramount for the human development of the technology, where adverse effects from plasmid delivery (such as integration due to tissue damage22 and pain or discomfort due to electroporation using high field intensity pulses23) can be a significant problem. Electroporation of cancer vaccines or drugs has been highlighted in recent reviews as an effective technique to either prevent tumor growth using intramuscular injections14 or when combined with injections of interleukin1224 or eliminate existing tumors using intra-tumoral injections.25,26 While the latter technology has yielded very promising results in tumors located on the skin surface, an intramuscular vaccination procedure that is able to induce long-term immunity to the tumors, which may develop within the tissue is a more effective technique. In the present study, two separate models of tumor development were studied: tumor implantation using TUBO cells and autochthonous tumor formation in BALB-neuT664VE mice. There are advantages and disadvantages to each method. TUBO cell implantation


is an effective method to quickly and accurately measure immunity to the vaccine candidate, however, it does not take into account the effects of the vaccine on spontaneous tumor formation. BALB-neuT664VE mice are a more clinically relevant model for tumor formation, but experiments may last for a year or more to delineate significant effects on tumor development. Mice that develop a tumor as a consequence of a gene alteration inserted in their genome constitute a relatively new experimental model. In these mice, tumors progress slowly and by stages, and the relationship of neoplastic lesions with the surrounding tissues is preserved mirroring a few additional features of human cancer.7 Constant current electroporation conditions were identified through previous experiments with reporter plasmids in mice.4 However, the current setting (0.1 A) required to elicit optimum expression of secreted embryonic alkaline phosphatase in mice is lower than the current setting (0.2 A) in cancer vaccine experiments, which induces effective levels of IgG2 antibodies in the present study. Higher current settings (0.3 and 0.4 A) yielded less efficient electroporations, possibly with more muscle damage, leading to a decrease in EC-TM plasmid expression and thus, specific antibody titers. This difference may be explained by a higher degree of transient inflammation that is usually correlated with higher current settings that may be beneficial for delivery of a plasmid encoding for some vaccines versus therapeutic proteins.27 The window of optimum settings is not wide in the case of vaccination. A setting that is too high may induce cellular damage and consequently lower vaccine expression, while a setting that is too low would not result in sufficient uptake and would actually lower vaccine expression. In wild-type BALB/c mice, rat p185neu is a xenogeneic protein that differs from mouse p185neu at multiple epitopes.28 In these mice, as little as 5 mg of EC-TM plasmid electroporated once using CCE affords a significant protection against a subsequent lethal challenge of neu þ carcinoma cells. Moreover, all BALB/c mice constant current electroporated with 10 mg were completely protected and developed a persistent immune memory. It is important to note that despite the relatively few animals per group in this portion of the study, the differences between the group treated with 10 mg (and above) were statistically different from controls. Rat neu transgenic BALB-neuT664VE female mice are genetically predestined to develop one of the most aggressive forms of multifocal mammary carcinogenesis with penetrance of all their mammary glands.20 In these mice, cancer progression is straightforwardly driven by the transgenic neu oncogene that both provides abnormal growth signals and inhibits cell-death pathways.29 Due to rat neu transgene expression in the thymus and overexpression in the mammary gland, the T-cell repertoire of these mice is markedly affected and CD8 þ T-cell clones reacting with dominant neu epitopes are deleted.19 Moreover, during the progression of the mammary lesions both suppressor CD4 þ CD25 þ Foxp3 þ GITR þ

T-reg cells18 and CD11b þ Gr1 þ immature myeloid cells,30 expand. Despite the presence of these multiple negative immunological controls, a complete protection is afforded by EC-TM plasmid CCE started when mice display diffuse atypical hyperplasia and multifocal in situ carcinomas in all mammary glands and repeated every 70 days. Even more significant is the finding that a single EC-TM plasmid CCE done on 100-day-old BALB-neuT664VE mice that already display multiple microscopic invasive carcinomas in all mammary glands markedly delays the progression of cancer lesion and keep about 50% of mice tumor free at one year of age. Numerous studies performed immunizing BALBneuT664VE mice with the EC-TM plasmids showed that the protection against neu-driven carcinogenesis in these mice mostly depends on the titer and isotype of antip185neu antibodies elicited by the vaccine.31,32 Present data showing that the protection afforded by EC-TM CCE is null in BALB-neuT664VE/FcgRI/III KO that are unable to mediate ADCC further endorse the indirect role of anti-p185neu antibodies in this experimental system. Despite the numerous studies performed with the same EC-TM plasmid administered to BALB-neuT664VE mice with different delivery systems,31 a direct comparison of the efficacy may not be perfect since, subtle technical variations and small deviations in the timing of vaccine administration may lead to major differences in efficacy. Nevertheless, EC-TM CCE appears to be the most efficacious and practical way of eliciting very significant protection. The enhancement in the efficacy and subsequently lowering of the effective DNA vaccine dose is beneficial from both a manufacturing (cost-effective) as well as a toxicological perspective. While plasmids manufactured under GMP have limited levels of endotoxins, residual genomic DNA or bacterial RNA, as plasmid-dose increases, the absolute levels of these contaminants increase and can negatively impact expression as well as increase the risk of adverse effects, both locally and systemically.33 Distant site distribution and integration of plasmids at the injection site is thought to be correlated to the plasmid-dose and electroporation conditions, with higher current and voltage settings associated to higher risk of unwanted effect.22 The ability to lower the dose and frequency of necessary vaccinations may allow this method to be both economically viable and safer by using less vaccine per human patient. The use of neu plasmids and CCE for long-term protection from breast carcinomas using lower doses and fewer vaccinations is an important breakthrough for the potential clinical relevance of this cancer vaccine regimen. Acknowledgements

This work was supported by grants from Italian Association for Cancer Research, the Italian Ministries for the Universities and Health, University of Torino and Compagnia di San Paolo, Turin.

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References 1 Brown PA, Davis WC, Draghia-Akli R. Immune enhancing effects of growth hormone releasing hormone delivered by plasmid injection and electroporation. Mol Ther 2004; 10: 644–651. 2 Draghia-Akli R, Fiorotto ML. A new plasmid-mediated approach to supplement somatotropin production in pigs. J Anim Sci 2004; 82: E264–E269. 3 Prud’homme GJ, Glinka Y, Khan AS, Draghia-Akli R. Electroporation-enhanced nonviral gene transfer for the prevention or treatment of immunological, endocrine and neoplastic diseases. Curr Gene Ther 2006; 6: 243–273. 4 Khan AS, Pope MA, Draghia-Akli R. Highly efficient constant-current electroporation increases in vivo plasmid expression. DNA Cell Biol 2005; 24: 810–818. 5 Bloquel C, Bessis N, Boissier MC, Scherman D, Bigey P. Gene therapy of collagen-induced arthritis by electrotransfer of human tumor necrosis factor-alpha soluble receptor I variants. Hum Gene Ther 2004; 15: 189–201. 6 Fattori E, Cappelletti M, Zampaglione I, Mennuni C, Calvaruso F, Arcuri M et al. Gene electro-transfer of an improved erythropoietin plasmid in mice and non-human primates. J Gene Med 2005; 7: 228–236. 7 Lollini PL, Cavallo F, Nanni P, Forni G. Vaccines for tumour prevention. Nat Rev Cancer 2006; 6: 204–216. 8 Stan R, Wolchok JD, Cohen AD. DNA vaccines against cancer. Hematol Oncol Clin North Am 2006; 20: 613–636. 9 Gothelf A, Mir LM, Gehl J. Electrochemotherapy: results of cancer treatment using enhanced delivery of bleomycin by electroporation. Cancer Treat Rev 2003; 29: 371–387. 10 Heller LC, Heller R. In vivo electroporation for gene therapy. Hum Gene Ther 2006; 17: 890–897. 11 Di CE, Diodoro MG, Boggio K, Modesti A, Modesti M, Nanni P et al. Analysis of mammary carcinoma onset and progression in HER-2/neu oncogene transgenic mice reveals a lobular origin. Lab Invest 1999; 79: 1261–1269. 12 Lollini PL, Nicoletti G, Landuzzi L, De GC, Rossi I, Di CE et al. Downregulation of major histocompatibility complex class I expression in mammary carcinoma of HER-2/neu transgenic mice. Int J Cancer 1998; 77: 937–941. 13 Ercolini AM, Machiels JP, Chen YC, Slansky JE, Giedlen M, Reilly RT et al. Identification and characterization of the immunodominant rat HER-2/neu MHC class I epitope presented by spontaneous mammary tumors from HER-2/ neu-transgenic mice. J Immunol 2003; 170: 4273–4280. 14 Quaglino E, Iezzi M, Mastini C, Amici A, Pericle F, Di CE et al. Electroporated DNA vaccine clears away multifocal mammary carcinomas in her-2/neu transgenic mice. Cancer Res 2004; 64: 2858–2864. 15 Rovero S, Amici A, Carlo ED, Bei R, Nanni P, Quaglino E et al. DNA vaccination against rat her-2/Neu p185 more effectively inhibits carcinogenesis than transplantable carcinomas in transgenic BALB/c mice. J Immunol 2000; 165: 5133–5142. 16 Ritchie DS, Hermans IF, Lumsden JM, Scanga CB, Roberts JM, Yang J et al. Dendritic cell elimination as an assay of cytotoxic T lymphocyte activity in vivo. J Immunol Methods 2000; 246: 109–117. 17 Nagata Y, Furugen R, Hiasa A, Ikeda H, Ohta N, Furukawa K et al. Peptides derived from a wild-type murine proto-oncogene c-erbB-2/HER2/neu can induce CTL and tumor suppression in syngeneic hosts. J Immunol 1997; 159: 1336–1343.

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18 Ambrosino E, Spadaro M, Iezzi M, Curcio C, Forni G, Musiani P et al. Immunosurveillance of Erb2 carcinogenesis in transgenic mice is concealed by a dominant regulatory T-cell self-tolerance. Cancer Res 2006; 66: 7734–7740. 19 Rolla S, Nicolo C, Malinarich S, Orsini M, Forni G, Cavallo F et al. Distinct and non-overlapping T cell receptor repertoires expanded by DNA vaccination in wild-type and HER-2 transgenic BALB/c mice. J Immunol 2006; 177: 7626–7633. 20 Pannellini T, Forni G, Musiani P. Immunobiology of her-2/ neu transgenic mice. Breast Dis 2004; 20: 33–42. 21 Babiuk S, Baca-Estrada M, Foldvari M, Storms M, Rabussay D, Widera G et al. Electroporation improves the efficacy of DNA vaccines in large animals. Vaccine 2002; 20: 3399. 22 Wang Z, Troilo PJ, Wang X, Griffiths TG, Pacchione SJ, Barnum AB et al. Detection of integration of plasmid DNA into host genomic DNA following intramuscular injection and electroporation. Gene Therapy 2004; 11: 711–721. 23 Gaudy C, Richard MA, Folchetti G, Bonerandi JJ, Grob JJ. Randomized controlled study of electrochemotherapy in the local treatment of skin metastases of melanoma. J Cutan Med Surg 2006; 10: 115–121. 24 Spadaro M, Ambrosino E, Iezzi M, Di CE, Sacchetti P, Curcio C et al. Cure of mammary carcinomas in Her-2 transgenic mice through sequential stimulation of innate (neoadjuvant interleukin-12) and adaptive (DNA vaccine electroporation) immunity. Clin Cancer Res 2005; 11: 1941–1952. 25 Heller R, Gilbert R, Jaroszeski MJ. Electrochemotherapy: an emerging drug delivery method for the treatment of cancer. Adv Drug Deliv Rev 1997; 26: 185–197. 26 Heller LC, Coppola D. Electrically mediated delivery of vector plasmid DNA elicits an antitumor effect. Gene Therapy 2002; 9: 1321–1325. 27 Babiuk S, Baca-Estrada ME, Foldvari M, Middleton DM, Rabussay D, Widera G et al. Increased gene expression and inflammatory cell infiltration caused by electroporation are both important for improving the efficacy of DNA vaccines. J Biotechnol 2004; 110: 1–10. 28 Nagata Y, Furugen R, Hiasa A, Ikeda H, Ohta N, Furukawa K et al. Peptides derived from a wild-type murine proto-oncogene c-erbB-2/HER2/neu can induce CTL and tumor suppression in syngeneic hosts. J Immunol 1997; 159: 1336–1343. 29 Muller WJ, Sinn E, Pattengale PK, Wallace R, Leder P. Single-step induction of mammary adenocarcinoma in transgenic mice bearing the activated c-neu oncogene. Cell 1988; 54: 105–115. 30 Melani C, Chiodoni C, Forni G, Colombo MP. Myeloid cell expansion elicited by the progression of spontaneous mammary carcinomas in c-erbB-2 transgenic BALB/c mice suppresses immune reactivity. Blood 2003; 102: 2138–2145. 31 Cavallo F, Offringa R, van der Burg SH, Forni G, Melief CJ. Vaccination for treatment and prevention of cancer in animal models. Adv Immunol 2006; 90: 175–213. 32 Park JM, Terabe M, Sakai Y, Munasinghe J, Forni G, Morris JC et al. Early role of CD4+ Th1 cells and antibodies in HER-2 adenovirus vaccine protection against autochthonous mammary carcinomas. J Immunol 2005; 174: 4228–4236. 33 Hebel H, Attra H, Khan A, Draghia-Akli R. Successful parallel development and integration of a plasmid-based biologic, container/closure system and electrokinetic delivery device. Vaccine 2006; 24: 4607–4614.