- Short paper
- Open Access
Regulatable systemic production of monoclonal antibodies by in vivo muscle electroporation
© Perez et al; licensee BioMed Central Ltd. 2004
- Received: 23 December 2003
- Accepted: 23 March 2004
- Published: 23 March 2004
The clinical application of monoclonal antibodies (mAbs) potentially concerns a wide range of diseases including, among others, viral infections, cancer and autoimmune diseases. Although intravenous infusion appears to be the simplest and most obvious mode of administration, it is very often not applicable to long-term treatments because of the restrictive cost of mAbs certified for human use and the side effects associated with injection of massive doses of antibodies. Gene/cell therapies designed for sustained and, possibly, regulatable in vivo production and systemic delivery of mAbs might permit to advantageously replace it. We have already shown that several such approaches allow month- to year-long ectopic antibody production by non-B cells in living organisms. Those include grafting of ex vivo genetically modified cells of various types, in vivo adenoviral gene transfer and implantation of encapsulated antibody-producing cells. Because intramuscular electrotransfer of naked DNA has already been used for in vivo production of a variety of proteins, we have wanted to test whether it could be adapted to that of ectopic mAbs as well. We report here that this is actually the case since both long-term and regulatable production of an ectopic mAb could be obtained in the mouse taken as a model animal. Although serum antibody concentrations obtained were relatively low, these data are encouraging in the perspective of future therapeutical applications of this technology in mAb-based immunotherapies, especially in developing countries where cost-effective and easily implementable technologies would be required for large-scale applications in the context of severe chronic viral diseases such as HIV and HCV infections.
- gene therapy / DNA electrotransfer / Muscle / Monoclonal antibody / Immunotherapy
The therapeutical potential of monoclonal antibodies (mAbs) is enormous. Twelve mAbs have already been approved by the US Food and Drug Administration for therapeutical use and 400 others are currently under clinical evaluation [1, 2]. However, a number of hurdles have still to be overcome before efficient therapeutical application of mAbs on large scales and at reasonable costs. This is particularly true in the case of chronic diseases where patients must be treated for years or even for their whole lifetime. If mAb intravenous injection is a suitable mode of administration for short-term treatments, this is often not the case for long-term ones mainly because of (i) the mild to severe side effects associated with infusion, (ii) the possible anti-idiotypic response resulting from repeated injections of massive doses of antibodies and (iii) the restrictive costs of in vitro produced proteins certified for human use. Moreover, injection of massive doses of mAbs results in great variations in the bioavaibility of these therapeutic agents that are often detrimental to the efficacy of treatments. It is, therefore, important to investigate whether in vivo production of therapeutic antibodies based on gene/cell therapy-based approaches can advantageously replace regular intravenous infusions. This would render long-term therapeutic antibody treatments cost-effective, eliminate side effects of infusions and lower, or delay, the antibody-neutralizing response of the host through continuous and sustained delivery of antibodies at relatively low, but therapeutic, levels (for a review, see ).
Several methods have already been used to achieve month- to year-long ectopic mAb production in the mouse taken as an animal model. Those include: (i) grafting of ex vivo modified myoblasts , skin fibroblasts  and skin patches , (ii) intravenous and intramuscular injection of recombinant adenoviral vectors , (iii) intramuscular injection of AAV vectors  and (iv) implantation of mAb-producing cells encapsulated in an immunoprotective matrix made of cellulose sulphate . Remarkably, the latter technology allowed to cure retrovirally-infected mice from a lethal neurodegenerative disease upon production of a neutralizing mAb  thereby demonstrating the therapeutical interest of the approach.
Neutralizing anti-HIV and anti-HCV antibodies, some of which are currently tested in the clinic [11, 12], might potentially be of great help to fight two major health concerns of developing countries, provided that cost-effective and easily implementable methods of administration are available. Because the above-mentioned techniques, even in optimized forms, would certainly not meet these criteria, we turned to gene transfer methods as simple and as inexpensive as possible in the perspective of long-term therapies. Taking into account that muscle cells are competent for synthesis and secretion of properly folded mAbs [4, 7, 8], we first tested intramuscular injection of naked plasmid vectors with, however, no success (not shown). We then considered skeletal muscle DNA electrotransfer, a physical method for DNA delivery based on intramuscular injection of plasmids followed by electric pulses delivery [13–15], since it was reported to be more efficient than injection of DNA alone for systemic production at a therapeutic level of a number of proteins such as erythropoietin, factor IX and cytokines (for reviews see [16, 17]). The higher efficiency of DNA electrotransfer appears to be a two component phenomenon involving, on one hand, cell permeabilization and, on the other hand, DNA electrophoresis [18–20]. Thus far, DNA electrotransfer has been used with success in different species including mouse, rat, dog and monkey, but has not been reported yet in humans. It is, however, important to underline that, in addition to its simplicity, this approach should also easily permit multivalent one-step treatments through the mixing of different expression vectors. In the specific case of mAb-based antiviral treatments, this would offer the advantage of limiting the risk of viral escape.
Because regulatable expression would eventually be desirable to adapt serum mAb levels to the patients' needs or to terminate treatments in case of adverse side effects, we next turned to the use of the tet-off and tet-on inducible systems developed by Bujard and collaborators (for a review, see ). In both of them, transgene transcription is under the cis-control of a minimal CMV promoter linked to multiple copies of the bacterial tetracycline operator (TetO). Expression is controlled by a transactivator (tTA) negatively regulated by tetracycline, or some of its derivatives such as doxycycline (Dox), in the tet-off system, whereas it is dependent on a transactivator (rtTA) positively regulated by tetracycline family antibiotics in the tet-on one. A monocistronic expression cassette carrying κTg10 and hTg10 cDNAs separated by the poliovirus IRES was thus cloned downstream of TetO in the pUHD10-3 vector  to give the tetO-Tg10 plasmid (Figure 1A). In a first series of experiments, four groups of 4 mice were subjected to intramuscular DNA electrotransfer with: (i) a saline solution, as a negative control, (ii) 25 μg of tetO-Tg10 alone, to detect possible leakiness of the expression system, (iii) 25 μg of both tetO-Tg10 and a constitutive expression vector for tTA (CMV-tTA; Figure 1A) and (iv) 25 μg of both tetO-Tg10 and a constitutive expression vector for rtTA (CMV-rtTA; Figure 1A). Tg10 was expressed in none of the mice treated with the saline solution or with tetO-Tg10 alone (not shown). Similarly, in the presence of Dox in the drinking water, Tg10 was detected in none of the mice electroporated with CMV-rtTA and tetO-Tg10 (not shown). In contrast, in the absence of doxycycline, mice injected with CVM-tTA plus tetO-Tg10, presented low, but detectable, serum levels (10–20 ng/ml) of Tg10 one month after electroporation, at which time the experiment was stopped. The better production observed with the tet-off system is consistent with in vitro transfection experiments previously performed with mouse myogenic C2.C12 cells (not shown). The tet-on system was therefore not considered any longer for further work. In a second series of experiments, we tested whether electroporation of higher quantities of DNA could lead to higher levels of mAb serum levels (Figure 1C). Three groups of 5 mice each were treated with either a saline solution, 100 μg of tetO-Tg10 alone or 100 μg of CVM-tTA plus 100 μg of tetO-Tg10. No Tg10 was detected in control mice (not shown). In the absence of doxycycline, the mice injected with CMV-tTA plus tetO-Tg10 showed a mAb production increasing for the first to weeks, at which time it was more or less stabilized at levels ranging from 10 to 110 ng/ml depending on the mouse. On day 42, Dox was added to the drinking water, which led to rapid Tg10 production shut-off, and removed on day 77, which permitted Tg10 reinduction albeit at a lower degree than in the first period of expression. A new cycle of repression/reinduction was attempted upon addition and removal of Dox on days 112 and 140, respectively, with, however, no success (Figure 1C). Taken together these data indicate that higher mAb production can be obtained upon electroporation of higher amounts of expression vectors and that regulated expression can be obtained, at least as long as the inducible system remains functional (see discussion below).
In conclusion, we report here that intramuscular electroporation of naked DNA allows both constitutive and regulatable in vivo production of ectopic mAbs, which constitutes a first step towards simplified, multivalent and cost-effective long-term mAb-based genetic immunotherapies. Although month-long mAb expressions could be observed, production levels remained low, indicating that improvements of the technology are required before efficacious and reliable human application. We and others have previously shown that muscle cells can achieve high antibody production when genetically modified by adenoviral or AAV vectors ([7, 8] and unpublished data). Thus, the low mAb serum levels observed here were not due to a limited ability of muscle cells to secrete mAb but rather to the electroporation method itself and/or to the poor efficiency of the expression vectors used. Optimizing electric pulses for better adaptation to muscle geometry as well as improving plasmid biodistribution will thus have to be considered to improve DNA electrotransfer. Similarly, the search for optimal DNA doses will have to be conducted as our own data also indicate that the quantity of expression vectors is critical with regard to the final mAb expression. Finally, further improvements might also come from the optimization of (i) the plasmid structure itself, to eliminate intrinsic immunostimulatory sequences as much as possible, and (ii) the expression cassette for better transcription, translation and secretion of antibodies by muscle cells [25, 26]. Along this line, rather than utilizing the CMV promoter, which is known to undergo progressive shut-off in vivo and to display relatively modest activity in a variety of tissues , using strong muscle-specific promoters such as the muscle-specific creatine kinase- , desmin-  or synthetic pSPc5-12 promoter developed by Li and collaborators  should reveal particularly rewarding. In this regard, preliminary in vitro studies showed that pSPc5-12 promoter allows 100-fold higher expression of a luciferase gene than the CMV promoter in differentiated muscle cells (NP, unpublished data). Concerning the regulatable tetracycline system used here, we were not able to reactivate Tg10 production a third time. The reasons for this are not yet clear. Whether this was due to an immune response mounted against the tetracycline-dependent transactivator as has already been reported elsewhere for rtTA ( and NP unpublished observations), to the instability of the DNA vectors used or to gene expression shut-off resulting from inactivation of the CMV promoter remains to be evaluated. It is, however, of note that longer term regulatable expression of other recombinant proteins such as erythropoietin upon MLV- and AAV-based transduction of muscle cells has been described [31–34]. In these experiments, transcription of tTA and rtTA genes was driven either by the muscle-specific desmin promoter or by a viral LTR promoter, suggesting that the choice of the promoter might be crucial for long-term expression of tetracycline-dependent transactivators in muscle electroporation settings. Testing other muscle-specific promoters for tTA and rtTA, as well as other inducible systems such as the rapamycin inducible system is currently underway towards this aim.
This work was supported by a grant from the Association Française contre les Myophaties (AFM) and the Association pour la Recherche contre le Cancer (ARC).
- Glennie MJ, Johnson PW: Clinical trials of antibody therapy. Immunol Today. 2000, 21: 403-410. 10.1016/S0167-5699(00)01669-8.View ArticlePubMedGoogle Scholar
- Gura T: Therapeutic antibodies: magic bullets hit the target. Nature. 2002, 417: 584-586. 10.1038/417584a.View ArticlePubMedGoogle Scholar
- Pelegrin M, Gros L, Dreja H, Piechaczyk M: Monoclonal antibody-based immunotherapies. Current gene therapy. 2004.Google Scholar
- Noel D, Pelegrin M, Marin M, Biard PM, Ourlin JC, Mani JC, Piechaczyk M: In vitro and in vivo secretion of cloned antibodies by genetically modified myogenic cells. Hum Gene Ther. 1997, 8: 1219-1229.View ArticlePubMedGoogle Scholar
- Noel D, Pelegrin M, Brockly F, Lund AH, Piechaczyk M: Sustained systemic delivery of monoclonal antibodies by genetically modified skin fibroblasts. J Invest Dermatol. 2000, 115: 740-745. 10.1046/j.1523-1747.2000.00106.x.View ArticlePubMedGoogle Scholar
- Noel D, Dazard JE, Pelegrin M, Jacquet C, Piechaczyk M: Skin as a potential organ for ectopic monoclonal antibody production. J Invest Dermatol. 2002, 118: 288-294. 10.1046/j.0022-202x.2001.01625.x.View ArticlePubMedGoogle Scholar
- Noel D, Pelegrin M, Kramer S, Jacquet C, Skander N, Piechaczyk M: High in vivo production of a model monoclonal antibody on adenoviral gene transfer. Hum Gene Ther. 2002, 13: 1483-1493. 10.1089/10430340260185111.View ArticlePubMedGoogle Scholar
- Lewis AD, Chen R, Montefiori DC, Johnson PR, Clark KR: Generation of neutralizing activity against human immunodeficiency virus type 1 in serum by antibody gene transfer. J Virol. 2002, 76: 8769-8775. 10.1128/JVI.76.17.8769-8775.2002.PubMed CentralView ArticlePubMedGoogle Scholar
- Pelegrin M, Marin M, Noël D, Del Rio M, Saller R, Günzburg W, Stange S, Steffen M, Piechaczyk M: Systemic long-term delivery of antibodies in immunocompetent animals using cellulose sulphate capsules containing antibody-producing cells. Gene Therapy. 1998, 5: 828-834. 10.1038/sj.gt.3300632.View ArticlePubMedGoogle Scholar
- Pelegrin M, Marin M, Oates A, Noel D, Saller R, Salmons B, Piechaczyk M: Immunotherapy of a viral disease by in vivo production of therapeutic monoclonal antibodies. Hum Gene Ther. 2000, 11: 1407-1415. 10.1089/10430340050057486.View ArticlePubMedGoogle Scholar
- Ilan E, Arazi J, Nussbaum O, Zauberman A, Eren R, Lubin I, Neville L, Ben-Moshe O, Kischitzky A, Litchi A, Margalit I, Gopher J, Mounir S, Cai W, Daudi N, Eid A, Jurim O, Czerniak A, Galun E, Dagan S: The hepatitis C virus (HCV)-Trimera mouse: a model for evaluation of agents against HCV. J Infect Dis. 2002, 185: 153-161. 10.1086/338266.View ArticlePubMedGoogle Scholar
- Stiegler G, Armbruster C, Vcelar B, Stoiber H, Kunert R, Michael NL, Jagodzinski LL, Ammann C, Jager W, Jacobson J, Vetter N, Katinger H: Antiviral activity of the neutralizing antibodies 2F5 and 2G12 in asymptomatic HIV-1-infected humans: a phase I evaluation. Aids. 2002, 16: 2019-2025. 10.1097/00002030-200210180-00006.View ArticlePubMedGoogle Scholar
- Aihara H, Miyazaki J: Gene transfer into muscle by electroporation in vivo. Nat Biotechnol. 1998, 16: 867-870.View ArticlePubMedGoogle Scholar
- Mathiesen I: Electropermeabilization of skeletal muscle enhances gene transfer in vivo. Gene Ther. 1999, 6: 508-514. 10.1038/sj.gt.3300847.View ArticlePubMedGoogle Scholar
- Mir LM, Bureau MF, Gehl J, Rangara R, Rouy D, Caillaud JM, Delaere P, Branellec D, Schwartz B, Scherman D: High-efficiency gene transfer into skeletal muscle mediated by electric pulses. Proc Natl Acad Sci U S A. 1999, 96: 4262-4267. 10.1073/pnas.96.8.4262.PubMed CentralView ArticlePubMedGoogle Scholar
- Bigey P, Bureau MF, Scherman D: In vivo plasmid DNA electrotransfer. Curr Opin Biotechnol. 2002, 13: 443-447. 10.1016/S0958-1669(02)00377-4.View ArticlePubMedGoogle Scholar
- Scherman D: [Towards non-viral gene therapy]. Bull Acad Natl Med. 2001, 185: 1683-1697.PubMedGoogle Scholar
- Klenchin VA, Sukharev SI, Serov SM, Chernomordik LV, Chizmadzhev Yu A: Electrically induced DNA uptake by cells is a fast process involving DNA electrophoresis. Biophys J. 1991, 60: 804-811.PubMed CentralView ArticlePubMedGoogle Scholar
- Bureau MF, Gehl J, Deleuze V, Mir LM, Scherman D: Importance of association between permeabilization and electrophoretic forces for intramuscular DNA electrotransfer. Biochim Biophys Acta. 2000, 1474: 353-359. 10.1016/S0304-4165(00)00028-3.View ArticlePubMedGoogle Scholar
- Golzio M, Teissie J, Rols MP: Direct visualization at the single-cell level of electrically mediated gene delivery. Proc Natl Acad Sci U S A. 2002, 99: 1292-1297. 10.1073/pnas.022646499.PubMed CentralView ArticlePubMedGoogle Scholar
- Noel D, Bernardi T, Navarro-Teulon I, Marin M, Martinetto JP, Ducancel F, Mani JC, Pau B, Piechaczyk M, Biard-Piechaczyk M: Analysis of the individual contributions of immunoglobulin heavy and light chains to the binding of antigen using cell transfection and plasmon resonance analysis. J Immunol Methods. 1996, 193: 177-187. 10.1016/0022-1759(96)00043-9.View ArticlePubMedGoogle Scholar
- Soubrier F, Cameron B, Manse B, Somarriba S, Dubertret C, Jaslin G, Jung G, Caer CL, Dang D, Mouvault JM, Scherman D, Mayaux JF, Crouzet J: pCOR: a new design of plasmid vectors for nonviral gene therapy. Gene Ther. 1999, 6: 1482-1488. 10.1038/sj.gt.3300968.View ArticlePubMedGoogle Scholar
- Gossen M, Bujard H: Studying gene function in eukaryotes by conditional gene inactivation. Annu Rev Genet. 2002, 36: 153-173. 10.1146/annurev.genet.36.041002.120114.View ArticlePubMedGoogle Scholar
- Gossen M, Bujard H: Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proc Natl Acad Sci U S A. 1992, 89: 5547-5551.PubMed CentralView ArticlePubMedGoogle Scholar
- Garmory HS, Brown KA, Titball RW: DNA vaccines: improving expression of antigens. Genet Vaccines Ther. 2003, 1: 2-10.1186/1479-0556-1-2.PubMed CentralView ArticlePubMedGoogle Scholar
- Bloquel C, Fabre E, Bureau MF, Scherman D: Plasmid DNA electrotransfer for intracellular and secreted proteins expression: new methodological developments and applications. J Gene Med. 2004, 6 (Suppl 1): S11-23. 10.1002/jgm.508.View ArticlePubMedGoogle Scholar
- Loser P, Jennings GS, Strauss M, Sandig V: Reactivation of the previously silenced cytomegalovirus major immediate-early promoter in the mouse liver: involvement of NFkappaB. J Virol. 1998, 72: 180-190.PubMed CentralPubMedGoogle Scholar
- Bartlett RJ, Secore SL, Singer JT, Bodo M, Sharma K, Ricordi C: Long-term expression of a fluorescent reporter gene via direct injection of plasmid vector into mouse skeletal muscle: comparison of human creatine kinase and CMV promoter expression levels in vivo. Cell Transplant. 1996, 5: 411-419. 10.1016/0963-6897(95)02026-8.View ArticlePubMedGoogle Scholar
- Loirat D, Li Z, Mancini M, Tiollais P, Paulin D, Michel ML: Muscle-specific expression of hepatitis B surface antigen: no effect on DNA-raised immune responses. Virology. 1999, 260: 74-83. 10.1006/viro.1999.9795.View ArticlePubMedGoogle Scholar
- Li X, Eastman EM, Schwartz RJ, Draghia-Akli R: Synthetic muscle promoters: activities exceeding naturally occurring regulatory sequences. Nat Biotechnol. 1999, 17: 241-245. 10.1038/6981.View ArticlePubMedGoogle Scholar
- Favre D, Blouin V, Provost N, Spisek R, Porrot F, Bohl D, Marme F, Cherel Y, Salvetti A, Hurtrel B, Heard JM, Riviere Y, Moullier P: Lack of an immune response against the tetracycline-dependent transactivator correlates with long-term doxycycline-regulated transgene expression in nonhuman primates after intramuscular injection of recombinant adeno-associated virus. J Virol. 2002, 76: 11605-11611. 10.1128/JVI.76.22.11605-11611.2002.PubMed CentralView ArticlePubMedGoogle Scholar
- Bohl D, Naffakh N, Heard JM: Long-term control of erythropoietin secretion by doxycycline in mice transplanted with engineered primary myoblasts. Nat Med. 1997, 3: 299-305.View ArticlePubMedGoogle Scholar
- Bohl D, Heard JM: Modulation of erythropoietin delivery from engineered muscles in mice. Hum Gene Ther. 1997, 8: 195-204.View ArticlePubMedGoogle Scholar
- Bohl D, Salvetti A, Moullier P, Heard JM: Control of erythropoietin delivery by doxycycline in mice after intramuscular injection of adeno-associated vector. Blood. 1998, 92: 1512-1517.PubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original URL.