Open Access

Short paper

Regulatable systemic production of monoclonal antibodies by in vivo

muscle electroporation

Norma Perez

1

, Pascal Bigey

2

, Daniel Scherman

2

, Olivier Danos

1

,

Marc Piechaczyk

3

and Mireia Pelegrin*

3

Address: 1Généthon & UMR 8115 CNRS, 91002 Evry, France, 2Unité de Pharmacologie Chimique et Génétique, FRE CNRS 2463 - INSERM U640,

Faculté de Pharmacie, Université René Descartes, 75270 PARIS, France and 3Institute of Molecular Genetics of Montpellier, UMR 5535 / IFR122

CNRS, 34293 Montpellier, France

Email: Norma Perez - [email protected]; Pascal Bigey - [email protected]; Daniel Scherman -
[email protected]; Olivier Danos - [email protected]; Marc Piechaczyk - [email protected]; Mireia Pelegrin* - [email protected]
* Corresponding author

gene therapy / DNA electrotransfer / Muscle / Monoclonal antibody / Immunotherapy

Abstract

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.

Findings

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
clin-ical evaluation [1,2]. However, a number of hurdles have
still to be overcome before efficient therapeutical
applica-tion of mAbs on large scales and at reasonable costs. This

Published: 23 March 2004

Genetic Vaccines and Therapy 2004, 2:2

Received: 23 December 2003
Accepted: 23 March 2004
This article is available from: www.gvt-journal.com/content/2/1/2

© 2004 Perez et al; licensee 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.

Genetic Vaccines and Therapy 2004, 2 www.gvt-journal.com/content/2/1/2

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
ther-apeutic 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
advan-tageously 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
anti-bodies at relatively low, but therapeutic, levels (for a
review, see [3]).

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)
graft-ing of ex vivo modified myoblasts [4], skin fibroblasts [5]
and skin patches [6], (ii) intravenous and intramuscular
injection of recombinant adenoviral vectors [7], (iii)
intramuscular injection of AAV vectors [8] and (iv)
implantation of mAb-producing cells encapsulated in an
immunoprotective matrix made of cellulose sulphate [9].
Remarkably, the latter technology allowed to cure
retrovi-rally-infected mice from a lethal neurodegenerative
dis-ease upon production of a neutralizing mAb [10] 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
con-cerns 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
crite-ria, 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
com-petent 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
electro-transfer, 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

produc-tion 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
elec-trotransfer 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.

In a first step, we tested whether long-term mAb
produc-tion could be obtained using constitutive expression
vec-tors. The cDNAs for heavy (hTg10) and light (κTg10)
chains of the Tg10 mouse mAb (IgG2a/κ; [21]) were
cloned downstream of a CMV promoter in the pCOR
vec-tor backbone [22] to give pCOR-κTg10 and pCOR-hTg10
plasmids, respectively (Figure 1A). Tg10 is directed against
human thyroglobulin and was selected because of its easy
assay in ELISA. The advantages of the pCOR vector are
multiple: (i) it contains a conditional replication origin
permitting its production only in specifically engineered
E. coli strains to reduce the risks of uncontrolled
propaga-tion in the environment and in treated patients, (ii) it can
be produced in high yields through fed-batch
fermenta-tion, (iii) it contains minimal amounts of procaryotic
sequences to minimize interferences with transgene
expression (by reducing the immune response) and (iv) it
already allowed substantial transgene expression upon
injection into mouse skeletal muscle [22]. Five C57Bl6
mice were subjected to intramuscular electroporation
with an equimolar mixture of pCOR-κTg10 and
pCOR-hTg10 and five others, taken as negative controls, with a
saline solution. Tg10 levels in the bloodstream were
sub-sequently followed up as a function of time. No Tg10 was
detected in control mice whereas all other mice expressed
it for at least 142 days (duration of the experiment)
(Fig-ure 1B). Although variations between animals were
observed, it is noteworthy that 3 of them expressed more
than 1 µg/ml in the initial production period under
non-optimized experimental conditions (see below).

Because regulatable expression would eventually be
desir-able 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 [23]). In both of them, transgene transcription
is under the cis-control of a minimal CMV promoter
linked to multiple copies of the bacterial tetracycline

Tg10 mAb production in mice subjected to intramuscular electrotransfer

Figure 1

Tg10 mAb production in mice subjected to intramuscular electrotransfer. (A) Tg10 mAb-expressing vectors used for

electroporation. pCOR-κTg10 and pCOR-hTg10, pCOR-derived vectors expressing the Tg10 light (κTg10) and Tg10 heavy
(hTg10) chains cDNAs under the control of the CMV promoter, respectively. CMV-tTA and CMV-rtTA express tTA and rtTA
transactivators under the transcriptional control of the CMV promoter, respectively. In tetO-Tg10, κTg10 and hTg10 are
expressed from a monocistronic expression cassette owing to the poliovirus internal ribosome entry sequence (IRES) placed
under the cis-control of a minimal CMV promoter linked to multiple copies of the bacterial tetO operator [24]. (B): in vivo
Tg10 production after electroporation of pCOR-derived vectors. Ten 4 week-old C57Bl6 mice were divided into 2 groups and
injected intramuscularly in the tibialis anterior with either 20 µg of pCOR-κTg10 plus 20 µg of pCOR-hTg10 (mice 1 to 5) or a
saline solution taken as a negative control. Electroporation was then performed as described in [15]. Blood samples were
with-drawn at the indicated time points post-electroporation and serum Tg10 levels were assayed by ELISA [4]. (C): Regulatable in
vivo Tg10 mAb production. 4 week-old C3H mice were divided into 3 groups of 5 animals and injected intramuscularly in the
tibi-alis anterior with either a saline solution (not shown), 100 µg of tetO-Tg10 (not shown) or 100 µg of CMV-tTA plus 100 µg of
tetO-Tg10 (mice 27 to 31). Electroporation was then performed as described in [15]. Doxycycline was added or removed
from mice drinking water at indicated times. Tg10 levels in serum samples taken at different time points post-electroporation
were assayed by ELISA.

B
C
pCOR-kTg10
3 ’ UTR
CMV
Tg10 k
pCOR-hTg10
Tg10 h 3 ’ UTR
CMV
tetO-Tg10
3 ’ UTR
CMV
tTA
CMV-tTA
rtTA 3 ’ UTR
CMV
CMV-rtTA
A

Days after electroporation

- dox + dox - dox + dox - dox

0
200
400
600
800
1000
1200
1400
1600
0 20 40 60 80 100 120 140 160
1
2
3
4
5
0
20
40
60
80
100
120
0 14 28 42 56 70 84 98 112 126 140 154
Tg10
(ng
/ml
)
Tg10
(ng
/ml
)

Days after electroporation

27
28
29
30
31
IRES Tg10 h
Tg10 k
tetO
3 ’ UTR
mCMV

Genetic Vaccines and Therapy 2004, 2 www.gvt-journal.com/content/2/1/2

operator (TetO). Expression is controlled by a
transactiva-tor (tTA) negatively regulated by tetracycline, or some of
its derivatives such as doxycycline (Dox), in the tet-off
sys-tem, whereas it is dependent on a transactivator (rtTA)
positively regulated by tetracycline family antibiotics in
the tet-on one. A monocistronic expression cassette
carry-ing κTg10 and hTg10 cDNAs separated by the poliovirus
IRES was thus cloned downstream of TetO in the
pUHD10-3 vector [24] to give the tetO-Tg10 plasmid
(Fig-ure 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
pro-duction 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 ctet-onsidered any ltet-onger for
fur-ther 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
doxycy-cline, 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
addi-tion and removal of Dox on days 112 and 140,
respec-tively, 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
electro-poration of naked DNA allows both constitutive and
reg-ulatable in vivo production of ectopic mAbs, which
constitutes a first step towards simplified, multivalent and
cost-effective long-term mAb-based genetic
immuno-therapies. 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
oth-ers 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.
Simi-larly, 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
struc-ture itself, to eliminate intrinsic immunostimulatory
sequences as much as possible, and (ii) the expression
cas-sette 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
rela-tively modest activity in a variety of tissues [27], using
strong muscle-specific promoters such as the
muscle-spe-cific creatine kinase- [28], desmin- [29] or synthetic
pSPc5-12 promoter developed by Li and collaborators
[30] should reveal particularly rewarding. In this regard,
preliminary in vitro studies showed that pSPc5-12
pro-moter 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
reacti-vate 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 ([31] 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
pro-teins 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
electro-poration 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.

Competing interests

None declared.

Authors' contributions

NP carried out and participated in the design of DNA
elec-trotransfer experiments allowing regulatable Tg10
expres-sion. PB constructed pCOR-derived vectors expressing
Tg10 and performed pCOR electrotransfer experiments.
DS and OD participated in the design of the study. MP
and MPelegrin participated in the design and
coordina-tion of the study and drafted the manuscript. MPelegrin
also performed in vitro transient transfection experiments
and Tg10 ELISA assays. All authors read and approved the
manuscript.

Acknowledgements

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).

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