Therapeutic Antibody Gene Transfer: An Active Approach to Passive Immunity

Abstract

Advances in gene transfer approaches are enabling the possibility of applying therapeutic antibodies using DNA. In particular gene transfer in combination with electroporation is promising and can result in generating in vivo antibody concentrations in the low therapeutic range. However, several important problems need to be dealt with before antibody gene transfer can become a valuable supplement to the current therapies. As antibody production following gene transfer is difficult to control, the danger of inducing autoimmune conditions or uncontrollable side effects occurs in cases in which autologous antigens are targeted. It is suggested that the most promising area of application therefore appears to be infectious disease in which heterologous antigens are targeted and concerns for long-term antibody exposure are minimal. Finally, genes encoding fully human antibodies will enhance long-term expression and decrease problems linked to immunogenicity.

Full text

Therapeutic Antibody Gene Transfer: An Active
Approach to Passive Immunity
Joost M. Bakker, Wim K. Bleeker, and Paul W.H.I. Parren
*
Genmab B.V., Yalelaan 60, P.O. Box 85199, 3508 AD Utrecht, The Netherlands
*To whom correspondence and reprint requests should be addressed. Fax: +31 30 2 123 196. E-mail: p.parren@nl.genmab.com.
Available online 4 August 2004
Advances in gene transfer approaches are enabling the possibility of applying therapeutic
antibodies using DNA. In particular gene transfer in combination with electroporation is promising
and can result in generating in vivo antibody concentrations in the low therapeutic range.
However, several important problems need to be dealt with before antibody gene transfer can
become a valuable supplement to the current therapies. As antibody production following gene
transfer is difficult to control, the danger of inducing autoimmune conditions or uncontrollable side
effects occurs in cases in which autologous antigens are targeted. It is suggested that the most
promising area of application therefore appears to be infectious disease in which heterologous
antigens are targeted and concerns for long-term antibody exposure are minimal. Finally, genes
encoding fully human antibodies will enhance long-term expression and decrease problems linked
to immunogenicity.
INTRODUCTION
In the past 5 years, antibodies have shown the promise to
become the most important class of therapeutic drugs [1].
Unlike any other therapeutic agent, antibodies combine
very high specificity with long half-life and an ability to
interact efficiently with the body’s immune system. The
newest generation of antibodies is fully human and
therefore does not elicit anti-antibody responses in
humans, which are known to limit the efficacy and
usability of antibodies of murine or chimeric origin. To
date, 17 therapeutic monoclonal antibodies are on the
market and are being successfully used for treatment of
diseases, including cancer, inflammation, and infectious
disease (for overview see [1]).
Despite their high efficacy and specificity, some prob-
lems slow down an even wider implementation of anti-
bodies in the practitioner’s arsenal of drugs. One
important limitation is cost; although the costs of devel-
opment of new antibodies are relatively low compared to
small-molecule drugs, the manufacturing of high amounts
of antibody is expensive [2]. For this reason, a lot of
research is being performed to improve efficiency of cells
to produce antibodies, to modify the antibodies to make
them more efficacious [3–5], or to move to production
systems in lower eukaryotes or prokaryotes [6,7].
METHODS OF GENE DELIVERY
An alternative approach would be to leave the production
of therapeutic antibodies to the body itself. A multitude
of studies have shown the possibility of in vivo gene
transfer into cells. Currently, adenoviral vectors are being
tested in phase II and III clinical trials for angiogenic or
cancer applications [8]. In vivo administration to mice of
adenoviral vectors expressing monoclonal antibodies has
been effective [9], and adeno-associated virus vectors that
transferred the genes for a broadly neutralizing antibody
against HIV-1 to mice induced significant HIV-1-neutral-
izing antibody titers in the mouse serum [10]. However,
the use of human adenoviruses for in vivo gene therapy is
hampered by the risk of accidental germ-line trans-
mission [11,12] or cancer [13], in addition to antiviral
immune responses [14,15], which may abrogate expres-
sion or induce side effects.
In vivo gene transfer can also be accomplished by use
of nonviral vectors, usually expression plasmids [16].
Nonviral vectors are easily produced and do not seem to
induce specific immune responses. Muscle tissue is most
often used as target tissue for transfection, because
muscle tissue is well vascularized and easily accessible,
and myocytes are long-lived cells. Intramuscular injec-
tion of naked plasmid DNA results in transfection of a
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small percentage of myocytes, although to date the
mechanism explaining the uptake of DNA from the
extracellular space into the cell nucleus remains unclear
[16]. Using this approach, plasmid DNA encoding cyto-
kines or cytokine/IgG1 chimeric proteins has been
introduced in vivo and has positively influenced (auto-
immune) disease outcome in several animal models [17–
23]. However, transfection efficiency remained low, with
serum protein levels within the 0.1–10 ng/ml range. A
method to increase efficiency may be via so-called intra-
vascular delivery in which increased gene delivery and
expression levels are achieved by inducing a short-lived
transient high pressure in the veins [24–26]. Special
blood-pressure cuffs that may facilitate localized uptake
by temporarily increasing vascular pressure might be
applicable for humans for this type of gene delivery [27].
GENE DELIVERY VIA ELECTROPORATION
Increased efficiency is to be expected from two other
techniques in which delivery of DNA is improved by use
of chemical carriers—cationic polymers or lipids—or via a
physical approach—gene gun delivery or electroporation
[28,29]. Electroporation is especially regarded as an
interesting technique for nonviral gene delivery [30,31].
With electroporation, pulsed electrical currents are
applied to a local tissue area to enhance cell permeability,
resulting in gene transfer across the membrane. Research
in the past 5 years has shown that in vivo gene delivery
can be 10–100 times more efficient with electroporation
than without [32–35]. In the field of oncology, electro-
poration or electrochemotherapy in combination with
bleomycin is promising in the treatment of cutaneous
and subcutaneous tumors [36], with several clinical phase
II trials under way [37].
Gene transfer of proteins by electroporation is still in a
preclinical phase, but seems to be promising. For exam-
ple, in a recent paper by Zhou et al., plasmids encoding a
murine soluble B7.1:IgG1 fusion protein were injected
intramuscularly into C57/BL6 mice, followed by electro-
poration [38]. This fusion protein is thought to enhance
immune effector functions, resulting in enhanced tumor
cell killing. After five treatments (100 Ag plasmid per
injection) with this plasmid, together with a plasmid
encoding the carcinoembryonic antigen, increased kill-
ing of tumor cells was indeed observed. Fusion protein
levels rose to approximately 1 Ag/ml, as measured
immediately after the last treatment.
In an elegant study by Kim et al., plasmids encoding
a human soluble TNF receptor:Fc fusion protein were
transferred to murine muscle by electroporation [39].
Mice (DBA/1) did not show long-term enhancement of
serum protein levels (approximately 7–10 days, with a
maximum of 2.3 ng/ml), probably due to the use of
human fusion proteins. The authors did detect recombi-
nant protein in knee joints, 5 days after electroporation.
Severity of collagen-induced arthritis was attenuated in
treated mice, as well as disease severity, synovitis, and
cartilage erosion. Electroporation also led to reduced IL-
12 and IL-1h protein levels in diseased ankle joints.
With respect to this latter finding, studies have shown
that site-specific injection of genes and subsequent
electroporation may prove useful in diseases such as
rheumatoid arthritis. To illustrate this point, a reporter
gene was injected into rat joints by intra-articular
injection [40]. Up to 40% of cells remained transfected
at 2 months following treatment. Not only joints can be
used as targets for electrogene therapy, but also liver and
skin,althoughtodatemuscletissueisstillmost
frequently targeted for this type of treatment [40–42].
Currently, studies are being performed to increase
further the efficacy of DNA vaccination by electro-
poration [43–46].
ELECTROPORATION OF GENES ENCODING
SPECIFIC ANTIBODIES
Experiments in which cells are transfected with immu-
noglobulin genes in vitro have been performed for over
20 years [47,48], but in two recent studies, plasmid DNA
was used to transfect muscle cells with immunoglobulin
genes in vivo. In one study, the authors made use of two
expression vectors for Ig H-chain genes (pLNOH2) and
L-chain genes (pLNOn), encoding murine MHC class II
antibodies [49]. After a single injection of naked plasmid
DNA into C57/BL6 mice, electroporation was applied to
the muscle. Antibodies of correct specificity were
detected in the serum. The immune-competent mice
developed antibody responses against xenogeneic
(human) parts of the antibodies, which markedly
reduced the persistence in plasma. However, when the
authors used plasmids coding for a fully murine anti-IgD
antibody, plasma concentrations were observed to build
up to 800 ng/ml in the course of 3 weeks, to decline
gradually thereafter over a 3-month period to 400 ng/ml
and to persist at that level for more than 7 months. Also
sheep were found to produce antibodies after electro-
poration, indicating that production can also be
achieved in larger animals. Importantly, in sheep (30
kg) relatively low amounts of DNA were required to
obtain significant monoclonal antibody production, and
electroporation with 100 or 200 Ag of DNA yielded
about the same antibody plasma levels as observed in
mice using the same amount of DNA (I. Mathiesen,
personal communication).
In another approach, antibody expression was con-
trolled by an inducible promoter (under the control of
the bacterial tetracycline operator) [34,50]. In this way,
antibody production was negatively regulated by addi-
tion of doxycycline. Indeed, adding doxycycline to the
drinking water on day 42 after electroporation led to
diminished antibody production. Removing doxycycline
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from the water again increased antibody production [50].
Maximum plasma mouse IgG2a/n anti-human thyroglo-
bulin concentrations between 800 and 1500 ng/ml were
reached after about 3 weeks. Although also in this study
expression was observed for more than 6 months, plasma
levels were low compared to a study in which a
recombinant adeno-associated vector was used for anti-
body gene transfer in immune-deficient mice [10]. In the
latter study the plasma concentrations of an HIV-neu-
tralizing human IgG1 monoclonal antibody continued to
rise in the course of 3 months up to 4 to 6 Ag/ml. This
suggests that by use of electroporation repeated admin-
istrations will be required to obtain prolonged therapeu-
tic antibody levels.
In summary, long-term antibody production can be
achieved by combining DNA immunization and electro-
poration. It is evident, however, that it is essential to
express a syngeneic antibody, as immune responses
against heterologous parts develop quickly, thus limiting
the buildup of antibody serum levels.
EFFECTIVE THERAPEUTIC ANTIBODY LEVELS
Before discussing whether plasma antibody levels
required for therapy can be achieved via gene transfer
in more detail, we will first take a closer look at the
potential mechanisms of action of therapeutic antibodies
and the required dose levels obtained by passive antibody
transfer.
The currently approved monoclonal antibodies are
used for widely varying clinical conditions, including
transplant rejection, cancer, rheumatoid arthritis, and
prevention of viral infection. These therapeutic anti-
bodies may exert their actions in several different ways
[51]. The primary biological role of antibodies is to
protect the body from invading micro-organisms via
opsonization and engagement of immune effector
systems, such as complement and Fc-receptor-bearing
cells. This may also be an important mechanism of
action of therapeutic antibodies that coat their target
cells, like CD52-specific antibodies in the treatment of
chronic lymphocytic leukemia [52] or anti-HER2/neu in
the treatment of breast cancer [53]. However, it is
becoming increasingly clear that an important feature
of many successful therapeutic antibodies is that they
can also interfere with signaling. Therapeutic antibodies
may block the activity of a growth factor, cytokine, or
other soluble mediator by binding directly to the soluble
factor itself or to its receptor. Similarly, antibodies can
prevent cell–cell cross talk by blocking receptor–ligand
interactions. This mechanism is important, for example,
for antibodies against cytokines, like anti-TNFa [54]and
IL-15 [55], or antibodies against growth factor receptors,
like anti-EGFR ([56]; Bleeker et al., [56a]). Finally, anti-
bodies may act by cross-linking the target molecules,
which in the case of targeting membrane receptors, may
activate signaling leading to cell activation, apoptosis, or
internalization of the receptor.
Generally speaking, the dose–effect relationship for
inhibition of signaling will correspond to the binding
curve of the antibody to the target molecule. However,
this is different for immune effector functions, which
may already become fully engaged at low antibody
occupancy levels of the target. For engagement of
immune effector systems a certain number of antibodies
are required in the proper arrangement for effective
interaction with either soluble C1q or Fc receptors on
effector cells. Maximum engagement might then already
occur before maximal antibody binding is achieved. This
is unlike blockade in ligand–receptor interactions, which
is not expected to be optimal before most target
molecules are occupied by the antibody. For most
therapeutic antibodies maximum binding occurs at
concentrations on the order of 1–10 Ag/ml, depending
on the affinity of the antibody in question and on its
binding valency. It should be taken into account that
most monoclonal antibodies are supposed to exert their
effect in the interstitial space where IgG concentrations
are on average two- to threefold below the plasma
concentration and even lower in solid tumors. This
means that for most antibodies plasma levels above 3–30
Ag/ml may be required for therapeutic efficacy. This level
is consistent with literature data from clinical studies
evaluating the efficacy of therapeutic antibodies. Kovarik
et al. [57] assessed the immunodynamics of basiliximab
(anti-CD25) in pediatric kidney allograft patients and
concluded that patients should receive two doses of 1
mg/kg to obtain plasma concentrations above 1 Ag/ml to
ensure the prolonged CD25 saturation needed for
prevention of rejection. Similarly, in the treatment of
rejections, muronomab-CD3 (Orthoclone) is given in
daily doses of 5 mg for 10–14 days [58]. Leyland-Jones et
al. [59] administered trastuzumab (anti-HER2/neu) to
breast cancer patients with multiple doses of about 8
mg/kg, which resulted in trough levels in plasma above
20 Ag/ml. St. Clair et al. [60] investigated the relationship
of serum infliximab (anti-TNFa ) concentrations to clin-
ical improvement in rheumatoid arthritis patients and
concluded that trough serum levels of infliximab main-
tained above 10 Ag/ml may approximate the best
possible efficacy of infliximab therapy. For palivizumab
(anti-respiratory syncytial virus (RSV)) a minimum
plasma level of 40 Ag/ml is targeted to protect at-risk
infants against RSV infections [61]. Maloney evaluated
the effect of rituximab (anti-CD20) in patients with
relapsed B cell lymphoma and concluded that optimal
efficacy was achieved at multiple doses of 375 mg/m
2
,
which results in trough plasma levels above 100 Ag/ml
[62]. In contrast, Morris et al. concluded from a study on
the use of alemtuzumab (anti-CD52) for donor T cell
depletion in allogeneic stem cell transplantation [63]
that plasma concentrations as low as 0.1 Ag/ml were
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effective. They explained this result by the fact that the
lympholytic action relies mainly on antibody-dependent
cellular cytotoxicity (ADCC), which does not seem to
require full saturation of the membrane target mole-
cules. This is consistent with our own preclinical
observations on a novel human anti-EGFR monoclonal
antibody that has anti-cancer effects both by interfering
with EGFR signaling and by inducing ADCC (Bleeker et
al., [56a]). We observed that ADCC induction already
may occur at less than 1–5% receptor occupancy,
whereas complete saturation is needed for maximal
receptor blockade.
PHARMACOKINETIC MODELING
The reported antibody levels achievable by gene transfer
were generated in mouse experiments and cannot be
simply transferred to humans. Therefore, we did some
pharmacokinetic modeling to estimate the production
levels underlying the observed plasma concentrations
and to extrapolate the mouse results to humans. By
performing simulations in a two-compartment model
adapted to IgG kinetics, which has been previously
described [64], we estimated that for mice a plasma level
of 1 Ag/ml corresponds to an IgG production of about 25
Ag/kg per day (see Fig. 1). Assuming the same production
level per kilogram of body weight we can expect higher
plasma levels in humans because of the five times longer
half-life of 21 days compared to 4 days for normal
laboratory mice [65,66]. The simulation further makes
clear that, assuming constant production, the concen-
tration gradually increases until a steady state is reached
between production and elimination. For mice this
process takes 1 to 2 weeks, but for humans, due to the
longer half-life, maximum levels will not be reached
before several months. Taking all data together, and
provided that the production level in humans can be
scaled up in proportion, we can expect that gene transfer
by in vivo electroporation can generate monoclonal
antibody concentrations in the low therapeutic range,
although buildup to steady-state concentrations may take
significant time. This means that this approach could be
effective for selected therapeutic antibodies in certain
applications.
LIMITATIONS AND DRAWBACKS
There are several important concerns about mAb gene
transfer that might hinder the fast introduction of this
technique into clinical practice. The major concern is
whether the IgG production will be sufficient to create
therapeutically effective plasma levels. Because of the
relatively slow buildup of antibody plasma levels, the
plasma concentrations remain low for prolonged peri-
ods of time. For most therapies, fast buildup is
required, and therefore administration of protein is
preferred. For maintenance therapy, one could switch
to gene transfer after protein has been administered
and buildup of antibody plasma levels has been
achieved.
Another concern about protein expression by gene
transfer might be the relative unpredictability of the
plasma levels that will be attained. First, concentrations
may be below or above the targeted therapeutic value. In
particular, the possibility of too high plasma concen-
trations is a major problem for therapy with hormones,
like insulin or erythropoietin. This has stimulated the
development of vectors with regulatory elements [34],
which has also been shown feasible for antibodies [50].
However, for most therapeutic antibodies toxicity is
related directly to binding to the target, which does not
increase above a certain dose level. This property of
monoclonal antibodies is, for example, becoming clear in
the application of antibodies in the field of cancer
therapy in which, traditionally, phase I clinical develop-
ment of chemotherapeutic agents is primarily directed on
the determination of the maximum tolerated dose [67].
Due to their specificity, anti-cancer antibodies are gene-
rally less toxic at clinically effective doses, and impor-
tantly, above saturating doses neither therapeutic nor
toxiceffectswillincrease.Uncontrolledlong-term,
chronic exposure, even to moderate side effects, however,
would be undesirable.
Second, the longevity of antibody production is
difficult to control. On the one hand, long-term
expression might be needed. To date, loss of expression
occurs over time, which might be due to promoter
inactivation via methylation, apoptosis, or immune
responses generated against prokaryotic plasmid sequen-
FIG. 1. Simulation of the plasma monoclonal antibody (mAb) concentrations
for mice and humans. Assumed is an IgG mAb production from day 0 to day
40 at a level of 25 Ag/kg per day. The mAb was assumed to be delivered into
the interstitial space and redistributed into the plasma compartment and from
there 50% into the interstitial space. Elimination from the plasma compart-
ment was set for mouse to a half-life of 4 days, for humans to a half-life of 21
days.
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ces [30]. On the other hand, potential anti-tumor
targets such as CD20 and EGFR are also expressed on
healthy cells and production of antibodies that is too
prolonged might negatively affect these healthy cells for
too long. In the case of anti-TNFa therapy, as in
inflammatory bowel disease and rheumatoid arthritis
[68,69], long-term blockade is associated with oppor-
tunistic infections, including infections caused by
Mycobacterium tuberculosis, which may necessitate inter-
ruption of treatment until the infection has been
effectively treated [70,71]. With antibody gene transfer
by electroporation such a blockade might persist for too
long a period and could then be potentially life-threat-
ening. Therefore, if application of antibodies against
autoantigens is contemplated, research on the develop-
ment of vectors with regulatory elements seems to be
crucial.
Currently, the long-term lack of control of expression
makes the application of therapeutic antibody gene
transfer in diseases such as cancer or inflammatory
diseases unlikely. The uncertainty of prolonged expo-
sure of healthy cells expressing the antibody target
seems too great. The most promising area of application
therefore appears to be infectious disease in which
heterologous antigens are targeted and concerns for
long-term antibody exposure are minimal. It seems
feasible that immunity against pathogens could be
induced by transferring the genes of neutralizing or
protective antibodies. Antibody-mediated immunity
could thus also be obtained against viruses such as
HIV-1 for which currently no vaccines are available, but
panels of potently neutralizing antibodies exist [72,73].
Antibody gene transfer would further simplify the
development of therapeutic antibody cocktails, which
are often considered essential for targeting genetically
diverse or rapidly mutating pathogens. The develop-
ment of antibody cocktails using conventional technol-
ogyishugelyexpensiveanddifficultbecauseof
regulatory hurdles.
CONCLUSIONS
Advances in gene-transfer approaches are enabling the
possibility of applying therapeutic antibodies using DNA.
In particular gene transfer in combination with electro-
poration is promising and can result in generating in vivo
antibody concentrations in the low therapeutic range.
Therapeutic monoclonal antibodies are primarily avail-
able for the treatment of cancer and inflammation
conditions. The use of antibody gene transfer in such
conditions is currently unlikely as the antibodies used
often recognize self-antigens. As antibody production
following gene transfer is difficult to control, the danger
of inducing autoimmune conditions or uncontrollable
side effects occurs. Promising applications might exist in
infectious diseases for which this technology makes it
possible to immunize with a focused antibody response
consisting of well-defined antibodies with potent activity
against the intended pathogen; a difficult feature to
attain even with successful vaccines. To enable long-term
expression and avoid problems linked to immunogenic-
ity, only genes encoding fully human antibodies should
be transferred.
A
CKNOWLEDGMENTS
We thank Dennis Burton, Iacob Mathiesen, and Jan van de Winkel for helpful
comments on the manuscript.
RECEIVED FOR PUBLICATION JUNE 2, 2004; ACCEPTED JUNE 18, 2004.
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REVIEW
doi:10.1016/j.ymthe.2004.06.865
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