Production of antibodies in plants and their use for global health
Rainer Fischer a,b,∗, Richard M. Twyman c, Stefan Schillberg a
a Fraunhofer Institute for Molecular Biology and Applied Ecology, IME, Grafschaft, Auf dem Aberg 1, 57392 Schmallenberg, Germany
b Institute for Molecular Biotechnology, Biology VII, RWTH Aachen, Worringerweg 1, 52074 Aachen, Germany
c Department of Biology, University of York, Heslington, York YO10 5DD, UK
Abstract
Recombinant antibodies can be used to diagnose, treat and prevent disease by exploiting their specific antigen-binding activities. A
large number of drugs currently in development are recombinant antibodies and most of these are produced in cultured rodent cells.
Although such cells produce authentic functional products, they are expensive, difficult to scale-up and may contain human pathogens.
Plants represent a cost-effective, convenient and safe alternative production system and are slowly gaining acceptance. Five plant-derived
therapeutic recombinant antibodies (plantibodies) are undergoing clinical evaluation, three of which can be used as prophylactics.
© 2002 Elsevier Science Ltd. All rights reserved.
Keywords: Recombinant antibody; Passive immunity; Transgenic plants
1. The importance of recombinant antibodies
to human health
Antibodies are complex glycoproteins, produced by the
vertebrate immune system, which recognize and bind to
target antigens with great specificity. This individual and
specific binding activity allows antibodies to be used for
a variety of applications, including the diagnosis, preven-
tion and treatment of disease [1–3]. It is estimated that
approximately 1000 therapeutic recombinant antibodies are
being developed by biopharmaceutical companies around
the world and over 200 of these are already in clinical tri-
als. A large proportion of these antibodies recognize cancer
antigens, but others have been developed for the diagnosis
and treatment of infectious diseases, autoimmune disorders,
cardiovascular disease, blood disorders, neurological disor-
ders, skin disorders, respiratory diseases, eye diseases and
transplant rejection [4].
2. Evolution of recombinant antibody technology
. Structure of naturally produced antibodies
Mammalian serum antibodies comprise two identical
heavy chains and two identical light chains joined by disul-
fide bonds (Fig. 1). Each heavy chain is folded into four
domains, two either side of a flexible ‘hinge’ which allows
∗ Corresponding author. Tel.: +49-241-8026631; fax: +49-241-871062.
E-mail address: fischer@ (R. Fischer).
the multimeric protein to adopt its characteristic Y-shape.
Each light chain is also folded into two domains. The
N-terminal domain of each of the four chains is variable,
. it differs among individual B-cells due to unique rear-
rangements of the germ-line immunoglobulin genes. This
part of the molecule is responsible for antigen recognition
and binding. The remainder of the antibody comprises a
series of constant domains, which are involved in effector
functions such as immune cell recognition and complement
fixation. Below the hinge, in what is known as the Fc por-
tion of the antibody, the constant domains are class specific.
Mammals produce five classes of immunoglobulins (IgG,
IgM, IgA, IgD and IgE) with different effector functions.
The Fc region also contains a conserved asparagine residue
at position 297 to which N-glycan chains are added. The
glycan chains play an important role both in the folding
of the protein and the performance of effector functions
[5]. Antibodies are found in mucosal secretions as well as
serum. These secretory antibodies have a more complex
structure than their serum counterparts. They are dimers of
the serum-type antibody, the two monomers being attached
by an additional component called the joining chain. There
is also a further polypeptide called the secretory component,
which protects the antibodies from proteases.
. Antibody derivatives
The constant regions of native immunoglobulins are not
required for antigen recognition, so it is possible to express
smaller derivative molecules and still retain antigen-binding
specificity [2,3]. Such derivatives include Fab and F(ab′)2
0264-410X/02/$ – see front matter © 2002 Elsevier Science Ltd. All rights reserved.
PII: S0 2 6 4 -410X(02 )00607 -2
R. Fischer et al. / Vaccine 21 (2003) 820–825 821
Fig. 1. Types of recombinant antibody expressed in plants: rAb, recombinant antibody; Fab, fragment antigen binding; scFv, single chain Fv fragment;
dAb, single domain antibody.
fragments (which contain only the sequences distal to the
hinge region) and single chain Fv fragments (scFvs) which
contain the variable regions of the heavy and light chains
joined by a flexible peptide chain. Such derivatives are often
more effective as drugs than full-length immunoglobulins,
because they show increased penetration of target tissues,
reduced immunogenicity and they are cleared from tissues
more rapidly. Other derivatives include bispecific scFvs,
which contain the antigen-recognition elements of two
different immunoglobulins and can bind to two different
antigens [6], and scFv-fusions, which are linked to proteins
with additional functions [7,8] (Fig. 1).
. Humanized antibodies
The traditional source of monoclonal antibodies is murine
B-cells. To provide a constant source of the antibody, B-cells
of appropriate specificity are fused to immortal myeloma
cells to produce a hybridoma cell line. The use of murine
hybridoma-derived antibodies as therapeutics is limited, be-
cause the murine components of the antibodies are immuno-
genic in humans. Therefore, numerous strategies have been
developed to humanize murine monoclonal antibodies [9]
culminating in the production of transgenic mice expressing
the human immunoglobulin repertoire [10]. An alternative
approach is to use phage display libraries based on the hu-
man immune repertoires for the production of scFvs. Phage
display is advantageous, because high-affinity antibodies can
be rapidly identified, novel combinations of heavy and light
chains can be tested and the DNA sequence encoding the
antibody is indirectly linked to the antibody itself [11,12].
This avoids the laborious isolation of cDNA or genomic im-
munoglobulin sequences from hybridoma cell lines.
3. Expression systems for recombinant antibodies
. Traditional expression systems
Most of the recombinant full-length immunoglobu-
lins being developed as pharmaceuticals are produced in
mammalian cell culture, predominantly in Chinese hamster
ovary cells or the murine myeloma cell line NS0 [13]. The
main reason for this is the belief that mammalian cells
yield authentic products, particularly in terms of glyco-
sylation patterns. However, there are minor differences in
glycan chain structure between rodent and human cells.
For example, human antibodies contain only the sialic acid
residue N-acetylneuraminic acid (NANA), while rodents
produce a mixture of NANA and N-glycosylneuraminic
acid (NGNA) [14]. There are many disadvantages to mam-
malian cell cultures, including the high set-up and running
costs, the limited opportunities for scale-up and the po-
tential contamination of purified recombinant antibodies
with human pathogens. Bacterial fermentation systems are
more cost-effective than mammalian cell cultures and are
therefore preferred for the production of Fab fragments and
scFvs since these derivatives are not glycosylated. Even so,
the yields of such products in bacteria are generally low,
because the proteins do not fold properly [2].
A more recent development is the production of antibod-
ies in the milk of transgenic animals [15,16]. Recombinant
proteins can be harvested periodically, and the yields are po-
tentially very high. However, the production of transgenic
farm animals is a difficult process and involves a long de-
velopment phase with many regulatory hurdles. Scale-up is
slow, being dependent on the animal’s natural breeding cy-
cle, and it is necessary to maintain a founder herd carrying
the transgene of interest. As with mammalian cell lines, there
are safety concerns about the transmission of pathogens or
oncogenic DNA sequences.
. The advantages of plants
Plants offer a unique combination of advantages for the
production of pharmaceutical antibodies [3,17–19]. The
main benefit is the low production costs, reflecting the fact
that traditional agricultural practices and unskilled labor
are sufficient for maintaining and harvesting transgenic
crops. Also, large-scale processing infrastructure is already
in place for most crops. Scale-up is rapid and efficient,
requiring only the cultivation of additional land. Unlike
transgenic animals, there is no need to maintain a founder
herd, because transgenic plant lines can be stored as seed.
822 R. Fischer et al. / Vaccine 21 (2003) 820–825
There are minimal risks of contamination with human
pathogens.
The general eukaryotic protein synthesis pathway is con-
served between plants and animals, so plants can efficiently
fold and assemble full-size serum immunoglobulins (as first
demonstrated by Hiatt et al. in 1989 [20]) and secretory IgAs
(first shown by Ma et al. in 1995 [21]). In the latter case, four
different subunits need to assemble in the same plant cell to
produce a functional product, even though two different cell
types are required in mammals. The post-translational mod-
ifications carried out by plants and animals are not identi-
cal. There are minor differences in the structure of complex
glycans, such as the presence of the plant-specific residues
�1,3-fucose and �1,2-xylose [22]. However, studies using
mice administered a recombinant IgG isolated from plants
showed that, while there were some differences in the gly-
can groups present on the recombinant antibody, neither the
antibody nor the glycans were immunogenic [23]. It there-
fore seems likely that many therapeutic antibodies could
be safely expressed in plants. As well as full-size antibod-
ies, various functional antibody derivatives have also been
produced successfully in plants, including Fab fragments,
scFvs, bispecific scFvs, single domain antibodies and anti-
body fusion proteins (reviewed in [3]).
4. Procedure for recombinant antibody expression in
transgenic plants
. Expression construct design
Once the appropriate cDNA has been isolated from a hy-
bridoma cell line or a phage display library, it must be in-
serted into a plant vector designed for high-level expression
(reviewed in [24]). A strong constitutive promoter is chosen
to maximize transcription. The cauliflower mosaic virus 35S
(CaMV 35S) promoter is widely used in dicot (broad leaf)
plants and the maize ubiquitin promoter is favored for use in
monocots (cereals). The presence of an intron often increases
the rate of transcription, so introns are also included in most
plant expression constructs. Seed-specific promoters can be
used to prevent recombinant antibodies accumulating in veg-
etative tissues and interfering with plant growth and develop-
ment, but such interference has generally not been a problem
when expressing antibodies raised against human antigens.
Protein synthesis can be optimized by making sure the trans-
lational start site conforms to the Kozak consensus for plants
and replacing any native untranslated sequence with transla-
tional enhancers such as the 5′ leader sequence derived from
tobacco mosaic virus (TMV) RNA (the omega sequence).
The most important consideration in expression construct
design is protein targeting. This is significant for several
reasons.
• Recombinant antibodies are more stable in some intra-
cellular compartments than others, and this contributes to
the overall yield.
• Full-length immunoglobulins must be targeted to the se-
cretory pathway to undergo glycosylation.
• Appropriate targeting can also facilitate protein isolation
and purification.
It is clear that targeting to the secretory pathway is the
best choice for maximizing yields of functional recombi-
nant antibodies [25–27]. The environment of the endoplas-
mic reticulum (ER) favors correct folding and assembly of
immunoglobulin chains, while glycosylation occurs in the
ER and Golgi apparatus. The highest yields are achieved by
retrieving antibodies from the Golgi apparatus and return-
ing them to the ER lumen in the manner of a resident ER
protein [28,29]. This is made possible by including in the
expression construct an N-terminal signal sequence (which
directs proteins to the secretory pathway) and a C-terminal
tetrapeptide signal, KDEL (which facilitates retrieval from
the Golgi apparatus). Recombinant antibodies accumulating
in the ER may reach levels 10,000-fold higher than those
expressed in the cytosol.
In the absence of a KDEL signal, the default destination
of a recombinant antibody is the apoplast (the space be-
tween the plasma membrane and the cell wall). This may
be an appropriate strategy in plant cell cultures, because it
facilitates extraction. Small molecules such as scFvs dif-
fuse through the wall and can be collected from the culture
medium, whereas larger molecules such as full-size antibod-
ies remain trapped in the apoplast, but can be released by
mild enzymatic digestion of the cell wall [29].
. Transgenic plants
The majority of plant-derived antibodies have been
expressed in transgenic plants. This requires stable transfor-
mation and is achieved either through the use of Agrobac-
terium tumefaciens or particle bombardment, depending on
the species [24]. Transformation results in the integration
of the antibody transgene into the plant’s genome and pro-
duces a permanent resource, a stably expressing transgenic
line. The transformation process itself is rapid, but it can
take several months to regenerate the first transgenic plants.
Once transgenics are available, they must be characterized
and scaled up for production. All together, about 2 years
may be required before routine production is possible [2].
. Choice of species
A large number of different crops can now be used to pro-
duce antibodies including tobacco (Nicotiana tabaccum and
N. benthamiana), cereals (rice, wheat, maize), legumes (pea,
soybean, alfalfa) and fruit and root crops (tomato, potato)
[24]. Many factors need to be considered before making a
choice and each case must be evaluated on its own merits
[30,31]. Leafy crops such as tobacco and alfalfa generally
have the greatest biomass yields per hectare, because they
can be cropped several times a year. Tomatoes also have
R. Fischer et al. / Vaccine 21 (2003) 820–825 823
a high biomass yield, but production costs are increased
because greenhouses are required. However, this may have
advantages in terms of containment compared to field
crops.
For valuable proteins, like antibodies, a more expensive
production system would be justified if other advantages
were apparent. For example, antibodies expressed in potato
tubers and cereal grains are stable at room temperature for
months or even years without loss of stability, while tobacco
leaves must be dried or frozen prior to transport or storage
to maintain the activity of recombinant proteins. However,
extraction of proteins from seeds is more expensive than
from watery tissue, such as tomatoes. The vast majority of
production costs for any recombinant protein reflect down-
stream processing, so this is likely to be one of the most
important economic considerations for the commercial pro-
duction of recombinant antibodies in plants. The presence
of toxic metabolites such as alkaloids in tobacco presents a
disadvantage in this context, so edible plants lacking such
compounds would be preferred expression hosts. Since most
pharmaceutical antibodies will be produced by industry, the
costs of production and processing in different crops will
have to be evaluated very carefully.
. Alternatives to transgenic plants
The main disadvantage of transgenic plants is the long
development phase and associated costs. Several alternative
approaches are available that reduce the development pe-
riod to a matter of days or weeks. The first is transient ex-
pression, which is generally used to evaluate the activity of
expression constructs or test the functionality of a recom-
binant protein before committing to transgenics. However,
if enough protein can be produced, transient expression can
also be used as a routine method for antibody production.
Agroinfiltration is a transient expression method involving
the infiltration of A. tumefaciens cells into tobacco leaf tis-
sue. The onset of expression is very rapid (days to weeks)
and milligram amounts of protein can be produced at each
‘harvest’. However, one disadvantage of this system is that
scale-up is uneconomical [32].
The second alternative strategy is the use of plant viruses
as vectors. Like agroinfiltration, virus infection does not re-
sult in stable transformation of the plant, but the onset of
recombinant antibody expression is rapid. The advantage of
viral vectors over agroinfiltration is that the yields are po-
tentially much higher (due to both the high level expres-
sion and the systemic spread of the virus) and that scale-up
is achieved easily by manual inoculation. TMV and potato
virus X (PVX) vectors have both been used for the produc-
tion of recombinant antibodies in tobacco, including scFv
fragments and a full-size immunoglobulins [33–36]. In the
latter case, two TMV vectors were required, one expressing
the heavy chain and one the light chain. Co-infection of to-
bacco plants resulted in the correct assembly of a functional
antibody [36].
Finally, plant cell cultures can also be used for the pro-
duction of therapeutic antibodies. In this case, the cells are
stably transformed with the antibody transgene but, since
regeneration is unnecessary, this is a relatively straightfor-
ward process and does not take very long. The onset of
protein synthesis is rapid and yields of up to 25 mg/l of
recombinant antibody are possible. The requirement for
fermentors or shaker flasks and skilled personnel makes
the set-up and running costs greater than for systems based
on whole plants, but extraction and purification is easier
and there is the added bonus of sterility and containment,
which may be particularly suitable for valuable pharmaceu-
tical products. Both tobacco and rice suspension cells have
been used for the production of recombinant antibodies
[29,37].
5. Case studies
Six plant-derived antibodies have been developed as hu-
man therapeutics, two of which have reached phase II clin-
ical trials. One of these is a full-length IgG specific for
EpCAM (a marker of colorectal cancer) developed as the
drug Avicidin by NeoRx and Monsanto. Although Avicidin
demonstrated some anti-cancer activity in patients with ad-
vanced colon and prostate cancers, it was withdrawn because
it also resulted in a high incidence of diarrhoea [4]. This
was possibly due to cross-reaction with related epitopes on
the cells lining the intestine. The five remaining antibodies
are discussed below.
. CaroRx
The other plant-derived antibody currently in phase II
clinical trials is CaroRx, a chimeric secretory IgA/G pro-
duced in transgenic tobacco plants [21,38,39]. Secretory
antibody production required the expression of the four sep-
arate components in four different plant lines, which were
crossed over two generations to eventually stack all the
transgenes in one line. The antibody is specific for the major
adhesin of Streptococcus mutans, the organism responsible
for tooth decay in humans. Topical application following
elimination of bacteria from the mouth helps to prevent
recolonization by S. mutans and leads to the replacement
of this pathogenic organism with harmless endogenous
flora.
. , and
is a monoclonal antibody that recognizes carci-
noembryonic antigen (CEA), a well-characterized tumor-
associated glycoprotein. This antigen is a widely used
marker for colorectal adenocarcinomas as well as lung,
breast and pancreatic carcinomas, and other carcinomas
of epithelial origin. is one of several anti-CEA
antibodies to have been evaluated for tumor imaging and
824 R. Fischer et al. / Vaccine 21 (2003) 820–825
therapy. The full-length IgG has been transiently expressed
in tobacco by agroinfiltration [32] and the scFv deriva-
tive has been expressed in transgenic tobacco, pea [40],
rice and wheat [41] as well as tobacco and rice cell sus-
pension cultures [37]. We have also produced a diabody,
by agroinfiltration of tobacco and in transgenic
tobacco plants [42] and a diabody fusion with interleukin-2
(IL-2).
. Anti-HSV
A full-length humanized IgG recognizing herpes simplex
virus 2 (HSV-2) has been expressed in transgenic soybean
[43]. Although not tested for efficacy in humans, topical ap-
plication of the antibody has been shown to prevent vaginal
HSV-2 transmission in a mouse model of the disease. The ac-
tivity of the plant-derived antibody both in vitro and in vivo
was indistinguishable from that of the cell culture-derived
monoclonal antibody on which it was based.
. 38C13
The production of anti-idiotype antibodies recognizing
malignant B-cells is a useful approach for the treatment of
diseases such as non-Hodgkin’s lymphoma. McCormick
et al. [35] produced a plant-derived scFv based on the
well-characterized mouse lymphoma cell line 38C13. When
administered to mice, the scFv stimulated the production
of anti-idiotype antibodies capable of recognizing 38C13
cells. This provided immunity against lethal challenge
with the lymphoma. It is envisaged that this strategy could
be used as a rapid production system for tumor-specific
vaccines customized for each patient and capable of rec-
ognizing unique markers on the surface of any malignant
B-cells. The rapid derivation of such prophylactic antibod-
ies would be desirable. Therefore, it is noteworthy that
the scFvs in this study were produced using viral vectors
and were isolated from virus-infected plants, not transgenic
plants.
. PIPP (anti-hCG)
We recently expressed three types of antibody specific
for human chorionic gonadotropin (hCG) in tobacco plants
transiently transformed by agroinfiltration. These were a
chimeric full-size IgG, an scFv fragment and a diabody,
each derived from the original monoclonal anti-hCG anti-
body known as PIPP [44]. Each of the antibodies was func-
tional in vitro and in vivo. The in vitro test demonstrated
inhibition of the hCG-stimulated production of testosterone
by cultured Leydig cells, while the in vivo test revealed in-
hibition of uterine weight increase in mice. The full-size
antibody was 1000 times more active than either derivative.
These antibodies could potentially be used for pregnancy de-
tection, (emergency) contraception and the diagnosis and/or
therapy of tumors that produce hCG.
6. Future prospects
The case studies discussed above show that efficacious
therapeutic antibodies can be produced rapidly and inexpen-
sively in transgenic plants or other plant expression systems.
The economical advantages of adopting plants as bioreac-
tors on a larger scale would reduce the cost of antibody
therapy and increase the number of patients with access to
these treatments. The widespread acceptance of plants will
be more likely when key bottlenecks are addressed such as
the yield after extraction, regulatory issues and plant-specific
glycans. It is therefore interesting to note that several strate-
gies have been developed to overcome these limitations, in-
cluding the co-expression in transgenic plants of mammalian
glycosyltransferases, to facilitate the production of antibod-
ies with authentic glycan chains [45].
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Production of antibodies in plants and their use for global health
The importance of recombinant antibodies to human health
Evolution of recombinant antibody technology
Structure of naturally produced antibodies
Antibody derivatives
Humanized antibodies
Expression systems for recombinant antibodies
Traditional expression systems
The advantages of plants
Procedure for recombinant antibody expression in transgenic plants
Expression construct design
Transgenic plants
Choice of species
Alternatives to transgenic plants
Case studies
CaroRx
, and
Anti-HSV
38C13
PIPP (anti-hCG)
Future prospects
References
