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European Biopharmaceutical Review
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Kathleen L Hefferon at Cornell
Research Foundation reviews the latest developments in plant expression
platforms for therapeutic proteins
Transgenic plants show great promise as a production platform for
pharmaceutical proteins. Plant-derived vaccines can be considered by
many to be an attractive alternative to non-conventional vaccines with
respect to safety and effectiveness, by facilitating oral delivery
through consumption of edible plant tissue (1). As the mode of entry
for many infectious diseases is through mucosal surfaces such as the
gut, plant-derived vaccines offer a select advantage since plant
tissues protect the antigen as it passes through the digestive tract.
Unlike bacteria, plants are capable of producing recombinant antigens
that undergo similar posttranslational modifications as their
mammalian-derived counterparts. Moreover, yields of plant-derived
biopharmaceuticals can be as high as 45 per cent of a plant cell’s
total soluble protein, depending on the specific production platform.
Indeed, quantities approaching 250mg of protein per litre have been
determined for foreign protein production in some plant cell culture
systems (2). After purification steps, the cost of producing
plant-derived proteins is only a fraction of the cost of proteins
produced from analogous mammalian cell culture systems.
An intrinsic difference between plantderived and traditional
vaccines made in mammalian cell cultures can be found in their
respective glycosylation motifs. Many mammalian therapeutic proteins
are in fact glycoproteins and possess specific N- and O- glycosylation
motifs which are not necessarily found in plants. In certain instances,
these changes may induce increased allergenicity or other adverse side
effects. Indeed, studies using various plant-type N-glycans attached to
a human monoclonal antibody evoked an IgE response in allergic
patients, providing evidence for the adverse potential of nonmammalian
N-glycan modifications (3). This emphasises the need for the use of
glyco-engineered plants lacking any potentially antigenic glycosylation
structures for the production of plantderived recombinant proteins
intended for human application. Further humanisation of plant-derived
therapeutic proteins and immunoglobulins has been achieved by altering
glycosylation pathways which predominate in plant but not mammalian
cells (4). This has been accomplished by incorporating the necessary
glycosylation pathways in transgenic plants, as well as producing
knockout plants which no longer produce the specific glycosylation
motifs which are prone to cause adverse effects in humans. Therapeutic
plant proteins can be further designed to accumulate in the endoplasmic
reticulum of plant cells; in this way, they can avoid undesired
translational modifications as well as retain their stability in a
plant cellular environment. Future work, such as the engineering of
plants to express glycoproteins which are correctly sialylated and
O-glycosylated, will further enhance the development and applications
of plant-derived proteins in medicine.
While plant nuclear or plastid transformation represents one
approach for the generation of biopharmaceutical proteins in plants,
other production systems are also routinely used. For example, plant
virus expression vector systems can be utilised to produce large
quantities of therapeutic proteins within a relatively short timeframe
(from a few days to a week or two, depending on the virus-host system).
Alternatively, a more recent technique known as agroinfection involves
dipping nontransformed plants into a solution of Agrobacteria
harbouring the pharmaceutical gene of interest. Plants infected in this
fashion are capable of rapidly producing larger quantities of the
desired protein. The choice of plant expression platform for
pharmaceutical protein production therefore becomes a matter of
determining the optimal plant species, whether it be whole plant or
cell culture, and whether stable transformation or transient expression
best fits the nature of the therapeutic protein under investigation and
its proposed applications.
One of the major driving forces behind the use of plants as
delivery systems for therapeutic protein production has been the need
to rapidly produce costeffective, safe and easily transportable
vaccines to combat infectious diseases, which are major causes of
mortality in developing countries. Enterotoxigenic E coli (ETEC)
and Norwalk Virus (NV) represent two of the most devastating diarrheal
diseases for children residing in the Third World. As a consequence,
initial clinical trials involving plantderived vaccines concerned
feeding healthy adult volunteers transgenic potato or corn expressing
either LT-B or NV in a randomised, double-blind fashion. Preliminary
studies in which antigens are eaten directly within edible plant
tissues indicated that both humoral and systemic immune responses can
be directly induced. Further studies using LT-B expressed in soybean
and cornseed respectively demonstrated that both IgG and IgA responses
could be evoked in mice and could partially protect them against a
challenge by LT (5).
In addition to this, LT-B and NV have been shown to successfully
act as carriers for other antigens. Norovirus capsid proteins expressed
in plant cells assemble into virus-like particles (NVLPs) that mimic
the antigenic structure of authentic virions. NVLPs are immunogenic
when delivered orally, suggesting that they would be suitable
candidates as vaccinedelivery vehicles. Since oral and nasal delivery
of NVLPs efficiently produce antibodies at distal mucosal sites, NVLPs
could be used to deliver any antigen in the form of a chimaeric fusion
protein (6).
Functional monoclonal antibodies (mAb) are also now routinely
produced in plants. Initially, this took place by crossing four
transgenic plant lines expressing both heavy and light immunoglobulin
domains, the J chain and the secretory component, so that all four
proteins were simultaneously produced and assembled into a single
immunoglobulin in the progeny plants. Studies using an antirabies human
mAb developed in tobacco plants have demonstrated that these
plant-derived mAbs exhibit anti-rabies virus neutralising activity and
affinity comparable to their mammalian-derived counterparts (7).
Tobacco-derived monoclonal IgG antibodies against the tumour-associated
antigen tenascin-C (TNC) were also shown to be biologically functional,
providing further evidence for the potential use of plant-derived
therapeutic products in medical settings (8). Another use in which
plant-derived immunoglobulins show promise is against the human
papillomavirus (HPV), which continues to be a major health problem in
developing countries. To date, several HPV genes have been expressed in
plants, including L1, which like NV capsid protein possesses the
ability to self-assemble into virus-like particles. Recently, tomato
plants expressing chimeric HPV 16 VLPs containing L1 fused to a string
of T-cell epitopes from HPV 16 E6 and E7 proteins were developed. The
particles were able to induce a significant antibody and cytotoxic
T-lymphocyte response (9).
Plant-derived vaccines can also be used to induce oral tolerance to
common allergies. As a proof-of-concept study of the ability of oral
tolerance to be induced by plant-derived antigens, transgenic rice
plants have been generated which accumulate mouse T-cell epitope
peptides corresponding to pollen allergens of Japanese Cedar (10). Mice
who consumed transgenic rice exhibited allergen-induced oral tolerance
prior to systemic challenge. This systemic unresponsiveness
corresponded with a reduction of pollen allergen-specific Th2-mediated
IgE response and histamine release.
On the other hand, there is some concern that orally administered
plantderived vaccines may in fact lead to the development of tolerance
to vaccines, or perhaps to allergies from co-administered food
proteins. If one were to accidently consume a plant-derived vaccine,
later exposure to the same vaccine antigen may result in an ineffective
response, and in turn, a reduced ability of the immune system to
eliminate infection. These concerns have been addressed in experiments
in which the maximum non-stimulatory dose of cornderived LT-B has been
determined in mice (11). Based on these studies, a threshold level of
orally administered plant-derived LT-B which did not stimulate
detectable levels of antibody, but could nonetheless induce immune
priming, was identified. The relationship between oral administration
of plant-derived antigens and the immune response continues to create
great interest and has currently become a focal point of research
regarding plant expression platform technologies.
Plant-derived vaccines have been produced in a variety of settings,
including cell culture, the field and the greenhouse. Since variations
in soil and weather in outdoor fields can compromise the good
manufacturing practice essential for commercial pharmaceutical
production, cell suspensions are often the method of choice as they can
be grown in precisely controlled environments. Continuous culture of
plant cell lines which secrete the specific protein product into the
surrounding media is one way to reduce expensive downstream processing.
As technologies advance and become even more sophisticated, expression
levels of plant-derived proteins will increase. In general, protein
purification from plant tissue requires fewer steps and is
significantly less expensive than purification from their mammalian and
bacterial counterparts. It is likely that in many cases, the
plant-derived biopharmaceutical will only need to be partially
purified, rendering it even less intensive in terms of labour and cost.
It is more than likely that, in many instances, these partially
purified therapeutic proteins will appear in the form of a capsule or
suspension, to keep the protein at consistent levels for oral
consumption. The first plant-derived vaccine, a poultry vaccine for
Newcastle disease which was produced in plant cell culture, is now
commercially available (12). Many other plant-derived therapeutic
proteins are completing clinical trials and approach market release.
Plant-derived vaccines provide an opportunity to develop safer, more
effective and less expensive vaccination strategies against mucosal
pathogens. This year, the GreenVax Project in the US is utilising a
plant-based expression system to produce the H1N1 swine flu vaccine
rapidly and in large quantities, to be stockpiled and on hand for
potential future pandemics (13). The time has come for plant-derived
biopharmaceuticals to make their mark.
References
- Thanavala Y, Huang Z and Mason H, Plant-derived vaccines: a look
back at the highlights and a view to the challenges on the road ahead, Expert Review on Vaccines 5(2): pp249-260, 2006
- McDonald KA, Hong LM, Trombly DM, Xie Q and Jackman AP,
Production of human alpha-1- antitrypsin from transgenic rice cell
culture in a membrane bioreactor, Biotechnology Progress 21(3): pp728-734, 2005
- Jin C, Altmann F, Strasser R, Mach L, Schähs M, Kunert R,
Rademacher T, Glössl J and Steinkellner H, A plant-derived human
monoclonal antibody induces an anti-carbohydrate immune response in
rabbits, Glycobiology 18(3): pp235-324, 2008
- Bardor M, Cabrera G, Rudd PM, Dwek RA, Cremata JA and Lerouge P,
Analytical strategies to investigate plant N-glycan profiles in the
context of plant-made pharmaceuticals, Current Opinion in Structural Biology 16(5): pp576-583, 2006
- Tacket CC, Plant based oral vaccines: results of human trials, Current Topics in Microbiology and Immunology 332: pp103-117, 2009
- Herbst-Kralovetz M, Mason HS and Chen Q, Norwalk virus-like particles as vaccines, Expert Review Vaccines 9(3): pp299-307, March 2010
- Ko K and Koprowski H, Plant biopharming of monoclonal antibodies, Virus Research 111(1): pp93-100, 2005
- Villan ME, Morgun B, Brunetti P, Marusic C, Lombardi R, Pisoni I,
Bacci C, Desiderio A, Benvenuto E and Donini M, Plant pharming of a
full-sized, tumour-targeting antibody using different expression
strategies, Plant Biotechnology Journal 7(1): pp59-72, 2009
- Paz De la Rosa G, Monroy-García A, de Lourdes Mora-García M,
Gehibie Reynaga Peña C, Hernández-Montes J, Weiss-Steider B and Gómez
MA, An HPV 16 L1-based chimeric human papilloma virus-like particles
containing a string of epitopes produced in plants is able to elicit
humoral and cytotoxic T-cell activity in mice, Virology Journal 6: p2, 2009
- Takagi H, Hirose S, Yasuda H and Takaiwa F, Biochemical safety
evaluation of transgenic rice seeds expressing T cell epitopes of
Japanese cedar pollen allergens, Journal of Agricultural and Food Chemistry 54(26): pp:9,901-9,905, 2006
- Beyer AJ, Wang K, Umble AN, Wolt JD and Cunnick JE, Low-dose
exposure and immunogenicity of transgenic maize expressing the
Escherichia coli heat-labile toxin B subunit, Environmental Health Perspectives 115(3): pp354-360, 2007
- www.dowagro.com/newsroom/ corporatenews/2006/20060131b.htm
- Naik G, Teasing Vaccines from Tobacco, Wall Street Journal, Feb 24, 2010, online.wsj.com/.../ SB1000142405274870350380457 5083611168442980.html
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