Transgenic Research 9: 279–299, 2000. © 2000 Kluwer Academic Publishers. Printed in the Netherlands.
279
Molecular farming of pharmaceutical proteins Rainer Fischer1,2,∗ & Neil Emans1 1 Institut
für Biologie I (Botanik/Molekulargenetik), RWTH Aachen, Worringerweg 1, D-52074 Aachen, Germany Department for Molecular Biotechnology, IUCT, Grafschaft, Auf dem Aberg 1, D-57392 Schmallenberg, Germany 2 Fraunhofer
Key words: molecular farming, recombinant protein, transgenic plant, antibody, expression, purification, biotechnology
Abstract Molecular farming is the production of pharmaceutically important and commercially valuable proteins in plants. Its purpose is to provide a safe and inexpensive means for the mass production of recombinant pharmaceutical proteins. Complex mammalian proteins can be produced in transformed plants or transformed plant suspension cells. Plants are suitable for the production of pharmaceutical proteins on a field scale because the expressed proteins are functional and almost indistinguishable from their mammalian counterparts. The breadth of therapeutic proteins produced by plants range from interleukins to recombinant antibodies. Molecular farming in plants has the potential to provide virtually unlimited quantities of recombinant proteins for use as diagnostic and therapeutic tools in health care and the life sciences. Plants produce a large amount of biomass and protein production can be increased using plant suspension cell culture in fermenters, or by the propagation of stably transformed plant lines in the field. Transgenic plants can also produce organs rich in a recombinant protein for its long-term storage. This demonstrates the promise of using transgenic plants as bioreactors for the molecular farming of recombinant therapeutics, including vaccines, diagnostics, such as recombinant antibodies, plasma proteins, cytokines and growth factors.
Introduction The use of plants in medicine stretches back to the earliest stages of civilization. As early as 1600 BC, the Egyptians compiled a list of more than 700 medicinal plants. The active ingredients in many of these plants have now been identified, and close to one quarter of prescription drugs are still of plant origin. Modern biotechnology is extending the use of plants in medicine well beyond its original boundaries. Plants are now a source of pharmaceutical proteins, such as mammalian antibodies (Hiatt et al., 1989; Düring et al., 1990; Hiatt, 1990; Whitelam et al., 1994; Ma et al., 1995; Conrad & Fiedler, 1998; Larrick et al., 1998; Zeitlin et al., 1998; Fischer et al., 1999a), blood substitutes (Magnuson et al., 1998) and vaccines (Arakawa et al., ∗ Author for correspondence: Institut für Biologie I Tel.: +49 241 806631; Fax: +49 241 871062; E-mail:
[email protected]
1998a; Haq et al., 1995; Kapusta et al., 1999; Mason & Arntzen, 1995; Mason et al., 1992; McGarvey et al., 1995; Walmsley & Arntzen, 2000). Molecular farming is the production of pharmaceutically important and commercially valuable proteins in plants (Franken et al., 1997). It harnesses heterologous protein expression systems, such as plants, for the large-scale production of recombinant proteins that are therapeutically valuable. Here, we briefly review its history, then discuss plant expression technology and how plant cells can be optimally used to produce recombinant proteins by molecular farming. In its simplest form, molecular farming is the expression of recombinant insulin in bacteria; in its most challenging form, molecular farming is the production of chimeric anti-tumor antibodies and multi-subunit protein complexes, like secretory IgA, in plants. This technology has now reached the point where it is commercially viable. Its further development will bring
280 many therapies that are now too expensive for wide use into the hands of most medical practitioners. We anticipate that molecular farming may be a major area of economic growth in agricultural biotechnology. In our definition, molecular farming encompasses the production of recombinant proteins in a heterologous expression system. In general, the recombinant proteins that we choose to express are pharmaceutically valuable, such as a tumor or pathogen specific antibody. Consequently, the expression of an antibody in tobacco is molecular farming while the purification of that antibody from its native source is not. Molecular farming a protein also implies that large-scale production of a recombinant protein is both possible and economically feasible. Thus, production of an antibody in a field of transgenic tobacco is closer to molecular farming than the production of the antibody in a hybridoma culture. Molecular farming is principally the production of a pharmaceutical protein and not the modification of the expression system. For example, the creation of a pathogen resistant plant by the expression of an anti-pathogen antibody is only an application of the molecular farming technology, but it is not molecular farming according to our definition. Molecular farming is thus best defined as the expression of pharmaceutically and commercially valuable proteins in plants. It was developed based on the pretext that many pharmaceutically active proteins have been identified but without the means to produce them, their therapeutic potential can not be evaluated. The purpose of molecular farming is to produce large amounts of an active, safe pharmaceutical protein at an affordable price. As such, there are two stages to molecular farming – the development of an optimized expression system and its scale up to economic levels of production. We feel that plants are the expression system that best meets these prerequisites. Further developments in protein expression in plants should make molecular farming an even more attractive alternative to animals, animal cells and microbial cells. In this review, after a brief historical overview of the development of molecular farming, we concentrate on discussing the expression of pharmaceutical proteins in plants through stable or transient expression in whole plants, leaves or seeds. The economic and practical advantages of plants are discussed, as is where molecular farming is likely to expand. We then speculate on how the use of plants as an expression system may be improved.
The demand for safe, recombinant pharmaceutical proteins is expanding rapidly. Both the amounts of proteins needed and protein complexity are increasing as novel pharmaceutical activities are identified or designed into macromolecules. We foresee that molecular farming in plants will become a preeminent method for the production of pharmaceutical molecules in the next 10 years.
Molecular farming Molecular farming is the production of recombinant pharmaceuticals outside their natural source. By definition, molecular farming is preceded by identification of a protein with a desirable therapeutic or diagnostic activity, its protein and DNA sequencing and finally its expression in a heterologous host. A classic example of molecular farming in microbes is the expression of recombinant insulin in bacteria. The anti-diabetic activity of insulin was first identified in 1921 and by 1951 the complete amino acid sequence had been determined. Since the standard source of the hormone was animal pancreas, a demand emerged for an alternative source of insulin that was safe, free of immunogenic contaminants and inexpensive. Human insulin was an attractive target for expression in microbes for it is a small polypeptide requiring only minimal post-translational processing to become functional. Expression in bacteria was successful and in 1982 it became the first recombinant protein to be approved for therapeutic use (Walsh, 1998). This released any restrictions on the amounts of insulin available, by producing a safe, active, recombinant human hormone at a low cost. The potential of using plants as a production system for recombinant pharmaceuticals was established between 1986 and 1990 with the successful expression of a human growth hormone fusion protein, an interferon and human serum albumin (Barta et al., 1986; De Zoeten et al., 1989; Sijmons et al., 1990). A crucial advance came with the successful expression of functional antibodies in plants in 1989 (Hiatt et al., 1989) and 1990 (Düring et al., 1990). This was a significant breakthrough for it showed that plants had the potential to produce complex mammalian proteins of medical importance. By analogy to the production of insulin in bacteria, the production of antibodies in plants had the potential to make large amounts of safe, inexpensive antibodies available. This was an impressive result because plants could produce
281
Figure 1. Forms of recombinant antibodies produced by antibody engineering. Ab – Antibody; rAb – recombinant antibody; Fab – fragment antigen binding; scFv – single chain antibody fragment; dAb – single domain antibody; IL-2 – interleukin-2.
functional full-length antibodies, indicating that all the post-translational modifications necessary for antibody activity occurred, which is not the case for full size antibody expression in E. coli. In the following 10 years, plants were shown to be able to produce a variety of antibody fragments (Figure 1), secretory IgA, blood substitutes and biological effectors including interleukins (Tables 2–4).
Plants as a production system Historically, bacteria were often the protein expression system of choice and yeast cells or baculovirusinfected insect cell systems were of minor importance (Skerra, 1993; Taticek et al., 1994). While bacteria are an inexpensive, convenient production system, they are incapable of most of the post-translational modifications necessary for the activity of many mammalian proteins. This limitation and the cost of expression of proteins in mammalian cells prompted the exploration of plants, as a cheap, safe and efficient alternative. Advances in recombinant DNA technology, plant transformation technology and antibody engineering
are major reasons why plants have emerged as an expression system. Antibody expression in plants showed proof that plants were capable of expressing functional mammalian proteins (Hiatt et al., 1989; Ma et al., 1995; Voss et al., 1995) and further progress has made it possible to produce chimeric mouse–human therapeutic antibodies in plants in sufficient quantities for pre-clinical trials (Zeitlin et al., 1998; Vaquero et al., 1999). Plant expression systems are attractive because they offer significant advantages over the classical expression systems based on bacterial, microbial and animal cells (Table 1). Firstly, they have a higher eukaryote protein synthesis pathway, very similar to animal cells with only minor differences in protein glycosylation (Cabanes-Macheteau et al., 1999). Contrastingly, bacteria cannot produce full size antibodies nor perform most of the important mammalian post-translational modifications. Secondly, proteins produced in plants accumulate to high levels (Verwoerd et al., 1995; Ziegler et al., 2000) and plantderived antibodies are functionally equivalent to those produced by hybridomas (Hiatt et al., 1989; Voss et al., 1995). Thirdly, concerns about contamination
282 Table 1. Comparison of features of recombinant protein production in plants, yeast and classical systems
Cost/storage Distribution Gene size Glycosylation Multimeric protein assembly (SIgA) Production cost Production scale Production vehicle Propagation Protein folding accuracy Protein homogeneity Protein yield Public perception of ‘risk’ Safety Scale up costs Therapeutic risk∗ Time required
Transgenic plants
Plant viruses
Yeast
Bacteria
Mammalian cell cultures
Transgenic animals
Cheap/RT Easy Not limited ‘Correct’ ? Yes
Cheap/−20◦ C Easy Limited ‘Correct’ ? No
Cheap/−20◦ C Feasible Unknown Incorrect No
Cheap/−20◦ C Feasible Unknown Absent No
expensive/N2 Difficult Limited ‘Correct’ No
Expensive Difficult Limited ‘Correct’ Yes
Low Worldwide Yes Easy High ?
Low Worldwide Yes Feasible High ?
Medium Limited Yes Easy Medium
Medium Limited Yes Easy Low
High Limited Yes Hard High
High Limited Yes Feasible high
High ? High High
Medium Very high High
Medium High Medium
Low Medium Low
Medium Medium-high Medium
Low high High
Unknown High∗∗
Low High∗∗
Medium High∗∗
High High
Unknown Medium
Yes Low
Yes High
Yes High
High High Low Low (unlimited biomass) Unknown Unknown Medium Low
∗ – residual viral sequences, oncogenes, endotoxins; ∗∗ – large, expensive fermenters etc; ? – unclear.
of expressed proteins with human or animal pathogens (HIV, hepatitis viruses) or the co-purification of blood-borne pathogens and oncogenic sequences, are entirely avoided by using plants. Classical methods of protein expression often require a significant investment in recombinant protein purification (bacteria) or require expensive growth media (animal cells). Bacteria produce contaminating endotoxins that are difficult to remove and bacterially expressed recombinant proteins often form inclusion bodies, making labour- and cost-intensive in vitro refolding necessary. Mammalian cell cultivation can be difficult, requires sophisticated equipment and expensive media supplements, such as foetal calf serum. With these classical expression systems, considerable care must be also taken during downstream processing of recombinant proteins to remove oncogenic sequences, protein or viral contaminants for in vivo therapeutic applications. In addition, the use of transgenic animals (Echelard, 1996) as a source of recombinant antibodies is becoming limited by legal and ethical constraints. Transgenic plants producing high levels of safe, functional recombinant proteins, can be cultivated on
an agricultural scale (Whitelam et al., 1993; Whitelam et al., 1994; Whitelam, 1996) and molecular farming requires only a virus-infected or a transgenic plant, water, mineral salts and sunlight. Current applications of the plant based expression systems in biotechnology include the production of recombinant antibodies (rAbs) (Ma and Hein, 1995a,b), enzymes (Hogue et al., 1990; Verwoerd et al., 1995), hormones, interleukins (Magnuson et al., 1998), plasma proteins (Sijmons et al., 1990) and vaccines (Mason & Arntzen, 1995; Walmsley & Arntzen, 2000). Chimeric plant viruses, produced in plants, can also be used for the presentation of vaccines on the viral surface (Johnson et al., 1997). The ease with which plants can be genetically manipulated, and grown in single cell suspension culture or scaled up for field scale production, is a great advantage over the more commonly used microbial methods, mammalian cell culture and transgenic animal technology. Importantly, antibody expression can also be used as a tool to modify the intrinsic properties of plants. For example, pathogen resistance can be increased by the expression of anti-pathogen antibodies (Tavladoraki et al., 1993; Voss et al., 1995; Fecker
283 Table 2. Selected pharmaceutical proteins expressed in transgenic plants Year
Protein
Transformed species
Reference
1986
Human growth hormone
(Barta et al., 1986)
1990
Human serum albumin
1993 1994 1994 1995
Human epidermal growth factor Trout growth factor Human α-interferon Hirudin
1995
Erythropoetin
1996
Glucocerebrosidase, human protein C serum protease Human α and β haemoglobin Human muscarinic cholinergic receptors Murine granulocyte-macrophage colony stimulating factor Interleukin-2 and Interleukin-4
N. tabacum H. annus N. tabacum S. tuberosum N. tabacum N. tabacum O. sativa N. tabacum Suspension cells N. tabacum Suspension cells N. tabacum N. tabacum N. tabacum
(Dieryck et al., 1997) (Mu et al., 1997)
N. tabacum
(Lee et al., 1997)
N. tabacum Suspension cells N. tabacum Rhizosecretion O. sativa Suspension cells N. tabacum seeds N. tabacum Chloroplasts
(Magnuson et al., 1998)
1997 1997 1997 1998 1999 1999 2000 2000
Human placental alkaline phosphatase Human α1-antitrypsin Human growth hormone (somatotrophin) Human growth hormone (somatotrophin)
et al., 1997; Le Gall et al., 1998; Zimmermann et al., 1998). Expressed antibodies can also be used to alter metabolic or hormonally regulated pathways by binding to intracellular substrates or hormones (Phillips et al., 1997).
Plant expression strategies Plant transformation involves the chromosomal integration of a heterologous gene and this is becoming straightforward. However, there are still technical and logistical hurdles to be overcome, such as developing efficient transformation techniques for all major crop species. Developing plant lines expressing recombinant proteins is time intensive and expensive. In the best case, 8–12 weeks are needed for transgenic plants to be available, but the time required depends on the plant species. Though this is slower
(Sijmons et al., 1990) (Higo et al., 1993) (Bosch et al., 1994) (Zhu et al., 1994) (Parmenter et al., 1995) (Matsumoto et al., 1995) (Cramer et al., 1996)
(Borisjuk et al., 1999) (Terashima et al., 1999) (Leite et al., 2000) (Staub et al., 2000)
than some classical expression systems, the development of transient expression systems (Kapila et al., 1996) means that the expression vectors and protein levels achieved can be checked before making this investment. Protein expression studies have demonstrated that many forms of recombinant antibody fragments (Figure 1) can be functionally expressed in plants and that the sub-cellular targeting of the protein is an important consideration for high level expression. These observations seem to be applicable to both transient and stable expression in plants and whether the protein is expressed in suspension cells, whole plants, or plant organs. Transient gene expression in plants Transient gene expression in plants has several advantages over the generation of stably transformed trans-
284 Table 3. Recombinant antibodies expressed in transgenic plants Year
Antibody format
Antigen
Plant organ
Cellular location
Transformed species
Reference
1989 1990 1991
IgG1 IgM VH domain
Leaf Leaf Leaf
(Hiatt et al., 1989) (Düring et al., 1990) (Benvenuto et al., 1991)
Leaf Leaf Leaf Leaf Root Leaf
Apoplast Cytosol Apoplast Apoplast
N. tabacum N. tabacum A. thaliana N. tabacum N. benthamiana N. tabacum N. tabacum
(Owen et al., 1992) (De Neve et al., 1993)
1993 1993 1994 1994
scFv IgG1 Fab scFv scFv IgG IgG1
ER ER chloroplast Intra- and extra-cellular Cytosol Nucleolus
N. tabacum N. tabacum N. benthamiana
1992 1993
(Firek et al., 1993a) (Tavladoraki et al., 1993) (van Engelen et al., 1994) (Ma et al., 1994)
1995
IgA/G
Leaf
Apoplast
N. tabacum
(Ma et al., 1995)
1995 1996 1996 1996 1996
IgG scFv IgM scFv scFv
Leaf Leaf Leaf root Leaf Leaf
(Voss et al., 1995) (Schouten et al., 1996) (Baum et al., 1996) (Fecker et al., 1996) (Bruyns et al., 1996)
IgG1 Fab scFv scFv scFv scFv scFv-IT
Leaf
Apoplast ER Apoplast Apoplast Cytoplasm ER Apoplast
N. tabacum N. tabacum N. tabacum N. benthamiana N. tabacum
1996
Phosphonate ester NP hapten Substance P (neuropeptide) Phytochrome Human creatine kinase Phytochrome AMCV Fungal cutinase Streptococcus mutans adhesin Streptococcus mutans adhesin TMV Cutinase RKN secretion BNYVV Human creatine kinase Human creatine kinase β-1,4-endoglucanase Oxazolone Abscisic acid Abscisic acid CD-40
A. thaliana
(De Wilde et al., 1996)
Root Leaf Leaf Seed Plant
Cytosol ER ER ER Apoplast
(Schouten et al., 1997) (Fiedler et al., 1997) (Fiedler et al., 1997) (Phillips et al., 1997) (Francisco et al., 1997)
Oxazolone HSV-2
tuber Plant
(Artsaenko et al., 1998) (Zeitlin et al., 1998)
Leaf
ER Secretory pathway Cytosol
S. tuberosum N. tabacum N. tabacum N. tabacum N. tabacum tissue culture S. tuberosum Glycine max P. hybrida
(De Jaeger et al., 1998)
Plant Leaf Plant Leaf
Apoplast Transient expression ER, apoplast ER, apoplast
Medicago sativa N. tabacum N. benthamiana N. tabacum Suspension cells N. tabacum O. sativa Suspension cells N. benthamiana
(Khoudi et al., 1999) (Vaquero et al., 1999) (Franconi et al., 1999) (Fischer et al., 1999d)
(McCormick et al., 1999)
O. sativa T. aestivum N. tabacum
(Stöger et al., 2000) (Schillberg et al., in press)
1997 1997 1997 1997 1997 1998 1998 1998
scFv Humanized IgG1 scFv
1999 1999 1999 1999
IgG scFv scFv bi-scFv
Dihydro-flavonol 4-reductase Human IgG CEA Tospoviruses TMV
1999 1999
scFv scFv
TMV CEA
Plant Cell
Cytosol ER, apoplast
1999
scFv
Leaf
Apoplast
2000 2000
scFv scFv
38C13 mouse B cell lymphoma CEA TMV
Plant Leaf
ER, apoplast Apoplast, membrane
(Zimmermann et al., 1998) (Torres et al., 1999)F
CEA – carcinoembryonic antigen; ER – endoplasmic reticulum; AMCV – Artichoke mottle crinkle virus; TMV – tobacco mosaic virus; RKN – root knot nematode; BNYVV – beet nectrotic yellow vein virus; HSV-2 – herpes simplex virus-2; scFv-IT – scFv-bryodin-immunotoxin.
285 Table 4. Recombinant vaccines expressed in plants Year
Vaccine antigen
Transformed species
Reference
1992 1995 1995 1995 1996
Hepatitis virus B surface antigen Malaria parasite antigen Rabies virus glycoprotein Escherichia coli heat-labile enterotoxin Human rhinovirus 14 (HRV-14) and human immunodeficiency virus type (HIV-1) epitopes Norwalk virus capsid protein Diabetes-associated autoantigen Hepatitis B surface proteins Mink Enteritis Virus epitope Rabies and HIV epitopes Foot and mouth disease virus VP1 structural protein Escherichia coli heat-labile enterotoxin Escherichia coli heat-labile enterotoxin Rabies virus Cholera toxin B subunit Human insulin-Cholera toxin B subunit fusion protein Foot and mouth disease virus VP1 structural protein Hepatitis B virus surface antigen Human cytomegalovirus glycoprotein B Diabetes-associated autoantigen
N. tabacum Virus particle L. esculentum N. tabacum, S. tuberosum Virus particle
(Mason et al., 1992) (Turpen et al., 1995) (McGarvey et al., 1995) (Haq et al., 1995) (Porta et al., 1996)
N. tabacum, S. tuberosum N. tabacum, S. tuberosum S. tuberosum Virus particle Virus particle A. thaliana
(Mason et al., 1996) (Ma et al., 1997) (Ehsani et al., 1997) (Dalsgaard et al., 1997) (Yusibov et al., 1997) (Carrillo et al., 1998)
S. tuberosum S. tuberosum Virus particle S. tuberosum S. tuberosum
(Mason et al., 1998) (Tacket et al., 1998) (Modelska et al., 1998) (Arakawa et al., 1998a) (Arakawa et al., 1998b)
Medicago sativa
(Wigdorovitz et al., 1999)
Lupinus luteus, Lactuca sativa N. tabacum N. tabacum, D. carota
(Kapusta et al., 1999) (Tackaberry et al., 1999) (Porceddu et al., 1999)
1996 1997 1997 1997 1997 1998 1998 1998 1998 1998 1998 1999 1999 1999 1999
genic plants. Transient expression is rapid and results on protein expression can be obtained in days (Kapila et al., 1996). This makes transient expression suitable for verifying that the gene product is functional before moving on to large-scale production in transgenic plants (Kapila et al., 1996). Stable plant transformation requires a considerable investment in time before the expressed proteins can be analysed. In contrast, transient gene expression systems are rapid, flexible and straightforward and often use either agrobacterial or viral vectors. Viral vectors (Scholthof et al., 1996) have attracted interest because high yields of protein can be produced rapidly, by comparison to stable plant transformation. Commercial field trials are proceeding for the production of recombinant proteins using viral vectors. For example, the Large Scale Biology Corporation (formerly BioSource Technologies, Vacaville, CA, USA) is using a tobacco mosaic virus-based vector for production of Hepatitis B surface antigen, scFvs and
other recombinant proteins (McCormick et al., 1999; Kumagai et al., 2000). There are three major transient expression systems to deliver a gene to plant cells: delivery of projectiles coated with ‘naked DNA’ by particle bombardment, infiltration of intact tissue with recombinant agrobacteria (agroinfiltration), or infection with modified viral vectors. The overall level of transformation varies between these three systems. Particle bombardment usually reaches only a few cells and for transcription the DNA has to reach the cell nucleus (Christou, 1996). Agroinfiltration targets many more cells than particle bombardment and the T-DNA harboring the gene of interest is actively transferred into the nucleus with the aid of several bacterial proteins (Kapila et al., 1996). A viral vector can systemically infect most cells in a plant and transcription of the introduced gene in RNA viruses is achieved by viral replication in the cytoplasm, which transiently generates many transcripts of the gene of interest.
286 Although particle bombardment can be used to test recombinant protein stability before stable transformation, it is unsuitable for the expression of larger amounts of foreign proteins. It is more important for the regeneration of transgenic cereal crops (Christou, 1996). Here, we concentrate on agroinfiltration as a method to test antibody production before producing stably transformed plants. Typically, agroinfiltration can provide milligram amounts of a recombinant protein within a week (Vaquero et al., 1999). This is an important issue because it dramatically accelerates the development of plant lines producing recombinant therapeutics. Agroinfiltration- expression with a bacterial vector In agroinfiltration, agrobacteria carrying the expression vector are delivered into leaf tissue by vacuum infiltration. Agrobacterial proteins then catalyze the transfer of the gene of interest into the host cells and protein expression can be detected three days after infiltration. As in conventional methods for generating transgenic plants, genes of interest are cloned into binary vectors that are transferred into suitable Agrobacterium strains. A bacterial suspension is then used for vacuum infiltration of leaves (An, 1985; Kapila et al., 1996) and no selection method to identify transformed cells is required since the leaf tissue is only used for transient protein production, which permits the use of smaller plasmid vectors. In agroinfiltration, the transferred T-DNA does not integrate into the host chromosome but is present in the nucleus, where it is transcribed and this leads to transient expression of the gene of interest (Kapila et al., 1996). A major advantage of this technique is that multiple genes present in different populations of agrobacteria can be simultaneously expressed. Thus, the assembly of complex multimeric proteins can be tested in planta (Vaquero et al., 1999). For transgenic plants, this can only be achieved by time consuming crossing experiments with individual transgenic plant lines, each expressing a single component of the multimer. Using agroinfiltration, we transiently expressed scFvs, individual heavy and light chains as well as full size mouse–human chimeric anti-carcinoembryonic antigen (CEA) antibodies in plant leaves (Vaquero et al., 1999). For full size chimeric antibody expression, the mouse–human chimeric heavy and light chain genes were integrated into two vectors in two separate recombinant Agrobacterium strains
and these two strains were simultaneously infiltrated into leaves. Functional full size chimeric antibodies were assembled in vivo by simultaneous expression of both genes in the host cells. This technique is also suitable for the expression of scFv-fusion proteins, diabodies and multi-component protein complexes (R. Fischer & C. Vaquero, unpublished results). Thus, agroinfiltration is rapid and yields sufficient quantities of protein for initial characterization of protein stability and protein function. Importantly, agroinfiltration can be scaled up to produce tens of milligrams of recombinant protein and may even prove suitable for pre-clinical trials without the need for production of stably transformed plants. Recombinant viral vectors Viral vectors (Scholthof et al., 1996) share several advantages with agroinfiltration. Here, the gene of interest is cloned into the genome of a viral plant pathogen under the control of a strong subgenomic promoter. Infectious recombinant viral transcripts are used to infect plants and produce the target protein. Target genes are expressed at high levels because of the high level of multiplication during virus replication (Porta & Lomonossoff, 1996). Some plant viruses have a wide host range, are easily transmissible by mechanical inoculation and can spread from plant to plant, making it possible to rapidly infect large numbers of plants with recombinant viruses. Viral vectors have been used to express single chain antibodies in plants (Franconi et al., 1999; Hendy et al., 1999; McCormick et al., 1999). We transiently expressed an scFv in plants, using a tobacco mosaic virus (TMV) based vector (Verch et al., 1999). The scFv coding region was inserted into the viral genome under the control of the strong subgenomic coat protein promoter. This promoter is duplicated and drives the transcription of the viral coat protein gene. The coat protein is essential for long distance, systemic viral infection and the scFv was expressed throughout the entire plant. This approach has been adapted to express the heavy and light chain of a full size antibody from two different viral vectors (Verch et al., 1998), but is generally limited to proteins smaller than 30 kDa. Improvement of this technique may involve the increase of inoculation efficiency by combining the cloned recombinant viral DNAs with particle bombardment or Agrobacterium
287 based techniques, like agroinfection (Shen & Hohn, 1995).
Table 5. Yields of several plant species in tonnes per hectare Crop
Crop yield (tonnes/hectare)∗
Reference
Banana Cabbage Eggplant Lettuce Maize Peanut Peas Potato Rapeseed Rice Tobacco Tobacco
16.6 24.3 26.9 33.1 8.4 2.9 9.1 124.5 1.5 6.6 2.2∗ 170–200∗∗
Tomato Wheat
59.4 2.7
FAO FAO FAO FAO FAO FAO FAO FAO FAO FAO FAO (Long, 1984; Sheen, 1983) FAO FAO
Stable plant transformation Stable transformation is defined by the integration of a target gene into the host plant genome. The generation of transgenic plants uses two principle technologies: Agrobacterium mediated gene transfer to dicots, such as tobacco and pea (Horsch et al., 1985), or biolistic delivery of genes to monocots, such as wheat and corn (Christou, 1993). Agrobacterium has a restricted host range and does not efficiently infect monocots but is the most widely used technique for dicot transformation. However, rice can be transformed by Agrobacterium (Chan et al., 1993; Hiei et al., 1994; Hiei et al., 1997) and methods have been developed for transforming other monocots. For transforming plants, the gene of interest is cloned into a binary vector that can be moved between E. coli and Agrobacterium. The transformed Agrobacterium itself delivers the target gene into the host cell genome. Transformation is followed by selection for cells with stably integrated copies of the target gene by following a selectable resistance gene that is introduced in the expression vector. Stable transformation of plants depends on the plant variety and it can take 3–9 months for plants to be available for testing the expressed protein. Clearly, transient expression is a prerequisite to stable transformation because it allows both the expression vectors and protein stability to be tested. Initial problems can be identified and eliminated so that the likelihood of regenerating the desired transgenic line is significantly improved. Expression in stably transformed plants When long term production of a recombinant antibody is necessary, stable transgenic plants are the most attractive strategy. The quantity of recombinant protein that can be harvested is only limited by the number of hectares that can be planted with transgenics. High intensity agriculture can produce suprisingly large amounts of biomass, for example intensive cultivation of tobacco plants can produce approximately 170 metric tonnes of biomass per hectare (Sheen, 1983; Cramer et al., 1996). Assuming that the levels of production seen on the laboratory scale could be at least kept constant in the field, and that for every 170 tonnes harvested, 100 tonnes are harvested leaves: a single hectare could yield 50 kg of secretory IgA
All yields refer to production in developed North America in 1999. Taken from the Food and Agriculture Organization’s on line database (http://apps.fao.org/).∗ : smoking tobacco; ∗∗ : close cropping and mowing.
(Ma et al., 1995) or 100 kg of recombinant glucocerebrosidase (Cramer et al., 1996). In Table 5, we summarize the yield per hectare of several crop species as a guide to what levels of biomass can be produced. In Table 1, we compare plants to the classical expression systems and show the advantages of plants, particularly their safety and low cost. Some recombinant proteins already reach very high expression levels, for example, apoplast targeted recombinant phytase accumulates to ≈14% total soluble protein (TSP) in tobacco leaves (Verwoerd et al., 1995), and a eubacterial glucanase has been reported to reach 26% TSP in the mouse eared cress, Arabidopsis thaliana (Ziegler et al., 2000). However, average expression levels of recombinant antibodies in stably transformed plants are usually on the level of 0.5–2% TSP (Conrad & Fiedler, 1998) but have reached 6.8% TSP (Fiedler et al., 1997). Importantly, plants have some unique features not found in bacterial or mammalian systems. Plants are the premier heterologous system for the production of secretory IgA antibodies (Ma et al., 1995). Planet Biotechnology (Mountain View, CA) are concentrating on using plants to produce the Streptococcus mutans specific Guy’s-13 antibody, which prevents dental caries
288 (Ma et al., 1995; Ma et al., 1998). Similar approaches are now underway to produce other antibodies under a collaboration between EPIcyte pharmaceuticals and ProdiGene. The low costs of producing recombinant antibodies in plants are as great a benefit as the increased safety and authentic post-translational modification pathways. It has been estimated that plant-expressed proteins are 10–50-fold less expensive than those made in E. coli and that these savings will be greater as production reaches agricultural cropping scales or as methods are developed to increase expression levels. This justifies the use of plants as an inexpensive source for producing recombinant proteins that eliminates the disadvantages associated with microbial or animal cell systems. Additionally, plant genetic material is readily stored in seeds or tubers, which are extremely stable, require no special maintenance and have a long shelf life (Conrad et al., 1998). This has the benefit that both the product and the production system itself can be stored almost indefinitely. Important considerations for pharmaceutical expression in plants In this section, we discuss the technical considerations that are important for high level pharmaceutical protein expression, which range from transcriptional modifications to changes in the sub-cellular destination of the newly synthesized recombinant protein. The plant pattern of protein glycosylation and Nlinked glycan processing differs from that of animals, and in some cases recombinant proteins could be immunogenic through their glycosylation. Therefore, the glycosylation of proteins intended for in vivo administration should be analysed in detail (Bardor et al., 1999). Sub-cellular protein targeting Expression levels of recombinant antibodies in plants can be enhanced by exploiting the innate protein sorting and targeting mechanisms that plant cells use to target host proteins to organelles. Significant increases in recombinant antibody yield have been observed when antibodies are targeted to the secretory pathway instead of the cytosol (Conrad & Fiedler, 1998). Targeting proteins for secretion to the intercellular space beneath the cell wall (apoplast) has advantages for downstream processing and also leads to significant levels of expression but ER retention can give
10–100-fold higher yields (Conrad & Fiedler, 1998). Recombinant antibodies have been targeted to the following compartments of plant cells: the intercellular space, chloroplasts and endoplasmic reticulum (ER) (Düring et al., 1990; Firek et al., 1993b; Ma et al., 1994; Artsaenko et al., 1995; Voss et al., 1995; Baum et al., 1996; De Wilde et al., 1996; Schouten et al., 1996; Conrad & Fiedler, 1998). Intracellular expression of rAbs in the cytoplasm has only been achieved using scFv fragments (Owen et al., 1992; Tavladoraki et al., 1993; Schouten et al., 1996; Zimmermann et al., 1998), presumably because scFv fragments require only minor post-translational processing. In the majority of transgenic plants expressing cytosolic scFvs, levels were found to be very low or at the detection limit (Owen et al., 1992; Fecker et al., 1996; Schouten et al., 1996; Schillberg et al., 1999). There is a report where cytosolic scFvs, isolated from a phage display library, have reached levels of up to 1.0% of total soluble protein (De Jaeger et al., 1998), but this is still an exception. The high cytosolic expression level may be because the in vitro antibody selection used in phage display naturally selects more stable antibody fragments. Overall expression levels of different antibodies in stably transformed plants vary, with expression of full size IgG under the control of the 35S promoter ranging from 0.35% (van Engelen et al., 1994) to 1.3% of the total soluble protein (TSP) in tobacco leaves (Hiatt et al., 1989). This is not an upper limit, because transgenic plants have been identified with expression levels of scFvs in leaves reaching 6.8% of the TSP (Fiedler et al., 1997) and levels of secretory IgA up to 500 µg per gram leaf material (Ma et al., 1995). In a recent report, Russell and colleagues at the Integrated Protein Technologies unit of Monsanto have reported that transformed chloroplasts can be used as a high capacity production system. Active human somatotrophin was expressed in transgenic plastids in tobacco and reached 7% of the total soluble protein (R. Bassuner, personal communication; Staub et al., 2000). This a remarkable result and indicates that chloroplast based expression, which is inherently biologically contained, may be useful for the expression of other pharmaceutical proteins. However, it is unclear to what extent the chloroplast protein synthesis pathway can accommodate eukaryotic proteins. It will be interesting to determine if chloroplasts will be capable of synthesizing complex eukaryotic proteins that require post-translational modifications or subunit assembly, such as sIgA.
289 Protein storage in tissues, seeds and tubers For plants to fulfill their potential as a production system, a prerequisite was that expressed proteins could be stored in harvested tissues, tubers and seeds. This has widely been shown to be the case and emphasizes the versatility of plants as an expression system. When leaves from transgenic tobacco plants expressing ERretained scFvs were dried and stored for more than three weeks, there were no losses in scFv specificity or antigen binding activity (Fiedler et al., 1997). Seeds are protein rich storage organs that can be stored almost indefinitely and can be exploited as storage containers for recombinant proteins (Fiedler and Conrad, 1995; Conrad and Fiedler, 1998; Conrad et al., 1998). As with expression in leaves, ER retention gave increases in scFv accumulation and scFvs can be stored for up to a year in seed at room temperature without losses. Potato tubers have also been used as storage containers with expression levels reaching 2% TSP and cold storage for 18 months resulting in only a 50% loss of functional antibody (Artsaenko et al., 1998). The most promising approach for protein expression and in field production is to target the protein to the ER and, if long-term storage is required, to target the protein for seed specific expression. Transformation of major crop plants is now becoming more straightforward. We foresee crop based expression systems (wheat, rice, corn, legumes) may be used because they have a lower content of toxic compounds than model species, like tobacco, and there is an existing infrastructure for crop cultivation, harvesting, distribution and processing. Optimization at the level of the gene The pattern of codon usage in plants differs to that of animals, but modifying the composition of the heterologous cDNA to meet the plant pattern can increase the rate of translation (Kusnadi et al., 1997). Further improvements in expression may come from using tissue specific promoters, improvement of transcript stability, translational enhancement with viral sequences (Gallie, 1998) and screens to identify stable cytosolic antibody scaffolds (Worn et al., 2000). Interestingly, the production of a bean arcelin in Arabidopsis thaliana seeds was increased by the expression of an antisense gene for the seed storage protein 2S albumin. Using this strategy, arcelin levels were enhanced and reached 24% of the total seed protein
(Goossens et al., 1999). Therefore, adroit selection of genes to suppress by anti sense expression may be a realistic method to dramatically increase the yield of co-introduced pharmaceutical protein genes in seed.
Pharmaceutically valuable proteins produced in plants As medical and biological knowledge of many diseases increases, through the sequencing of the human genome and medical research, many novel proteins that could be used for treatment have been identified. These include recombinant antibodies and it is clear this will expand to include more proteins in the future. Plants are likely to feature highly as an alternative to using transgenic animals to produce these proteins. Recombinant antibodies, plasma proteins and diagnostic reagents are targets for expression in plants because their conventional production or purification is often expensive and can bear risk of pathogen contamination. The number of mammalian proteins expressed in plants is expanding and include antibodies, plasma proteins, human enzymes and recombinant vaccines (Tables 2–4). The worldwide demand for some of these proteins is large, for example human serum albumin (HSA) is a non-glycosylated protein with a worldwide demand approaching 550 tonnes of purified protein a year. Conventionally, it is isolated from human blood donations and is therefore relatively inexpensive. However, isolated HSA does bear some risks of viral contamination and there is a market for sources of safer HSA. HSA can be safely made in plants as can interleukins, interferons and human enzymes (Table 2). Another application of molecular farming in plants is the production of vaccine antigens, and edible vaccines (Walmsley & Arntzen, 2000). This is likely to be important in the future, but here we focus on the more advanced fields of antibody expression and vaccine production. Therapeutic antibody production in plants Antibodies are an instructive example of the expression of pharmaceutically valuable proteins in plants (Hiatt, 1990; Hiatt, 1991; Hiatt et al., 1992; Hiatt & Ma, 1993). As described earlier, a key breakthrough in making molecular farming in plants a reality was the demonstration of functional antibody expression in
290 tobacco leaves (Hiatt et al., 1989; Düring et al., 1990). The importance of this is underscored by the fact that monoclonal antibodies (Koehler & Milstein, 1975) and recombinant antibodies are essential therapeutic and diagnostic tools used in medicine, human and animal health care, the life sciences and biotechnology (Winter & Milstein, 1991). Modern recombinant DNA techniques and antibody engineering have broadened the range of applications for recombinant antibodies. These advances have made possible the production of novel polypeptides with desirable properties (Figure 1). For example, smaller antibody fragments or antibody-fusion proteins linked to enzymes, biological response modifiers or toxins ( Shin et al., 1993; Gerstmayer et al., 1997; Bookman, 1998). The rapid development of combinatorial approaches, such as phage display, allow the isolation of rAbs recognizing almost any target antigen and the fine-tuning of these rAbs toward desired properties (Winter et al., 1994). Therefore, antibodies are likely to only become more important in the future and the demand for an economical, efficient expression system is likely to grow. After the demonstration that functional full size rAbs could be expressed in transgenic plants in 1989 (Hiatt et al., 1989) and 1990 (Düring et al., 1990), a wide range of different recombinant antibody formats have been successfully expressed in many plant species (Table 3). These formats include full-size antibodies (Ma et al., 1994; Voss et al., 1995; Baum et al., 1996; De Wilde et al., 1996), Fab fragments (De Neve et al., 1993), single chain antibody fragments (scFvs) (Owen et al., 1992; Tavladoraki et al., 1993; Firek et al., 1993b; Artsaenko et al., 1995; Fiedler & Conrad, 1995; Fecker et al., 1996; Schouten et al., 1996), bi-specific scFv fragments (Fischer et al., 1999d), membrane anchored scFv (Schillberg et al., in press) and chimeric antibodies (Vaquero et al., 1999). We have shown that it is possible to transiently express tumour (carcinoembryonic antigen) specific single chain and chimeric full size antibodies in tobacco leaves. A recombinant single-chain Fv antibody (scFvT84.66) and a full-size mouse/human chimeric antibody (cT84.66), derived from the parental murine mAbT84.66 specific for the human carcinoembryonic antigen, were engineered into a plant expression vector. Chimeric T84.66 heavy and light chain genes were constructed by exchanging the mouse light and heavy chain constant domain sequences with their human counterparts and cloned into two independent plant expression vectors. In vivo assembly of full-size
cT84.66 was achieved by simultaneous expression of the light and heavy chains after vacuum infiltration of tobacco leaves with two populations of recombinant Agrobacterium. Upscaling the transient system permitted purification of significant (milligram) amounts of functional recombinant antibodies from tobacco leaf extracts within a week (Vaquero et al., 1999). Secretory IgA Plants have a great advantage over animal cell expression systems, since single plant cells are capable of expressing recombinant secretory IgA (sIgA) (Ma et al., 1995). This has only recently become possible in single animal cells (Chintalacharuvu & Morrison, 1997). SIgA is a complex multi-subunit antibody with great potential for use in topical immunotherapy. It is the major antibody found in mucosal secretions and is made of two immunoglobulin chains, a joining chain and the secretory component. It was first expressed in tobacco by sequentially crossing plants expressing its individual polypeptide components. This permitted the production of high levels of recombinant sIgA (500 µg/gram) (Ma et al., 1995) and proved that plants could be used as a production system for sIgA suitable for use in passive immunotherapy. Three years later, the same group showed that recombinant sIgA specific for adhesion proteins from the oral pathogen Streptococcus mutans could prevent oral streptococcal colonization in human volunteers for up to four months (Ma et al., 1998). We anticipate that plant produced sIgA will become widely used in the future for the generation of passive immunity because of their stability in the mucosa. Thus, for antibodies at least, molecular farming has come virtually full circle, from proof of principle with the expression of model antibodies in 1989 (Hiatt et al., 1989) to the production of complex antibodies in plants and their use as pharmaceutical reagents. The circle will be closed when the first plant expressed antibodies are approved by the regulatory authorities and come onto the market as diagnostic or therapeutic products. Vaccines from plants Vaccination has been one of the greatest advances in medical science and has dramatically improved human life expectancy and quality of health. Vaccines are the most cost effective form of health care
291 (World Health Organisation: www.who.int/gpv/) but their world wide distribution is hampered, especially in developing countries. Plants can produce a range of immunogenic antigens (Table 4). It was proposed to induce B- and T-cell mediated immune responses using plants as a source of ‘edible vaccines’, where the vaccine antigens are eaten in a fruit or raw vegetable (Mason & Arntzen, 1995; Ma & Vine, 1999; Walmsley & Arntzen, 2000). Such vaccines would not require cold storage or sophisticated expertise for their distribution and use throughout the developing countries. The principle of edible vaccine activity was proven for transgenic potatoes producing the enterotoxigenic E. coli heat labile enterotoxin B subunit (Haq et al., 1995). Mice fed raw potato produced a serum and mucosal immune response to the antigen. Further work has gone on to show that edible vaccines may be feasible for a range of antigens, including rabies virus (McGarvey et al., 1995), foot and mouth disease (Wigdorovitz et al., 1999), Norwalk virus (Mason et al., 1996), autoimmune diabetes (Arakawa et al., 1998b) and cholera (Arakawa et al., 1998a). Furthermore, an effective edible hepatitis B vaccine has been generated using transgenic lupin and lettuce plants expressing the hepatitis B surface antigen. Mice and humans fed transgenic plant material produced hepatitis B specific antibodies (Kapusta et al., 1999). Many edible plants have been genetically transformed and tomatoes and bananas are good candidates for edible vaccine production for humans while cereals may be more suitable for animal immunisation. For edible vaccines to become widely used and useful, several issues have to be considered. Edible vaccines will need an infrastructure for their distribution and administration to the public to ensure they are as effective as current vaccines. Importantly, there have to be internal controls for the level of vaccine expression in every plant and their stability and efficacy need to be improved. Further, effective methods have to be developed for the biological containment of vaccine traits.
Alternative plant expression systems Intact tobacco plants are not the only expression system available for plant based molecular farming. There are options for the production of proteins in seeds, plant suspension cells and by ‘rhizosecretion’ from engineered plant roots. Cereals have advantages for
antibody production over ‘model’ species such as tobacco because they have a lower content of toxic secondary metabolites and there is a well-organised infrastructure for their production, distribution and processing. When recombinant proteins are expressed in cereal seed, such as for expression of avidin in corn, the protein can be collected and extracted directly from the kernels (Hood et al., 1997; Hood et al., 1999). Plant suspension cells are a model system that can be easily transformed and cultivated on a very large scale in fermenters (Fischer et al., 1999b), while rhizosecretion is the release of proteins from transgenic plant roots into a surrounding hydroponic medium (Borisjuk et al., 1999). Suspension cells can be used to produce and secrete proteins under carefully controlled certified conditions, but rhizosecretion has yet to be broadly used (Hooker et al., 1990; Bisaria & Panda, 1991; Nagata et al., 1992; Fischer et al., 1999b). Plant suspension cells can be grown in shake flasks or fermenters to produce recombinant proteins after transformation (Fischer et al., 1999b). Compared to the classical expression systems the number of applications is still relatively small (Kieran et al., 1997). A number of plant species has been used for the generation and propagation of cell suspension cultures, ranging from model systems like Arabidopsis (Desikan et al., 1996) to Catharanthus (Van Der Heijden et al., 1989), Taxus (Seki et al., 1997), to important monocot or dicot crop plants like rice (Chen et al., 1994), soybean (Hoehl et al., 1988), alfalfa (Daniell & Edwards, 1995) and tobacco (Nagata et al., 1992). Tobacco suspension cells have been used for the production of an scFv-fusion protein with a ribosome inactivating protein (Bryodin) with yields of 30 mg/l (Francisco et al., 1997). We focus on using suspension cells for the production of recombinant proteins and antibodies in plants and plant cell cultures. As discussed earlier, the expression of recombinant antibodies and antibody fragments in plants is well established (Hiatt et al., 1989; Whitelam et al., 1994). When clinical use of recombinant proteins is intended, their production under defined, controllable and sterile conditions with straightforward purification protocols may be advantageous. Therefore, we have expressed full-size antibodies, antibody fragments and fusion proteins in transgenic plant cell suspension systems including Nicotiana tabacum cv. Petite Havana SR-1 (Voss et al., 1995), Nicotiana tabacum BY-2 cells (Nagata
292 et al., 1992), pea, wheat and rice (Torres et al., 1999). Tobacco suspension cell lines for recombinant antibody production Transfer of a foreign gene into plant suspension cells can be performed using Agrobacterium-mediated transformation (Horsch et al., 1985; Koncz & Schell, 1986), particle bombardment (Christou, 1993), electroporation of protoplasts (Lindsey & Jones, 1987) or viral vectors (Porta & Lomonossoff, 1996). The BY-2 cell line can be directly transformed by co-cultivation of suspension cells and Agrobacterium (An, 1985). This has the advantage that transient expression of the foreign gene can be detected 2–3 days after cocultivation. Recombinant proteins expressed in plant cell suspension cultures are found in the culture supernatant or retained within the cells. This localisation is dependent on the presence of targeting/leader peptides (of plant or heterologous origin) in the recombinant protein and on the permeability of the plant cell wall to macromolecules (Carpita et al., 1979). Recombinant targeting signals can be used to direct the protein for secretion (Magnuson et al., 1998) or to intracellular organelles (ER, chloroplast, vacuole, intracellular membranes) (Moloney & Holbrook, 1997). The targeting signals can be used to retain recombinant proteins within distinct compartments of the cells to preserve integrity, protect them from proteolytic degradation and to increase accumulation levels (Kusnadi et al., 1997; Moloney & Holbrook, 1997). The cytosol is generally unsuitable as a recombinant protein storage compartment. Recombinant proteins smaller than 20–30 kDa can pass through the plant cell wall and are secreted into the culture medium but larger proteins tend to be retained in the apoplast. Intra-cellular protein retention makes the disruption of the cells necessary prior to protein purification, which has several drawbacks, since it causes release of phenolic substances or proteases that reduce protein yield. Thus, our preferred method is to target proteins for secretion and capture them from the culture supernatant or release them from the cell by mild enzymatic cell wall digestion (Fischer et al., 1999c). Using our standard plant expression vector, we could obtain expression levels between 2–20 µg of recombinant antibody per gram fresh cell weight. This level could be significantly increased by targeting of the protein to the ER as well as by optimizing cultivation condi-
tions with controlled amino-acid supplementation or elicitation.
Purification strategies for proteins expressed in plants Highly efficient purification schemes are a prerequisite for the use of recombinant proteins as pharmaceuticals (Baker & Harkonen, 1990; Mariani & Tarditi, 1992; Miele, 1997; Murano, 1997) and are an important consideration in designing molecular farming systems. Although there are established protocols available for purification of antibodies produced by animal or microbial sources, there is little available data on the purification of recombinant antibodies from plants, plant suspension culture cells, leaves or seeds (Moloney & Holbrook, 1997). We established a purification protocol for full-size antibodies produced in plant cell suspension cultures (Fischer et al., 1999c). Our data demonstrate that full-size antibodies can be purified from plant cell extracts on protein-A and protein-G based affinity matrices in a similar manner to antibodies purified from animal sources. Antibodies secreted to the intercellular space of plant cells were released by partial enzymatic lysis of the cell wall and this was the superior method for isolation of functional antibodies. Affinity chromatography using a Protein-A matrix as the first step efficiently removed contaminant plant proteins and gave a 100-fold concentration of the recombinant protein. Gel filtration served as a polishing step for the removal of rAb-dimers and for exchange of the rAbs into a suitable storage buffer. Using this method, more than 80% of expressed full size IgG can be recovered from suspension cultured plant cells (Fischer et al., 1999c). This shows that antibody purification from plants is essentially straightforward with no complications that could prevent the use of plants as an expression system. Downstream processing of full-length IgG antibodies is relatively straightforward because Protein-A and Protein-G are useful ligands for affinity chromatography. This approach is not applicable for the downstream processing of IgM antibodies, or most recombinant antibodies including scFvs and scFv fusion proteins. For most recombinant proteins, novel strategies that have a high processing speed, high capacity and that are inexpensive will need to be developed. To make large scale bioprocessing more efficient, the latest developments in downstream processing such as perfusion chromatography and expanded bed tech-
293 nology in combination with tangential flow filtration should be applied. This will overcome the diffusional limitations experienced with most chromatographic resins (Fahrner et al., 1999). Engineered affinity tags may enable improved handling of large clarified sample volumes, minimise processing time and avoid proteolytic and oxidative degradation of recombinant proteins. Here, we need to distinguish between the generation of N- or Cterminal gene fusions (tags) for affinity purification (Nilsson et al., 1997) and the development of stable synthetic peptides that reversibly bind the recombinant protein of interest. The peptides are immobilized on a solid support, to allow controlled capture and release during processing and multiple uses of a synthetic affinity matrix. Such peptides can be identified by epitope mapping using pepscan or phage peptide display technologies for the identification of linear peptide sequences. Modifications of the phage peptide display technology also permit the identification of mimotopes, either in a linear or a Cys-Cys-constrained library (McConnell et al., 1998; Zwick et al., 1998). The synthetic versions of identified binding peptides can be immobilized on an activated matrix (Sepharose, glass, silica) for the development of a specific affinity matrix for a given recombinant protein (Murray et al., 1997). Due to potential immunogenicity of certain tags (FLAG, MBP, GST) it may be particularly important to use specific, synthetic peptide affinity ligands for the purification of therapeutic proteins. The combination of the latest developments in downstream processing and affinity chromatography may lead to significant advances in the large scale production of inexpensive diagnostic and therapeutic proteins by molecular farming.
Commercial aspects of molecular farming The commercial interest in molecular farming is that it can produce recombinant proteins at a lower cost than alternatives, such as their production in mammalian tissue culture. An interesting case study for the farming of a recombinant protein was reported by Hood and co-workers for the production of recombinant avidin (Hood et al., 1997; Hood et al., 1999; Kusnadi et al., 1998). Avidin is widely used as a diagnostic reagent and is a relatively abundant eukaryotic protein found in egg white, from which it is routinely purified. The rationale was to produce avidin in transgenic corn
and determine if this could compete with egg white as a commercial source of the protein. A chicken avidin cDNA, codon optimised for the preferred maize codon usage pattern, was engineered in fusion to a barley α-amylase signal sequence. This targeted the recombinant protein to the secretory pathway and targeting was a crucial factor. Plants that expressed high levels of avidin in the secretory pathway were either partially or completely male sterile. In contrast, targeting the avidin to the cytosol was completely toxic to engineered maize. Avidin could be reproducibly produced at 230 mg per kg of maize seed (Hood et al., 1997). The authors estimated that plant produced avidin is 10-fold less expensive than avidin extracted from eggs. The maize avidin is functional and now commercially available (Sigma-Aldrich product # A8706). This illustrates how a relatively abundant protein with a rich natural source can still be produced less expensively in plants. β-Glucuronidase production in plants is also commercial (Witcher et al., 1998) and the costs of producing aprotinin in plants are comparable with extracting it from its natural source, bovine lung (Zhong et al., 1999). Considering that there are no natural sources of recombinant antibodies as inexpensive as using chicken eggs as a source of avidin, the savings from expressing antibodies in plants will be even higher. The demand for many pharmaceutical proteins is large, and any transgenic production system has to be capable of meeting the demand. As mentioned earlier, kilogram quantities of a recombinant protein can be obtained from as little as a hectare of transgenic tobacco. Transgenic animals are limited by the time needed to raise a herd of animals producing the recombinant protein. The demand for human serum albumin is in the range of 550 metric tons per year and it may take years to establish a herd of transgenic animals producing enough protein to meet this demand. In contrast, transgenic plants can be rapidly scaled up to field scale cultivation. It has been estimated that the worldwide demand for human serum albumin could be met by transgenic cultivation on 30,000 hectares of land (assuming an expression level of 1% TSP in tobacco), which is less than one thousandth of the total cultivated soil in the USA (32 million hectares). Clinical trials of plant produced pharmaceuticals The first clinical trial of plant-based immunotherapy was reported by Planet Biotechnology, Inc. (Mountain View, CA). The novel drug CaroRxTM is based
294 on sIgA antibodies produced in transgenic tobacco plants and is designed to prevent the oral bacterial infection that contributes to dental carries (Ma et al., 1998). Planet Biotechnology has demonstrated that CaroRxTM can effectively eliminate Streptococcus mutans, the bacteria that causes tooth decay in humans (Larrick et al., 1998). Planet Biotechnology is also engaged in the design and development of novel sIgA-based therapeutics to treat infectious diseases and toxic conditions affecting oral, respiratory, gastrointestinal, genital and urinary mucosal surfaces and skin. Monsanto (formerly Agracetus, Middleton, Wisconsin) created a corn line producing human antibodies at yields of 1.5 kg of pharmaceutical-quality protein per acre of corn. Given that the yield per acre of corn is on the range of 3.5 tonnes (Table 5), there is considerable room for improvement in yields. A pharmaceutical partner plans to begin injecting cancer patients with doses of up to 250 mg of the antibodybased cancer drug purified from corn seeds. The company is also cultivating transgenic soybeans that produce humanized antibodies against herpes simplex virus 2 (HSV-2). These antibodies were shown to be efficient in preventing vaginal HSV-2 transmission in mice. The ex vivo stability and in vivo efficacy of the plant and mammalian cell-culture produced antibodies were similar (Zeitlin et al., 1998). Plant-produced antibodies are likely to allow development of an inexpensive method for mucosal immuno-protection against sexually transmitted diseases. ProdiGene (College Station, Texas) and EPIcyte Pharmaceuticals (San Diego) have entered into a strategic partnership to produce antibodies in corn (www.prodigene.com/news.html). Their interest is the production of human mucosal antibodies for passive immunisation by exploiting ProdiGene’s expertise in protein expression (Hood et al., 1997; Kusnadi et al., 1998; Witcher et al., 1998; Hood et al., 1999; Zhong et al., 1999) together with EPIcyte’s academic and patent position. The collaborative research group at Biosource Technologies (now named the Large Scale Biology Corporation, Vacaville, CA) and Stanford University has developed a technology to produce a tumorspecific vaccine for the treatment of malignancies using a plant virus based transient expression system. The researchers created a modified tobacco mosaic virus vector that encodes the idiotype-specific scFv of the immunoglobulin from the 38C13 mouse B cell lymphoma. Infected Nicotiana benthamiana plants
secreted high levels of scFv protein to the apoplast. This antibody fragment reacted with an anti-idiotype antibody, suggesting that the plant-produced 38C13 scFv protein is properly folded. Mice vaccinated with the affinity-purified 38C13 scFv generated > 10 µg/ml anti-idiotype immunoglobulins. These mice were protected from challenge by a lethal dose of the 38C13 tumor, similar to mice immunized with the native 38C13 IgM-keyhole limpet hemocyanin conjugate vaccine (McCormick et al., 1999). This rapid production system for generating tumor-specific protein vaccines may provide a viable strategy for the treatment of non-Hodgkin’s lymphoma. The goal of the therapy is to create antibodies customized for each patient that will recognize unique markers on the surface of the malignant B-cells and target the cells for destruction.
Future directions The technical challenges that need to be solved for plants are essentially all related to expression levels of recombinant proteins. Higher expression levels will be reached by better control of gene silencing and the identification of novel, stronger promoters in intact plants, or better plant species as expression hosts. Large-scale fermentation may be a practical method for producing recombinant pharmaceutical proteins in suspension cells. The recent report of high level expression of human growth hormone (somatotrophin) in tobacco chloroplasts, which reached 7% of the total soluble protein, is an exciting result (R. Bassuner, Monsanto Integrated Protein Technologies unit, personal communication; Staub et al. 2000). This is highly interesting because through this technology, high level expression of many other pharmaceutical proteins may be possible and this approach is biologically selfcontained. Clearly, this plastid expression strategy may also be successful with mitochondria. The costs of licensing the technology for transforming plants or for using a promoter from its patent assignees are important aspects of molecular farming in plants. These costs are often large, close to the costs of development of the transgenic plant line or greater. If a laboratory develops its own proprietary transformation and expression system, this makes their molecular farming products less expensive and more attractive. It will also be valuable to screen a range of other plant species than used today for their use in molecular farming. The decision on what spe-
295 cies to use should be based on identifying a plant species that has a high annual yield and that is capable of high levels of protein expression in a background of low levels of noxious compounds. Concluding remarks We have discussed the advantages of plants as an expression system and described how they serve as an expression system for pharmaceutically important, commercially valuable proteins. At the current rate of progress, molecular farming may become the premier expression system for many pharmaceutical proteins in the next decade. This is partly because of the utility of plants, with their higher eukaryotic protein synthesis pathway, to make mammalian proteins. Importantly, the economic advantages brought by using plants, which can be grown with such a minimal investment in their cultivation, makes them attractive as an industrial scale expression system. It is unlikely that the consumer reaction against GM technology will extend to molecular farming because the public is reluctant to harshly criticize medically related research or attempts to provide safer supplies of medicines. In the near future, the human genome sequencing project will be completed. It is easy to foresee the causes of many diseases will be identified through it, leading to new therapies. It is possible many of the new therapeutics will be complex proteins requiring a eukaryotic production system and that plants will be the leading expression system. Moreover, the therapeutic proteins may become more complex and more demanding to produce and at that point, plants should dominate the field. We anticipate that plants will be the premier expression system for diagnostic and therapeutic proteins. Plant expression systems have the potential to make them as abundant tomorrow as prescription drugs are today. We foresee that molecular farming will provide a basket full of novel medicines for the diseases of the 21st century, just as plants were the source of medicines for the Egyptians 3600 years ago. Acknowledgements The authors would like to thank all the members of the Fischer group for their contribution to this work, in particular, Stefan Schillberg, Carmen Vaquero, Sabine Zimmermann, Markus Sack, Stefan Hellwig, Jürgen Drossard, Uli Commandeur and Flora Schuster. We
are indebted to Paul Christou’s research group at the JIC (Norwich, UK) for the ongoing, fruitful collaboration, in particular Eva Stöger, Esperanza Torres and Yolande Perrin. We thank Professor Fritz Kreuzaler (RWTH Aachen) for his long-term interest in the work of the group.
References An G (1985) High efficiency transformation of cultured tobacco cells. Plant Physiol 79: 568–570. Arakawa T, Chong DK and Langridge WH (1998a) Efficacy of a food plant-based oral cholera toxin B subunit vaccine [published erratum appears in Nat Biotechnol 1998 16: 478]. Nat Biotechnol 16: 292–297. Arakawa T, Yu J, Chong DK, Hough J, Engen PC and Langridge WH (1998b) A plant-based cholera toxin B subunit-insulin fusion protein protects against the development of autoimmune diabetes. Nat Biotechnol 16: 934–938. Artsaenko O, Kettig B, Fiedler U, Conrad U and Düring K (1998) Potato tubers as a biofactory for recombinant antibodies. Mol Breeding 4: 313–319. Artsaenko O, Peisker M, zur Nieden U, Fiedler U, Weiler EW, Müntz K and Conrad U (1995). Expression of a single-chain Fv antibody against abscisic acid creates a wilty phenotype in transgenic tobacco. Plant J 8: 745–750. Baker D and Harkonen W (1990) Regulatory agency concerns in the manufacturing and testing of monoclonal antibodies for therapeutic use. Targeted Diagn Ther 3: 75–98. Bardor M, Faye L and Lerouge P (1999) Analysis of the Nglycosylation of recombinant glycoproteins produced in transgenic plants. Trends Plants Sci 4: 376–380. Barta A, Sommergruber K, Thompson D, Hartmuth K, Matzke M and Matzke A (1986) The expression of a nopaline synthasehuman growth hormone chimaeric gene in transformed tobacco and sunflower callus tissue. Plant Mol Biol 6: 347–357. Baum TJ, Hiatt A, Parrott WA, Pratt LH and Hussey RS (1996) Expression in tobacco of a functional monoclonal antibody specific to stylet secretions of the root-knot nematode. Mol Plant Microbe In 9: 382–387. Benvenuto E, Ordas RJ, Tavazza R, Ancora G, Biocca S, Cattaneo A and Galeffi P (1991) ‘Phytoantibodies’: A general vector for the expression of immunoglobulin domains in transgenic plants. Plant Mol Biol 17: 865–874. Bisaria V and Panda A (1991) Large-scale plant cell culture: methods, applicatons and products. Curr Opin Biotech 2: 370–374. Bookman MA (1998) Biological therapy of ovarian cancer: current directions. Semin Oncol 25: 381–396. Borisjuk NV, Borisjuk LG, Logendra S, Petersen F, Gleba Y and Raskin I (1999) Production of recombinant proteins in plant root exudates. Nat Biotechnol 17: 466–469. Bosch D, Smal J and Krebber E (1994) A trout growth hormone is expressed, correctly folded and partially glycosylated in the leaves but not the seed of transgenic plants. Transgenic Res 3: 304–310. Bruyns AM, De Jaeger G, De Neve M, De Wilde C, Van Montagu M and Depicker A (1996) Bacterial and plant-produced scFv proteins have similar antigen-binding properties. FEBS Lett 386: 5–10. Cabanes-Macheteau M, Fitchette-Laine AC, Loutelier-Bourhis C, Lange C, Vine N, Ma J, et al. (1999) N-Glycosylation of a
296 mouse IgG expressed in transgenic tobacco plants. Glycobiology 9: 365–372. Carpita N, Sabularse D, Montezinos D and Delmer DP (1979). Determination of the pore size of cell walls of living plant cells. Science 205: 1144–1147. Carrillo C, Wigdorovitz A, Oliveros JC, Zamorano PI, Sadir AM, Gomez N, et al. (1998) Protective immune response to foot-andmouth disease virus with VP1 expressed in transgenic plants. J Virol 72: 1688–1690. Chan MT, Chang HH, Ho SL, Tong WF and Yu SM (1993) Agrobacterium-mediated production of transgenic rice plants expressing a chimeric alpha-amylase promoter/beta-glucuronidase gene. Plant Mol Biol 22: 491–506. Chen MH, Liu LF, Chen YR, Wu HK and Yu SM (1994) Expression of alpha-amylases, carbohydrate metabolism, and autophagy in cultured rice cells is coordinately regulated by sugar nutrient. Plant J 6: 625–636. Chintalacharuvu KR and Morrison SL (1997) Production of secretory immunoglobulin A by a single mammalian cell. Proc Natl Acad Sci USA 94: 6364–6368. Christou P (1993) Particle gun-mediated transformation. Curr Opin Biotech 4: 135–141. Christou P (1996) Transformation technology. Trends Plant Sci 1: 423–431. Conrad U and Fiedler U (1998). Compartment-specific accumulation of recombinant immunoglobulins in plant cells: An essential tool for antibody production and immunomodulation of physiological functions and pathogen activity. Plant Mol Biol 38: 101–109. Conrad U, Fiedler U, Artsaenko O and Phillips J (1998) High level and stable accumulation of single chain Fv antibodies in plant storage organs. J Plant Physiol 152: 708–711. Cramer CL, Weissenborn DL, Oishi KK, Grabau EA, Bennett S, Ponce E, Grabowski GA, and Radin DN (1996) Bioproduction of human enzymes in transgenic tobacco. Ann N Y Acad Sci. 792: 62–71. Dalsgaard K, Uttenthal A, Jones TD, Xu F, Merryweather A, Hamilton WD, Langeveld JP, Boshuizen RS, Kamstrup S, Lomonossoff GP, Porta C, Vela C, Casal JI, Meloen RH and Rodgers PB (1997) Plant-derived vaccine protects target animals against a viral disease. Nat Biotechnol 15: 248–252. Daniell T and Edwards R (1995) Changes in protein methylation associated with the elicitation response in cell cultures of alfalfa (Medicago sativa L.). FEBS Lett 360: 57–61. De Jaeger G, Buys E, Eeckhout D, De Wilde C, Jacobs A, Kapila J, Angenon G, Van Montagu M, Gerats T and Depicker A (1998) High level accumulation of single-chain variable fragments in the cytosol of transgenic Petunia hybrida. Eur J Biochem 259: 1–10. De Neve M, De Loose M, Jacobs A, Van Houdt H, Kaluza B, Weidle U, Van Montagu M and Depicker A (1993). Assembly of an antibody and its derived antibody fragment in Nicotiana and Arabidopsis. Transgenic Res 2: 227–237. De Wilde C, De Neve M, De Rycke R, Bruyns AM, De Jaeger G, Van Montagu M, Depicker A and Engler G (1996) Intact antigen-binding MAK33 antibody and Fab fragment accumulate in intercellular spaces of Arabidopsis thaliana. Plant Sci 114: 233–241. De Zoeten GA, Penswick JR, Horisberger MA, Ahl P, Schultze M and Hohn T (1989) The expression, localization, and effect of a human interferon in plants. Virology 172: 213–222. Desikan R, Hancock JT, Neill SJ, Coffey MJ and Jones OT (1996) Elicitor-induced generation of active oxygen in suspension cultures of Arabidopsis thaliana. Biochem Soc Trans 24: 199S.
Dieryck W, Pagnier J, Poyart C, Marden MC, Gruber V, Bournat P, et al. (1997) Human haemoglobin from transgenic tobacco [letter]. Nature 386: 29–30. Düring K, Hippe S, Kreuzaler F and Schell J (1990) Synthesis and selfassembly of a functional monoclonal antibody in transgenic Nicotiana tabacum. Plant Mol Biol 15: 281–293. Echelard Y (1996) Recombinant protein production in transgenic animals. Curr Opin Biotech 7: 536–540. Ehsani P, Khabiri A and Domansky NN (1997) Polypeptides of hepatitis B surface antigen produced in transgenic potato. Gene 190: 107–111. Fahrner RL, Blank GS, and Zapata GA (1999) Expanded bed protein A affinity chromatography of a recombinant humanized monoclonal antibody: Process development, operation, and comparison with a packed bed method. J Biotechnol 75: 273–280. Fecker L F, Kaufmann A, Commandeur U, Commandeur J, Koenig R and Burgermeister W (1996). Expression of single-chain antibody fragments (scFv) specific for beet necrotic yellow vein virus coat protein or 25kDa protein in Escherichia coli and Nicotiana benthamiana. Plant Mol Biol 32: 979–986. Fecker LF, Koenig R and Obermeier C (1997) Nicotiana benthamiana plants expressing beet necrotic yellow vein virus (BNYVV) coat protein-specific scFv are partially protected against the establishment of the virus in the early stages of infection and its pathogenic effects in the late stages of infection. Arch Virol 142: 1857–1863. Fiedler U and Conrad U (1995) High-level production and long-term storage of engineered antibodies in transgenic tobacco seeds. Bio/Technol 13: 1090–1093. Fiedler U, Philips J, Artsaenko O and Conrad U (1997) Optimisation of scFv antibody production in transgenic plants. Immunotechnology 3: 205–216. Firek S, Draper J, Owen MR, Gandecha A, Cockburn B and Whitelam GC (1993a) Secretion of a functional single-chain Fv protein in transgenic tobacco plants and cell suspension cultures [published erratum appears in Plant Mol Biol 1994 Mar; 24(5): 833]. Plant Mol Biol 23: 861–870. Firek S, Draper J, Owen MRL, Gandecha A, Cockburn B and Whitelam GC (1993b) Secretion of a functional single-chain Fv protein in transgenic tobacco plants and cell suspension cultures. Plant Mol Biol 23: 861–870. Fischer R, Drossard J, Commandeur U, Schillberg S and Emans N (1999a) Toward molecular farming in the future: Moving from diagnostic protein and antibody production in microbes to plants. Biotechnol Appl Biochem 30: 101–108. Fischer R, Emans N, Schuster F, Hellwig S and Drossard J (1999b) Toward molecular farming in the future: Using plant cell suspension cultures as bioreactors. Biotechnol Appl Biochem. 30: 113–116. Fischer R, Liao Y-C and Drossard J (1999c) Affinity-purification of a TMV-specific recombinant full-size antibody from a transgenic tobacco suspension culture. J Immunol Meth 226: 1–10. Fischer R, Schumann D, Zimmermann S, Drossard J, Sack M and Schillberg S (1999d) Expression and characterization of bispecific single chain Fv fragments produced in transgenic plants. European J Biochem 262: 810–816. Francisco JA, Gawlak SL, Miller M, Bathe J, Russell D, Chace D, et al. (1997) Expression and characterization of bryodin 1 and a bryodin 1-based single-chain immunotoxin from tobacco cell culture. Bioconjug Chem 8: 708–713. Franconi R, Roggero P, Pirazzi P, Arias FJ, Desiderio A, Bitti O, et al. (1999) Functional expression in bacteria and plants of an scFv antibody fragment against tospoviruses. Immunotechnology 4: 189–201.
297 Franken E, Teuschel U and Hain R (1997) Recombinant proteins from transgenic plants. Curr Opin Biotech 8: 411–416. Gallie D 1998. Controlling gene expression in transgenics. Curr Opin Plant Biol 1: 166–172. Gerstmayer B, Hoffmann M, Altenschmidt U and Wels W (1997) Costimulation of T-cell proliferation by a chimeric B7-antibody fusion protein. Cancer Immunol Immunother 45: 156–158. Goossens A, Van Montagu M and Angenon G (1999) Cointroduction of an antisense gene for an endogenous seed storage protein can increase expression of a transgene in Arabidopsis thaliana seeds. FEBS Lett 456: 160–164. Haq TA, Mason HS, Clements JD and Arntzen CJ (1995) Oral immunization with a recombinant bacterial antigen produced in transgenic plants. Science 268: 714–716. Hendy S, Chen ZC, Barker H, Santa Cruz S, Chapman S, Torrance L, Cockburn W and Whitelam GC (1999) Rapid production of single-chain Fv fragments in plants using a potato virus X episomal vector. J Immunol Meth 231: 137–146. Hiatt A (1990) Antibodies produced in plants. Nature 344: 469–470. Hiatt A, Cafferkey R and Bowdish K (1989) Production of antibodies in transgenic plants. Nature 342: 76–78. Hiatt A and Ma JK (1993) Characterization and applications of antibodies produced in plants. Int Rev Immunol 10: 139–152. Hiatt A, Tang Y, Weiser W and Hein MB (1992) Assembly of antibodies and mutagenized variants in transgenic plants and plant cell cultures. Genet Eng 14: 49–64. Hiatt AC (1991) Production of monoclonal antibody in plants. Transplant Proc 23: 147–151. Hiei Y, Komari T and Kubo T (1997) Transformation of rice mediated by Agrobacterium tumefaciens. Plant Mol Biol 35: 205–218. Hiei Y, Ohta S, Komari T and Kumashiro T (1994) Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. Plant J 6: 271–282. Higo K, Saito Y and Higo H (1993) Expression of a chemically synthesized gene for human epidermal growth factor under the control of cauliflower mosaic virus 35S promoter in transgenic tobacco. Biosci Biotech Biochem 57: 1477–1481. Hoehl U , Upmeier B and Barz W (1988) Growth and nicotinate biotransformation in batch cultured and airlift fermenter grown soybean cell suspension cultures. Appl Microbiol Biote 28: 319– 323. Hogue RS, Lee JM and An G (1990) Production of a foreign protein product with genetically modified plant cells. Enzyme Microb Technol 12: 533–538. Hood E, Witcher D Maddock S, Meyer T, Baszczynski C, Bailey M, et al. (1997) Commercial production of Avidin from transgenic maize: characterization of transformant, production, processing, extraction and purification. Mol Breed 3: 291–306. Hood EE, Kusnadi A, Nikolov Z and Howard JA (1999) Molecular farming of industrial proteins from transgenic maize. Adv Exp Med Biol 464: 127–147. Hooker BS, Lee JM and An G (1990) Cultivation of plant cells in a stirred tank reactor. Biotechnol Bioeng 35: 296–304. Horsch R, Fry JE, Hoffman N, Eicholtz D, Rogers S and Fraley R (1985) A simple and general method for transferring genes into plants. Science 227: 1229–1231. Johnson J, Lin T and Lomonossoff G (1997) Presentation of heterologous peptides on plant viruses- genetics, structure and function. Ann Rev Phytopathol 35: 67–86. Kapila J , De Rycke R, van Montagu M and Angenon G (1996) An Agrobacterium mediated transient gene expression system for intact leaves. Plant Sci 122: 101–108.
Kapusta J, Modelska A, Figlerowicz M, Pniewski T, Letellier M, Lisowa O, Yusibov V, Koprowski H, Plucienniczak A and Legocki AB (1999) A plant-derived edible vaccine against hepatitis B virus. FASEB J 13: 1796–1799. Khoudi H, Laberge S, Ferullo JM, Bazin R, Darveau A, Castonguay Y, Allard G, Lemieux R and Vezina LP (1999) Production of a diagnostic monoclonal antibody in perennial alfalfa plants. Biotechnol Bioeng 64: 135–143. Kieran PM, MacLoughlin PF and Malone DM (1997) Plant cell suspension cultures: some engineering considerations. J Biotechnol 59: 39–52. Koehler G and Milstein C (1975) Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256: 495– 497. Koncz C and Schell J (1986) The promoter of TL -DNA gene 5 controls the tissue specific expression of chimeric genes carried by a novel type of Agrobacterium binary vector. Mol Gen Genet 204: 383–396. Kumagai MH, Donson J, della-Cioppa G and Grill LK (2000) Rapid, high-level expression of glycosylated rice alpha-amylase in transfected plants by an RNA viral vector. Gene 245: 169–174. Kusnadi AR, Hood EE, Witcher DR, Howard JA and Nikolov ZL (1998) Production and purification of two recombinant proteins from transgenic corn. Biotechnol Prog 14: 149–155. Kusnadi AR, Nikolov ZL and Howard JA (1997) Production of recombinant proteins in transgenic plants: Practical considerations. Biotechnol Bioeng 56: 473–484. Larrick JW, Yu L, Chen J, Jaiswal S and Wycoff K (1998) Production of antibodies in transgenic plants. Res Immunol 149: 603–608. Le Gall F, Bove JM and Garnier M (1998) Engineering of a singlechain variable-fragment (scFv) antibody specific for the stolbur phytoplasma (Mollicute) and its expression in Escherichia coli and tobacco plants. Appl Environ Microbiol 64: 4566–4572. Lee JS, Choi SJ, Kang HS, Oh WG, Cho KH, Kwon TH, et al. (1997) Establishment of a transgenic tobacco cell suspension culture system for producing murine granulocyte-macrophage colony stimulating factor. Mol Cell 7: 783–787. Leite A, Kemper E, da Silva M, Luchessi A, Siloto R, Bonaccorsi E, et al. (2000) Expression of correctly processed human growth hormone in seeds of transgenic tobacco plants. Mol Breeding 6: 47–53. Lindsey K and Jones MGK (1987) Transient gene expression in electroporated protoplasts and intact cells of sugar beet. Plant Mol Biol 10: 43–52. Long R (1984) Edible tobacco protein. Crops and Soils Magazine. Feb.: 13–15. Ma J and Hein M (1995a) Immunotherpeuic potential of antibodies produced in plants. Trends Biotechnol 13: 522–527. Ma J and Hein M (1995b) Plant antibodies for Immunotherapy. Plant Physiol 109: 341–346. Ma JK, HiattA, Hein M, Vine ND, Wang F, Stabila P, et al. (1995) Generation and assembly of secretory antibodies in plants. Science 268: 716–719. Ma JK, Hikmat BY, Wycoff K, Vine ND, Chargelegue D, Yu L, et al. (1998) Characterization of a recombinant plant monoclonal secretory antibody and preventive immunotherapy in humans. Nat Med 4: 601–606. Ma JK and Vine ND (1999) Plant expression systems for the production of vaccines. Curr Top Microbiol Immunol 236: 275–292. Ma JKC, Lehner T, Stabila P, Fux CI and Hiatt A (1994) Assembly of monoclonal antibodies with IgG1 and IgA heavy chain domains in transgenic tobacco plants. Eur J Immunol 24: 131–138.
298 Ma SW, Zhao DL, Yin ZQ, Mukherjee R, Singh B, Qin HY, et al. (1997) Transgenic plants expressing autoantigens fed to mice to induce oral immune tolerance. Nat Med 3: 793–796. Magnuson NS, Linzmaier PM, Reeves R, An G, HayGlass K and Lee JM (1998) Secretion of biologically active human interleukin-2 and interleukin-4 from genetically modified tobacco cells in suspension culture. Protein Expr Purif 13: 45–52. Mariani M and L Tarditi (1992) Validating the preparation of clinical monoclonal antibodies. Bio/Technology 10: 394–396. Mason HS and CJ Arntzen (1995) Transgenic plants as vaccine production systems. Trends Biotechnol 13: 388–392. Mason HS, Ball JM, Shi JJ, Jiang X, Estes MK and Arntzen CJ (1996) Expression of Norwalk virus capsid protein in transgenic tobacco and potato and its oral immunogenicity in mice. Proc Natl Acad Sci USA 93: 5335–5340. Mason HS, Haq TA, Clements JD and Arntzen CJ (1998) Edible vaccine protects mice against Escherichia coli heat-labile enterotoxin (LT): potatoes expressing a synthetic LT-B gene. Vaccine 16: 1336–1343. Mason HS, Lam DM and Arntzen CJ (1992) Expression of hepatitis B surface antigen in transgenic plants. Proc Natl Acad Sci USA 89: 11745–11749. Matsumoto S, Ikura K, Ueda M and Sasaki R (1995) Characterisation of a human glycoprotein (erythropoetin) produced in cultured tobacco cells. Plant Mol Biol 27: 1163–1172. McConnell SJ, Dinh T, Le MH, Brown SJ, Becherer K, Blumeyer K, et al. (1998) Isolation of erythropoietin receptor agonist peptides using evolved phage libraries. Biol Chem 379: 1279–1286. McCormick AA, Kumagai MH, Hanley K, Turpen TH, Hakim I, Grill LK, et al. (1999) Rapid production of specific vaccines for lymphoma by expression of the tumor-derived single-chain Fv epitopes in tobacco plants. Proc Natl Acad Sci USA 96: 703–708. McGarvey PB, Hammond J, Dienelt MM, Hooper DC, Fu ZF, Dietzschold B, et al. (1995) Expression of the rabies virus glycoprotein in transgenic tomatoes. Bio/Technology 13: 1484–1487. Miele L (1997) Plants as bioreactors for pharmaceuticals: regulatory considerations. Trends Biotechnol 15: 45–50. Modelska A, Dietzschold B, Sleysh N, Fu ZF, Steplewski K, Hooper DC, Koprowski H and Yusibov V (1998) Immunization against rabies with plant-derived antigen. Proc Natl Acad Sci USA 95: 2481–2485. Moloney MM and Holbrook LA (1997) Subcellular targeting and purification of recombinant proteins in plant production systems. Biotechnol Genet Eng Rev 14: 321–336. Mu JH, Chua NH and Ross EM (1997) Expression of human muscarinic cholinergic receptors in tobacco. Plant Mol Biol 34: 357–362. Murano G (1997) FDA perspective on specifications for biotechnology products – from IND to PLA. Dev Biol Stand 91: 3–13. Murray A, Sekowski M, Spencer DI, Denton G and Price MR (1997) Purification of monoclonal antibodies by epitope and mimotope affinity chromatography. J Chromatogr A 782: 49–54. Nagata T, Nemoto Y and Seiichiro H (1992) Tobacco BY-2 cell line as the ‘HeLa’ cell in the cell biology of higher plants. Int Rev Cytol 132: 1–30. Nilsson J, Stahl S, Lundeberg J, Uhlen M and Nygren PA (1997) Affinity fusion strategies for detection, purification, and immobilization of recombinant proteins. Protein Expr Purif 11: 1–16. Owen M, Gandecha A, Cockburn B and Whitelam G (1992) Synthesis of a functional anti-phytochrome single-chain Fv protein in transgenic tobacco. Bio/Technology 10: 790–794.
Parmenter DL, Boothe JG, van Rooijen GJ, Yeung EC and Moloney MM (1995) Production of biologically active hirudin in plant seeds using oleosin partitioning. Plant Mol Biol 29: 1167–1180. Phillips J, Artsaencko O, Fiedler U, Horstmann C, Mock HP, Müntz K and Conrad U (1997) Seed-specific immunomodulation of abscisic acid activity induces a developmental switch. EMBO J 16: 4489–4496. Porceddu A, Falorni A, Ferradini N, Cosentino A, Calcinaro F, Faleri C, et al. (1999) Transgenic plants expressing human glutamic acid decarboxylase (GAD65), a mojor autoantigen in insulin-dependent diabetes mellitus. Mol Breeding 5: 553–560. Porta C and Lomonossoff GP (1996) Use of viral replicons for the expression of genes in plants. Mol Biotech 5: 209–221. Porta C, Spall VE, Lin T, Johnson JE and Lomonossoff GP (1996) The development of cowpea mosaic virus as a potential source of novel vaccines. Intervirology 39: 79–84. Schillberg S, Zimmermann S, Findlay K and R Fischer Plasma membrane display of anti-viral single chain Fv fragments confers resistance to tobacco mosaic virus. Mol. Breeding (in press). Schillberg S, Zimmermann S, Voss A and Fischer R (1999) Apoplastic and cytosolic expression of full-size antibodies and antibody fragments in Nicotiana tabacum. Transgenic Res 8: 255–263. Scholthof H, Scholthof K and Jackson A (1996) Plant virus gene vectors for transient expression of foreign proteins in plants. Annu Rev Phytopathol 34: 299–323. Schouten A, Roosien J, de Boer JM, Wilmink A, Rosso MN, Bosch D, Stiekema WJ, Gommers FJ, Bakker J and Schots A (1997) Improving scFv antibody expression levels in the plant cytosol. FEBS Lett 415: 235–241. Schouten A, Roosien J, van Engelen FA, de Jong GAM, BorstVrenssen AWM, Zilverentant JF, et al. (1996) The C-terminal KDEL sequence increases the expression level of a single-chain antibody designed to be targeted to both cytosol and the secretory pathway in transgenic tobacco. Plant Mol Biol 30: 781–793. Seki M, Ohzora C, Takeda M and Furusaki S (1997) Taxol (Paclitaxel) production using free and immobilized cells of taxus cuspidata. Biotechnol Bioengng 53: 214–219. Sheen S (1983) Biomass and chemical composition of tobacco plants under high density growth. Beitr Tabakforsch Int 12: 35–42. Shen WH and Hohn B (1995) Vectors based on maize streak virus can replicate to high copy numbers in maize plants. J Gen Virol 76: 965–969. Shin SU, Wright A and Morrison SL (1993) Hybrid antibodies. Int Rev Immunol 10: 177–186. Sijmons PC, Dekker BMM, Schrammeijer B, Verwoerd TC, van den Elzen PJM and Hoekema A (1990) Production of correctly processed human serum albumin in transgenic plants. Bio/Technol 8: 217–221. Skerra A (1993) Bacterial expression of immunoglobulin fragments. Curr Opin Biotech 5: 256–262. Staub J, Garcia B, Graves J, Hajdukiewicz P, Hunter P, Nehra N, Paradkar V, Schlittler M, Carroll J, Spatola L, Ward D, Ye G and Russell D (2000) High-yield production of a human therapeutic protein in tobacco chloroplasts. Nature Biotechnol 18: 333–338. Stöger E, Vaquero C, Torres E, Sack M, Nicholson L, Drossard J, Williams S, Keen D, Perrin Y, Christou P and Fischer R (2000) Cereal crops as viable production and storage systems for pharmaceutical scFv antibodies. Plant Mol Biol 42: 583–590. Tackaberry ES, Dudani AK, Prior F, Tocchi M, Sardana R, Altosaar I and Ganz PR (1999) Development of biopharmaceuticals in plant expression systems: cloning, expression and immun-
299 ological reactivity of human cytomegalovirus glycoprotein B (UL55) in seeds of transgenic tobacco. Vaccine 17: 3020–3029. Tacket CO, Mason HS, Losonsky G, Clements JD, Levine MM and Arntzen CJ (1998) Immunogenicity in humans of a recombinant bacterial antigen delivered in a transgenic potato. Nat Med 4: 607–609. Taticek RA, Lee CWT and Shukler ML (1994) Large scale insect and plant cell culture. Curr Opin Biotech 5: 165–174. Tavladoraki P, Benvenuto E, Trinca S, De Martinis D, Cattaneo A and Galeffi P (1993) Transgenic plants expressing a functional single-chain Fv antibody are specifically protected from virus attack. Nature 366: 469–472. Terashima M, Murai Y, Kawamura M, Nakanishi S, Stoltz T, Chen L, Drohan W, Rodriguez RL and Katoh S (1999) Production of functional human alpha 1-antitrypsin by plant cell culture. Appl Microbiol Biotechnol 52: 516–523. Torres E, Vaquero C, Nicholson L, Sack M, Stöger E, Drossard J, et al. (1999) Rice cell culture as an alternative production system for functional diagnostic and therapeutic antibodies. Transgenic Res 8: 441–449. Turpen TH, Reinl SJ, Charoenvit Y, Hoffman SL, Fallarme V and Grill LK (1995) Malarial epitopes expressed on the surface of recombinant tobacco mosaic virus. Bio/Technology 13: 53–57. Van Der Heijden R, Verpoorte R and Ten Hoopen HJG (1989) Cell and tissue cultures of Catharanthus-Roseus LG Don a literature survey. Plant Cell Tissue & Organ Culture 18: 231–280. van Engelen FA, Schouten A, Molthoff JW, Roosien J, Salinas J, Dirkse WG, et al. (1994) Coordinate expression of antibody subunit genes yields high levels of functional antibodies in roots of transgenic tobacco. Plant Mol Biol 26: 1701–1710. Vaquero C, Sack M, Chandler J, Drossard J, Schuster F, Schillberg S, et al. (1999) Transient expression of a tumor-specific single chain fragment and a chimeric antibody in tobacco leaves. Proc Natl Acad Sci 96: 11128–11133. Verch T, Lewandowski D and Fischer R (1999) Expression of a single chain antibody in plants using a viral vector (submitted). Verch T, Yusibov V and Koprowski H (1998) Expression and assembly of a full-length monoclonal antibody in plants using a plant virus vector. J Immunol Meth 220: 69–75. Verwoerd TC, van Paridon PA, van Ooyen AJJ, van Lent JWM, Hoekema A and Pen J (1995) Stable accumulation of Aspergillus niger phytase in transgenic tobacco leaves. Plant Physiol 109: 1199–1205. Voss A, Niersbach M, Hain R, Hirsch H, Liao Y, Kreuzaler F, et al. (1995) Reduced virus infectivity in N. tabacum secreting a TMVspecific full size antibody. Mol Breeding 1: 39–50. Walmsley A and Arntzen C (2000) Plants for delivery of edible vaccines. Curr Opin Biotech 11: 126–129. Walsh G (1998) Pharmaceuticals, biologics and biopharmaceuticals. In: Biopharmaceuticals: Biochemistry and Biotechnology, (pp. 1–35) Wiley, Chichester, UK.
Whitelam GC, Cockburn B, Gandecha AR and Owen MR (1993) Heterologous protein production in transgenic plants. Biotechnol Genet Eng Rev 11: 1–29. Whitelam GC, Cockburn W and Owen MR (1994) Antibody production in transgenic plants. Biochem Soc Trans 22: 940– 944. Whitelam GC, aG W (1996) Antibody expression in transgenic plants. Trends Plant Sci 1: 268–271. Wigdorovitz A, Carrillo C, Dus Santos MJ, Trono K, Peralta A, Gomez MC, et al. (1999) Induction of a protective antibody response to foot and mouth disease virus in mice following oral or parenteral immunization with alfalfa transgenic plants expressing the viral structural protein VP1. Virology 255: 347–353. Winter G, Griffiths AD, Hawkins RE and Hoogenboom HR (1994) Making antibodies by phage display technology. Annu Rev Immunol 12: 433–455. Winter G and Milstein C (1991) Man-made antibodies. Nature 349: 293–299. Witcher D, Hood E, Peterson D, Bailey M, Marchall L, Bond D, et al. (1998) Commercial production of b-glucuronidase (GUS): a model system for the production of proteins in plants. Mol Breeding 4: 301–312. Worn A, Auf Der Maur A, Escher D, Honegger A, Barberis A and Plückthun A (2000) Correlation between in vitro stability and in vivo performance of anti- GCN4 intrabodies as cytoplasmic inhibitors. J Biol Chem 275: 2795–2803. Yusibov V, Modelska A, Steplewski K, Agadjanyan M, Weiner D, Hooper DC, et al. (1997) Antigens produced in plants by infection with chimeric plant viruses immunize against rabies virus and HIV-1. Proc Natl Acad Sci USA 94: 5784–5788. Zeitlin L, Olmsted SS, Moench TR, Co MS, Martinell BJ, Paradkar VM, et al. (1998) A humanized monoclonal antibody produced in transgenic plants for immunoprotection of the vagina against genital herpes. Nat Biotechnol 16: 1361–1364. Zhong G-Y, Peterson D, Delaney D, Bailey M, Witcher D, Register J, et al. (1999) Commercial production of Aprotinin in transgenic maize seeds. Mol Breeding 5: 345–356. Zhu Z, Hughes K, Huang L, Sun B, Liu C, Li Y, et al. (1994) Expression of human alpha-interferon in plants. Virology 172: 213–222. Ziegler M, Thomas S and Danna K (2000) Accumulation of a thermostable endo-1,4-b-D-glucanase in the apoplast of Arabidopsis thaliana leaves. Mol Breeding 6: 37–46. Zimmermann S, Schillberg S, Liao YC and Fischer R (1998) Intracellular expression of TMV-specific single-chain Fv fragments leads to improved virus resistance in Nicotiana tabacum. Mol Breeding 4: 369–379. Zwick M, Shen J and Scott J (1998) Phage-displayed peptide libraries. Curr Opin Biotech 9: 427–436.