Transgenic Research9: 279–299, 2000.© 2000Kluwer Academic Publishers. Printed in the Netherlands.

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Molecular farming of pharmaceutical proteins

Rainer Fischer1,2,∗ & Neil Emans11Institut für Biologie I (Botanik/Molekulargenetik), RWTH Aachen, Worringerweg 1, D-52074 Aachen, Germany2Fraunhofer Department for Molecular Biotechnology, IUCT, Grafschaft, Auf dem Aberg 1, D-57392Schmallenberg, Germany

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 pharmaceuticalproteins. Complex mammalian proteins can be produced in transformed plants or transformed plant suspensioncells. Plants are suitable for the production of pharmaceutical proteins on a field scale because the expressedproteins are functional and almost indistinguishable from their mammalian counterparts. The breadth of therapeuticproteins produced by plants range from interleukins to recombinant antibodies. Molecular farming in plants hasthe potential to provide virtually unlimited quantities of recombinant proteins for use as diagnostic and therapeutictools in health care and the life sciences. Plants produce a large amount of biomass and protein production canbe increased using plant suspension cell culture in fermenters, or by the propagation of stably transformed plantlines 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 recombinanttherapeutics, including vaccines, diagnostics, such as recombinant antibodies, plasma proteins, cytokines andgrowth factors.

Introduction

The use of plants in medicine stretches back to theearliest stages of civilization. As early as 1600 BC, theEgyptians compiled a list of more than 700 medicinalplants. The active ingredients in many of these plantshave now been identified, and close to one quarter ofprescription drugs are still of plant origin. Modern bi-otechnology is extending the use of plants in medicinewell beyond its original boundaries. Plants are now asource of pharmaceutical proteins, such as mammalianantibodies (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; Zeitlinet al., 1998; Fischer et al., 1999a), blood substitutes(Magnuson et al., 1998) and vaccines (Arakawa et al.,

∗ Author for correspondence: Institut für Biologie ITel.: +49241 806631;Fax: +49 241 871062;E-mail: fischer@bio1.rwth-aachen.de

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 pharma-ceutically important and commercially valuable pro-teins in plants (Franken et al., 1997). It harnesses het-erologous protein expression systems, such as plants,for the large-scale production of recombinant proteinsthat are therapeutically valuable. Here, we brieflyreview its history, then discuss plant expression tech-nology and how plant cells can be optimally used toproduce recombinant proteins by molecular farming.

In its simplest form, molecular farming is the ex-pression of recombinant insulin in bacteria; in its mostchallenging form, molecular farming is the productionof chimeric anti-tumor antibodies and multi-subunitprotein complexes, like secretory IgA, in plants. Thistechnology has now reached the point where it is com-mercially viable. Its further development will bring

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many therapies that are now too expensive for wideuse into the hands of most medical practitioners. Weanticipate that molecular farming may be a major areaof economic growth in agricultural biotechnology.

In our definition, molecular farming encompassesthe production of recombinant proteins in a heterolog-ous expression system. In general, the recombinantproteins that we choose to express are pharmaceuti-cally valuable, such as a tumor or pathogen specificantibody. Consequently, the expression of an antibodyin tobacco is molecular farming while the purificationof that antibody from its native source is not. Mo-lecular farming a protein also implies that large-scaleproduction of a recombinant protein is both possibleand economically feasible. Thus, production of anantibody in a field of transgenic tobacco is closer tomolecular farming than the production of the antibodyin a hybridoma culture. Molecular farming is princip-ally the production of a pharmaceutical protein andnot the modification of the expression system. Forexample, the creation of a pathogen resistant plantby the expression of an anti-pathogen antibody isonly an application of the molecular farming techno-logy, but it is not molecular farming according to ourdefinition.

Molecular farming is thus best defined as the ex-pression of pharmaceutically and commercially valu-able proteins in plants. It was developed based onthe pretext that many pharmaceutically active proteinshave been identified but without the means to producethem, their therapeutic potential can not be evaluated.The purpose of molecular farming is to produce largeamounts of an active, safe pharmaceutical protein atan affordable price. As such, there are two stages tomolecular farming – the development of an optim-ized expression system and its scale up to economiclevels of production. We feel that plants are the ex-pression system that best meets these prerequisites.Further developments in protein expression in plantsshould make molecular farming an even more attract-ive alternative to animals, animal cells and microbialcells.

In this review, after a brief historical overview ofthe development of molecular farming, we concen-trate on discussing the expression of pharmaceuticalproteins in plants through stable or transient expres-sion in whole plants, leaves or seeds. The economicand practical advantages of plants are discussed, as iswhere molecular farming is likely to expand. We thenspeculate on how the use of plants as an expressionsystem may be improved.

The demand for safe, recombinant pharmaceut-ical proteins is expanding rapidly. Both the amountsof proteins needed and protein complexity are in-creasing as novel pharmaceutical activities are iden-tified or designed into macromolecules. We foreseethat molecular farming in plants will become a pre-eminent method for the production of pharmaceuticalmolecules in the next 10 years.

Molecular farming

Molecular farming is the production of recombinantpharmaceuticals outside their natural source. By defin-ition, molecular farming is preceded by identificationof a protein with a desirable therapeutic or diagnosticactivity, its protein and DNA sequencing and finally itsexpression in a heterologous host. A classic exampleof molecular farming in microbes is the expression ofrecombinant insulin in bacteria.

The anti-diabetic activity of insulin was first iden-tified in 1921 and by 1951 the complete amino acidsequence had been determined. Since the standardsource of the hormone was animal pancreas, a demandemerged for an alternative source of insulin that wassafe, free of immunogenic contaminants and inexpens-ive. Human insulin was an attractive target for expres-sion in microbes for it is a small polypeptide requiringonly minimal post-translational processing to becomefunctional. Expression in bacteria was successful andin 1982 it became the first recombinant protein tobe approved for therapeutic use (Walsh, 1998). Thisreleased any restrictions on the amounts of insulinavailable, by producing a safe, active, recombinanthuman hormone at a low cost.

The potential of using plants as a production sys-tem for recombinant pharmaceuticals was establishedbetween 1986 and 1990 with the successful expressionof a human growth hormone fusion protein, an inter-feron and human serum albumin (Barta et al., 1986;De Zoeten et al., 1989; Sijmons et al., 1990). A cru-cial advance came with the successful expression offunctional antibodies in plants in 1989 (Hiatt et al.,1989) and 1990 (Düring et al., 1990). This was asignificant breakthrough for it showed that plants hadthe potential to produce complex mammalian proteinsof medical importance. By analogy to the productionof insulin in bacteria, the production of antibodiesin plants had the potential to make large amountsof safe, inexpensive antibodies available. This wasan impressive result because plants could produce

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Figure 1. Forms of recombinant antibodies produced by antibody engineering. Ab – Antibody; rAb – recombinant antibody; Fab – fragmentantigen binding; scFv – single chain antibody fragment; dAb – single domain antibody; IL-2 – interleukin-2.

functional full-length antibodies, indicating that allthe post-translational modifications necessary for an-tibody activity occurred, which is not the case for fullsize antibody expression inE. coli. In the following 10years, plants were shown to be able to produce a vari-ety of antibody fragments (Figure 1), secretory IgA,blood substitutes and biological effectors includinginterleukins (Tables 2–4).

Plants as a production system

Historically, bacteria were often the protein expres-sion system of choice and yeast cells or baculovirus-infected insect cell systems were of minor importance(Skerra, 1993; Taticek et al., 1994). While bacteria arean inexpensive, convenient production system, theyare incapable of most of the post-translational modifi-cations necessary for the activity of many mammalianproteins. This limitation and the cost of expression ofproteins in mammalian cells prompted the explorationof plants, as a cheap, safe and efficient alternative.

Advances in recombinant DNA technology, planttransformation technology and antibody engineering

are major reasons why plants have emerged as anexpression system. Antibody expression in plantsshowed proof that plants were capable of expressingfunctional mammalian proteins (Hiatt et al., 1989; Maet al., 1995; Voss et al., 1995) and further progress hasmade it possible to produce chimeric mouse–humantherapeutic antibodies in plants in sufficient quantit-ies for pre-clinical trials (Zeitlin et al., 1998; Vaqueroet al., 1999).

Plant expression systems are attractive becausethey offer significant advantages over the classical ex-pression systems based on bacterial, microbial andanimal cells (Table 1). Firstly, they have a highereukaryote protein synthesis pathway, very similar toanimal cells with only minor differences in proteinglycosylation (Cabanes-Macheteau et al., 1999). Con-trastingly, bacteria cannot produce full size antibod-ies nor perform most of the important mammalianpost-translational modifications. Secondly, proteinsproduced in plants accumulate to high levels (Ver-woerd et al., 1995; Ziegler et al., 2000) and plant-derived antibodies are functionally equivalent to thoseproduced by hybridomas (Hiatt et al., 1989; Vosset al., 1995). Thirdly, concerns about contamination

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Table 1. Comparison of features of recombinant protein production in plants, yeast and classical systems

Transgenic Plant Yeast Bacteria Mammalian Transgenic

plants viruses cell cultures animals

Cost/storage Cheap/RT Cheap/−20◦C Cheap/−20◦C Cheap/−20◦C expensive/N2 Expensive

Distribution Easy Easy Feasible Feasible Difficult Difficult

Gene size Not limited Limited Unknown Unknown Limited Limited

Glycosylation ‘Correct’ ? ‘Correct’ ? Incorrect Absent ‘Correct’ ‘Correct’

Multimeric protein Yes No No No No Yes

assembly (SIgA)

Production cost Low Low Medium Medium High High

Production scale Worldwide Worldwide Limited Limited Limited Limited

Production vehicle Yes Yes Yes Yes Yes Yes

Propagation Easy Feasible Easy Easy Hard Feasible

Protein folding High ? High ? Medium Low High high

accuracy

Protein homogeneity High ? Medium Medium Low Medium Low

Protein yield High Very high High Medium Medium-high high

Public perception High High Medium Low Medium High

of ‘risk’

Safety High High Unknown Low Medium High

Scale up costs Low Low High∗∗ High∗∗ High∗∗ High

(unlimited biomass)

Therapeutic risk∗ Unknown Unknown Unknown Yes Yes Yes

Time required Medium Low Medium Low High High

∗ – residual viral sequences, oncogenes, endotoxins;∗∗ – large, expensive fermenters etc; ? – unclear.

of expressed proteins with human or animal patho-gens (HIV, hepatitis viruses) or the co-purification ofblood-borne pathogens and oncogenic sequences, areentirely avoided by using plants.

Classical methods of protein expression often re-quire a significant investment in recombinant proteinpurification (bacteria) or require expensive growth me-dia (animal cells). Bacteria produce contaminatingendotoxins that are difficult to remove and bacteriallyexpressed recombinant proteins often form inclusionbodies, making labour- and cost-intensivein vitro re-folding necessary. Mammalian cell cultivation can bedifficult, requires sophisticated equipment and expens-ive media supplements, such as foetal calf serum. Withthese classical expression systems, considerable caremust be also taken during downstream processing ofrecombinant proteins to remove oncogenic sequences,protein or viral contaminants forin vivo therapeuticapplications. In addition, the use of transgenic an-imals (Echelard, 1996) as a source of recombinantantibodies is becoming limited by legal and ethicalconstraints.

Transgenic plants producing high levels of safe,functional recombinant proteins, can be cultivated on

an agricultural scale (Whitelam et al., 1993; Whitelamet al., 1994; Whitelam, 1996) and molecular farmingrequires only a virus-infected or a transgenic plant,water, mineral salts and sunlight. Current applicationsof the plant based expression systems in biotechno-logy include the production of recombinant antibodies(rAbs) (Ma and Hein, 1995a,b), enzymes (Hogueet al., 1990; Verwoerd et al., 1995), hormones, in-terleukins (Magnuson et al., 1998), plasma proteins(Sijmons et al., 1990) and vaccines (Mason & Arntzen,1995; Walmsley & Arntzen, 2000). Chimeric plantviruses, produced in plants, can also be used for thepresentation of vaccines on the viral surface (John-son et al., 1997). The ease with which plants canbe genetically manipulated, and grown in single cellsuspension culture or scaled up for field scale produc-tion, is a great advantage over the more commonlyused microbial methods, mammalian cell culture andtransgenic animal technology.

Importantly, antibody expression can also beused as a tool to modify the intrinsic properties ofplants. For example, pathogen resistance can be in-creased by the expression of anti-pathogen antibodies(Tavladoraki et al., 1993; Voss et al., 1995; Fecker

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Table 2. Selected pharmaceutical proteins expressed in transgenic plants

Year Protein Transformed species Reference

1986 Human growth hormone N. tabacum (Barta et al., 1986)

H. annus

1990 Human serum albumin N. tabacum (Sijmons et al., 1990)

S. tuberosum

1993 Human epidermal growth factor N. tabacum (Higo et al., 1993)

1994 Trout growth factor N. tabacum (Bosch et al., 1994)

1994 Humanα-interferon O. sativa (Zhu et al., 1994)

1995 Hirudin N. tabacum (Parmenter et al., 1995)

Suspension cells

1995 Erythropoetin N. tabacum (Matsumoto et al., 1995)

Suspension cells

1996 Glucocerebrosidase, human N. tabacum (Cramer et al., 1996)

protein C serum protease

1997 Humanα andβ haemoglobin N. tabacum (Dieryck et al., 1997)

1997 Human muscarinic N. tabacum (Mu et al., 1997)

cholinergic receptors

1997 Murine granulocyte-macrophageN. tabacum (Lee et al., 1997)

colony stimulating factor

1998 Interleukin-2 and Interleukin-4 N. tabacum (Magnuson et al., 1998)

Suspension cells

1999 Human placental alkaline N. tabacum (Borisjuk et al., 1999)

phosphatase Rhizosecretion

1999 Humanα1-antitrypsin O. sativa (Terashima et al., 1999)

Suspension cells

2000 Human growth hormone N. tabacum (Leite et al., 2000)

(somatotrophin) seeds

2000 Human growth hormone N. tabacum (Staub et al., 2000)

(somatotrophin) Chloroplasts

et al., 1997; Le Gall et al., 1998; Zimmermann et al.,1998). Expressed antibodies can also be used to altermetabolic or hormonally regulated pathways by bind-ing to intracellular substrates or hormones (Phillipset al., 1997).

Plant expression strategies

Plant transformation involves the chromosomal integ-ration of a heterologous gene and this is becomingstraightforward. However, there are still technical andlogistical hurdles to be overcome, such as develop-ing efficient transformation techniques for all majorcrop species. Developing plant lines expressing re-combinant proteins is time intensive and expensive.In the best case, 8–12 weeks are needed for trans-genic plants to be available, but the time requireddepends on the plant species. Though this is slower

than some classical expression systems, the develop-ment of transient expression systems (Kapila et al.,1996) means that the expression vectors and proteinlevels achieved can be checked before making thisinvestment.

Protein expression studies have demonstrated thatmany forms of recombinant antibody fragments (Fig-ure 1) can be functionally expressed in plants and thatthe sub-cellular targeting of the protein is an importantconsideration for high level expression. These obser-vations seem to be applicable to both transient andstable expression in plants and whether the protein isexpressed in suspension cells, whole plants, or plantorgans.

Transient gene expression in plants

Transient gene expression in plants has several advant-ages over the generation of stably transformed trans-

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Table 3. Recombinant antibodies expressed in transgenic plants

Year Antibody Antigen Plant Cellular Transformed Reference

format organ location species

1989 IgG1 Phosphonate ester Leaf ER N. tabacum (Hiatt et al., 1989)

1990 IgM NP hapten Leaf ER chloroplast N. tabacum (Düring et al., 1990)

1991 VH domain Substance P Leaf Intra- and N. benthamiana (Benvenuto et al., 1991)

(neuropeptide) extra-cellular

1992 scFv Phytochrome Leaf Cytosol N. tabacum (Owen et al., 1992)

1993 IgG1 Human Leaf Nucleolus N. tabacum (De Neve et al., 1993)

Fab creatine kinase A. thaliana

1993 scFv Phytochrome Leaf Apoplast N. tabacum (Firek et al., 1993a)

1993 scFv AMCV Leaf Cytosol N. benthamiana (Tavladoraki et al., 1993)

1994 IgG Fungal cutinase Root Apoplast N. tabacum (van Engelen et al., 1994)

1994 IgG1 Streptococcus Leaf Apoplast N. tabacum (Ma et al., 1994)

mutansadhesin

1995 IgA/G Streptococcus Leaf Apoplast N. tabacum (Ma et al., 1995)

mutansadhesin

1995 IgG TMV Leaf Apoplast N. tabacum (Voss et al., 1995)

1996 scFv Cutinase Leaf ER N. tabacum (Schouten et al., 1996)

1996 IgM RKN secretion Leaf root Apoplast N. tabacum (Baum et al., 1996)

1996 scFv BNYVV Leaf Apoplast N. benthamiana (Fecker et al., 1996)

1996 scFv Human Leaf Cytoplasm N. tabacum (Bruyns et al., 1996)

creatine kinase ER

1996 IgG1 Human Leaf Apoplast A. thaliana (De Wilde et al., 1996)

Fab creatine kinase

1997 scFv β-1,4-endoglucanase Root Cytosol S. tuberosum (Schouten et al., 1997)

1997 scFv Oxazolone Leaf ER N. tabacum (Fiedler et al., 1997)

1997 scFv Abscisic acid Leaf ER N. tabacum (Fiedler et al., 1997)

1997 scFv Abscisic acid Seed ER N. tabacum (Phillips et al., 1997)

1997 scFv-IT CD-40 Plant Apoplast N. tabacum (Francisco et al., 1997)

tissue culture

1998 scFv Oxazolone tuber ER S. tuberosum (Artsaenko et al., 1998)

1998 Humanized HSV-2 Plant Secretory Glycine max (Zeitlin et al., 1998)

IgG1 pathway

1998 scFv Dihydro-flavonol Leaf Cytosol P. hybrida (De Jaeger et al., 1998)

4-reductase

1999 IgG Human IgG Plant Apoplast Medicago sativa (Khoudi et al., 1999)

1999 scFv CEA Leaf Transient expressionN. tabacum (Vaquero et al., 1999)

1999 scFv Tospoviruses Plant ER, apoplast N. benthamiana (Franconi et al., 1999)

1999 bi-scFv TMV Leaf ER, apoplast N. tabacum (Fischer et al., 1999d)

Suspension cells

1999 scFv TMV Plant Cytosol N. tabacum (Zimmermann et al., 1998)

1999 scFv CEA Cell ER, apoplast O. sativa (Torres et al., 1999)F

Suspension cells

1999 scFv 38C13 mouse Leaf Apoplast N. benthamiana (McCormick et al., 1999)

B cell lymphoma

2000 scFv CEA Plant ER, apoplast O. sativa T. aestivum (Stöger et al., 2000)

2000 scFv TMV Leaf Apoplast, N. tabacum (Schillberg et al., in press)

membrane

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.

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Table 4. Recombinant vaccines expressed in plants

Year Vaccine antigen Transformed species Reference

1992 Hepatitis virus B surface antigen N. tabacum (Mason et al., 1992)

1995 Malaria parasite antigen Virus particle (Turpen et al., 1995)

1995 Rabies virus glycoprotein L. esculentum (McGarvey et al., 1995)

1995 Escherichia coliheat-labile enterotoxin N. tabacum, S. tuberosum (Haq et al., 1995)

1996 Human rhinovirus 14 (HRV-14) Virus particle (Porta et al., 1996)

and human immunodeficiency

virus type (HIV-1) epitopes

1996 Norwalk virus capsid protein N. tabacum, S. tuberosum (Mason et al., 1996)

1997 Diabetes-associated autoantigen N. tabacum, S. tuberosum (Ma et al., 1997)

1997 Hepatitis B surface proteins S. tuberosum (Ehsani et al., 1997)

1997 Mink Enteritis Virus epitope Virus particle (Dalsgaard et al., 1997)

1997 Rabies and HIV epitopes Virus particle (Yusibov et al., 1997)

1998 Foot and mouth disease virus A. thaliana (Carrillo et al., 1998)

VP1 structural protein

1998 Escherichia coliheat-labile enterotoxin S. tuberosum (Mason et al., 1998)

1998 Escherichia coliheat-labile enterotoxin S. tuberosum (Tacket et al., 1998)

1998 Rabies virus Virus particle (Modelska et al., 1998)

1998 Cholera toxin B subunit S. tuberosum (Arakawa et al., 1998a)

1998 Human insulin-Cholera toxin B S. tuberosum (Arakawa et al., 1998b)

subunit fusion protein

1999 Foot and mouth disease virus Medicago sativa (Wigdorovitz et al., 1999)

VP1 structural protein

1999 Hepatitis B virus surface antigen Lupinus luteus, Lactuca sativa (Kapusta et al., 1999)

1999 Human cytomegalovirus glycoprotein BN. tabacum (Tackaberry et al., 1999)

1999 Diabetes-associated autoantigen N. tabacum, D. carota (Porceddu et al., 1999)

genic plants. Transient expression is rapid and resultson protein expression can be obtained in days (Kapilaet al., 1996). This makes transient expression suitablefor verifying that the gene product is functional be-fore moving on to large-scale production in transgenicplants (Kapila et al., 1996). Stable plant transforma-tion requires a considerable investment in time beforethe expressed proteins can be analysed. In contrast,transient gene expression systems are rapid, flexibleand straightforward and often use either agrobacterialor viral vectors.

Viral vectors (Scholthof et al., 1996) have attractedinterest because high yields of protein can be producedrapidly, by comparison to stable plant transformation.Commercial field trials are proceeding for the pro-duction 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 forproduction of Hepatitis B surface antigen, scFvs and

other recombinant proteins (McCormick et al., 1999;Kumagai et al., 2000).

There are three major transient expression systemsto deliver a gene to plant cells: delivery of projectilescoated with ‘naked DNA’ by particle bombardment,infiltration of intact tissue with recombinant agrobac-teria (agroinfiltration), or infection with modified viralvectors. The overall level of transformation variesbetween these three systems. Particle bombardmentusually reaches only a few cells and for transcrip-tion the DNA has to reach the cell nucleus (Christou,1996). Agroinfiltration targets many more cells thanparticle bombardment and the T-DNA harboring thegene of interest is actively transferred into the nuc-leus with the aid of several bacterial proteins (Kapilaet al., 1996). A viral vector can systemically infectmost cells in a plant and transcription of the introducedgene in RNA viruses is achieved by viral replicationin the cytoplasm, which transiently generates manytranscripts of the gene of interest.

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Although particle bombardment can be used totest recombinant protein stability before stable trans-formation, it is unsuitable for the expression of largeramounts of foreign proteins. It is more important forthe regeneration of transgenic cereal crops (Christou,1996). Here, we concentrate on agroinfiltration as amethod to test antibody production before producingstably transformed plants. Typically, agroinfiltrationcan provide milligram amounts of a recombinant pro-tein within a week (Vaquero et al., 1999). This isan important issue because it dramatically acceleratesthe development of plant lines producing recombinanttherapeutics.

Agroinfiltration- expression with a bacterial vector

In agroinfiltration, agrobacteria carrying the expres-sion vector are delivered into leaf tissue by vacuuminfiltration. Agrobacterial proteins then catalyze thetransfer of the gene of interest into the host cells andprotein expression can be detected three days after in-filtration. As in conventional methods for generatingtransgenic plants, genes of interest are cloned into bin-ary vectors that are transferred into suitableAgrobac-teriumstrains. A bacterial suspension is then used forvacuum infiltration of leaves (An, 1985; Kapila et al.,1996) and no selection method to identify transformedcells is required since the leaf tissue is only used fortransient protein production, which permits the use ofsmaller plasmid vectors.

In agroinfiltration, the transferred T-DNA does notintegrate into the host chromosome but is present inthe nucleus, where it is transcribed and this leads totransient expression of the gene of interest (Kapilaet al., 1996). A major advantage of this technique isthat multiple genes present in different populations ofagrobacteria can be simultaneously expressed. Thus,the assembly of complex multimeric proteins can betestedin planta (Vaquero et al., 1999). For transgenicplants, this can only be achieved by time consum-ing crossing experiments with individual transgenicplant lines, each expressing a single component of themultimer.

Using agroinfiltration, we transiently expressedscFvs, individual heavy and light chains as well as fullsize mouse–human chimeric anti-carcinoembryonicantigen (CEA) antibodies in plant leaves (Vaqueroet al., 1999). For full size chimeric antibody ex-pression, the mouse–human chimeric heavy andlight chain genes were integrated into two vectorsin two separate recombinantAgrobacteriumstrains

and these two strains were simultaneously infilt-rated into leaves. Functional full size chimeric anti-bodies were assembledin vivo by simultaneousexpression of both genes in the host cells. Thistechnique is also suitable for the expression ofscFv-fusion proteins, diabodies and multi-componentprotein complexes (R. Fischer & C. Vaquero, unpub-lished results).

Thus, agroinfiltration is rapid and yields suffi-cient quantities of protein for initial characteriza-tion of protein stability and protein function. Im-portantly, agroinfiltration can be scaled up to pro-duce tens of milligrams of recombinant proteinand may even prove suitable for pre-clinical trialswithout the need for production of stably transformedplants.

Recombinant viral vectors

Viral vectors (Scholthof et al., 1996) share severaladvantages with agroinfiltration. Here, the gene ofinterest is cloned into the genome of a viral plantpathogen under the control of a strong subgenomicpromoter. Infectious recombinant viral transcripts areused to infect plants and produce the target protein.Target genes are expressed at high levels because ofthe high level of multiplication during virus replica-tion (Porta & Lomonossoff, 1996). Some plant viruseshave a wide host range, are easily transmissible bymechanical inoculation and can spread from plant toplant, making it possible to rapidly infect large num-bers of plants with recombinant viruses. Viral vectorshave been used to express single chain antibodies inplants (Franconi et al., 1999; Hendy et al., 1999;McCormick et al., 1999).

We transiently expressed an scFv in plants, usinga tobacco mosaic virus (TMV) based vector (Verchet al., 1999). The scFv coding region was insertedinto the viral genome under the control of the strongsubgenomic coat protein promoter. This promoter isduplicated and drives the transcription of the viral coatprotein gene. The coat protein is essential for longdistance, systemic viral infection and the scFv wasexpressed throughout the entire plant. This approachhas been adapted to express the heavy and light chainof a full size antibody from two different viral vec-tors (Verch et al., 1998), but is generally limited toproteins smaller than 30 kDa. Improvement of thistechnique may involve the increase of inoculation ef-ficiency by combining the cloned recombinant viralDNAs with particle bombardment orAgrobacterium

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based techniques, like agroinfection (Shen & Hohn,1995).

Stable plant transformation

Stable transformation is defined by the integration of atarget gene into the host plant genome. The generationof transgenic plants uses two principle technologies:Agrobacteriummediated gene transfer to dicots, suchas tobacco and pea (Horsch et al., 1985), or biolisticdelivery of genes to monocots, such as wheat andcorn (Christou, 1993).Agrobacteriumhas a restric-ted host range and does not efficiently infect mono-cots but is the most widely used technique for dicottransformation. However, rice can be transformed byAgrobacterium(Chan et al., 1993; Hiei et al., 1994;Hiei et al., 1997) and methods have been developed fortransforming other monocots. For transforming plants,the gene of interest is cloned into a binary vector thatcan be moved betweenE. coli and Agrobacterium.The transformedAgrobacteriumitself delivers the tar-get gene into the host cell genome. Transformation isfollowed by selection for cells with stably integratedcopies of the target gene by following a selectableresistance gene that is introduced in the expressionvector.

Stable transformation of plants depends on theplant variety and it can take 3–9 months for plants tobe available for testing the expressed protein. Clearly,transient expression is a prerequisite to stable trans-formation because it allows both the expression vec-tors and protein stability to be tested. Initial problemscan be identified and eliminated so that the likeli-hood of regenerating the desired transgenic line issignificantly improved.

Expression in stably transformed plants

When long term production of a recombinant anti-body is necessary, stable transgenic plants are themost attractive strategy. The quantity of recombinantprotein that can be harvested is only limited by thenumber of hectares that can be planted with transgen-ics. High intensity agriculture can produce suprisinglylarge amounts of biomass, for example intensive cul-tivation of tobacco plants can produce approximately170 metric tonnes of biomass per hectare (Sheen,1983; Cramer et al., 1996). Assuming that the levelsof production seen on the laboratory scale could beat least kept constant in the field, and that for every170 tonnes harvested, 100 tonnes are harvested leaves:a single hectare could yield 50 kg of secretory IgA

Table 5. Yields of several plant species in tonnes perhectare

Crop Crop yield Reference

(tonnes/hectare)∗

Banana 16.6 FAO

Cabbage 24.3 FAO

Eggplant 26.9 FAO

Lettuce 33.1 FAO

Maize 8.4 FAO

Peanut 2.9 FAO

Peas 9.1 FAO

Potato 124.5 FAO

Rapeseed 1.5 FAO

Rice 6.6 FAO

Tobacco 2.2∗ FAO

Tobacco 170–200∗∗ (Long, 1984;

Sheen, 1983)

Tomato 59.4 FAO

Wheat 2.7 FAO

All yields refer to production in developed North Americain 1999.Taken from the Food and Agriculture Organization’s online database (http://apps.fao.org/).∗ : smoking tobacco;∗∗:close cropping and mowing.

(Ma et al., 1995) or 100 kg of recombinant gluco-cerebrosidase (Cramer et al., 1996). In Table 5, wesummarize the yield per hectare of several crop spe-cies as a guide to what levels of biomass can beproduced.

In Table 1, we compare plants to the classical ex-pression systems and show the advantages of plants,particularly their safety and low cost. Some recombin-ant proteins already reach very high expression levels,for example, apoplast targeted recombinant phytaseaccumulates to≈14% total soluble protein (TSP) intobacco leaves (Verwoerd et al., 1995), and a eubac-terial glucanase has been reported to reach 26% TSPin the mouse eared cress,Arabidopsis thaliana(Zie-gler et al., 2000). However, average expression levelsof recombinant antibodies in stably transformed plantsare usually on the level of 0.5–2% TSP (Conrad &Fiedler, 1998) but have reached 6.8% TSP (Fiedleret al., 1997).

Importantly, plants have some unique features notfound in bacterial or mammalian systems. Plants arethe premier heterologous system for the production ofsecretory IgA antibodies (Ma et al., 1995). Planet Bio-technology (Mountain View, CA) are concentrating onusing plants to produce theStreptococcus mutansspe-cific Guy’s-13 antibody, which prevents dental caries

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(Ma et al., 1995; Ma et al., 1998). Similar approachesare now underway to produce other antibodies undera collaboration between EPIcyte pharmaceuticals andProdiGene.

The low costs of producing recombinant antibod-ies in plants are as great a benefit as the increasedsafety and authentic post-translational modificationpathways. It has been estimated that plant-expressedproteins are 10–50-fold less expensive than thosemade inE. coliand that these savings will be greater asproduction reaches agricultural cropping scales or asmethods are developed to increase expression levels.This justifies the use of plants as an inexpensive sourcefor producing recombinant proteins that eliminates thedisadvantages associated with microbial or animal cellsystems. Additionally, plant genetic material is readilystored in seeds or tubers, which are extremely stable,require no special maintenance and have a long shelflife (Conrad et al., 1998). This has the benefit that boththe product and the production system itself can bestored almost indefinitely.

Important considerations for pharmaceuticalexpression in plants

In this section, we discuss the technical considera-tions that are important for high level pharmaceuticalprotein expression, which range from transcriptionalmodifications to changes in the sub-cellular destin-ation of the newly synthesized recombinant protein.The plant pattern of protein glycosylation and N-linked glycan processing differs from that of animals,and in some cases recombinant proteins could be im-munogenic through their glycosylation. Therefore, theglycosylation of proteins intended forin vivo admin-istration should be analysed in detail (Bardor et al.,1999).

Sub-cellular protein targeting

Expression levels of recombinant antibodies in plantscan be enhanced by exploiting the innate protein sort-ing and targeting mechanisms that plant cells use totarget host proteins to organelles. Significant increasesin recombinant antibody yield have been observedwhen antibodies are targeted to the secretory path-way instead of the cytosol (Conrad & Fiedler, 1998).Targeting proteins for secretion to the intercellularspace beneath the cell wall (apoplast) has advantagesfor downstream processing and also leads to signi-ficant levels of expression but ER retention can give

10–100-fold higher yields (Conrad & Fiedler, 1998).Recombinant antibodies have been targeted to the fol-lowing compartments of plant cells: the intercellularspace, 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; Baumet al., 1996; De Wilde et al., 1996; Schouten et al.,1996; Conrad & Fiedler, 1998). Intracellular expres-sion of rAbs in the cytoplasm has only been achievedusing scFv fragments (Owen et al., 1992; Tavladorakiet al., 1993; Schouten et al., 1996; Zimmermann et al.,1998), presumably because scFv fragments requireonly minor post-translational processing. In the ma-jority of transgenic plants expressing cytosolic scFvs,levels were found to be very low or at the detectionlimit (Owen et al., 1992; Fecker et al., 1996; Schoutenet al., 1996; Schillberg et al., 1999). There is a reportwhere cytosolic scFvs, isolated from a phage displaylibrary, have reached levels of up to 1.0% of totalsoluble protein (De Jaeger et al., 1998), but this isstill an exception. The high cytosolic expression levelmay be because thein vitro antibody selection used inphage display naturally selects more stable antibodyfragments.

Overall expression levels of different antibodies instably transformed plants vary, with expression of fullsize IgG under the control of the 35S promoter ran-ging 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, becausetransgenic plants have been identified with expressionlevels of scFvs in leaves reaching 6.8% of the TSP(Fiedler et al., 1997) and levels of secretory IgA upto 500µg per gram leaf material (Ma et al., 1995).

In a recent report, Russell and colleagues at the In-tegrated Protein Technologies unit of Monsanto havereported that transformed chloroplasts can be used asa high capacity production system. Active human so-matotrophin was expressed in transgenic plastids intobacco and reached 7% of the total soluble protein(R. Bassuner, personal communication; Staub et al.,2000). This a remarkable result and indicates thatchloroplast based expression, which is inherently bio-logically contained, may be useful for the expressionof other pharmaceutical proteins. However, it is un-clear to what extent the chloroplast protein synthesispathway can accommodate eukaryotic proteins. It willbe interesting to determine if chloroplasts will becapable of synthesizing complex eukaryotic proteinsthat require post-translational modifications or subunitassembly, such as sIgA.

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Protein storage in tissues, seeds and tubers

For plants to fulfill their potential as a production sys-tem, a prerequisite was that expressed proteins couldbe stored in harvested tissues, tubers and seeds. Thishas widely been shown to be the case and emphasizesthe versatility of plants as an expression system. Whenleaves from transgenic tobacco plants expressing ER-retained scFvs were dried and stored for more thanthree weeks, there were no losses in scFv specificityor antigen binding activity (Fiedler et al., 1997). Seedsare protein rich storage organs that can be storedalmost indefinitely and can be exploited as storagecontainers for recombinant proteins (Fiedler and Con-rad, 1995; Conrad and Fiedler, 1998; Conrad et al.,1998). As with expression in leaves, ER retentiongave increases in scFv accumulation and scFvs can bestored for up to a year in seed at room temperaturewithout losses. Potato tubers have also been used asstorage containers with expression levels reaching 2%TSP and cold storage for 18 months resulting in onlya 50% loss of functional antibody (Artsaenko et al.,1998).

The most promising approach for protein expres-sion and in field production is to target the pro-tein to the ER and, if long-term storage is re-quired, to target the protein for seed specific ex-pression. Transformation of major crop plants isnow becoming more straightforward. We foreseecrop based expression systems (wheat, rice, corn,legumes) may be used because they have a lowercontent of toxic compounds than model species,like tobacco, and there is an existing infrastruc-ture for crop cultivation, harvesting, distribution andprocessing.

Optimization at the level of the gene

The pattern of codon usage in plants differs to thatof animals, but modifying the composition of theheterologous cDNA to meet the plant pattern can in-crease the rate of translation (Kusnadi et al., 1997).Further improvements in expression may come fromusing tissue specific promoters, improvement of tran-script stability, translational enhancement with viralsequences (Gallie, 1998) and screens to identify stablecytosolic antibody scaffolds (Worn et al., 2000). Inter-estingly, the production of a bean arcelin inArabidop-sis thalianaseeds was increased by the expressionof an antisense gene for the seed storage protein 2Salbumin. Using this strategy, arcelin levels were en-hanced and reached 24% of the total seed protein

(Goossens et al., 1999). Therefore, adroit selectionof genes to suppress by anti sense expression maybe a realistic method to dramatically increase theyield of co-introduced pharmaceutical protein genes inseed.

Pharmaceutically valuable proteins produced inplants

As medical and biological knowledge of many dis-eases increases, through the sequencing of the humangenome and medical research, many novel proteinsthat could be used for treatment have been identified.These include recombinant antibodies and it is clearthis will expand to include more proteins in the future.Plants are likely to feature highly as an alternativeto using transgenic animals to produce these pro-teins. Recombinant antibodies, plasma proteins anddiagnostic reagents are targets for expression in plantsbecause their conventional production or purificationis often expensive and can bear risk of pathogencontamination.

The number of mammalian proteins expressed inplants is expanding and include antibodies, plasmaproteins, human enzymes and recombinant vaccines(Tables 2–4). The worldwide demand for some ofthese proteins is large, for example human serum al-bumin (HSA) is a non-glycosylated protein with aworldwide demand approaching 550 tonnes of puri-fied protein a year. Conventionally, it is isolated fromhuman blood donations and is therefore relatively in-expensive. However, isolated HSA does bear somerisks of viral contamination and there is a market forsources of safer HSA. HSA can be safely made inplants as can interleukins, interferons and human en-zymes (Table 2). Another application of molecularfarming in plants is the production of vaccine antigens,and edible vaccines (Walmsley & Arntzen, 2000).This is likely to be important in the future, but herewe focus on the more advanced fields of antibodyexpression and vaccine production.

Therapeutic antibody production in plants

Antibodies are an instructive example of the expres-sion of pharmaceutically valuable proteins in plants(Hiatt, 1990; Hiatt, 1991; Hiatt et al., 1992; Hiatt &Ma, 1993). As described earlier, a key breakthroughin making molecular farming in plants a reality wasthe demonstration of functional antibody expression in

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tobacco leaves (Hiatt et al., 1989; Düring et al., 1990).The importance of this is underscored by the fact thatmonoclonal antibodies (Koehler & Milstein, 1975)and recombinant antibodies are essential therapeuticand diagnostic tools used in medicine, human and an-imal health care, the life sciences and biotechnology(Winter & Milstein, 1991). Modern recombinant DNAtechniques and antibody engineering have broadenedthe range of applications for recombinant antibodies.These advances have made possible the productionof novel polypeptides with desirable properties (Fig-ure 1). For example, smaller antibody fragments orantibody-fusion proteins linked to enzymes, biologicalresponse modifiers or toxins ( Shin et al., 1993; Ger-stmayer et al., 1997; Bookman, 1998). The rapiddevelopment of combinatorial approaches, such asphage display, allow the isolation of rAbs recogniz-ing almost any target antigen and the fine-tuning ofthese rAbs toward desired properties (Winter et al.,1994). Therefore, antibodies are likely to only becomemore important in the future and the demand for aneconomical, efficient expression system is likely togrow.

After the demonstration that functional full sizerAbs could be expressed in transgenic plants in 1989(Hiatt et al., 1989) and 1990 (Düring et al., 1990), awide range of different recombinant antibody formatshave been successfully expressed in many plant spe-cies (Table 3). These formats include full-size anti-bodies (Ma et al., 1994; Voss et al., 1995; Baumet al., 1996; De Wilde et al., 1996), Fab fragments(De Neve et al., 1993), single chain antibody frag-ments (scFvs) (Owen et al., 1992; Tavladoraki et al.,1993; Firek et al., 1993b; Artsaenko et al., 1995;Fiedler & Conrad, 1995; Fecker et al., 1996; Schoutenet 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 transientlyexpress tumour (carcinoembryonic antigen) specificsingle chain and chimeric full size antibodies in to-bacco leaves. A recombinant single-chain Fv antibody(scFvT84.66) and a full-size mouse/human chimericantibody (cT84.66), derived from the parental murinemAbT84.66 specific for the human carcinoembryonicantigen, were engineered into a plant expression vec-tor. Chimeric T84.66 heavy and light chain geneswere constructed by exchanging the mouse light andheavy chain constant domain sequences with their hu-man counterparts and cloned into two independentplant expression vectors.In vivo assembly of full-size

cT84.66 was achieved by simultaneous expression ofthe light and heavy chains after vacuum infiltrationof tobacco leaves with two populations of recom-binant Agrobacterium. Upscaling the transient sys-tem permitted purification of significant (milligram)amounts of functional recombinant antibodies fromtobacco leaf extracts within a week (Vaquero et al.,1999).

Secretory IgA

Plants have a great advantage over animal cell ex-pression systems, since single plant cells are cap-able of expressing recombinant secretory IgA (sIgA)(Ma et al., 1995). This has only recently becomepossible in single animal cells (Chintalacharuvu &Morrison, 1997). SIgA is a complex multi-subunitantibody with great potential for use in topical im-munotherapy. It is the major antibody found in mu-cosal secretions and is made of two immunoglobulinchains, a joining chain and the secretory compon-ent. It was first expressed in tobacco by sequentiallycrossing plants expressing its individual polypeptidecomponents. This permitted the production of highlevels of recombinant sIgA (500µg/gram) (Ma et al.,1995) and proved that plants could be used as a pro-duction system for sIgA suitable for use in passiveimmunotherapy. Three years later, the same groupshowed that recombinant sIgA specific for adhesionproteins from the oral pathogenStreptococcus mutanscould prevent oral streptococcal colonization in hu-man volunteers for up to four months (Ma et al.,1998). We anticipate that plant produced sIgA willbecome widely used in the future for the generationof passive immunity because of their stability in themucosa.

Thus, for antibodies at least, molecular farminghas come virtually full circle, from proof of principlewith the expression of model antibodies in 1989 (Hiattet al., 1989) to the production of complex antibodiesin plants and their use as pharmaceutical reagents. Thecircle will be closed when the first plant expressedantibodies are approved by the regulatory authoritiesand come onto the market as diagnostic or therapeuticproducts.

Vaccines from plants

Vaccination has been one of the greatest advancesin medical science and has dramatically improvedhuman life expectancy and quality of health. Vac-cines are the most cost effective form of health care

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(World Health Organisation: www.who.int/gpv/) buttheir world wide distribution is hampered, especiallyin developing countries. Plants can produce a rangeof immunogenic antigens (Table 4). It was proposedto induce B- and T-cell mediated immune responsesusing plants as a source of ‘edible vaccines’, wherethe vaccine antigens are eaten in a fruit or raw ve-getable (Mason & Arntzen, 1995; Ma & Vine, 1999;Walmsley & Arntzen, 2000). Such vaccines wouldnot require cold storage or sophisticated expertise fortheir distribution and use throughout the developingcountries. The principle of edible vaccine activity wasproven for transgenic potatoes producing the entero-toxigenic E. coli heat labile enterotoxin B subunit(Haq et al., 1995). Mice fed raw potato produceda serum and mucosal immune response to the anti-gen. Further work has gone on to show that ediblevaccines may be feasible for a range of antigens, in-cluding rabies virus (McGarvey et al., 1995), footand mouth disease (Wigdorovitz et al., 1999), Nor-walk virus (Mason et al., 1996), autoimmune diabetes(Arakawa et al., 1998b) and cholera (Arakawa et al.,1998a). Furthermore, an effective edible hepatitis Bvaccine has been generated using transgenic lupinand lettuce plants expressing the hepatitis B surfaceantigen. Mice and humans fed transgenic plant mater-ial produced hepatitis B specific antibodies (Kapustaet al., 1999). Many edible plants have been ge-netically transformed and tomatoes and bananas aregood candidates for edible vaccine production for hu-mans while cereals may be more suitable for animalimmunisation.

For edible vaccines to become widely used anduseful, several issues have to be considered. Ediblevaccines will need an infrastructure for their distribu-tion and administration to the public to ensure theyare as effective as current vaccines. Importantly, therehave to be internal controls for the level of vaccineexpression in every plant and their stability and effic-acy need to be improved. Further, effective methodshave to be developed for the biological containment ofvaccine traits.

Alternative plant expression systems

Intact tobacco plants are not the only expression sys-tem available for plant based molecular farming. Thereare options for the production of proteins in seeds,plant suspension cells and by ‘rhizosecretion’ fromengineered plant roots. Cereals have advantages for

antibody production over ‘model’ species such as to-bacco because they have a lower content of toxicsecondary metabolites and there is a well-organisedinfrastructure for their production, distribution andprocessing. When recombinant proteins are expressedin cereal seed, such as for expression of avidin incorn, the protein can be collected and extracted dir-ectly from the kernels (Hood et al., 1997; Hood et al.,1999).

Plant suspension cells are a model system that canbe easily transformed and cultivated on a very largescale in fermenters (Fischer et al., 1999b), while rhizo-secretion is the release of proteins from transgenicplant roots into a surrounding hydroponic medium(Borisjuk et al., 1999). Suspension cells can be usedto produce and secrete proteins under carefully con-trolled certified conditions, but rhizosecretion has yetto 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 flasksor fermenters to produce recombinant proteins aftertransformation (Fischer et al., 1999b). Compared tothe classical expression systems the number of applic-ations is still relatively small (Kieran et al., 1997). Anumber of plant species has been used for the genera-tion and propagation of cell suspension cultures, ran-ging from model systems likeArabidopsis(Desikanet al., 1996) toCatharanthus(Van Der Heijden et al.,1989),Taxus(Seki et al., 1997), to important monocotor dicot crop plants like rice (Chen et al., 1994), soy-bean (Hoehl et al., 1988), alfalfa (Daniell & Edwards,1995) and tobacco (Nagata et al., 1992). Tobacco sus-pension cells have been used for the production of anscFv-fusion protein with a ribosome inactivating pro-tein (Bryodin) with yields of 30 mg/l (Francisco et al.,1997).

We focus on using suspension cells for the pro-duction of recombinant proteins and antibodies inplants and plant cell cultures. As discussed earlier,the expression of recombinant antibodies and anti-body fragments in plants is well established (Hiattet al., 1989; Whitelam et al., 1994). When clinicaluse of recombinant proteins is intended, their produc-tion under defined, controllable and sterile conditionswith straightforward purification protocols may be ad-vantageous. Therefore, we have expressed full-sizeantibodies, antibody fragments and fusion proteinsin transgenic plant cell suspension systems includ-ing Nicotiana tabacumcv. Petite Havana SR-1 (Vosset al., 1995),Nicotiana tabacumBY-2 cells (Nagata

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et al., 1992), pea, wheat and rice (Torres et al.,1999).

Tobacco suspension cell lines for recombinantantibody production

Transfer of a foreign gene into plant suspensioncells can be performed usingAgrobacterium-mediatedtransformation (Horsch et al., 1985; Koncz & Schell,1986), particle bombardment (Christou, 1993), elec-troporation of protoplasts (Lindsey & Jones, 1987) orviral vectors (Porta & Lomonossoff, 1996). The BY-2cell line can be directly transformed by co-cultivationof suspension cells andAgrobacterium(An, 1985).This has the advantage that transient expression ofthe foreign gene can be detected 2–3 days after co-cultivation.

Recombinant proteins expressed in plant cell sus-pension cultures are found in the culture supernatantor retained within the cells. This localisation is de-pendent on the presence of targeting/leader peptides(of plant or heterologous origin) in the recombin-ant protein and on the permeability of the plant cellwall to macromolecules (Carpita et al., 1979). Re-combinant targeting signals can be used to direct theprotein for secretion (Magnuson et al., 1998) or tointracellular organelles (ER, chloroplast, vacuole, in-tracellular membranes) (Moloney & Holbrook, 1997).The targeting signals can be used to retain recom-binant proteins within distinct compartments of thecells to preserve integrity, protect them from proteo-lytic degradation and to increase accumulation levels(Kusnadi et al., 1997; Moloney & Holbrook, 1997).The cytosol is generally unsuitable as a recombinantprotein storage compartment. Recombinant proteinssmaller than 20–30kDa can pass through the plantcell wall and are secreted into the culture mediumbut larger proteins tend to be retained in the apoplast.Intra-cellular protein retention makes the disruption ofthe cells necessary prior to protein purification, whichhas several drawbacks, since it causes release of phen-olic substances or proteases that reduce protein yield.Thus, our preferred method is to target proteins forsecretion and capture them from the culture super-natant or release them from the cell by mild enzymaticcell wall digestion (Fischer et al., 1999c). Using ourstandard plant expression vector, we could obtainexpression levels between 2–20µg of recombinant an-tibody per gram fresh cell weight. This level couldbe significantly increased by targeting of the proteinto the ER as well as by optimizing cultivation condi-

tions with controlled amino-acid supplementation orelicitation.

Purification strategies for proteins expressed inplants

Highly efficient purification schemes are a pre-requisite for the use of recombinant proteins as phar-maceuticals (Baker & Harkonen, 1990; Mariani &Tarditi, 1992; Miele, 1997; Murano, 1997) and are animportant consideration in designing molecular farm-ing systems. Although there are established protocolsavailable for purification of antibodies produced byanimal or microbial sources, there is little availabledata on the purification of recombinant antibodiesfrom plants, plant suspension culture cells, leaves orseeds (Moloney & Holbrook, 1997). We established apurification protocol for full-size antibodies producedin plant cell suspension cultures (Fischer et al., 1999c).Our data demonstrate that full-size antibodies can bepurified from plant cell extracts on protein-A andprotein-G based affinity matrices in a similar mannerto antibodies purified from animal sources. Antibodiessecreted to the intercellular space of plant cells werereleased by partial enzymatic lysis of the cell wall andthis was the superior method for isolation of functionalantibodies. Affinity chromatographyusing a Protein-Amatrix as the first step efficiently removed contamin-ant plant proteins and gave a 100-fold concentrationof the recombinant protein. Gel filtration served as apolishing step for the removal of rAb-dimers and forexchange of the rAbs into a suitable storage buffer. Us-ing this method, more than 80% of expressed full sizeIgG can be recovered from suspension cultured plantcells (Fischer et al., 1999c). This shows that antibodypurification from plants is essentially straightforwardwith no complications that could prevent the use ofplants as an expression system.

Downstream processing of full-length IgG anti-bodies is relatively straightforward because Protein-Aand Protein-G are useful ligands for affinity chromato-graphy. This approach is not applicable for the down-stream processing of IgM antibodies, or most recom-binant antibodies including scFvs and scFv fusion pro-teins. For most recombinant proteins, novel strategiesthat have a high processing speed, high capacity andthat are inexpensive will need to be developed. Tomake large scale bioprocessing more efficient, thelatest developments in downstream processing suchas perfusion chromatography and expanded bed tech-

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nology in combination with tangential flow filtrationshould be applied. This will overcome the diffusionallimitations experienced with most chromatographicresins (Fahrner et al., 1999).

Engineered affinity tags may enable improvedhandling of large clarified sample volumes, minimiseprocessing time and avoid proteolytic and oxidativedegradation of recombinant proteins. Here, we needto distinguish between the generation of N- or C-terminal gene fusions (tags) for affinity purification(Nilsson et al., 1997) and the development of stablesynthetic peptides that reversibly bind the recombin-ant protein of interest. The peptides are immobilizedon a solid support, to allow controlled capture andrelease during processing and multiple uses of a syn-thetic affinity matrix. Such peptides can be identifiedby epitope mapping using pepscan or phage peptidedisplay technologies for the identification of linearpeptide sequences. Modifications of the phage peptidedisplay technology also permit the identification ofmimotopes, either in a linear or a Cys-Cys-constrainedlibrary (McConnell et al., 1998; Zwick et al., 1998).The synthetic versions of identified binding peptidescan be immobilized on an activated matrix (Sepharose,glass, silica) for the development of a specific affinitymatrix for a given recombinant protein (Murray et al.,1997). Due to potential immunogenicity of certain tags(FLAG, MBP, GST) it may be particularly importantto use specific, synthetic peptide affinity ligands forthe purification of therapeutic proteins.

The combination of the latest developments indownstream processing and affinity chromatographymay lead to significant advances in the large scaleproduction of inexpensive diagnostic and therapeuticproteins by molecular farming.

Commercial aspects of molecular farming

The commercial interest in molecular farming is thatit can produce recombinant proteins at a lower costthan alternatives, such as their production in mam-malian tissue culture. An interesting case study forthe farming of a recombinant protein was reported byHood and co-workers for the production of recom-binant avidin (Hood et al., 1997; Hood et al., 1999;Kusnadi et al., 1998).

Avidin is widely used as a diagnostic reagent andis a relatively abundant eukaryotic protein found inegg white, from which it is routinely purified. Therationale was to produce avidin in transgenic corn

and determine if this could compete with egg whiteas a commercial source of the protein. A chickenavidin cDNA, codon optimised for the preferred maizecodon usage pattern, was engineered in fusion to abarley α-amylase signal sequence. This targeted therecombinant protein to the secretory pathway and tar-geting was a crucial factor. Plants that expressed highlevels of avidin in the secretory pathway were eitherpartially or completely male sterile. In contrast, tar-geting the avidin to the cytosol was completely toxicto engineered maize.

Avidin could be reproducibly produced at 230 mgper kg of maize seed (Hood et al., 1997). The authorsestimated that plant produced avidin is 10-fold lessexpensive than avidin extracted from eggs. The maizeavidin is functional and now commercially available(Sigma-Aldrich product # A8706). This illustrateshow a relatively abundant protein with a rich naturalsource can still be produced less expensively in plants.β-Glucuronidase production in plants is also commer-cial (Witcher et al., 1998) and the costs of producingaprotinin in plants are comparable with extracting itfrom its natural source, bovine lung (Zhong et al.,1999). Considering that there are no natural sourcesof recombinant antibodies as inexpensive as usingchicken eggs as a source of avidin, the savings fromexpressing antibodies in plants will be even higher.

The demand for many pharmaceutical proteins islarge, and any transgenic production system has tobe capable of meeting the demand. As mentionedearlier, kilogram quantities of a recombinant proteincan be obtained from as little as a hectare of trans-genic tobacco. Transgenic animals are limited by thetime needed to raise a herd of animals producing therecombinant protein. The demand for human serumalbumin is in the range of 550 metric tons per year andit may take years to establish a herd of transgenic an-imals producing enough protein to meet this demand.In contrast, transgenic plants can be rapidly scaled upto field scale cultivation. It has been estimated that theworldwide demand for human serum albumin couldbe met by transgenic cultivation on 30,000 hectaresof land (assuming an expression level of 1% TSP intobacco), which is less than one thousandth of the totalcultivated soil in the USA (32 million hectares).

Clinical trials of plant produced pharmaceuticals

The first clinical trial of plant-based immunotherapywas reported by Planet Biotechnology, Inc. (Moun-tain View, CA). The novel drug CaroRxTM is based

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on sIgA antibodies produced in transgenic tobaccoplants and is designed to prevent the oral bacterialinfection that contributes to dental carries (Ma et al.,1998). Planet Biotechnology has demonstrated thatCaroRxTM can effectively eliminateStreptococcusmutans, the bacteria that causes tooth decay in hu-mans (Larrick et al., 1998). Planet Biotechnologyis also engaged in the design and development ofnovel sIgA-based therapeutics to treat infectious dis-eases and toxic conditions affecting oral, respiratory,gastrointestinal, genital and urinary mucosal surfacesand skin.

Monsanto (formerly Agracetus, Middleton, Wis-consin) created a corn line producing human anti-bodies at yields of 1.5 kg of pharmaceutical-qualityprotein per acre of corn. Given that the yield per acreof corn is on the range of 3.5 tonnes (Table 5), thereis considerable room for improvement in yields. Apharmaceutical partner plans to begin injecting cancerpatients with doses of up to 250 mg of the antibody-based cancer drug purified from corn seeds. Thecompany is also cultivating transgenic soybeans thatproduce humanized antibodies against herpes simplexvirus 2 (HSV-2). These antibodies were shown to beefficient in preventing vaginal HSV-2 transmission inmice. Theex vivostability andin vivo efficacy of theplant and mammalian cell-culture produced antibodieswere similar (Zeitlin et al., 1998). Plant-produced anti-bodies are likely to allow development of an inexpens-ive method for mucosal immuno-protection againstsexually transmitted diseases.

ProdiGene (College Station, Texas) and EPIcytePharmaceuticals (San Diego) have entered into astrategic partnership to produce antibodies in corn(www.prodigene.com/news.html). Their interest is theproduction of human mucosal antibodies for passiveimmunisation by exploiting ProdiGene’s expertise inprotein expression (Hood et al., 1997; Kusnadi et al.,1998; Witcher et al., 1998; Hood et al., 1999; Zhonget al., 1999) together with EPIcyte’s academic andpatent position.

The collaborative research group at BiosourceTechnologies (now named the Large Scale BiologyCorporation, Vacaville, CA) and Stanford Univer-sity has developed a technology to produce a tumor-specific vaccine for the treatment of malignanciesusing a plant virus based transient expression system.The researchers created a modified tobacco mosaicvirus vector that encodes the idiotype-specific scFvof the immunoglobulin from the 38C13 mouse B celllymphoma. InfectedNicotiana benthamianaplants

secreted high levels of scFv protein to the apoplast.This antibody fragment reacted with an anti-idiotypeantibody, suggesting that the plant-produced 38C13scFv protein is properly folded. Mice vaccinated withthe affinity-purified 38C13 scFv generated> 10µg/mlanti-idiotype immunoglobulins. These mice were pro-tected from challenge by a lethal dose of the 38C13 tu-mor, similar to mice immunized with the native 38C13IgM-keyhole limpet hemocyanin conjugate vaccine(McCormick et al., 1999). This rapid production sys-tem for generating tumor-specific protein vaccinesmay provide a viable strategy for the treatment ofnon-Hodgkin’s lymphoma. The goal of the therapy isto create antibodies customized for each patient thatwill recognize unique markers on the surface of themalignant B-cells and target the cells for destruction.

Future directions

The technical challenges that need to be solved forplants are essentially all related to expression levelsof recombinant proteins. Higher expression levels willbe reached by better control of gene silencing andthe identification of novel, stronger promoters in in-tact plants, or better plant species as expression hosts.Large-scale fermentation may be a practical methodfor producing recombinant pharmaceutical proteins insuspension cells.

The recent report of high level expression ofhuman growth hormone (somatotrophin) in tobaccochloroplasts, which reached 7% of the total solubleprotein, is an exciting result (R. Bassuner, MonsantoIntegrated Protein Technologies unit, personal com-munication; Staub et al. 2000). This is highly in-teresting because through this technology, high levelexpression of many other pharmaceutical proteins maybe possible and this approach is biologically self-contained. Clearly, this plastid expression strategymay also be successful with mitochondria.

The costs of licensing the technology for trans-forming plants or for using a promoter from its patentassignees are important aspects of molecular farm-ing in plants. These costs are often large, close tothe costs of development of the transgenic plant lineor greater. If a laboratory develops its own propriet-ary transformation and expression system, this makestheir molecular farming products less expensive andmore attractive. It will also be valuable to screen arange of other plant species than used today for theiruse in molecular farming. The decision on what spe-

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cies to use should be based on identifying a plantspecies that has a high annual yield and that is capableof high levels of protein expression in a background oflow levels of noxious compounds.

Concluding remarks

We have discussed the advantages of plants as anexpression system and described how they serve asan expression system for pharmaceutically import-ant, commercially valuable proteins. At the currentrate of progress, molecular farming may become thepremier expression system for many pharmaceuticalproteins in the next decade. This is partly because ofthe utility of plants, with their higher eukaryotic pro-tein synthesis pathway, to make mammalian proteins.Importantly, the economic advantages brought by us-ing plants, which can be grown with such a minimalinvestment in their cultivation, makes them attractiveas an industrial scale expression system. It is unlikelythat the consumer reaction against GM technology willextend to molecular farming because the public is re-luctant to harshly criticize medically related researchor attempts to provide safer supplies of medicines.

In the near future, the human genome sequencingproject will be completed. It is easy to foresee thecauses of many diseases will be identified through it,leading to new therapies. It is possible many of thenew therapeutics will be complex proteins requiring aeukaryotic production system and that plants will bethe leading expression system. Moreover, the thera-peutic proteins may become more complex and moredemanding to produce and at that point, plants shoulddominate the field.

We anticipate that plants will be the premier ex-pression system for diagnostic and therapeutic pro-teins. Plant expression systems have the potentialto make them as abundant tomorrow as prescriptiondrugs are today. We foresee that molecular farmingwill provide a basket full of novel medicines for thediseases of the 21st century, just as plants were thesource of medicines for the Egyptians 3600 years ago.

Acknowledgements

The authors would like to thank all the members ofthe Fischer group for their contribution to this work, inparticular, Stefan Schillberg, Carmen Vaquero, SabineZimmermann, Markus Sack, Stefan Hellwig, JürgenDrossard, Uli Commandeur and Flora Schuster. We

are indebted to Paul Christou’s research group at theJIC (Norwich, UK) for the ongoing, fruitful collabor-ation, in particular Eva Stöger, Esperanza Torres andYolande Perrin. We thank Professor Fritz Kreuzaler(RWTH Aachen) for his long-term interest in the workof the group.

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