Production of antibodies in plants and their use for global health

Vaccine 21 (2003) 820–825

Production of antibodies in plants and their use for global health Rainer Fischer a,b,∗ , Richard M. Twyman c , Stefan Schillberg a a

Fraunhofer Institute for Molecular Biology and Applied Ecology, IME, Grafschaft, Auf dem Aberg 1, 57392 Schmallenberg, Germany b Institute for Molecular Biotechnology, Biology VII, RWTH Aachen, Worringerweg 1, 52074 Aachen, Germany c Department of Biology, University of York, Heslington, York YO10 5DD, UK

Abstract Recombinant antibodies can be used to diagnose, treat and prevent disease by exploiting their specific antigen-binding activities. A large number of drugs currently in development are recombinant antibodies and most of these are produced in cultured rodent cells. Although such cells produce authentic functional products, they are expensive, difficult to scale-up and may contain human pathogens. Plants represent a cost-effective, convenient and safe alternative production system and are slowly gaining acceptance. Five plant-derived therapeutic recombinant antibodies (plantibodies) are undergoing clinical evaluation, three of which can be used as prophylactics. © 2002 Elsevier Science Ltd. All rights reserved. Keywords: Recombinant antibody; Passive immunity; Transgenic plants

1. The importance of recombinant antibodies to human health Antibodies are complex glycoproteins, produced by the vertebrate immune system, which recognize and bind to target antigens with great specificity. This individual and specific binding activity allows antibodies to be used for a variety of applications, including the diagnosis, prevention and treatment of disease [1–3]. It is estimated that approximately 1000 therapeutic recombinant antibodies are being developed by biopharmaceutical companies around the world and over 200 of these are already in clinical trials. A large proportion of these antibodies recognize cancer antigens, but others have been developed for the diagnosis and treatment of infectious diseases, autoimmune disorders, cardiovascular disease, blood disorders, neurological disorders, skin disorders, respiratory diseases, eye diseases and transplant rejection [4].

2. Evolution of recombinant antibody technology 2.1. Structure of naturally produced antibodies Mammalian serum antibodies comprise two identical heavy chains and two identical light chains joined by disulfide bonds (Fig. 1). Each heavy chain is folded into four domains, two either side of a flexible ‘hinge’ which allows ∗

Corresponding author. Tel.: +49-241-8026631; fax: +49-241-871062. E-mail address: [email protected] (R. Fischer).

the multimeric protein to adopt its characteristic Y-shape. Each light chain is also folded into two domains. The N-terminal domain of each of the four chains is variable, i.e. it differs among individual B-cells due to unique rearrangements of the germ-line immunoglobulin genes. This part of the molecule is responsible for antigen recognition and binding. The remainder of the antibody comprises a series of constant domains, which are involved in effector functions such as immune cell recognition and complement fixation. Below the hinge, in what is known as the Fc portion of the antibody, the constant domains are class specific. Mammals produce five classes of immunoglobulins (IgG, IgM, IgA, IgD and IgE) with different effector functions. The Fc region also contains a conserved asparagine residue at position 297 to which N-glycan chains are added. The glycan chains play an important role both in the folding of the protein and the performance of effector functions [5]. Antibodies are found in mucosal secretions as well as serum. These secretory antibodies have a more complex structure than their serum counterparts. They are dimers of the serum-type antibody, the two monomers being attached by an additional component called the joining chain. There is also a further polypeptide called the secretory component, which protects the antibodies from proteases. 2.2. Antibody derivatives The constant regions of native immunoglobulins are not required for antigen recognition, so it is possible to express smaller derivative molecules and still retain antigen-binding specificity [2,3]. Such derivatives include Fab and F(ab )2

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Fig. 1. Types of recombinant antibody expressed in plants: rAb, recombinant antibody; Fab, fragment antigen binding; scFv, single chain Fv fragment; dAb, single domain antibody.

fragments (which contain only the sequences distal to the hinge region) and single chain Fv fragments (scFvs) which contain the variable regions of the heavy and light chains joined by a flexible peptide chain. Such derivatives are often more effective as drugs than full-length immunoglobulins, because they show increased penetration of target tissues, reduced immunogenicity and they are cleared from tissues more rapidly. Other derivatives include bispecific scFvs, which contain the antigen-recognition elements of two different immunoglobulins and can bind to two different antigens [6], and scFv-fusions, which are linked to proteins with additional functions [7,8] (Fig. 1). 2.3. Humanized antibodies The traditional source of monoclonal antibodies is murine B-cells. To provide a constant source of the antibody, B-cells of appropriate specificity are fused to immortal myeloma cells to produce a hybridoma cell line. The use of murine hybridoma-derived antibodies as therapeutics is limited, because the murine components of the antibodies are immunogenic in humans. Therefore, numerous strategies have been developed to humanize murine monoclonal antibodies [9] culminating in the production of transgenic mice expressing the human immunoglobulin repertoire [10]. An alternative approach is to use phage display libraries based on the human immune repertoires for the production of scFvs. Phage display is advantageous, because high-affinity antibodies can be rapidly identified, novel combinations of heavy and light chains can be tested and the DNA sequence encoding the antibody is indirectly linked to the antibody itself [11,12]. This avoids the laborious isolation of cDNA or genomic immunoglobulin sequences from hybridoma cell lines. 3. Expression systems for recombinant antibodies 3.1. Traditional expression systems Most of the recombinant full-length immunoglobulins being developed as pharmaceuticals are produced in mammalian cell culture, predominantly in Chinese hamster

ovary cells or the murine myeloma cell line NS0 [13]. The main reason for this is the belief that mammalian cells yield authentic products, particularly in terms of glycosylation patterns. However, there are minor differences in glycan chain structure between rodent and human cells. For example, human antibodies contain only the sialic acid residue N-acetylneuraminic acid (NANA), while rodents produce a mixture of NANA and N-glycosylneuraminic acid (NGNA) [14]. There are many disadvantages to mammalian cell cultures, including the high set-up and running costs, the limited opportunities for scale-up and the potential contamination of purified recombinant antibodies with human pathogens. Bacterial fermentation systems are more cost-effective than mammalian cell cultures and are therefore preferred for the production of Fab fragments and scFvs since these derivatives are not glycosylated. Even so, the yields of such products in bacteria are generally low, because the proteins do not fold properly [2]. A more recent development is the production of antibodies in the milk of transgenic animals [15,16]. Recombinant proteins can be harvested periodically, and the yields are potentially very high. However, the production of transgenic farm animals is a difficult process and involves a long development phase with many regulatory hurdles. Scale-up is slow, being dependent on the animal’s natural breeding cycle, and it is necessary to maintain a founder herd carrying the transgene of interest. As with mammalian cell lines, there are safety concerns about the transmission of pathogens or oncogenic DNA sequences. 3.2. The advantages of plants Plants offer a unique combination of advantages for the production of pharmaceutical antibodies [3,17–19]. The main benefit is the low production costs, reflecting the fact that traditional agricultural practices and unskilled labor are sufficient for maintaining and harvesting transgenic crops. Also, large-scale processing infrastructure is already in place for most crops. Scale-up is rapid and efficient, requiring only the cultivation of additional land. Unlike transgenic animals, there is no need to maintain a founder herd, because transgenic plant lines can be stored as seed.

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There are minimal risks of contamination with human pathogens. The general eukaryotic protein synthesis pathway is conserved between plants and animals, so plants can efficiently fold and assemble full-size serum immunoglobulins (as first demonstrated by Hiatt et al. in 1989 [20]) and secretory IgAs (first shown by Ma et al. in 1995 [21]). In the latter case, four different subunits need to assemble in the same plant cell to produce a functional product, even though two different cell types are required in mammals. The post-translational modifications carried out by plants and animals are not identical. There are minor differences in the structure of complex glycans, such as the presence of the plant-specific residues ␣1,3-fucose and ␤1,2-xylose [22]. However, studies using mice administered a recombinant IgG isolated from plants showed that, while there were some differences in the glycan groups present on the recombinant antibody, neither the antibody nor the glycans were immunogenic [23]. It therefore seems likely that many therapeutic antibodies could be safely expressed in plants. As well as full-size antibodies, various functional antibody derivatives have also been produced successfully in plants, including Fab fragments, scFvs, bispecific scFvs, single domain antibodies and antibody fusion proteins (reviewed in [3]). 4. Procedure for recombinant antibody expression in transgenic plants 4.1. Expression construct design Once the appropriate cDNA has been isolated from a hybridoma cell line or a phage display library, it must be inserted into a plant vector designed for high-level expression (reviewed in [24]). A strong constitutive promoter is chosen to maximize transcription. The cauliflower mosaic virus 35S (CaMV 35S) promoter is widely used in dicot (broad leaf) plants and the maize ubiquitin promoter is favored for use in monocots (cereals). The presence of an intron often increases the rate of transcription, so introns are also included in most plant expression constructs. Seed-specific promoters can be used to prevent recombinant antibodies accumulating in vegetative tissues and interfering with plant growth and development, but such interference has generally not been a problem when expressing antibodies raised against human antigens. Protein synthesis can be optimized by making sure the translational start site conforms to the Kozak consensus for plants and replacing any native untranslated sequence with translational enhancers such as the 5 leader sequence derived from tobacco mosaic virus (TMV) RNA (the omega sequence). The most important consideration in expression construct design is protein targeting. This is significant for several reasons. • Recombinant antibodies are more stable in some intracellular compartments than others, and this contributes to the overall yield.

• Full-length immunoglobulins must be targeted to the secretory pathway to undergo glycosylation. • Appropriate targeting can also facilitate protein isolation and purification. It is clear that targeting to the secretory pathway is the best choice for maximizing yields of functional recombinant antibodies [25–27]. The environment of the endoplasmic reticulum (ER) favors correct folding and assembly of immunoglobulin chains, while glycosylation occurs in the ER and Golgi apparatus. The highest yields are achieved by retrieving antibodies from the Golgi apparatus and returning them to the ER lumen in the manner of a resident ER protein [28,29]. This is made possible by including in the expression construct an N-terminal signal sequence (which directs proteins to the secretory pathway) and a C-terminal tetrapeptide signal, KDEL (which facilitates retrieval from the Golgi apparatus). Recombinant antibodies accumulating in the ER may reach levels 10,000-fold higher than those expressed in the cytosol. In the absence of a KDEL signal, the default destination of a recombinant antibody is the apoplast (the space between the plasma membrane and the cell wall). This may be an appropriate strategy in plant cell cultures, because it facilitates extraction. Small molecules such as scFvs diffuse through the wall and can be collected from the culture medium, whereas larger molecules such as full-size antibodies remain trapped in the apoplast, but can be released by mild enzymatic digestion of the cell wall [29]. 4.2. Transgenic plants The majority of plant-derived antibodies have been expressed in transgenic plants. This requires stable transformation and is achieved either through the use of Agrobacterium tumefaciens or particle bombardment, depending on the species [24]. Transformation results in the integration of the antibody transgene into the plant’s genome and produces a permanent resource, a stably expressing transgenic line. The transformation process itself is rapid, but it can take several months to regenerate the first transgenic plants. Once transgenics are available, they must be characterized and scaled up for production. All together, about 2 years may be required before routine production is possible [2]. 4.3. Choice of species A large number of different crops can now be used to produce antibodies including tobacco (Nicotiana tabaccum and N. benthamiana), cereals (rice, wheat, maize), legumes (pea, soybean, alfalfa) and fruit and root crops (tomato, potato) [24]. Many factors need to be considered before making a choice and each case must be evaluated on its own merits [30,31]. Leafy crops such as tobacco and alfalfa generally have the greatest biomass yields per hectare, because they can be cropped several times a year. Tomatoes also have

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a high biomass yield, but production costs are increased because greenhouses are required. However, this may have advantages in terms of containment compared to field crops. For valuable proteins, like antibodies, a more expensive production system would be justified if other advantages were apparent. For example, antibodies expressed in potato tubers and cereal grains are stable at room temperature for months or even years without loss of stability, while tobacco leaves must be dried or frozen prior to transport or storage to maintain the activity of recombinant proteins. However, extraction of proteins from seeds is more expensive than from watery tissue, such as tomatoes. The vast majority of production costs for any recombinant protein reflect downstream processing, so this is likely to be one of the most important economic considerations for the commercial production of recombinant antibodies in plants. The presence of toxic metabolites such as alkaloids in tobacco presents a disadvantage in this context, so edible plants lacking such compounds would be preferred expression hosts. Since most pharmaceutical antibodies will be produced by industry, the costs of production and processing in different crops will have to be evaluated very carefully. 4.4. Alternatives to transgenic plants The main disadvantage of transgenic plants is the long development phase and associated costs. Several alternative approaches are available that reduce the development period to a matter of days or weeks. The first is transient expression, which is generally used to evaluate the activity of expression constructs or test the functionality of a recombinant protein before committing to transgenics. However, if enough protein can be produced, transient expression can also be used as a routine method for antibody production. Agroinfiltration is a transient expression method involving the infiltration of A. tumefaciens cells into tobacco leaf tissue. The onset of expression is very rapid (days to weeks) and milligram amounts of protein can be produced at each ‘harvest’. However, one disadvantage of this system is that scale-up is uneconomical [32]. The second alternative strategy is the use of plant viruses as vectors. Like agroinfiltration, virus infection does not result in stable transformation of the plant, but the onset of recombinant antibody expression is rapid. The advantage of viral vectors over agroinfiltration is that the yields are potentially much higher (due to both the high level expression and the systemic spread of the virus) and that scale-up is achieved easily by manual inoculation. TMV and potato virus X (PVX) vectors have both been used for the production of recombinant antibodies in tobacco, including scFv fragments and a full-size immunoglobulins [33–36]. In the latter case, two TMV vectors were required, one expressing the heavy chain and one the light chain. Co-infection of tobacco plants resulted in the correct assembly of a functional antibody [36].

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Finally, plant cell cultures can also be used for the production of therapeutic antibodies. In this case, the cells are stably transformed with the antibody transgene but, since regeneration is unnecessary, this is a relatively straightforward process and does not take very long. The onset of protein synthesis is rapid and yields of up to 25 mg/l of recombinant antibody are possible. The requirement for fermentors or shaker flasks and skilled personnel makes the set-up and running costs greater than for systems based on whole plants, but extraction and purification is easier and there is the added bonus of sterility and containment, which may be particularly suitable for valuable pharmaceutical products. Both tobacco and rice suspension cells have been used for the production of recombinant antibodies [29,37]. 5. Case studies Six plant-derived antibodies have been developed as human therapeutics, two of which have reached phase II clinical trials. One of these is a full-length IgG specific for EpCAM (a marker of colorectal cancer) developed as the drug Avicidin by NeoRx and Monsanto. Although Avicidin demonstrated some anti-cancer activity in patients with advanced colon and prostate cancers, it was withdrawn because it also resulted in a high incidence of diarrhoea [4]. This was possibly due to cross-reaction with related epitopes on the cells lining the intestine. The five remaining antibodies are discussed below. 5.1. CaroRx The other plant-derived antibody currently in phase II clinical trials is CaroRx, a chimeric secretory IgA/G produced in transgenic tobacco plants [21,38,39]. Secretory antibody production required the expression of the four separate components in four different plant lines, which were crossed over two generations to eventually stack all the transgenes in one line. The antibody is specific for the major adhesin of Streptococcus mutans, the organism responsible for tooth decay in humans. Topical application following elimination of bacteria from the mouth helps to prevent recolonization by S. mutans and leads to the replacement of this pathogenic organism with harmless endogenous flora. 5.2. T84.66, scFvT84.66 and T84.66/GS8 T84.66 is a monoclonal antibody that recognizes carcinoembryonic antigen (CEA), a well-characterized tumorassociated glycoprotein. This antigen is a widely used marker for colorectal adenocarcinomas as well as lung, breast and pancreatic carcinomas, and other carcinomas of epithelial origin. T84.66 is one of several anti-CEA antibodies to have been evaluated for tumor imaging and

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therapy. The full-length IgG has been transiently expressed in tobacco by agroinfiltration [32] and the scFv derivative has been expressed in transgenic tobacco, pea [40], rice and wheat [41] as well as tobacco and rice cell suspension cultures [37]. We have also produced a diabody, T84.66/GS8, by agroinfiltration of tobacco and in transgenic tobacco plants [42] and a diabody fusion with interleukin-2 (IL-2). 5.3. Anti-HSV A full-length humanized IgG recognizing herpes simplex virus 2 (HSV-2) has been expressed in transgenic soybean [43]. Although not tested for efficacy in humans, topical application of the antibody has been shown to prevent vaginal HSV-2 transmission in a mouse model of the disease. The activity of the plant-derived antibody both in vitro and in vivo was indistinguishable from that of the cell culture-derived monoclonal antibody on which it was based. 5.4. 38C13 The production of anti-idiotype antibodies recognizing malignant B-cells is a useful approach for the treatment of diseases such as non-Hodgkin’s lymphoma. McCormick et al. [35] produced a plant-derived scFv based on the well-characterized mouse lymphoma cell line 38C13. When administered to mice, the scFv stimulated the production of anti-idiotype antibodies capable of recognizing 38C13 cells. This provided immunity against lethal challenge with the lymphoma. It is envisaged that this strategy could be used as a rapid production system for tumor-specific vaccines customized for each patient and capable of recognizing unique markers on the surface of any malignant B-cells. The rapid derivation of such prophylactic antibodies would be desirable. Therefore, it is noteworthy that the scFvs in this study were produced using viral vectors and were isolated from virus-infected plants, not transgenic plants. 5.5. PIPP (anti-hCG) We recently expressed three types of antibody specific for human chorionic gonadotropin (hCG) in tobacco plants transiently transformed by agroinfiltration. These were a chimeric full-size IgG, an scFv fragment and a diabody, each derived from the original monoclonal anti-hCG antibody known as PIPP [44]. Each of the antibodies was functional in vitro and in vivo. The in vitro test demonstrated inhibition of the hCG-stimulated production of testosterone by cultured Leydig cells, while the in vivo test revealed inhibition of uterine weight increase in mice. The full-size antibody was 1000 times more active than either derivative. These antibodies could potentially be used for pregnancy detection, (emergency) contraception and the diagnosis and/or therapy of tumors that produce hCG.

6. Future prospects The case studies discussed above show that efficacious therapeutic antibodies can be produced rapidly and inexpensively in transgenic plants or other plant expression systems. The economical advantages of adopting plants as bioreactors on a larger scale would reduce the cost of antibody therapy and increase the number of patients with access to these treatments. The widespread acceptance of plants will be more likely when key bottlenecks are addressed such as the yield after extraction, regulatory issues and plant-specific glycans. It is therefore interesting to note that several strategies have been developed to overcome these limitations, including the co-expression in transgenic plants of mammalian glycosyltransferases, to facilitate the production of antibodies with authentic glycan chains [45]. References [1] Andersen DC, Krummen L. Recombinant protein expression for therapeutic applications. Curr Opin Biotechnol 2002;13(2):117–23. [2] Chadd HE, Chamow SM. Therapeutic antibody expression technology. Curr Opin Biotechnol 2001;12(2):188–94. [3] Fischer R, Emans N. Molecular farming of pharmaceutical proteins. Transgenic Res 2000;9(4/5):279–99. [4] Gavilondo JV, Larrick JW. Antibody production technology in the millennium. Biotechniques 2000;29(1):128–45. [5] Jefferis R. Glycosylation of human IgG antibodies: relevance to therapeutic applications. Biopharma 2001;9:19–27. [6] Fischer R, Schumann D, Zimmermann S, Drossard J, Sack M, Schillberg S. Expression and characterization of bispecific single chain Fv fragments produced in transgenic plants. Eur J Biochem 1999;262(3):810–6. [7] Francisco JA, Gawlak SL, Miller M, Bathe J, Russell D, Chace D, et al. Expression and characterization of bryodin 1 and a bryodin 1-based single-chain immunotoxin from tobacco cell culture. Bioconjug Chem 1997;8(5):708–13. [8] Spiegel H, Schillberg S, Sack M, Holzem A, Nähring J, Monecke M, et al. Accumulation of antibody fusion proteins in the cytoplasm and ER of plant cells. Plant Sci 1999;149(1):63–71. [9] Kipriyanov SM, Little M. Generation of recombinant antibodies. Mol Biotechnol 1999;12(2):173–201. [10] Green L. Antibody engineering via genetic engineering of the mouse: xenomouse strains are a vehicle for the facile generation of therapeutic human monoclonal antibodies. J Immunol Methods 1999;231(12):11–23. [11] Griffiths A, Duncan A. Strategies for selection of antibodies by phage display. Curr Opin Biotechnol 1998;9(1):102–8. [12] Sidhu SS. Phage display in pharmaceutical biotechnology. Curr Opin Biotechnol 2000;11(6):610–6. [13] Chu L, Robinson DK. Industrial choices for protein production by large-scale cell culture. Curr Opin Biotechnol 2001;12(2):180–7. [14] Raju TS, Briggs J, Borge SM, Jones AJS. Species-specific variation in glycosylation of IgG: evidence for the species-specific sialylation and branch-specific galactosylation and importance for engineering recombinant glycoprotein therapeutics. Glycobiolgy 2000;10(5): 477–86. [15] Houdebaine LM. Transgenic animal bioreactors. Transgenic Res 2000;9(4/5):305–20. [16] Larrick JW, Thomas DW. Producing proteins in transgenic plants and animals. Curr Opin Biotechnol 2001;12(4):411–8. [17] Giddings G. Transgenic plants as protein factories. Curr Opin Biotechnol 2001;12(5):450–4.

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