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Antibody production in plants
Biotechnology Advances, Vol. 14, No. 3, pp. 267-281, 1996 Copyright © 1996 Elsevier Science Inc. Printed in the USA. All rights reserved 0734-9750/96 $32.00 ÷ .00
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ANTIBODY PRODUCTION IN PLANTS MATTHEW D. SMITH Department of Biology, University of Waterloo Waterloo, Ontario, Canada N2L 3G I
ABSTRACT Production of heterologous proteins in plants has become increasingly efficient due to recent advances in plant biotechnology. Heterologous proteins that have specifically attracted a great deal of attention are plant-produced monoclonal antibodies. A variety of applications for these so-called plantibodies have been explored since they were first expressed in tobacco seven years ago. Both full length antibodies and antibody fragments produced in transgenic plants offer many intriguing possibilities to plant molecular biologists and plant breeders. However, questions such as how cellular targeting influences the expression and accumulation of these proteins in plants still need to be answered before the technology can be used commercially, on a large-scale.
Key Words: Heterologous protein; Plant biotechnology; Plantibodies; lmmunotherapy; Immunomodulation; Single-chain Fv molecules.
INTRODUCTION
Microbial systems have traditionally
been used for the production of many
commercially important biomolecules (Glick and Pasternak, 1994). However, recent advances in plant biotechnology have made transgenic plants attractive, alternative systems for the production of lipids, carbohydrates and proteins of non-plant origin (Goddijn and Pen, 1995). Such biomolecules can be used to alter the phenotype of the plant itself. Alternatively, the unique properties of plants can be used for large-scale production of the desired biomolecule (Whitelam et al., 1993). 267
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Plant agriculture presents a number of advantages as a large-scale protein production system. Plants are relatively easy and inexpensive to produce, and the equipment already exists for harvesting and processing large volumes of plant material (Whitelam et al., 1993). In addition, plants are capable of targeting proteins to a number of different tissues for long-term storage.
However, plants have not been widely used for commercial protein production,
perhaps due to environmental concerns related to the production of non-plant products in crops or because of a preference for production in the controlled, sterile environment of a lab. There is also some concern about the potential costs of protein purification from plants, although Hiatt (1990) estimates that agriculturally-produced anitibodies are likely to cost less than antibodies derived from hybridoma cells.
The possibility of using plants for large-scale
production of non-protein products also exists. For example, researchers at Agrecetus Inc. are developing a cotton plant that produces a cotton-polyester blend fiber (Moffat, 1995). As well, efforts are being made to develop plants that produce biodegradable plastics, industrial lubricants, drugs and pharmaceuticals (Moffat, 1995). Within the next decade, phenotypic modification of plants using biotechnology will undoubtedly lead to transgenic crops with enhanced nutritional quality as a result of altered protein and lipid compositions (Flavell, 1995). Transgenic plants with altered agronomic traits will also be developed. To improve crop value, the heterologous protein may serve to modify metabolic pathways or could be used to supply the plant with novel pest, herbicide or viral resistance. Of the many heterologous proteins that can be produced in plants, monoclonal antibodies (tnAbs) may be the most intriguing. There are many potential uses for these socalled plantibodies. They may be used in basic plant research, or be produced on a large scale for therapeutic or diagnostic use (Whitelam et al., 1993). They also provide the opportunity to manipulate agronomic crop plant traits or reduce pathogenic infections through antibodymediated modifications of antigen activity in planta (Hiatt, 1990). The binding and retention capacity of antibodies could also be used for the isolation and processing of environmental contaminants, a process known as biofiltration (Hiatt, 1990). A plant-based strategy for antibody production has an advantage over a microbial strategy, as the largest antibody fragment that has been reliably produced in Escherichia coli is a monovalent Fab fragment (Ma and Hein, 1995a). Several groups have successfully produced full length antibodies in higher plants (Hiatt et al., 1989; D0ring et al., 1990; Ma et al., 1994; van Engelen et al., 1994; Ma et al., 1995; De Wilde et al., 1996), while a number of groups
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have had success producing smaller antibody fragments such as Fab and single-chain Fv molecules (Benvenuto et ai., 1991; Owen et al., 1992; Tavladoraki et al., 1993; Fiedler and Conrad, 1995; Schouten et al., 1996; De Wilde et al., 1996). The aim of this review is to provide an overview of developments made toward the production of full length antibodies and antibody fragments in transgenic plants. The problems and potential uses of these plantproduced antibodies will be identified and discussed.
FULL LENGTH ANTIBODIES
Assembly and Targeting Full length antibodies were first expressed in plants by Andrew Hiatt and co-workers in 1989. The monoclonal antibody 6D4 from a mouse hybridoma cell line was expressed in tobacco. Separate transgenic lines were produced which expressed either the light (t:, kappa:) or heavy (y, gamma) chains of the immunoglobulin G (IgG) molecule, both with and without their natural, mammalian leader sequence. These transgenic lines were then sexually crossed, to yield plants that expressed both K- and y-chains simultaneously. An ELISA assay, using the 6D4 antigen (P3-bovine serum albumin) was then used to assay the binding specificity of the plant-produced antibody, and hence confirm that the immunoglobulin chains were assembled into functional complexes.
In fact, assembly of immunoglobulin chains was found to be
extremely efficient (Hiatt et al., 1989). In addition, the plant-derived antibody bound antigen equally as well as the 6D4 hybridoma mAb. However, none of the plants transformed with the leaderless immunoglobulin chain genes contained any assembled gamma-kappa complexes. This indicated that leader sequences were necessary for the accumulation of functional antibodies in tobacco. The authors presumed that assembly took place in a similar manner as in B lymphocytes. While the mechanism of antibody assembly in these mammalian cells is only partially understood, it is known that both light and heavy chains are synthesized as precursor proteins. These precursors are targeted to the lumen of the endoplasmic reticulum (ER) where the signal sequences are cleaved and chaperonin molecules direct the assembly of functional immunoglobulins (Ma and Hein, 1995a).
Hiatt et al. (1989) suggested that the
association of immunoglobulin with the endoplasmic reticulum indicated the antibody was to be secreted from the plant cell. However, they did not discount the possibility that assembly
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could also take place spontaneously in other cellular compartments, given sufficient levels of each chain. To optimize secretion of antibodies from plant cells, a slightly different expression strategy was developed which utilized what is known about secretion pathways of other organisms (Hein et al., 1991). Secretion was desired to obtain higher levels of accumulation of the antibody in the extracellular environment of the plant, which may allow for gentler extraction and perhaps easier purification of the antibodies.
In order to evaluate the
dependence of assembly on the N-terminal leader sequence, expression of mAb in plants transformed with y- and ~:-chain genes, each containing their native mouse signal sequence, was compared to mAb expression in plants that were transformed with genes containing a yeast pre-pro sequence in place of the native signal (Hein et al., 1991). The aim of this study was to maximize antibody accumulation in plants.
Yet a potential advantage of using plants as
bioreactors is that a great many plants can be grown at one time, reducing the need for a single plant to produce a large amount of the heterologous protein. Therefore, while increasing perplant yields is desirable, more work on optimizing purification procedures for plant-produced antibodies is also needed.
A promising purification scheme uses the binding affinity of
staphylococcal protein A with the Fc region of IgG antibodies (Hein et al., 1991).
If this
powerful approach can be modified for efficient large-scale purification, it might reduce the need to increase antibody accumulation in individual plants. A slightly different strategy was later applied to obtain coordinate expression of light and heavy chain genes resulting in the accumulation of functional, full length immunoglobulin M (IgM) in tobacco. An expression cassette consisting of two chimeric genes encoding the light (~,) and heavy (It) chains of the B 1-8 antibody was used (DUring et al., 1990). This approach eliminated the need to perform sexual crosses of transgenic plants. The chimeric genes were constructed using the mature light and heavy chain genes of the antibody, each linked to the coding sequence for the barley tx-amylase signal peptide. The chimeric genes were each placed under the control of an active plant promoter, linked on a plasmid, and integrated into the genome of tobacco using Agrobacterium-mediated gene transfer (D~iring et al., 1990). This system differed from the strategy of Hiatt et al. (1989), which involved sexual crosses to obtain simultaneous expression and assembly of heavy and light chains in tobacco, and used a mammalian, rather than plant ER targeting sequence. Dtiring et al. (1990) used immunogold labeling to determine the cellular location of the IgM molecules. A few gold
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particles were detected in the cytoplasm, while the majority of the signal accumulated in the ER.
Surprisingly, considerable signal was also localized in the thylakoid membranes of
chloroplasts. The use of a plant-derived ER targeting sequence by DUring et al. (1990), and the fact that that they saw accumulation of antibody in the endoplasmic reticulum of transgenic tobacco, supports the contention that transport to the ER lumen is necessary for proper assembly of full length antibodies, as in mammalian systems (Hiatt et al., 1989).
ER
localization also supports the finding that antibodies are involved in the secretory pathway of plants (Hiatt et al., 1989; Dtiring et al., 1990). However, the cytoplasmic and chloroplastic antibody would not be part of the secretion pathway, and certainly this distribution pattern was not expected. Recently however, it was demonstrated that the light-harvesting chlorophyll a/bbinding protein and the small subunit of Rubisco (both synthesized in the cytoplasm) are targeted to the chloroplasts of Euglena gracilis, after first passing through the ER and Golgi apparatus (Sulli and Schwartzbach, 1995; Sulli and Schwartzbach, 1996). The precursors of these proteins are targeted to the ER, and are transported from the ER to the Golgi apparatus and from the Golgi apparatus to the chloroplast, where they are imported as membrane-bound precursors (Sulli and Schwartzbach, 1996). While this system may not directly parallel the targeting of antibody to chloroplasts of plant cells, it may provide an explanation for the cellular distribution of antibody seen by D0ring et al. (1990). Nevertheless, the most important factor in the accumulation of functional, full length antibody in transgenic tobacco appears to be the presence of an ER signal peptide, regardless of its origin (Dtiring et al., 1990; Ma and Hein, 1995b). Recently, De Wilde et ai. (1996) established that both full length IgG molecules and Fab fragments accumulate in the intercellular spaces of leaf mesophyll cells of transgenic
Arabidopsis thaliana. This immunolocalization study confirmed the assumption that full length antibody is secreted from plant cells (Hiatt et al., 1989; DUring et al., 1990; Ma and Hein, 1995b), and for the first time conclusively demonstrated that plant-produced antibodies accumulate in the intercellular spaces. As well, it was surprising that the antibodies were not retained by the cell walls which are believed to have exclusion limits of between 17 and 60 kDa (Carpita et al., 1979; Tepfer and Taylor, 1981; De Wilde et al., 1996). Yet the results of De Wilde et al. (1996) suggest that the mesophyll cell walls of Arabidopsis were permeable to
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intact IgG molecules, which have a molecular weight of 146 kDa. These findings are of particular interest to any application where extracellular antigen binding is desired. The finding that some plant-produced mAb accumulated in the cytoplasm of transgenic tobacco cells (DUring et al., 1990) led Stieger et al. (1991) to speculate that antibodies have the ability to self-assemble in the cytoplasm. Accumulation of antibody in the cytoplasm would certainly provide many interesting possibilities for use in planta. The immunoglobulin could be used to inhibit the activity of an endogenous enzyme, thus providing an alternative to antisense technology (Stieger et al., 1991). Intracellular accumulation would be desired for this type of immunomodulation approach to altering plant metabolism, since extracellular antibody would not be available to bind endogenous antigen. To obtain cytosolic expression, leaderless chimeric genes were injected into the nuclei of Acetabularia mediterranea (Stieger et al., 1991).
The heavy and light chain genes from the antibody B
1-8 were used.
Immunofluorescence confirmed that the antibodies formed correctly folded antigenic binding sites in the cytoplasm. However, the relatively high reducing conditions of the cytoplasm will not allow formation of disulfide bonds, which are a characteristic of all antibodies (Stieger et al., 1991). The authors speculated that the antibodies folded correctly in the cytoplasm without forming disulfide bonds, and relied simply on hydrogen bonds, van der Waals forces, ionic and hydrophobic interactions for proper folding. If indeed these antibodies are fully functional in the cytoplasm, this technology could be very important for plant molecular biologists and plant breeders. However, such results have not yet been reported in higher plants. It is possible that the cytoplasm of algae is less reducing and contains the chaperonins necessary for folding of full-size immunoglobulins.
Nevertheless, it appears that cytoplasmic expression and
accumulation of full length, functional antibodies is specific to algae Conrad and Fiedler, 1994).
Pathogen Protection The prospect of using whole antibodies to obtain disease resistance against plant pathogens has been examined by van Engelen et al. (1994). Heavy and light chain genes were placed under the control of promoters that provide expression in roots, and were introduced into tobacco on a single T-DNA using Agrobacterium-mediated transformation.
High
expression levels (1.1% of total soluble root protein) were achieved in the apoplast of roots
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(van Engelen et al., 1994). This system gives high expression levels of functional, full length antibodies in roots, and using the proper antibody could be used to provide pathogen resistance to plant roots by interfering with extracellular pathogenic antigens (van Engelen et al., 1994).
Human Immunotherapy
The immunotherapeutic potential of full length plant-produced antibodies has received much attention for a number of years (Ma and Hein, 1995a; Ma and Hein, 1995b). Plantibodies are more desirable for human use than microbially produced proteins because they undergo eukaryotic rather than prokaryotic post-translational processing.
For example,
glycosylation is a type of protein modification more closely matched by plants than by bacteria (Ma and Hein, 1995b) and is necessary for secretion of antibodies to the apoplastic space in transgenic plants (Stieger et al., 1991). Many of the N-linked glycans involved in glycosylation are the same with respect to both size and extent of branching in both plants and animals (Hein et al., 1991; Ma and Hein, 1995b).
As well, Hein et al. (1991) report that transgenic
plantibodies are processed in a similar manner as mammalian glycoproteins. However, there are some differences in the carbohydrates involved and the pattern of glycosylation in plants (Ma and Hein, 1995b). There is some concern that the presence of plant-specific glycans may increase the immunogenicity of plantibodies used for human immunotherapy, even though this seems unlikely, given humans' daily exposure to plant glycoproteins in food, pollen and personal care products (Ma and Hein, 1995a). Nevertheless, if these plant-produced antibodies are to be used for human immunotherapy, plant-specific glycosylation is a concern that needs to be addressed. If plant glycosylation does present a problem, it could be circumvented by removing the glycosylation site(s) from the antibody molecule, removing the glycan itself from the plant or perhaps by generating a mutant lacking the glycosylating enzyme (Ma and Hein, 1995b). The prospect of using plant-produced antibodies in passive immunization against a cellsurface antigen (SA ~
of Streptococcus mutans, the most common cause of tooth decay, has
been closely studied in recent years (Ma et al., 1994). Large doses of antibody are required in multiple applications for topical passive immunotherapy and transgenic plants have been explored as the production system that could provide the quantities of antibody needed. The predominant antibody in human mucosal secretions, such as saliva, is the secretory form of
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immunoglobulin A (SIgA) (Sigal and Ron, 1994). Secretory IgA is a good candidate for use in passive topical immunization against oral bacteria such as S. mutans, or other pathogens of the digestive tract, due to its resistance to proteolytic digestion (Ma et al., 1994; Stavnezer, 1996). However, a hybridoma-derived IgG antibody (Guy's 13) against the cell-surface antigen (SA I/II) of S. m u t a n s was first used successfully in a local passive immunization study on human subjects (Ma et al., 1987). This study suggested that passive immunization using mAb might be a good alternative approach for the prevention of colonization of teeth by S. mutans, and the subsequent development of tooth decay. Ma et al. (1994) later produced a hybrid IgA-G molecule in plants, and showed that it caused aggregation of S. m u t a n s in culture, which appears to be how the antibody prevents colonization of the bacteria in vivo.
Some non-
essential constant regions of the Guy's 13 heavy chain were replaced with IgA constant regions, making the hybrid less prone to proteolytic digestion in human secretions. Incorporation of IgA constant regions did not alter the antigen recognition capacity of the recombinant antibody (Ma et al., 1994). In an extension of the secretory antibody work, Ma et al. (1995) have achieved simultaneous expression and assembly of four separate protein chains to form functional, dimeric secretory antibody in transgenic tobacco. The resulting secretory antibody (SIgA-G) which binds the same cell-surface antigen of S. m u t a n s as IgA-G (Ma et al., 1994), consists of a monoclonal kappa chain, a hybrid IgG/IgA heavy chain, a joining (J) chain from mouse and a secretory component (SC) from rabbit (Ma et al., 1995). Successive sexual crosses between transgenic plants, each expressing one of the protein chains, resulted in the simultaneous expression and assembly of all four peptides. The resulting SIgA-G molecule matches the most common form of IgA found in mammals, which is a dimer associated with a small polypeptide J chain and a secretory component (Ma et al., 1995). In mammalian systems production of monoclonal SIgA requires two different cell types, while functional expression of this secretory molecule was achieved in single tobacco leaf cells. Moreover, the nature of the association of the J chain and SC with IgA-G (to form SIgA-G) was confirmed to be similar to their association with IgA in mammals. Binding specificity of the plant-produced SIgA-G was also similar to the native SIgA antibody (Ma et al., 1995). The yield of SIgA-G in tobacco was much higher than achieved for IgA-G in the previous study by Ma et al. (1994), which indicated SIgA-G was more resistant to proteolysis and suggested its production could be scaled up to agricultural proportions (Ma et al., 1995). Preliminary results also suggest that the
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development of plants capable of producing native SIgA may be possible, which could have significant impact on passive immunotherapeutic applications (Ma et al., 1995). Although this type of approach seems to have great potential for success, few antibodies have been approved for clinical use (Ma and Hein, 1995a). A major hurdle has been the cost of antibody production in plants. However, Hiatt (1990) estimated that antibodies expressed in soybean at a level of 1% of total protein, could be produced for $100 per kg of antibody. Surely, this cost can only go down as purification procedures are improved. Large quantities of antibody are required to overcome the rapid rate of clearance when used for passive topical immunotherapy and plant agriculture could provide the inexpensive, large-scale production system needed for such a treatment (Ma and Hein, 1995a).
ANTIBODY FRAGMENTS
Modification of Plant Metabolism
Small size and undemanding folding requirements make single-chain Fv molecules (scFvs) very attractive antibody derivatives for expression in plants. Single-chain Fv fragments consist of the variable regions of the light and heavy chains of an immunoglobulin linked together by a flexible peptide linker (Conrad and Fiedler, 1994; Ma and Hein, 1995a). The binding capacity of scFv molecules is similar to their parental antibodies, but their small size gives them an advantage over whole antibodies where the complement-binding (Fc) portion of the molecule is not needed (Owen et al., 1992). Owen et al. (1992) were able to successfully express an scFv in tobacco in an effort to modify plant metabolism. A synthetic gene was used to produce an scFv that binds to the plant regulatory molecule phytochrome. Seeds derived from plants producing the anti-phytochrome scFv had altered light-controlled germination. Specifically, germination was reduced in anti-phytochrome producing plants by approximately 40% (Owen et al., 1992). Presumably, the reduction in germination was due to binding of the scFv to phytochrome, which affected its ability to function properly. This was the first report of successfully altering plant metabolism using a plant-produced mAb against an endogenous antigen. This approach could also be applied to other metabolic pathways and could be used to study plant metabolism, or perhaps enhance crop value by blocking undesired pathways. Single-chain F~ fragments are more attractive than full length mAbs for the immunomodulation
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of plant cell components due to their less demanding assembly requirements in the reducing environment of the cytoplasm. In addition, full length antibodies must be secreted to achieve functional accumulation, which would make them unavailable for this type of intracellular immunomodulation. The production of full length antibodies in the ER of plant cells for secretion to the extracellular space is desirable for large-scale production because these molecules will undergo minimal hydrolytic processing in the extracellular environment and may be more easily extracted (Ma et al., 1995). However, if the antibody is to be used for altering or immunizing the plants themselves, targeting to the cytoplasm or other cellular compartment may be more desirable (Ma et al., 1995). Therefore, scFvs localized in the cytoplasm may be more valuable for use in planta, while secreted antibodies are best suited for large-scale production.
Pathogen Protection In an effort to confer viral resistance to plants, Tavladoraki et al. (1993) expressed an scFv antibody directed against the artichoke mottled crinkle virus in the cytoplasm of tobacco. These transgenic plants were specifically protected from virus attack, in that they had a reduced incidence of infection and showed delayed symptom development (Tavladoraki et al., 1993). The success of this approach to attenuate viral infection was dependent on stable antibody expression in the cytoplasm. This was achieved using scFvs rather than full length antibodies, which must be targeted to the ER for secretion in order to get stable accumulation in higher plants (Tavladoraki et al., 1993, Ma and Hein, 1995a). This "engineered intracellular immunization" approach seems to have great potential for supplying plants with viral and perhaps pest resistance (Tavladoraki et al., 1993). However, before it becomes a commercially useable or valuable approach, the mechanism(s) of action must be fully elucidated.
Targeting Schouten et al. (1996) believe cytosolic expression of functional scFv molecules is unlikely and suggest that such intracellular expression is not so straightforward. The objective of their study was to examine functional expression of antibodies targeted to different subcellular compartments. No transgenic plants containing scFv genes targeted for expression in
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the cytoplasm were found to accumulate any detectable scFv (Schouten et al., 1996). Whereas plants expressing scFv targeted to the ER accumulated significant amounts of the protein. Interestingly, when an ER retention signal (amino acids KDEL) was included in the antibody gene construct, accumulation of scFv was increased significantly (Schouten et al., 1996). This was true for both the secreted (ER targeted) and cytosolic forms of the scFv. The KDEL sequence may somehow protect the cytosolic scFv from proteolytic degradation, or may confer protection to the protein through an interaction with the cytosolic side of the ER (Schouten et al., 1996). However, Schouten et al. (1996) expressed a different scFv molecule than has been used in previous reports (Owen et al., 1992; Tavladoraki et al., 1993) and it may be possible that some antibody fragments are more prone to proteolytic degradation than others. Nevertheless, the work of Schouten et al. (1996) demonstrated that the exact requirements for antibody expression and accumulation in plants remain elusive. In a recent report, Fiedler and Conrad (1995) demonstrated that active scFv molecules can be targeted to an organ other than leaves. Specifically, scFv was shown to accumulate in developing and ripe tobacco seeds. The antibody accumulated to 0.67% of total soluble protein in the seeds, and was stably stored for one year at room temperature (Fiedler and Conrad, 1995). This system therefore offers high expression levels along with long-term storage of the protein and does not appear to influence plant growth rate or seed development. Exact cellular location was not determined for the antibody, although the authors felt it may have accumulated in protein bodies of the seeds (Fiedler and Conrad, 1995). Determining the exact cellular location of the stored antibody and transferring the system to another crop, such as corn, would make this strategy valuable for the commercial production of scFvs (Fiedler and Conrad, 1995). In fact, this system would be even more valuable if long-term stable expression could also be achieved for full length antibodies.
Specifically, such a system would be
valuable for delivering large quantities of full length antibodies for passive immunization. For instance, it has been shown that full length antibodies can be assembled and accumulated in the roots of transgenic tobacco (van Engelen et al., 1994). If this technology could be utilized to obtain stable accumulation of these antibodies in edible tissues such as potato tubers, it might allow for long-term storage and easy delivery of antibodies for immunotherapeutic applications. As well, these transgenic crops could be grown using existing knowledge and equipment, and be used to immunize large populations inexpensively without the need for specialized long-term storage facilities or purification procedures.
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SUMMARY
In recent years, plant biotechnology has made agriculture a more attractive alternative for the large-scale production of biomolecules such as proteins.
The ability to produce
antibodies and antibody fragments in plants presents some very interesting possibilities. Since the monoclonal antibody 6D4 was first expressed in tobacco by Hiatt et al. (1989), antibodies have been produced in plants for a variety of reasons.
Full length antibodies have been
expressed in plants with the aim of producing large quantities for use in human immunotherapy. Indeed, the production of multimeric secretory antibodies (Ma et al., 1995) and the ability to store antibodies long-term in seeds or other organs (Fiedler and Conrad, 1995) indicates this goal may soon be reached.
If the potential problem of plant-specific
glycosylation can be solved, passive immunotherapy using plant-produced antibodies against human mucosal pathogens may soon be possible. The use of antibodies in planta to alter plant phenotype is another exciting area of research. Plant-produced antibodies could be used to change the appearance of the plant, alter plant metabolism or confer pathogen resistance. For these purposes antigen binding in vivo is required, so cellular location of the plant-produced antibody is critical. Since the Fc portion of the antibody is not necessarily required, easily assembled scFv molecules have been used to achieve phenotypic modifications requiring intracellular accumulation. Phytochrome-mediated germination has been altered (Owen et al., 1992) and tobacco has been effectively immunized against virus attack (Tavladoraki et al., 1993) using intracellular scFvs. The possibility of using full length antibodies to interfere with pathogenesis in the apoplast has also been examined (van Engelen et al., 1994). Clearly though, the effects of cellular targeting on expression levels of antibodies in plants are far from resolved. Although some groups have reported successful expression of scFvs in the cytoplasm (Owen et al., 1992; Tavladoraki et al., 1993), others failed to get any antibody accumulation unless an ER retention signal was included (Schouten et al., 1996), and only recently has the exact extracellular location of secreted antibodies been determined (De Wilde et al., 1996). Surprisingly, it was also revealed that whole antibodies secreted from the ER are not retained by plant cell walls (De Wilde et al., 1996). As well, Dtiring et al. (1990) reported unexpected accumulation of full length antibodies in chloroplasts. Therefore, while the production of antibodies in plants offers many exciting possible
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applications, such as in passive immunization of large human populations and altering plant metabolism, there are still questions about the technology that need to be addressed.
REFERENCES
1.
Benvenuto, E., Ordhs, R. J., Tavazza, R., Ancora, G., Biocca, S., Cattaneo, A., and Galeffi, P. (1991), Plant Mol. Biol., 17, 865-874. "Phytoantibodies": a general vector for the expression of immunoglobulin domains in transgenic plants.
2.
Carpita, N., Sabularse, D., Montezinos, D., and Delmer, D. P. (1979), Science, 205, 1144-1147. Determination of the pore size of cell walls of living plant cells.
3.
Conrad, U., and Fiedler, U. (1994), Plant Mol. Biol., 26, 1023-1030. Expression of engineered antibodies in plant cells.
4.
De Wilde, C., De Neve, M., De Rycke, R., Bruyns, A.-M., De Jaeger, G., Van Montagu, M., Depicker, A., and Engler, G. (1996), Plant Sci., 114, 133-241. Intact antigen-binding MAK33 antibody and Fab fragment accumulate in intercellular spaces of Arabidopsis
thaliana. 5.
D~iring, K., Hippe, S., Kreuzaler, F., and Schell, J. (1990), Plant Mol. Biol., 15, 281-293. Synthesis and self-assembly of a functional monoclonal antibody in transgenic
Nicotiniana tabacum. 6.
Fiedler, U., and Conrad, U. (1995), Bio/Technol., 13, 1090-1093. High-level production and long-term storage of engineered antibodies in transgenic tobacco seeds.
7.
Flavell, R. B. (1995), Trends Biotech., 13, 313-319. Plant biotechnology R & D - the next ten years.
8.
Glick, B. R. and Pasternak, J. J. (1994), Molecular Biotechnology: Principles and
Applications of Recombinant DNA, ASM Press (Washington, D.C.), pp. 113, 155, 9.
Goddijn, O. J. M., and Pen, J. (1995), Trends Biotech., 13, 379-387. Plants as bioreactors.
10.
Hein, M. B., Tang, Y., McLeod, D. A., Janda, K. D., and Hiatt, A. (1991), Biotechnol.
Prog., 7, 455-461. Evaluation of immunoglobulins from plant cells. 11.
Hiatt, A. (1990), Nature, 344, 469-470. Antibodies produced in plants.
12.
Hiatt, A., Cafferkey, R., and Bowdish, K. (1989), Nature, 342, 76-78. Production of antibodies in transgenic plants.
280
13.
MATTHEWD. SMITH
Ma, J. K.-C., and Hein, M. B. (1995a), Plant Physiol., 109, 341-346. Plant antibodies for immunotherapy.
14.
Ma, J. K.-C., and Hein, M. B. (1995b), Trends Biotech., 13, 522-527. Immunotherapeutic potential of antibodies produced in plants.
15.
Ma, J. K.-C., Hiatt, A., Hein, M., Vine, N. D., Wang, F., Stabila, P., van Dolleweerd, C., Mostov, K., and Lehner, T. (1995), Science, 268, 716-719. Generation and assembly of secretory antibodies in plants.
16.
Ma, J. K.-C., Lehner, T., Stabila, P., Fux, C. I., and Hiatt, A. (1994), Eur. J. Immunol., 24, 131-138. Assembly of monoclonal antibodies with IgG1 and IgA heavy chain domains in transgenic tobacco plants.
17.
Ma, J. K.-C., Smith, R., and Lehner, T. (1987), Infect. Immun., 55, 1274-1278. Use of monoclonai antibodies in local passive immunization to prevent colonization of human teeth by Streptococcus mutans.
18.
Moffat, A. S. (1995), Science, 268, 658-660. Exploring transgenic plants as a new vaccine source.
19.
Owen, M., Gandecha, A., Cockburn, B., and Whitelam, G. (1992), Bio/Technol., 10, 790794. Synthesis of a functional anti-phytochrome single-chain Fv protein in transgenic tobacco.
20.
Schouten, A., Roosien, J., van Engelen, F. A., de Jong, G. A. M., Borst-Vrenssen, A. W. M., Zilverentant, J. F., Bosch, D., Steikema, W. J., Gommers, F. J., Schots A., and Bakker, J. (1996), Plant Mol. Biol., 30, 781-793. The C-terminal KDEL sequence increases the expression level of a single-chain antibody designed to be targeted to both the cytosol and the secretory pathway in transgenic tobacco.
21.
Sigal, L. H., and Ron, Y. (1994), Immunology and Inflammation,
McGraw-Hill
22.
Stavnezer, J. (1996), In Advances in Immunology, (Dixon, F. J., editor), Acedemic Press
(Toronto), pp. 48-51.
(Toronto), pp. 79-146. Antibody class Switching. 23.
Stieger, M., Neuhaus, G., Momma, T., Schell, J., and Kreuzaler, F. (1991), Plant Sci., 73, 181-190. Self assembly of immunoglobulins in the cytoplasm of the alga Acetabularia mediterranea.
ANTIBODYPRODUCTIONIN PLANTS
24.
281
Sulli, C., and Schwartzbach, S. D. (1995), J. Biol. Chem., 270, 13084-13090. The polyprotein precursor to the Euglena light harvesting chlorophyll a/b-binding protein is transported to the Golgi apparatus prior to chloroplast import and polyprotein processing.
25.
Sulli, C., and Schwartzbach, S. D. (1996), Plant Cell 8, 43-53. A soluble protein is imported into Euglena chloroplasts as a membrane-bound precursor.
26.
Tavladoraki, P., Benvenuto, E., Trinca, S., De Martinis, D., Cattaneo, A., and Galeffi, P. (1993), Nature, 366, 469-472. Transgenic plants expressing a functional single-chain Fv antibody are specifically protected from virus attack.
27.
Tepfer, M., and Taylor, I. E. P. (1981), Science, 213, 761-763. The permeability of plant cell wall as measured by gel filtration chromatography.
28.
van Engelen, F. A., Schouten, A., Molthoff, J. W., Roosien, J., Salinas, J., Dirkse, W. G., Schots, A., Bakker, J., Gommers, F. J., Jongsma, M. A., Bosch, D., and Stiekema, W. J. (1994), Plant Mol. Biol., 26, 1701-1710. Coordinate expression of antibody subunit genes yields high levels of functional antibodies in roots of transgenic tobacco.
29.
Whitelam, G. C., Cockburn, B., Gandecha, A. R., and Owen, M. R. L. (1993), In Biotechnology and Genetic Engineering Reviews, vol. 11, (Tombs, M. P., editor) Intercept Ltd. (Andover, UK), pp. 1-29. Heterologous protein production in transgenic plants.
; • ,4.~iv~i ELSEVIER
PII S0734-9750(96)00020-1
ANTIBODY PRODUCTION IN PLANTS MATTHEW D. SMITH Department of Biology, University of Waterloo Waterloo, Ontario, Canada N2L 3G I
ABSTRACT Production of heterologous proteins in plants has become increasingly efficient due to recent advances in plant biotechnology. Heterologous proteins that have specifically attracted a great deal of attention are plant-produced monoclonal antibodies. A variety of applications for these so-called plantibodies have been explored since they were first expressed in tobacco seven years ago. Both full length antibodies and antibody fragments produced in transgenic plants offer many intriguing possibilities to plant molecular biologists and plant breeders. However, questions such as how cellular targeting influences the expression and accumulation of these proteins in plants still need to be answered before the technology can be used commercially, on a large-scale.
Key Words: Heterologous protein; Plant biotechnology; Plantibodies; lmmunotherapy; Immunomodulation; Single-chain Fv molecules.
INTRODUCTION
Microbial systems have traditionally
been used for the production of many
commercially important biomolecules (Glick and Pasternak, 1994). However, recent advances in plant biotechnology have made transgenic plants attractive, alternative systems for the production of lipids, carbohydrates and proteins of non-plant origin (Goddijn and Pen, 1995). Such biomolecules can be used to alter the phenotype of the plant itself. Alternatively, the unique properties of plants can be used for large-scale production of the desired biomolecule (Whitelam et al., 1993). 267
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Plant agriculture presents a number of advantages as a large-scale protein production system. Plants are relatively easy and inexpensive to produce, and the equipment already exists for harvesting and processing large volumes of plant material (Whitelam et al., 1993). In addition, plants are capable of targeting proteins to a number of different tissues for long-term storage.
However, plants have not been widely used for commercial protein production,
perhaps due to environmental concerns related to the production of non-plant products in crops or because of a preference for production in the controlled, sterile environment of a lab. There is also some concern about the potential costs of protein purification from plants, although Hiatt (1990) estimates that agriculturally-produced anitibodies are likely to cost less than antibodies derived from hybridoma cells.
The possibility of using plants for large-scale
production of non-protein products also exists. For example, researchers at Agrecetus Inc. are developing a cotton plant that produces a cotton-polyester blend fiber (Moffat, 1995). As well, efforts are being made to develop plants that produce biodegradable plastics, industrial lubricants, drugs and pharmaceuticals (Moffat, 1995). Within the next decade, phenotypic modification of plants using biotechnology will undoubtedly lead to transgenic crops with enhanced nutritional quality as a result of altered protein and lipid compositions (Flavell, 1995). Transgenic plants with altered agronomic traits will also be developed. To improve crop value, the heterologous protein may serve to modify metabolic pathways or could be used to supply the plant with novel pest, herbicide or viral resistance. Of the many heterologous proteins that can be produced in plants, monoclonal antibodies (tnAbs) may be the most intriguing. There are many potential uses for these socalled plantibodies. They may be used in basic plant research, or be produced on a large scale for therapeutic or diagnostic use (Whitelam et al., 1993). They also provide the opportunity to manipulate agronomic crop plant traits or reduce pathogenic infections through antibodymediated modifications of antigen activity in planta (Hiatt, 1990). The binding and retention capacity of antibodies could also be used for the isolation and processing of environmental contaminants, a process known as biofiltration (Hiatt, 1990). A plant-based strategy for antibody production has an advantage over a microbial strategy, as the largest antibody fragment that has been reliably produced in Escherichia coli is a monovalent Fab fragment (Ma and Hein, 1995a). Several groups have successfully produced full length antibodies in higher plants (Hiatt et al., 1989; D0ring et al., 1990; Ma et al., 1994; van Engelen et al., 1994; Ma et al., 1995; De Wilde et al., 1996), while a number of groups
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have had success producing smaller antibody fragments such as Fab and single-chain Fv molecules (Benvenuto et ai., 1991; Owen et al., 1992; Tavladoraki et al., 1993; Fiedler and Conrad, 1995; Schouten et al., 1996; De Wilde et al., 1996). The aim of this review is to provide an overview of developments made toward the production of full length antibodies and antibody fragments in transgenic plants. The problems and potential uses of these plantproduced antibodies will be identified and discussed.
FULL LENGTH ANTIBODIES
Assembly and Targeting Full length antibodies were first expressed in plants by Andrew Hiatt and co-workers in 1989. The monoclonal antibody 6D4 from a mouse hybridoma cell line was expressed in tobacco. Separate transgenic lines were produced which expressed either the light (t:, kappa:) or heavy (y, gamma) chains of the immunoglobulin G (IgG) molecule, both with and without their natural, mammalian leader sequence. These transgenic lines were then sexually crossed, to yield plants that expressed both K- and y-chains simultaneously. An ELISA assay, using the 6D4 antigen (P3-bovine serum albumin) was then used to assay the binding specificity of the plant-produced antibody, and hence confirm that the immunoglobulin chains were assembled into functional complexes.
In fact, assembly of immunoglobulin chains was found to be
extremely efficient (Hiatt et al., 1989). In addition, the plant-derived antibody bound antigen equally as well as the 6D4 hybridoma mAb. However, none of the plants transformed with the leaderless immunoglobulin chain genes contained any assembled gamma-kappa complexes. This indicated that leader sequences were necessary for the accumulation of functional antibodies in tobacco. The authors presumed that assembly took place in a similar manner as in B lymphocytes. While the mechanism of antibody assembly in these mammalian cells is only partially understood, it is known that both light and heavy chains are synthesized as precursor proteins. These precursors are targeted to the lumen of the endoplasmic reticulum (ER) where the signal sequences are cleaved and chaperonin molecules direct the assembly of functional immunoglobulins (Ma and Hein, 1995a).
Hiatt et al. (1989) suggested that the
association of immunoglobulin with the endoplasmic reticulum indicated the antibody was to be secreted from the plant cell. However, they did not discount the possibility that assembly
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could also take place spontaneously in other cellular compartments, given sufficient levels of each chain. To optimize secretion of antibodies from plant cells, a slightly different expression strategy was developed which utilized what is known about secretion pathways of other organisms (Hein et al., 1991). Secretion was desired to obtain higher levels of accumulation of the antibody in the extracellular environment of the plant, which may allow for gentler extraction and perhaps easier purification of the antibodies.
In order to evaluate the
dependence of assembly on the N-terminal leader sequence, expression of mAb in plants transformed with y- and ~:-chain genes, each containing their native mouse signal sequence, was compared to mAb expression in plants that were transformed with genes containing a yeast pre-pro sequence in place of the native signal (Hein et al., 1991). The aim of this study was to maximize antibody accumulation in plants.
Yet a potential advantage of using plants as
bioreactors is that a great many plants can be grown at one time, reducing the need for a single plant to produce a large amount of the heterologous protein. Therefore, while increasing perplant yields is desirable, more work on optimizing purification procedures for plant-produced antibodies is also needed.
A promising purification scheme uses the binding affinity of
staphylococcal protein A with the Fc region of IgG antibodies (Hein et al., 1991).
If this
powerful approach can be modified for efficient large-scale purification, it might reduce the need to increase antibody accumulation in individual plants. A slightly different strategy was later applied to obtain coordinate expression of light and heavy chain genes resulting in the accumulation of functional, full length immunoglobulin M (IgM) in tobacco. An expression cassette consisting of two chimeric genes encoding the light (~,) and heavy (It) chains of the B 1-8 antibody was used (DUring et al., 1990). This approach eliminated the need to perform sexual crosses of transgenic plants. The chimeric genes were constructed using the mature light and heavy chain genes of the antibody, each linked to the coding sequence for the barley tx-amylase signal peptide. The chimeric genes were each placed under the control of an active plant promoter, linked on a plasmid, and integrated into the genome of tobacco using Agrobacterium-mediated gene transfer (D~iring et al., 1990). This system differed from the strategy of Hiatt et al. (1989), which involved sexual crosses to obtain simultaneous expression and assembly of heavy and light chains in tobacco, and used a mammalian, rather than plant ER targeting sequence. Dtiring et al. (1990) used immunogold labeling to determine the cellular location of the IgM molecules. A few gold
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particles were detected in the cytoplasm, while the majority of the signal accumulated in the ER.
Surprisingly, considerable signal was also localized in the thylakoid membranes of
chloroplasts. The use of a plant-derived ER targeting sequence by DUring et al. (1990), and the fact that that they saw accumulation of antibody in the endoplasmic reticulum of transgenic tobacco, supports the contention that transport to the ER lumen is necessary for proper assembly of full length antibodies, as in mammalian systems (Hiatt et al., 1989).
ER
localization also supports the finding that antibodies are involved in the secretory pathway of plants (Hiatt et al., 1989; Dtiring et al., 1990). However, the cytoplasmic and chloroplastic antibody would not be part of the secretion pathway, and certainly this distribution pattern was not expected. Recently however, it was demonstrated that the light-harvesting chlorophyll a/bbinding protein and the small subunit of Rubisco (both synthesized in the cytoplasm) are targeted to the chloroplasts of Euglena gracilis, after first passing through the ER and Golgi apparatus (Sulli and Schwartzbach, 1995; Sulli and Schwartzbach, 1996). The precursors of these proteins are targeted to the ER, and are transported from the ER to the Golgi apparatus and from the Golgi apparatus to the chloroplast, where they are imported as membrane-bound precursors (Sulli and Schwartzbach, 1996). While this system may not directly parallel the targeting of antibody to chloroplasts of plant cells, it may provide an explanation for the cellular distribution of antibody seen by D0ring et al. (1990). Nevertheless, the most important factor in the accumulation of functional, full length antibody in transgenic tobacco appears to be the presence of an ER signal peptide, regardless of its origin (Dtiring et al., 1990; Ma and Hein, 1995b). Recently, De Wilde et ai. (1996) established that both full length IgG molecules and Fab fragments accumulate in the intercellular spaces of leaf mesophyll cells of transgenic
Arabidopsis thaliana. This immunolocalization study confirmed the assumption that full length antibody is secreted from plant cells (Hiatt et al., 1989; DUring et al., 1990; Ma and Hein, 1995b), and for the first time conclusively demonstrated that plant-produced antibodies accumulate in the intercellular spaces. As well, it was surprising that the antibodies were not retained by the cell walls which are believed to have exclusion limits of between 17 and 60 kDa (Carpita et al., 1979; Tepfer and Taylor, 1981; De Wilde et al., 1996). Yet the results of De Wilde et al. (1996) suggest that the mesophyll cell walls of Arabidopsis were permeable to
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intact IgG molecules, which have a molecular weight of 146 kDa. These findings are of particular interest to any application where extracellular antigen binding is desired. The finding that some plant-produced mAb accumulated in the cytoplasm of transgenic tobacco cells (DUring et al., 1990) led Stieger et al. (1991) to speculate that antibodies have the ability to self-assemble in the cytoplasm. Accumulation of antibody in the cytoplasm would certainly provide many interesting possibilities for use in planta. The immunoglobulin could be used to inhibit the activity of an endogenous enzyme, thus providing an alternative to antisense technology (Stieger et al., 1991). Intracellular accumulation would be desired for this type of immunomodulation approach to altering plant metabolism, since extracellular antibody would not be available to bind endogenous antigen. To obtain cytosolic expression, leaderless chimeric genes were injected into the nuclei of Acetabularia mediterranea (Stieger et al., 1991).
The heavy and light chain genes from the antibody B
1-8 were used.
Immunofluorescence confirmed that the antibodies formed correctly folded antigenic binding sites in the cytoplasm. However, the relatively high reducing conditions of the cytoplasm will not allow formation of disulfide bonds, which are a characteristic of all antibodies (Stieger et al., 1991). The authors speculated that the antibodies folded correctly in the cytoplasm without forming disulfide bonds, and relied simply on hydrogen bonds, van der Waals forces, ionic and hydrophobic interactions for proper folding. If indeed these antibodies are fully functional in the cytoplasm, this technology could be very important for plant molecular biologists and plant breeders. However, such results have not yet been reported in higher plants. It is possible that the cytoplasm of algae is less reducing and contains the chaperonins necessary for folding of full-size immunoglobulins.
Nevertheless, it appears that cytoplasmic expression and
accumulation of full length, functional antibodies is specific to algae Conrad and Fiedler, 1994).
Pathogen Protection The prospect of using whole antibodies to obtain disease resistance against plant pathogens has been examined by van Engelen et al. (1994). Heavy and light chain genes were placed under the control of promoters that provide expression in roots, and were introduced into tobacco on a single T-DNA using Agrobacterium-mediated transformation.
High
expression levels (1.1% of total soluble root protein) were achieved in the apoplast of roots
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(van Engelen et al., 1994). This system gives high expression levels of functional, full length antibodies in roots, and using the proper antibody could be used to provide pathogen resistance to plant roots by interfering with extracellular pathogenic antigens (van Engelen et al., 1994).
Human Immunotherapy
The immunotherapeutic potential of full length plant-produced antibodies has received much attention for a number of years (Ma and Hein, 1995a; Ma and Hein, 1995b). Plantibodies are more desirable for human use than microbially produced proteins because they undergo eukaryotic rather than prokaryotic post-translational processing.
For example,
glycosylation is a type of protein modification more closely matched by plants than by bacteria (Ma and Hein, 1995b) and is necessary for secretion of antibodies to the apoplastic space in transgenic plants (Stieger et al., 1991). Many of the N-linked glycans involved in glycosylation are the same with respect to both size and extent of branching in both plants and animals (Hein et al., 1991; Ma and Hein, 1995b).
As well, Hein et al. (1991) report that transgenic
plantibodies are processed in a similar manner as mammalian glycoproteins. However, there are some differences in the carbohydrates involved and the pattern of glycosylation in plants (Ma and Hein, 1995b). There is some concern that the presence of plant-specific glycans may increase the immunogenicity of plantibodies used for human immunotherapy, even though this seems unlikely, given humans' daily exposure to plant glycoproteins in food, pollen and personal care products (Ma and Hein, 1995a). Nevertheless, if these plant-produced antibodies are to be used for human immunotherapy, plant-specific glycosylation is a concern that needs to be addressed. If plant glycosylation does present a problem, it could be circumvented by removing the glycosylation site(s) from the antibody molecule, removing the glycan itself from the plant or perhaps by generating a mutant lacking the glycosylating enzyme (Ma and Hein, 1995b). The prospect of using plant-produced antibodies in passive immunization against a cellsurface antigen (SA ~
of Streptococcus mutans, the most common cause of tooth decay, has
been closely studied in recent years (Ma et al., 1994). Large doses of antibody are required in multiple applications for topical passive immunotherapy and transgenic plants have been explored as the production system that could provide the quantities of antibody needed. The predominant antibody in human mucosal secretions, such as saliva, is the secretory form of
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immunoglobulin A (SIgA) (Sigal and Ron, 1994). Secretory IgA is a good candidate for use in passive topical immunization against oral bacteria such as S. mutans, or other pathogens of the digestive tract, due to its resistance to proteolytic digestion (Ma et al., 1994; Stavnezer, 1996). However, a hybridoma-derived IgG antibody (Guy's 13) against the cell-surface antigen (SA I/II) of S. m u t a n s was first used successfully in a local passive immunization study on human subjects (Ma et al., 1987). This study suggested that passive immunization using mAb might be a good alternative approach for the prevention of colonization of teeth by S. mutans, and the subsequent development of tooth decay. Ma et al. (1994) later produced a hybrid IgA-G molecule in plants, and showed that it caused aggregation of S. m u t a n s in culture, which appears to be how the antibody prevents colonization of the bacteria in vivo.
Some non-
essential constant regions of the Guy's 13 heavy chain were replaced with IgA constant regions, making the hybrid less prone to proteolytic digestion in human secretions. Incorporation of IgA constant regions did not alter the antigen recognition capacity of the recombinant antibody (Ma et al., 1994). In an extension of the secretory antibody work, Ma et al. (1995) have achieved simultaneous expression and assembly of four separate protein chains to form functional, dimeric secretory antibody in transgenic tobacco. The resulting secretory antibody (SIgA-G) which binds the same cell-surface antigen of S. m u t a n s as IgA-G (Ma et al., 1994), consists of a monoclonal kappa chain, a hybrid IgG/IgA heavy chain, a joining (J) chain from mouse and a secretory component (SC) from rabbit (Ma et al., 1995). Successive sexual crosses between transgenic plants, each expressing one of the protein chains, resulted in the simultaneous expression and assembly of all four peptides. The resulting SIgA-G molecule matches the most common form of IgA found in mammals, which is a dimer associated with a small polypeptide J chain and a secretory component (Ma et al., 1995). In mammalian systems production of monoclonal SIgA requires two different cell types, while functional expression of this secretory molecule was achieved in single tobacco leaf cells. Moreover, the nature of the association of the J chain and SC with IgA-G (to form SIgA-G) was confirmed to be similar to their association with IgA in mammals. Binding specificity of the plant-produced SIgA-G was also similar to the native SIgA antibody (Ma et al., 1995). The yield of SIgA-G in tobacco was much higher than achieved for IgA-G in the previous study by Ma et al. (1994), which indicated SIgA-G was more resistant to proteolysis and suggested its production could be scaled up to agricultural proportions (Ma et al., 1995). Preliminary results also suggest that the
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development of plants capable of producing native SIgA may be possible, which could have significant impact on passive immunotherapeutic applications (Ma et al., 1995). Although this type of approach seems to have great potential for success, few antibodies have been approved for clinical use (Ma and Hein, 1995a). A major hurdle has been the cost of antibody production in plants. However, Hiatt (1990) estimated that antibodies expressed in soybean at a level of 1% of total protein, could be produced for $100 per kg of antibody. Surely, this cost can only go down as purification procedures are improved. Large quantities of antibody are required to overcome the rapid rate of clearance when used for passive topical immunotherapy and plant agriculture could provide the inexpensive, large-scale production system needed for such a treatment (Ma and Hein, 1995a).
ANTIBODY FRAGMENTS
Modification of Plant Metabolism
Small size and undemanding folding requirements make single-chain Fv molecules (scFvs) very attractive antibody derivatives for expression in plants. Single-chain Fv fragments consist of the variable regions of the light and heavy chains of an immunoglobulin linked together by a flexible peptide linker (Conrad and Fiedler, 1994; Ma and Hein, 1995a). The binding capacity of scFv molecules is similar to their parental antibodies, but their small size gives them an advantage over whole antibodies where the complement-binding (Fc) portion of the molecule is not needed (Owen et al., 1992). Owen et al. (1992) were able to successfully express an scFv in tobacco in an effort to modify plant metabolism. A synthetic gene was used to produce an scFv that binds to the plant regulatory molecule phytochrome. Seeds derived from plants producing the anti-phytochrome scFv had altered light-controlled germination. Specifically, germination was reduced in anti-phytochrome producing plants by approximately 40% (Owen et al., 1992). Presumably, the reduction in germination was due to binding of the scFv to phytochrome, which affected its ability to function properly. This was the first report of successfully altering plant metabolism using a plant-produced mAb against an endogenous antigen. This approach could also be applied to other metabolic pathways and could be used to study plant metabolism, or perhaps enhance crop value by blocking undesired pathways. Single-chain F~ fragments are more attractive than full length mAbs for the immunomodulation
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of plant cell components due to their less demanding assembly requirements in the reducing environment of the cytoplasm. In addition, full length antibodies must be secreted to achieve functional accumulation, which would make them unavailable for this type of intracellular immunomodulation. The production of full length antibodies in the ER of plant cells for secretion to the extracellular space is desirable for large-scale production because these molecules will undergo minimal hydrolytic processing in the extracellular environment and may be more easily extracted (Ma et al., 1995). However, if the antibody is to be used for altering or immunizing the plants themselves, targeting to the cytoplasm or other cellular compartment may be more desirable (Ma et al., 1995). Therefore, scFvs localized in the cytoplasm may be more valuable for use in planta, while secreted antibodies are best suited for large-scale production.
Pathogen Protection In an effort to confer viral resistance to plants, Tavladoraki et al. (1993) expressed an scFv antibody directed against the artichoke mottled crinkle virus in the cytoplasm of tobacco. These transgenic plants were specifically protected from virus attack, in that they had a reduced incidence of infection and showed delayed symptom development (Tavladoraki et al., 1993). The success of this approach to attenuate viral infection was dependent on stable antibody expression in the cytoplasm. This was achieved using scFvs rather than full length antibodies, which must be targeted to the ER for secretion in order to get stable accumulation in higher plants (Tavladoraki et al., 1993, Ma and Hein, 1995a). This "engineered intracellular immunization" approach seems to have great potential for supplying plants with viral and perhaps pest resistance (Tavladoraki et al., 1993). However, before it becomes a commercially useable or valuable approach, the mechanism(s) of action must be fully elucidated.
Targeting Schouten et al. (1996) believe cytosolic expression of functional scFv molecules is unlikely and suggest that such intracellular expression is not so straightforward. The objective of their study was to examine functional expression of antibodies targeted to different subcellular compartments. No transgenic plants containing scFv genes targeted for expression in
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the cytoplasm were found to accumulate any detectable scFv (Schouten et al., 1996). Whereas plants expressing scFv targeted to the ER accumulated significant amounts of the protein. Interestingly, when an ER retention signal (amino acids KDEL) was included in the antibody gene construct, accumulation of scFv was increased significantly (Schouten et al., 1996). This was true for both the secreted (ER targeted) and cytosolic forms of the scFv. The KDEL sequence may somehow protect the cytosolic scFv from proteolytic degradation, or may confer protection to the protein through an interaction with the cytosolic side of the ER (Schouten et al., 1996). However, Schouten et al. (1996) expressed a different scFv molecule than has been used in previous reports (Owen et al., 1992; Tavladoraki et al., 1993) and it may be possible that some antibody fragments are more prone to proteolytic degradation than others. Nevertheless, the work of Schouten et al. (1996) demonstrated that the exact requirements for antibody expression and accumulation in plants remain elusive. In a recent report, Fiedler and Conrad (1995) demonstrated that active scFv molecules can be targeted to an organ other than leaves. Specifically, scFv was shown to accumulate in developing and ripe tobacco seeds. The antibody accumulated to 0.67% of total soluble protein in the seeds, and was stably stored for one year at room temperature (Fiedler and Conrad, 1995). This system therefore offers high expression levels along with long-term storage of the protein and does not appear to influence plant growth rate or seed development. Exact cellular location was not determined for the antibody, although the authors felt it may have accumulated in protein bodies of the seeds (Fiedler and Conrad, 1995). Determining the exact cellular location of the stored antibody and transferring the system to another crop, such as corn, would make this strategy valuable for the commercial production of scFvs (Fiedler and Conrad, 1995). In fact, this system would be even more valuable if long-term stable expression could also be achieved for full length antibodies.
Specifically, such a system would be
valuable for delivering large quantities of full length antibodies for passive immunization. For instance, it has been shown that full length antibodies can be assembled and accumulated in the roots of transgenic tobacco (van Engelen et al., 1994). If this technology could be utilized to obtain stable accumulation of these antibodies in edible tissues such as potato tubers, it might allow for long-term storage and easy delivery of antibodies for immunotherapeutic applications. As well, these transgenic crops could be grown using existing knowledge and equipment, and be used to immunize large populations inexpensively without the need for specialized long-term storage facilities or purification procedures.
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SUMMARY
In recent years, plant biotechnology has made agriculture a more attractive alternative for the large-scale production of biomolecules such as proteins.
The ability to produce
antibodies and antibody fragments in plants presents some very interesting possibilities. Since the monoclonal antibody 6D4 was first expressed in tobacco by Hiatt et al. (1989), antibodies have been produced in plants for a variety of reasons.
Full length antibodies have been
expressed in plants with the aim of producing large quantities for use in human immunotherapy. Indeed, the production of multimeric secretory antibodies (Ma et al., 1995) and the ability to store antibodies long-term in seeds or other organs (Fiedler and Conrad, 1995) indicates this goal may soon be reached.
If the potential problem of plant-specific
glycosylation can be solved, passive immunotherapy using plant-produced antibodies against human mucosal pathogens may soon be possible. The use of antibodies in planta to alter plant phenotype is another exciting area of research. Plant-produced antibodies could be used to change the appearance of the plant, alter plant metabolism or confer pathogen resistance. For these purposes antigen binding in vivo is required, so cellular location of the plant-produced antibody is critical. Since the Fc portion of the antibody is not necessarily required, easily assembled scFv molecules have been used to achieve phenotypic modifications requiring intracellular accumulation. Phytochrome-mediated germination has been altered (Owen et al., 1992) and tobacco has been effectively immunized against virus attack (Tavladoraki et al., 1993) using intracellular scFvs. The possibility of using full length antibodies to interfere with pathogenesis in the apoplast has also been examined (van Engelen et al., 1994). Clearly though, the effects of cellular targeting on expression levels of antibodies in plants are far from resolved. Although some groups have reported successful expression of scFvs in the cytoplasm (Owen et al., 1992; Tavladoraki et al., 1993), others failed to get any antibody accumulation unless an ER retention signal was included (Schouten et al., 1996), and only recently has the exact extracellular location of secreted antibodies been determined (De Wilde et al., 1996). Surprisingly, it was also revealed that whole antibodies secreted from the ER are not retained by plant cell walls (De Wilde et al., 1996). As well, Dtiring et al. (1990) reported unexpected accumulation of full length antibodies in chloroplasts. Therefore, while the production of antibodies in plants offers many exciting possible
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applications, such as in passive immunization of large human populations and altering plant metabolism, there are still questions about the technology that need to be addressed.
REFERENCES
1.
Benvenuto, E., Ordhs, R. J., Tavazza, R., Ancora, G., Biocca, S., Cattaneo, A., and Galeffi, P. (1991), Plant Mol. Biol., 17, 865-874. "Phytoantibodies": a general vector for the expression of immunoglobulin domains in transgenic plants.
2.
Carpita, N., Sabularse, D., Montezinos, D., and Delmer, D. P. (1979), Science, 205, 1144-1147. Determination of the pore size of cell walls of living plant cells.
3.
Conrad, U., and Fiedler, U. (1994), Plant Mol. Biol., 26, 1023-1030. Expression of engineered antibodies in plant cells.
4.
De Wilde, C., De Neve, M., De Rycke, R., Bruyns, A.-M., De Jaeger, G., Van Montagu, M., Depicker, A., and Engler, G. (1996), Plant Sci., 114, 133-241. Intact antigen-binding MAK33 antibody and Fab fragment accumulate in intercellular spaces of Arabidopsis
thaliana. 5.
D~iring, K., Hippe, S., Kreuzaler, F., and Schell, J. (1990), Plant Mol. Biol., 15, 281-293. Synthesis and self-assembly of a functional monoclonal antibody in transgenic
Nicotiniana tabacum. 6.
Fiedler, U., and Conrad, U. (1995), Bio/Technol., 13, 1090-1093. High-level production and long-term storage of engineered antibodies in transgenic tobacco seeds.
7.
Flavell, R. B. (1995), Trends Biotech., 13, 313-319. Plant biotechnology R & D - the next ten years.
8.
Glick, B. R. and Pasternak, J. J. (1994), Molecular Biotechnology: Principles and
Applications of Recombinant DNA, ASM Press (Washington, D.C.), pp. 113, 155, 9.
Goddijn, O. J. M., and Pen, J. (1995), Trends Biotech., 13, 379-387. Plants as bioreactors.
10.
Hein, M. B., Tang, Y., McLeod, D. A., Janda, K. D., and Hiatt, A. (1991), Biotechnol.
Prog., 7, 455-461. Evaluation of immunoglobulins from plant cells. 11.
Hiatt, A. (1990), Nature, 344, 469-470. Antibodies produced in plants.
12.
Hiatt, A., Cafferkey, R., and Bowdish, K. (1989), Nature, 342, 76-78. Production of antibodies in transgenic plants.
280
13.
MATTHEWD. SMITH
Ma, J. K.-C., and Hein, M. B. (1995a), Plant Physiol., 109, 341-346. Plant antibodies for immunotherapy.
14.
Ma, J. K.-C., and Hein, M. B. (1995b), Trends Biotech., 13, 522-527. Immunotherapeutic potential of antibodies produced in plants.
15.
Ma, J. K.-C., Hiatt, A., Hein, M., Vine, N. D., Wang, F., Stabila, P., van Dolleweerd, C., Mostov, K., and Lehner, T. (1995), Science, 268, 716-719. Generation and assembly of secretory antibodies in plants.
16.
Ma, J. K.-C., Lehner, T., Stabila, P., Fux, C. I., and Hiatt, A. (1994), Eur. J. Immunol., 24, 131-138. Assembly of monoclonal antibodies with IgG1 and IgA heavy chain domains in transgenic tobacco plants.
17.
Ma, J. K.-C., Smith, R., and Lehner, T. (1987), Infect. Immun., 55, 1274-1278. Use of monoclonai antibodies in local passive immunization to prevent colonization of human teeth by Streptococcus mutans.
18.
Moffat, A. S. (1995), Science, 268, 658-660. Exploring transgenic plants as a new vaccine source.
19.
Owen, M., Gandecha, A., Cockburn, B., and Whitelam, G. (1992), Bio/Technol., 10, 790794. Synthesis of a functional anti-phytochrome single-chain Fv protein in transgenic tobacco.
20.
Schouten, A., Roosien, J., van Engelen, F. A., de Jong, G. A. M., Borst-Vrenssen, A. W. M., Zilverentant, J. F., Bosch, D., Steikema, W. J., Gommers, F. J., Schots A., and Bakker, J. (1996), Plant Mol. Biol., 30, 781-793. The C-terminal KDEL sequence increases the expression level of a single-chain antibody designed to be targeted to both the cytosol and the secretory pathway in transgenic tobacco.
21.
Sigal, L. H., and Ron, Y. (1994), Immunology and Inflammation,
McGraw-Hill
22.
Stavnezer, J. (1996), In Advances in Immunology, (Dixon, F. J., editor), Acedemic Press
(Toronto), pp. 48-51.
(Toronto), pp. 79-146. Antibody class Switching. 23.
Stieger, M., Neuhaus, G., Momma, T., Schell, J., and Kreuzaler, F. (1991), Plant Sci., 73, 181-190. Self assembly of immunoglobulins in the cytoplasm of the alga Acetabularia mediterranea.
ANTIBODYPRODUCTIONIN PLANTS
24.
281
Sulli, C., and Schwartzbach, S. D. (1995), J. Biol. Chem., 270, 13084-13090. The polyprotein precursor to the Euglena light harvesting chlorophyll a/b-binding protein is transported to the Golgi apparatus prior to chloroplast import and polyprotein processing.
25.
Sulli, C., and Schwartzbach, S. D. (1996), Plant Cell 8, 43-53. A soluble protein is imported into Euglena chloroplasts as a membrane-bound precursor.
26.
Tavladoraki, P., Benvenuto, E., Trinca, S., De Martinis, D., Cattaneo, A., and Galeffi, P. (1993), Nature, 366, 469-472. Transgenic plants expressing a functional single-chain Fv antibody are specifically protected from virus attack.
27.
Tepfer, M., and Taylor, I. E. P. (1981), Science, 213, 761-763. The permeability of plant cell wall as measured by gel filtration chromatography.
28.
van Engelen, F. A., Schouten, A., Molthoff, J. W., Roosien, J., Salinas, J., Dirkse, W. G., Schots, A., Bakker, J., Gommers, F. J., Jongsma, M. A., Bosch, D., and Stiekema, W. J. (1994), Plant Mol. Biol., 26, 1701-1710. Coordinate expression of antibody subunit genes yields high levels of functional antibodies in roots of transgenic tobacco.
29.
Whitelam, G. C., Cockburn, B., Gandecha, A. R., and Owen, M. R. L. (1993), In Biotechnology and Genetic Engineering Reviews, vol. 11, (Tombs, M. P., editor) Intercept Ltd. (Andover, UK), pp. 1-29. Heterologous protein production in transgenic plants.