Molecular Farming of Antibodies

The applications for recombinant antibodies in healthcare are increasing. Being able to produce them on a large scale in plants will make them more affordable, which, in turn, will increase their availability to treat a greater number of diseases than is possible at present.

The contribution of Schillberg and Twyman focuses on the critical molecular factors for antibody expression in plant cells. Expression levels depend on both the structure of the chosen recombinant antibody and where it will be expressed within the organelles of eukaryotic cells. For example, most antibodies are poorly expressed in the cytoplasm of plant cells, but targeting them to the secretory pathway, and especially retaining them in the endoplasmic reticulum, results in higher production levels. The authors review both transgenic plants and cultured suspension cells as production systems for antibodies. The most interesting aspect of plant suspension cells is that they are a biologically contained system, which has advantages for the production of recombinant proteins under controlled conditions.

Antibodies are a diverse family of proteins and it is clear that some forms will have advantages over others in the context of plant-based expression. In addition to their role as pharmaceuticals, one attraction of antibodies is that they can be used to create disease-resistant plant lines. Schillberg and Twyman have pioneered the use of membrane-anchored antibodies to generate plant lines resistant to viral infection and—while this is not molecular farming per se—it demonstrates that antibody expression is, in itself, a useful tool for the improvement of plant characteristics.

Perspectives for Molecular Farming

In bringing these authors together, we have provided a snapshot of molecular farming. It is clear that there is still no consensus on the optimal production system for recombinant proteins in plants. This reflects, in part, the practicalities of the intellectual property situation in molecular farming. However, it is our opinion that consensus will eventually be determined by industry. We believe this to be the case because it will be industry that will commercialise molecular farming, not academic research laboratories. Industry will determine the commercial system that is the most appropriate and financially viable, and this decision will drive the progress of molecular farming.

Public acceptance of molecular farming and plant biotechnology is an issue that we have not discussed here, as our goal is to present an account of the state of the art in this field. We feel that success is the most powerful argument that can be used in favour of the technology. The contributions show that we have made significant progress towards that end. When the first protein from molecular farming is released into the market-place, and patients’ lives are seen to improve as a result, the public will then judge the technology on the basis of its benefits. We estimate that we are 2 to 4 years from that moment.

As with all new technologies, practical problems need to be overcome. Many of these, such as the difference between protein glycosylation patterns in plants and animals, have been discussed in detail in these contributions. We believe that, once defined, these challenges can all be solved. Advances in fundamental research, such as controlling gene silencing or chloroplast-targeted protein expression, will provide benefits for molecular farming. Although fundamental research remains the key tool available for improving the technology, molecular farming is already well advanced and close to product commercialisation.

The last decade has seen dramatic progress in plant biotechnology and this has led to the development of molecular farming. The next decade will see products approved as pharmaceuticals and once this happens molecular farming will finally have come of age.

Green Vaccines

Vaccination is one of the most successful developments in preventive human healthcare. In their contribution, Mor and Mason describe how edible vaccines that can be delivered in food plants may make that protection more widely accessible.

Despite their efficacy, most modern vaccines, which are inactivated or attenuated strains of the pathogen delivered by injection, have limitations. This creates constraints on the use of vaccines because many pathogens are difficult, and prohibitively expensive, to culture. Second, the majority of vaccines are delivered to patients by injection. This requires skilled staff and a sophisticated medical infrastructure, which limits their use in the developing world. Therefore, alternative routes for vaccination as well as alternate sources of vaccination antigens have been explored. Orally delivered vaccines that contain a subunit of the pathogen and can elicit a protective immune response are one such alternative. Using transgenic plants for both production and delivery is the major focus of this chapter.

The authors provide a historical overview of the development of strategies for production of recombinant antigens in plants, most of which have been achieved in the laboratories of Arntzen and Mason. They describe their success in producing immune responses in humans with plant-produced hepatitis B surface antigen, the labile toxin B subunit of enterotoxigenic Escherichia coli and the capsid protein of Norwalk virus. Success with these proteins in Phase I/II trials has now prompted larger scale clinical trials. What is compelling is that their approach is successful for proteins from widely different pathogens and indicates that orally delivered vaccines may be successful against a wide range of pathogens. Mor and Mason then discuss the rapid increase of research in the edible-vaccine field and point out that plants can be used to create multicomponent vaccines that can protect against several pathogens at once. This is an aspect of the edible-vaccine approach that further strengthens its impact.

After discussing strategies for vaccine expression, the authors turn their attention to the use of orally delivered antigens both as immunocontraceptive vaccines and in the treatment of autoimmune diseases. They end their chapter with a careful discussion of where the technical challenges in edible-vaccine technology lie, and how they may be solved.

We share their view that orally delivered vaccines are a proven technology, which holds great promise for development within mainstream pharmaceuticals. By their use, entire populations in the developing world will be able to share the same protection from disease that we take for granted in industrialised countries.

Harvesting Recombinant Proteins from Food Crops

Many recombinant proteins have been produced in those plants that are ideally suited for laboratory experiments, such as tobacco or Arabidopsis thaliana, but these are not suited to long-term or large-scale production. The global agricultural industry has focused on high-yield crops that have been optimised over generations to be cost-effective for the large-scale production of food, animal fodder and other products, such as fibres for clothing and pulp for paper-making. Therefore, research on molecular farming has moved towards evaluating these commercial species for the production of pharmaceuticals and other recombinant proteins.

In their contribution, Stoger et al. first discuss the nature of the recombinant proteins expressed in plants before progressing to a detailed discussion of the issues that are related to choosing the most suitable species for protein expression. These include a meticulous review of the decisions that need to be taken during protein-expression projects and an indication of where the potential advantages lie with each crop species. Their contribution provides a solid framework and entry point for researchers new to molecular farming.

Industrial ‘Plants’ of the Future

In her chapter, Hood describes the exploitation of transgenic plants as a production system for proteins that are of industrial importance. These include recombinant enzymes, the use of which would benefit industrial production processes that are currently based on synthetic chemistry. The high specificity and activity of enzymes is what makes them attractive to industry, and the argument presented in the contribution is that plants, with their high biomass and large-scale production potential, are ideal for mass production of these proteins. The central thesis is that using plants will reduce the cost of these proteins and make them available for a wide number of applications in industries where they were previously unaffordable.

The use of plants to produce industrially relevant enzymes has been shown to be practical in a number of species. For example, phytase and αα-amylase have been produced in tobacco. However, the author argues that the expression of proteins in leaf tissue is not ideally suited to molecular farming. This is primarily because of the expense and the difficulties involved in extracting the proteins from leaves. She discusses, therefore, using other plant species, such as alfalfa or oilseed crops, to produce industrially important proteins. The author makes the case for maize (Zea) as a production system, her well-reasoned argument being based on a comparison of the production costs of various crops. Although alfalfa has the greatest potential for the production of recombinant protein per hectare, this is offset by the need to extract the protein from leaf material. Of the crops surveyed, soybean has the lowest cost for protein production, but the methods required for transformation are labour intensive and expensive. The cereal crops, such as rice and wheat, are shown to have positive advantages for expression but, for pragmatic reasons, maize was their selected production platform. First, maize is the largest crop produced in North America and its two major advantages are the low cost and ease of large-scale production. The rationale for the use of maize rests on the sophisticated existing production and harvesting infrastructure and on the advantages of using a seed-based production system.

One contentious and difficult issue covered by Hood is that of containment, which is of serious concern given the large amounts of maize produced within the United States for both human and animal consumption. The author presents a series of measures that can be used to control inadvertent mixing of corn destined for food or fodder with the transgenic variety. It can be argued that this is where industry has to be at its most vigilant, because the consequences of transgenic crops entering the food chain are potentially very large. Overall, however, with close control over its use, maize could become an acceptable and profitable species for use as an ‘industrial plant’ of the future.

An Introduction to Industrial and Pharmaceutical Protein Production in Plants

Advances in plant molecular biotechnology do not merely mean that farmers and research scientists alike can now contemplate a more than adequate harvest from newly sown crops. During the last decade, transgenic plants have been shown to provide a practical and feasible method of producing recombinant proteins. This technology has now progressed to the point where plants can be used as a platform for the expression of proteins intended for use in the treatment, or diagnosis, of a number of diseases. Such proteins include recombinant antibodies, cytokines and blood substitutes.

This research area—the combination of molecular biotechnology and agriculture, which is referred to as ‘molecular farming’—focuses on producing valuable proteins in plants, and forms the subject of the following contributions to this handbook. Drawing on expertise from both industry and academia, we present reviews of both the plant species and strategies that are being used to transfer molecular farming from the research laboratory to the field.

It is widely predicted that the world capacity for recombinant protein production will soon be exceeded by the demand and that this demand will continue to increase. Yet, the justification for the use of plants for recombinant protein production may not be immediately obvious. While transgenic cell lines, animals and microbes will continue to have significant roles to play as expression hosts for recombinant pharmaceuticals, the future role of plants should not be underestimated. One reason for this is that plants constitute a mass-production platform that can be used for the economical, large-scale production of proteins for industrial use in processes that were previously unaffordable. This makes them particularly relevant for the production of the recombinant proteins that will be required to treat the diseases we shall be challenged by in the 21st century.

Molecular farming is a fast-developing research area where fundamental research into protein expression and purification is coupled to the practicalities of plant growth and harvesting. This is reflected in the following contributions of how to express proteins, where to express them and how to choose the most appropriate host plant for protein expression. At present, there is no consensus on either the ideal expression method or choice of species, which has, therefore, to be determined empirically in individual cases. It should be emphasised that crops producing recombinant proteins have been in commercial production since 1997 (see Chapter 41) and crops producing recombinant therapeutics have already entered clinical trials (see Chapter 40). The development of edible vaccines using plants, as described by Mor and Mason (see Chapter 39), will have a great impact on world health and protection from disease. It is certain that the impact of molecular farming will increase as the technique develops both scientifically and commercially.

Below, we briefly introduce the contributions to this section and focus on what we regard as the most interesting issues covered in each review.

Technical Proteins from Plants

While most plant-derived recombinant proteins are currently intended for human pharmaceutical applications, plants have also been used to produce veterinary products and feed additives. One application is the use of feed plants to produce vaccines against animal diseases. For example, vaccine trials have demonstrated that pigs fed on transgenic corn expressing transmissible gastroenteritis virus glycoprotein S are protected from the disease (Streatfield et al., 2000).

Plants engineered to express catabolic enzymes can be used to increase the nutritional value of feed. For example, transgenic canola expressing Aspergillus phytase is available as the feed additive PhytaSeed. A comparison of pigs fed on a diet substituted with Natuphos (a commercial phytase additive) and those fed on a diet supplemented with PhytaSeed showed similar levels of phosphate utilisation (Zhang et al., 2000).

Several plant species have been manipulated to express amylases and cellulases to help break down starch and cellulose after harvesting, therefore, increasing their utilisable carbohydrate content (Biesgen et al., 2002; Fischer et al., in press). In one of the most impressive examples, Zeigler et al. (2000) demonstrated that the thermostable endo-1,4-ββ-d-glucanase from Acidothermus cellulolyticus could be expressed at levels up to 26% total soluble protein in Arabidopsis thaliana, the highest level of recombinant protein ever achieved in a nuclear transgenic plant.

The protein was secreted to the apoplast, but because of its high temperature optimum (>80°C) it did not cause any damage to the host plant, though it was in direct contact with the cell wall. It would be very useful if this level of expression could be repeated in commercial crop species.

Plant-Derived Feed Additives and Processing Enzymes

While most plant-derived recombinant proteins are currently intended for human pharmaceutical applications, plants have also been used to produce veterinary products and feed additives. One application is the use of feed plants to produce vaccines against animal diseases. For example, vaccine trials have demonstrated that pigs fed on transgenic corn expressing transmissible gastroenteritis virus glycoprotein S are protected from the disease (Streatfield et al., 2000).

Plants engineered to express catabolic enzymes can be used to increase the nutritional value of feed. For example, transgenic canola expressing Aspergillus phytase is available as the feed additive PhytaSeed. A comparison of pigs fed on a diet substituted with Natuphos (a commercial phytase additive) and those fed on a diet supplemented with PhytaSeed showed similar levels of phosphate utilisation (Zhang et al., 2000). Several plant species have been manipulated to express amylases and cellulases to help break down starch and cellulose after harvesting, therefore, increasing their utilisable carbohydrate content (Biesgen et al., 2002; Fischer et al., in press).

In one of the most impressive examples, Zeigler et al. (2000) demonstrated that the thermostable endo-1,4-ββ-d-glucanase from Acidothermus cellulolyticus could be expressed at levels up to 26% total soluble protein in Arabidopsis thaliana, the highest level of recombinant protein ever achieved in a nuclear transgenic plant. The protein was secreted to the apoplast, but because of its high temperature optimum (>80°C) it did not cause any damage to the host plant, though it was in direct contact with the cell wall. It would be very useful if this level of expression could be repeated in commercial crop species.

High-Value Pharmaceutical Proteins in Plants

Most of the recombinant proteins that have been produced in plants to date are high-value pharmaceuticals intended for human use. These can be placed into three broad categories: (1) human or animal proteins used predominantly as replacement therapies (e.g. blood products, hormones, growth factors, enzymes); (2) recombinant antibodies used to prevent, diagnose and treat disease and (3) recombinant vaccines.

For the reasons stated above, many proteins within the first category of products have been expressed in tobacco. There are, however, some notable exceptions. For example, rice has been used for the production of human αα-interferon (Zhu et al., 1994) and αα-1-antitrypsin (Terashima et al., 1999), maize for bovine aprotinin (Zhong et al., 1999) and canola for hirudin (Parmenter et al., 1995).

The second category of products, recombinant antibodies, is unique in that many different antibodies have been expressed in a variety of plant-based expression systems, yet the molecules are, in general terms, relatively homogeneous in nature. For the first time, this has allowed the direct comparison of different plant species and expression systems using equivalent products, providing an objective measurement of efficiency. Much of our knowledge about the relative advantages of different plant-based expression systems comes from the study of recombinant antibodies produced in plants (Stoger et al., 2002b).

Recombinant antibody molecules range in complexity from simple polypeptides (e.g. single chain Fv, scFv, fragments) to secretory immunoglobulins (sIg), which comprise 10 individual polypeptide chains (some bearing glycans) covalently assembled through disulphide bridges. These represent the most complex recombinant proteins ever to have been successfully expressed in plants. The most advanced product is CaroRX, a secretory antibody produced in tobacco that can be used to prevent dental caries. The antibody recognises Streptococcus mutans adhesin and prevents colonisation of the oral cavity by this pathogen. The product has already proved to be successful in phase II clinical trials, and is likely to be the first plant-derived recombinant protein approved for use in humans (Larrick et al., 2001; Ma et al., 1998). Another ‘plantibody’ likely to be approved in the near future is a humanised full-length immunoglobulin against herpes simplex virus glycoprotein B.

This is a serum antibody, which has four components (two identical light chains and two identical glycosylated heavy chains). Both the folding of individual chains and the assembly of the protein’s quaternary structure are dependent on disulphide bridges. This protein has been produced in transgenic soybean and rice (Zeitlin et al., 1998; Briggs et al., 2001). The most clinically advanced scFv is an anti-idiotype antibody that can be used to target malignant B-cells. This has been produced in virus-infected tobacco plants (McCormick et al., 1999). In transgenic tobacco, both a full-length immunoglobulin and a single-chain Fv fragment have been produced recognising human chorionic gonadotropin. These antibodies, currently at the pre-clinical stage, may be useful for pregnancy detection, contraception and tumour diagnosis (Kathuria et al., 2002).

Turning to the third category of plant-derived recombinant proteins, a large number of vaccines are currently under development, many expressed in tobacco or potato (reviewed by Daniell et al., 2001b). Potato has been chosen as an alternative model system to tobacco because it is edible, an important consideration for the oral administration of vaccines without extensive purification. Several of these vaccines are undergoing clinical trials, including a recombinant E. coli enterotoxin (LT-B) and Norwalk virus capsid protein, both delivered in raw transgenic potatoes (Tacket and Mason, 1999; Walmsley and Arntzen, 2000). It is now necessary to transfer this technology from model varieties to edible crop plants.

For obvious reasons, it is preferable to use plants that are normally consumed raw for the production of edible vaccines. In this respect, it is interesting to note that vaccines against hepatitis B virus and rabies virus have been produced in transgenic lettuce and tomato, respectively (Kapusta et al., 1999; McGarvey et al., 1995). Similar considerations apply to the production of protein nutraceuticals (recombinant proteins incorporated into food that provide health benefit, but which are not extracted and used as refined drugs). For example, the milk proteins, lactoferrin and ββ-casein have been expressed in transgenic potatoes (Chong et al., 1997; Chong and Langridge, 2000).

Molecular Farming in Plants

The large-scale production of recombinant proteins in plants is known as molecular farming (Fischer and Emans, 2000). It is now widely recognised that plants provide a unique combination of advantages over more traditional expression systems, such as bacteria, yeast, mammalian cell lines and transgenic animals (Giddings et al., 2000; Giddings, 2001; Hood, 2002; Schillberg et al., 2002). From a commercial perspective, the major benefit is the anticipated cost savings since there are no requirements for expensive equipment or skilled labour.

Transgenic plants can be maintained, harvested and processed using normal agricultural practices and existing infrastructure. Other advantages include the ease of scale-up, the absence of human/animal pathogens and undesirable DNA sequences (oncogenes, endogenous retroviruses, etc.) and the structural and functional similarities between plant-derived recombinant proteins and their native counterparts.

Plants have therefore been used to produce a wide range of products, including therapeutic human proteins, recombinant antibodies, subunit vaccines, nutraceuticals, animal feed additives, biopolymers, molecular biology reagents and industrial enzymes. These range from simple polypeptides to complex proteins with multiple subunits, disulphide bonds and glycan chains. Some plant-derived recombinant proteins are now reaching commercial status, and a comprehensive list has been assembled in several recent reviews (Fischer and Emans, 2000; Daniell et al., 2001b; Giddings et al., 2000; Giddings, 2001; Hood, 2002; Stoger et al., 2002a).

Early demonstrations of recombinant protein production in plants involved model species and varieties due to the availability of well-established transformation protocols and suitable expression constructs. Model tobacco varieties have been used for the expression of most foreign proteins to date, beginning with the first human protein to be expressed in plants—growth hormone—which was produced as a fusion protein with the Agrobacterium nopaline synthase enzyme (Barta et al., 1986). However, for large-scale production it is necessary to transfer the technology from model varieties to commercial cultivars. Typically, the expression of a given protein (or protein class) is first optimised and evaluated in a model system, and then transferred to commercial varieties or other crop species. In this chapter, we discuss practical issues guiding the development of crop-based production systems for molecular farming in plants.