Antibody production by molecular farming in plants

"Molecular farming" is the production of pharmaceutical proteins in transgenic plants and has great potential for the production of therapeutic anti-cancer antibodies and recombinant therapeutic proteins. Plants make fully functional recombinant human or animal antibodies. Cultivating transgenic plants on an agricultural scale will produce almost unlimited supplies of recombinant proteins for uses in medicine. Combinatorial library technology is a key tool for the generation and optimisation of therapeutic antibodies ahead of their expression in plants. Optimised antibody expression can be rapidly verified using transient expression assays in plants before creation of transgenic suspension cells or plant lines. Subcellular targeting signals that increase expression levels and optimise protein stability can be identified and exploited using transient expression to create high expresser plant lines. When high expresser lines have been selected, the final step is the development of efficient purification methods to retrieve functional antibody. Antibody production on an industrial scale is then possible using plant suspension cell culture in fermenters, or by the propagation of stably transformed plant lines in the field. Recombinant proteins can be produced either in whole plants or in seeds and tubers, which can be used for the long-term storage of both the protein and its production system. The review will discuss these developments and how we are moving toward the molecular farming of therapeutic antibodies becoming an economic and clinical reality.     

Comparison of signal peptides for efficient protein secretion in the baculovirus-silkworm system

The baculovirus-silkworm expression system is widely used as a mass production system for recombinant secretory proteins. However, the final yields of some recombinant proteins are not sufficient for industrial use. In this study, we focused on the signal peptide as a key factor for improving the efficiency of protein production. Endoplasmic reticulum (ER) translocation of newly synthesized proteins is the first stage of the secretion pathway; therefore, the selection of an efficient signal peptide would lead to the efficient secretion of recombinant proteins. The Drosophila Bip and honeybee melittin signal peptides have often been used in this system, but to the best of our knowledge, there has been no study comparing secretion efficiency between exogenous and endogenous signal peptides. In this study we employed signal peptides from 30K Da and SP2 proteins as endogenous signals, and compared secretion efficiency with those of exogenous or synthetic origins. We have found that the endogenous secretory signal from the 30K Da protein is the most efficient for recombinant secretory protein production in the baculovirus-silkworm expression system.     

Technical enzymes produced in transgenic plants

Technical enzymes are used in many industrial applications. Nowadays technical enzymes are often produced in transgenic host organisms. The use of transgenic plants with respect to high level of expression at low costs as a prerequisite for successful commercial production of technical enzymes is discussed. This review summarises recently published examples for production of technical enzymes in plants. In addition, plastid transformation and viral vectors are discussed as methods which might be useful for obtaining high expression level of recombinant proteins in plants.     

Molecular farming of recombinant antibodies in plants.

'Molecular farming' is the production of recombinant proteins in plants. It is intended to harness the power of agriculture to cultivate and harvest transgenic plants producing recombinant therapeutics. Molecular farming has the potential to provide virtually unlimited quantities of recombinant antibodies for use as diagnostic and therapeutic tools in both health care and the life sciences. Importantly, recombinant antibody expression can be used to modify the inherent properties of plants, for example by using expressed antipathogen antibodies to increase disease resistance. Plant transformation is technically straightforward for model plant species and some cereals, and the functional expression of recombinant proteins can be rapidly analyzed using transient expression systems in intact or virally infected plants. Protein production can then be increased using plant suspension cell production in fermenters, or by the propagation of stably transformed plant lines in the field. Transgenic plants can be exploited to produce organs rich in a recombinant protein for its long-term storage. This demonstrates the promise of using transgenic plants as bioreactors for the 'molecular farming' of recombinant therapeutics, blood substitutes and diagnostics, such as recombinant antibodies.     

Antibody molecular farming in plants and plant cells

`Molecular Farming' is a novel approach to the production of pharmaceuticals, where valuable recombinant proteins can be produced in transgenic organisms on an agricultural scale. Plants have been traditionally used as a source of medicines, but the use of transgenic plants in molecular farming represents a novel source of molecular medicines that include plasma proteins, enzymes, growth factors, vaccines and recombinant antibodies. Until recently, the wide use of these molecular medicines was limited because of the difficulty in producing these proteins outside animals or animal cell cultures. The application of molecular biology and plant biotechnology in the 1990s showed that many molecular medicines could be synthesised in plants. The goal of this Molecular Farming technology is to produce pharmaceuticals that are safer, easier to produce and less expensive than those produced in animals or microbial cultures. Here, we examine the production of recombinant antibodies by Molecular Farming.     

Large-scale production of a therapeutic protein in transgenic tobacco plants: effect of subcellular targeting on quality of a recombinant dog gastric lipase

A recombinant dog gastric lipase with therapeutic potential for the treatment of exocrine pancreatic insufficiency was expressed in transgenic tobacco plants. We targeted the protein using two different signal sequences for either vacuolar retention or secretion. In both cases, an active glycosylated recombinant protein was obtained. The recombinant enzymes and the native enzyme displayed similar properties including acid resistance and acidic optimum pH. The proteolytic maturation and the specific activity of the recombinant proteins, however, were found to be dependent on subcellular compartmentalization. Expression levels of recombinant dog gastric lipase were about 5% and 7% of acid extractable plant proteins for vacuolar retention and secretion respectively. This expression system already has allowed the production of tens of grams of purified lipase through open-field culture of transgenic tobacco plants.     

Stable Expression of Adalimumab in <Emphasis Type="Italic">Nicotiana tabacum</Emphasis>

Production of monoclonal antibodies and pharmaceutical proteins in transgenic plants has been the focus of many research efforts for close to 30 years. Use of plants as bioreactors reduces large-scale production costs and minimizes risk for human pathogens contamination. Stable nuclear transformation of the plant genome offers a clear advantage in agricultural protein production platforms, limited only by the number of hectares that can be cultivated. We report here, for the first time, successful and stable expression of adalimumab in transgenic Nicotiana tabacum plants. The plant-derived adalimumab proved fully active and was shown to rescue L929 cells from the in vitro lethal effect of rhTNFα just as effectively as commercially available CHO-derived adalimumab (Humira). These results indicate that agricultural biopharming is an efficient alternative to mammalian cell-based expression platforms for the large-scale production of recombinant antibodies.     

Plant-based strategies aimed at expressing HIV antigens and neutralizing antibodies at high levels. Nef as a case study

The first evidence that plants represent a valid, safe and cost-effective alternative to traditional expression systems for large-scale production of antigens and antibodies was described more than 10 years ago. Since then, considerable improvements have been made to increase the yield of plant-produced proteins. These include the use of signal sequences to target proteins to different cellular compartments, plastid transformation to achieve high transgene dosage, codon usage optimization to boost gene expression, and protein fusions to improve recombinant protein stability and accumulation. Thus, several HIV/SIV antigens and neutralizing anti-HIV antibodies have recently been successfully expressed in plants by stable nuclear or plastid transformation, and by transient expression systems based on plant virus vectors or Agrobacterium-mediated infection. The current article gives an overview of plant expressed HIV antigens and antibodies and provides an account of the use of different strategies aimed at increasing the expression of the accessory multifunctional HIV-1 Nef protein in transgenic plants.     

Recombinant protein production in a variety of Nicotiana hosts: a comparative analysis

Although many different crop species have been used to produce a wide range of vaccines, antibodies, biopharmaceuticals and industrial enzymes, tobacco has the most established history for the production of recombinant proteins. To further improve the heterologous protein yield of tobacco platforms, transient and stable expression of four recombinant proteins (i.e. human erythropoietin and interleukin-10, an antibody against Pseudomonas aeruginosa, and a hyperthermostable α-amylase) was evaluated in numerous species and cultivars of Nicotiana. Whereas the transient level of recombinant protein accumulation varied significantly amongst the different Nicotiana plant hosts, the variety of Nicotiana had little practical impact on the recombinant protein concentration in stable transgenic plants. In addition, this study examined the growth rate, amount of leaf biomass, total soluble protein levels and the alkaloid content of the various Nicotiana varieties to establish the best plant platform for commercial production of recombinant proteins. Of the 52 Nicotiana varieties evaluated, Nicotiana tabacum (cv. I 64) produced the highest transient concentrations of recombinant proteins, in addition to producing a large amount of biomass and a relatively low quantity of alkaloids, probably making it the most effective plant host for recombinant protein production.     

Antibody-Based Resistance to Plant Pathogens

Plant diseases are a major threat to the world food supply, as up to 15% of production is lost to pathogens. In the past, disease control and the generation of resistant plant lines protected against viral, bacterial or fungal pathogens, was achieved using conventional breeding based on crossings, mutant screenings and backcrossing. Many approaches in this field have failed or the resistance obtained has been rapidly broken by the pathogens. Recent advances in molecular biotechnology have made it possible to obtain and to modify genes that are useful for generating disease resistant crops. Several strategies, including expression of pathogen-derived sequences or anti-pathogenic agents, have been developed to engineer improved pathogen resistance in transgenic plants. Antibody-based resistance is a novel strategy for generating transgenic plants resistant to pathogens. Decades ago it was shown that polyclonal and monoclonal antibodies can neutralize viruses, bacteria and selected fungi. This approach has been improved recently by the development of recombinant antibodies (rAbs). Crop resistance can be engineered by the expression of pathogen-specific antibodies, antibody fragments or antibody fusion proteins. The advantages of this approach are that rAbs can be engineered against almost any target molecule, and it has been demonstrated that expression of functional pathogen-specific rAbs in plants confers effective pathogen protection. The efficacy of antibody-based resistance was first shown for plant viruses and its application to other plant pathogens is becoming more established. However, successful use of antibodies to generate plant pathogen resistance relies on appropriate target selection, careful antibody design, efficient antibody expression, stability and targeting to appropriate cellular compartments.     

Microalgae as platforms for production of recombinant proteins and valuable compounds: progress and prospects

Over the last few years microalgae have gained increasing interest as a natural source of valuable compounds and as bioreactors for recombinant protein production. Natural high-value compounds including pigments, long-chain polyunsaturated fatty acids, and polysaccharides, which have a wide range of applications in the food, feed, cosmetics, and pharmaceutical industries, are currently produced with nontransgenic microalgae. However, transgenic microalgae can be used as bioreactors for the production of therapeutic and industrially relevant recombinant proteins. This technology shows great promise to simplify the production process and significantly decrease the production costs. To date, a variety of recombinant proteins have been produced experimentally from the nuclear or chloroplast genome of transgenic Chlamydomonas reinhardtii. These include monoclonal antibodies, vaccines, hormones, pharmaceutical proteins, and others. In this review, we outline recent progress in the production of recombinant proteins with transgenic microalgae as bioreactors, methods for genetic transformation of microalgae, and strategies for highly efficient expression of heterologous genes. In particular, we highlight the importance of maximizing the value of transgenic microalgae through producing recombinant proteins together with recovery of natural high-value compounds. Finally, we outline some important issues that need to be addressed before commercial-scale production of high-value recombinant proteins and compounds from transgenic microalgae can be realized.     

Plant-based production of xenogenic proteins.

Foreign protein production in transgenic plants has been successful, from the generation of transgenic plant lines to the marketing of purified proteins. Antigenic proteins from disease organisms, monoclonal antibodies raised against antigens of disease organisms, and proteins with industrial process applications have been produced and tested. For vaccines, clinical trials in humans and feeding trials in animals are in progress to demonstrate their efficacy. For industrial proteins, high expression and downstream processing efficiency are key concerns, with application and test market trials in progress.     

Sorting of Glycoprotein B from Human Cytomegalovirus to Protein Storage Vesicles in Seeds of Transgenic Tobacco

As part of ongoing studies into the use of plant expression systems for making human therapeutic proteins, we have successfully expressed the major glycoprotein, gB, of human cytomegalovirus (HCMV) in transgenic tobacco plants. Viral glycoprotein was detectable in the protein extracts of mature tobacco seeds using neutralizing and non-neutralizing monoclonal antibodies specific for gB. Although several mammalian proteins have been expressed in tobacco, localization of these proteins in transgenic tobacco tissue has not been extensively examined. The objective of this study was to identify the site(s) of recombinant gB deposition in mature tobacco seeds. Using immunogold labelling and electron microscopy, we found specific labelling for gB in the endosperm of transgenic seeds, with gB localized almost exclusively in protein storage vesicles (PSV). This occurred in seeds that were freshly harvested and in seeds that had been stored for several months. These data indicate that gB behaves like a plant storage protein when expressed in tobacco seeds, and provide further support for the suitability of plants for producing recombinant proteins of potential clinical relevance.     

Expression of modified gene encoding functional human α-1-antitrypsin protein in transgenic tomato plants

Transgenic plants offer promising alternative for large scale, sustainable production of safe, functional, recombinant proteins of therapeutic and industrial importance. Here, we report the expression of biologically active human alpha-1-antitrypsin in transgenic tomato plants. The 1,182 bp cDNA sequence of human AAT was strategically designed, modified and synthesized to adopt codon usage pattern of dicot plants, elimination of mRNA destabilizing sequences and modifications around 5' and 3' flanking regions of the gene to achieve high-level regulated expression in dicot plants. The native signal peptide sequence was substituted with modified signal peptide sequence of tobacco (Nicotiana tabacum) pathogenesis related protein PR1a, sweet potato (Ipomoea batatas) sporamineA and with dicot-preferred native signal peptide sequence of AAT gene. A dicot preferred translation initiation context sequence, 38 bp alfalfa mosaic virus untranslated region were incorporated at 5' while an endoplasmic reticulum retention signal (KDEL) was incorporated at 3' end of the gene. The modified gene was synthesized by PCR based method using overlapping oligonucleotides. Tomato plants were genetically engineered by nuclear transformation with Agrobacterium tumefaciens harbouring three different constructs pPAK, pSAK and pNAK having modified AAT gene with different signal peptide sequences under the control of CaMV35S duplicated enhancer promoter. Promising transgenic plants expressing recombinant AAT protein upto 1.55% of total soluble leaf protein has been developed and characterized. Plant-expressed recombinant AAT protein with molecular mass of around approximately 50 kDa was biologically active, showing high specific activity and efficient inhibition of elastase activity. The enzymatic deglycosylation established proper glycosylation of the plant-expressed recombinant AAT protein in contrast to unglycosylated rAAT expressed in E. coli ( approximately 45 kDa). Our results demonstrate feasibility for high-level expression of biologically active, glycosylated human alpha-1-antitrypsin in transgenic tomato plants.     

Towards industrial usefulness - cryo-cell-banking of transgenic BY-2 cell cultures

Plants have been established as an useful production system for commercially relevant proteins. Plant cell cultures show certain advantages compared to field-grown plants. However, one critical drawback for the long-term use of recombinant plant cell cultures is the instability of cell cultures concerning genetic background and productivity when maintained by subculturing. The ability to store recombinant cell lines stably by cryopreservation allows to maintain an efficient and stable production system. In this work, we describe the development of a cryopreservation protocol for a transgenic BY-2 cell culture expressing human serum albumin. In 1.75-L stirred-tank bioreactors growth and production kinetics of the transgenic cell line were compared after cryopreservation to the hitherto performed maintenance by subculturing. Growth and productivity of the cryopreserved cell culture remained stable after freezing for one week. Here, we show that we developed an efficient method which allows the storage of transgenic plant cell cultures, an important requirement for industrial processes.     

Transgenic Rice as a Vehicle for the Production of the Industrial Enzyme Transglutaminase

Transglutaminases have a range of catalytic activities, most of which concern the post-translational modification of proteins. The most important of these activities is the cross-linking of proteins into large supramolecular networks. The widespread use of transglutaminases has increased the demand for an inexpensive, efficient and safe source of recombinant enzyme. We explored the use of plant-based systems for the production of this important industrial enzyme. Transgenic rice plants engineered with a rat prostate transglutaminase (rTGp), driven by the strong constitutive maize-1 ubiquitin promoter and its first intron, were shown to express the recombinant enzyme at the mRNA and protein levels. The Ca2+ dependence of the recombinant enzyme was confirmed by the biotin-labelled cadaverine-incorporation assay. In this communication we report the molecular and biochemical characterisation of transgenic plants expressing rTGp and this sets the stage for establishing a bioreactor system for the production of transglutaminases in plants.     

Viral vectors for production of recombinant proteins in plants

Global demand for recombinant proteins has steadily accelerated for the last 20 years. These recombinant proteins have a wide range of important applications, including vaccines and therapeutics for human and animal health, industrial enzymes, new materials and components of novel nano-particles for various applications. The majority of recombinant proteins are produced by traditional biological "factories," that is, predominantly mammalian and microbial cell cultures along with yeast and insect cells. However, these traditional technologies cannot satisfy the increasing market demand due to prohibitive capital investment requirements. During the last two decades, plants have been under intensive investigation to provide an alternative system for cost-effective, highly scalable, and safe production of recombinant proteins. Although the genetic engineering of plant viral vectors for heterologous gene expression can be dated back to the early 1980s, recent understanding of plant virology and technical progress in molecular biology have allowed for significant improvements and fine tuning of these vectors. These breakthroughs enable the flourishing of a variety of new viral-based expression systems and their wide application by academic and industry groups. In this review, we describe the principal plant viral-based production strategies and the latest plant viral expression systems, with a particular focus on the variety of proteins produced and their applications. We will summarize the recent progress in the downstream processing of plant materials for efficient extraction and purification of recombinant proteins.     

From miracle fruit to transgenic tomato: mass production of the taste-modifying protein miraculin in transgenic plants

The utility of plants as biofactories has progressed in recent years. Some recombinant plant-derived pharmaceutical products have already reached the marketplace. However, with the exception of drugs and vaccines, a strong effort has not yet been made to bring recombinant products to market, as cost-effectiveness is critically important for commercialization. Sweet-tasting proteins and taste-modifying proteins have a great deal of potential in industry as substitutes for sugars and as artificial sweeteners. The taste-modifying protein, miraculin, functions to change the perception of a sour taste to a sweet one. This taste-modifying function can potentially be used not only as a low-calorie sweetener but also as a new seasoning that could be the basis of a new dietary lifestyle. However, miraculin is far from inexpensive, and its potential as a marketable product has not yet been fully developed. For the last several years, biotechnological production of this taste-modifying protein has progressed extensively. In this review, the characteristics of miraculin and recent advances in its production using transgenic plants are summarized, focusing on such topics as the suitability of plant species as expression hosts, the cultivation method for transgenic plants, the method of purifying miraculin and future advances required to achieve industrial use.