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.

Plants as a Green Pharmacopoeia

We all routinely consume plant secondary metabolites—such as caffeine—without necessarily considering the complex biochemistry that is behind their production. Secondary metabolites have been used as medicines or as components of industrial processes for millennia. The methods used to manipulate their production are discussed by Yazaki in Chapter 43. Some secondary metabolites are limited in supply because the natural source is either hard to cultivate or difficult to synthesise chemically. Therefore, an effort has been made to produce these compounds in vitro or increase their production through the metabolic engineering of plants.

Yazaki first discusses how in vitro culture can be exploited to produce secondary metabolites. Thereafter, the strategies for metabolic engineering—that is, the use of genetic engineering methods to manipulate metabolism—are discussed in detail. It is evident from the review that understanding the biochemistry of their biosynthesis is the first step towards engineering the production of metabolites. Metabolic engineering differs from most molecular farming techniques for therapeutic protein production, because the aim is to manipulate complex plant metabolic pathways and to express proteins that are unlikely to have an effect on the plant cell itself. The successes in metabolic engineering reported by Yazaki are impressive.

The most telling and inspiring result so far has been the creation of ‘Golden Rice’, a transgenic rice line that has been metabolically engineered to produce high levels of the vitamin-A precursor ββ-carotene. This result exemplifies the potential of metabolic engineering to turn plants into living pharmacies.

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.

Hijacking the Factory Management—Using Viral Vectors for Protein Expression

There are, of course, alternative ways of exploiting the protein synthesis machinery of plants for the production of foreign proteins. The earliest proponents of this process, however, were not plant molecular biologists, but rather the plant viruses that hijack plant cells and use them for self-reproduction. These viruses provide a direct method for the transient production of recombinant proteins in crop plants, and this is the subject of the chapter written by Grill (see Chapter 40). By inserting target genes into the viral genomes, the hijacker can be turned into a molecular farmer.

In discussing how plant expression can be achieved using stably transformed plants or transient viral expression, Grill uses as examples two plant-produced proteins that have already entered clinical trials in the United States. The protein made in stably transformed plants was a humanised version of the Avicidin antibody for the treatment of prostate cancer, while the transient viral expression approach was used for the production of personalised vaccines produced from the cancerous B cells of non-Hodgkins lymphoma patients. What these examples illustrate is the flexibility of the viral vector system. Essentially, using this approach, personalised plant-based production of treatments for individual patients becomes possible, which is not feasible using stably transformed plants.

Grill then gives a detailed description of how viral vectors work in practice. Most plant viruses have single-stranded positive sense RNA genomes, and those that are assembled into rod-like particles can accommodate large inserted genes because it is possible to increase the length of the virion. Using recombinant viruses to infect plants and then produce the chosen recombinant proteins is rapid, compared to standard transgenic approaches, and has been tested in the field since the early 1990s. Grill argues that the rapid high-level expression of proteins is what sets viral vectors apart from the classical methods. Scaling up the methods for plant inoculation with viral vectors from the laboratory to the field has involved innovative solutions. An example, shown in the review figures, illustrates plants being inoculated using high-pressure sprays containing an abrasive and the recombinant viruses. A further interesting aspect of viral vectors is that there is no requirement for the cultivation of transgenic plant lines in the field. However, proteins transiently produced by viral infection have to be harvested from green leafy tissue and cannot be stored as easily as the seeds produced by transgenic plants.

Grill puts the case, and we agree, that while viral vectors compare well with transgenic plants overall, their real advantage is the ability to produce proteins rapidly. Indeed, the use of viral vectors to produce patient-personalised vaccines against non-Hodgkins lymphoma is such a compelling prospect that we feel viral vectors must remain an important tool for farming molecular medicines in the future.

A Perennial Production System

In their contribution, D'Aoust et al. describe the use of perennial plants for the production of recombinant proteins. Perennial plants are interesting as production systems because, with several harvests possible from the same crop, the plants can be used for repeated extraction of the protein.

The authors argue the case for forage legumes and, in particular, discuss the merits of using alfalfa. Because of the difficulties that had to be overcome to enable protein expression and plant transformation, forage legumes are relatively new to molecular farming. The authors describe the advances in promoter technology that have led to the construction of efficient inducible or strong leaf-specific expression cassettes. The features of the available transformation methods are discussed for alfalfa, which was first transformed more than 15 years ago, with D'Aoust et al. stating that reliable and efficient Agrobacterium-mediated transformation has been made possible by the optimal choice of genetic background and the use of standardised transformation protocols.

Because alfalfa is a fodder crop for ruminants, high-protein-yield varieties have been obtained and large-scale protein extraction techniques have been established by the animal feed industry. The authors describe how they are adapting these techniques for the production of plant-derived vaccines and, through the description of a case study, illustrate successful antibody expression in alfalfa.

The Prospects of Developing Medicinal Plants (Present & Future)

Medicinal plants are the local heritage with global importance, World is endowed with a rich wealth of medicinal plants. Herbs have always been the principal form of medicine in India and presently they are becoming popular throughout the developed world, as people strive to stay healthy in the face of chronic stress and pollution, and to treat illness with medicines that work in concert with the body's own defense. People in Europe, North America and Australia are consulting trained herbal professionals and are using the plant medicines. Medicinal plants also play an important role in the lives of rural people, particularly in remote parts of developing countries with few health facilities.

The variety and sheer number of plants with therapeutic properties is quite astonishing. It is estimated that around 70,000 plant species, from lichens to towering trees, have been used at one time or another for medicinal purposes. The herbs provide the starting material for the isolation or synthesis of conventional drugs.

In Ayurveda about 2,000 plant species are considered to have medicinal value, while the Chinese Pharmacopoeia lists over 5,700 traditional medicines, most of which are of plant origin. About 500 herbs are still employed within conventional medicine, although whole plants are rarely used.

In India, medicinal plants have made a good contribution to the development of ancient Indian Material Medica. One of the earliest treatises on Indian medicine, the Charak Samhita (1000 B.C.), records the use of over 340 drugs of vegetable origin. Most of these continue to be gathered from wild plants to meet the demand of the medical profession. Thus, despite the rich heritage of knowledge on the use of plant drugs, little attention had been paid to grow them as field crops in the country till the latter part of the nineteenth century.

Medicinal plants help in alleviating human suffering. These plants "are being integrated to the field of foods as additives, beverages and cosmetics. They are widely used as sweeteners, as biters, as spices, as natural colouring agent and as insecticides. Mass selection recurrent selection, hybridization, clonal selection mutation and biotechnology are some of major techniques at their use for many proven medicinal plants. There are still several constraints.

During the past one century there has been a rapid extension of the allopathic system of medical treatment in India. It generated commercial demand for pharmacopoeial drugs and their products in India. Efforts have been made to introduce many of these drug plants to farmers. Several research institutes have undertaken studies on the cultivation practices of medicinal plants, which were found suitable and remunerative for commercial cultivation. The agronomic practices for growing poppy, isabgol, senna, cinchona, ipecac, belladonna, ergot and few others have been developed and there is now localized cultivation of these medicinal plants commercially.

Medicinal plants have curative properties due to the presence of various complex chemical substances of different composition, which are found as secondary plant metabolites in one or more parts of these plants. These plant metabolites, according to their composition, are grouped as alkaloids, glycosides, corticosteroids, essential oils, etc. The alkaloids form the largest group, which includes morphine and codeine (Poppy), strychnine and brucine (Nux vomica), quinine (Cinchona), ergotamine (Ergot), hyocyamine (Belladonna) scolapomine (Datura), emetine (Ipecac), cocaine (Coco), ephedrine (Ephedra), reserpine (Rauwolfia), caffeine (Tea dust), aconitine (Aconite), vascine (Vasaca) santonin (Artemisia), lobelin (Lobelia) and a large number of others. Glycosides form another important group represented by digoxin (Foxglove), stropanthin (Strophanthus), glycyrrhizin (Liquorice), barbolin (Aloe), sannocides (Senna), etc. Corticosteroids have come into sannocides (Senna), etc. Corticosteroids have come into prominence recently and diosgenin (Dioscorea), solasodin (Solanum sp.), etc. now command a large world demand. Some essential oils such as those of valerian kutch and peppermint also possess medicating properties and are used in the pharmaceutical industry. However, it should be stated in all fairness that our knowledge of the genetic and physiological make-up of most of the medicinal plants is poor and we know still less about the biosynthetic pathways leading to the formation of active constituents for which these crops are valued.

Medicinal and aromatic plants are found in forest areas throughout South Asia, from the plains to the high Himalayas, with the greatest concentration in the tropical and subtropical belts and arid region of Thar desert. India recognizes more than 2,500 plant species as having medicinal value, Sri Lanka about 1,400, and Nepal around 700. Some of these, found at high altitudes in particularly stressful environments, grow very slowly and cannot live elsewhere. Others are more broadly distributed and adapt more easily to different ecological conditions.

During the past decade, a dramatic increase in exports of medicinal plants attests to worldwide interest in these products as well as in traditional health systems. In the last 10 years, for example, India's exports of medicinal plants have trebled. But with most of these plants being taken from the wild, hundreds of species are now threatened with extinction because of over-harvesting, destructive collection techniques, and conversion of habitats to crop-based agriculture. For instance, the small coniferous Himalayan yew (Taxus baccata) has recently become a heavily traded species. Similarly, senna is being grown extensively in arid region of India.

The pharmaceutical industries have made massive investment on pharmacological, clinical and chemical researches all over the world in past five decades. Efforts have been made to discover still more potent plant drugs. In fact, a few new drug plants have successfully been passed the tests of commercial screening. The benefits of these efforts would reach to the masses in future if farmers initiate commercial cultivation of medicinal plants. In fact, agricultural studies on medicinal plants, by its very nature, demand an equally large investment and higher priority. India, in particular, has a big scope for the development of pharmaceutical and phytochemical industry.

The subcontinent, India is blessed with varieties of aromatic and medicinal plants. The agroclimatic conditions and rainfall favouring this bio-availability. More than 7,500 species of medicinal plants are grown in India. Owing to this India is considered as the botanical garden of the world and treasure house of the biodiversity. Ayurveda, our indigenous system of health care is accepted everywhere especially abroad. Vedas and other ancient scriptures give cleanout evidences of using herbs and medicinal plants. Ayurveda alone describes about 2000 species of plants, which constitutes more than 10,000 formulations.

Over the past 10 years there has been a considerable interest in the use of herbal medicines in the world. Regarding the export of medicinal plants India's contribution to the international market is comparatively very low. Utilizing our biodiversity and proper planning, Indian products can very well enter the overseas markets. This can be achieved only through proper development of medicinal plants, standardization of the extracts and keeping the quality. WHO has recognized the effectiveness of traditional system of medicine and its safety.

The Indian Pharmacopoeia (1966) recognized eighty five drug plants whose ingredients are used in various pharmaceutical preparations. The text is however; confine to a few important commercially grown medicinal plants whose cultivation deserves priority in out national economy.

According to R. B. S. Rawat and R. C. Uniyal, National Medicinal Plants Board Department of ISM&H (Agrobios News Letter, Vol. 1, No. 8, January 2003) the use of medicinal plants is as old as human civilization. India has a glorious tradition of health care system based on plants, which dates back to Vedic era. In Rig Veda which is the oldest known repository of human knowledge and wisdom (4500-2500 B.C.) mentions about hundred medicinal plants used by the Aryans while in Atharva Veda (2500-2000 B.C.) elaborate description of medicinal plants are given. Later in Samhita period the science of medicine systematically organized with clear concept and theories based on the treatises the Charak Samhita - 2000 B. C, Sushruta Samhita - 1000-800 B.C. Besides this there are other works on Ayurveda and medicinal plants by Nagarjun, Chakradatta, Sharangadhar and Bangasen - 1000-500 B.C. Vaghabhatta Junior - 800 A. D. complied most of the books on Ayurveda and wrote Ashtanga Hariday Samhita.

They further indicated that India is bestowed with unique diversity in culture and natural vegetation exhibiting rich plant diversity. It has all known types of agro-climatic, ecologic and edaphic conditions. It also have unique biogeographical positions having all known types of eco­systems ranging from coldest place, the dry cold desert of Ladakh (Nubra Valley with - 57°C), to temperate, alpine and sub-tropical regions of north-west and trans-Himalayas; rain forests with high rainfall; wet evergreen humid tropics of western ghats and arid and semi-arid regions of peninsular India; dry desert conditions of Rajasthan and Gujarat to the tidal mangroves of Sunderban. It harbors 17500 flowering plants out of which 2000 plants are used in various classical system of medicine like Ayurveda, Siddha and Unani. The tribal and other communities use about 8000 species of wild plants as traditional medicine. The drugs used in ISM are 90% based on plant material and are considered to be safe, cost effective and with minimal or no side effects when genuine ingredients are used.

Medicinal plants are living and irreparable resource, which is exhaustible if over used and sustainable if used with care and wisdom. The importance of medicinal plants has been overlooked in the past. However, at present medicinal plants are looked upon not only as a source of affordable health care but also as a source of income. According to WHO report, over 80% of the world population relies on traditional medicine largely plant based for their primary healthcare needs.

The forest areas have been the traditional source of medicinal plants and herbs. The position cannot be sustained much further because on the one hand the areas under forests have been steadily shrinking and on the other the requirement of medicinal plants and herbs has increased steeply. This has resulted in unscientific and over exploitation of medicinal plants in the forests. One indication of the scarcity of some medicinal plants is their steep prices. The Ministry of Environment and Forests have already banned 29 endangered species of medicinal plans from their natural habitat. While medicinal plants are being utilized in the preparation of a number of modern drugs, there is a new trend worldwide of using natural products. Besides medicinal values, Pharmaceuticals, herbal food supplements, toiletries and cosmetics are growing in consumption in the international market.