Revving up the Green Express

Courtesy of Large Scale Biology Agricultural researchers have designed a wide variety of genetically modified plants with traits deemed beneficial to those who grow, market, and consume them. But plants have another role in biotech: Members of the green kingdom also can be used--quite literally-- as manufacturing plants for large-scale, recombinant protein production.1-4 Think GM crops, with a twist. Such proteins have potential industrial, research, and clinical applications. Plant expression systems may ultimately help the pharmaceutical industry meet the rising demand for therapeutic proteins.1,2 "Monoclonal antibodies (mAbs) are one of the fastest expanding categories of protein drug," says Lee Quarles, spokesperson for Monsanto Protein Technologies. With analysts predicting that more than 70 therapeutic mAbs will be on the market by 2008, requiring production of over 10 metric tons of mAbs annually, plant-based expression could decrease manufacturing costs between four- and five-fold over traditional cell culture techniques, he says, depending on the scale of operation and the particular protein and expression host employed. Other advantages include easy scalability, high product yields, reduced risks of contamination with bacterial endotoxins or mammalian pathogens, proper folding and assembly of protein complexes, and the ability to perform most posttranslational modifications. But plants have downsides, too. Companies using plants for protein production must guard against a number of potential safety issues, including contamination with residual pesticides, herbicides, and toxic plant metabolites.5 In a broader context, using transgenic field crops to produce recombinant proteins invites the ire of those opposed to GM plants in general, who worry that transgenes and their encoded proteins will spread in the environment, ultimately affecting nontarget organisms. To address this latter concern, scientists are pursuing several options, including asexual reproduction, male sterility, "suicide" genes, plant host genomes incompatible with nearby related species, chloroplast genetic engineering, methods for removing or doing without selectable markers, and postharvest expression systems.5 As the US Food and Drug Administration and the US Department of Agriculture (USDA) establish a growing body of safety guidelines, several companies are begining to carefully move forward in developing, testing, and utilizing plants for protein expression. Nevertheless, it rarely hurts to hedge one's bets. "Many pharmaceutical companies are opting to prepare for projected increases in the demand for protein therapeutics by means other than, or in addition to, plant-based expression, including investing in additional infrastructure for producing proteins via already widely used methods such as cell culture, and/or exploring alternative approaches, such as protein production in transgenic animals," says Nate Cosper, industry manager for drug discovery and clinical diagnostics at Frost & Sullivan, a global market-consulting firm. FIELDS OF OPTIONS Plant researchers have developed a wide range of tools and strategies for protein expression in plant species such as corn, rice, wheat, tobacco, alfalfa, tomato, potato, banana, oilseed rape, and soybean. Transgenes can be stably introduced by using the soil pathogen Agrobacterium tumefaciens, or via direct transfer procedures such as electroporation, microinjection, or "biolistic" particle bombardment (biolistic is a coined word derived from biological and ballistic). Alternatively, plants can host transient gene expression driven by modified viral vectors. Protein expression can be cytosolic throughout the entire plant, or targeted to either specific plant organs or intracellular compartments. And proteins can be expressed directly within the plastids (for example, chloroplasts), which have their own separate genomes.6 Courtesy of Large Scale Biology HARVEST REPORT: Quality control scientists use HPLC to analyze recombinant biopharmaceuticals products. Each expression strategy has its own advantages and limitations. Green, leafy crops such as tobacco and alfalfa rank high with regard to sheer protein-producing biomass. Restricting protein expression to nonvegetative organs or compartmentalizing proteins within organelles is useful if the recombinant protein is toxic to its plant host, and targeting expression to seeds provides a convenient means for long-term storage of recombinant proteins prior to purification. Expressing orally administered pharmaceuticals such as "edible vaccines" in the fruits, leaves, or seeds of food crops can eliminate the need for postharvest purification. Stable chloroplast transformation in species such as tobacco and tomato can offer high protein yields, as well as an environmental plus: The chloroplasts of most field crops aren't transmitted by pollen.6 But chloroplasts also do not appear to support glycosylation. Nuclear expressed, complex proteins are generally targeted to the endoplasmic reticulum for folding, formation of disulfide bonds, assembly, and glycosylation. The different glycosylation patterns of proteins expressed in plants and animals may be problematic for proteins whose structural integrity, efficacy, and activity depend on human-style glycosylation. Also, because plant-produced glycoproteins contain some sugar moieties not found in their mammalian counterparts, the possibility exists that patients may manifest allergic reactions to plant-expressed recombinant antibodies or other glycosylated therapeutic proteins. Agricultural researchers could engineer plant hosts to produce the enzymes requisite for generating "humanized" glycoproteins,7 but such tinkering may not always be necessary. According to Debra Robertson, executive director of intellectual property at San Diego-based Epicyte Pharmaceutical, a company whose corn-grown mAbs are already in preclinical trials, "We have not observed immune responses to any of our drug candidates to date." Courtesy of Phytomedics MANUFACTURING PLANT: The roots of this small plant--essentially a "transgenic bioreactor"--can secrete protein into the hydroponic medium, simplifying purification. GREEN CORNUCOPIA Plant biotech workers have devised a variety of clever molecular strategies for different applications. SemBioSys of Calgary, Canada, for instance, has developed a system for expressing and purifying oleosin fusion proteins from oilseeds such as safflower. Oleosin is a membrane protein that specifically binds the lipid bodies of oil seeds (oilbodies), which are readily purified via flotation centrifugation. The transgenic construct can be modified to include a cleavage site so that the target protein can be removed from the oilbodies and further purified if necessary. Phytomedics of Dayton, NJ, employs a different tactic for simplifying purification: rhizosecretion. The company engineers tobacco plants to continuously secrete recombinant proteins from their roots into a liquid medium, thus obviating the need for extraction and purification from the plant biomass. "Because the roots excrete continuously, this method can be scaled up to remarkably high-volume production levels-and the transgenic plants never leave the greenhouse," explains Ira Pastor, executive vice president of Phytomedics. Pittsboro, NC-based Biolex also keeps its transgenic plants indoors, but this company utilizes Lemna, a fast-growing, green aquatic plant commonly known as duckweed. According to Lynn Dickey, Biolex's vice president of research, the company has successfully used Lemna to produce a variety of proteins, including antibodies, interferon, and human growth hormone. She adds, "We are optimistic that our transgenic system will achieve public confidence early on, largely because duckweed is not a crop plant, but also because biomass scale-up can be performed under highly controlled and contained conditions far more typical of cell-culture biomanufacturing than most transgenic systems employing whole plants." Like Phytomedics' tobacco, Biolex's Agrobacterium-transformed plants secrete recombinant proteins into a simple liquid medium for ready purification. Plant tissue culture systems are generally associated with higher production costs than agricultural methods, but they offer the benefits of growth under contained, environmentally controlled conditions as well as simplified downstream purification, particularly if the recombinant protein is secreted.8 Based in Freiburg, Germany, greenovation Biotech is using bioreactors for the scaled-up cultivation of recombinant protein-secreting protonema of the moss Physcomitrella patens. According to the company, this moss is well-suited for genetically engineering customized strains--such as those producing humanized glycoproteins--because its high rate of homologous recombination facilitates targeted gene knockouts and insertions. THE BIOPHARM REPORT Although plant-produced protein pharmaceuticals are not yet commercially available, a number of immunotherapeutic and vaccine proteins are currently moving through the drug-development pipeline. Epicyte Pharmaceutical specializes in developing plant-produced mAbs, or Plantibodies™. In most cases, the company employs corn as the production host, with recombinant protein expression restricted to the endosperm of the seeds (kernels). The sequences encoding the heavy and light chains of a candidate therapeutic monoclonal antibody are cloned, ligated into bacterial plasmids that contain plant regulatory sequences, and transferred into plant cells via biolistics. The vectors encoding the heavy and light chains are usually cointroduced into the same plant cell, which is cultured to regenerate a plant that produces correctly assembled antibody. Epicyte's first product likely will be EPI-19, an IgG mAb that neutralizes respiratory syncytial virus, a common cause of bronchiolitis and pneumonia in infants. EPI-19 is scheduled to enter Phase I trials by 2004, according to the company. In the meantime, preclinical studies of several other candidate therapeutics are underway, including HX8, a topical IgA antibody preparation for the treatment of herpes simplex virus; and novel antibodies to tumor necrosis factor-alpha, the amyloid beta peptide, and the diarrhea-causing enterotoxin produced by Clostridium difficile. Vacaville, Calif.-based Large Scale Biology Corp.'s individualized therapeutic vaccines for treatment of B-cell non-Hodgkin's lymphoma (NHL) have successfully completed Phase I clinical trials. "The results were so promising that we are currently drafting a proposal to the FDA for approval to proceed directly to Phase III," remarks Daniel Moriarty, Large Scale Biology's vice president of corporate affairs. These personalized vaccines employ single-chain antibody fragments specific for the immunologically unique, membrane-bound antibodies of NHL cells from a given patient. Courtesy of Large Scale Biology LIKE A JUICER, ONLY BIGGER: Large Scale Biology uses this process equipment to extract and purify biopharmaceuticals from plant material. The appropriate cDNA is ligated into a vector modified from tobacco mosaic virus for transient recombinant protein expression in the leaves of a tobacco cultivar. The vaccines, produced on a relatively small scale, are grown in greenhouses; but as the name of the company implies, it also has higher-volume applications in mind. Large Scale Biology has been conducting USDA-approved field trials for more than a decade to demonstrate that its technology is environmentally safe. The tobacco is harvested with equipment that is specially designed to avoid injuring the leaves, which are transferred to a high-capacity manufacturing facility for rapid homogenization and recombinant protein isolation. On the European front, Phase II trials are currently underway for Meristem Therapeutics' recombinant gastric lipase for the treatment of cystic fibrosis and pancreatitis, and the French company is optimistic that clinical testing in the United States ultimately will be approved. Meristem also is studying a variety of other drug candidates at the preclinical level, such as therapeutic antibodies for cancer. The company uses either stably transfected corn, with expression limited to seeds, or a tobacco host, depending on the market size and the protein to be expressed. CaroRx™, a topical preventative care formulation from Planet Biotechnology of Hayward, Calif., is currently in Phase II clinical trials. CaroRx employs sIgA specific for the bacterium that causes tooth decay, Streptococcus mutans, and is intended for regular preventative administration following teeth cleaning. The company also is developing other therapeutic candidates, including one employing secretory IgA specific for rhinovirus, a frequent culprit of the common cold, and another against the chemotherapeutic agent doxorubicin. The latter is intended to mitigate drug-induced hair loss without compromising chemotherapeutic action. Plant Biotechnology primarily employs stable nuclear transformation of a tobacco cultivar for protein production. Currently completing Phase I clinical trials is College Station, Texas-based ProdiGene's oral vaccine for enterotoxigenic Escherichia coli strains, which are a leading cause of morbidity and infant mortality in developing countries as well as traveler's diarrhea. Studies of ProdiGene's recombinant aprotinin, a protease inhibitor used in heart surgery, also are underway. In addition, the company has undertaken the large-scale production of trypsin, a workhorse protease used in research. ProdiGene uses Agrobacterium-mediated transformation of corn, with protein expression targeted to the seeds. Recombinant proteins can then be extracted and purified for subsequent use, or alternatively, the transgenic kernels can be ground into cornmeal for oral delivery. A number of other companies, including major players Dow Plant Biopharmaceuticals and Monsanto Protein Technologies, are driving the trend toward using plants as hosts for protein production by providing the needed development and manufacturing services, which include agricultural know-how, processing capabilities, and when applicable, drug-development expertise. "The science of plant-made pharmaceuticals represents a promising opportunity for the production of therapeutic proteins," comments Monsanto's Quarles. He explains, "Plants are a natural, renewable resource, and provide researchers with an environmentally sustainable production option that could ultimately result in a decreased reliance on bricks-and-mortar infrastructure. This less-costly approach to production may, in turn, mean that the medical industry will focus on new research--including alternative cures to life-threatening diseases." Deborah A. Fitzgerald can be contacted using the form posted at www.SciWriters.com. References 1. I. Raskin et al., "Plants and human health in the twenty-first century," Trends Biotechnol, 20:522- 31, 2002. 2. G. Giddings et al., "Transgenic plants as factories for biopharmaceuticals," Nature Biotechnol, 18:1151- 5, 2000. 3. H. Daniell, "Molecular farming for health--production of antibodies, biopharmaceuticals and edible vaccines in plants," in Business Briefing: Life Sciences Technology, London: World Markets Research Centre, pp. 34-8, 2002. 4. E. Stoger et al., "Plantibodies: Applications, advantages and bottlenecks," Curr Opin Biotechnol, 13:161-6, 2002. 5. U. Commandeur et al., "The biosafety of molecular farming in plants," AgBiotechNet, 5:ABN 110, April 5, 2003. 6. P. Maliga, "Progress towards commercialization of plastid transformation technology," Trends Biotechnol, 21:20-8, January 2003. 7. H. Bakker et al., "Galactose-extended glycans of antibodies produced by transgenic plants," Proc Natl Acad Sci, 98:2899-904, 2001. 8. P.M. Doran, "Foreign protein production in plant tissue cultures," Curr Opin Biotechnol, 11:199-204, 2000. function sendData() { document.frm.pathName.value = location.pathname; result = false if (document.frm.score[0].checked) result = true; if (document.frm.score[1].checked) result = true; if (document.frm.score[2].checked) result = true; if (document.frm.score[3].checked) result = true; if (document.frm.score[4].checked) result = true; if (!result) alert("Please select") return result; } .myradio{background-color : #94bee5} Please indicate on a 1 - 5 scale how strongly you would recommend this article to your colleagues? Not recommended 1 2 3 4 5 Highly recommended Please register your vote

Revving up the Green Express

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