Transgenic plants: A new biopharmaceutical manufacturing platform

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1 Transgenic plants: A new biopharmaceutical manufacturing platform Rachel K. Chikwamba 1, Hugh S. Mason 1, Richard Mahoney 2 and Charles J. Arntzen Center for Infectious Disease and 2 - Vaccinology Study of Translational Research The Biodesign Institute at Arizona State University School of Life Sciences P O Box Tempe, AZ Introduction The use of plants as a new platform for the large-scale production of biopharmaceutical proteins is a recent innovation that has its foundations in recombinant DNA techniques and molecular biology advances of the 1980s. Biopharmaceuticals are proteins or compounds manufactured in biological systems for diagnostic, therapeutic, or prophylactic use in human or animal health. Traditional methods of manufacturing biopharmaceuticals feature organisms or cell lines such as poxviruses, Chinese hamster ovary (CHO) cells, other mammalian cells, adenovirus, vaccinia virus, baculovirus, insect cells, E. coli, yeast and Salmonella. Today, however, more than a hundred protein-based drugs are in advanced clinical trials and yet just four molecules consume 75% of the biologics capacity (Burrill and Co., 2003). A uniform problem with current production systems is the high cost of the final product often so high as to make treatment expenses prohibitive. Clearly, there is a need for a new manufacturing system with a much larger production capacity at lower capital investment levels. Transgenic plants hold the promise to fill this need. Key advantages of producing biopharmaceuticals in plants include potentially lower production costs; reduced time, effort and cost to build the manufacturing system; freedom from contaminating pathogens of human or mammalian origin; ease of scalability using existing agricultural technologies; and the capability of plants to produce proteins that are similar in structure and function to their mammalian counterparts. Plant-derived pharmaceuticals are broadly classified as biologics, exemplified by subunit vaccines, or therapeutics such as monoclonal antibodies, growth hormones and cytokines. In this paper we discuss subunit vaccines as classical biologics, as well as monoclonal antibodies as the fastest growing category of therapeutics. Plant derived vaccines Key immunogenic proteins from pathogens are expressed in plants for use as subunit vaccines. Advantages of plant derived vaccines are the possibility of oral delivery, development of 144

2 multi-component vaccines, heat stability, lower cost of manufacture of active ingredient, and suitability of manufacturing and delivery technology in developing countries. The expression of vaccines in fruit and vegetables might allow oral administration in partially processed plant tissues. Oral delivery has unique advantages in vaccinology, with its capacity to stimulate strong mucosal immune responses. Development of immunity at the mucosa is seen as the most powerful means to prevent diseases that are caused by infections at the mucosal membranes, including global menaces such as TB, pneumonia, flu, diarrheal diseases, sexually transmitted diseases, HIV, and others. Challenges to oral delivery of protein subunit immunogens include high likelihood of degradation in the gut and poor recognition of most soluble proteins by the cells at mucosal immune effector sites in the gut, necessitating higher concentrations of immunogen for oral versus parenteral delivery. Plants allow the production of high amounts of immunogen at a much lower cost, and research data indicate that encapsulation of the immunogenic protein within the plant tissue matrix during administration provides protection from degradation by gastric enzymes and acids. Ideal subunit vaccines for use in oral vaccination will assemble into mucosally targeted multimeric complexes and aggregate or assemble into virus like particles (VLPs). Such proteins are most efficiently sampled by the cells of the mucosal immune system, triggering the required response. Oral vaccines have enormous potential for developing countries, where the procurement, distribution, use and disposal of syringes and needles present continuing impediments to the delivery of vaccines because of expense involved and potential of misuse. Heat stability during processing, transportation and storage is a key attribute of plant derived pharmaceuticals (PDPs), and is critical to elimination of the costly refrigeration currently needed in vaccine distribution. Co-expression of different antigens and crossing of crop lines expressing different antigens may allow production of combination vaccines. Blending of different components can also be done at processing. Combination vaccines are highly valued because they reduce the need for multiple injections or administrations. The early operation of the Global Fund for Children s Vaccines through GAVI (the Global Alliance for Vaccines and Immunization) has shown that developing countries accord very high priority to combination vaccines (Richard Mahoney, pers. comm). The results of several clinical trials with orally delivered plant-derived vaccines have been published. Tacket et al (1998), performed the first human clinical trial with plant derived vaccine, and demonstrated that a potato tuber expressing enterotoxigenic E. coli (ETEC) B subunit (LTB) is highly immunogenic in humans. Other human clinical trials followed with similar results. Data have been reported for potato expressed Norwalk virus capsid protein (NVCP), which assembled into Norwalk VLPs; potato and lettuce expressed hepatitis B surface antigen (HBsAg); and spinach expressed rabies virus epitopes from the rabies virus glycoprotein (G) and nucleoprotein (N) fused to the coat protein (CP) of alfalfa mosaic virus (AIMV) (Reviewed by Khalsa et al, in press). These data illustrate the functional feasibility of plant based vaccines. Plant derived antibodies Antibodies are versatile tools in the diagnosis and treatment of many diseases. Emerging clinical uses of recombinant antibodies (rabs) in diagnosis, prevention and therapy has generated a demand 145

3 for bulk quantities of these proteins. The market for monoclonal antibodies has grown from just under a US$1 billion in 1995 to about US$9 billion projected for 2005 (Burrill and Co, 2003). Established systems of rab production are based on fermentation systems using mammalian cells. While fast and efficient, bacterial systems do not fold and process proteins like eukaryotes do. Transgenic animals have also been used for production of high quality effective antibodies in their milk, but their utility is limited because production of transgenic farm animals is time consuming, has significant regulatory hurdles, and the presence of host IgA and IgG complicates the purification process. Full length humanized molecules, or derivatives that are smaller, dissected into minimal binding fragments and rebuilt into multivalent high avidity products can be designed and expressed in plants. The rabs can also be conjugated to other proteins and peptides, such as enzymes in prodrug therapy, toxins in cancer therapy, viruses in gene therapy and targeting molecules/ligands to guide them to target cells. To date, a variety of antibodies has been expressed in plants, ranging from full length IgGs, IgM and siga and various derivatives most as proof-of principle studies (Reviewed by Ma et al, 2003, Nolke et al, 2003). Current efforts are turning to the development of plant derived antibodies under good manufacturing practices (GMP) and as investigational new drugs (INDs). There are many plantderived antibodies in preclinical and clinical trials. The front runner is Guys 13, a mix of antibodies against Streptococcus mutans, the causative agent of tooth decay, which has gone through phase 1 human trials. Anti-epithelial cell adhesion molecule (EPCAM), an antibody against colon and prostate cancer, went through Phase I trials but has been recently been withdrawn from phase 2 trials for safety reasons that do not relate to means of manufacture. Anti-idiotypic scfv from individual lymphoma patients went through phase 1 trials, and many others are in preclinical trials (Burrill and Co, 2003, Ma et al, 2003, Nolke et al, 2003) Plant Expression systems Expression of proteins in plant systems can be achieved by two main methods: stable transformation, or transient expression. Stable transformation involves a DNA sequence encoding a PDP of choice stably integrated into the nuclear or chloroplast genome of the plant. Stable transformation has also been utilized to integrate viral genomes into plant chromosomes. Transcription of viral RNA initiates viral replication wherein an incorporated coding sequence for the pharmaceutical protein is co-expressed, and the protein accumulates in plant tissue from which it can be purified for formulation, or processed with the plant matrix as part of the formulation. In this case, gene expression can be controlled to occur in specific tissues, developmental stages or can be induced at an appropriate stage of plant growth. Alternatively, plant viruses have been used as a versatile system for expression of proteins in plants using various approaches. The most extensively used system utilizes the display of immunogenic peptides or epitopes on the surface of a virus. Plants are infected with chimeric viruses, which multiply with spread of infection. The chimeric viruses are then purified and administered.the second option utilizes cloned virus genomes to introduce pharmaceutical protein coding sequences driven by subviral promoters, resulting in expression of the antigen during the process of viral genome replication. Viral deconstruction is a recently developed 146

4 method and will be discussed in this meeting by Dr Gleba.The various expression systems have significant implications for containment of transgenes, and possibly regulatory requirements. Product Formulation Vaccines A key factor for success of plant-derived vaccines is the ability to formulate the active ingredient into homogeneous, temperature stable batches, and ultimately concentrated dosages that are appropriate for easy administration in unit doses. This ability will contribute to reduction in the cost of product delivery and administration. Current efforts at product formulation use food-processing technology such as freeze drying and air-drying.the processing stage allows the addition of fillers, adjuvants, antioxidants or protein stabilizers, and blending production runs will ensure lot-to-lot vaccine dose uniformity. Biosafety challenges PDPs have been expressed in a wide range of food and non-food crops, and choice of crops for pharmaceutical expression has been a subject for much debate. Food crops are preferred for oral delivery because of their history of safe consumption and therefore low risk for contaminants or toxins. One concern centers on the possible co-mingling of pharmaceutical and food crops. The perceived risks from such an event range from inherent toxicity of the novel protein or its products, capacity of the novel protein to alter existing levels of allergens to unintended effects arising from altered biochemical pathways, among others (Malarkey, 2003, Peterson and Arntzen, 2004). Dry cereal and legume seeds may be preferred because seed proteins are highly stable and can be stored for long periods at room temperature with no loss of activity. In a crop such as corn, which has a number of advantages for pharmaceutical production, there is concern about the spread of transgenes via pollen, wherein there may be contamination of commodity crops. These problems have been addressed by strict physical and temporal isolation of pharmaceutical and food crops, use of male sterile lines, and physical containment such as the use of special greenhouses. Because plant-derived pharmaceuticals are not intended for use as food products, the crops that produce them must have special stewardship to ensure crop genetic containment. In addition, the proteins they produce will have to be separated and purified in processing plants dedicated to that purpose. Good Manufacturing Practices (GMP) must be defined for pharmaceutical plant materials, and for processing and handling practices that utilize the raw product. The successful development of PDPs will depend on the development and licensure of candidates that exploit the unique properties of the plant platform such as scale and potentially lower costs of production References: Burrill and Company, Biotech 2003: 17th annual report on the industry. Burrill and Company (publishers). 147

5 Khalsa, G., et al. Plant-derived vaccines: progress and constraints. In: R Fischer and S Schillberg (eds.) Molecular Farming: Plant-made Pharmaceuticals and Technical Proteins. In press. John Wiley and Sons. Ma J. K-C., Drake, M.W., Christou, P. (2003). Nature Reviews Genetics 4: Malarkey, T. (2003). Mutation Research 544 (2-3): Nolke et al, (2003) Expert Opinion on Biological Therapy 3(7): Peterson, R.K.D. and Arntzen, C.J. (2004) Trends in Biotechnology 22: Tacket C.O. et al, (1998) Nature Medicine 4: