News & Views
Conference Scene Molecular pharming: manufacturing medicines in plants
EuroSciCon, Bioscience Catalyst campus, Stevenage, UK, 21 September 2012 Within the expanding area of molecular pharming, the development of plants for manufacturing immunoglobulins, enzymes, virus-like particles and vaccines has become a major focus point. On 21 September 2012, the meeting ‘Molecular Pharming – recent progress in manufacturing medicines in plants’, hosted by EuroSciCon, was held at the Bioscience Catalyst campus, Stevenage, UK. The scientific program of this eventful meeting covered diverse highlights of biopharming: monoclonal antibodies, virus-like particles from transient and chloroplast expression systems, for example, for Dengue and HPV, apolipoproteins from safflower seeds, and new production platforms, such as potato or hydroponics by rhizosecretion. This report summarizes the stimulating scientific presentations and fruitful panel discussions on the current topics in this promising research field. After approximately 20 years of academic resea rch work a nd development, plant-derived pharmaceutica ls are now passing clinical trials in Europe and the USA; Julian Ma gave an attention-grabbing keynote on the time frame for development of plantmade pharmaceuticals (PMPs). The idea to produce monoclonal antibodies (mABs) in Chinese hamster ovary cells started in the 1970s and took a long time to break through, but has finally succeeded. In the same way, it is merely a matter of time until PMPs will also become established as standard products on the world market. Investment of manufacturing pharmaceuticals in plants is on the rise globally. The first products are licensed mainly in the USA where they usually reach the market first. Taking a future perspective, this development additionally opens interesting oppor tunities for low-income countries, where larger quantities of medicines need to be provided on a regular basis. However, high-income countries need to nurture this technology. Regarding the capital investment in mammalian cell culture [1,2] , the costs are steadily increasing with fermenter volume in a hyperbolic tendency. Compared with investments in PMPs, these curves are expected to ascend considerably slower, due to the easy scalability by transgenic plants. In his opening lecture, Ma reviewed a series of major recent developments in the PMP sector, from concept to regulatory approval and clinical trials, discussed in the next section. 10.2217/IMT.12.146 © 2013 Future Medicine Ltd
Recent developments in the PMP sector The most promising milestone was achieved by Protalix Biotherapeutics Inc. (Carmiel, Israel) who received US FDA comm e rcial license for ELELYSO™, a plant cell-expressed form of the glucocerebrosidase enzyme [3]. The EU is yet a step behind, but the EMA has developed an important standard: a guideline for the qua lit y of biologically active substances produced in transgenic plants. On this basis, German authorities were able to grant a license for current good manufacturing practice of the plant-derived mAB P2G12 and for the UK to approve its Phase I clinical trial [4]. Companies in the USA and Europe invested in establishing new current good manufacturing practice facilities in North Carolina, Kentucky and Germany. For example, Synthon has acquired the assets of Biolex Therapeutics (Nijmegen, The Netherlands), and their proprietary LEX™ manufacturing platform. Bayer/Icon Genetics (Halle, Germany) has entered a Phase I trial with 20 patients for personalized idiotype vaccines against non-Hodgkin’s lymphoma. With funding from the US Defense Advanced Research Projects Agency, Medicago Inc. has developed a plant-derived H1N1 influenza vaccine. For this vaccine candidate, the company has surpassed a defined threshold of at least 10 million doses within 1 month. Medicago has already completed Phase II clinical trials with another PMP, an avian flu H5 pandemic vaccine candidate. Immunotherapy (2013) 5(1), 9–12
Andreas G Lössl*1 & Jihong L Clarke2 Department of Molecular Systems Biology, Faculty of Life Sciences, University of Vienna, Althanstrasse 14, 1090 Vienna, Austria 2 Plant Health & Protection Division, Bioforsk-Norwegian Institute for Agricultural & Environmental Research, Høgskoleveien 7, 1432 Ås, Norway *Author for correspondence:
[email protected] 1
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News & Views – Conference Scene Improved platforms In an informative talk by Andreas Lössl from the University of Natural Resources and Applied Life Sciences (Austria), a new approach to managing pleiotropic effects of PMPs was introduced. To date, transgenic plants allow for the production of various pharmaceuticals [5,6], vaccines [7,8] and adjuvants [9], which increase the immunogenicity of antigenic proteins [10]. However, phenotypical effects of constitutive transgene expression can occur if gene products interact with the metabolism of the transformed plant. These constraints can prevent regeneration of transformants or, in milder cases, transformants can be chlorotic. Regeneration of transplastomic plants was affected when they expressed a Dengue envelope protein domain (ED III), whereas chlorotic phenotypes have been observed in plastid transformants encoding vaccine candidates for HPV. In order to postpone the vaccine production in tobacco after its sensitive regeneration phase, Lössl’s group has constructed an inducible system to regulate expression of antigens in plastids. For this, a method with ethanol-inducible transcription was adapted [11]. After transformation, plant tissue which carried the ED III antigen under control of this system regenerated quickly and, following treatment with ethanol, the plants expressed the antigen. For transformants encoding the cassette for a HPV vaccine candidate, the system also allowed regeneration of healthy plants with green phenotypes without ethanol induction. In his presentation, Lössl described novel alternatives to constitutive expression of antigen subunit vaccines in the chloroplast genome. Chemical regulation offers the advantages that expression of transgenic protein can be triggered in a timely fashion and plants can regenerate and grow up safely, without stress.
Novel organelles Maurice Moloney from Rothamsted Research Ltd (UK), presented his new production technology involving targeting to subcellular organelles: oil bodies, which enable cost-effective recovery of PMPs [12–14]. By this system, his group expressed the cardiovascular therapeutic ApoAIMilano [15] as a fusion protein in transgenic 10
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safflower seeds [16]. Transformant plants achieved commercially interesting expression levels of up to 7 g per kg of seed. Extraction of ApoAIMilano from seeds was achieved through the use of an oil body-based process and its maturation was performed in vitro, which formed the product Des‑1,2-ApoAI Milano. The group characterized it biochemically and confirmed its correct protein structure by ApoAI antibodies, isoelectric point, N‑terminal sequencing and mass spectrometry. For full functionality, ApoAI needs to interact with phospholipids to form a lipoprotein particle. To test this property, the purified Des-1,2‑ApoAIMilano was further processed and a clearance assay was performed. Its biological activity was assessed by cholesterol efflux assays. By this expression system in safflower oil bodies, the investigators have produced high levels of biologically functional cardiovascular therapeutics [16].
PMPs from edible crops & plant synthetic biology A remarkable talk bridging the gap between molecular pharming and plant synthetic biology was given by Diego Orzaez from the Instituto de Biologia Molecular y Celular de Plantas, CSIC (Spain). He demonstrated the state-of-the-art production of immuno globulins in edible plant organs. As recently published [17], his group combined, in a single tomato plant, four transgenes encoding the transcription factors Rosea1 and Delila from Antirrihnum majus, and the heavy and light chains of a human IgA against rotavirus. This combination resulted in transgenic purple tomatoes producing high levels of a neutralizing human antibody (hIgA_2A1) against the diarrhea agent rotavirus. The amount of hIgA_2A1 protein reached approximately 4% of the total soluble protein. The immunogenicity of fruit-derived products was tested by antiVP8*-binding activity and they strongly inhibited virus infection in an in vitro virus neutralization assay. Orzaez stated that there are no significant changes in the metabolic profiles of these tomatoes, hence, their consumption could be considered safe. Their effect on the oral route is dependent on the formulation. Based on the current results, this human antibody has excellent prospects for use as an edible medicine. future science group
Conference Scene – In addition, Orzaez presented his group’s newest development [18]: GoldenBraid 2.0, a standardized assembly system based on type-IIS restriction enzymes, which allows the indefinite growth of reusable gene modules made of standardized DNA pieces. For this purpose, a set of binary plasmids was adapted to the GoldenBraid system for Agrobacterium tumefaciens-mediated plant transformation.
PMPs for nanobiotechnology In an exciting and inspiring lecture, George Lomonossoff from the John Innes Centre (UK), gave an overview on production of virus-like particles (VLPs) through transient expression in plants. Based on his prominent CPMV–HT expression system [19], he reviewed recent success stories on the production of VLPs: HPV and bovine papilloma viruses made from single proteins (L1) and VLPs that are assembled from a couple of different components, such as the capsid from the Bluetongue virus BTV‑8. These particles offer great applications in medical biotechnology as highly efficient immunogens [20]. However, they have also been explored as nanoparticles for use in nanotechnology, for example, to display functionalities on their outer surface or to encapsulate heterologous material [21]. The most fascinating approach currently consists of the development of so-called empty VLPs that can be filled via specific pores by other compounds, for example, cobalt chloride, which is magnetic. Such magnetic VLPs can be targeted to cancer cells to be selectively heated and to allow for a hyperthermia therapy. With these developments, Lomonossoff has pointed out the newest prospects for plant-made nanoparticles with a high relevance to medical application and nanotechnology.
Rhizosecretion Tim Szeto, a collaborator with Pascal Drake from St George’s (University of London, UK) gave an interesting talk on hydroponic cultivation and rhizosecretion for recombinant protein manufacture in transgenic plants. This technology has turned out to be a promising new platform, yet, the yields of PMP by this method have been too low to exploit it commercially; however, new studies provide a basis for resolving this
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problem [22]. Studies covered transgenic plant lines grown in hydroponic culture medium expressing the mABs Guy’s 13 and 4E10, and a microbicide, cyanovirin‑N. Rhizosecretion rates increased significantly with the addition of plant growth regulators, such as auxins and cytokinins. Interestingly, a‑naphthalene acetic acid and indole-butyric acid increased the root dry weight of hydroponics. On the other hand, the cytokinins 6‑benzylaminopurine and kinetin increased rhizosecretion without affecting root mass [22]. The authors achieved further optimization by enrichment of nitrate in the culture media, and the group recommends cosecretion of protease inhibitors that stabilize the mABs secreted to the medium. This technique offers the advantages of contained cultivation of PMP plants and the ease of downstream purification of recombinant proteins. This proof-of-concept paves the way for applications to produce antibodies by rhizosecretion and these data provide a good basis for patenting the new system [23].
Conclusion This was a reasonably well-organized meeting that enabled efficient interaction and networking between the participants in the biopharming area. The meeting, chaired by Julian Ma, addressed a wide spectrum of top ics within molecular pharming, ranging from mAB and vaccine production to the fields of synthetic biology and good manufacturing practice issues. Presentations covered numerous technological novelties, along with valuable contributions on safety issues and containment strategies. Delegates commented that the talks were inspiring and excellent, they liked the question and answer session, the quality of the speakers and talks was very high and also enjoyed the 1‑day format. Additional information regarding this and future EuroSciCon conferences can be found on their website [101]. Acknowledgements This meeting was organized by EuroSciCon (www. euroscicon.com). The authors also acknowledge the collaboration with the EU COST action FA0804 and the Norwegian Research Council on plant-derived vaccines.
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News & Views – Conference Scene Financial & competing interests disclosure The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the
References 1
2
3
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5
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8
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Farid SS. Process economics of industrial monoclonal antibody manufacture. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 848(1), 8–18 (2007). Pollock J, Ho SV, Farid SS. Fed-batch and perfusion culture processes: economic, environmental, and operational feasibility under uncertainty. Biotechnol. Bioeng. 110(1), 206–219 (2013). Shaaltiel Y, Bartfeld D, Hashmueli S et al. Production of glucocerebrosidase with terminal mannose glycans for enzyme replacement therapy of Gaucher’s disease using a plant cell system. Plant Biotechnol. J. 5, 579–590 (2007). Fischer R, Schillberg S, Hellwig S, Twyman RM, Drossard J. GMP issues for recombinant plant-derived pharmaceutical proteins. Rev. Biotechnol. Adv. 30, 434–439 (2012).
Waheed MT, Thönes N, Müller M et al. Plastid expression of a double-pentameric vaccine candidate containing human papillomavirus (HPV)-16 L1 antigen fused with LTB as adjuvant: transplastomic plants show pleiotropic phenotypes. Plant Biotechnol. J. 9, 651–660 (2011).
10 Waheed MT, Gottschamel J, Hassan SW,
Lössl A. Plant-derived vaccines. An approach for affordable vaccines against cervical cancer. Hum. Vaccin. Immunother. 8(3), 403–406 (2012). 11 Lössl A, Bohmert K, Harloff H, Eibl C,
Mühlbauer S, Koop HU. Inducible trans-activation of plastid transgenes: expression of the R. eutropha phb operon in transplastomic tobacco. Plant Cell Physiol. 46(9), 1462–1471 (2005). 12 Nykiforuk CL, Boothe JG, Murray EW et al.
Transgenic expression and recovery of biologically active recombinant human insulin from Arabidopsis thaliana seeds. Plant Biotechnol. J. 4, 77–85 (2006).
Ma JK, Drake PMW, Christou P. The production of recombinant pharmaceutical proteins in plants. Nat. Rev. Gen. 4, 794–805 (2003).
13 Markley N, Nykiforuk C, Boothe J,
Clarke JL, Daniell H. Plastid biotechnology for crop production: present status and future perspectives. Plant Mol. Biol. 76, 211–220 (2011).
14 Boothe J, Nykiforuk C, Shen Y et al.
Lössl A, Waheed MT. Chloroplast-derived vaccines against human diseases: achievements, challenges and scopes. Plant Biotechnol. J. 9, 527–539 (2011).
15
Waheed MT, Thönes N, Müller M et al. Expression of a modified human papillomavirus L1 protein leading to the assembly of capsomeres in tobacco: a step towards cost effective second generation vaccines. Transgenic Res. 20, 271–282 (2011).
16 Nykiforuk CL, Shen Y, Murray EW et al.
Moloney M. Producing proteins using transgenic oilbody oleosin technology. BioPharm Int. 19, 34–47 (2006). Seed-based expression systems for plant molecular pharming. Plant Biotechnol. J. 8, 588–606 (2010). Kuang-Yuh C, Yano J, Li X et al. Safflower seed-derived Des-1,2-Apo AIMilano reduces atherosclerosis in hypercholesterolemic apolipoprotein E (-/-) mice. J. Am. Coll. Cardiol. 55, A50 (2010). Expression and recovery of biologically active recombinant apolipoprotein AI Milano from transgenic safflower (Carthamus
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manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. No writing assistance was utilized in the production of this manuscript.
tinctorius) seeds. Plant Biotechnol. J. 9, 250–263 (2011). 17 Juárez P, Presa S, Espí J et al. Neutralizing
antibodies against rotavirus produced in transgenically labelled purple tomatoes. Plant Biotechnol. J. 10, 341–352 (2012). 18 Sarrion-Perdigones A, Falconi EE,
Zandalinas SI et al. GoldenBraid: an iterative cloning system for standardized assembly of reusable genetic modules. PLoS ONE 6(7), e21622 (2011). 19 Sainsbury F, Thuenemann EC,
Lomonossoff GP. pEAQ: versatile expression vectors for easy and quick transient expression of heterologous proteins in plants. Plant Biotechnol. J. 7, 682–693 (2009). 20 Love AJ, Chapman SN, Matic S, Noris E,
Lomonossoff GP, Taliansky M. In planta production of a candidate vaccine against bovine papillomavirus type 1. Planta 236, 1305–1313 (2012). 21 Sainsbury F, Saxena P, Geisler K, Osbourn A,
Lomonossoff GP. Using a virus-derived system to manipulate plant natural product biosynthetic pathways. Methods Enzymol. 517, 185–202 (2012). 22 Drake PMW, Barbi T, Sexton A et al.
Development of rhizosecretion as a production system for recombinant proteins from hydroponic cultivated tobacco. FASEB J. 23, 3581–3589 (2009). 23 Drake PMW, Thangaraj H. Molecular
farming, patents and access to medicines. Expert Rev. Vaccines 9(8), 811–819 (2010).
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