Molecular farming of pharmaceutical proteins

Molecular farming is the production of pharmaceutically important and commercially valuable proteins in plants. Its purpose is to provide a safe and inexpensive means for the mass production of recombinant pharmaceutical proteins. Complex mammalian proteins can be produced in transformed plants or transformed plant suspension cells. Plants are suitable for the production of pharmaceutical proteins on a field scale because the expressed proteins are functional and almost indistinguishable from their mammalian counterparts. The breadth of therapeutic proteins produced by plants range from interleukins to recombinant antibodies. Molecular farming in plants has the potential to provide virtually unlimited quantities of recombinant proteins for use as diagnostic and therapeutic tools in health care and the life sciences. Plants produce a large amount of biomass and protein production can be increased using plant suspension cell culture in fermenters, or by the propagation of stably transformed plant lines in the field. Transgenic plants can also 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, including vaccines, diagnostics, such as recombinant antibodies, plasma proteins, cytokines and growth factors. This revised version was published online in August 2006 with corrections to the Cover Date.     

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.     

抗FLAG标签抗体在植物中的高效表达

植物生物反应器是一种新兴的重组蛋白表达系统,是分子农业的核心内容之一。本研究在本氏烟草(Nicotiana benthamiana)中表达了抗八肽(DYKDDDDK, FLAG)标签抗体,并对其进行纯化与鉴定。通过多次免疫小鼠获得高效价抗FLAG抗体并测出其编码序列,然后亚克隆至植物DNA病毒表达载体,最后通过农杆菌介导转染烟草叶片。经Western blotting检测了转染后2−9 d抗体的表达情况：3 d后FLAG抗体开始在烟草叶片中表达,5 d后表达量达到峰值,每千克鲜叶估计可表达66 mg FLAG抗体。抗体经过分离纯化后浓缩为1 mg/mL,按1:10 000稀释仍可识别1 ng/mL的抗原,表明植物生产的FLAG抗体具有高亲和力。植物生物反应器可用于生产高亲和力抗体,并具有简易、成本低和生产周期短等特点,具有很高的应用价值。     

Production of Plant Made Pharmaceuticals: From Plant Host to Functional Protein

In the last two decades plants have emerged as valuable alternatives to mammalian cells for the production of pharmaceuticals and their potential as expression systems was shown by the commercial availability and acceptance of several plant made therapeuticals in clinical trials. Plants have many advantages over yeast, insect and bacterial expression systems such as the potential to properly fold the expressed proteins and the synthesis of more human-like N-glycans on the proteins. However, several constraints, such as expression yields, downstream processing and structural authenticity, currently limit the widespread use of plant expression systems. In this review, the focus is on the current limitations of plant systems for the production of pharmaceuticals and the possibilities to overcome these obstacles. A comparison is made with insect cell and yeast expression systems. Furthermore, the importance of glycosylation, in particular N-glycosylation for the biological function(s) of therapeutics in the human body will be discussed in detail and an overview of the state of art in the humanization of the N-glycosylation pathway in plants is provided.     

展望即到来的“分子农业”

在过去20余年里,植物生物技术领域中最值得重视的是多种转基因植物的建成,并已在一些国家大面积种植,转基因植物另一方面的进展是合成有医疗价值的多肽、蛋白质、包括抗体、抗体表位、复杂蛋白质等。这里主要介绍转基因植物生产的医疗用多肽、蛋白质,特别是作为口服疫苗在动物和临床试验的成功,预示这种新的用药途径将对第三世界人民的健康起到重大作用。由于转基因口服疫苗不需通过常规发展反应器,仅仅依赖于农业,因此有人提出“分子农业”一词,以显示其优越性。     

Public opinion toward the first,second, and third generations of plant biotechnology

Summary This paper considers public attitudes toward genetically modified plants in the fields or those soon to be planted. Analyzing a regional public opinion survey of 680 respondents in Arkansas, Texas, Louisiana, New Mexico, and Oklahoma carried out in the Spring—Summer of 2004, we look at the importance of public attitudes toward the three generations of agricultural biotechnology in light of the changing regulatory environment. Specifically, we ask questions concerning the first generation of plants with agronomic qualities, comparing our findings with previous studies, then look at perceptions of the second generation of crops with product quality characteristics, and the third generation, which expresses industrial products and pharmaceutical drugs. We look at perceived benefits, the likelihood, that these plants might accidentally enter the food supply, the likelihood that these plants might be eaten by the respondent, as well as how worried and angry the respondent would be as a result. Findings suggest that the public is still largely unaware of food biotechnology and genetically modified food products in their life. When compared with the first and second generation agricultural biotechnology products, survey respondents indicated that third generation products are not only likely to provide greater benefits, but are also potentially the source of more worry and anger if accidentally eaten.     

Characterization of a plant-produced recombinant human secretory IgA with broad neutralizing activity against HIV

Recombinant Secretory IgA (SIgA) complexes have the potential to improve antibody-based passive immunotherapeutic approaches to combat many mucosal pathogens. In this report, we describe the expression, purification and characterization of a human SIgA format of the broadly neutralizing anti-HIV monoclonal antibody (mAb) 2G12, using both transgenic tobacco plants and transient expression in Nicotiana benthamiana as expression hosts (P2G12 SIgA). The resulting heterodecameric complexes accumulated in intracellular compartments in leaf tissue, including the vacuole. SIgA complexes could not be detected in the apoplast. Maximum yields of antibody were 15.2 μg/g leaf fresh mass (LFM) in transgenic tobacco and 25 μg/g LFM after transient expression, and assembly of SIgA complexes was superior in transgenic tobacco. Protein L purified antibody specifically bound HIV gp140 and neutralised tier 2 and tier 3 HIV isolates. Glycoanalysis revealed predominantly high mannose structures present on most N-glycosylation sites, with limited evidence for complex glycosylation or processing to paucimannosidic forms. O-glycan structures were not identified. Functionally, P2G12 SIgA, but not IgG, effectively aggregated HIV virions. Binding of P2G12 SIgA was observed to CD209 / DC-SIGN, but not to CD89 / FcalphaR on a monocyte cell line. Furthermore, P2G12 SIgA demonstrated enhanced stability in mucosal secretions in comparison to P2G12 IgG mAb.     

Characterization of a plant-produced recombinant human secretory IgA with broad neutralizing activity against HIV

Recombinant Secretory IgA (SIgA) complexes have the potential to improve antibody-based passive immunotherapeutic approaches to combat many mucosal pathogens. In this report, we describe the expression, purification and characterization of a human SIgA format of the broadly neutralizing anti-HIV monoclonal antibody (mAb) 2G12, using both transgenic tobacco plants and transient expression in Nicotiana benthamiana as expression hosts (P2G12 SIgA). The resulting heterodecameric complexes accumulated in intracellular compartments in leaf tissue, including the vacuole. SIgA complexes could not be detected in the apoplast. Maximum yields of antibody were 15.2 μg/g leaf fresh mass (LFM) in transgenic tobacco and 25 μg/g LFM after transient expression, and assembly of SIgA complexes was superior in transgenic tobacco. Protein L purified antibody specifically bound HIV gp140 and neutralised tier 2 and tier 3 HIV isolates. Glycoanalysis revealed predominantly high mannose structures present on most N-glycosylation sites, with limited evidence for complex glycosylation or processing to paucimannosidic forms. O-glycan structures were not identified. Functionally, P2G12 SIgA, but not IgG, effectively aggregated HIV virions. Binding of P2G12 SIgA was observed to CD209 / DC-SIGN, but not to CD89 / FcalphaR on a monocyte cell line. Furthermore, P2G12 SIgA demonstrated enhanced stability in mucosal secretions in comparison to P2G12 IgG mAb.     

An improved method for the detection and quantification of recombinant protein in transgenic plants

A sensitive method has been developed for the detection of recombinant protein produced as a result of gene transfer into plants. This method is based upon antibody binding, which is then visualized using enhanced chemiluminescence and recorded on x-ray film for long-term storage. The technique is simple, rapid and reliable and can be used to screen large numbers of transgenic plants. Several plant species have been successfully tested in this way for a range of recombinant proteins.