58. 
Producing valuable pharmaceutical molecules in rice
E. Stoger1, C. VAQUERO2, E. Torres’, M. SACK2, L. Nicholson’, J. DROSSARD2, S. WILLIAMS’, D.
KEEN’, Y. PERRIN1, R. FISCHER2,3 and P. Christou1
1) 
John limes Center, Norwich Research Park, Colney Lane, Norwich NR4 7UH, UK
2) 
RWTH Aachen, Institute for Biology 1, Worringerweg 1, 52074 Aachen, Germany
3) 
Fraunhofer Department for Molecular Biotechnology, IUCT, Grafschaft, Auf dem Aberg 1, 57392 
Schmallenberg, Germany

 
     The use of plants as bioreactors for the production of valuable macromolecules is a relatively recent development, even though plants offer significant advantages over microbial expression systems and those based on animal cells or transgenic animals. We report in this communication the production of functional pharmaceutical recombinant antibodies in transgenic rice plants and rice cell culture. Antibodies are widely used as therapeutic and diagnostic tools due to their high specificity and affinity for their cognate antigens. However, their production in microbial culture is problematical due to incorrect folding and processing of higher eukaryotic proteins, while animal systems are labor-intensive and involve extra purification steps to avoid contamination by viruses and endotoxins. Plants offer a safe and reliable alternative, with added advantages including established processing capacity, ease of scale up and the need for only relatively unskilled personnel. Furthermore, crop plants such as rice are even more advantageous in that they lack the noxious secondary products found in model plant expression systems such as tobacco (King 1998).
     We report the use of transgenic rice plants and cell cultures to produce a recombinant single chain Fv (scFv) antibody against carcinoembryonic antigen, a well-characterized tumor-associated surface antigen. To optimize product yields, two different scFv constructs were engineered allowing antibody targeting to different cell compartments. An N-terminal murine leader sequence was encoded by both constructs to target scFvs to the secretory system. However, one construct also encoded a C-terminal KDEL retention signal, to retain scFvs in the endoplasmic reticulum (ER), while the other encoded a C-terminal His6 tag instead of the KDEL signal, and antibodies passed through the secretory system to the apoplast. Transgenic rice plants were generated with each of these constructs driven by two alternative promoters, the enhanced CaMV 35S promoter and the maize ubiquitin-1 promoter. We used competitive ELISA assays to determine the yeild of functional recombinant antibody. Up to 30 ug g1 fresh weight of antibody was detected in the leaves and seeds of transgenic rice plants (data not shown). The highest expression levels were achieved using the KDEL construct driven by the enhanced CaMV 35S promoter.
     We also explored the viability of rice cell lines, derived from transformed callus, as a system for stable and sustainable antibody production. In these experiments, we investigated the potential of a wider range of expression constructs in which the 5’ untranslated region (UTR) and leader sequence were also optimized for their effect on antibody expression levels. As in the transgenic plants, we compared alternative constructs encoding C-terminal His6 tag and KDEL retention sequences, although all constructs were driven by the maize ubiquitin- 1 promoter. Levels of functional antibody production were determined by competitive ELISA assay. We achieved the highest production level, approximately 3.5 ug g’ callus fresh weight, using the KDEL expression construct. We found that antibody levels were 6-14 times higher in cells transformed with the KDEL construct compared to those transformed with the His6 construct. We then generated an extended series of KDEL constructs with all possible combinations of alternative leader peptides (the murine immunoglobulin heavy and light chain leader peptides) and alternative 5’ UTRs (the chalcone synthase gene 5’ UTR and the tobacco mosaic virus omega sequence). The 3’ UTR comprised the tobacco mosaic virus pseudoknot region. There were no significant differences in antibody levels recorded in each of the resulting cell lines, showing that these sequences had little effect on the level of antibody production (Fig. 1).
     We have thus demonstrated the production of functional therapeutic antibodies in two rice expression systems, transgenic plants and cell culture. Each has its specific advantages: the cell culture system offers constant and sustainable antibody production; the transgenic plants are easy to propagate and harvest, and seed can be used as a long term storage vehicle. In all cases, the results we have achieved with the scFv antibody can be extrapolated to any conceivable macromolecule, thus rice could emerge as an important and beneficial production system for recombinant pharmaceuticals and other valuable products.


 

Reference
King, D.J., 1998. Applications and Engineering of Monoclonal Antibodies. Taylor & Francis, London.