E. Stoger1, C. VAQUERO2, E. Torres’, M. SACK2, L. Nicholson’,
J. DROSSARD2, S. WILLIAMS’, D.
KEEN’, Y. PERRIN1, R. FISCHER2,3 and P. Christou1
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.
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