161 Various strategies have been developed to exploit plants as bioreactors for the production of pharmaceutical antibodies, to engineer antibody-mediated pathogen resistance or to alter the plant phenotype by immunomodulation. Recent research developments focus on the fine-tuning of expression systems and the detailed characterisation of recombinant products, including the implications of plant-specific glycosylation. Meanwhile, the first of these plant-derived antibody products has successfully completed early phase clinical trials. Addresses *Molecular Biotechnology Unit, John Innes Centre, Norwich Research Park, Norwich NR4 7UH, UK; † e-mail: stoger@bbsrc.ac.uk; e-mail: stoger@molbiotech.rwth-aachen.de ‡ Department for Molecular Biotechnology, RWTH Aachen, Worringerweg 1, 52074 Aachen, Germany Current Opinion in Biotechnology 2002, 13:161–166 0958-1669/02/$ —see front matter © 2002 Elsevier Science Ltd. All rights reserved. Abbreviations ER endoplasmic reticulum Ig immunoglobulin rAbs recombinant antibodies scFv single-chain Fv fragment sIgA secretory immunoglobulin A Introduction Antibodies are bioactive molecules that, owing to their individual and specific binding properties, allow a large diversity of potential applications. These include medical diagnosis and therapy, the sensitive detection and removal of environmental contaminants, control of pathogens, and industrial purification processes. Antibodies provide an invaluable tool in fundamental research, because of their ability to interfere with metabolic processes within an organism. The concept of using plants as heterologous expression hosts for recombinant antibodies (plantibodies) is more than a decade old [1]. The combination of antibody and plant engineering, two rapidly advancing technologies, has resulted in the expression of a diversity of molecular forms in different plant species [2]. As we move closer to specific applications involving recombinant antibodies (rAbs), the focus of recent research activity has shifted towards strategies and decision making for achieving well-defined objectives (commercial or otherwise) involving specific forms of rAbs. Major targets include the improvement and comparison of different expression systems in terms of efficacy and feasibility. These encompass an assessment of the quality of the product and the use of antibody molecules with improved characteristics (e.g. fusion proteins with enhanced or novel functions). Contemporary applications in agronomic research include immunomodulation of physiological processes and engi- neering of antibody-mediated resistance to pathogen infection. The most advanced application, however, is the utilisation of plants as bioreactors to produce antibodies required for medical use or industrial processes. In this review, we concentrate on recent advances in expression technology and highlight emerging applications and constraints in the biopharming of plantibodies. Advances in transformation and expression technology Both Agrobacterium-mediated transformation and particle bombardment have been used to introduce antibody genes into plants [3]. Particle bombardment allows the simultaneous introduction of multiple constructs, thereby expediting the recovery of transgenic lines expressing multimeric antibodies such as secretory immunoglobulin A (sIgA) [4 • ]. The recombinant protein can be deposited throughout the plant or in specific organs. The deposition and storage of antibody molecules in seeds of various crop plants has been demonstrated [3,5,6]. As a recent example, high accumulation of a single-chain Fv fragment (scFv) antibody in pea seeds was reported using the seed- specific USP promoter [7]. The high stability of an scFv antibody was again confirmed in tobacco seeds over a period of 1.5 years [8]. Plant cell or organ culture in bioreactors is more expensive than agricultural production, but offers advantages as rAbs can be produced in containment and under controlled conditions. Recently, expression of a murine immuno- globulin (Ig) G1 in hairy root cultures [9–11], leading to secretion of the rAb into the medium, was reported. An alternative to nuclear gene transfer is the transforma- tion of organelles. Recent advances in chloroplast transformation methodology resulted in the plastidial expression of a multimeric vaccine [12]. Similarly, an rAb has also been expressed in chloroplasts [13]. Expression of recombinant proteins in the chloroplast genome has some advantages compared with nuclear gene transfer (e.g. high levels of expression and containment). Transient expression systems involving viral vectors [14–16] or agroinfiltration [17,18] are effective means for obtaining moderate quantities of recombinant product within a very short time frame (a few weeks). Such systems may prove to have advantages compared with routine small-scale bacterial expression systems for obtaining correctly folded, soluble proteins. Plantibodies: applications, advantages and bottlenecks Eva Stoger* † , Markus Sack ‡ , Rainer Fischer ‡ and Paul Christou*