Most of the recombinant proteins that have been produced in plants to date are high-value pharmaceuticals intended for human use. These can be placed into three broad categories: (1) human or animal proteins used predominantly as replacement therapies (e.g. blood products, hormones, growth factors, enzymes); (2) recombinant antibodies used to prevent, diagnose and treat disease and (3) recombinant vaccines.For the reasons stated above, many proteins within the first category of products have been expressed in tobacco. There are, however, some notable exceptions. For example, rice has been used for the production of human αα-interferon (Zhu et al., 1994) and αα-1-antitrypsin (Terashima et al., 1999), maize for bovine aprotinin (Zhong et al., 1999) and canola for hirudin (Parmenter et al., 1995).
The second category of products, recombinant antibodies, is unique in that many different antibodies have been expressed in a variety of plant-based expression systems, yet the molecules are, in general terms, relatively homogeneous in nature. For the first time, this has allowed the direct comparison of different plant species and expression systems using equivalent products, providing an objective measurement of efficiency. Much of our knowledge about the relative advantages of different plant-based expression systems comes from the study of recombinant antibodies produced in plants (Stoger et al., 2002b).
Recombinant antibody molecules range in complexity from simple polypeptides (e.g. single chain Fv, scFv, fragments) to secretory immunoglobulins (sIg), which comprise 10 individual polypeptide chains (some bearing glycans) covalently assembled through disulphide bridges. These represent the most complex recombinant proteins ever to have been successfully expressed in plants. The most advanced product is CaroRX, a secretory antibody produced in tobacco that can be used to prevent dental caries. The antibody recognises Streptococcus mutans adhesin and prevents colonisation of the oral cavity by this pathogen. The product has already proved to be successful in phase II clinical trials, and is likely to be the first plant-derived recombinant protein approved for use in humans (Larrick et al., 2001; Ma et al., 1998). Another ‘plantibody’ likely to be approved in the near future is a humanised full-length immunoglobulin against herpes simplex virus glycoprotein B.
This is a serum antibody, which has four components (two identical light chains and two identical glycosylated heavy chains). Both the folding of individual chains and the assembly of the protein’s quaternary structure are dependent on disulphide bridges. This protein has been produced in transgenic soybean and rice (Zeitlin et al., 1998; Briggs et al., 2001). The most clinically advanced scFv is an anti-idiotype antibody that can be used to target malignant B-cells. This has been produced in virus-infected tobacco plants (McCormick et al., 1999). In transgenic tobacco, both a full-length immunoglobulin and a single-chain Fv fragment have been produced recognising human chorionic gonadotropin. These antibodies, currently at the pre-clinical stage, may be useful for pregnancy detection, contraception and tumour diagnosis (Kathuria et al., 2002).
Turning to the third category of plant-derived recombinant proteins, a large number of vaccines are currently under development, many expressed in tobacco or potato (reviewed by Daniell et al., 2001b). Potato has been chosen as an alternative model system to tobacco because it is edible, an important consideration for the oral administration of vaccines without extensive purification. Several of these vaccines are undergoing clinical trials, including a recombinant E. coli enterotoxin (LT-B) and Norwalk virus capsid protein, both delivered in raw transgenic potatoes (Tacket and Mason, 1999; Walmsley and Arntzen, 2000). It is now necessary to transfer this technology from model varieties to edible crop plants.
For obvious reasons, it is preferable to use plants that are normally consumed raw for the production of edible vaccines. In this respect, it is interesting to note that vaccines against hepatitis B virus and rabies virus have been produced in transgenic lettuce and tomato, respectively (Kapusta et al., 1999; McGarvey et al., 1995). Similar considerations apply to the production of protein nutraceuticals (recombinant proteins incorporated into food that provide health benefit, but which are not extracted and used as refined drugs). For example, the milk proteins, lactoferrin and ββ-casein have been expressed in transgenic potatoes (Chong et al., 1997; Chong and Langridge, 2000).
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