Biotecnologia Aplicada Elfos Scientiae ISSN: 0684-4551 Vol. 17, Num. 1, 2000, pp. 45-46

James W Larrick, Lioyd Yu, Jun Chen, Sudhir Jaiswal, Keith Wycoff

Palo Alto Institute of Molecular Medicine, 2462 Wyandotte Street; and Planet Biotechnology Inc., 2438 Wyandotte Street, Mountain View, CA 94043 USA. Tel: 650-694-4996; fax: 650-694-7717; E-mail: jwlarrick@aol.com

Plant Bioreactors

The first transgenic plants were reported in 1983 [1,2]. Since then, many recombinant proteins have been expressed in several important agronomic species of plants including tobacco, corn, tomato, potato, banana, alfalfa, and canola [3]. Recent work suggests that plants will be an economic bioreactor for large-scale production of industrial and pharmaceutical recombinant proteins [3-6]. Perhaps most important are the cost benefits of plant production. For example, [3] calculated the cost of producing a recombinant protein in various agricultural crops. Although crops with more protein content (e.g. soybeans 400%, versus potatoes, 2%) are more cost effective. These costs are 10- to 50-fold less than protein produced at high-level in E.colí (i.e. 20% of total protein). Depending upon the use of the protein and the requirements for purification for in vivo pharmaceutical use, purification costs will obviously augment final product costs; however, at the hundred kilogram to metric ton level plant produced proteins will provide obvious savings.

To date three immunotherapeutic products produced in plants have entered the clinic: two antibodies and an oral vaccine (Table).

Table

Antibodies in plants: plantibodies

Although antibodies were first expressed in plants in the mid-1980s (Steiger, During) by two German graduate students, the first report was published in 1989 [7]. Since then a diverse group of "plantibody" types and forms have been prepared. Originally foreign antibody genes were introduced into plant cells by nonpathogenic strains of the natural plant pathogen Agrobacterium tumefaciens and regeneration in tissue culture resulted in the recovery of stable transgenic plants. Although this initial work to generate multichain proteins required crossing of plants expressing each chain, more recent studies have shown that multiple chains can be introduced via a single biolistic transformation event [8] (Wycoff et al., unpublished data), greatly reducing the time to final assembled plantibody.

SIgA: a novel antibody isotype

This laboratory has focused on the production of secretory IgA (SlgA) plantibodies [9]. At the present time plants offer the only large-scale commercially viable system for production of this unique form of antibody. SIgA is the most abundant antibody class produced by the body (>60% of total immunoglobulin). SIgA is secreted onto mucosal surfaces to provide local protection from toxins and pathogens. Dimeric IgA containing J chain derived from submucosal B cells binds to the epithelial cell polyimmunoglobulin receptor (PIG R) that transports the IgA to the mucosal surface. Binding triggers transcytosis to the mucosal surface where protease releases a portion of the PIGR called secretory component conveniently used to bind the SlgA. The secretory component protects the dimeric IgA from proteases and denaturation on the mucosal surface. Previously it was not possible to obtain therapeutic quantities of this class of immunoglobulin. The recent availability of large amounts of secretory IgA plantibodies opens up a number of novel therapeutic opportunities for disorders of the mucosal immune system. These include therapies for intestinal pathogens such as hepatitis viruses, Helicobacter pylori, and enterotoxigeníc E.coli, cholera etc., respiratory pathogens such as rhinovirus and influenza, and genitourinary sexually transmitted diseases and contraception.

Clinical studies of CaroRxTM anti-S.mutans SlgA to prevent dental caries

The most clinically advanced SlgA plantibody, called CaroRxTM, recognizes and inhibits the binding of the major oral pathogen, S.mutans to teeth. In preliminary work, a series of in vivo passive immunization experiments was carried out in 84 human subjects using murine anti-S.mutans antibodies [4-6]. Topical application of anti-S. mutans antigen SA l/ll MAbs prevented colonization of both artificially implanted exogenous strains of S.mutans, as well as natural recolonization by indigenous S.mutans. In these studies the pathogenic S.mutans was replaced by endogenous flora.

The presence of the complement-activating and phagocyte-binding sites on the Fc fragment of the MAb was not essential for activity, because the F(ab')2 portion of the MAb was as protective as the intact lgG; however, the Fab fragment failed to prevent recolonization of S.mutans. Prevention of recolonization was specifically restricted to S.mutans, as the proportion of other organisms, such as S.sanguis, did not change significantly. The surprising feature of these experiments was that protection from recolonization by S.mutans lasted up to 2 years (Ma J, personal communication), although MAb was applied for only 3 weeks and functional MAb was detected on the teeth for only 3 days following the final application of MAb. All studies indicated that this form of immunotherapy appears to be safe and well tolerated. The long-term protection could therefore not be accounted for by a persistence of MAb on the teeth, but may be due to a shift in the microbial balance in which other bacteria occupy the ecological niche vacated by S.mutans, resulting in resistance to recolonization by S. mutans.

The antigen-binding V regions of the best murine MAb identified by Ma and Lehner, Guy's 13, has been used to create an SlgA plantibody produced in tobacco designated CaroRxTM [9,14]. Levels of production of CaroRxTM in tobacco are up to 0.5mg/gram fresh weight. Future plans call for production of CaroRxTM in corn and other cereal grains. CaroRxTM has been produced and purified from tobacco under GMP conditions for clinical testing in the UK and USA. CaroRxTM was engineered with an additional IgG CH2 domain to facilitate purification of the antibody by protein G affinity chromatography. By protein G affinity purification CaroRxTM can be recovered with a high purity from green plant tissue.

Clinical evaluation of CaroRxTM in a pilot Phase ll trial has been completed at Guy's Hospital, London, UK [14]. In this trial a functional comparison was made between CaroRxTM and the parent IgG monoclonal antibody Guy's 13. BIACORE analysis revealed that the affinity of the antibodies for purified S.mutans SA l/ll was similar (Kd=0.5-1.3 x 10-9 M); however CaroRxTM had 4-fold higher avidity (functional affinity), a not unexpected result given the tetravalent binding of the SlgA.

Using an experimental design similar to that used to demonstrate activity of the parent MAb, CaroRxTM gave specific protection against colonization by oral streptococci for over four months [14]. In addition to this therapeutic endpoint, pharmacokinetics studies showed that in the human oral cavity, CaroRxTM survived for >3days versus 1day for the IgG antibody and multiple serum antibody samples were negative for human anti-mouse (HAMA) or anti-rabbit antibodies. There was no evidence of local or systemic toxicity of the topically applied plantibody.

These initial clinical studies demonstrate that topically applied anti-S. mutans SlgA plantibody (CaroRxTM) is safe (no HAMA, no local or systemic toxicity) and prevents colonization by S.mutans, the major cause of human dental caries [14]. Planet Biotechnology Inc. has submitted an IND (investigational new drug application) to the US FDA and Phase l/ll confirmatory clinical trials are underway at the School of Dentistry at the University of California in San Francisco.

References

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5. Austin S, et al. Ann. NY. Acad. Sci. 1994;721:235-44.

6. Krebbers E, et al. In: PR Shewry, S. Gutteridges, editors. Plant protein engineering. Cambridge University Press, London 1992;315-25.

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12. Ma JKC, Lehner T. Archs Oral Biol 1990;35:115S-22S.

13. Lehner T, Caldwell J, Smith R. lnfection and Immunity 1985;50-796.

14. Ma JKC, Hikmat BY, Wycoff K Vine, ND Chargelegue D, Yu L, Hein M, Lehner T. Nature Medicine 1998;4:601-6.

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