Antibodies from Hybrid GM Tobacco Plants
ISIS Press Release 09/07/07
Antibodies from Hybrid GM Tobacco Plants
Proposed release has not considered risks of antibodies to wild life and human beings, nor the horizontal transfer of antibiotic resistance marker genes to disease-causing bacteria. Prof. Joe Cummins and Dr. Mae-Wan Ho
This report has been submitted to the USDA, please circulate widely to your policy-makers
Antibody against tooth decay
The Animal and Plant Health Inspection Service (APHIS) of the United States Department of Agriculture (USDA) has prepared an environmental assessment in response to a request for a permit submitted by Planet Biotechnology for environmental release of a genetically engineered Nicotiana interspecies hybrid , and it is open for public comment by 13 July 2007 at http://www.regulations.gov/fdmspublic/component/main.
The interspecific hybrid line 06PBCarHG1 (Nicotiana tabacum x N. glauca) was obtained by crossing a transgenic N. tabacum (producing an antimicrobial monoclonal antibody that binds to the bacterium Streptococcus mutans associated with tooth decay in humans) with N. glauca. The application, submitted in 2005, requested permission to release the transgenic hybrid on 100 acres in Daviess County, Kentucky, beginning June 2007 and ending fall of 2007. Following harvest of the Nicotiana hybrid leaves, the company will extract and purify the CaroRx™ antibody intended to prevent tooth decay. In the United States, CaroRx™ is an Investigational New Drug (BB-IND # 7526) and in the European Union, it is a registered Medical Device .
Caro RX antibody used for passive immunization should not be presumed safe
The Nicotiana hybrid line 06PBCarHG1 produces the constituent parts of the secretory CaroRxTM antibody - light chain, heavy chain and J chain from mouse, and the secretory component from rabbit - all driven by the cauliflower mosaic virus (CaMV) promoter. These constituent parts were cloned and expressed in tobacco by independent transformation events. The events were combined into a single line by classical breeding methods. The monoclonal antibody produced in genetically modified (GM) tobacco is a synthetic mixture of gene sequences of mouse and rabbit. The application of the synthetic antibody leads to passive immunity to the bacterium Streptococcus mutans in humans treated with the antibody.
The production of monoclonal antibodies in plants has been described in a number of patents [2, 3]. This involves the assembly of monoclonal antibodies in transgenic tobacco plants  and the generation of secretory antibodies in plants . Recombinant plant monoclonal antibodies were tested in human subjects for preventive immunotherapy against dental caries . That small clinical study proved the concept, but was not sufficient to establish that the treatment was safe for a large heterogeneous population.
The APHIS environmental assessment for Caro RX antibody in transgenic hybrid tobacco presumes that the recombinant antibody will be contained on the 100-acre test-site because there will be little or no pollen produced by the hybrid tobacco plants. There is a good chance that the dust and debris from decaying plant parts will lead to surface and ground water being contaminated with the transgenic antibody, but no provisions were made to monitor for such contamination. As the genes and proteins produced in the transgenic hybrid tobacco are synthetic approximations of natural antibodies, the product should not be presumed safe for wild and protected animals before appropriate laboratory studies are conducted.
It is worth mentioning that the transgenic antibody may not be required for passive immunization to prevent dental caries. A mouth rinse of egg yolk from chickens immunized against Streptococcus mutans effectively prevented the bacterium from colonizing the human subjects .
In contrast, using transgenic probiotic bacteria for immunisation is riskier than transgenic tobacco antibodies. Lactobacilli modified to contain single chain antibodies against Streptococcus mutans can provide continuous release of recombinant antibody into the oral cavity, and is considered therapeutically superior to the mouth wash procedure . However, such transgenic probiotic bacteria may turn them into serious pathogens preadapted for invading the human gut, and we have recommended that Genetically Modified Probiotics Should Be Banned  (ISIS scientific publication).
DNA vaccines for active immunization
Passive immunity is not the only approach to controlling Streptococcus mutans. Antigens from the bacterium are delivered using oral or nasal applications. The preferred delivery of the antigen is through an anti-caries DNA plasmid carrying the gene that codes for the bacterial antigen. Intra-nasal application of the plasmid significantly reduced dental caries in rats . Genetic adjuvants, such as gene for interleukin-5 or the cholera toxin gene, enhanced protection against caries in mice . Intranasal immunization of rabbits and monkeys with an anti-caries vaccine proved effective in preventing dental caries . An anti-caries DNA found to produce extended immunity in mice was approved for human trials by the United States Food and Drug Administration . Active immunization may be inherently superior to passive immunization and the use of DNA vaccines will avoid potential environmental pollution associated with the production of passive vaccines in crop plants. However, large doubts remain over the safety of DNA vaccines  (How to Stop Bird Flu Instead, SiS 35), as acknowledged by the researchers themselves , pointing to risks that include autoimmune disease, contamination with bacterial toxins, immune cross reactions with human proteins, immune tolerance, integration of the nucleic acid into the genome of cells including germ cells and transmission to the next generations, recombination with host viruses and bacteria to create new pathogens, and transfer of antibiotic resistance marker genes to bacteria. The strong promoters from viruses may trigger cancer, and lastly, liver toxicity from small interference RNAs. Do these risks apply to the DNA vaccines used? It is important that post release safety monitoring should be put in place.
Risks from horizontal transfer of antibiotic resistance markers
The hybrid line 06PBCarHG1 contains two additional protein products expressed under the control of a plant recognized nos promoter (one of very few bacterial promoters known to be active in plants). These proteins are NPTII (from E.coli), an enzyme conferring resistance to kanamycin, used as a selectable marker, and NOS (from A. tumefaciens), an enzyme that forms nopaline from the amino acid arginine and alpha-ketoglutaric acid, but was not used as a selectable marker in the construction of 06PBCarHG1. Line 06PBCarHG1 also contains trfA (from E. coli) that encodes a DNA-binding protein important for plasmid DNA replication and add3 (from E. coli) that codes for resistance to the antibiotic streptomycin/spectinomycin. These genes are driven by bacterial promoters not recognized by plants, and are therefore not expressed in 06PBCarHG1. Additional non-coding sequences contained in the transformed plant, but not converted into protein products in the transgenic hybrid 06PBCarHG1 are colEI and rk2 origin of replication, both from E. coli and the nos terminator from A. tumefaciens . The antibiotic resistance genes pose dangers all the same to humans, plants and animals when transferred horizontally to pathogenic bacteria, whether they are active in the transgenic plants or not. The sequences trfA and rk2 increase the chance of replicating transgenic DNA and hence the likelihood for unintended horizontal gene transfer. The possibility of horizontal transfer of transgenic DNA appears not to have been addressed
The large field test release of transgenic tobacco modified with a mouse-rabbit hybrid antibody intended for passive immunization against dental caries entails environmental risks that outweigh the benefits. It has not been demonstrated that passive immunization offered by the transgenic tobacco is superior to using the egg yolk from chickens immunized with the bacteria. Furthermore, active immunization using DNA vaccines may provide long-term protection against the disease without the environmental risks, though the potential risks of the DNA vaccines also need to be addressed.
1. U.S. Department of Agriculture Animal and Plant Health Inspection Service Biotechnology Regulatory Services USDA/APHIS Environmental Assessment In response to the Planet Biotechnology permit application 05-354-03r for an environmental release to produce antibodies in genetically engineered N. tabacum X N. glauca hybrid plants 2007 http://www.regulations.gov/fdmspublic/component/main
2. Hiatt A, Ma J, Lehner T and Mostov K. Method for producing imunoglobulins containing protection proteins in plants and their use 2004 United States Patent 6,303,341
3. Hein M, Hiatt A and Ma J. Transgenic crops expressing assembled secretory antibodies 2006 Units States Patent 6,995,014
4. Ma JK, Lehner T, Stabila P, Fux CI and Hiatt A. Assembly of monoclonal antibodies with IgG1 and IgA heavy chain domains in transgenic tobacco plants. Eur J Immunol. 1994 Jan;24(1):131-8.
5. Ma JK, Hiatt A, Hein M, Vine ND, Wang F, Stabila P, van Dolleweerd C, Mostov K and Lehner T. Generation and assembly of secretory antibodies in plants. Science 1995, 268(5211), 716-9.
6. Ma JK, Hikmat BY, Wycoff K, Vine ND, Chargelegue D, Yu L, Hein MB and Lehner T. Characterization of a recombinant plant monoclonal secretory antibody and preventive immunotherapy in humans. Nat Med. 1998, 4(5), 601-6.
7. Hatta H, Tsuda K, Ozeki M, Kim M, Yamamoto T, Otake S, Hirasawa M, Katz J, Childers NK and Michalek SM. Passive immunization against dental plaque formation in humans: effect of a mouth rinse containing egg yolk antibodies (IgY) specific to Streptococcus mutans. Caries Res. 1997, 31(4), 268-74.
8. Kruger C, Hultberg A, van Dollenweerd C, Marcotte H and Hammarstrom L. Passive immunization by lactobacilli expressing single-chain antibodies against Streptococcus mutans. Mol Biotechnol. 2005, 31(3), 221-31.
9. Cummins J and Ho MW. Genetically modified probiotics should be banned. Microbial Ecology in Health and Disease 2005, 17, 66-68.
10. Xu QA, Yu F, Fan MW, Bian Z, Chen Z, Peng B, Jia R and Guo JH. Protective efficacy of a targeted anti-caries DNA plasmid against cariogenic bacteria infections. Vaccine 2007 25(7), 1191-5.
11. Han TK and Dao ML. Enhancement of salivary IgA response to a DNA vaccine against Streptococcus mutans wall-associated protein A in mice by plasmid-based adjuvants. J Med Microbiol. 2007, 56(5), 675-80.
12. Jia R, Guo JH, Fan MW, Bian Z, Chen Z, Fan B, Yu F and Xu QA.Immunogenicity of CTLA4 fusion anti-caries DNA vaccine in rabbits and monkeys. Vaccine 2006, 24(24):5192-200.
13. Xu QA, Yu F, Fan MW, Bian Z, Chen Z, Fan B, Jia R and Guo JH. Immunogenicity and persistence of a targeted anti-caries DNA vaccine. J Dent Res. 2006, 85(10), 915-8.