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[SANET-MG] Antibodies in Hybrid Tobacco Plants in Kentucky comments due by next friday
Comments on the EA for synthetic antibodies grown in tobacco plants in
Kentucky are due by next Friday. Even though APHIS seems to
automatically ignore comments criticizing field test releases of GM
crops it is worthwhile to keep commenting. I urge others to make , even
brief, comments on this important subject. Monocl;onal antibodies have
been prepared and tested in a number of food crops including corn and
rice. APHIS seems to be promoting release of such potential pollutants
of the food supply so it is unwise to fail to comment at the time
comments are sought, even though such comments are ignored by the
29 June 2007
Professor Joe Cummins
University of western Ontario and
Institute of Science in Society
Comments on the Environmental Assessment of the Environmental Release to
Antibodies in Hybrid Tobacco Plants in Kentucky
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 U.S. Department of Agriculture Animal and Plant Health
Inspection Service Biotechnology Regulatory Services APHIS-2007-0029
Planet Biotechnology, Inc.; Availability of an Environmental Assessment
for a Field Release of Nicotiana Hybrids Genetically Engineered To
Produce Antibodies Comments due by 07/13/2007
The Animal and Plant Health Inspection Service (APHIS) of the United
States Department of Agriculture, has prepared an environmental
assessment (EA) in response to a request for a permit (APHIS Number
05-354-03r) submitted by Planet Biotechnology for environmental release
of a genetically engineered Nicotiana interspecies hybrid. Plant
Biotechnology crossed a genetically engineered Nicotiana tabacum that
produces an antimicrobial antibody which binds to a bacterium
(Streptococcus mutans) associated with tooth decay in humans, with N.
glauca (N. tabacum X N. glauca) to generate an interspecific Nicotiana
hybrid line, 06PBCarHG1 On December 21, 2005, APHIS received a permit
application (05-354-03r) from Planet Biotechnology of Hayward, CA. The
application requests permission to release a genetically engineered N.
tabacum X N. glauca hybrid line, 06PBCarHG1, into the environment in a
confined field trial. The purpose of the environmental release is to
grow the genetically engineered Nicotiana hybrid line, 06PBCarHG1, which
produces an antibody, designated by Planet Biotechnology as CaroRx™. The
environmental release 100 acres ,in Daviess County, Kentucky, will begin
in June 2007 and will conclude in the fall of 2007. Following harvest of
the Nicotiana hybrid leaves, the permittee will extract and purify the
CaroRx™ antibody. Application of CaroRx™ to human teeth is 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 (1).
The Caro RX antibody in Genetically Modified Tobacco is used for passive
Plant Biotechnology generated the transformed Nicotiana interspecies
hybrid line, 06PBCarHG1, by first cloning and expressing the CaroRxTM
gene in N. tabacum line H8-105 and then crossing H8-105 with N. glauca.
The Nicotiana hybrid line, 06PBCarHG1,is genetically engineered with the
constituent parts of the secretory CaroRxTM antibody (light chain from
mouse, heavy chain from mouse, J chain from mouse, and 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 (Ma 1995). Hybrid line
06PBCarHG1 contains two additional protein products that are expressed
under the control of a plant recognized NOS promoter (one of very few
bacterial promoters known to be expressed in plants). These proteins are
NPTII (from E.coli), an enzyme that confers resistance to kanamycin,
which is used as a selectable marker, and NOS (from A. tumefaciens), an
enzyme that forms nopaline from the amino acid arginine and
alpha-ketoglutaric acid. NOS, although present in the plasmid vector
used in the production 06PBCarHG1, was not utilized as a selectable
marker in the construction of 06PBCarHG1. Line 06PBCarHG1 also contains
trfA (from E. coli) that encodes for a DNA-binding protein important for
plasmid DNA replication and add3 (from E. coli) that encodes for
resistance to the antibiotic streptomycin/spectinomycin. However,
because trfA and add3 are driven by bacterial promoters that are not
recognized by plants, these genes are not expressed in 06PBCarHG1.
Additional noncoding sequences (sequences contained in the transformed
plant, but not converted into protein products) present in 06PBCarHG1
are ColEI and RK2 origin of replication sequences (from E. coli) and the
nos terminator from A. Tumefaciens (1). The monoclonal antibody produced
in genetically modified (GM) tobacco is 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. Active immunity to Streptococcus
mutans has also been developed and that alternative will be discussed below.
A planet Biotechnology patent US Patent 6,303,341 deals with the
production of protective immune globulins in plants (2) US Patent
6,995014 transgenic plants expressing monoclonal antibodies (3). The
fundamental publications began with the assembly of monoclonal
antibodies in transgenic tobacco plants (4) followed by generation of
secretary antibodies in plants (5). Recombinant plant monoclonal
antibodies were tested on human subjects for preventive immunotherapy to
prevent caries (6). That small clinical study proved the concept but was
not sufficient to establish the that the treatment was safe for a large
The EA for Caro RX antibody in Genetically Modified 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 pars will lead to surface and ground water contamination
with the GM antibody and the surface and groundwater surrounding the
test acreage should be monitored. Since the genes and proteins produced
in the GM tobacco are synthetic approximations of natural antibodies the
product should not be presumed safe for wild and protected animals o
consume but rather laboratory studies should be conducted on the safety
of the GM antibody prior o the field tests.
It is worth mentioning that GM antibodies may not be required for
passive immunization to prevent dental caries. A mouth rinse of egg yolk
from chickens immunized against Streptococcus mutans to produce
antibodies that effectively controlled colonization of the bacteria in
human (7). In contrast, use of transgenic probiotic bacteria is riskier
than GM tobacco antibodies. Lactobacili modified to contain single chain
antibodies against Streptococcus mutans provide continous release of
recombinant antibody into the oral cavity is considered therapeutically
superior to the mouth wash procedure I8). However, such GM bacteria may
create untoward side effects.
DNA Vaccines for Active Immunization
Passive immunity is not the only approach to immunological control of
Streptococcus mutans. Antigens from the bacterium causing dental caries
is delivered using oral or nasal applications. The preferred delivery of
the antigen is through an anti-caries DNA plasmid Inter nasal
application of the plasmid significantly reduce3d dental caries in rats
(9). Genetic adjuvants such as gens for interleukin 5 or the cholera
toxin gene enhanced protection against caries in mice (10). Internasal
immunization of rabbits and monkeys with an anti-caries vaccine proved
effective in preventing dental caries (11). An anti-caries DNA found to
produce extended immunity in mice was approved for human trials by the
United States Food and Drug Administration (12). Active immunization may
be inherently superior to passive immunization and the use of DNA
vaccines will likely avoid potential environmental pollution associated
with the production of passive vaccines in crop plants.
Considering the large field test release of GM tobacco modified with a
mouse-rabbit hybrid antibody destined for passive immunization of human
to prevent dental caries it can be asked “is this release really
necessary?”. The environmental risks of the field release of the may
exceed the benefits of the release. First, it was not proved that the
benefits of the GM tobacco for passive immunization was superior to the
use of egg yolk from chickens immunized with the bacteria causing dental
caries. Second, active immunization against the pathogen causing dental
caries may prove to provide long term protection against disease using
DNA vaccines without posing the environmental risks associated with
production of passive vaccines in crop plants.
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
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 May 5;268(5211):716-9.
6. Ma JK, Hikmat BY, Wycoff K, Vine ND, Chargelegue D, Yu L, Hein MB and
Characterization of a recombinant plant monoclonal secretory antibody
and preventive immunotherapy in humans. Nat Med. 1998 May;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.
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
9. 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 Jan 26;25(7):1191-5
10. 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 May;56(Pt 5):675-80.
11. 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 Jun 12;24(24):5192-200.
12. 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 Oct;85(10):915-8
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