PLANTIBODIES

INTRODUCTION
The term "plantibodies" was created to describe the products of plants that have been genetically engineered to express antibodies and antibody fragments in planta, with many applications. (Antibodies were first produced in tobacco plants approximately twelve years ago, Smith, 1996). Plants are being used in this technology as antibody factories, using their endomembrane and secretory systems to produce large amounts of clinically viable proteins which can later be purified from the plant tissue. The purified antibodies have many possible applications, for example, as diagnostic tools, in immunochromatography or in medical therapy (Jaeger et. al., 2000) . This site will focus on the use of plantibdodies in MEDICAL THERAPY and IMMUNOMODULATION in plants.It has been shown that plants can produce fully functional antibodies, capable of binding and aggregating Streptococci bacteria. Both IgA and IgG have been successfully assembled and purified in tobacco plants. (Stabila et. al., 1994)A lot of research has been carried out in this field as molecular biology techniques are improving, the production of antibodies by plants for therapeutic purposes and plant pathogen resistance will be possible in the near future with much research currently in clinical trials. Inducing the production of specific antibodies in plants has many commercially viable applications not only to the pharmaceutical industry but also the plant breeder. Plantibodies reduce the risk of human disease contamination found with cell fermentation procedures & hence has the potential to forgo expensive screening for viruses and bacterial toxins
This is a completely novel technology as plants are being engineered to express proteins that are naturally, exclusively expressed in animal species. Antibodies were previously produced in mice and purified with a view to using the antibodies to treat disease. However, when administered to patients, they gave severe immune responses. Humanised monoclonal antibodies were then created with very similar gene sequence to those genes coding for human antibodies. These are created from murine immunoglobulins, the constant domains and framework regions of the variable domain are altered to give high sequence homology to human antibodies. This process was not only laborious, but also expensive and the mouse antibodies were very difficult to purify. The production of antibody fragments by plants is not only cheaper, but more efficient. Since plants do not produce antibodies naturally, the purification process is much simpler and plants are capable of producing unlimited amounts of the protein. It has been shown that plants are capable of correctly assembling and folding recombinant gene products, even full-length heavy and light chains into complete antibodies. (Ma and Hein, 1995)

HOW ARE ANTIBODIES EXPRESSED IN PLANTS?
Antibodies can be expressed in plants as either full length molecules or as smaller fragments. ScFv's (Single chain, Fragment variable) are produced by fusing variable light and variable heavy domains with a flexible linker peptide. This is a highly specific antibody fragment that binds to antigen with high affinity. ScFv's, unlike full length antibodies, can easily be targeted to sub-cellular compartments. This is very useful for the plant breeder when targeting specific pathogen proteins in plants, in order to engineer PATHOGEN RESISTANCE. The production of whole antibodies in planta has more therapuetic potential than fragmented antibodies, however, correct folding and assembly of the chains is more challenging for the plant cell. Fusion of cell signaling molecules e.g. the KDEL signal to the C terminus of the protein increases cytosolic expression. (Conrad and Fiedler, 1998) Each method has its advantages and disadvantages for antibody production. Smaller antibody fragments are both easier to produce and easier for the plant to assemble, however, full length antibodies are much more resistant to proteolysis in the plant. Plants are a favorable way of producing antibodies as they are capable of assembling a complete secretory antibody in one single cell. Mammals require numerous cell types to produce the different components of the antibody i.e. the plasma cells produce the dimerising J chain and epithelial cells synthesize the secretory component of the IgA antibody Firstly, the genes coding for the antibody fragments or full antibody must be engineered into the plant. This can be done using a variety of PLANT TRANSFORMATION METHODS In order to obtain high amounts of antibody production, it has been found that targeting the protein into the extracellular space (i.e. the apolplast) is the most efficient method. Plants, as animals, secrete antibodies after post-translational modification has occured. Proteins that are secreted into the apoplastic space undergo less hydrolysis due to the lack of hydrolytic enzymes in the apoplasm. Signal peptides can be used to target the light and heavy chains to the Endoplasmic Reticulum (ER), chaperones similar to those found in human cells are found here which ensure the antibody undergoes correct folding. Higher accumulation of antibody fragments has been observed in the ER and the apoplast compared to the cytosol
The addition of signal peptides to antibody fragments is obviously an advantage over the use of full antibodies. However, if full antibodies are used, certain domains could be altered to facilitate complement activation upon binding. This would significantly aid the therapeutic properties of the antibody if it were to be used in the treatment of human disease, helping to elicit an immune response. Plant seeds are an attractive organ for the genetic engineer to target protein expression to as they are natural storage organs capable of retaining high protein levels over long periods of time in the absence of degradation. This technique can be carried out by using seed-specific promoters. Plantibodies were first produced in tobacco plants, but it is now being developed in corn which is one of the most common crops grown worldwide. The corn seed kernel is capable of naturally storing plantibodies in a low moisture environment that has a high concentration of protective protease inhibitors. These can then be purified using simple milling techniques which easily separate the high molecular weight antibodies from other components of the kernel

PRODUCTION IN PLANTA
The availability of large amounts of cheaply produced purified antibodies could provide treatment to a wide range of pathogenic organisms. It is possible to raise antibodies to virtually any antigen in the laboratory, however, there are few systems available that facilitate the mass production of monoclonal antibodies. Plants provide an attractive system that could produce large amounts of antibody at a relatively low cost. The production of secretory IgA by plants has numerous therapeutic applications. Lehner et. al. have shown that antibodies produced in plants used to treat Rhesus monkeys for dental caries were devoid of systemic side effects. There have been many experiments carried out to investigate the stability of the antibody in the plant cell. It has been suggested that the difference in glycosylation in plant cells could affect the efficiency of the antibody produced. N-linked glycosylation occurs in all higher eukaryotes, including plants, however, it is thought that plant glycans are smaller, with different terminal sugar residues. It is speculated that the difference in glycosylation could cause lower affinity antigen binding and it may possibly cause the antibody to be immunogenic, raising concerns for systemic applications of plantibodies in humans.(Cabanes-Macheteau et. al., 1999)

PLANT TRANSFORMATION
Plant transformation is the introduction of a foreign piece of DNA, conferring a specific trait, into host plant tissue. The foreign gene (termed the "transgene") is incorporated into the host plant genome and stably inherited through future generations. The correct regulatory sequences are added to the gene of interest i.e. promoters and terminators, then the DNA is transferred to the plant cell culture using an appropriate vector. The gene is attached to a selectable marker which allows selection for the presence of the transgene. Genes conferring resistance to a specific antibiotic are often used to serve this purpose. Once the plant tissue has been transformed, the cells containing the transgene are selected and regeneration back into whole plants is carried out. This is possible as plant cells are totipotent, which means that they contain all the genetic information to control the development of that cell into a potentially fertile plant. Therefore, the gene is contained in every single plant cell, however, where it is switched on is determined by the promoter which is controlling the gene. Plant transformation can be carried out in a number of different ways depending on the species of plant in question. This is discussed in the sections below. Plant transformation was developed as an alternative to conventional breeding methods which are more laborious than this fairly simple (now routine) laboratory procedure.


WHY TRANSFORM PLANTS?
Plants are transformed in order to create transgenic plants that contain genes conferring traits that are desirable to the plant breeder. This includes resistance to specific plant diseases and pests, drought and saline tolerance. It also includes novel uses of plants, such as plantibody production and the production of edible vaccines in plant tissue. Plants can be engineered to express sub-unit vaccines which trigger oral immunity if plant tissues are consumed as food. Vaccines can be created to a huge number of pathogens, such as bacteria that cause diarrhea; responsible for 3 million infant deaths per year in the developing world.(Arntzen, 1998)Conventional breeding methods were seen to be slow and laborious, plant transformation techniques were developed to overcome these problems. The ability to introduce novel genes into plants, often across the species barrier, provides many commercially viable applications to the plant breeder
Two of the major techniques used to transform plants are :
AGROBACTERIUM TUMEFACIENS-MEDIATED TRANSFORMATION
BIOLISTICS

AGROBACTERIUM-MEDIATED TRANFORMATION

A major method of DNA transfer in plants is Agrobacterium mediated transformation. The natural living soil bacteria, Agrobacterium tumefaciens is capable of infecting a wide range of plant species, causing Crown Gall diseases (see image below). It has natural transformation abilities which can be exploited in plant biotechnology. When A. tumefaciens infects as a cell, it transfers a copy of its T-DNA, which is a small section of DNA carried on its Ti (Tumour Inducing) plasmid. The T-DNA is flanked by two (imperfect) 25 base pair repeats. Any DNA contained within these borders will be transferred to the host cell.(Zupan and Zambriski, 1995)
The T-DNA section on the Ti plasmid can be replaced by the transgene coding for the desirable trait attached to the appropriate regulatory sequences. The recombinant bacteria can then be used to infect both regenerating cell and protoplast cultures. (Protoplasts are wall-less special plant cells)
Marker genes such as those coding for antibiotic resistance are attached to the transgene so that it is possible to select those cells that have been transformed by the bacterium. Cell to plant regeneration is carried out on the selected cells and transgene expression is characterized i.e. it is necessary to check that the gene is expressed at the correct levels in the correct tissues.
Agrobacterium tumefaciens has been used to transform many dicotyledonous plant species with relative ease. However, it is, as yet, not possible to transform monocotyledonous species, which include commercially viable cereal crops such as maize and rice.
The image below shows a crown gall tumor on tomato. Tumors caused by Agrobacterium tumefaciens are often apparent on the plant at ground level

BIOLISTICS
Biolistics (other wise known as Particle Bombardment) involves directly "shooting" a piece of DNA into the recipient plant tissue. This is carried out using a gene gun. Tungsten or gold beads (which are smaller than the plant cells themselves) are coated in the gene of interest and fired through a stopping screen, accelerated by Helium, into the plant tissue. The particles pass through the plant cells, leaving the DNA inside.
This method can be used on both monocotyledonous and dicotyledonous species successfully. It is again a relatively simple laboratory procedure. The transformed tissue is selected using marker genes such as those that code for antibiotic resistance. Whole plants are then regenerated from the totipotent transformed cells in culture, containing a copy of the transgene in every single cell (Nottingham, 1998).

HUMAN APPLICATIONS OF PLANTIBODIES
Plantibodies have many applications. They can be used to protect plants against DISEASE by engineering the plant to express antibodies against a particular plant pathogen. However, this section will describe the use of plantibodies for the treatment, and more importantly, the prevention of human disease.It has been shown that large amounts of complex multimeric immunoglobulin protein can be successfully produced and assembled within the plant. These can be purified using simple affinity chromatography methods. Secretory antibodies survive longer in the body than serum antibodies due to protection from the secretory component present in the mucosa. The secretory IgA antibody protects the mucosal surfaces of the body as a primary defense against pathogen attack. Therefore the production of secretory antibodies in plants has a lot of therapeutic potential.When engineering plants to express antibodies intended for use in medical therapy, the following steps are taken:
Obtain the neutralizing human antibody and determine its sequence
Engineer the genes coding for the antibody into the plant germ line for field scale production
Harvest the crop, mill and purify the antibody product
Formulate the pharmaceutical product
Undergo clinical trials to test for safety and efficacy of product

TREATMENT OF DENTAL CARIES : Plantibodies were first produced in tobacco plants by the biotechnology company EPIcyte in California. However, the first clinical evidence for the effectiveness of plantibodies in the treatment of human disease was obtained at Guys Hospital (Hikmat et.al., 1998). Plantibodies were produced in tobacco against the bacteria Streptococcus mutans. This bacteria is responsible for causing the majority of tooth decay by producing lactic acid which erodes tooth enamel. The research carried out involved painting the purified secretory plantibodies onto teeth for a three week period. It was found that this provided protection from S. mutans for up to four months, with the plantibodies specifically inhibiting bacterial attachment to the tooth surface. It has been shown that it is possible to produce secretory monoclonal antibodies in plants that are capable of preventing bacterial colonization in the human body. The anti Streptococcus mutans plantibody which has been developed not only as a cure, but also a preventative treatment against dental caries. This is an alternative approach to local passive immunization without any systemic side effects. An IgG monoclonal antibody was raised to the cell surface streptococcal antigen I/II. The antibody was repeatedly applied to the teeth of Rhesus monkeys. The monkeys had decreased colonization of the antibody compared to control animals. It is thought that the antibody opsonises the S. mutans facilitating phagocytosis and killing by the gingival traffic of neutrophils.(Lehner et. al.)

TREATMENT OF HERPES: Sexually transmitted disease is one of the most worrying international epidemics of the modern day, therefore, the production of monoclonal antibodies in plants for the treatment of these diseases is promising. . The biotechnology company EPIctye holds the exclusive license for plantibody technology from the Scripps Research Institute in California. The company are currently in pre-clinical trials with a topical gel containing plantibodies against the Herpes Type I and II virus (Zeitlin et. al.). This has already been proved to be effective against the virus when applied to the vagina of mice. It is hoped in the future that a more potent plantibody will be produced that will be used to protect new born babies from transmission of the virus from infected mothers during birth.In October 2000, EPIctye Pharmaceutical Inc. were awarded a grant for Phase I and Phase II trials for the development of antibodies for sexual health products. This grant is to be used for the development of anti-HIV(Human Immunodeficiency Virus) and anti-HSV(Herpes Simplex Virus) plantbodies for the production of a microbicidal lubricant. News from the 2000 International AIDS conference in South Africa concluded that there is a dire need for products to prevent the spread of AIDS (this disease develops as a consequence of infections by the HIV virus.) EPIcyte's plantibody technology provides an attractive way of producing large quantities of monoclonal antibodies in plants for the treatment of this disease . Currently antibody production is too expensive and has limited therapeutic application due to the huge scale-up costs. The production of antibodies in planta could produce unlimited quantities of pharmaceutical grade antibodies at a significantly lower cost than those produced in animal cell culture.

THE FUTURE
The future of monoclonal antibodies is very promising. A lot of this technology is still in clinical trials, but the possibility of producing large amounts of monoclonal antibodies raised to a specific antigen at low cost surely has unlimited therapeutic applications. It is thought that disease prevention is the key application to this technology. Sexually transmitted diseases are becoming increasingly prevalent in todays society, costing the government $12 billion in the USA for treatment. The production of an over-the-counter topical gel derived from plantibodies capable of killing sexually transmitted pathogens may be available in the future. It is also thought that it may be possible to deliver the plantibodies via control release devices such as pessaries. These are not only relatively cheap to produce, they also provided a user-friendly way of delivering treatment to the patient.

PLANT APPLICATIONS OF PLANTIBODIES
Using proteins that belong exclusively to the realm of animals to help plants fight viruses and nematode infections in planta is an important application of plantibody technology. It is possible to modulate phytohormone activity and hence block pathogen infection, however, much research is being carried out in an attempt to achieve high level accumulation of antigen-specific antibodies and antibody fragments. It is possible to engineer specific recombinant antibodies against cellular pathogenic antigens. These are able to block regulatory functions of pathogens or block pathogens directly in specific target plant organs. It is possible to target antibodies to specific cellular compartments and hence block specific secreted antigens. This is termed INTRACELLULAR IMMUNISATION; modulating antigens inside the plant cell.Expressing antibodies in plants can also be used to study the actions of plant hormones such as Abscissic Acid (ABA). This has successfully been carried out by engineering anti-ABA ScFv's targeted directly to the Endoplasmic Reticulum (ER) of leaf cells. This blocks the action of ABA on stomatal functioning facilitating the study of the effects of this hormone on stomata

EXAMPLES
Cytosolic ScFv's were engineered against the Artichoke Mottled Crinkle Virus coat protein. These were found to give decreased viral infection and delayed symptoms.
Anti-Tobacco Mosaic Virus (TMV) transgenic plants containing full-length anti-TMV secreted antibodies were created. These showed decreased TMV necrotic lesions correlated with antibody expression.
Transgenic tobacco plants containing secreted ScFv's against a 25KDalton coat protein from the Beet Necrotic Yellow Vein Virus were created. The presence of the plantibodies gave partial protection against the viral infection and its associated symptoms.
Transgenic tomato plants were engineered to express antibodies against the Tomato Spotted Wilt Virus movement proteins with a view to assessing the levels of antibody mediated resistance to the virus.

PROTECTION AGAINST VIRUS INFECTION IN PLANTS: There are many examples of transgenic plants that express components of the viral genome, conferring resistance to specific viruses. It is thought that by expressing, for example, the viral coat protein genes, interference of viral replication occurs, hence rendering the plant resistant. However, this technique has many risk issues attached to it, due to the presence of viral DNA sequences present in human foodstuffs.It is now possible to express antibodies against viral proteins as an alternative approach. This paper describes the isolation of antibodies against the Artichoke Mottled Crinkle Virus (AMCV) coat protein... termed the F8 antibody. Variable (heavy) and Variable (light) chains were amplified using PCR ( the Polymerase Chain reaction) and their antigen binding affinity was tested using the ELISA staining technique. The ScFv fragments were expressed in the Nicotiana benthamiana plant; a natural host for AMCV. The plants showed reduced AMCV accumulation.This was the first example of a plant phenotype with attenuation of viral infection derived from a constitutively expressed virus-specific antibody
It can be seen that there is an UNLIMITED REPERTOIRE of antibody specificities that can be exploited by the plant breeder.. providing new and novel disease resistances.
ScFv's appear to be crucial as they are functionally stable in the cell cytoplasm and are suitable for immune modulation of selected cytoplasmic antigens
It is unknown exactly how plantibodies work. More information is required on virus-antibody interactions in early infection stages. It has been suggested that antibody binding to Calcium ion binding sites on the coat protein would uncoat the virus
The exact location of the antibody fragment within the cell is crucial, as it appears that antibody Compartmentalization plays a big part in determining protection for the plant.

CONCLUSIONS
Production of antibodies in plants has numerous applications not only to the pharmaceutical industry but also the plant breeder. If clinical trials are successful, plants could provide a "factory system" for the production of huge amounts of monoclonal antibodies for the treatment of disease. They could also be used an internal protection system for plants against their own specific pathogens
Plants can be engineered to express both antibody fragments and full length fragments, it has been shown that plants are capable of correctly assembling and folding complex antibody molecules. Antibody fragments such as ScFv's are often assembled in the plant more efficiently, however, full length antibodies are less prone to proteolysis in the plant cell
Problems have been encountered with regard to differential glycosylation within the plant cell. This is due to the presence of smaller plant glycans (thought to have different terminal sugar residues) used in N-linked glycosylation. When using plantibodies for therapeutic purposes and plant protection, it must be taken into account that differing glycosylation could decrease the antigen-binding affinity of the antibodies produced. It has also been suggested that the presence of foreign glycans could produce an allergenic effect on administration to humans.
There is currently a lot of research being carried out into the applications of plantibodies. In plants, protection against viral and nematode infection are at the primary targets of the technology. In humans, a lot of work has been carried out into treatment of dental caries, caused by the bacteria Streptococcus mutans and the treatment of the Herpes virus.
This technology holds a lot of promise for the future, hopefully contributing to the treatment of many diseases. EPIctye pharmaceuticals are currently developing a topical gel for the treatment of the Herpes Simplex Virus, with a view to using this to treat new born children born to infected mothers. It is hoped that subsequent antibody-based topical contraceptives will be formulated from antibodies produced by plants. The Respiratory Syncytial Virus which causes acute pulmonary infection and Clostridium difficile which is a major cause of diarrhea are also targets for plantibody technology. Eventually, it is hoped that it may be possible to treat the Human Immunodefficiency Virus (HIV) using antibodies derived from plants.