Plantibodies Approach

Kabeya J. Muamba Dept of Crop Physiology University of Agricultural Sciences, Bengaluru, India

Antibodies or antibody fragments produced in plants are often referred to as plantibodies, and they can be exploited for ex planta applications in the field of molecular farming. Many reports describe how very efficiently transgenic plants produce complete antibodies or antibody fragments, such as Fab and single-chain variable (scFv) fragments

The structure of antibody

 Single Chain Variable Fragment (scFv) is a fusion of the variable regions of the heavy and light chains of immunoglobulins, linked together with a short (usually serine, glycine) linker.

After isolation and purification from the plant tissue, the antibodies or antibody fragments can be used in :a. ex planta application/ industrial processes, as diagnostic tools, for immunochromatography, or in medical therapy b. in planta application , where the strategy is known as immunomodulation, in which antibodies or antibody fragments are produced to modulate the function of a corresponding antigen.

As such, immunomodulation can be used to study the function of the antigen or even of the epitope in plants, to change agronomic traits, or to immunize the plant against pathogen infection

Figure 1. In planta and ex planta applications of antibodies and antibody fragments produced in plants. Plants can be engineered to synthesize antibodies in order to obtain pathogen resistance (intra- and extracellular immunization) or to manipulate the plants metabolism, such as the immunomodulation of enzyme or signal molecule activity. Isolation and purification, if necessary, allows the use of plant-made antibodies for diagnosis, affinity-based purification, and therapy.

 In Tobacco plants accumulat scFv fragments in the ER that bind to abscisic acid (ABA) were shown to wilt under ambient conditions (Artsaenko et al., 1995). These plants exhibited an increased transpiration rate, indicating that they were unable to close their stomata.

This might occur because of the concentrated scFv antibodies in the ER provide an ABA sink and prevent the transport of the hormone and its interaction with ABA receptors in the guard cells of the stomata

 Transgenic lines that accumulate high levels of the scFv in the ER showed a dwarf phenotype and lower gibberellin A1 levels than wild type Owen et al. (1992) demonstrated receptor activity modulation with an scFv directed against the plant regulatory receptor protein, phytochrome. Seeds from transgenic plants expressing the scFv gene displayed aberrant phytochrome-dependent germination

INTRA- AND EXTRACELLULAR IMMUNIZATION AGAINST PLANT-PATHOGEN INFECTION

A major goal of molecular breeding is the generation of transgenic plants that show enhanced resistance to pathogen infection

Antibody-mediated virus resistance is seen as an attractive alternative to the various forms of pathogen-derived resistance
Most plant viruses are RNA viruses that replicate in the cytosol. As such, the highest resistance is expected by targeting the antibody to this cell compartment.

 Besides using immunomodulation to obtain virus resistance, production of a scFv directed against the major membrane protein of the stolbur phytoplasma proved to be an elegant strategy to control mycoplasma (phytoplasma and spiroplasma) infection
Plantibody-mediated resistance against complex eukaryotic, multicellular pathogens, such as nematodes, fungi, and insects, remains a major challenge.

Alternative methods to control pests, such as the plantibody approach, would reduce pollution from synthetic pesticides and make agriculture more sustainable.
Resistance against root-knot nematodes (Baum et al., 1996; Rosso et al., 1996), which is one of the worlds most damaging agricultural pests, has been tested upon accumulation of the IgM antibody 6D4. IgM 6D4 binds a glycoprotein of unknown function in the stylet secretions of the root-knot nematode, Meloidogyne incognita.

Isolation of antibody and antibody fragment-encoding sequences

Antibody genes are usually isolated from monoclonal hybridoma cell lines

However, hybridoma technology is expensive and time-consuming and, in general, plant laboratories do not have the facilities to work with animal cell cultures
Phage display technology, on the other hand, allows the isolation of antigen-binding scFv-encoding sequences directly from established libraries or from the spleen of immunized mice. Phage display makes use of generally applied recombinant DNA techniques

Monoclonal antibodies (mAb or moAb) are antibodies that are identical because they are produced by one type of immune cell that are all clones of a single parent cell

Antibody genes are usually isolated from monoclonal hybrid cell lines

 Phage display is a system in which a protein is

displayed on the surface of a phage as a fusion with one of the coat proteins of the virus. The DNA that encodes this protein is housed within the virion.
By cloning large numbers of DNA sequences into the phage, phage display libraries are produced with a repertoire of many billions of unique displayed proteins.

M13 Bacteriophage Structure

The antibody fragment are produced in the periplasm of E. coli and display on the pIII coat proteins of M13 bacteriophage
Through several rounds of panning, antigenbinding scFv clones are selected from the library. From the bacterial clones, antibody fragment-encoding sequences are available for further cloning in plant expression vectors.

M13 Bacteriophage Gene Functions

Gene II

Function DNA Replication

Amino Acids 410

Mol Wt 46137

X
V VIII

DNA Replication
Binding ssDNA Major capsid protein

111
87 50

12672
9682 5235

III
VI VII

Minor capsid protein

Minor capsid protein Minor capsid protein

406
112 33

42522
12342 3599

IX
I IV

Minor capsid protein

Assembly Assembly

32
348 405

3650
39502 43476

scFv isolated from phage display libraries

M13 phage
scFv

Conclusion With information on the life cycle of the pathogen at hand, a reasonable prediction can be made on the best way to approach engineering resistance The fungi secrete enzymes and toxins essential for fungal pathogenesis as part of an intimate relationship between the host plant and pathogen. These proteins and toxins are suitable targets that could be neutralized by expressed recombinant antibodies. It has found that viral infection can be blocked in the cytosol, which is expected because so many steps of viral reproduction require the cytosol

 The first successful use of antibodies to create a virus resistant plant was using single-chain antibody fragments specific for artichoke mottled crinkle virus (AMCV) (Tavladoraki et al. 1993).

The single chain antibody was constructed from a monoclonal antibody selected from a panel raised against the AMCV virion.
Antibody-based viral resistance is likely to be increased in effectiveness in the future as antibodies that target viral proteins crucial for replication (replicase), viral movement, and transmission are used in place of antibodies specific for structural components, like coat proteins.

Antibody-based resistance will benefit from technology for cytosolic antibody expression.

advances

in

the

Transgenic tomato plants expressing a scFv fragment derived from the F8 library are specifically protected from CMV infection

WT

scFv

Imagine a world in which any protein either naturally occurring or designed by man could be produced safely, inexpensively and in almost unlimited quantities using only simple nutrients, water and sunlight.. J. Ma et al. Nature Review Genetics, Oct 2003