Plants in Production of Recombinant Antibodies

A discussion of the techniques, advantages and disadvantages of the use of plants in production of recombinant antibodies for research and therapeutic use with named examples.
Introduction   
Application of plant for medical proposes goes back to thousand years ago. Our ancestors use plants and extract its beneficial substance to cure different illnesses and relief pain. This idea is continued with us and today we can manipulate the genetic information of plants to make them suitable for the production of recombinant protein and biopharmaceutical medicinal purposes [1]. Since the first generation of recombinant protein from tobacco cell culture, a variety of pharmaceutical products have been introduced such as vaccines, hormones, antibody, growth factors, and cytokines [1,4,9]. However, AB is the most common recombinant protein which is generated by plants and it has been called plantibody. Nowadays the development and the use of transgenic plants for production of recombinant ABs is an attractive subject among scientists because plants are easy to work with and also the cost of the production is considerably low. It is also beneficial because of the large-scale production [1].

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Production of high quality recombinant protein for research and therapeutic purposes from mammalian are quite expensive, therefore the idea of producing recombinant protein in transgenic animals and plants has formed recently [1]. In this essay, I try to summarise and highlight some of the most cutting-edge techniques in the use of transgenic plants for production of recombinant protein and antibody. I also discuss their advantages and disadvantages with the utilization of plants to produce antibody.
Plantibody
Plantibody made up of two words: plant and antibody. It means plantibody is an AB that is generated from plants. Antibody is a glycoprotein which mainly made by plasma cells and used by the immune system to neutralize any kind of foreign molecules inside the body such as bacteria and virus. Plantibody has this property to recognize and bind to its specific antigen. It can be generated from tobacco, rice cells culture, Lemna minor (duckweed), Arabidopsis thaliana seeds, Medicago Sativa (alfalfa), lettuce and maize [2] but tobacco is the most common source due to its high leaf biomass yield [3].
According to a report, recombinant protein level in tobacco stem is the same as this level in its leaf. That means to produce recombinant therapeutic protein, the whole plant biomass can be used [3]. Another advantage of tobacco is that it is not edible and this aspect of tobacco reduce biosafety concern but it contains toxic alkaloid and the plant should be purified from the toxic chemicals [3]. As tobacco is not an edible source, regulatory issue for production of recombinant protein is less controversial than food crops such as rice, soy bean and corn. Chinese cabbage has the highest amount of soluble protein among plants.

Production Techniques
Production of the recombinant protein includes utilizing the whole plant or plant cell culture in vitro [9]. The disadvantages of using the whole plant for production of recombinant AB are: time-consuming generation of transgenic plants, the risk of contamination with fertiliser, unstable quality and yield of the products, applying good manufacturing practice (GMP) to the whole-plant production pipeline [9]. Plant cell suspension culture has the benefits of both mammalian cell culture and whole plants. Undifferentiated plant calli can be developed under a proper condition in the liquid media environment and produce cell suspension culture. Plant cell culture can generate proteins which are more similar to human generated proteins. They can also grow rapidly in a simple media same as bacteria. Plants are eukaryote so they have fairly similar post-transitional modifications such as glycosylation that happen in human cells [9]. Glycosylation is an enzymatic process that glycan adds to organic molecules such as lipids and proteins. Correct pattern of protein folding is also essential for recombinant protein to function [1]. It is interesting to note that plant suspension cell culture lack fully functional plasmodesmata, therefore, systemic post-transcriptional gene silencing (PTGS) may be reduced because PTGS is transmitted through plasmodesmata and the vascular system [9].
Generally, three different methods are applied in the production of recombinant AB in plants: Agroinfiltration with recombinant agrobacteria, particle bombardment technology and Infection with modified viral vector [8,14]. The general technique for the production of genetically modified plants is agrobacterium-mediated transformation [2]. Agrobacterium Tumefaciens is a gram negative bacteria which is the cause of crown gall disease in plants [14]. These bacteria live in soil and attract to the plants with wounded parts. However, scientists use this bacteria as a tool for research and therapeutic purposes by introducing the gene with desired properties into the plant cells in plant genetic engineering. The gene of interest can be inserted into Ti plasmid (tumor inducing) then injected into the plants as a host. Plant cell divide out of control and the gene of interest proliferate as well [14]. There is a selectable marker on the T-DNA which is transferred into the host cells therefore it is possible to control if the gene is transferred successfully or not [2].
There are two transformation strategies for generation of recombinant antibody, Stable and transient expression. Stable expression is the stably insertion of cDNA encoding both heavy and light chains of AB into the genome of plants. The gene can be introduced into the chloroplast genome to produce chloroplast transgenic plants which can generate AB with correct folding and disulfide bonds. Some example of the transient expression is agroinfiltration and recombinant plant viruses for the production of antibody [3]. Agroinfiltration system has been used to produce multi-antennary N-glycan that mostly seen in mammalian derived glycoproteins [3]. Transient expressionmethod is fast and convenient for the production of recombinant antibody without generation of transgenic plant. The generation of transient expression is the precondition to stable transformation because it can test expression vectors and protein stability and also it is able to recognize any problem that may have happened [8]. Transient expression is better for low scale yield protein production yet transgenic plant are better approach for high yield production and also gives a better expression levels [7]. An important point to note is if the expression is targeted to the endoplasmic reticulum (ER), this results in higher yield [7].
Another approach for the insertion the gene of interest into the plant tissue is “particle bombardment technology”. The main idea of this technique is some microscopic golden bullet or tungsten bullet covered by the gene of interest. These particles are fired into the plant leaf. This technique used for all type of plants. The golden bullet preferably used because the tungsten bullets have the risk of toxicity for the plants. Then the bullet is placed at the end of plastic bullets and shoot with blasts of air or helium. There is a plastic mesh work shop on the way of the bullets which guide the bullet to move forward. An alternative technique used for this approach which can accelerate the beads with strong electrical discharge which results in a controlled penetration of beads into the plant tissue.
After penetration of the DNA dissolved into the cytoplasm of the leaf, the gene of interest can recombine with the chromosome of a plant. Finally, the leaf is transferred to media and let it grow and regenerated using tissue culture [8,11]. This technique does not use a lot due to its high cost and also as this method is physical so the insertion of the gene which is performed by gene gun may cause damage to plant without transferring the genetic material inside the plant and dose not give the precise or desirable results [14]. Production of ab transgenic plants can be generated by viral vectors. However low infectivity with this vectors needs to be considered as an obstacle [2]. One of the disadvantages of viral plant system is the injection of vector to leaf or stem every time which can result in gene mutation during replication of the virus. But we don’t face this problem in transgenic stable expression. Therefore, it is extremely important to choose the proper protocol for gene expression [3].
Advantages and Disadvantages
Plants paly an important role as a bioreactor for production of recombinant protein. Basically, the common systems use for the production of recombinant proteins is the manipulation of mammalian cells, bacterial systems, yeast and etc. However, recently due to some negative aspects of these systems many scientists prefer to work and study plant sources which have those benefits that they are looking for. There are several important benefits with the production of recombinant AB from plants. Firstly, is the large scale of production from cheap raw materials and the reduction of costs in comparison with other techniques of recombinant AB production such as yeast, mammalian and etc. [3,5].
Another advantage of using plants for production of AB is the flexibility of working with plants as it can be used both in vivo and in vitro [3]. In addition, introducing new transgenic plants is possible by sexual crosses and they are quite easy to work with. There is a very low risk of contamination by mammalian viruses when AB is generated from plants [5]. Another advantage is correct folding and assembly of produced AB for both single stranded peptides and multimeric protein with full size. Recombinant protein which generated from edible sources does not require purification. In terms of storage the enzymes which are produced by plants can be formulated to the seeds, so under the suitable condition they can be stored for long period of time and it is also possible to transport them to different locations easily. Plantibody have both avidity and affinity towards its specific antigen and its characteristics maintain the same after purification [1].
Although plants have lots of benefits but it is not 100% perfect source for production of antibody [3]. The most important disadvantage is the fact that Plant N-glycosylation is different from human and mammalian glycosylation. Another negative point is that plants has shown discrete yields due to low gene expression level [7]. There is also the problem with causing allergic and immunogenic reactions in humans, which is because of the difference in glycosylation pattern in humans and plant [7]. Moreover, there are some concerns regarding the activity of proteolytic degradation, which might influence fully assembled IgG that is secreted in the culture media [9]. Production of mycotoxin by impurities, limitation which caused by the environmental condition, and the possibilities of herbicides presence in the product are some other negative aspect of transgenic plants [1]. The controversy about plantibody generation is the presence of gene segments or marker segments in the produced drug and its effect on human body and the probability of allergic reaction to plant glycoprotein [1].
Although there are some disadvantages with the use of transgenic animals such as the risk of contamination of protein with animal viruses and also it takes a long time to produce recombinant protein from transgenic animals but, many biotechnologists prefer to produce AB from mammalian cell lines because the final ABs have a correct glycosylation pattern and protein folding [1].
Plant Antibody Application
The extracted AB from plants can be used for many different purposes such as vaccine production, clinical diagnosis protein, pharmaceutical and industrial proteins, biopolymer, biodiesel, food industry, tools for research, and diagnosis tool for chromatography and other immunoassays [1]. The application of AB in research is extremely wide, because of their transferability with the metabolic process in organism [1]. Protein pharmaceutical products are one of the most expensive and important products that human has managed to synthesis them in ways other than natural methods. In recent years, mAB has had an important role in the diagnosis and treatment of cancer-related research [3,12].
Each mAB influence cancer cells in 3 ways: it can signal to the immune system to kill cancerous cells, it can prevent the division of cancer cells or deliver drug to these cells [3]. mAB can attack tumour cells by complement system in cytotoxic reactions through complement system. They bound to the tumor cells which prevent tumor growth and finally result in apoptosis [3]. The ability of AB to prevent the pathogens and tumor cells is due to the affinity of the variable binding sites. This affinity of AB could have enhanced by modifying glycon structure and glycosylation patterns [3]. As we see mAB have many positive aspects for prevention of cancer but their application is not common which is duo to the risk of contamination with human pathogens, high cost and proliferation inability. However, these problems have been eliminated by the production of mAB from other bio-organism like bacteria, yeasts, insects, and plants [3]. The monoclonal AB expressed in plants by tobacco mosaic virus vectors [3].
Nimotozomab is a humanized anti-epidermal growth factor receptor recombinant AB which is produced in animal cell culture. This AB is used for treatment of different carcinoma cells. It seems that a mutation in the N297 position in the IgG1 FC region of this AB and apply it in a transgenic plant which result in producing a form of nomotozomab that is similar to mammalian-cell-produced AB. It also has the property to block the EGFR interaction and have antitumor effects [5,7]. Nicotiana tabacum were transformed by A.tumefaction-mediated gene transfer method. In order to infect the plant cells, recombinant pDEGF-R Agbacterium bearing the binary vector was applied [5,7]. According to experiments the mAB which was generated in plants was as effective as the one which was generated in mammalian (nude mice). In another experiment marine, mAB could prevent Brest cancer cell growth and mAB was generated from transgenic tobacco plant which had the same function as the murine mAB. Therefore, plants such as tobacco can produce two different mAB which can target two different types of cancer cells [3]. The most frequently chosen host cell lines used for recombinant protein expression are Tobacco BY-2 (Bright yellow-2) and NT-1 (Nicotiana tabacum-1) cells [9].
Generally, IgA, IgG and IgM are generated from plants. IgA and IgM have the potential for commercial production. They attach antigens in the first line defence at gastrointestinal mucosal surface, tears, saliva and milk [14]. IgG and IgA have been introduced in Nicotiana, Arabidopsis.
Plantibody have a high level of safety which rise the interest for production of mAB from plant Examples include the Guy’s 13 IgG1 (Fischer et al., 1999b; Sharp and Doran, 2001a, 2001b), a human mAB against hepatitis B virus surface antigen (HBsAg) (Yano et al., 2004), a human anti-rabies virus mAB (Girard et al., 2006), and most recently a human anti-HIV mAB (Holland et al., 2010) all of which have been reported to be expressed in tobacco cell suspension cultures [9]. Lots of effort have been done for production of these ABs in large scale but none of them sell in the market due to the high cost. Nonetheless, two plantibody is used in clinical CAROX which was expressed in transgenic tobacco that takes part in the prevention of tooth decay and the second one have an effect against non-Hodgkin-lymphoma(NHL) [2]. The following table demonstrates some IgA plantibodies which are generated in recent research.

Plantibody

Source

Target

Plantibody Characteristic

sIgA/G

Transgenic Tobacco Plant

S. Mutans

Prevention of tooth decay

Human IgA

Maize

Herpes Simplex Virus and saga 1 antigen

Herpes disease and sperm agglutination

Coccidia specific chicken IgA

Nocotiana Benthamiana

Eimeria Acervulina

Against the coccidiosis

Virus-specific IgA

Tomato and Nocotiana Benthamiana

Rota Virus

Development for passive immunisation against Diarrhoeal disease

Chimeric Enterotoxigenic Bacteria-Specific IgA (VHH-IgA)

Arabidopsis Thaliana seeds

Enterotoxigenic Escherichia Coli (ETEC)

Passive Mucosal Immunisation Against Enteric Infections

Chimeric Toxic-Specific IgA (Hybrid IgG/IgA)

A. Thaliana

Shiga Toxin From ETEC

Against Haemorrhagic colitis and Hemolytic-uremic Syndrome

Monomeric IgA1 K! Variants (Infliximab, Adalimumab, Ustekinumab)

N. Benthamiana

–

Against Autoimmune disease

2G12 sIgA

N. Benthamiana

Human Immunodeficiency Virus

Anti-HIV Human

This table shows IgA plantibodies, their sources, targets and characteristics.
Conclusion and future perspectives
Although there are problems with the generation of plantibody from mammalian cells, but they are the most common source for production of mABs. This is due to the correct folding and similar glycosylation patterns to human, complex type N-glycosyl, moieties and the presence of polypeptides with disulfide bonds. Using recombinant antibody fragment in research therapeutic purposes, biotechnology and pharmaceutical science is increasing because of the intrinsic properties of the components such as the ability to penetrate better and detect antigen with higher affinity, small size and easy production compared to AB full size [6,13]. More powerful tissue or inducible promoters, enhancement of transcript stability, translational improvement with cutting edge sequences or strategies and transgenic chloroplast system are some ways which are studied in order to raise the AB expression level in plants in the future [8]. Drug production seems to be one of the promising field in terms of commercial development in biotechnology [1].
In total, we can see a promising future for the production of drugs, vaccine, recombinant protein and biopharmaceuticals from plants. However, several bottlenecks including regulatory guidelines, ethical issues and public approval must be taken into account and solved [1].
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