An Insight Review on Application of Plantibodies

Tilahun et al. Available Ind. J. Pure online App. Biosci.at www.ijpab.com (2019) 7(6), 29 -41 ISSN: 2582 – 2845

DOI: http://dx.doi.org/10.18782/2582-2845.7900 ISSN: 2582 – 2845 Ind. J. Pure App. Biosci. (2019) 7(6), 29-41 Review Article

Hiwot Tilahun1, Guesh Negash1 and Haben Fesseha2*

1College of Veterinary Medicine, Mekele University, PO box 2084, Mekelle, Ethiopia 2School of Veterinary Medicine, Wolaita Sodo University, PO box 138, Wolaita Sodo, Ethiopia *Corresponding Author E-mail: [email protected] Received: 8.11.2019 | Revised: 14.12.2019 | Accepted: 20.12.2019

ABSTRACT A plantibody is an antibody produced by plants that have, as well as the advantage, challenge, and limitation of plantibodies, with animal genes. The term “Plantibodies” was created to describe the products of plants that have been genetically modified to express antibodies and antibody fragments in plants. Productions of plantibodies offer several advantages over other methods of antibody production, such as low cost of production, high yield of antibodies, safe and effective approach and bring the product to the market within a short time. Six plant-derived antibodies have been developed as human therapeutics, two of which have reached phase II clinical trials. Agricultural crops such as tobacco, tomato, potato, soya bean, alfalfa, rice, and wheat are commonly used for the production of plantibodies. These plantibodies are formed by various methods like conventional method, cell tissue culture method, breeding, and sexual crossing, transgenic seeds, targeting and compartmentalizing. Plantibodies are now used for

large scale medical and veterinary sectors for the treatment of immune disorders, cancers,

inflammatory diseases, for the production of vaccines and also for diagnostic purposes. This review highlights the methods of production and application of plantibodies as well as the various types of pharmaceutical antibodies produced in transgenic plants. Hence, scholars

should promote their application and use in the field of human and veterinary medicine.

Keywords: Antibody; Application; Plantibody; Production; Transgenic plant

INTRODUCTION for a variety of applications, including the Antibodies, also called immunoglobulins, are a diagnosis, prevention, and treatment of group of complex glycoproteins produced by disease. Typically, antibodies are composed of B-lymphocytes and present in the serum and a basic unit, comprising two identical 'heavy' tissue fluids of vertebrates. They constitute the polypeptide chains and two identical 'light' humoral arm of the adaptive immune system polypeptide chains, which is covalently linked and specifically recognize and bind to target together by intermolecular disulfide bonds antigens on pathogens or toxins produced by (Andersen & Krummen, 2002; Oluwayelu & such pathogens. This individual and specific Adebiyi, 2016). binding activity allows antibodies to be used

Cite this article: Tilahun, H., Negash, G., & Fesseha, H. (2019). An Insight Review on Application of Plantibodies, Ind. J. Pure App. Biosci. 7(6), 29-41. doi: http://dx.doi.org/10.18782/2582-2845.7900

Tilahun et al. Ind. J. Pure App. Biosci. (2019) 7(6), 29-41 ISSN: 2582 – 2845 Vaccines are traditionally produced in animals applied. In this regard, plants have several and administered to humans for their potential advantages over other production therapeutic value. Likewise, various strategies systems. Most importantly, Production of have been developed to exploit plants as recombinant proteins in plants and their bioreactors for the production of administration provides an added margin of pharmaceutical antibodies, to engineer safety when compared to that produced from antibody-mediated pathogen resistance or to transgenic animals (Koprowski, 2005). In alter the plant phenotype by addition, the capacity of plant cells to correctly immunomodulation (Stoger et al., 2002). Now fold and assemble, not only antibody a day’s antibody production in plants has fragments single-chain peptides, but also full- acquired significance as an emerging system length multimeric proteins, since they have for the production of many recombinant similar pathways of protein synthesis, proteins that can be used for therapeutic secretion, folding and post-translational purposes (Raja et al., 2014). modifications. Plants do not serve as hosts for A plantibody is an antibody produced human pathogens. Therefore, there is by plants that have been genetically increased safety (Raja et al., 2014). engineered with animal DNA. The term Six plant-derived antibodies have been “Plantibodies” was created to describe the developed as human therapeutics, two of products of plants that have been genetically which have reached phase II clinical trials. modified to express antibodies and antibody One of these is a full-length Immunoglobulin fragments in plants. Although plants do not G (IgG) specific for EpCAM (a marker of naturally make antibodies, plantibodies have colorectal cancer) developed as the drug been shown to function in the same way as Avicidin by NeoRx and Monsanto. The five normal antibodies. Plants are being used in this remaining antibodies are CaroRx, scFvT84.66, technology as antibody factories, using their Anti-HSV, 38C13 and PIPP (anti-hCG) endomembrane and secretory systems to (Doran, 1999). Though plantibodies have produce large amounts of clinically viable many advantages in the treatment of different proteins, which can later be purified from the pathologic conditions however, the technology plant tissue (Jain et al., 2011). is not well known and practiced in Africa in The combination of antibodies and general and in Ethiopia in particular especially plant engineering, two rapidly advancing in the veterinary sector. Hence, in this review, technologies, has led to emerging strategies in the application of plantibodies, the advantage, diversified plant species (Deepali et al., 2016). challenge, and limitation of plantibodies are Although antibodies were first expressed in described. plants in the mid-1980’s, (Steger and Duering) 2. CROPS AND PLANTS USED FOR by two German graduate students (During et PLANTIBODY PRODUCTION al, 1990), the first report was published in A large number of different crops can be used 1989 (Hiatt et al., 1989). Since then a diverse to produce antibodies including tobacco group of “plantibody” types and forms have (Nicotiana tabaccum and N. benthamiana), been prepared. Originally, foreign antibody cereals (rice, wheat, maize), legumes (pea, genes were introduced into plant cells by non- soybean, alfalfa) and fruit and root crops pathogenic strains of the natural plant (tomato, potato). Many factors should be pathogen Agrobacterium tumefaciens (Horsch considered before choosing the crop variety. et al., 1985) and regeneration in tissue culture Leafy crops like tobacco generally have the resulted in the recovery of stable transgenic greatest biomass yields per hectare, because plants (Duering et al., 1990; Deepali et al., they can be cropped several times a year. 2016). Tomatoes also have high biomass yield, but Nowadays, molecular farming of production costs are increased (Schillberg et antibodies using plants becomes commonly al., 2002). Copyright © Nov.-Dec., 2019; IJPAB 30

Tilahun et al. Ind. J. Pure App. Biosci. (2019) 7(6), 29-41 ISSN: 2582 – 2845 Antibodies expressed in potato tubers and recombinant antibodies. Alfalfa is a perennial cereal grains are stable at room temperature crop that can propagate easily and also have for months or even years without loss of good biomass yield (Bardor et al., 2003). One stability, while tobacco leaves must be dried or of the potential advantages of alfalfa is that frozen prior to transport or storage to maintain recombinant antibodies produced as a single the activity of recombinant proteins. However, glycoform rather than a heterogeneous the extraction of proteins from seeds is more collection of different glycoforms that are expensive than from watery tissue, such as found in other plant systems. Pea, a grain tomatoes (Stoger et al., 2002). legume also a useful production crop, the The presence of toxic metabolites such reason for its high protein content of the seed. as alkaloids in tobacco presents a Although at present only low yield is possible disadvantage, so edible plants lacking such with this species (Perrin et al., 2000). compounds would be preferred expression Cereals, Seeds, and Tubers: - Cereals, seeds, hosts. Since most pharmaceutical antibodies and tubers are better sources of plantibodies will be produced by industry, the costs of when we are mainly targeted for long term production and processing in different crops storage. They can be stored at room will have to be evaluated very carefully temperature and are easily transportable. The (Fischer et al., 2003). most commonly used cereals are rice, wheat, 2.1. Criterion for Selecting Host Plant and maize along with legumes such as pea, Before selecting a plant for the production of and soya bean (Schillberg et al., 2002). plantibodies, some points should be Antibodies expressed in potato tubers and considered. Most importantly they are other cereal grains are stable at room generalized by five main factors such as the temperature from months to years together, plant should be easily genetically engineered, while tobacco leaves must be dried or frozen capable of producing high level of specific for long term storage. However, the extraction proteins, should have a well-established of proteins from seeds is more expensive than technology for gene transfer and expression, watery tissue, such as tomatoes (Fischer et al., knowledge of agricultural techniques and 2003). management, physiology, pest and disease of Fruits and Vegetables: - Fruits and crop/ plant selected should be known and the vegetables can be consumed raw or as partially hosts plants should have no toxic or side processed material, which makes them suitable effects on the animal body (Poonam et al., for passive oral immune therapy. E.g. 2018). Tomatoes, bananas, Potato, etc. Tomatoes 2.2. Sources of Transgenic Plants have outstanding properties for pharmaceutical Tobacco: - Tobacco, among leafy crops, has protein production, such as high biomass yield the greatest biomass yield per hectare and and the advantage of contained growth in the allows rapid scale-up because it can crop greenhouse. Hence, tomatoes were first used to several times in a year (Fischer et al., 2003). produce a plant-derived rabies vaccine (Stoger Tobacco grows quickly and has been shown to et al., 2000). produce a comparatively large quantity of Potato has been used widely for the antibodies. Additionally, tobacco is a non- production of plant-derived vaccines and been food/non-feed crop if grown separately; there administered to humans in most of the clinical is less chance of cross-contamination food trials so far (Prasad et al., 2017). Transgenic chain by pharmaceuticals (Schillberg et al., tomato-based developed edible vaccine 2002). However, the presence of toxic expressed cholera toxin B against cholera in metabolites is hindrance for its use (Poonam et the ripening tomato fruit under the control of al., 2018). tomato fruit-specific E8 promoter using Alfalfa and other legumes: - Alfalfa and soya Agrobacterium-mediated transformation (Jiang bean are another leafy crop used to produce et al., 2007). Copyright © Nov.-Dec., 2019; IJPAB 31

Tilahun et al. Ind. J. Pure App. Biosci. (2019) 7(6), 29-41 ISSN: 2582 – 2845 3. PURIFICATION AND TECHNIQUE OF ultrafiltration steps (Gegenheimer, 1990; Priya PRODUCTION OF PLANTIBODIES et al., 2011). 3.1. Purification Alternative methods including the use The main reason for raising antibodies in of oleosin- or polymer-fusions to facilitate plants is its easy purification and low purification of recombinant proteins have been downstream processing. Easy Purification of discussed and may also be applicable to plantibodies makes biopharmaceutical antibody molecules (Daniell et al., 2001). The production more economic (Arntzen, 1998). main techniques used for the purification of Transgenic seeds assure excellent storage plantibodies are filtration, properties and thus added flexibility in immunofluorescence, chromatography, processing management and batch production. defiltration, polymer fusion and protein A- Separation of plantibody in seeds is less sepharose chromatography. Some other complicated because of the limited range of techniques such as RIA (Radioimmunoassay), endogenous proteins (Kusnadi et al., 1997). northern blot technique, ELISA (Enzyme- Efficient purification procedures for linked immune sorbent assay), western blot plant production systems are as important as analysis and immunofluorescence southern effective expression levels. In a plant blot analysis have been used of evaluation of expression system, the plant tissue and cells plantibody (Prasad et al., 2017). should be disrupted to release antibodies for 3.2. Methods for Plantibody Production purification since the antibody is expressed Various techniques have been developed to and localized within the cells. In addition, exploit plants as bioreactors for the production antibodies should be recovered with the of pharmaceutical antibodies (Prasad et al., removal of cell debris, noxious chemicals, and 2017). contaminants (Valdes et al., 2003). 3.2.1. Conventional method Although several affinity tagging It uses stable transformation and transient systems such as the histine tag and intein fusion expression have been applied for expression to introduce new genes into a host purification of recombinant proteins, these cell. Once the desired DNA from the systems do not resolve the problems caused by transformed host cell is isolated and purified, it plant cell debris. Therefore, oleaginous plants can be injected into the embryo of a maturing such as rapeseed oil are useful hosts for plant, which we want to use for plantibodies monoclonal antibody (mAb) production and production. After injecting the desired gene, purification since the oil bodies can be applied followed by the propagation of plants in open to simplify the first steps of antibody isolation fields allow large scale production of (Seon et al., 2002). plantibodies (Moffat, 1989). However, The absence of human pathogens in purification of these proteins is long and plants eliminates expensive validation of virus tedious, since, upon isolation of the antibody, removal steps during purification. But the several proteins, organic molecules glycans, probability of presence of a diverse burden on and herbicides must also be isolated, leading to plants grown outdoors in non-sterile a complex purification process (Kusnadi et al., conditions is high. So, the process for 1997). elimination or minimization of contamination 3.2.2. In vitro cell and tissue cultures with endotoxin and mycotoxins will be This is an economically important method for necessary in all commercial processes to producing plantibodies wherein plant cells in purify antibodies. Phenolics can interact with differentiated stages are grown under proteins in ways that can irreversibly alter the controlled conditions having desired genes/ properties of proteins but most of the proteins and are harvested either in the form of phenolics released during extraction are small biomass and culture liquid or combination of in size, water-soluble, and removable by both. This method offers large amounts of Copyright © Nov.-Dec., 2019; IJPAB 32

Tilahun et al. Ind. J. Pure App. Biosci. (2019) 7(6), 29-41 ISSN: 2582 – 2845 recombinant proteins produced in a shorter 4. APPLICATION OF PLANTIBODIES time (Doran, 1999). The use of transgenic plants for the expression This method has many advantages of molecules with therapeutic, diagnostic or over the conventional method of plantibodies veterinary applications has been documented production by overcoming the problem of in the last decade. The applications are extracting and purifying proteins but this increasing because recombinant DNA is very method has not used for edible vaccine useful in creating proteins that are identical production. In this method, no sexual when exposed to a plant's (Ayala et al., 2009). reproduction is needed, so transgenic stability This technology represents a great opportunity for the pharmaceutical industry since is increased because of the absence of crossing biological products now account for a large over, segregation and recombination and percentage of all pharmaceutical compounds. provides more chances in plantibodies Several plant-produced antibodies are production (Ferrante & David, 2001). presently undergoing clinical trials (Oluwayelu 3.2.3. Breeding and sexual crossing & Adebiyi, 2016). This method involves transformation to 4.1. Therapeutic Applications introduce kappa chains of either light or heavy Therapeutic applications of plantibody are the chains into the host plant. The same is done treatment of infectious disease, inflammation, with gamma chains of either light or heavy autoimmune disease or cancer. Tobacco regions. Upon crossing one plant with kappa produced mAb is a more viable alternative to chains and another plant with gamma chains, mAb produced in mouse ascites fluid for the an antibody is produced which expresses both large amounts needed for the purification of chains (Jain et al., 2011). This method the hepatitis B vaccine. Plant produced provides an easy way to produce plantibodies antibodies have also been investigated for without the need for double fertilization inflammatory disease and to induce tolerance (Ferrante & David, 2001). (Prasad et al., 2017). 3.2.4. Transgenic seeds A caries vaccine the first clinically Some researchers suggest the use of transgenic tested plantibody produced in the world that seeds in place of green plant tissue for the prevent and protects against tooth decay. production of plantibodies as green plants Streptococcus mutans has been identified as cannot store antibodies for a long period of the major etiological agent of human dental time. This is because they contain proteases caries. The development of a vaccine for tooth that degrade the recombinant protein. Thus, decay has been under investigation for more than 30 years. Planet Biotechnology developed transgenic seeds can store antibodies for an a monoclonal antibody against S. mutans, extended period without degradation because branded CaroRx, produced with transgenic they contain a low level of proteases tobacco plants. It is the first plant-derived (Oluwayelu & Adebiyi, 2016). antibody created from tobacco (Fischer et al., 3.2.5. Targeting and compartmentalization 2006). Targeting signals can be used to retain CaroRx is a Sig A secretory antibody. recombinant proteins within different It is a clinically advanced anti Streptococcus compartments of the cells such as endoplasmic mutans secretory immunoglobulin, a reticulum, chloroplasts, intracellular space, etc. plantibody that binds specifically to the This is done to preserve the integrity, protect bacterium, thus protecting humans from dental them from proteolytic degradation and to carries (Larrick et al., 2001). CaroRx® has increase accumulation levels of the been used in human trials and a tobacco recombinant protein. This is achieved by plantibody against a poultry virus (Newcastle tagging the antibodies with a small peptide disease) has been approved by the United sequence which can be targeted to a specific States Department of Agriculture (USDA) compartment of the cell (Jain et al., 2011). (Vermij & Waltz, 2006). Copyright © Nov.-Dec., 2019; IJPAB 33

Tilahun et al. Ind. J. Pure App. Biosci. (2019) 7(6), 29-41 ISSN: 2582 – 2845 Anti-tumor antibodies against Burkitt`s administered. There is no antigenic stimulus lymphoma was expressed in rice and wheat and the recipient’s immune system has no (Ghasempour et al., 2014). Antibodies active role. The immunity is transient usually engineered to bind to Bacillus anthracis was lasting only for days or weeks only till the extracted from transgenic strains of tobacco passively transmitted antibodies are and tested in mice in an experiment. The result metabolized and eliminated. The main of this study showed that the antibodies were advantage of passive immunization is it acts effective in fighting B. anthracis strain well for immediately and can be employed in the future in any anthrax epidemic will be a conditions requiring instant immunity (Stroger cheap and effective prevention against the et al., 2002; Raja et al., 2014). disease (Hull et a.l, 2005). The production of proteins in plants is Treatment or cure for rabies through a major task in producing pharmaceutical plantibodies has been investigated. A polypeptides. Potential proteins produced plantibody-based rabies vaccine produced in include cytokines, hormones, enzymes, tobacco was experimentally administered in epidermal growth factors, interferons, human hamsters to identify whether it could protein C, and pharmaceutical foodstuff which effectively target rabies. According to the are considered for oral immunization. plantibody proved to be a safe and Transgenic plants that express antigens in their economically feasible alternative to the current edible tissue might be used as an inexpensive methods of antibody production in animal oral vaccine production and delivery system. systems (Ko et al., 2003). Thus, immunization might be possible through Another study, tobacco-derived the consumption of an "edible vaccine" to plantibodies were experimentally administered provide passive immunization and disease in mice against the Lewis Y antigen found on prevention (Mason & Arntzen, 1995). tumor cells in mice and also in lung, breast, Genetically engineered plants and ovarian and colorectal cancer. According to plant viruses are also used to produce vaccines the plantibodies showed a definite positive against several human diseases for life- effect on the cancer-stricken mice by threatening infections such as diphtheria, preventing tumor formation in them (Brodzick cholera and Acquired immune deficiency et al., 2006). syndrome (AIDS) (Moffat, 1995). Some of the Antibodies against ovarian, testicular proteins are potent inducers of immune and colon cancer as well as melanoma, B-cell responses but some immunizing proteins may lymphoma, and human papillomavirus have not work well when taken orally. Oral already been expressed in transgenic tobacco. vaccines must be protected during passage These plantibodies are currently being through the hostile environment of the researched and are on their way to being stomach and intestine to the sites of immune approved for human use. Plantibodies called stimulation (Fisher et al., 2003). DoxoRx and RhinoRx for post-cancer therapy 4.3. Immunomodulation and rhinoviruses respectively are in various Immunomodulation is a molecular technique stages of completion (Fisher et al., 2003). that allows the interference with cellular 4.2. Immunization metabolism or pathogen infectivity by the Active immunization is the resistance ectopic expression of genes encoding developed in an individual as a result of an antibodies or antibody fragments (De Jaeger et antigenic stimulus which is otherwise known al., 2000). Applications that are relying on as adaptive immunity. Passive immunity is the modulating antigen levels in vivo are resistance transmitted to recipient in dependent on expression and accumulation in readymade form, preformed antibodies are specific subcellular compartments and specific

Tilahun et al. Ind. J. Pure App. Biosci. (2019) 7(6), 29-41 ISSN: 2582 – 2845 tissues. Development of crop resistance and carbohydrates, vitamins, minerals, passive immunization of plants by expression biopolymers and food (Sharma & Sharma, of pathogen-specific antibodies reduces 2009). Plant bioreactors are cost-effective and infection and symptoms caused by viruses and easy for agricultural scale-up. As a result, mollicutes, and significant progress has been certain expensive biopharmaceuticals such as made towards engineering residence against human lysosomal enzymes can be produced in insects (De Jaeger et al., 2000; Schillberg et plant bioreactors and this is particularly al., 2001). applicable in developing countries. Plant Immunomodulation is a powerful tool bioreactors have the advantages of having for studying or altering the function of an post-translational modiﬁcations and lacking of antigen in vivo. Antigen, which may be an contamination by animal pathogens (Giddings, enzyme or metabolite, can either be stabilized 2001; Lienard et al., 2007). In recent years, or blocked in its action. Physiological and many plant systems have been developed in morphological changes were observed in order to use plants as bioreactors for the plants when an artificial abscisic acid (ABA) production of recombinant antibodies for sink was created by the production of an ABA- many purposes (Stoger et al., 2002). specific scFv in the endoplasmic reticulum of 4.5. Treatment of Ebola patients tobacco and potato plants (Conrad & The production of anti-Ebola virus antibodies Manteuffel, 2001; Senger et al., 2001). has recently been explored in plants (Chen et Antibodies produced in plant-based al., 2011). A high yielding geminivirus-based expression systems are high-value products for expression system in the tobacco plant, pharmaceutical use. Plants represent cost- Nicotiana benthamiana, for the production of a effective systems for the large-scale mAb (6D8) that protected animals from Ebola production of pharmaceuticals. For complex virus infection. Using similar technology and molecular forms (secretary Immunoglobulin N. benthamiana, (Bhoo et al., 2011) produced A) sIgA, plants offer a commercially viable an Ebola immune complex (EIC) by fusing system for large-scale production (Ma et al, Ebola envelope glycoprotein (GP1) to the C- 1998). Moreover, agro filtration of tobacco terminus of the heavy chain (HC) of was used to produce a diabody against humanized 6D8 mAb that binds specifically to carcinoembryonic antigen (Vaquero et al., a linear epitope on GP1. Gemini virus vector- 2002). mediated co-expression of the GP1-HC fusion Transgenic plants are suitable for mAb and the 6D8 light chain in N. benthamiana production because they can be rapidly leaves produced assembled immunoglobulin, expanded in commercial products without the which was purified by protein G affinity high-capital investment associated with chromatography. The resultant recombinant traditional mAb bioreactor facilities (Priya et antibody bound the complement factor C1q, al., 2011). In addition, plantibodies may also indicating immune complex formation. prove useful as feed additives or for Thereafter, subcutaneous immunization of phytoremediation in human health care mice with purified EIC elicited high-level (Mason & Arntzen, 1995). production of anti-Ebola virus antibodies. This 4.4. Bioreactors was the first published account of an Ebola Antibodies produced in plants have virus candidate vaccine to be produced in applications such as the production of vaccine plants (Langreth et al., 2014; Daniel et al., antigens, protein for clinical diagnosis, 2016). pharmaceutical and industrial proteins,

Tilahun et al. Ind. J. Pure App. Biosci. (2019) 7(6), 29-41 ISSN: 2582 – 2845 Table 1: Pharmaceutical antibodies produced in transgenic plants Antigen Plant Antibody form Application Reference Human chorionic Tobacco scFv, diabody, Diagnostic/ Kathuria et al, 2002 Gonadotrophin chimeric, IgG1 contraceptive Streptococcus surface Tobacco SigA/G (CaroRx) Therapeutic Ma et al., 2003 antigen SAI/II (topical) Human IgG Alfalfa IgG Diagnostic Khoudi et al, 1999 Rabies Tobacco IgG Therapeutic Ko et al., 2003 Herpes simplex virus Soyabean, Rice IgG Therapeutic Zeitlin, 1998 (topical) Herpes simplex virus Algae One-chain antibody Therapeutic Mayfield et al, 2003 Chlamydomonas chloroplast Hepatitis B virus Lettuce IgG Vaccine Kapusta et al, 1999 New castle disease Corn Surface Vaccine Guerrero- Andrade et al, virus glycoprotein F 2006 Cholera Tomato Cholera toxin B Oral vaccine Jiang et al., 2007 subunit (ctb gene) Enterovirus Tomato Serum IgG VP1 Oral vaccine Chen et al, 2006 Porcine reproductive Banana IgG and IgA Oral Chan et al, 2013 and respiratory immunization syndrome virus

5. ADVANTAGE, CHALLENGES AND agents and they also produce a relatively high LIMITATION OF PLANTIBODY yield of antibodies in a comparatively shorter PRODUCTION time (Fisher et al., 2003). 5.1. Advantage and Challenges On the other hand, relative ease of Using plants for the production of recombinant genetic manipulation and reduced economic proteins has advantages compared with other constraints offer added advantage over expression systems such as animal systems, transgenic animals. Plants might also provide bacterial systems, yeast systems (Ghasempour an ideal vehicle for oral delivery of vaccine et al., 2007). Plantibodies work in a similar antigens owing to the presence of thick cell fashion to mammalian anti-bodies; however, walls composed of cellulose and sugars that compared to conventional methods using may provide protection against degradation by mammalian cells, the use of plants for the gastrointestinal tract (Koprowski & antibody production offers several unique Yusibov, 2001; Bindu, 2017). advantages. Firstly, plants are widespread, After lots of benefits, there are some abundant, and grow quickly; they usually remaining challenges are associated with mature after one season of growth and it is plantibodies production. During down possible to bring the product to the market streaming processes of plantibodies production within a short time. Therefore, the cost of such as extraction and purification of antibodies produced by plants is substantially plantibodies is an important step, covers more less than that from their animal counterparts than half of the total cost. Thus, the (Sharma et a.l, 2004). purification system during plantibodies Secondly, plants are less likely to production is very expensive. Currently, the introduce adventitious human or animal purification system in plant systems, an pathogens compared to mammalian cells or affinity purification protocol re-quires protein transgenic animals, thus reducing screening A-based matrix is mainly used (Valdes et al., costs for viruses, prions and bacterial toxins 2003). So, it is necessary to use alternative (Herbers & Sonnewald, 1999; Lai et al., 2010). economic methods that use oleosin or polymer Moreover, they do not trigger immune fusions for the purification of plantibodies responses in which animal antibodies are (Daniell et al., 2001). prone to do when exposed to foreign/non-self Copyright © Nov.-Dec., 2019; IJPAB 36

Tilahun et al. Ind. J. Pure App. Biosci. (2019) 7(6), 29-41 ISSN: 2582 – 2845 5.2. Limitations of Plantibodies as bioreactors on a larger scale would reduce Despite having so many advantages there are the cost of antibody therapy and increase the also some limitations to its application. number of patients with access to these Several studies have shown that people may treatments thus scholars should advocate the have negative reactions to plant-derived application of plantibodies in the field of allergens, fungal contaminations and veterinary medicine. Application of pesticides used during farming, inability to plantibodies is an important biotechnological perform post-translational modification of breakthrough in the field of medicine therefore produced proteins, insufficient expression in it should be encouraged. some plants, environmental restrictions, allergies or allergic reactions to plants REFERENCES glycoprotein and other plant antigens, Andersen, D., & Krummen, L. (2002). mycotoxins produced by impurities, herbicides Recombinant protein expression for and plant endogenous metabolites, gene therapeutic applications. Current silencing in some cases and different patterns Opinion in Biotechnology, 13, 117- of glycosylation and regulatory issues, 123. particularly for therapeutic proteins requiring Arntzen, C. (1998). Pharmaceutical food stuffs approval for human use. Plantibodies are not oral immunization with transgenic suitable for infants (Stoger et al., 2002; Doshi plants. Nature Medicine, 4, 502-503. et al., 2013; Rani & Usha, 2013; Hashemzade Ayala, M., Gavilondo, J., Rodríguez, M., et al., 2014). Fuentes, A., Enríquez, G., Pérez, L., Cremata, J., & Pujol, M. (2009. CONCLUSION Production of plantibodies in Nicotiana Transgenic plants have been shown to be the plants. Methods in Molecular Biology, most productive and economical system for 483, 103-34. making antibodies for human and animal use Bardor, M., Loutelier-Bourhis, C., Pac-calet, as they play a key role in providing T., Cosette, P., Fitchette, A., & Vezina, therapeutics and edible vaccines, which are L.P. (2003). Monoclonal C5-1 cheap and easy to administer. Plants have been antibody produced in transgenic alfalfa used for the large-scale production of several plants exhibits a N-glycosylation that is recombinant proteins, especially for homogenous and suitable for glycol- biopharmaceutical applications with particular engineering into human compatible emphasis on antibodies and vaccine structures. Plant Biotechnology components. The low-cost, high-scalability Journal, 1, 451-462. and safety characteristics of a plant-based Bhoo, S., Lai, H., Ma, J., Arntzen, C., Chen, production system offer an attractive Q., & Mason, H. (2011). Expression of alternative for both commercial an immunogenic Ebola immune pharmaceutical products and for complex in Nicotiana benthamiana. manufacturing products. However, purification Plant Biotechnology Journal, 9, 807- remains the most significant cost factor for 816. biopharmaceutical production. The technology Bindu, J. (2017). Plantibodies, Paving novel is not well-practiced in Africa in general and avenues for immunotherapy. in Ethiopia in particular, especially in the MedCrave online Journal of Surgery, veterinary sector. Even though, there is a great 4, 78. diversity of crops and plants that can be Brodzick, R., Glogowska, M., Bandurska, K., readily explored by the pharmaceutical Okulicz, M., Deka, D., & KO, K. industry for therapeutic, immune prophylactic, (2006). Plant-derived Anti-Lewis Y improved livestock productivity and other mAb exhibits biological activities for purposes. In conclusion, the adoption of plants efficient immunotherapy against Copyright © Nov.-Dec., 2019; IJPAB 37

Tilahun et al. Ind. J. Pure App. Biosci. (2019) 7(6), 29-41 ISSN: 2582 – 2845 human cancer cells. Proceedings of the Deepali, G., Nimisha, W., & Sayali, L. (2016). National Academy of Sciences United Pharming proteins, the future of States of America, 103, 8804-8809. Panacea. Journal of Chemical and Chan, H., Chia, M., Pang, V., Jeng, C., Do, Y., Pharmaceutical Research, 8, 133-138. & Huang, P. (2013). Oral Doran, P. (1999). Foreign protein production immunogenicity of porcine in plant tissue cultures. Current reproductive and respiratory syndrome Opinion in Biotechnology, 11, 199- virus antigen expressed in transgenic 204. banana. Journal of Plant Doshi, V., Rawat, H., & Mukhergee, S. Biotechnology, 11, 315-324. (2013). Edible vaccines from Chen, H., Chang, M., Chiang, B., & Jeng, S. Genetically Modified crops, current (2006). Oral immunization of mice status and future scope. Journal of using trans-genic tomato fruit Pharmaceutical and Scientific expressing VP1 protein from Innovation, 2, 1-6. enterovirus. Vaccine, 24, 2944-2951. Ferrante, E., & David, S. (2001). A review of Chen, Q., He, J., Phoolcharoen, W., & Mason, the progression of transgenic plants H. (2011). Gemini-viral vectors based used to produce plantibodies for on bean yellow dwarf virus for human usage. Biological and Bio- production of vaccine antigens and medical Sciences, 1, 1-6. monoclonal antibodies in plants. Fischer, R., Richard, M., & Schillberg, S. Human Vaccines, 7, 331-338. (2003). Production of antibodies in Conrad, U., & Manteuffel, R. (2001). plants and their use for global health. Immunomodulation of phytohormones Vaccine, 21, 820-825. and functional proteins in plant cells. Fischer, R., Twyman, M.R., Hellwig, S., Trends in Plant Science, 6, 399-402. Drossard, J., & Schillberg, S. (2006). Daniel, O., Oluwayelu, Adebowale, I., & Facing the Future with Adebiyi. (2016). Plantibodies in human Pharmaceuticals from Plants. and animal health, a review. African Biotechnology and Sustainable Health Sciences, 16, 511-534. Agriculture, 13-32. Daniell, H., Lee, S., Panchal, T., & Wiebe, P. Gegenheimer, P. (1990). Preparation of (2001). Expression of the native extracts from plants. Methods in cholera B toxin subunit gene and Enzymology, 182, 174-193. assembly as functional oligomers in Ghasempour, H., Kahrizi, D., & Mehdiah, N. transgenic tobacco chloroplasts. (2007). New Discussions in Journal of Molecular Biology, 311, Biotechnology. Razi University Press, 1001-1009. Pp. 274. Daniell, H., Streatfield, S., & Wycoff, K. Ghasempour, H., Kahrizi, D., & Mehdiah, N. (2001). Medical molecular farming, (2014). Application of transgenic production of antibodies, plants as factories for producing biopharmaceuticals and edible vaccines biopharmaceuticals. Journal of in plants. Trends in Plant Science, 6, Biodiversity and Environmental 219-226. Sciences, 4, 58-74. De Jaeger, G., De Wilde, C., Eeckhout, D., Giddings, G. (2001). Transgenic plants as Fiers, E., & Depicker, A. (2000). The protein factories. Current Opinion in plantibody approach, expression of Biotechnology, 12, 450-4. anti-body genes in plants to modulate Guerrero-Andrade, O., Loza-Rubio, E., plant metabolism or to obtain pathogen Olivera-Flores, T., Fehervari-Bone, T., resistance. Plant Molecular Biology, & Gomez-Lim, M. (2006). Expression 43, 419-428. of the Newcastle disease virus fusion Copyright © Nov.-Dec., 2019; IJPAB 38

Tilahun et al. Ind. J. Pure App. Biosci. (2019) 7(6), 29-41 ISSN: 2582 – 2845 protein in transgenic maize and Production of a diagnostic monoclonal immunological studies. Transgenic antibody in perennial alfalfa plants. Research, 15, 455-463. Biotechnology and Bioengineering, 64, Hashemzade, H., Zebarjade, A., Akhshi, N., & 135-43. Aghaee, K. (2014). Application of KO, K., Tekoah, Y., Rudd, P., Harvey, D., transgenic plants as factories for Dwek, R., & Spitsin, S. (2003). producing biopharmaceuticals. Journal Function and glycosylation of plant- of Biodiversity and Environmental derived antiviral monoclonal antibody. Sciences, 4, 58-74. Proceeding of the National Academy of Herbers, K., & Sonnewald, U. (1999). Science of the United States of Production of new/modified proteins in America, 100, 8013-8018. transgenic plants. Current Opinion in Koprowski, H. (2005). vaccines and sera Biotechnology, 10, 163-168. through plant biotechnology. Vaccine, Hiatt, A., Cafferkey, R., & Bowdish, K. 23, 1757–1763. (1989). Production of antibodies in Koprowski, H., & Yusibov, V. (2001). The transgenic plants. Nature, 342, 76-78. green revolution, plants as Horsch, R., Fry, J., Hoffmann, N., Eichholtz, heterologous expression vectors. D., Rogers, S., & Fraley, R. (1985). A Vaccine, 19, 2735-2741. simple and general method for Kusnadi, A., Nikolov, Z., & Howard, J. transferring genes into plants. Science, (1997). Production of recombinant 227, 1229-1231. proteins in transgenic plants, practical Hull, A., Criscuolo, C., Mett, V., Groen, H., considerations. Biotechnology and Steeman, W., & Westra, H. (2005). Bioengineering, 56, 473-484. Human-derived plant-produced Lai, H., Engle, M., Fuchs, A., Keller, T., monoclonal antibody for the treatment Johnson, S., & Gorlatov, S. (2010). of anthrax. Vaccine, 23, 2082-2086. Monoclonal antibody produced in Jain, P., Pandey, P., Jain, D., & Dwivedi, P. plants efficiently treats West Nile virus (2011). Plantibody, An Overview. infection in mice. Proceeding of the Asian Journal of Pharmacy and Life National Academy of Science of the Science, 1, 87-94. United States of America, 107, 2419– Jiang, X., He, Z., Peng, Z., Qi, Y., Chen, Q., & 2424. Su, S. (2007). Cholera toxin B protein Larrick, J., Yu, L., Naftzger, C., Jaiswal, S., & in transgenic tomato fruit induces Wycoff, K. (2001). Production of systemic response in mice. Transgenic secretory IgA antibodies in plants. research, 16, 169-175. Biomolecular Engineering, 18, 87–94. Kapusta, J., Modelska, A., Figlerowicz, M., Lienard, D., Sourrouille, C., Gomord, V., & Pniewski, T., Letellier, M., Faye, L. (2007). Pharming and Plucienniczak, A., & Legocki, A. transgenic plants. Biotechnology (1999). A plant-derived edible vaccine Annual Review, 13, 115-47. against hepatitis B virus. Federation of Ma, J., Drake, P., & Christou, P. (2003). The American Societies for Experimental production of recombinant Biology Journal, 13, 1796-1799. pharmaceutical proteins in plants. Kathuria, S., Sriraman, R., Nath, R., Sack, M., Nature Review Genetics, 4, 794-805. Pal, R., & Artsaenko, O. (2002). Ma, J.K.C., Hikmat, B., Wycoff, K., Vine, N., Efficacy of plant produced Yu, L., Hein, M., & Lehner, T. (1998). recombinant antibodies against HCG. Characterization of a recombinant plant Human Reproduction, 17, 2054-61. monoclonal secretory antibody and Khoudi, H., Laberge, S., Ferullo, J., Bazin, R., preventive immunotherapy in humans. Darveau, A., & Castonguay, Y. (1999). Natural. Medicine, 4, 601-606. Copyright © Nov.-Dec., 2019; IJPAB 39

Tilahun et al. Ind. J. Pure App. Biosci. (2019) 7(6), 29-41 ISSN: 2582 – 2845 Mason, H., & Arntzen, C. (1995). Transgenic Schillberg, S., Emans, N., & Fischer, R. plants as vaccine production systems. (2002). Antibody molecular farming in Trends in Biotechnology, 13, 388-392. plants and plant cells. Phytochemistry Mayfield, S., Franklin, S., & Lerner, R. Reviews, 1, 45–54. (2003). Expression and assembly of a Schillberg, S., Zimmermann, S., Zhang, M., & fully active antibody in algae. Fischer, R. (2001). Anti-body-based Proceedings of the National Academy resistance to plant pathogens. of Sciences United States of America, Transgenic Research, 10, 1-12. 100, 438-442. Senger, S., Mock, H., Conrad, U., & Moffat, A. (1989). Exploring transgenic plants Manteuffel, R. (2001). as a new vaccine source. Science, 268, Immunomodulation of ABA function 658-660. affects early events in somatic embryo Moffat, A. (1995). Exploring transgenic plants development. Plant Cell Reports, 20, as a new vaccine source. Science, 268, 112-120. 658-660. Seon, J., Szarka, S., & Moloney, M. (2002). A Oluwayelu, D., & Adebiyi, A. (2016). unique strategy for recovering Plantibodies in human and animal recombinant proteins from molecular health a review. African health farming, affinity capture on engineered Sciences, 16, 640-645. oil bodies. Journal of Plant Perrin, Y., Vaquero, C., Gerrard, I., Sack, M., Biotechnology, 4, 95–101. Christou, P., & Fischer, R. (2000). Sharma, A., & Sharma, K. (2009). Plants as Transgenic pea seeds as bioreactors bioreactors, recent developments and for the production of a single-chain Fv emerging opportunities. Biotechnology fragment (scFV) antibody used in Advances, 27, 811-832. cancer diagnosis and therapy. Sharma, A., Jani, D., Raghunath, C., & Tyagi, Molecular Breeding, 6, 345–352. A. (2004). Transgenic plants as Poonam, S., Varsha, S., Anju, N., Joycee, J., bioreactors. Indian Journal of Vandana, G., Ajay, R., & Smita, B. Biotechnology, 3, 274 –290. (2018). Plantibodies as Stoger, E., Sack, M., Fischer, R., & Christou, biopharmaceuticals, A review. Journal P. (2002). Plantibodies, applications, of Pharmacognosy and advantages and bottlenecks. Current Phytochemistry, 7, 2072-2074. Opinion in Biotechnology, 13, 161– Prasad, M., Basanti, B., Manimegalai, J., 166. Ikbal, K., Ranjan, U., Pradeep, L., & Stoger, E., Sack, M., Perrin, Y., Vaquero, C., Gaya, P. (2017). Plantibodies for Global Health, Challenges and Torres, E., & Twyman, R.M. (2000). Perspectives. Biotechnology Practical considerations for Sustainability, 7, 305-321. pharmaceutical antibody production in Priya, J., Prasoon, P., Dheeraj, J., & Pankaj, D. different crop systems. Molecular (2011). Plantibody, An Overview. Breeding, 9, 149-158. Asian journal of Pharmacy and Life Stoger, E., Sack, M., Perrin, Y., Vaquero, C., Science, 1, 563-589. Torres, E., & Twyman, R. (2002). Raja, P., Tejaswi, C., Vidya, S., Practical considerations for &Satyanarayana, D. (2014). pharmaceutical antibody production in Plantibodies. Indian journal of dental different crop systems. Molecular advancements, 6, 1543-1545. Breeding, 9, 149–58. Rani, S., & Usha, R. (2013). Transgenic plants Valdes, R., Gomez, L., Padilla, S., Brito, J., Types, benefits, public concerns and Reyes, B., Alvarez, T., Mendoza, O., future. Journal of Pharmacy Research, Herrera, O., Ferro, W., Luis Vega, J., 6, 879-883. & Borroto, C. (2003). Large-scale Copyright © Nov.-Dec., 2019; IJPAB 40

Tilahun et al. Ind. J. Pure App. Biosci. (2019) 7(6), 29-41 ISSN: 2582 – 2845 purification of an antibody directed Vermij, P., & Waltz, E. (2006). United States against hepatitis B surface antigen Department of Agriculture approves from transgenic tobacco plants. the first plant-based vaccine. Nature Biochemical Biophysical Research Biotechnology, 24, 233-234. Communications, 308, 94-100. Zeitlin, L., Olmsted, S., Moench, T., Co, M., Vaquero, C., Sack, M., Schuster, F., Finnern, Martinell, B., & Paradkar, V. (1998). A R., Drossard, J., & Schumann, D. humanized monoclonal antibody (2002). A carcinoembryonic antigen- produced in transgenic plants for specific diabody produced in tobacco. immunoprotection of the vagina Federation of American Societies for against genital herpes. Nature Experimental Biology Journal, 16, Biotechnology, 16, 1361-1364. 408-410.