**2. Transgenic plants in farming**

Since the discovery of genetic mechanisms of reproduction and biodiversity, plant breeders methodically try to exploit agronomically desired traits for more profitable crop production in many aspects. In the last quarter of the twentieth century, genome manipulation techniques known as genetic engineering were introduced into various organisms. In the early years of this novel technology in plant science, desired traits were related to higher yield, resistance to various biotic and abiotic stress factors. Considerable success has been obtained in enhancing photosynthetic capacity, increasing root and leaf size, stimulating vegetative growth, improving biomass and more. Following transgenic approaches aimed nutritional quality through enhancing various biomolecule production and increased shelf life as well [15]. In recent days, transgenic technology may even offer some solutions to the global energy crisis through improvements in biofuel production. A total of 17 million farmers from 29 different countries surged in biotech crops in the period from 1996 to 2019 after the first successful commercialized release. During these 24 years, global biotech crop planting increased 112- fold from 1.7 million to 190.4 hectares. In the second third of this period, trends in leading biotech cropproducing countries started to change as well. Since 2011, 24 developing countries produced 56% of total biotech crops while 5 leading industrial countries share the rest of the 44%. This trend tends to accelerate as long as other developing countries realize the potential. A total of 19 countries among 29 are considered as "biotech mega" with at least 50,000 hectares planting. United States, Brazil, Argentina, Canada and India are the leading planters. Soybean, maize, cotton and canola are the most planted biotech crops worldwide (**Figure 1**). Plants as alfalfa, sugar beet,

#### **Figure 1.**

*Worldwide biotech crop production [16].*

potato, apples, squash, papaya and eggplant share only 1.8% of total production besides these four plants.

Biotech crops, which have reached 224.9 billion US dollar global value have also contributed to conserving biodiversity by preventing deforestation, reducing pesticide requirement, decreasing CO2 emissions in agricultural applications and alleviating socio-economic conditions of small farmers.

Transgenic plants are generally classified into three generations. First-generation GM crops were developed against various biotic and abiotic stress factors, while the second generation targeted better nutritional quality [15]. The remarkable amount of progress has been achieved in countless laboratory practices with many different plant species. On the other hand, commercial GM varieties are considerably limited compared to these laboratory practices. As it can be seen in **Table 2**, all commercial GM traits belong to the first and second generations of transgenic approaches. In laboratory practices, abiotic stress tolerance is one of the key aspects of desired crops. However, abiotic stress tolerance mechanisms and pathways are extremely intricate. Defining a particular gene target for an abiotic stress factor and introducing it to a susceptible variety is usually an insufficient strategy due to these complex responses within and between species. Plants have developed stress signal perception and transduction pathways that regulate stress-inducible genes through transcription factors (NAC, WRKY, MYB, bZIP, DREB/CBF), kinases and phosphatases. Main stress-inducible genes are kinases, molecular chaperones, osmoprotectants, transcription factors [17–20].

Many biomolecules are identified with their known protective roles against abiotic stresses. Therefore, these molecules are potential gene targets for transgenic abiotic stress tolerance approaches. Transgenic regulations of solutes such as glycine betaine, mannitol, trehalose and proline which acts as an osmoprotectant, metal chelator, antioxidative defense molecule and signal molecule have been used to enhance stress tolerance in many plants. For an instance, codA expressing GM indica rice plant which has enhanced glycine betaine production through increased choline oxidase activity, present induced water stress tolerance [21]. Abiotic stress factors usually cause misfolding and precipitation of crucial proteins. Heat shock proteins (HSPs) act as molecular chaperones and mediate folding, assembly, translocation and degradation of misfolded proteins. As a molecular chaperon,

#### *Next Generation of Transgenic Plants: From Farming to Pharming DOI: http://dx.doi.org/10.5772/intechopen.102004*



#### *Genetically Modified Plants and Beyond*

**Table 2.**

*Registered commercial GM traits and related crops [16].*

#### *Next Generation of Transgenic Plants: From Farming to Pharming DOI: http://dx.doi.org/10.5772/intechopen.102004*

transgenic *Trichoderma harzianum* hsp70 is shown to increase heat and other abiotic stress resistance in *Arabidopsis thaliana* plants [22]. Also, late embryogenesis abundant (LEA) proteins, aquaporins and calcineurin B-like proteins with antioxidant, membrane protection and ion binding functions are widely preferred targets [1]. Rab28 LEA gene over-expressing maize plants are reported to have improved desiccation tolerance under a constitutive maize promoter [23]. Likewise, transgenic expression of halophilic fungus *Aspergillus glaucus* AgGlpF gene encoding an aquaporin protein confers extreme salt tolerance in soybean [24].

Other commercially registered GM traits are altered insect and disease resistance, growth/yield, herbicide tolerance, modified product quality and pollination control system. Insect and disease resistance is mainly obtained through introducing natural pest genes from insects, fungi and bacteria to target plants. The most known example of the application is δ-endotoxin insecticidal protein-expressing cry gene transfer from *Bacillus thuringiensis* (Bt) which is extremely effective against lepidopterans, dipterans and coleopterans. In recent days, there are several alternatives to Bt toxins including lectins, protease inhibitors, antibodies, peptide hormones [15]. In particular, protease inhibitors (PIs) have devastating effects on insect digestive systems since most insects facilitate serine-type proteinase enzymes in digestion. Serine-type mustard trypsin inhibitor −2 (MTI-2) expressing *Brassica napus (L*.) plant are proven to present insecticidal properties on *Pterostichus madidus* beetle. It was also found effective on *Plutella diamondback* moth xylostella as the intermediary pest species [25]. As an alternative to Bt toxin alone, lectin genes were also introduced for insecticidal properties. Codon-optimized synthetic Bt Vip3Aa gene under CaMV35S promoter and *Allium sativum* leaf agglutinin gene under phloem-specific promoter transformation significantly improved *Helicoverpa armigera* resistance. As mentioned in environmental stress factors earlier, transcription factors are known to play important roles in plant resistance to environmental stress factors. MYB4L transcription factors were shown to induce ethylene pathway and enhance tobacco mosaic virus (TMV) resistance in *Nicotiana benthamiana*. On the other hand, silencing the transcription factor intensified the susceptibility [26]. Similarly, the WRKY17 transcription factor was proven to both enhance artemisinin biosynthesis in a traditional Chinese medicinal plant *Artemisia annua,* and provide resistance against *Pseudomonas syringae*. Two defense marker genes, pathogenesisrelated 5 (PR5) and NDR1/HIN1-LIKE 10 (NHL10), were significantly increased in AaWRKY17-overexpressing transgenic *A. annua* plants as well as AaWRKY17 directly bound to the promoter region of the artemisinin biosynthetic pathway gene amorpha-4,11-diene synthase (ADS) and promoted its expression.

Herbicide resistance can be maintained through two transgenic approaches. The first involves the modification of the target enzyme to overcome herbicide sensitivity. In the second approach herbicide, detoxifying pathways are introduced to the susceptible target plants. Glyphosate (N-phosphonomethylglycine) is a highly efficient, low-toxicity, broad-spectrum and nonselective herbicide that has been widely applied. Glyphosate specifically inhibits the activity of 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), a key enzyme in the shikimate pathway and prevents aromatic amino acid synthesis unselectively in any plants. Therefore, fortification of target enzymes in intended plants is crucial. Recently, cotransformation of a codon-optimized glyphosate oxidase gene WBceGO-B3S1 from a variant BceGO-B3S1 and a glyphosate-tolerant gene I. variabilis-EPSPS from the bacterium *Isoptericola variabilis* into an *O. sativa* variety by *Agrobacterium*-mediated genetic transformation resulted in high glyphosate tolerance [27]. In the second approach, the herbicidal effects of glufosinate, which is a nonselective, glutamine synthetase (GS) inhibitor, are targeted. The GS enzyme produces glutamine amino acids from ammonia and glutamate. Glufosinate causes glutamine deficiency in

susceptible plants through ammonia and glyoxylate accumulation, inhibition of photosynthesis due to defected chloroplast structure. Transformation of the pat gene from *Streptomyces viridochromogenes* to susceptible plants leads to expression of phosphinothricin acetyltransferase (PAT) enzyme in plants which metabolize glufosinate into N-acetyl-L-glufosinate (NAG). This non-toxic compound does not inhibit GS enzymes [28].

The first two generations of transgenic plants in farming applications consistently enhanced food supply and essential traits. Following the improvements within this technology, it became more friendly to the environment, less risky for human health, more profitable to small scale farmers and more regulated around the world. The third generation will be the era of recombinant pharmaceuticals as plant-based vaccines against pathogens in human health and beneficial health products as therapeutic proteins, monoclonal antibodies, hormones, enzymes along with transgenic bioenergy plants which are not going to be discussed in this chapter.

#### **3. Transgenic plants in pharming**

Excluding transgenic farming applications which were summarized in the previous section, the term of molecular farming through transgenic plants refers utilization of plants for recombinant protein production instead of microorganisms and animal cell in fermenter systems. When we narrow this definition from recombinant proteins to pharmaceutical products still in plants basis, we define the alternative term "molecular pharming". Before the revolutionary contribution of Stieger et al. [29] in which they presented the capability of plant cells to produce multimeric assembled mammalian antibodies in *Acetabularia mediterranea*, the general opinion was that functional full-length mammalian antibodies would not assemble in plants without mammalian chaperones. Before this accomplishment, there were various well-established production systems as *Escherichia coli* in prokaryotes, *Saccharomyces cerevisiae* in single-cell eukaryotes, Chinese Hamster Ovary (CHO) cells, non-secreting murine myeloma (NS0) cells, Sp2/0 HEK293 mammalian cells for recombinant protein production in which there were boundaries of the expensive fermenter and good manufacturing practice (GMP) required for pharmaceutical production. After successful production practices of mammalian IgG1 antibody in tobacco [30] and human serum albumin (HSA) in transgenic potato and tobacco plants [31], the molecular pharming approach rapidly accelerated and production systems like tobacco, potato, tomato, alfalfa, safflower, carrot, lettuce, strawberry, moss, duckweed, maize, wheat and rice were emerged. Besides the plant species, there are various approaches differing in plant tissues (whole plant, hairy roots, cell suspension etc.), expression type (stable, transient, transplastomic, tissue-specific, inducible) and product targeting (post-translational modifications and accumulation targets) [4]. Today, there are thousands of different recombinant proteins produced in plant systems in which we can also include antibody, vaccine, hormone or enzyme type pharmaceuticals. There is also an increasing number of companies producing commercial plant-based therapeutics (**Table 3**). Along with the obvious advantage of pharming in scale-up production, this approach also has a downside on downstream processing (including maintaining product quality, extraction and purification) due to the wide range of plant metabolites.

#### **3.1 Monoclonal antibodies, viral antigens and vaccines**

Recombinant monoclonal antibody (mAbs) production in pharming applications has grown rapidly since the first reported IgG1 antibody in transgenic tobacco by


*Next Generation of Transgenic Plants: From Farming to Pharming DOI: http://dx.doi.org/10.5772/intechopen.102004*


**Table 3.**

*Plant-based therapeutic producing companies and commercial products.*

Andrew Hiatt [30]. In the beginning, it was not an easy task due to the complicated nature of antibodies on basis of post-translational modifications, folding patterns and structural assembly. IgG is the simplest immunoglobulin structure, therefore only two plant genes are required to produce fully functional IgG in plant systems. On the other hand, IgA which has four heavy chains and four light chains requires the expression of four genes at the same time. Early antibody studies also considered producing antibody fragments, mini bodies, large single chains, single-chain variable fragments (scFvs), bispecific scFvs, diabodies and fusion proteins as well [47]. Some

#### *Next Generation of Transgenic Plants: From Farming to Pharming DOI: http://dx.doi.org/10.5772/intechopen.102004*

achievements on targeting the mAbs into different plant cell parts for accumulation followed in a short time. The first plant-derived IgM was produced in *N. tabacum* and targeted into chloroplasts. Constructed chimeric genes and the barley aleurone α-amylase signal peptide coding sequence which had already been used successfully to transport bacteriophage T4 lysozyme from transgenic tobacco cells to the intercellular spaces was introduced to *N. tabacum* to initiate the secretory pathway of chimeric IgM in transgenic plants. Subcellular localization of IgM, presented the assembly of the antibody in the endoplasmic reticulum and the targeted accumulation in chloroplasts. Assembly and targeting of complex foreign protein in the transgenic plant were shown through fusing the individual chains to a plant signal peptide [48]. Secretory IgA was also expressed recombinantly. Four transgenic *N. tabacum* plants were generated that expressed a murine monoclonal antibody kappa chain, a hybrid immunoglobulin A-G heavy chain, a murine joining chain and a rabbit secretory component, respectively. Sexual crosses were achieved among the transgenic tobaccos resulting expression of all four protein chains simultaneously. These chains were assembled into a functional, high molecular weight secretory IgA which recognized the native streptococcal antigen I/II cell surface adhesion molecule. Transgenic plants were suggested as suitable systems for large-scale production of assembled recombinant secretory IgA for passive mucosal immunotherapy since plants require a single cell to assemble secretory antibodies while mammalian cells require two different cell types [49]. Transgenic plant-derived antibodies (plantibodies) are thought to be particularly effective in topical immunotherapies which are based on the antigenic competition by using immunomodulators to induce hypersensitivity. In recent days, mABs are designed for various purposes as chemotherapeutics for cancer, antibody-mediated passive immunization against highly contagious infectious diseases as SARS and COVID-19, curing or slowing down disease progression, active immunization through antigens (**Table 4**).

mABs may aim cancer cells in different mechanisms. They can directly bind and flag cancer cells for immune cells prevent angiogenic properties, stimulate disruption of the cell membrane, block immune system inhibitors, retard cancer growth, act as chemotherapy or radiotherapy agent carrier. Targeted antigens are generally related to growth and differentiation including epidermal growth factor receptor (EGFR), carcinoembryonic antigen (CEA), human epidermal growth factor receptor 2 (HER2), vascular endothelial growth factor (VEGF), VEGF receptor (VEGFR), fibroblast activation protein (FAP). mAbs are not native products of plant tissues. However, the transgenic plant approach provides the capacity to induce production and modification of mAbs through posttranslational modifications as glycosylation to enhance therapeutic efficacy. Pembrolizumab which is an anti-human PD-1 monoclonal mAb has been produced in wild-type *Nicotiana benthamiana* through transient expression. 344.12 ± 98.23 μg/g fresh leaf weight Pembrolizumab accumulation was obtained after 4 days of agroinfiltration. Molecular characterization of plant-based Pembrolizumab was compared to mammalian cell-based commercial counterpart Keytruda®. Physicochemical properties of plant-based Pembrolizumab were found comparable to Keytruda® with similar secondary and tertiary structures. Both products presented no aggregation differences and binding efficacy to PD-1 protein and inhibitory activity between programmed cell death 1 (PD-1) and programmed cell death ligand 1 (PD-L1) interaction. In this respect, researchers concluded that plant-produced Pembrolizumab could induce IL-2 and IFN-γ production and plant-based production of functional Pembrolizumab can be utilized for immunotherapy purposes [50]. In another anticancer approach, heavy and light chains of mAb BR55-2 were expressed separately and assembled in plant cells of transgenic tobacco plants (*N. tabacum* cv. LAMD609). Production was as high as 30 mg/kg of fresh leaves in the first generation of plants. Like in mammalian counterpart, the Fc


#### *Genetically Modified Plants and Beyond*


*Next Generation of Transgenic Plants: From Farming to Pharming DOI: http://dx.doi.org/10.5772/intechopen.102004*

> **Table 4.**

*Monoclonal antibodies produced from transgenic plants.*

domain of the plant mAbP presented a similar binding to FcγRI receptor (CD64) and bound to both SK-BR3 breast cancer cells and SW948 colorectal cancer cells, specifically. This plant-derived BR55-2 also inhibited SW948 tumor growth in nude mice, efficiently and was suggested as a possible immunotherapy option [65]. Both samples represent IgG-type plant-derived antibodies. However, more complex IgA-type plant-derived products are also presented with better efficacy, recently. The secretory component (SC) of immunoglobulin A (SIgA), which is an efficient therapeutic antibody against mucosal pathogens, was successfully expressed in *A. thaliana*. The expression level of SC was increased in the plant system through the insertion of endoplasmic reticulum retention signal peptide, KDEL (Lys-Asp-Glu-Leu), into a binary vector with translational enhancer and an efficient terminator [66]. This approach was also reported as useful against food poisoning causing *E. coli* virulence factor Shiga toxin through recombinant IgA (S-hyIgA) produced in transgenic *A. thaliana* plants [67].

Plant-derived antibodies and viral antigens were also targeted for highly contagious infectious diseases as SARS, Ebola, Zica, Hepatitis B, AIDS and even the most recent COVID-19 (**Tables 5** and **6**). SARS-CoV-2 is a single-stranded RNAenveloped virus, which has 29,881 bp genome encoding 9860 amino acids belonging to structural proteins, spike (S), envelope (E), membrane (M) and nucleocapsid (N) and 16 non-structural proteins (such as 3-chymotrypsin-like protease, papainlike protease and RNA-dependent RNA polymerase) and 9 accessory proteins. N protein which is the most abundant viral protein shown to be highly conserved in CoV families. It is highly immunogenic during CoV infections. It is a major target for antibody responses and also contains T cell epitopes [82, 83]. Posttranscriptional gene silencing suppressor p19 protein from tomato bushy stunt virus substantially demonstrated the transient expression of recombinant SARS-CoV nucleocapsid (rN) protein in *Nicotiana benthamiana*. The rN protein accumulated up to a concentration of 79 μg per g fresh leaf weight in the agrobacteria-infiltrated plant leaf after the third day of infiltration. BALB/c mice were intraperitoneally vaccinated with pre-treated plant extract emulsified in Freund's adjuvant and plantexpressed recombinant SARS-CoV N protein-induced strong humoral and cellular responses in mice [74]. Also, iBio company developed a plant-derived vaccine targeting the N protein in their IBIO-202 program which is under pre-clinical trials. M and E proteins contribute very low on protection owing to their small ectodomains for immune cell recognition and small molecular sizes and poorly immunogenic for humoral responses. Glycosylated S proteins cover the SARS-CoV-2 surface and bind to the host cell receptor angiotensin-converting enzyme 2 (ACE2) during viral cell entry. Hence, S protein is the main viable vaccine target against the ongoing pandemic for the time being [84]. Rapid production of SARS-CoV-2 receptor-binding domain (RBD) and spike-specific monoclonal antibody CR3022 were achieved in *Nicotiana benthamiana*. Both RBD and mAb CR3022 were transiently produced with the highest expression level of 8 μg/g and 130 μg/g leaf fresh weight respectively at 3 days post-infiltration. The plant-produced RBD exhibited specific binding to the SARS-CoV-2 receptor, angiotensin-converting enzyme 2 (ACE2) [52]. In July 2020, phase 1 study was initiated for a plant-derived virus-like particle (VLP) vaccine candidate called CoVLP which expresses the SARS-CoV-2 spike glycoprotein (CoVLP: NCT04450004). It has been evaluated through 21 days apart from two doses of 3.75 μg, 7.5 μg or 15μg vaccine alone or with AS03 or CpG1018 adjuvants in healthy adults 18–55 years of age. Based on the available data two-dose schedule of CoVLP at 3.75μg per dose adjuvanted with AS03 has been carried forward into ongoing phase 2/3 studies in Canada and the United States, with planned expansion to additional countries in Latin America and Europe [85]. In December 2020, the Kentucky BioProcessing company announced its phase 1 plant-derived


#### *Next Generation of Transgenic Plants: From Farming to Pharming DOI: http://dx.doi.org/10.5772/intechopen.102004*

**Table 5.**

*Plant-based vaccines for non-infectious diseases.*


