**6. Soybeans as bioreactors for pharmaceuticals**

Recombinant proteins are widely used in medicine, research laboratories, food and nutri‐ tion, and play a key role in important agriculture and biopharmaceutical industries. Since the development of recombinant DNA technology in the early 1970's, the commercial pro‐ duction of recombinant proteins has traditionally relied on a variety of protein expression systems, each with intrinsic advantages and disadvantages. Over the years several methods have been used to produce recombinant proteins. Traditionally, prokaryotic systems based on fermentation have been used for the production of biopharmaceuticals and enzymes. The bacteria *Escherichia coli*, being one of the earliest and most widely used host for this method, has been used in the production of human insulin since the 1970's [59]. Other platforms in‐ clude the use of fungal cells and yeast, which have been used as an expression system that is able to perform many of the post-translational modifications required by recombinant pro‐ tein production. Recombinant proteins that require more complex modifications can be pro‐ duced using insect or mammalian cells, or transgenic animals. However, major disadvantages associated with these platforms include the inability to perform complex post-translational modifications, the alteration in glycosylation patterns affecting protein ac‐ tivity, the high overall cost associated with manufacturing, the potential for contamination, and long time commitments associated with production in transgenic animals making these platforms impractical for the production of most proteins.

Enhanced amounts of α-tocopherol in soybean have proven easier to metabolically engineer. Expression of genes for two enzymes responsible for methylation of tocopherol head groups (VTE3 and VTE4) from *Arabidopsis* were co-expressed within the seed and generated plants with α-tocopherol levels greater than 90% of total tocopherol content [54]. The total levels of tocopherol remained the same in these seeds, showing a shift in tocopherol to mainly the αform yielding a 5-fold increase in vitamin E activity. This research can lead to soybean oil

A Comprehensive Survey of International Soybean Research - Genetics, Physiology, Agronomy and Nitrogen

Soy is also deficient in several essential dietary amino acids, most notably methionine and cysteine due to their high sulfur contents. Albumins from Brazil nuts, sunflowers, and corn have been expressed in soybean and although they resulted in increased methionine and cysteine levels, they are not adequate enough to avoid supplementation of these amino acids in animal feed and human diets. The physical synthesis of cysteine is carried out by the en‐ zyme O-acetylserinesulfhydrylase (OASS). In an attempt to increase sulfur containing amino acids in soybean, Kim et al., overexpressed cytosolic OASS and found that transgenic seeds contained elevated levels of both protein bound cysteine (58-74%) and free cysteine (22-32%) [55]. Another approach used to increase sulfur amino acid content in soybean is the use of a maize zein gene which gives rise to several species of insoluble proteins containing high lev‐ els of methionine. Dinkins et al., overexpressed maize zein in soybean seeds and observed a 12-20% increase in methionine and 15-35% increase in cysteine without adverse effects on

The essential amino acids lysine and threonine have also been explored for their potential to create nutritionally enhanced soybean. An increased level of lysine was observed by geneti‐ cally engineering the lysine biosynthetic pathway to circumvent the normal feedback regu‐ lation of the enzymes aspartokinase and dihydrodipicolinic acid synthase in soybean [57]. In this case, a >100-fold increase of free lysine and 5-fold increase of total seed lysine content was observed. More recently, soybeans showing enhanced threonine levels have been engi‐ neered using seed-specific expression of lysine-insensitive variants of aspartate kinases from bacteria [58]. This strategy produced transgenic soybeans with a 100-fold increase in threo‐ nine levels and 3.5-fold increase in total free amino acid content without negative impacts on seed morphology or germination. While enhancement of essential amino acids in soy‐ bean seeds has clear potential for commercial applications, it will be important to demon‐ strate that transgenic soybean with increased nutritional enhancement traits also maintain optimal agronomic characteristics when grown in the field under a variety of conditions.

Recombinant proteins are widely used in medicine, research laboratories, food and nutri‐ tion, and play a key role in important agriculture and biopharmaceutical industries. Since the development of recombinant DNA technology in the early 1970's, the commercial pro‐

with enhanced vitamin E and more nutritional value for consumers.

**6. Soybeans as bioreactors for pharmaceuticals**

**5.3. Dietary amino acids**

Relationships

460

protein composition [56].

Over the past two decades, there has been a push for recombinant protein technologies to move towards more effective expression systems. These systems must be safe, cost-effective, and conducive to post-translational modifications and processing methods on a large scale. Transgenic plants represent an economical system for accurate expression of complex re‐ combinant proteins on a large scale. Plant cells combine the potential for full post transla‐ tional modifications and correct protein folding with simple growth requirements. The use of plants as a platform for recombinant protein production has a low risk of contamination with prions, viruses, and other pathogens that infect mammalian cells, and therefore offer advantages that are not associated with existing expression systems. An important advant‐ age of plants as a bioreactor is that recombinant proteins and biopharmaceuticals may be ex‐ pressed in multiple plant organs including seeds which naturally accumulate high amounts of stored proteins. In general, crops that have higher protein content are more cost-effective for molecular farming. Among recombinant systems that utilize seeds, soybeans present an exceptionally high endogenous protein content, which can reach up to 40% of the dry seed weight. Soybeans are an ideal source of protein for food and feed thus occupying a unique position as a premier target for genetic engineering, and as a platform for the production of recombinant protein. An important characteristic favoring expression in soybean seeds is that these organs have evolved as specialized compartments to store proteins for embryo nutrition. Based on this, soybean seeds offer an environment with metabolic adaptations that permit the stable and long-term storage of proteins, reducing the requirement for so‐ phisticated and expensive conditions for storage. This makes it possible to stockpile harvest‐ ed seeds so that the downstream processing can be made available based on the demands of the industry. Similarly, soybean seeds provide a compact compartmentalization biomass, which can considerably reduce overall production costs since purification expenses are typi‐ cally inversely proportional to the final concentration in the plant biomass.

The concept of a soy-derived pharmaceutical was tested back in 1995 when Cho et al., devel‐ oped a transformation expression cassette using a soybean seed-specific lectin promoter to

test for potential expression of the *β*-glucuronidase reporter gene [60]. This same expression cassette was used to produce bovine β-casein in soybean which accumulated to 0.1-0.4% of seed total soluble protein (TSP) [61]. A follow-up paper characterized post-translational processing, subcellular localization to the PSV, and purification of transgenic β-casein [62]. These proof-of-concept studies showed that a seed-specific promoter could be used to target stable expression of proteins with commercial value in soybean seed.

### **6.1. Antibodies**

Monoclonal antibodies (mAbs) have played a major role in the advancement of biotechnolo‐ gy and development of mAb-based therapeutics and diagnostics. Plants have great potential to serve as a platform for the production of antibodies for therapeutic use. One of the first reports of a functional plant-based antibody was developed in soybean. In an effort to ex‐ plore cost effective methods of mucosal immunoprotection against sexually transmitted dis‐ eases, Zeitlin et al., expressed a humanized monoclonal anti-herpes simplex virus 2 (HSV-2) antibody in leaf tissue [63]. That study compared purified soy-derived and mammalian cellderived HSV-2 mAbs and found that both were similar with respect to stability in human semen and cervical mucus over a 24 hour period. Both antibodies were also able to diffuse in human cervical mucus, and were efficacious in preventing vaginal HSV-2 infection in a murine model.

### **6.2. Vaccines**

When plants are mentioned as a platform for the production of pharmaceuticals, the concept of edible vaccines often comes to mind. Edible vaccines are desirable since they would elimi‐ nate the use of needles and specialized personnel to administer shots, which may have broad applicability in developing nations. Soybean seeds represent an ideal target for the production of vaccines since soymilk-based formulations are safe and can be easily adminis‐ tered orally. Furthermore, soybean seeds are capable of storing vaccine antigens for many years at ambient temperatures without loss or degradation of the antigen [64-66]. Such fea‐ tures can reduce the need for a cold chain therefore reducing costs.

Vaccines that can be administered at mucosal surfaces offer systemic immunity. Subunit an‐ tigens used to vaccinate orally or nasally are often ineffective and require formulation with a mucosal adjuvant for increased efficacy. The heat labile toxin (LT) of *E*. *coli* is comprised of a single A subunit (LTA) and pentameric B subunit (LTB) and has been shown to act as both a strong mucosal adjuvant as well as an antigen [67]. Moravec et al., targeted LTB expression to the endoplasmic reticulum of seed storage parenchyma cells where it accumulated to lev‐ els up to 2.4% of seed TSP [68]. Mice orally immunized with seed extracts containing LTB induced both, systemic IgG and mucosal IgA anti-LTB antibody responses. The soybean de‐ rived LTB also increased an antibody response against a co-administered bacterial FimHt antigen by 500-fold demonstrating that soy-derived LTB may function as an oral adjuvant.

Several subunit antigens have been expressed in soybeans that are important to the agricul‐ tural industry and could lead to effective vaccines. FanC is a specialized adhesion protein located on the bacterial surfaces of Enterotoxigenic *E*. *coli* (ETEC). K99 and other ETEC strains cause acute diarrhea in humans and livestock and can be severe and even cause death if left untreated. ETEC vaccinations are routinely administered parenterally to preg‐ nant farm animals in order to stimulate systemic immunity and offer protection in new‐ borns. An edible form of this vaccine has the potential to increase efficacy by conferring mucosal immunity at sites of pathogen invasion. Piller et al., constitutively overexpressed the bacterial FanC antigen in soybeans and reported stable accumulation to levels represent‐ ing ~0.4% TSP in both leaves and seeds [69]. Mice immunized with adjuvanted soymilk for‐ mulations containing FanC elicited FanC-specific systemic and cellular immune responses demonstrating immunogenicity of the soy-derived antigen.

In another study a soybean-based vaccine was developed against the virus that causes por‐ cine reproductive and respiratory syndrome (PRRS) [70]. PRRS is a serious health problem among breeding swine herds and the current vaccine is not efficacious when applied in the field. Vimolmangkang et al., overexpressed a nucleocapsid protein (PRRSV-ORF7) that ac‐ cumuolated to 0. 64% of seed TSP. Intragastric immunization of mice with transgenic seed extract, in the absence of adjuvant, induced specific humoral and mucosal immune respons‐ es against PRRSV-ORF7 [70].

#### **6.3. Therapeutics**

test for potential expression of the *β*-glucuronidase reporter gene [60]. This same expression cassette was used to produce bovine β-casein in soybean which accumulated to 0.1-0.4% of seed total soluble protein (TSP) [61]. A follow-up paper characterized post-translational processing, subcellular localization to the PSV, and purification of transgenic β-casein [62]. These proof-of-concept studies showed that a seed-specific promoter could be used to target

A Comprehensive Survey of International Soybean Research - Genetics, Physiology, Agronomy and Nitrogen

Monoclonal antibodies (mAbs) have played a major role in the advancement of biotechnolo‐ gy and development of mAb-based therapeutics and diagnostics. Plants have great potential to serve as a platform for the production of antibodies for therapeutic use. One of the first reports of a functional plant-based antibody was developed in soybean. In an effort to ex‐ plore cost effective methods of mucosal immunoprotection against sexually transmitted dis‐ eases, Zeitlin et al., expressed a humanized monoclonal anti-herpes simplex virus 2 (HSV-2) antibody in leaf tissue [63]. That study compared purified soy-derived and mammalian cellderived HSV-2 mAbs and found that both were similar with respect to stability in human semen and cervical mucus over a 24 hour period. Both antibodies were also able to diffuse in human cervical mucus, and were efficacious in preventing vaginal HSV-2 infection in a

When plants are mentioned as a platform for the production of pharmaceuticals, the concept of edible vaccines often comes to mind. Edible vaccines are desirable since they would elimi‐ nate the use of needles and specialized personnel to administer shots, which may have broad applicability in developing nations. Soybean seeds represent an ideal target for the production of vaccines since soymilk-based formulations are safe and can be easily adminis‐ tered orally. Furthermore, soybean seeds are capable of storing vaccine antigens for many years at ambient temperatures without loss or degradation of the antigen [64-66]. Such fea‐

Vaccines that can be administered at mucosal surfaces offer systemic immunity. Subunit an‐ tigens used to vaccinate orally or nasally are often ineffective and require formulation with a mucosal adjuvant for increased efficacy. The heat labile toxin (LT) of *E*. *coli* is comprised of a single A subunit (LTA) and pentameric B subunit (LTB) and has been shown to act as both a strong mucosal adjuvant as well as an antigen [67]. Moravec et al., targeted LTB expression to the endoplasmic reticulum of seed storage parenchyma cells where it accumulated to lev‐ els up to 2.4% of seed TSP [68]. Mice orally immunized with seed extracts containing LTB induced both, systemic IgG and mucosal IgA anti-LTB antibody responses. The soybean de‐ rived LTB also increased an antibody response against a co-administered bacterial FimHt antigen by 500-fold demonstrating that soy-derived LTB may function as an oral adjuvant.

Several subunit antigens have been expressed in soybeans that are important to the agricul‐ tural industry and could lead to effective vaccines. FanC is a specialized adhesion protein

stable expression of proteins with commercial value in soybean seed.

tures can reduce the need for a cold chain therefore reducing costs.

**6.1. Antibodies**

Relationships

462

murine model.

**6.2. Vaccines**

Protein therapeutic use is limited by the shortfalls in manufacturing capacity and the high cost of production. While an aging population is a key driver of the protein therapeutics market, the potential for future growth is dependent largely on the industry overcoming drug delivery challenges and cost issues. Plants are cost-effective systems that excel at pro‐ ducing complex therapeutic proteins and therefore could help address some of these issues. The high protein content of soybean seeds, low costs associated with growth, simplified pu‐ rification methods, and safety, make soybean a unique platform for the production of pro‐ tein-based therapeutics.

Russell et al., expressed human growth hormone (hGH) in soybean with transformation cas‐ settes using both the constitutive 35S promoter as well as a soybean seed-specific promoter 7S *β-*conglycyinin [71]. The resulting expression of hGH, both constitutively and within the seed, was detected at low levels of 0.0008% TSP. More recently hGH was expressed in soy‐ bean seeds with a more effective expression cassette utilizing the 7S α' subunit of β-congly‐ cinin promoter and α-coixin signal peptide. In this case, hGH was directed to protein storage vacuoles within the seed and accumulated to 2.9% TSP. Bioassays demonstrated that the soy-derived hGH was fully active [66]. The cost of recombinant *E*. *coli*-derived hGH is still a very expensive therapy. Having such a high level of bioactive hGH protein expression in soybean seeds demonstrates the potential for high-yield production of recombinant pro‐ teins in soybean seeds and could lead to reduce costs for large-scale production of therapeu‐ tic molecules.

Human basic fibroblast growth factor (bFGF) is another high value therapeutic that has been expressed in soybean seeds [72]. This therapeutic was expressed under the control of the soybean seed specific G1 promoter and endogenous signal sequence from soybean. The

bFGF protein accumulated to levels of ~2.3% of seed TSP and biological activity of the trans‐ genic protein was confirmed by its mitogenic activity in mice.

Recombinant expression of Insulin was first reported using *E*. *coli* [59] and has since been commercialized. Like many pharmaceuticals derived from other expression systems, the po‐ tential for contamination along with high costs associated with production remain consider‐ able for this hormone. To show that a soybean expression system could address some of these issues Cunha et al., used a sorghum γ-kafirin seed storage protein promoter and αcoixin PSV signal peptide to target recombinant proinsulin expression to soybean seeds [65]. Transgenic protein was stably expressed in seeds though accumulation levels were not re‐ ported. Transgenic seeds containing proinsulin were stable for up to seven years when stor‐ ed under ambient storage conditions.

The soybean platform has also been used to produce a therapeutic for reducing systolic blood pressure. Novokinin is a hypotensive peptide that has vasorelaxing activity [73]. No‐ vakinin was expressed in soybean seeds under the control of a modified β-conglycinin pro‐ moter and accumulated to 0.5% of seed TSP. A purified soy-derived formulation, as well as a less pure defatted flour formulation, was orally administered to groups of spontaneously hypertensive rats. Both the purified and partially purified formulations successfully reduced systolic blood pressure after a single dose [74].

Haemophilia B is a bleeding disorder that results from a deficiency of human coagulation factor IX (hFIX). The current treatment for this disease is intravenous infusion of plasma-de‐ rived or recombinant hFIX protein. While this treatment is effective at preventing and ar‐ resting hemorrhage, it is very costly and the protein is difficult to produce in large quantities. Using a biolistic transformation approach, hFIX expression was targeted to soy‐ bean seeds using the soy 7S promoter and coixin signal peptide [75]. Recombinant hFIX pro‐ tein accumulated to 0.23% of seed TSP, and purified protein exhibited blood-clotting activity up to 1. 4% of normal plasma demonstrating functionality and efficacy of the soy-derived protein. The recombinant protein was stable for 6 years when stored at room temperature.

Soybeans are also capable of supporting expression and stable accumulation of large and complex proteins that can be difficult or impossible to express using current expression sys‐ tems. Human thyroglobulin (hTG) is a 660 kDa homodimeric protein that is used as a pro‐ tein standard and diagnostic for the detection of thyroid disease. To date, no expression system has been capable of producing a recombinant form of hTG which is likely due to strict requirements for correct post-translational modification and proper folding during protein synthesis. As a result, commercial hTG supplied to manufacturers for their assay kits is derived from cadaver and surgically removed thyroid tissue. The heterogeneity and lack of uniformity of commercially-purified hTG preparations is a major factor of variation between kits of different manufacturers. To explore the potential of soybean as a platform for production of large and complex proteins, Powell et al., used the 7S promoter and en‐ dogenous hTG signal peptide to target recombinant expression of hTG to soybean seeds [76]. Transgenic lines showed stable expression of full length hTG dimeric protein over mul‐ tiple generations, and accumulated the protein to levels approaching 1.5% of seed TSP. Functionality of soy-derived hTG was demonstrated with commercial ELISA kits developed specifically for the detection of hTG in patient sera. The expression of 660 kDa dimerich TG appears to be the largest functional recombinant protein expressed in any plant system to date, and demonstrates the practicality of soy as an alternative system for the expression of proteins that are recalcitrant to expression in traditional systems [76].

Soybeans have a high intrinsic capacity for protein production and storage. Other than vari‐ ability caused by nutrient modulation or environmental effects, the relative distribution of seed protein is primarily determined by genetics. Several groups have been able to achieve recombinant protein expression in soybean seeds at respectable levels (approaching 3% of seed TSP). However, knowledge of protein distribution in soybeans may help to further maximize expression levels. Schmidt and Herman tested this theory by overexpressing Green Fluorescent Protein (GFP) in soybeans with a β-conglycinin suppression background, to observe whether proteome rebalancing would result in a higher GFP yield [77]. They found that the rebalancing of intrinsic proteins could be exploited to obtain protein yields which increased ~4-fold in suppression backgrounds and approached levels representing >7% seed TSP. Thus, proteome rebalancing may represent a strategy that can be used to de‐ velop soybean lines capable of producing high levels of recombinant proteins in the future.
