Expression of Colorectal Cancer Antigenic Protein Fused to IgM Fc in Chinese Cabbage (Brassica rapa)

The epithelial cell adhesion molecule (EpCAM) is a tumor-associated antigen and a potential target for tumor vaccine. The EpCAM is a cell-surface glycoprotein highly expressed in colorectal carcinomas. The objective of the present study is to develop an edible vaccine system through Agrobacterium-mediated transformation in Chinese cabbage (Brassica rapa). For the transformation, two plant expression vectors containing genes encoding for the EpCAM recombinant protein along with the fragment crystallizable (Fc) region of immunoglobulin M (IgM) and Joining (J)-chain tagged with the KDEL endoplasmic reticulum retention motif (J-chain K) were constructed. The vectors were successfully transformed and expressed in the Chinese cabbage individually using Agrobacterium. The transgenic Chinese cabbages were screened using genomic polymerase chain reaction (PCR) in T0 transgenic plant lines generated from both transformants. Similarly, the immunoblot analysis revealed the expression of recombinant proteins in the transformants. Further, the T1 transgenic plants were generated by selfing the transgenic plants (T0) carrying EpCAM–IgM Fc and J-chain K proteins, respectively. Subsequently, the T1 plants generated from EpCAM–IgM Fc and J-chain K transformants were crossed to generate F1 plants carrying both transgenes. The presence of both transgenes was validated using PCR in the F1 plants. In addition, the expression of Chinese cabbage-derived EpCAM–IgM Fc × J-chain K was evaluated using immunoblot and ELISA analyses in the F1 plants. The outcomes of the present study can be utilized for the development of a potential anti-cancer vaccine candidate using Chinese cabbage.


Introduction
Plant expression systems, such as those in tomatoes [1,2], carrots [3], bananas [4], spinach [5], lettuce [6], and tobacco [7,8] have been established to produce valuable recombinant proteins, including therapeutic enzymes, vaccines, and antibodies. Among horticultural crops, Chinese cabbage has been recently recognized as a potential candidate to produce such valuable recombinant proteins, because it has a reasonable total soluble protein capacity relative to plant biomass [9]. In general, a large amount of soluble protein in plant biomass can provide better production of recombinant proteins, thereby enhancing the possibility of an efficient plant-based bioreactor system [10]. Chinese cabbage 2D,E). The shoots were isolated from the explants and transferred onto the regeneration medium with 6 mg/L phosphinotricin (PPT) when they were at least 4-5 mm in length ( Figure 2E,F). Most shoots showed vigorous regenerative growth on the new selective medium. However, some of the shoots became etiolated and died one week later. These results suggested that there were escapees from the selective medium culture. In some cases, the leaves of regenerated plants exhibited chlorosis, indicating that these plants might be chimeras.

Agrobacterium-Mediated Transformation and Regeneration of T 0 Transgenic Chinese Cabbage
Agrobacterium-mediated transformation was conducted to transfer genes encoding EpCAM-IgM Fc or J-chain K to Chinese cabbage ( Figure 2). Three hundred hypocotyl pieces of Chinese cabbage were applied to transform plant expression vectors (pH2GW EpCAM-IgM Fc, and J-chain K) ( Figure 1A,B). After co-cultivation with Agrobacterium, the hypocotyl pieces were transferred onto regeneration media ( Figure 2C,D). A callus began to form at the cut ends of 30-40% of the hypocotyls within 2 weeks ( Figure 2D). Adventitious shoots were observed from calli after 3-4 weeks ( Figure 2D,E). The shoots were isolated from the explants and transferred onto the regeneration medium with 6 mg/L phosphinotricin (PPT) when they were at least 4-5 mm in length ( Figure 2E,F). Most shoots showed vigorous regenerative growth on the new selective medium. However, some of the shoots became etiolated and died one week later. These results suggested that there were escapees from the selective medium culture. In some cases, the leaves of regenerated plants exhibited chlorosis, indicating that these plants might be chimeras.

Presence of Transgenes Encoding EpCAM-IgM Fc and J-Chain K in T0 Transgenic Plants
A polymerase chain reaction (PCR) analysis of the genomic DNA was conducted to confirm the presence of transgenes in transgenic plants (EpCAM-IgM Fc) and (J-chain K) randomly selected Chinese cabbage regenerants obtained by transforming each vector ( Figure 3A,B, respectively). All tested regenerants obtained from transformations using plant expression vectors carrying the gene encoding EpCAM-IgM Fc had amplified bands at 837 bp (EpCAM), 1053 bp (IgM Fc), and 1023 bp (hygromycin phosphotransferase (HTP)) ( Figure 3A). All tested regenerants obtained from transformation using the plant expression vector carrying the gene encoding the J-chain K had the expected amplified J-chain K band at 543 bp ( Figure 3B). The left shows newly regenerated shoots from the callus. The right shows shoots after further growth following transfer to new regeneration media. (F) Root induction from the regenerants in root-inducing media. (G) In Vivo T 0 transgenic Chinese cabbage growth after transplanting into pots with soil from the in vitro root-induced transgenic plants.

Presence of Transgenes Encoding EpCAM-IgM Fc and J-Chain K in T 0 Transgenic Plants
A polymerase chain reaction (PCR) analysis of the genomic DNA was conducted to confirm the presence of transgenes in transgenic plants (EpCAM-IgM Fc) and (J-chain K) randomly selected Chinese cabbage regenerants obtained by transforming each vector ( Figure 3A,B, respectively). All tested regenerants obtained from transformations using plant expression vectors carrying the gene encoding EpCAM-IgM Fc had amplified bands at 837 bp (EpCAM), 1053 bp (IgM Fc), and 1023 bp (hygromycin phosphotransferase (HTP)) ( Figure 3A). All tested regenerants obtained from transformation using the

Expression of EpCAM-IgM Fc in T0 Transgenic Plants
Expression of the EpCAM-IgM Fc protein in leaf tissues of transgenic plants was analyzed using Western blotting ( Figure 4). In the IgM Fc detection immunoblot, all randomly selected transgenic lines (2,6,7,9,11) showed an approximately 75 kDa protein band similar to that of the positive control ( Figure 4A black arrowhead). In the EpCAM detection immunoblot, all lines (2,6,7,9,11)

Expression of EpCAM-IgM Fc in T 0 Transgenic Plants
Expression of the EpCAM-IgM Fc protein in leaf tissues of transgenic plants was analyzed using Western blotting ( Figure 4). In the IgM Fc detection immunoblot, all randomly selected transgenic lines (2,6,7,9,11) showed an approximately 75 kDa protein band similar to that of the positive control ( Figure 4A black arrowhead). In the EpCAM detection immunoblot, all lines (2,6,7,9,11)

Expression of EpCAM-IgM Fc in T0 Transgenic Plants
Expression of the EpCAM-IgM Fc protein in leaf tissues of transgenic plants was analyzed using Western blotting ( Figure 4). In the IgM Fc detection immunoblot, all randomly selected transgenic lines (2,6,7,9,11) showed an approximately 75 kDa protein band similar to that of the positive control ( Figure 4A black arrowhead). In the EpCAM detection immunoblot, all lines (2,6,7,9,11)

Presence of Transgenes Encoding EpCAM-IgM Fc and J-Chain K in T 1 Transgenic Plants
The T 0 transgenic line #2, with the highest expression of EpCAM-IgM Fc, was self-crossed to generate a T 1 line #2 (Figure 1; Figure 4). PCR was performed for the presence of transgenes in eight (EpCAM-IgM Fc) T 1 plants ( Figure 5). All tested T 1 plants obtained from the transformation using the plant expression vector carrying the gene encoding EpCAM-IgM Fc had the expected amplified bands at 837 bp (EpCAM), 1053 bp (IgM-Fc), and 1023bp (HTP) ( Figure 5A). In the transgenic plant carrying the J-chain K transgene, the transgenic line 1 was self-crossed to generate T 1 . Transgenic plants in T 1 carrying the J-chain K transgene were randomly selected for PCR testing ( Figure 5B). All of the tested T 1 plants obtained from selfing had the expected amplified band at 543 bp (J-chain K) ( Figure 5B).

Presence of Transgenes Encoding EpCAM-IgM Fc and J-Chain K in T1 Transgenic Plants
The T0 transgenic line #2, with the highest expression of EpCAM-IgM Fc, was self-crossed to generate a T1 line #2 ( Figure 1; Figure 4). PCR was performed for the presence of transgenes in eight (EpCAM-IgM Fc) T1 plants ( Figure 5). All tested T1 plants obtained from the transformation using the plant expression vector carrying the gene encoding EpCAM-IgM Fc had the expected amplified bands at 837 bp (EpCAM), 1053 bp (IgM-Fc), and 1023bp (HTP) ( Figure 5A). In the transgenic plant carrying the J-chain K transgene, the transgenic line 1 was self-crossed to generate T1. Transgenic plants in T1 carrying the J-chain K transgene were randomly selected for PCR testing ( Figure 5B). All of the tested T1 plants obtained from selfing had the expected amplified band at 543 bp (J-chain K) ( Figure 5B).

Presence of Transgenes Encoding EpCAM-IgM Fc and J-Chain K in F1 Transgenic Plants Obtained between EpCAM-IgM Fc and J-Chain K T1 Plants
The

Expression of EpCAM-IgM Fc in F1 Transgenic Plants Obtained by Crossing EpCAM-IgM Fc and J-Chain K T1 Plants
In the F1 transgenic lines with positive PCR results for EpCAM, IgM Fc, and J-chain K transgenes, the expression of EpCAM-IgM Fc was confirmed by Western blotting (Figure 8A). In the IgM Fc detection immunoblot, all tested F1 transgenic lines (#1-9) showed the 75 kDa size protein band. Furthermore, in the EpCAM detection immunoblot, all tested F1 transgenic lines (#1-9) showed the 75 kDa size protein band ( Figure 8B). No bands were observed in the non-transgenic plants ( Figure  8).  hygromycin phosphotransferase (HTP).

Presence of Transgenes Encoding EpCAM-IgM Fc and J-Chain K in F1 Transgenic Plants Obtained between EpCAM-IgM Fc and J-Chain K T1 Plants
The

Expression of EpCAM-IgM Fc in F1 Transgenic Plants Obtained by Crossing EpCAM-IgM Fc and J-Chain K T1 Plants
In the F1 transgenic lines with positive PCR results for EpCAM, IgM Fc, and J-chain K transgenes, the expression of EpCAM-IgM Fc was confirmed by Western blotting ( Figure 8A). In the IgM Fc detection immunoblot, all tested F1 transgenic lines (#1-9) showed the 75 kDa size protein band. Furthermore, in the EpCAM detection immunoblot, all tested F1 transgenic lines (#1-9) showed the 75 kDa size protein band ( Figure 8B). No bands were observed in the non-transgenic plants ( Figure  8).

Expression of EpCAM-IgM Fc in F 1 Transgenic Plants Obtained by Crossing EpCAM-IgM Fc and J-Chain K T 1 Plants
In the F 1 transgenic lines with positive PCR results for EpCAM, IgM Fc, and J-chain K transgenes, the expression of EpCAM-IgM Fc was confirmed by Western blotting ( Figure 8A). In the IgM Fc detection immunoblot, all tested F 1 transgenic lines (#1-9) showed the 75 kDa size protein band. Furthermore, in the EpCAM detection immunoblot, all tested F 1 transgenic lines (#1-9) showed the 75 kDa size protein band ( Figure 8B). No bands were observed in the non-transgenic plants (Figure 8).

Discussion
This study demonstrated the successful expression of EpCAM-IgM Fc as anti-colorectal cancer IgM Fc fusion antigenic proteins, creating a candidate for a cancer vaccine in transgenic T 0 and T 1 Chinese cabbage, including F 1 plants from a crossing between EpCAM-IgM Fc and J-chain K T 1 transgenic plants. Transgenic Chinese cabbage expressing EpCAM-IgM Fc and J-chain K were obtained from Agrobacterium-mediated transformation. PCR analysis revealed that all tested T 0 transgenic plants carrying EpCAM-IgM Fc and J-chain K transgenes had PCR bands of both transgenes, indicating that these genes were properly embedded in the plant genome. Western blotting showed variable expression of the EpCAM-IgM Fc transgene in the T 0 transgenic plants. In EpCAM-IgM Fc, among the tested T 0 transgenic plants, transgenic line #2 had the highest level of EpCAM-IgM Fc. Thus, EpCAM-IgM Fc transgenic line #2 was selected for self-crossing to generate T 1 plants. Regarding the J-chain K, according to Western blot analysis, its expression was not observed in the T 0 transgenic plants.
In both EpCAM-IgM Fc and J-chain K T 1 transgenic plants, the existence of EpCAM-IgM Fc and J-chain K transgenes was confirmed by PCR, respectively. However, Western blotting revealed that only the EpCAM-IgM Fc protein expression was detected in the T 1 plants, and the J-chain K protein was not detected. It appeared that J-chain protein expression is difficult to detect. Currently, there are no reports demonstrating J-chain expression in transgenic plants. However, the function of the J-chain is active when it is co-expressed with IgM or immunoglobulin A (IgA) antibodies [36]. Despite a barely detectable J-chain protein level, it has vital activity when it assembles with IgM Fc or IgA Fc to generate pentameric or dimeric structures, respectively [38].
The T 1 transgenic line highly expressing EpCAM-IgM Fc proteins was selected to cross with the T 1 transgenic line carrying the J-chain K transgene to generate F 1 plants carrying two transgenes. Since transgenic Chinese cabbage plants have the ability of self-fertilization and cross-fertilization, this crop has been efficiently bred to build useful characteristics in terms of growth physiology, taste, and disease resistance [39][40][41]. In this study, we used self-and cross-fertilization of Chinese cabbage to obtain the F 1 plants having two different transgenes encoding EpCAM-IgM Fc and J-chain K. The immunoglobulin Fc-fusion protein is the protein linked to the immunoglobulin Fc fragment. The Fc-fused proteins obtain beneficial biological and pharmacological effects from the Fc fragment [42,43]. The Fc fragment remarkably enhances the plasma half-life of their fused proteins or peptides through its FcRn interaction, eventually prolonging their therapeutic activities [43]. Additionally, the Fc fragment interacts with Fc receptors on immune cells to provoke antigen presentation, inducing immune responses [44,45]. Furthermore, regarding protein expression, the Fc-fused proteins in general exhibit better protein expression [29]. The Fc-fused protein can be purified by Protein-A affinity chromatography [42]. In general, there are three major Ig Fc fragment forms, IgG, IgA, and IgM. The IgG and IgA-Fc fused proteins can be assembled in monomeric and dimeric forms, respectively [46]. The IgM Fc can be assembled to polymerize through disulfide bonding localized to the junction portions of CH2 and CH3 of the Fc fragment [35]. The J-chain has important functions in the assembly with IgA and IgM, acting as a glue between the Fc regions of each antibody [35]. In IgA, the J-chain enhances the dimerization of IgA and its secretion process [47]. In IgM, J-chain forms the pentameric structure from the hexameric structure [35]. The pentameric structures are less effective at activating complements than the hexameric structures, reducing damage to epithelial membranes. The J-chain on IgM might sterically hinder the binding of complement component 1q (C1q) and thereby decrease the cytolytic activity of pentameric IgM [48]. Therefore, the J-chain should be co-expressed with the IgM-Fc fusion anti-colorectal cancer vaccine candidate. The two expression cassettes can be combined and transferred into one Agrobacterium-mediated transformation. However, in this study, each transgene was individually transferred to each Chinese cabbage plant, and these plants were successfully cross-fertilized to obtain both transgenes. To confirm whether an anti-human IgM Fc µ-chain antibody can recognize Chinese cabbage-derived EpCAM-IgM Fc × J-chain K, an ELISA analysis was conducted. The binding reaction between EpCAM-IgM Fc × J-chain K and the anti-human IgM Fc µ-chain antibody showed an absorbance signal indicating that the EpCAM-IgM Fc × J-chain K was structurally formed to be recognized by the anti-IgM Fc µ-chain antibody. In previous studies, EpCAM alone and fused with IgG Fc have been expressed in diverse plants [26][27][28], however, the IgM-Fc fused EpCAM is the first report in this study. In this current study, a glycosylation study with the EpCAM-IgM × J-chain K was not conducted, which affects its immunogencitiy. It has been reported that the Brasscia species has glycosylation apparatus for protein glycosylation [49,50]. In the current study, it is speculated that the EpCAM-IgM Fc assembled with J-chain K, retaining it inside endoplasmic reticulum (ER), harboring mainly an oligomannose type. Thus, in the future, glycosylation should be further investigated.
All considered, we confirmed that each EpCAM-IgM Fc and J-chain K transgene expression cassette was successfully transferred to the Chinese cabbage, and cross-fertilization between both transgenic plants carrying one of the transgenes generated F 1 transgenic seedlings carrying both transgenes. These results revealed that both transgenes were stably inserted, and the EpCAM-IgM Fc protein gene was expressed in F 1 transgenic plants reproduced from crossing between EpCAM-IgM Fc and J-chain K T 1 transgenic plants. Furthermore, these results suggested that two genes can co-exist through cross-breeding in Chinese cabbage T 1 transgenic plants. In conclusion, the transgenic Chinese cabbage expressing EpCAM-IgM Fc can be applied to express anti-colorectal cancer IgM Fc fusion recombinant vaccine candidate proteins.

Plant Expression Vector and Agrobacterium Strain
The gene encoding the human EpCAM protein (Thr17-Lys265, GenBank accession no. BC014785) was fused to the gene encoding human IgM Fc polypeptides (Leu103-Tyr453, GenBank accession no. X57086) to construct the EpCAM-IgM Fc fusion protein, and this was cloned under the CaMV promoter in the plant expression vector pRCV2 to generate pRCV2 EpCAM-IgM Fc carrying the hygromycin phosphotransferase (hpt) gene for Chinese cabbage (B. rapa L. ssp. pekinensis) transformation [45]. The 30 amino acids (MATQRRANPSSLHLITVFSLLAAVVSAEVD) as an ER signal peptide from Nicotiana plumbaginifolia was fused to N-terminus of the cleavage site (Thr17-Ala23). Chinese cabbage was transformed with the plant expression vector pRCV2 EpCAM-IgM Fc ( Figure 1A). This vector was constructed by ligating pCAMBIA 1301 with pBluescript II KS (+) (Stratagene, San Diego, CA, USA) according to a previous study [12]. The Agrobacterium tumefaciens strain LBA 4404 carrying pRCV2 EpCAM-IgM Fc was applied for plant transformation. Agrobacteria were grown in a yeast extract peptone (YEP) medium.

Plant Material and Preparation
Chinese cabbage Seoul (Dong-bu Seed, Seoul, Korea) was used for Agrobacterium-mediated transformation to express EpCAM-IgM Fc and the J-chain tagged with the KDEL endoplasmic reticulum retention motif (J-chain K) ( Figure 1B). The seeds were submerged in 70% ethanol for 1 min. Then, the seeds were vigorously shaken in 30% commercial Clorox (The Clorox company, Oakland, CA, USA) (1.6% hypochlorite) plus 0.1% Tween-20 (USB, Cleveland, OH, USA) for 20 min. Then, they were rinsed three times with water. The seeds were germinated on an MS medium [51] and cultivated in vitro to grow hypocotyls 4-5 cm long for 7 days under a 16 h photoperiod. Following this, the hypocotyls were dissected, avoiding the shoot apex, and quickly cut into 7-8 mm segments for Agrobacterium-mediated transformation.

Transformation and Selection Procedures
One mL of Agrobacterium cell stock was cultured in 50 mL of a YEP medium containing kanamycin (50 mg/L) and acetosyringone (5 mg/L) until an optical density (OD) 600 value of 1.0 was obtained. The Agrobacterium cells were pelleted and washed. Then, they were resuspended in 50 mL of the YEP medium. Agrobacterium cells were applied to infect hypocotyl explants by immersing them in the bacterial inoculum for 10 min ( Figure 1B). The hypocotyl explants were blotted on sterile filter paper and placed on a co-cultivation medium containing acetosyringone (5 mg/L) at pH 5.2-5.7. After 3 days of co-cultivation, the explants were washed with an MS liquid medium supplemented with cefotaxime (200 mg/L) and transferred to an MS selection medium containing 3% sucrose, IBA (4 mg/L), NAA (3 mg/L), AgNO 3 (4 mg/L), acetosyringone (5 mg/L), cefotaxime (200 mg/L), hygromycin (10 mg/L), and 0.8% plant agar (pH 5.6). After cultivation for 3 and 8 weeks, the number of calli and shoots that formed on the explants were determined, respectively ( Figure 1B). Then, the regenerated shoots were transferred to a rooting medium that consisted of 1/2-strength MS medium, 3% sucrose, cefotaxime (200 mg/L), and 0.7% plant agar (pH 5.8).

PCR Amplification from Genomic DNA of Plant Leaf
To confirm the existence of transgene encoding EpCAM-IgM Fc, genomic DNA was prepared from the fresh leaves of transgenic and non-transgenic Chinese cabbage plants according to the DNA extraction kit (RBC Bioscience, Taipei, Taiwan) using the mini-prep method and protocol. DNA concentration was measured using a Nanovue TM plus spectrophotometer (GE Healthcare, Boston, MA, USA) and adjusted to 20 ng/µL, and the DNA was used for PCR amplification. The status of transgenic plants was confirmed by PCR using HTP (hygromycine) and target gene (EpCAM, IgM, and J-Chain K) specific primers. The reaction solution for PCR analysis contained 20 ng of gDNA, 5.0 µL of Takara buffer mixture buffer (Takara, Kusatsu, Japan), 1.0 µL each of 10 pmol/µL of primer set, and autoclaved distilled water, to reach a total volume of 20 µL. The primer set for EpCAM (837 bp, forward primer (F)-GCA GGC TAT GGC TAC TCA ACG AAG G/reverse primer (R)-CTC GAG TTT TAG ACC CTG CAT TGA G), IgM (1053 bp, F-CTC GAG CTT CCA GTG ATT GCT GAG C/R-CTC GAG TCA GTA GCA GGT GCC AGC TGT G), J-chain K (543 bp, F-GCA GGC TAT GGC CAA GAA CCA TTT GC/R-AGT TCA TCT TTG TCA GGA TAG CAG G), and hygromycine (HPT, 1023 bp, F-CTA TTC CTT TGC CCT CGG ACG GC/R-ATG AAA AAG CCT GAA CTC ACC GCG ACG) genes were used for DNA amplification to confirm the presence of the transgene in the plants. The PCR reaction was performed as follows: denaturation at 95°C for 10 min in the beginning, a repeated cycling step of denaturation at 95°C for 1 min, annealing at 56°C for 40 s, and extension at 72°C for 1 min (35 times). This was followed by a final denaturation at 72°C for 10 min. A non-transgenic Chinese cabbage plant was used as a negative control, whereas plant expression vectors containing the EpCAM-IgM Fc and J-chain K genes were used as positive controls.

Western Blot
To confirm the expression of EpCAM-IgM Fc in transgenic Chinese cabbage, 100 mg of leaf tissue was harvested from in vitro tissue culture and homogenized to generate leaf extracts in 1× PBS. A volume of 20 µL of leaf extract samples (100 mg of leaf/300 µL) mixed with 5 µL of protein loading buffer (1 M Tris-HCl, 50% glycerol, 10% SDS, 5% 2-mercaptoethanol, and 0.1% bromophenol blue) was loaded on 10% SDS-PAGE and transferred to a nitrocellulose membrane (Millipore, Billerica, MA, USA). The membrane was incubated in blocking buffer (5% skim milk (Fluka, Buchs, Switzerland) in Tris-Buffered Saline (TBS) plus 0.5% (v/v) Tween 20). The blot was incubated for 1 h 30 min at room temperature (RT) with either mouse anti-human EpCAM antibody # MAB960-500 (R&D Systems, Minneapolis, MN, USA) diluted in blocking buffer at 1:500 or goat anti-human IgM Fc µ-chain conjugated to horseradish peroxidase (HRP) (Jackson ImmunoResearch, West Grove, PA, USA) diluted in blocking buffer at 1:5000 and then incubated for 1 h and 30 min at RT with a secondary antibody goat anti-mouse IgG 2a heavy chain conjugated to horseradish peroxidase (Abcam, Cambridge, UK) diluted in blocking buffer at 1:5000. The goat anti-human IgM Fc µ-chain antibody recognized the IgM Fc portion of EpCAM-IgM Fc, whereas the anti-human EpCAM antibody detected EpCAM. Protein bands were visualized by exposing the membrane to X-ray film (Fuji, Tokyo, Japan) using a chemiluminescence substrate (Pierce). Non-transgenic plants and tobacco-derived human EpCAM-IgM Fc (50 ng) [52] were used as negative and positive controls, respectively.

Cross-Fertilization
After T 0 transgenic plant validation by PCR and Western blot analysis, the transgenic plantlets (T 0 ) with well-developed roots were transplanted into 10 cm pots containing soil and transferred to a glasshouse at the National Institute of Horticultural and Herbal Science (NIHHS), Wanju, Korea from 2015 to 2017 ( Figure 1B). The plants were grown under normal daylight conditions with a set temperature of 22 • C and 60-70% relative humidity (RH). The T 0 plants were grown in 2015 and selfed to produce seeds (T 1 ). In 2016, the T 1 seeds were sown and the plants were grown for 4-6 weeks with vernalization treatment ( Figure 1C). The T 1 plants containing the EpCAM-IgM Fc genes were crossed with the T 1 plants with J-chain K during the blooming stage for the generation of the F 1 transgenic lines in 2017. The crossing resulted in the generation of the F 1 transgenic line ( Figure 1D).

Statistical Analysis
Statistical analysis consisted of using Student's t-test to determine the differences in absorbance of each group (EpCAM-IgG Fc T , EpCAM-IgM Fc T × J-Chain K T , and EpCAM-IgM Fc C × J-Chain K C ) using Microsoft Excel software (Microsoft Office Excel; Microsoft Corporation, Redmond, WA, USA). The difference between each group (EpCAM-IgG Fc T , EpCAM-IgM Fc T × J-Chain K T , and EpCAM-IgM Fc C × J-Chain K C ) was compared for statistical significance at 0.05 and 0.01 probabilities (* p < 0.05, ** p < 0.01).

Conflicts of Interest:
The authors declare no conflict of interest.