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plants

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

Ye-Rin Lee 1, Chae-Yeon Lim 1, Sohee Lim 2 , Se Ra Park 2 , Jong-Pil Hong 1, Jinhee Kim 1, Hye-Eun Lee 1, Kisung Ko 2,* and Do-Sun Kim 1,*

1 Vegetable Research Division, National Institute of Horticultural and Herbal Science, Rural Development Administration, Wanju-gun 55365, Korea; [email protected] (Y.-R.L.); [email protected] (C.-Y.L.); [email protected] (J.-P.H.); [email protected] (J.K.); [email protected] (H.-E.L.) 2 Department of Medicine, College of Medicine, Chung-Ang University, Seoul 06974, Korea; [email protected] (S.L.); [email protected] (S.R.P.) * Correspondence: [email protected] (K.K.); [email protected] (D.-S.K.); Tel.: +82-63-238-6670 (K.K.); +82-63-238-6670 (D.-S.K.) Received: 18 September 2020; Accepted: 23 October 2020; Published: 30 October 2020

Abstract: 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.

Keywords: Agrobacterium; colorectal carcinoma; EpCAM; plant molecular biopharming; recombinant vaccine; transgenic chinese cabbage

1. 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

Plants 2020, 9, 1466; doi:10.3390/plants9111466 www.mdpi.com/journal/plants Plants 2020, 9, 1466 2 of 15 transformation protocols are well established for the overexpression of transgenes, which can impart resistance to plant pathogenesis [11–13] and tolerance to harsh environments, such as drought and salinity [14–16], and it can also improve physiological traits, such as tolerance to calcium deficiency [17]. There are several advantages of using Chinese cabbage for the production of therapeutic proteins. Chinese cabbage leaf biomass can be directly used in oral application without the purification of recombinant vaccines. Isolates of the targeted recombinant proteins can be easily purified from its leaf biomass. Additionally, Chinese cabbage is easy to manipulate by breeding and establishing mass seed production with self-compatibility or self-incompatibility [18]. However, the application of Chinese cabbage for therapeutic protein production has not yet been actively studied. Among the valuable recombinant proteins expressed in plants, an epithelial cell adhesion molecule (EpCAM) is a tumor-associated antigen (TAA) that has been shown to be highly expressed on cancer cells over the last several decades and expressed using diverse heterologous expression including plants such as Swiss chard (Beta vulgaris subsp. Vulgaris), Nicotiana tabaccum, and N. Benthamiana [19–26]. Plants have glutaminyl cyclases to catalyze the formation of pyroglutamic acid at the N-terminus of proteins, which is important for its immunogenicity [27,28]. Thus, it is assumed that N-terminal glutamine could be modified to pyroglutamate in diverse transgenic plants species, including Chinese cabbage. The EpCAM cancer antigenic protein can be used to prevent and inhibit cancer; hence, it is a vaccine candidate [23]. The EpCAM protein is often fused to the fragment crystallizable (Fc) region of immunoglobulin G (IgG Fc), having the advantages of Fc fusion, including enhanced protein stability, ease of application for affinity chromatography, and enhanced biofunctions [24,29,30]. Therefore, the Fc-fusion strategy has also been applied to the EpCAM-Fc fusion cancer vaccine expressed in insect and plant systems [31,32]. The plant-derived EpCAM fused to IgG Fc induced a humoral immune response in BALB/c mice [29,30]. In previous studies, the Fc fused to EpCAM originated from IgG, eventually forming IgG monomeric structures [33,34]. Recently, the fusion of Fc originating from IgA and IgM, for therapeutic recombinant proteins, has been applied to form dimeric or polymeric structures that have improved biofunctions [30,31]. In particular, IgM-like protein structures can provide hexameric or pentameric structures that have advantages, such as an ease of recognition of dendritic cells, thereby efficiently presenting an antigen for an induction of the immune response [35]. The co-expression of both IgM and Joining (J)-chain could avoid the hexameric structures that cause cytosolic responses through IgM-C1q complement-dependent cytotoxicity [36]. Indeed, such an antibody-like protein complex could have beneficial biological and chemical properties as a vaccine by targeting the antigen-presenting cells (APCs), protein–A/G-derived affinity purification, and protein in vivo stability [37]. Additionally, the Fc-fusion strategy could provide higher expression levels and stability in heterologous expression systems, eventually leading to better yield in a plant expression system [29,31,33]. In the present study, the Fc of IgM was fused to EpCAM to generate polymeric protein structures as a cancer vaccine in the Chinese cabbage expression system. Additionally, the J-chain was expressed in Chinese cabbage. Both transgenic plants carrying EpCAM fused to IgM Fc, and J-chain recombinant protein transgenes were crossed to generate F1 transgenic plants and investigate the potential of Chinese cabbage as a plant bioreactor to produce highly valuable antigen–Fc complex proteins for cancer immunotherapy (Figure1). Plants 2020, 9, x FOR PEER REVIEW 3 of 15

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 Plantsfrom2020 the, selective9, 1466 medium culture. In some cases, the leaves of regenerated plants exhibited chlorosis,3 of 15 indicating that these plants might be chimeras.

FigureFigure 1.1. Schematic diagram for Agrobacterium-mediated-mediated Chinese Chinese cabbag cabbagee transformation transformation and and generationgeneration ofof transgenic plants plants carrying carrying both both ep epithelialithelial cell cell adhesion adhesion molecule–immunoglobulin molecule–immunoglobulin M Mfragment fragment crystallizable crystallizable (EpCAM–IgM (EpCAM–IgM Fc) Fc) and and Joining Joining (J)-chain (J)-chain K. K. (A (A) )Plant Plant expression expression vectors vectors to to expressexpress EpCAM–IgMEpCAM–IgM FcFc andand J-chainJ-chain KK forfor AgrobacteriumAgrobacterium-mediated-mediated transformation.transformation. The The expected expected quaternary structure of EpCAM–IgM Fc in T0 transgenic plants carrying the transgene encoding quaternary structure of EpCAM–IgM Fc in T0 transgenic plants carrying the transgene encoding EpCAM–IgM Fc is hexameric. The expected protein structure of the J-chain K in T0 transgenic plants EpCAM–IgM Fc is hexameric. The expected protein structure of the J-chain K in T0 transgenic plants carrying the transgene encoding J-chain K is hook-like. (B) Generation of T0 transgenic plants carrying carrying the transgene encoding J-chain K is hook-like. (B) Generation of T0 transgenic plants carrying EpCAM–IgM Fc and J-chain K obtained from Agrobacterium-mediated transformation. (C) T1 EpCAM–IgM Fc and J-chain K obtained from Agrobacterium-mediated transformation. (C)T1 transgenic transgenic plants with EpCAM–IgM Fc and J-chain K obtained from self-crossing of T0 transgenic plants with EpCAM–IgM Fc and J-chain K obtained from self-crossing of T0 transgenic plants with plants with EpCAM–IgM Fc and J-chain K. Cross-fertilization between T1 transgenic plants carrying EpCAM–IgM Fc and J-chain K. Cross-fertilization between T1 transgenic plants carrying EpCAM–IgM EpCAM–IgM Fc and J-chain K transgenes after selfing of T0 plant generation. (D) F1 transgenic plants Fc and J-chain K transgenes after selﬁng of T0 plant generation. (D)F1 transgenic plants carrying both carrying both transgenes encoding EpCAM–IgM Fc and J-chain K obtained from crossing between T1 transgenes encoding EpCAM–IgM Fc and J-chain K obtained from crossing between T1 transgenic

plants carrying transgenes encoding EpCAM–IgM Fc and T1 transgenic plants carrying transgenes encoding J-chain K. The expected quaternary structure of EpCAM–IgM Fc J-chain K in transgenic × plant F1 is pentameric. Plants 2020, 9, 1466 4 of 15

2. Results

2.1. Agrobacterium-Mediated Transformation and Regeneration of T0 Transgenic Chinese Cabbage Agrobacterium-mediated transformation was conducted to transfer genes encoding EpCAM–IgM Fc or J-chain K to Chinese cabbage (Figure2). Three hundred hypocotyl pieces of Chinese cabbage were applied to transform plant expression vectors (pH2GW EpCAM–IgM Fc, and J-chain K) (Figure1A,B). After co-cultivation with Agrobacterium, the hypocotyl pieces were transferred onto regeneration media (Figure2C,D). A callus began to form at the cut ends of 30–40% of the hypocotyls within 2 weeks (Figure2D). Adventitious shoots were observed from calli after 3–4 weeks (Figure2D,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 (Figure2E,F). Most shoots showed Plantsvigorous 2020, 9 regenerative, x FOR PEER REVIEW growth on the new selective medium. However, some of the shoots became4 of 15 etiolated and died one week later. These results suggested that there were escapees from the selective mediumtransgenic culture. plants In somecarrying cases, transgen the leaveses encoding of regenerated EpCAM–IgM plants Fc and exhibited T1 transgenic chlorosis, plants indicating carrying that thesetransgenes plants might encoding be chimeras. J-chain K. The expected quaternary structure of EpCAM–IgM Fc × J-chain K in transgenic plant F1 is pentameric.

FigureFigure 2. AgrobacteriumAgrobacterium-mediated-mediated Chinese Chinese cabbage cabbage transformati transformationon and and generation generation of of transgenic ChineseChinese cabbage cabbage carrying carrying transgenes transgenes enco encodingding EpCAM–IgM EpCAM–IgM Fc Fc and and J-chain J-chain K. K. ( (AA)) In In Vitro Vitro seed seed germinationgermination to to generate generate Chinese Chinese cabbage cabbage seedlings. seedlings. (B (B) )In In Vitro Vitro preculture preculture of of hypocotyls hypocotyls in in media. media. ((C)) AgrobacteriumAgrobacterium-inoculated-inoculated hypocotyls hypocotyls incubated incubated in in in vitro in regenerationvitro regeneration media. (Dmedia.) Callus (D generated) Callus generatedfrom in vitro fromcallus-inducing in vitro callus-inducing media. (E) Shoot media. regeneration (E) Shoot from regeneration calli under thefrom regeneration calli under media. the regenerationThe left shows media. newly The regenerated left shows shoots newly from regenerated the callus. shoots The from right showsthe callus. shoots The after right further shows growthshoots afterfollowing further transfer growth to new following regeneration transf media.er to new (F) Rootregeneration induction media. from the (F regenerants) Root induction in root-inducing from the

regenerantsmedia. (G) Inin Vivo root-inducing T0 transgenic media. Chinese (G cabbage) In Vivo growth T0 transgenic after transplanting Chinese intocabbage pots withgrowth soil after from transplantingthe in vitro root-induced into pots with transgenic soil from plants. the in vitro root-induced transgenic plants.

2.2. Presence of Transgenes Encoding EpCAM–IgM Fc and J-Chain K in T0 Transgenic Plants 2.2. 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 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 presence of transgenes in transgenic plants (EpCAM–IgM Fc) and (J-chain K) randomly selected cabbage regenerants obtained by transforming each vector (Figure3A,B, respectively). All tested Chinese cabbage regenerants obtained by transforming each vector (Figure 3A,B, respectively). All regenerants obtained from transformations using plant expression vectors carrying the gene encoding tested regenerants obtained from transformations using plant expression vectors carrying the gene EpCAM–IgM Fc had ampliﬁed bands at 837 bp (EpCAM), 1053 bp (IgM Fc), and 1023 bp (hygromycin encoding EpCAM–IgM Fc had amplified bands at 837 bp (EpCAM), 1053 bp (IgM Fc), and 1023 bp phosphotransferase (HTP)) (Figure3A). All tested regenerants obtained from transformation using the (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).

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plantPlants expression2020, 9, x FOR vector PEER REVIEW carrying the gene encoding the J-chain K had the expected ampliﬁed J-chain5 of K15 bandPlants at 2020 543, 9 bp, x FOR (Figure PEER3 REVIEWB). 5 of 15

Figure 3. A polymerase chain reaction (PCR) analysis to confirm the transgenes encoding EpCAM– IgMFigure Fc and3. A J-chainpolymerase K in chain T0 transgenic reaction (PCR)lines. analysis(A) Transgenic to confirm lines the (#1–12) transgenes carrying encoding the transgene EpCAM– Figure 3. A polymerase chain reaction (PCR) analysis to conﬁrm the transgenes encoding EpCAM–IgM encodingIgM Fc andEpCAM–IgM J-chain K Fcin wereT0 transgenic tested by lines.PCR using(A) Transgenic three different lines primer (#1–12) sets carrying for EP, the IgM transgene Fc, and Fc and J-chain K in T0 transgenic lines. (A) Transgenic lines (#1–12) carrying the transgene encoding HTP.encoding PC is EpCAM–IgM a positive control; Fc were M, testedsize marker; by PCR Ep, using EpCAM three (837 different bp); IgM, primer Fc ofsets IgM for (1053 EP, IgM bp); Fc, HTP, and EpCAM–IgM Fc were tested by PCR using three diﬀerent primer sets for EP, IgM Fc, and HTP. PC is a hygromycinHTP. PC is aphosphotransferase positive control; M, size(1023 marker; bp); non-transgenic Ep, EpCAM (837 plant bp); (NT). IgM, (B Fc) Transgenic of IgM (1053 lines bp); (#1–5) HTP, positive control; M, size marker; Ep, EpCAM (837 bp); IgM, Fc of IgM (1053 bp); HTP, hygromycin carryinghygromycin a transgene encoding J-chain (1023 K. J-K, bp); J-cha non-transgenicin fused to KDEL plant endoplasmic (NT). (B) Transgenic reticulum lines retention (#1–5) phosphotransferasephosphotransferase (1023 bp); non-transgenic plant (NT). (B) Transgenic lines (#1–5) carrying a transgene signal; J-K, J-chain K (543 bp); hygromycin phosphotransferase (HTP) (1023 bp); non-transgenic encodingcarrying J-chain a transgene K. J-K, encoding J-chain fusedJ-chain to K. KDEL J-K, J-cha endoplasmicin fused reticulumto KDEL endoplasmic retention signal; reticulum J-K, J-chain retention K plant (NT). (543signal; bp); J-K, hygromycin J-chain K phosphotransferase (543 bp); hygromycin (HTP) phosphotransferase (1023 bp); non-transgenic (HTP) plant (1023 (NT). bp); non-transgenic plant (NT). 2.3.2.3. ExpressionExpression ofof EpCAM–IgMEpCAM–IgM FcFc inin TT00 Transgenic PlantsPlants 2.3. Expression of EpCAM–IgM Fc in T0 Transgenic Plants ExpressionExpression ofof thethe EpCAM–IgMEpCAM–IgM FcFc proteinprotein inin leafleaf tissuestissues ofof transgenictransgenic plantsplants waswas analyzedanalyzed usingusing WesternWesternExpression blottingblotting (Figureof(Figure the EpCAM–IgM4 ).4). In In the the IgM IgM Fc Fc proteinFc detection detection in leaf immunoblot, immunoblot, tissues of transgenic all all randomly randomly plants selected selected was analyzed transgenic transgenic using lineslinesWestern (2,6,7,9,11)(2,6,7,9,11) blotting showedshowed (Figure anan 4). approximatelyapproximately In the IgM Fc 7575 detection kDa kDa proteinprotein immunoblot, bandband similarsimilar all totorandomly thatthat ofof thethe selected positivepositive transgenic controlcontrol (Figure(Figurelines (2,6,7,9,11)4 A4A black black arrowhead). arrowhead).showed an approximately In In the the EpCAM EpCAM detection75 detection kDa protein immunoblot, immunoblot, band similar all all lines lines to that (2,6,7,9,11) (2,6,7,9,11) of the positive of of transgenic transgenic control plantsplants(Figure showedshowed 4A black thethe arrowhead). expectedexpected bandsbands In the forfor EpCAM EpCAM–IgMEpCAM–IgM detection FcFc immunoblot, (Figure(Figure4 A)4A) with allwith lines an an EpCAM–IgM (2,6,7,9,11)EpCAM–IgM of Fc-sizedtransgenic Fc-sized proteinproteinplants bandshowedband (Figure(Figure the expected4 B4B white white bands arrowhead). arrowhead). for EpCAM–IgM NoNo band band Fc was was (Figure observed observed 4A) inwith in the the an non-transgenic non-transgenicEpCAM–IgM plantsFc-sized plants (Figure(Figureprotein4 ).4). band (Figure 4B white arrowhead). No band was observed in the non-transgenic plants (Figure 4).

Figure 4. Immunoblot analysis to conﬁrm the expression of EpCAM–IgM Fc in T0 transgenic lines. 0 (FigureA)T0 transgenic 4. Immunoblot plants analysis #2, 6, 7, 9,to andconfirm 11 were the testedexpression by goat of anti-IgMEpCAM–IgM Fc µ-chain Fc in conjugatedT transgenic to lines. HRP. M,(AFigure) protein T0 transgenic 4. marker;Immunoblot plants+, tobacco #2,analysis 6, 7, plant 9, to and confirm EpCAM–IgM 11 were the tested expression Fc by as goat a positive of anti-IgM EpCAM–IgM control; Fc µ-chain Fc, non-transgenic in conjugated T0 transgenic to plantHRP. lines. − asM,(A a protein) negative T0 transgenic marker; control. plants +, (tobaccoB)T #2,0 6,transgenic plant7, 9, and EpCAM–IgM 11 plants were #2,tested Fc 6, 7,as by 9,a goat andpositive anti-IgM 11 were control; testedFc µ-chain−, non-transgenic by mouse conjugated anti-human plant to HRP. as EpCAMaM, negative protein as control. a marker; primary (B +,)ﬁrst T tobacco0 transgenic antibody plant plants and EpCAM–IgM goat #2, anti-mouse6, 7, 9, andFc as 11 IgGa werepositive 2a tested heavy control; by chain mouse − conjugated, non-transgenic anti-human to HRP EpCAM plant as aas secondaryasa anegative primary antibody. control. first antibody ( M,B) T protein0 transgenic and goat marker; anti-mouseplants+, tobacco #2, 6, Ig 7,G plant9, 2a and heavy EpCAM–IgM 11 were chain tested conjugated Fc by as mouse a positive to HRPanti-human control as a secondary (50EpCAM ng); antibody.,as non-transgenic a primary M, proteinfirst plant antibody marker; as a negativeand +, goattobacco control;anti-mouse plant hygromycin EpCAM–IgM IgG 2a heavy phosphotransferase Fc chain as a conjugatedpositive control (HTP). to HRP (50 as ng); a secondary −, non- − transgenicantibody. plantM, protein as a nega marker;tive control; +, tobacco hygromycin plant EpCAM–IgM phosphotransferase Fc as a positive (HTP). control (50 ng); −, non- transgenic plant as a negative control; hygromycin phosphotransferase (HTP).

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2.4. Presence of Transgenes Encoding EpCAM–IgM Fc and J-Chain K in T1 Transgenic Plants 2.4. 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 generateThe a T 0Ttransgenic1 line #2 (Figure line #2,1; Figure with the 4). highestPCR was expression performed of for EpCAM–IgM the presence Fc, of wastransgenes self-crossed in eight to generate(EpCAM–IgM a T1 line Fc) #2T1 (Figureplants 1(Figure; Figure 5).4). All PCR tested was performedT1 plants obtained for the presence from the of transformation transgenes in eightusing (EpCAM–IgMthe plant expression Fc) T1 plantsvector (Figurecarrying5). the All gene tested encoding T 1 plants EpCAM–IgM obtained from Fc thehad transformation the expected amplified using the plantbands expression at 837 bp (EpCAM), vector carrying 1053 thebp gene(IgM–Fc), encoding and 1023 EpCAM–IgMbp (HTP) Fc(Figure had the 5A). expected In the transgenic amplified bandsplant atcarrying 837 bp the (EpCAM), J-chain 1053K transgene, bp (IgM–Fc), the andtransgenic 1023bp line (HTP) 1 was (Figure self-crossed5A). In the to transgenic generate plantT1. Transgenic carrying theplants J-chain in T1 K carrying transgene, the theJ-chain transgenic K transgene line 1 were was self-crossedrandomly selected to generate for PCR T1. Transgenictesting (Figure plants 5B). in All T1 carryingof the tested the J-chainT1 plants K transgeneobtained from were randomlyselfing had selected the expect for PCRed amplified testing (Figure band at5B). 543 All bp of (J-chain the tested K) T(Figure1 plants 5B). obtained from selfing had the expected amplified band at 543 bp (J-chain K) (Figure5B).

Figure 5. A polymerasepolymerase chainchain reaction reaction (PCR) (PCR) analysis analysis to to conﬁrm confirm the the transgenes transgenes encoding encoding EpCAM–IgM EpCAM– IgM Fc and J-chain K in the T1 transgenic lines. (A) T1 transgenic lines (#2–9, 2–14, 2–17, 2–20, 2–21, 2– Fc and J-chain K in the T1 transgenic lines. (A)T1 transgenic lines (#2–9, 2–14, 2–17, 2–20, 2–21, 2–23, 2–25,23, 2–25, and and 2–27) 2–27) carrying carrying the the transgene transgene encoding encoding EpCAM–IgM EpCAM–IgM Fc Fc were were tested tested byby PCRPCR usingusing three didifferentﬀerent primer sets for EP, IgMIgM Fc,Fc, andand HTP. PCPC isis aa positivepositive control;control; M,M, sizesize marker;marker; Ep,Ep, EpCAM (837 bp);bp); IgM, IgM, Fc Fc of IgMof IgM (1053 (1053 bp); bp); HTP, HTP, hygromycine hygromycine phosphotransferase phosphotransferase (1023 bp); (1023 non-transgenic bp); non- transgenic plant (NT). (B) T1 transgenic lines (#1–1, 1–2, 1–3, 1–4, and 1–5) carrying the transgene plant (NT). (B)T1 transgenic lines (#1–1, 1–2, 1–3, 1–4, and 1–5) carrying the transgene encoding J-chainencoding K. J-chain J-K, J-chain K. J-K, fused J-chain to KDEL fused endoplasmicto KDEL endoplasmic reticulum reticulum retention retention signal; J-K, signal; J-chain J-K, K J-chain (543 bp); K hygromycin(543 bp); hygromycin phosphotransferase phosphotransferase (HTP) (1023 (HTP) bp); non-transgenic (1023 bp); non-transgenic plant (NT). plant (NT).

2.5. Expression of EpCAM–IgM Fc in T1 TransgenicTransgenic Plants

In all testedtested TT11 transgenictransgenic lines, lines, expression expression of of the the EpCAM–IgM Fc Fc protein was confirmed conﬁrmed by Western blotting (Figure 66).). In the the IgM IgM Fc Fc detection detection immunoblot, immunoblot, all all tested tested transgenic transgenic lines lines (#2–9, (#2–9, 2– 2–14,14, 2–28, 2–28, 2–35, 2–35, and and 2–54), 2–54), except except for for lines lines 2–17, 2–17, show showeded a astrong strong protein protein band band (75 (75 kDa) kDa) (Figure (Figure 66A).A). In the EpCAMEpCAM detectiondetection immunoblot, the #2–9, 2–1 2–14,4, and 2–21 lines showed the strongest protein band density, whereas the #2–17, 2–23, and 2–27 line liness had weak protein bands (75 (75 kDa) kDa) (Figure (Figure 66B).B). Additionally, 5050 kDakDa sizesize bandsbands werewere alsoalso observedobserved in all lines, including the non-transgenic control line. No No band was observed in the non-transgenic plants (Figure 66).). ItIt isis speculatedspeculated thatthat thethe 5050 kDakDa protein bands are RubiscoRubisco proteins,proteins, whichwhich areare oftenoften detecteddetected inin plantplant totaltotal solublesoluble proteins.proteins.

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Plants 2020, 9, x FOR PEER REVIEW 7 of 15

Figure 6. Immunoblot analysis to confirm the expression of EpCAM–IgM Fc in representative T1 Figure 6. Immunoblot analysis to confirm the expression of EpCAM–IgM Fc in representative T1 transgenic lines. (A) T1 transgenic plants #2–9, 2–14, 2–17, 2–21, 2–23, and 2–27 were tested by goat transgenic lines. (A) T1 transgenic plants #2–9, 2–14, 2–17, 2–21, 2–23, and 2–27 were tested by goat anti-humanFigure IgM6. Immunoblot Fc µ-chain conjugatedanalysis to to confirm HRP. M, the protein expressi marker;on of +,EpCAM–IgM tobacco plant Fc EpCAM–IgM in representative Fc T1 anti-human IgM Fc µ-chain conjugated to HRP. M, protein marker; +, tobacco plant EpCAM–IgM Fc as as a positivetransgenic control; lines. − ,( non-transgenicA) T1 transgenic plant plants as a#2–9, negative 2–14, control. 2–17, 2–21, (B) T1 2–23, transgenic and 2–27 plants were #2–9, tested 2–14, by goat a positiveanti-human control; IgM, non-transgenic Fc µ-chain conjugated plant as to a negativeHRP. M, control.protein marker; (B) T1 transgenic +, tobacco plantsplant EpCAM–IgM #2–9, 2–14, Fc 2–17, 2–21, 2–23, and− 2–27 were tested by mouse anti-human EpCAM as the first antibody and goat 2–17, 2–21, 2–23, and 2–27 were tested by mouse anti-human EpCAM as the first antibody and goat anti-mouseas a positive IgG 2a control; conjugated −, non-transgenic to HRP as the plant second as a negativeantibody. control. M, protein (B) T1 marker; transgenic +, tobacco plants #2–9,plant 2–14, anti-mouse IgG 2a conjugated to HRP as the second antibody. M, protein marker; +, tobacco plant EpCAM–IgM2–17, 2–21, Fc 2–23, as a and positive 2–27 werecontrol tested (50 by ng); mouse −, non-transgenicanti-human EpCAM plant asas the a firstnegative antibody control; and goat EpCAM–IgM Fc as a positive control (50 ng); , non-transgenic plant as a negative control; hygromycin hygromycinanti-mouse phosphotransferase IgG 2a conjugated ( HTP).to HRP − as the second antibody. M, protein marker; +, tobacco plant phosphotransferaseEpCAM–IgM (HTP).Fc as a positive control (50 ng); −, non-transgenic plant as a negative control; hygromycin phosphotransferase (HTP). 2.6. Presence Presence of Transgenes Encoding EpCAM–IgM FcFc andand J-ChainJ-Chain KK inin FF11 Transgenic PlantsPlants ObtainedObtained between EpCAM–IgM Fc and J-Chain K T1 Plants 2.6. Presence of Transgenes Encoding EpCAM–IgM Fc and J-Chain K in F1 Transgenic Plants Obtained Thebetween F 1 plantsplants EpCAM–IgM were were obtained Fc and from J-Chain crossing K T1 Plants EpCAM–IgM FcFc andand J-chainJ-chain KK TT11 plants. The The presence presence of transgenes encoding encoding EpCAM–IgM EpCAM–IgM Fc Fc and and J-chain J-chain K Kwas was confirmed confirmed in inthe the F1 Fplants1 plants using using PCR. PCR. In The F1 plants were obtained from crossing EpCAM–IgM Fc and J-chain K T1 plants. The presence randomlyIn randomly selected selected F1 plants, F1 plants, all tested all tested F1 lines F1 lines showed showed the presence the presence of the of transgenes the transgenes EpCAM EpCAM (837 (837bp), bp),IgMof transgenes IgMFc (1053 Fc (1053 bp),encoding bp), and and J-chain EpCAM–IgM J-chain K (543 K (543 bp),Fc bp),and except exceptJ-chain for for KM209-8, M209-8,was confirmed in in which which in only onlythe theF the1 plants J-chain J-chain using K K gene PCR. In was detectedrandomly without selected the F 1 EpCAM plants, all and tested IgM FcFcF1 genesgeneslines showed (Figure(Figure7 the7).). presence of the transgenes EpCAM (837 bp), IgM Fc (1053 bp), and J-chain K (543 bp), except for M209-8, in which only the J-chain K gene was detected without the EpCAM and IgM Fc genes (Figure 7).

Figure 7. A polymerase chain reaction (PCR) analysis to confirm the transgenes encoding EpCAM– Figure 7. A polymerase chain reaction (PCR) analysis to confirm the transgenes encoding EpCAM–IgM IgM Fc and J-chain K in F1 transgenic lines obtained by crossing between T1 EpCAM Fc transgenic Fc andFigure J-chain 7. KA inpolymerase F1 transgenic chain lines reaction obtained (PCR) by crossinganalysis betweento confirm T1 theEpCAM transgenes Fc transgenic encoding plants EpCAM– plants (#2) and T1 J-chain transgenic lines (#1). F1 transgenic lines (#M209-1, M209-2, M209-3, M209-4, (#2) and T1 J-chain transgenic lines (#1). F1 transgenic lines (#M209-1, M209-2, M209-3, M209-4, M209-5, M209-5,IgM M209-6, Fc and M209-7, J-chain M209-9,K in F1 transgenic and M209-10) lines carry obtaineding a transgeneby crossing encoding between EpCAM–IgM T1 EpCAM FcFc weretransgenic M209-6, M209-7, M209-9, and M209-10) carrying a transgene encoding EpCAM–IgM Fc were tested plants (#2) and T1 J-chain transgenic lines (#1). F1 transgenic lines (#M209-1, M209-2, M209-3, M209-4, testedby PCR by using PCR threeusing di threefferent different primer primer sets for sets Ep, fo IgMr Ep, Fc, IgM and Fc, HTP. and positive HTP. positive control (PC);control size (PC); marker size M209-5, M209-6, M209-7, M209-9, and M209-10) carrying a transgene encoding EpCAM–IgM Fc were (M);marker Ep, (M); EpCAM Ep, EpCAM (837 bp); (837 IgM, bp); Fc of IgM, IgM Fc (1053 of IgM bp); (1053 J-K, J-chain bp); J-K, K (543J-chain bp); K non-transgenic (543 bp); non-transgenic plant (NT); tested by PCR using three different primer sets for Ep, IgM Fc, and HTP. positive control (PC); size planthygromycine (NT); hygromycine phosphotransferase phosphotransferase (HTP). (HTP). marker (M); Ep, EpCAM (837 bp); IgM, Fc of IgM (1053 bp); J-K, J-chain K (543 bp); non-transgenic plant (NT); hygromycine phosphotransferase (HTP). 2.7. Expression Expression of EpCAM–IgM Fc in F 1 TransgenicTransgenic Plants Obtained by Crossing EpCAM–IgM Fc and J- ChainJ-Chain K KT1 T Plants1 Plants 2.7. Expression of EpCAM–IgM Fc in F1 Transgenic Plants Obtained by Crossing EpCAM–IgM Fc and J- In the F1 transgenic lines with positive PCR results for EpCAM, IgM Fc, and J-chain K transgenes, InChain the FK1 Ttransgenic1 Plants lines with positive PCR results for EpCAM, IgM Fc, and J-chain K transgenes, the expression of EpCAM–IgM Fc Fc was confirmed confirmed by by Western blotting (Figure 88A).A). InIn thethe IgMIgM FcFc In the F1 transgenic lines with positive PCR results for EpCAM, IgM Fc, and J-chain K transgenes, detection immunoblot, all tested F 1 transgenictransgenic lines lines (#1–9) (#1–9) showed showed the the 75 75 kDa kDa size size protein protein band. band. the expression of EpCAM–IgM Fc was confirmed by Western blotting (Figure 8A). In the IgM Fc Furthermore, in the EpCAM detectiondetection immunoblot, all tested F11 transgenic lines (#1–9) showed the 75 kDadetection size proteinprotein immunoblot, bandband (Figure(Figure all 8tested B).8B). No No F bands1 transgenicbands were were observedlines observed (#1–9) in in the showed the non-transgenic non-transgenic the 75 kDa plants plantssize (Figure protein (Figure8). band. 8). 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).

Plants 2020, 9, 1466 8 of 15 Plants 2020, 9, x FOR PEER REVIEW 8 of 15

Plants 2020, 9, x FOR PEER REVIEW 8 of 15

Figure 8. Immunoblot analysis to confirm the expression of EpCAM–IgM Fc in representative F1 Figure 8. Immunoblot analysis to conﬁrm the expression of EpCAM–IgM Fc in representative F1 Figure 8. Immunoblot analysis to confirm the expression of EpCAM–IgM Fc in representative F1 transgenic lines. ( (AA))F F1 transgenictransgenic plants plants M209-1–209-10 M209-1–209-10 were were test testeded by by goat goat anti-IgM Fc µµ-chain-chain transgenic lines. (A1) F1 transgenic plants M209-1–209-10 were tested by goat anti-IgM Fc µ-chain conjugated to HRP. protein marker (M); +, tobacco plant EpCAM–IgM Fc as a positive control; − non- conjugatedconjugated to HRP. to HRP. protein protein marker marker (M); +,+ tobacco, tobacco plant plant EpCAM–IgM EpCAM–IgM Fc as a positive Fc as a control; positive − non- control; transgenicnon-transgenic plant as plant a negative as a negative control. control.(B) F1 transgenic (B)F transgenic plants M209-1–209-10 plants M209-1–209-10 were tested were by mouse tested − transgenic plant as a negative control. (B) F1 transgenic1 plants M209-1–209-10 were tested by mouse byanti-human mouseanti-human anti-human EpCAM EpCAM as EpCAMthe as thefirst first antibody as antibody the ﬁrst and and antibody goat goat an anti-mouse andti-mouse goat IgGIgG anti-mouse 2a 2a conjugated conjugated IgG to 2a toHRP conjugated HRP as the as thesecond tosecond HRP antibody. 2–9, T1 transgenic plant #2–9 (Figure 6); +, tobacco plant EpCAM–IgM Fc as a positive as theantibody. second antibody.2–9, T1 transgenic 2–9, T 1planttransgenic #2–9 (Fig planture 6); #2–9 +, tobacco (Figure plant6); EpCAM–IgM+, tobacco plant Fc as EpCAM–IgMa positive Fccontrol ascontrol a (50 positive ng); (50 −ng);,control non-transgenic −, non-transgenic (50 ng); plant ,plant non-transgenic (NT) (NT) as as a anegative negative plant control; (NT) ashygromycin hygromycin a negative phosphotransferase phosphotransferase control; hygromycin − phosphotransferase(HTP)(HTP). . (HTP).

2.8. Binding 2.8. Binding Affinity Aﬃnity Affinity of the of Anti-IgMAntithe Anti-IgM-IgM Fc Fcµµ-Chain-Chain µ-Chain Antibody Antibody toto ChineseChineseinese Cabbage-Derived Cabbage-DerivedCabbage-Derived EpCAM-IgM EpCAM-IgMEpCAM-IgM Fc × Fc × Fc J-ChainJ-Chain KK (EpCAM-IgM FcC FcC × J-ChainJ-Chain KC). KC). J-Chain× K (EpCAM-IgM FcC × J-Chain× KC). Enzyme-linkedEnzyme-linked immunosorbent immunosorbent assay assay (ELISA) (ELISA) waswas conducted for for the the recognition recognition of Chinese of Chinese Enzyme-linked immunosorbent assay (ELISA) was conducted for the recognition of Chinese cabbage-derivedcabbage-derived EpCAM–IgM EpCAM–IgM FcC FcCJ-chain × J-chain KC KC using using an an anti-IgM anti-IgM Fc Fcµ µ-chain-chain antibody antibody (Figure(Figure 99).). In In the cabbage-derived EpCAM–IgM FcC × J-chain KC using an anti-IgM Fc µ-chain antibody (Figure 9). In the anti-IgM Fc µ-chain antibody× treatment group, both EpCAM–IgMT FcT × J-ChainKT T and EpCAM– anti-IgM Fc µ-chain antibody treatment group, both EpCAM–IgM Fc J-ChainKT andT EpCAM–IgM the anti-IgMIgM FcC Fc× J-ChainK µ-chainC antibodyshowed high treatment absorbance group, levels both (0.367 EpCAM–IgM and 0.315, respectively),× Fc × J-ChainK whereas and EpCAM– EpCAM– FcC J-ChainKC showed high absorbance levels (0.367 and 0.315, respectively), whereas EpCAM–IgG IgM× FcIgGC × J-ChainKFcT had almostC showed no absorbance high absorbance signal (0.02) levels (Figure (0.367 9).and However, 0.315, respectively), in the anti-IgG whereas Fc treatment EpCAM– FcT had almost no absorbance signal (0.02) (Figure9). However, in the anti-IgG Fc treatment group, IgG Fcgroup,T had bothalmost EpCAM–IgM no absorbance FcT × J-ChainKsignal (0.02)T and (Figure EpCAM–IgM 9). However, FcC × J-ChainK in theC anti-IgGshowed almostFc treatment no both EpCAM–IgM FcT J-ChainKT and EpCAM–IgM FcC J-ChainKC showed almost no absorbance group,absorbance both EpCAM–IgM signal ×(0.01 FcandT ×0.018, J-ChainK respectively),T and EpCAM–IgM whereas× EpCAM–IgG FcC × J-ChainK FcT showedC showed an absorbance almost no signal (0.01 and 0.018, respectively), whereas EpCAM–IgG FcT showed an absorbance signal (0.165) absorbancesignal (0.165)signal (Figure (0.01 and 9). In 0.018, the 1X respectively),PBS treatment group,whereas all three EpCAM–IgG samples lacked FcT showedan absorbance an absorbance signal (Figuresignal(Figure (0.165)9). In 9). the(Figure 1X PBS 9). In treatment the 1X PBS group, treatment all three group, samples all three lacked samples an absorbance lacked an signal absorbance (Figure signal9). (Figure 9).

FigureFigure 9. Enzyme-linked 9. Enzyme-linked immunosorbent immunosorbent assay assay (ELISA) (ELISA) was performed was performed to confirm to confirm binding binding interaction betweeninteraction anti-IgM between Fc µ-chain anti-IgM antibody Fc µ-chain and antibody EpCAM–IgM and EpCAM–IgM Fc J-ChainK Fc × J-ChainK purified purified from F fromtransgenic F1 × 1 Chinesetransgenic cabbage Chinese plant. cabbage Each plant. ELISA Each plate ELISA well plate was well coatedwas coated with with 10 10 ng ng of the the purified purified Chinse Chinse cabbage-derived EpCAM–IgM Fc × J-ChainK (EpCAM–IgM FcC × J-ChainKC), tobacco-derived cabbage-derivedFigure 9. Enzyme-linked EpCAM–IgM immunosorbent Fc J-ChainK assay (EpCAM–IgM (ELISA) was Fc CperformedJ-ChainK toC), confirm tobacco-derived binding EpCAM–IgM Fc × J-ChainK (EpCAM–IgM× FcT x J-ChainKT), or tobacco-derived× EpCAM–IgG Fc EpCAM–IgMinteraction between Fc J-ChainK anti-IgM (EpCAM–IgMFc µ-chain antibody FcT x and J-ChainK EpCAM–IgMT), or tobacco-derived Fc × J-ChainK EpCAM–IgGpurified from FcF1 (EpCAM–IgG× Fc T). Each anti-IgM Fc µ-chain antibody and anti-IgG Fc antibody conjugated to HRP (EpCAM–IgGtransgenic Chinese Fc T). cabbage Each anti-IgM plant. Ea Fcchµ ELISA-chain antibodyplate well and was anti-IgG coated with Fc antibody 10 ng of the conjugated purified toChinse HRP was applied to the coated well. Error bars indicate standard deviation (* p < 0.05); hygromycin C C wascabbage-derived appliedphosphotransferase to the EpCAM–IgM coated (HTP) well. .Fc Error × J-ChainK bars indicate (EpCAM–IgM standard deviationFc × J-ChainK (* p < ),0.05); tobacco-derived hygromycin T T phosphotransferaseEpCAM–IgM Fc × J-ChainK (HTP). (EpCAM–IgM Fc x J-ChainK ), or tobacco-derived EpCAM–IgG Fc (EpCAM–IgG Fc T). Each anti-IgM Fc µ-chain antibody and anti-IgG Fc antibody conjugated to HRP was applied to the coated well. Error bars indicate standard deviation (* p < 0.05); hygromycin phosphotransferase (HTP).

Plants 2020, 9, 1466 9 of 15

3. 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 T0 and T1 Chinese cabbage, including F1 plants from a crossing between EpCAM–IgM Fc and J-chain K T1 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 T0 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 T0 transgenic plants. In EpCAM–IgM Fc, among the tested T0 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 T1 plants. Regarding the J-chain K, according to Western blot analysis, its expression was not observed in the T0 transgenic plants. In both EpCAM–IgM Fc and J-chain K T1 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 T1 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 T1 transgenic line highly expressing EpCAM–IgM Fc proteins was selected to cross with the T1 transgenic line carrying the J-chain K transgene to generate F1 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 [39–41]. In this study, we used self- and cross-fertilization of Chinese cabbage to obtain the F1 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 Plants 2020, 9, 1466 10 of 15 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 [26–28], 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 F1 transgenic seedlings carrying both transgenes. These results revealed that both transgenes were stably inserted, and the EpCAM–IgM Fc protein gene was expressed in F1 transgenic plants reproduced from crossing between EpCAM–IgM Fc and J-chain K T1 transgenic plants. Furthermore, these results suggested that two genes can co-exist through cross-breeding in Chinese cabbage T1 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.

4. Materials and Methods

4.1. 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 (Figure1A). 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.

4.2. 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) (Figure1B). 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.

4.3. 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 Plants 2020, 9, 1466 11 of 15 the bacterial inoculum for 10 min (Figure1B). 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), AgNO3 (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 (Figure1B). 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).

4.4. 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 NanovueTM 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.

4.5. 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. Plants 2020, 9, 1466 12 of 15

4.6. Cross-Fertilization

After T0 transgenic plant validation by PCR and Western blot analysis, the transgenic plantlets (T0) 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 (Figure1B). The plants were grown under normal daylight conditions with a set temperature of 22 ◦C and 60–70% relative humidity (RH). The T0 plants were grown in 2015 and selfed to produce seeds (T1). In 2016, the T1 seeds were sown and the plants were grown for 4–6 weeks with vernalization treatment (Figure1C). The T 1 plants containing the EpCAM–IgM Fc genes were crossed with the T1 plants with J-chain K during the blooming stage for the generation of the F1 transgenic lines in 2017. The crossing resulted in the generation of the F1 transgenic line (Figure1D).

4.7. Enzyme-Linked Immunosorbent Assay (ELISA) The Maxisorp 96-well immuno plates (Sigma-Aldrich, St. Louis, MO, USA) were coated and incubated overnight at 4 °C with 10 ng per well of puriﬁed tobacco plant-derived EpCAM–IgG Fc (EpCAM–IgG FcT)[44], tobacco plant-derived EpCAM–IgM Fc (EpCAM–IgM FcT) J-Chain KT [48], × and Chinese cabbage-derived EpCAM–IgM Fc (EpCAM–IgM FcC) J-Chain KC proteins diluted in × 0.05 M carbonate/bicarbonate buﬀer (Sigma-Aldrich, St. Louis, MO, USA). After overnight incubation, the plates were washed with 1X PBS plus 0.5% (v/v) Tween 20 (1X PBS-T) four times and blocked with 5% skim milk (Sigma-Aldrich, St. Louis, MO, USA) in 1X PBS-T for 1 h at RT. Then, the plates were treated with rabbit anti-human IgG Fcγ antibody conjugated to HRP (Jackson Immunolab, West Grove, PA, USA) or goat anti-human IgM Fc µ-chain antibody conjugated to HRP (Abcam, Cambridge, UK) diluted 1:5000 in 1X PBS-T or 1X PBS and incubated for 2 h at RT. After washing four times with 1X PBS-T, the plates were detected using 3,30,5,50-Tetramethylbenzidine (TMB) substrate and TMB stop solution (SeraCare, Milford, CT, USA). The plates were read using an Epoch Microplate Spectrophotometer (BioTek, Winooski, VT, USA) at 450 nm.

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

Author Contributions: Conceptualization, Y.-R.L., K.K., and D.-S.K.; methodology, Y.-R.L., C.-Y.L., J.K., H.-E.L., S.L., K.K., and D.-S.K.; software, S.R.P., S.L., H.-E.L., and C.-Y.L.; validation, Y.-R.L., K.K., and D.-S.K.; formal analysis, Y.-R.L., C.-Y.L., S.L., J.-P.H., and H.-E.L.; investigation, Y.-R.L., K.K., and D.-S.K.; resources, Y.-R.L., and C.-Y.L.; data curation, Y.-R.L., C.-Y.L., S.R.P., S.L., K.K., and D.-S.K.; writing—original draft preparation, Y.-R.L., D.-S.K., and K.K.; writing—review and editing, Y.-R.L., S.R.P., S.L., K.K., and D.-S.K.; visualization, Y.-R.L., C.-Y.L., S.R.P., S.L., S.K., and K.K.; supervision K.K. and D.-S.K.; project administration, J.K., K.K., and D.-S.K. All authors have read and agreed to the published version of the manuscript. Funding: This work was carried out with the support of “Cooperative Research Program for Agriculture Science and Technology Development (Project No. PJ01343703)” Rural Development Administration, Republic of Korea. Conﬂicts of Interest: The authors declare no conﬂict of interest.

Abbreviations

ELISA Enzyme-linked immunosorbent assay ER Endoplasmic reticulum EpCAM Epithelial cell adhesion molecule Fc Fragment crystallizable IgM Immunoglobulin M Plants 2020, 9, 1466 13 of 15

J-chain Joining-chain HRP Horseradish peroxidase MS Murashige and skoog PBS Phosphate-buﬀered saline PCR Polymerase chain reaction TAA Tumor-associated antigen

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