Introduction

The GA733-2 antigen is present in the majority of human epithelia and is highly expressed in gastrointestinal carcinomas (Ross et al. 1986). The GA733-2 gene encodes for a 314 amino acid polypeptide that includes a 23 amino acid signal sequence, a 242 amino acid extracellular domain, a 23 amino acid transmembrane domain, and a 26 amino acid cytoplasmic domain (Szala et al. 1990). The extracellular domain of GA733-2 antigen has three potential N-linked glycosylation sites and an N-terminal cysteine-rich region containing 12 cysteines. The extracellular domain of antigen GA733-2 has been used as a target of cancer vaccination in several clinical trials (Birebent et al. 2001; Herlyn et al. 1994; Staib et al. 2001). Expression of recombinant GA733-2 has been reported in many heterologous expression systems (Strassburg et al. 1992; Schmetzer et al. 2005; Seok et al. 2010). Plants offer unique advantages for production of recombinant antigens in terms of scale, speed, costs, and safety (Fisher et al. 2004). Recently, tumor-associated colorectal cancer antigen EpCAM (GA733-2) was expressed in plants and the recombinant plant-derived antigen induced a humoral immune response (Brodzik et al. 2008). However, plants are not an ideal expression system for producing therapeutic proteins because N-glycan processing differs in plants and humans. Plant-derived specific N-glycans contain antigenic and/or allergenic β(1,2)-xylose and α(1,3)-fucose, which are absent in mammalian glycans (Fötisch and Vieths 2001). To avoid the plant-derived specific N-glycan structure, we attempted to generate an oligomannose glycan structure by retaining the recombinant protein (GA733-2-Fc) in the endoplasmic reticulum. Also, we have previously reported that fusion of GA733-2 peptides with the immunoglobulin Fc fragment facilitates the purification process in an insect-cell expression system (Seok et al. 2010). The ability of fusion molecules containing the Fc fragment to assemble together probably confers some structural advantages for protein stability.

Therefore, in this study, we describe the expression of human colorectal cancer antigen GA733-2 fused to Fc fragment of antibody and KDEL (the ER retention motif) (GA733-2-Fc/KDEL) in infiltrated Nicotiana benthamiana leaves. We also examine the immunogenic response of recombinant GA733-2-Fc/KDEL in mice. Immunization with a plant-derived GA733-2-Fc/KDEL fusion protein induces a strong serum antibody response and regresses tumor volumes in colorectal CT-26 mouse model.

Materials and methods

DNA constructions

A GA733-2-Fc sequence was amplified from pGEM-T/GA733-2-Fc (Seok et al. 2010) via PCR using sense (5′-GTCGACGCGGTTCGAAGGTCTTCC-3′) and antisense (5′-ACTAGTTTTGACAAGCATGCT-3′) primers. The product was cloned into the pGEM-T vector (Promega, Madison, WI, USA), and recloned into plasmid pImpactVector-1.3tag (Plant Research International, Wageningen, The Netherlands) comprised of the RbcS1 promoter, C-terminal tags (c-myc and His6) and the KDEL ER retention signal. The GA733-2-Fc/KDEL sequences were amplified from pImpatVector-1.3tag/GA733-2-Fc via PCR using sense (5′-GTCGACGCGGTTCGAAGGTCTTCC-3′) and antisense (5′-ACTAGTTTTGACAAGCATGCT-3′) primers and subcloned into the SalI and SpeI sites of pBCTVR, thereby yielding pCsVMV-BCTVR-GA733-2-Fc/KDEL (Fig. 1a). PCR was conducted using a Thermal Cycler (PE Biosystems, Foster City, CA, USA) with PCR mix (Takara, Shiga, Japan) in a volume of 50 μl. All of the constructs were verified via both restriction enzyme map** and DNA sequencing.

Fig. 1
figure 1

a Schematic diagram of the vector construct (pCsVMV-BCTVR-GA733-2-Fc/KDEL). Ovals mark the duplicated viral origin of replication, and REP indicates the viral gene block containing the viral L1 (REP), L2, L3 (REn), and L4 genes of Beet curly top virus. The region between the right (TR) and left (TL) borders also carries GA733-2-Fc (the sequence of the GA733-2 fused to the Fc fragment of human IgG), the CsVMV promoter from the Cassava vein mosaic virus, the nopaline synthase (NOS) transcription terminator, the signal peptide, C-terminal tag (c-myc-His6), the ER retention signal (KDEL), and the selection marker cassette (HPT) conferring resistance to hygromycin. T-DNA regions are not drawn to scale. b Replication of the pCsVMV-BCTVR-GA733-2-Fc/KDEL vector in Agrobacterium-infiltrated N. benthamiana leaves. A sample of 10 μg of total DNA was separated on 0.9% agarose gel, transferred to a nylon membrane, and then hybridized with mixed 32P-labled probes specific for GA733-2 and λ/Hind III marker. Lane M contains λ/Hind III molecular weight markers. Lane N contains DNA sample isolated from non-inoculated N. benthamiana leaves. DNA isolated from leaves 3 and 6 days after infiltration with recombinant Agrobacterium containing pCsVMV-BCTVR-GA733-2-Fc/KDEL was either untreated (lanes 1 and 2) or restricted with Sal I (lanes 3 and 4). The arrow indicates replicating DNA molecules. c Expression of recombinant GA733-2/Fc/KDEL from a BCTV vector. Western blot of extracts from Agrobacterium-infiltrated leaves of N. benthamiana is shown. A sample of 20 μg of proteins from extracts of infiltrated leaves were separated on 10% SDS-polyacrylamide gel, transferred to a nitrocellulose membrane, and incubated with goat anti-human IgG (Fc specific) horseperoxidase. Lanes 1 and 2 contains total protein isolated from leaves 3 and 6 days after infiltration with recombinant Agrobacterium containing pCsVMV-BCTVR-GA733-2-Fc/KDEL. Lanes 3, 4, and 5 contain 50, 10, and 300 ng of purified GA733-2-Fc protein, respectively. The arrow indicates recombinant GA733-2-Fc/KDEL

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Recombinant Agrobacterium tumefaciens strains infiltration

The recombinant plant expression vector pCsVMV-BCTVR-GA733-2-Fc/KDEL was introduced into A. tumefaciens GV3101 by electroporation, as described by Ainsworth et al. (1996). Recombinant A. tumefaciens GV3101 strains harboring pCsVMV-BCTVR-GA733-2-Fc/KDEL were infiltrated into the abaxial air spaces of 2- to 4-week-old Nicotiana benthamiana plants (Kim et al. 2007). Western blot analysis was performed at 3 and 6 days post-infiltration.

Southern blot analysis

Total DNA was extracted from leaf-disks using DNeasy Plant Mini Kit (Qiagen, Valencia, CA, USA) and 20 μg samples were analyzed by gel blot hybridization. Samples were electrophoresed on 0.8% agarose gel in 0.5× TBE buffer, transferred to a nylon membrane (Hybond-N+; Amersham Pharmacia Biotech, Buckinghamshire, UK), and UV-crosslinked. Hybridization was carried out in 6× SSC, 5× Denhardt’s solution, 0.5% SDS, and 0.1 mg ml−1 of herring sperm DNA (Gibco BRL, Grand Island, NY, USA) using a mixed probes specific for GA733-2 and λ/HindIII marker. The DNA probes were prepared by random primer labeling in the presence of [α-32P]dCTP.

Western blot analysis

Proteins were extracted from tobacco leaves, as described by Ainsworth et al. (1996), followed by electrophoresis on 10% SDS-polyacrylamide gel. The fractionated proteins were transferred to nitrocellulose membrane (Hybond-C extra; Amersharm Pharmacia Biotech) The membranes were pre-incubated with blocking solution [5% (w/v) non-fat dry milk in TBS containing 0.1% Tween-20] for 1 h, and then probed with goat anti-human IgG (Fc specific)-alkaline phosphatase conjugate (diluted in blocking solution to 1:10,000). After washing, a BCIP/NBT solution (Amresco, Solon, OH, USA) was added and the reaction was quenched with distilled water.

Purification of recombinant GA733-2-Fc/KDEL

All steps were performed at 4°C. Recombinant GA733-2-Fc/KDEL was purified using affinity fractionation with Protein A Sepharose 4 Fast Flow (GE Healthcare, Sweden) according to the manufacturer’s recommendations. Protein extracts were dialyzed with binding buffer (20 mM sodium phosphate buffer, pH 7.0) and applied to a Protein A Sepharose 4 Fast Flow column. Weakly bound contaminating proteins were washed from the beads using binding buffer. Recombinant GA733-2-Fc/KDEL proteins were then eluted with 0.1 M glycine (pH 3.0) and dialyzed in phosphate-buffered saline (PBS, pH 7.4). Protein concentrations were determined using a Bradford Protein Assay Kit (Bio-Rad, Hercules, CA, USA) with BSA as a standard.

Enzyme-linked immunosorbent assay of recombinant GA733-2-Fc/KDEL proteins in Agrobacterium infiltrated tobacco leaves

For quantification of recombinant GA733-2-Fc/KDEL proteins, we used the general enzyme-linked immunosorbent assay (ELISA) protocol by DuoSet® ELISA Development System Human EpCAM/TROP1 (R&D Systems, Minneapolis, MN, USA). Aliquots of total soluble proteins from N. benthamiana leaf disks and samples of known amounts of recombinant GA733-2-Fc/KDEL purified from transformed S2 cells comprising a standard curve were added to the wells. The absorbance at 450 nm was measured using an ELISA reader.

Deglycosylation of recombinant GA-733-2-Fc/KDEL

Purified recombinant GA733-2-Fc/KDEL was digested using N-glycosidase (PNGase) F and O-glycosidase (Roche, Basel, Switzerland). PNGase F can cleave all types of N-glycans bound to asparagine residues of proteins except the glycans containing α(1,3)-fucose attached to the innermost GlcNAc. O-glycosidase releases the Gal β(1–3) GalNAc unit from O-glycans, which is bound to either serine or threonine as a core unit. For reaction with one of the enzymes, 5 μg of purified recombinant GA733-2-Fc/KDEL was deglycosylated for 18 h at 37°C with either 1 U of PNGase F in 50 mM sodium phosphate buffer (pH 7.5) or 1.5 mU of O-glycosidase in 50 mM sodium acetate buffer (pH 5.0). For reaction with both enzymes, 5 μg of purified recombinant GA733-2-Fc/KDEL was treated with a cocktail of 1 U of PNGase F and 1.5 mU of O-glycosidase in 50 mM sodium phosphate buffer (pH 7.5) for 18 h at 37°C. Reactions were stopped by addition of SDS sample buffer. Samples were subjected to SDS-PAGE and western blotting as described previously.

Matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy for analyzing N-glycans

N-glycans were released from purified recombinant GA733-2-Fc/KDEL by the treatment of peptide N-glycosidase (PNGase) F after the digestion into glycopeptides with 0.1 μg of pepsin in 10 mM HCl buffer (pH 2.2). The purification of the cleaved glycans was carried out as previously described (Lee et al. 2009). After deglycosylation, an aliquot of the obtained glycans was digested in 20 mM ammonium acetate buffer (pH 5.3) with exoglycosidases including Aspergillus saitoi α(1,2)-mannosidase (Prozyme, Hayward, CA, USA) for the structure analysis. Glycans treated with and without exoglycosidase were dried and then permethylated using solid-phase permethylation method (Goetz et al. 2009). After the process previously described, the permethylated glycans were resuspended in 50% methanol solution. Then, this solution was mixed equally with matrix (2,5-dihydroxybenzoic acid) prepared in 1 mM sodium acetate solution. The resulting mixtures were applied onto a matrix-assisted laser desorption/ionization (MALDI) MSP 96 polished steel Chip (Bruker Daltonik, Bremen, Germany) and dried. MALDI-time-of-flight (TOF) mass spectrometry was performed in the reflector positive ion mode using a microflex (Bruker Daltonik). All mass spectra were acquired at a 20-kV accelerating voltage using the method recommended by the manufacturer.

Immunization experiments

This study protocol was approved by the Institutional Animal Care and Use Committee of Kyung Hee University, and animal care and experimental procedures followed university guidelines for the care and use of laboratory animals. Five-week-old female BALB/c mice (ten mice per group) were divided into three groups and immunized intraperitoneally three times at 2-week intervals with (1) recombinant GA733-2-Fc/KDEL purified from tobacco leaves (45 μg) plus adjuvant, (2) recombinant GA733-2-Fc/KDEL purified from stably transfected insect S2 cells (45 μg) plus adjuvant, and (3) PBS plus adjuvant. As an adjuvant, Freund’s complete adjuvant (FCA) was used in the first immunization and Freund’s incomplete adjuvant (FIA) was used for subsequent boost injections. Blood samples were collected from the retro-orbital plexus at 1 week after the last immunization. Sera were separated and stored at −70°C until use.

On day 42, serum samples were collected and tested for specific anti-GA733-2 IgG. The levels of anti-GA733-2 IgG were assayed by ELISA using purified recombinant GA733-2 protein from the S2 cell as the antigen. A 96-well ELISA plate was coated with purified GA733-2 protein from the S2 cell in coating buffer overnight at 4°C. The plate was washed three times with PBST. Wash buffer was discarded and diluted mouse serum samples were added to the wells, and the plate was incubated for 1 h at room temperature. After three washes, goat anti-mouse IgG-HRP conjugate (Sigma, St. Louis, MO, USA) was used as the second antibody to detect anti-GA733-2 IgG. Finally, the plate was washed and developed for 30 min with TMB (3,3′,5,5′-tetramethylbenzidine) in phosphate-citrate buffer (pH 5.0) containing 0.003% hydrogen peroxide. The reaction was then stopped by addition of 50 μl of 2 M H2SO4 to each well. The absorbance was determined at 450 nm using an ELISA reader.

For animal protection studies, mice were immunized intraperitoneally three times at 2-week intervals with recombinant GA733-2-Fc/KDEL purified from tobacco leaves (45 μg) plus adjuvant and PBS plus adjuvant, and inoculated subcutaneously with mouse colorectal cancer CT-26 cells (5 × 105 cells) on the right flank. Tumor volumes were measured with a caliper, and calculated according to the formular [(l × w 2)/2], where l and w are length and width, respectively. All mice were killed 19 days after tumor inoculation and the tumors were excised and weighted.

Cell binding assay of recombinant GA733-2-Fc/KDEL-induced antibodies

Human colorectal carcinoma HCT-116 cells were seeded into 96-well plates at 10,000 cells per well. After incubation for 18 h, the plates were washed with 200 μl PBS, fixed with 0.05% glutaraldehyde for 15 min and blocked with 100 mM glycine in PBS at 4°C until use. Sera from immunized mice were diluted to 1:1,000 and applied to the plates. After probing with goat anti-mouse IgG-HRP conjugate, the plates were developed as described above.

Flow cytometry

Cell suspensions of spleen, inguinal lymph node, and tumor infiltrating lymphocyte were prepared by standard protocols and stained for analysis by flow cytometry using PBS containing 1% FBS and 0.01% sodium azide (NaN3). The following antibodies were used: fluorescein isothiocyanate (FITC)-conjugated anti-γδTCR, anti-CD69, anti-NK1.1, and phycoerythrin (PE)-conjugated anti-CD3. All flow cytometric agents were purchased from BD Biosciences (Oxford, UK). Cell surface staining was done as described by Krieg et al. (2005). In some instances, cells were pre-incubated with purified monoclonal antibody to the Fc receptor (2.4G2) to block Fc receptors. At least 100,000 viable cells were live-gated on FACSCalibur and analyzed using CellQuest Software (BD Biosciences).

Results

Expression of recombinant GA733-2-Fc/KDEL in Agrobacterium-infiltrated leaves using a replicating BCTV vector

Nicotiana benthamiana leaves were infiltrated with recombinant A. tumefaciens GV3101 harboring pCsVMV-BCTVR-GA733-2-Fc/KDEL and were harvested 3 or 6 days after infiltration. The pCsVMV-BCTVR-GA733-2-Fc/KDEL vector system includes two BCTV replication origins flanking the intact viral Rep and REn genes and GA733-2-Fc cDNA under control of the strong and constitutive CsVMV promoter (Fig. 1a).

Southern hybridization analysis using a GA733-2-specific probe indicated that, in Agrobacterium-infiltrated leaves receiving pCsVMV-BCTVR-GA733-2-Fc, a smaller DNA species containing GA733-2-Fc and corresponding in size to sequences between the duplicated replication origins (i.e. replicating BCTV vector) was present at similar levels both 3 and 6 days after infiltration (Fig. 1b). Restriction endonuclease digestion confirmed the identity of the smaller species as a covalently closed circular, double-stranded replicative form DNA, and a single cleavage had the effect of producing a slower-migrating, linear dsDNA form (Fig. 1b).

Western blot analysis was performed using extracts prepared from leaves infiltrated with pCsVMV-BCTVR-GA733-2-Fc/KDEL. Recombinant GA733-2-Fc/KDEL, with an apparent molecular mass of ~68 kDa, was present in leaves 3 or 6 days after infiltration (Fig. 1c). The molecular weight of recombinant GA733-2-Fc/KDEL is higher than predicted (61.56 kDa), as a consequence of glycosylation. The level of expression of GA733-2-Fc/KDEL retention signal in the expression vector system was 0.96% of total soluble proteins.

Deglycosylation of recombinant GA733-2-Fc/KDEL using glycosidases and analysis on SDS-PAGE were performed to examine the reduction in apparent molecular mass (Fig. 2). The apparent molecular mass for purified recombinant GA733-2-Fc/KDEL was approximately 68 kDa. Recombinant GA733-2-Fc/KDEL digested using PNGase F and O-glycosidase resulted in a single band of approximately 62 kDa. The results shown in Fig. 2 demonstrate that recombinant GA733-2-Fc/KDEL does not contain any O-linked glycans, which can be removed by O-glycosidase. Analysis of the chimeric GA733-2-Fc/KDEL peptide sequence using the NetNGlyc 3.1 server (http://www.cbs.dk/services) and the OGPET 1.0 server (http://ogpet.utep.edu/OGPET) indicates that the chimeric GA733-2-Fc/KDEL protein has three potential sites (N 100, N 137, and N 379) for N-linked glycosylation, but does not contain any consensus sites for O-linked glycosylation.

Fig. 2
figure 2

Deglycosylation of recombinant GA733-2-Fc/KDEL. Purified recombinant GA733-2-Fc/KDEL was treated with PNGase F (lane 2), O-glycosidase (lane 3), and with a cocktail of PNGase F and O-glycosidase (lane 4). Lane M molecular mass markers

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To elucidate the N-glycan structure of recombinant GA733-2-Fc/KDEL, glycan profile and exoglycosidase digestion analysis was performed using MALDI-TOF mass spectrometry (Fig. 3). ER retention signal of GA733-2-Fc/KDEL directs its location into ER so that the attached glycans were expected to comprise oligomannose type glycans. As expected, N-glycan profile analysis shows that GA733-2-Fc/KDEL has only oligomannose-type glycans (Man5-8GlcNAc2). Further, all these glycan structures were converted to Man5GlcNAc2 glycan after α(1,2)-mannosidase digestion. These results indicate that GA733-2-Fc/KDEL was only attached with ER-type glycans without any complex-type glycan structures which are decorated in Golgi apparatus.

Fig. 3
figure 3

N-glycan analysis of GA733-2-Fc by MALDI-TOF. a Mass-spectrometric profile of glycans released by PNGase A treatment. b Glycan profile was further analyzed after the α(1,2)-mannosidase digestion. Symbols used for glycans structures are those suggested by the consortium for functional glycomics (http://www.functionalglycomics.org). Gray circles mannose, black squares N-acetylglucosamine (GlcNAc)

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Purification of recombinant GA733-2-Fc/KDEL

A GA733-2-Fc/KDEL chimeric protein (~68 kDa in size) was highly expressed in Agrobacterium-infiltrated leaves. Recombinant GA733-2-Fc/KDEL was rapidly purified to near homogeneity using a protein A sepharose affinity purification procedure. The purity of the protein was analyzed using SDS-PAGE and silver staining (Fig. 4a, b). Western blot analysis using an anti-human IgG (Fc specific)-alkaline phosphatase conjugate further confirmed the identity of the purified protein (Fig. 4b). No contaminating protein was visible on silver nitrate-stained SDS-PAGE gel (Fig. 4a). We obtained approximately 22 μg of recombinant purified GA733-2-Fc Fc/KDEL by purification using Protein A Sepharose 4 Fast Flow column from 1 g of tobacco leaf material. Using non-reducing conditions, recombinant GA733-2-Fc/KDEL migrated at a size consistent with its assembly into dimers (Fig. 4c, d).

Fig. 4
figure 4

SDS-PAGE (a) and western blot analysis (b) of the recombinant GA733-2-Fc/KDEL purified from tobacco leaves. M molecular weight markers. Before and after affinity purification is shown in lanes 1 and 2, respectively. The arrows indicate the recombinant GA733-2-Fc/KDEL. SDS-PAGE (c) and western blot analysis (d) of dimerization of recombinant GA733-2-Fc/KDEL. Recombinant GA733-2-Fc/KDEL protein purified under reducing (lane 1) or non-reducing (lane 2) conditions was resolved on 8% SDS polyacrylamide gels and analyzed by western blot analysis

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Measurement of antibody response

The sera from the BALB/c mice immunized with recombinant GA733-2-Fc/KDEL were applied to ELISA plates coated with recombinant antigen GA733-2. Absorbance values of plant-derived GA733-2-Fc/KDEL were significantly greater than that of the PBS-control, but were similar to those of insect cell-derived GA733-2-Fc (Fig. 5). The values for the sera of plant-derived and insect cell-derived GA733-2-Fc were slightly lower than that of commercial mAb (Santa Cruz Biotech, Santa Cruz, CA, USA).

Fig. 5
figure 5

GA733-2-specific IgG antibodies in sera from mice immunized i.p. with recombinant either plant-derived GA733-2-Fc or insect-derived GA733-2-Fc. Blood samples were collected from immunized BALB/c mice 1 week after the final immunization. Serum samples were serially diluted and titrated using ELISA. Filled circles PBS + FA, empty squares plant-derived GA733-2-Fc + FA, filled squares insect-derived GA733-2-Fc + FA, empty circles mouse anti-EpCAM mAb (Santa Cruz Biotech). Data represent the mean ± SD from ten mice per group

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Immune response with plant-derived GA733-2-Fc/KDEL or insect cell-derived GA733-2-Fc antigen mounted a strong GA733-2-specific serum antibody response, with comparable titers after immunization as tested in ELISA against insect cell-derived antigen.

To confirm reactivity of sera samples from immunized mice on GA733-2 positive colon cancer cells, in vitro cell ELISA was performed. Mouse serum samples were diluted to 1:1,000 and applied to ELISA plates coated with human colorectal carcinoma HCT-116 cells, and plant-derived GA733-2-Fc/KDEL-induced antibodies showed higher binding activities to HCT-116 cells (Fig. 6). The binding activity of plant-derived GA733-2-Fc/KDEL-induced antibodies was similar to that of insect cell-derived GA733-2-Fc and slightly lower than that of commercial EpCAM mAb. This indicates that antibodies produced by immunization with recombinant GA733-2-Fc/KDEL were highly reactive with human colorectal carcinoma cells.

Fig. 6
figure 6

Reactivity of serum samples from immunized mice on GA733-2 positive colorectal carcinoma HCT-116 cells. Blood samples were collected from immunized BALB/c mice 1 week after the final immunization, diluted to 1:1,000 for ELISA analysis. Open square PBS + FA, filled square insect-derived GA733-2-Fc + FA, hatched square plant-derived GA733-2-Fc + FA, dotted square mouse anti-EpCAM mAb. Data represent the mean ± SD from ten mice per group

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Anti-tumor response elicited by chimeric proteins GA733-2-Fc/KDEL in CT-26 tumor model

We examined tumor growth inhibition in mice immunized with recombinant GA733-2-Fc/KDEL. Tumor cells were inoculated to the mice 2 weeks after last immunization. At 13, 15, 17, and 18 days after tumor injection, recombinant GA733-2-Fc/KDEL regressed tumor volume in mice (Fig. 7).

Fig. 7
figure 7

Suppression of tumor growth in BALB/c mice immunized by plant-derived GA733-2-Fc/KDEL. Mice were immunized intraperitoneally three times at 2-week intervals with recombinant GA733-2-Fc/KDEL purified from tobacco leaves (45 μg) plus adjuvant and PBS plus adjuvant and inoculated subcutaneously with mouse colorectal cancer CT-26 cells (5 × 105 cells) on the right flank. On data, tumor volumes (mm3) were measured 13, 15, 17, and 18 days after initial inoculation with cancer cells. Data are given as mean ± SD from five mice per group. Student’s t test was used to compare different groups of data (*p < 0.05)

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To determine if the recombinant-expressed cancer antigens stimulate specific T cell proliferation in a distinct or similar manner, we did flow cytometry analysis using the cells of lymph node, spleen, and TIL derived from mice immunized with recombinant GA733-2-Fc/KDEL (Fig. 8). Data on the cells of lymph node and spleen derived from mice indicate that recombinant GA733-2-Fc/KDEL activated the proliferation of T cells. Also, data on TIL indicate that recombinant GA733-2-Fc/KDEL activated the proliferation of NK-T cells.

Fig. 8
figure 8

Flow cytometry analysis of spleen, inguinal lymph node and tumor infiltrating lymphocyte in tumor-inoculating mice immunized by plant-derived GA733-2-Fc/KDEL. Cell suspensions of spleen, inguinal lymph node and tumor infiltrating lymphocyte were prepared by standard protocols and stained for analysis by flow cytometry using PBS containing 1% FBS and 0.01% sodium azide (NaN3). At least 100,000 viable cells were live-gated on FACSCalibur and analyzed using CellQuest Software

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Discussion

Recently, we described the feasibility of a BCTV-based, transient expression vector system by using it to express recombinant green fluorescent protein (GFP) in Agrobacterium-inoculated leaf-disks prepared from Nicotiana benthamiana and infiltrated N. benthamiana leaves (Kim et al. 2007). In this study, we used this system to express colorectal cancer antigen GA733-2 in Agrobacterium-infiltrated N. benthamiana leaves. Fusion of Fc fragment of antibody, as well as the ER retention signal KDEL to the C-terminus of GA733-2, facilitated detection and purification of antigens. The expression level of GA7332-Fc was determined by ELISA to be approximately 0.96% of total soluble proteins from tobacco leaf material, which is similar to the level estimated on a comparison with purified GA733-2-Fc/KDEL standards on a western blot (Fig. 1); however, this level was lower than the level (1.5%) of GFP expression previously reported (Kim et al. 2007). Lower levels of expression of human papillomavirus major CP L1 using a vector based on Bean yellow dwarf virus, as compared to EGFP have also been reported (Regnard et al. 2010).

N-glycan structure analysis of GA733-2-Fc/KDEL clearly showed that it has only oligomannose type glycans (Man5-8GlcNAc2), demonstrating ER localization. It is noteworthy that we could not detect any plant-specific glycan structures such as bisected β(1,2)-xylose and α(1,3)-fucose linked to innermost GlcNAc of N-glycan. These glycans have not been observed in human or mammals and are therefore regarded as a potentially allergic epitope (Tomiya et al. 2004). Absence of allergic glycan structure in GA733-2-Fc/KDEL would be a great advantage for the development of effective cancer vaccine.

The plant-derived recombinant GA733-2-Fc/KDEL was readily extracted from the pooled leaf material in soluble form. One-step, immunoaffinity-based purification procedure resulted in an efficient recovery of recombinant antigen from plant tissue. We believe that Fc-tag can be used as single-step purification of recombinant proteins from other plant expression systems. Using non-reducing conditions, we have confirmed that recombinant GA733-2-Fc/KDEL is assembled into dimers. The ability of these molecules to dimerize might confer some structural advantage in terms of recombinant protein stability expressed in tobacco leaves. The assembly status of a chimeric peptide has been suggested to be a determinant of stability for a recombinant HIV-1 p24-immunoglobulin fusion molecule (Obregon et al. 2006).

The immune response to plant-derived GA733-2-Fc/KDEL appeared to be the same or only slightly weaker than that of an equal dose of insect cell-derived GA733-2-Fc. It seems unlikely that differential reactivity, e.g., due to different glycosylation patterns in plant-derived GA733-2-Fc/KDEL and insect cell-derived GA733-2-Fc, underlies this observation, since similar results were obtained in ELISA with either antigen. In fact, both antigens evoked a similar antibody response upon immunization of mice. Also, cell binding assay showed that antibodies produced by immunization with recombinant GA733-2-Fc/KDEL were highly reactive with human colorectal carcinoma cells. These antibodies should be the subject of research regarding their anti-colon cancer immunotherapeutic potential.

Mice immunized with plant-derived GA733-2-Fc/KDEL demonstrated an inhibition of tumor growth in comparison to PBS control in colorectal carcinoma CT-26 model. In flow cytometry analysis regarding mice experiments, data on TIL indicate that recombinant GA733-2-Fc/KDEL activated the proliferation of NK-T cells. NK-T cells have a capacity to produce immune cytokines, including IL-4, IL-10, IL-13, and IFN-γ, that can influence the nature of adaptive immune responses (Smyth and Godfrey 2000). NKT cells also express a wide variety of cell-death-inducing effector molecules, and have been demonstrated to kill tumor target cells in vitro (Mark et al. 2002). The activation of invariant NKT (iNKT) cells via the CD1d glycolipid ligand, α-galactosylceramide (α-GalCer), protects the host from tumor cell metastases in several experimental mouse models (Nakagawa et al. 2000; Smyth et al. 2002). iNKT cell activation by soluble α-GalCer leads to rapid downstream activation of other immune cells, including NK cells, which express CD69, secrete IFN-γ, become more cytotoxic, and proliferate (Carnaud et al. 1999). iNKT cells are potent immunoregulatory T cells that can be activated after interaction with DCs to stimulate innate and adaptive tumor immunity (Mark et al. 2005). Considering the increase in IFN-γ expression by activated NKT cells, our data indicate a potential Th1 response due to plant-derived GA733-2 in mice. The Th1 response can lead to a cytotoxic T cell response and tumor regression. IFN-γ plays an important role in the GA733-2-Fc-mediated T cell response since elevation of the IFN-γ level is considered to be related to Th1-type immunity. Our data also show that GA733-2-Fc induces a typical Th2 response in mice, which can eventually lead to a B cell response and production of specific antibodies. Hence, plant-derived GA733-2-Fc has a potent effect, inducing both cellular and humoral immunity. This could be extremely useful feature for a plant-derived vaccine candidate targeted against cancer.

Our in vitro and in vivo data clearly suggest that plant-derived recombinant GA733-2-Fc stimulated an anti-tumor response and induced tumor regression in colorectal carcinoma CT-26 model. A chimeric protein of colorectal cancer antigen and Fc expressed in plant leaves had similar immunogenic properties to the same chimeric protein produced in insect cell expression systems.