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ORIGINAL ARTICLE
Year : 2018  |  Volume : 14  |  Issue : 10  |  Page : 748-757

Human colorectal cancer antigen GA733-2-Fc fused to endoplasmic reticulum retention motif KDEL enhances its immunotherapeutic effects


1 Department of Genetic Engineering, Graduate School of Biotechnology, Kyung Hee University, Yongin, Republic of Korea
2 Department of Horticultural Biotechnology, Institute of Life Science and Resources, Kyung Hee University, Yongin, Republic of Korea
3 URISEED Inc., Icheon, Republic of Korea

Date of Web Publication24-Sep-2018

Correspondence Address:
In Sik Chung
Department of Genetic Engineering, Graduate School of Biotechnology, Kyung Hee University, Yongin 446-701
Republic of Korea
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0973-1482.199445

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 > Abstract 


Objective: The aim of this is to compare the immunotherapeutic effects of human colorectal cancer antigen GA733-2 fused to the Fc fragment of antibody (GA733-2-Fc) and to Fc and endoplasmic reticulum (ER) retention motif KDEL (GA733-2-Fc-KDEL).
Materials and Methods: Recombinant GA733-2-Fc and GA733-2-Fc-KDEL were produced from infiltrated Nicotiana benthamiana leaves and purified by affinity chromatography. Glycan structures were determined by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry. The allergic and immunogenic responses of recombinant GA733-2-Fc and GA733-2-Fc-KDEL were estimated in an intraperitoneally immunized mouse. The tumor regression effect of recombinant GA733-2-Fc and GA733-2-Fc-KDEL was examined using a colorectal carcinoma CT-26 animal model.
Results: Recombinant GA733-2-Fc contained plant-specific glycan structures including β(1,2)-xylose and α(1,3)-fucose whereas recombinant GA733-2-Fc-KDEL contained oligomannose type glycan structures. Mice immunized intraperitoneally with recombinant GA733-2-Fc and GA733-2-Fc-KDEL elicited strong GA733-2-Fc-specific immunoglobulin G (IgG) and IgA serum antibody responses. Recombinant GA733-2-Fc-KDEL reduced the production of GA733-2-Fc-specific IgE. Recombinant GA733-2-Fc-KDEL increased the production of interferon-γ. Intraperitoneal preimmunization with recombinant GA733-2-Fc and GA733-2-Fc-KDEL regressed tumor growth in a colorectal carcinoma CT-26 animal model. The tumor regression effect induced by recombinant GA733-2-Fc-KDEL was greater than that induced by recombinant GA733-2-Fc. The human and mouse colorectal carcinoma cell binding activities of recombinant GA733-2-Fc-KDEL-immunized sera were higher than those of recombinant GA733-2-Fc.
Conclusions: Our results suggest that GA733-2-Fc conjugated to ER-retention motif KDEL is a more efficient antigen to prevent tumor growth induced by colorectal carcinoma and minimize an allergic response.

Keywords: Colorectal cancer, endoplasmic reticulum retention motif, GA733-2, immunotherapeutic effect


How to cite this article:
Fu YY, Zhao J, Park JH, Choi GW, Park KY, Lee YH, Chung IS. Human colorectal cancer antigen GA733-2-Fc fused to endoplasmic reticulum retention motif KDEL enhances its immunotherapeutic effects. J Can Res Ther 2018;14, Suppl S3:748-57

How to cite this URL:
Fu YY, Zhao J, Park JH, Choi GW, Park KY, Lee YH, Chung IS. Human colorectal cancer antigen GA733-2-Fc fused to endoplasmic reticulum retention motif KDEL enhances its immunotherapeutic effects. J Can Res Ther [serial online] 2018 [cited 2020 Oct 26];14:748-57. Available from: https://www.cancerjournal.net/text.asp?2018/14/10/748/199445




 > Introduction Top


GA733-2 antigen is a calcium-independent epithelial cell adhesion molecule presented in the majority of human epithelia and highly expressed in gastrointestinal and colorectal carcinomas.[1] GA733-2, therefore, represents an important immunotherapeutic target for common human cancers.[2],[3] GA733-2 is a Type I membrane glycoprotein composed of 314 amino acids, of which only 26 amino acids face toward the cytoplasm. Its extracellular domain contains two epidermal growth factor-like domains, one of which is a thyroglobulin repeat domain. Thyroglobulin domain can potently inhibit cathepsin, a family of cysteine proteases frequently produced by tumor cells and involved in metastasis.[4] The extracellular domain of GA733-2 antigen has been used as an immunotherapeutic target to vaccinate gastrointestinal, colorectal, and other carcinomas in clinical trials.[5],[6],[7]

Recombinant GA733-2 has been expressed in several heterologous expression systems including epithelial cell and insect cell.[8],[9],[10] GA733-2 has been also produced in plants, and the plant-derived recombinant GA733-2 antigen has been proved to induce protective anti-cancer immune responses.[7],[11] Plant contains a different glycosylation modification mechanism with mammalian. N-glycans of plant-derived glycoprotein contain antigenic and/or allergic β(1,2)-xylose and α(1,3)-fucose glycans, which are absent in mammalian.[12] Glycoproteins retained in the lumen of plant endoplasmic reticulum (ER) contain high-mannose type N-glycans with structures common to mammals.[13],[14] These glycoproteins that do not contain allergic β(1,2)-xylose and α(1,3)-fucose glycans have been expected to reduce allergenic responses in humans. ER retention motif KDEL has been successfully used to avoid the plant-derived specific N-glycan structures without affecting the biological activities of recombinant proteins.[15] In our previous work, ER retention motif KDEL-fused GA733-2 has been established to produce recombinant GA733-2-Fc containing high-mannose type N-glycans, eventually minimizing allergenic immune responses in mammalian. Immunoglobulin Fc (IgFc) fragment has been confirmed to facilitate a purification process in insect-cell and plant expression systems.[10],[11] Although, in our previous work, Fc- and KDEL-fused GA733-2 have been proved to successfully regress tumor volumes in mice,[11] the allergic and immunotherapeutic effects of this protein have not fully determined, compared to plant-derived GA733-2-Fc containing normal plant-specific N-glycan structures.

In this study, we produced human colorectal cancer antigen GA733-2 fused to Fc (GA733-2-Fc) and GA733-2 fused to Fc and KDEL (GA733-2-Fc-KDEL) in infiltrated Nicotiana benthamiana leaves. We examined the allergic and immunogenic responses of recombinant GA733-2-Fc and GA733-2-Fc-KDEL and compared their immunotherapeutic effects using colorectal carcinoma CT-26 animal model.


 > Materials and Methods Top


DNA constructions

A GA733-2-Fc DNA fragment was amplified from pGEM-T/GA733-2-Fc[10] by polymerase chain reaction (PCR) using sense (5′-CCATGGATATGGCGCCCCCGCAGGTCCTCGCG-3′) and anti-sense (5′-GCGGCCGCTGCATTGAGTTCCCTATGCATCTC-3′) primers. The amplified GA733-2-Fc DNA fragment was cloned into a T/A cloning vector, pGEM-T vector (Promega, Madison, WI, USA). The identity of the PCR fragment was confirmed by restriction mapping and DNA sequence analysis. The Nco I-Not I fragment (GA733-2-Fc) of pGEM-T/GA733-2-Fc was inserted between the Nco I and Not I sites of ImpactVector1.3-tag (Plant Research International, Wageningen, The Netherlands) comprising the RbcS1 promoter, signal sequence, C-terminal tags (c-myc and His6), and the ER retention signal KDEL. The GA733-2-Fc DNA fragment containing the N-terminal signal sequence and the C-terminal c-myc-His tag was amplified from ImpactVector1.3-tag/GA733-2-Fc-KDEL by PCR using sense (5′-GATATCATGTCTCTTAGCCAGAACCAGGCCAAG-3′) and anti-sense (5′-ACTAGTTTAGTGATGGTGATGGTGATGA AGATC-3′) primers. The GA733-2-Fc-KDEL DNA fragment containing N-terminal signal sequence and C-terminal c-myc-His tag and KDEL was also amplified from ImpactVector1.3-tag/GA733-2-Fc-KDEL by PCR using sense (5′-GATATCATGTCTCTTAGCCAGAACCAGGCCAAG-3′) and anti-sense (5′-ACTAGTTTAAAGTTCGTCCTTGTGATGGTGATG-3′) primers. The PCR fragments were separately cloned into pGEM-T vector and the identities of PCR fragments were confirmed by restriction mapping and DNA sequence analysis. The EcoRV-SpeI fragments (GA733-2-Fc and GA733-2-Fc-KDEL) of pGEM-T/GA733-2-Fc and pGEM-T/GA733-2-Fc-KDEL were inserted between the SmaI and SpeI sites of pCsVMV-BCTVR, respectively, yielding pCsVMV-BCTVR/GA733-2-Fc and pCsVMV-BCTVR/GA733-2-Fc-KDEL [Figure 1]a and [Figure 1]b.
Figure 1: Schematic diagrams of the vector constructs used in the production of recombinant GA733-2-Fc and GA733-2-Fc-KDEL: (a) pCsVMV-BCTVR/GA733-2-Fc, (b) pCsVMV-BCTVR/GA733-2-Fc/KDEL

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Infiltration of recombinant Agrobacterium tumefaciens

The recombinant plant expression vectors pCsVMV-BCTVR/GA733-2-Fc and pCsVMV-BCTVR/GA733-2-Fc-KDEL were introduced into Agrobacterium tumefaciens strain GV3101 by electroporation, as described elsewhere.[16] Recombinant A. tumefaciens GV3101 strains harboring pCsVMV-BCTVR/GA733-2-Fc or pCsVMV-BCTVR/GA733-2-Fc-KDEL were infiltrated into the abaxial air spaces of 2–4-week-old N. benthamiana plants.[17] Protein extracts were collected from tobacco leaves at 4 days postinfiltration, as described elsewhere[16] and were used in Western blot analysis and purification experiment.

Western blot analysis

Protein extracts were separated by electrophoresis on 10% (v/v) sodium dodecyl sulfate (SDS) polyacrylamide gels and transferred onto polyvinylidene difluoride membranes (PALL Life Science, Port Washington, NY, USA). The membranes were preincubated with blocking solution (5% [w/v] nonfat dried milk in TBS-T [TBS with 0.1% (v/v) Tween-20]) for 1 h at room temperature, and then probed with horseradish peroxidase (HRP)-conjugated goat anti-human IgG (Fc specific, diluted in blocking solution to 1:10,000; Sigma-Aldrich, St. Louis, MO, USA). After the membranes were washed with TBS-T, the protein bands were detected with enhanced chemiluminescence using Supersignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific Inc., Rockford, IL, USA).

Purification of recombinant GA733-2-Fc and GA733-2-Fc-KDEL

Recombinant GA733-2-Fc and GA733-2-Fc-KDEL were purified by affinity chromatography using Protein A Sepharose 4 Fast Flow resin (GE Healthcare, Uppsala, 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 column containing Protein A Sepharose 4 Fast Flow resin. Weakly bound contaminating proteins were washed from the beads using binding buffer. Recombinant GA733-2-Fc and GA733-2-Fc-KDEL were then eluted with 0.1 M glycine (pH 2.9) 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 bovine serum albumin as a standard.

Deglycosylation of recombinant GA733-2-Fc and GA733-2-Fc-KDEL

Purified recombinant GA733-2-Fc and GA733-2-Fc-KDEL were digested with peptide N-glycosidase (PNGase) F (Roche, Basel, Switzerland), cleaving all types of N-glycans bound to asparagine residues of proteins. For the reaction with PNase F, 5 μg of purified recombinant GA733-2-Fc and GA733-2-Fc-KDEL was deglycosylated for 18 h at 37°C with 1 U of PNGase F in 50 mM sodium phosphate buffer (pH 7.5). Reactions were stopped by the addition of SDS sample buffer. Samples were subjected to SDS-polyacrylamide gel electrophoresis (PAGE) followed by sliver staining.

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

N-glycans were released from purified recombinant GA733-2-Fc and GA733-2-FC-KDEL by treatment with PNGase F after digestion into glycopeptides with 0.1 μg of pepsin in 10 mM HCl buffer (pH 2.2). The cleaved glycans were purified as described elsewhere.[18] Glycans were permethylated using a solid-phase permethylation method.[19] 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, Bremen, Germany). All mass spectra were acquired at a 20-kV accelerating voltage using the method recommended by the manufacturer.

Immunization experiments

5-week-old female Balb/c mice were purchased from Orient Bio Inc., (Seongnam, Korea). The mice were provided with water and food ad libitum, and quarantined in a specific-pathogen-free environment with a 12 h light and 12 h dark photoperiod in an animal care facility accredited by the Kyung Hee University Institutional Animal Care and Use Committee. Animal care and experimental procedures followed the Kyung Hee University guidelines for the care and use of laboratory animals. The mice were divided into three groups (10 mice per group) and immunized intraperitoneally four times at 2-week intervals with 45 μg of purified recombinant GA733-2-Fc, GA733-2-Fc-KDEL, or PBS (as a control) as a 1:1 emulsion with Freund's adjuvant. Freund's complete adjuvant was used in the first immunization and Freund's incomplete adjuvant was used for subsequent booster injections. Blood was collected from the retro-orbital plexus at 1 week after every injection of recombinant GA733-2-Fc and recombinant GA733-2-Fc-KDEL. Blood samples were allowed to clot at room temperature for 30 min, and sera were collected by centrifugation at 10,000 ×g for 15 min and stored at −70°C until use. GA733-2 specific IgG, IgA, and IgE antibodies in sera were analyzed by enzyme-linked immunosorbent assay (ELISA) using purified recombinant GA733-2 protein from Drosophila S2 cells as the antigen. A 96-well ELISA plate was coated overnight at 4°C with purified GA733-2 protein (0.2 μg/well) in coating buffer (0.05 M carbonate-bicarbonate buffer, pH 9.6). The plate was washed three times with PBS-T (PBS with 0.1% Tween 20). Sera were diluted (1:100 dilutions or serial dilutions in PBS-T), added to each well, and incubated for 1 h at room temperature. After washing with PBS-T, goat anti-mouse IgG-HRP, goat anti-mouse IgA-HRP, and goat anti-mouse IgE-HRP conjugates (1:10,000 dilutions in PBS-T; Sigma-Aldrich, St. Louis, MO, USA) were added as the second antibodies to detect GA733-2 specific IgG, IgA and IgE, respectively, and incubated for 1 h at room temperature. Finally, the plate was washed and developed for 30 min with 100 μl of 3,3′, 5, 5′-tetramethylbenzidine One Component HRP Microwell Substrate (SurModics, Edan Prairie, MN, USA) in phosphate-citrate buffer (pH 5.0) containing 0.002% (v/v) hydrogen peroxide. The reaction was stopped by the addition of 50 μl of 2 M H2 SO4 to each well. The absorbance was determined at 450 nm using an ELISA reader (Bio-Tek Inc., Winooski, VT, USA).

For in vivo tumor regression analysis, mice were immunized intraperitoneally four times at 2 week intervals with recombinant GA733-2-Fc, GA733-2-Fc-KDEL and PBS (as a control) as described above. One week after the last immunization, mice were inoculated subcutaneously with mouse colorectal carcinoma CT-26 cells (5 × 105) on the right flank. Tumor volumes were measured with a caliper, and calculated according to the formula ([l × w2]/2), where l and w are length and width, respectively.

Cell binding assay of recombinant GA733-2-Fc-and GA733-2-Fc-KDEL-induced sera

Mouse colorectal carcinoma CT-26 cells and human colorectal carcinoma HCT-116 cells were seeded into 96-well plates at 50,000 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. After washes with PBS, the plates were incubated for 1 h at room temperature with sera (1:1,000 dilutions in PBS) from mice immunized with recombinant GA733-2-Fc and GA733-2-Fc-KDEL. After probing with goat anti-mouse IgG-HRP conjugate, the plates were developed as described above.

Splenocyte culture and cytokine detection

Spleens were surgically removed from mice and gently crushed using the plunger of a disposable syringe on a Falcon CELL strainer (BD Biosciences, San Jose, CA, USA). Red blood cells were removed by the addition of 0.2 μm filter sterilized-ACK buffer (0.15 M NH4 Cl, 10 mM KHCO3, 0.1 mM ethylenediaminetetraacetic acid, pH 7.3) and incubated for 2 min at room temperature. After washing three times with Roswell Park Memorial Institute (RPMI)-1640 medium (Thermo Scientific Hyclone, South Logan, UT, USA), spleen cells were resuspended with RPMI-1640 medium containing 20% fetal bovine serum (Thermo Scientific Hyclone, South Logan, UT, USA), and seeded at a density of 5 × 106 cells/well onto 24-well culture plates. The cells were cultured for 24 h in the presence of recombinant GA733-2-Fc, GA733-2-Fc-KDEL (25 μg/well) and PBS (as a control). Cell culture supernatants were collected by centrifugation at 10,000 ×g for 5 min and applied to a sandwich ELISA system (BD Biosciences, San Jose, CA, USA) for detection of interferon (IFN)-γ and interleukin (IL)-4 according to the manufacturer's instructions.

Statistical analysis

Data are presented as the mean ± standard deviation or standard error. Student's t-test was used to compare different data groups, and all the data were analyzed using Microsoft Excel 2010 software (Microsoft Corporation, Redmond, WA, USA).


 > Results Top


Expression and purification of recombinant GA733-2-Fc and GA733-2-Fc-KDEL in Agrobacterium-infiltrated leaves

N. benthamiana leaves were infiltrated with recombinant A. tumefaciens GV3101 harboring either pCsVMV-BCTVR/GA733-2-Fc or pCsVMV-BCTVR/GA733-2-Fc-KDEL, and then harvested 4 days after infiltration. Recombinant GA733-2-Fc and GA733-2-Fc-KDEL were successfully expressed in Agrobacterium-infiltrated leaves [Figure 2]a. Their apparent molecular masses were ~65 and ~68 kDa, respectively. Recombinant GA733-2-Fc and GA733-2-Fc-KDEL were rapidly purified to near homogeneity using a protein A Sepharose affinity purification procedure and the purity of the proteins was analyzed using SDS-PAGE followed by silver staining [Figure 2]b. No contaminating protein was visible on silver nitrate-stained SDS-PAGE gel. The identities of the purified proteins were further confirmed by Western blot analysis using an anti-human IgG (Fc specific)-HRP conjugate (data not shown).
Figure 2: Expression (a) and purification (b) of recombinant GA733-2-Fc and GA733-2-Fc-KDEL in Nicotiana benthamiana leaves infiltrated with recombinant Agrobacterium harboring pCsVMV-BCTVR/GA733-2-Fc or pCsVMV-BCTVR/GA733-2-Fc/KDEL; (a) 1: Protein extract of normal leaves, 2: Protein extract of leaves infiltrated with recombinant Agrobacterium harboring pCsVMV-BCTVR/GA733-2-Fc, 3: Protein extract of leaves infiltrated with recombinant Agrobacterium harboring pCsVMV-BCTVR/GA733-2-Fc-KDEL. (b) M: molecular weight marker, 1: Purified recombinant GA733-2-Fc, 2: Purified recombinant GA733-2-Fc-KDEL

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Glycosylation analysis of recombinant GA733-2-Fc and GA733-2-Fc-KDEL

Recombinant GA733-2-Fc and GA733-2-Fc-KDEL were deglycosylated with PNGase F and analyzed on SDS-PAGE to examine the reduction in apparent molecular mass [Figure 3]a. The treatment of PNGase F changed the migration of recombinant GA733-2-Fc and GA733-2-Fc-KDEL. The molecular weight of recombinant GA733-2-Fc and GA733-2-Fc-KDEL treated with PNGase F was approximately 62 kDa. To further compare the structural features of recombinant GA733-2-Fc and GA733-2-Fc-KDEL, the glycan structures were determined by MALDI-TOF mass spectrometry after affinity purification [Figure 3]b. Recombinant GA733-2-Fc contained plant-specific glycan structures including β(1,2)-xylose and α(1,3)-fucose. Whereas, recombinant GA733-2-Fc-KDEL contained an oligomannose-type glycan structure (Man5-8GlcNAc2).
Figure 3: Deglycosylation (a) and glycan analysis (b) of recombinant GA733-2-Fc and GA733-2-Fc-KDEL; (a) M: molecular weight marker, 1: Recombinant GA733-2-Fc, 2: Recombinant GA733-2-Fc treated with PNGase F, 3: Recombinant GA733-2-Fc-KDEL, 4: Recombinant GA733-2-Fc-KDEL treated with PNGase F. (b) Square: N-acetylglucosamine, circle: Mannose, triangle: Fucose, star: Xylose

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Immune response analysis of recombinant GA733-2-Fc and GA733-2-Fc-KDEL

Balb/c mice (10 mice/group) were intraperitoneally immunized with purified recombinant GA733-2-Fc and GA733-2-Fc-KDEL or PBS (used as a control) four times at 2 week intervals. Sera were collected 1 week after the last immunization. The presence of GA733-2-Fc specific IgG, IgA and IgE in the sera of the mice immunized with recombinant GA733-2-Fc and GA733-2-Fc-KDEL was determined by ELISA. Absorbance values of GA733-2-Fc specific IgG and IgA significantly increased in sera from mice immunized with recombinant GA733-2-Fc and GA733-2-Fc-KDEL, compared with PBS-immunized control mice [Figure 4]a,[Figure 4]b,[Figure 4]c,[Figure 4]d. Absorbance value of GA733-2-Fc specific IgE also increased in sera from mice immunized with recombinant GA733-2-Fc [Figure 4]e and [Figure 4]f. Immunization with recombinant GA733-2-Fc-KDEL reduced the production of GA733-2-Fc-specific IgE, compared to recombinant GA733-2-Fc.
Figure 4: Production of GA733-2-Fc specific immunoglobulin G (a and b), immunoglobulin A (c and d) and immunoglobulin E (e and f) antibodies in sera (a, c, and e, 1:100 dilutes of sera collected at 1 week after immunization; b, d, and f, serial dilutes of sera collected at 1 week after the last immunization) from mice intraperitoneally immunized with recombinant GA733-2-Fc and GA733-2-Fc-KDEL; *P < 0.05, **P < 0.01, ***P < 0.001, between treated and control groups

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Effect of recombinant GA733-2-Fc and GA733-2-Fc-KDEL on the production of interferon-γ and interlekuin-4

Splenocytes were collected from normal mice and cultured with recombinant GA733-2-Fc and GA733-2-Fc-KDEL. The levels of IFN-γ and IL-4 in culture media were determined by ELISA. Recombinant GA733-2-Fc-KDEL increased the production of IFN-γ, but this effect was not seen with recombinant GA733-2-Fc [Figure 5]a. By contrast, both recombinant GA733-2-Fc and GA733-2-Fc-KDEL increased the production of IL-4 [Figure 5]b, with recombinant GA733-2-Fc inducing slightly higher IL-4 production than recombinant GA733-2-Fc-KDEL. The relative ratio of IFN-γ/IL-4 was high in recombinant GA733-2-Fc-KDEL-treated splenocytes [Figure 5]c.
Figure 5: Production of cytokines interferon-γ and interleukin-4 from splenocytes treated with recombinant GA733-2-Fc and GA733-2-Fc-KDEL; (a) levels of interferon-γ, (b) levels of interleukin-4, (c) relative ratio of interferon-γ/interleukin-4; *P < 0.05, **P < 0.01, ***P < 0.001, between treated and control groups

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Anti-tumor response elicited by recombinant GA733-2-Fc and GA733-2-Fc-KDEL in colorectal carcinoma CT-26 animal model

Balb/c mice were intraperitoneally preimmunized with recombinant GA733-2-Fc and GA733-2-Fc-KDEL four times at 2 week intervals. Mouse colorectal carcinoma CT-26 cells were inoculated into mice 1 week after the last immunization. At 3, 7, 11, 15, 19, and 23 days after inoculation, tumor volumes were measured as described in Materials and Methods. Intraperitoneal pre-immunization with recombinant GA733-2-Fc and GA733-2-Fc-KDEL reduced the volumes of tumor induced by CT-26 cells [Figure 6]. The tumor regression effect of recombinant GA733-2-Fc-KDEL was greater than that of recombinant GA733-2-Fc.
Figure 6: Effect of recombinant GA733-2-Fc and GA733-2-Fc-KDEL on tumor growth induced by CT-26 cells; *P < 0.05, **P < 0.01, between treated and control groups

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In vitro cell binding analysis of sera collected from recombinant GA733-2-Fc-or GA733-2-Fc-KDEL-immunized mice

Mouse serum samples collected from recombinant GA733-2-Fc-or GA733-2-Fc-KDEL-immunized mice were diluted to 1:1,000 and applied to ELISA plates coated with human colorectal carcinoma HCT-116 or mouse colorectal carcinoma CT-26 cells. Recombinant GA733-2-Fc-and GA733-2-Fc-KDEL-immunized sera showed higher binding activities to HCT-116 and CT-26 cells [Figure 7]a and [Figure 7]b. The binding activities of recombinant GA733-2-Fc-KDEL-immunized sera were higher than those of recombinant GA733-2-Fc.
Figure 7: Carcinoma cell binding assay of serum samples from mice immunized with recombinant GA733-2-Fc or GA733-2-Fc-KDEL; (a) human colorectal carcinoma HCT-116 cell binding assay, (b) mouse colorectal carcinoma CT-26 cell binding assay, *P < 0.05, ***P < 0.001, between treated and control groups

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 > Discussion Top


Cancer vaccines can either be designed to stimulate the immune system to cause tumor regression in a patient with cancer, a therapeutic vaccine or they can prepare the immune system before getting cancer for cancer prevention, so-called prophylactic vaccines. Prophylactic vaccines based on hepatitis B virus and human papilloma virus antigens have been established to prevent liver and cervical cancer.[20] In several studies, cancer prophylactic vaccines based on human tumor-associated antigens are immunogenic and safe and capable of eliciting long-term memory in clinical studies performed in patients without cancer but with a history of premalignant lesion and in patients with metastatic colorectal cancer.[21],[22] Here, we produced human colorectal cancer antigen GA733-2 fused to Fc and KDEL in infiltrated N. benthamiana leaves. The allergic and immunogenic responses of recombinant GA733-2 proteins (GA733-2-Fc and GA733-2-Fc-KDEL) and immunotherapeutic effects using colorectal carcinoma CT-26 animal model were determined to estimate a possibility for developing prophylactic vaccine candidates against colorectal cancer.

GA733-2-Fc and ER retention signal-fused GA733-2-Fc-KDEL DNA fragments were, respectively, cloned into the pCsVMV-BCTVR vector system including a constitutive CsVMV promoter, a nopaline synthase terminator, and two Beet curly top virus replication origins flanking intact viral Rep and REn genes [Figure 1]a and [Figure 1]b. Recombinant GA733-2-Fc and GA733-2-Fc-KDEL were successfully expressed in N. benthamiana leaves infiltrated with Agrobacterium harboring pCsVMV-BCTVR/GA733-2-Fc or pCsVMV-BCTVR/GA733-2-Fc-KDEL. Their apparent molecular masses are higher than predicted (61.5 kDa), probably due to glycosylation. The molecular weight of recombinant GA733-2-Fc-KDEL is higher than recombinant GA733-2-Fc [Figure 2]a, indicating that recombinant GA733-2-Fc and GA733-2-Fc-KDEL contain different glycan structures. Recombinant GA733-2-Fc and GA733-2-Fc-KDEL contain three potential sites (N100, N137 and N379) for N-linked glycosylation (http://www.cbs.dtu.dk/services). Digestion of recombinant GA733-2-Fc and GA733-2-Fc-KDEL using PNGase F resulted in a single band of approximately 62 kDa [Figure 3]a. Digestion using O-glycosylase did not change the migration of recombinant GA733-2-Fc and GA733-2-Fc-KDEL on SDS-PAGE gels (data not shown). These results indicate that recombinant GA733-2-Fc and GA733-2-Fc-KDEL contain only N-linked glycans. Glycoproteins in the lumen of plant ER contain high-mannose type N-glycans with structures common to mammals.[13],[14] The addition of KDEL motif at the C-terminal end of a secreted protein is sufficient for its retention in the plant ER.[23] Recombinant GA733-2-Fc-KDEL contained an oligomannose-type glycan structure (Man5-8GlcNAc2) [Figure 3]b, indicating that recombinant GA733-2-Fc-KDEL was retained in the ER and attached by ER-type glycans without any complex-type glycan structures. The addition of KDEL motif at the C-terminal end of a secreted protein enhances the accumulation of tagged proteins in plants.[24],[25] However, the fusion of KDEL did not enhance the expression level of recombinant GA733-2-Fc-KDEL.

To evaluate the immune response ability of recombinant GA733-2-Fc and GA733-2-Fc-KDEL, Balb/c mice were immunized with recombinant GA733-2-Fc and GA733-2-Fc-KDEL and the production of GA733-2-Fc specific IgG, IgA and IgE was determined by ELISA. Mice were intraperitoneally immunized with recombinant GA733-2-Fc and GA733-2-Fc-KDEL based on our previous experiments. The intraperitoneal immunization of recombinant human colorectal cancer antigen GA733-2 efficiently induces both cellular and humoral immune responses and activates the proliferation of natural killer (NK)-T cells.[11] Recombinant GA733-2-Fc and GA733-2-Fc-KDEL induced strong GA733-2-specific serum antibody responses [Figure 4]. In addition, recombinant GA733-2-Fc-KDEL reduced the production of GA733-2-Fc-specific IgE, compared to recombinant GA733-2-Fc. Glycoepitopes containing β(1,2)-xylose and α(1,3)-fucose glycans are important IgE binding carbohydrate determinants on plant allergens.[26] These epitopes have been regarded as allergic epitopes because β(1,2)-xylose and α(1,3)-fucose glycans have not been observed in human or mammals.[27] The production of GA733-2-Fc-specific IgE antibodies was significantly reduced in sera from mice immunized with recombinant GA733-2-Fc-KDEL. This implies that recombinant GA733-2-Fc-KDEL induces a lower allergic response because of the absence of β(1,2)-xylose and α(1,3)-fucose glycans. The absence of allergic glycans in recombinant GA733-2-Fc-KDEL could be a great advantage for developing an effective cancer vaccine.

The production of IgE induced by IL-4 has been suppressed by INF-γ in a dose-dependent way.[28],[29] To further evaluate the lower allergic response of recombinant GA733-2-Fc-KDEL, the production of cytokine IFN-γ and IL-4 by recombinant GA733-2-Fc and GA733-2-Fc-KDEL was determined using mouse splenocytes. Recombinant GA733-2-Fc-KDEL highly increased the production of IFN-γ, compared to recombinant GA733-2-Fc [Figure 5]. The relative ratio of IFN-γ/IL-4 was high in recombinant GA733-2-Fc-KDEL-treated splenocytes. These indicate that recombinant GA733-2-Fc-KDEL highly increases the production of IFN-γ, and can reduce allergic responses caused by plant-derived GA733-2-Fc.

Intraperitoneal pre-immunization with recombinant GA733-2-Fc and GA733-2-Fc-KDEL suppressed the tumor growth induced by mouse colorectal cancer CT-26 cells [Figure 6]. The tumor regression by the preimmunization of recombinant GA733-2-Fc-KDEL was greater than that by recombinant GA733-2-Fc. Sera from mice immunized with recombinant GA733-2-Fc-KDEL contained higher binding activities to HCT-116 and CT-26 cells, compared to recombinant GA733-2-Fc [Figure 7]. These indicate that recombinant GA733-2-Fc-KDEL is more suitable to regress tumor growth induced by colorectal carcinoma, compared to recombinant GA733-2-Fc, probably higher binding activities of antibodies produced from recombinant GA733-2-Fc-KDEL-pre-immunized mice to colorectal carcinoma.

Recombinant GA733-2-Fc-KDEL activates the proliferation of NK-T cells,[11] which have the capacity to produce immune cytokines (i.e., IL-10 and IFN-γ). In particularly, IFN-γ plays an important role in the detection and elimination of tumor cells and shows a potent anti-tumor activity.[30],[31] Our in vitro data showed that recombinant GA733-2-Fc-KDEL stimulated normal mouse splenocytes to secrete higher levels of IFN-γ, compared to recombinant GA733-2-Fc [Figure 5]. Tumor-specific antigen conjugated with KDEL motif at the C-terminus promotes MHC (major histocompatibility complex) molecules synthesis and prolongs MHC Class I-associated presentation of exogenous peptides.[32] Taken together, these suggest that ER retention signal-mediated antigen delivery system has an important clinical application for tumor immune therapy.


 > Conclusions Top


Recombinant GA733-2-Fc-KDEL was produced to avoid plant-derived specific N-glycan structures containing β(1,2)-xylose and α(1,3)-fucose. Recombinant GA733-2-Fc-KDEL contained an oligomannose-type glycan structure. Mice immunized intraperitoneally with recombinant GA733-2-Fc-KDEL elicited strong GA733-2-Fc specific IgG and IgA serum antibody responses. Recombinant GA733-2-Fc-KDEL reduced the production of GA733-2-Fc-specific IgE antibody, compared to recombinant GA733-2-Fc. Recombinant GA733-2-Fc-KDEL greatly increased the production of IFN-γ. In a colorectal carcinoma CT-26 animal model, the tumor regression effect induced by recombinant GA733-2-Fc-KDEL was greater than that induced by recombinant GA733-2-Fc. Taken together, our results suggest that GA733-2-Fc conjugated to ER-retention motif KDEL is a more efficient antigen to prevent tumor growth induced by colorectal carcinoma and minimize an allergic response.

Acknowledgments

This work was supported by the Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries (IPET) through Agri-Bio Industry Technology Development Program, funded by Ministry of Agriculture, Food and Rural Affairs, Republic of Korea (112019-5).

Financial support and sponsorship

This work was supported by the Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries (IPET) through Agri-Bio Industry Technology Development Program, funded by Ministry of Agriculture, Food and Rural Affairs, Republic of Korea (112019-5).

Conflicts of interest

There are no conflicts of interest.



 
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  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7]



 

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