The recombinant antibody protein expressed in pIRES2-ZSGreen1-rAbs-APN-CHO cells and monoclonal antibodies produced using traditional hybridoma technology can recognize and bind to the porcine aminopeptidase N (APN) protein.
Porcine aminopeptidase N (APN), a membrane-bound metallopeptidase abundantly present in small intestinal mucosa, can initiate a mucosal immune response without any interference such as low protein expression, enzyme inactivity, or structural changes. This makes APN an attractive candidate in the development of vaccines that selectively target the mucosal epithelium. Previous studies have shown that APN is a receptor protein for both enterotoxigenic Escherichia coli (E. coli) F4 and transmissible gastroenteritis virus. Thus, APN shows promise in the development of antibody-drug conjugates or novel vaccines based on APN-specific antibodies. In this study, we compared production of APN-specific monoclonal antibodies (mAbs) using traditional hybridoma technology and recombinant antibody expression method. We also established a stably transfected Chinese hamster ovary (CHO) cell line using pIRES2-ZSGreen1-rAbs-APN and an E. coli expression BL21(DE3) strain harboring the pET28a (+)-rAbs-APN vector. The results show that antibodies expressed in pIRES2-ZSGreen1-rAbs-APN-CHO cells and mAbs produced using hybridomas could recognize and bind to the APN protein. This provides the basis for further elucidation of the APN receptor function for the development of therapeutics targeting different APN-specific epitopes.
Aminopeptidase N (APN), a moonlighting enzyme that belongs to the metalloproteinase M1 family, acts as a tumor marker, receptor, and signaling molecule via enzyme-dependent and enzyme-independent pathways1,2. In addition to cleaving the N-terminal amino-acid residues of various bioactive peptides for the regulation of their biological activity, APN plays an important role in the pathogenesis of various inflammatory diseases. APN participates in antigen processing and presentation by trimmed peptides that bind tightly to major histocompatibility complex class II molecules2,3. APN also exerts anti-inflammatory effects by binding with G protein-coupled receptors participating in multiple signal transduction, modulating cytokine secretion, and contributing to Fc gamma receptor-mediated phagocytosis in the immune response4,5,6,7.
As a widely distributed membrane-bound exopeptidase, APN is abundant in the porcine small intestinal mucosa and is closely associated with receptor-mediated endocytosis1,5,8. APN recognizes and binds the spike protein of the transmissible gastroenteritis virus for cell entry, and directly interacts with the FaeG subunit of enterotoxigenic Escherichia coli F4 fimbriae to affect bacterial adherence with host cells9,10,11. Thus, APN is a potential therapeutic target in the treatment of viral and bacterial infectious diseases.
Since the development of hybridoma technology and other strategies for monoclonal antibodies (mAbs) production in 1975, mAbs have been widely used in immunotherapy, drug delivery, and diagnosis12,13,14. Currently, mAbs are successfully used to treat diseases, such as cancer, inflammatory bowel disease, and multiple sclerosis12,15. Because of their strong affinity and specificity, mAbs can be ideal targets in the development of antibody-drug conjugates (ADC) or new vaccines16,17. The APN protein is critical for selectively delivering antigens to specific cells, and can elicit a specific and strong mucosal immune response against pathogens without any interference including low protein expression, enzyme inactivity, or structural changes5,8,18. Therefore, therapeutic products based on APN-specific mAbs show promise against bacterial and viral infections. In this study, we describe the production of APN-specific mAbs using hybridoma technology, and expression of anti-APN recombinant antibodies (rAbs) using prokaryotic and eukaryotic vectors. The result indicates that the APN protein was recognized by both rAbs expressed in pIRES2-ZSGreen1-rAbs-APN-CHO cells and hybridoma-derived mAbs.
All animal experiments in this study were approved by the Yangzhou University Institutional Animal Care and Use Committee (SYXK20200041).
1. Preparation of porcine APN protein antigen
NOTE: The pET28a (+)-APN-BL21 (DE3) strain and the APN stably expressed cells pEGFP-C1-APN-IPEC-J2 were constructed in a previous study11.
2. Animal immunization
3. Hybridoma technology to produce monoclonal antibodies against APN
4. Characterization of mAbs against APN protein
5. Expression of rAbs against APN
In this study, the purified soluble APN protein (2.12 mg/mL) was used for mouse immunization. Mice immunized with the APN protein four times at 14-day intervals exhibited a higher antibody titer against APN in their sera. Although 14 hybridomas were obtained using the fusion experiments, only 9 hybridomas survived the three continuous freeze-thaw cycles, resulting in 9 stable clones that secreted antibodies against APN. All these cells are round, bright, and clear (Figure 1). The purified mAbs possessing heavy and light chains (50 kDa and 25 kDa, respectively) were confirmed by SDS-PAGE and found in the purified ascites (Figure 2). The titers of these anti-APN mAbs in culture supernatants and ascites are shown in Table 2.
The result of mouse mAbs isotyping revealed that mAbs derived from clones 5B31, 5B36, 3C48, 5C51, and 6C56 possessed IgG2b subclasses, while APN-2A20 was an IgG2a κappa- (κ) type antibody, and mAbs APN-3FD9, -3F10, and -10F3 belonged to IgM type and processed κ light chains (Table 3). As shown in Table 4, most of these mAbs showed AV values of over 50%, indicating that they targeted different epitopes in the APN, while the APN-5C51 antibody recognized antigenic epitopes similar to those recognized by APN-3C48, -5B31, and -6C56 mAbs.
APN-5B36 showed considerably higher antibody titer compared with those of other mAbs. Therefore, the APN-5B36 VH-VL gene was amplified and ligated into a pET28a (+) or pIRES2-ZsGreen1 vector to construct the recombinant expression plasmids pET28a (+)-rAbs-APN and pIRES2-ZsGreen1-rAbs-APN, respectively (Figure 3). The antibodies expressed by both pET28a (+)-rAbs-APN-BL21 (DE3) and pIRES2-ZsGreen1-rAbs-APN-CHO cells were purified and analyzed using ELISA and IFA assays. However, as shown in Figure 4, only the antibody expressed in the supernatant of pIRES2-ZsGreen1-rAbs-APN-CHO cells recognized the APN protein, as did hybridoma-derived mAbs. This recombinant antibody consisted of IgG2b heavy chains and lambda light chains, and showed a titer of 2.56 × 105 as determined using ELISA. The binding of APN-5B36 mAbs to APN proteins reached an equilibrium earlier than rAbs did (Figure 5), showing KD value of (4.232±0.475) × 10-9 and (2.201±0.367) × 10-8 mol/L, respectively.
Primer | Sequence (5’-3’) | ||
VH-VL-F | CCGGGTGGGCCGGATAGACMGATGGGGCTG | ||
VH-VL-R | CCGGCCACATAGGCCCCACTTGACATTGATGT | ||
pET28a (+)-F | TCCACCAGTCATGCTAGCCATAACAACGGTCGTGATTCGA | ||
pET28a (+)-R | CTGGTGCCGCGCGGCAGCCAGTGGGATACCCGTATTACCC | ||
pIRES2-ZsGreen1-F | CGACGGTACCGCGGGCCCGGTAACAACGGTCGTGATTCGA | ||
pIRES2-ZsGreen1-R | GGGGGGGAGGGAGAGGGGCGGTGGGATACCCGTATTACCC |
Table 1. The specific primers used in this study.
Cells | Titers of supernatants (U/mL) | Titers of ascites (U/mL) |
2A20 | 0.64×104 | 3.20×105 |
5B31 | 1.28×104 | 1.60×105 |
5B36 | 0.64×104 | 1.28×106 |
3C48 | 0.16×104 | 0.80×105 |
5C51 | 0.16×104 | 0.80×104 |
6C56 | 0.80×103 | 0.80×104 |
3FD9 | 0.80×103 | 0.80×104 |
3F10 | 0.16×104 | 0.16×105 |
10F3 | 0.80×103 | 0.32×105 |
Table 2. The ELISA titers of APN mAbs.
Ig | IgA | IgM | IgG1 | IgG2a | IgG2b | IgG3 | Kappa | Lambda | Özet | |
2A20 | 1.735 | 0.023 | 0.011 | 0.006 | 0.903 | 0.044 | 0.015 | 0.137 | 0.073 | IgG2a, Kappa |
5B31 | 1.199 | 0.006 | 0.003 | 0.005 | 0.005 | 1.731 | 0.004 | 0.004 | 0.413 | IgG2b, Lambda |
5B36 | 1.652 | 0.012 | 0.013 | 0.01 | 0.008 | 2.41 | 0.002 | 0.003 | 0.707 | IgG2b, Lambda |
3C48 | 0.951 | 0.063 | 0.068 | 0.104 | 0.062 | 1.785 | 0.059 | 0.065 | 0.51 | IgG2b, Lambda |
5C51 | 1.064 | 0.008 | 0.007 | 0.008 | 0.008 | 1.87 | 0.004 | 0.004 | 0.415 | IgG2b, Lambda |
6C56 | 0.78 | 0.062 | 0.06 | 0.063 | 0.063 | 1.516 | 0.062 | 0.061 | 0.387 | IgG2b, Lambda |
3FD9 | 1.474 | 0.007 | 1.678 | 0.003 | 0.016 | 0.081 | 0.002 | 0.519 | 0.059 | IgM, Kappa |
3F10 | 1.21 | 0.002 | 1.454 | 0.009 | 0.008 | 0.054 | 0.003 | 0.414 | 0.096 | IgM, Kappa |
10F3 | 1.179 | 0.058 | 1.562 | 0.152 | 0.131 | 0.179 | 0.044 | 0.359 | 0.049 | IgM, Kappa |
Table 3. Isotypes of hybridoma-derived APN mAbs.
mAbs | AV (100 %) | ||||||||
2A20 | 5B31 | 5B36 | 3C48 | 5C51 | 6C56 | 3FD9 | 3F10 | 10F3 | |
2A20 | – | 0.601 | 0.905 | 0.889 | 0.804 | 0.884 | 1.009 | 1.047 | 0.914 |
5B31 | 0.601 | – | 0.871 | 0.754 | 0.464 | 0.694 | 0.613 | 0.88 | 0.989 |
5B36 | 0.905 | 0.871 | – | 0.794 | 0.684 | 0.934 | 0.91 | 1.07 | 0.959 |
3C48 | 0.889 | 0.754 | 0.794 | – | 0.461 | 0.709 | 0.428 | 1 | 0.787 |
5C51 | 0.804 | 0.464 | 0.684 | 0.461 | – | 0.301 | 0.601 | 0.594 | 0.852 |
6C56 | 0.884 | 0.694 | 0.934 | 0.709 | 0.301 | – | 1.216 | 0.583 | 0.389 |
3FD9 | 1.009 | 0.613 | 0.91 | 0.428 | 0.601 | 1.216 | – | 1.737 | 0.744 |
3F10 | 1.047 | 0.88 | 1.07 | 1 | 0.594 | 0.583 | 1.737 | – | 0.682 |
10F3 | 0.914 | 0.989 | 0.959 | 0.787 | 0.852 | 0.389 | 0.744 | 0.682 | – |
Table 4. Discrimination of antigen-epitope specificity of APN-specific mAbs. AV values greater than 0.5 indicate that these two mAbs recognize different antigenic sites; AV values less than 0.5 indicate that these two mAbs recognize a similar antigenic site.
Figure 1. Image of hybridomas. Under microscopic analysis, hybridomas are round, bright, and clear. Please click here to view a larger version of this figure.
Figure 2. Recombinant antibody expression levels and ascites analyzed using SDS-PAGE. (A) Lane M, protein marker; lane 1, purified pET28a (+)-rAbs-APN-BL21 (DE3) lysate; lane 2, pET28a (+)-rAbs-APN-BL21 (DE3) supernatant; lane 3, ascites fluid purified by 33% (NH4)2SO4 precipitation. (B) Lane M, protein marker; lane 1, ascites fluid purified using protein A agarose. In this assay, 3-5 µg of total protein was loaded into each lane of the gel. Please click here to view a larger version of this figure.
Figure 3. Recombinant expression plasmids pET28a (+)-rAbs-APN and pIRES2-ZsGreen1-rAbs-APN analyzed using agarose gel electrophoresis. Lane M, Trans 2K plus DNA marker; lane 1, pET28a (+) vector (5369 bp); lane 2 and 5, VH-VL gene combined with APN-5B36 leader sequence; lane 3, pET28a (+)-rAbs-APN plasmid expressed in BL21 (DE3) E. coli; lane 4, pIRES2-ZsGreen1 vector (5283 bp); lane 6, pIRES2-ZsGreen1-rAbs-APN plasmid expressed in DH5α E. coli. Please click here to view a larger version of this figure.
Figure 4. Expression of recombinant antibody protein and ascites analyzed using indirect immunofluorescence. pEGFP-C1-APN-IPEC-J2 cells (green fluorescence) stably expressing APN were treated with (A) PBS, used as control treatment; (B) purified protein expressed by pET28a (+)-rAbs-APN-BL21 (DE3); (C) APN polyclonal antibody (1:500); (D) purified ascites fluid (1:500); E) purified supernatant obtained from pIRES2-ZsGreen1-rAbs-APN-CHO cells (1:500). DAPI was used as nuclear counterstain in confocal microscopy. The cells incubated with the DyLight 549-conjugated goat anti-mouse IgG secondary antibody (1:200) and treated with purified ascites and purified supernatant from pIRES2-ZsGreen1-rAbs-APN-CHO cells showed a robust red-fluorescence signal indicative as the APN polyclonal antibody did. Please click here to view a larger version of this figure.
Figure 5. Determination of antibody relative binding affinities using the ELISA22. Absorption of samples containing APN-5B36 mAbs or rAbs, in the absence and presence of APN protein, was measured at the wavelength of 450 nm. Binding curve was plotted using a four-parameter logistic curve fit; x-axis shows the logarithmic concentration of antibodies, and y-axis shows the absorbance. Please click here to view a larger version of this figure.
Induction of mucosal immunity is one of the most effective approaches in counteracting pathogens and in prevention and treatment of various diseases. APN, a highly expressed membrane-bound protein in the intestinal mucosa, is involved in the induction of adaptive immune response and in receptor-mediated viral and bacterial endocytosis1,5,8. APN is used as antigen particulate in many formats of antigen loading and vaccine delivery. The oral administration of APN-targeted antibodies can also elicit effective immune responses18,24,25. However, monoclonal antibodies targeting different APN-specific epitopes require further investigation.
The methods described here were employed to produce monoclonal antibodies against APN using both traditional hybridoma and recombinant technologies. This approach can be used in the production of other mAbs. First, we followed a previously mentioned protocol to obtain nine mAbs derived from different hybridoma clones. Although the titers of these mAbs in cell supernatants and ascites were different, all the mAbs contained the 50 kDa heavy chain and 25 kDa light chain and showed specific binding with the porcine APN protein. The results of isotyping and identification of antigen epitopes showed that most of the mAbs targeted different epitopes and belonged to different antibody types. These results indicate that traditional hybridoma technology remains an effective choice in the production of mAbs.
Approaches used for recombinant-antibody production can increase mAb production efficiency and minimize labor- and time-associated costs. Therefore, these approaches have grown in popularity, especially in the development of antibodies for diagnostic and therapeutic applications16,17,26. rAbs show several advantages over hybridoma-derived mAbs. First, rAbs can be produced in vitro by cloning antibody genes into expression vectors, thereby eliminating animal use in antibody production. Additionally, using eukaryotic or prokaryotic expression systems to produce rAbs results in low batch-to-batch variations and increases reliability and stability of the final product. In contrast, mAbs produced using hybridomas often do not recognize or bind to the targeted antigen epitopes, and are affected by hybridoma cell-line drift, contamination, and gene loss and mutations. Presently, hybridoma-derived mAbs are mostly used in diagnostic or therapeutic immune-reagents, highlighting the need to produce stable and reliable antibodies in a limited production period. For diagnostic and therapeutic applications, recombinant antibody technology is a better choice than traditional hybridoma-based approach. This is because recombinant antibody technology allows us to modify the sequence of rAbs to switch immunoglobulin isotypes, thereby increasing the binding specificity of the antibody14,16,17.
In this study, we used antibody-engineering technology to obtain APN-targeted recombinant antibodies. We found that both the spleens of immunized mice and hybridoma cells were similarly effective for amplifying the mAb heavy- and light-chain sequences. The antibody expressed by pET28a (+)-rAbs-APN-BL21 (DE3) did not effectively recognize APN in either ELISA or indirect immunofluorescence assays. However, rAbs expressed by the pIRES2-ZsGreen1-rAbs-APN-CHO cell suspension and hybridoma-derived mAbs did recognize and effectively bind the APN protein. The methods described in this study can be used to develop APN antibody-based ADC and other therapeutic products targeting different APN-specific epitopes. This study will also aid in further clarifying the role of APN in the prevention and treatment of various diseases. However, strategies to improve the affinity and yield of rAbs require further investigation.
The authors have nothing to disclose.
This study was supported by the Chinese National Science Foundation Grant (No. 32072820, 31702242), grants from Jiangsu Government Scholarship for Overseas Studies (JS20190246) and High-level Talents of Yangzhou University Scientific Research Foundation, a project founded by the Priority Academic Program of Development Jiangsu High Education Institution.
Complete Freund’s adjuvant | Sigma-Aldrich | F5881 | Animal immunization |
DAPI | Beyotime Biotechnology | C1002 | Nuclear counterstain |
DMEM | Gibco | 11965092 | Cell culture |
DMEM-F12 | Gibco | 12634010 | Cell culture |
Dylight 549-conjugated goat anti-mouse IgG secondary antibody | Abbkine | A23310 | Indirect immunofluorescence analysis |
Enhanced Cell Counting Kit-8 | Beyotime Biotechnology | C0042 | Measurement of cell viability and vitality |
Fetal bovine serum | Gibco | 10091 | Cell culture |
Geneticin™ Selective Antibiotic | Gibco | 11811098 | Selective antibiotic |
HAT Supplement (50X) | Gibco | 21060017 | Cell selection |
HT Supplement (100X) | Gibco | 11067030 | Cell selection |
Incomplete Freund’s adjuvant | Sigma-Aldrich | F5506 | Animal immunization |
isopropyl β-d-1-thiogalactopyranoside | Sigma-Aldrich | I5502 | Protein expression |
kanamycin | Beyotime Biotechnology | ST102 | Bactericidal antibiotic |
Leica TCS SP8 STED confocal microscope | Leica Microsystems | SP8 STED | Fluorescence imaging |
Lipofectamine® 2000 Reagent | Thermofisher | 11668019 | Transfection |
LSRFortessa™ fluorescence-activated cell sorting | BD | FACS LSRFortessa | Flow cytometry |
Microplate reader | BioTek | BOX 998 | ELISA analysis |
Micro spectrophotometer | Thermo Fisher | Nano Drop one | Nucleic acid concentration detection |
NaCl | Sinopharm Chemical Reagent | 10019308 | Culture broth |
(NH4)2SO4 | Sinopharm Chemical Reagent | 10002917 | Culture broth |
Opti-MEM | Gibco | 31985088 | Cell culture |
Polyethylene glycol 1500 | Roche Diagnostics | 10783641001 | Cell fusion |
PrimeScript™ 1st strand cDNA Synthesis Kit | Takara Bio | RR047 | qPCR |
protein A agarose | Beyotime Biotechnology | P2006 | Antibody protein purification |
Protino® Ni+-TED 2000 Packed Columns | MACHEREY-NAGEL | 745120.5 | Protein purification |
SBA Clonotyping System-HRP | Southern Biotech | May-00 | Isotyping of mouse monoclonal antibodies |
Seamless Cloning Kit | Beyotime Biotechnology | D7010S | Construction of plasmids |
Shake flasks | Beyotime Biotechnology | E3285 | Cell culture |
Sodium carbonate-sodium bicarbonate buffer | Beyotime Biotechnology | C0221A | Cell culture |
Trans-Blot SD Semi-Dry Transfer Cell | Bio-rad | 170-3940 | Western blot |
Tryptone | Oxoid | LP0042 | Culture broth |
Ultrasonic Homogenizer | Ningbo Xinzhi Biotechnology | JY92-IIN | Sample homogenization |
Yeast extract | Oxoid | LP0021 | Culture broth |
96-well microplate | Corning | 3599 | Cell culture |