Cre-LoxP-regulated expression of monoclonal antibodies driven by an ovalbumin promoter in primary oviduct cells
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A promoter capable of driving high-level transgene expression in oviduct cells is important for developing transgenic chickens capable of producing therapeutic proteins, including monoclonal antibodies (mAbs), in the whites of laid eggs. Ovalbumin promoters can be used as oviduct-specific regulatory sequences in transgenic chickens, but their promoter activities are not high, according to previous reports.
In this study, while using a previously characterized ovalbumin promoter, we attempted to improve the expression level of mAbs using a Cre/loxP-mediated conditional excision system. We constructed a therapeutic mAb expression vector, pBS-DS-hIgG, driven by the CMV and CAG promoters, in which the expression of the heavy and light chains of humanized immunoglobulin G (hIgG) is preceded by two floxed stuffer reporter genes. In the presence of Cre, the stuffer genes were precisely excised and hIgG expression was induced in pBS-DS-hIgG-transfected 293T cells. In chicken oviduct primary culture cells, hIgG was expressed after transfection of pBS-DS-hIgG together with the ovalbumin promoter-driven Cre expression vector. The expression level of hIgG in these cells was increased 40-fold over that induced directly by the ovalbumin promoter. On the other hand, hIgG was not induced by the ovalbumin promoter-driven Cre in chicken embryonic fibroblast cells.
The Cre/loxP-based system could significantly increase ovalbumin promoter-driven production of proteins of interest, specifically in oviduct cells. This expression system could be useful for producing therapeutic mAbs at high level using transgenic chickens as bioreactors.
KeywordsTrastuzumab Therapeutic mAbs Oviduct Cell Transgenic Chicken Chicken Embryonic Fibroblast Cell
The market for therapeutic monoclonal antibodies (mAbs) has dramatically expanded over the past decade because of their high clinical efficacy. In the U.S., around 30 mAbs are currently approved for therapeutic use in cancers, autoimmune disorders, and infectious diseases, and the number of available mAb products is predicted to increase [1, 2]. Although therapeutic mAbs have become a major class of drugs, their high production cost is a major obstacle. This is mainly due to the use of cultured mammalian cells in the manufacturing of mAbs, which requires a complex industrial bioreactor system. To reduce the cost of mAb production, a more convenient method to replace mammalian cell culture is required.
One alternative method involves generating transgenic farm animals as living bioreactors that produce high-yield therapeutic mAbs in milk or other secretory fluids, such as egg whites. The production of recombinant pharmaceutical proteins has been demonstrated in transgenic animals including sheep, goats, cattle, rabbits, and chickens (reviewed in [3, 4]). Among these animals, the use of transgenic chickens as bioreactors is expected to have several advantages, including a shorter timescale for setup, ease of scaling up, and small space requirements (reviewed in [5, 6]). Several groups reported the production of therapeutic proteins, such as cytokines, mini-antibodies, and mAbs using transgenic chickens [7, 8, 9, 10, 11]. In these transgenic chickens, ubiquitous promoters were used to express the transgenic products; thus, tissue-restricted expression of exogenous proteins was not demonstrated.
Compared to tissue-restricted expression, ubiquitous expression of therapeutic mAbs in transgenic chickens will increase the heterogeneity of oligosaccharide structure of mAbs due to the glycosylation in various type of cells [11, 12]. In addition, depending on the antigen recognition, whole-body expression of foreign mAb could be the risk of negatively affecting the development and health of the transgenic chickens. Therefore, oviduct-specific mAb expression is desirable to synthesize mAbs as a component of egg whites. Using chicken ovalbumin promoters, two groups demonstrated oviduct-specific expression of therapeutic proteins in transgenic chickens and secretion of these proteins into the egg whites [12, 13] However, expression levels of exogenous proteins in the egg whites driven by ovalbumin promoters were not high (<0.5 mg/ml, egg whites) compared to their expression in the mammalian cell culture bioreactor (1-13 mg/ml, culture media) [13, 14]. Thus, a highly efficient oviduct promoter is demanded but such a promoter has not been developed .
In an attempt to increase the expression level of therapeutic mAbs in chicken oviduct cells, we developed a Cre-loxP-regulated exogenous immunoglobulin G (IgG) expression vector. The vector consists of two tandem expression units, each containing a strong promoter, a fluorescent gene flanked by loxP or mutant loxP as a stuffer fragment, and the gene for the heavy chain or light chain of humanized IgG (hIgG) encoding the human therapeutic mAb, trastuzumab. Trastuzumab recognizes human epidermal growth facter receptor 2 (HER2), and is clinically used to treat breast cancer. Cre-dependent hIgG induction was observed in mammalian cultured cells as well as laying hen-derived oviduct primary cultured cells following vector transfection. We quantified the expression level of hIgG and observed the 40-fold enhancement of hIgG expression compared to that induced by the ovalbumin promoter as a result of Cre-dependent transcriptional activation.
Results and Discussion
The concentration of hIgG in the cell media was analyzed using ELISA (Figure 3D). Consistent with the immunoblotting results, hIgG was more abundant in the culture media of oviduct cells co-transfected with pBS-DS-hIgG and pBS-Ova2.8-Cre (20 ng/ml) than in cells transfected with pBS-Ova2.8-hIgG alone (0.5 ng/ml). Thus, in chicken oviduct cells, pBS-DS-hIgG and pBS-Ova2.8-Cre co-transfection induces a 40-fold greater expression of hIgG than direct induction of hIgG by the ovalbumin regulatory sequence Ova2.8.
In a previous report, Ova2.8 was shown to be the regulatory sequence required for chicken oviduct-restricted expression in vivo . Transgenic hens carrying Ova2.8-driven transgenes specifically express the transgenic products in oviduct cells, and the products are secreted into the egg white (>1.5 mg/egg). However, more efficient production of exogenous protein is required for the commercial use of transgenic chickens as bioreactors to synthesize therapeutic mAbs. In this study, we succeeded in enhancing hIgG expression regulated by Ova2.8 in chicken oviduct cells. Although it is not clear whether the Cre/loxP-based gene induction system will induce a 40-fold increase in exogenous gene expression upon its introduction into transgenic chickens, it is expected that strong promoters will induce higer hIgG expression in an oviduct-specific manner.
The size of the transgene unit in pBS-DS-hIgG is over 11 kb. This is larger than the size limitation of the inserted DNA into viral vectors which used to produce transgenic chickens, such as avian leukosis virus vector (size limit 4-5 kb), murine stem cell virus vector (3-4 kb), and lentiviral vector (≈10 kb). Recently, through the use of chicken embryonic stem cells and chicken primordial germ cells, nonviral chicken transgenesis has been developed [12, 15, 16]. With these new tools, transgenic chickens that carry pBS-DS-hIgG and pBS-Ova2.8-Cre could be established. The creation of transgenic hens carrying these transgenes will demonstrate whether the strategy we have developed can produce living bioreactors that generate therapeutic mAbs at high levels in egg whites.
It is essential to develop gene expression system that allows high level expression of quality proteins both in research and therapeutic purposes. One promising method is to use chickens as bioreactors where production of proteins of interest is tightly controlled in tissue specific manner to allow accumulation of the gene product only in egg whites. We describe a new method to produce therapeutic mAbs in chicken oviduct cells. This method is based on Cre/loxP-mediated conditional excision system, regulated by oviduct specific ovalbumin promoter. In oviduct primary cultured cells, we achieved a 40-fold increase in therapeutic mAb production with this method compared to the mAb induction directly by the ovalbumin promoter. High level Oviduct-specific expression of proteins is required to produce therapeutic mAbs in the egg white. Therefore, this method offers a novel strategy practically useful for producing therapeutic proteins in transgenic chickens.
Sequences for PCR primers and synthetic oligo DNAs
Sequences for PCR primers
Sequences for synthetic oligo DNAs
pBS-DS-hIgG consists of 2xHS4, the CAG promoter of pCAGGS , the synthesized loxP sequence, the synthesized prokaryotic promoter em7 sequence, the neomycin resistance gene PCR amplified from pEGFP-N1 (Clontech, Mountain View, CA), an IRES sequence followed by the EGFP gene and SV40 polyA of pIRES2-EGFP (Clontech), a second loxP sequence, a hIgG light chain, the bovine growth hormone (BGH) polyA PCR fragment amplified from pcDNA3 (Invitrogen, Carlsbad, CA), a second 2xHS4, the CMV promoter of pEGFP-N1, the synthesized loxP511 sequence, the mCherry gene (Clontech), a rabbit β-globin (RGB) polyA PCR fragment amplified from pCAGGS, a second loxP511 sequence, a hIgG heavy chain, a herpes simplex virus thymidine kinase (HSVTK) polyA sequence amplified from pEGFP-N1, and a third 2xHS4, in this specific order. Each primer sequence for PCR amplification and DNA synthesis is indicated in Table 1. pBS-Ova2.8-Cre consists of 2xHS4, Ova2.8, Cre gene, BGH polyA, a second 2xHS4, the SV40 promoter PCR amplified from pEGFP-N1, the hygromycin resistance gene, HSVTK polyA, and a third 2xHS4, in this specific order (Table 1). pBS-Ova2.8-hIgG consists of 2xHS4, Ova2.8, a hIgG light chain, BGH polyA, another 2xHS4, a second Ova2.8, a hIgG heavy chain, HSVTK polyA, and a third 2xHS4, in this specific order. pCMV-Cre was constructed by ligating the Cre gene into the EcoRI site of pcDNA3 (Invitrogen).
Cell culture and transfection
293T cells were maintained continuously in DMEM (Invitrogen) supplemented with 10% (v/v) fetal calf serum (FCS). For transfection analysis, cells were plated onto 6-well plates at 3 × 105 cells/well, and 1.5 μg of plasmid was transfected using the FuGene 6 (Roch Diagnostics GmbH, Mannheim, Germany ) transfection reagent according to the manufacturer's protocols. Oviduct cells were dissociated from 18-24-month-old laying hens, as described previously [18, 19]. The cells were suspended in DMEM with 10% FCS containing 1 × 10-7 M 17β-estradiol (Nacalai, Tokyo, Japan), 1 × 10-6 M corticosterone (Wako, Osaka, Japan), and 50 ng/ml insulin (Nacalai) and plated onto gelatin-coated 12-well plates. After 24-48 h of culture, cells were grown to 50% confluence and utilized for transfection studies. Before transfection, culture media was replaced with Opti-MEM containing 17β-estradiol, corticosterone, and insulin in the same concentrations as described above. Plasmids were transfected into oviduct primary cultured cells using FuGene6 with 0.5 μg of DNA for 48 h. The total amount of DNA was kept constant with that of the empty vector.
Cell culture supernatants and recombinant hIgG protein (Trastuzumab, Roche) was eluted with non-reducing or reducing Laemmli sample buffer, separated by SDS-PAGE, and transferred to polyvinylidene difluoride membrane filters (Immobilon, Millipore, Bedford, MA). The membranes were immunoblotted with anti-human IgG Fc antibody (I-124, Leinco Technologies, St. Louis, MO) and horseradish peroxidase (HRP)-conjugated anti-human IgG (H+L) antibody (Jackson ImmunoResearch Laboratories, West Grove, PA) for the non-reduced and reduced conditions, respectively. Bound anti-human IgG Fc antibody was visualized with anti-mouse HRP-conjugated antibody using a chemiluminescence reagent (ImmunoStar, WAKO). Bound anti-human IgG (H+L) antibody was directly visualized by the chemiluminescence reagent. To detect ovalbumin expression, anti-ovalbumin antibody (Millipore) was utilized for immunoblotting experiments.
The concentration of human IgG in culture media was measured by a sandwich ELISA with trastuzumab as the standard. Ninety-six-well plates (Nunc Maxisorb, Thermo Fisher Scientific, Rochester, NY) were coated with 100 μl of 10 μg/ml anti-human IgG (I-124) per well overnight at 4°C. Plates were washed twice with 200 μl of Tris-buffered saline (TBS)/0.05% Tween 20 and blocked with 4 × Block Ace (DS Pharma Biomedical, Osaka, Japan) for 1 h at room temperature. After three washes, 100 μl of culture media and trastuzumab standards with 0.02 × Block Ace were added and incubated for 2 h at room temperature. The plates were washed three times and then incubated with 100 μl of 100 ng/ml HRP-conjugated anti-human IgG for 1 h. After three washes, plates were incubated with 100 μl per well of 3,3',5,5'-tetramethylbenzidine solution (TMB One Solution, Promega, Madison, Wisconsin) for 10 min. The reactions were stopped with the addition of 100 μl of 1 M HCl. The optical density of the samples was measured at 450 nm using a microplate reader (Molecular Devices, Sunnyvale, CA)
We thank Dr. M Morita for help and advice. This study was supported by Industrial Technology Research Grant Program in 2006 from New Energy and Industrial Technology Development Organization (NEDO) of Japan, by JSPS and NRF under the Japan-Korea Basic Scientific Cooperation Program, by The Mochida Memorial Foundation for Medical and Pharmaceutical Research, by Kowa Life Science Foundation, by National Research Foundation of Korea (NRF) Grant (NRF-2010-616-C00036), MICINN, CIBER and Fundacion Cellex.
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