Stable Expression of Adalimumab in Nicotiana tabacum
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Production of monoclonal antibodies and pharmaceutical proteins in transgenic plants has been the focus of many research efforts for close to 30 years. Use of plants as bioreactors reduces large-scale production costs and minimizes risk for human pathogens contamination. Stable nuclear transformation of the plant genome offers a clear advantage in agricultural protein production platforms, limited only by the number of hectares that can be cultivated. We report here, for the first time, successful and stable expression of adalimumab in transgenic Nicotiana tabacum plants. The plant-derived adalimumab proved fully active and was shown to rescue L929 cells from the in vitro lethal effect of rhTNFα just as effectively as commercially available CHO-derived adalimumab (Humira). These results indicate that agricultural biopharming is an efficient alternative to mammalian cell-based expression platforms for the large-scale production of recombinant antibodies.
KeywordsPlant-derived antibody Plant-made pharmaceuticals Adalimumab Humira TNF-α Nicotiana tabacum
Plants have long been proposed as an attractive platform for the production of recombinant proteins. The most advanced biopharming application utilizes the plants as a bioreactor to generate antibodies required for medical use or industrial processes [1, 2, 3, 4, 5, 6, 7, 8]. The key advantages of plant expression systems, as compared to traditional approaches, such as bacterial or mammalian cells expression systems, include their low costs, particularly when considering large-scale production, and the limited risk of contamination by human pathogens . Moreover, bacteria can neither produce full-size antibodies nor perform most of the important mammalian posttranslational modifications [4, 10, 11]. Plants, like other eukaryotes, can perform posttranslational modifications, including N-glycosylation, which is substantially similar to that found in mammalian cells . In addition, disulfide bonds, which are required for folding and assembly of complex multimeric proteins such as mAbs  can be formed in proteins expressed in plants. Thus, molecular pharming presents an unprecedented opportunity to manufacture affordable modern medicines and to make them available on a global scale .
Today, the majority of therapeutic antibodies are generated in mammalian cell lines or by their transient expression in Nicotiana benthamiana. Stable nuclear transformation of Nicotiana tabacum has been established as an excellent alternative production platform, that can yield high levels of complex proteins [10, 15, 16], while providing marked advantages in upscaling processes, requiring conventional agricultural practices [17, 18]. More importantly, unlike transient expression systems, transgenic plant lines involving stable transformation require neither costly fermenters, nor vacuum infiltration equipment, nor sterile conditions . Moreover, N. tabacum is a relatively easy-to-manipulate organism, with very high leaf biomass, compared with N. benthamiana.
Molecular pharming designs must determine the subcellular compartment and posttranslational modifications most appropriate for expression of the target proteins. To date, most antibodies produced in plants accumulate to higher levels when expressed in the apoplast or endoplasmic reticulum (ER), as compared to the cytosol. In addition, protein assembly and posttranslational modifications in the ER are important for the synthesis of full-size antibodies and Fab fragments .
Management of many autoimmune and inflammatory disorders is achieved via antibody-based neutralization of pro-inflammatory cytokines. Neutralization of TNF-α is the most common therapy for such conditions and has been reported to elicit clinically relevant immune response suppression . Adalimumab is the first fully human therapeutic monoclonal antibody approved for clinical use by the FDA. It is a TNF-α-neutralizing mAb, generated by recombinant DNA techniques, and its structure is indistinguishable from its human counterpart. TNF-α is a potent pro-inflammatory cytokine expressed as a surface type II polypeptide on activated macrophages and lymphocytes, among other cell types. It exerts its biological functions, including cytotoxic activity and polyclonal B cell activation, in a cell-to-cell contact-dependent manner [20, 21]. The soluble form of TNF-α mediates its biological activities through type I and type II TNF receptors [21, 22].
Adalimumab is a tetramer composed of two light kappa chains and two heavy immunoglobulin G1 (IgG1) chains, each of the latter containing one N-glycosylation site. The drug was first discovered by means of the “phage display” technique and was named D2E7. Currently, adalimumab is produced by cell culture using Chinese hamster ovary (CHO) cells .
This study aimed to achieve stable transgenic adalimumab expression in N. tabacum plants and to demonstrate that plant-derived mAb is fully functional, having similar bioactivity as its CHO-derived counterpart.
Accumulation of Adalimumab in Nicotiana tabacum cv. Samsun Plants
Stable Apoplastic Adalimumab Expression
Two vectors were constructed for the purpose of stable adalimumab expression in the apoplast: one for RuBisCO promoter-driven overexpression of the adalimumab heavy chain, tagged with the apoplast signal peptide, and the other for RuBisCO promoter-driven overexpression of the adalimumab light chain, tagged with the apoplast signal peptide. A. tumefaciens-mediated co-transformation of the two T vector into sterile N. tabacum cv. Samsun leaf tissue was performed.
Purification and Characterization of the Plant-Expressed Adalimumab
Plant-Derived Adalimumab Target-Binding Activity
Adalimumab Neutralization Bioactivity
Ongoing efforts to advance the production of mAbs in plants have shown that plant-produced mAbs are as functionally active as their mammalian counterparts produced using traditional and well-established mammalian cell culture techniques [25, 26]. In this study, we, for the first time, demonstrate the production of fully active adalimumab, a human anti-TNF-α mAb, in transgenic N. tabacum plants. Plant-derived adalimumab exhibited in vitro activity (Fig. 5) and neutralization bioactivity similar to those measured for commercially available Humira (Fig. 6). At high PDA concentrations (2 μg/ml), a slight toxic effect was observed, likely due to plant-derived protein contaminants eluting out with the mAb. Therefore, for future production, further purification steps should be included, such as an additional protein A chromatography purification or ion-exchange chromatography step to remove impurities.
This study provides further evidence of the utility of plants for stable expression of human therapeutic antibodies. Plants can serve as cost-effective bioreactors, with enormous scalability potential. Furthermore, plant-derived adalimumab is free of mammalian pathogen contaminants. The N. tobacco-expressed adalimumab exhibited a sequence identical to that produced in CHO cell lines. In addition, PDA seemingly folded into its fully active form, as was shown by its functional activities and binding. The last was confirmed on the one hand by PDA binding to hrTNF-α through its Fab region, and on the other hand to protein A through its FC domain. The described heterozygous plants produced up to 10 mg adalimumab per kg fresh weight (data not shown). Further optimization of the purification process and homozygote plant production are expected to increase yields further.
Several expression systems are available for the expression of foreign proteins in plants. Agrobacterium-mediated plant tissue transformation is the most traditional technique, producing genetically modified plants, stably expressing the desired protein. Thus far, stable expression is the most promising approach, as it significantly cuts costs by requiring traditional agriculture for most of the production process. In contrast, transient expression systems require costly bioreactors for the production of the transformation vector, vacuum infiltration equipment, and sterile conditions , and fail to exploit the advantages embodied in plant expression systems. Yet, although production costs of plant-based mAbs are two orders of magnitude lower than mammalian cell-based mAbs [14, 27], there is still an urgent need to address two major limitations of plant-derived therapeutic mAbs. In mammalian proteins, the N-glycosylation structure is a central determinant of antibody half-life in the circulation and of its ability to be recognized by Fc receptors . Recent works have shown that mAbs modified with plant N-glycans exhibit a half-live and antigen-binding properties similar to those decorated with mammalian N-glycans [6, 12, 28]. Yet, immunogenic effects ascribed to the plant-specific core N-glycans β1,2 xylose and α1,3/4 fucose have been noted in laboratory mammals and in humans [29, 30]. Therefore, plants N-glycans may render plant-derived antibodies immunogenic in humans when delivered parenterally , especially with repeated immunotherapy . Thus, future research on the plant-produced adalimumab will require implementation of strategies to removing β1,2 xylose and α1,3/4 fucose immunogenic residues without impacting the bi-antennary complex glycan structure.
In addition, in order to comply with the demand for plant-derived mAb, hundreds of thousands of tons of green biomass will have to be processed and purified. This will present an enormous bottleneck and will demand effective downstream process strategies.
In this work, we established that a therapeutic mAb (PDA) can be produced in a plant-based expression system. PDA showed identical primary structure and similar in vitro bioactivities to those of the clinically approved CHO-derived mAb. We therefore provided further evidence that agricultural biopharming is a practical alternative to mammalian cell systems for the production of therapeutic mAbs.
In the future, the production of biological drugs will shift rapidly toward plant-based expression system, where plant stable nuclear transformation play a key role in making modern medicines affordable for all.
Materials and Methods
Coding gene sequences of human monoclonal D2E7 adalimumab heavy chain (AH) and light chain (AL), were synthesized with desired flanking regions, and optimized for expression in tobacco plants by GenScript Biotech (Piscataway, NJ, USA).
AH and AL were also cloned into a plasmid containing the CaMV 35S promoter, Agrobacterium nopaline synthase (NOS) terminator, and either the vacuolar-targeting SP (35sVac) or the apoplast-targeting SP (35sCel1) (Fig. 7a). The complete expression cassettes were cloned into the multiple cloning site of pBINPLUS vectors, using the EcoRI and SacI restriction enzymes (Fig. 7b).
Vacuole SP sequence of barley thiol protease aleurain precursor (NCBI accession P05167 GI:113603): MAHARVLLLALAVLATAAVAVASSSSFADSNPIRPVTDRAASTLA.
Apoplast signal of Arabidopsis thaliana endo-1,4-β-glucanase (Cel-1, NCBI accession CAA67156.1 GI:2440033): RKSLIFPVILLAVLLFSPPIYSAGHDYRDALRKSSMA.
Transgenic Tobacco Plant Production
Tobacco plants were grown under sterile conditions for approximately 4–5 weeks. Agrobacterium tumefaciens bacteria were grown in 25 ml LB medium with 50 mg/ml kanamycin, for 48 h, on a shaker, at 28 °C, to a stationary stage. Starters were then centrifuged for 10 min, 4000×g, at room temperature. The supernatant was aspirated and the bacterial pellet was diluted with sterile liquid MS medium (4.4 g/L Murashige and Skoog medium including vitamins (Duchefa (cat#M0222.0050)) and 30 g/L sucrose (J.T.Baker (cat#4072-05), pH = 5.8) and grown to a final O.D600 of 0.5. Bacteria (10 ml) were then applied to tobacco leaf sections and incubated for 5 min. Inoculated explants were then transferred to Petri dishes containing solid MS medium (liquid MS medium with 0.7% plant agar (Duchefa (cat# P1001.1000)), supplemented with 0.8 ml/L indole-3-acetic acid (IAA) and 2 ml/L kinetin. Plates were incubated at 28 °C, in the dark, for 48 h, AND then explants were transferred to new Petri dishes containing selective MS medium (0.8 ml/L IAA, 2 ml/L kinetin, 400 mg/L carbenicillin and 100 mg/L kanamycin). Petri dishes were incubated at 25 °C, in the light, for 3 weeks; medium was exchanged every 10 days. During this period, shoots formed. The shoots were detached and transferred to rooting medium (MS medium with 100 mg/L kanamycin and 400 mg/L carbenicillin), under the same light conditions. Shoots that produced roots were transferred to soil pots in the greenhouse, equipped with an upper irrigation system, and grown to mature transgenic plants. Transgenic plants were grown in pots in the greenhouse in the controlled environment. Temperature was 25 °C under a natural photoperiod. The plants were watered in a frequency of three daily 5-min irrigation events. Liquid fertilizer (Shefer 7 – 3 – 7 + 3, ICL Fertilizers, Israel) was injected into the irrigation water.
SDS–PAGE and Western Blot Analysis
SDS–PAGE analysis was performed on 12.5% SDS gels, using a “mini-protein gel system” (Hercules, CA, USA). Western blot analysis was performed as previously described . Then, the Membrane was blocked with 4% skim milk for 0.5 h, at room temperature (RT). The membrane was incubated with a primary antibody (alkaline phosphatase-conjugated goat anti-human IgG heavy and light chain antibody or goat anti-human kappa light chain antibody) overnight at RT, washed three times with tris-buffered saline (TBS) Tween 20 (TBST), and then incubated with an alkaline phosphatase-conjugated secondary antibody, for 2 h, followed by three washes with TBST. Finally, 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and nitro blue tetrazolium (NBT) substrates (Sigma) diluted in substrate buffer (100 mM Tris, 100 mM sodium chloride, and 5 mM MgCl2, pH 9.5. adjusted with HCl), were added.
Plant tissue Preparation for ELISA Assay
For sample preparation, pre-weighed Eppendorf tubes were prepared, and 100 μl grinding buffer (100 mM Tris–HCl pH 8, 25 mM NaCl, 1 mM phenylmethylsulfonyl fluoride (PMSF), 10 mM EDTA, and 1 mM potassium metabisulfite (PMBS)) were added to each Eppendorf. Six leaf disks from each plant were sampled by clipping with the Eppendorf lid directly into pre-weighed Eppendorf and immediately placed on ice. Samples were taken from lower, middle, and upper plant sections. The samples were then weighed and ground for 30 s with a plastic pistol on mortar homogenizer, at 500 RPM. Soluble fraction was extracted by centrifugation for 15 min, at 12,000 × g, in 4 °C. Samples were then 500-fold-diluted in TBS.
The average concentration in each set of PDA samples was calculated from the calibration curve. Then, the PDA concentration in the sample was multiplied by the dilution factor. Ab yield in the transgenic plant was calculated by dividing PDA concentration in the sample by fresh tissue weight. The obtained result represents mg PDA per kg fresh leaf tissue.
Plant Tissue Preparation for mAb Purification
Leaves (1 kg) from PDA-expressing plants were screw-pressed with homogenizing buffer (100 mM Tris–HCl pH 8, 25 mM NaCl, 1 mM PMSF, 10 mM EDTA), (w/v 1:1). The soluble mAb fraction was obtained by centrifugation for 30 min, 10,000 × g at 4 °C and then incubated with 3% polyvinylpolypyrrolidone (PVPP), for 4 h, at 4 °C, with shaking, to remove polyphenols. Samples were then centrifuged three times at 10,000 × g for 30 min, at 4 °C, and then filtrated through 0.8- and 0.45-µm filters.
Soluble fraction obtained from plants expressing adalimumab was filtered through a 0.2-µm filter and passed through a HiTrap Protein A high-performance 5-ml column was prepacked with Protein A Sepharose, on an ÄKTA design chromatography system. Samples were diluted in 20 mM sodium phosphate buffer pH7.4, which was also used to wash the column. Elution was performed under acidic conditions, with 100 mM citric acid, pH 3. Fractions were collected and analyzed by SDS–PAGE. Protein concentration was measured using ELISA assay.
Protein Sequence Analysis
Samples were digested with trypsin and analyzed by liquid chromatography coupled to a LTQ mass spectrometer (Hybrid-2D-Linear Quadrupole Ion Trap—mass spectrometer, Thermo Scientific or Thermo Electron, Bremen, Germany) and identified using the Discoverer software, using the adalimumab D2E7 heavy and light chain sequences as reference (performed in The Smoler Proteomics Center, Department of Biology, Technion, Israel). Peptide contig was produced with BioEdit software CAP feature, peptides were assembled to a protein, and CLUSTALW protein alignment was performed in comparison with the adalimumab sequence (D2E7).
The adalimumab activity assay was performed by Harlan Biotech Ltd., Israel. Briefly, activity was tested by antibody-driven neutralization of TNFα-mediated cytotoxicity in L929 fibroblast cells. Two 96-well tissue culture plates were filled with 100 μl of a L929 cell suspension, prepared at a density of 3.5 × 105 cells/ml, and incubated overnight at 37 °C, 5% CO2, in a humidified incubator. After 12-h incubation, rhTNF-α and actinomycin D were added to a final concentration of 1 ng/ml rhTNF-α [LC50 (lethal concentration 50%) as determined in preliminary experiments (data not shown)] and 1 µg/ml actinomycin D, and incubated for 2 h at 37 °C, 5% CO2, in a humidified incubator. The first plate (experiment plate) was then treated with PDA (1.95–1000 ng/ml). The second plate (control plate) was treated with Humira (1.95–1000 ng/ml). MTT solution (tetrazolium dye MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was added to each well, to a final concentration of 0.5 mg/ml, and incubated for approximately 4 h, at 37 °C. MTT solution was then aspirated, and 100 µl isopropanol was added to each well for no less than 30 min. Absorbance was measured using a microplate spectrophotometer (Multiscan® FC; Thermo Scientific), with 570–650 nm wavelength filters.
We thank Dr. Meirav Blanka for her help in the development of the quantitative ELISA and Dr. Yehudit Posen for critically reviewing this manuscript. This study was supported by the Minerva Center for bio-hybrid complex system.
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