Rapid transient expression of functional human vascular endothelial growth factor in Nicotiana benthamiana and characterization of its biological activity

Bulaon, Christine Joy I.; Shanmugaraj, Balamurugan; Oo, Yamin; Rattanapisit, Kaewta; Chuanasa, Taksina; Chaotham, Chatchai; Phoolcharoen, Waranyoo

doi:10.1016/j.btre.2020.e00514

Cited by 13 publications

(11 citation statements)

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“…Because the investment cost and time for phase 1 cGMP manufacturing of plant expression system are comparatively cost-effective (7.5- to 10-fold) and faster (<6 months) than mammalian expression system, the plant-production platform might be an alternative platform for the production of many biopharmaceutical products. The use of plant expression systems for the production of pharmaceutically important proteins ( Bulaon et al, 2020 ; Hanittinan et al, 2020 ), vaccines ( Marsian et al, 2017 ; Rosales-Mendoza et al, 2017 ), diagnostic reagents ( Rattanapisit et al, 2021 ), and antibodies ( Kopertekh et al, 2019 ; Hurtado et al, 2020 ; Rattanapisit et al, 2020 ) were documented. In addition, the prior studies reported that the generation of plant-made protective immunogen and therapeutic antibodies ( Dent et al, 2016 ; Rattanapisit et al, 2019a , b ; Nessa et al, 2020 ; Yiemchavee et al, 2021 ).…”

Section: Discussionmentioning

confidence: 99%

Functional Characterization of Pembrolizumab Produced in Nicotiana benthamiana Using a Rapid Transient Expression System

et al. 2021

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The striking innovation and clinical success of immune checkpoint inhibitors (ICIs) have undoubtedly contributed to a breakthrough in cancer immunotherapy. Generally, ICIs produced in mammalian cells requires high investment, production costs, and involves time consuming procedures. Recently, the plants are considered as an emerging protein production platform due to its cost-effectiveness and rapidity for the production of recombinant biopharmaceuticals. This study explored the potential of plant-based system to produce an anti-human PD-1 monoclonal antibody (mAb), Pembrolizumab, in Nicotiana benthamiana. The transient expression of this mAb in wild-type N. benthamiana accumulated up to 344.12 ± 98.23 μg/g fresh leaf weight after 4 days of agroinfiltration. The physicochemical and functional characteristics of plant-produced Pembrolizumab were compared to mammalian cell-produced commercial Pembrolizumab (Keytruda®). Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analysis results demonstrated that the plant-produced Pembrolizumab has the expected molecular weight and is comparable with the Keytruda®. Structural characterization also confirmed that both antibodies have no protein aggregation and similar secondary and tertiary structures. Furthermore, the plant-produced Pembrolizumab displayed no differences in its binding efficacy to PD-1 protein and inhibitory activity between programmed cell death 1 (PD-1) and programmed cell death ligand 1 (PD-L1) interaction with the Keytruda®. In vitro efficacy for T cell activation demonstrated that the plant-produced Pembrolizumab could induce IL-2 and IFN-γ production. Hence, this proof-of-concept study showed that the plant-production platform can be utilized for the rapid production of functional mAbs for immunotherapy.

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Section: Discussionmentioning

confidence: 99%

Functional Characterization of Pembrolizumab Produced in Nicotiana benthamiana Using a Rapid Transient Expression System

et al. 2021

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“…Alternatively, plant expression systems have gained major interests in the making of recombinant proteins due to the economical production costs in upstream processes, rapid scalability employing versatile growth conditions, pathogen free with low risks of contamination [3,17]. Nicotiana genus has been widely used establishing a history in the production of heterologous recombinant proteins that include antibodies [18][19][20], vaccines [21,22], enzymes [23,24], growth factors [25][26][27], diagnostics [28], anti-microbial peptides [29] and other value-added products [30] because of the ease in genome manipulation and fast growth rate [31,32]. The transient expression by agroinfiltration was adopted in plants to produce various proteins for therapeutic use.…”

Section: Introductionmentioning

confidence: 99%

Expression of plant-produced anti-PD-L1 antibody with anoikis sensitizing activity in human lung cancer cells via., suppression on epithelial-mesenchymal transition

Phetphoung

Malla²,

Rattanapisit³

et al. 2022

PLoS ONE

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Immune checkpoint antibodies in cancer treatment are receptor-ligand pairs that modulate cancer immunity. PD-1/PD-L1 pathway has emerged as one of the major targets in cancer immunotherapy. Atezolizumab, the first anti-PD-L1 antibody approved for the treatment of metastatic urothelial, non-small cell lung, small cell lung and triple-negative breast cancers, is produced in Chinese Hamster Ovary (CHO) cells with several limitations i.e., high-production costs, low-capacity yields, and contamination risks. Due to the rapid scalability and low production costs, the transient expression in Nicotiana benthamiana leaves was investigated by co-infiltration of Agrobacterium tumefaciens GV3101 cultures harboring the nucleic acid sequences encoding for Atezolizumab heavy chain and light chain in this study. The transient expression of Atezolizumab in transformed N. benthamiana accumulated up to 86.76 μg/g fresh leaf weight after 6 days of agroinfiltration (OD 600 nm: 0.4) with 1:1 ratio of heavy chain to light chain. The structural and functional characteristics of plant-produced Atezolizumab was compared with commercially available Tecentriq® from CHO cells with similar binding efficacies to PD-L1 receptor. The direct anti-cancer effect of plant-produced anti-PD-L1 was further performed in human lung metastatic cancer cells H460 cultured under detachment condition, demonstrating the activity of anti-PD-L1-antibody on sensitizing anoikis as well as the suppression on anti-apoptosis proteins (Bcl-2 and Mcl-1) and modulation of epithelial to mesenchymal regulating proteins (E-cadherin, N-cadherin, Snail and Slug). In conclusion, this study manifests plants as an alternative cost-effective platform for the production of functional monoclonal antibodies for use in cancer therapy.

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“…Over the last few decades, abundant literature reveals that plant-based expression of recombinant proteins is a promising technology (Shanmugaraj and Phoolcharoen, 2021) and was successful in the expression of virus-like-particles (VLP's) (Ward et al, 2021), monoclonal antibodies (Shanmugaraj et al, 2020b;Swope et al, 2022), antigens (Rattanapisit et al, 2020b), growth factors (Bulaon et al, 2020;Hanittinan et al, 2020;Rattanapisit et al, 2020a), enzymes (Fox, 2012) due to the advantages in terms of high reproducibility, scalability in short time with lower contamination menace (Shanmugaraj et al, 2020a). Accordingly, the present study focuses on the recombinant production of anti-IgE antibod y by Agrobacterium mediated transformation in N. benthamiana host system.…”

Section: Discussionmentioning

confidence: 99%

Feasibility of plant-expression system for production of recombinant anti-human IgE: An alternative production platform for therapeutic monoclonal antibodies

Hanittinan

Rattanapisit²,

Malla³

et al. 2022

Front. Plant Sci.

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Omalizumab, the anti-immunoglobulin IgE antibody is the only approved and available monoclonal antibody as an auxiliary medicament for the severe respiratory allergic reactions. It forms small size immune complexes by binding to free IgE, thereby inhibiting the interaction of IgE with its receptors. Additionally, the anti-IgE can also differently shape the airflow by impeding the stimulation of IgE receptors present on structural cells in the respiratory tract. The present study aimed to use plants as an expression system for anti-human IgE antibody production, using Nicotiana benthamiana as hosts. Recombinant Agrobacterium tumefaciens containing heavy chain (HC) and light chain (LC) domains of anti-human IgE were co-transformed in N. benthamiana. The assembling of the antibody and its expression was detected by SDS-PAGE and Western blot analysis. The functional ability of the anti-IgE antibody was determined via its binding capacity with target IgE by ELISA and the inhibition of basophil activation. The anti-human IgE mAb generated in plants was shown to be effective in binding to its target IgE and inhibit the IgE-crosslink in RS-ATL8 reporter cells. Although, antibody yield and purification process have to be further optimized, this study demonstrates the use of plant expression system as a promising platform for the production of Omalizumab which showed a comparable in vitro function to that of commercial Omalizumab (Xolair) in the inhibition of basophil activation.

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Rapid transient expression of functional human vascular endothelial growth factor in Nicotiana benthamiana and characterization of its biological activity

Abstract: Highlights Growth factors play a crucial role in wound healing. Plant-produced human vascular endothelial growth factor (hVEGF) induces the keratinocyte cells migration. The plant-produced hVEGF has shown potential as a wound healing agent in drug and cosmetic industry.

Cited by 13 publications

References 49 publications

Functional Characterization of Pembrolizumab Produced in Nicotiana benthamiana Using a Rapid Transient Expression System

Functional Characterization of Pembrolizumab Produced in Nicotiana benthamiana Using a Rapid Transient Expression System

Expression of plant-produced anti-PD-L1 antibody with anoikis sensitizing activity in human lung cancer cells via., suppression on epithelial-mesenchymal transition

Feasibility of plant-expression system for production of recombinant anti-human IgE: An alternative production platform for therapeutic monoclonal antibodies

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