Nicotiana benthamiana as a potential source for producing anti-dengue virus D54 neutralizing therapeutic antibody.

Dengue virus (DENV), transmitted by mosquitoes, is classified into four serotypes (DENV1-4) and typically causes mild, self-limiting symptoms upon initial infection. However, secondary infection can lead to severe symptoms due to antibody-dependent enhancement (ADE). To address this, anti-DENV antibodies are being developed with the goal of neutralizing infection without ADE activity. Previous attempts using a 54_hG1 antibody from CHO-K1 mammalian cells resulted in ADE induction, increasing viral infection. This study aimed to express the D54 monoclonal antibody in Nicotiana benthamiana. The plant-produced antibody had a similar neutralizing profile to the previous 54_hG1 antibody. Notably, the ADE activities of the plant-derived antibody were successfully eliminated, with no sign of viral induction. These findings suggest that N. benthamiana could be a source of therapeutic DENV antibodies. The method offers several advantages, including lower ADE, cost-effectiveness, simple facility requirements, scalability, and potential industrial-scale production in GMP facilities.

Characterization of plant produced VHH antibodies against cobra venom toxins for antivenom therapy.

Cobra (Naja kaouthia) venom contains many toxins including α-neurotoxin (αNTX) and phospholipase A2 (PLA2), which can cause neurodegeneration, respiratory failure, and even death. The traditional antivenom derived from animal serum faces many challenges and limitations. Heavy-chain-only antibodies (HCAb), fusing VHH with human IgG Fc region, offer advantages in tissue penetration, antigen binding, and extended half-life. This research involved the construction and transient expression of two types of VHH-FC which are specific to α-Neurotoxin (VHH-αNTX-FC) and Phospholipase A2 (VHH-PLA2-FC) in Nicotiana benthamiana leaves. The recombinant HCAbs were incubated for up to six days to optimize expression levels followed by purification by affinity chromatography and characterization using LC/Q-TOF mass spectrometry (MS). Purified proteins demonstrated over 92 % sequence coverage and an average mass of around 82 kDa with a high-mannose N-glycan profile. An antigen binding assay showed that the VHH-αNTX-Fc has a greater ability to bind to crude venom than VHH-PLA2-Fc.

Functionalizable bacterial cellulose composite membrane for guided tissue regeneration.

This research aims to develop guided tissue regeneration (GTR) membranes from bacterial cellulose (BC), a natural polysaccharide-based biopolymer. A double-layered BC composite membrane was prepared by coating the BC membrane with mixed carboxymethyl cellulose/poly(ethylene oxide) (CMC/PEO) fibers via electrospinning. The CMC/PEO-BC membranes were then characterized for their chemical and physical characteristics. The 8 % (wt/v) CMC/PEO (1:1) aqueous solution yielded well-defined electrospun CMC/PEO nanofibers (125 ± 10 nm) without beads. The CMC/PEO-BC membranes exhibited good mechanical and swelling properties as well as good cytocompatibility against human periodontal ligament cells (hPDLs). Its functionalizability via carboxyl entities in CMC was tested using the calcium-binding domain of plant-derived recombinant human osteopontin (p-rhOPN-C122). As evaluated by enzyme-linked immunosorbent assay, a 98-99 % immobilization efficiency was achieved in a concentration-dependent manner over an applied p-rhOPN-C122 concentration range of 7.5-30 ng/mL. The biological function of the membrane was assessed by determining the expression levels of osteogenic-related gene transcripts using quantitative real-time reverse-transcriptase polymerase chain reaction. Mineralization assay indicated that the p-rhOPN-C122 immobilized CMC/PEO-BC membrane promoted hPDLs osteogenic differentiation. These results suggested that the developed membrane could serve as a promising GTR membrane for application in bone tissue regeneration.

Quality control in SARS-CoV-2 RBD-Fc vaccine production using LC-MS to confirm strain selection and detect contaminations from other strains.

Coronavirus disease of 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is an ongoing outbreak, disrupting human life worldwide. Vaccine development was prioritized to obtain a biological substance for combating the viral pathogen and lessening disease severity. In vaccine production, biological origin and relevant materials must be carefully examined for potential contaminants in conformity with good manufacturing practice. Due to fast mutation, several SARS-CoV-2 variants and sublineages have been identified. Currently, most of COVID-19 vaccines are developed based on the protein sequence of the Wuhan wild type strain. New vaccines specific for emerging SARS-CoV-2 strains are continuously needed to tackle the incessant evolution of the virus. Therefore, in vaccine development and production, a reliable method to identify the nature of subunit vaccines is required to avoid cross-contamination. In this study, liquid chromatography-mass spectrometry using quadrupole-time of flight along with tryptic digestion was developed for distinguishing protein materials derived from different SARS-CoV-2 strains. After analyzing the recombinantly produced receptor-binding domain (RBD) of the SARS-CoV-2 spike protein, nine characteristic peptides were identified with acceptable limits of detection. They can be used together to distinguish 14 SARS-CoV-2 strains, except Kappa and Epsilon. Plant-produced RBD-Fc protein derived from Omicron strains can be easily distinguished from the others with 4-5 unique peptides. Eventually, a peptide key was developed based on the nine peptides, offering a prompt and precise flowchart to facilitate SARS-CoV-2 strain identification in COVID-19 vaccine manufacturing.

Neutralizing activity of anti-respiratory syncytial virus monoclonal antibody produced in Nicotiana benthamiana.

Respiratory syncytial virus (RSV) is a highly contagious virus that affects the lungs and respiratory passages of many vulnerable people. It is a leading cause of lower respiratory tract infections and clinical complications, particularly among infants and elderly. It can develop into serious complications such as pneumonia and bronchiolitis. The development of RSV vaccine or immunoprophylaxis remains highly active and a global health priority. Currently, GSK's Arexvy™ vaccine is approved for the prevention of lower respiratory tract disease in older adults (>60 years). Palivizumab and currently nirsevimab are the approved monoclonal antibodies (mAbs) for RSV prevention in high-risk patients. Many studies are ongoing to develop additional therapeutic antibodies for preventing RSV infections among newborns and other susceptible groups. Recently, additional antibodies have been discovered and shown greater potential for development as therapeutic alternatives to palivizumab and nirsevimab. Plant expression platforms have proven successful in producing recombinant proteins, including antibodies, offering a potential cost-effective alternative to mammalian expression platforms. Hence in this study, an attempt was made to use a plant expression platform to produce two anti-RSV fusion (F) mAbs 5C4 and CR9501. The heavy-chain and light-chain sequences of both these antibodies were transiently expressed in Nicotiana benthamiana plants using a geminiviral vector and then purified using single-step protein A affinity column chromatography. Both these plant-produced mAbs showed specific binding to the RSV fusion protein and demonstrate effective viral neutralization activity in vitro. These preliminary findings suggest that plant-produced anti-RSV mAbs are able to neutralize RSV in vitro.

Production of monoclonal antibodies against botulinum neurotoxin in Nicotiana benthamiana.

Botulism is a fatal neurologic disease caused by the botulinum toxin (BoNT) produced by Clostridium botulinum. It is a rare but highly toxic disease with symptoms, such as cramps, nausea, vomiting, diarrhea, dysphagia, respiratory failure, muscle weakness, and even death. Currently, two types of antitoxin are used: equine-derived heptavalent antitoxin and human-derived immunoglobulin (BabyBIG®). However, heptavalent treatment may result in hypersensitivity, whereas BabyBIG®, has a low yield. The present study focused on the development of three anti-BoNT monoclonal antibodies (mAbs), 1B18, C25, and M2, in Nicotiana benthamiana. The plant-expressed mAbs were purified and examined for size, purity and integrity by SDS-PAGE, western blotting and size-exclusion chromatography. Analysis showed that plant-produced anti-BoNT mAbs can fully assemble in plants, can be purified in a single purification step, and mostly remain as monomeric proteins. The efficiency of anti-BoNT mAbs binding to BoNT/A and B was then tested. Plant-produced 1B18 retained its ability to recognize both mBoNT/A1 and ciBoNT/B1. At the same time, the binding specificities of two other mAbs were determined: C25 for mBoNT/A1 and M2 for ciBoNT/B1. In conclusion, our results confirm the use of plants as an alternative platform for the production of anti-BoNT mAbs. This plant-based technology will serve as a versatile system for the development botulism immunotherapeutics.

Development of Plant-Derived Bispecific Monoclonal Antibody Targeting PD-L1 and CTLA-4 against Mouse Colorectal Cancer.

Checkpoint blockade immunotherapy has revolutionized cancer treatment, with monoclonal antibodies targeting immune checkpoints, yielding promising clinical benefits. However, with the advent of resistance to immune checkpoint inhibitor treatment in clinical trials, developing next-generation antibodies with potentially increased efficacy is critical. Here, we aimed to generate a recombinant bispecific monoclonal antibody for dual inhibition of programmed cell death protein 1/programmed cell death ligand 1 and cytotoxic T-lymphocyte-associated protein 4 axes. The plant system was used as an alternative platform for bispecific monoclonal antibody production. Dual variable domain immunoglobulin atezolizumab × 2C8 is a plant-derived bispecific monoclonal antibody that combines both programmed cell death ligand 1 and cytotoxic T-lymphocyte-associated protein 4 blockade into a single molecule. Dual variable domain immunoglobulin atezolizumab × 2C8 was transiently expressed in Nicotiana benthamiana and the expression level was determined to be the highest after 4 days of infiltration. The size and assembly of the purified bispecific monoclonal antibody were determined, and its function was investigated in vitro and in vivo. The molecular structures of plant-produced dual variable domain immunoglobulin atezolizumab × 2C8 are as expected, and it was mostly present as a monomer. The plant-produced dual variable domain immunoglobulin atezolizumab × 2C8 showed in vitro binding to programmed cell death ligand 1 and cytotoxic T-lymphocyte-associated protein 4 proteins. The antitumor activity of plant-produced bispecific monoclonal antibody was tested in vivo by treating humanized Balb/c mice bearing a CT26 colorectal tumor. Plant-produced dual variable domain immunoglobulin atezolizumab × 2C8 significantly inhibited tumor growth by reducing tumor volume and weight. Body weight changes indicated that the plant-produced bispecific monoclonal antibody was safe and tolerable. Overall, this proof of concept study demonstrated the viability of plants to produce functional plant-based bispecific immunotherapy.

Antitumor effect of plant-produced anti-CTLA-4 monoclonal antibody in a murine model of colon cancer.

Cytotoxic T lymphocyte-associated protein 4 (CTLA-4) is an immune checkpoint regulator exclusively expressed on T cells that obstructs the cell's effector functions. Ipilimumab (Yervoy®), a CTLA-4 blocking antibody, emerged as a notable breakthrough in modern cancer treatment, showing upfront clinical benefits in multiple carcinomas. However, the exhilarating cost of checkpoint blockade therapy is discouraging and even utmost prominent in developing countries. Thereby, affordability of cancer care has become a point of emphasis in drug development pipelines. Plant expression system blossomed as a cutting-edge platform for rapid, facile to scale-up, and economical production of recombinant therapeutics. Here, we describe the production of an anti-CTLA-4 2C8 antibody in Nicotiana benthamiana. ELISA and bio-layer interferometry were used to analyze antigen binding and binding kinetics. Anticancer responses in vivo were evaluated using knocked-in mice implanted with syngeneic colon tumor. At 4 days post-infiltration, the antibody was transiently expressed in plants with yields of up to 39.65 ± 8.42 µg/g fresh weight. Plant-produced 2C8 binds to both human and murine CTLA-4, and the plant-produced IgG1 also binds to human FcγRIIIa (V158). In addition, the plant-produced 2C8 monoclonal antibody is as effective as Yervoy® in inhibiting tumor growth in vivo. In conclusion, our study underlines the applicability of plant platform to produce functional therapeutic antibodies with promising potential in cancer immunotherapy.

Efficiency Comparative Approach of Plant-Produced Monoclonal Antibodies against Rabies Virus Infection.

Rabies encephalitis is a fatal zoonotic viral disease caused by the neurotropic rabies virus. It remains a major public health concern as it causes almost 100% fatality and has no effective medication after the onset of the disease. However, this illness is preventable with the timely administration of effective post-exposure prophylaxis (PEP) consisting of the rabies vaccine and passive immune globulins (HRIG and ERIG). Recently, conventional PEP has been shown to have many limitations, resulting in little support for these expensive and heterologous globulins. Monoclonal antibody (mAb) production via recombinant technology in animal and human cell cultures, as well as a plant-based platform, was introduced to overcome the costly and high-tech constraints of former preparations. We used transient expression technology to produce two mAbs against the rabies virus in Nicotiana benthamiana and compared their viral neutralizing activity in vitro and in vivo. The expression levels of selective mAbs E559 and 62-71-3 in plants were estimated to be 17.3 mg/kg and 28.6 mg/kg in fresh weight, respectively. The plant-produced mAbs effectively neutralized the challenge virus CVS-11 strain in a cell-based RFFIT. In addition, the combination of these two mAbs in a cocktail protected hamsters from rabies virus infection more effectively than standard HRIG and ERIG. This study suggests that the plant-produced rabies antibody cocktail has promising potential as an alternative biological to polyclonal RIG in rabies PEP.

In vitro and in vivo studies of plant-produced Atezolizumab as a potential immunotherapeutic antibody.

Immune checkpoint inhibitors are a well-known class of immunotherapeutic drugs that have been used for effective treatment of several cancers. Atezolizumab (Tecentriq) was the first antibody to target immune checkpoint PD-L1 and is now among the most commonly used anticancer therapies. However, this anti-PD-L1 antibody is produced in mammalian cells with high manufacturing costs, limiting cancer patients' access to the antibody treatment. Plant expression system is another platform that can be utilized, as they can synthesize complex glycoproteins, are rapidly scalable, and relatively cost-efficient. Herein, Atezolizumab was transiently produced in Nicotiana benthamiana and demonstrated high expression level within 4-6 days post-infiltration. After purification by affinity chromatography, the purified plant-produced Atezolizumab was compared to Tecentriq and showed the absence of glycosylation. Furthermore, the plant-produced Atezolizumab could bind to PD-L1 with comparable affinity to Tecentriq in ELISA. The tumor growth inhibitory activity of plant-produced Atezolizumab in mice was also found to be similar to that of Tecentriq. These findings confirm the plant's capability to serve as an efficient production platform for immunotherapeutic antibodies and suggest that it could be used to alleviate the cost of existing anticancer products.

Effect of plant produced Anti-hIL-6 receptor antibody blockade on pSTAT3 expression in human peripheral blood mononuclear cells.

As a response to invasion by pathogens, the secretion of interleukin 6 (IL-6) which is a cytokine, activates IL-6/JAKs/STAT3 intracellular signaling via., phosphorylation. Over expression of pSTAT3 induces IL-6 positive feedback loop causing cytokine release syndrome or cytokine storm. Plants have gained momentum as an alternative expression system. Hence, this study aims to produce mAb targeting human IL-6 receptor (hIL-6R) in Nicotiana benthamiana for down regulating its cellular signaling thus, decreasing the expression of pSTAT3. The variable regions of heavy and light chains of anti-hIL-6R mAb were constructed in pBYK2e geminiviral plant expression vector and transiently co-expressed in N. benthamiana. The results demonstrate the proper protein assembly of anti-hIL-6R mAb with highest expression level of 2.24 mg/g FW at 5 dpi, with a yield of 21.4 µg/g FW after purification. The purity and N-glycosylation of plant produced antibody was analyzed, including its specificity to human IL-6 receptor by ELISA. Additionally, we investigated the effect to pSTAT3 expression in human PBMC's by flow cytometry wherein, the results confirmed lower expression of pSTAT3 with increasing concentrations of plant produced anti-hIL-6R mAb. Although, further in vivo studies are key to unveil the absolute functionality of anti-hIL-6R, we hereby show the potential of the plant platform and its suitability for the production of this therapeutic antibody.

Development of SARS-CoV-2 neutralizing antibody detection assay by using recombinant plant-produced proteins.

Detecting immunity against SARS-CoV-2 is vital for evaluating vaccine response and natural infection, but conventional virus neutralization test (cVNT) requires BSL3 and live viruses, and pseudo-virus neutralization test (pVNT) needs specialized equipment and trained professionals. The surrogate virus neutralization test (sVNT) was developed to overcome these limitations. This study explored the use of angiotensin converting enzyme 2 (ACE2) produced from Nicotiana benthamiana for the development of an affordable neutralizing antibodies detection assay. The results showed that the plant-produced ACE2 can bind to the receptor binding domain (RBD) of the SARS-CoV-2, and was used to develop sVNT with plant-produced RBD protein. The sVNT developed using plant-produced proteins showed high sensitivity and specificity when validated with a group of 30 RBD vaccinated mice sera and the results were correlated with cVNT titer. This preliminary finding suggests that the plants could offer a cost-effective platform for producing diagnostic reagents.

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

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.

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

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.

Plant-Produced S1 Subunit Protein of SARS-CoV-2 Elicits Immunogenic Responses in Mice.

SARS-CoV-2 is responsible for the ongoing COVID-19 pandemic. The virus spreads rapidly with a high transmission rate among humans, and hence virus management has been challenging owing to finding specific therapies or vaccinations. Hence, an effective, low-cost vaccine is urgently required. In this study, the immunogenicity of the plant-produced S1 subunit protein of SARS-CoV-2 was examined in order to assess it as a potential candidate for SARS-CoV-2. The SARS-CoV-2 S1-Fc fusion protein was transiently produced in Nicotiana benthamiana. Within four days of infiltration, the SARS-CoV-2 S1-Fc protein was expressed in high quantities, and using protein A affinity column chromatography, plant-produced S1-Fc protein was purified from the crude extracts. The characterization of plant-produced S1-Fc protein was analyzed by SDS-PAGE and Western blotting. Immunogenicity of the purified S1-Fc protein formulated with alum induced both RBD specific antibodies and T cell immune responses in mice. These preliminary results indicated that the plant-produced S1 protein is immunogenic in mice.

A recombinant subunit vaccine candidate produced in plants elicits neutralizing antibodies against SARS-CoV-2 variants in macaques.

Since the outbreak of the coronavirus disease (COVID) pandemic in 2019, the development of effective vaccines to combat the infection has been accelerated. With the recent emergence of highly transmissible severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOC), there are concerns regarding the immune escape from vaccine-induced immunity. Hence an effective vaccine against VOC with a potent immune response is required. Our previous study confirmed that the two doses of the plant-produced receptor-binding domain (RBD) of SARS-CoV-2 fused with the Fc region of human IgG1, namely Baiya SARS-CoV-2 Vax 1, showed high immunogenicity in mice and monkeys. Here, we aimed to evaluate the immunogenicity of a three-dose intramuscular injection of Baiya SARS-CoV-2 Vax 1 on days 0, 21, and 133 in cynomolgus monkeys. At 14 days after immunization, blood samples were collected to determine RBD-specific antibody titer, neutralizing antibody, and pseudovirus neutralizing antibody titers. Immunized monkeys developed significantly high levels of antigen-specific antibodies against SARS-CoV-2 compared to the control group. Interestingly, the sera collected from immunized monkeys also showed a neutralizing antibody response against the SARS-CoV-2 VOCs; Alpha, Beta, Gamma, Delta, and Omicron. These findings demonstrate that a three-dose regimen of Baiya SARS-CoV-2 Vax 1 vaccine elicits neutralizing immune response against SARS-CoV-2 variants.

Receptor binding domain proteins of SARS-CoV-2 variants produced in Nicotiana benthamiana elicit neutralizing antibodies against variants of concern.

The constantly emerging severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) variants of concerns (VOCs) with mutations in the receptor-binding domain (RBD) spread rapidly and has become a severe public health problem worldwide. Effective vaccines and optimized booster vaccination strategies are thus highly required. Here, the gene encoding six different RBD (Alpha, Beta, Gamma, Kappa, Delta, and Epsilon variants) along with the Fc fragment of human IgG1 (RBD-Fc) was cloned into plant expression vector and produced in Nicotiana benthamiana by transient expression. Further, the immunogenicity of plant-produced variant RBD-Fc fusion proteins were tested in cynomolgus monkeys. Each group of cynomolgus monkeys was immunized three times intramuscularly with variant RBD-Fc vaccines at Day 0, 21, 42, and neutralizing antibody responses were evaluated against ancestral (Wuhan), Alpha, Beta, Gamma, and Delta variants. The results showed that three doses of the RBD-Fc vaccine significantly enhanced the immune response against all tested SARS-CoV-2 variants. In particular, the vaccines based on Delta and Epsilon mutant RBD elicit broadly neutralizing antibodies against ancestral (Wuhan), Alpha, and Delta SARS-CoV-2 variants whereas Beta and Gamma RBD-Fc vaccines elicit neutralizing antibodies against their respective SARS-CoV-2 strains. The Delta and Epsilon RBD-Fc based vaccines displayed cross-reactive immunogenicity and might be applied as a booster vaccine to induce broadly neutralizing antibodies. These proof-of-concept results will be helpful for the development of plant-derived RBD-Fc-based vaccines against SARS-CoV-2 and its variants.

Osteopontin induces osteogenic differentiation by human periodontal ligament cells via calcium binding domain-ALK-1 interaction.

BACKGROUND: Recently we have generated recombinant human osteopontin (rhOPN) using a plant platform (Nicotiana benthamiana) and demonstrated, when coated on culture plates, its osteogenic induction capacity of human periodontal ligament (PDL) cells. The aim of this study is to elucidate the molecular mechanism underlying the rhOPN-induced osteogenic differentiation of human PDL cells. METHODS: Full length rhOPN (FL-OPN) and three constructs of OPN containing integrin binding domain (N142), calcium binding domain (C122) and mutated calcium-binding domain (C122δ) were generated from N. benthamiana. Human PDL cells were isolated from extracted third molars and cultured on FL-OPN, N142, C122, or C122δ-coated surfaces. Real-time PCR and Western blot analyses were used to determine mRNA and protein expression. In vitro calcification was determined by Alizarin red staining. A chemical inhibitor and RNAi silencing were used to elucidate signaling pathways. In silico analyses were performed to predict the protein-protein interaction. In vivo analysis was performed using a rat calvaria defect model. RESULTS: Human PDL cells seeded on FL-OPN and C122-coated surfaces significantly increased both mRNA and protein expression of osterix (OSX) and enhanced in vitro calcification. Soluble FL-OPN as well as a surface coated with N142 did not affect OSX expression. Inhibition of activin receptor-like kinase (ALK-1) abolished the induction of osterix expression. In silico analysis suggested a possible interaction between the calcium binding domain (CaBD) of OPN and ALK-1 receptor. C122, but not C122δ coated surfaces, induced the expression of p-Smad-1 and this induction was inhibited by an ALK-1 inhibitor and RNAi against ALK-1. In vivo data showed that 3D porous scaffold containing C-122 enhanced new bone formation as compared to scaffold alone. CONCLUSION: The results suggest that next to full length OPN, the CaBD of OPN, if coated to a surface, induces osteogenic differentiation via interaction with ALK-1 receptor.

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

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.