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.

Molecular Farming Strategy for the Rapid Production of Protein-Based Reagents for Use in Infectious Disease Diagnostics.

Recombinant proteins are a major breakthrough in biomedical research with a wide range of applications from diagnostics to therapeutics. Strategic construct design, consistent expression platforms, and suitable upstream and downstream techniques are key considerations to produce commercially viable recombinant proteins. The recombinant antigenic protein production for use either as a diagnostic reagent or subunit vaccine formulation is usually carried out in prokaryotic or eukaryotic expression platforms. Microbial and mammalian systems dominate the biopharmaceutical industry for such applications. However, there is no universal expression system that can meet all the requirements for different types of proteins. The adoptability of any expression system is likely based on the quality and quantity of the proteins that can be produced from it. The huge demand of recombinant proteins for different applications requires an inexpensive production platform for rapid development. The molecular farming scientific community has been promoting the plant system for nearly 3 decades as a cost-effective alternative to produce high-quality proteins for research, diagnostic, and therapeutic applications. Here, we discuss how plant biotechnology could offer solutions for the rapid and scalable production of protein antigens as low-cost diagnostic reagents for use in functional assays.

Plant molecular farming-derived epidermal growth factor revolutionizes hydrogels for improving glandular epithelial organoid biofabrication.

Epidermal growth factor (EGF) is a known signaling cue essential towards the development and organoid biofabrication particularly for exocrine glands. This study developed an in vitro EGF delivery platform with Nicotiana benthamiana plant-produced EGF (P-EGF) encapsulated on hyaluronic acid/alginate (HA/Alg) hydrogel to improve the effectiveness of glandular organoid biofabrication in short-term culture systems. Primary submandibular gland epithelial cells were treated with 5 - 20 ng/mL of P-EGF and commercially available bacteria-derived EGF (B-EGF). Cell proliferation and metabolic activity were measured by MTT and luciferase-based ATP assays. P-EGF and B-EGF 5 - 20 ng/mL promoted glandular epithelial cell proliferation during 6 culture days on a comparable fashion. Organoid forming efficiency and cellular viability, ATP-dependent activity and expansion were evaluated using two EGF delivery systems, HA/Alg-based encapsulation and media supplementation. Phosphate buffered saline (PBS) was used as a control vehicle. Epithelial organoids fabricated from PBS-, B-EGF-, and P-EGF-encapsulated hydrogels were characterized genotypically, phenotypically and by functional assays. P-EGF-encapsulated hydrogel enhanced organoid formation efficiency and cellular viability and metabolism relative to P-EGF supplementation. At culture day 3, epithelial organoids developed from P-EGF-encapsulated HA/Alg platform contained functional cell clusters expressing specific glandular epithelial markers such as exocrine pro-acinar (AQP5, NKCC1, CHRM1, CHRM3, Mist1), ductal (K18, Krt19), and myoepithelial (α-SMA, Acta2), and possessed a high mitotic activity (38-62% Ki67 cells) with a large epithelial progenitor population (∼70% K14 cells). The P-EGF encapsulation strikingly upregulated the expression of pro-acinar AQP5 cells through culture time when compared to others (B-EGF, PBS). Thus, the utilization of Nicotiana benthamiana in molecular farming can produce EGF biologicals amenable to encapsulation in HA/Alg-based in vitro platforms, which can effectively and promptly induce the biofabrication of exocrine gland organoids.

Plant-made antibody against miroestrol: a new platform for expression of full-length immunoglobulin G against small-molecule targets in immunoassays.

KEY MESSAGE: Plant expression platform is the new source of immunoglobulin G (IgG) toward small low-molecular-weight targets. The plant-made monoclonal antibody-based immunoassay exhibits comparable analytical performance with hybridoma antibody. Immunoassays for small molecules are efficiently applied for monitoring of serum therapeutic drug concentration, food toxins, environmental contamination, etc. Immunoglobulin G (IgG) is usually produced using hybridoma cells, which requires complicated procedures and expensive equipment. Plants can act as alternative and economic hosts for IgG production. However, the production of free hapten (low-molecular-weight target)-recognizing IgG from plants has not been successfully developed yet. The current study aimed at creating a plant platform as an affordable source of IgG for use in immunoassays and diagnostic tools. The functional IgG was expressed in Nicotiana benthamiana leaves infiltrated with Agrobacterium tumefaciens strain GV3101 with recombinant geminiviral vectors (pBY3R) occupying chimeric anti-miroestrol IgG genes. The appropriate assembly between heavy and light chains was achieved, and the yield of expression was 0.57 µg/g fresh N. benthamiana leaves. The binding characteristics of the IgG to miroestrol and binding specificity to related compounds, such as isomiroestrol and deoxymiroestrol, were similar to those of hybridoma-produced IgG (monoclonal antibody, mAb). The plant-based mAbs exhibited high sensitivity for miroestrol (IC50, 23.2 ± 2.1 ng/mL), precision (relative standard deviation ≤ 5.01%), and accuracy (97.8-103% recovery), as determined using quantitative enzyme-linked immunosorbent assay. The validated enzyme-linked immunosorbent assay was applicable to determine miroestrol in plant samples. Overall, the plant-produced functional IgG conserved the binding activity and specificity of the parent IgG derived from mammalian cells. Therefore, the plant expression system may be an efficient and affordable platform for the production of antibodies against low-molecular-weight targets in immunoassays.

Correction to: Enzyme-linked immunosorbent assay for the quantitative/qualitative analysis of plant secondary metabolites.

The article Enzyme-linked immunosorbent assay for the quantitative/qualitative analysis of plant secondary metabolites, written by Seiichi Sakamoto, Waraporn Putalun, Sornkanok Vimolmangkang, Waranyoo Phoolcharoen, Yukihiro Shoyama, Hiroyuki Tanaka and Satoshi Morimoto, was originally published electronically on the publisher's internet portal (currently SpringerLink) on 21 November 2017 without open access.

Enzyme-linked immunosorbent assay for the quantitative/qualitative analysis of plant secondary metabolites.

Immunoassays are antibody-based analytical methods for quantitative/qualitative analysis. Since the principle of immunoassays is based on specific antigen-antibody reaction, the assays have been utilized worldwide for diagnosis, pharmacokinetic studies by drug monitoring, and the quality control of commercially available products. Berson and Yalow were the first to develop an immunoassay, known as radioimmunoassay (RIA), for detecting endogenous plasma insulin [1], a development for which Yalow was awarded the Nobel Prize in Physiology or Medicine in 1977. Even today, after half a century, immunoassays are widely utilized with some modifications from the originally proposed system, e.g., radioisotopes have been replaced with enzymes because of safety concerns regarding the use of radioactivity, which is referred to as enzyme immunoassay/enzyme-linked immunosorbent assay (ELISA). In addition, progress has been made in ELISA with the recent advances in recombinant DNA technology, leading to increase in the range of antibodies, probes, and even systems. This review article describes ELISA and its applications for the detection of plant secondary metabolites.

Rapid Transient Production of a Monoclonal Antibody Neutralizing the Porcine Epidemic Diarrhea Virus (PEDV) in Nicotiana benthamiana and Lactuca sativa.

Porcine epidemic diarrhea virus (PEDV) causes acute diarrhea, vomiting, dehydration, weight loss, and high mortality rate in neonatal piglets. Porcine epidemic diarrhea (PED) has been reported in Europe, America, and Asia including Thailand. The disease causes substantial losses to the swine industry in many countries. Presently, there is no effective PEDV vaccine available. In this study, we developed a plant-produced monoclonal antibody (mAb) 2C10 as a prophylactic candidate to prevent the PEDV infection. Recently, plant expression systems have gained interest as an alternative for the production of antibodies because of many advantages, such as low production cost, lack of human and animal pathogen, large scalability, etc. The 2C10 mAb was transiently expressed in Nicotiana benthamiana and lettuce using geminiviral vector. After purification by protein A affinity chromatography, the antibody was tested for the binding and neutralizing activity against PEDV. Our result showed that the plant produced 2C10 mAb can bind to the virus and also inhibit PEDV infection in vitro. These results show excellent potential for a plant-expressed 2C10 as a PEDV prophylaxis and a diagnostic for PEDV infection.

Molecular analysis of Vitex species using candidate DNA barcoding and PCR-RFLP of the matK gene for authentication of Vitex glabrata.

Several species of the Vitex genus, family Lamiaceae, are used in folk medicine for a variety of remedies. V. glabrata is unique among Vitex species because its main effect is sexual enhancement. However, crude drugs derived from different Vitex species might not be easily distinguishable, which could lead to their misidentification and misuse. Therefore, the accurate authentication of V. glabrata is critical for its effective medicinal use. In this study, the matK gene and the psbA-trnH intergenic spacer candidate DNA barcodes were sequenced and analyzed to identify five different Vitex species that are medicinally used in Thailand: V. negundo, V. trifolia, V. rotundifolia, V. limonifolia, and V. glabrata. Each region was successfully amplified from the leaves of the five species using a single set of primers, and the sequences determined. The size difference in PCR products of psbA-trnH and PCR restriction fragment length polymorphism (PCR-RFLP) of the matK gene sequences were used to differentiate V. glabrata from other Vitex species. These results indicate both the matK gene and the psbA-trnH intergenic spacer as candidate DNA barcodes of Vitex species and suggest that the difference of psbA-trnH PCR products and PCR-RFLP analysis based on the matK gene are effective for the authentication of V. glabrata.

Antioxidant, antimicrobial and wound healing activities of Boesenbergia rotunda.

The ethanolic extract of Boesenbergia rotunda (L.) Mansf was studied for its wound-healing potential. Since wound healing is interrelated with microbial infection and reactive oxygen species (ROS), this study was conducted to evaluate the antimicrobial and antioxidant activity of B. rotunda. The antimicrobial activity of B. rotunda was studied against six bacterial and two yeast strains using disc diffusion, minimum inhibitory concentration (MIC), and minimum microbicidal concentration (MMC). The B. rotunda extract displayed potential antimicrobial and antifungal activities by inhibiting the Gram-positive bacteria Staphylococcus aureus (ATCC 25923), S. epidermidis, and Bacillus subtilis (ATCC 6633), and the yeasts Candida albicans (ATCC 10231), and Saccharomyces cerevisiae. MIC and MMC values varied from 0.04 to 25 mg/mL and from 0.16 to 25 mg/mL, respectively. The antioxidant activity of B. rotunda was evaluated by measuring the Ferric Reducing/Antioxidant Power (FRAP) and DPPH free radical scavenging activity. The FRAP and DPPH values were 22.2 microM/microg and 76.3 mg/mL, respectively. In the wound-healing studies, the topical application of the B. rotunda extract indicated a significantly increased percentage of wound contraction on day 12 compared with the control group. Histological studies showed the complete epidermis and found collagen fibers and hair follicles in the dermis. The results of the present study support the continued and expanded utilization of B. rotunda in Thai folk medicine.