An overview of the current status of engineered therapeutic monoclonal antibodies

Since the commercialization of the first therapeutic monoclonal antibody (mAb) product in 1986, this class of biopharmaceutical products have grown significantly. Due to the enhanced antigen binding and reduced cellular toxicity, they result in more efficacy in treatment of variety of diseases. The global sales of mAbs which was 95.1 b$in 2017 have grown annually due to the dramatic increase in cancer and severe diseases rates and are estimated to reach 131.33 b$ by 2023, this represents a clear accelerating trend with more than 5.53% growth. In this review, we discuss some of these mAbs which have been approved by the FDA as well as others that are experiencing or being evaluated in clinical phases. Global sales of some monoclonal antibodies in 2016 are also considered, suggesting a significant increase in sales of mAbs over the years ahead. &nbsp

Isolation and characterization of novel phage displayed scFv antibody for human tumor necrosis factor alpha and molecular docking analysis of their interactions

Introduction: Tumor necrosis factor alpha (TNF-α) expression amplifies to excess amounts in several disorders such as rheumatoid arthritis and psoriasis. Although, Anti-TNF biologics have revolutionized the treatment of these autoimmune diseases, formation of anti-drug antibodies (ADA) has dramatically affected their use. The next generation antibodies (e.g. Fab, scFv) have not only reduced resulted immunogenicity, but also proved several benefits including better tumor penetration and more rapid blood clearance.This study highlights the use of phage display for identification of human single chain fragment antibody against disulfide-bonded TNF-α using phage display technology. Methods and Results: Using affinity selection procedures in this study, a scFv antibody clone was isolated from naïve Tomlinson I phage display library that specifically recognizes and binds to TNF-α. The TNF-α recombinant protein was expressed in genetically engineered Escherichia coli SHuffle® T7 Express, for the first time, which is able to express disulfide-bonded recombinant proteins into their correctly folded states. Conclusions: ELISA-based affinity characterization results indicated that the isolated novel 29.2 kDa scFv binds TNF-α with suitable affinity. In silico homology modeling study using ‘ModWeb’ as well as molecular docking study using Hex program confirmed the scFv and TNF-α interactions with a scFv-TNF- α binding energy of around -593 kj/mol which is well in agreement with our ELSIA results. The cloned scFv antibody may potentially be useful for research and therapeutic applications in the future

Use of a recombinant protein for development of a DAS-ELISA serological kit for sensitive detection of witches' broom disease of lime

Abstract Witches' broom disease of lime (WBDL), associated with 'Candidatus Phytoplasma aurantifolia' is the most devastating disease of acid lime in Southern Iran. Lack of an efficient approach for control of the disease has resulted in application of quarantine measures for protection of healthy plants and to limit the spread of disease to uninfected areas. Toward this aim, development of a rapid and efficient method for detection of infected plants is a major focus. The present study introduces application of recombinant DNA technology for development of a sensitive serological technique (DAS-ELISA) for detection of infected plants. The immunodominant membrane protein (IMP), as a major protein present on the surface of phytoplasma cells, was selected as a target for generating specific antibody molecules. The gene encoding IMP of 'Ca. P. aurantifolia' was obtained from infected plants. The region encoding the IMP fragment was isolated by PCR amplification followed by insertion into the pZ57R/T cloning vector. Intact clones containing the right sequences were selected and sub-cloned into the pET28a bacterial expression vector. Large scale expression of recombinant protein was performed in E. coli and purification was carried out through affinity chromatography in Ni-agarose columns. To obtain specific polyclonal antibodies against WBDL, the purified recombinant IMP was used for rabbit immunization. The antisera titer was determined after each boosting via indirect ELISA. When the titer reached 1:100,000, the animal was sacrificed, blood was collected and serum was separated from blood cells. The IgG molecules were purified from serum content by affinity chromatography using protein A columns followed by conjugation to alkaline phosphatase (AP) enzyme. The purified specific antibodies and conjugate were used for detection of the corresponding antigen, IMP in infected plants in DAS-ELISA and dot-blot methods. The results confirmed the capability of this technique for efficient detection of infected plants, while no reaction was observed in negative controls. The detection limit of the DAS-ELISA method was determined at 70 µg IMP/ml leaf extract

Production of a phage-displayed single chain variable fragment antibody against infectious bursal disease virus

Purpose: To develop specific single chain variable fragments (scFv) against infectious bursal disease virus (IBDV) via phage display technology.Methods: Purified viruses were initially applied for iterative panning rounds of scFv phage display libraries. The binding ability of the selected scFv antibody fragments against the IBDV particles was analyzed by indirect enzyme-linked immunosorbent assay (ELISA) followed by blotting assays. Threedimensional (3D) structure of the selected scFv antibody fragment and VP3 protein were predicted through in silico analysis. Structural characterization of the antibody-antigen complexes was carried out by computational docking analysis.Results: The serological results obtained from the ELISA and blotting analysis showed that the selected clones produced specific scFv antibody fragments that were capable of effectively detecting infectious bursal disease (IBD) in the infected animal tissue. Biodiversity analysis by BstNI finger printing and nucleotide sequencing revealed that there was no major difference in nucleotide sequences of the selected clones. Further analysis demonstrated that this recombinant fragment of the antibody was able to bind to VP3 structural protein of IBDV with a molecular weight of ~30 kDa. Molecular docking results revealed that the binding energy of scFv to IBDV-VP3 was 545 kj/mol.Conclusion: The developed scFv antibody fragments possess great potentials for the diagnosis of IBD. The findings of the present study confirm the feasibility of using phage display technology for rapid production of antibodies against IBD diseases by applying naïve scFv libraries. Keywords: Antibody, Molecular docking, Phage display technology, Single chain variable fragment

Development of recombinant antibody mediated resistance against Tomato yellow leaf curl virus

In this study, we exploited the expression of specific scFv fragment in plant cells for suppression of disease symptoms caused by TYLCV infection. The C1, V1 and V2 genes encoding Rep, CP and MP, respectively, were cloned into the TOPO vector and subsequently into pGEX-5x3 and pMALc2x expression vectors. Recombinant proteins were expressed in E. coli as C-terminal fusion with GST or MBP and purified proteins obtained by affinity chromatography method. In addition, the amino terminal part of CP and Rep proteins were also cloned and expressed as fusion proteins with GST and MBP. Using phage display technology one scFv fragments against Rep (scFv-ScRep1) was isolated through panning of naïve Tomlinson I scFv phage library. In addition, an ARep phage display library constructed from total spleen RNA of a mouse immunized with MBP-Rep was analyzed by panning and another scFv fragment (scFv-ScRep2) was selected and characterized. The scFv fragments were expressed in pHENHI bacterial expression vector and purified by immobilized metal affinity chromatography. ELISA and Western blot analyses were used to analyze binding activities of bacterially expressed scFv. The observed high binding activity of bacterially expressed scFv-ScRep1 to both full length and C- terminal truncated of Rep protein indicated specific binding to the amino terminal end of Rep while scFv-ScRep2 interacted exclusively to intact Rep protein. Four more scFv fragments were developed from total RNA of murine hybridoma cells secreting specific MAbs against TYLCV virions. The pTRAkt plant expression vector was used to clone the generated scFv fragment genes individually and/or as an amino terminal fusion to GFP. Expression of scFv fragment constructs and their functionality within transiently transformed plant cells were analyzed. Blotting analyses showed detectable amounts of scFv-ScRep1, scFv-ScRep2, and NLS-scFv-ScRep1 presented in crude leaf extract of transformed plants. Further analyses proved binding ability of these scFv extracted from leaves against recombinant Rep. Fluorescence microscopy results confirmed expression and localization of scFv-ScRep1-GFP, scFv-ScRep2-GFP and scFv-NLS-ScRep1-GFP fusion protein within the cytoplasm and nucleus. Selected constructs were also used to generate stable transformations in entire N. benthamiana plants through leaf disc transformation. To determine the protection ability of transgenically expressed proteins, independent T0 progenies expressing different scFvs were challenged with pBIN19-2TYLCV-Ir construct. Early symptoms including leaf curling and size reduction of newly emerged leaves were observed on non-transgenic and sensitive transgenic plants 3-4 weeks after inoculation. Southern hybridization analyses confirmed reduction or complete suppression of viral DNA replication in the symptomless plants. Assessment of the resistance status within T0 inoculated plants was evaluated 5 weeks after inoculation. Infectivity assays of T0 progenies revealed that all inoculated NSR, HSC2, HSC3 and HSC4 lines as well as wild type plants are susceptible for the TYLCV challenge inoculation. However, SRG, SR and RW lines presented varying degrees of resistance from 8-28 percent. Inoculated plants were observed for disease symptoms development and assayed for presence of TYLCV DNA through molecular hybridization methods. Early symptoms in non-transgenic and sensitive plants appeared 2-3 wpi and developed further during the next weeks. Resistance response was evaluated at 4-5 wpi bases of symptom observation and DNA hybridization assays. A heterogeneous response was obtained within inoculated T1 progenies. These results indicated that all T1 progenies raised from RW14, RW22 and SR27 reveal typical TYLCV symptoms whereas SRG T1 plants expressing ScRep1-GFP recombinant protein showed a spectrum of symptoms ranging from a severely diseased to mild ones and the complete absence of any symptoms. The resistance phenotype was characterised by absence or remarkable reducing of disease symptoms and a concomitant substantial reduction or complete suppression of viral DNA replication. T1 plants developed from SRG28 and SRG18 lines revealed highest resistance. Further analyses indicated that individual SRG transgenic plants emit varying intensity of fluorescence under excitation by UV light. Usually, elevated amounts of ScRep1-GFP transcripts are directly correlated with higher fluorescence intensity emitted from transgenic lines. Together comparative Q-PCR and fluorescence intensity analyses with data obtained from virus resistant assays indicated that mostly plants with higher transcripts level consistently exhibited a higher degree of virus resistance

Development of recombinant antibody mediated resistance against Tomato yellow leaf curl virus

In this study, we exploited the expression of specific scFv fragment in plant cells for suppression of disease symptoms caused by TYLCV infection. The C1, V1 and V2 genes encoding Rep, CP and MP, respectively, were cloned into the TOPO vector and subsequently into pGEX-5x3 and pMALc2x expression vectors. Recombinant proteins were expressed in E. coli as C-terminal fusion with GST or MBP and purified proteins obtained by affinity chromatography method. In addition, the amino terminal part of CP and Rep proteins were also cloned and expressed as fusion proteins with GST and MBP. Using phage display technology one scFv fragments against Rep (scFv-ScRep1) was isolated through panning of naïve Tomlinson I scFv phage library. In addition, an ARep phage display library constructed from total spleen RNA of a mouse immunized with MBP-Rep was analyzed by panning and another scFv fragment (scFv-ScRep2) was selected and characterized. The scFv fragments were expressed in pHENHI bacterial expression vector and purified by immobilized metal affinity chromatography. ELISA and Western blot analyses were used to analyze binding activities of bacterially expressed scFv. The observed high binding activity of bacterially expressed scFv-ScRep1 to both full length and C- terminal truncated of Rep protein indicated specific binding to the amino terminal end of Rep while scFv-ScRep2 interacted exclusively to intact Rep protein. Four more scFv fragments were developed from total RNA of murine hybridoma cells secreting specific MAbs against TYLCV virions. The pTRAkt plant expression vector was used to clone the generated scFv fragment genes individually and/or as an amino terminal fusion to GFP. Expression of scFv fragment constructs and their functionality within transiently transformed plant cells were analyzed. Blotting analyses showed detectable amounts of scFv-ScRep1, scFv-ScRep2, and NLS-scFv-ScRep1 presented in crude leaf extract of transformed plants. Further analyses proved binding ability of these scFv extracted from leaves against recombinant Rep. Fluorescence microscopy results confirmed expression and localization of scFv-ScRep1-GFP, scFv-ScRep2-GFP and scFv-NLS-ScRep1-GFP fusion protein within the cytoplasm and nucleus. Selected constructs were also used to generate stable transformations in entire N. benthamiana plants through leaf disc transformation. To determine the protection ability of transgenically expressed proteins, independent T0 progenies expressing different scFvs were challenged with pBIN19-2TYLCV-Ir construct. Early symptoms including leaf curling and size reduction of newly emerged leaves were observed on non-transgenic and sensitive transgenic plants 3-4 weeks after inoculation. Southern hybridization analyses confirmed reduction or complete suppression of viral DNA replication in the symptomless plants. Assessment of the resistance status within T0 inoculated plants was evaluated 5 weeks after inoculation. Infectivity assays of T0 progenies revealed that all inoculated NSR, HSC2, HSC3 and HSC4 lines as well as wild type plants are susceptible for the TYLCV challenge inoculation. However, SRG, SR and RW lines presented varying degrees of resistance from 8-28 percent. Inoculated plants were observed for disease symptoms development and assayed for presence of TYLCV DNA through molecular hybridization methods. Early symptoms in non-transgenic and sensitive plants appeared 2-3 wpi and developed further during the next weeks. Resistance response was evaluated at 4-5 wpi bases of symptom observation and DNA hybridization assays. A heterogeneous response was obtained within inoculated T1 progenies. These results indicated that all T1 progenies raised from RW14, RW22 and SR27 reveal typical TYLCV symptoms whereas SRG T1 plants expressing ScRep1-GFP recombinant protein showed a spectrum of symptoms ranging from a severely diseased to mild ones and the complete absence of any symptoms. The resistance phenotype was characterised by absence or remarkable reducing of disease symptoms and a concomitant substantial reduction or complete suppression of viral DNA replication. T1 plants developed from SRG28 and SRG18 lines revealed highest resistance. Further analyses indicated that individual SRG transgenic plants emit varying intensity of fluorescence under excitation by UV light. Usually, elevated amounts of ScRep1-GFP transcripts are directly correlated with higher fluorescence intensity emitted from transgenic lines. Together comparative Q-PCR and fluorescence intensity analyses with data obtained from virus resistant assays indicated that mostly plants with higher transcripts level consistently exhibited a higher degree of virus resistance

Genetic Diversity Assessment in Several Barley (Hordeum vulgare L.) Cultivars Using Microsatellite Markers

In the present study, genetic diversity in seven cultivars of cultivated barley (Hordeum vulgare) populations was evaluated using 10 microsatellite markers. Genomic DNA was extracted from fresh leaves and amplification reactions were done by PCR. The amplification products were separated on 6% denaturing polyacrylamide gels containing 7M urea and visualized via silver staining method. High level of polymorphism was observed among populations. Polymorphic bands ranged from 100 to 300 bp. Altogether 65 alleles were observed among all genotypes, with an average of 9.2 alleles per locus for all loci. Polymorphic information content (PIC) ranged from 0.80 to 0.88 with an average of 0.84. Sahand populations showed the lowest mean of gene diversity whereas the highest mean of heterozygosity observed in Rayhan populations that can prepare a powerful resource of genetic diversity for breeding programs. The genotypes were clustered using unweight pair-group method on arithmetic average by POPGEN32 software. The dendrogram discriminated all the genotypes in several groups. The results showed that SSR markers have a high ability to reveal most of the information in a single locus and can be used for genetic analysis in molecular levels determination of genetic similarity and clustering barley cultivars

Antibody-mediated resistance against plant pathogens

Plant diseases have a significant impact on the yield and quality of crops. Many strategies have been developed to combat plant diseases, including the transfer of resistance genes to crops by conventional breeding. However, resistance genes can only be introgressed from sexually-compatible species, so breeders need alternative measures to introduce resistance traits from more distant sources. In this context, genetic engineering provides an opportunity to exploit diverse and novel forms of resistance, e.g. the use of recombinant antibodies targeting plant pathogens. Native antibodies, as a part of the vertebrate adaptive immune system, can bind to foreign antigens and eliminate them from the body. The ectopic expression of antibodies in plants can also interfere with pathogen activity to confer disease resistance. With sufficient knowledge of the pathogen life cycle, it is possible to counter any disease by designing expression constructs so that pathogen-specific antibodies accumulate at high levels in appropriate sub-cellular compartments. Although first developed to tackle plant viruses and still used predominantly for this purpose, antibodies have been targeted against a diverse range of pathogens as well as proteins involved in plant-pathogen interactions. Here we comprehensively review the development and implementation of antibody-mediated disease resistance in plants