Phage and Yeast Display

Sheehan, Jared; Marasco, Wayne A.

doi:10.1128/microbiolspec.aid-0028-2014

Cited by 51 publications

(18 citation statements)

References 118 publications

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“…More recently, in vitro production of mAbs revolutionized the way to isolate and produce mAbs and, to date, these methods represent the default way to perform screenings against new targets 3 . The most powerful way to isolate recombinant mAbs is the application of phage/yeast display of single-chain variable fragment (scFv) libraries 4 . These systems allow, potentially, the identification of specific scFv fusions for any target of interest, from a huge scFv repertoire, through biopanning.…”

Section: Introductionmentioning

confidence: 99%

A long non-coding SINEUP RNA boosts semi-stable production of fully human monoclonal antibodies in HEK293E cells

Sasso

Latino

Froechlich

et al. 2018

mAbs

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Use of monoclonal antibodies is emerging as a highly promising and fast-developing scenario for innovative treatment of viral, autoimmune and tumour diseases. The search for diagnostic and therapeutic antibodies currently depends on in vitro screening approaches, such as phage and yeast display technologies. Antibody production still represents a critical step for preclinical and clinical evaluations. Accordingly, improving production of monoclonal antibodies represents an opportunity, to facilitate downstream target validations. SINEUP RNAs are long non-coding transcripts, possessing the ability to enhance translation of selected mRNAs. We applied SINEUP technology to semi-stable production of monoclonal antibodies in HEK293E cells, which allows for episomal propagation of the expression vectors encoding the heavy and light chains of IgGs. Co-expression of SINEUP RNA with mRNAs encoding heavy and light chains of IgG4s was able to increase the production of different anti-CLDN1 antibodies up to three-fold. Improved production of monoclonal antibodies was achieved both in transiently transfected HEK293E cells and in cellular clones with stable expression of the SINEUP. Compared to antibody preparations obtained under standard conditions, the anti-CLDN1 IgG4s produced in the presence of the SINEUP transcript showed unaltered post-translational modifications, and retained the ability to recognize their target. We thus propose SINEUP technology as a valuable tool to enhance semi-stable antibody production in human cell lines.

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Section: Introductionmentioning

confidence: 99%

A long non-coding SINEUP RNA boosts semi-stable production of fully human monoclonal antibodies in HEK293E cells

Sasso

Latino

Froechlich

et al. 2018

mAbs

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“…We are building a platform that takes advantage of fundamental molecular biology principles to enable the rapid identification of specific Abs. Our screening platform is being developed with single-chain variable fragment Abs (scFvs) but the technology is applicable to other scaffolds, including both Fabs (4) and yeast display libraries (12,(25)(26)(27). Here, we describe a vector system that enables high-throughput conversion of Ab fragments to full length immunoglobulin G (IgG) molecules that can be directly validated in standard immunoassays.…”

Section: Discussionmentioning

confidence: 99%

pMINERVA: A donor–acceptor system for the in vivo recombineering of scFv into IgG molecules

Batonick

Kiss

Fuller

et al. 2016

Journal of Immunological Methods

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Phage display is the most widely used method for selecting binding molecules from recombinant antibody libraries. However, validation of the phage antibodies often requires early production of the cognate full-length immunoglobulin G (IgG). The conversion of phage library outputs to a full immunoglobulin via standard subcloning is time-consuming and limits the number of clones that can be evaluated. We have developed a novel system to convert scFvs from a phage display vector directly into IgGs without any in vitro subcloning steps. This new vector system, named pMINERVA, makes clever use of site-specific bacteriophage integrases that are expressed in E. coli and intron splicing that occurs within mammalian cells. Using this system, a phage display vector contains both bacterial and mammalian regulatory regions that support antibody expression in E. coli and mammalian cells. A single-chain variable fragment (scFv) antibody is expressed on the surface of bacteriophage M13 as a genetic fusion to the gpIII coat protein. The scFv is converted to an IgG that can be expressed in mammalian cells by transducing a second E. coli strain. In that strain, the phiC31 recombinase fuses the heavy chain constant domain from an acceptor plasmid to the heavy chain variable domain and introduces controlling elements upstream of the light chain variable domain. Splicing in mammalian cells removes a synthetic intron containing the M13 gpIII gene to produce the fusion of the light chain variable domain to the constant domain. We show that phage displaying a scFv and recombinant IgGs generated using this system are expressed at wild-type levels and retain normal function. Use of the pMINERVA completely eliminates the labor-intensive subcloning and DNA sequence confirmation steps currently needed to convert a scFv into a functional IgG Ab.

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“…Methods to generate binders are briefly summarized in Table 2. For more detailed information, interested readers are referred to reviews on technologies for generating binders, such as display technologies including phage display, yeast display, ribosome display and bacterial display [24][25][26][27][28][29]. Among the group of recombinant binders, there are differences in species and in biochemical properties, which might be relevant for their use as intrabodies or other purposes.…”

Section: Sources For Bindersmentioning

confidence: 99%

Applying Antibodies Inside Cells: Principles and Recent Advances in Neurobiology, Virology and Oncology

et al. 2020

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To interfere with cell function, many scientists rely on methods that target DNA or RNA due to the ease with which they can be applied. Proteins are usually the final executors of function but are targeted only indirectly by these methods. Recent advances in targeted degradation of proteins based on proteolysis-targeting chimaeras (PROTACs), ubiquibodies, deGradFP (degrade Green Fluorescent Protein) and other approaches have demonstrated the potential of interfering directly at the protein level for research and therapy. Proteins can be targeted directly and very specifically by antibodies, but using antibodies inside cells has so far been considered to be challenging. However, it is possible to deliver antibodies or other proteins into the cytosol using standard laboratory equipment. Physical methods such as electroporation have been demonstrated to be efficient and validated thoroughly over time. The expression of intracellular antibodies (intrabodies) inside cells is another way to interfere with intracellular targets at the protein level. Methodological strategies to target the inside of cells with antibodies, including delivered antibodies and expressed antibodies, as well as applications in the research areas of neurobiology, viral infections and oncology, are reviewed here. Antibodies have already been used to interfere with a wide range of intracellular targets. Disease-related targets included proteins associated with neurodegenerative diseases such as Parkinson's disease (α-synuclein), Alzheimer's disease (amyloid-β) or Huntington's disease (mutant huntingtin [mHtt]). The applications of intrabodies in the context of viral infections include targeting proteins associated with HIV (e.g. HIV1-TAT, Rev, Vif, gp41, gp120, gp160) and different oncoviruses such as human papillomavirus (HPV), hepatitis B virus (HBV), hepatitis C virus (HCV) and Epstein-Barr virus, and they have been used to interfere with various targets related to different processes in cancer, including oncogenic pathways, proliferation, cell cycle, apoptosis, metastasis, angiogenesis or neo-antigens (e.g. p53, human epidermal growth factor receptor-2 [HER2], signal transducer and activator of transcription 3 [STAT3], RAS-related RHO-GTPase B (RHOB), cortactin, vascular endothelial growth factor receptor 2 [VEGFR2], Ras, Bcr-Abl). Interfering at the protein level allows questions to be addressed that may remain unanswered using alternative methods. This review addresses why direct targeting of proteins allows unique insights, what is currently feasible in vitro, and how this relates to potential therapeutic applications.Therapeutic antibodies are valuable drugs, which mostly act outside of cells.Reaching the numerous drug targets that reside inside cells by antibodies is possible in vitro and allows unique insights compared with other methods.Applying antibodies inside cells for therapeutic purposes has been explored in animal models and promises specific therapeutic benefits in neurobiology, virology and oncology.

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Phage and Yeast Display

Cited by 51 publications

References 118 publications

A long non-coding SINEUP RNA boosts semi-stable production of fully human monoclonal antibodies in HEK293E cells

A long non-coding SINEUP RNA boosts semi-stable production of fully human monoclonal antibodies in HEK293E cells

pMINERVA: A donor–acceptor system for the in vivo recombineering of scFv into IgG molecules

Applying Antibodies Inside Cells: Principles and Recent Advances in Neurobiology, Virology and Oncology

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