Selection of Functional Intracellular Nanobodies

Woods, James H.

doi:10.1177/2472555219853235

Cited by 17 publications

(13 citation statements)

References 74 publications

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“…Such frameworks may be selected antibody scaffolds that are functional even in the absence of disulfide bonds [ 90 ] or non-antibody scaffolds [ 35 , 36 ]. The properties of nanobodies render them more suitable for expression in the cytosol [ 91 ], but various tendencies for aggregation may still occur [ 92 , 93 ]. Non-antibody scaffolds may also not always be functional; only 10–20% of a fibronectin-derived alternative scaffold called FingR (Fibronectin intrabody generated with messenger RNA display) that was target-specific in vitro also co-localized with its target intracellularly [ 94 ].…”

Section: Strategies To Use Antibodies Inside Living Cellsmentioning

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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Section: Strategies To Use Antibodies Inside Living Cellsmentioning

confidence: 99%

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

et al. 2020

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“…In terms of selecting nanobody phage libraries, the process is similar to the phage display libraries used to select for antibodies. Interestingly, these nanobody libraries have recently been used to select for cell surface receptors in situ as well as intracellular targets [62,63], which does open up possibilities for selecting nanobodies against unknown targets on particular sub-populations of cells. Additionally, nanobodies have been generated that are cross-reactive to human and murine proteins [64] or all Trypanosoma species [65].…”

Section: Towards a Better System Of Detecting Proteome Signaturesmentioning

confidence: 99%

Antibodies, Nanobodies, or Aptamers—Which Is Best for Deciphering the Proteomes of Non-Model Species?

Dhar

Samarasinghe

Shigdar

2020

IJMS

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This planet is home to countless species, some more well-known than the others. While we have developed many techniques to be able to interrogate some of the “omics”, proteomics is becoming recognized as a very important part of the puzzle, given how important the protein is as a functional part of the cell. Within human health, the proteome is fairly well-established, with numerous reagents being available to decipher cellular pathways. Recent research advancements have assisted in characterizing the proteomes of some model (non-human) species, however, in many other species, we are only just touching the surface. This review considers three main reagent classes—antibodies, aptamers, and nanobodies—as a means of continuing to investigate the proteomes of non-model species without the complications of understanding the full protein signature of a species. Considerations of ease of production, potential applications, and the necessity for producing a new reagent depending on homology are presented.

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“…Several strategies allow to target and manipulate POIs in vivo via the use of protein binders. Binders against proteins can be isolated using existing platforms and/or libraries, functionalized in a desired manner and expressed in cells or organisms upon transfection, viral transduction or from transgenes inserted into the genome (Dong et al, 2019;Dreier and Pluckthun, 2012;Fridy et al, 2014;McMahon et al, 2018;Moutel et al, 2016;Roder et al, 2017;Woods, 2019). Alternatively, binders against fluorescent tags can be used to manipulate a POI that has been fused to a fluorescent protein (FP).…”

Section: Introductionmentioning

confidence: 99%

Protein manipulation using single copies of short peptide tags in cultured cells and in Drosophila melanogaster

et al. 2021

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Cellular development and function rely on highly dynamic molecular interactions among proteins distributed in all cell compartments. Analysis of these interactions has been one of the main topics in cellular and developmental research and has been mostly achieved by the manipulation of proteins of interest (POIs) at the genetic level. Although genetic strategies significantly contributed to our current understanding, targeting specific interactions of POIs in a time- and space-controlled manner or analyzing the role of POIs in dynamic cellular processes such as cell migration or cell division would profit from more direct approaches. The recent development of specific protein binders, which can be expressed and function intracellularly, along with advancement in synthetic biology, have contributed to the creation of a new toolbox for direct protein manipulations. Here, we selected a number of short tag epitopes for which protein binders from different scaffolds have been generated and showed that single copies of these tags allowed efficient POIs binding and manipulation in living cells. Using Drosophila, we also find that single short tags can be utilized for POI manipulation in vivo.

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Selection of Functional Intracellular Nanobodies

Cited by 17 publications

References 74 publications

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

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

Antibodies, Nanobodies, or Aptamers—Which Is Best for Deciphering the Proteomes of Non-Model Species?

Protein manipulation using single copies of short peptide tags in cultured cells and in Drosophila melanogaster

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