Recent progress in generating intracellular functional antibody fragments to target and trace cellular components in living cells

Kaiser, Philipp; Maier, Julia; Traenkle, Bjoern; Emele, Felix Emele; Rothbauer, Ulrich

doi:10.1016/j.bbapap.2014.04.019

Cited by 73 publications

(62 citation statements)

References 114 publications

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“…Over the last few years, genetically encoded protein binders have been introduced to basic biological research and provide novel means for protein manipulation in vivo. While protein function was largely studied by genetic manipulation at the DNA or RNA levels in the past, protein binders allow direct, specific and acute modification and interference of protein function in vivo (Kaiser et al, 2014; Bieli et al, 2016) and might therefore represent valid tools to study protein localization.…”

Section: Introductionmentioning

confidence: 99%

A nanobody-based toolset to investigate the role of protein localization and dispersal in Drosophila

Harmansa

Alborelli

Bieli

et al. 2017

eLife

121

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The role of protein localization along the apical-basal axis of polarized cells is difficult to investigate in vivo, partially due to lack of suitable tools. Here, we present the GrabFP system, a collection of four nanobody-based GFP-traps that localize to defined positions along the apical-basal axis. We show that the localization preference of the GrabFP traps can impose a novel localization on GFP-tagged target proteins and results in their controlled mislocalization. These new tools were used to mislocalize transmembrane and cytoplasmic GFP fusion proteins in the Drosophila wing disc epithelium and to investigate the effect of protein mislocalization. Furthermore, we used the GrabFP system as a tool to study the extracellular dispersal of the Decapentaplegic (Dpp) protein and show that the Dpp gradient forming in the lateral plane of the Drosophila wing disc epithelium is essential for patterning of the wing imaginal disc.DOI: http://dx.doi.org/10.7554/eLife.22549.001

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Section: Introductionmentioning

confidence: 99%

A nanobody-based toolset to investigate the role of protein localization and dispersal in Drosophila

Harmansa

Alborelli

Bieli

et al. 2017

eLife

121

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“…In the following section, the main applications in developmental biology are briefly described and classified. For a more thorough description of these applications and studies in cultured cells, we refer the reader to several recent reviews covering this topic (Aguilar, Matsuda, Vigano, & Affolter, ; Beghein & Gettemans, ; Bieli et al, ; Harmansa & Affolter, ; Helma, Cardoso, Muyldermans, & Leonhardt, ; Kaiser, Maier, Traenkle, Emele, & Rothbauer, ; Pluckthun, ).…”

Section: Overview Of Protein Binder Applications In Developmental Biomentioning

confidence: 99%

Reflections on the use of protein binders to study protein function in developmental biology

Aguilar

Viganò

Affolter

et al. 2019

WIREs Developmental Biology

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Studies in the field of developmental biology aim to unravel how a fertilized egg develops into an adult organism and how proteins and other macromolecules work together during this process. With regard to protein function, most of the developmental studies have used genetic and RNA interference approaches, combined with biochemical analyses, to reach this goal. However, there always remains much room for interpretation on how a given protein functions, because proteins work together with many other molecules in complex regulatory networks and it is not easy to reveal the function of one given protein without affecting the networks. Likewise, it has remained difficult to experimentally challenge and/or validate the proposed concepts derived from mutant analyses without tools that directly manipulate protein function in a predictable manner. Recently, synthetic tools based on protein binders such as scFvs, nanobodies, DARPins, and others have been applied in developmental biology to directly manipulate target proteins in a predicted manner. Although such tools would have a great impact in filling the gap of knowledge between mutant phenotypes and protein functions, careful investigations are required when applying functionalized protein binders to fundamental questions in developmental biology. In this review, we first summarize how protein binders have been used in the field, and then reflect on possible guidelines for applying such tools to study protein functions in developmental biology.This article is categorized under:Technologies > Analysis of ProteinsEstablishment of Spatial and Temporal Patterns > GradientsInvertebrate Organogenesis > Flies

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“…80 A second technique to enhance cellular delivery, similar to cationization-enhanced adsorptive-mediated transcytosis across the blood-brain barrier (BBB), is to increase protein net positive charge such as that described for +36 GFP-mediated small interfering RNA (siRNA) delivery. 81,82 Of particular interest is a subset of lupus autoantibodies reacting against host DNA, which can penetrate living cells, even localizing in the nucleus. A particular lupus antidouble-stranded DNA (dsDNA) antibody 3E10, which unusually is not harmful to cells, was isolated from a mouse model of systemic lupus erythematosus and penetrated cells via an equilibrative nucleoside salvage transporter, which is ubiquitous on human cells.…”

Section: Intracellular Deliverymentioning

confidence: 99%

“…25 ScFv can be problematic as intrabodies because disulfide bonds cannot form in the reducing environment of the cell, potentially resulting in incorrect folding, insolubility, and instability. 82,85 Intracellular antibody capture (IAC) technology enables direct selection of antibodies that can fold correctly and bind their target in the reducing environment of the cell cytoplasm. The method involves a modified two-hybrid system where antibody libraries are screened directly in yeast using an antibody-antigen interaction assay.…”

Section: Intracellular Deliverymentioning

confidence: 99%

“…While delivery of proteins themselves is difficult, nucleic acid expression vectors are being explored for direct expression in target cells. 82,86 Options include viral delivery, antibody-coupled vector delivery via antigen internalization, and liposome nanoparticles using antibodies for cell surface antigen targeting, with encapsulated single domains or vectors able to express them.…”

Section: Intracellular Deliverymentioning

confidence: 99%

See 1 more Smart Citation

New Horizons in Therapeutic Antibody Discovery: Opportunities and Challenges versus Small-Molecule Therapeutics

Smith¹

2015

SLAS Discovery

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Antibody drugs have become an increasingly significant component of the therapeutic landscape. Their success has been driven by some of their unique properties, in particular their very high specificity and selectivity, in contrast to the offtarget liabilities of small molecules (SMs). Antibodies can bring additional functionality to the table with their ability to interact with the immune system, and this can be further manipulated with advances in antibody engineering. This review summarizes what antibody therapeutics have achieved to date and what opportunities and challenges lie ahead. The target landscape for large molecules (LMs) versus SMs and some of the challenges for antibody drug development are discussed. Effective penetration of membrane barriers and intracellular targeting is one challenge, particularly across the highly resistant blood-brain barrier. The expanding pipeline of antibody-drug conjugates offers the potential to combine SM and LM modalities in a variety of creative ways, and antibodies also offer exciting potential to build bi-and multispecific molecules. The ability to pursue more challenging targets can also be further exploited but highlights the need for earlier screening in functional cell-based assays. I discuss how this might be addressed given the practical constraints imposed by high-throughput screening sample type and process differences in antibody primary screening.

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Recent progress in generating intracellular functional antibody fragments to target and trace cellular components in living cells

Cited by 73 publications

References 114 publications

A nanobody-based toolset to investigate the role of protein localization and dispersal in Drosophila

A nanobody-based toolset to investigate the role of protein localization and dispersal in Drosophila

Reflections on the use of protein binders to study protein function in developmental biology

New Horizons in Therapeutic Antibody Discovery: Opportunities and Challenges versus Small-Molecule Therapeutics

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