Optimizing recombinant antibodies for intracellular function using hitchhiker-mediated survival selection

Waraho-Zhmayev, Dujduan; Meksiriporn, Bunyarit; Portnoff, Alyse D.; DeLisa, Matthew P.

doi:10.1093/protein/gzu038

Cited by 25 publications

(28 citation statements)

References 44 publications

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“…To date, isolating functional intrabodies is still a big challenge in academic and pharmaceutical researches. Besides the development of selection methods such as IACT, FLI‐TRAP, and the growth sensor which can be conducted directly in live cells, a suitable library with sufficiently high diversity and large library size is crucial. Indeed, the capabilities of IACT and FLI‐TRAP were maximized by developing SPLINT and ISELATE intrabody libraries, respectively .…”

Section: Discussionmentioning

confidence: 99%

A Suicide Switch Directly Eliminates Intracellular scFv Oligomers in the Cytoplasm of Mammalian Cells

Nguyen

Nagamune

Kawahara

2018

Biotechnology Journal

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As intracellular antibodies (intrabodies) are highly promising tools for drug discovery, an innovative antibody screening platform in mammalian cells was previously developed by a single-chain Fv (scFv)-c-kit growth sensor, which successfully selected rabies nucleoprotein and phosphoprotein-specific intrabodies from a synthetic scFv library. Since the scFv-c-kit growth sensor releases a growth signal after forming oligomers due to binding to an oligomeric antigen, it is critical to use a library which does not contain self-oligomeric scFvs to avoid the off-target signal of the growth sensor. Here, a novel method to eliminate self-oligomeric scFvs directly in the cytoplasm of mammalian cells is presented. A suicide switch by fusing an scFv with a cell-death signaling domain to eliminate scFv oligomers is developed. It is found that among four cell-death signaling domains, a suicide switch by fusing scFv with Fas-associated death domain (FADD) can selectively reduce oligomeric scFvs. Furthermore, the library after eliminating scFv oligomers results in higher efficiency in the intrabody selection platform with a growth sensor. Collectively, the scFv-FADD suicide switch can be applied to eliminate oligomeric scFvs from a library, which can consequently improve the quality of intracellular scFv libraries and accelerate the discovery of intrabodies in the future.

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Section: Discussionmentioning

confidence: 99%

A Suicide Switch Directly Eliminates Intracellular scFv Oligomers in the Cytoplasm of Mammalian Cells

Nguyen

Nagamune

Kawahara

2018

Biotechnology Journal

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“…The development of new tools for a successful delivery of Ab fragments into the brain and the design of fusion peptides/proteins for targeting them to the cells/cell compartments of interest might enhance immunotherapy efficacy [ 146 , 147 ]. Also, novel methods for optimizing the properties of Ab fragments such as affinity, stability and solubility merit further research [ 110 , 148 ]. Finally, application of elegant tools such as the use of cellular implants for controlled and continuous delivery might be of interest [ 149 , 150 ].…”

Section: Discussionmentioning

confidence: 99%

Recombinant Antibody Fragments for Neurodegenerative Diseases

Manoutcharian

Perez-Garmendia

Gevorkian

2017

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Background:Recombinant antibody fragments are promising alternatives to full-length immunoglobulins and offer important advantages compared with conventional monoclonal antibodies: extreme specificity, higher affinity, superior stability and solubility, reduced immuno-genicity as well as easy and inexpensive large-scale production.Objective:In this article we will review and discuss recombinant antibodies that are being evaluated for neurodegenerative diseases in pre-clinical models and in clinical studies and will summarize new strategies that are being developed to optimize their stability, specificity and potency for advancing their use.Methods:Articles describing recombinant antibody fragments used for neurological diseases were selected (PubMed) and evaluated for their significance.Results:Different antibody formats such as single-chain fragment variable (scFv), single-domain antibody fragments (VHHs or sdAbs), bispecific antibodies (bsAbs), intrabodies and nanobodies, are currently being studied in pre-clinical models of cancer as well as infectious and autoimmune diseases and many of them are being tested as therapeutics in clinical trials. Immunotherapy approaches have shown therapeutic efficacy in several animal models of Alzheimer´s disease (AD), Parkinson disease (PD), dementia with Lewy bodies (DLB), frontotemporal dementia (FTD), Huntington disease (HD), transmissible spongiform encephalopathies (TSEs) and multiple sclerosis (MS). It has been demonstrated that recombinant antibody fragments may neutralize toxic extra- and intracellular misfolded proteins involved in the pathogenesis of AD, PD, DLB, FTD, HD or TSEs and may target toxic immune cells participating in the pathogenesis of MS.Conclusion:Recombinant antibody fragments represent a promising tool for the development of antibody-based immunotherapeutics for neurodegenerative diseases.

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“…coli strain MC4100 containing both pDD18 and pDD322Kan plasmids were subcultured in fresh LB media at 37°C until the cells reached an OD600 ~0.4-0.5 then induced by adding arabinose to a final concentration of 0.01% wt/vol and protein expression was continued at 30°C and harvested 3 h after induction. Samples were normalized by OD 600 = 75 before being subjected to subcellular fractionation, which was performed using the ice-cold osmotic shock procedure to release periplasmic proteins, followed by sonication to release cytoplasmic proteins [12]. Each fraction was separated by SDS/PAGE and Western blotting was performed according to standard protocols.…”

Section: Protein Analysismentioning

confidence: 99%

Application of the Synthetic Escherichia coli’s Twin-Arginine Translocation Pathway for the Detection of Intracellular Antibodies against Hepatitis C Viral Protease

Kamthong¹,

Boonyalekha²,

Leelawattanachai³

et al. 2020

IJBBB

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Hepatitis C is a liver disease caused by hepatitis C virus (HCV) infection. Protease inhibitor (PI) is included in the current oral direct-acting antiviral (DAA) combination therapy. However, these HCV PIs are small molecule drugs; therefore, they could have serious off-target adverse effects. New types of treatment such as antibody drugs that have very high specificity and selectivity would be a better alternative. Our study reports the application of the PROTECT (Protease inhibitor Recognition based On Tat Export after Cleavage Tampering) assay, an in vivo detection method for protease inhibiting intracellular antibodies (intrabodies) based on the bacterial twin-arginine translocation (Tat) pathway to the HCV NS3 protease system. This assay was designed such that when protease is co-expressed inside the bacterial cytoplasm, Tat transportation of the uncleaved protease substrate due to protection of the cleavage site by a specific intrabody will result in β-lactam antibiotic resistance. Using the anti-NS3 intrabodies isolated previously, we demonstrated that PROTECT assay could distinguish between the inhibitory and non-inhibitory anti-NS3 intrabodies resulting in selective growth in the presence of β-lactam antibiotics. This method has potential for the screening of agents that inhibit proteolytic cleavage in a bacterial cell-based assay, which may find use in identifying, reconstituting, and characterizing protease inhibitors, identifying mutations on the substrate that can inhibit proteolytic cleavage, and drug screening.

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Optimizing recombinant antibodies for intracellular function using hitchhiker-mediated survival selection

Cited by 25 publications

References 44 publications

A Suicide Switch Directly Eliminates Intracellular scFv Oligomers in the Cytoplasm of Mammalian Cells

A Suicide Switch Directly Eliminates Intracellular scFv Oligomers in the Cytoplasm of Mammalian Cells

Recombinant Antibody Fragments for Neurodegenerative Diseases

Application of the Synthetic Escherichia coli’s Twin-Arginine Translocation Pathway for the Detection of Intracellular Antibodies against Hepatitis C Viral Protease

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