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Nanobody
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Nanobody

A special issue of Antibodies (ISSN 2073-4468).

Deadline for manuscript submissions: closed (30 November 2018) | Viewed by 142321

Printed Edition Available!
A printed edition of this Special Issue is available here.

Special Issue Editors

Prof. Dr. Ulrich Rothbauer

E-Mail Website
Guest Editor
Pharmaceutical Biotechnology, Eberhard Karls University Tuebingen, 72076 Tuebingen, Germany
Interests: nanobodies; antibodies; recombinant antibody-technologies; phage display; live cell imaging; high content analysis; molecular imaging; theranostic
Dr. Patrick Chames

E-Mail Website
Guest Editor
Aix-Marseille Université, CNRS, Inserm, Institut Paoli-Calmettes, CRCM, F-13009 Marseille, France
Interests: onco immunology; antibody engineering

Special Issue Information

Dear Colleagues,

Since their first description 25 years ago, single-domain antibody fragments, derived from heavy-chain-only antibodies of camelids, so-called nanobodies, have emerged as attractive alternatives to conventional antibodies for multiple applications in biomedical research. Compared to other small antibody fragments like Fab or scFv, nanobodies have numerous advantages. First, only one domain has to be cloned and expressed to generate a fully functional binding molecule. Secondly, nanobodies are highly soluble and stable, can be easily genetically or chemically modified, and produced in various cells and/or organisms. Microbial expression systems enable the production of purified nanobodies in the mg–g range per liter of culture, thereby offering an unlimited supply of consistent binding molecules.

To date nanobodies have become outstanding tools for in vitro and in vivo imaging, as well as structural and proteome analysis. As genetically encoded intrabodies, they open new possibilities for visualization or functional studies on proteins in living cells. The recently described advances in identification of antigen-specific nanobodies from synthetic gene libraries now makes nanobody-based approaches broadly available to many researches in the field.

This Special Issue is aimed to provide an up-to-date overview of the rising field of nanobodies including generation and functionalization of nanobodies as well as their application for immunoassays, proteomics, protein crystallization and in vitro and in vivo imaging.

Prof. Ulrich Rothbauer
Dr. Patrick Chames
Guest Editor

Manuscript Submission Information

Manuscripts should be submitted online at www.mdpi.com by registering and logging in to this website. Once you are registered, click here to go to the submission form. Manuscripts can be submitted until the deadline. All submissions that pass pre-check are peer-reviewed. Accepted papers will be published continuously in the journal (as soon as accepted) and will be listed together on the special issue website. Research articles, review articles as well as short communications are invited. For planned papers, a title and short abstract (about 100 words) can be sent to the Editorial Office for announcement on this website.

Submitted manuscripts should not have been published previously, nor be under consideration for publication elsewhere (except conference proceedings papers). All manuscripts are thoroughly refereed through a single-blind peer-review process. A guide for authors and other relevant information for submission of manuscripts is available on the Instructions for Authors page. Antibodies is an international peer-reviewed open access quarterly journal published by MDPI.

Please visit the Instructions for Authors page before submitting a manuscript. The Article Processing Charge (APC) for publication in this open access journal is 1800 CHF (Swiss Francs). Submitted papers should be well formatted and use good English. Authors may use MDPI's English editing service prior to publication or during author revisions.

Keywords

  • Nanobodies
  • Nanobody Display Technologies
  • Functionalization of nanobodies
  • Unique application of nanobodies
  • Live cell imaging
  • Super resolution microscopy
  • In vivo imaging
  • Protein crystallization chaperones

Published Papers (17 papers)

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Editorial

Jump to: Research, Review

4 pages, 191 KiB  
Editorial
Special Issue: Nanobody
by Patrick Chames and Ulrich Rothbauer
Antibodies 2020, 9(1), 6; https://0-doi-org.brum.beds.ac.uk/10.3390/antib9010006 - 06 Mar 2020
Cited by 9 | Viewed by 5736
Abstract
Since their first description in 1993 [...] Full article
(This article belongs to the Special Issue Nanobody)

Research

Jump to: Editorial, Review

19 pages, 4062 KiB  
Article
Super Potent Bispecific Llama VHH Antibodies Neutralize HIV via a Combination of gp41 and gp120 Epitopes
by Nika M. Strokappe, Miriam Hock, Lucy Rutten, Laura E. Mccoy, Jaap W. Back, Christophe Caillat, Matthias Haffke, Robin A. Weiss, Winfried Weissenhorn and Theo Verrips
Antibodies 2019, 8(2), 38; https://0-doi-org.brum.beds.ac.uk/10.3390/antib8020038 - 18 Jun 2019
Cited by 24 | Viewed by 8258
Abstract
Broad and potent neutralizing llama single domain antibodies (VHH) against HIV-1 targeting the CD4 binding site (CD4bs) have previously been isolated upon llama immunization. Here we describe the epitopes of three additional VHH groups selected from phage libraries. The 2E7 group binds to [...] Read more.
Broad and potent neutralizing llama single domain antibodies (VHH) against HIV-1 targeting the CD4 binding site (CD4bs) have previously been isolated upon llama immunization. Here we describe the epitopes of three additional VHH groups selected from phage libraries. The 2E7 group binds to a new linear epitope in the first heptad repeat of gp41 that is only exposed in the fusion-intermediate conformation. The 1B5 group competes with co-receptor binding and the 1F10 group interacts with the crown of the gp120 V3 loop, occluded in native Env. We present biophysical and structural details on the 2E7 interaction with gp41. In order to further increase breadth and potency, we constructed bi-specific VHH. The combination of CD4bs VHH (J3/3E3) with 2E7 group VHH enhanced strain-specific neutralization with potencies up to 1400-fold higher than the mixture of the individual VHHs. Thus, these new bivalent VHH are potent new tools to develop therapeutic approaches or microbicide intervention. Full article
(This article belongs to the Special Issue Nanobody)
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13 pages, 2850 KiB  
Article
VHH-Photosensitizer Conjugates for Targeted Photodynamic Therapy of Met-Overexpressing Tumor Cells
by Raimond Heukers, Vida Mashayekhi, Mercedes Ramirez-Escudero, Hans de Haard, Theo C. Verrips, Paul. M.P. van Bergen en Henegouwen and Sabrina Oliveira
Antibodies 2019, 8(2), 26; https://0-doi-org.brum.beds.ac.uk/10.3390/antib8020026 - 04 Apr 2019
Cited by 27 | Viewed by 8080
Abstract
Photodynamic therapy (PDT) is an approach that kills (cancer) cells by the local production of toxic reactive oxygen species upon the local illumination of a photosensitizer (PS). The specificity of PDT has been further enhanced by the development of a new water-soluble PS [...] Read more.
Photodynamic therapy (PDT) is an approach that kills (cancer) cells by the local production of toxic reactive oxygen species upon the local illumination of a photosensitizer (PS). The specificity of PDT has been further enhanced by the development of a new water-soluble PS and by the specific delivery of PS via conjugation to tumor-targeting antibodies. To improve tissue penetration and shorten photosensitivity, we have recently introduced nanobodies, also known as VHH (variable domains from the heavy chain of llama heavy chain antibodies), for targeted PDT of cancer cells overexpressing the epidermal growth factor receptor (EGFR). Overexpression and activation of another cancer-related receptor, the hepatocyte growth factor receptor (HGFR, c-Met or Met) is also involved in the progression and metastasis of a large variety of malignancies. In this study we evaluate whether anti-Met VHHs conjugated to PS can also serve as a biopharmaceutical for targeted PDT. VHHs targeting the SEMA (semaphorin-like) subdomain of Met were provided with a C-terminal tag that allowed both straightforward purification from yeast supernatant and directional conjugation to the PS IRDye700DX using maleimide chemistry. The generated anti-Met VHH-PS showed nanomolar binding affinity and, upon illumination, specifically killed MKN45 cells with nanomolar potency. This study shows that Met can also serve as a membrane target for targeted PDT. Full article
(This article belongs to the Special Issue Nanobody)
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Graphical abstract

20 pages, 2224 KiB  
Article
Cross-Reactive and Lineage-Specific Single Domain Antibodies against Influenza B Hemagglutinin
by Walter Ramage, Tiziano Gaiotto, Christina Ball, Paul Risley, George W. Carnell, Nigel Temperton, Chung Y. Cheung, Othmar G. Engelhardt and Simon E. Hufton
Antibodies 2019, 8(1), 14; https://0-doi-org.brum.beds.ac.uk/10.3390/antib8010014 - 10 Feb 2019
Cited by 13 | Viewed by 6455
Abstract
Influenza B virus (IBV) circulates in the human population and causes considerable disease burden worldwide, each year. Current IBV vaccines can struggle to mount an effective cross-reactive immune response, as strains become mismatched, due to constant antigenic changes. Additional strategies which use monoclonal [...] Read more.
Influenza B virus (IBV) circulates in the human population and causes considerable disease burden worldwide, each year. Current IBV vaccines can struggle to mount an effective cross-reactive immune response, as strains become mismatched, due to constant antigenic changes. Additional strategies which use monoclonal antibodies, with broad reactivity, are of considerable interest, both, as diagnostics and as immunotherapeutics. Alternatives to conventional monoclonal antibodies, such as single domain antibodies (NanobodiesTM) with well-documented advantages for applications in infectious disease, have been emerging. In this study we have isolated single domain antibodies (sdAbs), specific to IBV, using alpacas immunised with recombinant hemagglutinin (HA) from two representative viruses, B/Florida/04/2006 (B/Yamagata lineage) and B/Brisbane/60/2008 (B/Victoria lineage). Using phage display, we have isolated a panel of single domain antibodies (sdAbs), with both cross-reactive and lineage-specific binding. Several sdAbs recognise whole virus antigens, corresponding to influenza B strains included in vaccines spanning over 20 years, and were capable of neutralising IBV pseudotypes corresponding to prototype strains from both lineages. Lineage-specific sdAbs recognised the head domain, whereas, sdAbs identified as cross-reactive could be classified as either head binding or stem binding. Using yeast display, we were able to correlate lineage specificity with naturally occurring sequence divergence, at residue 122 in the highly variable 120 loop of the HA1 domain. The single domain antibodies described, might have applications in IBV diagnostics, vaccine potency testing and as immunotherapeutics. Full article
(This article belongs to the Special Issue Nanobody)
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18 pages, 2570 KiB  
Article
A Strategy to Optimize the Generation of Stable Chromobody Cell Lines for Visualization and Quantification of Endogenous Proteins in Living Cells
by Bettina-Maria Keller, Julia Maier, Melissa Weldle, Soeren Segan, Bjoern Traenkle and Ulrich Rothbauer
Antibodies 2019, 8(1), 10; https://0-doi-org.brum.beds.ac.uk/10.3390/antib8010010 - 10 Jan 2019
Cited by 17 | Viewed by 8919
Abstract
Single-domain antibodies have emerged as highly versatile nanoprobes for advanced cellular imaging. For real-time visualization of endogenous antigens, fluorescently labelled nanobodies (chromobodies, CBs) are introduced as DNA-encoded expression constructs in living cells. Commonly, CB expression is driven from strong, constitutively active promoters. However, [...] Read more.
Single-domain antibodies have emerged as highly versatile nanoprobes for advanced cellular imaging. For real-time visualization of endogenous antigens, fluorescently labelled nanobodies (chromobodies, CBs) are introduced as DNA-encoded expression constructs in living cells. Commonly, CB expression is driven from strong, constitutively active promoters. However, high expression levels are sometimes accompanied by misfolding and aggregation of those intracellular nanoprobes. Moreover, stable cell lines derived from random genomic insertion of CB-encoding transgenes bear the risk of disturbed cellular processes and inhomogeneous CB signal intensities due to gene positioning effects and epigenetic silencing. In this study we propose a strategy to generate optimized CB expressing cell lines. We demonstrate that expression as ubiquitin fusion increases the fraction of intracellularly functional CBs and identified the elongation factor 1α (EF1-α) promoter as highly suited for constitutive CB expression upon long-term cell line cultivation. Finally, we applied a CRISPR/Cas9-based gene editing approach for targeted insertion of CB expression constructs into the adeno-associated virus integration site 1 (AAVS1) safe harbour locus of human cells. Our results indicate that this combinatorial approach facilitates the generation of fully functional and stable CB cell lines for quantitative live-cell imaging of endogenous antigens. Full article
(This article belongs to the Special Issue Nanobody)
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17 pages, 1658 KiB  
Article
Selection and Characterization of a Nanobody Biosensor of GTP-Bound RHO Activities
by Laura Keller, Nicolas Bery, Claudine Tardy, Laetitia Ligat, Gilles Favre, Terence H. Rabbitts and Aurélien Olichon
Antibodies 2019, 8(1), 8; https://0-doi-org.brum.beds.ac.uk/10.3390/antib8010008 - 09 Jan 2019
Cited by 24 | Viewed by 7780
Abstract
RHO (Ras HOmologous) GTPases are molecular switches that activate, in their state bound to Guanosine triphosphate (GTP), key signaling pathways, which involve actin cytoskeleton dynamics. Previously, we selected the nanobody RH12, from a synthetic phage display library, which binds the GTP-bound active conformation [...] Read more.
RHO (Ras HOmologous) GTPases are molecular switches that activate, in their state bound to Guanosine triphosphate (GTP), key signaling pathways, which involve actin cytoskeleton dynamics. Previously, we selected the nanobody RH12, from a synthetic phage display library, which binds the GTP-bound active conformation of RHOA (Ras Homologous family member A). However, when expressed as an intracellular antibody, its blocking effect on RHO signaling led to a loss of actin fibers, which in turn affected cell shape and cell survival. Here, in order to engineer an intracellular biosensor of RHOA-GTP activation, we screened the same phage nanobody library and identified another RHO-GTP selective intracellular nanobody, but with no apparent toxicity. The recombinant RH57 nanobody displays high affinity towards GTP-bound RHOA/B/C subgroup of small GTPases in vitro. Intracellular expression of the RH57 allowed selective co-precipitation with the GTP-bound state of the endogenous RHOA subfamily. When expressed as a fluorescent fusion protein, the chromobody GFP-RH57 was localized to the inner plasma membrane upon stimulation of the activation of endogenous RHO. Finally, the RH57 nanobody was used to establish a BRET-based biosensor (Bioluminescence Resonance Energy Transfer) of RHO activation. The dynamic range of the BRET signal could potentially offer new opportunities to develop cell-based screening of RHOA subfamily activation modulators. Full article
(This article belongs to the Special Issue Nanobody)
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19 pages, 11718 KiB  
Article
A Collection of Single-Domain Antibodies that Crowd Ricin Toxin’s Active Site
by Siva Krishna Angalakurthi, David J. Vance, Yinghui Rong, Chi My Thi Nguyen, Michael J. Rudolph, David Volkin, C. Russell Middaugh, David D. Weis and Nicholas J. Mantis
Antibodies 2018, 7(4), 45; https://0-doi-org.brum.beds.ac.uk/10.3390/antib7040045 - 17 Dec 2018
Cited by 9 | Viewed by 4445
Abstract
In this report, we used hydrogen exchange-mass spectrometry (HX-MS) to identify the epitopes recognized by 21 single-domain camelid antibodies (VHHs) directed against the ribosome-inactivating subunit (RTA) of ricin toxin, a biothreat agent of concern to military and public health authorities. The [...] Read more.
In this report, we used hydrogen exchange-mass spectrometry (HX-MS) to identify the epitopes recognized by 21 single-domain camelid antibodies (VHHs) directed against the ribosome-inactivating subunit (RTA) of ricin toxin, a biothreat agent of concern to military and public health authorities. The VHHs, which derive from 11 different B-cell lineages, were binned together based on competition ELISAs with IB2, a monoclonal antibody that defines a toxin-neutralizing hotspot (“cluster 3”) located in close proximity to RTA’s active site. HX-MS analysis revealed that the 21 VHHs recognized four distinct epitope subclusters (3.1–3.4). Sixteen of the 21 VHHs grouped within subcluster 3.1 and engage RTA α-helices C and G. Three VHHs grouped within subcluster 3.2, encompassing α-helices C and G, plus α-helix B. The single VHH in subcluster 3.3 engaged RTA α-helices B and G, while the epitope of the sole VHH defining subcluster 3.4 encompassed α-helices C and E, and β-strand h. Modeling these epitopes on the surface of RTA predicts that the 20 VHHs within subclusters 3.1–3.3 physically occlude RTA’s active site cleft, while the single antibody in subcluster 3.4 associates on the active site’s upper rim. Full article
(This article belongs to the Special Issue Nanobody)
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Graphical abstract

12 pages, 3092 KiB  
Article
Selection of Single-Domain Antibodies towards Western Equine Encephalitis Virus
by Jinny L. Liu, Lisa C. Shriver-Lake, Dan Zabetakis, Ellen R. Goldman and George P. Anderson
Antibodies 2018, 7(4), 44; https://0-doi-org.brum.beds.ac.uk/10.3390/antib7040044 - 15 Dec 2018
Cited by 11 | Viewed by 6054
Abstract
In this work, we describe the selection and characterization of single-domain antibodies (sdAb) towards the E2/E3E2 envelope protein of the Western equine encephalitis virus (WEEV). Our purpose was to identify novel recognition elements which could be used for the detection, diagnosis, and perhaps [...] Read more.
In this work, we describe the selection and characterization of single-domain antibodies (sdAb) towards the E2/E3E2 envelope protein of the Western equine encephalitis virus (WEEV). Our purpose was to identify novel recognition elements which could be used for the detection, diagnosis, and perhaps treatment of western equine encephalitis (WEE). To achieve this goal, we prepared an immune phage display library derived from the peripheral blood lymphocytes of a llama that had been immunized with an equine vaccine that includes killed WEEV (West Nile Innovator + VEWT). This library was panned against recombinant envelope (E2/E3E2) protein from WEEV, and seven representative sdAb from the five identified sequence families were characterized. The specificity, affinity, and melting point of each sdAb was determined, and their ability to detect the recombinant protein in a MagPlex sandwich immunoassay was confirmed. Thus, these new binders represent novel recognition elements for the E2/E3E2 proteins of WEEV that are available to the research community for further investigation into their applicability for use in the diagnosis or treatment of WEE. Full article
(This article belongs to the Special Issue Nanobody)
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14 pages, 2314 KiB  
Article
Properties of Fluorescent Far-Red Anti-TNF Nanobodies
by Ekaterina N. Gorshkova, Grigory A. Efimov, Ksenia D. Ermakova, Ekaterina A. Vasilenko, Diana V. Yuzhakova, Marina V. Shirmanova, Vladislav V. Mokhonov, Sergei V. Tillib, Sergei A. Nedospasov and Irina V. Astrakhantseva
Antibodies 2018, 7(4), 43; https://0-doi-org.brum.beds.ac.uk/10.3390/antib7040043 - 15 Dec 2018
Cited by 2 | Viewed by 5337
Abstract
Upregulation of the expression of tumor necrosis factor (TNF-α, TNF) has a significant role in the development of autoimmune diseases. The fluorescent antibodies binding TNF may be used for personalized therapy of TNF-dependent diseases as a tool to predict the response to anti-TNF [...] Read more.
Upregulation of the expression of tumor necrosis factor (TNF-α, TNF) has a significant role in the development of autoimmune diseases. The fluorescent antibodies binding TNF may be used for personalized therapy of TNF-dependent diseases as a tool to predict the response to anti-TNF treatment. We generated recombinant fluorescent proteins consisting of the anti-TNF module based on the variable heavy chain (VHH) of camelid antibodies fused with the far-red fluorescent protein Katushka (Kat). Two types of anti-TNF VHH were developed: one (BTN-Kat) that was bound both human or mouse TNF, but did not neutralize their activity, and a second (ITN-Kat) that was binding and neutralizing human TNF. BTN-Kat does not interfere with TNF biological functions and can be used for whole-body imaging. ITN-Kat can be evaluated in humanized mice or in cells isolated from humanized mice. It is able to block human TNF (hTNF) activities both in vitro and in vivo and may be considered as a prototype of a theranostic agent for autoimmune diseases. Full article
(This article belongs to the Special Issue Nanobody)
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Graphical abstract

15 pages, 4669 KiB  
Article
Isolation and Characterization of Nanobodies against a Zinc-Transporting P-Type ATPase
by Elena Longhin, Christina Grønberg, Qiaoxia Hu, Annette Susanne Duelli, Kasper Røjkjær Andersen, Nick Stub Laursen and Pontus Gourdon
Antibodies 2018, 7(4), 39; https://0-doi-org.brum.beds.ac.uk/10.3390/antib7040039 - 07 Nov 2018
Cited by 4 | Viewed by 6918
Abstract
P-type ATPases form a large and ubiquitous superfamily of ion and lipid transporters that use ATP (adenosine triphosphate) to carry out their function. The IB subclass (PIB-ATPases) allows flux of heavy metals and are key players in metal detoxification, critical for [...] Read more.
P-type ATPases form a large and ubiquitous superfamily of ion and lipid transporters that use ATP (adenosine triphosphate) to carry out their function. The IB subclass (PIB-ATPases) allows flux of heavy metals and are key players in metal detoxification, critical for human health, crops, and survival of pathogens. Nevertheless, PIB-ATPases remain poorly understood at a molecular level. In this study, nanobodies (Nbs) are selected against the zinc-transporting PIB-ATPase ZntA from Shigella sonnei (SsZntA), aiming at developing tools to assist the characterization of the structure and function of this class of transporters. We identify six different Nbs that bind detergent stabilized SsZntA. We further assess the effect of the Nbs on the catalytic function of SsZntA, and find that five nanobodies associate without affecting the function, while one nanobody significantly reduces the ATPase activity. This study paves the way for more refined mechanistical and structural studies of zinc-transporting PIB-ATPases. Full article
(This article belongs to the Special Issue Nanobody)
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12 pages, 2064 KiB  
Article
Genetic Fusion of an Anti-BclA Single-Domain Antibody with Beta Galactosidase
by George P. Anderson, Lisa C. Shriver-Lake, Scott A. Walper, Lauryn Ashford, Dan Zabetakis, Jinny L. Liu, Joyce C. Breger, P. Audrey Brozozog Lee and Ellen R. Goldman
Antibodies 2018, 7(4), 36; https://0-doi-org.brum.beds.ac.uk/10.3390/antib7040036 - 29 Sep 2018
Cited by 8 | Viewed by 5490
Abstract
The Bacillus collagen-like protein of anthracis (BclA), found in Bacillus anthracis spores, is an attractive target for immunoassays. Previously, using phage display we had selected llama-derived single-domain antibodies that bound to B. anthracis spore proteins including BclA. Single-domain antibodies (sdAbs), the recombinantly expressed [...] Read more.
The Bacillus collagen-like protein of anthracis (BclA), found in Bacillus anthracis spores, is an attractive target for immunoassays. Previously, using phage display we had selected llama-derived single-domain antibodies that bound to B. anthracis spore proteins including BclA. Single-domain antibodies (sdAbs), the recombinantly expressed heavy domains from the unique heavy-chain-only antibodies found in camelids, provide stable and well-expressed binding elements with excellent affinity. In addition, sdAbs offer the important advantage that they can be tailored for specific applications through protein engineering. A fusion of a BclA targeting sdAb with the enzyme Beta galactosidase (β-gal) would enable highly sensitive immunoassays with no need for a secondary reagent. First, we evaluated five anti-BclA sdAbs, including four that had been previously identified but not characterized. Each was tested to determine its binding affinity, melting temperature, producibility, and ability to function as both capture and reporter in sandwich assays for BclA. The sdAb with the best combination of properties was constructed as a fusion with β-gal and shown to enable sensitive detection. This fusion has the potential to be incorporated into highly sensitive assays for the detection of anthrax spores. Full article
(This article belongs to the Special Issue Nanobody)
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Review

Jump to: Editorial, Research

25 pages, 685 KiB  
Review
Single-Domain Antibodies as Therapeutic and Imaging Agents for the Treatment of CNS Diseases
by Kasandra Bélanger, Umar Iqbal, Jamshid Tanha, Roger MacKenzie, Maria Moreno and Danica Stanimirovic
Antibodies 2019, 8(2), 27; https://0-doi-org.brum.beds.ac.uk/10.3390/antib8020027 - 05 Apr 2019
Cited by 36 | Viewed by 7662
Abstract
Antibodies have become one of the most successful therapeutics for a number of oncology and inflammatory diseases. So far, central nervous system (CNS) indications have missed out on the antibody revolution, while they remain ‘hidden’ behind several hard to breach barriers. Among the [...] Read more.
Antibodies have become one of the most successful therapeutics for a number of oncology and inflammatory diseases. So far, central nervous system (CNS) indications have missed out on the antibody revolution, while they remain ‘hidden’ behind several hard to breach barriers. Among the various antibody modalities, single-domain antibodies (sdAbs) may hold the ‘key’ to unlocking the access of antibody therapies to CNS diseases. The unique structural features of sdAbs make them the smallest monomeric antibody fragments suitable for molecular targeting. These features are of particular importance when developing antibodies as modular building blocks for engineering CNS-targeting therapeutics and imaging agents. In this review, we first introduce the characteristic properties of sdAbs compared to traditional antibodies. We then present recent advances in the development of sdAbs as potential therapeutics across brain barriers, including their use for the delivery of biologics across the blood–brain and blood–cerebrospinal fluid (CSF) barriers, treatment of neurodegenerative diseases and molecular imaging of brain targets. Full article
(This article belongs to the Special Issue Nanobody)
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11 pages, 1266 KiB  
Review
Using Nanobodies to Study Protein Function in Developing Organisms
by Gustavo Aguilar, Shinya Matsuda, M. Alessandra Vigano and Markus Affolter
Antibodies 2019, 8(1), 16; https://0-doi-org.brum.beds.ac.uk/10.3390/antib8010016 - 12 Feb 2019
Cited by 31 | Viewed by 7307
Abstract
Polyclonal and monoclonal antibodies have been invaluable tools to study proteins over the past decades. While indispensable for most biological studies including developmental biology, antibodies have been used mostly in fixed tissues or as binding reagents in the extracellular milieu. For functional studies [...] Read more.
Polyclonal and monoclonal antibodies have been invaluable tools to study proteins over the past decades. While indispensable for most biological studies including developmental biology, antibodies have been used mostly in fixed tissues or as binding reagents in the extracellular milieu. For functional studies and for clinical applications, antibodies have been functionalized by covalently fusing them to heterologous partners (i.e., chemicals, proteins or other moieties). Such functionalized antibodies have been less widely used in developmental biology studies. In the past few years, the discovery and application of small functional binding fragments derived from single-chain antibodies, so-called nanobodies, has resulted in novel approaches to study proteins during the development of multicellular animals in vivo. Expression of functionalized nanobody fusions from integrated transgenes allows manipulating proteins of interest in the extracellular and the intracellular milieu in a tissue- and time-dependent manner in an unprecedented manner. Here, we describe how nanobodies have been used in the field of developmental biology and look into the future to imagine how else nanobody-based reagents could be further developed to study the proteome in living organisms. Full article
(This article belongs to the Special Issue Nanobody)
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21 pages, 3129 KiB  
Review
Nanobody Engineering: Toward Next Generation Immunotherapies and Immunoimaging of Cancer
by Timothée Chanier and Patrick Chames
Antibodies 2019, 8(1), 13; https://0-doi-org.brum.beds.ac.uk/10.3390/antib8010013 - 21 Jan 2019
Cited by 84 | Viewed by 18966
Abstract
In the last decade, cancer immunotherapies have produced impressive therapeutic results. However, the potency of immunotherapy is tightly linked to immune cell infiltration within the tumor and varies from patient to patient. Thus, it is becoming increasingly important to monitor and modulate the [...] Read more.
In the last decade, cancer immunotherapies have produced impressive therapeutic results. However, the potency of immunotherapy is tightly linked to immune cell infiltration within the tumor and varies from patient to patient. Thus, it is becoming increasingly important to monitor and modulate the tumor immune infiltrate for an efficient diagnosis and therapy. Various bispecific approaches are being developed to favor immune cell infiltration through specific tumor targeting. The discovery of antibodies devoid of light chains in camelids has spurred the development of single domain antibodies (also called VHH or nanobody), allowing for an increased diversity of multispecific and/or multivalent formats of relatively small sizes endowed with high tissue penetration. The small size of nanobodies is also an asset leading to high contrasts for non-invasive imaging. The approval of the first therapeutic nanobody directed against the von Willebrand factor for the treatment of acquired thrombotic thrombocypenic purpura (Caplacizumab, Ablynx), is expected to bolster the rise of these innovative molecules. In this review, we discuss the latest advances in the development of nanobodies and nanobody-derived molecules for use in cancer immunotherapy and immunoimaging. Full article
(This article belongs to the Special Issue Nanobody)
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21 pages, 946 KiB  
Review
Targeted Nanobody-Based Molecular Tracers for Nuclear Imaging and Image-Guided Surgery
by Pieterjan Debie, Nick Devoogdt and Sophie Hernot
Antibodies 2019, 8(1), 12; https://0-doi-org.brum.beds.ac.uk/10.3390/antib8010012 - 11 Jan 2019
Cited by 77 | Viewed by 10531
Abstract
Molecular imaging is paving the way towards noninvasive detection, staging, and treatment follow-up of diseases such as cancer and inflammation-related conditions. Monoclonal antibodies have long been one of the staples of molecular imaging tracer design, although their long blood circulation and high nonspecific [...] Read more.
Molecular imaging is paving the way towards noninvasive detection, staging, and treatment follow-up of diseases such as cancer and inflammation-related conditions. Monoclonal antibodies have long been one of the staples of molecular imaging tracer design, although their long blood circulation and high nonspecific background limits their applicability. Nanobodies, unique antibody-binding fragments derived from camelid heavy-chain antibodies, have excellent properties for molecular imaging as they are able to specifically find their target early after injection, with little to no nonspecific background. Nanobody-based tracers using either nuclear or fluorescent labels have been heavily investigated preclinically and are currently making their way into the clinic. In this review, we will discuss different important factors in nanobody-tracer design, as well as the current state of the art regarding their application for nuclear and fluorescent imaging purposes. Furthermore, we will discuss how nanobodies can also be exploited for molecular therapy applications such as targeted radionuclide therapy and photodynamic therapy. Full article
(This article belongs to the Special Issue Nanobody)
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17 pages, 2773 KiB  
Review
Current Approaches and Future Perspectives for Nanobodies in Stroke Diagnostic and Therapy
by Larissa Jank, Carolina Pinto-Espinoza, Yinghui Duan, Friedrich Koch-Nolte, Tim Magnus and Björn Rissiek
Antibodies 2019, 8(1), 5; https://0-doi-org.brum.beds.ac.uk/10.3390/antib8010005 - 03 Jan 2019
Cited by 19 | Viewed by 11700
Abstract
Antibody-based biologics are the corner stone of modern immunomodulatory therapy. Though highly effective in dampening systemic inflammatory processes, their large size and Fc-fragment mediated effects hamper crossing of the blood brain barrier (BBB). Nanobodies (Nbs) are single domain antibodies derived from llama or [...] Read more.
Antibody-based biologics are the corner stone of modern immunomodulatory therapy. Though highly effective in dampening systemic inflammatory processes, their large size and Fc-fragment mediated effects hamper crossing of the blood brain barrier (BBB). Nanobodies (Nbs) are single domain antibodies derived from llama or shark heavy-chain antibodies and represent a new generation of biologics. Due to their small size, they display excellent tissue penetration capacities and can be easily modified to adjust their vivo half-life for short-term diagnostic or long-term therapeutic purposes or to facilitate crossing of the BBB. Furthermore, owing to their characteristic binding mode, they are capable of antagonizing receptors involved in immune signaling and of neutralizing proinflammatory mediators, such as cytokines. These qualities combined make Nbs well-suited for down-modulating neuroinflammatory processes that occur in the context of brain ischemia. In this review, we summarize recent findings on Nbs in preclinical stroke models and how they can be used as diagnostic and therapeutic reagents. We further provide a perspective on the design of innovative Nb-based treatment protocols to complement and improve stroke therapy. Full article
(This article belongs to the Special Issue Nanobody)
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Review
Single-Domain Antibodies and Their Formatting to Combat Viral Infections
by Dorien De Vlieger, Marlies Ballegeer, Iebe Rossey, Bert Schepens and Xavier Saelens
Antibodies 2019, 8(1), 1; https://0-doi-org.brum.beds.ac.uk/10.3390/antib8010001 - 20 Dec 2018
Cited by 62 | Viewed by 10319
Abstract
Since their discovery in the 1990s, single-domain antibodies (VHHs), also known as Nanobodies®, have changed the landscape of affinity reagents. The outstanding solubility, stability, and specificity of VHHs, as well as their small size, ease of production and formatting flexibility favor [...] Read more.
Since their discovery in the 1990s, single-domain antibodies (VHHs), also known as Nanobodies®, have changed the landscape of affinity reagents. The outstanding solubility, stability, and specificity of VHHs, as well as their small size, ease of production and formatting flexibility favor VHHs over conventional antibody formats for many applications. The exceptional ease by which it is possible to fuse VHHs with different molecular modules has been particularly explored in the context of viral infections. In this review, we focus on VHH formats that have been developed to combat viruses including influenza viruses, human immunodeficiency virus-1 (HIV-1), and human respiratory syncytial virus (RSV). Such formats may significantly increase the affinity, half-life, breadth of protection of an antiviral VHH and reduce the risk of viral escape. In addition, VHHs can be equipped with effector functions, for example to guide components of the immune system with high precision to sites of viral infection. Full article
(This article belongs to the Special Issue Nanobody)
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