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      A guide to: generation and design of nanobodies

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          Abstract

          Immune or naïve nanobody (Nb) repertoires are cloned from B lymphocytes of camelids. Alternatively, synthetic Nb libraries are generated in vitro by randomising codons forming antigen‐binding loops of a Nb scaffold. These libraries serve to retrieve target‐specific Nbs by phage display pannings, yeast surface display or other selection methods. After identification of the lead Nb and possible engineering efforts, multiple applications have been explored in research, diagnosis or therapy.

          Abstract

          A nanobody (Nb) is a registered trademark of Ablynx, referring to the single antigen‐binding domain of heavy chain‐only antibodies (HCAbs) that are circulating in Camelidae. Nbs are produced recombinantly in micro‐organisms and employed as research tools or for diagnostic and therapeutic applications. They were – and still are – also named single‐domain antibodies (sdAbs) or variable domain of the heavy chain of HCAbs (VHH). A variety of methods are currently in use for the fast and efficient generation of target‐specific Nbs. Such Nbs are produced at low cost and associate with high affinity to their cognate antigen. They are robust, strictly monomeric and easy to tailor into more complex entities to meet the requirements of their application. Here, we review the various sources and different strategies that have been developed to identify rapidly, target‐specific Nbs. We further discuss a variety of engineering technologies that have been explored to broaden the application range of Nbs and summarise those applications where designed Nbs might offer a marked advantage over other affinity reagents.

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          Most cited references148

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          UCSF Chimera--a visualization system for exploratory research and analysis.

          The design, implementation, and capabilities of an extensible visualization system, UCSF Chimera, are discussed. Chimera is segmented into a core that provides basic services and visualization, and extensions that provide most higher level functionality. This architecture ensures that the extension mechanism satisfies the demands of outside developers who wish to incorporate new features. Two unusual extensions are presented: Multiscale, which adds the ability to visualize large-scale molecular assemblies such as viral coats, and Collaboratory, which allows researchers to share a Chimera session interactively despite being at separate locales. Other extensions include Multalign Viewer, for showing multiple sequence alignments and associated structures; ViewDock, for screening docked ligand orientations; Movie, for replaying molecular dynamics trajectories; and Volume Viewer, for display and analysis of volumetric data. A discussion of the usage of Chimera in real-world situations is given, along with anticipated future directions. Chimera includes full user documentation, is free to academic and nonprofit users, and is available for Microsoft Windows, Linux, Apple Mac OS X, SGI IRIX, and HP Tru64 Unix from http://www.cgl.ucsf.edu/chimera/. Copyright 2004 Wiley Periodicals, Inc.
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            Nanobodies: natural single-domain antibodies.

            Sera of camelids contain both conventional heterotetrameric antibodies and unique functional heavy (H)-chain antibodies (HCAbs). The H chain of these homodimeric antibodies consists of one antigen-binding domain, the VHH, and two constant domains. HCAbs fail to incorporate light (L) chains owing to the deletion of the first constant domain and a reshaped surface at the VHH side, which normally associates with L chains in conventional antibodies. The genetic elements composing HCAbs have been identified, but the in vivo generation of these antibodies from their dedicated genes into antigen-specific and affinity-matured bona fide antibodies remains largely underinvestigated. However, the facile identification of antigen-specific VHHs and their beneficial biochemical and economic properties (size, affinity, specificity, stability, production cost) supported by multiple crystal structures have encouraged antibody engineering of these single-domain antibodies for use as a research tool and in biotechnology and medicine.
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              Origin and evolution of the adaptive immune system: genetic events and selective pressures.

              The adaptive immune system (AIS) in mammals, which is centred on lymphocytes bearing antigen receptors that are generated by somatic recombination, arose approximately 500 million years ago in jawed fish. This intricate defence system consists of many molecules, mechanisms and tissues that are not present in jawless vertebrates. Two macroevolutionary events are believed to have contributed to the genesis of the AIS: the emergence of the recombination-activating gene (RAG) transposon, and two rounds of whole-genome duplication. It has recently been discovered that a non-RAG-based AIS with similarities to the jawed vertebrate AIS - including two lymphoid cell lineages - arose in jawless fish by convergent evolution. We offer insights into the latest advances in this field and speculate on the selective pressures that led to the emergence and maintenance of the AIS.
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                Author and article information

                Contributors
                serge.muyldermans@vub.be
                Journal
                FEBS J
                FEBS J
                10.1111/(ISSN)1742-4658
                FEBS
                The Febs Journal
                John Wiley and Sons Inc. (Hoboken )
                1742-464X
                1742-4658
                28 August 2020
                April 2021
                : 288
                : 7 ( doiID: 10.1111/febs.v288.7 )
                : 2084-2102
                Affiliations
                [ 1 ] Cellular and Molecular Immunology Vrije Universiteit Brussel Belgium
                [ 2 ] Liaoning Key Laboratory of Molecular Recognition and Imaging School of Bioengineering Dalian University of Technology China
                Author notes
                [*] [* ] Correspondence

                S. Muyldermans, Cellular and Molecular Immunology, Vrije Universiteit Brussel, Pleinlaan 2, 1050 Brussels, Belgium

                Tel: + 32‐2‐629‐1969

                E‐mail: serge.muyldermans@ 123456vub.be

                Article
                FEBS15515
                10.1111/febs.15515
                8048825
                32780549
                ac80946a-35ad-421b-85c9-b55827ef11aa
                © 2020 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies

                This is an open access article under the terms of the http://creativecommons.org/licenses/by/4.0/ License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

                Page count
                Figures: 3, Tables: 1, Pages: 19, Words: 13901
                Product
                Funding
                Funded by: Agentschap voor Innovatie door Wetenschap en Technologie , open-funder-registry 10.13039/501100003132;
                Funded by: Fonds Wetenschappelijk Onderzoek , open-funder-registry 10.13039/501100003130;
                Funded by: Innoviris , open-funder-registry 10.13039/501100004744;
                Categories
                A Guide To…
                A Guide To…
                Custom metadata
                2.0
                April 2021
                Converter:WILEY_ML3GV2_TO_JATSPMC version:6.0.2 mode:remove_FC converted:15.04.2021

                Molecular biology
                immune libraries,naïve libraries,nanobodies,single‐domain antibodies,synthetic libraries

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