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      NaLi-H1: A universal synthetic library of humanized nanobodies providing highly functional antibodies and intrabodies

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          Abstract

          In vitro selection of antibodies allows to obtain highly functional binders, rapidly and at lower cost. Here, we describe the first fully synthetic phage display library of humanized llama single domain antibody (NaLi-H1: Nanobody Library Humanized 1). Based on a humanized synthetic single domain antibody (hs2dAb) scaffold optimized for intracellular stability, the highly diverse library provides high affinity binders without animal immunization. NaLi-H1 was screened following several selection schemes against various targets (Fluorescent proteins, actin, tubulin, p53, HP1). Conformation antibodies against active RHO GTPase were also obtained. Selected hs2dAb were used in various immunoassays and were often found to be functional intrabodies, enabling tracking or inhibition of endogenous targets. Functionalization of intrabodies allowed specific protein knockdown in living cells. Finally, direct selection against the surface of tumor cells produced hs2dAb directed against tumor-specific antigens further highlighting the potential use of this library for therapeutic applications.

          DOI: http://dx.doi.org/10.7554/eLife.16228.001

          eLife digest

          Antibodies are proteins that form part of an animal’s immune system and can identify and help eradicate infections. These proteins are also needed at many stages in biological research and represent one of the most promising tools in medical applications, from diagnostics to treatments.

          Traditionally, antibodies have been collected from animals that had been previously injected with a target molecule that the antibodies must recognize. An alternative strategy that uses bacteria and bacteria-infecting viruses instead of animals was developed several decades ago and allows researchers to obtain antibodies more quickly. However, the majority of the scientific community view these “in vitro selected antibodies” as inferior to those produced via the more traditional approach.

          Moutel, Bery et al. set out to challenge this widespread opinion, using a smaller kind of antibody known as nanobodies. The proteins were originally found in animals like llamas and camels and are now widely used in biological research. One particularly stable nanobody was chosen to form the backbone of the in vitro antibodies, and the DNA that encodes this nanobody was altered to make the protein more similar to human antibodies. Moutel, Bery et al. then changed the DNA sequence further to make billions of different versions of the nanobody, each one slightly different from the next in the region that binds to the target molecules.

          Transferring this DNA into bacteria resulted in a library (called the NaLi-H1 library) of bacterial clones that produce the nanobodies displayed at the surface of bacteria-infecting viruses. Moutel, Bery et al. then screened this library against various target molecules, including some from tumor cells, and showed that the fully in vitro selected antibodies worked just as well as natural antibodies in a number of assays. The in vitro antibodies could even be used to track, or inactivate, proteins within living cells.

          The NaLi-H1 library will help other researchers obtain new antibodies that bind strongly to their targets. The approaches developed to create the library could also see more people decide to create their own synthetic libraries, which would accelerate the identification of new antibodies in a way that is cheaper and requires fewer experiments to be done using animals. These in vitro selected antibodies could help to advance both fundamental and medical research.

          DOI: http://dx.doi.org/10.7554/eLife.16228.002

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          Most cited references50

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          The small GTP-binding protein rho regulates the assembly of focal adhesions and actin stress fibers in response to growth factors.

          Actin stress fibers are one of the major cytoskeletal structures in fibroblasts and are linked to the plasma membrane at focal adhesions. rho, a ras-related GTP-binding protein, rapidly stimulated stress fiber and focal adhesion formation when microinjected into serum-starved Swiss 3T3 cells. Readdition of serum produced a similar response, detectable within 2 min. This activity was due to a lysophospholipid, most likely lysophosphatidic acid, bound to serum albumin. Other growth factors including PDGF induced actin reorganization initially to form membrane ruffles, and later, after 5 to 10 min, stress fibers. For all growth factors tested the stimulation of focal adhesion and stress fiber assembly was inhibited when endogenous rho function was blocked, whereas membrane ruffling was unaffected. These data imply that rho is essential specifically for the coordinated assembly of focal adhesions and stress fibers induced by growth factors.
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            Modulation of protein properties in living cells using nanobodies.

            Protein conformation is critically linked to function and often controlled by interactions with regulatory factors. Here we report the selection of camelid-derived single-domain antibodies (nanobodies) that modulate the conformation and spectral properties of the green fluorescent protein (GFP). One nanobody could reversibly reduce GFP fluorescence by a factor of 5, whereas its displacement by a second nanobody caused an increase by a factor of 10. Structural analysis of GFP-nanobody complexes revealed that the two nanobodies induce subtle opposing changes in the chromophore environment, leading to altered absorption properties. Unlike conventional antibodies, the small, stable nanobodies are functional in living cells. Nanobody-induced changes were detected by ratio imaging and used to monitor protein expression and subcellular localization as well as translocation events such as the tamoxifen-induced nuclear localization of estrogen receptor. This work demonstrates that protein conformations can be manipulated and studied with nanobodies in living cells.
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              Single domain antibodies: promising experimental and therapeutic tools in infection and immunity

              Antibodies are important tools for experimental research and medical applications. Most antibodies are composed of two heavy and two light chains. Both chains contribute to the antigen-binding site which is usually flat or concave. In addition to these conventional antibodies, llamas, other camelids, and sharks also produce antibodies composed only of heavy chains. The antigen-binding site of these unusual heavy chain antibodies (hcAbs) is formed only by a single domain, designated VHH in camelid hcAbs and VNAR in shark hcAbs. VHH and VNAR are easily produced as recombinant proteins, designated single domain antibodies (sdAbs) or nanobodies. The CDR3 region of these sdAbs possesses the extraordinary capacity to form long fingerlike extensions that can extend into cavities on antigens, e.g., the active site crevice of enzymes. Other advantageous features of nanobodies include their small size, high solubility, thermal stability, refolding capacity, and good tissue penetration in vivo. Here we review the results of several recent proof-of-principle studies that open the exciting perspective of using sdAbs for modulating immune functions and for targeting toxins and microbes.
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                Author and article information

                Contributors
                Role: Reviewing editor
                Journal
                eLife
                Elife
                eLife
                eLife
                eLife
                eLife Sciences Publications, Ltd
                2050-084X
                19 July 2016
                2016
                : 5
                Affiliations
                [1 ]Institut Curie, PSL Research University , Paris, France
                [2 ]CNRS UMR144 , Paris, France
                [3 ]deptTranslational Research Department , Institut Curie , Paris, France
                [4 ]Inserm, UMR 1037-CRCT , Toulouse, France
                [5 ]Faculté des Sciences Pharmaceutiques, Université Toulouse III-Paul Sabatier , Toulouse, France
                [6 ]Institut Claudius Regaud , Toulouse, France
                [7 ]Le Pôle Technologique du Centre de Recherches en Cancérologie de Toulouse, plateau de protéomique , Toulouse, France
                [8 ]Hybrigenics Service , Paris, France
                [9]Utrecht University , Netherlands
                [10]Utrecht University , Netherlands
                Author notes
                [†]

                These authors contributed equally to this work.

                Article
                16228
                10.7554/eLife.16228
                4985285
                27434673
                8a8ee3a5-d82b-4f64-b61d-6b6e5900f405
                © 2016, Moutel et al

                This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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                Funding
                Funded by: FundRef http://dx.doi.org/10.13039/501100001665, Agence Nationale de la Recherche;
                Award ID: ANR-09-BIOT-05
                Award Recipient :
                Funded by: FundRef http://dx.doi.org/10.13039/501100001677, Institut National de la Santé et de la Recherche Médicale;
                Award Recipient :
                Funded by: Groupe de Recherche of the Claudius Regaud Institute;
                Award Recipient :
                Funded by: FundRef http://dx.doi.org/10.13039/501100001677, Institut National de la Santé et de la Recherche Médicale;
                Award ID: ITS-201103
                Award Recipient :
                Funded by: IDEX Paris Sciences Lettres;
                Award ID: ANR-10-IDEX-0001-02 PSL
                Award Recipient :
                Funded by: LABEX CellTisPhyBio;
                Award ID: 11-LBX-0038
                Award Recipient :
                Funded by: FundRef http://dx.doi.org/10.13039/501100004794, Centre National de la Recherche Scientifique;
                Award Recipient :
                Funded by: Institut Curie;
                Award Recipient :
                Funded by: FundRef http://dx.doi.org/10.13039/501100002915, Fondation pour la Recherche Médicale;
                Award ID: DEQ20120323723
                Award Recipient :
                Funded by: Aviesan;
                Award ID: Project TAbIP
                Award Recipient :
                The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
                Categories
                Cell Biology
                Immunology
                Tools and Resources
                Custom metadata
                2.5
                A fully synthetic library of humanized single domain antibodies yields in vitro high affinity antibodies usable in cell biology and translational projects.

                Life sciences
                recombinant antibodies,phage display,intrabodies,synthetic library,e. coli,human,mouse
                Life sciences
                recombinant antibodies, phage display, intrabodies, synthetic library, e. coli, human, mouse

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