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.

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Most cited references 50

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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.

A J Ridley, A. Hall (1992)

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.

Axel Kirchhofer, Jonas Helma, Katrin Schmidthals … (2010)

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

Janusz Wesolowski, Vanina Alzogaray, Jan Reyelt … (2009)

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

Journal

Journal ID (iso-abbrev): Elife

Title: eLife

Publisher: eLife Sciences Organisation, Ltd.

ISSN (Electronic): 2050-084X

ISSN (Print): 2050-084X

Publication date (Electronic): Jul 19 2016

Volume: 5

Affiliations

[1 ] Institut Curie, PSL Research University, Paris, France.

[2 ] CNRS UMR144, Paris, France.

[3 ] Translational 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.

Article

DOI: 10.7554/eLife.16228

PMC ID: 4985285

PubMed ID: 27434673

SO-VID: 8a8ee3a5-d82b-4f64-b61d-6b6e5900f405

History

Keywords: E. coli,cell biology,human,immunology,intrabodies,mouse,phage display,recombinant antibodies,synthetic library

Data availability:

Keywords: E. coli, cell biology, human, immunology, intrabodies, mouse, phage display, recombinant antibodies, synthetic library

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

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Most cited references 50

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

Modulation of protein properties in living cells using nanobodies.

Single domain antibodies: promising experimental and therapeutic tools in infection and immunity

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