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

  1. Sandrine Moutel
  2. Nicolas Bery
  3. Virginie Bernard
  4. Laura Keller
  5. Emilie Lemesre
  6. Ario de Marco
  7. Laetitia Ligat
  8. Jean-Christophe Rain
  9. Gilles Favre
  10. Aurélien Olichon  Is a corresponding author
  11. Franck Perez  Is a corresponding author
  1. Institut Curie, France
  2. Inserm, UMR 1037-CRCT, France
  3. CRCT, France
  4. Hybrigenics Service SA, France

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.

Article and author information

Author details

  1. Sandrine Moutel

    Institut Curie, Paris, France
    Competing interests
    Sandrine Moutel, Co-inventor on a patent application (filled under ref: WO/2015/063331) that covers the hs2dAb scaffold and the commercial use of the library. The library has been licensed to Hybrigenics Service SA, which will perform screens on a fee-for-service basis.A consultant for Hybrigenics Service SA.
  2. Nicolas Bery

    Inserm, UMR 1037-CRCT, Toulouse, France
    Competing interests
    No competing interests declared.
  3. Virginie Bernard

    Institut Curie, Paris, France
    Competing interests
    No competing interests declared.
  4. Laura Keller

    Inserm, UMR 1037-CRCT, Toulouse, France
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-1786-9760
  5. Emilie Lemesre

    Institut Curie, Paris, France
    Competing interests
    No competing interests declared.
  6. Ario de Marco

    Institut Curie, Paris, France
    Competing interests
    No competing interests declared.
  7. Laetitia Ligat

    plateau de protéomique, CRCT, Toulouse, France
    Competing interests
    No competing interests declared.
  8. Jean-Christophe Rain

    Hybrigenics Service SA, Paris, France
    Competing interests
    Jean-Christophe Rain, Employed by, and a stockholder in, Hybrigenics Service SA.
  9. Gilles Favre

    Inserm, UMR 1037-CRCT, Toulouse, France
    Competing interests
    No competing interests declared.
  10. Aurélien Olichon

    Inserm, UMR 1037-CRCT, Toulouse, France
    For correspondence
    aurelien.olichon@inserm.fr
    Competing interests
    Aurélien Olichon, Co-inventor on a patent application (filled under ref: WO/2015/063331) that covers the hs2dAb scaffold and the commercial use of the library. The library has been licensed to Hybrigenics Service SA, which will perform screens on a fee-for-service basis.
  11. Franck Perez

    Institut Curie, Paris, France
    For correspondence
    Franck.Perez@curie.fr
    Competing interests
    Franck Perez, Co-inventor on a patent application (filled under ref: WO/2015/063331) that covers the hs2dAb scaffold and the commercial use of the library. The library has been licensed to Hybrigenics Service SA, which will perform screens on a fee-for-service basis.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-9129-9401

Funding

Institut Curie

  • Sandrine Moutel
  • Virginie Bernard
  • Ario de Marco
  • Franck Perez

Centre National de la Recherche Scientifique

  • Sandrine Moutel
  • Franck Perez

Institut National de la Santé et de la Recherche Médicale

  • Nicolas Bery
  • Laura Keller
  • Laetitia Ligat
  • Gilles Favre
  • Aurélien Olichon

Agence Nationale de la Recherche (ANR-09-BIOT-05)

  • Sandrine Moutel
  • Jean-Christophe Rain
  • Franck Perez

Institut National de la Santé et de la Recherche Médicale (ITS-201103)

  • Ario de Marco
  • Franck Perez

Fondation pour la Recherche Médicale (DEQ20120323723)

  • Franck Perez

Groupe de Recherche of the Claudius Regaud Institute

  • Gilles Favre
  • Aurélien Olichon

LABEX CellTisPhyBio (11-LBX-0038)

  • Sandrine Moutel
  • Virginie Bernard
  • Ario de Marco
  • Franck Perez

IDEX Paris Sciences Lettres (ANR-10-IDEX-0001-02 PSL)

  • Sandrine Moutel
  • Virginie Bernard
  • Ario de Marco
  • Franck Perez

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Reviewing Editor

  1. Anna Akhmanova, Utrecht University, Netherlands

Publication history

  1. Received: March 20, 2016
  2. Accepted: July 18, 2016
  3. Accepted Manuscript published: July 19, 2016 (version 1)
  4. Version of Record published: August 15, 2016 (version 2)

Copyright

© 2016, Moutel et al.

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

Metrics

  • 17,632
    Page views
  • 3,337
    Downloads
  • 147
    Citations

Article citation count generated by polling the highest count across the following sources: Crossref, Scopus, PubMed Central.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Sandrine Moutel
  2. Nicolas Bery
  3. Virginie Bernard
  4. Laura Keller
  5. Emilie Lemesre
  6. Ario de Marco
  7. Laetitia Ligat
  8. Jean-Christophe Rain
  9. Gilles Favre
  10. Aurélien Olichon
  11. Franck Perez
(2016)
NaLi-H1: A universal synthetic library of humanized nanobodies providing highly functional antibodies and intrabodies
eLife 5:e16228.
https://doi.org/10.7554/eLife.16228

Further reading

    1. Cell Biology
    2. Structural Biology and Molecular Biophysics
    Ritvija Agrawal et al.
    Research Article Updated

    Dynein harnesses ATP hydrolysis to move cargo on microtubules in multiple biological contexts. Dynein meets a unique challenge in meiosis by moving chromosomes tethered to the nuclear envelope to facilitate homolog pairing essential for gametogenesis. Though processive dynein motility requires binding to an activating adaptor, the identity of the activating adaptor required for dynein to move meiotic chromosomes is unknown. We show that the meiosis-specific nuclear-envelope protein KASH5 is a dynein activating adaptor: KASH5 directly binds dynein using a mechanism conserved among activating adaptors and converts dynein into a processive motor. We map the dynein-binding surface of KASH5, identifying mutations that abrogate dynein binding in vitro and disrupt recruitment of the dynein machinery to the nuclear envelope in cultured cells and mouse spermatocytes in vivo. Our study identifies KASH5 as the first transmembrane dynein activating adaptor and provides molecular insights into how it activates dynein during meiosis.

    1. Cell Biology
    2. Developmental Biology
    Juan Lu et al.
    Research Article Updated

    Phosphatidylinositol 4-phosphate (PI4P) and phosphatidylinositol 4,5-biphosphate (PIP2) are key phosphoinositides that determine the identity of the plasma membrane (PM) and regulate numerous key biological events there. To date, mechanisms regulating the homeostasis and dynamic turnover of PM PI4P and PIP2 in response to various physiological conditions and stresses remain to be fully elucidated. Here, we report that hypoxia in Drosophila induces acute and reversible depletion of PM PI4P and PIP2 that severely disrupts the electrostatic PM targeting of multiple polybasic polarity proteins. Genetically encoded ATP sensors confirmed that hypoxia induces acute and reversible reduction of cellular ATP levels which showed a strong real-time correlation with the levels of PM PI4P and PIP2 in cultured cells. By combining genetic manipulations with quantitative imaging assays we showed that PI4KIIIα, as well as Rbo/EFR3 and TTC7 that are essential for targeting PI4KIIIα to PM, are required for maintaining the homeostasis and dynamic turnover of PM PI4P and PIP2 under normoxia and hypoxia. Our results revealed that in cells challenged by energetic stresses triggered by hypoxia, ATP inhibition and possibly ischemia, dramatic turnover of PM PI4P and PIP2 could have profound impact on many cellular processes including electrostatic PM targeting of numerous polybasic proteins.