NaLi-H1: A universal synthetic library of humanized nanobodies providing highly functional antibodies and intrabodies
- Institut Curie, France
- Inserm, UMR 1037-CRCT, France
- CRCT, France
- 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
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.
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
-
- 23,810
- views
-
- 4,414
- downloads
-
- 252
- citations
Views, downloads and citations are aggregated across all versions of this paper published by eLife.
Download links
Further reading
-
- Cell Biology
Niemann–Pick disease type C (NPC) is a devastating lysosomal storage disease characterized by abnormal cholesterol accumulation in lysosomes. Currently, there is no treatment for NPC. Transcription factor EB (TFEB), a member of the microphthalmia transcription factors (MiTF), has emerged as a master regulator of lysosomal function and promoted the clearance of substrates stored in cells. However, it is not known whether TFEB plays a role in cholesterol clearance in NPC disease. Here, we show that transgenic overexpression of TFEB, but not TFE3 (another member of MiTF family) facilitates cholesterol clearance in various NPC1 cell models. Pharmacological activation of TFEB by sulforaphane (SFN), a previously identified natural small-molecule TFEB agonist by us, can dramatically ameliorate cholesterol accumulation in human and mouse NPC1 cell models. In NPC1 cells, SFN induces TFEB nuclear translocation via a ROS-Ca2+-calcineurin-dependent but MTOR-independent pathway and upregulates the expression of TFEB-downstream genes, promoting lysosomal exocytosis and biogenesis. While genetic inhibition of TFEB abolishes the cholesterol clearance and exocytosis effect by SFN. In the NPC1 mouse model, SFN dephosphorylates/activates TFEB in the brain and exhibits potent efficacy of rescuing the loss of Purkinje cells and body weight. Hence, pharmacological upregulating lysosome machinery via targeting TFEB represents a promising approach to treat NPC and related lysosomal storage diseases, and provides the possibility of TFEB agonists, that is, SFN as potential NPC therapeutic candidates.
-
- Cell Biology
- Developmental Biology
A study in mice reveals key interactions between proteins involved in fibroblast growth factor signaling and how they contribute to distinct stages of eye lens development.
