Sandrine Moutel | Institut Curie (original) (raw)

Papers by Sandrine Moutel

Les anticorps (Ac) monoclonaux sont maintenant des outils couramment utilises aussi bien pour le ... more Les anticorps (Ac) monoclonaux sont maintenant des outils couramment utilises aussi bien pour le diagnostic que pour la recherche fondamentale dans l’etude ou la purification des proteines. Avec le developpement de l’ingenierie genetique des Ac, il a ete possible de cloner des regions variables d’anticorps humains et d’obtenir des banques de grande diversite. Au laboratoire, un investissement particulier a ete fait ces dernieres annees pour adapter les methodes de selection d'anticorps recombinants (rAc) aux questions de la biologie cellulaire. La technique du phage display que nous utilisons pour cribler notre banque de scFv, nous a permis d’obtenir de nombreux anticorps notamment contre des echantillons complexes comme des membranes intactes de Golgi ou encore des rAc sensibles aux changements conformationnels indispensables pour nos recherches. Ces scFv ont ete selectionnes rapidement a moindre frais et sans avoir recours au passage par l’animal. Malgre le nombre incroyable de publications decrivant des scFv ou d’autres formats d’rAc, ils n’ont toujours pas reussi a s’imposer comme veritables outils alternatifs aux Ac naturels dans les laboratoires de recherche academiques. Nous avons donc developpe notre propre systeme afin de simplifier et d’ameliorer la production d’rAc. Nous avons choisi de les remettre dans un contexte d’Immunoglobuline en leur ajoutant une portion Fc pour les dimeriser et de les produire dans des cellules de mammiferes. Nous avons choisi le format appele minibody, il differe de la structure d’une IgG classique par l’abscence des domaines CH1 et Ckappa, permettant de facilement manipuler l’rAc qui reste sous forme monocatenaire. Pour tous les scFv obtenus au laboratoire, le format minbody devient tres robuste. Outre la dimerisation, ce systeme est versatile, on a pu aisement remplacer le Fc humain par un Fc de lapin ou un Fc de souris. Nous avons aussi developpe une nouvelle methode de selection d'rAc totalement in vitro. Cette methode de selection in vitro d’rAc offre tout d’abord l’avantage d’obtenir des Ac difficiles voire impossibles a selectionner par immunisation d’animaux , avec des quantites d’Ag de l’orde du µg. L’Ag peut etre une proteine tres conservee au cours de l’evolution, toxique, etre sous une forme native ou dans une conformation d’interet. Cette methode nous a permis de preparer les Ag cibles sans passer par l'expression et la purification chez E. Coli. Deux cribles ont ete realises avec succes, un contre la GFP pour demontrer la faisabilite du systeme et un en collaboration avec l’equipe de Philippe Benaroch contre Tsg101. Pour ces etudes, nous avons developpe des methodes permettant d'obtenir des outils uniques, dont nous etendons maintenant les applications, notamment au domaine therapeutique. En effet, un avantage lie a ces techniques de selection in vitro est le fait de disposer simultanement de la sequence nucleotidique codant pour ces rAc entierement humains. Dans le cadre d’un travail collaboratif, nous nous sommes interesses aux applications therapeutiques qui peuvent decouler de telles molecules. Le but du projet est d’evaluer l’efficacite de notre rAc chimerique anti-Tn en immunotherapie passive in vivo dans des modeles precliniques de souris greffees par des lignees epitheliales tumorales et d’evaluer la potentialite de son utilisation chez l’homme. C’est un tres bon marqueur diagnostic en histologie et aussi pronostic car son expression est correlee avec le grade de la tumeur Dans tous les tissus Tn est masque, et il est present sur la membrane cellulaire dans la majorite des carcinomes (90%), ce qui en fait une cible de choix pour l'immunotherapie. .

bioRxiv (Cold Spring Harbor Laboratory), Oct 24, 2019

Human mononuclear phagocytes comprise several subsets of dendritic cells (DCs), monocytes and mac... more Human mononuclear phagocytes comprise several subsets of dendritic cells (DCs), monocytes and macrophages. Distinguishing one population from another is challenging, especially in inflammed tissues, due to the promiscuous expression of phenotypic markers. Using a synthetic library of humanized llama single domain antibodies, we identified a novel surface marker for human naturally-occuring monocyte-derived DCs. Our antibody targets an extra-cellular domain of LSP-1, specifically on monocyte-derived DCs, but not on monocytes, macrophages or classical DCs. Using this antibody, we provide evidence that the recently described blood DC3 population does not correspond to circulating monocyte-derived DCs. Our findings will pave the way for a better characterization of human mononuclear phagocytes in pathological settings. .

Science, Nov 28, 2008

Microtubules display dynamic instability, with alternating phases of growth and shrinkage separat... more Microtubules display dynamic instability, with alternating phases of growth and shrinkage separated by catastrophe and rescue events. The guanosine triphosphate (GTP) cap at the growing end of microtubules, whose presence is essential to prevent microtubule catastrophes in vitro, has been difficult to observe in vivo. We selected a recombinant antibody that specifically recognizes GTP-bound tubulin in microtubules and found that GTP-tubulin was indeed present at the plus end of growing microtubules. Unexpectedly, GTP-tubulin remnants were also present in older parts of microtubules, which suggests that GTP hydrolysis is sometimes incomplete during polymerization. Observations in living cells suggested that these GTP remnants may be responsible for the rescue events in which microtubules recover from catastrophe.

iScience, Apr 1, 2020

Human mononuclear phagocytes comprise several subsets of dendritic cells (DCs), monocytes, and ma... more Human mononuclear phagocytes comprise several subsets of dendritic cells (DCs), monocytes, and macrophages. Distinguishing one population from another is challenging, especially in inflamed tissues, owing to the promiscuous expression of phenotypic markers. Using a synthetic library of humanized llama single domain antibodies, we identified a novel surface marker for human naturally occurring monocyte-derived DCs. Our antibody targets an extra-cellular domain of LSP-1, specifically on monocyte-derived DCs, but not on other leukocytes, in particular monocytes, macrophages, classical DCs, or the recently described blood DC3 population. Our findings will pave the way for a better characterization of human mononuclear phagocytes in pathological settings.

eLife, Jul 19, 2016

In vitro selection of antibodies allows to obtain highly functional binders, rapidly and at lower... more 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.

Rhabdomyosarcoma (RMS) is an aggressive pediatric soft tissue sarcoma and is classified into two ... more Rhabdomyosarcoma (RMS) is an aggressive pediatric soft tissue sarcoma and is classified into two main histopathological subtypes: embryonal RMS (eRMS), characterized by different genomic changes or alveolar RMS (aRMS), driven by the oncogenic fusion protein PAX3-FOXO1. The significant toxicity associated with conventional chemotherapies represents a major complication in pediatric oncology. To improve current therapies, we adopted two different strategies targeting fibroblast growth factor receptors (FGFR) in RMS. FGFR1-4 are a family of transmembrane receptor tyrosine kinases. Their activation upon binding of fibroblast growth factors (FGF) triggers pro-survival and proliferative signals. Our goal is to deliver drugs specifically to the tumor site by taking advantage of FGFR4 overexpression in RMS. To this end, we will covalently link FGFR4 specific nanobodies to the surface of liposomal vincristine in order to actively target RMS cells. We have established the optimal conditions to formulate liposomes loaded with vincristine. Following nanobody phage display selection on recombinant FGFR4 we focused on the top scoring candidates. Flow cytometry analysis on FGFR4-expressing versus FGFR4 knock-out RMS cell lines showed receptor-specific binding of three nanobodies. In activation assays with the FGFR4 specific growth factor FGF19, we demonstrated that the three binding candidates also blocked downstream ERK activation in RMS cells. We will now assess their theranostic potential on drug-loaded and fluorescently labeled nanovesicles on RMS tumor cells in vitro and in xenografts in vivo. Surprisingly, we observed change in cell morphology followed by cell death upon exposure to FGF2 in a subset of cultured cells established from eRMS patient-derived xenografts. Inappropriate expression of FGFRs and FGF signaling is implicated in tumor progression and therefore our findings appear contradictory. Dose-response experiments have shown that FGFR inhibition with small molecule inhibitors completely rescued FGF2 toxicity. In contrast, however, we detected high expression levels of FGFR1, 2 and 4 as well as activating mutations of FGFR4 in FGF2-sensitive eRMS cells. Therefore, our results are of upmost clinical relevance since genetically-based drug selection could lead to an inappropriate treatment inducing tumor promoting conditions. Hence, our second goal is to further unravel the molecular mechanism underlying the toxic effect of FGF-2 in a subset of eRMS tumors to avoid potentially harmful treatments. In summary, we have identified FGFR4 specific nanobodies that bind to the receptor and block downstream signaling in RMS cells. Active drug delivery of liposomal vincristine to the tumor site has the potential to enhance the therapeutic impact and decrease side effects. Moreover, we discovered a toxic effect of FGF2 in a subgroup of eRMS patient derived xenograft cells which might open new avenues for treatment. Citation Format: Nagjie Alijaj, Sandrine Moutel, Maxim Gray, Maurizio Roveri, Gabriele Manzella, Marco Wachtel, Franck Perez, Beat Schäfer, Michele Bernasconi. Targeting fibroblast growth factor receptors in rhabdomyosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3116.

Biotechnology Journal, Mar 1, 2008

Meeting report: “Antibodies‐Europe. Engineering the Next Generation of Antibodies”News from acade... more Meeting report: “Antibodies‐Europe. Engineering the Next Generation of Antibodies”News from academia:FEBS/EMBO Women in Science Award 2008Teaching biotech: The stem cell game

BMC Biotechnology, Feb 26, 2009

Background: Genomic, transcriptomic and proteomic projects often suffer from a lack of functional... more Background: Genomic, transcriptomic and proteomic projects often suffer from a lack of functional validation creating a strong demand for specific and versatile antibodies. Antibody phage display represents an attractive approach to select rapidly in vitro the equivalent of monoclonal antibodies, like single chain Fv antibodies, in an inexpensive and animal free way. However, so far, recombinant antibodies have not managed to impose themselves as efficient alternatives to natural antibodies. Results: We developed a series of vectors that allow one to easily fuse single chain Fv antibodies to Fc domains of immunoglobulins, improving their sensitivity and facilitating their use. This series enables the fusion of single chain Fv antibodies with human, mouse or rabbit Fc so that a given antibody is no longer restricted to a particular species. This opens up unlimited multiplexing possibilities and gives additional value to recombinant antibodies. We also show that this multi-Fc species production system can be applied to natural monoclonal antibodies cloned as single chain Fv antibodies and we converted the widely used 9E10 mouse anti-Myc-tag antibody into a human and a rabbit antibody. Conclusion: Altogether, this new expression system, that brings constant quality, sensitivity and unique versatility, will be important to broaden the use of recombinant and natural monoclonal antibodies both for laboratory and diagnosis use.

British Journal of Haematology, Mar 1, 1998

Neutrophils from 13 children who received G‐CSF for the collection of peripheral blood progenitor... more Neutrophils from 13 children who received G‐CSF for the collection of peripheral blood progenitors while they were in haematological steady state were studied at various times after G‐CSF injection for FcγR expression (FcγRI or CD 64, FcγRII or CD32, and FcγRIII or CD16) and for their ability to exert antibody‐dependent cell cytotoxicity (ADCC) through FcγRI. Changes in IFNγ, IL8, IL10, MCP1 and TNFα mRNA levels in peripheral blood cells were also studied 4 h and 24 h after the first G‐CSF injection. FcγRI expression increased strongly after 24 h and then remained at the same level throughout treatment. In contrast, FcγRIII expression sharply decreased at day 1 and diminished even further thereafter. No change in FcγRII was observed. ADCC exerted by neutrophils through FcγRI started to increase after 24 h with the peak level at day 5. Cytokine mRNA analyses indicated a reproducible and strong increase of IL8 mRNA (11/13 children) after 24 h, whereas the changes in the mRNA levels of the other cytokines tested were more heterogenous (IFNγ: three; IL10: six; MCP1: five; TNFα: four, of the 13 children). Therefore this study opens the way to an optimized therapeutic schedule for the combined use of G‐CSF and monoclonal antibodies in adjuvant immuno‐intervention.

Humana Press eBooks, 2012

Intracellularly expressed recombinant antibodies, or intrabodies, are powerful tools for cell bio... more Intracellularly expressed recombinant antibodies, or intrabodies, are powerful tools for cell biology studies as well as therapeutic applications. Cell biologists use them to either block the intracellular antibody target or to image endogenous target dynamics. We describe here methods to select recombinant antibodies from antibody phage display libraries and to subsequently express them as fluorescent intrabodies.

Biotechnology Journal, 2009

Antibodies are essential for the identification and characterization of proteins. In the current ... more Antibodies are essential for the identification and characterization of proteins. In the current postgenomic era the need for highly specific antibodies has further increased not only for research applications but also because they represent one of the most promising therapeutic options, especially in the field of cancer treatment. One appealing approach for rapid and inexpensive antibody generation is the use of phage display. This technique allows for a fast and animal‐free selection of highly functional alternatives to classical antibodies. However, one strong limitation of this recombinant approach has been the difficulty in producing and purifying antigens. These steps have to be adjusted for each new target, are time consuming and sometimes present an insurmountable obstacle. Here we report the development of new antibody selection approach where antigens are produced through in vitro translation and are used directly and without the need for purification. With this approach we were able to rapidly select recombinant antibodies directed against GFP and the mammalian protein tsg101, respectively. We believe that our method greatly facilitates antigen preparation and thus may broaden the use of the recombinant approach for antibody generation, especially since the technique could in the future be adapted to a high‐throughput technology, thus further accelerating antibody selection.

Biochemical and Biophysical Research Communications, Dec 1, 2017

Whole-cell biopanning with a synthetic phage display library of nanobodies enabled the recovery o... more Whole-cell biopanning with a synthetic phage display library of nanobodies enabled the recovery of follicle-stimulating hormone receptor inhibitors,

Microbial Cell Factories, 2014

Background: The isolation of recombinant antibody fragments from displayed libraries represents a... more Background: The isolation of recombinant antibody fragments from displayed libraries represents a powerful alternative to the generation of IgGs using hybridoma technology. The selected antibody fragments can then be easily engineered into (multi)-tagged constructs of variable mass and complexity as well as reconstituted into Camelidae IgG-like molecules when expressed fused to Fc domains. Nevertheless, all antibody constructs depend on an oxidizing environment for correct folding and consequently still belong to the proteins difficult to express in bacteria. In such organisms they are mostly produced at low yields in the periplasmic space. Results: We demonstrate that fusion constructs of recombinant antibodies in combination with multiple tags can be produced at high yields and totally functional in the cytoplasm of bacteria expressing sulfhydryl oxidase. The method was applied to structurally demanding molecules such as VHHs fused to SNAP and Fc domains and was validated using the antibody-derived reagents in a variety of immune techniques (FACS, ELISA, WB, IP, SPR, and IF). Conclusions: The collected data demonstrate the feasibility of a method that establishes a totally new approach for producing rapidly and inexpensively functional Camelidae IgG-like monoclonal antibodies and antibody-based reagents containing multiple disulfide bonds and suitable for both basic research and clinical applications.

M S-medecine Sciences, Dec 1, 2009

Methods in molecular biology, 2018

ABSTRACT Intracellularly expressed recombinant antibodies, or intrabodies, are powerful tools for... more ABSTRACT Intracellularly expressed recombinant antibodies, or intrabodies, are powerful tools for cell biology studies as well as therapeutic applications. Cell biologists use them to either block the intracellular antibody target or to image endogenous target dynamics. We describe here methods to select recombinant antibodies from antibody phage display libraries and to subsequently express them as fluorescent intrabodies.

Science, May 9, 2003

Here we report an approach, based on antibody phage display, to generate molecular conformation s... more Here we report an approach, based on antibody phage display, to generate molecular conformation sensors. Recombinant antibodies specific to the guanosine triphosphate (GTP)–bound conformation of the small guanosine triphosphatase (GTPase) Rab6, a regulator of membrane traffic, were generated and used to locate Rab6·GTP in fixed cells, and, after green fluorescent protein (GFP) tagging and intracellular expression, to follow Rab6·GTP in vivo. Rab6 was in its GTP-bound conformation on the Golgi apparatus and transport intermediates, and the geometry of transport intermediates was modulated by Rab6 activity. More generally, the same approach could be applied to other molecules that can be locked in a particular conformation in vitro.