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Article ; Online: A cytoplasmic protein-protein interaction detection method based on reporter translation.

Renaut, Laurence / Bouayadi, Khalil / Kharrat, Hakim / Mondon, Philippe

2009 Volume 384, Issue 2, Page(s) 362–364

Abstract: One approach to drug discovery involves the targeting of abnormal protein-protein interactions that lead to pathology. We present a new technology allowing the detection of such interactions within the cytoplasm in a yeast-based system. The interaction ... ...

Abstract	One approach to drug discovery involves the targeting of abnormal protein-protein interactions that lead to pathology. We present a new technology allowing the detection of such interactions within the cytoplasm in a yeast-based system. The interaction detection is based on the sequestration of a translation termination factor involved in stop codon recognition. This sequestration inhibits the activity of the factor, thereby permitting the translation of a reporter gene harboring a premature stop codon. This novel cytoplasmic protein-protein interaction (CPPI) detection system should prove to be useful in the characterization of proteins as well as in partner identification, interaction mapping, and drug discovery applications.
MeSH term(s)	Cytoplasm/metabolism ; Genes, Reporter/genetics ; Lac Operon/genetics ; Models, Biological ; Peptide Chain Termination, Translational/genetics ; Protein Interaction Mapping/methods ; Saccharomyces cerevisiae/genetics ; Saccharomyces cerevisiae/metabolism
Language	English
Publishing date	2009-01-15
Publishing country	United States
Document type	Journal Article
ZDB-ID	1110-1
ISSN	1096-0309 ; 0003-2697
ISSN (online)	1096-0309
ISSN	0003-2697
DOI	10.1016/j.ab.2008.10.011
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Article: Human antibody libraries: a race to engineer and explore a larger diversity.

Mondon, Philippe / Dubreuil, Olivier / Bouayadi, Khalil / Kharrat, Hakim

Frontiers in bioscience : a journal and virtual library

2008 Volume 13, Page(s) 1117–1129

Abstract: Several recombinant antibody libraries associated with different screening technologies have been generated since the first steps of antibody engineering 15 years ago, in order to isolate human monoclonal antibodies. In this race to isolate antibody with ...

Abstract	Several recombinant antibody libraries associated with different screening technologies have been generated since the first steps of antibody engineering 15 years ago, in order to isolate human monoclonal antibodies. In this race to isolate antibody with virtually any specificity, innovative strategies have been developed to clone natural antibody repertoires or to increase library diversity beyond the scope of the immune system. After the in vitro transfer of the natural diversity, the second generation of partly or completely man-designed libraries was based on the available structural data of the antibody binding. Efficient selection strategies have proven critical in exploiting the potential of a library's diversity. The development and improvement of screening methods such as phage display, yeast display, ribosome display and robotic platforms have provided innovative tools to efficiently screen and sort out the desired binding specificities of billions of antibodies. Efforts to improve diversity exploration have been mainly focused on screening conditions of display techniques and the new emerging techniques. Here we review some of these prominent approaches in the field of human recombinant antibody libraries.
MeSH term(s)	Animals ; Antibodies, Monoclonal/chemistry ; Antibody Affinity ; Binding Sites, Antibody ; Biochemistry/methods ; Fungal Proteins/chemistry ; Humans ; Hybridomas/metabolism ; Immune System/pathology ; Immunoglobulin Fab Fragments ; Peptide Library ; Protein Engineering/methods ; Ribosomes/chemistry
Chemical Substances	Antibodies, Monoclonal ; Fungal Proteins ; Immunoglobulin Fab Fragments ; Peptide Library
Language	English
Publishing date	2008-01-01
Publishing country	United States
Document type	Journal Article ; Review
ZDB-ID	2141320-4
ISSN	1093-9946
ISSN	1093-9946
DOI	10.2741/2749
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Article: Selection of IgG Variants with Increased FcRn Binding Using Random and Directed Mutagenesis: Impact on Effector Functions.

Monnet, Céline / Jorieux, Sylvie / Urbain, Rémi / Fournier, Nathalie / Bouayadi, Khalil / De Romeuf, Christophe / Behrens, Christian K / Fontayne, Alexandre / Mondon, Philippe

Frontiers in immunology

2015 Volume 6, Page(s) 39

Abstract: Despite the reasonably long half-life of immunoglogulin G (IgGs), market pressure for higher patient convenience while conserving efficacy continues to drive IgG half-life improvement. IgG half-life is dependent on the neonatal Fc receptor (FcRn), which ... ...

Abstract	Despite the reasonably long half-life of immunoglogulin G (IgGs), market pressure for higher patient convenience while conserving efficacy continues to drive IgG half-life improvement. IgG half-life is dependent on the neonatal Fc receptor (FcRn), which among other functions, protects IgG from catabolism. FcRn binds the Fc domain of IgG at an acidic pH ensuring that endocytosed IgG will not be degraded in lysosomal compartments and will then be released into the bloodstream. Consistent with this mechanism of action, several Fc-engineered IgG with increased FcRn affinity and conserved pH dependency were designed and resulted in longer half-life in vivo in human FcRn-transgenic mice (hFcRn), cynomolgus monkeys, and recently in healthy humans. These IgG variants were usually obtained by in silico approaches or directed mutagenesis in the FcRn-binding site. Using random mutagenesis, combined with a pH-dependent phage display selection process, we isolated IgG variants with improved FcRn-binding, which exhibited longer in vivo half-life in hFcRn mice. Interestingly, many mutations enhancing Fc/FcRn interaction were located at a distance from the FcRn-binding site validating our random molecular approach. Directed mutagenesis was then applied to generate new variants to further characterize our IgG variants and the effect of the mutations selected. Since these mutations are distributed over the whole Fc sequence, binding to other Fc effectors, such as complement C1q and FcγRs, was dramatically modified, even by mutations distant from these effectors' binding sites. Hence, we obtained numerous IgG variants with increased FcRn-binding and different binding patterns to other Fc effectors, including variants without any effector function, providing distinct "fit-for-purpose" Fc molecules. We therefore provide evidence that half-life and effector functions should be optimized simultaneously as mutations can have unexpected effects on all Fc receptors that are critical for IgG therapeutic efficacy.
Language	English
Publishing date	2015
Publishing country	Switzerland
Document type	Journal Article
ZDB-ID	2606827-8
ISSN	1664-3224
ISSN	1664-3224
DOI	10.3389/fimmu.2015.00039
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Article ; Online: Mutagen: a random mutagenesis method providing a complementary diversity generated by human error-prone DNA polymerases.

Mondon, Philippe / Grand, David / Souyris, Nathalie / Emond, Stéphane / Bouayadi, Khalil / Kharrat, Hakim

Methods in molecular biology (Clifton, N.J.)

2010 Volume 634, Page(s) 373–386

Abstract: Random mutagenesis is one of the most effective methodologies to generate variant libraries for directed protein evolution. Indeed, this approach requires no structural or mechanistic information and can uncover unexpected beneficial mutations. Here, we ... ...

Abstract	Random mutagenesis is one of the most effective methodologies to generate variant libraries for directed protein evolution. Indeed, this approach requires no structural or mechanistic information and can uncover unexpected beneficial mutations. Here, we describe a new random mutagenesis method based on the use of human error-prone DNA polymerases (pol beta, pol eta and pol iota). This approach allows the random introduction of mutations through a single replication step followed by a selective PCR amplification of the replicated mutated sequences. The libraries generated using this methodology display different mutation rates and complementary mutational spectra. By taking advantage of the mutation bias of naturally highly error-prone DNA polymerases, MutaGen thus appears as a very useful tool for gene and protein randomization.
MeSH term(s)	DNA-Directed DNA Polymerase/genetics ; Humans ; Mutagenesis
Chemical Substances	DNA-Directed DNA Polymerase (EC 2.7.7.7)
Language	English
Publishing date	2010
Publishing country	United States
Document type	Journal Article
ISSN	1940-6029
ISSN (online)	1940-6029
DOI	10.1007/978-1-60761-652-8_26
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Article ; Online: Affinity maturation of antibodies: optimized methods to generate high-quality ScFv libraries and isolate IgG candidates by high-throughput screening.

Renaut, Laurence / Monnet, Céline / Dubreuil, Olivier / Zaki, Ouafa / Crozet, Fabien / Bouayadi, Khalil / Kharrat, Hakim / Mondon, Philippe

Methods in molecular biology (Clifton, N.J.)

2012 Volume 907, Page(s) 451–461

Abstract: As a growing number of therapeutic antibodies are developed, robust methods to efficiently improve the affinity and/or specificity of antibody candidates are needed. Here we describe our powerful platform that combines scFv affinity maturation and IgG ... ...

Abstract	As a growing number of therapeutic antibodies are developed, robust methods to efficiently improve the affinity and/or specificity of antibody candidates are needed. Here we describe our powerful platform that combines scFv affinity maturation and IgG high-throughput screening. After creating diversity with our random mutagenesis technology (MutaGen™), the scFv libraries are fully cleaned using a fusion system introducing the beta-lactamase gene to select in-frame and stop codon free variants on the basis of ampicillin resistance. The high-quality scFv libraries thereby constructed are then selected on the target in vitro using phage display technology. Contrary to standard procedures, instead of producing a limited number of affinity matured scFv as IgG molecules, we developed a cloning system to directly transfer the entire pool of selected scFv into an IgG expression vector permitting rapid IgG small-scale production (96 wells) in mammalian cells. Our integrated process allows us to generate high-quality scFv libraries and test numerous IgG variants, increasing the chances to select the best therapeutic antibody candidate.
MeSH term(s)	Ampicillin/pharmacology ; Antibody Affinity/drug effects ; Antibody Affinity/immunology ; Cell Line ; Cloning, Molecular ; Genetic Vectors/genetics ; High-Throughput Screening Assays/methods ; Humans ; Immunoglobulin G/biosynthesis ; Immunoglobulin G/genetics ; Immunoglobulin G/isolation & purification ; Open Reading Frames/genetics ; Peptide Library ; Single-Chain Antibodies/biosynthesis
Chemical Substances	Immunoglobulin G ; Peptide Library ; Single-Chain Antibodies ; Ampicillin (7C782967RD)
Language	English
Publishing date	2012
Publishing country	United States
Document type	Journal Article
ISSN	1940-6029
ISSN (online)	1940-6029
DOI	10.1007/978-1-61779-974-7_26
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Article ; Online: Method for generation of human hyperdiversified antibody fragment library.

Mondon, Philippe / Souyris, Nathalie / Douchy, Laurent / Crozet, Fabien / Bouayadi, Khalil / Kharrat, Hakim

Biotechnology journal

2007 Volume 2, Issue 1, Page(s) 76–82

Abstract: The selection of antibody fragments from libraries using in vitro screening technologies has proven to be a very good alternative to the classical hybridoma technology, and has overcome the laborious process of antibody humanization. However, the ... ...

Abstract	The selection of antibody fragments from libraries using in vitro screening technologies has proven to be a very good alternative to the classical hybridoma technology, and has overcome the laborious process of antibody humanization. However, the complexity of the library is critical in the probability of being able to directly isolate a high affinity antibody specific to a target. We report a method to make hyperdiversified antibody fragment libraries, based on human immunoglobulin variable genes mimicking the somatic hypermutation process. This mutagenesis technology, MutaGen, was used for the first time on the entire variable domain (frameworks and CDRs) of large repertoires of human variable antibody domains. Our MutaGen process uses low-fidelity human polymerases, known as mutases, suggested to be involved in the somatic hypermutation process of immunoglobulin genes. Depending on the mutases used, we generated complementary mutation patterns with randomly distributed mutations. The libraries were generated with an average of 1.8 mutations per 100 amino acids. The hyperdiversified antibody fragment libraries constructed with our process should enable the selection of antibody fragments specific to virtually any target.
MeSH term(s)	Antibodies/genetics ; Antibodies/immunology ; Antibodies/metabolism ; B-Lymphocytes/immunology ; Drug Delivery Systems/methods ; Humans ; Immunoglobulin Fragments/genetics ; Immunoglobulin Fragments/immunology ; Immunoglobulin Fragments/metabolism ; Mutation ; Peptide Library ; Protein Engineering/methods
Chemical Substances	Antibodies ; Immunoglobulin Fragments ; Peptide Library
Language	English
Publishing date	2007-01
Publishing country	Germany
Document type	Journal Article ; Research Support, Non-U.S. Gov't ; Technical Report
ZDB-ID	2221885-3
ISSN	1860-7314 ; 1860-6768
ISSN (online)	1860-7314
ISSN	1860-6768
DOI	10.1002/biot.200600205
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Article ; Online: Combined glyco- and protein-Fc engineering simultaneously enhance cytotoxicity and half-life of a therapeutic antibody.

Monnet, Céline / Jorieux, Sylvie / Souyris, Nathalie / Zaki, Ouafa / Jacquet, Alexandra / Fournier, Nathalie / Crozet, Fabien / de Romeuf, Christophe / Bouayadi, Khalil / Urbain, Rémi / Behrens, Christian K / Mondon, Philippe / Fontayne, Alexandre

mAbs

2014 Volume 6, Issue 2, Page(s) 422–436

Abstract: While glyco-engineered monoclonal antibodies (mAbs) with improved antibody-dependent cell-mediated cytotoxicity (ADCC) are reaching the market, extensive efforts have also been made to improve their pharmacokinetic properties to generate biologically ... ...

Abstract	While glyco-engineered monoclonal antibodies (mAbs) with improved antibody-dependent cell-mediated cytotoxicity (ADCC) are reaching the market, extensive efforts have also been made to improve their pharmacokinetic properties to generate biologically superior molecules. Most therapeutic mAbs are human or humanized IgG molecules whose half-life is dependent on the neonatal Fc receptor FcRn. FcRn reduces IgG catabolism by binding to the Fc domain of endocytosed IgG in acidic lysosomal compartments, allowing them to be recycled into the blood. Fc-engineered mAbs with increased FcRn affinity resulted in longer in vivo half-life in animal models, but also in healthy humans. These Fc-engineered mAbs were obtained by alanine scanning, directed mutagenesis or in silico approach of the FcRn binding site. In our approach, we applied a random mutagenesis technology (MutaGen™) to generate mutations evenly distributed over the whole Fc sequence of human IgG1. IgG variants with improved FcRn-binding were then isolated from these Fc-libraries using a pH-dependent phage display selection process. Two successive rounds of mutagenesis and selection were performed to identify several mutations that dramatically improve FcRn binding. Notably, many of these mutations were unpredictable by rational design as they were located distantly from the FcRn binding site, validating our random molecular approach. When produced on the EMABling(®) platform allowing effector function increase, our IgG variants retained both higher ADCC and higher FcRn binding. Moreover, these IgG variants exhibited longer half-life in human FcRn transgenic mice. These results clearly demonstrate that glyco-engineering to improve cytotoxicity and protein-engineering to increase half-life can be combined to further optimize therapeutic mAbs.
MeSH term(s)	Animals ; Antibodies, Monoclonal/genetics ; Antibodies, Monoclonal/pharmacokinetics ; Antibody-Dependent Cell Cytotoxicity/genetics ; Cell Surface Display Techniques ; Cytotoxicity, Immunologic/genetics ; Glycosylation ; Half-Life ; Histocompatibility Antigens Class I/genetics ; Histocompatibility Antigens Class I/metabolism ; Humans ; Immunoglobulin G/genetics ; Immunoglobulin G/metabolism ; Immunotherapy/methods ; Immunotherapy/trends ; Mice ; Mice, Transgenic ; Mutagenesis, Site-Directed ; Mutation/genetics ; Protein Engineering/methods ; Receptors, Fc/genetics ; Receptors, Fc/metabolism ; Receptors, IgG/antagonists & inhibitors ; Receptors, IgG/immunology ; Receptors, IgG/metabolism
Chemical Substances	Antibodies, Monoclonal ; FCGR3A protein, human ; Fc gamma receptor IIA ; Histocompatibility Antigens Class I ; Immunoglobulin G ; Receptors, Fc ; Receptors, IgG ; Fc receptor, neonatal (TW3XAW0RCY)
Language	English
Publishing date	2014-01-15
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2537838-7
ISSN	1942-0870 ; 1942-0870
ISSN (online)	1942-0870
ISSN	1942-0870
DOI	10.4161/mabs.27854
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Article ; Online: Alternative-splicing-based bicistronic vectors for ratio-controlled protein expression and application to recombinant antibody production.

Fallot, Stéphanie / Ben Naya, Raouia / Hieblot, Corinne / Mondon, Philippe / Lacazette, Eric / Bouayadi, Khalil / Kharrat, Abdelhakim / Touriol, Christian / Prats, Hervé

Nucleic acids research

2009 Volume 37, Issue 20, Page(s) e134

Abstract: In the last decade polycistronic vectors have become essential tools for both basic science and gene therapy applications. In order to co-express heterologous polypeptides, different systems have been developed from Internal Ribosome Entry Site (IRES) ... ...

Abstract	In the last decade polycistronic vectors have become essential tools for both basic science and gene therapy applications. In order to co-express heterologous polypeptides, different systems have been developed from Internal Ribosome Entry Site (IRES) based vectors to the use of the 2A peptide. Unfortunately, these methods are not fully suitable for the efficient and reproducible modulation of the ratio between the proteins of interest. Here we describe a novel bicistronic vector type based on the use of alternative splicing. By modifying the consensus sequence that governs splicing, we demonstrate that the ratio between the synthesized proteins could easily vary from 1 : 10 to 10 : 1. We have established this system with luciferase genes and we extended its application to the production of recombinant monoclonal antibodies. We have shown that these vectors could be used in several typical cell lines with similar efficiencies. We also present an adaptation of these vectors to hybrid alternative splicing/IRES constructs that allow a ratio-controlled expression of proteins of interest in stably transfected cell lines.
MeSH term(s)	Alternative Splicing ; Animals ; Antibodies, Monoclonal/biosynthesis ; Antibodies, Monoclonal/genetics ; Cricetinae ; Genetic Vectors ; Humans ; Luciferases/analysis ; Polyribosomes/metabolism ; RNA Splice Sites ; RNA, Messenger/metabolism ; Recombinant Proteins/biosynthesis ; Recombinant Proteins/genetics ; Transfection
Chemical Substances	Antibodies, Monoclonal ; RNA Splice Sites ; RNA, Messenger ; Recombinant Proteins ; Luciferases (EC 1.13.12.-)
Language	English
Publishing date	2009-09-03
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	186809-3
ISSN	1362-4962 ; 1362-4954 ; 0301-5610 ; 0305-1048
ISSN (online)	1362-4962 ; 1362-4954
ISSN	0301-5610 ; 0305-1048
DOI	10.1093/nar/gkp716
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Zs.A 1067: Show issues

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Article ; Online: Combinatorial engineering to enhance thermostability of amylosucrase.

Emond, Stéphane / André, Isabelle / Jaziri, Kais / Potocki-Véronèse, Gabrielle / Mondon, Philippe / Bouayadi, Khalil / Kharrat, Hakim / Monsan, Pierre / Remaud-Simeon, Magali

Protein science : a publication of the Protein Society

2008 Volume 17, Issue 6, Page(s) 967–976

Abstract: Amylosucrase is a transglucosidase that catalyzes amylose-like polymer synthesis from sucrose substrate. About 60,000 amylosucrase variants from two libraries generated by the MutaGen random mutagenesis method were submitted to an in vivo selection ... ...

Abstract	Amylosucrase is a transglucosidase that catalyzes amylose-like polymer synthesis from sucrose substrate. About 60,000 amylosucrase variants from two libraries generated by the MutaGen random mutagenesis method were submitted to an in vivo selection procedure leading to the isolation of more than 7000 active variants. These clones were then screened for increased thermostability using an automated screening process. This experiment yielded three improved variants (two double mutants and one single mutant) showing 3.5- to 10-fold increased half-lives at 50 degrees C compared to the wild-type enzyme. Structural analysis revealed that the main differences between wild-type amylosucrase and the most improved variant (R20C/A451T) might reside in the reorganization of salt bridges involving the surface residue R20 and the introduction of a hydrogen-bonding interaction between T451 of the B' domain and D488 of flexible loop 8. This double mutant is the most thermostable amylosucrase known to date and the only one usable at 50 degrees C. At this temperature, amylose synthesis by this variant using high sucrose concentration (600 mM) led to the production of amylose chains twice as long as those obtained by the wild-type enzyme at 30 degrees C.
MeSH term(s)	Base Sequence ; Combinatorial Chemistry Techniques ; DNA Primers ; Enzyme Stability ; Glucosyltransferases/chemistry ; Glucosyltransferases/genetics ; Glucosyltransferases/metabolism ; Hot Temperature ; Models, Molecular ; Mutagenesis ; Protein Conformation ; Protein Engineering
Chemical Substances	DNA Primers ; Glucosyltransferases (EC 2.4.1.-) ; amylosucrase (EC 2.4.1.4)
Language	English
Publishing date	2008-04-25
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	1106283-6
ISSN	1469-896X ; 0961-8368
ISSN (online)	1469-896X
ISSN	0961-8368
DOI	10.1110/ps.083492608
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Article: Alternative-splicing-based bicistronic vectors for ratio-controlled protein expression and application to recombinant antibody production

Fallot, StÃ©phanie / Ben Naya, Raouia / Hieblot, Corinne / Mondon, Philippe / Lacazette, Eric / Bouayadi, Khalil / Kharrat, Abdelhakim / Touriol, Christian / Prats, HervÃ©

Nucleic acids research. 2009 Nov., v. 37, no. 20

2009

Abstract	In the last decade polycistronic vectors have become essential tools for both basic science and gene therapy applications. In order to co-express heterologous polypeptides, different systems have been developed from Internal Ribosome Entry Site (IRES) based vectors to the use of the 2A peptide. Unfortunately, these methods are not fully suitable for the efficient and reproducible modulation of the ratio between the proteins of interest. Here we describe a novel bicistronic vector type based on the use of alternative splicing. By modifying the consensus sequence that governs splicing, we demonstrate that the ratio between the synthesized proteins could easily vary from 1 : 10 to 10 : 1. We have established this system with luciferase genes and we extended its application to the production of recombinant monoclonal antibodies. We have shown that these vectors could be used in several typical cell lines with similar efficiencies. We also present an adaptation of these vectors to hybrid alternative splicing/IRES constructs that allow a ratio-controlled expression of proteins of interest in stably transfected cell lines.
Keywords	alternative splicing ; antibody formation ; consensus sequence ; gene therapy ; genes ; hybrids ; luciferase ; monoclonal antibodies ; nucleic acids ; polypeptides ; protein synthesis ; recombinant antibodies ; ribosomes
Language	English
Dates of publication	2009-11
Size	p. e134.
Document type	Article
ZDB-ID	186809-3
ISSN	0301-5610 ; 0305-1048
ISSN	0301-5610 ; 0305-1048
DOI	10.1093/nar/gkp716
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