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Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Anticorpos são moléculas de grande interesse científico e farmacêutico, principalmente, devido a sua alta especificidade contra antígenos determinados. Atualmente, anticorpos monoclonais estão entre os medicamentos (biofármacos) mais vendidos do mundo. São utilizados para o tratamento das mais diversas doenças, como câncer, retinopatias, doenças inflamatórias e do sistema imune, entre outras. Nos últimos 30 anos, as tecnologias para a obtenção de anticorpos monoclonais evoluíram muito, desde a tecnologia do hibridoma, até os processos de humanização de anticorpos murinos. Entre os métodos mais utilizados para a produção de anticorpos humanos, destaca-se a tecnologia do Phage Display. Nesta técnica, os genes que codificam as regiões variáveis de imunoglobulinas são inseridos no genoma de um bacteriófago, resultando na produção de partículas virais híbridas que contém fragmentos de anticorpos em fusão com uma das proteínas do capsídeo viral. Neste trabalho, desenvolvemos novos vetores para a apresentação de fragmentos ScFv em fusão com duas proteínas das proteínas do capsídeo viral, a pIII e pVIII. Os oligonucleotídeos utilizados para amplificar os genes de imunoglobulinas foram redesenhados e para minimizar a perda do repertório durante a produção da biblioteca, avaliamos em bancos de dados enzimas de restrição que não apresentam sítios de restrição nas sequencias gênicas. Esses sítios de restrição foram utilizados para construir as regiões de clonagem do vetor Phagemid. Outra etapa crítica na produção de bibliotecas de anticorpos é a reação do PCR de overlap, que pode restringir a diversidade de anticorpos e resultar na produção de amplicons codificando anticorpos truncados. Por isso, nossos vetores foram desenhados para permitir a clonagem direta das regiões variáveis das imunoglobulinas humanas ou murinas, sem a necessidade do PCR de overlap. Nossa expectativa, é que estes novos reagentes serão mais efetivos para a produção de novas bibliotecas de anticorpos pelo sistema do Phage Display. / Antibodies are molecules of great scientific and pharmaceutical interest, mainly because of their high specificity against certain antigens. Currently, monoclonal antibodies are among the best selling drugs (biopharmaceuticals) in the world. They are used for the treatment of the most diverse disorders, such as cancer, retinopathies, inflammatory and immune system diseases, among others. In the past 30 years, technologies for obtaining monoclonal antibodies has greatly evolved from hybridoma technology to the humanization processes of murine antibodies. Among the methods used for the production of human antibodies, the technology of Phage Display stands out. In this technique, the genes encoding the immunoglobulin variable regions are inserted into the genome of a bacteriophage, resulting in the production of hybrid virus particles which contain fragments of antibodies in fusion with one of the viral capsid proteins. In this work, we developed new vectors for the presentation of ScFv fragments in fusion with two proteins of viral capsid proteins, pIII and pVIII. The oligonucleotides used to amplify the immunoglobulin genes were redesigned and to minimize repertory loss during library production, we evaluated restriction enzymes in databases that lack restriction sites in the gene sequences. These restriction sites were used to construct the cloning regions of the Phagemid vector. Another critical step in the production of antibody libraries is the overlap PCR reaction, which may restrict the diversity of antibodies and result in the production of amplicons encoding truncated antibodies. Therefore, our vectors were designed to allow the direct cloning of human or murine Immunoglobulins variable regions without the need for overlap PCR. Our expectation is that these new reagents will be more effective for the production of new antibody libraries by the Phage Display system.

Les interactions protéine-protéine (IPPs), complexes et dynamiques, sont le cœur des réseaux protéiques cellulaires. Au niveau des synapses excitatrices, la densité post-synaptique (PSD) est un exemple typique de réseau protéique dont la structure et la composition à l’échelle nanoscopique détermine la fonction cellulaire. Ainsi, la régulation dynamique de la composition de la PSD et des mouvements des récepteurs au glutamate dans ou hors de la PSD constitue la base des théories moléculaires actuelles sur l’apprentissage et la mémoire. Dans ce contexte, durant ma thèse, j’ai étudié une classe d’IPPs faisant intervenir les domaines PDZ. En effet, durant ces dernières années, de nombreuses études ont démontré l’implication de ces interactions impliquant les domaines PDZ de la famille de PSD95 dans le ciblage synaptique et l’ancrage des récepteurs au glutamate. Cependant, en partie dû au manque d’outils adaptés, les mécanismes moléculaires sous-jacents qui contrôlent de façon dynamique leur rétention à la synapse restent mal compris. Dans le but d’étudier ces interactions impliquant des domaines PDZ, j’ai développé plusieurs stratégies de sélection par phage display basées sur l’utilisation du dixième domaine de type III de la fibronectine humaine (10Fn3) dans le but de cibler les motifs d’interaction aux domaines PDZ des récepteurs (Stargazin pour les rAMPA et GluN2A pour les rNMDA) ou les domaines PDZ eux-mêmes. En utilisant une approche multidisciplinaire, mes objectifs principaux ont été de concevoir de petits anticorps synthétiques qui nous permettront de rompre ou de stabiliser spécifiquement ces complexes protéiques, ainsi que d’observer les interactions endogènes. / Complex and dynamic protein-protein interactions are the core of protein-based networks in cells. At excitatory synapses, the postsynaptic density (PSD) is a typical example of protein-based network whose nanoscale structure and composition determines the cellular function. For instance, the dynamic regulation of PSD composition and glutamate receptors movements into or out of the PSD are the base of current molecular theories of learning and memory. In this context, during my PhD, I focused on a class of protein-protein interactions mediated by PDZ domains. Indeed, over the last decade, numerous studies have shown the critical implication of PDZ domain-mediated interactions from the PSD95 scaffolding protein family in the synaptic targeting and anchoring of glutamate receptors. However, in part due to the lack of adapted tools, the molecular mechanisms that dynamically govern their respective synaptic retention remain poorly understood. In order to investigate these PDZ domain-mediated interactions, I developed several selection strategies by phage-display based on the fibronectin type III (FN3) scaffold in order to either target the PDZ domain-binding motifs of the receptors complexes (e.g., stargazin for AMPARs and GluN2A for NMDARs) or the PDZ domains themselves. Using a multidisciplinary approach, my main objectives were to engineer small synthetic antibodies that will allow us to acutely and specifically disrupt or stabilize these protein complexes, as well as monitor endogenous interactions.

Noroviruses are the major cause of acute viral gastroenteritis worldwide. Thus, rapid and reliable pathogen detection and control are crucial to avoid epidemic outbreaks. Peptides which bind to these viruses with high specificity and affinity could serve as small and stable recognition elements in biosensing applications for a point-of-care diagnostic of noroviruses. They can be identified by screening large phage display libraries using the biopanning technique. In the present study, this method was applied to identify norovirus-binding peptide motifs. For this purpose, a biopanning based on column chromatography was established, and three rounds of selections were performed. After the second round, the cosmix-plexing recombination technique was implemented to enhance the chance of obtaining peptides with very high affinity. Biopanning data evaluation was based on next-generation sequencing (NGS), to show that this innovative method can enable a detailed analysis of the complete sequence spectrum obtained during and after biopanning. Highly enriched motifs could be characterized by their large proportion of the amino acids W, K, R, N, and F. Neighbourhood analysis was exemplarily performed for selected motifs, showing that the motifs FAT, RWN, and KWF possessed the fingerprints with the largest differences relative to the original library. This thesis thus presents next-generation sequencing-based analysis tools, which could now be transferred to any other biopanning project. The identified peptide motifs represent promising candidates for a future examination of their norovirus-specific binding. A new option for testing such phage-target interactions in the context of biopanning selections was studied in the second part of the thesis. For this purpose, a phage-based microarray was developed as a miniaturized binding assay. As a prerequisite, the different immobilization behaviour of phages on positively and negatively charged surfaces was studied, and a non-contact printing technique for bacteriophages was developed. Subsequently, the interaction of phages and antibodies directed against phage coat proteins was characterized in enzyme-linked immunosorbent assays, and the protocol was successfully transferred to the non-contact printed phage spots. At the proof-of-concept level, the phage array could finally be integrated into a microfluidic setup, showing a higher signal-to-background ratio relative to the static phage array. These results point the way towards a microfluidic phage array, allowing online monitoring, automation, and parallelisation of the phage array analysis. / Noroviren gelten als Hauptursache akuter viraler Magen-Darm-Erkrankungen. Nur eine zeitnahe und verlässliche Detektion und Kontrolle dieser Pathogene kann epidemische Ausbrüche vermeiden. Um dies zu ermöglichen, könnten Peptide, die an diese Viren mit hoher Spezifität und Affinität binden, als kleine und stabile Erkennungselemente in biosensorischen Anwendungen eingesetzt werden. Solche Peptide können mithilfe der Biopanning-Technik identifiziert werden, die auf dem Screening großer Phagen-Display-Bibliotheken beruht. In der vorliegenden Arbeit wurde diese Methode genutzt, um Norovirus-bindende Peptidmotive zu identifizieren. Dazu wurde ein auf Säulenchromatographie basierendes Biopanning entwickelt und drei Selektionsrunden durchgeführt. Die Cosmix-Plexing-Rekombinationstechnik wurde nach der zweiten Runde eingesetzt, um die Wahrscheinlichkeit der Gewinnung hochaffiner Binder zu erhöhen. Die Auswertung der Biopanningdaten erfolgte mittels Hochdurchsatzsequenzierung (Next-Generation Sequencing). Es konnte gezeigt werden, dass diese innovative Methode die detailierte Analyse des kompletten Sequenzspektrums während und nach dem Biopanning ermöglicht. Stark angereicherte Motive konnten durch ihren hohen Anteil an den Aminosäuren W, K, R, N und F charakterisiert werden. Eine Nachbarschaftsanalyse wurde exemplarisch für ausgewählte Motive durchgeführt. Dabei wurden die stärksten Unterschiede im Fingerprint im Vergleich zur Ausgangsbibliothek bei den Motiven FAT, RWN und KWF gefunden. Diese Dissertation stellt damit auf Next-Generation Sequencing basierende Analysetechniken vor, die für weitere Biopanningprojekte übernommen werden können. Die identifizierten Peptidmotive könnten als vielversprechende Kandidaten zukünftig auf ihre Norovirus-spezifische Bindung hin getestet werden. Eine neue Möglichkeit, solche Phagen-Analyt-Interaktionen zu untersuchen, wurde im zweiten Teil der Dissertation untersucht. Dafür wurde als miniaturisierter Bindungsassay ein Phagen-basiertes Mikroarray entwickelt. Als Voraussetzung wurde zunächst das unterschiedliche Immobilisierungsverhalten von Bakteriophagen auf positiv und negativ geladenen Oberflächen untersucht und eine kontaktfreie Drucktechnik für Bakteriophagen etabliert. Anschließend wurde die Interaktion von Phagen und gegen sie gerichteten Antikörpern in Enzym-gekoppelten Immunadsorptionstests charakterisiert und das Protokoll erfolgreich auf die kontaktfrei gedruckten Phagenspots übertragen. Schließlich wurde erstmals die grundsätzliche Möglichkeit gezeigt, das Array in ein mikrofluidisches Setup zu integrieren, was zu einem höheren Signal-zu-Hintergrund-Verhältnis im Vergleich zum statischen Array führte. Diese Ergebnisse zeigen damit den Weg zu einem mikrofluidischen Phagen-Array auf, das sowohl die Möglichkeit des Online-Monitorings als auch der Automatisierung und Parallelisierung der Phagen-Array-Analyse bietet.

La recherche de nouvelles molécules à visée thérapeutique ou diagnostic ciblant les récepteurs membranaires incluant les RCPGs, les récepteurs à tyrosine kinase et les canaux ioniques sont au coeur des recherches investies par les entreprises pharmaceutiques. Dans ce projet nous avons étudié et caractérisé des domaines variables de chaîne lourde d'anticorps de lamas (sdAbs) qui peuvent contourner certaines limites liées à l'utilisation des anticorps monoclonaux ou des petites molécules. En effet, de par leurs particularités structurales qui les rendent particulièrement intéressants en termes de stabilité, d'affinité et de reconnaissance d'antigène, les sdAbs représentent etre une alternative prométeuse. Dans ce manuscrit sont exposés les travaux effectués sur les récepteurs aux tyrosines kinases appartenant à la famille des ErbBs et les récepteurs au glutamate mGluRs (RCPG). En plus d'avoir sélectionné des sdAbs hautement spécifiques de ces différents antigènes, ces molécules ont également montré des caractéristiques très étonnantes et inattendues. En effet, la majeure partie des sdAbs sélectionnés présentent une spécificité pour une conformation du récepteur particulière (forme active ou inactive). Cette particularité très pertinente nous ouvre un spectre d'application très diversifié, car elle permet de cibler et d'analyser les récepteurs dans ces différents états d'activation. Ajouté à cela, certains sdAbs possèdent une activité de modulateur allostérique, voir même présentent un effet agoniste. Ces résultats très encourageants nous ouvrent de nouvelles perspectives, et ces molécules représentent une nouvelle approche pour la modulation et l'étude des ces récepteurs. / The research for new therapeutic or diagnostic molecules targeting membrane receptors, including GPCRs, tyrosine kinase receptors and ion channels are the heart of the research invested by pharmaceutical companies. In this project we used the variable domain of llama antibody heavy chain also called single domain antibodies (sdAbs) that can bypass some limitations to the use of monoclonal antibodies or small molecules. Indeed, because of their structural features that make them particularly interesting in terms of stability, affinity and antigen recognition, sdAbs represent a very promising candidates that can be used in various fiels of application: as fluorescent probes , screening tools , or therapeutic molecules. In this manuscript are exposed the work performed on the tyrosine kinases receptor belonging to the ErbBs family and metabotropic glutamate receptors, mGluRs (GPCRs).We selected very highly specific sdAbs directed against antigen of interest, but these molecules have also shown very surprising and unexpected particularities. Indeed, most of the selected sdAbs exhibit specificity for a particular conformation of the receptor (active or inactive form). This very relevant feature opens an highly diversified application spectrum, because it allows to identify and analyze these receptors in different states of activation. Added to this, some sdAbs present an allosteric modulator activity, or even present an agonist effect. These encouraging results open up new perspectives, and these molecules represent a new approach for modulation and study of these membrane receptors.

Thèse numérisée par la Division de la gestion de documents et des archives de l'Université de Montréal.

Le VIH-1 est l'agent infectieux qui cause le SIDA. De nombreuses thérapies existent pour combattre le SIDA mais aucune ne permet son éradication et des résistances apparaissent. Le développement de nouvelles thérapies est donc nécessaire. Les anticorps simple-domaine (sdAb) de lamas présentent les propriétés idéales pour le développement de molécules neutralisantes. Des lamas ont donc été immunisés avec Vpr et les formes native, ou induite par un miniCD4, du trimère de gp140 (partie extracellulaire de l'enveloppe (Env)). Des banques de sdAbs ont ensuite été construites et des sélections par phage display et par double hybride ont été réalisées. Trois sdAbs se liant au site de liaison du co- récepteur de l'Env et un sdAb se liant au site de liaison du CD4 ont ainsi été sélectionnés. Ces sites sont conservés mais difficile d'accès pour des immunoglobulines conventionnelles. Ces sdAbs ont ensuite été caractérisés par ELISA, SPR et cytométrie de flux pour leur capacité de liaison à différentes Env, et en « single round assay » pour leur capacité de neutralisation d'un large spectre (LS) de pseudovirus. Des protéines multidomaines (plusieurs sdAbs reliés par un linker) ont ensuite été construites et testées pour leur neutralisation. Plusieurs de ces molécules, neutralisant un LS de virus, pourraient être utilisées dans des microbicides. La stabilité caractéristique des sdAbs, même en absence de formation de pont disulfure, par exemple dans un environnement réducteur tel que le cytoplasme, est primordiale dans le développement d'anticorps intracellulaires (intrabodies). / HIV-1 is the infectious agent of AIDS. Numerous therapies exist to fight AIDS, but they are not able to eradicate it, and resistances appear. So, new therapy development is necessary. Single-domain antibodies (sdAb) of llamas have ideal properties to develop neutralizing molecules. So, llamas have been immunized with Vpr and with free or miniCD4 induced trimeric gp140 (extracellular part of the envelope (Env)). SdAb libraries have been built and selections were done by phage display and yeast two hybrid. Three sdAbs targeting the co-receptor binding site of the Env and one sdAb targeting the CD4 binding site have been selected. These sites are conserved but inaccessible by conventional immunoglobulins. These sdAbs have been characterized by ELISA, SPR and FACS for their ability to bind different Env and by single-round assay for their neutralization ability. Multimeric proteins (linked sdAbs) have been built and tested for their neutralization ability. Several of these molecules are able to neutralize a broad spectrum of pseudoviruses. They can be used in microbicides. The characteristic stability of these sdAbs, even without disulfide bound formation, ie into reducing environment, as the cytoplasm, is primordial for intracellular antibody (intrabody) development. One sdAb anti-Vpr has been selected using the Sos Recruitment System (SRS), an yeast two-hybrid system allowing detection of cytoplasmic protein-protein interactions. This sdAb is able to alter the localization of its antigen into eukaryotic cells. It is a proof of concept ot the use of SRS in the selection of intracellularly functional sdAbs.

O conceito de heterogeneidade vascular é bem aceito pela comunidade cientifica, desempenhando papel essencial em processos fisiológicos e patológicos. Uma vez que os vasos sanguíneos são importantes na organogênese, diferenciação e morfogênese de tecidos e órgãos, torna-se interessante desvendar a diversidade vascular cerebral, identificando novos marcadores moleculares para este órgão tão importante. Utilizando tecnologia combinatorial de phage display in vivo, identificamos um novo motivo peptídico, na qual os aminoácidos FenilalaninaArginina-Triptofano (Phe-Arg-Trp; FRW) predominam. Este motivo peptídico é um ligante seletivo para vasos sanguíneos cerebrais e não se acumula em outros órgãos, incluíndo tecidos como intestinos e gônadas, que também apresentam barreiras endoteliais especificas. No entanto, mais surpreendente foi a observação de que o motivo FRW não se liga aos vasos sanguíneos da retina, o que implica em uma diferença até então desconhecida entre duas barreiras: a barreira hematoencefálica e a barreira hematoretiniana. Combinando phage display in vivo e microscopia eletrônica de transmissão, observamos a presença de partículas de fago ligadas à vasculatura cerebral em um nível supramolecular: aglomerados de fagos filamentosos expressando o motivo FRW foram visualizados ligados às regiões de contato entre as células endoteliais. Por fim, a utilização do peptídeo CFFWKFRWMC permite imageamento in vivo, demonstrando que novas ferramentas para estudar e visualizar o cérebro podem surgir deste motivo. / The concept of vascular heterogeneity is well accepted by the scientific community, playing an essential role in physiological and pathological processes. Since blood vessels are important in organogenesis, differentiation, and morphogenesis of tissues and organs, it becomes interesting to unveil the cerebral vascular diversity, identifying new molecular markers for such important organ. Using in vivo phage display, we show that a new peptide motif that emerged from our combinatorial screening of the vasculature binds selectively to blood vessels in the brain in vivo but not to vessels in other organs. Peptides containing a conserved motif in which amino acids Phenylalanine-Arginine-Tryptophan (Phe-Arg-Trp; FRW) predominate could be visualized by transmission electron microscopy bound to the junctions between endothelial in all areas of the brain, including the optic nerve but not in other barrier containing tissues, such as intestines and testis. Remarkably, peptides containing the motif do not bind to vessels in the retina, implying an important molecular difference between these two vascular barriers. Furthermore, the peptide allows for in vivo imaging, demonstrating that new tools for studying and imaging the brain are likely to emerge from this motif.