Dissertations / Theses on the topic 'Nanobody'

The myc family of oncogenic transcription factors, which includes c-myc, N-myc and L-myc, control major cellular processes such as proliferation and differentiation by integrating upstream signals and orchestrating global gene transcription. They do this largely through dimerising with Max, which together bind to enhancer (E)-box elements in DNA. Myc proteins function similarly but differ in potency and tissue distribution. For instance, N-myc is expressed predominantly during development in undifferentiated cells of the nervous system, whereas c-myc is ubiquitously expressed in all proliferating cells. Myc proteins, when deregulated, are major drivers of tumourigenesis. Myc deregulation occurs in up to 70% of all human cancers and is often associated with the most aggressive forms. For example, MYCN, the gene encoding N-myc, is amplified in 20-30% of neuroblastomas, and amplification strongly correlates with advanced stage and poor prognosis. Myc proteins are therefore considered “most wanted” targets for cancer therapy, but have long been considered undruggable mainly due to challenges in nuclear drug delivery and physically targeting myc directly given that it is a largely disordered protein that lacks discernible clefts and pockets for small molecules to inhabit. Furthermore, c-myc is important in normal tissue maintenance so the effect of its inhibition in humans is difficult to predict. However, recent in vivo studies showed that systemic myc inhibition (using the peptide pan myc inhibitor Omomyc) has mild and reversible side effects and induces tumour regression. This has alleviated concerns about the side effects that myc inhibition might have, and reinforced the promise of myc as a powerful drug target. However, the translation of Omomyc into the clinic has been hindered by poor cellular delivery. In fact, no direct myc inhibitor has yet been approved, indicating that novel approaches are needed. Moreover, inhibitors in development tend to inhibit all myc family proteins. An inhibitor that could specifically target N-myc might improve safety through bypassing c-myc inhibition. This could be used for the treatment of N-myc-driven cancers such as MYCN-amplified neuroblastoma. Nanobodies, camelid-derived single-domain antibodies, are a relatively new drug class. Whilst some are already in clinical trials for a wide range of diseases, these are specific for cell-surface or extracellular targets. However, their properties also make them ideal for use as intracellular antibodies or ‘intrabodies’. For example, they are small (just 12-15 kDa) and highly soluble due to naturally occurring hydrophobic to hydrophilic amino acid substitutions. Their small size and convex shape makes them advantageous in capturing structures in intrinsically disordered proteins and allows them to reach hidden epitopes not accessible to conventional antibodies, which could improve biological activity. Importantly, nanobodies retain the high specificities and affinities of conventional antibodies. Their small, single-domain nature also means they can be engineered with ease to modify aspects of their localisation and/or function. For example, they can be coupled to carrier molecules to facilitate cellular entry, and a nuclear localisation signal (NLS) can be added to drive them into the nucleus. Also, it was recently shown that an F-box domain could also be incorporated into nanobodies to recruit degradation machinery to its antigen, which depletes the antigen from cells via the proteasomal degradation pathway. Due to their highly advantageous properties, nanobodies raised against N-myc might overcome the barriers to targeting N-myc, providing potent and specific means of directly inhibiting N-myc therapeutically, which has not yet been achieved. In this thesis, nine unique nanobodies were raised against N-myc. These included three against the basic helix-loop-helix leucine zipper (bHLH-LZ) domain where Max dimerises, and six against the transactivation domain where numerous regulatory and cofactor proteins bind, such as the E3 ubiquitin ligase Skp2. Nanobodies against the transactivation domain were more specific for N-myc and were shown to inhibit its Skp-2-mediated ubiquitylation. This could provide novel means of eradicating tumours based on a study showing that inhibition of ubiquitylation at this domain triggers a transcriptional ‘switch’ that induces a non-canonical target gene Egr1, leading to p53-independent apoptosis. A nanobody against the bHLH-LZ (Nb C2) was shown to bind both N- and c-myc to similar magnitudes. Its affinity for N-myc bHLH-LZ was superior to that of the small molecule myc inhibitor 10058-F4, which prolongs survival in a MYCN-dependent mouse model of high-risk neuroblastoma. Nb C2 spontaneously transduced cell membranes and its coupling to a novel small molecule carrier (SMoC) enhanced its cellular uptake. Furthermore, the addition of a NLS increased its nuclear localisation. Preliminary experiments showed that Nb C2 might slow proliferation and induce apoptosis in cancer cell lines expressing c-myc, suggesting that Nb C2 might also be effective against cancers characterised by deregulated c-myc. Taken together, data generated in this thesis have revealed intriguing findings that provide a basis for the development of these nanobodies for the treatment of N-myc- and c-myc-driven cancers.

Current single molecule localisation microscopy methods allow for multicolour imaging of macromolecules in cells, and for a degree quantification on molecule numbers in one colour. However, that has not yet been an attempt to develop tools capable of quantitative imaging with multiple colours in cells. This work addressed this challenge by designing linker peptides with chemospecific groups to allow attachment of activator and emitter dyes for STORM imaging, and a targeting module. The design ensured a stoichiometric ratio of targeting module to activator and emitter dyes. Peptides with HaloTag ligands attached were labelled with various activator and emitter pairs and used to label HaloTag fusions of S. pombe and mouse embryonic stem cells. These peptides were found to bind non-specifically to various areas of both cell types, and did not localise to HaloTag protein, whereas controls did. Another peptide was also labelled with activator-emitter pairs and attached to expressed anti-GFP and ant-mCherry nanobodies via native chemical ligation. The labelled anti-GFP nanobody was to demonstrate ensemble and single molecule imaging in S. pombe, as well as characterisation on single molecule surfaces in comparison to a conventional randomly labelled antibody. The stoichiometrically labelled nanobody had a more consistent number of photons detected per localisation, number of localisation per molecule and number of blinks per molecule, which implied that it could be more useful than randomly labelled nanobodies for counting experiments. It was also shown to be capable of specific laser activation for STORM imaging with both an Alexa405Cy5 and Cy3Cy5 pairs. These anti-GFP and anti-mCherry nanobodies and peptide linker are new tools for both counting and multicolour imaging in super-resolution, which could be widely applied to constructs that are already tagged with GFP or mCherry.

I have been successful in improving and developing a homemade device based on a three-electrodes electrochemical redout setup thanks to it, we are able to perform measurements of DNA-hybridization, in real-time, from probe ssDNA-SAMs coupled to gold coated sensor surfaces. The measurements were carried out, in pure saline buffer solution, on a large range of concentrations of complementary-DNA strands (from 1 pM to 100 nM), monitoring the differential capacitance at the Working Electrode versus the incubation time. The studies on kinetics, modeled using the Langmuir adsorption model, not only give us important information on the kinetics itself but they allow us to detect eventual mismatches along the DNA-sequence target proving to be sensitive to the position of the mismatch with respect to the surface of the device or to define, in human extract and plasma, the unknown concentration of a specific miRNA-target connected to the heart failure taking into account the hindrances carried by the Argonaute proteins in which the miRNA are inglobed. This goal was achieved by performing a calibration curve on experiments of DNA/DNA hybridization performed in a simple saline buffer. The results were then confirmed using a real time qPCR by our partners in MD D. Cesselli's and MD A.P. Beltrami's group at University of Udine. Another strand of my PhD project concerns the detection of more complex components such as proteins or single-domain antibodies (e.g. VHH fragments)–DNA conjugates, with the final purpose of the detection of circulating tumor cells (CTCs) not only in pure saline buffer but also in human serum. In particular, we have focused on the detection of the protein HER2 whose overexpression is connected to certain aggressive types of breast cancer. In addition, the systematic characterization of the device caught our attention, and it was developed by performing measurements of Self Assembled Monolayer (SAM) detection, carried out in different physiological buffers (KCl, NaCl, MgCl2, PBS, etc.), in different probe-density conditions and applying different potential in order to have a more comprehensive understanding of the phenomena occurring at the electrode/electrolyte interface. Studies that have led to the implementation of a theoretical model, able to provide an acceptable physical explanation of the biorecognition events of interest.

Les camélidés possèdent une caractéristique immunologique particulière parmi les mammifères. En plus des anticorps conventionnels tétramériques composés de 2 chaînes lourdes et de 2 chaînes légères, on retrouve dans des proportions variant de 25 à 50% des anticorps dépourvus de chaînes légères. Le paratope de ces anticorps est dès lors constitué de la partie variable monomérique des chaînes lourdes. Ce domaine d’environ 15 kDa représente le plus petit fragment capable de lier un antigène et est communément appelé nanobody de par sa petite taille. Les nanobodies possèdent des propriétés uniques considérables comparés aux anticorps conventionnels, comme leur capacité à reconnaître des épitopes cryptiques mais aussi la possibilité de les modifier et les assembler facilement afin d’améliorer leurs propriétés. Ces dernières années, les nanobodies ont connu un intérêt grandissant tant au niveau de la recherche fondamentale qu’au niveau du développement de nouvelles solutions diagnostiques et thérapeutiques. Grâce à leur utilisation, la biologie structurale des RCPGs a connu des avancées significatives avec notamment l’obtention de la structure du récepteur β2-adrénergique dans une conformation active et complexé à une protéine G hétérotrimérique. Les RCPGs représentent la plus grande famille de récepteurs membranaires avec près de 800 récepteurs différents. Ils sont exprimés dans toutes les cellules de l’organisme et répondent à une large variété de ligands, les rendant indispensables dans la régulation de nombreux processus physiologiques. Ce rôle central dans la modulation des fonctions biologiques fait des RCPGs des cibles thérapeutiques de premier choix, comme en atteste le pourcentage élevé (30 à 40%) de médicaments dirigés contre cette classe de récepteurs et actuellement sur le marché. Depuis quelques années maintenant, la biologie structurale des RCPGs a connu un essor sans précédent avec à ce jour, près de 190 structures tridimensionnelles expérimentales résolues. Ces avancées ont permis de mieux comprendre les mécanismes d’action de ces récepteurs ainsi que le mode de liaison de ligands, ouvrant notamment de nouvelles perspectives thérapeutiques par le développement rationnel de nouvelles molécules.Au cours de ce travail, nous nous sommes efforcés de développer des outils et une méthodologie nous permettant de résoudre la structure expérimentale de 2 récepteurs :ChemR23 et VPAC1. Pour cela, nous avons développé et caractérisé des nanobodies dirigés contre ces 2 récepteurs. Nous avons montré que les nanobodies dirigés contre le récepteur ChemR23 possèdent des propriétés antagonistes en inhibant partiellement la libération calcique de cellules CHO surexprimant ChemR23 ainsi que le chimiotactisme de cellules dendritiques induit par la chémérine. Profitant de la modularité offerte par les nanobodies, nous avons conçu un nanobody bivalent, dont les propriétés antagonistes sont significativement améliorées. Concernant le récepteur VPAC1, nous avons identifié que les nanobodies générés reconnaissent un épitope présent au niveau du large domaine amino-terminal et distinct du site orthostérique du peptide VIP. Bien que dépourvu de propriétés fonctionnelles, 2 de ces nanobodies voient leur affinité augmentée en présence d’un agoniste, et diminué en présence d’un agoniste inverse. Enfin, nous montrons qu’ils sont utilisables pour la détection du récepteur endogène présent à la surface de leucocytes mais également au niveau de coupes de tissus gastro-intestinaux sains.En parallèle, nous avons mis au point la production de ces récepteurs dans des cellules d’insecte, permettant de produire les quantités nécessaires à des études structurales. Nous avons également apporté et validé diverses modifications à la structure de ces récepteurs, en vue d’augmenter leur stabilité une fois extraits de leur environnement natif. Un processus itératif nous a permis de déterminer les conditions optimales de solubilisation de ces récepteurs afin de maximiser l’obtention d’une forme monomérique et de minimiser la présence de formes multimériques ou dégradées. Nos premiers essais de purification par chromatographie d’affinité sur colonnes de nickel, ainsi que par chromatographie d’exclusion de taille, nous ont permis d’isoler des récepteurs entiers. Cependant, les chromatogrammes issus des purifications par chromatographie d’exclusion de taille suggèrent la présence de récepteurs en partie agrégés. De plus, nous n’avons pu déterminer précisément à ce jour si les récepteurs purifiés maintenaient une conformation native, prérequis indispensable pour réaliser des études cristallographiques.Bien que nous n’ayons pas résolu la structure expérimentale de ces 2 récepteurs, le travail réalisé dans le cadre de notre thèse de doctorat a permis de développer des nanobodies qui représentent des outils innovants pour l’études des RCPGs ainsi que de mettre au point des protocoles de production et de purification préliminaire des récepteurs ChemR23 et VPAC1 en vue de leur étude cristallographique.
Doctorat en Sciences biomédicales et pharmaceutiques (Médecine)
info:eu-repo/semantics/nonPublished

L’identité et le devenir de chaque cellule dépend du contenu en protéines et, en particulier, des réseaux d'interactions protéine-protéine (IPP, également appelés interactomes). Les protéines ont la propriété générale de s'engager dans des assemblages macromoléculaires très variés, chacun ayant des fonctions bien distinctes. Par conséquent, identifier les IPP et les lier à des complexes particuliers est un enjeu crucial mais difficile en biologie. Cette problématique a été au cœur de mon travail de doctorat. Une première partie de mon travail est dédiée à l'amélioration d'une méthode existante pour capturer de nouvelles IPP dans le contexte de fonctions biologiques définies. Ce travail a été réalisé avec ERK1, un régulateur clé en aval de plusieurs voies de signalisation impliquées dans de nombreux cancers. Les nouveaux outils ont été testés dans le contexte de fonctions de ERK1 sensibles à deux molécules inhibitrices dans les cellules humaines HEK293T. Une interaction a été confirmée aux niveaux fonctionnel et moléculaire, ainsi qu’en utilisant une stratégie d'imagerie originale pour accéder à la dynamique des IPP dans les cellules vivantes. La deuxième partie de mon travail de doctorat est dédiée à l'établissement d'une méthodologie pionnière pour capturer les IPP endogènes établies par un complexe protéique dimérique spécifique dans les cellules humaines vivantes. Cette méthodologie couple la Complémentation de Fluorescence Bimoléculaire (BiFC) et les technologies démarquage par la biotine de proximité. Plus précisément, elle repose sur l’utilisation d’un petit anticorps (appelé aussi « nanobody ») dirigé contre le complexe BiFC et fusionné à la ligase biotine TurboID. Ces outils ont été établis avec les complexes TAZ/14-3-3e et TAZ/TEAD2, qui traduisent respectivement l'activité de la voie de signalisation Hippo dans le cytoplasme et le noyau. Notre approche a permis de capturer les interactomes spécifiques de ces deux complexes protéiques et d'identifier un nouveau régulateur clé du complexe TAZ/14-3-3e pour contrôler ses fonctions de prolifération cellulaire. Dans son ensemble, mon travail de doctorat a introduit deux méthodologies complémentaires pour déchiffrer les réseaux d'IPP au niveau de fonctions biologiques spécifiques ou pour un complexe protéique spécifique en contexte cellulaire vivant. Ces approches offrent une nouvelle dimension pour comprendre les fonctions des protéines et les interactomes sous-jacents dans des contextes cellulaires normaux ou pathologiques
Cell fate and fitness depend on the protein content, and in particular on the interaction networks (also called interactomes) connecting the different proteins. Proteins have the general property to engage in diverse and occasionally overlapping macromolecular assemblies, each serving distinct purposes. Therefore, identifying protein-protein interactions (PPIs) and linking them to complexes is a crucial yet challenging issue in biology. This issue was at the core of my PhD work. The first part of my work was dedicated to the improvement of an existing method for capturing novel PPIs in the context of defined biological functions. This work was established with ERK1, which is a key downstream regulator of several signaling pathways involved in many different cancers. The new tools were tested in the context of two different inhibitory molecules to capture drug-sensitive interactions of ERK1 in human HEK293T cells. One such interaction was confirmed at the functional and molecular levels, by using an original imaging strategy to access the PPI dynamics in live cells. The second part of my PhD work was dedicated to the establishment of a pioneer methodology to capture endogenous PPIs established by a specific dimeric protein complex in human live cells. This methodology couples Bimolecular Fluorescence Complementation (BiFC) and proximity biotin labelling technologies. More specifically, it is based on a GFP-nanobody directed toward the BiFC complex and fused to the TurboID biotin ligase. Tools were established to map TAZ/14-3-3 and TAZ/TEAD complexes interactome, which translate the activity of the Hippo signaling pathway in the cytoplasm and nucleus, respectively. Our approach allowed capturing specific interactomes of the two dimeric protein complexes and identifying a novel key regulator of TAZ/14-3-3 complexes in a cancer cell context. Collectively, my PhD work introduced two complementary methodologies for deciphering PPI networks in the context of specific biological functions or in the context of a specific protein complex in human live cells. These approaches provide a novel dimension for understanding protein functions and the underlying interactomes in normal or pathological cell contexts

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biology, May, 2020
Cataloged from the official PDF of thesis.
Includes bibliographical references.
Nuclear pore complexes (NPCs) are the main conduits for molecular exchange across the nuclear envelope. The NPC is a modular assembly of ~500 individual proteins, called nucleoporins or nups, that can be classified into three categories: 1. Stably associated scaffolding nups, 2. Peripheral nups, and 3. Phenylalanine-glycine (FG) repeat containing nups that form the permeability barrier of the NPC. Most scaffolding nups are organized in two multimeric subcomplexes, the Nup84 or Y complex and the Nic96 complex. Working in S. cerevisiae to study the assembly of these two essential subcomplexes, we developed a suite of twelve nanobodies that recognize seven constituent nucleoporins of the Y and Nic96 complexes. The nanobodies bind their targets specifically and with high affinity, albeit with varying kinetics. We mapped the epitope of eight members of the nanobody library via crystal structures of nup-nanobody co-complexes.
Nuclear pore complexes (NPCs) are the main conduits for molecular exchange across the nuclear envelope. The NPC is a modular assembly of ~500 individual proteins, called nucleoporins or nups, that can be classified into three categories: 1. Stably associated scaffolding nups, 2. Peripheral nups, and 3. Phenylalanine-glycine (FG) repeat containing nups that form the permeability barrier of the NPC. Most scaffolding nups are organized in two multimeric subcomplexes, the Nup84 or Y complex and the Nic96 complex. Working in S. cerevisiae to study the assembly of these two essential subcomplexes, we developed a suite of twelve nanobodies that recognize seven constituent nucleoporins of the Y and Nic96 complexes. The nanobodies bind their targets specifically and with high affinity, albeit with varying kinetics. We mapped the epitope of eight members of the nanobody library via crystal structures of nup-nanobody co-complexes.
In two cases, the nanobodies facilitated the crystallization of novel nup structures, namely the full-length Nup84-Nup133 [alpha]-helical domain structure and the Nup133 [beta]-propeller domain structure. Together these two structures completely characterize the S. cerevisiae Y complex molecular assembly. Further, the Nup133 [beta]-propeller domain contains a structurally conserved amphipathic lipid packing sensor (ALPS) motif thought to anchor the Y complex to the nuclear envelope, which we confirmed by liposome interaction studies. An additional nanobody facilitated the structure of Nic96 at an improved resolution, revealing previously missing helices. In addition to the utility of these nanobodies for in vitro characterization of NPC assemblies, we also show that expression of nanobody-fluorescent protein fusions reveals details of the NPC assembly in their native, in vivo environment, and possibly of NPC heterogeneity within the nuclear envelope.
Overall, this suite of nanobodies provides a unique and versatile toolkit for the study of the NPC.
by Sarah Ann Nordeen.
Ph. D.
Ph.D. Massachusetts Institute of Technology, Department of Biology

Directed evolution is a powerful method for engineering of specific affinity proteins such as antibodies and alternative scaffold proteins. For selections from combinatorial protein libraries, robust and high-throughput selection platforms are needed. An attractive technology for this purpose is cell surface display, offering many advantages, such as the quantitative isolation of high-affinity library members using flow-cytometric cell sorting. This thesis describes the development, evaluation and use of bacterial display technologies for the engineering of affinity proteins. Affinity proteins used in therapeutic and diagnostic applications commonly aim to specifically bind to disease-related drug targets. Angiogenesis, the formation of new blood vessels from pre-existing vasculature, is a critical process in various types of cancer and vascular eye disorders. Vascular Growth Factor Receptor 2 (VEGFR2) is one of the main regulators of angiogenesis. The first two studies presented in this thesis describe the engineering of a biparatopic Affibody molecule targeting VEGFR2, intended for therapeutic and in vivo imaging applications. Monomeric VEGFR2-specific Affibody molecules were generated by combining phage and staphylococcal display technologies, and the engineering of two Affibody molecules, targeting distinct epitopes on VEGFR2 into a biparatopic construct, resulted in a dramatic increase in affinity. The biparatopic construct was able to block the ligand VEGF-A from binding to VEGFR2-expressing cells, resulting in an efficient inhibition of VEGFR2 phosphorylation and angiogenesis-like tube formation in vitro. In the third study, the staphylococcal display system was evaluated for the selection from a single-domain antibody library. This was the first demonstration of successful selection from an antibody-based library on Gram-positive bacteria. A direct comparison to the selection from the same library displayed on phage resulted in different sets of binders, and higher affinities among the clones selected by staphylococcal display. These results highlight the importance of choosing a display system that is suitable for the intended application. The last study describes the development and evaluation of an autotransporter-based display system intended for display of Affibody libraries on E. coli. A dual-purpose expression vector was designed, allowing efficient display of Affibody molecules, as well as small-scale protein production and purification of selected candidates without the need for sub-cloning. The use of E. coli would allow the display of large Affibody libraries due to a high transformation frequency. In combination with the facilitated means for protein production, this system has potential to improve the throughput of the engineering process of Affibody molecules. In summary, this thesis describes the development, evaluation and use of bacterial display systems for engineering of affinity proteins. The results demonstrate great potential of these display systems and the generated affinity proteins for future biotechnological and therapeutic use.

QC 20141203

Le transfert d'un cargo, contenu dans une vésicule, vers des cellules d'intérêt reste un défi pour les thérapies ciblées. Les glycoprotéines virales, se liant à un récepteur cellulaire et induisant la fusion membranaire, constituent des outils prometteurs pour ce type d'approche. La glycoprotéine G du VSV est la glycoprotéine virale la plus utilisée pour pseudotyper des lentivirus en thérapie génique. Il y a néanmoins des limites à l'utilisation de G : les récepteurs de G (membres de la famille du LDLR) sont ubiquitaires et présents à la surface de cellules non cibles. Les travaux de l'équipe sur la structure du complexe VSVG/LDLR avaient identifié des mutants de G ne liant plus le LDLR mais conservant leur activité de fusion. Ce découplage entre reconnaissance du récepteur et fusion ouvrait la possibilité de recibler spécifiquement la glycoprotéine vers des récepteurs d'intérêt. Nous avons construit une glycoprotéine chimérique en fusion avec un nanobody dirigé contre la mCherry en position aminoterminale de G. L'insertion de ce nanobody dans G est délétère. Par évolution expérimentale, nous avons identifié deux mutations dans G améliorant le repliement de la chimére. Ces mutations favorisent le repliement des G chimériques quel que soit le nanobody inséré en position amino-terminale. Les virus pseudotypés (VSV et lentivirus) avec ces G chimériques ont un titre 10 fois plus élevé lorsque les mutations d'optimisations sont présentes. Nous avons ensuite construit des glycoprotéines chimériques avec plusieurs nanobodies ciblant le récepteur HER2. Dans ces glycoprotéines, nous avons introduit les mutations abolissant la reconnaissance des récepteurs naturels de G. Les virus pseudotypés par ces glycoprotéines n'infectent plus que des cellules exprimant le récepteur HER2. Nous avons donc identifié des mutations de G permettant de conférer un nouveau tropisme à G par insertion aminoterminale de nanobodies. Ceci ouvre la voie à des thérapies ciblées personnalisées
The transfer of vesicles containing cargos toward cells of interest remains a challenge for targeted therapy. Viral fusion glycoproteins having the property of receptor recognition and fusion activity constitute promising tools for this kind of approach. VSV glycoprotein (G) is the most used viral glycoprotein to pseudotype lentiviruses in gene therapy. However, we encounter limits to the use of G: G cellular receptors (from LDLR family) are ubiquitous and expressed at the surface of non-target cells. The work of the team on VSVG/LDLR structure enabled us to identify G mutants that no longer bind the LDLR without affecting its fusion activity. This uncoupling between the recognition of the receptor and the fusion capacity opened up the possibility of retargeting G towards receptors of interest. A chimeric glycoprotein fused with a nanobody directed against the mCherry protein, in N-terminal, of G has been constructed. The insertion of a nanobody in G is deleterious for its activity. Using experimental evolution, we identified two mutations on G enhancing the chimera folding. Remarkably, these mutations improve the folding of chimeric Gs, regardless of the sequence of the nanobody inserted in amino-terminal. Pseudotyped viruses (both VSV and lentiviruses) with these chimeric Gs at their surface show 10 times higher titers with these mutations of optimisation. We then constructed chimeric Gs with several nanobodies targeting the receptor HER2. We introduced the mutations abolishing LDLR recognition in these Gs. Viruses pseudotyped with these glycoproteins only infected cells expressing HER2. We therefore identified G mutations conferring a new tropism of G thanks to the N-terminal insertion of a nanobody. All this work opens the way to personalised targeted therapies

Par leur stabilité, leur petite taille et leur nature monomérique, les domaines variables des immunoglobulines à chaînes lourdes, ou Nanobodies (Nb), sont incontournables en diagnostic et recherche médicale. Pourtant, leur utilisation en agro-biotechnologies demeure confidentielle.Dans l'idée de les utiliser pour étudier et combattre le Grapevine fanleaf virus (GFLV), responsable de la maladie du court-noué très préjudiciable à l'économie viticole mondiale, j'ai produit une collection de Nb spécifiques du GFLV.Fusionné à une protéine fluorescente et exprimé en plante de façon stable, un de ces Nb (alors appelé Chromobody, Cb) a conféré une haute résistance au GFLV inoculé mécaniquement ou transmis par nématodes.Le potentiel du Cb comme biocapteur a été validé par le suivi in vivo d’un isolat contournant la résistance mais toujours reconnu par le Cb. La structure du complexe Nb/GFLV a été résolue à 2,8 Å et révèle la zone occupée par le Nb à la surface de la capside.Ces résultats ouvrent des perspectives innovantes pour la compréhension du cycle infectieux d'un phytovirus et l'élaboration de nouvelles stratégies de lutte antivirale
Due to their small size, high stability and strict monomeric nature, Nanobodies (Nbs) deriving from camelids heavy chain only antibodies have proven very valuable as diagnostic and therapeutic tools. However their use in agro biotechnology remains limited.In order to apply Nbs to the study and the control of grapevine fanleaf degeneration, I produced acollection of Nbs against Grapevine fanleaf virus (GFLV), the causal agent of this devastating disease worldwide.When fused to a fluorescent protein and stably expressed in plants, one of these Nbs (calledChromobody, Cb) conferred high resistance to GFLV, whether inoculated mechanically or by vector-mediated transmission.The identification of an isolate overcoming the resistance but still bound by the Cb allowed real-time tracking of the infection showing the high potential of Cbs as biosensors.The cryoEM structure of the Nb/GFLV complex was obtained at 2,8 Å and provides a clear picture of the footprint of the Nb on the surface of the GFLV capsid.These results pave the way for the innovative use of Nbs to unravel viral life cycle and to counter viral diseases

La conoscenza della struttura tridimensionale di un potenziale target farmacologico apre la via a nuove strategie terapeutiche (ad esempio tramite structure-based drug design (SBDD)) ed è requisito fondamentale per la bioinformatica strutturale. In questo contesto, durante la mia tesi di dottorato, sono state studiate due proteine di interesse biomedico. La prima è una proteina di membrana, l’isoforma 1 dell’Acid Sensing Ion Channel (ASIC), implicata in diverse malattie neurodegenerative. In studi precedenti il diminazene aceturato (DA) si era dimostrato un potente inibitore del canale. Diversi analoghi di DA sono stati progettati su base strutturale e la loro affinità per ASIC analizzata tramite docking molecolare. Le molecole migliori sono state testate in saggi cellulari per valutarne l’efficacia. Per caratterizzare la capacità inibitoria degli analoghi sintetizzati in vitro, è stato messo a punto un protocollo per la produzione della proteina ASIC1, utilizzando diversi sistemi di espressione eterologa. La purificazione della proteina è stata effettuata usando un approccio high-throughput per supportare successivamente la cristallizzazione della proteina, al fine di ottenere informazioni più dettagliate sul meccanismo d’azione degli analoghi del DA e, di conseguenza, disegnare nuovi farmaci, isoforma-selettivi e in grado di attraversare la barriera emato-encefalica. In secondo luogo, ho studiato la proteina Gelsolin (GSN), responsabile di una malattia familiare degenerativa (detta amiloidosi AGel). Lo scopo di questo progetto era quello di investigare l'effetto della mutazione D187N sulla struttura di GSN e la sua propensione ad aggregare e/o degradarsi in maniera anomala. Il D187N GSN è stato il primo mutante ad essere identificato, ma, ad oggi, non si avevano informazioni sulla sua struttura. In uno studio precedente, era stato identificato un nanobody (Nb11) in grado di proteggere la proteina dalla degradazione, ma il meccanismo di protezione non era stato chiarito. Nel mio lavoro ho risolto la struttura del complesso Nb11:D187N a 1.9 Å, permettendo la caratterizzazione molecolare del meccanismo di azione del Nb11. I dati strutturali ottenuti sono stati completati con una caratterizzazione biofisica e biochimica, estesa anche ad altre due varianti patologiche della GSN, recentemente identificate (G167R e N184K).
Knowledge of the three-dimensional structure of therapeutically relevant proteins paves the way for novel strategies in pharmacological research (such as the structure-based drug design (SBDD) method) and establishes the foundations for structural bioinformatics. In this context, during my PhD Thesis, two therapeutically relevant proteins have been studied. First, a membrane protein, Acid Sensing Ion Channel (ASIC) isoform 1, a validated target in neurodegenerative disorders, was selected. Previous studies showed that diminazene aceturate (DA) is a potent small-molecule inhibitor of ASIC channels. Here, several DA analogues were screened by molecular docking and the best binders were tested in cell-based assays to further assess their efficacy. In order to determine the inhibitory capability of the synthesized analogues in vitro on the purified protein, the expression of ASIC1 was undertaken, using different organisms of expression. The protein purification was performed in a high-throughput approach in order to recover enough protein for crystallization, with the final aim of studying the mechanism of action of DA analogs, and support the design of new, isoform-selective and brain-penetrant drugs. Secondly, the soluble protein Gelsolin (GSN), responsible for a familial degenerative disease (AGel amyloidosis) was studied. Aim of this project was to understand the impact of the D187N mutation on GSN structure and its propensity to aberrant aggregation and/or degradation. D187N GSN mutant was the first identified in man, but its crystal structure had until now eluded any characterization. Conversely, a nanobody (Nb11) was shown to protect GSN from aberrant proteolysis, but its mechanism of protection remained unclear. Here, the structure of the Nb11:D187N complex was solved at 1.9 Å resolution, enabling the characterization of the Nb11action mechanism. The structural data were complemented with biophysical and biochemical characterisations. These studies were then extended to two recently identified pathological variants of GSN (G167R and N184K).

The tumour suppressor protein p16INK4a (p16) is a cyclin-dependent kinase (CDK) inhibitor that plays a key role in the regulation of the cell cycle by controlling the progression of cells through the G1 to S phase transition. Dysregulation of the protein through deletion, silencing or mutation of the gene encoding p16 is implicated in a range of different cancers including melanoma, cervical and oesophageal to name a few. p16 is composed of four ankyrin repeats and it has a very low thermodynamic and kinetic stability and rapidly unfolds even in the absence of denaturants. This low stability means that the protein is highly vulnerable to point mutations, which can result in functional inactivation through a range of different mechanisms such as deletion of key binding contacts, disruption of secondary or tertiary structure and consequent destabilisation leading to unfolding or aggregation. Heavy-chain antibodies are a unique form of antibody devoid of light chains found in the serum of the Camelid family (camels and llamas). Despite the absence of light chains, heavy-chain antibodies have evolved to complement traditional antibodies and retain the full binding capacity seen in canonical IgG antibodies. The single variable domain, known as a nanobody, is, at 15 kDa, the smallest antigen binding fragment, a tenth the size of a standard IgG antibody. The small size and relative ease of production, coupled with an unusually high stability, makes nanobodies useful tools as biological reagents, crystallography chaperones and therapeutics. The research contained within this PhD looks at the development of nanobodies to target p16. By leveraging the high stability of selected nanobodies, the aim was to obtain binders that could stabilise and reactivate a range of unstable cancer-associated mutants. The initial stages of the project focused on generating and optimising the expression and purification of p16 constructs prior to immunisation of animals to raise nanobodies. A high-throughput approach was taken to generate forty-five different p16 constructs with a range of different solubility and purification tags. These constructs were assessed in a multi-factorial expression screen, which resulted in the identification of a p16 construct with a ten-fold improvement in soluble expression levels compared with previous studies. A range of biophysical techniques, including circular dichroism and chemical denaturation, were performed to characterise this protein fully prior to immunisation. The second part of this project utilised a phage display library of two immune nanobody libraries generated against p16 and a p16 variant stabilised by previously published second-site mutations. This process yielded a large number of diverse nanobodies. Biophysical characterisation of these nanobodies was first performed, and they were found to have a range of chemical and thermal stabilities. Assays were then developed to test the ability of the nanobodies to stabilise p16. Two nanobodies were found to dramatically stabilise wild-type p16, with an increase in stability of approximately 44 % and 60 %, respectively. Furthermore, these nanobodies were also able to stabilise a subset of cancer-associated point mutants. Although there are NMR structures of p16, as well as a crystal structure of p16 bound to CDK6, the resolution of is very low, most likely due to the high backbone flexibility of p16. The last part of the project aimed to obtain a higher-resolution structure of p16 by using the two stabilising nanobodies as crystallisation chaperones. The more stabilising of the two nanobodies resulted in crystals that diffracted to a resolution of less than 2 $\AA$, a significant improvement compared with the previously published structure. In conclusion, a number of nanobodies were generated against tumour-associated p16 and shown to be capable of stabilising p16, allowing structure determination to high resolution and restoration of the stability of cancer-associated mutants to wild-type levels. In the project, a range of different approaches for nanobody production were explored, and these will be important for future applications. Moreover, the crystal structure of the p16-nanobody complex showed that the nanobody binds on the opposite face of p16, to the face involved in binding to CDKs; thus, this nanobody could potentially be exploited as a pharmacological chaperone to stabilise and restore the activity of cancer-associated mutant p16 in the cell.

Les parasites de la classe des Kinetoplastidae, comprenant notamment les trypanosomes et les leishmanies, sont responsables pour plusieurs maladies d’importance socio-économique et de santé publique. La maladie du sommeil, la maladie de Chagas et la leishmaniose, classées comme maladies tropicales négligées (NTD) par l’Organisation mondiale de la santé (OMS) et la Surra, reportée par l’Organisation pour l’alimentation et l’agriculture, des Nations Unies (FAO). La Trypanosomiase Animale Africain sub-saharienne entraîne la mort de 3 millions bovins par an accompagné d'une perte annuelle de l'économie de 4,5 milliards de dollars américains. La leishmaniose cutanée, une maladie zoonose, présente 1,5 millions de nouveaux cas chaque année.Trypanosoma brucei (T. brucei) est un ancien eucaryote, utilisé comme organisme modèle dans le laboratoire pour l’étude des cils et des flagelles. Le remodelage du cytosquelette des trypanosomes est essentiel pour la morphologie cellulaire, le positionnement et la division des organites. L’étude des protéines essentielles du cytosquelette permet de mieux comprendre les processus cellulaires. Ces protéines pourraient également constituer des cibles potentielles pour des traitements thérapeutiques. Les trypanosomes échappent au système immunitaire de l’hôte en modifiant périodiquement les antigènes de présent à leur surface. En effet ces antigènes de surface sont endocytés, ainsi que les anticorps de l’hôte qui y sont attachés, au niveau d’une structure appelée la poche flagellaire (FP). TbBILBO1 est une protéine structurelle du collier de la poche flagellaire (FPC), essentielle à la biogenèse du FPC et à la survie du parasite. En raison du rôle majeur de la protéine TbBILBO1 dans le parasite, des partenaires de TbBILBO1 ont été recherchés.Dans ce travail, j’ai pu caractériser une nouvelle protéine essentielle du cytoskelette, la protéine FPC6, partenaire de TbBILBO1, qui se situe au niveau du complexe FPC/Complexe du Hook de T. brucei. L’ARN interférence de FPC6 conduit à une mort rapide des formes sanguines des trypanosomes, accompagnée d’un blocage de l’endocytose. Ensuite, j’ai produit un nanobody (Nb48), dirigé contre TbBILBO1, dans le système d’expression bactérien. Je l’ai également exprimé dans les lignées de trypanosomes. Le Nb48 reconnait TbBILBO1 sur les trypanosomes fixés par immunofluorescence et dans les extraits totaux de protéines dénaturées. L’analyse par résonance plasmonique de surface (SPR) a confirmé une haute affinité du Nb48 pour TbBILBO1. L’expression de Nb48 dans le parasite T. brucei en tant qu’intrabody demontrant que ce nanobody pouvait être exprimé de manière fonctionnelle, capable de reconnaitre spécifiquement sa cible protéique, TbBILBO1, intra-cellulaire et de bloquer sa fonction conduit à un effet trypanocide rapide. Ces études ouvrant ainsi la voie pour de nouvelles utilisations potentielles thérapeutiques dans le traitement des trypanosomiases
Kinetoplastid parasites, including trypanosomes and leishmania, are responsible for several diseases of socio-economic and public health importance worldwide. These include the Neglected Tropical Diseases: Sleeping Sickness, Chagas disease and Leishmaniasis, as classified by the World Health Organisation (WHO) and the global wasting disease of animals, Surra, as reported by the Food and Agricultural Organisation of the United Nations (FAO). Animal African Trypanosomiais (AAT) causes the death of 3 million cattle per year in sub-Saharan Africa, with an annual loss of 4.5 billion US dollars to the African economy. Cutaneaous leishmaniasis is a zoonotic disease, with 1.5 million new cases reported globally each year.Trypanosoma brucei is an ancient, early diverging eukaryote, used as a model organism in the laboratory for studying eukaryotic cilia and flagella. Remodelling of the trypanosome cytoskeleton is essential for cell morphology, organelle positioning and division. Study of essential proteins of the cytoskeleton provides insight into intracellular processes and could provide potential targets for therapeutic interventions. Trypanosomes evade the host immune system by periodically changing their external surface coat, which is endocytosed, along with any attached host antibodies, via a structure called the flagellar pocket. TbBILBO1 is a structural protein of the Flagellar Pocket Collar (FPC) that is essential for FPC biogenesis and parasite survival. Due to the importance of TbBILBO1 for the parasite, protein partners were investigated.In my thesis, I describe, firstly, the characterisation of a novel and essential cytoskeletal protein, FPC6, of the FPC/Hook complex of T. brucei; FPC6 is a partner of TbBILBO1. RNAi Knock-down of FPC6 protein leads to rapid cell death in the blood-stream form of the parasite accompanied with a block in endocytosis. Secondly, I describe the purification and intracellular expression of a nanobody (Nb48), raised against TbBILBO1. The purified Nb is able to identify TbBILBO1 in fixed trypanosomes and denatured protein. Surface Plasmon Resonance analysis confirmed a high affinity of Nb48 to TbBILBO1. Expression of Nb48 as an intrabody in T. brucei, reveals that it binds precisely to its target, TbBILBO1 and leads to rapid cell death. Further exploration of the potential uses of this trypanocidal nanobody is warranted

L'ADN est constamment la cible de dommages, provenant aussi bien de sources endogènes (propriétés intrinsèques de la macromolécule, métabolisme cellulaire. . . ) qu'exogènes (radiations, contaminants alimentaires. . . ). Parmi ces dommages, les cassures double-brin de l'ADN (CDB) représentent une des lésions les plus cytotoxiques. En réponse à ce danger, la cellule dispose de voies de détection et signalisation impliquant le recrutement de protéines. Au cours de cette réponse des modifications post-traductionnelles de protéines de signalisation et de réparation sont produites au niveau du site de cassure comme, par exemple, la phosphorylation de H2AX, un variant de l'histone H2A. Cette voie de signalisation permet d'une part d'activer les points de contrôle du cycle cellulaire pour stopper la prolifération et, d'autre part, de stimuler les systèmes de réparation de CDB afin de restaurer l'intégrité de l'ADN. Une réparation infidèle des CDB peut aboutir à des additions/délétions de bases, voir des réarrangements chromosomiques, à l'origine de cancers. Ainsi, élucider les causes et les mécanismes responsables de la formation des CDB en réponse à des stress génotoxiques et suivre leur prise en charge par la cellule sont des éléments importants pour la compréhension de la génotoxicité. Des techniques permettant d'analyser la formation des CDB (immunofluorescence, électrophorèse en champs pulsé, COMET neutre. . . ) existent, mais elles ne permettent d'analyser l'état de l'ADN qu'à un instant fixe. La première partie de mon travail de thèse a été de créer un outil innovant, permettant de détecter et suivre la formation de CDB en temps réel, sur cellules vivantes. Cet outil repose sur la technologie des nanobodies, anticorps monochaines produits uniquement chez les camélidés et certains requins. Nous avons fait exprimer un nanobody intracellulaire dirigé contre H2AX phosphorylé (appelé gammaH2AX), qui semble se relocaliser aux CDB créées par microirradiation. La création de cet outil a nécessité l'immunisation d'un lama avec le peptide phosphorylé et l'isolement/le clonage des séquences codantes des nanobodies afin de produire une banque. Les nanobodies spécifiques de gammaH2AX ont été sélectionnés par phage display et leurs séquences ont été exprimées en cellules humaines, fusionnées à un fluorophore afin d'observer leur relocalisation aux CDB en temps réel. La seconde partie de ma thèse a permis de mieux comprendre le mécanisme d'action d'une génotoxine bactérienne, responsable de cancers en modèle murin : la Cytolethal Distending Toxin (CDT). Cette toxine, sécrétée par des bactéries commensales et pathogènes, se localise au noyau des cellules cibles et provoque des CDB. Si le fonctionnement de cette toxine était préalablement décrit comme celui d'une nucléase produisant des CDB directes, mon travail a montré que pour des concentrations équivalentes à la Dose Létale 50, CDT produit d'abord des cassures simple-brin qui dérivent en CDB au cours de la réplication de l'ADN. De plus, la réparation de ces CDB par recombinaison homologue est cruciale pour la survie des cellules exposées à CDT. En conclusion, mon travail de thèse a permis d'une part de développer un outil innovant permettant d'analyser la dynamique en temps réel des CDB en cellules humaine et, d'autre part, d'éclaircir le mécanisme menant à la génotoxicité de CDT, toxine représentant un risque potentiellement cancérigène chez les mammifères. Les apports de ces travaux sont discutés ici
Human DNA is constantly damaged by endogenous (cellular metabolism) or exogenous (radiations, food contaminants) sources. Among these lesions, DNA double-strand breaks (DSB) are the most cytotoxic. To survive to these lesions, a cellular pathway is in charge for the detection and the signaling of DSB. This pathway involves recruitment and post-translationnal modifications of several proteins around the DSB site (like the phosphorylation of a H2A histone variant called H2AX). This signalization pathway elicits cellular checkpoints in order to stop proliferation, and stimulates DSB repair systems in order to restore DNA initial integrity. An error-prone repair of DSB can lead to base additions/deletions, or chromosomal aberrations that can induce cancer. In order to understand genotoxicity, it is important to elucidate causes and mechanisms responsible for DSB formation and to follow their management by the cell. Techniques allowing DSB formation analysis (immunofluorescence, pulse-field gel electrophoresis, neutral COMET assay. . . ) exist, but can only show DNA state for a given point. During the first part of my thesis work, I created a new tool to detect and follow DSB formation in real time, in human cells. This tool rely on nanobody technology, which are miniatures antibodies produced by camelidae species and some sharks. An intracellular nanobody directed against phosphorylated H2AX (gammaH2AX) has been expressed, and seems to relocate to microirradiation-induced DSB. In order to build this tool, anti-gammaH2AX peptides were designed to immunize a llama, and nanobodies coding sequences were isolated/cloned and gathered as a library. Nanobodies specific for gammaH2AX were selected by phage display. Fused to a fluorophore these nanobodies were expressed in human cells in order to analyze their relocalization to DSB in real time. The second part of my phD shed a new light on the mechanism of action of a bacterial génotoxine causing cancers in mouse models: the Cytolethal Distending Toxin (CDT). This toxin is secreted by commensal and pathogenous bacteria, translocate into the nucleus of targeted cells and induces DSB. CDT mechanism of action was previously described as those of a nuclease inducing DSB. But my work demonstrated for lower doses (equivalent to lethal dose 50), that CDT induced first single-strand breaks leading to double-strand breaks through DNA replication. Moreover, homologous recombination repair of these DSB is crucial in order for cells exposed to CDT to survive. In conclusion, thanks to my thesis work, I developed a new tool to analyze real time dynamic of DSB in human cells in one hand. And in another hand, my work shed a new light on the mechanism of action of CDT genotoxicity, a toxin displaying cancer hazard in mammalians. Contributions brought by this work are discussed here

Les amyloïdoses sont des maladies caractérisées par l’agrégation structurée de protéines, sous forme de fibres amyloïdes. Le diagnostic précoce de ces maladies représente un enjeu important, pour la prise en charge de nombreuses pathologies associées. Dans ce travail, nous avons montré le ciblage et la détection de plusieurs fibres amyloïdes, de l’in vitro à l’in vivo. Pour cela, nous avons utilisé des nanoparticules multimodales pour l’imagerie médicale (TEP, IRM), greffées avec diverses molécules ciblant les fibres. Trois types de fibres amyloïdes ont été testées, formées à partir du peptide amyloïde β (maladie d’Alzheimer), d’amyline (diabète de type 2), et de la transthyrétine (polyneuropathie familiale). Comme montré par des techniques de spectroscopie et de résonance plasmonique de surface (fluorescence et Biacore), des nanoparticules génériques (dû au greffage du Pittsburgh compound B ou d’un nanocorps) ciblent avec une bonne affinité les trois types de fibres in vitro, tandis que des nanoparticules spécifiques (dû au greffage de peptides) ciblent avec une affinité moindre les fibres d’amyloïde β ou de transthyrétine. Le ciblage et la détection des dépôts amyloïdes par les nanoparticules ont été confirmés par microscopie à fluorescence, sur des tissus de souris présentant chacune des trois maladies. Le suivi par imageries in vivo (par IRM) et post-mortem (par microscopie optique), après injection de nanoparticules génériques chez la souris Alzheimer, supposent un ciblage des dépôts amyloïdes intracérébraux. Par ailleurs, nous avons détecté les fibres amyloïdes sans aucun marquage. Des études spectroscopiques in vitro ont permis de montrer des propriétés luminescentes intrinsèques des fibres amyloïdes, dans l’UV-visible et le proche infrarouge. Ces caractéristiques ont été observées sur des coupes de tissus de cerveau de souris Alzheimer par microscopie à fluorescence, et les études in vivo en cours semblent prometteuses (par imagerie photo-acoustique, et en temps résolu). Que cela soit par l’utilisation de nanoparticules fonctionnalisées multimodales, ou de propriétés intrinsèques des fibres amyloïdes suggérant une détection complètement non-invasive, ces deux stratégies innovantes semblent adaptées pour le diagnostic précoce des amyloïdoses chez l’Homme
Amyloidosis are diseases characterised by self-agregation of misfolded proteins in fibrillary forms, called amyloid fibrils. They are associated with many diseases, and their early diagnosis remains a clinical challenge. In this work, we show the targeting and the detection of amyloid fibrils, from in vitro to in vivo experiments in mice models, useful for early diagnostic of amyloidosis. For that purpose, we use multimodal nanoparticles, further functionalized with specific molecules against amyloid fibrils. This multimodality for imaging (fluorescence, MRI, PET) represent a breakthrough in modern medicine, to add structural and functional informations. After negative toxicity tests on various cell lines, these nanoparticles have been tested on three different amyloid fibrils, formed with amyloid β(1-42) peptide (Alzheimer’s disease), amylin (type II diabetes mellitus), or mutated Val30Met transthyretin (familial polyneuropathy). As it is shown by spectroscopy and surface plasmon resonance experiments, nanoparticles grafted with generic targeters (Pittsburgh compound B or a nanobody) target the three amyloid fibrils, with good affinity, whereas nanoparticles vectorised with peptides show specific targeting for amyloid β(1-42) or Val30Met mutated transthyretin, but with lower affinity. The targeting by nanoparticles have been confirmed ex vivo on pathological tissues with each amyloid burden, thanks to fluorescence microscopy. Generic nanoparticles have been injected in Alzheimer’s mice model, and the monitoring in vivo by IRM and post-mortem by optical microscopy supposed a targeting of amyloid β(1-42) aggregates in brain. Furthermore, we demonstrate the possibility to detect amyloid fibrils without labelling. Spectroscopic measurements show original, and specific optical properties of amyloid fibrils, around the UV-visible and the near infra-red regions. Interestingly, these properties are also observed on pathological tissues by ex vivo fluorescence microscopy, and in vivo ongoing analysis by photoacoustic imagery and real-time imaging seems to be very promising. By multimodality of non-toxic grafted nanoparticles or by intrinsic properties of amyloid fibrils suggesting completely non-invasive tests, these two strategies can be useful for early diagnostic of amyloidoses in humans

Scanning ion conductance microscopy (SICM) is a technique for non-contact topographic imaging. In this thesis, a biophysical investigation into Alzheimer's Disease (AD) was carried, with toxic oligomers dosed locally and quantitatively on to single astrocytes using SICM and simultaneously monitoring the response of the target cell. Examination of the effectiveness of antibodies that bind to Abeta or alpha-synuclein (Asyn)peptides depends on the measurement of oligomer-induced abnormal calcium homeostasis in single astrocytes. The method was shown to work at physiological concentrations of oligomers. A series of experiments measuring the reduction in calcium inux in mixtures of antibodies and cerebrospinal fluid (CSF) of AD patients suggested that the binding to co-oligomers composed of Abeta and Asyn may be crucial in the treatment of AD. Furthermore, it may be beneficial to test antibodies before the clinical trial using this assay. The mechanism of this entry of calcium is hypothesised to be the result of the formation of oligomer-induced transient pores in the cell membrane. To verify this hypothesis, a new SICM instrument was built with two nanopipettes; one for dosing and one for detection of the adenosine triphosphate (ATP) release from these pores. A variety of different ATP sensors were made. The best had a sensitivity of 10 micro molar and works as a hexokinase-cofunctioned electrolyte-gated organic field-effect-transistor. However no statistically significant results for ATP release have been obtained in the experiments performed to date. Overall this thesis describes new biophysical methods to study the effect of protein aggregates on live cells and the effectiveness of potential therapies, such as antibodies and nanobodies, to reduce these aggregate induced effects. It can be applied to synthetic aggregates of Abeta or the aggregates present in human CSF.

Cette thèse a pour vocation de contribuer au développement de différents immunocapteurs ampérométriques associés aux électrodes sérigraphiées (SPE). Les immunocapteurs sont des dispositifs simples associant un anticorps ou un antigène qui assurent la sélectivité à un transducteur (ici une SPE) ;ce dernier transforme la liaison anticorps/antigène en un signal mesurable (ici ampérométrique).

Le travail est divisé en deux volets principaux.

Le premier est consacré à la mise en œuvre de différents modèles d’immunocapteurs ampérométriques pour le dosage d’anticorps anti-tetani. La vaccination contre le tétanos est généralisée, toutefois pour maintenir un taux d’anticorps suffisant, il est indispensable d’administrer un rappel tous les 10 ans. Ce schéma vaccinal n’est pas toujours respecté, ce qui a pour conséquence qu’une partie de la population n’est plus protégée. Afin de déterminer le statut immunitaire du patient, il est indispensable de pouvoir déterminer le taux d’anticorps. Les immunocapteurs ampérométriques répondent à cet objectif. Plusieurs stratégies d’immobilisation de l’anatoxine tétanique (antigène) sur une SPE ont été mises en œuvre et comparées. L’une d’elles repose sur l’utilisation de microparticules superparamagnétiques pour la réaction immunologique et d’une SPE rendue magnétique par un support aimanté pour la mesure. D’autres reposent sur l’immobilisation de l’antigène et les réactions immunologiques directement à la surface de la SPE. L’utilisation de plans d’expérience, pour l’optimisation des immunoessais sur SPE est également exploitée dans ce travail. Les immunocapteurs développés ont permis de doser les anticorps anti-tetani dans le sérum de cobaye en dessous des valeurs considérées comme protectrices.

Dans le second volet, un immunocapteur basé sur l’utilisation de nanobodies® (NB) a été mis au point. Nous avons qualifié ce type d’immunocapteur original de nanoimmunocapteur. Le récepteur de facteur de croissance épidermique humain (HER2) a été utilisé comme cible. La protéine HER2 est considérée comme un biomarqueur important car sa surexpression provoque un type agressif de cancer du sein. Les NB sont des fragments à domaine unique dérivés d'anticorps à chaînes lourdes de camélidés. La stratégie de dosage immunologique en sandwich développée a tiré profit de la petite taille des NB pour la détection du marqueur électroactif d’oxydoréduction. La stabilité élevée des NB immobilisés a permis une durée de stockage des SPE modifiées supérieure à 3 semaines. De très courtes durées d'incubation étaient suffisantes pour obtenir une réponse satisfaisante. Le nanoimmunoessai a permis de déterminer le taux d’HER2 dopé dans des lysats cellulaires.

Doctorat en Sciences biomédicales et pharmaceutiques
info:eu-repo/semantics/nonPublished

Les méthodes traditionnelles de décontamination restent confrontées à des défis importants, tels que la difficulté d'accès aux espaces complexes ou confinés, la quantité élevée de déchets, etc. Il est encore nécessaire aujourd'hui d'explorer de nouvelles méthodes permettant d'atteindre des géométries complexes avec des processus de décontamination efficaces. Même si les macrorobots se révèlent utiles dans les tâches de décontamination à grande échelle, leur taille limite leur capacité à naviguer dans des environnements complexes et confinés. Les micro ou nano robots, quant à eux, pourraient traverser de tels espaces et cibler des sites de contamination spécifiques, ce qui les rendrait plus adaptés aux travaux de décontamination en milieu confinés et complexes. Dans ce contexte, cette thèse porte sur la synthèse à grande échelle et l'étude de la mobilité de colloïdes actifs, en particulier de particules Janus, susceptibles d'être utilisés comme brique dans de nouvelles méthodes de décontamination. À cette fin, différents assemblages de particules d'or sur silice (assemblage isotrope ou Janus, nanoparticules discrètes ou couche d'or continue) ont été préparés, caractérisés et comparés. Leurs mouvements 3D et 2D ont été suivis dans différents environnements. Un élément clé de ce travail a consisté en particulier à développer un dispositif microfluidique capable de générer des gradients stables d'eau oxygénée, produit de la radiolyse de l'eau. Ce dispositif s'est révélé essentiel pour l'analyse directionnelle du mouvement des différentes particule. Ces travaux ont montré que les assemblages or silice pouvaient se déplacer de manière autonome vers une source d'H₂O₂, ce qui pourrait les rendre efficaces pour cibler les sites de contamination radioactives. Nous avons également montré que des assemblages isotropes, plus simples à préparer, pouvaient également présenter un mouvement directionnel
Traditional methods of decontamination face significant challenges, such as difficulty in accessing complex or confined spaces, high amount of waste, etc. There is still a need for the development of new methods to reach complex geometries with effective decontamination processes. While macro-robots have been useful in large-scale decontamination tasks, their size limits their ability to navigate in intricate environments. Micro or nano robots, on the other hand, can traverse small, complex spaces and target specific contamination sites, making them more suitable for detailed decontamination work. In this context, this thesis studies the capacities of micro/nanoparticles to move towards contaminated spot in complex geometries, by mimicing the chemotaxism guided by H₂O₂ (product of water radiolysis). To this end, the large-scale synthesis and mobility of active colloids, in particular Janus particles is described. A set of different assemblies of gold particles on silica (isotropic or Janus assembly, discrete nanoparticles or continuous gold layer) were prepared, characterized and compared. Their movements were monitored in different environments. A key part of this work was the developement of a microfluidic device capable of generating stable hydrogen peroxide gradients. This device was essential to study the directionnal orientation of the different particles. This work showed that silica-gold assemblies could move autonomously towards a source of H₂O₂, which could make them effective for targeting radioactive contamination sites. We have also shown that isotropic assemblies, which are simpler to prepare, can also exhibit directional movement

La maladie d’Alzheimer, principale forme de démence dans le monde, est devenue un problème de santé publique majeur. Cette pathologie est caractérisée par la présence de deux lésions principales dans le cerveau : la dégénérescence neurofibrillaire composée de protéine Tau hyperphosphorylée et des plaques amyloïdes, agrégats denses de peptides β amyloïdes (Aβ). Le peptide Aβ provient du clivage de la protéine précurseur de l’amyloïde (APP) par des β- et ɣ-sécrétases. Un clivage dit non-amyloïdogène de l’APP existe également, au cours duquel la protéine est clivée par une α-sécrétase (principalement ADAM10) ce qui conduit à la production d'un fragment extracellulaire connu pour être neuroprotecteur et neurotrophique, le sAPPα. Le récepteur sérotoninergique 5-HT4 est un récepteur couplé aux protéines G (RCPG) qui interagit avec l’APP et ADAM10, module leur trafic et leur maturation, et dont l’activation pharmacologique favorise le clivage non-amyloïdogène de l’APP. Le récepteur 5-HT6 est un autre récepteur sérotoninergique dont la modulation pharmacologique induit des effets pro-cognitifs. Les récepteurs 5-HT4 et 5-HT6 apparaissent donc comme des cibles thérapeutiques d'intérêt dans le contexte de la maladie d’Alzheimer. Néanmoins, les mécanismes moléculaires et cellulaires sous-jacents responsables des effets liés à l’expression et l’activation du récepteur 5 HT4 ne sont pas encore élucidés. Les objectifs de cette thèse ont été d’une part de développer une technique de criblage des nanocorps (fragments d’anticorps à domaine unique provenant des camélidés) contre les récepteurs 5-HT4 et 5-HT6 en utilisant notamment des approches de TR-FRET et de cytométrie de flux. Dans un second temps, l’utilisation d’une approche de protéomique combinant la purification des partenaires du récepteur 5-HT4 par affinité suivie de leur identification systématique par spectrométrie de masse a permis l’identification de partenaires protéiques potentiels associés au récepteur 5-HT4 dans les cellules HEK-293N. Parmi les protéines recrutées par le récepteur 5-HT4, nous avons notamment identifié la protéine IRS4 (Insulin Receptor Substrate 4). La protéine IRS4 est un transducteur de la voie de la signalisation de l’insuline. Étant donné qu’une dérégulation de la voie de signalisation de l’insuline est l’un des évènements majeurs impliqués dans le développement et la progression de la maladie d’Alzheimer, nous nous sommes focalisés sur ce partenaire. Après avoir validé cette interaction par Western Blot, nous avons montré une co-localisation de IRS4 avec le récepteur 5-HT4 dans les cellules HEK-293. Par ailleurs, des études fonctionnelles ont montré qu’une inhibition de l’expression de IRS4 induit une surexpression du récepteur 5-HT4 dans les cellules HEK-293, associée à une augmentation de la signalisation canonique du récepteur via la voie de la protéine Gs.Ces résultats suggèrent une modulation du trafic du récepteur 5-HT4 et donc du complexe associé incluant l’APP et l’ADAM10 par la protéine IRS4. Ce travail de thèse dévoile pour la première fois un lien fonctionnel entre la signalisation du récepteur sérotoninergique 5-HT4 et la signalisation de la voie de l’insuline via IRS4 qui pourrait être d’intérêt dans le contexte de la maladie d’Alzheimer. Il pose également les bases à une identification rapide de nanocorps dirigés contre les récepteurs 5-HT4 et 5-HT6 dont les outils immunohistologiques manquent pour approfondir la compréhension mécanistique
Alzheimer’s Disease is the most common form of dementia worldwide and has become a major public health problem. This pathology is characterized by the presence of two main features in the brain: neurofibrillary tangles (NFTs) composed of hyperphosphorylated Tau and amyloid plaques, dense aggregates of hydrophobic β-amyloid peptide (Aβ). Aβ peptide results from the amyloidogenic degradation of the Amyloid Precursor Protein (APP) by β- and ɣ-secretases. On the other hand, the non-amyloidogenic proteolysis of APP, within the Aβ sequence by an α-secretase (mainly ADAM10), releases the extracellular fragment of APP (sAPPα), which is neurotrophic and neuroprotective. The 5-HT4 receptor (5-HT4R) is a G protein coupled receptor (GPCR) which interacts with APP and ADAM10, modulates their trafficking and maturation, and which pharmacological activation promotes the non-amyloidogenic cleavage of APP. The 5-HT6 receptor (5-HT6R) is another serotoninergic receptor which pharmacological modulation induces pro-cognitive effects. Thus, 5-HT4R and 5 HT6R constitute interesting therapeutic targets in the context of Alzheimer’s Disease. Nevertheless, the molecular and cellular mechanisms underlying these effects linked to 5-HT4R expression and activation remain elusive. This thesis aimed, in a first part, to develop a screening technique to isolate nanobodies (single domain antibody fragment from camelids) directed against 5-HT4R and 5 HT6R, by using TR FRET and flow cytometry approaches. In a second part, the use of a proteomic approach combining affinity purification of 5-HT4R partner proteins followed by their systematic identification by mass spectrometry (AP-MS) lead to the identification of potential partner proteins associated with 5-HT4R in HEK-293N cells. Amongst proteins recruited by 5 HT4Rs, we identified IRS4 (Insulin Receptor Substrate 4) protein. IRS4 protein is a transducer of the insulin signaling pathway. Given that a dysregulation of the insulin signaling pathway is one of the major events involved in Alzheimer’s Disease development, we decided to focus on this partner. We validated the interaction by Western blotting and showed a colocalization of endogenous IRS4 with 5 HT4R in HEK 293 cells. Then, functional studies showed that IRS4 downregulation induced 5 HT4R overexpression in HEK-293 cells, associated with an increase in the canonical signaling of the receptor through its Gs protein pathway.These results suggest a modulation of 5-HT4R trafficking and of its associated complex including APP and ADAM10 by IRS4 protein. This thesis work reveals for the first time a functional link between 5-HT4R signaling and insulin signaling pathway via IRS4 protein which could be of interest in the context of Alzheimer’s disease. It also establishes the preliminary basis for a fast identification of nanobodies targeting 5-HT4 and 5-HT6 receptors for which immunohistological tools are lacking to explore their underlying mechanisms

An ambitious long-term goal of medicine is to make analyses and deliver drugs selectively at cell level. In particular such "ideal" drugs should be able to travel through the vasculature, reach the intended target at full concentration, and there act selectively on diseased cells and tissues only, without creating undesired side effects. The state of the art in pursuing this goal is represented by \emph{nanoparticles} \cite{Ferrari2010}, which can be roughly defined as the combination of a drug molecule with a suitable vector of nanoscale dimensions. One of the main limitations of nanoparticles is that they do not have an onboard control system. Their ``program'' has to be defined in the drug design phase. A different program means the development of a new nanoparticle. Moreover the lack of onboard control means also that the complexity of the actions to be performed by nanoparticles is quite restricted. The basic idea sustaining this goal is inspired by the immune system. That the immune system is a fantastic machine for surveilling and fighting against exogenous diseases is too well known to deserve discussion. However, it is not perfect. A drawback of the immune system system is the poor recognition of endogenous pathological cells like those responsible for cancer or self-immune diseases. In this context it should be interesting to develop artificial devices (\emph{nanobots}) working as blood white cells but addressed to the recognition and eventually the destruction of endogenous pathological states. A nanobot \cite{Requicha2003} can be defined as any artificial machine with overall size of the order of a few microns or less ($\simeq$ red cells), constituted by nanoscopic components with individual dimensions in the interval $1-10^2$ nm, and able to perform sophisticated functions like navigation, cell recognition, data collection and transmission. To the best of my knowledge no attempt toward the definition of an auxiliary immune system is however known. Of course, this goal is extremely ambitious and is expected to require decades. Nonetheless, sketching a scenario is not a mere speculation, but rather is useful to identify the nature of problems posed by the development of nanobots for real applications. One of the possible reasons for the ineffectiveness of the immune system in detecting malignant cells is that the common features associated with cancer genesis and growth (hyperthermia, hypoxia and acidification) are the same as those characteristic of muscle under physical exercise. My program is thus that of \emph{endowing the immune system with an artificial surveillance system devoted to detecting the simultaneous occurrence of hyperthermia, hypoxia and excessive acidity due to localized cancerous states without confusing it with the similar conditions produced under physiological conditions}. Considering the diagnosis of cancer as a crime detection, the search of the perpetrator is not addressed to the identification of the smoking gun but rather to a frame evidence resulting from the simultaneous occurrence of three events (hyperthermia, hypoxia and excessive acidity) and their persistence in time. The aim of this thesis is twofold. On one side I want to describe an architecture of nanobots which is not only able to embed sophisticated functions, but also suitable for being manufactured by processes compatible with today and likely tomorrow semiconductor industries. On the other side, I want also to analyze and develop the algorithms needed for successfully tackling the nanobot tasks. In particular, since the nanobots would be devices with very limited computational resources and the interaction between them could only happen locally, the algorithms to control them could be based on swarm intelligence; i.e. it could be inspired by the collective behavior of social-insect colonies and other animal societies.

Le GPR54 et le RFSH régulent la reproduction en agissant sur l’axe hypothalamus-hypophyse-gonades (HPG). En agissant dans l’hypothalamus, GPR54 est un régulateur proximal de l’axe tandis que le RFSH contrôle la gamétogenèse dans les deux sexes. Ils représentent deux cibles thérapeutiques majeures pour le traitement des problèmes liés à la fertilité. GPR54 et RFSH appartiennent à la superfamille des récepteurs couplés aux protéines G (GPCR). GPR54 active préférentiellement la voie Gαq/PLC/Ca2+ alors que le RFSH induit principalement la voie Gαs/PKA/cAMP. Les deux récepteurs recrutent et activent les β-arrestines. Un nombre croissant de profils pharmacologiques ont été décrits pour agir sur des GPCR. En effet, des ligands biaisés, capables d’activer préférentiellement un sous-ensemble du répertoire de signalisation par rapport aux ligands endogènes, sont découverts à un taux élevé. Les ligands orthostériques et allostériques peuvent induire des biais en stabilisant des conformations particulières des récepteurs. Sur le plan thérapeutique, les ligands biaisés ont démontré leur potentiel à induire des effets thérapeutiques tout en évitant les effets indésirables. De plus, les ligands allostériques permettent le modulation positive ou négative d’un récepteur tout en conservant l’information temporelle associée au ligand endogène. Jusqu’il y a peu, des outils pharmacologiques aussi divers et intéressant n’existaient pas pour GPR54 ni pour RFSH. L’objectif de cette thèse a donc été d’identifier des ligands allostériques de GPR54 et du RFSH puis, de caractériser leur signalisation biaisée. Dans la première partie, nous avons caractérisé les propriétés pharmacologique d’un panel de composés de faible poids moléculaire, décrits récemment pour être des modulateurs allostériques du FSHR et appartenant à deux classes chimiques différentes. Nous avons profilé leurs actions sur différentes voies de signalisation dans des cellules HEK293 vivantes exprimant différents biosenseurs. Nous avons démontré que chacun de ces composés induisait des biais par comparaison à la FSH. En comparant différents modèles cellulaires, nous avons confirmé que les biais de système représentent un facteur de confusion crucial pour la détermination des biais. Nous avons également identifié des cas limites pour lesquels le modèle opérationnel n’a pas permis de calculer les facteurs de biais. En parallèle, nous avons caractérisé deux nouveaux composés appartenant à des classes chimiques qui n’avaient jamais été décrites pour activer le RFSH. Nous avons démontré qu’ils étaient allostériques et que leurs profils de biais étaient distincts de ceux des autres composés caractérisés. Dans la seconde partie, nous avons sélectionné and caractérisé des nanobodies ciblant GPR54 et RFSH. Nous avons identifié un nanobody exerçant un effet de modulateur allostérique positif (PAM) sur GPR54. Nous avons aussi identifié un nanobody ciblant le RFSH. Ce dernier présentait des propriétés biaisées originales puisqu’il était modulateur allostérique négatif (NAM) pour la voie AMPc mais PAM pour le recrutement de β-arrestine 2. Dans la dernière partie, nous avons essayé de développer des conjugués nanobody-drogue (NDC) en liant nos nanobodies à des agonistes – soit kisspeptin, soit un des composés agonistes que nous avons caractérisés dans la première partie – à l’aide d’un linker flexible. Bien que nous n’ayons pas eu le temps d’obtenir une preuve de concept pour les NDC, cette stratégie nous semble au minimum prometteuse en tant qu’outil de recherche. Dans sa globalité, cette thèse propose de nouveaux outils pharmacologiques qui vont permettre de comprendre les contributions relatives des différentes voies de couplage de GPR54 et du RFSH, in vivo, sur la fonction de reproduction. Ce travail ouvre également de nouvelles pistes thérapeutiques pour contrôler la reproduction chez les animaux d’élevage et en médecine de la reproduction
GPR54 and FSHR regulate reproduction by acting on the hypothalamus-pituitary-gonads (HPG) axis. Acting in the hypothalamus, GPR54 is an upstream regulator of the axis whereas FSHR controls gametogenesis in both sexes. They represents two major pharmacological targets for the treatment of fertility-related problems. Both GPR54 and FSHR belong to the G protein-coupled receptor (GPCR) superfamilly. GPR54 preferentially activates the Gαq/PLC/Ca2+ pathway whereas FSHR mainly activates the Gαs/PKA/cAMP pathway. Both receptors recruit and activate β-arrestins. Increasing number pharmacological profiles have been reported to act on GPCR. Indeed biased ligands capable of preferentially eliciting a subset of the full signalling repertoire, compared to the endogenous ligand are discovered at a high rate. Orthosteric and allosteric ligands can both induce biased signalling by stabilizing specific receptor conformations. Therapeutically, biased ligand have demonstrated the potential to avoid side effects while still activating the signalling pathways leading to therapeutic effects. Moreover, allosteric ligands allow positive or negative modulation of a receptor while keeping the temporal information provided by the endogenous ligand. Until recently, such diverse and valuable pharmacological tools were not available for FSHR and GPR54. The aim of this thesis was to identify allosteric ligands at the FSHR and GPR54 and to characterize their biased signalling. In the first section, we pharmacological characterized a panel of low molecular weight ligands, recently reported to allosterically activate the FSHR and belonging to two chemical classes. We profiled their actions on different signalling pathways in living HEK293 cells expressing different biosensors. We demonstrated each of these compounds induced biased signalling at the FSHR compared to FSH. Using different cell models, we confirmed that system bias is a crucial confounding factor in bias determination. We also identified limit cases in which the operational model did not allow to calculated bias factors. In parallel, we characterized two novel compounds belonging to chemical classes that were not yet reported to activate FSHR. We demonstrated that they were allosteric and that their biased profiles were distinct from the compounds characterized in the first study. In second section of the thesis, we selected and pharmacological characterized nanobodies targeting GPR54 and FSHR. We identified a nanobody that behaved as a positive allosteric modulator (PAM) at the GPR54. We also identified a nanobody against FSHR. This nanobody displayed striking biased properties as it was negative allosteric modulator (NAM) for cAMP production but PAM for β-arrestin 2 recruitment. In the last section of the thesis, we attempted to develop nanobody-drug conjugates (NDC) by linking our nanobodies to agonists - either kisspeptin or one of the low molecular weight agonist of the FSHR - through a flexible linker. Though we did not have time to achieve a proof of concept for NDC, we believe that such hybrid compound could represent at minimum a promising research tools. As a whole, this thesis provides novel pharmacological tools that should allow deciphering the relative contributions of the different transduction mechanisms operating at the FSHR and GPR54, in vivo, in the reproductive function. This work also opens possible avenues for future therapeutic strategies in the control of reproduction in farm animals and in reproductive medicine

Thesis (Ph. D.)--Materials Science and Engineering, Georgia Institute of Technology, 2007.
Wang, Zhong Lin, Committee Chair ; Wong, C.P., Committee Member ; Summers, Christopher J., Committee Member ; Degertekin, F. Levent, Committee Member ; Bottomley, Lawrence A., Committee Member.

Tese de doutoramento em Biologia Experimental e Biomedicina, no ramo de Biotecnologia e Saúde, apresentada ao Instituto de Investigação Interdisciplinar da Universidade de Coimbra
Cancer is nowadays the second leading cause of death and current therapeutic approaches still reveal ineffective in several cases. Therefore, there is a need to develop more efficacious therapeutic agents, especially for subtypes of cancer where targeted therapies are still missing, and, as such, unmet medical needs are evident. Limited drug penetration into tumors limits the efficacy of therapies targeting cancer cells. One of the strategies to overcome this problem is upon targeting the more accessible tumor vasculature. In this context, nucleolin arises as a target of extreme relevance, as it is overexpressed at the surface of cancer and angiogenic endothelial cells thus enabling a dual cellular targeting strategy. Antibodies have been extensively studied and some have been approved for the treatment of different types of tumors. Antibodies act by several mechanisms, from direct cell death to immune-mediated mechanisms (through the Fc region of the antibody). The latter include antibody-dependent cell-mediated cytotoxicity (ADCC), which plays a central role in the clinical efficacy of some antibodies. However, the high molecular weight of these proteins (≈150 kDa) limits effective penetration in solid tumors. Therefore, smaller units for antigen recognition have been developed. Nanobodies or VHHs are the variable domains of the heavy chains from camelid antibodies that lack light chains, presenting small size (≈15 kDa), high affinity to their antigens and reduced immunogenicity in humans. In addition, they are also versatile platforms for the development of different antibody formats. The main aim of this project was to develop and characterize new nanobody-based antibody formats against nucleolin. These consisted of (i) nanobodies generated by a grafting strategy using a nucleolin-binding sequence and (ii) an anti-nucleolin nanobody- Fc antibody, aiming at exploring immune mechanisms, namely ADCC. In addition, this ii work aimed also at exploring the use of nanobodies as bispecific proteins, where nucleolin and tumor necrosis factor alpha (TNF-α) were selected as models of target proteins. An anti-TNF-α VHH was used as a scaffold for grafting F3 peptide-derived nucleolin-binding sequences onto either complementarity determining region 1 (CDR1) or 3 (CDR3), the two most relevant antigen-binding regions. The grafted sequences consisted on a 10-amino acid sequence, with or without flanking linkers at each end, aiming at improving CDR flexibility. The generated nanobodies revealed binding to both purified nucleolin and nucleolin-overexpressing cells, as well as to the original target, TNF-α. These nanobodies presented cytotoxic effects in the micromolar range against these nucleolin-overexpressing cells. Grafting of the F3 peptide-derived sequence onto CDR3 enabled improved binding and cytotoxicity relative to grafting of the same sequence onto CDR1. However, the presence of flanking linkers in the grafted sequence did not alter the binding and cytotoxicity patterns. These results led to the selection of the nanobody presenting the F3 peptidederived sequence (without linkers) in CDR3 to generate a nanobody-Fc, upon fusion to the Fc region of a human IgG1. This anti-nucleolin nanobody-Fc antibody presented increased cytotoxic effects (in the nanomolar range) and also capacity of triggering a nucleolin-mediated ADCC effect against a cancer cell line. In conclusion, in this work, new nucleolin-targeting entities with cytotoxic activity against nucleolin-overexpressing cells were developed. In addition, the nanobodies presented bispecific properties, as evidenced by their binding to two different antigens. Notably, the nanobody-Fc antibody presented nucleolin-mediated ADCC capacity, which had not been described for this target yet. Therefore, the antibodies here described might contribute for the development of novel therapies targeting nucleolin.
O cancro é atualmente a segunda principal causa de morte e as abordagens terapêuticas atuais ainda se revelam ineficazes em muitos casos. Assim, há necessidade de desenvolver agentes terapêuticos mais eficazes, principalmente para subtipos de cancro para os quais não existem terapias direcionadas e para os quais são evidentes as necessidades médicas não satisfeitas. A limitada penetração de fármacos nos tumores compromete a eficácia das terapias direcionadas para as células cancerígenas. Uma das estratégias para ultrapassar este problema é o direcionamento da terapia para a vasculatura tumoral, que se encontra mais acessível. Neste contexto, a nucleolina surge como um alvo de extrema relevância, uma vez que é sobre-expressa à superfície de células cancerígenas e células endoteliais angiogénicas, permitindo uma estratégia de direcionamento multicelular. Os anticorpos têm sido extensivamente estudados e alguns foram aprovados para o tratamento de diferentes tipos de tumores. Os anticorpos atuam através de diversos mecanismos, desde morte celular direta a mecanismos imunitários (através da região Fc do anticorpo). Nestes últimos, inclui-se a citotoxicidade celular dependente de anticorpos (ADCC), que desempenha um papel central na eficácia clínica de alguns destes agentes terapêuticos. No entanto, o elevado peso molecular destas proteínas (≈150 kDa) limita a penetração eficaz em tumores sólidos. Consequentemente, foram desenvolvidas unidades mais pequenas de reconhecimento de antigénio. Os nanobodies ou VHH são os domínios variáveis das cadeias pesadas dos anticorpos de camelídeos sem cadeias leves, apresentando tamanho reduzido (≈15 kDa), elevada afinidade para os seus antigénios e imunogenicidade reduzida em humanos. Além iv disso, os nanobodies são também plataformas versáteis para o desenvolvimento de diferentes formatos de anticorpos. O principal objetivo deste projeto foi o desenvolvimento e caracterização de novos formatos de anticorpos baseados em nanobodies contra a nucleolina. Estes formatos consistiram em (i) nanobodies obtidos por uma estratégia de inserção de um peptídeo de 10 de aminoácidos, derivado de um outro (designado por F3) com demonstrada ligação à nucleolina sobre-expressa à superfície de células cancerígenas e (ii) um anticorpo nanobody-Fc anti-nucleolina, com o objetivo de explorar mecanismos imunitários, nomeadamente ADCC. Além disso, este trabalho teve também o objetivo de explorar o uso de nanobodies como proteínas bi-específicas, tendo a nucleolina e o fator de necrose tumoral alfa (TNF-α) sido selecionados como modelos de proteínas alvo. Um nanobody anti-TNF-α foi usado como base para inserção de sequências derivadas do péptido F3 na região determinante de complementaridade 1 (CDR1) ou 3 (CDR3), as duas regiões mais importantes na ligação ao antigénio. As sequências inseridas consistiram na referida sequência de 10 aminoácidos, com ou sem linkers em cada extremidade, os quais visavam aumentar a flexibilidade da CDR. Os nanobodies gerados apresentaram ligação a nucleolina purificada e a células que sobre-expressam nucleolina, assim como ao alvo original, TNF-α. Estes nanobodies apresentaram efeitos citotóxicos na ordem dos micromolar contra células que sobre-expressam nucleolina. A inserção da sequência derivada do péptido F3 em CDR3, resultou em ligação e citotoxicidade superiores às observadas com a inserção da mesma sequência em CDR1. Contudo, a presença de linkers nas extremidades da sequência inserida não alterou os padrões de ligação e citotoxicidade. Estes resultados levaram à seleção do nanobody com a sequência derivada do péptido F3 no CDR3 (sem linkers) para gerar um nanobody-Fc, após fusão a uma região Fc de uma IgG1 humana. Este anticorpo nanobody-Fc anti-nucleolina apresentou efeitos citótoxicos mais pronunciados (na ordem dos nanomolar) e também a v capacidade de promover um efeito de ADCC mediado por nucleolina, contra uma linha celular de cancro. Em conclusão, neste trabalho foram desenvolvidas entidades direcionadas para a nucleolina com atividade citotóxica contra células que sobre-expressam nucleolina. Além disso, os nanobodies apresentaram propriedades bi-específicas, como evidenciado pela sua ligação a dois antigénios diferentes. De notar que o anticorpo nanobody-Fc apresentou capacidade de ADCC mediada por nucleolina, um efeito que ainda não tinha sido descrito para este alvo. Desta forma, os anticorpos apresentados neste trabalho poderão contribuir para o desenvolvimento de novas terapias direcionadas para a nucleolina sobre-expressa em tumores.

Although phage display-based enrichments became a standard procedure, they still suffer from some drawbacks. Nonspecific phage binding limits the enrichment that can be achieved per se-lection round and therefore, in most cases, at least three or four rounds are required to identify the antigen binding nanobodies (Nbs) from the library. Moreover, the release of the phages captured via their antigen-specific Nb on the immobilized antigen is accomplished most often, by a pH shock that will also release the a-specific absorbed phages, thereby generating an unwanted back-ground. However, the biggest shortcoming is the difficulty to identify the Nb of best binding affinity. We now have to ferment and purify all individual Nb clones to homogeneity and measure their affinity parameters one after the other. This is very tedious, certainly, as the identified Nbs often share a high degree of amino acid sequence identity, which makes it impossible to predict the one having the best affinity. The objective of this thesis aimed to modify the phage display vector so that one round of selection will be sufficient, while the affinity of all antigen-positive Nbs can be compared immediately after ELISA. Firstly, a vector containing the Calmodulin Binding Peptide (CBP) tag was created, by substitut-ing the hemagglutinin (HA) tag present in a pMECS-GG plasmid by the CBP tag. Moreover, 6 Nbs with well-known and variable kinetic binding rates were also inserted into the vector pMECS-CBP. Next, the vector was used in a phage display setting, where two mini libraries comprising the 6 Nbs were made. One library had the conventional pMECS vector while the other was made using the pMECS-CBP vector. Although an enrichment of 1000 times was achieved using pMECS, pMECS-CBP failed to show any enrichment, supporting the hypothesis that Nb-CBP encountered serious expression problems. To investigate the validity of this hypothesis, periplasmic expression of Nb-CBP was performed and compared with that of the Nb without the tag. While the Nb yielded almost 4 mg per liter of culture media, the Nb-CBP could only be obtained at 0.63 mg per liter culture. Moreover, the Nb-CBP couldn’t be detected on a western blot. Several methods were used, such as adding lysozyme to improve the periplasmic extraction step, expressing 5 different colonies to check if there was a homogeneity of expression between them. In all meth-ods, the amount produced always remained below 1 mg per liter and Nb-CBP protein couldn’t be detected in any step of expression by Coomassie stained SDS-PAGE or western blot. Additionally, a high yield expressing-protein (SIRPα) was cloned into the CBP vector to identify whether the expression problem was caused by the peptide tag itself or by the Nb-CBP combina-tion. Even though SIRPα was obtained at a yield of 8.7 mg/L of culture when expressed from a vector not containing the CBP, its expressed yield using the CBP vector, dropped to 0.7 mg/L of culture, indicating that the CBP tag is incompatible with good periplasmic expression. Lastly, a nucleotide alignment of our CBP tag with a previously reported one, revealed differences in two Arginine codons, two Alanine codons and one Lysine codon. Moreover, our sequence employed two codons, AGA and CGG, rarely used in E. coli, which might be linked to the re-duced expression levels that we observed during this thesis.

One of the key components of the innate immune system are natural killer (NK) cells. The task of these cells is to induce apoptosis in target cells (e.g., cancer or virally infected cells). The target cells are identified by their interaction with surface receptors of the NK cells. On the surface of the NK cells, there are activating and inhibiting receptors. One of the activating receptors is the natural cytotoxicity receptor NKp46. Several ligands of this receptor have been identified, one of them being the epithelial adhesin Epa1 of yeast Candida glabrata. The invasive candidiasis caused by this yeast is a feared complication for patients with haematological diseases. The use of the NK cells in immunotherapy includes bispecific fusion proteins which can bind to the NK receptor with one part and to tumour antigen with the other part. This work focuses on recombinant preparation of the NKp46 protein. To facilitate a study of the effects of O-glycosylation on the binding of the ligands, a mutation of the glycosylation site NKp46 T225A was prepared. A stably transfected HEK293S GnTI- and HEK293T cells had been prepared and these proteins were then extracellularly secreted. The Epa1 protein had been produced in E. coli bacterial expression system and purified. The binding ability of the Epa1 protein...

The functions of the immune system include immunosurveillance of transformed cells, i.e., the ability to eliminate these cells before they become harmful to the organism. If the transformed cells succeed to escape the immune system surveillance, an oncological disease develops. The tumour immunotherapy aims to stimulate the immune system mechanisms to fight against the tumour. Lately, there's an interest in using NK cells in the immunotherapy of tumours. These cells appertain to the innate immune system and participate in immunosurveillance. When an NK cell encounters a target cell, its activation depends on the integration of signals from the surface activating and inhibiting receptors which bind ligands on the surface of the target cell. Upon activation, NK cell exhibits a cytotoxic response against the target cell. The use of NK cells in immunotherapy includes, among others, the testing of bispecific fusion proteins which can bind a tumour surface antigen by one part and NK cell activating receptor by the other part. Thus, these fusion proteins mediate a contact between both cells and trigger the cytotoxic response. This work presents a preparation of bispecific fusion proteins which consist of an activating ligand MICA (for the receptor NKG2D) or B7H6 (for the receptor NKp30), and a nanobody...

The emergence of multi-drug resistance in bacteria is a global health care challenge. One of the effective means of gaining antimicrobial resistance, among superbugs, is through expression of efflux pumps. Quaternary ammonium compound transporters, QacA/B, that are observed in Staphylococcus aureus strains are capable of transporting 30 chemically dissimilar monovalent and divalent cationic antibacterial compounds and dyes. The 14-TM (transmembrane) helix containing transporter QacA, belongs to the drug/H+ antiporter 2 (DHA2) family which is a part of major facilitator superfamily (MFS). MFS transporters are the largest superfamily of secondary active transporters. QacA utilizes the H+ gradient across the bacterial cell membrane for the uphill efflux of the cytotoxic compounds. QacA has two distinct TM domains, each of which consists of 6-TM helices that retain a pseudo 2-fold symmetry amongst them. During the transport process, the domains move in rocker-switch mechanism to allow alternating-access to either side of the membrane in order to transport the substrates. The expression of this efflux pump is regulated by a trans-acting regulatory DNA binding protein QacR. Under normal condition, QacR blocks the transcription of qacA gene by binding to the operator DNA but under antibacterial stress, the substrates of QacA binds to QacR, causing dissociation of QacR from the operator DNA and QacA gets expressed. In this thesis, structural and functional investigation of QacA was carried out and some fundamental questions about this multi-drug efflux transporter are addressed. In the first part, wild-type QacA was purified and the transport activity of the transporter both in native membrane and in isolation was analyzed using substrate-induced H+-release assay and a reconstitution-based assay. The binding studies with the cytotoxic substrates (TPP, Pm, Dq) displayed sub-millimolar binding affinity with the purified transporter and substrate/H+ competition assay suggested the presence of substrate-protonation site interactions in QacA. Further, survival assays done in the presence of TPP and Pm and whole cell ethidium efflux assay illustrated that ΔpH provides primary driving energy to the transporter. In the second part of the thesis, six protonatable acidic residues D34, D61, D323, E406, E407 and D411, lining the transport vestibule were identified using a homology model of QacA and each of the residue was characterized using mutagenesis. The binding studies and the transport assays illustrated D34, D323, E407 and D411 are crucial for the transport activity of the transporter either as substrate recognition sites or indirectly facilitating the transport process as protonation sites. The findings of the study suggested the inherent residue level promiscuity for different substrates of QacA, that can explain broad substrate specificities of QacA and other related multi-drug efflux transporters. The third part of the thesis described single-domain Indian camelid antibody (ICab) library generation and isolation of high affinity binders against QacA, in order to stabilize the transporter to facilitate the structural studies. The sorting of the binding population was done using yeast surface display coupled with flow-cytometry and 7 unique ICab binders were isolated. The last part of the thesis is focused on the heterologous expression and purification of two of the camelid antibodies in E. coli through refolding and cytosolic protein preparation. The binding studies using FSEC (fluorescence detection size exclusion chromatography), flow-cytometry and microscale thermophoresis suggested that the purified ICabs bind to the transporter with nanomolar affinity. Furthermore, 2D classes from cryo-electron microscopy of QacA-ICab complex clearly displayed the presence of the transporter in detergent micelles bound to the ICab. Moreover, the effect of ICab on QacA-substrate interactions indicated that ICabs can block substrate binding to the transporter. The results provide an interesting prospect of using the ICabs as efflux pump inhibitors (EPI).

With the remarkable advancement in the nanoparticles (NPs)-based drug delivery systems (DDS) over the past several decades, the pharmacological properties associated with conventional free drugs delivery are improved. In this thesis, we report potential candidates for the next-generation NP-based DDS. While natural DDS are promising as they possess exceptional delivery mechanisms and selective targeting, synthetic DDS are more favorable for their low immunogenicity. Our developed natural DDS called magnetotactic bacterial cages (MBC), which is based on magnetotactic bacteria (MTB) as a guidable delivery vehicle for DNA functionalized gold nanoparticles (AuNPs). Loading DNA functionalized AuNPs in MTB aided in increasing the maximum-tolerated dose of DNA functionalized AuNPs and tackled issues related to DNA functionalized AuNPs stability and systemic delivery. Natural DDS hold great advantages; however, it is difficult to make complete prediction about their immunogenicity and toxicity on the basis of preclinical trials. Thus, we assessed the efficacy of synthetic NP-based DDS. Using inorganic platforms, we were able to develop the first visual monitoring system of bacteria-NPs interaction. The system offers simultaneous sensing and inhibition of bacteria in infected cells. The system is comprised of Au nanoclusters @lysozyme (AuNC@lys) colloids MSN loaded with antibacterial agents. The applicability of the inorganic DDS in the biomedical field has been limited by the high bioaccumulation risks. Hybrid materials combine the advantages of organic, inorganic and natural carriers, offering opportunities for enhanced stability, manipulating release behavior and combine two or more functions in a single platform. To further enhance the properties our inorganic DDS, we incorporated light-responsive organic ligands to silicabased NPs. Plasmid DNA was loaded on the light-responsive bridged silsesquioxane nanocomposites (BS NPs). Light irradiation was performed to reverse the surface charge of NPs via a photoreaction of the organic fragments (silsesquioxane) within the NPs, that resulted in the release of plasmid DNA in HeLa cancer cells. Finally, we assessed a new class of organic-inorganic DDS composed of inorganic metal ions and organic linkers, zeolite imidazolate frameworks-8 (ZIF-8). These NPs showed exceptional ability to entrap large cargo due to their tunable porosity and structural flexibility.