Academic literature 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.<br>Doctorat en Sciences biomédicales et pharmaceutiques (Médecine)<br>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<br>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<br>Cataloged from the official PDF of thesis.<br>Includes bibliographical references.<br>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.<br>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.<br>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.<br>Overall, this suite of nanobodies provides a unique and versatile toolkit for the study of the NPC.<br>by Sarah Ann Nordeen.<br>Ph. D.<br>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.<br><p>QC 20141203</p>

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<br>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