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This thesis is focused on determining the structures of two anti-carbohydrate antibodies to understand how they achieve their specificity toward antigen. First, the structure of the antigen-binding fragment from the monoclonal antibody S64-4 in complex with a pentasaccharide bisphosphate fragment from chlamydial lipopolysaccharide (LPS) has been determined by x-ray diffraction to 2.6 Å resolution. Like the well-characterized antibody S25-2, S64-4 displays a pocket formed by the residues of germline sequence corresponding to the heavy and light chain V gene segments that binds the terminal Kdo (3-deoxy-α-D-manno-oct-2-ulopyranosonic acid) residue of the antigen; however, although S64-4 shares the same heavy chain V gene segment as S25-2, it has a different light chain V gene segment. The new light chain V gene segment codes for a combining site that displays greater avidity, different specificity, and allows a novel antigen conformation that brings a greater number of antigen residues into the combining site than possible in S25-2. Further, while antibodies in the S25-2 family use complementarity determining region (CDR) H3 to discriminate among antigens, S64-4 achieves its specificity via the new light chain V gene segment and resulting change in antigen conformation. These structures reveal an intriguing parallel strategy where two different combinations of germline-coded V gene segments can act as starting points for the generation of germline antibodies against chlamydial antigens and show how anti-carbohydrate antibodies can exploit the conformational flexibility of this class of antigens to achieve high avidity and specificity independently of CDR H3. Second, the structure of a rabbit, single chain variable fragment against terminal mannose-6-phosphate (Man6P) residues, termed scFv M6P-1, has been determined by x-ray diffraction to 2.7 Å resolution with Man6P in the binding site. The Man6P pathway is the predominant pathway that transports acid hydrolases from the trans-Golgi to endosomes. Newly synthesized hydrolases first require the generation of Man6P markers before they can be transported. Maintaining a full complement of hydrolases within lysosomes is essential as failure to do so results in a number of different lysosomal storage diseases. Due to its specificity, scFv M6P-1 is able to diagnose lysosomal storage diseases mucolipidosis II and mucolipidosis III. scFv M6P-1 is also able to purify Man6P containing proteins which may be useful for enzyme replacement therapies. Additionally, scFv M6P-1 is one of the first structures of an antibody fragment that exhibits high specificity for a single carbohydrate residue and is one of the first structures of a rabbit antibody fragment. The specificity of scFv M6P-1, which gives it these unique attributes, is revealed in the structure where multiple hydrogen bonds are seen between the antibody’s heavy chain and the mannose ring while two salt bridges are observed between the antibody’s light chain and the phosphate moiety. Finally, scFv M6P-1 binds in such a way as to allow binding to proteins possessing terminal Man6P residues. Crystallographic challenges that arose during this research included poor crystal growth as well as twinning and these are explored while the structure of scFv M6P-1 complex with Man6P is analysed. / Graduate / 0487 / 0982 / 0307 / dyl.w.evans@gmail.com

Cystic Fibrosis (CF) is one of the most common heriditary diseases. This congenital condition, caused by CFTR gene mutation, affects gastrointestinal and respiratory system especially. The affection of respiratory system is considered the most serious life-threatening symptom. Pacients suffer from reccurent infections proceeding to the development of chronic inflammation and progressive pulmonary tissue destruction. A typical specific microoganism colonizating pulmonary tissue of thouse suffering from CF is recognised as Pseudomonas aeruginosa. Pulmonary infections caused by this microorganism are the most often cause of death in patients suffering from CF. Antiobiotics are the first-line therapy of this condition currently. Nevertheless, the need to find alternatives occurs due to antibiotics resistance development. Passive immunization by specific hen-egg-yolk antibodies against P. aeruginosa is a possible alternative. An observation of IgY influence on a bacterial adhesion to pulmonary epithelial cells required an appropriate model. In this context an appropriate adhesion testing method based on P. aeruginosa and pulmonary epithelial cells visualisation was searched. At first bacterial cells labelling was tested by CellTracker, resazurin, FITC and consequently PKH 26. P. aeruginosa fluorescent...

De part, leur implication dans la prolifération cellulaire, l'invasion et leur surexpression dans de nombreux cancers, les récepteurs à tyrosine kinase de la famille HER constituent des cibles de choix en oncologie. Parmi ces récepteurs, le récepteur HER3 semble pertinent car il est impliqué dans la tumorigenèse de nombreux cancers (sein, ovaire, pancréas, mélanome…) et il est associé à un mauvais pronostic. De plus, la surexpression du récepteur HER3 est souvent associée à l'apparition de résistance aux thérapies ciblées. Nous avons sélectionné plusieurs anticorps anti-HER3 humains et murins respectivement par « phage display » et fusion cellulaire. Ces derniers reconnaissent spécifiqument le récepteur HER3. Parmi les nombreux anticorps découvert, nous avons sélectionné un anticorps anti-HER3 humain H4B-121 et 2 anticorps anti-HER3 murins 9F7-F11 et 16D3-C1. Ces derniers ont la capacité à faire régresser des tumeurs épidermoide, pancréatique et triple négative chez la souris, en présence ou en abscence de neuréguline et indépendamment du status HER2 et P53/PTEN. Cette inhibition est possible grâce à un blocage du cycle cellulaire en phase G1, une inhibition de la prolifération ainsi qu'une induction de l'apoptose. Ces trois anticorps sont capables de bloquer l'hétérodimérisation des récepteurs HER2/HER3 ainsi que la phosphorylation du récepteur HER3. Ils sont aussi capables d'inhiber la phosphorylation de la protéine AKT ainsi que de ses cibles (MDM2, FOXO et XIAP). De plus, nous avons montré que nos anticorps sont capables de lyser les cellules tumorales par ADCC (Antibody-dependent cell-mediated cytotoxicity). Cette étude démontre que ces anticorps anti-HER3 représentent une nouvelle thérapie pour les cancers du pancréas et du sein triple négatif. / Due to their implication in the cellular proliferation, the invasion and their surexpression in numerous cancers, the tyrosine kinase receptors of HER family constitute one of the best targets in oncology. Within this family, the human epidermal growth factor receptor 3 (HER3) plays a role in tumorigenesis of different cancers (Breast, melanoma, pancreas and ovary). This receptor is implicated in drug resistance and he is over expressed in cancers that are not eligible for the currently approved targeted therapies. To this end, we generated specific antibodies (Abs) against domain 1 (D1) and domain 3 (D3) of HER3 that recognize epitopes that do not overlap with the neuregulin-binding site. The fully human H4B-121 Ab and the mouse monoclonal Abs 16D3-C1 and 9F7-F11 inhibited tumor growth in nude mice xenografted with epidermoid, pancreatic, or triple-negative breast cancer cells independently of NRG addiction, HER2 status and p53/PTEN mutations. The combination of one anti-HER3 Ab and trastuzumab improved tumor growth inhibition in mice xenografted with HER2(low) cancer cell lines, for which trastuzumab alone shows no or moderate efficiency. Ab-induced disruption of tumor growth was associated with G1 cell cycle arrest, proliferation inhibition, and apoptosis of cancer cells. Anti-HER3 Abs blocked HER2/HER3 heterodimerization and HER3 phosphorylation at the cell membrane, leading to inhibition of phosphorylation of the downstream AKT targets murine double minute 2, X-linked inhibitor of apoptosis, and forkhead box O1. Anti-HER3 Abs can also induice antibody dependant cell-mediated cytotoxicity. This study demonstrates that anti-HER3 D1 and D3 Abs could represent a new option for immunotherapy of pancreatic and triple-negative breast cancers.

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Immunoglobulin (Ig) gene diversification plays an essential role in adaptive immunity. Faced with a continuous yet varied stream of self, non-self, and possibly harmful molecules, many organisms have mechanisms in their arsenal that have evolved to match the diversity of the antigens they encounter. In humans and mice, developing B and T lymphocytes go through a first round of genomic alteration — V(D)J recombination — in the bone marrow and the thymus, respectively. B cells can subsequently undergo two additional Ig gene diversification processes in secondary lymphoid tissues. Through somatic hypermutation (SHM), Ig variable regions of stimulated germinal center (GC)-forming B cells are mutated and further diversified, enabling affinity maturation. During class-switch recombination (CSR), on the other hand, B cells in the GC or prior to entering the GC recombine Ig constant regions, swapping the IgM-encoding locus for another isotype constant regions gene (e.g., IgG1, IgG3, IgE, IgA) to allow for different effector functions. Both B cell-specific genomic alterations are initiated when the single-stranded DNA (ssDNA) mutator enzyme activation-induced cytidine deaminase (AID) catalyzes the removal of the amino group off deoxycytidine residues, resulting in deoxyuridines and dU:dG mismatches. Low-fidelity cellular responses to the presence of dU, including the mismatch repair (MMR) and the base-excision repair (BER) pathways, are then thought to introduce mutations in SHM and CSR, as well as cause double-strand breaks (DSBs) repaired through canonical and alternative non-homologous end-joining in CSR. Though necessary for proper physiological function, these lymphocyte genome diversification processes are rife with danger for B cells and there is strong selective pressure to carefully orchestrate and target them so as not to threaten the genomic integrity of the cells through breaks or other mutations at non-Ig loci. Yet, these events can still occur, as demonstrated by the implication of AID with translocations found in some cancers (e.g., c- MYC:IGH in Burkitt’s lymphoma). Therefore, the mechanisms underlying AID mutagenic activity targeting to physiological deamination substrates have been the focus of several studies. Protein kinase A (PKA)-dependent phosphorylation of AID at its serine 38 residue has been shown to enable its interaction with replication protein A (RPA) before binding to ssDNA. Others have reported that SPT5 helps target AID to sites of RNA polymerase II (Pol II) stalling, such as the Ig switch sequences. Another cofactor, the RNA exosome complex, helps target the ssDNA mutator AID to both strands of DNA in vivo. The RNA exosome had hitherto been described in the context of RNA processing and degradation as 3’ → 5’ exoribonuclease. Sterile transcript-generating transcription at Ig loci was known to be required for proper AID catalytic activity; the newly described link between the RNA exosome and AID activity raised the prospect that RNA processing, and not mere transcription, might be playing a role in shaping the diversification of the immune repertoire in B lymphocytes. During CSR, transient three-strand structures called R loops are generated. R loops are formed as the nascent transcript invades the DNA duplex, hybridizing to the template strand, and displacing the non-template one. The G-rich nature of the non-template strand is posited to help stabilize the R loop, which allows the ssDNA mutator AID to use the exposed, non-template strand as a substrate. AID must then access the template strand. Here, we investigate the role that the RNA exosome and a potential cofactor, the putative RNA/DNA helicase senataxin (SETX), play in the sequence of biological events that result in CSR while protecting the cell from R-loop accumulation-associated genomic instability.