Avec quatorze anticorps utilisés en oncologie, les anticorps monoclonaux ont enfin une place de choix dans l'arsenal thérapeutique anticancéreux. Cependant, l'un des modes d'action les plus importants de ces molécules, la cytotoxicité à médiation cellulaire dépendante des anticorps (ADCC), souffre de plusieurs limites en raison de la nécessité d'une interaction optimale avec le FcγRIIIA. L'équipe du Dr. Daniel Baty a conçu de nouveaux formats d'anticorps bispécifiques basés sur l'utilisation de domaine unique d'anticorps de lamas (sdAb) et capables de contourner la plupart de ces limites. Des banques de phages issus de lamas immunisés ont permis de sélectionner deux sdAbs (d'affinités différentes) dirigés contre le FcγRIIIA exprimé par des cellules NK et les macrophages, ainsi qu'un sdAb dirigé contre l'antigène tumoral carcino-embryonnaire (ACE). En utilisant les domaines CH1 et Ck d'IgG1 humaine comme motif d'hétérodimérisation et les sdabs anti-ACE et anti-FcγRIII précédemment sélectionnés, nous avons développé des formats innovants d'anticorps bispécifiques Fab-like nommés bsFabs. Ces molécules sont faciles à produire, très stables et peuvent déclencher la lyse des cellules tumorales par les cellules NK humaines à des concentrations de l'ordre du picomolaire. Elles ne se lient pas au récepteur inhibiteur FcγRIIB, n'entrent pas en compétition avec des IgG sériques pour la liaison aux récepteurs, et leur activité cytotoxique est indépendante de la glycosylation du Fc et du polymorphisme du FcγRIIIA. / With fourteen antibodies used in oncology, monoclonal antibodies finally have a place in the therapeutic arsenal against cancer. However one of the modes of action of the most important of these molecules, cell-mediated cytotoxicity antibody-dependent (ADCC), suffers from several limitations due to the need for optimal interaction with the FcγRIIIA. Daniel Baty's team has developed new formats of bispecific antibodies based on the use of single domain llama antibodies (SdAb) that are capable of circumventing most of the these limitations. Phage libraries from immunized llamas were used to select two sdAbs directed against the FcγRIIIA expressed by NK cells and macrophages (with different affinities for FcγRIII), as well as one SdAb directed against the tumor antigen carcinoembryonic (CEA). Using CH1 and Ck domains of human IgG1 as a motif for heterodimerization and sdabs previously selected, we have developed innovative anti-CEA and anti-FcγRIII formats of bispecific antibodies Fab-like named bsFabs. These molecules are easy to produce, very stable and can trigger tumor cell lysis by human NK cells at picomolar concentrations. They do not bind FcγRIIB inhibitory receptor, do not compete with serum IgG and their cytotoxic activity is independent of FcγRIIIA polymorphism. In addition, they slow tumor growth in mouse model. In terms of pharmacokinetics, although bsFabs have a reasonable tumor retention, one of the limitations of these formats is size, which leads to rapid renal elimination. Such findings led us to consider the creation of new antibody formats to both increase tumor retention and secondly the half-life of antibodies in the body.

A Artrite reumatóide (AR) é uma doença inflamatória crônica e sistêmica, de etiologia desconhecida, que pode dificultar ou impossibilitar as funções habituais das articulações devido à destruição da cartilagem, edema e dor. A patogênese da AR envolve uma complexa inter-relação de fatores imunológicos, ambientais e genéticos, dificultando assim a descoberta de terapias eficientes. Na AR, o influxo de neutrófilos para a cavidade sinovial é predominante e contínuo de tal forma que, os neutrófilos são as células mais abundantes no sítio inflamatório. Uma vez ativados, os neutrófilos são capazes de produzir espécies reativas de oxigênio que, juntamente com enzimas proteolíticas, podem estar envolvidos na lesão articular observada nos pacientes com AR. A ativação dos neutrófilos pode ser mediada por receptores para IgG (Fc?R) e para o complemento (CR) presentes nas membranas dessas células, através da interação com complexos imunes (ICs). Esses receptores são também importantes moduladores das reações inflamatórias mediadas por ICs. Entretanto, a função do sistema complemento e dos Fc?R, bem como a importância de cada um na manifestação da AR ainda não está clara. Assim, neste estudo avaliou-se o metabolismo oxidativo de neutrófilos estimulados através de receptores Fc?R e Fc?R/CR em ambos os grupos de pacientes com AR, ativa e inativa, e em controles saudáveis. Esta função celular foi avaliada por quimioluminescência (QL) dependente de luminol e de lucigenina. Os resultados mostraram que não houve diferenças na produção de QL, tanto dependente de luminol quanto de lucigenina, quando mediada por Fc?R ou pela cooperação Fc?R/CR, em ambos os grupos de pacientes com AR comparados entre si e comparados com neutrófilos de indivíduos saudáveis. No entanto, a comparação do padrão da resposta de QL dentro de ambos os grupos de pacientes com AR, mostrou que a resposta celular aos IC opsonizados por soro humano de pacientes com AR (IC-IgG/SHAR) não foi significativamente significantemente maior em relação à observada para os IC não opsonizados, refletindo uma ausência da cooperação Fc?R/CR nestas células. Vale ressaltar que a atividade hemolítica do complemento sérico, tanto da via clássica/lectina quanto da alternativa, não foi diferente entre os grupos estudados. Quanto à expressão dos CR, somente no grupo de pacientes com AR ativa observou-se diferenças, sendo que CR1 estava aumentado em relação ao grupo controle (p<0,05) e CR3 aumentado quando comparado ao grupo de pacientes com AR inativa (p<0,05). A expressão dos Fc?R, CD32 e CD16, não foi diferente entre os grupos estudados. Ainda, as análises de correlação da expressão dos diferentes receptores de membrana mostraram: (i) correlação positiva CD16 vs CR1 somente nos grupos com AR, ativa e inativa; (ii) correlação positiva CD16 vs CR3 no grupo com AR ativa; (iii) ausência de correlação entre a expressão de CD32 e o número de neutrófilos CD32+ no grupo com AR ativa; e (iv) correlação positiva entre a expressão de CR3 e o número de neutrófilos CR3+ no grupo com AR ativa. O conjunto de resultados sugere que estas diferenças ocorrem apenas durante a atividade da doença, reforçando a hipótese que estas alterações sejam uma característica adquirida da doença. Desta forma, este estudo pode contribuir para esclarecer mecanismos envolvidos na patogênese da AR, que possam ser alvos em potencial para o desenvolvimento de agentes terapêuticos específicos para esta doença. / Rheumatoid arthritis (RA) is a chronic and systemic inflammatory disease of unknown etiology that can impair the usual joint functions, due to cartilage damage, edema and pain. The RA pathogenesis involves multiple interacting immunological, environmental and genetic factors, making it difficult to find effective therapies. Neutrophils are the most abundant cells at AR inflammatory sites, since they migrate continuously to the synovial cavity. The activated neutrophils generate reactive oxygen species and release a variety of proteases that seem to be involved in the joint lesions of RA patients. Neutrophil activation can be mediated by membrane receptors for IgG (Fc?R) and for complement (CR) through interaction with immune complexes (IC). These receptors are also very important modulators of IC-mediated inflammatory reactions. However, the role of the complement system and Fc?R and the hierarchy of them in the manifestation of RA are still unclear. In this study, we evaluated the neutrophil oxidative burst induced by Fc?R and Fc?R/CR, from RA patients at active and inactive disease stages, and from healthy controls. This cellular function was assessed by luminol- and lucigenin-enhanced chemiluminescence (CL) systems. When neutrophils were stimulated via Fc?R or Fc?R/CR cooperation, we found that the cellular responses of active and inactive RA patients were not significantly different when compared to each other and to the healthy controls, by using both CL systems. However, the comparison between the active and inactive RA groups revealed that the CL responses triggered by IC opsonized with RA serum were not significantly higher than those observed for neutrophils stimulated only via Fc?R (IC-IgG), reflecting an absence of Fc?R/CR cooperation in these cells. In addition, the hemolytic activities of serum complement, classical/lectin and alternative pathways, were not different among the groups studied. With regard to the CR expression, only neutrophils from the active RA group showed an increased number of CR1 and CR3 on their surfaces when compared with the control (p<0.05) and the inactive RA groups (p<0.05), respectively. The Fc?R expressions, CD32 and CD16, were not different among the groups studied. In addition, correlation analysis of the expression among the different receptors showed: (i) a positive correlation CD16 vs CR1 in both RA groups, active and inactive; (ii) a positive correlation CD16 vs CR3 in the active RA group; (iii) an absence of correlation between the CD32/neutrophil expression and the number of this cell bearing CD32 in the active RA groups; and (iv) a positive correlation between the CR3/neutrophil expression and the number of this cell bearing CR3 in the active RA group. These results suggest that such differences could be occurring just during the activity of the RA, supporting once again an acquired characteristic of the disease. This study can contribute for the understanding of the mechanisms involved in the pathogenesis of the RA, which might become potential targets for the development of specific therapeutic agents for this disease.

Plus de 40 ans après la découverte de la technologie des hybridomes, une soixantaine d’anticorps monoclonaux thérapeutiques IgG ou assimilés sont aujourd’hui commercialisés. Leur succès découle de leur humanisation, en particulier celle de la portion Fc qui dérive de différents variants humains naturels, isotypes et allotypes. Depuis quelques années, apparaissent sur le marché de nombreux anticorps comportant des portions Fc artificiellement modifiées dans le but de moduler diverses propriétés pharmacologiques (propriétés cytolytiques, demi-vie, stabilité, etc.), dont certaines ont été particulièrement étudiées suite aux travaux de notre équipe. Les variants Fc sont protégés par des technologies brevetées dont on connaît mal l’étendue, qui ne font pas nécessairement l’objet de publications scientifiques, et dont la raison d’être reste méconnue des chercheurs et plus encore des professionnels de santé. Nous avons donc entrepris de réaliser une cartographie et une analyse fine des brevets traitant des modifications dans la portion Fc des IgG. Cette analyse a été menée de front avec une étude bibliographique détaillée, car les données scientifiques décrites dans les brevets sont toujours à considérer avec prudence, les demandes de brevets n’étant pas revues par des pairs. Nous avons eu l’occasion d’ailleurs d’épingler certaines dérives, comme celle de considérer qu’il pourrait y avoir plus de 4 sous-classes d’IgG dans l’espèce humaine (jusqu’à 19 dans certaines revendications…). / More than 40 years after the discovery of the hybridoma technology, around sixty therapeutic monoclonal antibodies based on IgG or assimilated are marketed today. Their success comes from their humanization, especially of the Fc portion derived from various natural human variants, isotypes and allotypes. For some years, many antibodies artificially modified in their Fc portions have emerged, in order to alter various pharmacological properties (cytolitic properties, half-life, stability, etc.), some of them having been particularly studied following the works of our team. Fc variants are covered by patented technologies of which little is known about the extent, which are not necessarily the subject of scientific publications, and whose purpose remains unknown for researchers and even more for health professionals. We therefore undertook to realize a landscape and a detailed analysis of patents dealing with modifications in the Fc portion of IgG. This analysis has been conducted in front with a detailed literature survey, since the scientific data described in patents must be treated with caution, as patent application are not peer reviewed. We actually point certain abuses, such as to consider that there might be more than four human IgG subclasses (up to 19 in some claims…).

Um die Wirkung herpesviral-kodierter FcgammaRezeptoren auf wirtskodierte zelluläre FcgammaRezeptoren und IgG-vermittelten Effektorfunktionen untersuchen zu können, einen methodisch neuen Ansatz wurde entwickelt, der die Detektion FcgammaR-aktivierender Antikörper ermöglicht. Dieses neuartige Assay beinhaltet die Kokultivierung virusinfizierter Zellen, die mit virusspezifischen IgG-Antikörpern opsoniert sind, mit FcgammaR-zeta BW5147-Transfektanten als Reporterzellen. Diese stabilen Transfektanten exprimieren chimäre Rezeptoren, die aus der extrazellulären Domäne der zellulären FcgammaRezeptoren bestehen, welche mit der TM und intrazellulären Domäne der murinen CD3zeta-Kette fusioniert wurden. Die Aktivierung der CD3zeta-Kette führt zu einer IgG-dosisabhängigen mIL-2 Sekretion, die im ELISA gemessen werden kann. Die FcgammaR-spezifische immune IgG könnte eine wichtige biologische Rolle in der antiviralen Immunabwehr spielen. Herpesviren exprimieren auf der Oberfläche infizierter Zellen viral-kodierte Fc-bindende Glykoproteine. Um zu bestimmen, ob virale FcgammaRezeptoren die IgG-abhängige Aktivierung von wirtskodierten FcgammaRezeptoren beeinflussen können, wurde das oben beschriebene Assay angewandt. Es wurde festgestellt, dass der HCMV-kodierte FcgammaR gp68 die Aktivierung und die nachfolgende Signalkaskade von CD16>CD32=CD64 inhibiert, während der HCMV-kodierte FcgammaR gp34 die Aktivierung von CD16>CD64>CD32 inhibiert. In klarem Kontrast dazu wirkt der HSV-kodierte FcgammaR gE, der CD16 Aktivierung vermindert, CD32 hingegen nur sehr schwach und CD64 gar nicht beeinflußt. Der MCMV-kodierte FcgammaR m138/fcr-1 vermindert die Aktivierung des murinenCD16. Zusammenfassend betrachtet zeigen die ermittelten Daten, dass es sich bei den herpesviral-kodierten FcgammaRezeptoren um hierarchische und redundante Antagonisten der wirtskodierten zellulären FcgammaRezeptoren handelt. Herpesviral-kodierte FcgammaRezeptoren wirken somit der Aktivierung des Immunsystems entgegen. / To study the possible interference of the herpesviral vFcgammaRs with the host FcgammaRs and IgG-mediated effector functions, a new methodological approach to detect FcgammaR activating antibodies was developed. The novel assay comprises the co-cultivation of virus infected cells upon opsonization with immune IgG antibodies and the stably transfected FcgammaR-zeta BW5147 transfectants as responder cells. The transfectants express chimeric receptors bearing the extracellular domain of the host FcgammaRs fused to the transmembrane and tail domains of the murine CD3zeta chain. Triggering the CD3zeta chain is sufficient to elicit IL-2 secretion in a dose dependent manner which is measured in an ELISA. The setup of the new assay provides a defined effector cell population bearing one Fcgamma receptor on the surface, which becomes activated in the presence of immune IgG antibodies bound to the native viral antigens displayed on the surface of infected cells. The assay system allows us to detect and quantify Fc gamma receptor-activating immune IgG in an FcgammaR-specific way, which is thought to have an important biological function in antiviral defense. Several alpha- and beta- herpesviruses express on the surface of infected cells virally encoded Fc binding glycoproteins. The assay described above was applied to determine if the viral FcgammaRs are able to impair IgG-mediated activation of host FcgammaRs. In a systematic approach, the effect on each host FcgammaR by each of the herpesviral FcgammaR was investigated. It was found that HCMV FcgammaR gp68 affects activation and downstream signaling of CD16 > CD32 = CD64, while gp34 attenuates CD16 > CD64 > CD32. In clear contrast, HSV gE impairs CD16 activation and weakly CD32, but has no effect on CD64. Furthemore, MCMV m138/fcr-1 diminishes activation of mouse CD16. Taken together, this data uncover herpesviral FcgammaRs as hierarchical and redundant antagonists precluding host FcgammaRs from triggering immune responses.

A Artrite reumatóide (AR) é uma doença inflamatória crônica e sistêmica, de etiologia desconhecida, que pode dificultar ou impossibilitar as funções habituais das articulações devido à destruição da cartilagem, edema e dor. A patogênese da AR envolve uma complexa inter-relação de fatores imunológicos, ambientais e genéticos, dificultando assim a descoberta de terapias eficientes. Na AR, o influxo de neutrófilos para a cavidade sinovial é predominante e contínuo de tal forma que, os neutrófilos são as células mais abundantes no sítio inflamatório. Uma vez ativados, os neutrófilos são capazes de produzir espécies reativas de oxigênio que, juntamente com enzimas proteolíticas, podem estar envolvidos na lesão articular observada nos pacientes com AR. A ativação dos neutrófilos pode ser mediada por receptores para IgG (Fc?R) e para o complemento (CR) presentes nas membranas dessas células, através da interação com complexos imunes (ICs). Esses receptores são também importantes moduladores das reações inflamatórias mediadas por ICs. Entretanto, a função do sistema complemento e dos Fc?R, bem como a importância de cada um na manifestação da AR ainda não está clara. Assim, neste estudo avaliou-se o metabolismo oxidativo de neutrófilos estimulados através de receptores Fc?R e Fc?R/CR em ambos os grupos de pacientes com AR, ativa e inativa, e em controles saudáveis. Esta função celular foi avaliada por quimioluminescência (QL) dependente de luminol e de lucigenina. Os resultados mostraram que não houve diferenças na produção de QL, tanto dependente de luminol quanto de lucigenina, quando mediada por Fc?R ou pela cooperação Fc?R/CR, em ambos os grupos de pacientes com AR comparados entre si e comparados com neutrófilos de indivíduos saudáveis. No entanto, a comparação do padrão da resposta de QL dentro de ambos os grupos de pacientes com AR, mostrou que a resposta celular aos IC opsonizados por soro humano de pacientes com AR (IC-IgG/SHAR) não foi significativamente significantemente maior em relação à observada para os IC não opsonizados, refletindo uma ausência da cooperação Fc?R/CR nestas células. Vale ressaltar que a atividade hemolítica do complemento sérico, tanto da via clássica/lectina quanto da alternativa, não foi diferente entre os grupos estudados. Quanto à expressão dos CR, somente no grupo de pacientes com AR ativa observou-se diferenças, sendo que CR1 estava aumentado em relação ao grupo controle (p<0,05) e CR3 aumentado quando comparado ao grupo de pacientes com AR inativa (p<0,05). A expressão dos Fc?R, CD32 e CD16, não foi diferente entre os grupos estudados. Ainda, as análises de correlação da expressão dos diferentes receptores de membrana mostraram: (i) correlação positiva CD16 vs CR1 somente nos grupos com AR, ativa e inativa; (ii) correlação positiva CD16 vs CR3 no grupo com AR ativa; (iii) ausência de correlação entre a expressão de CD32 e o número de neutrófilos CD32+ no grupo com AR ativa; e (iv) correlação positiva entre a expressão de CR3 e o número de neutrófilos CR3+ no grupo com AR ativa. O conjunto de resultados sugere que estas diferenças ocorrem apenas durante a atividade da doença, reforçando a hipótese que estas alterações sejam uma característica adquirida da doença. Desta forma, este estudo pode contribuir para esclarecer mecanismos envolvidos na patogênese da AR, que possam ser alvos em potencial para o desenvolvimento de agentes terapêuticos específicos para esta doença. / Rheumatoid arthritis (RA) is a chronic and systemic inflammatory disease of unknown etiology that can impair the usual joint functions, due to cartilage damage, edema and pain. The RA pathogenesis involves multiple interacting immunological, environmental and genetic factors, making it difficult to find effective therapies. Neutrophils are the most abundant cells at AR inflammatory sites, since they migrate continuously to the synovial cavity. The activated neutrophils generate reactive oxygen species and release a variety of proteases that seem to be involved in the joint lesions of RA patients. Neutrophil activation can be mediated by membrane receptors for IgG (Fc?R) and for complement (CR) through interaction with immune complexes (IC). These receptors are also very important modulators of IC-mediated inflammatory reactions. However, the role of the complement system and Fc?R and the hierarchy of them in the manifestation of RA are still unclear. In this study, we evaluated the neutrophil oxidative burst induced by Fc?R and Fc?R/CR, from RA patients at active and inactive disease stages, and from healthy controls. This cellular function was assessed by luminol- and lucigenin-enhanced chemiluminescence (CL) systems. When neutrophils were stimulated via Fc?R or Fc?R/CR cooperation, we found that the cellular responses of active and inactive RA patients were not significantly different when compared to each other and to the healthy controls, by using both CL systems. However, the comparison between the active and inactive RA groups revealed that the CL responses triggered by IC opsonized with RA serum were not significantly higher than those observed for neutrophils stimulated only via Fc?R (IC-IgG), reflecting an absence of Fc?R/CR cooperation in these cells. In addition, the hemolytic activities of serum complement, classical/lectin and alternative pathways, were not different among the groups studied. With regard to the CR expression, only neutrophils from the active RA group showed an increased number of CR1 and CR3 on their surfaces when compared with the control (p<0.05) and the inactive RA groups (p<0.05), respectively. The Fc?R expressions, CD32 and CD16, were not different among the groups studied. In addition, correlation analysis of the expression among the different receptors showed: (i) a positive correlation CD16 vs CR1 in both RA groups, active and inactive; (ii) a positive correlation CD16 vs CR3 in the active RA group; (iii) an absence of correlation between the CD32/neutrophil expression and the number of this cell bearing CD32 in the active RA groups; and (iv) a positive correlation between the CR3/neutrophil expression and the number of this cell bearing CR3 in the active RA group. These results suggest that such differences could be occurring just during the activity of the RA, supporting once again an acquired characteristic of the disease. This study can contribute for the understanding of the mechanisms involved in the pathogenesis of the RA, which might become potential targets for the development of specific therapeutic agents for this disease.

Syftet med studien var att studera effekterna av några miljöfaktorer och genetiskt predisponerande faktorer för reumatoid artrit (RA). RA är en inflammatorisk sjukdom där immunsystemet bryter ner kroppsegen vävnad. En signifikant ökad risk för RA påvisades vid exponering för spannmålsdamm och rökning. Generna som studerades var GSTM1, GSTT1 och Fc-gamma-RII. Generna i sig var ingen predisponerande faktor för RA, men rökning visade sig vara en signifikant högre riskfaktor för de som hade en deletion av GSTM1 eller Fc-gamma-RII genen samt för de som inte hade deletion av GSTT1 genen. Exponering för spannmålsdamm visade sig vara en signifikant större riskfaktor för RA för de som inte hade deletion av GSTM1, GSTT1 eller Fc-gamma-RII genen. Vidare visade individer som inte hade en deletion av Fc-gamma-RII genen en ökad risk för RA vid exponering för stendamm. / The aim of this study was to evaluate the effects of some environmental and genetic predisposing factors of Rheumatoid Arthritis (RA). RA is an inflammatory disease where the immune system decomposes body tissue. A significant increased risk of RA was shown when exposed to grain and smoking. The genes that were studied were GSTM1, GSTT1 and Fcgamma- RII. The genes themselves were no predisposing factors of RA, but smoking showed to be a significant greater riskfactor for RA for individuals having a deletion of the GSTM1 or the Fc-gamma-RII gene, or not having a deletion of the GSTT1 gene. Exposure to grain showed to be a significant greater riskfactor for RA for individuals not having a deletion of the GSTM1, GSTT1 or the Fc-gamma-RII gene. Also individuals not having a deletion of the Fc-gamma-RII gene showed an increased risk for RA when exposed to stonedust.

La pharmacocinétique (PK) des anticorps monoclonaux (mAbs) est sujette à d’importantes variations interindividuelles. Le récepteur néonatal au Fc des IgG (FcRn) et le statut immun à l’encontre de ces mAbs sont des déterminants de cette PK. La bioconjugaison des mAbs à des cytotoxiques entraîne une altération de leur PK. Nous montrons que le taux de couplage modifie l’affinité de ces espèces pour le FcRn à pH6. La proportion d’agrégats au sein des solutions d’anticorps armés augmente avec le taux de couplage et pourrait entraîner une altération de leur PK. Par ailleurs, cette agrégation est impliquée dans l’immunogénicité des mAbs, et nous avons donc cherché à identifier des acides aminés impliqués dans l’agrégation de mAbs indiqués en clinique. Il apparait que la nature biochimique de résidus des paratopes pourrait augmenter cette agrégation. Les anti-TNF- présentent très peu d'agrégats et figurent pourtant parmi les plus immunogènes chez l’Homme. Nous avons donc exploré le rôle des complexes immuns dans leur immunogénicité chez la souris. Il apparait que la présence du FcRn n’est pas à l’origine de l’immunisation contre ces mAbs, contrairement à celle des complexes immuns. Ces résultats donnent des pistes pour la production de mAbs plus efficients et mieux tolérés. / The pharmacokinetic (PK) profile of monoclonal antibodies (mAbs) shows interindividudal variability. The neonatal Fc receptor (FcRn) and the immounogenicity of these mAbs are determinative factors of mAb PK. Generation of antibody-drug-conjugates alters their PK profile. We show that the the affinity for FcRn at pH6 increases with the drug-to-mAb ratio, as does the amount of aggregates inside the mAb-drug-conjugate. The amount of aggregates could be responsible for an avidity effect towards FcRn. These aggregates are known to cause immunogenicity, so we studied biochemical determinants inside the aminoacid sequence of marketed mAbs. We show that the biochemical nature of some aminoacids inside the paratope has an impact on the amount of aggregation. Anti-TNF- mAbs show very little aggregation but are very immunogenic in humans. We studied the role of the formation of immune complexes in the immunization against anti-TNF- mAbs in mice, and showed that immune complexes, but not FcRn are essential in the immunization process against anti- TNF- mAbs. These results give leads towards the generation of more efficient, better tolerated mAbs.

Les anticorps monoclonaux ont révolutionné le traitement de nombreuses pathologies. Cependant, leur pharmacocinétique (PK) et l’influence de leur concentration sur la réponse clinique restent mal connues. Nous avons étudié les sources de variabilité interindividuelle de la PK du cetuximab, un anticorps anti- EGFR, ainsi que l’influence de l’exposition à cet anticorps sur la réponse. Nous avons validé une méthode ELISA de dosage du cetuximab. Dans un modèle murin, nous avons étudié l’absorption pulmonaire du cetuximab. Nous avons étudié la PK du cetuximab chez un patient hémodialysé. Nous avons décrit la PK du cetuximab chez des patients traités pour cancer colorectal métastatique, à l’aide d’un modèle combinant des éliminations d’ordre 0 et 1. Enfin, nous avons identifié la clairance globale du cetuximab, paramètre pouvant être estimé précocement par la concentration résiduelle à J14, comme un facteur influençant la survie sans progression des patients. Nos travaux montrent qu’une description de la PK d’un anticorps par approche compartimentale permet d’identifier les sources de variabilité et d’étudier l’impact de la PK sur la réponse clinique. / Monoclonal antibodies have profoundly modified the treatment of many diseases. However, their pharmacokinetics (PK) and the influence of their concentrations on the clinical response are poorly known. We studied the sources of the interindividual variability of PK of cetuximab, an anti-EGFR, and the influence of the exposure to this antibody on the response. We validated an ELISA technique to measure cetuximab concentrations. We studied the pulmonary absorption of cetuximab in a murine model. We studied cetuximab PK in a hemodialysed patient. In metastatic colorectal cancer patients, we described cetuximab PK with the help of a model combining zero- and first-order eliminations. Finally, we identified the global clearance of cetuximab, a parameter which can be estimated by residual concentration on day 14, as a factor influencing progression-free survival of the patients. Our work shows that the description of the PK of an antibody by compartmental approach allows to identify sources of variability and to study the impact of PK on the clinical response.

Cette thèse analyse la fonction politique et identitaire du football en Espagne pendant le franquisme et la transition démocratique, en se focalisant particulièrement sur deux clubs : le Real Madrid et le FC Barcelone. L’un est considéré comme le meilleur ambassadeur de l’Espagne à l’étranger. L’autre, un instrument pour la mobilisation de l’opposition démocratique, et surtout un refuge pour les revendications de types identitaires à cette époque. Réaliser une étude sur le football peut sembler a priori ne pas s’inscrire dans une démarche scientifique. Pourtant, au-delà du sport et du divertissement, le football est un fait social, dont l’analyse est essentielle à la compréhension des sociétés contemporaines. Il mérite de ce fait une attention particulière, notamment lorsque l’on s’intéresse à l’histoire de l’Espagne, laquelle est extrêmement révélatrice des enjeux socioculturels et politiques que revêt le football dans ce pays / This thesis analyzes the political and social function of football in Francoist Spain and during the democratic transition, focusing particularly on two clubs: Real Madrid and FC Barcelona. One is considered the best ambassador of Spain abroad. And the other is an instrument for the mobilization of the democratic opposition, and above all a hub for ethno-social identity related claims at that time. A study about football may at first seem to not to belong within the scientific approach. Yet, beyond sport and entertainment, football is a social phenomenon, the analysis of which is essential to the understanding of contemporary societies. It deserves attention, especially when one is interested in the history of Spain, which is extremely revealing of the socio-cultural and political stakes that football has in this country

[pt] Nanopartículas são importantes ferramentas utilizadas em medicina, tanto para diagnóstico como para tratamento de diversas doenças. Seus tamanhos podem ser controlados, variando de dezenas até centenas de nanômetros, tornando-as menores ou comparáveis às dimensões de células, bactérias e vírus. As nanopartículas magnéticas possuem um núcleo de material magnético recoberto por camadas de diferentes materiais, incluindo sílica ou um polímero. Esta cobertura é responsável pela funcionalização, de forma que elas realizem tarefas específicas, seja para funcionar como um marcador com fins diagnósticos e/ou como um transportador de fármacos. É muito importante no processo de fabricação e utilização das nanopartículas o conhecimento de suas propriedades magnéticas. Com este objetivo, construímos um magnetômetro baseado em um criorefrigerador com capacidade para medir propriedades magnéticas em função da temperatura desde ambiente até 6 K. Como sensor magnético utilizamos um elemento Hall de GaAs de baixo custo. O magnetômetro construído tem uma configuração diferente dos magnetômetros Hall tradicionais, já que neste caso a amostra se movimenta na região do sensor. De forma a aumentar a exatidão do momento magnético obtido, foi desenvolvido um modelo que leva em consideração a geometria da amostra. A resolução está limitada pelo sensor utilizado em 10-7 Am2. O magnetômetro foi calibrado de forma independente e seu desempenho foi comparado a magnetômetros de amostra vibrante (VSM) comerciais, apresentando erros menores que 2 porcento na magnetização obtida de diversas amostras. Todos os equipamentos envolvidos na operação do magnetômetro a baixas temperaturas são controlados utilizando a linguagem LabVIEW. Na versão atual do programa, curvas M x H e ZFC-FC podem ser obtidas. Como exemplo de aplicação, fabricamos nanopartículas magnéticas com núcleo de oxido de ferro pelo processo de coprecipitação em meio alcalino e recobrimos com surfactantes e SiO2. As propriedades magnéticas das nanopartículas foram obtidas utilizando o magnetômetro construído. As nanopartículas apresentaram comportamento superparamagnético e grande potencial para liberação controlada de drogas. / [en] Nanoparticles are important tools used in medicine, for diagnosis as well as for treatment of various diseases. Their sizes can be controlled, ranging from tens to hundreds of nanometers, enabling them to interact with cells, bacteria, and viruses. Magnetic nanoparticles have a core of magnetic material coated with layers of different materials, including silica or a polymer. This coating is responsible for their functionalization, so they can carry out specific tasks serving as a marker for diagnostic purposes and / or as a carrier for drugs. The knowledge of the magnetic properties of nanoparticles is very important in the manufacturing process and their use. With this aim, we built a magnetometer based on a cryorefrigerator capable of measuring their magnetic properties as a function of temperature from room temperature to 6 K. We used a low cost GaAs Hall element as its magnetic sensor. The magnetometer built has a different configuration from the traditional Hall magnetometers, since in this case the sample moves in the region of the sensor. A model which takes into consideration the geometry of the sample was developed in order to increase the accuracy of the magnetic moment obtained. The magnetometer resolution is limited by the Hall sensor used in 10-7 Am2. The magnetometer was calibrated independently and its performance was compared to commercial vibrating sample magnetometers (VSM) showing errors smaller than 2 percent in the magnetization obtained from various samples. All the equipment involved in the operation of magnetometers at low temperatures is controlled by using the LabVIEW language. The M x H e ZFC-FC curves can be obtained in the current version. We manufactured the core with magnetic nanoparticles of iron oxide by coprecipitation process in an alkaline medium, coated with surfactants and SiO2. The magnetic properties of the nanoparticles were obtained using the magnetometer built. The nanoparticles showed superparamagnetic behavior and great potential for controlled drug release.