EG2-hFc is an ≈ 80 kDa chimeric heavy-chain antibody comprised of human IgG1 hinge and fragment crystallisable bivalently linked to EG2; a camelid-derived, heavy chain antibody variable domain specific for the human epithelial growth factor receptor 1 and its associated EGFRvIII mutant. Though previous work revealed EG2-hFc to demonstrate impressive in-vivo tumour accumulation, it’s therapeutic potential, as well as that of chimeric heavy-chain antibodies in general, remains largely unexplored. With this in mind, our current study was successful in showing that EG2-hFc could facilitate in-vitro antibody-dependent cell-mediated cytotoxicity of epithelial growth factor receptor-positive breast cancer cells. Additionally, EG2-hFc’s intrinsic cytotoxicity was augmented following the implementation of engineering strategies that are currently being explored in the context of conventional anti-cancer monoclonal antibodies: including the modification of a conserved N-linked CH2 glycan, as well as the alteration of EG2-hFc’s hinge length. Collectively, these findings contribute to the growing body of research that has revealed chimeric heavy-chain antibodies to be a promising class of novel anti-tumour therapeutics.

L’objet de cette thèse est d’étudier la dynamique des prix des matières premières, à travers l’étude du degré d’efficience de ces marchés et la dynamique de la prime de risque. A cette fin, ce travail de recherche a été autour de trois principaux chapitres : le premier étant d’ordre théorique tandis que les deux autres chapitres proposent deux essais empiriques. En particulier, le premier chapitre dresse le cadre conceptuel de cette étude, définit les concepts, rappelle les enjeux des stratégies de d’investissement et de diversification sur les marchés des matières premières. Il discute également l’état de la littérature relatif à nos questions de recherche. Le second chapitre applique des tests économétriques paramétriques et non paramétriques pour tester l’hypothèse d’efficience informationnelle à court et à long terme et montre que le degré d’efficience varie selon l’horizon temporel, la région et la commodité.En outre, il propose des simulations de portefeuilles bivariés pour illustrer plus concrètement les gains de diversification et repérer les stratégies d’investissement optimales. Dans le troisième chapitre, le modèle DCC-GARCH (1,1) est estimé pour étudier la dynamique de la prime de risque et expliquer ainsi les sources d’inefficience des marchés de matières premières. Nos résultats ne rejettent pas l’hypothèse de variabilité de prime de risque dans le temps appuyant l’alternance des marchés énergétiques entre l’inefficience à court terme et l’efficience à long terme. / This thesis aims at studying the dynamics of energy price through the investigation of their efficiency degree and the dynamics of risk premium.To this end, this study has been structured into three chapters : The first one is theoretical while the two others are empirical. In particular, the first chapter develops the conceptual framework for this study, defines the concepts, and recalls the issues related to investment strategies and diversification opportunities on energy markets. It also discusses the related literature review. The second chapter focuses on the informational efficiency hypothesis for commodity markets in the short and long terms using several parametric and non-parametric tests. It shows that the efficiency degree varies with commodity, region and temporal horizon. Further, it carries out bivariate portfolio simulations in order to illustrate diversification opportunities and identify optimal investment strategies. In the third chapter, we look at the dynamics of risk premium in order to explain the inefficient character of commodity markets using a DCC-GARCH (1,1) model. Our findings do not reject the hypothesis of time-varying risk premium, which helps to better understand the fact that commodity markets alternate between inefficiency in the short term and efficiency in the long term.

CD20 is a 35kDa surface antigen expressed on B cells from the early pre-B stage through the mature B stage. Moreover, the CD20 antigen is found on a majority of B cell malignancies. Rituximab is a chimeric anti-CD20 monoclonal antibody which has been extensively used alone or in combination in the treatment of CD20+ B cell malignancies including acute lymphoblastic leukemia (ALL), non-Hodgkin’s lymphomas (NHL) and chronic lymphocytic leukemia (CLL), as well as in the treatment of numerous autoimmune disorders. Despite its emerging use in the clinic, 30% to 50% of patients with low-grade NHL exhibit no clinical response to Rituximab. Previous work to elucidate the mechanisms of Rituximab resistance has established that antibody dependent cellular cytotoxicity (ADCC) is important as a predominant mechanism of lymphoma cell clearance and that Fcγ receptors (FcγRs) are critical for the in vivo actions of Rituximab in NHL. Natural killer group 2D, NKG2D is a major activating receptor on T lymphocytes and natural killer cells. The NKG2D–ligand (NKG2D-L) interaction triggers an activating signal which results in cytotoxic lysis of the cell expressing the ligand. One potential ligand for murine NKG2D is the retinoic acid early 1β (Rae-1β) protein which is expressed during cellular stress and has a high affinity for the NKG2D receptor in mice. We have recently shown that an anti-HER2-IgG3 fused to murine NKG2D Ligand, Rae1β inhibited HER2+ tumor growth significantly more than Herceptin alone. Similarly, our objective is to enhance the performance of anti-CD20 directed therapy through activation of NK cells by an anti-CD20 antibody encoding the same NK activation ligand. Previous results with anti-HER2-IgG3-Raelβ led us to hypothesize that a CD20 specific fusion protein will bind to CD20 expressing tumor cells and deliver an activation signal to local NKG2D receptors on effector cells triggering a non-FcγR dependent anti-tumor response. Here we show that anti-CD20-NKG2D-L can be synthesized and tested for its ability to bind human CD20 and activate NK cells through the NKG2D receptor in vitro.

Le développement d’un cancer chez un individu immunocompétent témoigne, en partie, d’un échappement tumoral au système d’immunosurveillance. Par conséquent, la restauration ou l’induction de ces mécanismes de défense anti-tumorale est une des stratégies thérapeutiques actuelles. Un des principes de l’immunothérapie est basé sur l’injection d’anticorps ayant pour cible la cellule tumorale ou les cellules effectrices de l’immunité. L’efficacité anti-tumorale de ces anticorps a été considérablement améliorée par une meilleure compréhension des modes d’action et des effets modulateurs de ces anticorps. Ainsi, afin d’optimiser l’action des effecteurs immunitaires sur les cellules tumorales, un anticorps bispécifique, trifonctionnel, le catumaxomab, capable de se lier à la molécule d'adhérence des cellules épithéliales (EpCAM) exprimée par les cellules tumorales et à l'antigène CD3 des lymphocytes T, a été développé, essentiellement en traitement intra-péritonéal des ascites néoplasiques réfractaires.L’objectif de cette étude était de déterminer les effets immunomodulateurs du catumaxomab sur des cellules néoplasiques exprimant EpCAM, à partir de deux modèles expérimentaux (allogénique et autologue), de rechercher une cytotoxicité induite par la catumaxomab, et de la caractériser, notamment en analysant la présence ou non de signaux de stress inducteurs d’une mort immunogène tels que l’exposition membranaire de la calréticuline par les cellules tumorales pré-apoptotiques, la libération d’HMGB1 et d’adénosine triphosphate (ATP) dans le milieu extra-cellulaire, responsables d’une activation des lymphocytes T.En présence de cellules EpCAM+, le catumaxomab entrainait une activation majeure des lymphocytes T (expression de CD69, CD107a, HLA-DR et PD1), stimulait une réponse inflammatoire de type Thelper 1(Th1), et provoquait la synthèse d’interféron-gamma par les lymphocytes T CD8. Le catumaxomab engageait le CD16 (FcR) des cellules monocytaires et NK. De plus, sur des modèles allogéniques, le catumaxomab, provoquait une mort cellulaire associée à la libération d’ATP et induisait une mort immunogène après pré-incubation dans de l’oxaliplatine.Par conséquent, le catumaxomab permet de moduler l’environnement immunitaire dans les ascites néoplasiques, et de convertir une inflammation chronique et immunosuppressive (Th2) en une inflammation aigüe et immunogène (Th1). En revanche, dans ces conditions, l’administration seule de catumaxomab ne semble pas déclencher de mort immunogène.Différents moyens pourraient permettre d’améliorer la cytotoxicité de cet anticorps bispécifique : (1) le combiner avec un agent anti-néoplasique tel que l’oxaliplatine afin de promouvoir une mort immunogène, (2) affiner son action sur le CD3 des lymphocytes en modifiant sa configuration spatiale (anticorps BiTE), (3) amplifier son affinité pour le récepteur Fcdes cellules accessoires (Fc défucosylé), (4) augmenter sa cytotoxicité en modifiant la cible dirigée contre la molécule du système immunitaire (anti-PD-1…). Enfin, l’utilisation clinique pourrait être facilitée en humanisant cet anticorps chimérique murin afin d’éviter la formation d’anticorps anti-murins, dirigés contre le catumaxomab.Un essai thérapeutique de phase II dont le but est d’évaluer l’efficacité du catumaxomab intrapéritonéal après chirurgie de cytoréduction complète d’une carcinose gastrique, chez des patients ayant reçu en préopératoire une chimiothérapie systémique à base d’oxaliplatine vient de débuter. Au cours de cette étude, nous allons valider la capacité du catumaxomab 1) à induire un stress cellulaire immunogène et la mort des cellules cancéreuses, 2) à modifier la polarisation des cellules effectrices vers une maladie inflammatoire Th1, 3) à promouvoir l'expression des molécules de costimulation et TRAIL sur les cellules NK et monocytes, et corréler ces biomarqueurs immunitaires à l’efficacité du traitement. / The development of cancer in an immunocompetent individual reflects, in part, a tumor escape from the immunosurveillance. The tumor escape is a complex, multifactorial, in which tumor cells will evade the defense mechanisms of the host by changing their microenvironment. Therefore, restoration or induction of these defense mechanisms is one of the therapeutic strategies against cancer. One of the principles of immunotherapy is based on the injection of antibodies that target tumor cells or effector cells of immunity. The anti-tumor efficacy of these antibodies has been greatly improved by a better understanding of modes of action and modulatory effects of these antibodies.Thus, to optimize the action of immune effectors to tumor cells, a bispecific antibody, trifunctional: catumaxomab, capable of binding to the adhesion molecule of the epithelial cells (EpCAM), expressed by tumor cells and the CD3 antigen of T cells, has been developed mainly in intraperitoneal treatment of refractory malignant ascites.The objective of this study was to determine the immunomodulatory effects of catumaxomab on tumoral cells expressing EpCAM, from two experimental models (allogeneic and autologous), evaluate and characterize cytotoxicity induced by catumaxomab, and analyze the presence of stress signals inducing immunogenic cell death such as membrane exposure of calreticulin by pre-apoptotic tumor cells, release of HMGB1 and of adenosine triphosphate (ATP) in the extracellular medium, inducing a T cell activation.In the presence of EpCAM + cells, catumaxomab induced a major the activation of T cells (expression of CD69, CD107a, HLA-DR and PD1), stimulated an inflammatory response Thelper type 1 (Th1) and the synthesis of interferon-gamma by CD8 T cells. Catumaxomab committed CD16 NK cells and monocytes. More, in models allogeneic catumaxomab, caused cell death associated with ATP release and induced an immunogenic cell death after pre-incubation of oxaliplatin.Therefore, catumaxomab modulates the immune environment in malignant ascites, and convert chronic and immunosuppressive inflammation (Th2) in acute and immunogenic inflammation (Th1). However, in these conditions, catumaxomab alone does not seem to trigger immunogenic cell death.the cytotoxicity of this bispecific antibody could be enhance by different techniques: (1) combining with chemotherapy such as oxaliplatin to promote immunogenic cell death, (2) refining its action on CD3 lymphocytes by changing its spatial configuration (BiTE antibody), (3) increasing its affinity for the FcR of accessory cells (Fc aglycosylated), (4) increasing its cytotoxicity by changing the target directed against the immune molecule (anti-PD-1 ...). Finally, the clinical use could be facilitated by this humanizing murine chimeric antibody to prevent the formation of anti-murine antibodies directed against catumaxomab.A phase II clinical trial aimed to evaluate the efficacy of intraperitoneal catumaxomab after complete cytoreductive surgery of gastric carcinomatosis in patients who received preoperative systemic chemotherapy with oxaliplatin have just started. In this study, we will validate the ability of catumaxomab 1) to induce immunogenic cell stress and death of cancer cells, 2) to change the polarization of effector cells to Th1 inflammatory disease, 3) to promote the expression of costimulatory molecules and TRAIL on NK cells and monocytes, and we will correlate these immune biomarkers to treatment efficacy.

Le trastuzumab est un anticorps monoclonal déjà utilisé dans le traitement du cancer du sein sur-exprimant la protéine HER2. Malgré son efficacité, la résistance est souvent apparue. Ici, nous avons étudié l'impact des cellules adipocytaires sur la cytotoxicité cellulaire dépendante de l'anticorps (ADCC), une des mécanismes d'action principaux du trastuzumab. Nous avons trouvé que les adipocytes, ainsi que les pré- adipocytes, inhibent l'ADCC médié par le trastuzumab. Nous avons montré que les factors dérivées d'adipocytes, comme des protéines ou des exosomes, causent l'inhibition d'ADCC. Aucune séquestration ou dégradation de l'anticorps a été observée. Des analyses phénotypiques n'ont pas révélé de modification des récepteurs des cellules NK, ni du niveau de HER2 sur les cellules cancéreuses en présence d'adipocytes. La pré-incubation des cellules cancéreuses avec le surnageant des adipocytes réduit la sensitivité tumorale à l'ADCC. Nous avons trouvé que le factor de croissance et de différenciation GDFI5 est rapidement induit dans les cellules cancéreuses exposées au surnageant d'adipocytes. Une diminution de l'expression de GDFI5 par les siRNA réverse l'inhibition d'ADCC induite par les adipocytes. En conclusion, nous avons démontré que les adipocytes jouent un rôle dans la résistance des cellules de cancer du sein à l'ADCC médié par le trastuzumab, et nous suggérons que cibler GDFI5 ou l'interaction entre les adipocytes et les cellules cancéreuses pourrait sensibiliser les cellules cancéreuses au traitement par l'anticorps monoclonal / Trastuzumab is a monoclonal antibody already approved in the treatment of HER2-expressing breast cancer. Despite its efficacy, resistance often occurs. Here, we investigated the impact of adipocytes on antibody dependent cellular cytotoxicity (ADCC), one of the main mechanisms of action of trastuzumab. We found that adipocytes, as well as preadipocytes, inhibited trastuzumab-mediated ADCC. We showed that adipocyte-derived factors, likely proteins or exosomes, mediated the inhibition of ADCC. No titration or degradation of the antibody was detected. Analysis of cell phenotype did not reveal any modification of NK cell receptors, nor of HER2 levels on cancer cells in the presence of adipocytes. Pre-incubation of cancer cells with adipocyte-conditioned medium reduced sensitivity of cancer cells to ADCC. We found that growth differentiation factor l5 (GDFl5) was rapidly induced in cancer cells exposed to adipocyte-conditioned medium. Down-regulation of GDFl5 by siRNA reversed the adipocyte-induced inhibition of ADCC. In conclusion, we demonstrated that adipocytes play a role in the resistance of breast cancer to trastuzumab-mediated ADCC, and suggested that targeting GDFl5 or the crosstalk between adipocytes and cancer cells may sensitize cancer cells to monoclonal antibody treatment

Disorders of the corpus callosum (ADCC) present developmental challenges to children and adults. These disorders are characterized by symptoms of abnormal behaviors and/or thinking patterns. Because ADCC may exist in combination with other disabilities, individual IQs and the severity and problems vary from individual to individual. Using the double ABCx model of family adaptation to stress related to a family member with a disability, the purpose of this cross-sectional study was to provide the first evaluation of parental adaptation among parents of children with ADCC. The final sample, 265 mothers of children with ADCC, was recruited through online support groups for ADCC parents. Parent adaptation was operationally defined as quality of life and operationalized by scores on the World Health Organization (WHO) Quality of Life Questionnaire (QOL). The predictors were measured by the Questionnaire on Resources and Stress (QRS), Family Empowerment Scale (FES), Sense of Coherence Scale (SOC), and Coping Health Inventory for Parents (CHIP). Linear regressions were used to evaluate the predictors in the 4-factor double ABCx prediction model of parent adaptation. Except for parent stress level, family empowerment, sense of coherence, and coping styles were statistically significant predictors of parental quality of life. That is, mothers who reported experiences of empowerment, coherence, and positive coping also have high self-reported quality of life. The findings, the first for experiences of parents of children with ADCC, provide valuable information for further research, but also for other parents and those who may be instrumental in the development of supportive services for this population.

The properties of human immunodeficiency virus type 1 (HIV-1) and its simian counterpart SIV that enable persistent replication in the face of robust cellular, antibody, and innate immune responses have complicated efforts to develop a safe and effective vaccine. Vaccine protection against HIV-1 infection may require a combination of immune mechanisms. However, the types of immune responses that can be induced by vaccination to prevent HIV-1 infection remain unclear. The features of the viral envelope glycoprotein (Env) that confer inherent resistance to neutralization by antibodies also interfere with the development of antibody responses. We therefore vaccinated rhesus macaques with single-cycle SIV (scSIV) strains expressing Env proteins mutated to remove features that interfere with the induction of antibody responses. Antibodies capable of neutralizing Env-modified but not wild-type SIV were selectively enhanced. Identifying the immune responses underlying complete protection by live-attenuated SIV against pathogenic SIV challenge may provide guidance for HIV-1 vaccine design. To test the hypothesis that antibodies not measurable by assays for virus neutralization correlate with protection by live-attenuated SIV, we developed a novel assay for antibody-dependent cell-mediated cytotoxicity (ADCC). ADCC activity increased progressively over time after inoculation, and was measurable against viruses expressing heterologous Env proteins from independent SIV isolates when neutralization was undetectable. Two separate pathogenic \(SIV_{mac}251\) challenge experiments took advantage of either the strain specificity or the time-dependent development of immunity to overcome complete protection by live-attenuated SIV. In both experiments, macaques inoculated with live-attenuated SIV that remained uninfected by \(SIV_{mac}251\) had significantly higher ADCC activity than those that became infected. We also measured ADCC for the primary immune correlates analysis of a recent HIV-1 vaccine clinical trial in Thailand (RV144) that reported modest vaccine protection (31%). There was a nonsignificant trend towards lower risk of infection among vaccinees with high versus low relative ADCC activity. However, Env-specific IgA correlated with risk, prompting an analysis stratified by IgA levels. Among vaccinees with low Env-specific IgA, there was lower risk of infection among those with higher ADCC activity. These observations suggest that antibodies that direct ADCC may contribute to vaccine protection against SIV and HIV-1 infection.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22887号 / 医博第4681号 / 新制||医||1048(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 武藤 学, 教授 小川 誠司, 教授 藤田 恭之 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

No description available.

Natural Killer (NK) cells are cytotoxic lymphocytes that can recognize and kill virally infected or tumour transformed cells by the secretion of cytolytic granules containing perforin. An individual NK cell can kill several target cells sequentially. Each target cell can trigger NK cell activation via different activating ligands and here we report that the order in which ligands are encountered affects the NK cell response. When NK cells are repeatedly activated via their Fc receptor CD16, with the therapeutic antibody rituximab, perforin secretion decreases with each stimulation. However, perforin secretion is restored to its initial level upon subsequent activation by MICA, which ligates NKG2D. Repeated stimulation of NK cells via MICA also decreases the degranulation capacity of NK cells but, strikingly, this effect cannot be rescued by a subsequent stimulation with rituximab. The strength of perforin secretion is also translated to killing of Daudi target cells, expressing different ligands. When Daudi, opsonised with rituximab is the first target NK cell encounters, the sequential killing of another opsonised rituximab or Daudi, expressing MICA will not be affected. But, when Daudi-MICA is met first, the consecutive killing of Daudi-MICA as well as Daudi-rituximab will be impaired. We found that the mechanism underlying these differential outcomes involves shedding of CD16, which occurs upon NK cell activation through both, CD16 and NKG2D. Shedding of CD16 renders the cells insensitive to further activation via that receptor but they remain competent for further activation through NKG2D. Interestingly, however, we also identified the beneficial role of CD16 shedding for NK cell serial killing. NK cells are more motile on rituximab-coated surfaces than on MICA-coated surfaces and their migration speed decreases upon inhibition of CD16 shedding. Moreover, the inhibition of CD16 shedding also prevents the NK cell detachment from rituximab opsonised Daudi cells. Thus, the shedding of the receptor can serve to augment NK cell motility to move between target cells. Efficient NK cell detachment also correlated with their increased survival. Finally, we report that CD16 is constitutively organised in small, dense nanoclusters and that the ligation with rituximab does not affect their spatial distribution. Despite the shedding of the receptor, leading to less protein molecules at the surface, the area of these clusters remains the same. Together these data suggest that CD16 shedding hinders NK cell cytotoxicity against opsonised targets, but promotes their movements between different targets. Thus, receptor shedding is important for efficient NK cell serial killing. Manipulation of CD16 shedding, perhaps by boosting its recovery, might therefore represent an important target for NK cell-based therapies including treatments with therapeutic antibodies.