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The antibody response has evolved under constant pressure to recognize common pathogens and also remain adaptable to novel threats. Given the limited size of the germline antibody repertoire, adaptability requires that some antibodies must be polyspecific for multiple distinct antigens. Despite the profound importance of polyspecificity in the antibody response, the structural features that allow it are not well understood. Antibodies raised against glycoconjugates of Chlamydiaceae LPS oligosaccharides of the inner-core sugar Kdo (3-deoxy-d-manno-oct-2-ulosonic acid) have been shown to cross-react with several inner-core oligosaccharides through conserved recognition of single Kdo residues in a germline-encoded pocket, with additional sugars accommodated by flexible side-chains. Two of these antibodies, S25-2 and S25-39, were observed to bind several Kdo oligosaccharides with an identical binding site conformation, but adopted unique conformations of the heavy chain complementarity determining region loop 3 (CDR H3) in the absence of ligand. Conformational flexibility of germline antibodies is believed to facilitate polyspecificity by generating multiple unique binding sites in a single antibody. This thesis research further explores the conformational flexibility of the antibodies S25-2 and S25-39 to gain insight into mechanisms of antigen recognition and how this feature may allow polyspecificity. This was achieved first by solving structures of S25-39 from crystals grown in unique conditions to observe alternate CDR H3 conformations, and second by designing synthetic Kdo-based antigens so as both to inhibit interaction with the previously observed liganded conformation of S25-2 and S25-39 and to be accommodated by their observed unliganded conformations. These structures reveal an unprecedented level of structural diversity of CDR H3, notably including the exact ‘liganded’ conformation in the absence of ligand. This is the first direct structural evidence that CDR H3 can exist in a conformational equilibrium with antigen binding through a selection mechanism, as opposed to induced fit where antigen causes the observed conformational change. Definitive evidence for binding the synthetic antigens was not obtained, however the resulting structures revealed several additional unique conformations of CDR H3 suggesting that ligands can alter conformational equilibria during crystallization. A unique conformation was also observed with CDR H3 coordinating multiple iodide ions, revealing another potential source of polyspecificity with unique binding paratopes generated by ion coordination. Finally, the unparalleled level of conformational diversity observed for these antibodies highlights the challenges of antibody structure classification and prediction, and stresses the need for additional in-depth studies of conformational diversity and binding mechanisms to advance these fields for therapeutic application. This is the first targeted structural study of flexibility in antibodies and provides insight into their conformational dynamics and antigen-binding mechanisms. These are of fundamental importance in understanding antibody structure and function, a critical consideration in practical applications such as modelling and design of therapeutic or diagnostic antibodies. / Graduate / 2019-11-27

<p>The most common primary malignant brain tumor, glioblastoma, is uniformly fatal. Current therapy provides only incremental benefits in survival and is often incapacitating owing to limits defined by nonspecific toxicity. By contrast, immunotherapy offers a particularly promising approach, and has the theoretical potential to target and eliminate malignant cells with unprecedented specificity. The goal of this dissertation is to apply recombinant technologies to develop a new immune-based therapy for patients with malignant glioma. This work will span the design, production, and preclinical testing of a novel bispecific antibody designed to redirect T cells against a tumor-specific mutant of the epidermal growth factor receptor, EGFRvIII.</p><p>Chapters 1 and 2 will provide an overview of broad topics in antitumor immunotherapy and immune biology, with special focus on concepts as they relate to tumors of the central nervous system. In addition, the history and current state of bispecific antibodies, particularly those of the bispecific T-cell engager (BiTE) subclass, as well as their potential role in the treatment of malignant disease, will be considered in detail. Data presented in Chapter 3 will describe our approach to generating novel bispecific tandem single-chain antibody reagents, while experiments in Chapter 4 will demonstrate the capacity of one of these molecules, an EGFRvIII-specific BiTE, to achieve antitumor efficacy both <italic>in vitro</italic> and <italic>in vivo</italic> using murine models of glioma. Addressing a major barrier to the translation of immune therapies for cancer, chapter 5 will establish a potential role for BiTEs in overcoming cell-mediated immune suppression associated with malignant disease. Lastly, Chapter 6 and 7 will report on emerging areas of study, including the use of syngeneic, transgenic murine systems, and strategies by which BiTEs may be propelled rapidly into early phase clinical trials. </p><p>In summary, separating BiTEs from other available immunotherapeutic approaches, our work in this field suggests that BiTEs are (1) highly-specific molecules that greatly reduce the risk of toxicity, (2) have the ability to penetrate the blood-brain barrier and accumulate in intracerebral tumors, and (3) may potentially overcome multiple mechanisms of immunosuppression present in patients with glioblastoma. Together, these studies have the potential to improve the clinical management of patients with glioblastoma through the generation of a novel therapeutic.</p> / Dissertation

T cells are a cornerstone of the mammalian adaptive immune system. A range of T-cell subsets exist that can orchestrate the overall immune response to pathogens or cancers, either by directly killing infected cells or licensing other cells to do so. Dysregulation of this important process can result in immunodeficiency or autoimmunity. Although T cells have been studied extensively over many decades, the detailed mechanisms underlying T-cell activation remain to be fully resolved. This thesis describes the development of new optogenetic approaches for the modulation of T-cell signalling dynamics and the interrogation of key events in T-cell activation to help investigate this question. Optogenetics is a rapidly emerging technique whereby light can be used to control the spatial and temporal activation, or inactivation of signalling pathways at unprecedented resolution. The methods described in this work utilise the blue light-responsive LOV2 photo-domain from the common oat A. Sativa, which is the foundation of the both the ‘LOVTRAP’ and ‘TULIPs’ optogenetic toolkits. T-cell antigen receptor (TCR) microclusters arise early during the interaction between T cells and antigen presenting cells (APCs). These TCR signalling platforms contain the proteins necessary for sustained T-cell activation, yet the processes underlying their formation and dissociation are still not fully characterised as they have been difficult to investigate with current chemical and genetic manipulations of T cells. Using two optogenetics systems combining either LOVTRAP or TULIPs and the microcluster- scaffolding protein LAT (Linker for the Activation of T cells), it was possible to modulate early T-cell signalling events and measure functional outputs in real-time. Unfortunately, the biological limitations of these LAT-based systems meant that they could not be used to quantitatively investigate microcluster formation. However, in an alternative approach, a drug-inducible, light-controllable chimeric antigen receptor was successfully developed that yielded important new insights into the rapid rate of signal decay within the TCR signalling pathway and the temporal dynamics of T-cell activation over several timescales. T cell-dependent bispecific antibodies (TDBs) are a new class of immuno-therapeutics that can specifically direct a T-cell response towards tumours, by crosslinking the TCR complex to a surface- expressed target on the cancerous cells. However, their mechanism of action has not been studied in detail. The close apposition of the T cell and target cells driven by the TDB interaction can result in the steric exclusion of phosphatases, such as CD45, away from the TCR at the TDB-generated cell-cell interface due to their large, rigid extracellular domains. Using the myeloma-expressed antigen, FcRH5, it was found that membrane-proximal epitopes of FcRH5 drive more robust TCR clustering and increased CD45 exclusion than membrane distal epitopes, which strongly correlated with effective killing of the target cell. These findings have important implications for therapeutic design and implementation of TDBs.

abstract: Flaviviruses (FVs) are among the most medically important arboviruses of the world with the Dengue virus (DENV) accounting for a large percentage of infections observed in tropical and subtropical regions of the world. Globalization, travel, and the expanding range of mosquito vectors, such as Aedes aegypti, have increased the potential of infection rates and illnesses associated with FVs. The DENV and the Zika (ZIKV) FVs frequently co-circulate and generally cause mild self-liming febrile illnesses. However, a secondary infection with a heterologous DENV serotype may lead to life threatening dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). DHF/DSS have been linked to antibody dependent enhancement of infection (ADE), a phenomenon that occurs when antibodies (Abs) formed against an initial infection with one serotype of DENV cross-reacts but does not neutralize a heterologous DENV serotype in a secondary infection. Furthermore, Abs raised against the ZIKV have been observed to cross-react with the DENV and vice versa, which can potentially cause ADE and lead to severe DENV disease. The ZIKV can be transmitted vertically and has been linked to devastating congenital defects such as microcephaly in newborns. FDA approved treatments do not exist for DENV and ZIKV illnesses. Thus, there is a need for safe and effective treatments for these co-circulating viruses. Here, a tetravalent bispecific antibody (bsAb) targeting the ZIKV and all four serotypes of the DENV was expressed in the Nicotiana benthamiana (N. benthamiana) plant. Functional assays of the DENV/ZIKV bsAb demonstrated binding, neutralization, and a significant reduction in ADE activity against both the DENV and the ZIKV. A single chain variable fragment (scFv) and a diabody based on an antibody directed against the immune checkpoint inhibitor PD-L1, were also expressed in N. benthamiana leaves. The smaller sizes of the scFv and diabody confers them with the ability to penetrate deeper tissues making them beneficial in diagnostics, imaging, and possibly cancer therapy. The past few decades has seen long strives in recombinant protein production in plants with significant improvements in production, safety, and efficacy. These characteristics make plants an attractive platform for the production of recombinant proteins, biologics, and therapeutics. / Dissertation/Thesis / Doctoral Dissertation Molecular and Cellular Biology 2019

Stimuler la réponse immunitaire anti-tumorale constitue une voie d’avenir indiscutable pour le traitement des cancers. Aujourd'hui, les thérapies ciblées à base d'anticorps ont une place majeure dans l’immunothérapie des cancers du sein de par leur impact positif sur le pronostic des patientes. Cependant, les cancers du sein triple négatifs (TNBC) résistent aux innovations thérapeutiques actuelles, et, par défaut de traitement ciblé efficace, restent de sombre pronostic. Notre équipe développe des stratégies d’immunothérapie à base d'anticorps bispécifiques (bsFabs) conçus à partir de fragments d'anticorps de camélidés qui présentent la particularité de cibler simultanément les cellules immunitaires et tumorales. Ainsi, mon projet visait à évaluer le potentiel anti-tumoral de deux bsFabs sur des modèles précliniques de TNBC à travers leur capacité à activer et à rediriger le système immunitaire contre les cellules tumorales. La finalité du projet est de proposer un nouvel axe de thérapie ciblée susceptible d'améliorer le pronostic des patientes atteintes de TNBC. / Mounting evidence of the key contribution of NK cells in immunity against cancer has boosted the investigations on NK cell-based therapies. Among these strategies, monoclonal antibody-based therapeutics (mAbs) are currently the fastest growing segment of the medicine market. Despite therapeutic innovations, triple negative breast cancers (TNBC) remain insensitive to the current targeted or hormono-therapies. Our objective is to manipulate NK cell functions and tumor targets using an original format of nanobody-based bispecific antibodies (bsFab) to revert the dampened immune response for treating TNBC. Thus, we generate two bsFabs able to crosslink NK and tumor cells. NK antitumor effects driven by mAbs and bsFabs, alone or in combination, were investigated in vitro and in vivo on preclinical TNBC models. Here, we demonstrate the potential of bsFabs to enlarge the number of patients eligible for breast cancer immunotherapy and prompt to consider combination strategies.

Apoptoseresistenzmechanismen spielen bei der Pathogenese maligner Lymphome eine zentrale Rolle. So konnte bei Hodgkin/Reed-Sternbergzellen eine Deregulation des Transkriptionsfaktors NF-_B beobachtet werden, die zu verstärkter Apotoseresistenz führt und so zum malignen Wachstum dieser Zellen wahrscheinlich entscheidend beiträgt. Es konnte gezeigt werden, dass die selektive Blockade von NF-_B sowohl zu erhöhter Apoptosesensitivität als auch zur Inhibition der Zellzyklusprogression in kultivierten Hodgkinzellen führt. Der genaue molekulare Mechanismus der NF-_B-Deregulation in Hodgkinzellen ist jedoch noch unklar. Apoptoseresistenzmechanismen sind nicht nur bei der Pathogenese maligner Lymphome, sondern auch bei der Entstehung von Therapieresistenz von Bedeutung. So konnte gezeigt werden, dass die Überexpression proapoptotischer Gene der bcl-2 Familie in resistenten malignen Zellen sowohl die Empfindlichkeit gegenüber Zytostatika als auch gegenüber Antikörperbehandlung wiederherstellen kann. Neben der bcl-2 Familie spielt wahrscheinlich auch das Apo-I/Fas-System eine wichtige Rolle bei der Entstehung von Zytostatikaresistenz und immunologischer Resistenz. Somit stellt neben der Überexpression des P-Glykoproteins (MDR1), das als transmembranes "Pumpenprotein" Zytostatika aus der Tumorzelle heraustransportieren kann, die Deregulation Apoptose-steuernder Gene einen weiteren wichtigen Therapie-Resistenzmechanismus dar. Eine Möglichkeit, intrazelluläre Resistenzmechanismen zu umgehen, stellt die indirekte Induktion von Zelltod mit Hilfe bispezifische Antikörper dar. Durch diese Moleküle kann eine T-Zell-vermittelte Zellyse von Lymphomzellen erreicht werden. / Resistance towards apoptosis plays an important role in the pathogenesis of malignant lymphomas. It could be demonstrated that deregulation of the transcription factor NF-kB is a common molecular defect of Hodgkin/Reed-Sternberg cells that leads to enhanced resistance towards apoptosis and therefore probabaly contributes to the malignant growth of these cells. It couldbe shown that blocking of NF-kB leads to increased sensitivity towards apoptosis and decreased cell cycle progression. The precise molecular mechanism that leads deregulation of NF-kB is still unknown. Besides its role in the pathogenesis of malignant lymphoma resistance towards apoptosis plays an important role in the development of drug resistance. It could be shown that overexpression of pro-apoptotic members of the bcl-2 family in resistant tumor cells can restore sensitivity towards both cytotoxic drugs as well as antibody treatment. In addition to the bcl-2 family the Apo-I/Fas system is also involved in the development of drug resistance. Thus, besides overexpression of p-glycoprotein (MDR-1) that might pump cytotoxic drugs out of a malignant cell deregulation of apoptosis regulating genes is another important mechanisms of drug resistance development. One possibility to overcome drug resistance is the induction of cell death via bispecific antibodies. These molecules can induce T cell mediated lysis of lymohoma cells.