High resolution x-ray structures of allergens for designing hypoallergenic derivatives for specific immunotherapy

IgE-vermittelte Typ-I-Allergien sind eines der wichtigsten Gesundheitsprobleme in den Industrienationen, wo sie fast 25% der Bev\uf6lkerung betreffen. Die einzige kausale Behandlung von Typ-I-Allergien ist die spezifische Immunotherapie (SIT). Die vorliegende Dissertation besch\ue4ftigt sich mit der strukturellen Charakterisierung des Gruppe-3-Hauptallergens Phl p 3. Mit Hilfe des Vergleichs struktureller Eigenschaften mit verwandten, kreuzreaktiven Allergenen und nicht-kreuzreaktiven Allergenen wurden strukturelle Epitope des Phl p 3 abgrenzend beschrieben. Im Zuge der in-silico-Epitop-Vorhersage wurde eine neuartige Strategie zur Ber\ufccksichtigung immunologischer Daten verfolgt, wodurch die Genauigkeit der Vorhersagen gesteigert werden konnte. Mittels dieser Methode wurden zwei konformationelle Epitope mit l\uf6sungsmittelzug\ue4nglichen Oberfl\ue4chen von ca. 500 \uc52 lokalisiert und innerhalb dieser Regionen einzelne Aminos\ue4ure-Reste mit einer m\uf6glichen Schl\ufcsselrolle identifiziert. Eine nichtmeroedrische Verzwillingung wurde bereits w\ue4hrend des Kristallwachstums beobachtet und die entsprechenden Beugungsbilder wiesen ebenfalls auf das Vorhandensein eines zweiten Kristallgitters hin. Zahlreiche Kristalle wurden getestet und Datensammlungen vollf\ufchrt um einen Datensatz ohne Verzwillingungs-Effekte zu erhalten. Zus\ue4tzlich zur Verzwillingung wurde Pseudotranslation entlang der c-Achse beobachtet - ein Ph\ue4nomen dass bekannterma fen die Strukturverfeinerung behindern kann. Ein hochaufgel\uf6ster Datensatz (1.8 \uc5) erm\uf6glichte dank der bei dieser Aufl\uf6sung ausreichend gro fen Zahl unabh\ue4ngiger Reflexe eine zufriedenstellende Verfeinerung. Es wurden dabei signifikante strukturelle Abweichungen zwischen den \ufcber Pseudotranslation verkn\ufcpften Molek\ufclen beobachtet, so dass ihre Modelle unabh\ue4ngig voneinander erstellt werden mussten.IgE mediated Type I allergies represent a major health concern in the industrialized countries as they affect almost 25% of the population. Sensitized atopic individuals on re-exposure to an allergen suffer from rhinoconjunctivitis, dermatitis, bronchial asthma and life threatening anaphylactic shock. The only causative treatment for type I allergies is specific immunotherapy. This Ph.D thesis deals with the structural characterization of major group 3 allergen Phl p 3. With the help of comparison of structural features to related cross-reactive allergens and non cross-reactive allergens, structural epitopes have been delineated. In this work of in-silico prediction a novel strategy of including immunological data has been employed to increase the accuracy of the predictions. Based on these strategies two conformational epitopes having solvent accessible areas in range of ~ 500 \uc52 have been identified. Key residues have been identified in these epitopes for point mutations. Intact Phl p 3 with these point mutations will act as hypoallergenic derivative for specific immunotherapy. Non-merohedral twinning was observed during crystal growth, and subsequent diffraction patterns also indicated the presence of a second lattice in the crystal. Numerous crystals have been screened and data collection was performed to obtain a data set with no twinning effects. In addition to the twinning, pseudo-translation was observed along the c-axis. A high resolution data set (1.8 \uc5) lead to the satisfactory refinement due to the high amount of unique data generated at this resolution. We have observed gross deviations among pseudo-translated molecules, which had to modelled independently.Venkata Siva Charan DevanaboyinaAbweichender Titel laut cbersetzung der Verfasserin/des VerfassersZsfassung in dt. SpracheGraz, Univ., Diss., 200

Targeting FcRn for the modulation of antibody dynamics

The MHC class I-related receptor, FcRn, is a multitasking protein that transports its IgG ligand within and across cells of diverse origins. The role of this receptor as a global regulator of IgG homeostasis and transport, combined with knowledge of the molecular details of FcRn-IgG interactions, has led to opportunities to modulate the in vivo dynamics of antibodies and their antigens through protein engineering. Consequently, the generation of half-life extended antibodies has shown a rapid expansion over the past decade. Further, FcRn itself can be targeted by inhibitors to induce decreased levels of circulating IgGs, which could have applications in multiple clinical settings. The engineering of antibody-antigen interactions to reduce antibody-mediated buffering of soluble ligand has also developed into an active area of investigation, leading to novel antibody platforms designed to result in more effective antigen clearance. Similarly, the target-mediated elimination of antibodies by internalizing, membrane bound antigens (receptors) can be decreased using novel engineering approaches. These strategies, combined with subcellular trafficking analyses of antibody/antigen/FcRn behavior in cells to predict in vivo behavior, have considerable promise for the production of next generation therapeutics and diagnostics.</p

Engineered clearing agents for the selective depletion of antigen-specific antibodies

Here we have designed a novel class of engineered antibody-based reagents ('Seldegs') that induce the selective degradation of antigen-specific antibodies. We demonstrate the rapid and specific clearance of antibodies recognizing the autoantigen, myelin oligodendrocyte glycoprotein and tumour target, HER2. Seldegs have considerable potential in multiple areas, including the treatment of antibody-mediated autoimmunity and diagnostic imaging.</p

Myelin oligodendrocyte glycoprotein-specific antibodies from multiple sclerosis patients exacerbate disease in a humanized mouse model

Myelin oligodendrocyte glycoprotein (MOG) is exposed on the outer surface of the myelin sheath, and as such, represents a possible target antigen for antibodies in multiple sclerosis (MS) and other demyelinating diseases. However, despite extensive analyses, whether MOG-specific antibodies contribute to pathogenesis in human MS remains an area of uncertainty. In the current study we demonstrate that antibodies derived from adult MS patients exacerbate experimental autoimmune encephalomyelitis (EAE) in ‘humanized’ mice that transgenically express human FcγRs (hFcγRs). Importantly, this exacerbation is dependent on MOG recognition by the human-derived antibodies. The use of mice that express hFcγRs has allowed us to also investigate the contribution of these receptors to disease in the absence of confounding effects of cross-species differences. Specifically, by engineering the Fc region of MOG-specific antibodies to modulate FcγR and complement (C1q) binding, we reveal that FcγRs but not complement activation contribute to EAE pathogenesis. Importantly, selective enhancement of the affinities of these antibodies for specific FcγRs reveals that FcγRIIA is more important than FcγRIIIA in mediating disease exacerbation. These studies not only provide definitive evidence for the contribution of MOG-specific antibodies to MS, but also reveal mechanistic insight that could lead to new therapeutic targets.</p

Targeting phosphatidylserine with calcium-dependent protein-drug conjugates for the treatment of cancer

In response to cellular stress, phosphatidylserine is exposed on the outer membrane leaflet of tumor blood vessels and cancer cells, motivating the development of phosphatidylserine-specific therapies. The generation of drug-conjugated phosphatidylserine-targeting agents represents an unexplored therapeutic approach, for which antitumor effects are critically dependent on efficient internalization and lysosomal delivery of the cytotoxic drug. In the current study, we have generated phosphatidylserine-targeting agents by fusing phosphatidylserine-binding domains to a human IgG1-derived Fc fragment. The tumor localization and pharmacokinetics of several phosphatidylserine-specific Fc fusions have been analyzed in mice and demonstrate that Fc-Syt1, a fusion containing the synaptotagmin 1 C2A domain, effectively targets tumor tissue. Conjugation of Fc-Syt1 to the cytotoxic drug monomethyl auristatin E results in a protein-drug conjugate (PDC) that is internalized into target cells and, due to the Ca2+ dependence of phosphatidylserine binding, dissociates from phosphatidylserine in early endosomes. The released PDC is efficiently delivered to lysosomes and has potent antitumor effects in mouse xenograft tumor models. Interestingly, although an engineered, tetravalent Fc-Syt1 fusion shows increased binding to target cells, this higher avidity variant demonstrates reduced persistence and therapeutic effects compared with bivalent Fc-Syt1. Collectively, these studies show that finely tuned, Ca2+-switched phosphatidylserine-targeting agents can be therapeutically efficacious.</p

Rapid depletion of “catch-and-release” anti-ASGR1 antibody in vivo

Targeting antigens with antibodies exhibiting pH/Ca2+-dependent binding against an antigen is an attractive strategy to mitigate target-mediated disposition and antigen buffering. Studies have reported improved serum exposure of antibodies exhibiting pH/Ca2+-binding against membrane-bound receptors. Asialoglycoprotein receptor 1 (ASGR1) is a membrane-bound receptor primarily localized in hepatocytes. With a high expression level of approximately one million receptors per cell, high turnover, and rapid recycling, targeting this receptor with a conventional antibody is a challenge. In this study, we identified an antibody exhibiting pH/Ca2+-dependent binding to ASGR1 and generated antibody variants with increased binding to neonatal crystallizable fragment receptor (FcRn). Serum exposures of the generated anti-ASGR1 antibodies were analyzed in transgenic mice expressing human FcRn. Contrary to published reports of increased serum exposure of pH/Ca2+-dependent antibodies, the pH/Ca2+-dependent anti-ASGR1 antibody had rapid serum clearance in comparison to a conventional anti-ASGR1 antibody. We conducted sub-cellular trafficking studies of the anti-ASGR1 antibodies along with receptor quantification analysis for mechanistic understanding of the rapid serum clearance of pH/Ca2+-dependent anti-ASGR1 antibody. The findings from our study provide valuable insights in identifying the antigens, especially membrane bound, that may benefit from targeting with pH/Ca2+-dependent antibodies to obtain increased serum exposure

The effect of pH dependence of antibody-antigen interactions on subcellular trafficking dynamics

A drawback of targeting soluble antigens such as cytokines or toxins with long-lived antibodies is that such antibodies can prolong the half-life of the target antigen by a "buffering" effect. This has motivated the design of antibodies that bind to target with higher affinity at near neutral pH relative to acidic endosomal pH (∼pH 6.0). Such antibodies are expected to release antigen within endosomes following uptake into cells, whereas antibody will be recycled and exocytosed in FcRn-expressing cells. To understand how the pH dependence of antibody-antigen interactions affects intracellular trafficking, we generated three antibodies that bind IL-6 with different pH dependencies in the range pH 6.0-7.4. The behavior of antigen in the presence of these antibodies has been characterized using a combination of fixed and live cell fluorescence microscopy. As the affinity of the antibody:IL-6 interaction at pH 6.0 decreases, an increasing amount of antigen dissociates from FcRn-bound antibody in early and late endosomes, and then enters lysosomes. Segregation of antibody and FcRn from endosomes in tubulovesicular transport carriers (TCs) into the recycling pathway can also be observed in live cells, and the extent of IL-6 association with TCs correlates with increasing affinity of the antibody:IL-6 interaction at acidic pH. These analyses result in an understanding, in spatiotemporal terms, of the effect of pH dependence of antibody-antigen interactions on subcellular trafficking and inform the design of antibodies with optimized binding properties for antigen elimination.</p