Untersuchung der Interaktion humaner IgG Antikörper mit humanen FcgR in vitro und in vivo

Immunoglobulin G (IgG) antibodies are an essential part of the adaptive immune system. Genetic defects leading to a reduced production or a complete lack of this antibody isotype are associated with an increased susceptibility to infectious diseases. Studies of the murine immune system suggest that the biological activity of IgG is mainly mediated by their binding to Fcg receptors (FcgR) expressed on almost all cells of the innate immune system. The aim of the thesis was to analyse which factors influence the biological activity of IgG in vitro and in vivo. We performed in vitro interaction studies of human immune complexes and human FcgR which suggested a correlation of immune complex size and binding to FcgRIIA, FcgRIIB and FcgRIIIA, but not FcgRIA. In addition, we could show that a lack of glycosylation on asparagine 297 impairs binding to low affinity FcgR. However, this reduced binding could be overcome by increasing the size of the immune complexes. Polymorphisms of the human FcgR pose another factor influencing the interaction with IgG antibodies. To study this, we analysed genetic variants of the activating FcgR, FcgRIIA131H/R and FcgRIIIA158F/V. In vitro binding studies showed an enhanced affinity of FcgRIIA131H for all human IgG subclasses. To study IgG induced effector functions in vivo we developed a humanized mouse model which mirrors the genetic complexity of the human immune system. Immunodeficient Rag2/gc/Fcg-/- mice transplanted with human hematopoetic stem cells developed a human immune system as shown by the appearance of B and T lymphocytes, NK cells, myeloid cells and dendritic cells. Effector cells such as monocytes and NK cells displayed expression levels of human FcgRs comparable to their human counterparts. This new humanized mouse model was used to study the influence of FcgRIIA131H/R and FcgRIIIA158F/V polymorphisms on the biological activity of IgG antibodies such as the induction of antibody induced cell mediated cytotoxicity (ADCC). B cell depletion induced by the injection of anti-CD20 IgG antibodies greatly depended on the constant region of the antibody. Furthermore, IgG subclasses showed a distinct in vivo activity. Preliminary data also suggest an enhanced IgG2 mediated B cell depletion in the presence of FcgRIIA131H and FcgRIIIA158V.Immunglobulin G (IgG) Antikörper sind ein essentieller Bestandteil des adaptiven Immunsystems. Genetische Defekte, die zu einer verminderten Bildung bzw. dem vollständigen Fehlen dieses Antikörper-Isotyps führen, gehen mit einer erhöhten Infektionsanfälligkeit einher. Eine Vielzahl von Studien in der Maus deuten darauf hin, dass insbesondere Fcg Rezeptoren (FcgR), die auf fast allen Zellen des angeborenen Immunsystems exprimiert werden, eine wichtige Rolle für die Aktivität von IgG Antikörpern spielen. In der vorliegenden Arbeit sollte daher untersucht werden, ob die Aktivität von humanen IgG Antikörpern von ähnlichen Faktoren in vitro und in vivo beeinflusst wird. Durch in vitro Bindungsstudien von humanen Immunkomplexen an humane FcgR konnte im ersten Teil dieser Arbeit ein Zusammenhang zwischen der Größe von Immunkomplexen und der Bindung an FcgRIIA, FcgRIIB und FcgRIIIA, nicht aber an FcgRIA, gezeigt werden. Weiterhin konnte nachgewiesen werden, dass Immunkomplexe ohne die Glykosylierung am Asparagin 297 eine verminderte Bindung an die niedrigaffinen FcgR aufweisen. Große Immunkomplexe konnten die durch Deglykosylierung bedingte reduzierte Affinität teilweise kompensieren. Einen weiteren möglichen Einflussfaktor auf die IgG-FcgR Interaktion stellen Polymorphismen in den humanen FcgR dar. In dieser Arbeit wurden genetische Varianten der aktivierenden FcgR, FcgRIIA131H/R und FcgRIIIA158F/V, untersucht. In in vitro Analysen wies FcgRIIA131H für alle humanen IgG Isotypen eine erhöhte Affinität auf. Für die in vivo Analyse IgG-vermittelter Effektorfunktionen wurde ein humanisiertes Mausmodell etabliert, in dem die genetische Komplexität des humanen Immunsystems abgebildet wird. Hierfür wurden immundefiziente Rag2/gc/Fcg-/- Mäuse verwendet, die nach Transplantation mit humanen hämatopoetischen Stammzellen ein humanes Immunsystem mit B- und T-Lymphozyten sowie NK-Zellen, Monozyten und dendritischen Zellen entwickelten. Weiterhin zeigten die humanen Effektorzellen, wie etwa Monozyten und NK-Zellen, ein dem Menschen vergleichbares Expressionsmuster der humanen FcgR. In diesem neuen humanisierten Mausmodell wurde der Zusammenhang zwischen den FcgRIIA131H/R und FcgRIIIA158F/V Polymorphismen und der biologischen Wirksamkeit von IgG beispielhaft durch die Induktion der Antikörper-induzierten Zell-vermittelten Zytotoxizität (ADCC) analysiert. Es konnte gezeigt werden, dass die durch Injektion von anti-CD20 IgG verursachte Depletion von humanen B-Zellen vom konstanten Teil des IgG Antikörpers abhängt. Zudem zeigten die verschiedenen IgG Subklassen eine unterschiedliche Aktivität und es konnten erste Hinweise gewonnen werden, die vermuten lassen, dass die IgG2-vermittelte Depletion in Gegenwart der hochaffinen Varianten der aktivierenden Fc Rezeptoren FcgRIIA131H bzw. FcgRIIIA158V verstärkt war

Medien und Asyl - Methodenausbildung mit Anwendungsbezug als Chance und Herausforderung

Das Lehr-Lern-Projekt “Medien und Asyl” kombinierte Forschungs- und Anwendungsbezug in der universitären Hochschullehre. Der Beitrag diskutiert die didaktischen Herausforderungen eines solchen Lehrforschungsprojekts mit Service Learning- Orientierung, und wie diese adressiert wurden. In der Darstellung des Ablaufs wird der konsekutive Charakter des dreisemestrigen Projekts deutlich. Die finale Phase der Medienkonzeption wird dabei ausführlicher vorgestellt. Abschließend wird die Bedeutung fortlaufender Evaluation und Anpassung des Projektablaufs als methodischer Schlüssel zum Gelingen identifiziert

Minimal B Cell Extrinsic IgG Glycan Modifications of Pro- and Anti-Inflammatory IgG Preparations in vivo

Select residues in the biantennary sugar moiety attached to the fragment crystallizable of immunoglobulin G (IgG) antibodies can modulate IgG effector functions. Thus, afucosylated IgG glycovariants have enhanced cytotoxic activity, whereas IgG glycovariants rich in terminal sialic acid residues can trigger anti-inflammatory effects. More recent evidence suggests that terminal α2,6 linked sialic acids can be attached to antibodies post IgG secretion. These findings raise concerns for the use of therapeutic antibodies as they may change their glycosylation status in the patient and hence affect their activity. To investigate to what extent B cell extrinsic sialylation processes modify therapeutic IgG preparations in vivo, we analyzed changes in human intravenous IgG (IVIg) sialylation upon injection in mice deficient in B cells or in mice lacking the sialyltransferase 1, which catalyzes the addition of α2,6 linked sialic acid residues. By performing a time course of IgG glycan analysis with HILIC-UPLC-FLR (plus MS) and xCGE-LIF our study suggests that therapeutic IgG glycosylation is stable upon injection in vivo. Only a very small fraction of IgG molecules acquired sialic acid structures predominantly in the Fab- but not the Fc-portion upon injection in vivo, suggesting that therapeutic antibody glycosylation will remain stable upon injection in vivo

Presentation and medical management of peripheral arterial disease in general practice: rationale, aims, design and baseline results of the PACE-PAD Study

Background: Peripheral arterial disease (PAD) is highly prevalent among individuals of higher age or those with one or more cardiovascular risk factors. Screening for PAD is recommended, since it is often linked to atherothrombotic manifestations in the coronary or carotid circulation and associated with a substantial increase in all-cause and cardiovascular mortality. We aimed to assess patients with newly diagnosed, suspected and confirmed PAD in the primary care setting with regards to clinical characteristics, diagnostic and therapeutic management (including referral to specialists), and medium-term outcomes. Methods: This was a multicentre, prospective, observational cohort study with a cross-sectional and a longitudinal part. A total of 2,781 general practitioners across Germany were cluster randomised to document five consecutive patients each in one of the strata: (1) patients with intermittent claudication (IC) or other typical PAD-related complaints (group A) or (2) patients >55 years of age with one or more risk factors (group B) for PAD (current smoking, diabetes, previous myocardial infection and/or previous stroke). Patients with confirmed PAD will be followed up for diagnostic procedures, therapy and vascular events over 18 months. Results: In group A, a total of 2,131 patients with suspected PAD (80.1% confirmed, 75.9% with referral to specialists) and in group B 9,921 patients were included (44.6% confirmed, 54.6% referral). The ankle-brachial index was calculated in 41.3% and 33.5% only. Mean age was 66.6 years (group A) and 68.4 years (group B), respectively. Vascular risk factors were prevalent in both groups, in particular smoking (group A 44.6%, group B 44.4%), hypertension (73.2 and 78.1%), hypercholesterolaemia (64.6 and 70.6%) and diabetes mellitus (41.7 and 60.6%). Concomitant atherothrombotic morbidities were frequent in both groups. In patients with the respective diseases, antihypertensive, antidiabetic, lipid-lowering and antithrombotic therapies were prescribed in group A in 96.6, 96.0, 91.1 and 89.7% and in group B in 98.3, 97.4, 94.1 and 91.2%. Conclusion: The cross-sectional part of the study indicates a substantial burden of disease in PAD patients in primary care. Treatment rates appear to have improved compared to earlier surveys. In the follow-up period, outcomes of these patients and their association with disease stages, guideline-oriented treatment or patient compliance and disease-coping strategies, among other factors, will be determined

Outcomes of medical management of peripheral arterial disease in general practice: follow-up results of the PACE-PAD Study

Aim: Peripheral arterial disease (PAD), a marker of elevated vascular risk, is highly prevalent in general practice. We aimed to investigate patient characteristics and outcomes of PAD patients treated according to the guidelines versus those who were not. Methods: The Patient Care Evaluation-Peripheral Arterial Disease Study (PACE-PAD) was a multicenter, cluster randomized, prospective, longitudinal cohort study of patients with PAD in primary care, who were followed up for death or vascular events over 18 months. Guideline orientation was assumed if patients received anticoagulant/antiplatelet therapy, exercise training, and (if applicable) advice for smoking cessation and therapy of diabetes mellitus, hypertension, or hypercholesterolemia, respectively. Results: Of the 5,099 PAD patients (mean age 68.0 ± 9.0 years, 68.5% male subjects) who were followed up, 22.5, 34.6, 30.1, 7.8, and 3.5% (1.5% not specified) were in Fontaine stages I, IIa, IIb, III, and IV. Comprehensive guideline orientation was reported in 28.4% only; however, patients in lower Fontaine stages received guideline-oriented therapy more often (I: 30.3%, IIa: 31.6%, IIb: 29.1%, III: 9.8%, IV: 18.0%). During 18 months, 457 patients died (224 due to cerebrovascular or coronary deaths), 319 had unstable angina pectoris, 116 myocardial infarction, and 140 an ischemic stroke event. In total, 24% of patients had experienced any vascular event (19.1% a first event). Event rates did not differ between patients treated according to guidelines and those who were not. Conclusion: The present PAD cohort was a high-risk sample with an unexpectedly high rate of deaths and vascular events. While physicians appear to focus on the treatment of individual risk factors, rates of comprehensive PAD management in line with guideline recommendations are still suboptimal. Factors contributing to the lacking difference between outcomes in the guideline-oriented and non-guideline-oriented groups may comprise low treatment intensity or other reasons for unsatisfactory effect of treatment, misclassification of events, and patient’s noncompliance with therapy

E‐ARK Dissemination Information Package (DIP) Final Specification

The primary aim of this report is to present the final version of the E-ARK Dissemination Information Package (DIP) formats. The secondary aim is to describe the access scenarios in which these DIP formats will be rendered for use

Expression Profiling and Glycan Engineering of IgG Subclass 1–4 in Nicotiana benthamiana

IgG, the main serum immunoglobulin isotype, exists in four subclasses which selectively appear with distinctive glycosylation profiles. However, very little is known about the biological consequences mainly due to the difficulties in the generation of distinct IgG subtypes with targeted glycosylation. Here, we show a comprehensive expression and glycan modulation profiling of IgG variants in planta that are identical in their antigen binding domain but differ in their subclass appearance. While IgG1, 2, and 4 exhibit similar expression levels and purification yields, IgG3 is generated only at low levels due to the in planta degradation of the heavy chain. All IgG subtypes are produced with four distinct N-glycosylation profiles, differing in sugar residues previously shown to impact IgG activities, i.e., galactosylation, sialylation and core fucosylation. Affinity purified IgG variants are shown to be fully assembled to heterodimers but display different biochemical/physical features. All subtypes are equally well amenable to targeted glycosylation, except sialylated IgG4 which frequently accumulates substantial fractions of unusual oligo-mannosidic structures. IgG variants show significant differences in aggregate formation and endotoxin contamination which are eliminated by additional polishing steps (size exclusion chromatography, endotoxin removal treatments). Collectively we demonstrate the generation of 16 IgG variants at high purity and large glycan homogeneity which constitute an excellent toolbox to further study the biological impact of the two main Fc features, subclass and glycosylation

Fc-engineering significantly improves the recruitment of immune effector cells by anti-ICAM-1 antibody MSH-TP15 for myeloma therapy

Despite several therapeutic advances, patients with multiple myeloma (MM) require additional treatment options since no curative therapy exists yet. In search of a novel therapeutic antibody, we previously applied phage display with myeloma cell screening and developed TP15, a scFv targeting intercellular adhesion molecule 1 (ICAM-1/CD54). To more precisely evaluate the antibody's modes of action, fully human IgG1 antibody variants were generated bearing wild-type (MSH-TP15) or mutated Fc to either enhance (MSH-TP15 Fc-eng.) or prevent (MSH-TP15 Fc k.o.) Fc gamma receptor binding. Especially MSH-TP15 Fc-eng. induced potent antibody-dependent cell-mediated cytotoxicity (ADCC) against malignant plasma cells by efficiently recruiting NK cells and engaged macrophages for antibody-dependent cellular phagocytosis (ADCP) of tumor cells. Binding studies with truncated ICAM-1 demonstrated MSH-TP15 binding to ICAM-1 domain 1-2. Importantly, MSH-TP15 and MSH-TP15 Fc-eng. both prevented myeloma cell engraftment and significantly prolonged survival of mice in an intraperitoneal xenograft model. In the subcutaneous model MSH-TP15 Fc-eng. was superior to MSH-TP15, whereas MSH-TP15 Fc k.o. was not effective in both models - reflecting the importance of Fc-dependent mechanisms of action also in vivo. The efficient recruitment of immune cells and the potent anti-tumor activity of the Fc-engineered MSH-TP15 antibody hold significant potential for myeloma immunotherapy

E‐ARK Search, Access and Display Interfaces

The aim of this report is to describe the “Search, Access and Display Interfaces” that have been developed in the Access component of the E-ARK project. The deliverable associated with this report is mainly a software deliverable and therefore this document provides only underpinning descriptions of and links to the software itself. The tools that are described and provided allow Consumers (ie. end-users and archivists) to: 1. Search and order records (primarily end-users, but also archivists) 2. Manage orders of records and manage the records themselves, including the AIP to DIP conversion (archivists only) 3. Access ordered records as DIPs (primarily end-users, but also archivists) In addition to the the introductory remarks in chapter 2, the functionality of the tools that allow the Consumers to search, manage, and access records is described in chapter 3. After the description of each tool, links are provided to code and documentation