Zielgerichtete Eliminierung autoreaktiver B-Lymphozyten mithilfe Antigen-basierter Fusionsproteine
| Autor: | Klose, Diana |
|---|---|
| Přispěvatelé: | Fischer, Rainer, Bernhagen, Jürgen |
| Jazyk: | němčina |
| Rok vydání: | 2017 |
| Předmět: | |
| Zdroj: | Aachen 1 Online-Ressource (ix, 121 Seiten) : Illustrationen, Diagramme (2017). doi:10.18154/RWTH-2017-00555 = Dissertation, RWTH Aachen University, 2017 |
| DOI: | 10.18154/rwth-2017-00555 |
| Popis: | RWTH Aachen University, Diss., 2017; 1 Online-Ressource (ix, 121 Seiten) : Illustrationen, Diagramme(2017). Autoantibodies play an import role in diagnostic of autoimmune diseases and significantly contribute to diseases pathogenesis. In the last years, several B-cell depletion strategies including monoclonal antibodies were used in clinical trials with patients of different autoimmune disorders. In case of systemic lupus erythematosus (SLE), the placebo-controlled randomized trials did not achieve the endpoints. Nevertheless, the antigen-specific modification of autoreactive B-lymphocytes via their specific B cell receptor (BCR) is the basis for a novel treatment approach of autoimmune diseases. Therefore, fusion proteins build of a target-cell specific binding domain (autoantigen) recombinant fused to an effector domain, a bacterial toxin or a human pro-apoptotic enzyme, were used to eliminate the target cell population. The project was initially funded by the DFG (Deutsche Forschungsgemeinschaft) and carried out in close cooperation with the Münster University Hospital (Department of Rheumatology and Medical Immunology). The major goal of the thesis was the targeting of antigen-specific B cells via their unique BCR by either using first a model antigen and second a disease-relevant autoantigen. To successfully develop an active fusion protein, an appropriate cell targeting domain had to be identified. For the proof-of-concept we chose the well-established model-antigen tetanus toxoid fragment C (TTC). Vaccinated individuals have a frequency of 0.1-0.2% TTC reactive memory B cells in the periphery. Different TTC-based fusion proteins were cloned and subsequently produced using prokaryotic or eukaryotic expression systems followed by in vitro characterization. The specific targeting, binding and dose-dependent elimination of the recombinant fusion protein of TTC-ETA' was confirmed on the murine TTC-reactive hybridoma cell line 5E4 and on freshly isolated human antigen-specific CD27+ memory B cells. Subsequently, TTC-based human cytolytic fusion proteins consisting of a human effector domain, the microtubule-associated protein Tau (MAPTau) or granzyme B (GrB) mutant (R201K) were generated. For the in vitro characterization of these TTC-based fusion proteins, a human lymphocytic cell line was necessary. Here, the Transpo-mAb technology (NBE Therapeutics, Basel, CH), depending on transposon-based gene transfer, was used for the generation of a novel TTC-reactive REH cell line. Therefore, the variable fragments of the α-TTC antibody from the mouse hybridoma cell line 5E4 was rescued and cloned into the transposon-based expression vectors. After successful generation and enrichment of the TTC-reactive REH cell population the characterization of the TTC-based human cytolytic fusion proteins was performed. GrB(R201K) based human cytolytic fusion protein induced apoptosis in a dose-dependent manner with a half maximal effective concentration (EC50) of 0,77 ± 0,25 nM in contrast to the MAPTau based fusion proteins. According to these results, the GrB(R201K) effector domain could be a promising candidate for the generation of human cytolytic fusion proteins for autoimmune diseases treatment. In the next step, the established methods to generate cytotoxic fusion proteins were transferred on a SLE -relevant autoantigen using the laminin α1-chain. Different protein variants of the C-terminal laminin globular domains (LG3-5) of the laminin α1-chain were produced using prokaryotic or eukaryotic expression systems, respectively. By in vitro analysis of the novel human laminin-fusion proteins autoantibodies against the laminin α1-chain fragments could be detected in serum and urine samples of patients with SLE. Using the murine derived laminin-based fusion proteins anti-laminin autoantibodies could be detected in serum and urine samples of K14-CD40ligand (L) transgenic (tg) mice, which develop SLE-like symptoms. These results indicates that the laminin α1-chain could be used for further in vivo studies or generation of cytotoxic fusion proteins. In summary a new approach of the direct elimination of autoreactive B-lymphocytes using antigen-based fusion proteins was developed. Such novel antigen-based fusion proteins could be used for future development of tailor-made therapeutics in autoimmune diseases. Published by Aachen |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|