Charakterisierung von B-Zell-Liganden im Kontext maligner und allergener Erkrankungen

Abstract

Gegenstand der vorliegenden Dissertation ist die Charakterisierung humaner Antigen- Rezeptoren von Lymphom- und Allergie-Patienten. Dabei werden erstmalig zugrundeliegende Rezeptor-Ligand-Interaktionen beschrieben und Epitope definiert. B-Zell-Rezeptoren (BZR) sind hochspezifische Schlüsselmoleküle der Antigen- Erkennung und lösen bei lymphatischen Zellen schützende Immunreaktionen aus. Um neue Erkenntnisse zur pathophysiologischen Transformation der B-Lymphozyten zu erhalten, erfolgt in dieser Arbeit die Analyse einzelner repräsentativer Rezeptoren von entarteten Zellen hinsichtlich ihrer Bindungs-Spezifität und -Modalität. Das Verständnis dieser Interaktion von Rezeptor gegen spezifischen Ligand ist essentiell für ein tiefergehendes Wissen über die Entwicklung Rezeptor-abhängiger B-Zell-basierter Erkrankungen. Außerdem kann es einer erfolgreichen therapeutischen Intervention dienen. In den folgenden Abschnitten werden einleitend die Mechanismen der B-Zell- Entwicklung mit besonderem Fokus auf den B-Zell-Rezeptor erläutert. Anschließend wird dargestellt wie die Dysbalance dieser physiologischen Prozesse in einer pathogenen Transformation von B-Zellen resultiert und zur Ausprägung von unterschiedlichen Erkrankungen führen kann. Zu den möglichen Krankheiten zählen die chronische lymphatische Leukämie, das Burkitt-Lymphom und die Entwicklung einer Allergie.

A milestone in the research on chronic lymphocytic leukemia (CLL) was the discovery that about a third of all cases of CLL express practically identical, stereotype B-cell receptors. These receptors show high similarities in the CDR3 region of the heavy chain domain in immunoglobulins. These homologies in immunogenetic parameters lead to the assumption that certain groups of antigens are recognized by the B-cell receptors of lymphoma cells, thereby contributing decisively to their survival and proliferation. In this dissertation, this hypothesis was examined to test for receptors in selected lymphoma patients, identify previously unknown epitopes, and define patterns of binding. For this purpose, monoclonal antibodies were generated based on receptor sequence data. These receptor molecules were used to select phage display peptide libraries in order to identify mimotopes. These are synthetic peptides that mimic the conformation of the epitope. The sequence and database analysis with mimotopes led to the identification of essential interaction structures and potential natural antigens that are bound by lymphoma cells. Autoantigen binders were found in the form of the beta chain of T-cell receptors, and in a sequence pattern in the FR2 of the VL domain of the immunoglobulin chain, whereby the latter binding reaction resulted in self-recognition of the receptor. Interaction analyses using SPR-based methods also documented for the first time the high affinity of B-cell receptors to their ligands. This finding suggests that there is comparatively high receptor specificity. This insight is important for preserving the crucial interaction molecules between receptors and ligands. It supports the successful selection of synthetic peptide ligands and thereby a method for determining antigenic structures. An extraordinary receptor specificity and affinity is a unifying element for both diseases, also seen in the context of Type I allergies. Here the cross-linking of receptor-bound IgE antibodies by allergens is the key event in allergic reactions. To identify an IgE epitope, a human antibody was used that has specificity to the major allergen from grass pollen, Phl p 5. A recombinant construction of different allergen variants made it possible to determine essential structures of receptor-ligand interaction. In this way, an original Phl p 5 IgE epitope was identified for the first time. Finally, structure-lending techniques were applied to attain a fine definition of the epitope. For this purpose, the generation/production of Fab fragments by means of proteolytic cleavage of intact immunoglobulins as well as prokaryotic and eukaryotic expression was established and evaluated. Furthermore, affinity matrices were generated for the specific purification of target molecules, and peptide ligands were synthesized. The analysis of receptor epitopes and receptor interactions carried out during the work on this dissertation contributes significantly to understanding the diseases. Furthermore, the insights gained offer great potential for new and highly specific medical treatment. Due to their high ligand affinity and specificity, the B-cell receptors of the abnormal lymphoma cells that were studied are optimal therapeutic target structures for toxic peptides. Regarding allergies, the identification of IgE epitopes makes it possible to design allergen variants for safer and more effective immunotherapy than was previously possible.