Natürlich vorkommende Autoantikörper – Charakterisierung, Klonierung und Testung rekombinanter Antikörper am Beispiel von Amyloid-β-reaktiven Autoantikörpern

Die weltweit häufigste neurodegenerative Erkrankung ist die Alzheimer-Erkrankung. Sie ist durch das Absterben von Neuronen gekennzeichnet mit der Folge einer chronisch fortschreitenden kognitiven Beeinträchtigung. Durch bisher ungeklärte Mechanismen werden in der Pathogenese vorrangig Amyloid-β (Aβ)...

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Bibliographic Details
Main Author: Albus, Alexandra
Contributors: Dodel, Richard (Prof. Dr. med.) (Thesis advisor)
Format: Dissertation
Language:German
Published: Philipps-Universität Marburg 2021
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Table of Contents: Alzheimer’s Disease is the most common neurodegenerative disease and characterized by neuronal degeneration, which leads to cognitive impairment. Due to yet unknown mechanisms misfolded Amyloid-β (Aβ) accumulates and deposits on neurons, followed by destabilization of neurons and pro-inflammatory processes within the brain. Until now, there is no effective treatment to prevent or to slow down Alzheimer’s Disease progression. In this context, passive immunization trials come up to fore as most promising approaches. Pathological peptides are marked by antibodies and mediated to surrounding phagocytozing macrophages, which are represented by microglia in the CNS. However, clinical studies on passive immunization trials failed due to ineffective cognitive stabilization. In this context, the aim of the here presented study was a physiological approach of passive immunization using already existing naturally occurring autoantibodies (nAbs). NAbs detect autoantigens, such as misfolded Aβ and mediate them to macrophages (nAbs-Aβ). Until now, they were enriched from commercially available intravenous immunoglobulins of class G (IVIg). However, their availability is restricted to blood donations. Therefore, we investigated on the recombinant production of nAbs-Aβ. By the single isolation of Aβ-reactive B1 cells from healthy donors, we were able to identify the antibodies’ Ig-genes. Thereby, we could produce three recombinant human antibodies in IgG format as well as one antibody in an altered single chain (scFv-Fc) construct. Using different binding and functional assays we identified the altered B07 scFv-Fc antibody as most promising candidate for a passive immunization approach as it showed the most positive results in all used assays. Further investigation on the glycosylation pattern of the recombinant antibodies showed a possible deficiency of recombinant antibodies in general. The antibody’s stability as well as the Fc-receptor mediated functions could massively be altered due to the specific glycosylation of the used HEK cells. For further immunization trials with recombinant antibodies the glycosylation pattern should be considered and potentially modified by specific “glycoengeneering”. However, as we used murine cell culture systems, the study is limited by the species barrier between human and mice. Therefore, the results could be influenced using murine cells, which could induce further immunological reactions. In conclusion, by the enrichment of B1 cells from healthy blood donations four recombinant Aβ-reactive antibodies were produced and tested. Thereby, the multimeric B07 scFv-Fc antibody expressed the most positive outcome in comparison to nAbs-Aβ and could be investigated in further passive immunization approaches to slow down the progression of Alzheimer’s Disease or even prevent its occurrence. By the application of the potential antibody as early as possible Aβ molecules could be led to phagocytosis before they deposit and get pathological. As the multimerized B07 antibody showed reactivity against monomeric as well as oligomeric Aβ both aggregation states could be phagocytized. In addition, due to its multimerization the B07 showed a higher avidity and thereby reflects a more efficient Aβ clearance. However, a general disadvantage of passive immunization is the half-life of antibodies, which make applications at regular intervals necessary to be effective. By the change of the glycosylation pattern the antibody stability could be increased, whereby the antibody would be longer available. Thus, the used antibody concentration could be decreased and side-effects, such as edema, will decreased as well. Nevertheless, it is not clarified if the antibodies enter the brain to bind Aβ or the mechanism function in the periphery. By the combination of the multimerized Aβ-reactive antibody B07 and the modifications of the glycans a potential antibody could be used against AD.