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Gewählte Doctoral Thesis:

Michael Traxlmayr (2012): Stability engineering of IgG1-Fc by random mutagenesis and in vitro directed evolution.
Doctoral Thesis - Abteilung für Biochemie (DCH/BC), BOKU-Universität für Bodenkultur, pp 105. UB BOKU obvsg

Abstract:

Antibodies represent a rapidly growing class of therapeutic proteins that are used for the treatment of a variety of diseases, including immunological disorders and cancer. An IgG1 protein (immunoglobulin G class 1) can be dissected into two antigen binding fragments (Fabs) and one crystallizable fragment (Fc). Except for antigen binding, the Fc mediates all other antibody functions and the long in vivo half life. Thus, the introduction of an antigen binding site into IgG1-Fc would enable the construction of an Fc protein that possesses all functions, but only one third of the mass of a full-size antibody. In the first project of this thesis it was demonstrated that it is possible to construct such an antigen binding site by mutating the C-terminal structural loops of the CH3 domains of human IgG1-Fc. However, those loop mutations caused destabilization, partial misfolding and aggregation. Prompted by those observations a new directed evolution-based method suitable for protein stability engineering was developed. This strategy enabled the selection of a set of stabilizing point mutations in the CH3 domains of IgG1-Fc. Those mutations will serve as a toolkit for the reparation of destabilized Fcabs. Next, this selection method was adopted for optimizing the antigen binding loops of Fcabs, resulting in stable, wild-type like folded and aggregation-resistant Fcab clones that also showed high affinities to the tumor antigen Her2/neu. Finally, a stability landscape of the CH3 domain of human IgG1 was constructed. This landscape shows the impact of each amino acid residue on the fold and stability of this domain. Apart from new insights into the relationship between sequence and stability/folding of proteins in general, this stability landscape provides valuable information for Fcab engineering. By choosing mutation-tolerant loop regions for the construction of binding sites the negative impact of the mutations on protein fold and stability will be reduced.

Betreuer: Obinger Christian

1. Berater: Rüker Florian

2. Berater: Oostenbrink Chris