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Gewählte Doctoral Thesis:

Patrick Mayrhofer (2016): Engineering of critical antibody sequences for humanization, deimmunization and germinalization with detailed analysis of their influence on production cells.
Doctoral Thesis - Institut für Biotechnologie, BOKU-Universität für Bodenkultur, pp 168. UB BOKU obvsg FullText

Data Source: ZID Abstracts
Antibody engineering allows the rational optimization of beneficial protein properties defined by the primary amino acid sequence. Non-human monoclonal antibodies (mAb) require modification in the variable region to remove immunogenic sequences, but simultaneously need to retain specificity and affinity. Our model protein, the mouse anti-idiotypic mAb Ab2/3H6, was used for humanization strategies by introducing rationally designed framework mutations. Novel 3H6 variants were expressed and evaluated for binding affinity. The gained information was supplied to develop an upgraded humanization workflow using a prospective design cycle supported by molecular dynamics (MD) simulations. Remaining non-human residues were additionally investigated in detail using T cell epitope prediction tools. Expression of some 3H6 variants in cell culture yielded only moderate production levels and in one case the mAb could not be secreted into the cell culture supernatant. This emphasized the common assumption that the primary amino acid sequence influences not only protein properties but also the cell biology and expression machinery. To investigate these impacts we engineered two different mammalian host cell lines (CHO) for targeted gene integration using recombinase-mediated cassette exchange (RMCE). Expression in RMCE-competent cells enabled us to test different mAb variants under isogenic conditions, eliminating positioning effects resulting from disparate integration loci. We designed and expressed a set of naturally occurring antibodies and germline-derived antibodies by comparing mature antibody sequences to their closest human germline genes. Isogenic RMCE subclones were subjected to proteomics and metabolomics analyses to study the influence of the protein sequence on cellular properties. The presented results demonstrate how different in-silico and in-vitro methods from different scientific fields can be merged to develop novel concepts for antibody engineering.

Betreuer: Kunert Renate
1. Berater: Oostenbrink Chris
2. Berater:

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