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Transfer of engineered biophysical properties between different antibody formats and expression systems

Schaefer, Jonas V. ; Plückthun, Andreas

In: Protein Engineering, Design & Selection, 2012, vol. 25, no. 10, p. 485-506

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    Title
    • Transfer of engineered biophysical properties between different antibody formats and expression systems
    Author
    • Schaefer, Jonas V.. Department of Biochemistry, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland
    • Plückthun, Andreas. E-mail: plueckthun@bioc.uzh.ch
    Document Type
    • Postprint
    Language
    • English
    Published in
    • Protein Engineering, Design & Selection, 2012, vol. 25, no. 10, p. 485-506. Oxford University Press
    Other Version
    OAI-PMH Identifier
    • oai:doc.rero.ch:301956
    Summary
    Recombinant antibodies and their derivatives are receiving ever increasing attention for many applications. Nevertheless, they differ widely in biophysical properties, from stable monomers to metastable aggregation-prone mixtures of oligomers. Previous work from our laboratory presented the combination of structure-based analysis with family consensus alignments as being able to improve the properties of immunoglobulin variable domains. We had identified a series of mutations in the variable domains that greatly influenced both the stability and the expression level of single-chain Fv (scFv) fragments produced in the periplasm of Escherichia coli. We now investigated whether these effects are transferable to Fab fragments and immunoglobulin G (IgG) produced in bacteria, Pichia pastoris, and mammalian cells. Taken together, our data indicate that engineered mutations can increase functional expression levels only for periplasmic expression in prokaryotes. In contrast, stability against thermal and denaturant-induced unfolding is improved by the same mutations in all formats tested, including scFv, Fab and IgG, independent of the expression system. The mutations in VH also influenced the structural homogeneity of full-length IgG, and the reducibility of the distant CH1-CL inter-chain disulfide bond. These results confirm the potential of structure-based protein engineering in the context of full-length IgGs and the transferability of stability improvements discovered with smaller antibody fragments