Derivatization of antibody fab fragments: A designer enzyme for native protein modification

Sandra Liebscher, Petra Kornberger, Gerhard Fink, Eva Maria Trost-Gross, Eva Höss, Arne Skerra, Frank Bordusa

Research output: Contribution to journalArticlepeer-review

23 Scopus citations

Abstract

Bioconjugates, such as antibody-drug conjugates, have gained recent attention because of their increasing use in therapeutic and diagnostic applications. Commonly used conjugation reactions based upon chemoselective reagents exhibit a number of drawbacks: most of these reactions lack regio- and stereospecificity, thus resulting in loss of protein functionality due to random modifications. Enzymes provide an obvious solution to this problem, but the intrinsic (natural) substrate specificities of existing enzymes pose severe limitations to the kind of modifications that can be introduced. Here we describe the application of the novel trypsin variant trypsiligase for site-specific modification of the C terminus of a Fab antibody fragment via a stable peptide bond. The suitability of this designed biocatalyst was demonstrated by coupling the Her2-specific Fab to artificial functionalities of either therapeutic (PEG) or diagnostic (fluorescein) relevance. In both cases we obtained homogeneously modified Fab products bearing the artificial functionality exclusively at the desired position. Trypsin in reverse: Trypsiligase (trypsin K60E/N143H/E151H/D189K) was used for highly selective PEGylation of the anti-Her2 Fab fragment under native conditions. The approach resulted in a fully functional antibody fragment with good product yield.

Original languageEnglish
Pages (from-to)1096-1100
Number of pages5
JournalChemBioChem
Volume15
Issue number8
DOIs
StatePublished - 26 May 2014

Keywords

  • antibody conjugation
  • biocatalysis
  • proteases
  • protein modifications
  • transamidation

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