TY - JOUR
T1 - Derivatization of antibody fab fragments
T2 - A designer enzyme for native protein modification
AU - Liebscher, Sandra
AU - Kornberger, Petra
AU - Fink, Gerhard
AU - Trost-Gross, Eva Maria
AU - Höss, Eva
AU - Skerra, Arne
AU - Bordusa, Frank
PY - 2014/5/26
Y1 - 2014/5/26
N2 - 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.
AB - 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.
KW - antibody conjugation
KW - biocatalysis
KW - proteases
KW - protein modifications
KW - transamidation
UR - http://www.scopus.com/inward/record.url?scp=84901491331&partnerID=8YFLogxK
U2 - 10.1002/cbic.201400059
DO - 10.1002/cbic.201400059
M3 - Article
C2 - 24782039
AN - SCOPUS:84901491331
SN - 1439-4227
VL - 15
SP - 1096
EP - 1100
JO - ChemBioChem
JF - ChemBioChem
IS - 8
ER -