Derivatization of antibody Fab fragments: a designer enzyme for native protein modification.
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.
Institute of Biochemistry/Biotechnology, Martin Luther University Halle-Wittenberg, Kurt-Mothes-Strasse 3, 06120 Halle/Saale (Germany).
Sandra
S
Liebscher
Petra
P
Kornberger
Gerhard
G
Fink
Eva-Maria
EM
Trost-Gross
Eva
E
Höss
Arne
A
Skerra
Frank
F
Bordusa
D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000378:metabolism; D055162:Biocatalysis; D045744:Cell Line, Tumor; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry* / Q000378:metabolism*; D015394:Molecular Structure; D014357:Trypsin / Q000737:chemistry / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D007140:Immunoglobulin Fab Fragments; D014357:Trypsin
antibody conjugation; biocatalysis; proteases; protein modifications; transamidation