Comparative study of inhibitory antibody derivatives towards thrombin activatable fibrinolysis inhibitor.
Thrombin activatable fibrinolysis inhibitor (TAFI) attenuates fibrinolysis and is considered as an attractive drug target. We generated two different antibody fragments, an antigen-binding fragment (Fab) and a single-chain variable fragment (scFv), derived from three distinct monoclonal antibodies (MAs) that inhibit the activation of TAFI by the thrombin/thrombomodulin complex (T/TM) and plasmin (MA-T1C10 and MA-T94H3) or by T/TM alone (MA-T12D11). The Fabs were obtained by papain digestion of the purified MAs, whereas the scFvs were cloned and subsequently expressed in bacteria. All antibody fragments revealed similar or slightly decreased affinities compared to those of the respective MAs, except scFv-T94H3. In the presence of a 16-fold molar excess of all antibody fragments, activation of TAFI by T/TM was completely blocked. Furthermore, Fab and scFv-derivatives from MA-T1C10 and MA-T94H3 were capable of interfering with the plasmin-mediated activation of TAFI. Addition of 850 nM of MA, Fab or scFv to an in-vitro clot lysis assay caused a significant reduction of clot lysis time (except for scFv-T94H3) and this effect was comparable to that of potato tuber carboxypeptidase inhibitor, a well-known TAFIa inhibitor. Dose-response experiments with the antibody (derivatives) in clot lysis and chromogenic assay revealed that the inhibitory capacity of the Fabs was comparable to that of the MAs, whereas the scFvs had a more reduced potency. In conclusion, these highly specific TAFI inhibitors are interesting tools to further evaluate the concept of TAFI inhibition in various in-vitro and in-vivo models.
Develter
Jan
J
Laboratory for Pharmaceutical Biology, Faculty of Pharmaceutical Sciences, Katholieke Universiteit Leuven, Belgium.
Dewilde
Maarten
M
Gils
Ann
A
Declerck
Paul J
PJ
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology*; D025901:Carboxypeptidase B2 / Q000276:immunology* / Q000378:metabolism; D003001:Cloning, Molecular; D004306:Dose-Response Relationship, Immunologic; D015195:Drug Design; D005342:Fibrinolysis / Q000502:physiology*; D007140:Immunoglobulin Fab Fragments / Q000235:genetics / Q000276:immunology* / Q000494:pharmacology; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000276:immunology* / Q000494:pharmacology; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000276:immunology / Q000378:metabolism
D000911:Antibodies, Monoclonal; D007140:Immunoglobulin Fab Fragments; D007135:Immunoglobulin Variable Region; D011994:Recombinant Proteins; D025901:Carboxypeptidase B2