Comparative study of inhibitory antibody derivatives towards thrombin activatable fibrinolysis inhibitor

Jan Develter; Maarten Dewilde; Ann Gils; Paul J. Declerck

doi:10.1160/TH08-09-0834

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 2009; 102(01): 69-75
DOI: 10.1160/TH08-09-0834

Blood Coagulation, Fibrinolysis and Cellular Haemostasis

Schattauer GmbH

Comparative study of inhibitory antibody derivatives towards thrombin activatable fibrinolysis inhibitor

Jan Develter

¹Laboratory for Pharmaceutical Biology, Faculty of Pharmaceutical Sciences, Katholieke Universiteit Leuven, Belgium

,

Maarten Dewilde

¹Laboratory for Pharmaceutical Biology, Faculty of Pharmaceutical Sciences, Katholieke Universiteit Leuven, Belgium

,

Ann Gils

¹Laboratory for Pharmaceutical Biology, Faculty of Pharmaceutical Sciences, Katholieke Universiteit Leuven, Belgium

,

Paul J. Declerck

¹Laboratory for Pharmaceutical Biology, Faculty of Pharmaceutical Sciences, Katholieke Universiteit Leuven, Belgium

› Author Affiliations

Abstract

Summary

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.

Keywords

Thrombin activatable fibrinolysis inhibitor (TAFI) / carboxypeptidases - drug design - fibrinolysis - antibody - single-chain Fv

Full Text

References

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