Cloning and characterization of an antibody-based inhibitor of TAFI

Introduction: Thrombin Activatable Fibrinolysis Inhibitor (TAFI) counteracts fibrinolysis after being activated by thrombin, plasmin or the thrombin-thrombomodulin (T/TM) complex. Even though it is generally accepted that T/TM is the main physiological activator of TAFI in vivo, a recent study demonstrated that MA-TCK26D6, a monoclonal antibody (MA) mainly inhibiting the plasmin-mediated activation of human, mouse and rat TAFI, exerts a pronounced profibrinolytic effect in vivo in a mouse thromboembolism model. Objective: To avoid disadvantages typically associated with the therapeutic use of antibodies (size, immunogenicity, production costs), the size of MA-TCK26D6 was reduced to a single-chain variable fragment (scFv) and this scFv was characterized. Methods and results: ScFv-TCK26D6 was constructed by cloning and assembling the variable domains of MA-TCK26D6. This scFv was then produced in E.coli, purified and its functional properties were compared to those of the hybridoma-produced parental MA. The scFv effectuated a dose-dependent neutralizing response on the plasmin-mediated activation of human, mouse and rat TAFI with a maximal inhibition of 96±2%, 84±4% and 85±1%, respectively. In accordance with the two-fold lower valency of a scFv compared to that of a MA, the IC50 value of scFv-TCK26D6 was slightly increased compared to that of the MA (48±15 nM vs 25±4 nM, 119±31 nM vs 89±30 nM and 106±25 nM vs 36±2 nM for human, mouse and rat TAFI, respectively). Evaluation in clot lysis experiments using human and rat plasma revealed profibrinolytic effects of scFv-TCK26D6 comparable to those of MA-TCK26D6 (98±4% and 90±8% for scFv in human and rat plasma, respectively, compared to the MA). Conclusion: An antibody-based inhibitor of TAFI was generated, retaining the inhibitory characteristics of the parental MA and allowing its in vivo evaluation in thrombosis models.

Jaar van publicatie:2011