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Evaluation of the profibrinolytic properties of a bispecific antibody‐based inhibitor against human and mouse thrombin‐activatable fibrinolysis inhibitor and plasminogen activator inhibitor‐1

To date, no profibrinolytic agent has reached the market, although such an agent would be particularly useful in certain disease states associated with impaired endogenous fibrinolysis, e.g. severe sepsis and atherosclerosis [1– 4]. In cases of occlusive thrombi, the only approved pharmacologic treatment to obtain acute revascularization consists of intravenous infusion of plasminogen activators, which is limited by low efficiency (low recanalization and high reocclusion rates) and a high bleeding risk [5]. Elevated levels of antifibrinolytic proteins, such as thrombin-activatable fibrinolysis inhibitor (TAFI) and plasminogen activator inhibitor-1 (PAI-1), lead to an increased risk of thrombosis, and are therefore interesting drug targets [1]. TAFI and PAI-1 are intimately linked proteins, as activated TAFI only suppresses fibrinolysis when its level is above a certain threshold that is proportional to the tissue-type plasminogen activator (t-PA) concentration [6,7]. Consequently, this threshold value increases in the presence of an inhibitor of PAI-1. In this study, we developed a dual targeting strategy towards TAFI and PAI-1 to robustly enhance fibrinolysis. This would allow a lower dosage of t-PA, which would constitute a safer treatment without compromising thrombolytic efficiency. In a previous study, Develter et al. [8] created a bispecific antibody fragment, a diabody, which enables simultaneous targeting of TAFI and PAI-1. Although this diabody stimulates fibrinolysis of model thrombi to a higher extent than the combination of the two parental mAbs, a clear limitation was the lack of cross-reactivity to the respective antigens from other species, thereby ruling out in vivo studies. In the present study, a new diabody was designed after the selection of two parental mAbs, MA-TCK26D6, directed towards human TAFI [9], and MA-33H1F7, directed towards human PAI-1 [10], which are both cross-reactive with their murine counterparts. The inhibitory properties of this novel diabody, Db-TCK26D6x33H1F7, were extensively characterized. Briefly, maximal inhibition of TAFI by MA-TCK26D6 was 97% 2% and 86% 6% for human and mouse TAFI, respectively. Similar maximal inhibitory values were observed for Db-TCK26D6x33H1F7 (100% 2% and 86% 3% for human and mouse TAFI, respectively). For both human and mouse TAFI, IC50 values were approximately two-fold higher than the respective IC50 value of the parental mAb (0.2 0.1 vs. 0.4 0.1 for human TAFI, P < 0.02; and 0.5 0.2 vs. 0.8 0.3 for mouse TAFI, P < 0.05; expressed as molar ratio over TAFI, mean standard deviation [SD], n = 3–6). This two-fold increase corresponds with the two-fold lower valency of the diabody compared to that of the parental mAb. Maximal inhibition of PAI-1 activity by MA-33H1F7 was 42% 2% and 63% 3% for human and mouse PAI-1, respectively. A slight reduction of 10– 20% in maximal inhibition was observed for the diabody, which was only significant (P < 0.02) for mouse PAI-1 (31% 9% and 46% 6% for human and mouse PAI-1, respectively). IC50 values of the diabody were similar to those of the parental mAb (1.7 0.6 vs. 1.5 1.3 for human PAI-1; and 0.8 0.3 vs. 1.2 0.9 for mouse PAI-1; expressed as molar ratio over PAI-1, mean SD, n = 3, P > 0.05). In addition, the inhibitory properties against rat TAFI and rat PAI-1 were similar to those of the parental antibodies (data not shown). Overall, the functional properties of MA-TCK26D6 and MA-33H1F7 were almost fully retained within the diabody construct. Profibrinolytic properties in human whole blood were assessed in vitro with thromboelastometry on a ROTEM Delta analyzer (Pentapharm, Munich, Germany). Citrated whole blood (250 lL) from four healthy donors was Correspondence: Paul Declerck, Department of Pharmaceutical and Pharmacological Sciences, Laboratory for Therapeutic and Diagnostic Antibodies, KU Leuven, Campus Gasthuisberg, O&N2, PB 820, Herestraat 49, B-3000 Leuven, Belgium. Tel.: +32 16 32 34 31. E-mail: paul.declerck@pharm.kuleuven.be