Construction and characterization of a recombinant plasminogen activator composed of an anti-fibrin single-chain antibody and low-molecular-weight urokinase

Background: Targeting of plasminogen activators to the fibrin component of a thrombus by antibodies directed against human fibrin can enhance their thrombolytic potency and clot specificity. Objectives: To overcome the disadvantages of chemical conjugation, we investigated whether the recombinant fusion of a single-chain antibody and a plasminogen activator results in an active bifunctional molecule that might be useful as a therapeutic agent. Methods: The cDNA of low-molecular-weight single-chain urokinase-type plasminogen activator, comprising amino acids Leu144-Leu411 (scuPALMW), was cloned from human endothelial cells and fused to a single-chain antibody specific for the 7 N-terminal amino acids (?15-22) in the ?-chain of human fibrin (scFv59D8). The fusion protein was purified using affinity chromatography with the ?15-22-peptide of human fibrin. Results: Purified scFv59D8-scuPALMW migrated as a 60-kDa band, which is consistent with a molecule composed of one scFv59D8 and one scuPALMW moiety. Both functions of the fusion molecule, fibrin-specific binding and plasminogen activation, were fully preserved. In human plasma clots, thrombolysis by scFv59D8-scuPALMW is significantly faster and more potent compared with the clinically used urokinase. Conclusions: ScFv59D8-scuPALMW constitutes a new recombinant chimeric plasminogen activator with a significantly enhanced thrombolytic potency and relative fibrin selectivity, that can be produced with modern methods at low cost, large quantities and reproducible activity in Escherichia coli.