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

• Published in: Journal of thrombosis and haemostasis Vol. 2; no. 5; pp. 797 - 803
• Main Authors: Hagemeyer, C. E., Tomic, I., Weirich, U., Graeber, J., Nordt, T., Runge, M. S., Bode, C., Peter, K.
• Format: Journal Article
• Language: English
• Published: Oxford, UK Blackwell Science Inc 01.05.2004
• Subjects: Antibodies, Monoclonal - genetics; Antibodies, Monoclonal - therapeutic use; Cloning, Molecular; Drug Delivery Systems - methods; fibrin; Fibrin - immunology; Fibrinolysis - drug effects; Fibrinolytic Agents - administration & dosage; Humans; Kinetics; plasminogen activators; Plasminogen Activators - administration & dosage; Plasminogen Activators - genetics; Protein Binding; Protein Engineering - methods; Recombinant Fusion Proteins; thrombolysis; Urokinase-Type Plasminogen Activator - administration & dosage; Urokinase-Type Plasminogen Activator - genetics
• ISSN: 1538-7933; 1538-7836; 1538-7836
• DOI: 10.1111/j.1538-7836.2004.00697.x

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 (β15−22) in the β‐chain of human fibrin (scFv59D8). The fusion protein was purified using affinity chromatography with the β15−22‐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.