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Construction and characterization of a recombinant plasminogen activator composed of an anti‐fibrin single‐chain antibody and low‐molecular‐weight urokinase
Author(s) -
Hagemeyer C. E.,
Tomic I.,
Weirich U.,
Graeber J.,
Nordt T.,
Runge M. S.,
Bode C.,
Peter K.
Publication year - 2004
Publication title -
journal of thrombosis and haemostasis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.947
H-Index - 178
eISSN - 1538-7836
pISSN - 1538-7933
DOI - 10.1111/j.1538-7836.2004.00697.x
Subject(s) - recombinant dna , single chain , fibrin , urokinase , antibody , plasminogen activator , chemistry , microbiology and biotechnology , medicine , immunology , biochemistry , biology , gene
Summary. 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 (scuPA LMW ), was cloned from human endothelial cells and fused to a single‐chain antibody specific for the 7 N‐terminal amino acids (β 15−22 ) in the β‐chain of human fibrin (scFv 59D8 ). The fusion protein was purified using affinity chromatography with the β 15−22 ‐peptide of human fibrin. Results: Purified scFv 59D8 –scuPA LMW migrated as a 60‐kDa band, which is consistent with a molecule composed of one scFv 59D8 and one scuPA LMW moiety. Both functions of the fusion molecule, fibrin‐specific binding and plasminogen activation, were fully preserved. In human plasma clots, thrombolysis by scFv 59D8 –scuPA LMW is significantly faster and more potent compared with the clinically used urokinase. Conclusions : ScFv 59D8 –scuPA LMW 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.