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 (beta(15-22)) in the beta-chain of human fibrin (scFv(59D8)). The fusion protein was purified using affinity chromatography with the beta(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.
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 (beta(15-22)) in the beta-chain of human fibrin (scFv(59D8)). The fusion protein was purified using affinity chromatography with the beta(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.
Authors: Sergei Zaitsev; Kristina Danielyan; Juan-Carlos Murciano; Kumkum Ganguly; Tatiana Krasik; Ronald P Taylor; Steven Pincus; Steven Jones; Douglas B Cines; Vladimir R Muzykantov Journal: Blood Date: 2006-05-30 Impact factor: 22.113
Authors: Bi-Sen Ding; Claudia Gottstein; Andrea Grunow; Alice Kuo; Kumkum Ganguly; Steven M Albelda; Douglas B Cines; Vladimir R Muzykantov Journal: Blood Date: 2005-09-06 Impact factor: 22.113
Authors: Jan David Hohmann; Xiaowei Wang; Stefanie Krajewski; Carly Selan; Carolyn A Haller; Andreas Straub; Elliot L Chaikof; Harshal H Nandurkar; Christoph E Hagemeyer; Karlheinz Peter Journal: Blood Date: 2013-02-04 Impact factor: 22.113
Authors: Bi-Sen Ding; Nankang Hong; Juan-Carlos Murciano; Kumkum Ganguly; Claudia Gottstein; Melpo Christofidou-Solomidou; Steven M Albelda; Aron B Fisher; Douglas B Cines; Vladimir R Muzykantov Journal: Blood Date: 2007-11-28 Impact factor: 22.113