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.


OBJECTIVE
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.
Hagemeyer
C E
CE
Department of Cardiology, University of Freiburg, Freiburg, Germany.
Tomic
I
I
Weirich
U
U
Graeber
J
J
Nordt
T
T
Runge
M S
MS
Bode
C
C
Peter
K
K
D000911:Antibodies, Monoclonal / Q000235:genetics / Q000627:therapeutic use*; D003001:Cloning, Molecular; D016503:Drug Delivery Systems / Q000379:methods*; D005337:Fibrin / Q000276:immunology*; D005342:Fibrinolysis / Q000187:drug effects; D005343:Fibrinolytic Agents / Q000008:administration & dosage; D006801:Humans; D007700:Kinetics; D010960:Plasminogen Activators / Q000008:administration & dosage* / Q000235:genetics; D011485:Protein Binding; D015202:Protein Engineering / Q000379:methods*; D011993:Recombinant Fusion Proteins; D014568:Urokinase-Type Plasminogen Activator / Q000008:administration & dosage* / Q000235:genetics
D000911:Antibodies, Monoclonal; D005343:Fibrinolytic Agents; D011993:Recombinant Fusion Proteins; D005337:Fibrin; D010960:Plasminogen Activators; D014568:Urokinase-Type Plasminogen Activator