Background-Recombinant technology was used to produce a new anticoagulant that is preferentially localized and active at the site of the clot. Methods and Results-The variable regions of the heavy and light chains of a fibrin-specific antibody were amplified by polymerase chain reaction (PCR) with hybridoma cDNA. To obtain a functional single-chain antibody (scFv), a linker region consisting of (Gly 4 Ser) 3 was introduced by overlap PCR. After the scFv clones were ligated with DNA encoding the pIII protein of the M13 phage, high-affinity clones were selected by 10 rounds of panning on the B␤15-22 peptide of fibrin (␤-peptide). Hirudin was genetically fused to the C-terminus of the variable region of the light chain. To release the functionally essential N-terminus of hirudin at the site of a blood clot, a factor Xa recognition site was introduced between scFv 59D8 and hirudin. The fusion protein was characterized by its size on SDS-PAGE (36 kDa), by Western blotting, by its cleavage into a 29-kDa (single chain alone) and 7-kDa (hirudin) fragment, by its binding to ␤-peptide, and by thrombin inhibition after Xa cleavage. Finally, the fusion protein inhibited appositional growth of whole blood clots in vitro more efficiently than native hirudin. Conclusions-A fusion protein was constructed that binds to a fibrin-specific epitope and inhibits thrombin after its activation by factor Xa. This recombinant anticoagulant effectively inhibits appositional clot growth in vitro. Its efficient and fast production at low cost should facilitate a large-scale evaluation to determine whether an effective localized antithrombin activity can be achieved without systemic bleeding complications.

To cite this article: Hagemeyer CE, Tomic I, Weirich U, Graeber J, Nordt T, Runge MS, Bode C, Peter K. Construction and characterization of a recombinant plasminogen activator composed of an anti-fibrin single-chain antibody and low-molecular-weight urokinase. J Thromb Haemost 2004; 2: 797-803.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 (b 15)22 ) in the b-chain of human fibrin (scFv 59D8 ). The fusion protein was purified using affinity chromatography with the b 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.