CONJUGATION TO ANTIFIBRIN FAB' ENHANCES FIBRINOLYTIC POTENCY OF SINGLE-CHAIN UROKINASE PLASMINOGEN-ACTIVATOR

Single-chain urokinase plasminogen activator (scu-PA) that had been modified with N-succinimidyl-3-(2-pyridyldithio)propionate was covalently linked by disulfide bonds to the Fab' of a monoclonal antibody specific for the β-chain of fibrin (antibody 59D8). scu-PA-59D8 Fab′ conjugate was separated from free scu-PA and two-chain urokinase coupled to 59D8 Fab′ by two-step affinity chromatography. First, the reaction mixture was chromatographed on a column containing Sepharose linked to the peptide that had been used as immunogen for antibody 59D8; scu-PA-59D8 Fab′ conjugate and unconjugated 59D8 Fab′ were retained but not unconjugated scu-PA. Then, the eluate from the peptide-Sepharose column was chromatographed on a column containing Sepharose linked to benzamidine, which acts as a ligand for two-chain urokinase. The molecular weight of the scu-PA-59D8 Fab′ conjugate was approximately 100 kDa when electrophoresed on a nonreducing sodium dodecylsulfate-polyacrylamide gel. Enzymatic assay after purification revealed that more than 97% of the scu-PA present in the conjugate retained the single-chain form. The Fab′ portion of the conjugate functioned in a manner indistinguishable from that of native antibody 59D8. In an in vitro assay for lysis of fibrin monomer, the fibrinolytic potency of scu-PA-59D8 Fab′ was 33-fold more than that of tissue plasminogen activator (p <0.001), 230-fold more than that of unconjugated scu-PA (p <0.0001), and 420-fold more than that of urokinase (p <0.0001). In a human plasma clot assay, scu-PA-59D8 Fab′ was significantly more potent than native scu-PA in clot lysis and consumed less fibrinogen at equipotent fibrinolytic concentrations. Potency in vivo, measured in the rabbit jugular vein thrombus model, increased by 29-fold. Thus, conjugation to a fibrin-specific Fab′ appears to increase the fibrin affinity and fibrinolytic potency of scu-PA.