A recombinant chimeric plasminogen activator with high affinity for fibrin has increased thrombolytic potency in vitro and in vivo.

Abstract

A recombinant plasminogen activator with high fibrin affinity and specificity was expressed by transfecting hybridoma cells with a plasmid that combines sequence coding for low molecular mass (32 kDa) single-chain urokinasetype plasminogen activator [scuPA(32kDa)J and anti-fibrin monoclonal antibody 59D8. The expression of the recombinant molecule [r-scuPA(32kDa)-59D8] was optimized by replacing the 3' untranslated region (initially that of high molecular mass scuPA) in the plasmid with the 3' untranslated region of either 13-globin or mouse immunoglobulin. This modification resulted in a >100-fold improvement in the level of protein expression.The 103-kDa r-scuPA(32kDa)-59D8 protein displayed catalytic activity indistinguishable from that of high molecular mass scuPA and fibrin binding comparable to that of native antibody 59D8. r-scuPA(32kDa)-59D8 was 6 times more potent than high molecular mass scuPA in lysing a human plasma clot in vitro and was 20 times more potent than high molecular mass scuPA in the rabbit jugular vein model of thrombolysis.Molecules of this type may serve as prototypes for highly specific, antibody-targeted enzymes suitable for human use.Acute thrombotic occlusion of a major epicardial coronary artery results in myocardial infarction, the single most common cause of death in industrialized societies. The use of thrombolytic therapy in patients with acute myocardial infarction has resulted in a significant reduction in mortality (1-3). At its present stage of development, however, thrombolytic therapy is limited by (i) significant bleeding at high doses (intracranial hemorrhage causes stroke or death in 0.1-0.5% of patients who receive plasminogen activators)(1-4), (ii) the failure to restore blood flow in 20%o of patients or the fact that thrombotic reocclusion after cessation of therapy occurs in 15-25% of patients (5), and (iii) the lag between initiation of therapy and clot lysis, which averages about 60 min (5).These limitations have prompted generation (by recombinant DNA technology) of hundreds of plasminogen activator mutants, which have produced, at best, only modest improvements in thrombolytic efficacy. Most investigators have either rearranged (or duplicated or deleted) various plasminogen activator functional domains or altered their posttranslational modification (6-10). We and others (11-16) have pursued an alternative strategy, the generation ofchemical conjugates or recombinant molecules with domains for both plasminogen activator activity and high-affinity fibrin binding [conferred by a monoclonal antibody (17) that binds to fibrin, the principal component of a thrombus, but not fibrinogen, its circulating precursor]. Here we report the generation and characterization (in vitro and in vivo) of a recombinant molecule that is fibrin selective by two different mechanisms. It combines a high-affinity anti-fibrin antibody, 59D8, with a low molecular mass (32 kDa) single-chain urokinase-type plasminogen activator [scuPA(32kDa)], a fibrin-selective plasminogen activato...

Construction and Functional Evaluation of a Single-Chain Antibody Fusion Protein With Fibrin Targeting and Thrombin Inhibition After Activation by Factor Xa

“…10,11 Furthermore, a chemical conjugate of the fibrinspecific mAb 59D8 and the direct thrombin inhibitor hirudin inhibited fibrin deposition on experimental clots 12 and demonstrated an increase in antithrombotic potency in baboons. 13 To increase the yield and activity of antibody-targeted hirudin and to further improve the risk/benefit ratio of anticoagulation, we have developed a recombinant fusion molecule consisting of an antifibrin single-chain antibody and hirudin.…”

“…9 Coupling of mAb 59D8 to plasminogen activators resulted in enhanced thrombolytic potency and specificity in vitro and in vivo. 10,11 A chemical conjugate between hirudin and 59D8 effectively inhibited fibrin deposition on experimental clots 12 and demonstrated potent antithrombotic activity in nonhuman primates. 13 Nevertheless, chemical coupling of hirudin to mAbs has several limitations, the major ones being low yield and loss of hirudin activity.…”

Endothelial targeting of a recombinant construct fusing a PECAM-1 single-chain variable antibody fragment (scFv) with prourokinase facilitates prophylactic thrombolysis in the pulmonary vasculature

“…[38][39][40][41][42][43][44][45] However, delivery systems targeting determinants appearing after the thrombotic event are likely to be less suitable for prophylactic usage (in part because of their masking by thrombi) versus those targeting stably expressed endothelial determinants, such as PECAM-1, which bind to at-risk vasculature. In addition, masking the adhesion Anti-PECAM scFv-uPA circulates in a form of a prodrug, single-chain uPA, binds to PECAM-1, and remains anchored on the luminal surface of endothelium for at least several hours.…”

Prophylactic thrombolysis by thrombin-activated latent prourokinase targeted to PECAM-1 in the pulmonary vasculature

“…8,9 Attempts to improve the effectiveness of PAs by increasing clot affinity have further diminished permeation into occluding thrombi due to retention on the clot surface 5 and have yet to provide decisively better clinical outcomes. [10][11][12][13][14] Thrombi often recur due to underlying procoagulation states, vascular damage, disturbance in blood flow, inflammation, or/and patients' immobility. 15 Rethromboses often occur within hours to days after acute myocardial infarction, transient ischemic attack, ischemic stroke, and pulmonary embolism.…”