Conjugation to antifibrin Fab' enhances fibrinolytic potency of single-chain urokinase plasminogen activator.

Abstract

Single-chain urokinase plasminogen activator (scu-PA) that had been modified with Nsuccinimidyl-3-(2-pyridyldithio)propionate was covalently linked by disulfide bonds to the Fab' of a monoclonal antibody specific for the P-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. (Circulation 1990:81;1974-1980 W hen plasminogen activator therapy is instituted within 4-6 hours of the onset of symptoms of acute myocardial infarction, there is a significant reduction in morbidity and mortality.1 2 The first generation of plasminogen activators, streptokinase and urokinase, are effective but lack. fibrin and hence clot specificity. Secondgeneration plasminogen activators tissue-type plasminogen activator (t-PA) and single-chain urokinase plasminogen activator (scu-PA) are relatively fibrin One approach to improving the specificity of currently available thrombolytic agents is to increase their fibrin affinity by conjugation to an antifibrin antibody. We have studied conjugates between antifibrin monoclonal antibody 59D8 and urokinase8,9 and between 59D8 and t-PA.l0,1l Conjugation markedly enhances the thrombolytic potency of the two plasminogen activators, both in vitro and in vivo.scu-PA is perhaps the most promising plasminogen activator to which direct fibrin specificity could be added. Native scu-PA is fibrin selective, but this selectivity derives from the activation of fibrin-bound plasminogen. Although the mechanism of activ...

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

“…Most investigators have either rearranged (or duplicated or deleted) various plasminogen activator functional domains or altered their posttranslational modification (6)(7)(8)(9)(10). We and others (11)(12)(13)(14)(15)(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.…”

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.…”

“…10 Columns containing ␤-peptide-conjugated sepharose were loaded and washed with TA buffer. Bound protein was eluted by 0.1 mol/L glycine, pH 2.8, and 1-mL fractions were collected and adjusted to pH 7.0 with 0.5 mol/L Tris buffer.…”

“…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.…”

Biochemical characterization of single‐chain chimeric plasminogen activators consisting of a single‐chain Fv fragment of a fibrin‐specific antibody and single‐chain urokinase

“…MA-I 5C5, inurine monoclonal antibody specific for human fibrin fragment D-dimer; VH, variable heavy-chain domain of IgG; VL, variable light-chain domain of IgG; Fv, variable-domain antigen-binding fragment of IgG; scFv, single-chain Fv analogue with the VL and Vl, domains connected through a synthetic peptidc linker; S C F V -K~~G~, recombinant single-chain molecule consisting of the variable domains ol' MA-15C5 (Argl08 of VL linked to Clnl of VH via a synthetic Ala-Gly-Gln-Gly-Ser-Ser-Val linker); u-PA, human urokinase-type plasminogen activator; scu-PA, 54-kDa single-chain u-PA, consisting of amino acids Serl -Leu41 1 ; rscu-PA, recombinant scu-PA, expressed in Spodoptera frugiperda insect cells; rscu-PA(M), a variant of rscu-PA in which the Argl56-Phel57 thrombin-cleavage sitc is removed by substitution of Phe157 with Asp; rtcu-PA, recombinant two-chain u-PA, dcrived from rscu-PA by partial digestion with plasmin; u-PA-32k, low-molecular-mass u-PA consisting of amino acids Leu144-Leu41 1 ; u-PA-33k, low-molecular-mass u-PA consisting of amino acids Alal32 ~ Leu41 I ; u-PA-45k, low-molecular-mass u-PA consisting of amino acids Asp45 -Leu41 1 ; K1 2GOS32r single-chain chimeric plasminogen activator consisting of scFv-Kl2Go and u-PA-33k, in which the Argl56-Phel57 thrombincleavage site is rcmoved by substitution of Phel57 with Asp, and in which the Lysl35-Lysl36-cleavage site is removed by substitution of Targeting of thrombolytic agents to a thrombus by combining the antigen-binding properties of a fibrin-specific antibody with the catalytic activity of a plasminogen activator in a single molecule, has resulted in a significant increase of both their in vitro fibrinolytic and their in uivo thrombolytic potencies [l, 21. Conjugation of the antibody and plasminogen-activator molecules has been achieved by chemical coupling of single-chain urokindse-type plasminogen activator (scu-PA) and a monoclonal antibody directed against the Nterminus of the p chain of fibrin, 59D8 [3], or a monoclonal antibody specific for fragment D-dimer of cross-linked human fibrin (MA-1 5C5) [4 -71. Alternatively, the recombinant single-chain chimeric plasminogen activator, rscu-PA-32k/ 59D8, in which intact 59D8 was spliced to scu-PA-32k (a lowmolecular-mass truncated scu-PA), yielded a 20-fold higher thrombolytic potency than scu-PA in a rabbit jugular-vein thrombosis model [8].…”