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...

We conclude that antibody targeting of scuPA to fibrin increases thrombolytic and antithrombotic potencies with less impairment of hemostasis compared with rTPA and rscuPA.

Two distinct spontaneous variants of the murine anti-digoxin hybridoma 26-10 were isolated by fluorescenceactivated cell sorting for reduced affinity of surface antibody for antigen. Nucleotide and partial amino acid sequencing of the variant antibody variable regions revealed that 1 variant had a single amino acid substitution: Lys for Asn at heavy chain position 35. The second variant antibody had 2 heavy chain substitutions: Tyr for Asn at position 35, and Met for Arg at position 38. Mutagenesis experiments confirmed that the position 35 substitutions were solely responsible for the markedly reduced affinity of both variant antibodies. Several mutants with more conservative position 35 substitutions were engineered to ascertain the contribution of Asn 35 to the binding of digoxin to antibody 26-10. Replacement of Asn with Gln reduced affinity for digoxin 10-fold relative to the wild-type antibody, but maintained wild-type fine specificity for cardiac glycoside analogues. All other substitutions (Val, Thr, Leu, Ala, and Asp) reduced affinity by at least 90-fold and caused distinct shifts in fine specificity. The Ala mutant demonstrated greatly increased relative affinities for 16-acetylated haptens and haptens with a saturated lactone.The X-ray crystal structure of the 26-10 Fab in complex with digoxin (Jeffrey PD et al., 1993, Proc NUN Acud Sci USA 90: 10310-10314) reveals that the position 35 Asn contacts hapten and forms hydrogen bonds with 2 other contact residues. The reductions in affinity of the position 35 mutants for digoxin are greater than expected based upon the small hapten contact area provided by the wild-type Asn. We therefore performed molecular modeling experiments which suggested that substitution of Gln or Asp can maintain these hydrogen bonds whereas the other substituted side chains cannot. The altered binding of the Asp mutant may be due to the introduction of a negative charge. The similarities in binding of the wild-type and Gln-mutant antibodies, however, suggest that these hydrogen bonds are important for maintaining the architecture of the binding site and therefore the affinity and specificity of this antibody. The Ala mutant eliminates the wild-type hydrogen bonding, and molecular modeling suggests that the reduced side-chain volume also provides space that can accommodate a congener with a 16-acetyl group or saturated lactone, accounting for the altered fine specificity of this antibody.

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