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

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Abstract
K12G0S32 is a 57‐kDa recombinant single‐chain chimeric plasminogen activator consisting of scFv‐K12G0, a single‐chain variable‐region antigen‐binding fragment (Fv) of the monoclonal anti‐body MA‐15C5, which is specific for fragment D‐dimer of human cross‐linked fibrin, and a low‐molecular‐mass (33 kDa) urokinase‐type plasminogen activator (u‐PA‐33k) containing amino acids Ala132‐Leu411 (Holvoet, P., Laroche, Y., Lijnen, H. R., Van Cauwenberghe, R., Demarsin, E., Brouwers, E., Matthyssens, G. & Collen D. (1991) J. Biol. Chem. 266, 19717–19724). In addition, the Arg156‐Phe157 thrombin‐cleavage site in the u‐PA moiety of K12G0S32 is removed by substitution of Phe157 with Asp. In the present study, the fibrinolytic potency of K12G0S32, determined in a system composed of a 125I‐fibrin‐labeled human plasma clot submerged in citrated plasma, was found to be only twofold higher than that of intact single‐chain u‐Pa (rscu‐PA), but 17‐fold higher than that of rscu‐PA(M), a variant of rscu‐PA in which the thrombin‐cleavage site was removed by substitution of Phe157 with Asp. The fibrinolytic potency of K12G0S32T, with an intact thrombin‐cleavage site, was 6–15‐fold higher than that of rscu‐PA. Conversion of 1 μM single‐chain K12G0S32 or rscu‐PA(M) into their two‐chain derivatives with plasmin occurred at a rate of 1.0 ± 0.15 nmol · min−1· nmol plasmin−1 and 0.85 ± 0.074 nmol · min−1· nmol plasmin−1, compared to 14 ± 2.3 nmol · min−1· nmol plasmin−1 and 18 ± 2.6 nM · min−1· nmol plasmin−1 for K12G0S32T and rscu‐PA, respectively. Purified fragment D‐dimer of human cross‐linked fibrin inhibited the fibrinolytic potency of single‐chain K12G0S32T, but not of two‐chain K12G0S32T, in a dose‐dependent manner. Furthermore, the fibrinolytic potencies of two‐chain K12G0S32 and K12G0S32T were not significantly higher than those of recombinant two‐chain u‐PA (rtcu‐PA) or of rtcu‐PA(M). These findings suggest that the 50‐fold increase in fibrinolytic potency of K12G0S32T, relative to that of rscu‐PA(M), is due both to targeting of the activator to the clot via the single‐chain Fv fragment (sixfold increase) and to a more efficient conversion of single‐chain K12G0S32T to its two‐chain derivative (eightfold increase). Thus, targeting to clots by means of fibrin‐specific antibodies results in a significant increase of the fibrinolytic potency of single‐chain but not of two‐chain u‐PA. K12G0S45 which contains the scu‐PA kringle domain, and K12G0S54 which contains intact scu‐PA, had a similar fibrinolytic potency as K12G0S32, indicating that the fibrin‐targeting in the Fv domain of K12G0S32 is not hampered by spatial constraints in the molecule.

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