Characterization of a recombinant chimeric plasminogen activator composed of a fibrin fragment-D-dimer-specific humanized monoclonal antibody and a truncated single-chain urokinase.

A recombinant chimeric plasminogen activator, MA-15C5Hu/scu-PA-32k, composed of a humanized fibrin fragment-D-dimer-specific monoclonal antibody (MA-15C5Hu) and a recombinant low-molecular-mass single-chain urokinase-type plasminogen activator, comprising amino acids Leu144-Leu411 (scu-PA-32k), was produced by cotransfecting Chinese hamster ovary (CHO) cells with the cDNA encoding the MA-15C5Hu light-chain sequence and the cDNA encoding the MA-15C5Hu heavy-chain sequence fused with the cDNA encoding scu-PA-32k. Purified MA-15C5Hu/scu-PA-32k migrated as a 215-kDa band on non-reducing SDS/PAGE, which is consistent with a molecule composed of one antibody and two scu-PA-32k moieties. However, the chimera was obtained as a mixture of single-chain u-PA-32k (37%) and amidolytically inactive (50%) and active (13%) two-chain u-PA-32k, the latter of which was removed by immunoadsorption on a monoclonal antibody specific for two-chain urokinase. The fragment-D-dimer affinity and enzymatic properties of MA-15CHu/scu-PA-32k were similar to those of MA-15C5Hu or of scu-PA-32k. In an in vitro system composed of a 125I-fibrin-labeled human plasma clot submerged in citrated human plasma, MA-15C5Hu/scu-PA-32k had a 12-fold higher fibrinolytic potency than scu-PA-32k: 50% lysis in 2 h required 0.43 +/- 0.12 micrograms u-PA-32k equivalent of the chimera/ml versus 5.4 +/- 0.3 micrograms/ml of scu-PA-32k (mean +/- SEM, n = 4). Addition of purified fibrin fragment-D dimer reduced the fibrinolytic potency of MA-15C5Hu/scu-PA-32k in a concentration-dependent way, indicating that the increased potency is the result of antibody targeting. Thus, a recombinant humanized antifibrin antibody/u-PA chimera has been obtained in which only the variable domains of the antibody moiety are of non-human origin. The chimera has intact antigen-binding capacity, u-PA enzymatic activity and a significantly increased fibrinolytic potency in a plasma medium in vitro.