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

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Abstract
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‐15C5Hu/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 μg u‐PA‐32k equivalent of the chimera/ml versus 5.4 ± 0.3 μg/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.

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