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.
Vandamme
A M
AM
Center for Thrombosis and Vascular Research, University of Leuven, Belgium.
Dewerchin
M
M
Lijnen
H R
HR
Bernar
H
H
Bulens
F
F
Nelles
L
L
Collen
D
D
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics*; D001483:Base Sequence; D015153:Blotting, Western; D016466:CHO Cells; D006224:Cricetinae; D004247:DNA / Q000235:genetics; D004226:Dithioerythritol / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D005337:Fibrin / Q000235:genetics* / Q000378:metabolism; D005341:Fibrinolysin / Q000494:pharmacology; D005822:Genetic Vectors; D006801:Humans; D006868:Hydrolysis; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000235:genetics; D010960:Plasminogen Activators / Q000235:genetics* / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011956:Receptors, Cell Surface / Q000378:metabolism; D018000:Receptors, Peptide; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000378:metabolism; D014568:Urokinase-Type Plasminogen Activator / Q000235:genetics*
D000911:Antibodies, Monoclonal; D010446:Peptide Fragments; D011956:Receptors, Cell Surface; D018000:Receptors, Peptide; D011993:Recombinant Fusion Proteins; C048650:fibrin-fibrinogen degradation products receptor; D004226:Dithioerythritol; D005337:Fibrin; D004247:DNA; D010960:Plasminogen Activators; D005341:Fibrinolysin; D014568:Urokinase-Type Plasminogen Activator