Pharmacokinetic and thrombolytic properties of chimeric plasminogen activators consisting of a single-chain Fv fragment of a fibrin-specific antibody fused to single-chain urokinase.
The pharmacokinetic and thrombolytic properties were determined of two recombinant single-chain chimeric plasminogen activators (PA) consisting of u-PA-33k, a low-molecular weight derivative of single-chain urokinase-type PA (scu-PA) comprising amino acids Ala132 through Leu411, and of either a single-chain variable region fragment (Fv) derived from the fibrin fragment D-dimer-specific monoclonal antibody MA-15C5 (K12G0S32) or of the deglycosylated single-chain Fv fragment obtained by substitution of Asn88 with Glu (K12G2S32). Following bolus injection in hamsters, clearances of recombinant scu-PA (rscu-PA) and of K12G0S32 were similar. In contrast, clearance of K12G2S32 was fourfold slower than that of rscu-PA. The thrombolytic potency (percent lysis per u-PA administered in milligrams per kilogram body weight) and specific thrombolytic activity (percent lysis per microgram per milliliter steady-state plasma u-PA antigen level) of these compounds were studied in hamsters with an experimental pulmonary embolus consisting of a human plasma clot injected via the jugular vein. The doses of K12G0S32 and K12G2S32 required to obtain maximal rate of clot lysis were sixfold and 11-fold lower than that of rscu-PA. The steady-state u-PA-related plasma antigen levels of K12G0S32 and K12G2S32 required to obtain maximal rate of clot lysis were 10-fold and fourfold lower than that of rscu-PA. Thus, targeting of K12G0S32 to the clot surface by means of its glycosylated Fv fragment results in a 10-fold increase of its specific thrombolytic activity and sixfold increase of its thrombolytic potency as compared with those of rscu-PA. Targeting of K12G2S32 to the clot surface by means of its deglycosylated Fv fragment results in only a twofold increase of its thrombolytic activity. However, its fourfold slower clearance, combined with its twofold higher specific thrombolytic activity, results in an 11-fold increase of its thrombolytic potency over that of rscu-PA. These findings indicate that the thrombolytic potency of chimeric antibody-targeted PA may be increased by increasing the specific thrombolytic activity, reducing the clearance, or both.
Holvoet
P
P
Center for Thrombosis and Vascular Research, University of Leuven, Belgium.
Laroche
Y
Y
Stassen
J M
JM
Lijnen
H R
HR
Van Hoef
B
B
De Cock
F
F
Van Houtven
A
A
Gansemans
Y
Y
Matthyssens
G
G
Collen
D
D
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism / Q000627:therapeutic use; D001483:Base Sequence; D002460:Cell Line; D006224:Cricetinae; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D005337:Fibrin / Q000276:immunology*; D005343:Fibrinolytic Agents / Q000493:pharmacokinetics* / Q000627:therapeutic use*; D006801:Humans; D007313:Insecta; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D011655:Pulmonary Embolism / Q000188:drug therapy*; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000493:pharmacokinetics* / Q000627:therapeutic use*; D011994:Recombinant Proteins / Q000493:pharmacokinetics / Q000627:therapeutic use; D014568:Urokinase-Type Plasminogen Activator / Q000235:genetics / Q000493:pharmacokinetics* / Q000494:pharmacology*
D000911:Antibodies, Monoclonal; D005343:Fibrinolytic Agents; D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D005337:Fibrin; D004247:DNA; D014568:Urokinase-Type Plasminogen Activator