Conjugation to antifibrin Fab' enhances fibrinolytic potency of single-chain urokinase plasminogen activator.
Single-chain urokinase plasminogen activator (scu-PA) that had been modified with N-succinimidyl-3-(2-pyridyldithio)propionate was covalently linked by disulfide bonds to the Fab' of a monoclonal antibody specific for the beta-chain of fibrin (antibody 59D8). scu-PA-59D8 Fab' conjugate was separated from free scu-PA and two-chain urokinase coupled to 59D8 Fab' by two-step affinity chromatography. First, the reaction mixture was chromatographed on a column containing Sepharose linked to the peptide that had been used as immunogen for antibody 59D8; scu-PA-59D8 Fab' conjugate and unconjugated 59D8 Fab' were retained but not unconjugated scu-PA. Then, the eluate from the peptide-Sepharose column was chromatographed on a column containing Sepharose linked to benzamidine, which acts as a ligand for two-chain urokinase. The molecular weight of the scu-PA-59D8 Fab' conjugate was approximately 100 kDa when electrophoresed on a nonreducing sodium dodecylsulfate-polyacrylamide gel. Enzymatic assay after purification revealed that more than 97% of the scu-PA present in the conjugate retained the single-chain form. The Fab' portion of the conjugate functioned in a manner indistinguishable from that of native antibody 59D8. In an in vitro assay for lysis of fibrin monomer, the fibrinolytic potency of scu-PA-59D8 Fab' was 33-fold more than that of tissue plasminogen activator (p less than 0.001), 230-fold more than that of unconjugated scu-PA (p less than 0.0001), and 420-fold more than that of urokinase (p less than 0.0001).(ABSTRACT TRUNCATED AT 250 WORDS)
Bode
C
C
Medizinische Klinik III (Kardiologie), Universität Heidelberg, FRG.
Runge
M S
MS
Schönermark
S
S
Eberle
T
T
Newell
J B
JB
Kübler
W
W
Haber
E
E
D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology*; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D005337:Fibrin / Q000494:pharmacology; D005340:Fibrinogen / Q000378:metabolism; D005343:Fibrinolytic Agents / Q000138:chemical synthesis*; D008970:Molecular Weight; D010960:Plasminogen Activators / Q000494:pharmacology*; D011817:Rabbits; D013388:Succinimides / Q000494:pharmacology*; D010959:Tissue Plasminogen Activator / Q000494:pharmacology; D014568:Urokinase-Type Plasminogen Activator / Q000494:pharmacology*
D000911:Antibodies, Monoclonal; D005343:Fibrinolytic Agents; D013388:Succinimides; C018151:N-succinimidyl 3-(2-pyridyldithio)propionate; D005337:Fibrin; D005340:Fibrinogen; D010960:Plasminogen Activators; D010959:Tissue Plasminogen Activator; D014568:Urokinase-Type Plasminogen Activator