Characterization of an antibody-urokinase conjugate. A plasminogen activator targeted to fibrin.
The plasminogen activator urokinase was linked covalently to a monoclonal antibody specific for the amino terminus of the beta chain of human fibrin by means of the unidirectional cross-linking reagent N-succinimidyl-3-(2-pyridyldithio)propionate. N-Succinimidyl-3-(2-pyridyldithio)propionate allowed the amino groups on urokinase to be coupled to the sulfhydryl groups on iminothiolane (which had been introduced into the antibody before the coupling reaction). The inter-heavy chain sulfhydryl of the Fab' of this antibody was also linked to N-succinimidyl-3-(2-pyridyldithio)propionate-substituted urokinase. The antibody- or Fab'-urokinase complexes were purified by two affinity chromatography steps. In the first, benzamidine was used as ligand for urokinase, in the second, a heptapeptide consisting of the 7 amino-terminal residues of the beta chain of fibrin (beta peptide) was used as ligand for the antibody. The activity of the purified conjugates was compared with that of urokinase alone in an assay measuring lysis of 125I-fibrin monomer covalently linked to Sepharose CL-4B. For any concentration of either urokinase alone or urokinase-antifibrin antibody conjugate, an equivalent amount of lysis (release of labeled peptide from fibrin monomer-Sepharose) was obtained with 1/250 the concentration (with respect to urokinase content) of antifibrin antibody-urokinase conjugate. The antifibrin Fab'-urokinase conjugate exhibited a similar enhancement of activity in comparison with urokinase. Enhanced fibrinolysis was fully inhibited by beta peptide. These results suggest that antibody targeting enhances the concentration of urokinase in the vicinity of immobilized fibrin monomer, thereby also increasing the local conversion of plasminogen to plasmin, which in turn degrades its substrate, fibrin. Univalent antigen-antibody binding is sufficient for optimal efficiency.
D000911:Antibodies, Monoclonal; D055598:Chemical Phenomena; D002621:Chemistry; D002846:Chromatography, Affinity; D003432:Cross-Linking Reagents; D005337:Fibrin / Q000276:immunology* / Q000378:metabolism; D005342:Fibrinolysis; D006801:Humans; D007140:Immunoglobulin Fab Fragments; D013388:Succinimides; D014568:Urokinase-Type Plasminogen Activator / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D003432:Cross-Linking Reagents; D007140:Immunoglobulin Fab Fragments; D013388:Succinimides; C018151:N-succinimidyl 3-(2-pyridyldithio)propionate; D005337:Fibrin; D014568:Urokinase-Type Plasminogen Activator