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)

[1]  Sol Sherry,et al.  Thrombolytic Therapy: Current Status , 1988 .

[2]  G. Schuler,et al.  Efficacy of intravenous prourokinase and a combination of prourokinase and urokinase in acute myocardial infarction. , 1988, The American journal of cardiology.

[3]  E. Haber,et al.  Conjugation to an antifibrin monoclonal antibody enhances the fibrinolytic potency of tissue plasminogen activator in vitro. , 1988, Biochemistry.

[4]  E. Haber,et al.  Antibody-enhanced thrombolysis: targeting of tissue plasminogen activator in vivo. , 1987, Proceedings of the National Academy of Sciences of the United States of America.

[5]  E. Haber,et al.  Characterization of an antibody-urokinase conjugate. A plasminogen activator targeted to fibrin. , 1987, The Journal of biological chemistry.

[6]  E. Haber,et al.  Thrombolysis by a fibrin-specific antibody Fab'-urokinase conjugate. , 1987, Journal of molecular and cellular cardiology.

[7]  V. Gurewich,et al.  Pro-urokinase: a study of its stability in plasma and of a mechanism for its selective fibrinolytic effect. , 1986, Blood.

[8]  M. Hoylaerts,et al.  Activation of plasminogen by pro-urokinase. II. Kinetics. , 1986, The Journal of biological chemistry.

[9]  M. Blaber,et al.  Activation of plasminogen by pro-urokinase. I. Mechanism. , 1986, The Journal of biological chemistry.

[10]  E. Haber,et al.  Antibody-directed urokinase: a specific fibrinolytic agent. , 1985, Science.

[11]  P F Davison,et al.  Preparation of bispecific antibodies by chemical recombination of monoclonal immunoglobulin G1 fragments. , 1985, Science.

[12]  D. Collen,et al.  In Vitro Fibrinolytic Activity of Recombinant Tissue-Type Plasminogen Activator in the Plasma of Various Primate Species , 1984, Thrombosis and Haemostasis.

[13]  E. Braunwald,et al.  Thrombolytic therapy. A new strategy for the treatment of acute myocardial infarction (1). , 1984, The New England journal of medicine.

[14]  D. Collen,et al.  Biological and Thrombolytic Properties of Proenzyme and Active Forms of Human Urokinase – I. Fibrinolytic and Fibrinogenolytic Properties in Human Plasma In Vitro of Urokinases Obtained from Human Urine or by Recombinant DNA Technology , 1984, Thrombosis and Haemostasis.

[15]  V. Gurewich,et al.  Effective and fibrin-specific clot lysis by a zymogen precursor form of urokinase (pro-urokinase). A study in vitro and in two animal species. , 1984, The Journal of clinical investigation.

[16]  E. Haber,et al.  Monoclonal antibodies to a synthetic fibrin-like peptide bind to human fibrin but not fibrinogen. , 1983, Science.

[17]  P. Parham.  On the fragmentation of monoclonal IgG1, IgG2a, and IgG2b from BALB/c mice. , 1983, Journal of immunology.

[18]  M. Verstraete,et al.  Thrombolysis with human extrinsic (tissue-type) plasminogen activator in rabbits with experimental jugular vein thrombosis. Effect of molecular form and dose of activator, age of the thrombus, and route of administration. , 1983, The Journal of clinical investigation.

[19]  O. Matsuo,et al.  Comparison of the Relative Fibrinogenolytic, Fibrinolytic and Thrombolytic Properties of Tissue Plasminogen Activator and Urokinase in Vitro , 1981, Thrombosis and Haemostasis.

[20]  O. Matsuo,et al.  Thrombolysis by human tissue plasminogen activator and urokinase in rabbits with experimental pulmonary embolus , 1981, Nature.

[21]  L. Holmberg,et al.  Purification of urokinase by affinity chromatography. , 1976, Biochimica et biophysica acta.

[22]  M. Shipton,et al.  A reporter group delivery system with both absolute and selective specificity for thiol groups and an improved fluorescent probe containing the 7-nitrobenzo-2-oxa-1,3-diazole moiety. , 1975, The Biochemical journal.

[23]  D. Deutsch,et al.  Plasminogen: Purification from Human Plasma by Affinity Chromatography , 1970, Science.

[24]  U. K. Laemmli,et al.  Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 , 1970, Nature.

[25]  A. Clauss,et al.  Gerinnungsphysiologische Schnellmethode zur Bestimmung des Fibrinogens , 1957 .

[26]  O. H. Lowry,et al.  Protein measurement with the Folin phenol reagent. , 1951, The Journal of biological chemistry.