Bispecific Monoclonal Antibodies Produced by Somatic Cell Fusion Increase the Potency of Tissue Plasminogen Activator

Summary

Bispecific monoclonal antibodies that bind simultaneously to human fibrin and tissue plasminogen activator (tPA) enhance the fibrinolytic potency of tPA. Two bispecific antibodies (F36.23 and F32.1) were generated by somatic cell fusion. Antibody F36.23 derives its tPA binding from monoclonal anti-tPA antibody TCL8 and its fibrin binding from monoclonal antifibrin antibody 59D8. After purification from cell supernatants and ascites by two steps of affinity chromatography, hybrid-hybridoma bispecific antibody F36.23 simultaneously bound tPA and fibrin in solution and in solid-phase assays. In an assay for the lysis of human fibrin monomer, F36.23 increased the fibrinolytic potency of tPA by 5 to 10 fold, regardless of whether the bispecific antibody had been combined with the tPA before or during the assay. Bispecific F36.23 F(ab′)₂ also bound tPA and fibrin simultaneously, and the enhancement in fibrinolysis in the presence of F36.23 F(ab′)₂ was identical to that in the presence of intact F36.23. The second bispecific antibody, F32.1, was produced by an alternative strategy that has a wider potential for applicaton in other systems. Hybridoma bispecific antibody F32.1 was derived from the fusion of immune splenocytes (in mice immunized with a synthetic oligopeptide representing the amino terminus of the α-chain of human fibrin) with the anti-tPA cell line TCL8. The properties of hybridoma bispecific antibody F32.1 and its F(ab′)₂ were indistinguishable from those of hybrid-hybridoma bispecific antibody F36.23 in solid-phase binding assays and in assays of fibrinolysis. Bispecific antibodies produced by somatic cell fusion, particularly in the form of F(ab′)₂, may have potential for use in clinical thrombolysis.

• References

• 1 Runge MS, Quertermous T, Haber E. Plasminogen activators: The old and the new. Circulation 1989; 79: 217-224
• 2 Marder VJ, Sherry S. Thrombolytic therapy: Current status (parts I and II). N Engl J Med 1988; 318: 1512-1520 and 1585-1595
• 3 Bode C, Matsueda GR, Hui KY, Haber E. Antibody-directed urokinase: A specific fibrinolytic agent. Science 1985; 229: 765-767
• 4 Schnee JM, Runge MS, Matsueda GR, Hudson NW, Seidman JG, Haber E, Quertermous T. Construction and expression of a recombinant antibody-targeted plasminogen activator. Proc Natl Acad Sci USA 1987; 84: 6904-6908
• 5 Runge MS, Huang P, Savard CE, Schnee JM, Love TW, Bode C, Matsueda GR, Haber E, Quertermous T. A recombinant molecule with antifibrin antibody and single-chain urokinase activities has increased fibrinolytic potency. Circulation 1988; 78 (Suppl I) 11-509 (abstract)
• 6 Runge MS, Bode C, Matsueda GR, Haber E. Antibody-enhanced thrombolysis: Targeting of tissue plasminogen activator in vivo. Proc Natl Acad Sci USA 1987; 84: 7659-7662
• 7 Bode C, Runge MS, Branscomb EE, Newell JB, Matsueda GR, Haber E. Antibody-directed fibrinolysis: An antibody specific for both fibrin and tissue plasminogen activator. J Biol Chem 1989; 264: 944-948
• 8 Greenwood FC, Hunter WH, Glover JS. The preparation of ¹³¹I-labelled human growth hormone of high specific radioactivity. Biochem J 1963; 89: 1142-1123
• 9 Hui KY, Haber E, Matsueda GR. Monoclonal antibodies to a synthetic fibrin-like peptide bind to human fibrin but not fibrinogen. Science 1983; 222: 1129-1132
• 10 Köhler G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature (London) 1975; 256: 495-497
• 11 Parham P. On the fragmentation of monoclonal IgG₁, IgG_2a, and IgG_2b from BALB/c mice. J Immunol 1983; 131: 2895-2902
• 12 Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 1970; 227: 680-685
• 13 Collen D, Stassen JM, Verstraete M. 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. J Clin Invest 1983; 71: 368-376
• 14 Clauss A. Gerinnungsphysiologische Schnellmethode zur Bestimmung des Fibrinogens. Acta Haematol 1957; 17: 237-246
• 15 Rampling MW, Gaffney PJ. The sulphite precipitation method for fibrinogen measurement; its use on small samples in the presence of fibrinogen degradation products. Clin Chim Acta 1976; 67: 43-52
• 16 Edy J, De Cock F, Collen D. Inhibition of plasmin by normal and antiplasmin depleted plasma. Thromb Res 1976; 8: 513-518
• 17 Friberger P, Kiros M. Plasminogen determinaton in human plasma. In: Skully MF, Kakker VV. (Eds) Chromogenic Peptide Substrates. Churchill Livingstone; Edinburgh, Scotland: 1979. pp 128-140
• 18 Milstein C, Cuello AC. Hybrid hybridomas and their use in immunohistochemistry. Nature (London) 1983; 305: 537-540
• 19 Staerz UD, Bevan MJ. Hybrid hybridoma producing a bispecific monoclonal antibody that can focus effector T-cell activity. Proc Natl Acad Sci USA 1986; 83: 1453-1457
• 20 Lanzaveccia A, Scheidegger D. The use of hybrid hybridomas to target human cytotoxic T-lymphocytes. Eur J Immunol 1987; 17: 105-111
• 21 Suresh MR, Cuello AC, Milstein C. Bispecific monoclonal antibodies from hybrid hybridomas. Meth Enzymol 1986; 121: 210-228