Thrombolytic and pharmacokinetic properties of a conjugate of recombinant single-chain urokinase-type plasminogen activator with a monoclonal antibody specific for cross-linked fibrin in a baboon venous thrombosis model.

Chemical conjugates between recombinant single-chain urokinase-type plasminogen activator (rscu-PA) and a murine monoclonal antibody directed against fragment D-dimer of cross-linked human fibrin (MA-15C5), rscu-PA/MA-15C5, and between rscu-PA and a control monoclonal antibody (MA-1C8), rscu-PA/MA-1C8, were produced by cross-linking with N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP). In an in vitro system composed of a [125 I]fibrin-labeled baboon plasma clot immersed in autologous citrated plasma, dose- and time-dependent lysis was obtained with a ratio of the potencies of free and conjugated rscu-PA similar to that in human plasma: 50% lysis in 2 hours required 4.3 micrograms/ml rscu-PA, 1.0 microgram/ml urokinase-type plasminogen activator (u-PA) equivalent rscu-PA/MA-15C5, or 15 micrograms/ml u-PA equivalent rscu-PA/MA-1C8. The thrombolytic and pharmacokinetic properties of rscu-PA and of rscu-PA/MA-15C5 were compared in baboons with a 0.8-1.0 ml [125 I]fibrin-labeled autologous blood clot produced in a femoral vein. Continuous intravenous infusion of these compounds during a 2-hour period resulted in dose- and time-dependent lysis. The thrombolytic potency of rscu-PA/MA-15C5 was 3.0 +/- 0.5 times higher (50% lysis with 0.3 +/- 0.02 mg u-PA equivalent/kg body wt) than that of rscu-PA measured by ex vivo isotope recovery from the femoral vein segment (p less than 0.001) and was 2.7 +/- 0.5 times higher (50% lysis with 0.35 +/- 0.02 mg/kg rscu-PA/MA-15C5) by external radioisotope counting (p less than 0.001). A dose of 0.5 mg/kg of rscu-PA/MA-1C8 was much less active than rscu-PA. After the end of the infusion, u-PA-related antigen disappeared from plasma in a biphasic manner with an initial half-time of 2.7 +/- 0.5 for rscu-PA, 24 +/- 1.2 for rscu-PA/MA-15C5, and 21 +/- 0.5 minutes for rscu-PA/MA-1C8 with corresponding plasma clearances of 340 +/- 40, 20 +/- 3, and 24 +/- 2 ml/min, respectively. In conclusion, the increased thrombolytic potency of rscu-PA/MA-15C5 is the result of a reduction of the thrombolytic potency due to coupling of rscu-PA to the antibody molecule, which is counter-balanced by an enhancement of the thrombolytic potency due to fibrin targeting by the specific idiotype.