A recombinant antibody-targeted plasminogen activator with high affinity for activated platelets increases thrombolytic potency in vitro and in vivo

To increase thrombolytic specificity of urokinase (uPA), we engineered a re combinant chimeric plasminogen activator SZ51Hu-scuPA, which consists of a humanized monoclonal antibody (SZ-51Hu) specifically against P-selectin on activated human platelet and a single-chain urokinase (scuPA). The cDNA, en coding scuPA amino acids 1-411, was inserted in 5' end to 3' end orientatio n immediately after the CH3 of SZ-51Hu heavy-chain sequence in the expressi on vector alpha Lys30. The resulting construct alpha Lys30-SZ51VH/Hu -scuPA was used to transfect into SP2/0 murine myeloma cell line, which was pretr ansfected with SZ51Hu light chain. The fusion protein SZ51Hu-scuPA was expr essed at 5 mg/L in the supernatant of cell culture. The fusion protein puri fied by affinity chromatography had a molecular weight of 160 kDa with fibr inolytic activity of 39000 IU/mg and its affinity to activated human platel et was 67% of the parent murine mAb SZ-51, The thrombolytic property of the fusion protein was first characterized in an in vitro system, which consis ts of a I-125-fibrin-labeled human plasma clot containing different concent rations of human platelets suspended in citrated human plasma. Fifty percen t lysis was reached with SZ51Hu-scuPA in 1 hour at a concentration of 20 IU /mL or in 2 hours at a concentration of 10 IU/mL, which was much faster tha n uPA at the same concentration. The maximal lysis of the clots by SZ51Hu-s cuPA was 4.1 to 8.4 times more potent than that by uPA. The fusion protein was further characterized in the hamster pulmonary embolism model with clot s prepared from fresh platelet-rich human plasma containing I-125-labeled f ibrinogen. The thrombolytic activity of SZ51-scuPA was 3.9 times more poten t than that of uPA at 2000 IU/kg in this model, Almost no significant fibri nogen breakdown was observed either in vitro and in vivo. (C) 2000 Elsevier Science Ltd. All rights reserved.