Hy. Wan et al., A recombinant antibody-targeted plasminogen activator with high affinity for activated platelets increases thrombolytic potency in vitro and in vivo, THROMB RES, 97(3), 2000, pp. 133-141
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
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