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 recombinant 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, encoding scuPA amino acids 1-411, was inserted in 5' end to 3' end orientation immediately after the CH3 of SZ-51Hu heavy-chain sequence in the expression vector alphaLys30. The resulting construct alphaLys30-SZ51VH/Hu-scuPA was used to transfect into SP2/0 murine myeloma cell line, which was pretransfected with SZ51Hu light chain. The fusion protein SZ51Hu-scuPA was expressed at 5 mg/L in the supernatant of cell culture. The fusion protein purified by affinity chromatography had a molecular weight of 160 kDa with fibrinolytic activity of 39,000 IU/mg and its affinity to activated human platelet 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 consists of a 125I-fibrin-labeled human plasma clot containing different concentrations of human platelets suspended in citrated human plasma. Fifty percent 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 than uPA at the same concentration. The maximal lysis of the clots by SZ51Hu-scuPA 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 clots prepared from fresh platelet-rich human plasma containing 125I-labeled fibrinogen. The thrombolytic activity of SZ51-scuPA was 3.9 times more potent than that of uPA at 2,000 IU/kg in this model. Almost no significant fibrinogen breakdown was observed either in vitro and in vivo.
Wan
H
H
Jiangsu Institute of Haematology, First Affiliated Hospital of Suzhou Medical College, People's Republic of China.
Liu
Z
Z
Xia
X
X
Gu
J
J
Wang
B
B
Liu
X
X
Zhu
M
M
Li
P
P
Ruan
C
C
D000818:Animals; D000911:Antibodies, Monoclonal / Q000627:therapeutic use*; D000915:Antibody Affinity; D001777:Blood Coagulation / Q000187:drug effects; D001792:Blood Platelets / Q000276:immunology*; D006224:Cricetinae; D004195:Disease Models, Animal; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D005337:Fibrin / Q000187:drug effects; D005343:Fibrinolytic Agents / Q000627:therapeutic use; D005822:Genetic Vectors / Q000138:chemical synthesis; D006801:Humans; D007457:Iodine Radioisotopes; D051379:Mice; D019007:P-Selectin / Q000276:immunology; D015539:Platelet Activation; D015202:Protein Engineering; D011655:Pulmonary Embolism / Q000188:drug therapy; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification; D015912:Thrombolytic Therapy; D014407:Tumor Cells, Cultured; D014568:Urokinase-Type Plasminogen Activator / Q000235:genetics / Q000276:immunology
D000911:Antibodies, Monoclonal; D005343:Fibrinolytic Agents; D007457:Iodine Radioisotopes; D019007:P-Selectin; D011993:Recombinant Fusion Proteins; D005337:Fibrin; D014568:Urokinase-Type Plasminogen Activator