Expression and characterization of a chimeric bispecific antibody against fibrin and against urokinase-type plasminogen activator.
We have produced a chimeric bispecific antibody that has dual specificity of human fibrin and urokinase-type plasminogen activator (u-PA). Complementary DNAs for variable regions of both anti-fibrin and anti-u-PA antibodies were cloned from two murine hybridomas secreting respective antibodies using polymerase chain reaction (PCR) techniques, and joined to cDNAs for human constant regions to form chimeric antibody genes. Both of two expression vectors for chimeric anti-fibrin and chimeric anti-u-PA antibodies were sequentially introduced into Chinese hamster ovary cells, and stable transfectants secreting the chimeric bispecific antibody were obtained. The highest producer transfectant (SULF/C2-30) secreted high level (about 40 micrograms ml-1) of total chimeric IgG and about 2% of the IgG had the bispecific activity of binding with both antigens. The chimeric bispecific antibody was purified by a combination of affinity chromatographies employing antigen-coupled columns and hydroxyapatite high-performance liquid chromatography. The purified chimeric bispecific antibody significantly enhanced the thrombolytic potency of single chain u-PA in an in vitro clot lysis assay as well as the original murine bispecific antibody.
Tada
H
H
DDS Research Laboratories, Pharmaceutical Research Division, Takeda Chemical Industries, Ltd., Osaka, Japan.
Kurokawa
T
T
Seita
T
T
Watanabe
T
T
Iwasa
S
S
D000595:Amino Acid Sequence; D000818:Animals; D018033:Antibodies, Bispecific / Q000096:biosynthesis* / Q000276:immunology / Q000302:isolation & purification; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D005337:Fibrin / Q000276:immunology*; D051379:Mice; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000276:immunology / Q000302:isolation & purification; D014162:Transfection; D014568:Urokinase-Type Plasminogen Activator / Q000276:immunology*
D018033:Antibodies, Bispecific; D011993:Recombinant Fusion Proteins; D005337:Fibrin; D014568:Urokinase-Type Plasminogen Activator