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 specific ity of human fibrin and urokinase-type plasminogen activator (u-PA). C omplementary DNAs for variable regions of both anti-fibrin and anti-u- PA antibodies were cloned from two murine hybridomas secreting respect ive antibodies using polymerase chain reaction (PCR) techniques, and j oined to cDNAs for human constant regions to form chimeric antibody ge nes. Both of two expression vectors for chimeric anti-fibrin and chime ric anti-u-PA antibodies were sequentially introduced into Chinese ham ster ovary cells, and stable transfectants secreting the chimeric bisp ecific antibody were obtained. The highest producer transfectant (SULF /C2-30) secreted high level (about 40 mug ml-1) of total chimeric IgG and about 2% of the IgG had the bispecific activity of binding with bo th antigens. The chimeric bispecific antibody was purified by a combin ation of affinity chromatographies employing antigen-coupled columns a nd 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 a s the original murine bispecific antibody.