CONSTRUCTION AND EXPRESSION OF MOUSE-HUMAN CHIMERIC ANTIBODY SZ-51 SPECIFIC FOR ACTIVATED PLATELET P-SELECTIN

A murine monoclonal (mAb) SZ-51 specific for human P-selectin may be u sed for in vivo thrombus imaging and for the targeting of fibrinolytic agents to thrombi. In order to reduce the immunogenicity of the murin e mAb SZ-51 in humans, we cloned and sequenced the cDNAs encoding the variable region of mAb SZ-51 in order to develop mouse/human chimeric reagents. The E, coli expression vector pHEN1-SZ51Fab/Hu was construct ed by fusing the variable regions of mAb SZ-51 with human IgG gamma 1C H1 and CK genes. The constructs were introduced into E. coli HB2151 fo r expression of soluble chimeric Fab fragment. We also constructed two fusion products by joining the variable regions of mouse antibody to the appropriate constant regions of human Ig gamma 1 and K. These chim eras were cloned into two eukaryotic selectable expression vectors sep arately which were then cotransfected into a non-Ig secreting murine m yeloma line SP2/0 with lipofectin reagent. Six cell lines remained pos itive for Ig secretion. The highest producing cell line, which showed stable integration and expression at 5 mg/l of culture, was selected f or the large scale production of chimeric antibody. Immunoblotting ana lysis demonstrated that both of the chimeric antibodies (SZ51Fab/Hu, S Z51/Hu) in the culture supernatants, like the native mAb SZ-51, bind P -selectin. In addition, the whole chimeric antibody can compete for bi nding to activated platelets with murine SZ-51. Therefore, the SZ-51 c himeric antibody may be a potential agent for diagnosis and treatment of thrombotic diseases in the future.