Construction and Expression of a Recombinant Antibody-targeted Plasminogen Activator Composed of a Humanized Monoclonal Antibody against Activated Human Platelets and a Single-chain Urokinase.

To obtain a thromblytic agent with high efficacy and specifity, we have engineered a recombinant chimeric plasminogen activator SZ51Hu-scuPA, which was composed of a humanized monoclonal antibody (SZ51Hu) directed against activated human platelets and a single-chain urokinase (scuPA). The cDNA sequence encoding scuPA was fused through 5' end to 3' end of the CH(3) of SZ51Hu heavy-chain sequence in the expression vector alphalys30(alphalys30-SZ51VH/Hu-scuPA) by PCR. This construct was transfected into a murine myeloma line secreting SZ51Hu light chain with Lipofectin reagent and three lines which showed stable integration and high level expression with concentration of 5 mg/L in supernatant, were selected in the end.

Construction and Expression of Mouse-human Chimeric Antibody SZ-51 Specific for Human Activated Platelets.

SZ-51, a murine monoclonal antibody (McAb) specific for alpha-granule membrane protein (GMP-140) on the surface of the activated human platelets, has shown promise for thrombus imaging and thrombolysis. In order to reduce the immunogenicity of the murine McAb SZ-51 in man and to obtain a high level of antibody production, we constructed two chimeras (alpha-Lys17-51BVK/Hu, alpha-Lys30-51VH/Hu) by joining the variable regions gene of mouse antibody to the constant regions gene of human immunoglobulin (Ig)(gamma1,k). Both chimeric genes were cloned into two selectable expression vectors separately, which were co-transfected into a non-Ig secreting murine myeloma cell line SP2/0 with the Lipofectin reagent.