title: Construction and expression of a recombinant antibody-targeted plasminogen activator.
abstract: Covalent linkage of tissue-type plasminogen activator (t-PA) to a monoclonal antibody specific for the fibrin beta chain (anti-fibrin 59D8) results in a thrombolytic agent that is more specific and more potent than t-PA alone. To provide a ready source of this hybrid molecule and to allow tailoring of the active moieties for optimal activity, we have engineered a recombinant version of the 59D8-t-PA conjugate. The rearranged 59D8 heavy chain gene was cloned and combined in the expression vector pSV2gpt with sequence coding for a portion of the gamma 2b constant region and the catalytic beta chain of t-PA. This construct was transfected into heavy chain loss variant cells derived from the 59D8 hybridoma. Recombinant protein was purified by affinity chromatography and analyzed with electrophoretic transfer blots. These revealed a 65-kDa heavy chain-t-PA fusion protein that is secreted in association with the 59D8 light chain in the form of a 170-kDa disulfide-linked dimer. Chromogenic substrate assays showed the fusion protein to have 70% of the peptidolytic activity of native t-PA and to activate plasminogen as efficiently as t-PA. In a competitive binding assay, reconstituted antibody was shown to have a binding profile similar to that of native 59D8. Thus, by recombinant techniques, we have produced a hybrid protein capable of high-affinity fibrin binding and plasminogen activation.
mesh_terms: D000911:Antibodies, Monoclonal; D005337:Fibrin / Q000276:immunology; D005342:Fibrinolysis; D005796:Genes; D005803:Genes, Immunoglobulin; D014644:Genetic Variation; D005822:Genetic Vectors; D007143:Immunoglobulin Heavy Chains; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism*; D010959:Tissue Plasminogen Activator / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D000911:Antibodies, Monoclonal; D007143:Immunoglobulin Heavy Chains; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D005337:Fibrin; D010959:Tissue Plasminogen Activator