CLONING AND EXPRESSION IN ESCHERICHIA-COLI OF A HUMAN GELATINASE B-INHIBITORY SINGLE-CHAIN IMMUNOGLOBULIN VARIABLE FRAGMENT (SCFV)

The murine monoclonal antibody REGA-3G12 selectively and specifically inhibits the activity of human gelatinase B. The cDNA fragments which encode the variable regions of the Light and heavy chains were isolate d by PCR-mediated cloning and sequenced, Single-chain Fv expression co nstructs for Escherichia coli were generated in which c-myc tag sequen ces were encoded, Inducible expression of the scFv and secretion to th e periplasm were obtained with higher fields when the c-myc tag sequen ce was positioned at the amino-terminal side, The inhibitory activity of purified scFv on neutrophil gelatinase B was tested in a gelatin de gradation assay and it was found to possess a similar specific activit y as that of the intact monoclonal antibody and of the pepsin-clipped F(ab')(2) derivative, This shows for the first time that inhibition of soluble enzymes with scFv is possible and opens new perspectives for the treatment of diseases with excessive and detrimental enzyme produc tion in the host. (C) 1997 Federation of European Biochemical Societie s.