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 isolated by PCR-mediated cloning and sequenced. Single-chain Fv expression constructs for Escherichia coli were generated in which c-myc tag sequences were encoded. Inducible expression of the scFv and secretion to the periplasm were obtained with higher yields when the c-myc tag sequence was positioned at the amino-terminal side. The inhibitory activity of purified scFv on neutrophil gelatinase B was tested in a gelatin degradation assay and it was found to possess a similar specific activity 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 production in the host.
Zhou
N
N
Laboratory of Molecular Immunology, Rega Institute for Medical Research, University of Leuven, Belgium.
Paemen
L
L
Opdenakker
G
G
Froyen
G
G
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics*; D006801:Humans; D007128:Immunoglobulin Fragments / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology*; D007135:Immunoglobulin Variable Region / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology; D020780:Matrix Metalloproteinase 9; D061965:Matrix Metalloproteinase Inhibitors; D051379:Mice; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017421:Sequence Analysis
D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D061965:Matrix Metalloproteinase Inhibitors; D011994:Recombinant Proteins; D020780:Matrix Metalloproteinase 9