title: Mammalian expression and secretion of functional single-chain Fv molecules.
abstract: Single-chain Fv (sFv) proteins are genetically engineered molecules that consist of the two variable domains of an antibody connected by a polypeptide linker; they contain the antigen binding function of the parental protein in a single 30-kDa polypeptide chain. sFvs are usually produced in bacteria where they are insoluble and therefore require extensive refolding in vitro. In this report we followed the processing of three antibody sFvs (145-2C11 directed against murine CD3 epsilon chain, OKT9 against the human transferrin receptor, and U7.6 against dinitrophenyl groups) by transfected mammalian (COS-7) cells to determine whether the mammalian protein folding machinery can produce and secrete active sFv with high efficiency. The sFvs contained an immunoglobulin light chain leader sequence, which directed them to the endoplasmic reticulum and allowed secretion into the medium. We found that the sFvs were secreted at different rates, with the rate-limiting step of secretion being their exit from the endoplasmic reticulum. We increased the secretion rate of one of the sFvs by introducing an asparagine-linked glycosylation site in FR1 of the heavy chain, and by using tunicamycin (an inhibitor of glycosylation) we found that glycosylated antibody sFvs were secreted faster than their nonglycosylated counterparts. All secreted sFvs specifically bound their antigens; where tested, at least 90% of the secreted sFv was functional. Therefore, mammalian cells can effectively fold and secrete sFv antibody and can provide a convenient system for testing and producing sFv proteins.
authors.affiliation: Experimental Immunology Branch, NCI, National Institutes of Health, Bethesda, Maryland 20892.
authors.forename: C R
authors.initials: CR
authors.lastname: Jost
authors.forename: I
authors.initials: I
authors.lastname: Kurucz
authors.forename: C M
authors.initials: CM
authors.lastname: Jacobus
authors.forename: J A
authors.initials: JA
authors.lastname: Titus
authors.forename: A J
authors.initials: AJ
authors.lastname: George
authors.forename: D M
authors.initials: DM
authors.lastname: Segal
mesh_terms: D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D004721:Endoplasmic Reticulum / Q000378:metabolism; D006031:Glycosylation; D000882:Haplorhini; D006801:Humans; D007128:Immunoglobulin Fragments / Q000096:biosynthesis* / Q000378:metabolism; D007135:Immunoglobulin Variable Region / Q000096:biosynthesis*; D051379:Mice; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000378:metabolism
chemical_list: D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D011994:Recombinant Proteins; C055966:immunoglobulin Fv