title: Two amino acid mutations in an anti-human CD3 single chain Fv antibody fragment that affect the yield on bacterial secretion but not the affinity.
abstract: Recombinant antibody fragments directed against cell surface antigens have facilitated the development of novel therapeutic agents. As a first step in the creation of cytotoxic immunoconjugates, we constructed a single-chain Fv fragment derived from the murine hybridoma OKT3, that recognizes an epitope on the epsilon-subunit of the human CD3 complex. Two amino acid residues were identified that are critical for the high level production of this scFv in Escherichia coli. First, the substitution of glutamic acid encoded by a PCR primer at position 6 of VH framework 1 by glutamine led to a more than a 30-fold increase in the production of soluble scFv. Second, the substitution of cysteine by a serine in the middle of CDR-H3 additionally doubled the yield of soluble antibody fragment without any adverse effect on its affinity for the CD3 antigen. The double mutant scFv (Q,S) proved to be very stable in vitro: no loss of activity was observed after storage for 1 month at 4 degrees C, while the activity of scFv containing a cysteine residue in CDR-H3 decreased by more than half. The results of production yield, affinity, stability measurements and analysis of three-dimensional models of the structure suggest that the sixth amino acid influences the correct folding of the VH domain, presumably by affecting a folding intermediate, but has no effect on antigen binding.
authors.affiliation: Department of Molecular Immunology, German Cancer Research Center (DKFZ), Heidelberg, Germany.
authors.forename: S M
authors.initials: SM
authors.lastname: Kipriyanov
authors.forename: G
authors.initials: G
authors.lastname: Moldenhauer
authors.forename: A C
authors.initials: AC
authors.lastname: Martin
authors.forename: O A
authors.initials: OA
authors.lastname: Kupriyanova
authors.forename: M
authors.initials: M
authors.lastname: Little
mesh_terms: D000595:Amino Acid Sequence; D004926:Escherichia coli; D006801:Humans; D007128:Immunoglobulin Fragments / Q000737:chemistry* / Q000235:genetics; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry / Q000235:genetics; D007135:Immunoglobulin Variable Region / Q000737:chemistry / Q000235:genetics; D019169:Jurkat Cells; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016853:Muromonab-CD3 / Q000737:chemistry* / Q000235:genetics; D009154:Mutation; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D007128:Immunoglobulin Fragments; D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region; D016853:Muromonab-CD3; D011994:Recombinant Proteins; C055966:immunoglobulin Fv