title: Construction and characterization of a humanized single chain Fv antibody fragment against the main immunogenic region of the acetylcholine receptor.
abstract: The single chain Fv fragment of mAb198 (scFv198) directed against the main immunogenic region (MIR) of the nicotinic acetylcholine receptor (AChR), can efficiently protect the AChR in muscle cell cultures against the destructive activity of human myasthenic autoantibodies. Humanization of the scFv198 antibody fragment should prove useful for therapeutic application by reducing its immunogenicity. Framework sequences from human immunoglobulins homologous to the rat scFv198 sequences were selected and a totally synthetic humanized scFv198 antibody fragment was constructed in vitro. Humanized VH and VL domains were synthesized using two overlapping sets of 225 bases long oligonucleotides overlap extension and polymerase chain reaction (PCR), then assembled into a full-length gene by overlap extension of single-stranded DNA (ssDNA) fragments and PCR. The initial humanized antibody fragment had a very low affinity for the AChR. Molecular modeling was then performed and four residues from the framework regions (FR) of the humanized VH domain were selected to be replaced by the corresponding amino acid from the rat sequence. Three mutants were constructed by overlap extension, using PCR. The humanized variant containing replacements at VH residues 27, 29, 30 and 71 showed very good recovery of AChR binding activity; its binding affinities for Torpedo or human AChR (K(D): 8.5 or 323 nM, respectively) being only four times lower than those of the parental scFv198 (K(D): 2 or 80 nM, respectively). This variant was able to protect the human AChR against the binding of anti-MIR mAb and anti-alpha autoantibodies from a myasthenic patient. It was also able to protect AChR against antigenic modulation induced by the anti-MIR mAb198.
authors.affiliation: Department of Biochemistry, Hellenic Pasteur Institute, Athens, Greece.
authors.forename: D
authors.initials: D
authors.lastname: Papanastasiou
authors.forename: A
authors.initials: A
authors.lastname: Mamalaki
authors.forename: E
authors.initials: E
authors.lastname: Eliopoulos
authors.forename: K
authors.initials: K
authors.lastname: Poulas
authors.forename: C
authors.initials: C
authors.lastname: Liolitsas
authors.forename: S J
authors.initials: SJ
authors.lastname: Tzartos
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal; D001483:Base Sequence; D001667:Binding, Competitive / Q000276:immunology; D017931:DNA Primers; D000939:Epitopes; D015870:Gene Expression / Q000276:immunology; D006801:Humans; D006825:Hybridomas; D007128:Immunoglobulin Fragments / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D007147:Immunoglobulin Light Chains / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D007135:Immunoglobulin Variable Region / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000737:chemistry / Q000276:immunology; D016296:Mutagenesis / Q000276:immunology; D009157:Myasthenia Gravis / Q000276:immunology*; D016133:Polymerase Chain Reaction; D017433:Protein Structure, Secondary; D051381:Rats; D011917:Rats, Inbred Lew; D011950:Receptors, Cholinergic / Q000276:immunology*; D017386:Sequence Homology, Amino Acid; D014101:Torpedo
chemical_list: D000911:Antibodies, Monoclonal; D017931:DNA Primers; D000939:Epitopes; D007128:Immunoglobulin Fragments; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; D011950:Receptors, Cholinergic; C055966:immunoglobulin Fv