title: Production of a single-chain variable fragment antibody recognizing type III mutant epidermal growth factor receptor.
abstract: The type III deletion mutant of the epidermal growth factor receptor (EGFR) is a potential target in diagnostic and therapeutic approaches for those glioblastomas characterized by its expression. We previously raised a mouse monoclonal antibody, 3C10 (IgG2b) specifically recognizing this mutant EGFR. In this study, a single-chain variable fragment (scFv) antibody was produced. Partial determination of its N-terminal amino acid sequence and preparation of adequate primers for variable heavy chain (V(H)) and variable light chain (V(L)) genes were performed to allow cloning by means of reverse transcriptase-polymerase chain reaction. The genes cloned were assembled with a linker, (Gly4Ser)3, and ligated into a bacterial expression vector to express the scFv as cytoplasmic inclusion bodies. After appropriate refolding, the antibody activity of the V(H)-V(L) scFv was examined in an enzyme-linked immunosorbent assay. 3C10 scFv showed a selective reactivity with the mutant peptide, similarly to the parental 3C10 antibody. A mouse transfectant expressing the type III mutant EGFR and a glioblastoma with type III deletion-mutant EGFR were positively stained by immunofluorescence. By Biacore analysis, the affinity (K(A)) of the parental 3C10 for the mutant peptide was 9.7 x 10(7) M(-1), while that of 3C10 scFv was 2.45 - 2.48 x 10(7) M(-1), being approximately 4-fold weaker. The results together suggested that the scFv antibody retained the appropriate structure to recognize a conformational epitope of the mutant receptor, similarly to the parental antibody.
authors.affiliation: Second Department of Pathology, Aichi Medical University School of Medicine, Nagakute, Aichi 480-1195, Japan.
authors.forename: N
authors.initials: N
authors.lastname: Nakayashiki
authors.forename: K
authors.initials: K
authors.lastname: Yoshikawa
authors.forename: K
authors.initials: K
authors.lastname: Nakamura
authors.forename: N
authors.initials: N
authors.lastname: Hanai
authors.forename: K
authors.initials: K
authors.lastname: Okamoto
authors.forename: S
authors.initials: S
authors.lastname: Okamoto
authors.forename: M
authors.initials: M
authors.lastname: Mizuno
authors.forename: T
authors.initials: T
authors.lastname: Wakabayashi
authors.forename: S
authors.initials: S
authors.lastname: Saga
authors.forename: J
authors.initials: J
authors.lastname: Yoshida
authors.forename: T
authors.initials: T
authors.lastname: Takahashi
mesh_terms: D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D000917:Antibody Formation; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D018076:DNA, Complementary / Q000032:analysis; D066246:ErbB Receptors / Q000235:genetics / Q000276:immunology*; D017353:Gene Deletion; D015870:Gene Expression; D007128:Immunoglobulin Fragments / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D051379:Mice; D008969:Molecular Sequence Data; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology
chemical_list: D000911:Antibodies, Monoclonal; D017931:DNA Primers; D018076:DNA, Complementary; D007128:Immunoglobulin Fragments; D011994:Recombinant Proteins; D066246:ErbB Receptors