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. 2000 Oct;91(10):1035–1043. doi: 10.1111/j.1349-7006.2000.tb00882.x

Production of a Single‐chain Variable Fragment Antibody Recognizing Type III Mutant Epidermal Growth Factor Receptor

Norihisa Nakayashiki 1,2, Kazuhiro Yoshikawa 1,✉, Kazuyasu Nakamura 3, Nobuo Hanai 3, Kenta Okamoto 2, Sho Okamoto 2, Masaaki Mizuno 2, Toshihiko Wakabayashi 2, Sinsuke Saga 1, Jun Yoshida 2, Toshitada Takahashi 4
PMCID: PMC5926257  PMID: 11050475

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 (VH) and variable light chain (VL) 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 VH‐VL 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 (KA) of the parental 3C10 for the mutant peptide was 9.7x107M‐1, while that of 3C10 scFv was 2.45‐2.48x107M‐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.

Keywords: scFv, Epidermal growth factor receptor, Type III deletion mutant, Glioblastoma

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