Production of a single-chain variable fragment antibody recognizing type III mutant epidermal growth factor receptor

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.