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 g lioblastomas 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 produ ced. Partial determination of its N-terminal amino acid sequence and prepar ation 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 tran scriptase-polymerase chain reaction. The genes cloned were assembled with a linker, (Gly(4)Ser)(3), and ligated into a bacterial expression vector to express the scFv as cytoplasmic inclusion bodies. After appropriate refoldi ng, the antibody activity of the V-H-V-L scFv was examined in an enzyme-lin ked immunosorbent assay. 3C10 scFv showed a selective reactivity with the m utant peptide, similarly to the parental 3C10 antibody. A mouse transfectan t expressing the type III mutant EGFR and a glioblastoma with type III dele tion-mutant EGFR were positively stained by immunofluorescence, By Biacore analysis, the affinity (K-A) of the parental 3C10 for the mutant peptide wa s 9.7x10(7) M-1, while that of 3C10 scFv was 2.45-2.48x10(7) M-1, being app roximately 4-fold weaker. The results together suggested that the scFv anti body retained the appropriate structure to recognize a conformational epito pe of the mutant receptor, similarly to the parental antibody.