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, (Gly 4 Ser) 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 × × × ×10, while that of 3C10 scFv was 2.45-2.48× × × ×10, 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.Key words: scFv -Epidermal growth factor receptor -Type III deletion mutant -GlioblastomaThe epidermal growth factor receptor (EGFR) gene is amplified and overexpressed in about 40% of glioblastoma cases. This amplification is frequently related to structural rearrangement, resulting in in-frame deletion mutations in the extracellular domain. Such deletions in EGFR have been classified into three types, based on size and location.1-7) Type III has been identified in about 17% of glioblastomas, and is characterized by an 801 bp in-frame deletion, which creates a unique sequence with a glycine residue at the fusion junction between amino acid residues 5 and 274. Since the sequence around the fusion junction is expressed only in glioblastoma cells, it is a potential target for diagnostic and therapeutic approaches. Accordingly, several laboratories including our own [8][9][10][11] have attempted to produce mouse monoclonal antibodies with specificity for the type III mutant. Wikstrand et al. 8) and Hills et al. 9) obtained antibodies by immunization with a synthetic peptide, named Pep3, covering the fusion junction of the type III deletion mutant which had been used by Humphrey et al. 10) for production of polyclonal rabbit anti-serum. Using the same peptide, we also succeeded in producing an antibody, 3C10, showing specificity for type III mutant EGFR. 11)With the recent advances in technology involving cloning of immunoglobulin (Ig) genes, generation of recombinant/chimeri...
The type III mutant epidermal growth factor receptor (EGFR) is expressed on the cell surface of a subset of glioma, but not of normal tissues. In this study, we investigated the in vivo kinetics of 3C10 mouse monoclonal antibody (mAb), specifically recognizing the type III mutant EGFR (EGFRvIII), using athymic nude mice bearing the intracranial glioma xenograft overexpressing the EGFRvIII. Human glioma cell line, U87MG, expressing the wild type EGFR and the transfectant, named U87MG x deltaEGFR, expressing the EGFRvIII, were transplanted subcutaneously or intracranially to nude mice. 3C10 mAb labeled with a technetium-99m (99mTc) was intravenously injected into these nude mice and then the mice were sacrificed at 24 h later, and the 99mTc-uptake by xenografts and major normal organs was measured to determine the biodistribution of mAb. Furthermore, at 3, 6 and 24 h after injection of 99mTc-labeled 3C10 mAb, whole-body scintigraphy was obtained with a gamma camera to localize the tumor site. 3C10 mAb significantly accumulated to U87MG x deltaEGFR xenografts transplanted subcutaneously or intracranially in nude mice, showing high tumor-to-blood ratio of 10.30 and 4.01, respectively. In contrast, uptake of control antibody in the intracranial tumor was as low as 0.43. In scintigrams, intracranially transplanted U87MG x deltaEGFR xenografts were detectable at 3 h after injection of 99mTc-labeled 3C10 mAb. These results suggest that intravenously injected 3C10 mAb specifically accumulated to the subcutaneous or intracranial glioma xenograft expressing the EGFRvIII and 3C10 mAb is a potential diagnostic and therapeutic agent for patients with gliomas expressing the EGFRvIII.
Progressive nodular histiocytosis (PNH) is a rare benign self-limiting histiocytic disorder of the skin that is characterised by the progressive appearance of widespread xanthomatous lesions with no spontaneous remission. We operated on a 13-year-old girl with PNH four times with a result that had stabilised the condition at 1 year.
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