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, (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...

Lenalidomide enhances the function of chimeric antigen receptor T cells against the epidermal growth factor receptor variant III by enhancing immune synapses

“…16,17 Our group generated the mouse monoclonal antibody 3C10 and a recombinant single-chain variable fragment (scFv) antibody that specifically recognizes the de novo glycine residue of EGFRvIII. 18,19 Further, we constructed human T cells that expressed chimeric antigen receptor (CAR) targeting the EGFRvIII antigen (3C10-CAR). 20,21 CARs are genetically constructed from scFvs connected by a transmembrane hinge region, the T-cell signaling complex, and costimulatory signaling domains (e.g.…”

Recognition of Glioma Stem Cells by Genetically Modified T Cells Targeting EGFRvIII and Development of Adoptive Cell Therapy for Glioma

“…Based on a review of scientific literature and publicly available databases, the amino acid sequences for seven monoclonal antibodies (mAbs) (four murine and three human) specific for EGFRvIII were obtained and used to synthesize single-chain variable fragment (scFv) genes (Wikstrand et al, 1995;Lorimer et al, 1996;Reist et al, 1997;Nakayashiki et al, 2000;Weber, 2005). The amino acid sequences for variable light and heavy chains (VL and VH, respectively) of the antibodies were combined in the following order: amino acid leader sequence (from the human GM-CSFR protein), VL, a peptide linker peptide sequence (GSTSGSGKPGSGEGS), and VH, which were reverse-translated and produced as a codon-optimized synthetic DNA (Invitrogen).…”

Phage Display Cloning and Characterization of Monoclonal Antibody Genes and Recombinant Fab Fragment against the CD98 Oncoprotein

“…Recently, phage display cloning has become a general strategy for preparing recombinant antibodies originating from humans, [16][17][18][19][20][21][22][23] mice, 24,25) and other species. 29,30) Antibody genes are expressed as the Fab or single chain Fv (scFv) form using suitable vectors and cloning systems.…”

“…bone marrow cells, lymph node cells, peripheral blood lymphocytes, spleen cells, and hybridoma cells. It has been reported that recombinant human mAb fragments against viral pathogens [16][17][18][19] and autoantigens, [20][21][22][23] and recombinant murine mAb fragments against various tumor-associated antigens 24,25) were successfully isolated by means of the phage display system. In this study, among the anti-CD98 h.c. mAbs, we selected HBJ127 5,7,8) as a starting material because it showed not only antigen binding activity, but also characteristic bioactivity including an inhibitory effect on cell growth 7,9,10) and lymphocyte proliferation.…”