Monoclonal antibody against the fusion junction of a deletion-mutant epidermal growth factor receptor

Okamoto, Sho; Yoshikawa, Kazuhiro; Obata, Yuichi; Shibuya, Masabumi; Aoki, Seishiro; Yoshida, Jun; Takahashi, Toshitada

doi:10.1038/bjc.1996.260

Cited by 33 publications

(22 citation statements)

References 20 publications

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“…Although a wide variety of antibodies to the wtEGFR are available (19,20), only a few reagents to its mutant form have been generated (11,(21)(22)(23). Some of the currently available anti-⌬EGFR reagents are polyclonal antibodies (11,21,22), but these are not useful for therapeutic applications.…”

Section: Figmentioning

confidence: 99%

A monoclonal antibody recognizing human cancers with amplification/overexpression of the human epidermal growth factor receptor

Jungbluth

Stockert

Huang

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

159

130

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CorrectionsArai, and Glenn D. Prestwich, which appeared in number 1, January 7, 2003, of Proc. Natl. Acad. Sci. USA (100, 131-136; First Published December 26, 2002; 10.1073͞pnas.0135855100), Fig. 4 should have appeared in color. The correct figure and its legend appear below. Fig. 4.LPA stimulates lipid accumulation, CD36 expression, and oxidized LDL uptake through a PPAR-responsive element. (a) LPA stimulates monocyte uptake of oxidized LDL. Freshly elutriated human monocytes were allowed to interact with an anti-ICAM3-coated well, which leads to rapid PPAR␥ expression (13), and then stimulated, or not (negative, oxLDL), with oleoyl LPA. Some cells were then briefly exposed to oxidized LDL before intracellular lipid stores were visualized with oil red O stain. (b) LPA increases the expression of CD36 on the surface of primary human monocytes. Monocytes engaging anti-ICAM3 were treated or not with LPA, and then recovered by gentle scraping and washing by centrifugation before their surface CD36 was assessed by flow cytometry. (c) LPA and the LPA analogs XY4 and XY8 stimulate CD36 promoter function only when the PPRE is present. RAW264.7 cells were transfected with the human CD36 promoter containing the PPRE (CD36 Ϫ273 ) or a reporter that lacks only this element (CD36 Ϫ261 ) and then stimulated with oleoyl LPA, azPC, XY4, or XY8. Expression of luciferase normalized to ␤-galactosidase was determined as above. (d) Anti-CD36 blocks LPA-stimulated accumulation of cholesterol from oxidized LDL. Freshly isolated human monocytes were treated as in a, but after being preincubated with a blocking anti-CD36 antibody before exposure to oxidized LDL. G rowth factor receptors are promising targets for antibodybased cancer therapies. Because of their cell-surface location, they are readily accessible, and therapeutic antibodies can exert their inhibitory effects by either interfering with cellular signaling or targeting toxic molecules or biological effectors to the tumor site (1).The epidermal growth factor receptor (EGFR) is expressed in normal tissues and neoplastic lesions of most organs, and its expression level has been associated with biological characteristics of tumors (2). Elevated levels of EGFR have been demonstrated in many different types of cancer including glioblastoma, and EGFR overexpression seems to be associated with poor prognosis in several neoplasms (3). EGFR overexpression is often associated with gene amplification (4-8). In glioblastoma, EGFR amplification has been shown to be accompanied by gene rearrangement (9-11), frequently with deletions in the coding region. Several mutant forms have been found (12, 13), and among these the most common mutation is the ⌬2-7 deletion (⌬EGFR), which lacks exons 2-7 of the external EGFR domain, resulting in the loss of an 801-bp fragment of the wild-type (wt) gene (14). Several studies have indicated that the presence of ⌬EGFR enhances the tumorigenic behavior of cancer cells (15-17). ⌬EGFR has only been found in neoplastic lesions and not in any normal tissue. ...

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Section: Figmentioning

confidence: 99%

A monoclonal antibody recognizing human cancers with amplification/overexpression of the human epidermal growth factor receptor

Jungbluth

Stockert

Huang

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

159

130

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“…4), similarly to the parental 3C10 whole antibody. 11) In order to obtain the same degree of reactivity with Pep3, the 3C10 scFv required about 10 times the concentration of whole 3C10 antibody. However, this does not necessarily mean that 3C10 scFv has one-tenth of the binding activity, because the binding of the second antibody must be taken into account.…”

Section: Resultsmentioning

confidence: 99%

“…Serological assays An enzyme-linked immunosorbent assay (ELISA), a mixed hemadsorption assay (MHA), fluorescence-activated cell sorter (FACS) analysis and immunohistological staining were employed to detect antigens with 3C10 scFv and parental whole antibodies using the methods described previously. 11) In the immunostaining analysis, Ab-1 antibody which reacts with intact EGFR (Oncogene Science Diagnostic, Cambridge, MA) was used as the positive control. In the case of scFv, a mouse antiHis-tag antibody (RGS⋅His Antibody, QIAGEN, San Diego, CA) was used as the second antibody after incubation of scFv with target peptides or cells, followed by further reaction with peroxidase-or fluorescein isothiocyanate (FITC)-conjugated anti-mouse Ig or MHA indicator cells.…”

Section: Methodsmentioning

confidence: 99%

“…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/chimeric Ig genes, and their expression in a variety of systems, clinical application of a variety of antibody molecules appears feasible. One advance has been the development of a recombinant single-chain variable fragment (scFv) antibody composed of a variable heavy chain (V H ) amino acid sequence tethered to a variable light chain (V L ) sequence by a designed peptide which links the carboxyl terminus of the V H to the amino terminus of the V L or vice versa.…”

mentioning

confidence: 99%

“…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.…”

mentioning

confidence: 99%

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

Nakayashiki

Yoshikawa

Nakamura

et al. 2000

Japanese Journal of Cancer Research

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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...

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Novel monoclonal antibody specific for the de2‐7 epidermal growth factor receptor (EGFR) that also recognizes the EGFR expressed in cells containing amplification of the EGFR gene

Johns

Stockert

Ritter

et al. 2002

Intl Journal of Cancer

118

139

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In some respects, the EGFR appears to be an attractive target for tumor-targeted antibody therapy: it is overexpressed in many types of epithelial tumor and inhibition of signaling often induces an anti-tumor effect. The use of EGFR specific antibodies, however, may be limited by uptake in organs that have high endogenous levels of the wild type EGFR such as the liver. The de2-7 EGFR (or EGFRvIII) is a naturally occurring extracellular truncation of the EGFR found in a number of tumor types including glioma, breast, lung and prostate. Antibodies directed to this tumor specific variant of the EGFR provide an alternative targeting strategy, although the lower proportion of tumors that express the de2-7 EGFR restricts this approach. We describe a novel monoclonal antibody (MAb 806 The delta 2-7 epidermal growth factor receptor (de2-7 EGFR) is a naturally occurring extracellular truncation of the EGFR found in a number of tumor types including glioma, breast, lung and prostate. [1][2][3][4] In adults, the expression of the de2-7 EGFR appears restricted to tumors exhibiting amplification of the EGFR gene. 5 Prostate cancer maybe an exception to this observation, as the expression level of de2-7 and wild type EGFR appear to be inversely related in this tumor type. 2 The rearrangement seen in the de2-7 EGFR results in an in-frame mature mRNA lacking 801 nucleotides spanning exons 2-7. 6 -9 The corresponding EGFR protein contains a 267 amino acid deletion and a novel glycine residue at the fusion junction. 9 Although this truncated receptor does not bind ligand, it does possess low constitutive activity and imparts a significant growth advantage to glioma cells grown as tumor xenografts in nude mice 10 and is able to transform NIH3T3 cells. 11 The cellular mechanisms utilized by the de2-7 EGFR in glioma cells are not fully defined but include a decrease in apoptosis 12 and a small enhancement of proliferation. 12 The aberrant splicing event that generates the de2-7 EGFR leads to the formation of an unique junctional peptide in the N-terminal of the extracellular domain. 13 As this peptide sequence is not found in the wild type EGFR, its expression is restricted to tumor cells making it an attractive target for antibody therapy. Indeed, the production of both polyclonal 14 and monoclonal 3,15,16 antibodies specific to this unique peptide have been described. One potential drawback in targeting of the de2-7 EGFR is that it is only expressed in a portion of tumors containing EGFR gene amplification. Expression of the de2-7 EGFR has been analyzed in glioma by a number of groups with most reporting that approximately 30% of gliomas express the mutant receptor 13 with expression being lowest in anaplastic astrocytomas and highest in glioblastoma multiforme. 17 The proportion of positive cells within de2-7 EGFR expressing gliomas has been reported to range from 37-86%. 1 A very recent report found that all high-grade prostate carcinomas expressed the de2-7 EGFR 2 although this observation has not been confirmed. Expression in ...

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Monoclonal antibody against the fusion junction of a deletion-mutant epidermal growth factor receptor

Cited by 33 publications

References 20 publications

A monoclonal antibody recognizing human cancers with amplification/overexpression of the human epidermal growth factor receptor

A monoclonal antibody recognizing human cancers with amplification/overexpression of the human epidermal growth factor receptor

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

Novel monoclonal antibody specific for the de2‐7 epidermal growth factor receptor (EGFR) that also recognizes the EGFR expressed in cells containing amplification of the EGFR gene

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