Anti-synthetic peptide antibody reacting at the fusion junction of deletion-mutant epidermal growth factor receptors in human glioblastoma.

Abstract

We have investigated human gliomas that amplify and rearrange the epidermal growth factor receptor gene, with generation of an in-frame deletion mutation of 802 nucleotides in the external domain. This in-frame deletion mutation generates a local amino acid sequence at the fusion junction of what normally were distant polypeptide sequences in the intact epidermal growth factor receptor. This 14-amino acid peptide was chemically synthesized, coupled to keyhole limpet hemocyanin, and used as an immunogen in rabbits. The elicited antibody reacted specifically with the fusion peptide in ELISA. The anti-fusion junction peptide antibody was purified by passage of the antiserum over a peptide affinity column with acidic elution. The purified antibody selectively bound the glioma deletion mutant as compared to the intact epidermal growth factor receptor as assessed by immunocytochemistry, immunofluorescence, immunoprecipitation with gel electrophoresis, and binding experiments using radioiodinated antibody. These data indicate that it is feasible to generate sitespecific anti-peptide antibodies that are highly selective for mutant proteins in human tumors. The anti-peptide antibody described here, and other mutation site-specific antibodies, should be ideal candidates for tumor immunoimaging and immunotherapy.

A phase I clinical trial with monoclonal antibody ch806 targeting transitional state and mutant epidermal growth factor receptors

“…The most common EGFR mutation is an extracellular truncation of the EGFR known as the de2-7 EGFR (or EGFRvIII), which is frequently expressed in glioblastoma and possibly some other tumor types including prostate and breast cancer (2,16). Inhibition of the EGFR by monoclonal antibodies and tyrosine kinase inhibitors is a rational strategy for the development of new cancer therapeutics, because of the high expression on epithelial tumors, and the role of EGFR signaling in maintaining the neoplastic phenotype of cancer cells (2,4,5,(18)(19)(20). A number of antibodies directed to the extracellular domain of the EGFR have now been tested in the clinic including Abbreviations: EGFR, EGF receptor; HACA, human antichimeric antibodies; DLT, doselimiting toxicity; ALP, alkaline phosphatase; GGT, ␥-glutamyl-transferase; MTD, maximum tolerated dose; SPECT, single-photon emission computed tomography; V1, volume of central compartment; Cmax, maximum serum concentration; Cmin, minimum serum concentration; AUC, area under the serum concentration curve extrapolated to infinite time; CL, total serum clearance; 111 In, Indium-111; wt, wild type.…”

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

“…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. Also, most ⌬EGFR reagents have been tested on only a limited number of tissues (22,(24)(25)(26)(27), and no comprehensive analysis of ⌬EGFR expression in normal and tumor tissue has been performed.…”

“…EGFR overexpression is often associated with gene amplification (4)(5)(6)(7)(8). In glioblastoma, EGFR amplification has been shown to be accompanied by gene rearrangement (9)(10)(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).…”

Mutant epidermal growth factor receptor displays increased signaling through the phosphatidylinositol-3 kinase/AKT pathway and promotes radioresistance in cells of astrocytic origin

“…Mutant forms of EGFR are commonly associated with amplification in GBMs. The most common and best characterized EGFR mutant (EGFRvIII) results from the deletion of exon 2-7 leading to expression of a truncated receptor that has lost roughly half of its extracellular, ligand-binding domain (Humphrey et al, 1990;Wong et al, 1992). EGFRvIII no longer binds ligand and displays ligandindependent, constitutive activity (Wong et al, 1992).…”