title: ERG monoclonal antibody in the diagnosis and biological stratification of prostate cancer: delineation of minimal epitope, critical residues for binding, and molecular basis of specificity.
abstract: Recently we reported the development of a highly specific murine monoclonal antibody (ERG MAb 9FY) against the ERG oncoprotein. ERG is expressed in over half of all prostate cancers (CaP) as a result of specific gene fusions involving ERG and the androgen regulated TMPRSS2 promoter. ERG MAb 9FY has been extensively used in the evaluations of CaP. Increasing use of ERG MAb in CaP has prompted us to characterize the precise ERG epitope it binds to and to define the molecular basis of its specificity to ERG. The 9FY antibody binds to an epitope formed by amino acid residues 42-66 of the ERG protein. To determine the key residues involved in 9FY binding, experiments were carried out using a combination of approaches including overlapping peptides, alanine scanning mutagenesis, ELISA, and immunoblot assays. Analysis of both overlapping and variant peptides harboring truncations of amino acids revealed that a minimal epitope of eight residues (RVPQQDWL) is sufficient for binding to the 9FY antibody. In order to further identify key residues that mediate the binding of the antibody to ERG protein, a 14-residue peptide (P23) with optimal reactivity was subjected to alanine scanning mutagenesis. Alterations to residues QQDW were found to eliminate binding to the antibody, while residues (R50 and L57) were found to contribute to the binding of the antibody. Further experiments showed that peptide P23 competed effectively with ERG protein for binding 9FY. On the other hand, peptides with alanine substitutions for residues Q53 and W56 (P27 and P30, respectively) failed to interfere with binding. These data provide new information about a minimal epitope (RVPQQDWL) within amino acid residues 42-66 of the ERG protein that is recognized by MAb 9FY, which may aid in the diagnosis and also development of antibody based therapeutics against prostate and other cancers showing ERG overexpression.
authors.affiliation: 1 Center for Prostate Disease Research, Department of Surgery, Uniformed Services University of the Health Sciences , Bethesda, Maryland.
authors.forename: Anshu
authors.initials: A
authors.lastname: Rastogi
authors.forename: Shyh-Han
authors.initials: SH
authors.lastname: Tan
authors.forename: Sreedatta
authors.initials: S
authors.lastname: Banerjee
authors.forename: Shashwat
authors.initials: S
authors.lastname: Sharad
authors.forename: Jacob
authors.initials: J
authors.lastname: Kagan
authors.forename: Sudhir
authors.initials: S
authors.lastname: Srivastava
authors.forename: David G
authors.initials: DG
authors.lastname: McLeod
authors.forename: Shiv
authors.initials: S
authors.lastname: Srivastava
authors.forename: Alagarsamy
authors.initials: A
authors.lastname: Srinivasan
mesh_terms: D058846:Antibodies, Monoclonal, Murine-Derived / Q000235:genetics / Q000276:immunology*; D000918:Antibody Specificity; D001667:Binding, Competitive; D015153:Blotting, Western; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000235:genetics*; D006801:Humans; D015151:Immunoblotting; D008297:Male; D016296:Mutagenesis; D010455:Peptides / Q000235:genetics / Q000378:metabolism; D011471:Prostatic Neoplasms / Q000175:diagnosis* / Q000276:immunology*; D011485:Protein Binding; D015534:Trans-Activators / Q000235:genetics / Q000276:immunology*; D000071230:Transcriptional Regulator ERG
chemical_list: D058846:Antibodies, Monoclonal, Murine-Derived; C072312:ERG protein, human; D000939:Epitopes; D010455:Peptides; D015534:Trans-Activators; D000071230:Transcriptional Regulator ERG