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.