title: Selection of peptide ligands for the antimucin core antibody C595 using phage display technology: definition of candidate epitopes for a cancer vaccine.
abstract: Aims-To further define the specificity of the antimucin core antibody C595 by fitting it with a family of hexapeptide ligands by immunoselection of filamentous bacteriophage from a gene III display library of approximately 6.4 x 10(7) random hexapeptides.Methods-Three rounds of immuno-selection were used to enrich for C595 binding phage. DNA sequencing revealed the hexapeptides expressed. Bacteriophage and corresponding synthetic hexapeptides were used in ELISA assay to determine binding affinities.Results-Twenty nine clones from this selected population were analysed. Seven contained the natural epitope RPAP, encoded by two different DNA sequences; 17/29 contained the motif RLPP. In all, 28/29 clones contained the motif RXXP and one clone (RVRPAP) contained the motif RXXP in two peptidic registers; 24/28 clones (6/8 DNA sequences) contained a hydrophobic residue (V or I) at position 1 relative to the RXXP motif. In addition the proximity of RXXP to glycine (position 5) suggests that this contributes in the natural epitope to antibody/antigen binding, which was not detected by chemical synthetic methods. One clone, KSKAGV, bears no obvious relationship to the natural epitope and therefore qualifies as a weakly binding mimotope.Conclusions-This approach has rapidly defined the specificity of this antibody in unprecedented detail, and provides a more comprehensive molecular basis for exploring the immune recognition of the MUC1 mucin by the C595 antibody. Importantly, the novel but related epitopes seen provide peptide specificities and a strategy which may prove useful in generating cancer vaccine candidates.
authors.affiliation: Division of Molecular and Clinical Immunology, Clinical Laboratory Science Dept, University Hospital, Queens Medical Centre, Nottingham NG7 2UH.
authors.forename: P
authors.initials: P
authors.lastname: Laing
authors.forename: P
authors.initials: P
authors.lastname: Tighe
authors.forename: E
authors.initials: E
authors.lastname: Kwiatkowski
authors.forename: J
authors.initials: J
authors.lastname: Milligan
authors.forename: M
authors.initials: M
authors.lastname: Price
authors.forename: H
authors.initials: H
authors.lastname: Sewell