CHARACTERIZATION OF EPITOPES ON HUMAN P53 USING PHAGE-DISPLAYED PEPTIDE LIBRARIES - INSIGHTS INTO ANTIBODY PEPTIDE INTERACTIONS

We previously described the use of a phage-displayed library of random hexapeptides to define and localise the epitope on the human tumor su ppressor protein p53 recognised by the monoclonal antibody PAb240. Her e we have extended these results to a further eight anti-p53 monoclona l antibodies and to two further libraries, which display 12-mer and 20 -mer peptides, respectively First, we showed that selection of PAb240 binding clones from the 12-mer and 20-mer libraries gives essentially identical results to those obtained by screening the 6-mer library. Se cond, we used the 6-mer and 12-mer libraries to define the derailed sp ecificity profiles of six antibodies (DO-1, DO-2, DO-7, Bp53-11, Bp53- 12 and Bp53-19), which recognise the same short, highly immunogenic N- terminal segment of p53. Finally, we employed all three libraries to r eveal the distinct mechanisms by which PAb421 and PAb122, two monoclon al antibodies that allosterically activate sequence-specific DNA bindi ng by p53, react specifically with the same positively-charged C-termi nal segment. In each case the epitope locations inferred from the sele cted sequences were confirmed by probing an array of overlapping synth etic peptides representing the primary sequence of p53. The results em phasise the consequences for epitope mapping of screening random, as o pposed to antigen-derived, peptide libraries; specifically (1) that co mparison of selected sequences reveals the contribution of individual residues to binding energy and specificity; (2) that heteroclitic reac tions call lead to definition of a consensus that is related to but di stinct from the immunising epitope and (3) that isolation of non-immun ogen-homologous ''mimotope'' sequences reveals discrete, alternative l igand structures. The results with PAb421 and PAb122 provide examples where, while selection from the 12-mer and 20-mer libraries leads to i solation of immunogen-homologous sequences, selection from the 6-mer l ibrary results in the isolation either of no binding clones (PAb122) o r solely of ''mimotope'' sequences with no discernible homology to the original antigen (PAb421). In addition the results with PAb421 reveal that linear epitopes can be longer than previously thought and can be formally discontinuous, consisting of independent contact motifs, whi ch show promiscuous relative positioning.