Identification of the human melanoma-associated chondroitin sulfate proteoglycan antigen epitope recognized by the antitumor monoclonal antibody 763.74 from a peptide phage library

To identify the epitope of the melanoma-associated chondroitin sulfate prot eoglycan (MCSP) recognized by the monoclonal antibody (mAb) 763.74, we firs t expressed random DNA fragments obtained from the complete ending sequence of the MCSP core glycoproteins in phages and selected without success for binders to the murine mAb 763.74. We then used a library of random heptapep tides displayed at the surface of the filamentous M13 phage as fusion prote in to the NH2-terminal portion of the minor coat protein III. After three r ounds of selection on the bound mAb, several phages displaying related bind ing peptides were identified, yielding the consensus sequence Val-His-Leu-A sn-Tyr-Glu-His. Competitive ELISA experiments showed that this peptide can be specifically prevented from binding to mAb 763.74 by an anti-idiotypic M K2-23 mouse:human chimeric mAb and by A375 melanoma cells expressing the an tigen MCSP. We screened the amino acid sequence of the MCSP molecule for a region of homology to the consensus sequence and found that the amino acid sequence Val-His-Ile-Asn-Ala-His spanning positions 289 and 294 has high ho mology, Synthetic linear peptides corresponding to the consensus sequence a s well as to the MCSP-derived epitope inhibit the binding of mAb 763.74 to the phages displaying the consensus amino acid sequence. Finally, the bioti nylated consensus peptide absorbed to streptavidin-microtiter plates can be used for the detection of mAb 763.74 in human serum. These results show cl early that the MCSP epitope defined by mAb 763.74 has been identified.