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 proteoglycan (MCSP) recognized by the monoclonal antibody (mAb) 763.74, we first expressed random DNA fragments obtained from the complete coding 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 heptapeptides displayed at the surface of the filamentous M13 phage as fusion protein to the NH2-terminal portion of the minor coat protein III. After three rounds of selection on the bound mAb, several phages displaying related binding peptides were identified, yielding the consensus sequence Val-His-Leu-Asn-Tyr-Glu-His. Competitive ELISA experiments showed that this peptide can be specifically prevented from binding to mAb 763.74 by an anti-idiotypic MK2-23 mouse:human chimeric mAb and by A375 melanoma cells expressing the antigen 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 homology. Synthetic linear peptides corresponding to the consensus sequence as 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 biotinylated consensus peptide absorbed to streptavidin-microtiter plates can be used for the detection of mAb 763.74 in human serum. These results show clearly that the MCSP epitope defined by mAb 763.74 has been identified.
Novartis Pharma AG, Core Technology Area, Basel, Switzerland. martin.geiser@pharma.novartis.com
M
M
Geiser
D
D
Schultz
A
A
Le Cardinal
H
H
Voshol
C
C
García-Echeverría
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000912:Antibodies, Neoplasm / Q000276:immunology*; D001435:Bacteriophages / Q000235:genetics; D011508:Chondroitin Sulfate Proteoglycans / Q000032:analysis* / Q000737:chemistry / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes; D015723:Gene Library; D006801:Humans; D008545:Melanoma / Q000276:immunology*; D008565:Membrane Proteins / Q000032:analysis* / Q000737:chemistry / Q000276:immunology; D051379:Mice; D017386:Sequence Homology, Amino Acid; D014407:Tumor Cells, Cultured; D000099216:Chondroitin Sulfate Proteoglycan 4
D000911:Antibodies, Monoclonal; D000912:Antibodies, Neoplasm; D011508:Chondroitin Sulfate Proteoglycans; D000939:Epitopes; D008565:Membrane Proteins; C101390:CSPG4 protein, human; D000099216:Chondroitin Sulfate Proteoglycan 4