Identification of a Mu-9 (anti-colon-specific antigen-p)-reactive peptide having homology to CA125 (MUC16).
Mu-9, an anti-colon specific antigen-p (CSAp) monoclonal antibody (MAb), has shown excellent gastrointestinal cancer targeting in both pre-clinical and clinical trials. With the recent development of the humanized version of this MAb, Mu-9 will receive further attention as a potential therapeutic agent for colorectal cancer. Hence, we have undertaken studies to examine the nature of the CSAp antigen and the structure of the Mu-9 epitope. M13 phage displaying random 12-mer peptides were used to isolate a peptide that binds both murine and humanized Mu-9 MAbs. The peptide, Mu-9-p16, was synthesized and found to inhibit binding of Mu-9 to CSAp with a Ka for the humanized antibody of 4.28 x 10(-7) +/- 0.91 x 10(-7) M. Control peptides did not bind Mu-9, nor did they inhibit the Mu-9-CSAp interaction. Three overlapping peptides were synthesized and used to demonstrate that the last six residues were sufficient to inhibit the Mu-9-CSAp interaction. A search of GenBank revealed that the peptide sequence IHPRP, was also present within CA125, a very high molecular weight ovarian cancer-associated antigen. The sequence is present outside of the known antigenic regions identified by the Oc125, M-11 and Ov197 anti-CA125 antibodies. To demonstrate that CSAp and CA125 may be the same protein, a heterologous sandwich enzyme immunoassay was developed with anti-CA125 MAbs used as capture reagents and Mu-9 as probe. By use of this ELISA system, we were able to specifically identify CSAp. In conclusion, our results indicate that the Mu-9-p16 peptide isolated through our screen identifies a peptide epitope shared by CSAp and CA125 and suggest that these proteins are related.
Modrak
David E
DE
Garden State Cancer Center, Belleville, NJ 07109, USA. dmodrak@gscancer.org
Karacay
Habibe
H
Cardillo
Thomas M
TM
Newsome
Guy
G
Goldenberg
David M
DM
Gold
David V
DV
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000951:Antigens, Neoplasm / Q000032:analysis* / Q000737:chemistry / Q000276:immunology; D001667:Binding, Competitive; D018394:CA-125 Antigen / Q000032:analysis* / Q000737:chemistry / Q000276:immunology; D006801:Humans; D008969:Molecular Sequence Data; D019151:Peptide Library
D000911:Antibodies, Monoclonal; D000951:Antigens, Neoplasm; D018394:CA-125 Antigen; D019151:Peptide Library; C030139:colon-specific antigen