CHARACTERIZATION OF PEPTIDES THAT BIND THE TUMOR-ASSOCIATED THOMSEN-FRIEDENREICH ANTIGEN SELECTED FROM BACTERIOPHAGE DISPLAY LIBRARIES

Peptides with high affinities and specificities for numerous proteins and nucleic acids have been previously identified from random peptide bacteriophage display libraries. Here, random peptide bacteriophage di splay libraries were used to identify sequences that bound the cancer- associated Thomsen-Friedenreich glycoantigen (T antigen). The T antige n, present on most malignant cells, contains an immunodominant Gal bet a 1 --> 3GalNAc alpha disaccharide unmasked on the surfaces of most ca rcinomas. This antigen has been postulated to be involved in tumor cel l aggregation and metastasis. Two 15 amino acid random peptide bacteri ophage display libraries were affinity selected with glycoproteins dis playing T antigen on their surfaces. Sequence analysis revealed that m any of the peptides shared homology with sugar recognition sites in se veral carbohydrate-binding proteins. A comparison of affinity selected sequences from both libraries yielded a common motif (W-Y-A-W/F-S-P) rich in aromatic amino acids. Four peptides, corresponding to the affi nity selected sequences, were chemically synthesized and characterized for their carbohydrate recognition properties. The synthetic peptides exhibited high specificities and affinities to T antigen displayed on asialofetuin or conjugated to bovine serum albumin (K-d=5 nM for MAP- P30 binding to asialofetuin) as well as free T-antigen disaccharide in solution (K-d=10 mu M for MAP-P30, 20 mu M for P10). Two peptides, P3 0 and P10, demonstrated high affinities and specificities for both asi alofetuin and T antigen in solution. Iodination of a lone tyrosine res idue in each sequence dramatically reduced their abilities to bind T a ntigen, suggesting that the tyrosine residue plays an important role i n carbohydrate recognition. That these peptides are of functional sign ificance is evidenced by the ability of both P30 and P10 to inhibit as ialofetuin-mediated melanoma cell aggregation in vitro and to compete with peanut lectin for binding to T antigen displayed on the surface o f MDA-MB-435 breast carcinoma cells in situ. (C) 1997 Academic Press L imited.