Characterization of Peptides that Bind the Tumor-Associated Thomsen-Friedenreich Antigen Selected from Bacteriophage Display Libraries Elena N. Peletskaya 1 , Vladislav V. Glinsky 2 , Gennadi V. Glinsky 1,2 Susan L. Deutscher 1 and Thomas P. Quinn 1 * 1 Department of Biochemistry University of Missouri Columbia, MO 65211, USA 2 Cancer Research Center 350 Berrywood Drive Columbia, MO 65201, USA Peptides with high af®nities and speci®cities for numerous proteins and nucleic acids have been previously identi®ed from random peptide bacteriophage display libraries. Here, random peptide bacteriophage dis- play libraries were used to identify sequences that bound the cancer-as- sociated Thomsen-Friedenreich glycoantigen (T antigen). The T antigen, present on most malignant cells, contains an immunodominant Galb1 ! 3GalNAca disaccharide unmasked on the surfaces of most carci- nomas. This antigen has been postulated to be involved in tumor cell aggregation and metastasis. Two 15 amino acid random peptide bacterio- phage display libraries were af®nity selected with glycoproteins display- ing T antigen on their surfaces. Sequence analysis revealed that many of the peptides shared homology with sugar recognition sites in several carbohydrate-binding proteins. A comparison of af®nity 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 af®nity selected sequences, were chemically synthesized and characterized for their carbohydrate recog- nition properties. The synthetic peptides exhibited high speci®cities and af®nities 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 mM for MAP-P30, 20 mM for P10). Two peptides, P30 and P10, demonstrated high af®nities and speci®cities for both asialofetuin and T antigen in solution. Iodina- tion of a lone tyrosine residue in each sequence dramatically reduced their abilities to bind T antigen, suggesting that the tyrosine residue plays an important role in carbohydrate recognition. That these peptides are of functional signi®cance is evidenced by the ability of both P30 and P10 to inhibit asialofetuin-mediated melanoma cell aggregation in vitro and to compete with peanut lectin for binding to T antigen displayed on the surface of MDA-MB-435 breast carcinoma cells in situ. # 1997 Academic Press Limited Keywords: phage display peptide libraries; T antigen; synthetic peptides; tumor cell binding; aggregation inhibition *Corresponding author Introduction Peptides and their analogs have been success- fully used in a number of important medical appli- cations, including roles as regulators of blood pressure (Maack, 1992), cancer cell proliferation (Kvols et al., 1986), and viral replication (Collier et al., 1996). Until recently, the use of peptides and peptide analogs as diagnostic or therapeutic agents was limited to a small set of sequences that bound well characterized receptors or targets, such as Abbreviations used: MAP, multiple antigenic peptide; FITC, ¯uorescein isothiocyanate; BSA, bovine serum albumin; T, Thomsen-Friedenreich; PBS, phosphate buffered saline (pH 7.4); TPBS, 0.5% Tween-20 (v/v) in phosphate buffered saline (pH 7.4); PNA, peanut lectin; HRP, horseradish peroxidase; GlcNAc, N- acetylglucosamine; DAB, diaminobenzidine. J. Mol. Biol. (1997) 270, 374±384 0022±2836/97/280374±11 $25.00/0/mb971107 # 1997 Academic Press Limited