A PEPTIDE ISOLATED BY PHAGE DISPLAY BINDS TO ICAM-1 AND INHIBITS BINDING TO LFA-1

A mixed phage Library containing random peptides from four to eight re sidues in length flanked by cysteine residues was screened using a rec ombinant soluble, form of human ICAM-1, which included residues 1-453, (ICAM(1-453)), Phage bound to immobilized ICAM-1(1-453) were eluted b y three methods: (1) soluble ICAM-1(1-453), (2) neutralizing murine mo noclonal antibody, (anti-ICAM-1, M174F5B7), (3) acidic conditions, Aft er three rounds of binding and elution, a single, unique ICAM-1 bindin g phage bearing the peptide EWCEYLGGYLRYCA was isolated; the identical phage was selected with each method of elution, Attempts to isolate p hage from nonconstrained (i.e., not containing cysteines) libraries di d not yield a phage that bound to ICAM-1, Phage displaying EWCEYLGGYLR CYA bound to immobilized ICAM-1(1-453) and to ICAM-1(1-185) a recombin ant ICAM-1, which contains only the two amino-terminal immunoglobulin domains residing within residues 1-185. This is the region of the ICAM -1 that is bound by LFA-1, The phage did not bind to proteins other th an ICAM-1. The phage bound to two ICAM-1 mutants, which contained amin o acid substitutions that dramatically decreased or eliminated the bin ding to LFA-1. Studies were also performed with the corresponding synt hetic peptide, The linear form of the synthetic EWCEYLGGYLCYA peptide was found to inhibit LFA-1 binding to immobilized ICAM(1-453) in a pro tein-protein binding assay. By contrast, the disulfide, cyclized, form of the peptide was inactive. The EWCEYL portion of the sequence is ho mologous to the EWPEYL sequence found within rhinovirus coat protein 1 4, a nonintegrin protein that binds to ICAM-1. Tab;en together, the re sults suggests that the EWCEYLGGYLRCYA sequence is capable to binding to immobilized ICAM-1. Phage display appears to represent a new approa ch for the identification of peptides that interfere with ICAM-1 bindi ng to beta 2 integrins. (C) 1996 Wiley-Liss, Inc.