Identification of biologically active peptides that inhibit binding of human CXCL8 to its receptors from a random phage-epitope library

A random bacteriophage peptide library was used to map structural features of human (h) CXCR1 and hCXCR2 by determining the epitopes of neutralizing mAb 5A12 anti-hCXCR1 and mAb 6C6 anti-hCXCR2. After three rounds of biopanning, five mAb5 A12- and four mAb 6C6-binding peptides were identified from a 6-mer peptide library. Consensus sequences (S/T)(1)(F/A/N/D)(2)(I/M)(3)W(4)D(5)F(6) and F/L/M)(1)W(2)(D/N/L)(3)D(4)F(5)W(6) were deduced from sequences of these peptides. They correspond to a highly conserved N-domain sequence (9)MWDF(12) of hCXCR1 and (13)DFW(15) of hCXCR2. The phage bearing the peptides showed specific binding to immobilized mAb 5A12 or mAb 6CC, and over 86% of phages bound were competitively inhibited by free synthetic peptides. In FACS-can analysis, all selected phage peptides were able to strongly inhibit the binding of mAb 5A12 and mAb 6C6 to hCXCR1- and hCXCR2-transfected preB 300-19 murine cells. Furthermore, synthetic peptides of the corresponding phage epitopes were effective in blocking the antibody-CXCR1/2 interactions and to inhibit the binding of hCXCL8 to hCXCR1 and hCXCR2 transfectants. Peptides 5A12/2 (SAMWDF) and 6C6/1 (FWDDFW) competed effectively for (125)I-hCXCL8 binding to hCXCR1 and hCXCR2 with IC(50), respectively, equal to 10 mu M and 5.4 mu M. Calcium release and chemotaxis of hCXCR1/2 transfectants or human neutrophils were inhibited by all peptides in a dose-dependent manner. Furthermore, the peptide 6C6/1 FWDDFW showed inhibitory effects on chemotaxis of human netrophils induced by hCXCR2 chemokines such as hCXCL1-3 and hCXCL5. Specificities of peptides 5A12/2 and 6C6/1 were assessed with hCXCR3, hCXCR4, hCXCR5, hCCR3, and hCCR5 receptors. In vivo, peptides 5A12/2 and p6C6/1 blockade hCXCL8-induced neutrophil recruitment in skin inflammation in rabbits. Taken together, these data demonstrate that phage-display analysis provides information about the relative location of amino acids on the N-domain surfaces of hCXCR1 and hCXCR2 proteins using antibody imprints of the receptor-surface structure. The derived mimotopes could be used as inhibitors of hCXCL8-induced activities related to its interaction with the N-domain of hCXCR1 and hCXCR2. J. Leukoc. Biol. 85: 728-738; 2009.