Searching For Novel Human ICAM-1 Targeting Peptide Using A Phage Displayed Peptide Library

Objective: The screening of phage-displayed random peptide library has been used to identify a novel human ICAM-1 targeting peptide. The ultimate goal is to develop peptide guided anti-inflammation drug. Methods: Two different methods were used to isolate the human ICAM-1 targeting peptides from phage-displayed random 15-peptide library. One is by using 96-well high-binding plates that coated with human recombinant ICAM-1; the other is by using cryosections form patient with acute or chronic inflammation. Binding phages were eluted, titrated, amplified, and purified respectively. Amplified phages were used for further selection rounds. After several rounds, the enriched phage clones were chosen and the plasmid DNA was isolated from single positive colonies. DNA sequencing of the plasmids was conducted using a standard Ml3 primer. The binding affinity of the selected peptides for human was measured using enzyme-linked immunosorbent assay (ELISA) method, and the affinity constant (Ka) values of the targeting displayed phage clone with ICAM-1 was calculated. The space conformations of the peptides that have the consensus sequence was analyzed by computer-guided homologous modeling techniques. The target peptide was synthesized, purified and labeled with biotin in vitro. To investigate the binding of the synthesized target peptide for human ICAM-1, we compared the distribution of synthesized target peptide and monoclonal antibody to human ICAM-1 in sections of acute and chronicinflammatory tissue by immunohistochemical staining. Furthermore, the synthesized target peptide was labeled with FITC and injected intravenously into mice with unilateral ureteral obstruction (UUO) and the distribution of the peptide in the kidney was determined. A FITC labeled unrelated peptide was used as control in vivo experiments. Results: Six positive phage clones screened out of 80 clones showed specific binding with human ICAM-1, but no conserved motif was found in these peptides. The DNA sequences of these positive clones were determined and amino acid sequences of the 15-peptide were deduced. All the results of ELISA and computer-guided homologous modeling techniques showed that No. 14 peptide has the strongest affinity to the ICAM-1 in the six peptides. The amino acid sequence of the target peptide (No. 14) is GRGEFRGRDNSVSVV and the Ka of the peptide was 7.78+10E7 L/mol. The purity of the synthesized target peptides was greater than 99.9% as assessed by high performance liquid chromatography. The binding of the target peptide for human ICAM-1 was in a dose-dependent manner and the binding was not blocked by polyclonal antibody to ICAM-1. The binding position of the synthetic target peptide is the same as that of the monoclonal antibody to human ICAM-1 in inflammatory tissue sections. The synthetic target peptide was detected on the renal tubules in the damaged kidney of the mice with UUO. In contrast, the unrelated peptide was not detected in the kidney. Conclusion: A novel human ICAM-1 targeting peptide was obtained by biopanning the phage-displayed random 15-peptide library. This result is a significant step toward developing the peptide guiding anti-inflammation drug to the inflammatory site with ICAM-1 highest expression ultimately. Although the target peptide cannot block the effect of ICAM-1, it should be useful in elucidation of ICAM-1 in vivo function, which will contribute our better understanding of the biology of inflammation in the future. Our results alsodemonstrated that library selection by phage display technology could be a rapid and efficient way to discover potent and specific peptides for the diagnosis and therapy of diseases.