Phage Display-Derived Cyclic Peptides as Ligand-Specific Modulators for β ₂ -Integrin Receptors

The leukocyte-specific β ₂ -integrin receptor family exerts a wide range of functions: β ₂ -integrins are involved in leukocyte trafficking, where they mediate cell adhesion during inflammatory responses via binding to ICAM-1, ICAM-2, or JAM-C. Furthermore, they are essential for the recognition and phagocytosis of pathogens opsonized by complement. Accordingly, the β ₂ -integrin family is known to be involved in autoimmune and inflammatory diseases, such as systemic lupus erythematosus. Owing to their complex biology, involving multiple conformational transitions, different signaling pathways, and a broad spectrum of ligands, the development of β ₂ -integrin-targeted probes and therapeutics has remained challenging.

We aimed to develop macrocyclic peptides, derived from phage display screening, which can be used to unravel ligand binding profiles of β ₂ -integrins with an emphasis on the αI domain. The selection of suitable lead peptides, and the characterization of their interaction profiles with different αI domains, was enabled by an established in-vitro assay platform. Various peptide sequences were enriched during several rounds of phage display against the αI-domain of CR3, of which two peptides with particularly low micromolar binding affinity were further characterized. Both peptides showed direct binding to β ₂ -integrin αI-domains and, in a competitive assay, dose-dependent inhibition of the αI-domains’ interactions with their main ligands iC3b and ICAM-1, respectively. These ligand-interfering properties were confirmed in bead- and cell-based adhesion assays.

The modulators developed here are expected to provide valuable insight into the (patho-)physiology of CR3 and the other members of the β ₂ -integrin family, as the two peptides were able to compete with different ligands. In the future, this may help to identify potential therapeutic approaches for autoimmune, inflammatory, and age-related diseases.