Mechanism of Binding and Internalization of ICAM-1-derived Cyclic Peptides by LFA-1 on the Surface of T-cells: A Potential Method for Targeted Drug Delivery

Abstract

Purpose Peptides derived from the Domain 1 of the adhesion molecule ICAM-1(1-21) are being developed as targeting ligands for LFA-1 receptors expressed on activated T-cells. This work aims to elucidate the binding and internalization of ICAM-1-derived cyclic peptides (cIBL, cIBC, and cIBR) to LFA-1. Methods 96-well plates coated with soluble LFA-1 (sLFA-1) were used to characterize the binding of FITC-labeled peptide. An anti-CD11a antibody to the I-domain of LFA-1 was used to inhibit the binding of these peptides, which was quantified using a fluorescence plate reader. An unrelated FITC-labeled cyclic peptide was used as a negative control and PE-labeled anti-CD11a antibodies (PE-R3.2 and PE-R7.1) were used as positive controls. Peptide binding to cell surface LFA-1 was visualized using co-localization of FITC-cIBR peptide and PE-labeled anti-CD18 antibody (LFA-1 β-subunit) on SKW-3 T-cells by fluorescent microscopy. Inhibition of ICAM-1-binding to LFA-1 by peptides was evaluated using a Biacore assay. Binding and internalization of FITC-labeled peptides was evaluated by flow cytometry and confocal microscopy at 4 and 37ºC. Results These FITC-labeled cyclic peptides bind to sLFA-1 and can be blocked by an anti-CD11a antibody to the I-domain, suggesting that their binding site is on the I-domain of LFA-1. The FITC-cIBR peptide was localized with an anti-CD18 antibody on the surface of T-cells, indicating that the FITC-cIBR peptide binds to LFA-1 on the cell surface. Flow cytometry and confocal microscopy demonstrated that FITC-labeled peptides were internalized in temperature dependent manner. Biacore analysis, demonstrated these peptides did not inhibit sICAM-1 from binding to immobilized sLFA-1. However, the binding properties of the soluble forms of LFA-1 and ICAM-1 may not correlate to their interaction at the cell surface. Conclusion Cyclic ICAM-1 derived peptides (cIBL, cIBC, and cIBR) bind to the I-domain of LFA-1, and are internalized by LFA-1 receptors on the surface of T-cells. Therefore, these peptides could be utilized to target and deliver drugs to the cytoplasmic domain of T-cells.