Work

Improving the in vivo activity of glycosaminoglycan-binding immunomodulatory drugs by fusion to human serum albumin

Year: 2025

Type: article

Abstract: Chemokines are essential immune regulators that interact with G protein-coupled receptors (GPCRs) on leukocytes and with glycosaminoglycans (GAGs) on the surface of endothelial cells to form a chemotactic gradient, essential for directional leukocyte migration. The chemokine CXCL9 contains multiple C-terminal GAG-binding motifs, responsible for the high affinity of the protein to endothelial GAGs. Through chemical synthesis of CXCL9's C-terminal domain, composed of the last 30 amino acids of the sequence, we generated the GAG-binding chemotaxis antagonist CXCL9(74-103). This peptide is able to bind GAGs on the endothelial surface, but fails to elicit a chemotactic response. As such, it appeared to be an effective anti-inflammatory compound, dampening leukocyte infiltration and tissue damage without increasing pathogen load. However, the therapeutic applications of CXCL9(74-103) are limited due to its rapid renal clearance, a well-known drawback of peptide-based therapeutics. This study explores the use of human serum albumin (HSA) as a fusion scaffold to extend the serum half-life and thus improve the anti-inflammatory properties of CXCL9(74-103). This HSA-CXCL9(74-103) fusion protein was produced in Pichia pastoris for biochemical, pharmacological and functional characterization. The GAG-binding affinity was assessed by isothermal fluorescence titration and surface plasmon resonance experiments. Bioavailability experiments following i.v. injection in C57BL/6JRj mice demonstrated prolonged blood half-life of our fusion protein, as well as stronger reduction of leukocyte recruitment to inflamed joints, both acutely and over an extended period of time. These findings show that HSA-CXCL9(74-103) has substantial potential as a long-lasting anti-inflammatory therapeutic, offering improved pharmacokinetics and sustained efficacy.

Source: International Journal of Biological Macromolecules

DOI: 10.1016/j.ijbiomac.2025.149825