Designing a mutant CCL2-HSA chimera with high glycosaminoglycan-binding affinity and selectivity.

Chemokines like CCL2 mediate leukocyte migration to inflammatory sites by binding to G-protein coupled receptors on the target cell as well as to glycosaminoglycans (GAGs) on the endothelium of the inflamed tissue. We have recently shown that the dominant-negative Met-CCL2 mutant Y13A/S21K/Q23R with improved GAG binding affinity is highly bio-active in several animal models of inflammatory diseases. For chronic indications, we have performed here a fusion to human serum albumin (HSA) in order to extend the serum half-life of the chemokine mutant. To compensate a potential drop in GAG-binding affinity due to steric hindrance by HSA, a series of novel CCL2 mutants was generated with additional basic amino acids which were genetically introduced at sites oriented towards the GAG ligand. From this set of mutants, the Met-CCL2 variant Y13A/N17K/S21K/Q23K/S34K exhibited high GAG-binding affinity and a similar selectivity as wild type (wt) CCL2. From a set of different HSA-chemokine chimeric constructs, the linked HSA(C34A)(Gly)4Ser-Met-CCL2(Y13A/N17K/S21K/Q23K/S34K) fusion protein was found to show the best overall GAG-binding characteristics. Molecular modeling demonstrated an energetically beneficial fold of this novel protein chimera. This was experimentally supported by GdmCl-induced unfolding studies, in which the fusion construct exhibited a well-defined secondary structure and a transition point significantly higher than both the wt and the unfused CCL2 mutant protein. Unlike the wt chemokine, the quaternary structure of the HSA-fusion protein is monomeric according to size-exclusion chromatography experiments. In competition experiments, the HSA-fusion construct displaced only two of seven unrelated chemokines from heparan sulfate, whereas the unfused CCL2 mutant protein displaced five other chemokines. The most effective concentration of the HSA-fusion protein in inhibiting CCL2-mediated monocyte attachment to endothelial cells, as detected in the flow chamber, was 8.6 µg/ml. This novel HSA-fusion protein exhibits not only high affinity but also selective displacement of chemokines from GAGs binding. HSA is therefore proposed to be a highly promising scaffold candidate for therapeutic, GAG-targeting chemokine mutants.

Rationally evolving MCP-1/CCL2 into a decoy protein with potent anti-inflammatory activity in vivo.

Leukocyte recruitment from the blood into injured tissues during inflammatory diseases is the result of sequential events involving chemokines binding to their GPC receptors as well as to their glycosaminoglycan (GAG) co-receptors. The induction and the crucial role of MCP-1/CCL2 in the course of diseases that feature monocyte-rich infiltrates have been validated in many animal models, and several MCP-1/CCL2 as well as CCR2 antagonists have since been generated. However, despite some of them being shown to be efficacious in a number of animal models, many failed in clinical trials, and therapeutically interfering with the activity of this chemokine is not yet possible. We have therefore generated novel MCP-1/CCL2 mutants with increased GAG binding affinity and knocked out CCR2 activity, which were designed to interrupt the MCP-1/CCL2-related signaling cascade. We provide evidence that our lead mutant MCP-1(Y13A/S21K/Q23R) exhibits a 4-fold higher affinity toward the natural MCP-1 GAG ligand heparan sulfate and that it shows a complete deficiency in activating CCR2 on THP-1 cells. Furthermore, a significantly longer residual time on GAG ligands was observed by surface plasmon resonance. Finally, we were able to show that MCP-1(Y13A/S21K/Q23R) had a mild ameliorating effect on experimental autoimmune uveitis and that a marginal effect on oral tolerance in the group co-fed with Met-MCP-1(Y13A/S21K/Q23R) plus immunogenic peptide PDSAg was observed. These results suggest that disrupting wild type chemokine-GAG interactions by a chemokine-based antagonist can result in anti-inflammatory activity that could have potential therapeutic implications.