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.
Piccinini
Anna Maria
AM
ProtAffin Biotechnologie AG, Reininghausstrasse 13a, A-8020 Graz, Austria.
Knebl
Kerstin
K
Rek
Angelika
A
Wildner
Gerhild
G
Diedrichs-Möhring
Maria
M
Kungl
Andreas J
AJ
D000893:Anti-Inflammatory Agents / Q000494:pharmacology*; D018932:Chemokine CCL2 / Q000737:chemistry* / Q000378:metabolism; D018925:Chemokines / Q000378:metabolism; D002633:Chemotaxis; D006801:Humans; D007700:Kinetics; D007962:Leukocytes / Q000378:metabolism; D008024:Ligands; D009000:Monocytes / Q000378:metabolism; D009154:Mutation; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D015398:Signal Transduction; D013050:Spectrometry, Fluorescence / Q000379:methods; D020349:Surface Plasmon Resonance
D000893:Anti-Inflammatory Agents; D018932:Chemokine CCL2; D018925:Chemokines; D008024:Ligands; D010455:Peptides