A Novel Recombinant Fusion Protein Encoding a 20-Amino Acid Residue of the Third Extracellular (E3) Domain of CCR2 Neutralizes the Biological Activity of CCL2

Abstract

CCL2 is a key CC chemokine that has been implicated in a variety of inflammatory autoimmune diseases and in tumor progression and it is therefore an important target for therapeutic intervention in these diseases. Soluble receptor-based therapy is a known approach for neutralizing the in vivo functions of soluble mediators. Owing to the complexity of seven-transmembrane G protein-coupled receptors, efforts to generate neutralizing soluble chemokine receptors have so far failed. We developed a strategy that is based on the generation of short recombinant proteins encoding different segments of a G protein-coupled receptor, and tested the ability of each of them to bind and neutralize its target chemokine. We show that a fusion protein comprised of as few as 20 aa of the third extracellular (E3) domain of the CCL2 receptor, stabilized by the IgG H chain Fc domain (E3-IgG or BL-2030), selectively binds CCL2 and CCL16 and effectively neutralizes their biological activities. More importantly, E3-IgG (BL-2030) could effectively suppress the in vivo biological activity of CCL2, attenuating ongoing experimental autoimmune encephalomyelitis, as well as the development of human prostate tumor in SCID mice.

A Fusion Protein Encoding the Second Extracellular Domain of CCR5 Arrests Chemokine-Induced Cosignaling and Effectively Suppresses Ongoing Experimental Autoimmune Encephalomyelitis

“…In addition, the neutralizing dose of CCR5(E2)-Ig required to inhibit 50% of the chemokine-induced migration (ND 50 ) was calculated. M 6 30% for each detected chemokine, which is comparable to the one of CCL2 to CCR2 (E3-Ig) (33), and is ∼200-fold lower than the binding affinity of each reciprocal mAb (data not shown). This may explain, in part, the functional differences in the ability to block chemokine induced attraction between mAb and CCR5(E2)-Ig (Fig.…”

“…One possible explanation for this paradigm is that beyond their function as chemoattractants, which could be overcome in the absence of a single chemokine, chemokines also execute other biological activities that direct the biological function of their target cells, and these features are fundamental in the dynamics of the inflammatory process. For example, the CCL2-CCR2 interaction is essential for monocyte emigration from bone marrow during inflammatory processes (37), which may explain why CCR2 2/2 or CCL2 2/2 mice develop an attenuated form of EAE (8,38), and why targeted neutralization of CCL2 suppresses the disease so effectively (33). Chemokines are also involved in T cell and monocytic cell polarization.…”

“…A fusion protein encoding 30 aa of the second extracellular domain of CCR5 selectively binds all CCR5 ligands and neutralizes their biological function As G protein-coupled receptors, chemokine receptors span the plasma membrane seven times, generating three extra cellular domains, aside from the N-terminal domain (i.e., first, second, and third extracellular domains [E1, E2, and E3]). We have recently mapped the binding sites of CCR2 to CCL2 and generated a functionally binding CCR2-Ig soluble receptor (33). In this study, we used this strategy once again; first, we have cloned the extracellular loops of murine CCR5 (Fig.…”

Predominant Expression of CCL2 at the Tumor Site of Prostate Cancer Patients Directs a Selective Loss of Immunological Tolerance to CCL2 That Could Be Amplified in a Beneficial Manner

“…Chemotaxis assays of RAW cells (1 3 10 6 ) were performed in a TransWell system (5-mm pore size, Corning Costar Corporation, Cambridge, MA) (23). Chemotaxis assays of PC-3 and TRAMP-C1 cell lines (1 3 10 6 ) were conducted using the CytoSelect TM cell migration assay (8-mm pore size) (Cell Biolabs, San Diego, CA) (23).…”

“…Chemotaxis assays of PC-3 and TRAMP-C1 cell lines (1 3 10 6 ) were conducted using the CytoSelect TM cell migration assay (8-mm pore size) (Cell Biolabs, San Diego, CA) (23).…”