An Engineered GM-CSF-CCL2 Fusokine Is a Potent Inhibitor of CCR2-Driven Inflammation As Demonstrated in a Murine Model of Inflammatory Arthritis

Abstract

CCR2 is a chemokine receptor widely expressed by lymphomyeloid cells involved in maladaptive autoimmune ailments. Therefore CCR2 is of great interest as a biological target for immune suppression due to its direct implication in autoimmune diseases such as rheumatoid arthritis. We have generated a novel fusion protein using GM-CSF and an N-terminal truncated version of MCP-1/CCL2 (6–76, GMME1) and investigated its utility as a CCR2-specific immune suppressor. Using BRET studies, we found that distinct to CCL2, GMME1 binding to CCR2 led to altered conformational changes in the CCR2 homodimer and did not induce the recruitment of β-arrestin 2 to the receptor. However, CCR2-dependent calcium mobilization, BAX induction and caspase-3 activation followed by cell death was observed. Using Th17 cells harvested from DBA/1 mice ill with bovine collagen-induced arthritis, we demonstrate that GMME1 is capable of blocking their production of IL-17 in vitro. Upon its delivery to mice symptomatic with inflammatory arthritis, a robust clinical recovery occurred with decreased paw thickness to normal levels and a significant reduction in anti-collagen Ab titer and rheumatoid factor titer, as well as reduction of proinflammatory cytokines levels both intraarticular and systemic. Our data demonstrate that GMME1 is a powerful synthetic suppressor cytokine that coopts CCR2-dependent cellular signaling and blunts the effects of CCR2-expressing lymphomyeloid cells causative of autoimmune arthritis.

Pan–CC Chemokine Neutralization Restricts Splenocyte Egress and Reduces Inflammation in a Model of Arthritis

“…These levels coincide with the recruitment of, for example, CCR5 + monocytes and T cells into human synovial tissues (28). The functional importance of individual CC chemokines in eliciting inflammation has been assessed using murine models of RA in conjunction with neutralizing agents, such as blocking Abs (26,29), low-molecular mass compounds (30,31), or a fusion protein (32). However, the heterogeneity of the results and marginal potency of the effect suggest that redundancy in chemokine biology may compensate for neutralizing a specific ligand or receptor.…”

GM-CSF–Based Fusion Cytokines as Ligands for Immune Modulation

“…In vitro recall-response experiments were performed, and the MSC GMME1-treated mice had significantly less myelin oligodendrocyte glycoprotein and type II collagen-reactive T cells. Further experiments also found significantly less cellular infiltrates in the CNS and joints of mice treated for EAE and rheumatoid arthritis, respectively (62,63). Further research remains to be done with GMME1 against cancer to evaluate whether it can be a receptor-specific chemotherapeutic drug to interfere with the migration of monocytes to cancer and their differentiation into tumor-associated macrophages (60,64).…”

“…When fusing GM-CSF with CCL2 (GMME1), Rafei et al (62) found that, in contrast to CCL2, GMME1's interaction with CCR2 induced robust intracellular calcium mobilization and the upregulation of Bax, but it did not induce STAT3 phosphorylation. Bioluminescence resonance energy transfer (BRET) studies of GMME1's interaction with CCR2 found that GMME1 reduced the BRET signal below background resting levels, akin to an inverse agonist and while failing to recruit b-arrestin 2 to CCR2.…”

CCL2: A potential prognostic marker and target of anti‐inflammatory strategy in HIV/AIDS pathogenesis

“…From a clinical point of view, CCL2 is one of the most studied pro-inflammatory molecules among the C-C family members and a potential intervention point for the treatment of various inflammatory and autoimmune diseases. These include multiple sclerosis [6], atherosclerosis [7], insulin-resistant diabetes [8], rheumatoid arthritis [9][10][11] and lupus nephritis [12]. CCL2 blockade is also a promising target of cancer immunotherapy in various cancer treatments [13] including breast cancer [14] and prostate cancer [15] among others.…”