Therapeutic Targeting of the G-CSF Receptor Reduces Neutrophil Trafficking and Joint Inflammation in Antibody-Mediated Inflammatory Arthritis.

G-CSF is a hemopoietic growth factor that has a role in steady state granulopoiesis, as well as in mature neutrophil activation and function. G-CSF- and G-CSF receptor-deficient mice are profoundly protected in several models of rheumatoid arthritis, and Ab blockade of G-CSF also protects against disease. To further investigate the actions of blocking G-CSF/G-CSF receptor signaling in inflammatory disease, and as a prelude to human studies of the same approach, we developed a neutralizing mAb to the murine G-CSF receptor, which potently antagonizes binding of murine G-CSF and thereby inhibits STAT3 phosphorylation and G-CSF receptor signaling. Anti-G-CSF receptor rapidly halted the progression of established disease in collagen Ab-induced arthritis in mice. Neutrophil accumulation in joints was inhibited, without rendering animals neutropenic, suggesting an effect of G-CSF receptor blockade on neutrophil homing to inflammatory sites. Consistent with this, neutrophils in the blood and arthritic joints of anti-G-CSF receptor-treated mice showed alterations in cell adhesion receptors, with reduced CXCR2 and increased CD62L expression. Furthermore, blocking neutrophil trafficking with anti-G-CSF receptor suppressed local production of proinflammatory cytokines (IL-1ß, IL-6) and chemokines (KC, MCP-1) known to drive tissue damage. Differential gene expression analysis of joint neutrophils showed a switch away from an inflammatory phenotype following anti-G-CSF receptor therapy in collagen Ab-induced arthritis. Importantly, G-CSF receptor blockade did not adversely affect viral clearance during influenza infection in mice. To our knowledge, we describe for the first time the effect of G-CSF receptor blockade in a therapeutic model of inflammatory joint disease and provide support for pursuing this therapeutic approach in treating neutrophil-associated inflammatory diseases.

Production of a human neutralizing monoclonal antibody and its crystal structure in complex with ectodomain 3 of the interleukin-13 receptor α1.

Gene deletion studies in mice have revealed critical roles for IL (interleukin)-4 and -13 in asthma development, with the latter controlling lung airways resistance and mucus secretion. We have now developed human neutralizing monoclonal antibodies against human IL-13Rα1 (IL-13 receptor α1) subunit that prevent activation of the receptor complex by both IL-4 and IL-13. We describe the crystal structures of the Fab fragment of antibody 10G5H6 alone and in complex with D3 (ectodomain 3) of IL-13Rα1. Although the structure showed significant domain swapping within a D3 dimer, we showed that Arg(230), Phe(233), Tyr(250), Gln(252) and Leu(293) in each D3 monomer and Ser(32), Asn(102) and Trp(103) in 10G5H6 Fab are the key interacting residues at the interface of the 10G5H6 Fab-D3 complex. One of the most striking contacts is the insertion of the ligand-contacting residue Leu(293) of D3 into a deep pocket on the surface of 10G5H6 Fab, and this appears to be a central determinant of the high binding affinity and neutralizing activity of the antibody.