Background Collectively, the cell surface Fc region of IgG receptors (FcγRs) engage soluble IgG and IgG containing immune complexes and trigger activation or inhibtory signals that play a critical role in the regulation of immune responses. The low affinity FcγRIIA (CD32A) is the most widely expressed activating FcγR in humans and appears to drive autoantibody and immune complex mediated autoimmune disorders. So far a therapeutic targeting this receptor has not been developed. Objectives To generate and characterize a novel humanized effector-deficient FcγRIIA antibody (MEDI9600) for clinical development. Methods The mode of action of MEDI9600 was assessed by confocal microscopy, whole blood internalization, and binding competition assays. Multiple cell based assays were used to measure autoantibody and immune complex mediated responses. The safety of MEDI9600 was assessed in in vitro by neutrophil migration, activation and opsonophagocytic killing assays. Safety and pharmacokinetics were examined in vivo in a single-dose PK/PD study in cynomolgus monkey. Results We generated a humanized effector-deficient FcγRIIA antibody (MEDI9600) that potently blocks both autoantibody and immuno complex-mediated proinflammatory responses from a variety of cell types. This includes the inhibition of Toll-like receptor stimulatory immune complexes that induce type I Interferons from pDC, and the inhibition of anti-neutrophil cytoplasmic antibody (ANCA) induced production of reactive oxygen species from neutrophils, which are associated with the pathogenesis of systemic lupus and ANCA vasculitis respectively. MEDI9600 specifically binds FcγRIIA and its suppressive activity is attributed to its capacity to block ligand engagement and to internalize the receptor from the cell surface. Moreover, in vivo studies indicate that MEDI9600 has a favorable pharmacokinetic and safety profile. Conclusions We have generated MEDI9600, a specific humanized antibody antagonist of FcγRIIA with null effector function that may provide a novel therapeutic approach in the treatment of immune complex mediated diseases. Disclosure of Interest None declared

Interleukin-1 beta (IL-1 beta) is a central mediator of inflammation and connective tissue destruction in rheumatoid arthritis. IL-1 beta activates articular chondrocytes to produce matrix metalloproteinase-1 (MMP-1), an enzyme capable of dismantling the collagen scaffold of articular cartilage. To define the transcription factors and signaling intermediates that activate MMP-1 transcription in chondrocytes, we performed transient transfection of MMP-1 promoter constructs followed by reporter assays. These Studies identified an IL-1 beta-responsive region of the human MMP-1 promoter that contains a consensus CCAAT enhancer-binding protein (C/EBP) binding site. Deletion of this site reduced overall transcriptional activity of the MMP-1 promoter, as well as decreased fold induction by IL-1 beta. IL-1 beta stimulation of chondrocytes increased binding of C/EBP-beta to the MMP-1 C/EBP site. Extracellular signal regulated kinase (ERK) pathway-dependent phosphorylation of C/EBP-beta on threonine 235 activates this transcription factor. Here we show that IL-1 beta stimulation of chondrocytes induced phosphorylation of C/EBP-beta on threonine 235, and that the ERK pathway inhibitor PD98059 reduced this phosphorylation. We further show that PD98059 reduces IL-1 beta-induced MMP-1 mRNA expression in chondrocytes. Moreover, inhibition of the ERK pathway by expression of dominant-negative forms of ERK1 and ERK2 impaired the ability of IL-1 beta to transactivate the MMP-1 promoter. Our findings demonstrate a novel role for C/EBP-beta in IL-1 beta-induced connective tissue disease and define a new nuclear target for the ERK pathway in MMP-1 gene activation.

Objective: Interleukin-1 beta (IL-1 beta) stimulates collagenase-1 (Matrix Metalloproteinase-1 (MMP-1)) expression in articular chondrocytes, leading to cleavage of type II collagen and irreversible cartilage degradation. The nuclear factor-kappa B (NF-kappa B) pathway is potently activated in IL-1 beta-stimulated cells and has been implicated as an intermediate in MMP-1 gene expression. However, the roles of individual NF-kappa B family members during IL-1 beta-induced MMP-1 gene expression have not been defined.Results: To address the relationship between the NF-kappa B pathway and MMP-1 gene activation in chondrocytes, primary cultured human articular chondrocyte cultures (HAC) and SW-1353 cells were stimulated with IL-1 beta over a 24-h time course and MMP-1, NF-kappa B1, NF-kappa B2 and ReIA gene expression was assayed. IL-1 beta-induced MMP-1 expression was comparable in HAC and SW-1353 cells both temporally and quantitatively. MMP-1 gene expression was mirrored by increases in NF-kappa B gene expression, and inhibition of NF-kappa B nuclear translocation with dominant-negative I kappa B alpha reduced IL-1 beta-dependent MMP-1 gene expression. IL-1 beta activated the NF-kappa B pathway in chondrocytes, both through phosphorylation and transient degradation of I kappa B alpha, as well as through sustained phosphorylation of ReIA. Small inhibitory RNAs (siRNA) specific for ReIA resulted in significant reduction of MMP-1 mRNA, whereas siRNA for NF-kappa B1 and NF-kappa B2 augmented IL-1 beta-induced MMP-1 expression.Conclusions: Our data demonstrate that IL-1 beta activation of the NF-kappa B pathway is required for IL-1 beta induction of MMP-1 in chondrocytes and that ReIA can work independently of NF-kappa B1 or NF-kappa B2 to activate this gene expression program. Published by Elsevier Ltd on behalf of Osteoarthritis Research Society International.