Elucidating the interplay between IgG-Fc valency and FcγR activation for the design of immune complex inhibitors.

Autoantibody immune complex (IC) activation of Fcγ receptors (FcγRs) is a common pathogenic hallmark of multiple autoimmune diseases. Given that the IC structural features that elicit FcγR activation are poorly understood and the FcγR system is highly complex, few therapeutics can directly block these processes without inadvertently activating the FcγR system. To address these issues, the structure activity relationships of an engineered panel of multivalent Fc constructs were evaluated using sensitive FcγR binding and signaling cellular assays. These studies identified an Fc valency with avid binding to FcγRs but without activation of immune cell effector functions. These observations directed the design of a potent trivalent immunoglobulin G-Fc molecule that broadly inhibited IC-driven processes in a variety of immune cells expressing FcγRs. The Fc trimer, Fc3Y, was highly efficacious in three different animal models of autoimmune diseases. This recombinant molecule may represent an effective therapeutic candidate for FcγR-mediated autoimmune diseases.

Pathways Responsible for Human Autoantibody and Therapeutic Intravenous IgG Activity in Humanized Mice.

Immunoglobulin G (IgG) antibodies are major drivers of autoimmune pathology, but they are also used in the form of intravenous IgG (IVIg) therapy to suppress autoantibody activity. To identify the pathways underlying human autoantibody and IVIg activity, we established a humanized mouse model of an autoantibody-dependent autoimmune disease responding to treatment with IVIg preparations. We show that the human IgG subclass strongly impacts autoantibody activity and that the Fc-receptor genotype of the human donor immune system further modulates autoantibody activity. Human mononuclear phagocytes were responsible for autoantibody activity, and IVIg therapy was able to suppress disease pathology in an Fc-fragment-dependent manner. While highly sialylated IgG glycovariants were essential for IVIg activity, it was independent of the Fc-receptor genotype and did not result in a general block of activating or the neonatal Fc-receptor. These findings may help in the development of strategies to block autoantibody and enhance therapeutic IVIg activity in humans.

Siglec-G Deficiency Leads to Autoimmunity in Aging C57BL/6 Mice.

Siglec-G, a member of the sialic acid-binding Ig-like lectin (Siglec) family, is expressed on B cell and dendritic cell surfaces. It acts as an inhibitory coreceptor and modulates B cell activation, especially on B1 cells, as Siglec-G-deficient mice show mainly a B1 cell-restricted phenotype resulting in increased B1 cell numbers. Although higher B1 cell numbers are discussed to be associated with autoimmunity, loss of Siglec-G does not result in autoimmune disease in BALB/c mice. However, there is evidence from Siglec-G × CD22 double-deficient mice and Siglec-G(-/-) mice on an autoimmune-prone MRL/lpr background that Siglec-G is important to maintain tolerance in B cells. In this study, we analyzed the role of Siglec-G in induction and maintenance of B cell tolerance on C57BL/6 background and in the FcγRIIb-deficient background. We find that aging Siglec-G-deficient and Siglec-G × FcγRIIb double-deficient mice develop an autoimmune phenotype with elevated autoantibody levels and mild glomerulonephritis. Aging Siglec-G-deficient mice have elevated numbers of plasma cells and germinal center B cells, as well as a higher number of activated CD4 T cells, which likely all contribute to autoantibody production. Additional loss of the inhibitory receptor FcγRIIb in Siglec-G(-/-) mice does not result in exacerbation of disease. These results indicate that Siglec-G is important to maintain tolerance in B cells and prevent autoimmunity.

Controlled tetra-Fc sialylation of IVIg results in a drug candidate with consistent enhanced anti-inflammatory activity.

Despite the beneficial therapeutic effects of intravenous immunoglobulin (IVIg) in inflammatory diseases, consistent therapeutic efficacy and potency remain major limitations for patients and physicians using IVIg. These limitations have stimulated a desire to generate therapeutic alternatives that could leverage the broad mechanisms of action of IVIg while improving therapeutic consistency and potency. The identification of the important anti-inflammatory role of fragment crystallizable domain (Fc) sialylation has presented an opportunity to develop more potent Ig therapies. However, translating this concept to potent anti-inflammatory therapeutics has been hampered by the difficulty of generating suitable sialylated products for clinical use. Therefore, we set out to develop the first, to our knowledge, robust and scalable process for generating a well-qualified sialylated IVIg drug candidate with maximum Fc sialylation devoid of unwanted alterations to the IVIg mixture. Here, we describe a controlled enzymatic, scalable process to produce a tetra-Fc-sialylated (s4-IVIg) IVIg drug candidate and its qualification across a wide panel of analytic assays, including physicochemical, pharmacokinetic, biodistribution, and in vivo animal models of inflammation. Our in vivo characterization of this drug candidate revealed consistent, enhanced anti-inflammatory activity up to 10-fold higher than IVIg across different animal models. To our knowledge, this candidate represents the first s4-IVIg suitable for clinical use; it is also a valuable therapeutic alternative with more consistent and potent anti-inflammatory activity.

A humanized mouse identifies the bone marrow as a niche with low therapeutic IgG activity.

Genetic differences between humans and in vivo model systems, including mice and nonhuman primates, make it difficult to predict the efficacy of immunoglobulin G (IgG) activity in humans and understand the molecular and cellular mechanisms underlying that activity. To bridge this gap, we established a small-animal model system that allowed us to study human IgG effector functions in the context of an intact human immune system without the interference of murine Fcγ receptors expressed on mouse innate immune effector cells in vivo. Using a model of B cell depletion with different human IgG variants that recognize CD20, we show that this humanized mouse model can provide unique insights into the mechanism of human IgG activity in vivo. Importantly, these studies identify the bone marrow as a niche with low therapeutic IgG activity.

Targeting B cells and autoantibodies in the therapy of autoimmune diseases.

B cells and B cell-derived autoantibodies play a central role in the pathogenesis of many autoimmune diseases. Thus, depletion of B cells via monoclonal antibodies such as Rituximab is an obvious therapeutic intervention and has been used successfully in many instances. More recently, novel therapeutic options targeting either the autoantibody itself or resetting the threshold for B cell activation have become available and show promising immunomodulatory and anti-inflammatory effects in a variety of animal models. The aim of this review is to summarize these results and to provide an insight into the underlying molecular and cellular pathways of these novel therapeutic interventions targeting autoantibodies and B cells and to discuss their value for human therapy.

Broad requirement for terminal sialic acid residues and FcγRIIB for the preventive and therapeutic activity of intravenous immunoglobulins in vivo.

Intravenous immunoglobulin (IVIg) therapy is widely used to treat a variety of autoimmune diseases including immunothrombocytopenia, chronic inflammatory demyelinating polyneuropathy, and more recently autoimmune skin blistering diseases. Despite this well-documented clinical success, the precise molecular and cellular mechanisms underlying this immunomodulatory activity are discussed controversially. In particular, the clinically relevant therapeutic pathway of IVIg-mediated immune modulation has not been studied in detail. In the present study, we use four independent in vivo model systems of auto-Ab-mediated autoimmune disease to identify a common pathway explaining IVIg activity under therapeutic conditions in vivo. We show that irrespective of the in vivo model system, IVIg activity is strictly dependent on the presence of terminal sialic acid residues and the inhibitory FcγRIIB under preventive as well as therapeutic treatment conditions. In contrast, specific ICAM3 grabbing nonintegrin related 1, previously demonstrated to be essential under preventative treatment conditions, showed a disease-specific impact on IVIg-mediated resolution of established autoimmune disease.

Intravenous immunoglobulin therapy: how does IgG modulate the immune system?

Intravenous immunoglobulin (IVIG) preparations comprise pooled IgG antibodies from the serum of thousands of donors and were initially used as an IgG replacement therapy in immunocompromised patients. Since the discovery, more than 30 years ago, that IVIG therapy can ameliorate immune thrombocytopenia, the use of IVIG preparations has been extended to a wide range of autoimmune and inflammatory diseases. Despite the broad efficacy of IVIG therapy, its modes of action remain unclear. In this Review, we cover the recent insights into the molecular and cellular pathways that are involved in IVIG-mediated immunosuppression, with a particular focus on IVIG as a therapy for IgG-dependent autoimmune diseases.

B cells and CD22 are dispensable for the immediate antiinflammatory activity of intravenous immunoglobulins in vivo.

Intravenous immunoglobulins (IVIgs) efficiently suppress a variety of autoimmune diseases. Over the past few years several potential mechanisms underlying this antiinflammatory activity have become apparent. Among these, terminal sialic acid residues in the sugar moiety of the immunoglobulin G constant fragment have been shown to be critical for the antiinflammatory activity of IVIgs in models of rheumatoid arthritis and immunothrombocytopenia (ITP). More recently, B cells and the sialic acid-binding protein CD22 were suggested to be involved in this IVIg-dependent immunomodulatory pathway. To study whether B cells are directly involved in IVIg-mediated suppression of acute autoimmune diseases, we tested the activity of IVIgs in mice deficient in B cells or CD22. We show that neither B cells nor CD22 are critical for the immediate antiinflammatory activity of IVIgs in mouse models of rheumatoid arthritis and ITP.

FcγRIIB: a modulator of cell activation and humoral tolerance.

An immune response needs to be tightly regulated to prevent excessive inflammation, which may result in the destruction of healthy tissues. At the molecular level, the strength of an immune response is determined by the integration of a multitude of positive and negative signals. This review will focus on IgG-dependent immune responses and discuss how the inhibitory receptor FcγRIIB may be involved in regulating both the afferent and efferent phases of such a response. Furthermore, we will discuss recent evidence suggesting that FcγRIIB may have important functions beyond the negative regulation of signals transduced by the B-cell receptor or activating FcγRs and could be responsible for the activity of agonistic antibodies in vivo.

The role of sialic acid as a modulator of the anti-inflammatory activity of IgG.

Immunoglobulin G (IgG) molecules can have two completely opposing activities. They can be very potent pro-inflammatory mediators on the one hand, directing the effector functions of the innate immune system towards infected cells, tumor cells or healthy tissues in the case of autoimmune diseases. On the other hand, a mixture of IgG molecules purified from the blood of ten thousands of healthy donors is used as an anti-inflammatory treatment for many autoimmune diseases since several decades. It has become evident only recently that certain residues in the sugar moiety attached to the IgG constant fragment can dramatically alter the pro- and anti-inflammatory activities of IgG. This review will focus on sialic acid residues as a modulator of the anti-inflammatory activity and provide an overview of situations where serum IgG glycosylation and sialylation is altered and which molecular and cellular pathways may be involved in this immunomodulatory pathway.

IVIg-mediated amelioration of ITP in mice is dependent on sialic acid and SIGNR1.

Intravenous immunoglobulin G (IVIg) therapy is widely used to treat autoimmune and inflammatory diseases. Recent evidence suggests that in mice, splenic resident cells might be important for the anti-inflammatory activity of IVIg in a model of serum transfer arthritis. Splenectomized human immunothrombocytopenia (ITP) patients, however, still respond to IVIg therapy. To investigate whether the requirement of the spleen is essential for mouse ITP, we used a passive model of induced ITP and demonstrated that IVIg activity was functional in splenectomized animals. Further analysis showed that the IVIg-mediated amelioration of platelet phagocytosis was fully dependent on terminal sialic acid residues in the IVIg preparation and could be blocked with a specific ICAM3 grabbing nonintegrin-related 1 (SIGNR1) specific antibody. These results suggest that, similar to the human system, a spleen-independent but sialic acid- and SIGNR1-dependent pathway is responsible for IVIg-mediated suppression of autoantibody-dependent platelet depletion in mice.

Monocyte subsets responsible for immunoglobulin G-dependent effector functions in vivo.

Immunoglobulin G (IgG) antibodies confer protection against pathogenic microorganisms, serve as therapeutics in tumor therapy, and are involved in destruction of healthy tissues during autoimmune diseases. Understanding the molecular pathways and effector cell types involved in antibody-mediated effector functions is a prerequisite to modulate these activities. In this study we used two independent model systems to identify innate immune effector cells required for IgG activity in vivo. We first defined the precise repertoire of receptors for the IgG Fc fragment (FcγR) on innate immune effector cells in the blood and on tissue-resident macrophage populations. Despite expression of relevant activating FcγRs on various phagocyte populations, our data indicate that the majority of these cell types are dispensable for IgG activity in vivo. In contrast, IgG-dependent effector functions were selectively impaired in animals lacking the CX(3)CR1(hi)Ly6C(lo)CD11c(int) monocyte subset, which expressed the full set of FcγRs required for IgG activity.