Inessa Schwab

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.

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.

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.

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

Significance IgG fragment crystallizable domain (Fc) sialylation has emerged as an important but controversial concept for regulating anti-inflammatory activity of antibodies. Moreover, translating this concept to potent anti-inflammatory therapeutics has been hampered by the difficulty of generating suitable sialylated products for human use. We describe for the first time, to our knowledge, the development of a robust, scalable process to generate a sialylated intravenous immunoglobulin (IVIg) drug candidate with maximum Fc sialylation devoid of unwanted modifications. By using a wide panel of physicochemical analytics and in vivo models, we have validated the quality and potent anti-inflammatory activity of this clinical candidate. This report not only confirms the controversial anti-inflammatory activity of IgG-Fc sialylation, it also represents the first sialylated IVIg preparation, to our knowledge, with consistent anti-inflammatory potency suitable for clinical development. 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.

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.

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.

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.

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.

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.

Role of sialylation in the anti‐inflammatory activity of intravenous immunoglobulin – F(ab′)2 versus Fc sialylation

Immunoglobulin (Ig)G antibodies play an important role in the defence against pathogenic microorganisms, but are also responsible for tissue destruction and inflammation during autoimmune diseases such as rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). Paradoxically, pooled IgG preparations from thousands of donors [intravenous immunoglobulin (IVIg)] are also an efficient treatment to suppress several autoimmune diseases and chronic inflammation. Research has highlighted the role of the sugar domain attached to the IgG Fc-fragment as a molecular switch, which can enhance or block the pro- and anti-inflammatory effector functions of the antibody molecule. Indeed, deglycosylation of serum IgG in mice with active autoimmune diseases results in an amelioration of autoantibody-dependent inflammation 1. Of note, altered IgG glycosylation patterns, containing low levels of terminal galactose and sialic acid residues, and single nucleotide polymorphisms in glycosyltransferase genes have been associated with active autoimmune disease in RA, SLE and Crohns disease, for example 2–5. Furthermore, IVIg infusion was shown to induce sialylated IgG glycovariants in patients with Kawasaki disease and correlated with a prolonged treatment response 6. Together with the results of several studies showing that sialylated IgG glycovariants are critical for the anti-inflammatory activity of IVIg in several in vivo model systems, this has led to proposing a model in which one possible mechanism of IVIg therapy may be to replenish these active anti-inflammatory and immunomodulatory IgG glycovariants, thereby re-establishing immune homeostasis. However, sialic acid-containing sugar moieties can attach to the IgG Fc- or F(ab′)2-fragment, and evidence showing the relevance of both these sialylated sugar domains for IVIg activity has been reported recently.