Antibody glycosylation in neuroimmune diseases

Abstract

Background

Glycosylation, a critical post-translational modification of proteins, is particularly evident in immunoglobulins (Ig), also known as antibodies, and plays a significant role in various neuroimmune diseases. Of particular importance is the N-glycosylation of the crystallizable fragment (Fc) of IgG. It has a straightforward structure and directly impacts antibody effector functions, thus making it a focal point in antibody glycosylation studies.

Methods

This review systematically summarizes characteristic IgG glycosylation patterns in neuroimmune disorders, including multiple sclerosis (MS), neuromyelitis optica spectrum disorders (NMOSD), Guillain–Barré syndrome (GBS), chronic inflammatory demyelinating polyradiculoneuropathies (CIDP), and myasthenia gravis (MG).

Results

A prevailing pattern of reduced IgG galactosylation, sialylation, and core fucosylation has been consistently observed across these diseases, and these alterations are intricately linked to a pro-inflammatory shift in antibody functionality. Beyond pathological mechanisms, this review further explores the potential clinical value of IgG glycosylation, emphasizing its applications as a biomarker and as a target for innovative therapeutic strategies, such as the design of engineered therapeutic monoclonal antibodies and the use of glycosidases.

Conclusions

The specific alteration of IgG glycosylation is a hallmark of neuroimmune diseases, contributing to their pathogenesis and offering substantial translational promise. Harnessing this knowledge for the development of glycosylation-based diagnostics and therapeutics represents a significant frontier in the management of neuroimmune diseases.

Introduction

Glycosylation, a crucial post-translational modification of proteins, is present in about 50% of genes encode proteins [1]. This modification plays a fundamental role in various biological functions. Glycoproteins consist of glycans and protein components, with the glycan portion being a key player in numerous biological processes and implicated in various diseases such as autoimmune conditions, tumors, and infections. However, due to the intricate nature of glycans and the lack of suitable analytical tools, research on glycans lags that of other biomolecules [2]. Immunoglobulin (Ig), also known as an antibody, is a glycoprotein produced by the adaptive immune system. Based on its heavy chain characteristics, Ig can be categorized into five types: IgG, IgM, IgA, IgE, and IgD. In serum, IgG accounts for approximately 75% of circulating Ig, IgA 15%, IgM 10%, and there are also very small amounts of IgD and IgE [3]. Two types of glycans are found attached to protein part of Ig: N-glycans and O-glycans. N-glycans typically attach to the nitrogen atom of asparagine (Asn, N) within the conserved Asn-Xaa-[Ser/Thr] sequence (where Xaa can be any amino acid except proline), while O-glycans typically link to the oxygen atom of serine (Ser, S) or threonine (Thr, T) without reliance on a specific amino acid sequence [4, 5].

IgG, the predominant immunoglobulin in humans, exhibits the longest half-life period among all Ig classes. Engaging in crucial biological activities, IgG stands out as one of the prevalent N-glycosylated proteins in serum. It comprises four subclasses (IgG1-4), with IgG1 emerging as the principal subclass [6]. Among all Ig classes, IgG exhibits the most straightforward glycosylation pattern. Each IgG subclass (IgG1-4), except IgG3, contains a single highly conserved N-glycosylation site where glycans attach specifically at position Asn 297 within the constant regions 2 (CH2) of both heavy chains [7]. The effector functions of IgG are predominantly influenced by the N-glycans present on its heavy chains [8]. These glycans are oligosaccharides comprising around 15 monosaccharide residues, constituting approximately 15% of the total IgG weight. They exist in three main forms, including complex, high-mannose and hybrid, with complex-form N-glycan IgG being the most prevalent, representing over 98% in healthy individuals [9] (Fig. 1). The glycans found in the antigen-binding fragment (Fab) and the crystallizable fragment (Fc) predominantly consist of complex-type N-glycans [10–13]. These glycans share core structures comprising heptasaccharides consisting of four N-acetylglucosamine (GlcNAc) and three mannose (Man) residues [14]. Moreover, they feature fucose (Fuc), bisecting GlcNAc, galactose (Gal), and sialic acid (Sial) residues [14]. Given the high prevalence of IgG in the body, its significant functional role, and the relatively simple nature of IgG glycosylation, it has garnered considerable attention in antibody glycosylation research [6, 7].

Fig. 1.

Glycosylation of IgG. The Asn297 site in the IgG Fc region can carry more than 30 different N-glycan structures, mainly complex forms, with only a very small amount of high-mannose and hybrid forms. All N-glycans have the same pentasaccharide core structure, containing 2 GlcNAc and 3 Man. N-linked glycans are attached to the nitrogen atom of Asn within the conserved Asn-Xaa-[Ser/thr] sequence (where Xaa can be any amino acid except proline). Asn, N, asparagine; GlcNAc, N-acetylglucosamine; Man, mannose; Ser, S, serine; Thr, T, threonine

IgG plays a crucial role in the pathogenesis of various neuroimmune diseases, including myasthenia gravis (MG) [15], Guillain–Barré syndrome (GBS) [16, 17], chronic inflammatory demyelinating polyradiculoneuropathies (CIDP) [16, 17], neuromyelitis optica spectrum disorders (NMOSD) [18, 19], multiple sclerosis (MS) [20, 21], anti-myelin oligodendrocyte glycoprotein IgG antibody-associated disease (MOGAD) [22], and autoimmune encephalitis (AE) [23]. For example, acetylcholine receptor (AChR) antibodies can be detected in over 80% of MG patients [24]. AChR antibodies are mainly IgG antibodies, among which the IgG1 and IgG3 subclasses are the main ones. The Fab fragment of these antibodies identifies antigenic epitopes, exhibiting neutralizing and opsonizing properties, while the Fc fragment facilitates antibody effector functions by engaging with complement or Fc receptors, leading to complement activation, phagocytosis, and cytotoxicity. Among these, complement activation is the most important [15, 25]. The maintenance of the half-life of IgG in the blood depends on the neonatal Fc receptor (FcRn). FcRn is widely expressed in human vascular endothelial cells, antigen-presenting cells, intestinal epithelial cells, etc. By enhancing or weakening the interaction between FcRn and IgG, the half-life of IgG in the circulation can be controlled [26]. The pathogenic IgG antibody is closely related to the onset of the above-mentioned neuroimmune diseases, but the antibody titer not completely parallel to the disease severity [15, 19, 27, 28]. Considering the important role of IgG glycosylation in its downstream effector functions and its close relationship with various autoimmune diseases, we have reason to infer that IgG glycosylation is likely to play an important role in the pathogenesis of neuroimmune diseases.

This paper outlines the roles of IgG glycosylation in neuroimmune diseases, elucidates the potential clinical applications of IgG glycosylation in neuroimmune diseases, and highlights the extensive treatment possibilities based on IgG glycosylation regulation.

Antibody glycosylation - glycan biosynthesis and glycan structures

Antibody glycosylation can be classified into N-linked glycosylation and O-linked glycosylation based on the anchoring position of glycans. In N-linked glycosylation, N-glycans are typically attached to the nitrogen atom of Asn in the consensus sequence, consisting of Asn-Xaa-[Ser/Thr] (where Xaa can be any amino acid except for proline), and the sugar directly attached to Asn is a GlcNAc [4, 29]. The N-glycosylation process can be divided into two steps. First, precursor glycans are co-translationally or post-translationally attached to the Asn residues in the endoplasmic reticulum (ER). Then, the protein enters the Golgi apparatus (GA), where the precursor glycans with high-mannose structures (glycans with five to nine mannose residues) can be trimmed down and further extended through the interaction of glycosidases and glycosyltransferases to form complex N-glycans, thus completing the synthesis of antibody N-glycosylation [30–33]. The Asn297 site in the IgG Fc region can carry more than 30 different N-glycan structures, mainly complex forms, with only a very small amount of high-mannose and hybrid forms [11–13]. All N-glycans have the same pentasaccharide core structure, containing 2 GlcNAc and 3 Man. In complex-form N-glycans, GlcNAc residues are further attached to the core Man residues of each branch, forming a common core structure containing 4 GlcNAc and 3 Man, and Gal and Sial can be further attached on this basis. In addition, the core Fuc attached to the first GlcNAc and the bisecting GlcNAc attached to the core Man are also common in IgG complex-form N-glycans [34, 35]. The glycans on each Asn 297 may vary among different IgGs, mainly being biantennary complex-form N-glycans [36]. Under physiological conditions, the different glycans carried by IgG are relatively stable at the individual level. Approximately 35% are agalactosylated, nearly 40% are monogalactosylated, 15% are digalactosylated, and about 5%–20% are sialylated. More than 90% of IgG has core fucosylation, and about 10%-15% of IgG contains bisecting GlcNAc [36–40]. There are also N-glycosylation sites in the variable region of the IgG Fab segment. These sites are acquired during the somatic hypermutation process and are not present in all IgGs, only in 15%–25% of circulating IgGs [10, 41]. In O-linked glycosylation, O-glycans are exclusively attached post-translationally in the GA to Ser or Thr residues and do not depend on a specific amino acid sequence [4, 7]. Compared with N-glycans, O-glycan chains are generally short (1–3-oses) and variable, and are only found in specific antibody classes such as IgG3, IgA, and IgD [4].

Impact of glycosylation on antibody effector functions

IgG can mediate a variety of effector functions, including pro/anti-inflammatory effects, antibody dependent cellular cytotoxicity (ADCC), antibody dependent cellular phagocytosis (ADCP), complement dependent cytotoxicity (CDC), etc. The glycosylation profile of IgG plays a crucial role in modulating the effector functions facilitated by IgG [42, 43].

Fucosylation

Under physiological conditions, more than 90% of IgG exhibits core fucosylation. Core fucosylation significantly influences the effector functions of IgG, especially ADCC. In ADCC, the Fc region of IgG binds to FcγRs (including FcγRI, FcγRII, FcγRIIIa, and FcγRIIIb) on the surface of killer cells (including natural killer (NK) cells, macrophages, neutrophils, etc.), mediating the killing of target cells by killer cells [44]. The core fucosylation at the Asn297 site of IgG significantly affects the affinity between the IgG Fc region and FcγRIIIa (commonly expressed on the surface of NK cells and macrophages) and FcγRIIIb (commonly expressed on the surface of neutrophils). This phenomenon is observed in all IgG subtypes (including IgG1, IgG2, IgG3, IgG4), and the degree of increased affinity when core fucosylation is removed depends on the IgG subtype [45–49]. Studies have shown that non-core-fucosylated IgG can significantly enhance the NK cell-mediated ADCC effect through increased affinity with FcγRIIIa [45, 50, 51]. However, IgG core fucosylation does not affect its binding to other Fcγ receptors (including FcγRI and FcγRII), nor does it affect the effector functions mediated by these receptors [52]. Some studies have also shown that IgG core fucosylation has no significant effect on complement activation [53]. In terms of treatment, since removing core fucosylation can enhance the ADCC effect mediated by IgG, therapeutic monoclonal antibodies often utilize this modification to improve treatment efficacy. Currently, some non-fucosylated therapeutic antibodies have been approved for marketing or are in the clinical research stage [54].

Bisecting N-acetylglucosamine

Under physiological conditions, approximately 10–15% of IgG carries bisecting GlcNAc. Studies have shown that IgG with a higher proportion of bisecting GlcNAc has a higher affinity for FcγRIIIa, which can enhance the ADCC effect [55–57]. However, the presence of bisecting GlcNAc may restrict the addition of core fucose by the corresponding fucosyltransferase enzyme through steric effects. Therefore, the presence of bisecting GlcNAc often coincides with the absence of core fucose, and the enhanced ADCC effect may also be related to this [57–60]. Some studies have also shown that when the level of core fucose is fixed, increasing the level of bisecting GlcNAc can still enhance the ability of IgG to mediate ADCC [56, 61]. In addition, current studies suggest that bisecting GlcNAc has no significant effect on complement activation [61, 62].

Galactosylation

Under physiological conditions, approximately 35% of IgG is agalactosylated (G0), nearly 40% is monogalactosylated (G1), and 15% is digalactosylated (G2). Studies have shown that galactosylation can increase the affinity of IgG for C1q and enhance the CDC effect, which is mainly observed in the two IgG subtypes, IgG1 and IgG3 [63–67]. Additionally, research has found that galactosylation also enhances the affinity of IgG for FcγRIIa/b and FcγRIIIa [46, 53, 63, 68, 69]. The enhanced affinity of IgG for FcγRIIa and FcγRIIIa can improve ADCC activity, although this effect may be related to the absence of core fucose [69, 70]. On the other hand, the enhanced affinity of IgG for FcγRIIb can exert an anti - inflammatory effect. Specifically, after galactosylation-rich IgG immune complexes (ICs) bind to Galectin-3 (a galactose-binding lectin), they can promote the cross-linking of ICs with FcγRIIb and Dectin-1 (a C-type lectin expressed by neutrophils) [70–72]. This effect can synergistically inhibit the C5a-mediated inflammatory response in neutrophils, thereby exerting an anti-inflammatory effect [71, 72]. Multiple studies have shown that galactosylated IgG has an anti-inflammatory effect, and in inflammatory and autoimmune diseases, the level of IgG galactosylation decreases significantly [37, 73–76]. However, as mentioned above, IgG galactosylation can promote IgG-C1q binding and downstream activation, which seems contradictory. Existing research has provided a reasonable explanation for this. During the onset stage of autoimmune diseases, the total IgG galactosylation is relatively low. The reduced binding of non-specific low-galactosylated IgG to C1q lowers the activation threshold of C1q, resulting in a relative increase in complement activation after pathogenic IgG binds to its corresponding antigen [65]. Correspondingly, under physiological conditions or during the remission stage of autoimmune diseases, the total IgG galactosylation is relatively high. Non-specific high-galactosylated IgG binds more C1q, but non-specific IgG does not bind to pathogenic antigens to form highly active IgG hexamers and activate downstream complement [65, 77, 78].

Sialylation

Under physiological conditions, approximately 5%-20% of IgG is sialylated. The impact of sialylation on IgG effector functions has been widely studied, but the specific mechanism is not yet fully understood, and the results of different studies also have certain controversies. Studies suggest that IgG sialylation can influence the binding of IgG to C1q and subsequent complement activation [53, 67, 78]. However, divergent outcomes have been reported. For instance, one study demonstrated that sialylation of monoclonal IgG1 led to a notable decrease in both C1q binding and complement activation [67]. In contrast, another study indicated that the presence of sialylated IgG resulted in enhanced complement activation compared to IgG with only galactosylation [53]. The binding of IgG to FcγRs and its downstream effector functions are also affected by sialylation, but the results are also controversial. Most existing studies suggest that the binding ability of sialylated IgG to FcγRs is similar to that of galactosylated IgG [67, 69]. Sialylation plays a crucial role in modulating the inflammatory response and is pivotal in regulating the dual anti-inflammatory and pro-inflammatory functions of IgG antibodies. Reduced levels of IgG sialylation have been observed in autoimmune conditions such as rheumatoid arthritis (RA) [74, 79–81], systemic lupus erythematosus (SLE) [82], inflammatory bowel disease (IBD) [75, 83, 84], antiphospholipid syndrome (APS) [85], among others. Conversely, elevated levels of IgG sialylation have been reported in certain tumor contexts [86–88].

Mannosylation

Under physiological conditions, high-mannose form IgG accounts for only about 1% of the total IgG glycan chains. However, therapeutic monoclonal antibodies may contain a relatively high proportion of high-mannose chains [10, 11, 89]. Studies have shown that compared with complex and hybrid form IgG, high-mannose form IgG has a higher affinity for FcγRIIIa and can enhance ADCC activity. Nevertheless, this effect may be related to the fact that high-mannose form IgG also lacks core fucose [63, 90, 91]. Additionally, compared with complex form IgG, high-mannose and hybrid form IgG have a lower affinity for C1q and a weaker ability to induce CDC [63, 91].

Antibody glycosylation in neuroimmune diseases

Glycosylation plays a crucial role in modulating the downstream effector functions of IgG antibodies, thereby impacting the pathogenesis and progression of diseases. This phenomenon offers a novel perspective for investigating the pathophysiological mechanisms of diseases and identifying potential targets for disease diagnosis and treatment. In a seminal study dating back to 1985, Parekh et al. initially documented variations in IgG glycosylation associated with diseases, noting a reduction in galactosylation levels in RA [92]. Subsequent research has corroborated these findings, revealing altered glycosylation profiles of IgG antibodies in autoimmune conditions such as RA, SLE, IBD, and APS. These glycosylation modifications are intricately linked to the initiation, activity, and progression of these diseases. Notably, there is a growing body of literature investigating antibody glycosylation in neuroimmune disorders.

Mechanisms of antibody glycosylation in neuroimmune diseases

In antibody-mediated neuroimmune diseases, there are common pathogenic mechanisms, including multiple steps such as antibody production, antibody circulation, and antibody effects [93] (Fig. 2). During the antibody production stage, due to defects in B-cell tolerance checkpoints, various triggering factors such as infection and tissue injury lead to the transport of autoantigens to lymph nodes. With the help of T cells, autoreactive B cells are stimulated by antigens to form germinal centers. B cells further transform into plasma blasts and plasma cells, secreting a large amount of autoantibodies. A study in a human B-cell line derived from germinal centers showed that ɣ-heavy chain (ɣHC) glycans do not affect the membrane expression of IgG-B-cell receptor (BCR), moreover, antigen binding and other BCR-related mechanisms are also unaffected, including BCR downmodulation and BCR-mediated signaling [94]. During the antibody circulation stage, the binding of IgG to FcRn is particularly crucial for maintaining the half-life of IgG in the blood circulation. Currently, there is controversy regarding whether IgG glycosylation affects the binding of IgG to FcRn. Some studies have shown that glycosylation affects the interaction between IgG1 and FcRn to a certain extent, and deglycosylated IgG exhibits lower affinity [68]. Other studies have found that although both the IgG N-glycosylation site and the IgG-FcRn binding site are located in the Fc segment, they are relatively far apart, and the interaction between IgG and FcRn is independent of glycosylation [95–98]. During the antibody effect stage, the Fab segment of autoantibodies recognizes antigenic epitopes, and the Fc segment interacts with Fc receptors or complement, mediating multiple downstream effector functions including ADCC and CDC, thereby exerting pathogenic effects. Numerous studies have shown that N-glycosylation of the IgG Fc segment significantly affects the downstream effector functions of IgG. The affinity of IgG lacking N-glycosylation in the Fc segment for Fcγ receptors and complement C1q is significantly weakened, which in turn greatly reduces its ability to induce downstream effector functions such as ADCC and CDC [68, 99]. Meanwhile, as mentioned earlier, the specific glycan structure of N-glycosylation in the IgG Fc segment also significantly affects the effector functions of IgG.

Fig. 2.

Pathogenic mechanisms in antibody-mediated neuroimmune diseases: the role of glycosylation. Antibody-mediated neuroimmune diseases share common pathogenic mechanisms involving antibody production, circulation, and effects. Glycosylation primarily impacts antibody effects, and there is still controversy about the effect on antibody circulation. Current research indicates minimal impact of antibody glycosylation on antibody production

Antibody glycosylation in multiple sclerosis

MS is an immune-mediated autoimmune disease of the central nervous system (CNS), characterized by inflammatory demyelination of neuronal axonal fibers in the brain and spinal cord. This alteration results from the complex and dynamic interactions among the immune system, glial cells, and neurons [20, 21]. An important feature of CNS inflammation in MS is the intrathecal synthesis of IgG and the presence of cerebrospinal fluid (CSF)-specific oligoclonal bands (OCB), which can be detected in over 90% of patients [100]. Current studies generally suggest that the IgG glycosylation pattern in CSF differs from that in serum, and significant changes in the IgG glycosylation pattern occur in the CSF of MS patients. However, it remains unclear whether the IgG glycosylation pattern changes in the serum of MS patients, and most studies indicate no obvious alterations [101–103]. Compared with healthy controls, the levels of IgG galactosylation and sialylation in the CSF of MS patients are decreased, while the levels of bisecting GlcNAc and mannosylation are increased. These changes can enhance the effector functions of IgG and promote the inflammatory response in the disease state [101–104]. Some studies suggest that the level of IgG core fucosylation is decreased in the CSF of MS patients, but this remains controversial [101, 104]. Another study has found that the specific IgG glycans in the serum of MS patients changes, with a decrease in H3N4F1 glycan content and an increase in H3N5F1 and H5N4F1 glycan content. In these individuals, reduced levels of bisecting GlcNAc and sialylation were also observed [105]. Based on the above studies, changes in the IgG glycosylation pattern may serve as potential biomarkers for MS, facilitating early disease diagnosis, prediction of relapse, and disease activity. Additionally, some studies have found that the level of IgG Fab fragment glycosylation is increased in MS, and this change is not affected by B-cell depletion therapy, showing potential as a predictive biomarker [106].

Antibody glycosylation in neuromyelitis optica spectrum disorders

NMOSD is a chronic inflammatory autoimmune disease of the central nervous system, characterized by acute optic neuritis and myelitis. More than 80% of patients have autoantibody IgG against aquaporin-4 (AQP4) in their bodies [18, 19]. Existing studies have found through lectin enzyme-linked immunosorbent assay (ELISA) that the galactosylation of IgG in the serum of NMOSD patients decreases during relapse, and it is significantly correlated with the severity of the disease [107]. However, there is currently no study systematically characterizing the N-glycosylation pattern of antibody IgG in NMOSD by methods such as mass spectrometry. In addition, other studies have shown that endoglycosidase S (EndoS) can reduce the CDC and ADCC in the serum of NMO patients by 95% without affecting the binding of AQP4-IgG to AQP4. The non-pathogenic IgG treated with EndoS competitively replaces the pathogenic IgG that can bind to AQP4, preventing NMO pathology in spinal cord slice culture and mouse models of NMO [108]. Therefore, using EndoS to neutralize pathogenic AQP4-IgG provides a feasible new idea for the treatment of NMO.

Antibody glycosylation in immune-mediated peripheral neuropathies

Immune-mediated peripheral neuropathies (IMPN) are a complex and diverse group of neurological disorders that can be caused by the immune response of the peripheral nervous system (PNS) to self-antigens, encompassing various clinical types such as GBS, CIDP, and multifocal motor neuropathies (MMN) [16, 17]. Studies have found that in patients with CIDP, inducing an increase in the sialylation level of IgG Fc can reduce the level of complement activation, which is associated with clinical disease remission [67]. Another study investigated the relationship between serum IgG N-glycosylation and clinical course and outcome in GBS patients before and after intravenous immunoglobulin (IVIg) treatment. It was found that untreated GBS patients had lower galactosylation levels of IgG1 and IgG2. In addition, in some patients, despite receiving IVIg treatment, the levels of IgG galactosylation and sialylation remained low, and these patients had the most severe symptoms, and a higher proportion required ventilator support [109]. In conclusion, in peripheral neuropathies, IgG glycosylation is related to disease severity and clinical recovery, and it may be promising for monitoring disease progression and treatment efficacy.

Antibody glycosylation in myasthenia gravis

MG is a T cell-dependent, antibody-mediated, and complement-involved autoimmune disease of the nervous system, and antibodies are the key pathogenic factors. Glycosylation, as an important post-translational modification, significantly affects antibody function. However, current research on antibody glycosylation in MG is relatively scarce. Currently, only one study has preliminarily characterized the N-glycosylation changes in the Fc region of IgG in patients with Lambert Eaton syndrome (LEMS) and MG. It was found that the glycosylation levels of IgG1 and IgG2 in the disease group were lower than those in the control group, and the galactosylation level in the disease group was also low. However, this study did not deeply explore the possible pathogenic mechanisms of glycosylation changes and the guiding role in drug efficacy [110]. In addition, some literature has reported the glycosylation changes in the Fab region of IgG in MG. Studies have shown that the N-glycosylation in the V region of IgG in MG patients increases, and this change is mainly observed in muscle-specific kinase (MuSK) antibody IgG, while no change in glycosylation level is observed in the total antibody IgG and AChR antibody IgG of patients [111, 112]. Further investigation revealed that in MG patients positive for MuSK antibodies, the level of sialylated IgG4 subtype antibodies in the Fab region significantly increases [112]. It is worth noting that in MG patients, the titer of pathogenic AChR antibody IgG is not completely parallel to the severity of MG, which may be caused by multiple factors, including differences in antibody binding and cross-linking with complement, differences in immunoglobulin classes, and the presence of more than one specific pathogenic antibody in the same patient [113]. These factors may lead to high antibody titers in some MG patients with relieved symptoms or only ocular symptoms, while some MG patients with severe symptoms have low antibody titers. As mentioned above, IgG antibody glycosylation plays an important role in various antibody effector functions, including complement activation, and the IgG antibody glycosylation profile changes in MG. Therefore, IgG glycosylation is very likely to cause the difference between antibody titer and disease severity to some extent and play a key role in the pathogenesis of MG.

Clinical potential of antibody glycosylation

The clinical potential of antibody glycosylation encompasses three key areas: serving as a biomarker for disease surveillance, a crucial parameter for engineering therapeutic antibodies, and a direct therapeutic target (Fig. 3). This section delves deeply into these applications to emphasize the growing clinical significance of antibody glycoengineering.

Fig. 3.

Clinical potential of antibody glycosylation. For pathogenic antibodies, reduced galactosylation, sialylation, and core fucosylation may be effective disease markers for neuroimmune diseases. Modulating antibody glycosylation presents a novel approach for the effective treatment of autoimmune diseases, including neuroimmune diseases. In therapeutic antibodies, manipulating the N-glycosylation of the Fc segment can optimize safety and improve efficacy

Clinical potential of antibody glycosylation as a biomarker

Research suggests that the glycosylation profiles of specific antibodies can function as effective biomarkers for early disease detection, monitoring disease progression, predicting relapse, and evaluating disease prognosis in various conditions, including autoimmune diseases [73, 76, 83, 114–121]. Utilizing the nano hydrophilic interaction chromatography (HILIC)-LC-MS/MS method, a recent study has delineated the glycosylation profiles of human IgG subclasses (IgG1, IgG2, IgG3, IgG4), the analysis revealed that IgG1 and IgG3 display predominant galactosylation of the 6-branched antenna, IgG2 instead of the 3-branched antenna, while IgG4 displays a balance [122]. Notably, these glycosylation patterns exhibit stability in healthy individuals but diverge between recombinant IgG and plasma IgG. These observations bear significant implications for antibody-based therapeutics by enhancing our comprehension of the impact of glycosylation on IgG functionality and offering promise for biomarker discovery. Alterations in the glycosylation patterns of IgG have been observed in multiple autoimmune diseases before the onset of clinical symptoms, and these modifications tend to remain consistent over time. For instance, reduced galactosylation and sialylation of IgG have been linked to the eventual diagnosis of RA [73]. Moreover, changes in IgG glycosylation are related to alterations in the inflammatory state and disease activity of RA patients [1]. In Crohn’s disease (CD), researchers analyzed the IgG glycosylation profiles of patients six years before diagnosis and found a stable decrease in the galactosylation level of the IgG Fc segment. Additionally, these IgG glycan changes are associated with the recognition of mannan glycans by pathogenic anti-Saccharomyces cerevisiae (ASCA) antibodies [115]. Furthermore, other studies have found that IgG glycosylation characteristics can be used to predict the active transformation of tuberculosis, the severity of influenza, future cardiovascular events, and cancer, etc. [117–121]. While the integration of antibody glycosylation-based biomarker detection into clinical practice is currently lacking, advancements in diagnostic tools, such as lectin detection and high-throughput screening methods, suggest that the specific IgG glycosylation profiles of patients could serve as effective markers for disease diagnosis and detection in clinical settings in the foreseeable future.

Clinical potential of glycosylation of therapeutic monoclonal antibodies

In recent years, the research, development, and launch of various novel targeted biological agents have provided more options for the treatment of neuroimmune diseases [15]. Optimizing therapeutic monoclonal antibodies through post-translational modifications such as glycosylation has become a new direction in drug research and development [54, 123]. The exploration of the therapeutic potential of glycosylation of therapeutic monoclonal antibodies is manifested in multiple aspects, including improving efficacy, optimizing safety, and prolonging the half-life. Adjusting the antibody glycosylation pattern can enhance efficacy [124, 125]. For example, reducing core fucosylation enables antibodies to activate immune cells more effectively, enhancing the ADCC effect and thereby more potently killing target cells [54]. The antibody glycosylation pattern also affects drug safety [126]. Abnormal glycan structures may erroneously activate the human immune system, affect drug efficacy, and even trigger adverse reactions. Adjusting the antibody glycosylation pattern can reduce the risk of immunogenicity [127]. Moreover, adjusting the antibody glycosylation pattern allows antibodies to better bind to the FcRn receptor in the body, prolonging the half-life of antibody drugs in the blood circulation, reducing the frequency of administration, and improving patient compliance [128, 129]. Therefore, optimizing the performance of therapeutic monoclonal antibodies by adjusting the antibody glycosylation pattern is particularly important for the treatment of neuroimmune diseases and can also serve as a new therapeutic strategy for various diseases such as cancer and infections.

FcRn inhibitors have attracted extensive attention as innovative treatment modalities for IgG-related neuroimmune diseases. By blocking IgG circulation, FcRn inhibitors can reduce the levels of pathogenic IgG antibodies and improve patients’ symptoms. Representative drugs include efgartigimod, rozanolixizumab, batoclimab, and nipocalimab [26]. Among them, nipocalimab is a high-affinity FcRn inhibitor with an aglycosylated design, which eliminates its immune effector functions, avoids CDC and ADCC effects, and further optimizes safety [126]. Cluster of differentiation (CD) 20 monoclonal antibodies, as a targeted B-cell therapy, can specifically bind to CD20 on the surface of pre-B cells and mature B cells, leading to B-cell depletion. Representative drugs include rituximab, obinutuzumab, etc. [130]. CD20 monoclonal antibodies can be used to treat various neuroimmune diseases and are also commonly used in tumor treatment [130–132]. Obinutuzumab is a humanized anti-CD20 monoclonal antibody. The glycosylation modification of its Fc segment (reduced core fucosylation) can enhance its affinity with immune effector cells, thereby enhancing the ADCC effect and enabling it to kill B cells more potently. Its B-cell depletion activity and efficacy are significantly superior to those of rituximab [54, 124, 133]. In addition, some studies have attempted to optimize the glycosylation patterns of therapeutic monoclonal antibodies such as bevacizumab, adalimumab, and palivizumab to improve treatment efficacy for diseases such as autoimmune diseases, cancers, and infections [125, 134, 135]. In general, the glycosylation of therapeutic monoclonal antibodies has broad clinical application prospects, providing new breakthroughs for the development and clinical application of antibody drugs.

Developing efficient glycosylation technologies for therapeutic monoclonal antibodies holds great value. Sotomayor et al. discovered an oligosaccharyltransferase (OST) in Escherichia coli that can add N-glycans to the N297 site of the IgG Fc region. This enzyme can catalyze the glycosylation of the natural receptor site in the Fc region. By means of chemoenzymatic methods, the bacterial glycans can be “upgraded” to functional glycans, enabling the E. coli-derived Fc protein to bind to human FcγRIIIa/CD16a and mediate the ADCC effect [136]. In summary, this study for the first time established a complete pathway from antibody expression, antibody glycosylation to glycan function restoration in a bacterial system, offering the potential to produce “functional therapeutic monoclonal antibodies” in a prokaryotic system that is cost-effective and easy to scale up.

Clinical potential of antibody glycosylation for therapy

Glycosylation modification of IgG antibodies significantly affects the downstream effector functions of antibodies and is closely associated with the onset, activity, and progression of diseases. Therapeutic approaches based on antibody glycosylation regulation hold great promise. In recent years, numerous studies have attempted to specifically alter the glycosylation traits of antibodies through various means and develop new glycan-based therapeutic approaches, providing novel insights into the treatment of autoimmune diseases, including neuroimmune diseases.

Research has shown that the EndoS secreted by Streptococcus pyogenes can specifically hydrolyze the core of the N-glycan in the Fc region of IgG, leading to the loss of the pro-inflammatory activity of IgG and interfering with the IgG-mediated pro-inflammatory processes in various autoimmune disease models [137, 138]. EndoS has a significant impact on the effector functions of IgG (such as complement activation and Fc receptor binding) through hydrolyzing IgG glycans and can serve as an immunomodulator for the treatment of IgG-mediated diseases [139]. However, traditional EndoS and multi-domain glycosidases in its family are large, difficult to engineer, and face obstacles in clinical development. Sastre et al. discovered a class of IgG-specific single - domain endoglycosidases (CU43) secreted by Corynebacterium diphtheriae and constructed an Fc fusion protein with endoglycosidase activity (CU43-Fc) based on it. CU43-Fc can alleviate various IgG-mediated diseases in vivo, and its in -vivo efficacy is at least 4000 times that of efgartigimod [97]. In addition, another study attempted to convert endogenous IgG into anti-inflammatory IgG through glycosyltransferases [140]. Specifically, in a mouse model of collagen-induced arthritis, researchers tried to convert endogenous IgG into an anti-inflammatory mediator by administering recombinant soluble β-1,4-galactosyltransferase1 (B4GALT1) and β-galactoside-α-2,6-sialyltransferase1 (ST6GAL1), resulting in a significant attenuation of autoimmune inflammation in mice. These enzymes act through a pathway similar to that of IVIG, and this process requires the participation of inhibitory FcγRIIB, STAT6, and type II FcγRs. Sialylation is highly specific to pathogenic IgG at the inflammatory site, and mouse platelets can locally release Gal or Sial as sugar donors without additional supplementation. These research results all emphasize the great therapeutic potential of in-vivo glycosylation regulation.

Another recent study has investigated the efficacy of a glyco-engineered IgG Fc variant as a cost-effective alternative to IVIG for treating autoimmune diseases [141]. The research findings indicate that the glyco-engineered IgG1 Fc variant, FcF241A, demonstrates significant anti-inflammatory properties in autoimmune models comparable to high-dose IVIG but with increased efficacy at lower dosages. Its mechanism of action is not reliant on sialylation, although sialylation can improve its serum half-life and bioavailability. When combined with efgartigimod, FcF241A exhibits synergistic anti-inflammatory effects. From a clinical perspective, FcF241A presents a promising and cost-effective substitute for IVIG in the treatment of autoimmune diseases, with the potential for enhanced therapeutic outcomes through combination therapies. However, most of the current treatment methods based on antibody glycosylation remain in the basic research stage and have not been integrated into clinical practice, which requires further in-depth exploration.

Conclusions

Glycosylation is a common antibody modification. Changes in glycan composition are directly related to various diseases, especially autoimmune diseases. Numerous studies have demonstrated a strong association between antibody glycosylation and autoimmune diseases. Recently, there has been a growing body of research focusing on antibody glycosylation in neuroimmune diseases, uncovering deviations in antibody glycosylation patterns in these conditions. Patients with neuroimmune diseases have IgGs with aberrant Fc glycan forms that lack galactose, sialic acid, and core fucose. Some of these atypical changes have the potential to serve as predictive markers for the onset, progression, and prognosis of neuroimmune diseases. Manipulating antibody glycosylation modifications can impact the functional properties of antibodies and modify the pathological mechanisms of diseases. Nonetheless, in comparison to autoimmune diseases like RA, investigations into antibody glycosylation in neuroimmune diseases remain relatively limited. Therefore, delving into the role of antibody glycosylation in the pathogenesis of neuroimmune diseases holds significant research value and necessitates further comprehensive exploration.

The emergence of novel targeted biological agents has significantly expanded the treatment options for neuroimmune diseases. These agents primarily target pathogenic antibodies and focus on three key stages: antibody production, circulation, and effects. For instance, rituximab, a CD20 monoclonal antibody, targets B cells to inhibit antibody production. Efgartigimod, an FcRn inhibitor, targets FcRn to impede antibody circulation. Eculizumab, a complement C5 inhibitor, targets the complement pathway to counteract antibody effects. Modifying the glycosylation pattern of therapeutic monoclonal antibodies and enhancing antibody functions are crucial strategies to enhance efficacy and safety, representing a novel direction in drug research and development. Moreover, the majority of the aforementioned targeted biological agents target a single step, resulting in limited efficacy in certain patient populations. Recent research efforts have focused on novel treatment strategies centered around antibody glycosylation, offering innovative avenues for addressing neuroimmune diseases. For instance, the glycosidase-Fc fusion protein discussed earlier functions as an FcRn inhibitor by competitively binding to the FcRn site with its Fc segment. By facilitating the removal of antibody glycosylation, it can modulate downstream effector mechanisms, leading to enhanced therapeutic outcomes. This approach demonstrates improved efficacy and is applicable to a wide range of patients, thus presenting promising clinical implications. Consequently, it is poised to introduce a novel therapeutic paradigm for neuroimmune diseases.

Abbreviations

AChR

Acetylcholine receptor

ADCC

Antibody dependent cellular cytotoxicity

ADCP

Antibody dependent cellular phagocytosis

AE

Autoimmune encephalitis

APS

Antiphospholipid syndrome

AQP4

Aquaporin-4

ASCA

Anti-Saccharomyces cerevisiae

Asn, N

Asparagine

B4GALT1

β-1,4-Galactosyltransferase1

BCR

B-Cell receptor

CD

Cluster of differentiation

CD

Crohn’s disease

CDC

Complement dependent cytotoxicity

CH2

Constant regions 2

CIDP

Chronic inflammatory demyelinating polyradiculoneuropathies

CNS

Central nervous system

CSF

Cerebrospinal fluid

ELISA

Enzyme-linked immunosorbent assay

EndoS

Endoglycosidase S

ER

Endoplasmic reticulum

Fab

Antigen-binding fragment

Fc

Crystallizable fragment

FcRn

Neonatal Fc receptor

Fuc

Fucose

GA

Golgi apparatus

Gal

Galactose

GBS

Guillain–Barré syndrome

GlcNAc

N-Acetylglucosamine

HILIC

Hydrophilic interaction chromatography

IBD

Inflammatory bowel disease

ICs

Immune complexes

Ig

Immunoglobulins

IMPN

Immune-mediated peripheral neuropathies

IVIg

Intravenous immunoglobulin

LEMS

Lambert Eaton syndrome

Man

Mannose

MG

Myasthenia gravis

MMN

Multifocal motor neuropathies

MOGAD

Anti-myelin oligodendrocyte glycoprotein IgG antibody-associated disease

MS

Multiple sclerosis

MuSK

Muscle-specific kinase

NK

Natural killer

NMOSD

Neuromyelitis optica spectrum disorders

OCB

Oligoclonal bands

OST

Oligosaccharyltransferase

PNS

Peripheral nervous system

RA

Rheumatoid arthritis

Ser, S

Serine

Sial

Sialic acid

SLE

Systemic lupus erythematosus

ST6GAL1

β-Galactoside-α-2,6-sialyltransferase1

Thr, T

Threonine

ɣHC

ɣ-Heavy chain

Author contributions

KL, ZL and HY defined the scope of the review. KL consulted related data and wrote this manuscript. KL, ML, LX, YO, ZL and HY contributed to the revision of the manuscript. All authors contributed to the article and approved the submitted version.

Funding

This work was supported by grants from the National Natural Science Foundation of China (No.82171399, 82101474, 82371413, 82571597), the Key Research and Development Program of Hunan Province (No.2024JK2115).

Data availability

Not applicable.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.