Harnessing IgG Fc glycosylation for clinical benefit

Abstract

The effector activity of IgG antibodies is regulated at several levels, including IgG subclass, modifications of the Fc glycan, and the distribution of Type I and II Fcγ receptors (FcγR) on effector cells. Here, we explore how Fc glycosylation, particularly sialylation and fucosylation, tunes cellular responses to immune complexes. We review the current understanding of the pathways and mechanisms underlying this biology, address FcγR in antigen presentation, and discuss aspects of the clinical understanding of Fc glycans in therapies and disease.

Introduction

Immune cells use a number of receptors to identify pathogens based on their expression of conserved antigens or pathogen-associated molecular patterns. Importantly, they also leverage aspects of the adaptive immune response, IgG antibodies in particular, to assist in pathogen recognition and internalization, and to enhance or suppress cellular activation and subsequent immune effector functions. The multivalent interaction between cells expressing Fc-gamma receptors (FcγRs) and IgG-opsonized pathogens (immune complexes; IC) enables crosstalk between the innate and adaptive immune systems to refine the overall response. Significant structural and functional heterogeneity exists within both IgG antibodies and FcγRs, which modulates the response of effector cells towards a pro-inflammatory or anti-inflammatory phenotype. In this review, we present the current understanding of how immune effector cells interact with IgG antibodies using Type I and Type II FcγRs, with a focus on fucosylation and sialylation of the Fc glycan to modulate pro- and anti-inflammatory effects.

IgG antibody glycosylation

IgG antibodies are among the most abundant immunomodulatory proteins in human serum and are mostly generated in response to infection, vaccination, or autoimmunity. The IgG isotype is comprised of four distinct subclasses of varying serum abundance, with IgG1 > IgG2 > IgG3 > IgG4 [1,2]. Differences in the amino acids within the constant region (Fc) of each IgG subclass influence binding to FcγRs, and the relative affinity of each subclass to activating versus inhibitory FcγRs, or A/I ratio, leads to differences in biological function of IgGs [2]. For example, IgG1 and IgG3 subclasses preferentially bind activating FcγRs and trigger complement activation compared to IgG2 and IgG4. The activating or inhibitory potential of each IgG subclass is further refined by a posttranslational modification of the Fc at asparagine-297 (N297) [3]. The combined presence or absence of galactose, sialic acid, bisecting N-acetylglucosamine, and core fucose within the Fc N-glycan can further modulate the interactions with FcγRs, thereby fine-tuning the activating and inhibitory signaling (Figure 1) [4–6]. Although the Fc glycan is highly conserved across IgG subclasses, the effect of IgG1 glycosylation on FcγR affinity is most well studied. Of the Fc glycan modifications, the presence or absence of the core fucose and the terminal sialic acids have the greatest impact on IgG1 Fc-mediated activity through modification of Type I and Type II FcγR affinity, respectively [7].

Figure 1.

Cartoon representation of the complex, biantennary, N-linked Fc glycan.

The regulation of Fc glycosylation is an important topic of ongoing investigation. Regulatory mechanisms of both intra-and inter-IgG Fc glycan patterns/repertoires exist, with intra-repertoire mechanisms driving common shifts in Fc glycoforms that are observed during vaccination or infection and inter-repertoire mechanisms resulting in broad distinctions in Fc domain glycosylation among individuals. One mechanism for intra-repertoire regulation occurs via activation of distinct B cell populations. For example, antibody responses likely generated by memory cell-derived plasmablasts are reduced in Fc sialylation and fucosylation relative to responses observed during a primary response [9–10]. The type of antigen encountered also impacts Fc glycoforms on newly produced IgGs. For example, T cell-independent antigens elicit IgGs enriched for Fc sialylation [11]. A recent study suggests that enveloped viruses, non-enveloped viruses, and soluble antigens each elicit IgGs with distinct Fc glycoforms, indicating that differences in antigen recognition and BCR signaling can impact regulatory pathways [12]. A variety of extrinsic factors that can be present during B cell activation and differentiation, such as CpG oligodeoxynucleotide or IL-21, may also drive selective Fc glycoform production [13].

Inter-repertoire regulation is likely driven by a combination of heritable and non-heritable factors. One of the better characterized pathways involved in both intra- and inter-repertoire regulation of Fc glycosylation is regulation by sex hormones to modulate the pro- or anti-inflammatory activity of IgG. Fc glycosylation changes with physiologic shifts in estrogen levels, such as in puberty [14] and menopause [15,16]. Sialylation, an active anti-inflammatory modification of the Fc, decreases during therapeutic estrogen suppression [17] and increases during pregnancy, directly supporting a role for estrogen in positive regulation of Fc sialylation [18,19]. The regulation of sialyation by estrogen has clinical implications for many autoimmune diseases, particularly those that occur predominantly in women. This is illustrated by studies of patients with rheumatoid arthritis (RA), whose baseline sialylation and galactosylation are decreased relative to healthy controls [20]. RA symptoms frequently go into remission during the high estrogen state of pregnancy [21] when sialylation and galactosylation are elevated [22,23]. A potential mechanism underlying this regulation was demonstrated by increased expression of St6Gal1, the relevant sialylatransferase, in RA patients who received exogenous estrogen treatment [24]. In a mouse model of RA, St6Gal1 was down-regulated by the well-characterized IL-23-activation of Th17 cells that occurs in arthritis [25]. Age and sex are generally associated with distinct patterns of Fc glycosylation with male sex or increasing age often correlating with decreased Fc sialylation and fucosylation [26]. More work is needed to define additional mechanisms for regulation of Fc glycosylation, but the potential to manipulate this determinant of IgG activity for clinical benefit is promising.

Type I receptors and IgG1 fucosylation

The Type I FcγRs are a small family of receptors that are critical in inflammatory homeostasis and appropriate maturation of antigen-presenting cells (Figure 2 and Table 1) [2,27–29]. Nearly all the Type I FcγRs exhibit low affinity for IgG and require multivalent interactions with IC to stabilize binding through avidity-based interactions [30]. FcγRI (CD64) is the sole exception, capable of monomeric binding to IgG1, IgG3, and IgG4. The majority of Type I FcγRs are activating through an activation signaling motif (ITAM) [31]. FcγRIIb (CD32b) and FcγRIIIb (CD16b) are exceptions: FcγRIIb has an inhibitory signaling motif (ITIM) and FcγRIIIb has no signaling motif. Crosslinking of activating FcγRs promotes ITAM phosphorylation and activation through the recruitment of Src family kinases and subsequent Syk activation, which initiates a variety of signaling cascades that contribute to cytoskeletal remodeling, calcium flux, and pro-inflammatory gene up-regulation. FcγRIIb crosslinking induces ITIM signaling that directly counteracts ITAM signaling through the recruitment of tyrosine and inositol phosphatases that dephosphorylate activated ITAMs and associated adapter molecules, overriding an activating response [32].

Figure 2.

Type I and Type II FcγRs. The Type I FcγRs are members of the immunoglobulin super family. The Type II FcγRs are members of the C-type lectin family.

Table 1.

Type I and II FcγR. Table includes various designations for each FcγR, relative affinity of each FcγR for IgG1 (fucosylated and not sialylated), and expression by distinct immune cell subsets.

	Type I FcγRs - immunoglobulin superfamily					Type II FcγRs - C-type lectins
FcγR designations	FcγRI	FcγRIIa	FcγRIIb	FcγRIIIa	FcγRIIIb	DC-SIGN	FcεRII
CD designation	CD64	CD32a	CD32b	CD16a	CD16b	CD209	CD23a/b
Affinity for IgG1	high	low	low	low	low	low	low
Expression of Type 1 and Type II FcγRs on immune cells
Dendritic cell	−/*	+	+	−/*	−	+	*
Macrophage	+	+	+	+	−	+	*
Non-classical monocyte	+	+	+/−	+	−	−/*	*
Classical monocyte	+	+	+/−	−	−	−/*	*
B cell	−	−	+	−	−	−	+
T cell	−	−	−	−/*	−	−	+/−
NK cell	−	−	−	+	−	−	−
Neutrophil	*	+	+/−	−	+	−	−

⁺represents positive expression,

⁻represents negative or unappreciable expression,

^+/−represents mixed expression,

^*represents inducible expression,

^−/*represents negative expression with some evidence of inducible expression.

Type I FcγRs are expressed in various combinations on immune cells [33] (Table 1). In instances of FcγR coexpression, the net signal received is a function of the combination of FcγR expression along with IgG subclasses and Fc glycans within an IC. In particular, the presence or absence of a core fucose within the IgG1 Fc N-linked glycan has a pronounced impact on FcγR affinity for IgG1, particularly that of the activating FcγRIIIa and the non-signaling FcγRIIIb; afucosylation (absence of the fucose) enhances affinity for these receptors by ~10-fold. Overall, afucosylation of the IgG1 Fc is a strongly pro-inflammatory modification. Afucosylated IC-FcγRIIIa interactions, in particular, are well characterized and mediate a variety of activating cellular activities, including enhanced cytotoxicity in tumor models, maturation of myeloid cells and NK cell activation [34,35].

The increased inflammatory potential of afucosylated IgG1 is typically offset by a low serum frequency of approximately 8 ± 5% [36]. Increased abundance of afucosylated IgG1 has been observed in a number of disease settings including fetal/neonatal alloimmunity, severe secondary dengue infection, and COVID-19 [12,37–43]. In the context of secondary dengue infections, where reactive but non-neutralizing IgG antibodies are present, afucosylated anti-dengue IgGs are enriched in people who progress to more severe disease [39,41,44]. Mechanistically, afucosylated dengue IC have increased infection potential in cells that express both FcγRIIa and FcgRIIIa in a pathway that requires both receptors: FcγRIIa was shown to mediate a majority of virus IC entry, while FcγRIIIa signaling enhanced a post-entry step in infection [44]. In studies related to COVID-19, SARS-CoV-2 reactive, afucosylated IgGs were also found to be enriched in people who would later progress to more severe disease [12,42]. In the lungs of humanized mice, afucosylated SARS-CoV-2 spike IC triggered cellular infiltration and inflammatory cytokine production similar to what is observed in severe COVID-19, including monocyte and neutrophil infiltration with elevated TNFα and IL-6 in the bronchoalveolar lavage fluid [42,43]. Fc fucosylation has been manipulated for clinical benefit with the introduction of obinutuzumab, an anti-CD20 monoclonal antibody that is enriched for an afucosylated (and bisected) Fc glycans. Obinutuzumab showed superior progression-free survival in leukemia and lymphoma [45–47]. Whether levels of Fc fucosylation can be regulated to prevent severe inflammatory sequelae in some diseases or to enhance maturation of innate cells for adjuvant-like purposes during vaccination remain important areas for future investigation.

Type II receptors and IgG1 sialylation

Two functionally distinct Type II FcγRs have been described: FcεRII (CD23) and dendritic cell-specific intracellular adhesion molecule-3-grabbing non-integrin (DC-SIGN, CD209) (Figure 2). These are multifunctional C-type lectins that were initially characterized based on their binding of IgE and ICAM3 [48–50]. Type II FcγR affinity for IgG1 is dependent on Fc sialylation [51] and Type II FcγRs are required for sialylated IgG1-mediated cellular effects in vivo [7,52,53]. The combination of Fc sialylation and core fucosylation promotes a more flexible, ‘closed’ conformation of the CH2 region of the Fc [54]. This conformation is thought to reduce the binding of IgG Fc by many Type I FcγRs, but enable binding by the Type II receptors [55,56].

DC-SIGN and its murine homolog SIGN-R1 are expressed on dendritic cells and macrophages and play a role in innate and adaptive immunity as pattern recognition receptors of carbohydrates with endocytic capability [3,49]. The signaling pathway downstream of sialylated IC-DC-SIGN interactions is an area of ongoing investigation, but observations in mice suggest that engagement of the murine orthologue, SIGN-R1, by sialylated IC ultimately promotes a Th2 response through the production of IL-33, which stimulates the release of the cytokine IL-4 by basophils [57,58]. Elevated IL-4, in turn, triggers increased expression of the inhibitory FcγR CD32b on myeloid cells, reducing inflammatory activity, including suppressing serum-induced arthritis in an animal model [58,59].

Clinically, this pathway is harnessed in the use of intravenous immunoglobulin (IVIg), which is made from purified IgG pooled from thousands of donors and is used broadly as a treatment for inflammatory and autoimmune diseases. Multiple Fab- and Fc-mediated mechanisms have been proposed for the anti-inflammatory properties of IVIg, and we focus here on the role of Fc sialylation. The fraction of sialylated IgG in individuals varies with age and sex but is generally less than 20% [60], and in commercial preparations, the percentage of sialylated IVIg is relatively constant at ~16% [61]. However, individuals can differ by up to 30% in their abundance of sialylated antibodies [62], and abundance within an individual can vary over time and in response to antigens. IVIg is given at supraphysiologic doses (1–2 g/kg) such that the total circulating sialylated IgG in an individual is increased, regardless of pre-treatment abundance. A number of in vivo studies in which the Fc sialylated component of IVIg was either removed or enriched have demonstrated a direct correlation between the abundance of sialylated Fc and the therapeutic efficacy of IVIg [63–65].

The second Type II FcγR, CD23, has two variants, CD23a and CD23b, that differ in their cell-type expression: low levels of CD23a are constitutively expressed by B cells, whereas expression of CD23b is induced by IL-4 on myeloid cells, B cells, and several subsets of T cells [66,67]. In B cells, CD23 ligation by sialylated IC also promotes increased expression of FcγRIIb, effectively raising the threshold for ITAM-mediated cell activation [52]. The outcome of this in B cells is an increased threshold for cell activation, based on the affinity of the B cell receptor for the antigen within an IC. Thus, sialylated IC vaccines have been shown to elicit higher affinity antibody responses [10,68]. It is not yet clear what IgG-CD23-dependent mechanisms exist in myeloid cells.

FcγRs and antigen presentation

Type I FcγRs play a crucial role in presentation of soluble protein antigen by uptake of IgG-antigen IC [69]. FcγR-bound IC are sorted in a FcγR-ITAM dependent manner to lysosomal compartments for proteolytic processing of antigen and FcγR, with subsequent presentation of antigen-derived peptides and degradation of FcγR [70]. Evidence for Ig/HLA-DM interaction in MHCII/peptide loading compartments of B cells raises the possibility that a similar interaction mediates lysosomal association of FcγR-IC with a class II peptideloading complex in DCs or monocyte/macrophages [71]. In addition, in certain APCs, such as murine CD8⁺ DC, or human CD141⁺ DCs, FcγRs function in association with the neonatal Fc Receptor to regulate endosomal transport of IC, allowing cross-presentation of antigen via MHCI [72]. Antigen internalized via cross-linked FcγRs is enhanced for cross-presentation by MHC class I compared to fluid phase uptake without opsonization [73].

A focused investigation into the effect of IgG1 fucosylation on antigen presentation has yet to be performed. In vitro stimulation of human monocytes and macrophages with afucosylated IC has been shown to promote greater IC internalization and pro-inflammatory cytokine production [37,42,74,75]. It is noteworthy that human DCs, the paramount APC, do not typically express FcγRIIIa, and thus would largely be agnostic to differences in IgG fucosylation. While FcγRIIIa-ITAM signaling is clearly not necessary for professional antigen presentation in humans, the murine ortholog, FcγRIV, is expressed on mouse DCs, raising the question of separate evolutionary pressures leading to distinct patterns of expression.

For Type II FcγR, high affinity targeting of the carbohydrate-recognition domain of DC-SIGN has served as an efficient mechanism for the direct delivery of antigens (and adjuvants) to DCs. This approach promotes DC activation and enhances antigen presentation, including cross-presentation in vitro [76–80].

Direct targeting of vaccine antigens to FcγR pathways to improve antigen processing and presentation may be a useful strategy to improve vaccine responses. This could be particularly pertinent for poorly immunogenic antigens or for populations that mount sub-optimal vaccine responses [81].

Conclusions

Antibody interactions with effector cells are influenced by multiple factors, including IgG subclass, modifications to the Fc glycan, and the expression of various FcγRs. The diversity of FcγRs expressed and the highly variable activities that they transduce, represent multiple levers to modulate activating and inhibitory signaling on immune effector cells. Specific modifications to the IgG1 Fc glycan, such as the absence of fucose or the presence of sialic acid, further polarize the inflammatory effects of IgG. Studies of IVIg and sialylated IgG1, specifically, in inflammatory diseases have led to new understandings of Fc sialylation in anti-inflammatory processes, although much remains to be learned. The role of Fc fucosylation in modulating inflammatory responses to IC and the diversity of mechanisms by which afucosylated IC may contribute to modulation of various diseases is another area of active discovery. Our hope is that continued investigations into the regulation of antibody-mediated effector functions through IgG Fc glycosylation will yield breakthroughs in the purposeful manipulation of relevant pathways for clinical benefit.