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Rhesus macaques are a common non-human primate model used in the evaluation of human monoclonal antibodies, molecules whose effector functions depend on a conserved N-linked glycan in the Fc region. This carbohydrate is a target of glycoengineering efforts aimed at altering antibody effector function by modulating the affinity of Fcγ receptors. For example, a reduction in the overall core fucose content is one such strategy that can increase antibody-mediated cellular cytotoxicity by increasing Fc-FcγRIIIa affinity. While the position of the Fc glycan is conserved in macaques, differences in the frequency of glycoforms and the use of an alternate monosaccharide in sialylated glycan species add a degree of uncertainty to the testing of glycoengineered human antibodies in rhesus macaques. Using a panel of 16 human IgG1 glycovariants, we measured the affinities of macaque FcγRs for differing glycoforms via surface plasmon resonance. Our results suggest that macaques are a tractable species in which to test the effects of antibody glycoengineering.

It has long been appreciated that immunoglobulins are not just the effector endpoint of humoral immunity, but rather have a complex role in regulating antibody responses themselves. Donor derived anti-RhD IgG has been used for over 50 years as an immunoprophylactic to prevent maternal alloimmunization to RhD. Although anti-RhD has dramatically decreased rates of hemolytic disease of the fetus and newborn (for the RhD alloantigen), anti-RhD also fails in some cases, and can even paradoxically enhance immune responses in some circumstances. Attempts to generate a monoclonal anti-RhD have largely failed, with some monoclonals suppressing less than donor derived anti-RhD and others enhancing immunity. These difficulties likely result, in part, because the mechanism of anti-RhD remains unclear. However, substantial evidence exists to reject the common explanations of simple clearance of RhD + RBCs or masking of antigen. Donor derived anti-RhD is a mixture of 4 different IgG subtypes. To the best of our knowledge an analysis of the role different IgG subtypes play in immunoregulation has not been carried out; and, only IgG1 and IgG3 have been tested as monoclonals. Multiple attempts to elicit alloimmune responses to human RhD epitopes in mice have failed. To circumvent this limitation, we utilize a tractable animal model of RBC alloimmunization using the human Kell glycoprotein as an antigen to test the effect of IgG subtype on immunoregulation by antibodies to RBC alloantigens. We report that the ability of an anti-RBC IgG to enhance, suppress (at the level of IgM responses), or have no effect is a function of the IgG subclass in this model system.

The binding strength between IgG and Fc gamma R is influenced by the composition of the N-linked glycan at position N297 in the Fc-domain of IgG. Particularly, afucosylation increases the binding affinity of human IgG1 to human Fc gamma RIIIa up to similar to 20 fold, and additional galactosylation of the afucosylated IgG increases the affinity up to similar to 40 fold. The increase in affinity for afucosylated IgG has previously been shown to depend on direct carbohydrate carbohydrate interactions between the IgG-Fc glycan with an N-linked glycan at position 162 unique to hFc gamma RIIIa and hFc gamma RIIIb. Here we report that the N162 glycosylation site is also found in the orthologous mouse Fc gamma R, mFc gamma RIV. The N162-glycan in mFc gamma RIV was also responsible for enhancing the binding to mouse IgG with reduced fucose similar to hFc gamma RIIIa. However, unlike hFc gamma RIIIa, mFc gamma RIV did not bind more avidly to IgG with increased galactose and reduced fucose. Overall, these results suggest the N162-glycan in the human Fc gamma RIII family and its orthologous mouse Fc gamma RIV to be functionally conserved

Ab-dependent cellular cytotoxicity (ADCC) is one of the most important effector mechanisms of tumor-targeting Abs in current immunotherapies. In ADCC and other Ab-dependent activation of myeloid effector cells, close cell–cell contact (between effector and target cell) and formation of immunological synapses are required. However, we still lack basic knowledge on the principal factors influencing ADCC potential by therapeutic Abs. In this study we investigated the combined roles of five factors affecting human NK cell–mediated ADCC, namely: 1) Ag density, 2) target cell membrane composition, 3) IgG FcγR polymorphism, 4) FcγR-blocking cytophilic Abs, and 5) Ab fucosylation. We demonstrate that the magnitude of NK cell–mediated ADCC responses is predominantly influenced by Ag density and Ab fucosylation. Afucosylation consistently induced efficient ADCC, even at very low Ag density, where fucosylated target Abs did not elicit ADCC. On the side of the effector cell, the FcγRIIIa–Val/Phe158 polymorphism influenced ADCC potency, with NK cells expressing the Val158 variant showing more potent ADCC. In addition, we identified the sialic acid content of the target cell membrane as an important inhibitory factor for ADCC. Furthermore, we found that the presence and glycosylation status of aspecific endogenous Abs bound to NK cell FcγRIIIa (cytophilic Abs) determine the blocking effect on ADCC. These five parameters affect the potency of Abs in vitro and should be further tested as predictors of in vivo capacity.

The precise mechanisms underlying anti-inflammatory effects of intravenous immunoglobulin (IVIg) therapies remain elusive. The sialylated IgG fraction within IVIg has been shown to be therapeutically more active in mouse models. Functionally, it has been suggested that IgG undergoes conformational changes upon Fc-sialylation which sterically impede binding to conventional FcγRs, but simultaneously allow binding to human DC-SIGN (SIGN-R1 in mice) and also CD23. These latter C-type lectins have been proposed responsible for the immunomodulatory effects in mouse models. However, there is conflicting evidence supporting direct interactions between sialylated human IgG and CD23/DC-SIGN. While cells expressing human CD23 and DC-SIGN in their native configuration bound their natural ligands IgE and ICAM-3, respectively, no IgG binding was observed, regardless of Fc-glycan sialylation in any context (with or without bisection and/or fucosylation) or presence of sialylated Fab-glycans. This was tested by both by FACS and a novel cellular Surface Plasmon Resonance imaging (cSPRi) approach allowing for monitoring low-affinity but high-avidity interactions. In summary, we find no evidence for human CD23 or DC-SIGN being bona fide receptors to human IgG, regardless of IgG Fc- or Fab-glycosylation status. However, these results do not exclude the possibility that either IgG glycosylation or C-type lectins affect IVIg therapies.

The composition of the conserved N297 glycan in immunoglobulin G (IgG) has been shown to affect antibody effector functions via C1q of the complement system and Fc gamma receptors (FcγR) on immune cells. Changes in the general levels of IgG-glycoforms, such as lowered total IgG galactosylation observed in many autoimmune diseases have been associated with elevated disease severity. Agalactosyslated IgG has therefore been regarded and classified by many as pro-inflammatory. However, and somewhat counterintuitively, agalactosylation has been shown by several groups to decrease affinity for FcγRIII and decrease C1q binding and downstream activation, which seems at odds with this proposed pro-inflammatory nature. In this review, we discuss these circumstances where altered IgG galactosylation/glycosylation is found. We propose a novel model based on these observations and current biochemical evidence, where the levels of IgG galactosylation found in the total bulk IgG affect the threshold required to achieve immune activation by autoantibodies through either C1q or FcγR. Although this model needs experimental verification, it is supported by several clinical observations and reconciles apparent discrepancies in the literature, and suggests a general mechanism in IgG-mediated autoimmune diseases.

Immunoglobulin G (IgG) is one of the most abundant proteins present in human serum and a fundamental component of the immune system. IgG3 represents ∼8% of the total amount of IgG in human serum and stands out from the other IgG subclasses because of its elongated hinge region and enhanced effector functions. This study reports partial O-glycosylation of the IgG3 hinge region, observed with nanoLC-ESI-IT-MS(/MS) analysis after proteolytic digestion. The repeat regions within the IgG3 hinge were found to be in part O-glycosylated at the threonine in the triple repeat motif. Non-, mono- and disialylated core 1-type O-glycans were detected in various IgG3 samples, both poly- and monoclonal. NanoLC-ESI-IT-MS/MS with electron transfer dissociation fragmentation and CE-MS/MS with CID fragmentation were used to determine the site of IgG3 O-glycosylation. The O-glycosylation site was further confirmed by the recombinant production of mutant IgG3 in which potential O-glycosylation sites had been knocked out. For IgG3 samples from six donors we found similar O-glycan structures and site occupancies, whereas for the same samples the conserved N-glycosylation of the Fc CH2 domain showed considerable interindividual variation. The occupancy of each of the three O-glycosylation sites was found to be ∼10% in six serum-derived IgG3 samples and ∼13% in two monoclonal IgG3 allotypes.

Glycosylation of the immunoglobulin G (IgG)-Fc tail is required for binding to Fc-gamma receptors (FcγRs) and complement-component C1q. A variety of IgG1-glycoforms is detected in human sera. Several groups have found global or antigen-specific skewing of IgG glycosylation, for example in autoimmune diseases, viral infections, and alloimmune reactions. The IgG glycoprofiles seem to correlate with disease outcome. Additionally, IgG-glycan composition contributes significantly to Ig-based therapies, as for example IVIg in autoimmune diseases and therapeutic antibodies for cancer treatment. The effect of the different glycan modifications, especially of fucosylation, has been studied before. However, the contribution of the 20 individual IgG glycoforms, in which the combined effect of all 4 modifications, to the IgG function has never been investigated. Here, we combined six glyco-engineering methods to generate all 20 major human IgG1-glycoforms and screened their functional capacity for FcγR and complement activity. Bisection had no effect on FcγR or C1q-binding, and sialylation had no- or little effect on FcγR binding. We confirmed that hypo-fucosylation of IgG1 increased binding to FcγRIIIa and FcγRIIIb by ~17-fold, but in addition we showed that this effect could be further increased to ~40-fold for FcγRIIIa upon simultaneous hypo-fucosylation and hyper-galactosylation, resulting in enhanced NK cell-mediated antibody-dependent cellular cytotoxicity. Moreover, elevated galactosylation and sialylation significantly increased (independent of fucosylation) C1q-binding, downstream complement deposition, and cytotoxicity. In conclusion, fucosylation and galactosylation are primary mediators of functional changes in IgG for FcγR- and complement-mediated effector functions, respectively, with galactose having an auxiliary role for FcγRIII-mediated functions. This knowledge could be used not only for glycan profiling of clinically important (antigen-specific) IgG but also to optimize therapeutic antibody applications.

Human IgG is the main antibody class used in antibody therapies because of its efficacy and longer half-life, which are completely or partly due to Fc gamma R-mediated functions of the molecules. Preclinical testing in mouse models are frequently performed using human IgG, but no detailed information on binding of human IgG to mouse Fc gamma Rs is available. The orthologous mouse and human Fc gamma Rs share roughly 60-70% identity, suggesting some incompatibility. Here, we report binding affinities of all mouse and human IgG subclasses to mouse Fc gamma R. Human IgGs bound to mouse Fc gamma R with remarkably similar binding strengths as we know from binding to human ortholog receptors, with relative affinities IgG3 > IgG1 > IgG4 > IgG2 and Fc gamma RI >> Fc gamma RIV > Fc gamma RIII > Fc gamma RIIb. This suggests human IgG subclasses to have similar relative FcR-mediated biological activities in mice

Abs of the IgG isotype are glycosylated in their Fc domain at a conserved asparagine at position 297. Removal of the core fucose of this glycan greatly increases the affinity for FcγRIII, resulting in enhanced FcγRIII-mediated effector functions. Normal plasma IgG contains ∼94% fucosylated Abs, but alloantibodies against, for example, Rhesus D (RhD) and platelet Ags frequently have reduced fucosylation that enhances their pathogenicity. The increased FcγRIII-mediated effector functions have been put to use in various afucosylated therapeutic Abs in anticancer treatment. To test the functional consequences of Ab fucosylation, we produced V-gene–matched recombinant anti-RhD IgG Abs of the four different subclasses (IgG1–4) with and without core fucose (i.e., 20% fucose remaining). Binding to all human FcγR types and their functional isoforms was assessed with surface plasmon resonance. All hypofucosylated anti-RhD IgGs of all IgG subclasses indeed showed enhanced binding affinity for isolated FcγRIII isoforms, without affecting binding affinity to other FcγRs. In contrast, when testing hypofucosylated anti-RhD Abs with FcγRIIIa-expressing NK cells, a 12- and 7-fold increased erythrocyte lysis was observed with the IgG1 and IgG3, respectively, but no increase with IgG2 and IgG4 anti-RhD Abs. Notably, none of the hypofucosylated IgGs enhanced effector function of macrophages, which, in contrast to NK cells, express a complex set of FcγRs, including FcγRIIIa. Our data suggest that the beneficial effects of afucosylated biologicals for clinical use can be particularly anticipated when there is a substantial involvement of FcγRIIIa-expressing cells, such as NK cells.

Objectives Recent reports describe interactions between the two most prominent RA-related autoantibodies, RFs and ACPAs. The main aim of the present study was to investigate whether RFs preferentially interact with ACPA-IgG over non-ACPA IgG. Additionally, interactions of RFs with IgG with altered galactose content in the Fc domain were examined, since ACPA-IgGs have been shown to have decreased Fc galactose content in RF+ patients. Methods (Auto)antibody interactions were studied in a surface plasmon resonance imaging assay and with ELISA. Target antibodies were isolated from RA patient plasma (polyclonal ACPA- and non-ACPA-IgG) or recombinantly produced to obtain monoclonal IgG with well-defined Fc galactose content. Interacting autoantibodies were studied using autoantibody positive patient sera and two recombinantly produced IgM-RFs. Results The sera from 41 RF+ RA patients showed similar RF binding to ACPA- and non-ACPA-IgG and no differences in binding to IgG with normal, high or low levels of Fc galactosylation. Two monoclonal IgM-RFs, one interacting with the CH2-CH3 interface and one binding close to the C-terminal end of the CH3 domain showed no influence of the Fc glycan on IgG binding by IgM-RF. Conclusion Although interactions between RF and ACPA may play a role in inflammatory processes in RA, RFs do not preferentially interact with ACPA-IgG over non-ACPA-IgG nor with agalatosylated IgG over IgG with normal or high galactosylation.

Fetal or neonatal alloimmune thrombocytopenia (FNAIT) is a potentially life-threatening disease where fetal platelets are destroyed by maternal antiplatelet IgG alloantibodies. The clinical outcome varies from asymptomatic, to petechiae or intracranial haemorrhage, but no marker has shown reliable correlation with severity, making screening for FNAIT impractical and highly inefficient. We recently found IgG Fc-glycosylation towards platelet and red blood cell antigens to be skewed towards decreased fucosylation, increased galactosylation and sialylation. The lowered core-fucosylation increases the affinity of the pathogenic antibodies to Fc gamma RIIIa and Fc gamma RIIIb, and hence platelet destruction. Here we analysed the N-linked glycans of human platelet antigen (HPA)-1a specific IgG1 with mass spectrometry in large series of FNAIT cases (n = 166) including longitudinal samples (n = 26). Besides a significant decrease in Fc-fucosylation after the first pregnancy (P = 0.0124), Fc-glycosylation levels remained stable during and after pregnancy and in subsequent pregnancies. Multiple logistic regression analysis identified anti-HPA-1a - fucosylation (P = 0.006) combined with galactosylation (P = 0.021) and antibody level (P = 0.038) correlated with bleeding severity, making these parameters a feasible marker in screening for severe cases of FNAIT.

In immune thrombocytopenia and warm autoimmune hemolytic anemia, circulating immunoglobulin G (IgG)-opsonized blood cells are cleared from the circulation by macrophages. Administration of intravenous immunoglobulin (IVIg) can prevent uptake, but the exact working mechanism is not known. The prevailing theory from murine studies, which states that Fc-sialylated IgG alters the balance between activating and inhibitory Fc-gamma receptors (FcγRs) by inducing upregulation of the inhibitory FcγRIIb on effector macrophages, is currently debated. We studied phagocytosis of IgG-opsonized blood cells in a human system, assessing the effect of IVIg and blocking anti-FcγR F(ab')2 fragments on uptake by monocyte-derived macrophages (both M1 and M2 macrophages). Phagocytosis was remarkably sensitive to administration of IVIg, but unexpectedly, recombinant Fc-sialylated IgG or sialic acid-enriched IVIg were equally active as unsialylated IgG fractions in mediating this inhibition, independent of FcγRIIb expression. Instead, IVIg inhibited phagocytosis by direct blockade of FcγRs. IgG fractions enriched for IgG dimers with enhanced avidity for FcγRs showed increased inhibition compared with monomeric IgG fractions. Together, our data demonstrate that inhibition of IgG-mediated phagocytosis in human macrophages by IVIg is dependent on the capacity to directly bind FcγRs but is independent of FcγRIIb or sialylation of the Fc fragment in the human setting.

Of the five immunoglobulin isotypes, immunoglobulin G (IgG) is most abundant in human serum. The four subclasses, IgG1, IgG2, IgG3, and IgG4, which are highly conserved, differ in their constant region, particularly in their hinges and upper CH2 domains. These regions are involved in binding to both IgG-Fc receptors (Fc gamma R) and C1g. As a result, the different subclasses have different effector functions, both in terms of triggering Fc gamma R-expressing cells, resulting in phagocytosis or antibody-dependent cell-mediated cytotoxicity, and activating complement. The Fc-regions also contain a binding epitope for the neonatal Fc receptor (FcRn), responsible for the extended half-life, placental transport, and bidirectional transport of IgG to mucosal surfaces. However, FcRn is also expressed in myeloid cells, where it participates in both phagocytosis and antigen presentation together with classical Fc gamma R and complement. How these properties, IgG-polymorphisms and post-translational modification of the antibodies in the form of glycosylation, affect IgG-function will be the focus of the current review