Structural Characterization of Anti-Inflammatory Immunoglobulin G Fc Proteins

Immunoglobulin G (IgG) is a central mediator of host defense due to its ability to recognize and eliminate pathogens. The recognition and effector responses are encoded on distinct regions of IgGs. The diversity of the antigen recognition Fab domains accounts for IgG's ability to bind with high specificity to essentially any antigen. Recent studies have indicated that the Fc effector domain also displays considerable heterogeneity, accounting for its complex effector functions of inflammation, modulation, and immune suppression. Therapeutic anti-tumor antibodies, for example, require the pro-inflammatory properties of the IgG Fc to eliminate tumor cells, while the anti-inflammatory activity of intravenous IgG requires specific Fc glycans for activity. In particular, the anti-inflammatory activity of intravenous IgG is ascribed to a small population of IgGs in which the Asn297-linked complex N-glycans attached to each Fc C_H2 domain include terminal α2,6-linked sialic acids. We used chemoenzymatic glycoengineering to prepare fully disialylated IgG Fc and solved its crystal structure. Comparison of the structures of asialylated Fc, sialylated Fc, and F241A Fc, a mutant that displays increased glycan sialylation, suggests that increased conformational flexibility of the C_H2 domain is associated with the switch from pro-inflammatory to anti-inflammatory activity of the Fc.     

Immunoglobulin G1 Fc Domain Motions: Implications for Fc Engineering

The fragment crystallizable (Fc) region links the key pathogen identification and destruction properties of immunoglobulin G (IgG). Pathogen opsonization positions Fcs to activate pro-inflammatory Fcγ receptors (FcγRs) on immune cells. The cellular response and committal to a damaging, though protective, immune response are tightly controlled at multiple levels. Control mechanisms are diverse and in many cases unclear, but one frequently suggested contribution originates in FcγR affinity being modulated through shifts in Fc conformational sampling. Here, we report a previously unseen IgG1 Fc conformation. This observation motivated an extensive molecular dynamics investigation of polypeptide and glycan motions that revealed greater amplitude of motion for the N-terminal Cγ2 domains and N-glycan than previously observed. Residues in the Cγ2/Cγ3 interface and disulfide-bonded hinge were identified as influencing the Cγ2 motion. Our results are consistent with a model of Fc that is structurally dynamic. Conformational states that are competent to bind immune-stimulating FcγRs interconverted with Fc conformations distinct from those observed in FcγR complexes, which may represent a transient, nonbinding population.     

Immunoglobulin G Fc fragment-binding proteins in Mycoplasma salivarium cells.

Cell proteins of Mycoplasma salivarium were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to membranes, then examined for reactivity with human IgG molecules, the Fc fragment of human IgG, and concanavalin A (ConA). Multiple protein bands bound IgG, and most of them also bound ConA. One (corresponding to a molecular mass of 90 kDa) of the IgG- and ConA-binding bands intensely interacted with the Fc fragment of IgG. The reactivity of proteins eluted from the band with the Fc fragment, tested by dot-blotting and ELISA, was inhibited (90%) by pre-incubation with IgG and to a lesser extent (50%), with IgM. Thus, M. salivarium contained a cellular protein with a molecular mass of 90 kDa, that bound the Fc fragment of human IgG.     

Loci Associated with N-Glycosylation of Human Immunoglobulin G Show Pleiotropy with Autoimmune Diseases and Haematological Cancers

Glycosylation of immunoglobulin G (IgG) influences IgG effector function by modulating binding to Fc receptors. To identify genetic loci associated with IgG glycosylation, we quantitated N-linked IgG glycans using two approaches. After isolating IgG from human plasma, we performed 77 quantitative measurements of N-glycosylation using ultra-performance liquid chromatography (UPLC) in 2,247 individuals from four European discovery populations. In parallel, we measured IgG N-glycans using MALDI-TOF mass spectrometry (MS) in a replication cohort of 1,848 Europeans. Meta-analysis of genome-wide association study (GWAS) results identified 9 genome-wide significant loci (P<2.27×10⁻⁹) in the discovery analysis and two of the same loci (B4GALT1 and MGAT3) in the replication cohort. Four loci contained genes encoding glycosyltransferases (ST6GAL1, B4GALT1, FUT8, and MGAT3), while the remaining 5 contained genes that have not been previously implicated in protein glycosylation (IKZF1, IL6ST-ANKRD55, ABCF2-SMARCD3, SUV420H1, and SMARCB1-DERL3). However, most of them have been strongly associated with autoimmune and inflammatory conditions (e.g., systemic lupus erythematosus, rheumatoid arthritis, ulcerative colitis, Crohn''s disease, diabetes type 1, multiple sclerosis, Graves'' disease, celiac disease, nodular sclerosis) and/or haematological cancers (acute lymphoblastic leukaemia, Hodgkin lymphoma, and multiple myeloma). Follow-up functional experiments in haplodeficient Ikzf1 knock-out mice showed the same general pattern of changes in IgG glycosylation as identified in the meta-analysis. As IKZF1 was associated with multiple IgG N-glycan traits, we explored biomarker potential of affected N-glycans in 101 cases with SLE and 183 matched controls and demonstrated substantial discriminative power in a ROC-curve analysis (area under the curve = 0.842). Our study shows that it is possible to identify new loci that control glycosylation of a single plasma protein using GWAS. The results may also provide an explanation for the reported pleiotropy and antagonistic effects of loci involved in autoimmune diseases and haematological cancer.     

Recombinant Human Cytomegalovirus (HCMV) RL13 Binds Human Immunoglobulin G Fc

The human cytomegalovirus (HCMV) protein RL13 has recently been described to be present in all primary isolates but rapidly mutated in culture adapted viruses. Although these data suggest a crucial role for this gene product in HCMV primary infection, no function has so far been assigned to this protein. Working with RL13 expressed in isolation in transfected human epithelial cells, we demonstrated that recombinant RL13 from the clinical HCMV isolates TR and Merlin have selective human immunoglobulin (Ig)-binding properties towards IgG1 and IgG2 subtypes. An additional Fc binding protein, RL12, was also identified as an IgG1 and IgG2 binding protein but not further characterized. The glycoprotein RL13 trafficked to the plasma membrane where it bound and internalized exogenous IgG or its constant fragment (Fcγ). Analysis of RL13 ectodomain mutants suggested that the RL13 Ig-like domain is responsible for the Fc binding activity. Ligand-dependent internalization relied on a YxxL endocytic motif located in the C-terminal tail of RL13. Additionally, we showed that the tyrosine residue could be replaced by phenylalanine but not by alanine, indicating that the internalization signal was independent from phosphorylation events. In sum, RL13 binds human IgG and may contribute to HCMV immune evasion in the infected host, but this function does not readily explain the instability of the RL13 gene during viral propagation in cultured cells.     

Engineering of Immunoglobulin Fc Heterodimers Using Yeast Surface-Displayed Combinatorial Fc Library Screening

Immunoglobulin Fc heterodimers, which are useful scaffolds for the generation of bispecific antibodies, have been mostly generated through structure-based rational design methods that introduce asymmetric mutations into the CH3 homodimeric interface to favor heterodimeric Fc formation. Here, we report an approach to generate heterodimeric Fc variants through directed evolution combined with yeast surface display. We developed a combinatorial heterodimeric Fc library display system by mating two haploid yeast cell lines, one haploid cell line displayed an Fc chain library (displayed Fc_CH3A) with mutations in one CH3 domain (CH3A) on the yeast cell surface, and the other cell line secreted an Fc chain library (secreted Fc_CH3B) with mutations in the other CH3 domain (CH3B). In the mated cells, secreted Fc_CH3B is displayed on the cell surface through heterodimerization with the displayed Fc_CH3A, the detection of which enabled us to screen the library for heterodimeric Fc variants. We constructed combinatorial heterodimeric Fc libraries with simultaneous mutations in the homodimer-favoring electrostatic interaction pairs K370-E357/S364 or D399-K392/K409 at the CH3 domain interface. High-throughput screening of the libraries using flow cytometry yielded heterodimeric Fc variants with heterodimer-favoring CH3 domain interface mutation pairs, some of them showed high heterodimerization yields (~80–90%) with previously unidentified CH3 domain interface mutation pairs, such as hydrogen bonds and cation-π interactions. Our study provides a new approach for engineering Fc heterodimers that could be used to engineer other heterodimeric protein-protein interactions through directed evolution combined with yeast surface display.     

Conformational Destabilization of Immunoglobulin G Increases the Low pH Binding Affinity with the Neonatal Fc Receptor

Crystallographic evidence suggests that the pH-dependent affinity of IgG molecules for the neonatal Fc receptor (FcRn) receptor primarily arises from salt bridges involving IgG histidine residues, resulting in moderate affinity at mildly acidic conditions. However, this view does not explain the diversity in affinity found in IgG variants, such as the YTE mutant (M252Y,S254T,T256E), which increases affinity to FcRn by up to 10×. Here we compare hydrogen exchange measurements at pH 7.0 and pH 5.5 with and without FcRn bound with surface plasmon resonance estimates of dissociation constants and FcRn affinity chromatography. The combination of experimental results demonstrates that differences between an IgG and its cognate YTE mutant vary with their pH-sensitive dynamics prior to binding FcRn. The conformational dynamics of these two molecules are nearly indistinguishable upon binding FcRn. We present evidence that pH-induced destabilization in the CH2/3 domain interface of IgG increases binding affinity by breaking intramolecular H-bonds and increases side-chain adaptability in sites that form intermolecular contacts with FcRn. Our results provide new insights into the mechanism of pH-dependent affinity in IgG-FcRn interactions and exemplify the important and often ignored role of intrinsic conformational dynamics in a protein ligand, to dictate affinity for biologically important receptors.     

Comparison of the Fc glycosylation of fetal and maternal immunoglobulin G

Human immunoglobulin G (IgG) molecules are composed of two Fab portions and one Fc portion. The glycans attached to the Fc portions of IgG are known to modulate its biological activity as they influence interaction with both complement and various cellular Fc receptors. IgG glycosylation changes significantly with pregnancy, showing a vast increase in galactosylation and sialylation and a concomitant decrease in the incidence of bisecting GlcNAc. Maternal IgGs are actively transported to the fetus by the neonatal Fc receptor (FcRn) expressed in syncytiotrophoblasts in the placenta, providing the fetus and newborn with immunological protection. Two earlier reports described significant differences in total glycosylation between fetal and maternal IgG, suggesting a possible glycosylation-selective transport via the placenta. These results might suggest an alternative maternal transport pathway, since FcRn binding to IgG does not depend on Fc-glycosylation. These early studies were performed by releasing N-glycans from total IgG. Here, we chose for an alternative approach analyzing IgG Fc glycosylation at the glycopeptide level in an Fc-specific manner, providing glycosylation profiles for IgG1 and IgG4 as well as combined Fc glycosylation profiles of IgG2 and 3. The analysis of ten pairs of fetal and maternal IgG samples revealed largely comparable Fc glycosylation for all the analyzed subclasses. Average levels of galactosylation, sialylation, bisecting GlcNAc and fucosylation were very similar for the fetal and maternal IgGs. Our data suggest that the placental IgG transport is not Fc glycosylation selective.     

Immunoglobulin G1 Fc fragment-tagged human opioid receptor-like receptor retains the ability to inhibit cAMP accumulation

The human opioid receptor-like (ORL(1)) receptor was tagged with the immunoglobulin G1 Fc fragment at the carboxy-terminus and expressed in human embryonic kidney 293 cells. Expression of the ORL(1)-Fc receptor was confirmed by immunohistochemical staining. The fusion protein was enriched by affinity chromatography and then verified by immunodetection. The function of the ORL(1)-Fc receptor was determined by examining nociceptin/OFQ-induced inhibition of cAMP accumulation. The ORL(1)-Fc receptor inhibited the forskolin-stimulated cAMP accumulation. The inhibitory response was selectively induced by nociceptin/OFQ in a dose-dependent and pertussis toxin-sensitive manner. Our results indicate that the carboxy-terminal Fc-tagged ORL(1) receptor retained the ability to interact with G(i) proteins to inhibit adenylyl cyclase.     

In Vivo Immune Evasion Mediated by the Herpes Simplex Virus Type 1 Immunoglobulin G Fc Receptor

Herpes simplex virus (HSV) glycoproteins gE and gI form an immunoglobulin G (IgG) Fc receptor (FcγR) that binds the Fc domain of human anti-HSV IgG and inhibits Fc-mediated immune functions in vitro. gE or gI deletion mutant viruses are avirulent, probably because gE and gI are also involved in cell-to-cell spread. In an effort to modify FcγR activity without affecting other gE functions, we constructed a mutant virus, NS-gE₃₃₉, that has four amino acids inserted into gE within the domain homologous to mammalian IgG FcγRs. NS-gE₃₃₉ expresses gE and gI, is FcγR⁻, and does not participate in antibody bipolar bridging since it does not block activities mediated by the Fc domain of anti-HSV IgG. In vivo studies were performed with mice because the HSV-1 FcγR does not bind murine IgG; therefore, the absence of an FcγR should not affect virulence in mice. NS-gE₃₃₉ causes disease at the skin inoculation site comparably to wild-type and rescued viruses, indicating that the FcγR⁻ mutant virus is pathogenic in animals. Mice were passively immunized with human anti-HSV IgG and then infected with mutant or wild-type virus. We postulated that the HSV-1 FcγR should protect wild-type virus from antibody attack. Human anti-HSV IgG greatly reduced viral titers and disease severity in NS-gE₃₃₉-infected animals while having little effect on wild-type or rescued virus. We conclude that the HSV-1 FcγR enables the virus to evade antibody attack in vivo, which likely explains why antibodies are relatively ineffective against HSV infection.     

Engineering a Monomeric Fc Domain Modality by N-Glycosylation for the Half-life Extension of Biotherapeutics

Human IgG is a bivalent molecule that has two identical Fab domains connected by a dimeric Fc domain. For therapeutic purposes, however, the bivalency of IgG and Fc fusion proteins could cause undesired properties. We therefore engineered the conversion of the natural dimeric Fc domain to a highly soluble monomer by introducing two Asn-linked glycans onto the hydrophobic C_H3-C_H3 dimer interface. The monomeric Fc (monoFc) maintained the binding affinity for neonatal Fc receptor (FcRn) in a pH-dependent manner. We solved the crystal structure of monoFc, which explains how the carbohydrates can stabilize the protein surface and provides the rationale for molecular recognition between monoFc and FcRn. The monoFc prolonged the in vivo half-life of an antibody Fab domain, and a tandem repeat of the monoFc further prolonged the half-life. This monoFc modality can be used to improve the pharmacokinetics of monomeric therapeutic proteins with an option to modulate the degree of half-life extension.     

静脉注射免疫球蛋白制备中的病毒灭活

在静脉注射免疫球蛋白（ＩＶＩＧ）的制备中,采用有机溶剂结合表面活性剂（Ｓ／Ｄ）处理或６０℃１０小时液态加热（巴氏灭活法）的方法对组分Ⅱ（ＩｇＧ）进行病毒灭活处理,灭活效果用指示病毒进行了评价,结果表明：Ｓ／Ｄ法可有效灭活脂包膜病毒ＶＳＶ和Ｓｉｎｄｂｉｓ,巴氏灭活法则对上述病毒以及Ｖａｃｃｉｎｉａ和Ｅｃｈｏ病毒均有较好的灭活作用。经病毒灭活处理的免疫球蛋白,在理化及生物学特性上基本未受到不利影响,用两种方法分别处理组分Ⅱ后制备的ＩＶＩＧ,其主要特性指标均符合该制品的有关质量规定。     

Conformational Plasticity of the Immunoglobulin Fc Domain in Solution

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Cross-species Binding Analyses of Mouse and Human Neonatal Fc Receptor Show Dramatic Differences in Immunoglobulin G and Albumin Binding

The neonatal Fc receptor (FcRn) regulates the serum half-life of both IgG and albumin through a pH-dependent mechanism that involves salvage from intracellular degradation. Therapeutics and diagnostics built on IgG, Fc, and albumin fusions are frequently evaluated in rodents regarding biodistribution and pharmacokinetics. Thus, it is important to address cross-species ligand reactivity with FcRn, because in vivo testing of such molecules is done in the presence of competing murine ligands, both in wild type (WT) and human FcRn (hFcRn) transgenic mice. Here, binding studies were performed in vitro using enzyme-linked immunosorbent assay and surface plasmon resonance with recombinant soluble forms of human (shFcRn^WT) and mouse (smFcRn^WT) receptors. No binding of albumin from either species was observed at physiological pH to either receptor. At acidic pH, a 100-fold difference in binding affinity was observed. Specifically, smFcRn^WT bound human serum albumin with a K_D of ∼90 μm, whereas shFcRn^WT bound mouse serum albumin with a K_D of 0.8 μm. shFcRn^WT ignored mouse IgG1, and smFcRn^WT bound strongly to human IgG1. The latter pair also interacted at physiological pH with calculated affinity in the micromolar range. In all cases, binding of albumin and IgG from either species to both receptors were additive. Cross-species albumin binding differences could partly be explained by non-conserved amino acids found within the α2-domain of the receptor. Such distinct cross-species FcRn binding differences must be taken into consideration when IgG- and albumin-based therapeutics and diagnostics are evaluated in rodents for their pharmacokinetics.     

Immunoglobulin G Fc N-glycan profiling in patients with gastric cancer by LC-ESI-MS: relation to tumor progression and survival

The IgG Fc glycans strongly influence the Fcγ receptor interactions and Fc-mediated effector mechanisms. Changes in the structure of IgG glycans are associated with various diseases, such as infections and autoimmunity. However, the possible role of Fc glycans in tumor immunity is not yet fully understood. The aim of this study was to profile the Fc N-glycans of IgG samples from patients with gastric cancer (n = 80) and controls (n = 51) using LC-ESI-MS method to correlate the findings with stage of cancer and patients survival. Analysis of 32 different IgG N-glycans revealed significant increase of agalactosylated (GnGnF, GnGn(bi)F), and decrease of galactosylated (AGn(bi), AGn(bi)F, AA(bi), AAF) and monosialylated IgG glycoforms (NaAF, NaA(bi)) in cancer patients. A statistically significant increase of Fc fucosylation was observed in tumor stage II and III whereas reverse changes were found for the presence of bisecting GlcNAc. Higher level of fully sialylated glycans and elevated expression of glycans with bisecting GlcNAc were associated with better survival rate. Our findings provide the first evidence that the changes in Fc glycan profile may predict the survival of patients with gastric cancer. Cancer stage-dependent changes in Fc fucosylation and the bisecting N-acteylglucosamine expression as well as an association of several IgG glycoforms with the survival suggest that IgG glycosylation is related to pathogenesis of cancer and progression of the disease.     

N-Glycosylation at the SynCAM (Synaptic Cell Adhesion Molecule) Immunoglobulin Interface Modulates Synaptic Adhesion

Select adhesion molecules connect pre- and postsynaptic membranes and organize developing synapses. The regulation of these trans-synaptic interactions is an important neurobiological question. We have previously shown that the synaptic cell adhesion molecules (SynCAMs) 1 and 2 engage in homo- and heterophilic interactions and bridge the synaptic cleft to induce presynaptic terminals. Here, we demonstrate that site-specific N-glycosylation impacts the structure and function of adhesive SynCAM interactions. Through crystallographic analysis of SynCAM 2, we identified within the adhesive interface of its Ig1 domain an N-glycan on residue Asn⁶⁰. Structural modeling of the corresponding SynCAM 1 Ig1 domain indicates that its glycosylation sites Asn⁷⁰/Asn¹⁰⁴ flank the binding interface of this domain. Mass spectrometric and mutational studies confirm and characterize the modification of these three sites. These site-specific N-glycans affect SynCAM adhesion yet act in a differential manner. Although glycosylation of SynCAM 2 at Asn⁶⁰ reduces adhesion, N-glycans at Asn⁷⁰/Asn¹⁰⁴ of SynCAM 1 increase its interactions. The modification of SynCAM 1 with sialic acids contributes to the glycan-dependent strengthening of its binding. Functionally, N-glycosylation promotes the trans-synaptic interactions of SynCAM 1 and is required for synapse induction. These results demonstrate that N-glycosylation of SynCAM proteins differentially affects their binding interface and implicate post-translational modification as a mechanism to regulate trans-synaptic adhesion.