Inhibitory Fc-Gamma IIb Receptor Signaling Induced by Multivalent IgG-Fc Is Dependent on Sialylation.

Immunoglobulin (IgG) Fc glycosylation has been shown to be important for the biological activity of antibodies. Fc sialylation is important for the anti-inflammatory activity of IgGs. However, evaluating the structure-activity relationship (SAR) of antibody Fc glycosylation has been hindered using simplified in vitro models in which antibodies are often displayed in monomeric forms. Presenting antibodies in monomeric forms may not accurately replicate the natural environment of the antibodies when binding their antigen in vivo. To address these limitations, we used different Fc-containing molecules, displaying their Fc domains in monovalent and multivalent fashion. Given the inhibitory role of Fc gamma receptor IIb (FcγRIIb) in autoimmune and inflammatory diseases, we focused on evaluating the impact of Fc sialylation on the activation of FcγRIIb. We report for the first time that in human cellular systems, sialic acid mediates the induction of FcγRIIb phosphorylation by IgG-Fc when the IgG-Fc is displayed in a multivalent fashion. This effect was observed with different types of therapeutic agents such as sialylated anti-TNFα antibodies, sialylated IVIg and sialylated recombinant multivalent Fc products. These studies represent the first report of the specific effects of Fc sialylation on FcγRIIb signaling on human immune cells and may help in the characterization of the anti-inflammatory activity of Fc-containing therapeutic candidates.

Engineered IgG1-Fc Molecules Define Valency Control of Cell Surface Fcγ Receptor Inhibition and Activation in Endosomes.

The inhibition of Fcγ receptors (FcγR) is an attractive strategy for treating diseases driven by IgG immune complexes (IC). Previously, we demonstrated that an engineered tri-valent arrangement of IgG1 Fc domains (SIF1) potently inhibited FcγR activation by IC, whereas a penta-valent Fc molecule (PentX) activated FcγR, potentially mimicking ICs and leading to Syk phosphorylation. Thus, a precise balance exists between the number of engaged FcγRs for inhibition versus activation. Here, we demonstrate that Fc valency differentially controls FcγR activation and inhibition within distinct subcellular compartments. Large Fc multimer clusters consisting of 5-50 Fc domains predominately recruited Syk-mScarlet to patches on the plasma membrane, whereas PentX exclusively recruited Syk-mScarlet to endosomes in human monocytic cell line (THP-1 cells). In contrast, SIF1, similar to monomeric Fc, spent longer periods docked to FcγRs on the plasma membrane and did not accumulate and recruit Syk-mScarlet within large endosomes. Single particle tracking (SPT) of fluorescent engineered Fc molecules and Syk-mScarlet at the plasma membrane imaged by total internal reflection fluorescence microscopy (SPT-TIRF), revealed that Syk-mScarlet sampled the plasma membrane was not recruited to FcγR docked with any of the engineered Fc molecules at the plasma membrane. Furthermore, the motions of FcγRs docked with recombinant Fc (rFc), SIF1 or PentX, displayed similar motions with D ~ 0.15 µm2/s, indicating that SIF1 and PentX did not induce reorganization or microclustering of FcγRs beyond the ligating valency. Multicolor SPT-TIRF and brightness analysis of docked rFc, SIF1 and PentX also indicated that FcγRs were not pre-assembled into clusters. Taken together, activation on the plasma membrane requires assembly of more than 5 FcγRs. Unlike rFc or SIF1, PentX accumulated Syk-mScarlet on endosomes indicating that the threshold for FcγR activation on endosomes is lower than on the plasma membrane. We conclude that the inhibitory effects of SIF1 are mediated by stabilizing a ligated and inactive FcγR on the plasma membrane. Thus, FcγR inhibition can be achieved by low valency ligation with SIF1 that behaves similarly to FcγR docked with monomeric IgG.

M281, an anti-FcRn antibody, inhibits IgG transfer in a human ex vivo placental perfusion model.

BACKGROUND: The transfer of pathogenic immunoglobulin G antibodies from mother to fetus is a critical step in the pathophysiology of alloimmune and autoimmune diseases of the fetus and neonate. Immunoglobulin G transfer across the human placenta to the fetus is mediated by the neonatal Fc receptor, and blockade of the neonatal Fc receptor may provide a therapeutic strategy to prevent or minimize pathological events associated with immune-mediated diseases of pregnancy. M281 is a fully human, aglycosylated monoclonal immunoglobulin G1 antineonatal Fc receptor antibody that has been shown to block the neonatal Fc receptor with high affinity in nonclinical studies and in a phase 1 study in healthy volunteers. OBJECTIVE: The objective of the study was to determine the transplacental transfer of M281 and its potential to inhibit transfer of immunoglobulin G from maternal to fetal circulation. STUDY DESIGN: To determine the concentration of M281 required for rapid cellular uptake and complete saturation of the neonatal Fc receptor in placental trophoblasts, primary human villous trophoblasts were incubated with various concentrations of M281 in a receptor occupancy assay. The placental transfer of M281, immunoglobulin G, and immunoglobulin G in the presence of M281 was studied using the dually perfused human placental lobule model. Immunoglobulin G transfer was established using a representative immunoglobulin G molecule, adalimumab, a human immunoglobulin G1 monoclonal antibody, at a concentration of 270 µg/mL. Inhibition of immunoglobulin G transfer by M281 was determined by cotransfusing 270 µg/mL of adalimumab with 10 µg/mL or 300 µg/mL of M281. Concentrations of adalimumab and M281 in sample aliquots from maternal and fetal circuits were analyzed using a sandwich enzyme-linked immunosorbent assay and Meso Scale Discovery assay, respectively. RESULTS: In primary human villous trophoblasts, the saturation of the neonatal Fc receptor by M281 was observed within 30-60 minutes at 0.15-5.0 µg/mL, suggesting rapid blockade of neonatal Fc receptor in placental cells. The transfer rate of adalimumab (0.23% ± 0.21%) across dually perfused human placental lobule was significantly decreased by 10 µg/mL and 300 µg/mL of M281 to 0.07 ± 0.01% and 0.06 ± 0.01%, respectively. Furthermore, the transfer rate of M281 was 0.002% ± 0.02%, approximately 100-fold lower than that of adalimumab. CONCLUSION: The significant inhibition of immunoglobulin G transfer across the human placental lobule by M281 and the minimal transfer of M281 supports the development of M281 as a novel agent for the treatment of fetal and neonatal diseases caused by transplacental transfer of alloimmune and autoimmune pathogenic immunoglobulin G antibodies.

High-resolution physicochemical characterization of different intravenous immunoglobulin products.

Intravenous immunoglobulin (IVIg) is a complex mixture drug comprising diverse immunoglobulins and non-IgG proteins purified from the plasma of thousands of healthy donors. Approved IVIg products on the market differ regarding source of plasma, isolation process, and formulation. These products are used widely, and often interchangeably, for the treatment of immunodeficiency and autoimmune and inflammatory diseases, but their mechanisms of action in different indications are not well understood. A primary limitation to understanding the therapeutic relevance of specific components within IVIg has been the limited resolution of analytics historically implemented to characterize its complex mixture. In this study, high-resolution analytics were applied to better understand the composition of IVIg and product variations. We characterized three approved IVIg products: Gammagard®, Privigen®, and Octagam®. Differences in the distribution of molecular weight species, IgG sequence variants, isoforms, glycoforms, and the repertoire of previously reported antibody specificities were identified. We also compared the effect of aging on these products to identify changes in size distribution and posttranslational modifications. This type of characterization may provide insights into the specific factors and components of IVIg that may influence its activity and ultimately lead to optimization of IVIg products for use in autoimmune diseases.

Characterization of heparin-protein interaction by saturation transfer difference (STD) NMR.

The binding affinity and specificity of heparin to proteins is widely recognized to be sulfation-pattern dependent. However, for the majority of heparin-binding proteins (HBPs), it still remains unclear what moieties are involved in the specific binding interaction. Here, we report our study using saturation transfer difference (STD) nuclear magnetic resonance (NMR) to map out the interactions of synthetic heparin oligosaccharides with HBPs, such as basic fibroblast growth factor (FGF2) and fibroblast growth factor 10 (FGF10), to provide insight into the critical epitopes of heparin ligands involved. The irradiation frequency of STD NMR was carefully chosen to excite the methylene protons so that enhanced sensitivity was obtained for the heparin-protein complex. We believe this approach opens up additional application avenues to further investigate heparin-protein interactions.

Identification of a novel structure in heparin generated by sequential oxidative-reductive treatment.

Unfractionated heparin is isolated from animal organs, predominantly porcine intestinal mucosa, and goes through an extensive process of purification before it can be used for pharmaceutical purposes. While the structural microheterogeneity of heparin is predominantly biosynthetically imprinted in the Golgi, subsequent steps involved in the purification and manufacture of commercial heparin can lead to the introduction of additional modifications. Postheparin crisis of 2008, it has become increasingly important to identify what additional structural diversity is introduced as a function of the purification process and thus can be determined as being heparin-related, as opposed to being an adulterant or contaminant, e.g., oversulfated chondroitin sulfate. Our study focuses on the identification of a previously unreported structure in heparin that arises due to specific steps used in the manufacturing process. This structure was initially observed as a disaccharide peak in a complete enzymatic digest of heparin, but its presence was later identified in the NMR spectra of intact heparin as well. Structural elucidation experiments involved isolation of this structure and analysis based on multidimensional NMR and liquid chromatography coupled with mass spectrometry (LC-MS). Heparin was also subjected to specific chemical reactions to determine which steps in the manufacturing process are responsible for this novel structure. Our results allowed for the definitive assignment of the structure of this novel process-related modification and enabled an identification of the putative steps in the process that give rise to the structure.