An engineered immunomodulatory IgG1 Fc suppresses autoimmune inflammation through pathways shared with i.v. immunoglobulin
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ABSTRACT:
SUBMITTER: Sneed S
PROVIDER: S-EPMC10866649 | biostudies-literature | 2024 Feb
REPOSITORIES: biostudies-literature
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Project description:Multiple sclerosis (MS) is a neurodegenerative disease that is estimated to affect over 2.3 million people worldwide. The exact cause for this disease is unknown but involves immune system attack and destruction of the myelin protein surrounding the neurons in the central nervous system. One promising class of compounds that selectively prevent the activation of immune cells involved in the pathway leading to myelin destruction are bifunctional peptide inhibitors (BPIs). Treatment with BPIs reduces neurodegenerative symptoms in experimental autoimmune encephalomyelitis (EAE), a mouse model of MS. In this work, as an effort to further improve the bioactivity of BPIs, BPI peptides were conjugated to the N- and C-termini of the fragment crystallizable (Fc) region of the human IgG1 antibody. Initially, the two peptides were conjugated to IgG1 Fc using recombinant DNA technology. However, expression in yeast resulted in low yields and one of the peptides being heavily proteolyzed. To circumvent this problem, the poorly expressed peptide was instead produced by solid phase peptide synthesis and conjugated enzymatically using a sortase-mediated ligation. The sortase-mediated method showed near-complete conjugation yield as observed by SDS-PAGE and mass spectrometry in small-scale reactions. This method was scaled up to obtain sufficient quantities for testing the BPI-Fc fusion in mice induced with EAE. Compared to the PBS-treated control, mice treated with the BPI-Fc fusion showed significantly reduced disease symptoms, did not experience weight loss, and showed reduced de-myelination. These results demonstrate that the BPI peptides were highly active at suppressing EAE when conjugated to the large Fc scaffold in this manner.
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Project description:The crystallizable fragment (Fc) of the immunoglobulin class G (IgG) is a very attractive scaffold for the design of novel therapeutics due to its quality of uniting all essential antibody functions. This article reviews the functionalization of this homodimeric glycoprotein by diversification of structural loops of CH3 domains for the design of Fcabs, i.e. antigen-binding Fc proteins. It reports the design of libraries for the selection of nanomolar binders with wildtype-like in vivo half-life and correlation of Fc receptor binding and ADCC. The in vitro and preclinical biological activity of selected Fcabs is compared with that of clinically approved antibodies. Recently, the great potential of the scaffold for the development of therapeutics for clinical use has been shown when the HER2-binding Fcab FS102 entered clinical phase I. Furthermore, methods for the engineering of biophysical properties of Fcabs applicable to proteins in general are presented as well as the different approaches in the design of heterodimeric Fc-based scaffolds used in the generation of bispecific monoclonal antibodies. Finally, this work critically analyzes and compares the various efforts in the design of highly diverse and functional libraries that have been made in the engineering of IgG1-Fc and structurally similar scaffolds.
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Project description:Due to the long serum half-life provided by the neonatal Fc receptor (FcRn) recycling, the IgG1 Fc has been pursued as the fusion partner to develop therapeutic Fc-fusion proteins, or as the antibody-derived scaffold that could be engineered with antigen-binding capabilities. In previous studies, we engineered the monomeric Fc by mutating critical residues located on the IgG1 Fc dimerization interface. Comparing with the wild-type dimeric Fc, monomeric Fc might possess substantial advantages conferred by its smaller size, but also suffers the disadvantage of non-specific binding to some unrelated antigens, raising considerable concerns over its potential clinical development. Here, we describe a phage display-based strategy to examine the effects of multiple mutations of IgG1 monomeric Fc and, simultaneously, to identify new Fc monomers with desired properties. Consequently, we identified a novel monomeric Fc that displayed significantly decreased non-specificity. In addition, it exhibited higher thermal stability and comparable pH-dependent FcRn binding to the previous reported monomeric Fc. These results provide baseline to understand the mechanism underlying the generation of soluble IgG1 Fc monomers and warrant the further clinical development of monomeric Fc-based fusion proteins as well as antigen binders.
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Project description:Intravenous immunoglobulin (IVIG) is used as an immunomodulatory agent in the treatment of various autoimmune/inflammatory diseases although its mechanism of action remains elusive. Recently, nonfucosylated IgG has been shown to be preferentially bound to Fcγ receptor IIIa (FcγRIIIa) on circulating natural killer cells; therefore, we hypothesized that nonfucosylated IVIG may modulate immune responses through FcγRIIIa blockade. Here, homogeneous fucosylated or nonfucosylated glycoforms of normal polyclonal IgG bearing sialylated, galactosylated or nongalactosylated Fc oligosaccharides were generated by chemoenzymatic glycoengineering to investigate whether the IgG glycoforms can inhibit antibody-dependent cellular cytotoxicity (ADCC). Among the six IgG glycoforms, galactosylated, nonfucosylated IgG [(G2)2] had the highest affinity to FcγRIIIa and 20 times higher potency to inhibit ADCC than native IgG. A pilot study of IVIG treatment in mice with collagen antibody-induced arthritis highlighted the low-dose (G2)2 glycoform of IVIG (0.1 g/kg) as an effective immunomodulatory agent as the 10-fold higher dose of native IVIG. These preliminary results suggest that the anti-inflammatory activity of IVIG is in part mediated via activating FcγR blockade by galactosylated, nonfucosylated IgG and that such nonfucosylated IgG glycoforms bound to FcγRs on immune cells play immunomodulatory roles in health and disease. This study provides insights into improved therapeutic strategies for autoimmune/inflammatory diseases using glycoengineered IVIG and recombinant Fc.
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Project description:Understanding the underlying mechanisms of Fc aggregation is an important prerequisite for developing stable and efficacious antibody-based therapeutics. In our study, high resolution two-dimensional nuclear magnetic resonance (NMR) was employed to probe structural changes in the IgG1 Fc. A series of (1)H-(15)N heteronuclear single-quantum correlation NMR spectra were collected between pH 2.5 and 4.7 to assess whether unfolding of C(H)2 domains precedes that of C(H)3 domains. The same pH range was subsequently screened in Fc aggregation experiments that utilized molecules of IgG1 and IgG2 subclasses with varying levels of C(H)2 glycosylation. In addition, differential scanning calorimetry data were collected over a pH range of 3-7 to assess changes in C(H)2 and C(H)3 thermostability. As a result, compelling evidence was gathered that emphasizes the importance of C(H)2 stability in determining the rate and extent of Fc aggregation. In particular, we found that Fc domains of the IgG1 subclass have a lower propensity to aggregate compared with those of the IgG2 subclass. Our data for glycosylated, partially deglycosylated, and fully deglycosylated molecules further revealed the criticality of C(H)2 glycans in modulating Fc aggregation. These findings provide important insights into the stability of Fc-based therapeutics and promote better understanding of their acid-induced aggregation process.
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Project description:We report the stabilization of the human IgG1 Fc fragment by engineered intradomain disulfide bonds. One of these bonds, which connects the N-terminus of the CH3 domain with the F-strand, led to an increase of the melting temperature of this domain by 10°C as compared to the CH3 domain in the context of the wild-type Fc region. Another engineered disulfide bond, which connects the BC loop of the CH3 domain with the D-strand, resulted in an increase of T(m) of 5°C. Combined in one molecule, both intradomain disulfide bonds led to an increase of the T(m) of about 15°C. All of these mutations had no impact on the thermal stability of the CH2 domain. Importantly, the binding of neonatal Fc receptor was also not influenced by the mutations. Overall, the stabilized CH3 domains described in this report provide an excellent basic scaffold for the engineering of Fc fragments for antigen-binding or other desired additional or improved properties. Additionally, we have introduced the intradomain disulfide bonds into an IgG Fc fragment engineered in C-terminal loops of the CH3 domain for binding to Her2/neu, and observed an increase of the T(m) of the CH3 domain for 7.5°C for CysP4, 15.5°C for CysP2 and 19°C for the CysP2 and CysP4 disulfide bonds combined in one molecule.
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Project description:Antibody fragments are emerging as promising biopharmaceuticals because of their relatively small size and other unique properties. However, compared with full-size antibodies, these antibody fragments lack the ability to bind the neonatal Fc receptor (FcRn) and have reduced half-lives. Fc engineered to bind antigens but preserve interactions with FcRn and Fc fused with monomeric proteins currently are being developed as candidate therapeutics with prolonged half-lives; in these and other cases, Fc is a dimer of two CH2-CH3 chains. To further reduce the size of Fc but preserve FcRn binding, we generated three human soluble monomeric IgG1 Fcs (mFcs) by using a combination of structure-based rational protein design combined with multiple screening strategies. These mFcs were highly soluble and retained binding to human FcRn comparable with that of Fc. These results provide direct experimental evidence that efficient binding to human FcRn does not require human Fc dimerization. The newly identified mFcs are promising for the development of mFc fusion proteins and for novel types of mFc-based therapeutic antibodies of small size and long half-lives.
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Project description:Intravenous immunoglobulin (IVIG) is an established treatment for numerous autoimmune conditions. Although Fc fragments derived from IVIG have shown efficacy in controlling immune thrombocytopenia in children, the mechanisms of action are unclear and controversial. The aim of this study was to dissect IVIG effector mechanisms using further adapted Fc fragments on demyelination in an ex vivo model of the central nervous system-immune interface. Using organotypic cerebellar slice cultures (OSCs) from transgenic mice, we induced extensive immune-mediated demyelination and oligodendrocyte loss with an antibody specific for myelin oligodendrocyte glycoprotein (MOG) and complement. Protective effects of adapted Fc fragments were assessed by live imaging of green fluorescent protein expression, immunohistochemistry and confocal microscopy. Cysteine- and glycan-adapted Fc fragments protected OSC from demyelination in a dose-dependent manner where equimolar concentrations of either IVIG or control Fc were ineffective. The protective effects of the adapted Fc fragments are partly attributed to interference with complement-mediated oligodendroglia damage. Transcriptome analysis ruled out signatures associated with inflammatory or innate immune responses. Taken together, our findings show that recombinant biomimetics can be made that are at least two hundred-fold more effective than IVIG in controlling demyelination by anti-MOG antibodies.
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Project description: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.
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