Aglycosylated immunoglobulin G1 variants productively engage activating Fc receptors. Proc. Natl. Acad. Sci. USA. 105: 20167-20172

Department of Biological Engineering, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 01/2009; 105(51):20167-72. DOI: 10.1073/pnas.0809257105
Source: PubMed


Immunoglobulin G plays a vital role in adaptive immunity and antibody-based therapy through engagement of its Fc region by the Fc gamma receptors (Fc gammaRs) on immune cells. In addition to specific protein-protein contacts, N-linked glycosylation of the IgG Fc has been thought to be essential for the recognition of Fc by Fc gammaR. This requirement for the N-linked glycan has limited biomanufacture of therapeutic antibodies by restricting it to mammalian expression systems. We report here aglycosylated Fc domain variants that maintain engagement to Fc gammaRs, both in vitro and in vivo, demonstrating that Fc glycosylation is not strictly required for the activation of immune cells by IgG. These variants provide insight into how the N-linked glycan is used biologically in the recognition of Fc by Fc gammaRs, as well as represent a step toward the production in alternative expression systems of antibody-based therapeutics capable of eliciting immune effector functions.

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Available from: Bruce Tidor, Oct 02, 2015
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    • "In this study, we evaluate the contribution of Fc N-glycosylation to Fc structure and FcgRIIIa affinity to determine how the Fc N-glycan contributes to Fc activity and, in so doing, define the mechanism behind a previously unknown role for N-glycans in biology. We utilized two well-described Fc variants that are reported to show no measurable affinity for FcgRIIIa: (glycosyl) Fc D265A (Baudino et al., 2008; Bournazos et al., 2014; Clynes et al., 2000; Lund et al., 1995, 1996) and (aglycosyl) Fc T299A (Lazar et al., 2009; Sazinsky et al., 2008; Subedi et al., 2014). Here, we report the identification of IgG1 Fc features that are stabilized by N-glycosylation, and in the stabilized state contribute to FcgRIIIa binding. "
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    ABSTRACT: Asparagine(N)297-linked glycosylation of immunoglobulin G (IgG) Fc is required for binding to FcγRIIa, IIb, and IIIa, although it is unclear how it contributes. We found the quaternary structure of glycosylated Fc was indistinguishable from aglycosylated Fc, indicating that N-glycosylation does not maintain relative Fc Cγ2/Cγ3 domain orientation. However, the conformation of the C'E loop, which contains N297, was significantly perturbed in the aglycosylated Fc variant. The conformation of the C'E loop as measured with a range of Fc variants shows a strong correlation with FcγRIIIa affinity. These results indicate that the primary role of the IgG1 Fc N-glycan is to stabilize the C'E loop through intramolecular interactions between carbohydrate and amino acid residues, and preorganize the FcγRIIIa interface for optimal binding affinity. The features that contribute to the capacity of the IgG1 Fc N-glycan to restrict protein conformation and tune binding affinity are conserved in other antibodies including IgG2-IgG4, IgD, IgE, and IgM. Copyright © 2015 Elsevier Ltd. All rights reserved.
    Structure 09/2015; 23(9):1-11. DOI:10.1016/j.str.2015.06.015 · 5.62 Impact Factor
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    • "Igawa). number of reports about engineering the heavy chain Fc region to improve the binding affinity for Fc␥Rs by methods such as afucosylation of the N-linked glycan attached to Asn297 (Shields et al., 2002; Shinkawa et al., 2003) or symmetrically introducing amino acid substitutions into the heavy chain Fc region with or without the N-linked glycan attached to Asn297 (Green et al., 2002; Jung et al., 2010; Lazar et al., 2006; Richards et al., 2008; Sazinsky et al., 2008; Stavenhagen et al., 2007). These engineered Fc variants enhanced the binding affinity for Fc␥RIIa and Fc␥RIIIa or improved the ratio of activating Fc␥R binding to inhibitory Fc␥R binding (A/I ratio), which resulted in enhanced ADCP and ADCC activity. "
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    ABSTRACT: Enhancing the effector function by optimizing the interaction between Fc and Fcγ receptor (FcγR) is a promising approach to enhance the potency of anticancer monoclonal antibodies (mAbs). To date, a variety of Fc engineering approaches to modulate the interaction have been reported, such as afucosylation in the heavy chain Fc region or symmetrically introducing amino acid substitutions into the region, and there is still room to improve FcγR binding and thermal stability of the CH2 domain with these approaches. Recently, we have reported that asymmetric Fc engineering, which introduces different substitutions into each Fc region of heavy chain, can further improve the FcγR binding while maintaining the thermal stability of the CH2 domain by fine-tuning the asymmetric interface between the Fc domain and FcγR. However, the structural mechanism by which the asymmetrically engineered Fc improved FcγR binding remained unclear. In order to elucidate the mechanism, we solved the crystal structure of a novel asymmetrically engineered Fc, asym-mAb23, in complex with FcγRIIIa. Asym-mAb23 has enhanced binding affinity for both FcγRIIIa and FcγRIIa at the highest level of previously reported Fc variants. The structural analysis reveals the features of the asymmetrically engineered Fc in comparison with symmetric Fc and how each asymmetrically introduced substitution contributes to the improved interaction between asym-mAb23 and FcγRIIIa. This crystal structure could be utilized to enable us to design a more potent asymmetric Fc.
    Molecular Immunology 12/2013; 58(1):132-138. DOI:10.1016/j.molimm.2013.11.017 · 2.97 Impact Factor
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    • "Together with the development of new glycosylation strategies in non-mammalian hosts, two recent reports on the development of aglycosylated IgG, which can bind to specific Fc receptors without glycosylation , suggest a new solution for the glycosylation problem. Using yeast mutants in which glycosylation does not occur, Wittrup and coworkers [18] isolated aglycosylated IgG1 variants that had affinities for FcγRs similar to those of the wild-type glycosylated IgG. The aglycosylated IgG variant contains mutations of two amino acids adjacent to Asn 297 . "
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    ABSTRACT: After the appearance of the first FDA-approved antibody 25 years ago, antibodies have become major therapeutic agents in the treatment of many human diseases, including cancer and infectious diseases, and the use of antibodies as therapeutic/diagnostic agents is expected to increase in the future. So far, a variety of strategies have been devised for engineering of these fascinating molecules to develop superior properties and functions. Recent progress in systems biology has provided more information about the structures and cellular networks of antibodies, and, in addition, recent development of biotechnology tools, particularly in regard to high-throughput screening, has made it possible to perform more intensive engineering on these substances. Based on a sound understanding and new technologies, antibodies are now being developed as more powerful drugs. In this review, we highlight the recent, significant progress that has been made in antibody engineering, with a particular focus on Fc engineering and glycoengineering for improved functions, and cellular engineering for enhanced production of antibodies in yeast and bacterial hosts.
    Biotechnology Journal 01/2011; 6(1):16-27. DOI:10.1002/biot.201000381 · 3.49 Impact Factor
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