Immunoglobulin G1 Fc Domain Motions: Implications for Fc Engineering

Frank, Martin; Walker, Ross C.; Lanzilotta, William N.; Prestegard, James H.; Barb, Adam W.

doi:10.1016/j.jmb.2014.01.011

Cited by 73 publications

(102 citation statements)

References 51 publications

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“…These residues were not observed in structures of the Fc determined by X-ray crystallography. [31][32][33][34][35] The G0 preparation revealed an additional, less abundant form that was consistent with proteolysis at the C-terminus that removes an additional Ser residue plus GluProLysSer residues at the N-terminus (Fig. 4C).…”

Section: Fc G Receptor Expression and Homogenous Igg1 Fc Preparationmentioning

confidence: 53%

The immunoglobulin G1 N-glycan composition affects binding to each low affinity Fc γ receptor

Subedi

Barb

2016

mAbs

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161

169

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Immunoglobulin G1 (IgG1) is the most abundant circulating human antibody and also the scaffold for many therapeutic monoclonal antibodies (mAbs). The destruction of IgG-coated targets by cell-mediated pathways begins with an interaction between the IgG Fc region and multiple varieties of membranebound Fc g receptors (FcgRs) on the surface of leukocytes. This interaction requires the presence of an asparagine-linked (N-)glycan on the Fc, and variations in the N-glycan composition can affect the affinity of CD16A binding (an FcgR). Contemporary efforts to glycoengineer mAbs focus on increasing CD16A affinity, and thus treatment efficacy, but it is unclear how these changes affect affinity for the other FcgRs. Here, we measure binding of the extracellular Fc-binding domains for human CD16A and B, CD32A, B and C, and CD64 to 6 well-defined IgG1 Fc glycoforms that cover »85% of the pool of human IgG1 Fc glycoforms. Core a1-6 fucosylation showed the greatest changes with CD16B (8.5-fold decrease), CD16A (3.9-fold decrease) and CD32B/C (1.8-fold decrease), but did not affect binding to CD32A. Adding galactose to the non-reducing termini of the complex-type, biantennary glycan increased affinity for all CD16s and 32s tested by 1.7-fold. Sialylation did not change the affinity of core-fucosylated Fc, but increased the affinity of afucosylated Fc slightly by an average of 1.16-fold for all CD16s and CD32s tested. The effects of fucose and galactose modification are additive, suggesting the contributions of these residues to Fc g receptor affinity are independent.

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Section: Fc G Receptor Expression and Homogenous Igg1 Fc Preparationmentioning

confidence: 53%

The immunoglobulin G1 N-glycan composition affects binding to each low affinity Fc γ receptor

Subedi

Barb

2016

mAbs

Self Cite

161

169

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“…20 Furthermore, the difference in the relative orientation of CH2 with respect to CH3 highlights the dynamic nature of the CH2-CH3 domains at different pHs (Supplementary Figure 1). 22 Recently, deuterium exchange studies of human Fc were performed in the absence or presence of FcRn. The studies were performed at both acidic and neutral pHs and showed that FcRn binding to the Fc domain limits exchange on contacting Fc residues.…”

Section: Resultsmentioning

confidence: 99%

Extending human IgG half-life using structure-guided design

Booth

Ramakrishnan

Narayan

et al. 2018

mAbs

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Engineering of antibodies for improved pharmacokinetics through enhanced binding to the neonatal Fc receptor (FcRn) has been demonstrated in transgenic mice, non-human primates and humans. Traditionally, such approaches have largely relied on random mutagenesis and display formats, which fail to address related critical attributes of the antibody, such as effector functions or biophysical stability. We have developed a structure- and network-based framework to interrogate the engagement of IgG with multiple Fc receptors (FcRn, C1q, TRIM21, FcγRI, FcγRIIa/b, FcγRIIIa) simultaneously. Using this framework, we identified features that govern Fc-FcRn interactions and identified multiple distinct pathways for enhancing FcRn binding in a pH-specific manner. Network analysis provided a novel lens to study the allosteric impact of half-life-enhancing Fc mutations on FcγR engagement, which occurs distal to the FcRn binding site. Applying these principles, we engineered a panel of unique Fc variants that enhance FcRn binding while maintaining robust biophysical properties and wild type-like binding to activating receptors. An antibody harboring representative Fc designs demonstrates a half-life improvement of > 9 fold in transgenic mice and > 3.5 fold in cynomolgus monkeys, and maintains robust effector functions such as antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity.

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“…Interestingly, crystallography and small-angle x-ray scattering studies of this Fc fragment show that it has a more closed C H 2-C H 2 conformation than does a similarly aglycosylated WT fragment (65). A recent study (built on crystallization, model building, and simulation of Fc fragments) highlights that the Fc is flexible and can adapt a range of different conformations that are distinct from those observed in FcgR complexes (66). The same study also predicts that mutations at the C H 2-C H 3 interface may have a large impact on the flexibility of the domains, which may fit well with the data presented in the current study.…”

Section: Discussionmentioning

confidence: 95%

Fc Engineering of Human IgG1 for Altered Binding to the Neonatal Fc Receptor Affects Fc Effector Functions

Grevys

Bern

Foss

et al. 2015

The Journal of Immunology

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Engineering of the constant Fc part of monoclonal human IgG1 (hIgG1) Abs is an approach to improve effector functions and clinical efficacy of next-generation IgG1-based therapeutics. A main focus in such development is tailoring of in vivo half-life and transport properties by engineering the pH-dependent interaction between IgG and the neonatal Fc receptor (FcRn), as FcRn is the main homeostatic regulator of hIgG1 half-life. However, whether such engineering affects binding to other Fc-binding molecules, such as the classical FcγRs and complement factor C1q, has not been studied in detail. These effector molecules bind to IgG1 in the lower hinge–CH2 region, structurally distant from the binding site for FcRn at the CH2–CH3 elbow region. However, alterations of the structural composition of the Fc may have long-distance effects. Indeed, in this study we show that Fc engineering of hIgG1 for altered binding to FcRn also influences binding to both the classical FcγRs and complement factor C1q, which ultimately results in alterations of cellular mechanisms such as Ab-dependent cell-mediated cytotoxicity, Ab-dependent cellular phagocytosis, and Ab-dependent complement-mediated cell lysis. Thus, engineering of the FcRn–IgG1 interaction may greatly influence effector functions, which has implications for the therapeutic efficacy and use of Fc-engineered hIgG1 variants.

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Immunoglobulin G1 Fc Domain Motions: Implications for Fc Engineering

Cited by 73 publications

References 51 publications

The immunoglobulin G1 N-glycan composition affects binding to each low affinity Fc γ receptor

The immunoglobulin G1 N-glycan composition affects binding to each low affinity Fc γ receptor

Extending human IgG half-life using structure-guided design

Fc Engineering of Human IgG1 for Altered Binding to the Neonatal Fc Receptor Affects Fc Effector Functions

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