Structure of the Fc fragment of the NIST reference antibody RM8671

Gallagher, D.T.; Galvin, Connor; Karageorgos, Ioannis

doi:10.1107/s2053230x18009834

Cited by 15 publications

(11 citation statements)

References 14 publications

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“…Potential Iron-Binding Site of NISTmAb. The crystal structure of the fragment crystallizable (Fc) region of the NISTmAb (PDB 5VGP) 47 was visualized with PyMOL as shown in Figure 11A. Interestingly, negatively charged oxygen atoms of the side chain carboxylate groups of Asp 252 and Glu 261 are located rather close to the side chain hydroxyl oxygen atom of Thr 259 , with distances of 7.4 and 5.8 Å, respectively.…”

Section: ■ Discussionmentioning

confidence: 99%

Visible Light Induces Site-Specific Oxidative Heavy Chain Fragmentation of a Monoclonal Antibody (IgG1) Mediated by an Iron(III)-Containing Histidine Buffer

Zhang

Schöneich

2022

Mol. Pharmaceutics

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Fragmentation of therapeutic monoclonal antibodies represents a critical quality attribute. Here, we report a novel visible light-induced heavy chain fragmentation of IgG1 mediated by an Fe(III)-containing histidine (His) buffer. Based on non-reducing sodium dodecylsulfate–polyacrylamide gel electrophoresis and mass spectrometry analysis, IgG1 fragments with apparent molecular weights of ∼130, ∼110, and ∼22 kDa were detected in photo-irradiated samples and were mechanistically rationalized with an oxidative cleavage at Thr259. Specifically, the reactions are proposed to involve the generation of an intermediary alkoxyl radical, which undergoes β-cleavage to yield a glycyl radical. The latter either converts into Gly or adds oxygen and follows a peroxyl radical chemistry. The cleavage process requires the presence of His, while only negligible yields of cleavage products are formed when His is replaced by acetate, succinate, or phosphate buffer. Importantly, the fragmentation can be prevented by ethylenediaminetetraacetic acid (EDTA) only when the EDTA concentrations are in significant excess over the concentrations of Fe(III) and proteins, suggesting a strong binding between Fe(III) and IgG1.

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Section: ■ Discussionmentioning

confidence: 99%

Visible Light Induces Site-Specific Oxidative Heavy Chain Fragmentation of a Monoclonal Antibody (IgG1) Mediated by an Iron(III)-Containing Histidine Buffer

Zhang

Schöneich

2022

Mol. Pharmaceutics

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“…However, CPA samples for M255 and M431 trend more closely to the 0.3 % Nox regression line ( Figures 7B,D ), indicating decreased protein A binding is more closely associated with oxidation at these sites. Previous reports have indeed demonstrated that oxidation in the Fc region disrupts protein A binding as it binds to a region where M255 and M431 are located and where the C H 2 and C H 3 domains interact ( Deis et al, 2015 ; Gallagher et al, 2018 ). In the above example, it is our position that if a specific residue is a causal factor in reducing binding, then alteration of the kinetics of that oxidation (e.g., slowed via ligand-bound protection) will not alter the trend of relative binding vs. site-specific %Met oxidation.…”

Section: Resultsmentioning

confidence: 99%

Ligand-Bound Forced Degradation as a Strategy to Generate Functionally Relevant Analytical Challenge Materials for Assessment of CQAs

Giddens

Schiel

2022

Front. Mol. Biosci.

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Therapeutic monoclonal antibodies (mAbs) contain a variety of amino acids that are susceptible to enzymatic, chemical, and physical modifications. These modifications can happen throughout production, purification, formulation, and storage and many are known to affect the biological activity of a mAb. Methods that are able to characterize and evaluate these attributes are critical in order to understand how they might alter biological activity. Methods capable of site-specific monitoring of these critical quality attributes are extremely valuable to biopharmaceutical research but also require well-defined materials with site-specific attribute modifications. Here, we describe the development and application of a strategy to generate functionally relevant analytical challenge materials that have unique site-specific attributes. This method involves the use of a ligand that is bound to the mAb during oxidative stress resulting in unique oxidation patterns with some methionine residues protected while others are exposed to oxidation. These unique materials were used to develop a rapid surface plasmon resonance (SPR) assay that could detect methionine oxidation in both the Fab and Fc regions using specific molecular probes. The addition of uniquely oxidized materials to our data set enabled us to determine specific methionine residues vital to binding. Further analysis showed that antibody oxidation could also be rapidly detected in multiple domains from qualitative thermal melting using intrinsic tryptophan fluorescence. Methionine oxidation of an antibody was explored in this study, but we envision this method could be useful to explore structure function relationships of a variety of antibody modifications and modifications to other biologically relevant protein drugs.

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“…g Representative 2D class averages of Tie2 dimeric mutant/hTAAB IgG1 complex (Top). Cyclical higher-order structure of the Tie2 dimer/hTAAB IgG1 complex in 4-to-4, 5-to-5, and 6-to-6 assemblies were modeled based on the 2D class averages of tetragonal, pentagonal, and hexagonal closed-ring structures, respectively, using crystal structures of the Tie2 Fn2–3/chimeric hTAAB Fab complex, Tie2 Ig1–Fn1 (PDB: 4K0V 11 ) and Fc fragment of human IgG1 (PDB: 5VGP 57 ) (bottom). Scale bars, 20 nm.…”

Section: Resultsmentioning

confidence: 99%

Structural insights into the clustering and activation of Tie2 receptor mediated by Tie2 agonistic antibody

Bae

Hong

et al. 2021

Nat Commun

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Angiopoietin (Angpt)-Tie receptor 2 (Tie2) plays key roles in vascular development and homeostasis as well as pathological vascular remodeling. Therefore, Tie2-agonistic antibody and engineered Angpt1 variants have been developed as potential therapeutics for ischemic and inflammatory vascular diseases. However, their underlying mechanisms for Tie2 clustering and activation remain elusive and the poor manufacturability and stability of Angpt1 variants limit their clinical application. Here, we develop a human Tie2-agonistic antibody (hTAAB), which targets the membrane proximal fibronectin type III domain of Tie2 distinct from the Angpt-binding site. Our Tie2/hTAAB complex structures reveal that hTAAB tethers the preformed Tie2 homodimers into polygonal assemblies through specific binding to Tie2 Fn3 domain. Notably, the polygonal Tie2 clustering induced by hTAAB is critical for Tie2 activation and are resistant to antagonism by Angpt2. Our results provide insight into the molecular mechanism of Tie2 clustering and activation mediated by hTAAB, and the structure-based humanization of hTAAB creates a potential clinical application.

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Structure of the Fc fragment of the NIST reference antibody RM8671

Abstract: The structure of the Fc fragment of the NIST reference antibody RM8671 is described in an orthorhombic crystal form. The molecular conformation is compared with those of precedents using a CH3-based reference frame and the pronounced asymmetry is linked to packing interactions.

Cited by 15 publications

References 14 publications

Visible Light Induces Site-Specific Oxidative Heavy Chain Fragmentation of a Monoclonal Antibody (IgG1) Mediated by an Iron(III)-Containing Histidine Buffer

Visible Light Induces Site-Specific Oxidative Heavy Chain Fragmentation of a Monoclonal Antibody (IgG1) Mediated by an Iron(III)-Containing Histidine Buffer

Ligand-Bound Forced Degradation as a Strategy to Generate Functionally Relevant Analytical Challenge Materials for Assessment of CQAs

Structural insights into the clustering and activation of Tie2 receptor mediated by Tie2 agonistic antibody

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