3D Structural Fluctuation of IgG1 Antibody Revealed by Individual Particle Electron Tomography

Zhang, Xing; Zhang, Lei; Tong, Huimin; Peng, Bo; Rames, Matthew J.; Zhang, Shengli; Ren, Gang

doi:10.1038/srep09803

Cited by 110 publications

(138 citation statements)

References 57 publications

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“…For example, in solution IgG can sample not only the canonical Y-shaped conformation, but also T- shaped and intermediate conformations. 52,53 Due to the proximity of the membrane bilayer when Fc is bound to FcRn, the “standing up” model likely restricts binding of IgG molecules to the T-shaped conformation, as this would allow minimal steric hindrance with the membrane bilayer. In contrast, other conformations would likely lead to severe steric hindrance (Figure 6).…”

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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Section: Resultsmentioning

confidence: 99%

Extending human IgG half-life using structure-guided design

Booth

Ramakrishnan

Narayan

et al. 2018

mAbs

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“…The resulting structure shows that the two predicted hu11E6 epitopes and the single known hu1B7 epitope are located on different faces of PTx and are oriented in opposing directions. The midpoints of any two epitopes are approximately 50 Å apart, which is much narrower than the typical ϳ130-Å wingspan of a cross-linking antibody (35). A line normal to the best-fit plane calculated from the solvent-exposed residues was used to approximate the orientation of an antibody bound to the PTx surface.…”

Section: Resultsmentioning

confidence: 99%

Synergistic Neutralization of Pertussis Toxin by a Bispecific Antibody In Vitro and In Vivo

Wagner

Wang

Bui

et al. 2016

Clin Vaccine Immunol

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Bispecific antibodies are a rapidly growing class of therapeutic molecules, originally developed for the treatment of cancer but recently explored for the treatment of autoimmune and infectious diseases. Bordetella pertussis is a reemerging pathogen, and several of the key symptoms of infection are caused by the pertussis toxin (PTx). Two humanized antibodies, hu1B7 and hu11E6, bind distinct epitopes on PTx and, when coadministered, mitigate disease severity in murine and baboon models of infection. Here we describe the generation of a bispecific human IgG1 molecule combining the hu1B7 and hu11E6 binding sites via a knobs-in-holes design. The bispecific antibody showed binding activity equivalent to that of the antibody mixture in a competition enzyme-linked immunosorbent assay (ELISA). A CHO cell neutralization assay provided preliminary evidence for synergy between the two antibodies, while a murine model of PTx-induced leukocytosis definitively showed synergistic neutralization. Notably, the bispecific antibody retained the synergy observed for the antibody mixture, supporting the conclusion that synergy is due to simultaneous blockade of both the catalytic and receptor binding activities of pertussis toxin. These data suggest that a hu1B7/hu11E6 bispecific antibody is a viable alternative to an antibody mixture for pertussis treatment. Despite vaccination, pertussis infection continues to cause ϳ195,000 deaths worldwide, primarily of infants (1). Of the estimated 16 million cases of pertussis each year, ϳ95% occur in the developing world. Even in developed countries, the disease incidence has increased dramatically over the last decade, reaching prevaccination levels in some countries (2, 3). This rise has been attributed to shortcomings of the current acellular vaccine (4) as well as pathogen adaptation (5). In both cases, high levels of circulating disease place young infants at risk, as this population is the most susceptible to severe disease. An antibody therapeutic could be used to treat seriously ill infants in the developing world and to prevent disease in high-risk areas.Pertussis toxin (PTx) is one of several virulence factors secreted by the Gram-negative bacterium Bordetella pertussis. PTx is directly responsible for suppression of the innate immune system (6) and for systemic leukocytosis, which is the key clinical indicator of severe disease and appears to be directly responsible for pulmonary hypertension and organ failure (7). In addition, low titers of PTx-neutralizing antibodies correlate with susceptibility to clinical infection (8). We previously developed a binary mixture of two humanized anti-pertussis toxin antibodies which was able to mitigate whooping cough in mouse and baboon models of infection (9). The antibody hu11E6 blocks binding of the toxin to host cells, while the antibody hu1B7 interferes with the catalytic pathway. At a dose selected to demonstrate efficacy but not synergy, the individual antibodies and the mixture were able to completely suppress leukocytosis in a murine ...

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“…When generating a model-derived map, it has been common practice in cryo-EM to consider all atoms equally weighted (20)(21)(22). Because our model strives to be a faithful representation of the original experimental density map, it is important that we correctly account for the map densities present at individual atom locations.…”

Section: Resultsmentioning

confidence: 99%

Accurate model annotation of a near-atomic resolution cryo-EM map

Hryc

Chen

Afonine

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

113

118

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Electron cryomicroscopy (cryo-EM) has been used to determine the atomic coordinates (models) from density maps of biological assemblies. These models can be assessed by their overall fit to the experimental data and stereochemical information. However, these models do not annotate the actual density values of the atoms nor their positional uncertainty. Here, we introduce a computational procedure to derive an atomic model from a cryo-EM map with annotated metadata. The accuracy of such a model is validated by a faithful replication of the experimental cryo-EM map computed using the coordinates and associated metadata. The functional interpretation of any structural features in the model and its utilization for future studies can be made in the context of its measure of uncertainty. We applied this protocol to the 3.3-Å map of the mature P22 bacteriophage capsid, a large and complex macromolecular assembly. With this protocol, we identify and annotate previously undescribed molecular interactions between capsid subunits that are crucial to maintain stability in the absence of cementing proteins or cross-linking, as occur in other bacteriophages.ecently, cryo-EM maps with associated atomic coordinates have been reported at resolutions better than 4 Å (1, 2). However, few have been subjected to rigorous evaluation of the reliability of the observed features or of the correlation between the experimental map and its corresponding model at the residue level. Typically, such correlation is reported in terms of a curve known as the Fourier shell correlation (FSC), which is a function of spatial frequency (3, 4). Although informative, it does not assure the authenticity of local features, nor does it indicate which features in the model agree or disagree with observed density. Ideally, a molecular model can be used to generate a map that replicates the experimental map in most or all of its details, and thus constitutes a trustworthy and informative model for the specimen's structure at the reported resolution. This study examines the agreement and/or disagreement between the model and the experimental map density, determined at nearatomic resolution. These efforts establish the groundwork for a quantitative assessment of a cryo-EM structure. The methods described here were applied to the capsid of P22 bacteriophage, which infects Salmonella and has been extensively studied through biochemistry, genetics, and biophysics (5-8). ResultsCryo-EM Images and Reconstructions. To study the P22 structure, we used a 300-keV electron cryomicroscope (JEM-3200FSC; JEOL Ltd.) and a Direct Electron detector (DE-20; operated in integrating mode) to collect frozen, hydrated P22 bacteriophage images (Fig. 1A and Table S1). Signal was detectable out to 3-Å resolution ( Fig. S1 and Movie S1). A total exposure of 37.5 e − /Å 2 was fractionated into 24 frames during a 1.5-s exposure. All frames were dose-weighted (4) and used to refine particle orientation parameters; empirically, we found that using image frames 1 to 6 (a cumulative exposu...

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3D Structural Fluctuation of IgG1 Antibody Revealed by Individual Particle Electron Tomography

Cited by 110 publications

References 57 publications

Extending human IgG half-life using structure-guided design

Extending human IgG half-life using structure-guided design

Synergistic Neutralization of Pertussis Toxin by a Bispecific Antibody In Vitro and In Vivo

Accurate model annotation of a near-atomic resolution cryo-EM map

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