An Expanded Conformation of an Antibody Fab Region by X-Ray Scattering, Molecular Dynamics, and smFRET Identifies an Aggregation Mechanism

Codina, Nuria; Hilton, David; Zhang, Cheng; Chakroun, Nesrine; Ahmad, Shahina S.; Perkins, Stephen J.; Dalby, Paul A.

doi:10.1016/j.jmb.2019.02.009

Cited by 20 publications

(26 citation statements)

References 71 publications

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“…Alignments of the C L domain at pH 7.0 and pH 3.5 revealed a slight displacement at low pH, especially visible in the loop regions (Fig 2F). These findings agreed with previous experimental work, which combined SAXS, atomistic modelling and smFRET to reveal the displacement of the C L domain in Fab A33 at low pH [14].…”

Section: Resultssupporting

confidence: 92%

“…At low pH, only one APR (residues 387-402), was found to increase its solvent accessibility significantly at pH 3.5, with an increase of 57 ± 25 Å 2 (10 % increase) (Table 2), consistent with previous experimental findings [14]. This APR is located in the C H 1 domain and its exposure can be explained by the C L domain displacement observed at low pH (Fig 8A).…”

Section: Resultssupporting

confidence: 90%

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Insights into the stability of a therapeutic antibody Fab fragment by molecular dynamics and its stabilization by computational design

Codina

Zhang

Chakroun

et al. 2019

Preprint

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23Successful development of protein therapeutics depends critically on achieving stability 24 under a range of conditions, while retaining their specific mode of action. Gaining a deeper 25 understanding of the drivers of instability across different stress conditions, will potentially enable 26 the engineering of protein scaffolds that are inherently manufacturable and stable. Here, we 27 compared the structural robustness of a humanized antibody fragment (Fab) A33 using atomistic 28 molecular dynamics simulations under two different stresses of low pH and high temperature.29 RMSD calculations, structural alignments and contact analysis revealed that low pH unfolding 30 was initiated through loss of contacts at the constant domain interface (C L -C H 1), prior to C L domain 31 unfolding. By contrast, thermal unfolding began with loss of contacts in both the C L -C H 1 and 32 variable domain interface (V L -V H ), followed by domain unfolding of C L and also of V H , thus 33 revealing divergent unfolding pathways. FoldX and Rosetta both agreed that mutations at the C L -34 C H 1 interface have the greatest potential for increasing the stability of Fab A33. Additionally, 35 packing density calculations found these residues to be under-packed relative to other inter-36 domain residues. Two salt bridges were identified that possibly drive the conformational change 37 at low pH, while at high temperature, salt bridges were lost and reformed quickly, and not always 38 with the same partner, thus contributing to an overall destabilization. Sequence entropy analysis 39 of existing Fab sequences revealed considerable scope for further engineering, where certain 40 natural mutations agreed with FoldX and Rosetta predictions. Lastly, the unfolding events at the 41 two stress conditions exposed different predicted aggregation-prone regions (APR), which would 42 potentially lead to different aggregation mechanisms. Overall, our results identified the early 43 stages of unfolding and stability-limiting regions of Fab A33, which provide interesting targets for 44 future protein engineering work aimed at stabilizing to both thermal and pH-stresses 45 simultaneously. 46 3 47 Author Summary 48 49Currently, antibody-based products are the most rapidly growing class of pharmaceuticals 50 due to their high specificity towards their targets, such as biomarkers on the surface of cancer 51 cells. However, they tend to aggregate at all stages of product development, which leads to 52 decreased efficiency and could elicit an immunological response. Improvements in the stability of 53 therapeutic antibodies are generally made during the development phase, by trial and error of the 54 composition of the formulated product, which is both costly and time consuming. There is great 55 demand and potential for identifying the drivers of instability across different stress conditions, 56 early in the discovery phase, which will enable the rational engineering of protein scaffolds. This 57 work elucidated the stability-limiting regions of the ant...

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Section: Resultssupporting

confidence: 92%

Section: Resultssupporting

confidence: 90%

Insights into the stability of a therapeutic antibody Fab fragment by molecular dynamics and its stabilization by computational design

Codina

Zhang

Chakroun

et al. 2019

Preprint

Self Cite

View full text Add to dashboard Cite

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“…of a Fab region at low pH by a combination of SAXS, molecular dynamics, and FRET analyses (57). A polydisperse mixture of monomeric and dimeric bovine IgG, IgA, and IgM has also been studied, also going to very high concentrations but with examples of bead modeling and fits to the human IgG1 b12 crystal structure that provided some insight into the scattering data (58).…”

Section: Discussionmentioning

confidence: 99%

Atomistic Modeling of Scattering Curves for Human IgG1/4 Reveals New Structure-Function Insights

Wright

Elliston

Hui

et al. 2019

Biophysical Journal

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Small angle x-ray and neutron scattering are techniques that give solution structures for large macromolecules. The creation of physically realistic atomistic models from known high-resolution structures to determine joint x-ray and neutron scattering best-fit structures offers a, to our knowledge, new method that significantly enhances the utility of scattering. To validate this approach, we determined scattering curves for two human antibody subclasses, immunoglobulin G (IgG) 1 and IgG4, on five different x-ray and neutron instruments to show that these were reproducible, then we modeled these by Monte Carlo simulations. The two antibodies have different hinge lengths that connect their antigen-binding Fab and effector-binding Fc regions. Starting from 231,492 and 190,437 acceptable conformations for IgG1 and IgG4, respectively, joint x-ray and neutron scattering curve fits gave low goodness-of-fit R factors for 28 IgG1 and 2748 IgG4 structures that satisfied the disulphide connectivity in their hinges. These joint best-fit structures showed that the best-fit IgG1 models had a greater separation between the centers of their Fab regions than those for IgG4, in agreement with their hinge lengths of 15 and 12 residues, respectively. The resulting asymmetric IgG1 solution structures resembled its crystal structure. Both symmetric and asymmetric solution structures were determined for IgG4. Docking simulations with our best-fit IgG4 structures showed greater steric clashes with its receptor to explain its weaker FcgRI receptor binding compared to our best-fit IgG1 structures with fewer clashes and stronger receptor binding. Compared to earlier approaches for fitting molecular antibody structures by solution scattering, we conclude that this joint fit approach based on x-ray and neutron scattering data, combined with Monte Carlo simulations, significantly improved our understanding of antibody solution structures. The atomistic nature of the output extended our understanding of known functional differences in Fc receptor binding between IgG1 and IgG4.

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“…Second, a plethora of in vitro assays has been developed which probe the different biophysical properties of a mAb, such as: biolayer interferometry and self‐interaction nanoparticle spectroscopy to probe self‐association; cross‐interaction chromatography or the use of polyspecificity reagent to detect cross‐association; dynamic light scattering and standup monolayer adsorption chromatography to assess colloidal stability; as well as various spectroscopic methods to determine conformational stability . The existence of so many methods highlights the fact that no one assay is able to identify the specific aggregation‐prone sequences that drive aggregation, especially from partially unfolded states . Furthermore, the ability of these methods to predict long‐term stability remains unclear and, until recently, the relationship between these techniques was unknown …”

Section: Introductionmentioning

confidence: 99%

The uniqueness of flow in probing the aggregation behavior of clinically relevant antibodies

Willis

Kumar

Jain

et al. 2020

Engineering Reports

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The development of therapeutic monoclonal antibodies (mAbs) can be hindered by their tendency to aggregate throughout their lifetime, which can illicit immunogenic responses and render mAb manufacturing unfeasible. Consequently, there is a need to identify mAbs with desirable thermodynamic stability, solubility, and lack of self-association. These behaviors are assessed using an array of in silico and in vitro assays, as no single assay can predict aggregation and developability. We have developed an extensional and shear flow device (EFD), which subjects proteins to defined hydrodynamic forces which mimic those experienced in bioprocessing. Here, we utilize the EFD to explore the aggregation propensity of 33 IgG1 mAbs, whose variable domains are derived from clinical antibodies. Using submilligram quantities of material per replicate, wide-ranging EFD-induced aggregation (9-81% protein in pellet) was observed for these mAbs, highlighting the EFD as a sensitive method to assess aggregation propensity. By comparing the EFD-induced aggregation data to those obtained previously from 12 other biophysical assays, we show that the EFD provides distinct information compared with current measures of adverse biophysical behavior. Assessing a candidate's liability to hydrodynamic force thus adds novel insight into the rational selection of developable mAbs that complements other assays. K E Y W O R D S aggregation, developability, extensional flow, monoclonal antibody, shear flowThis is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

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An Expanded Conformation of an Antibody Fab Region by X-Ray Scattering, Molecular Dynamics, and smFRET Identifies an Aggregation Mechanism

Cited by 20 publications

References 71 publications

Insights into the stability of a therapeutic antibody Fab fragment by molecular dynamics and its stabilization by computational design

Insights into the stability of a therapeutic antibody Fab fragment by molecular dynamics and its stabilization by computational design

Atomistic Modeling of Scattering Curves for Human IgG1/4 Reveals New Structure-Function Insights

The uniqueness of flow in probing the aggregation behavior of clinically relevant antibodies

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