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Crystal structures and molecular dynamics simulations of a humanised antibody fragment at acidic to basic pH

Tang, Jiazhi; Zhang, Cheng; Castillo, Nuria Codina; Lalaurie, Christophe J; Gao, Xin; Dalby, Paul A; Kozielski, Frank; (2023) Crystal structures and molecular dynamics simulations of a humanised antibody fragment at acidic to basic pH. Scientific Reports , 13 , Article 16281. 10.1038/s41598-023-42698-7. Green open access

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Abstract

Antibody-fragment (Fab) therapy development has the potential to be accelerated by computational modelling and simulations that predict their target binding, stability, formulation, manufacturability, and the impact of further protein engineering. Such approaches are currently predicated on starting with good crystal structures that closely represent those found under the solution conditions to be simulated. A33 Fab, is an undeveloped immunotherapeutic antibody candidate that was targeted to the human A33 antigen homogeneously expressed in 95% cases of primary and metastatic colorectal cancers. It is now used as a very well characterised testing ground for developing analytics, formulation and protein engineering strategies, and to gain a deeper understanding of mechanisms of destabilisation, representative of the wider therapeutic Fab platform. In this article, we report the structure of A33 Fab in two different crystal forms obtained at acidic and basic pH. The structures overlapped with RMSD of 1.33 Å overall, yet only 0.5 Å and 0.76 Å for the variable- and constant regions alone. While most of the differences were within experimental error, the switch linker between the variable and the constant regions showed some small differences between the two pHs. The two structures then enabled a direct evaluation of the impact of initial crystal structure selection on the outcomes of molecular dynamics simulations under different conditions, and their subsequent use for determining best fit solution structures using previously obtained small-angle x-ray scattering (SAXS) data. The differences in the two structures did not have a major impact on MD simulations regardless of the pH, other than a slight persistence of structure affecting the solvent accessibility of one of the predicted APR regions of A33 Fab. Interestingly, despite being obtained at pH 4 and pH 9, the two crystal structures were more similar to the SAXS solution structures obtained at pH 7, than to those at pH 4 or pH 9. Furthermore, the P65 crystal structure from pH 4 was also a better representation of the solution structures at any other pH, than was the P1 structure obtained at pH 9. Thus, while obtained at different pH, the two crystal structures may represent highly (P65) and lesser (P1) populated species that both exist at pH 7 in solution. These results now lay the foundation for confident MD simulations of A33 Fab that rationalise or predict behaviours in a range of conditions.

Type: Article
Title: Crystal structures and molecular dynamics simulations of a humanised antibody fragment at acidic to basic pH
Location: England
Open access status: An open access version is available from UCL Discovery
DOI: 10.1038/s41598-023-42698-7
Publisher version: https://doi.org/10.1038/s41598-023-42698-7
Language: English
Additional information: This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
Keywords: Humans, Molecular Dynamics Simulation, X-Ray Diffraction, Protein Conformation, Scattering, Small Angle, Immunoglobulin Fab Fragments
UCL classification: UCL
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences
UCL > Provost and Vice Provost Offices > UCL BEAMS
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences
UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Engineering Science
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > UCL School of Pharmacy
UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Engineering Science > Dept of Biochemical Engineering
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > UCL School of Pharmacy > Pharma and Bio Chemistry
URI: https://discovery.ucl.ac.uk/id/eprint/10178293
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