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

Abstract

Successful development of protein therapeutics depends critically on achieving stability under a range of conditions, while retaining their specific mode of action. Gaining a deeper understanding of the drivers of instability across different stress conditions, will potentially enable the engineering of protein scaffolds that are inherently manufacturable and stable. Here, we compared the structural robustness of a humanized antibody fragment (Fab) A33 using atomistic molecular dynamics simulations under two different stresses of low pH and high temperature. RMSD calculations, structural alignments and contact analysis revealed that low pH unfolding was initiated through loss of contacts at the constant domain interface (CL-CH1), prior to CL domain unfolding. By contrast, thermal unfolding began with loss of contacts in both the CL-CH1 and variable domain interface (VL-VH), followed by domain unfolding of CL and also of VH, thus revealing divergent unfolding pathways. FoldX and Rosetta both agreed that mutations at the CL-CH1 interface have the greatest potential for increasing the stability of Fab A33. Additionally, packing density calculations found these residues to be under-packed relative to other inter-domain residues. Two salt bridges were identified that possibly drive the conformational change at low pH, while at high temperature, salt bridges were lost and reformed quickly, and not always with the same partner, thus contributing to an overall destabilization. Sequence entropy analysis of existing Fab sequences revealed considerable scope for further engineering, where certain natural mutations agreed with FoldX and Rosetta predictions. Lastly, the unfolding events at the two stress conditions exposed different predicted aggregation-prone regions (APR), which would potentially lead to different aggregation mechanisms. Overall, our results identified the early stages of unfolding and stability-limiting regions of Fab A33, which provide interesting targets for future protein engineering work aimed at stabilizing to both thermal and pH-stresses simultaneously.\n\nAuthor SummaryCurrently, antibody-based products are the most rapidly growing class of pharmaceuticals due to their high specificity towards their targets, such as biomarkers on the surface of cancer cells. However, they tend to aggregate at all stages of product development, which leads to decreased efficiency and could elicit an immunological response. Improvements in the stability of therapeutic antibodies are generally made during the development phase, by trial and error of the composition of the formulated product, which is both costly and time consuming. There is great demand and potential for identifying the drivers of instability across different stress conditions, early in the discovery phase, which will enable the rational engineering of protein scaffolds. This work elucidated the stability-limiting regions of the antibody fragment Fab A33 using several computational tools: atomistic molecular dynamics simulations, in-silico mutational analysis by FoldX and Rosetta, packing density calculators, analysis of existing Fab sequences and predictors of aggregation-prone regions. Results identified particular regions in which mutagenesis has the potential to stabilize Fab against both thermal and pH-stresses simultaneously. Overall, the methodology used here could improve the developability screening of candidate antibody products for many diseases, such as cancer, chronic inflammatory diseases and infectious diseases.