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Computational Design To Reduce Conformational Flexibility and Aggregation Rates of an Antibody Fab Fragment

Zhang, C; Samad, M; Yu, H; Chakroun, N; Hilton, D; Dalby, PA; (2018) Computational Design To Reduce Conformational Flexibility and Aggregation Rates of an Antibody Fab Fragment. Molecular Pharmaceutics , 15 (8) pp. 3079-3092. 10.1021/acs.molpharmaceut.8b00186. Green open access

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Abstract

Computationally-guided semi-rational design has significant potential for improving the aggregation kinetics of protein biopharmaceuticals. While improvement in the global conformational stability can stabilise proteins to aggregation under some conditions, previous studies suggest that such an approach is limited because thermal transition temperatures (Tm) and the fraction of protein unfolded (fT) tend to only correlate with aggregation kinetics where the protein is incubated at temperatures approaching the Tm. This is because under these conditions, aggregation from globally unfolded protein becomes dominant. However, under native conditions, the aggregation kinetics are presumed to be dependent on local structural fluctuations or partial unfolding of the native state, that reveal regions of high propensity to form protein-protein interactions that lead to aggregation. In this work, we have targeted the design of stabilising mutations to regions of the A33 Fab surface structure, that were predicted to be more flexible. This Fab already has high global stability, and global unfolding is not the main cause of aggregation under most conditions. Therefore, the aim was to reduce the conformational flexibility and entropy of the native protein at various locations, and thus identify which of those regions has the greatest influence on the aggregation kinetics. Highly dynamic regions of structure were identified through both molecular dynamics simulation, and B-factor analysis of related X-ray crystal structures. The most flexible residues were mutated into more stable variants, as predicted by Rosetta, which evaluates the ΔΔGND for each potential point mutation. Additional destabilising variants were prepared as controls to evaluate the prediction accuracy, and also to assess the general influence of conformational stability on aggregation kinetics. The thermal conformational stability, and aggregation rates of eighteen variants at 65 °C, were each examined at pH 4, 200 mM ionic strength, under which conditions the initial wild-type protein was <5% unfolded. Variants with decreased Tm values led to more rapid aggregation due to an increase in the fraction of protein unfolded under the conditions studied. As expected, no significant improvements were observed in the global conformational stability as measured by Tm. However, six of the twelve stable variants led to an increase in the cooperativity of unfolding, consistent with lower conformational flexibility and entropy in the native ensemble. Three of these had 5-11% lower aggregation rates, and their structural clustering indicated that the local dynamics of the C-terminus of the heavy chain had a role in influencing the aggregation rate.

Type: Article
Title: Computational Design To Reduce Conformational Flexibility and Aggregation Rates of an Antibody Fab Fragment
Location: United States
Open access status: An open access version is available from UCL Discovery
DOI: 10.1021/acs.molpharmaceut.8b00186
Publisher version: https://doi.org/10.1021/acs.molpharmaceut.8b00186
Language: English
Additional information: This version is the author accepted manuscript. For information on re-use, please refer to the publisher’s terms and conditions.
Keywords: Fab, mutagenesis, aggregation, thermal stability, melting temperature (Tm), global unfolding, molecular dynamics, protein engineering, cooperativity, entropy
UCL classification: UCL
UCL > Provost and Vice Provost Offices > UCL BEAMS
UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Engineering Science
UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Engineering Science > Dept of Biochemical Engineering
UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Maths and Physical Sciences
UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Maths and Physical Sciences > Dept of Chemistry
URI: https://discovery.ucl.ac.uk/id/eprint/10050886
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