Engineered Antigen-Binding Fragments for Enhanced Crystallisation of Antibody:Antigen Complexes

Bruce, H. A.; Singer, A. U.; Filippova, E. V.; Blazer, L. L.; Adams, J. J.; Enderle, L.; Ben-David, M.; Radley, E. H.; Mao, D. Y.; Pau, V.; Orlicky, S.; Sicheri, F.; Kourinov, I.; Atwell, S.; Kossiakoff, A. A.; Sidhu, S. S.

doi:10.1101/2023.07.06.548021

bioRxiv · 10.1101/2023.07.06.548021

Engineered Antigen-Binding Fragments for Enhanced Crystallisation of Antibody:Antigen Complexes

Bruce, H. A.·Singer, A. U.·Filippova, E. V.·Blazer, L. L.·Adams, J. J.·Enderle, L.·Ben-David, M.·Radley, E. H.·Mao, D. Y.·Pau, V.·Orlicky, S.·Sicheri, F.·Kourinov, I.·Atwell, S.·Kossiakoff, A. A.·Sidhu, S. S.

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Abstract

The atomic-resolution structural information that X-ray crystallography can provide on the binding interface between a Fab and its cognate antigen is highly valuable for understanding the mechanism of interaction. However, many Fab:antigen complexes are recalcitrant to crystallisation, making the endeavour a significant effort with no guarantee of success. Consequently, there have been significant steps taken to increase the likelihood of Fab:antigen complex crystallisation by altering the Fab framework. In this investigation, we applied the surface entropy reduction strategy coupled with phage-display technology to identify a set of surface substitutions that improve the propensity of a human Fab framework to crystallise. In addition, we showed that combining these surface substitutions with previously reported Crystal Kappa and elbow substitutions results in a striking improvement in Fab and Fab:antigen complex crystallisability, revealing a synergistic relationship between these sets of substitutions. Through comprehensive Fab and Fab:antigen complex crystallisation screenings followed by structure determination and analysis, we defined the roles that each of these substitutions play in facilitating crystallisation and how they complement each other in the process.

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Bruce, H. A., Singer, A. U., Filippova, E. V., Blazer, L. L., Adams, J. J., Enderle, L., Ben-David, M., Radley, E. H., Mao, D. Y., Pau, V., Orlicky, S., Sicheri, F., Kourinov, I., Atwell, S., Kossiakoff, A. A., Sidhu, S. S.. 2023-07-06. Engineered Antigen-Binding Fragments for Enhanced Crystallisation of Antibody:Antigen Complexes. https://doi.org/10.1101/2023.07.06.548021

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