Unique Binding and Stabilization Mechanisms Employed By and Engineered Into Nanobodies.

Ketaren, N. E.; Fridy, P. C.; Malashkevich, V.; Sanyal, T.; Brilliantes, M.; Thompson, M. K.; Orens, D. A.; Bonanno, J. B.; Sali, a.; Almo, S. C.; Chait, B. T.; Rout, M. P.

doi:10.1101/2023.10.22.563475

bioRxiv · 10.1101/2023.10.22.563475

Unique Binding and Stabilization Mechanisms Employed By and Engineered Into Nanobodies.

Ketaren, N. E.·Fridy, P. C.·Malashkevich, V.·Sanyal, T.·Brilliantes, M.·Thompson, M. K.·Orens, D. A.·Bonanno, J. B.·Sali, a.·Almo, S. C.·Chait, B. T.·Rout, M. P.

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Abstract

Nanobodies are single domain antibody variants that bind an antigen with the precision and affinity of a conventional antibody at only a fraction of their size. In solving the crystal structures of our nanobody-GFP complexes and compared with other available structures, we uncover mechanism that enable nanobodies to function so efficiently and effectively as single-domain antibodies. We show that unlike conventional antibodies, a nanobody repertoire maximizes sampling of their antigen surface by binding a single antigen in at least three different orientations which can be predicted by their paratope composition. We also structurally reengineering these nanobodies to improve their antigen affinity, their stability, or both - results which also revealed the strong connection between nanobody stability and affinity. We achieved this by either directly modifying the paratope, or by altering a particular region within their third framework, which is a highly conserved area that we determined plays a role in controlling nanobody stability. Our study suggests that these unique characteristics of nanobodies allow them to interact with antigens as effectively as conventional antibodies, despite their smaller size. This understanding provides methods to facilitate optimizing, humanizing and functionalizing nanobodies, thus paving the way for their utilization in diverse areas such as research, diagnostics, and therapeutic development. Significance StatementNanobodies are a unique type of antibody fragment found in select animals, containing all its antigen binding ability reduced to a single [~]15 kDa protein. There is increasing development of nanobodies for research, diagnostics, and therapeutics, yet how nanobodies function so effectively as single domain antigen binders with the precision and affinity of conventional antibodies is unclear. In this study, we present key observations to help answer this question, where one key finding is the strong relationship between nanobody stability and antigen affinity aided by the identification of a highly conserved region in nanobodies essential for maintaining nanobody stability. This region may have been retained in nanobodies in lieu of stabilizing mechanisms induced by dimerization as seen in conventional antibodies.

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Ketaren, N. E., Fridy, P. C., Malashkevich, V., Sanyal, T., Brilliantes, M., Thompson, M. K., Orens, D. A., Bonanno, J. B., Sali, a., Almo, S. C., Chait, B. T., Rout, M. P.. 2023-10-22. Unique Binding and Stabilization Mechanisms Employed By and Engineered Into Nanobodies.. https://doi.org/10.1101/2023.10.22.563475

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