Designing high affinity therapeutic nanobodies through the incorporation of unnatural a mino acids

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Protein engineering and design principles employing the twenty standard amino acids have been extensively used in the production of stable protein scaffolds and in the affinity maturation of therapeutic proteins. While this confers some advantages, it often restricts the sequence, chemical space, and ultimately the functional diversity of proteins. Besides, current experimental methods often exclude the use of nnAAs due to their enormous library size and infinite possibility of combinations. To address this, we have developed an integrated computational pipeline employing structure-based protein design methodologies, molecular dynamics simulations, free energy calculations and in cerebro approaches, for the prediction of the binding affinity of proteins incorporated with nnAA toward their target and for the selection of the potent binders. To test the applicability of our approach, the extracellular region of epidermal growth factor receptor (EGFR) was targeted by 9G8 nanobody with tyrosine residues substituted with 3-chloro-L-tyrosine (3MY), as they are frequently found in the variable domain and responsible for antigen binding. Owing to this, several 3MY-incorporated nanobody designs were shortlisted that improve the affinity towards the extracellular region of EGFR, a crucial target for many cancers. Our results demonstrate that the structure-based computational pipeline can be used to improve the affinity of therapeutic proteins incorporated with nnAAs, and may prove to be useful in other biologically important protein complexes in light of the growing demand of nnAAs.