Exploiting sequence and stability information for directing nanobody stability engineering

[Background] Variable domains of camelid heavy-chain antibodies, commonly named nanobodies, have highbiotechnological potential. In view of their broad range of applications in research, diagnostics and therapy,engineering their stability is of particular interest. One important aspect is the improvement of thermostability,because it can have immediate effects on conformational stability, protease resistance and aggregation pro-pensity of the protein
[Methods] We analyzed the sequences and thermostabilities of 78 purified nanobody binders. From this data,potentially stabilizing amino acid variations were identified and studied experimentally.Results:Some mutations improved the stability of nanobodies by up to 6.1 °C, with an average of 2.3 °C acrosseight modified nanobodies. The stabilizing mechanism involves an improvement of both conformational stabilityand aggregation behavior, explaining the variable degree of stabilization in individual molecules. In some in-stances, variations predicted to be stabilizing actually led to thermal destabilization of the proteins. The reasonsfor this contradiction between prediction and experiment were investigated.
[Conclusions] The results reveal a mutational strategy to improve the biophysical behavior of nanobody bindersand indicate a species-specificity of nanobody architecture
[General significance] This study illustrates the potential and limitations of engineering nanobody thermostabilityby merging sequence information with stability data, an aspect that is becoming increasingly important with therecent development of high-throughput biophysical methods