Our study suggests strategies to engineer nanobodies that exhibit optimal performance parameters and gives insights into general mechanisms which evolved to prevent protein aggregation. Nanobodies with two disulfide bonds, for example, are prone to lose their functionality in the cytosol. The effect of such variations depends on environmental conditions, however. This effect is further enhanced by increasing the length of a complementarity determining loop which, although expected to destabilize, contributes to nanobody stability. However, by deriving aggregation propensities from apparent melting temperatures, we show that an optional disulfide bond suppresses nanobody aggregation. By characterizing nearly 70 nanobodies, we show that irreversible aggregation does occur upon heat denaturation for the large majority of binders, potentially affecting application-relevant parameters like stability and immunogenicity. This view, however, is contrasted by studies which involve irreversibly aggregating nanobodies, asking for a quantitative analysis that clearly defines nanobody thermoresistance and reveals the determinants of unfolding reversibility and aggregation propensity. Their success is based on unique properties including their reported ability to reversibly refold after heat-induced denaturation. Nanobodies represent the variable binding domain of camelid heavy-chain antibodies and are employed in a rapidly growing range of applications in biotechnology and biomedicine.
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