Abstract
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. Their success is based on unique properties including their reported ability to reversibly refold after heat-induced denaturation. 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. 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. However, by deriving aggregation propensities from apparent melting temperatures, we show that an optional disulfide bond suppresses nanobody aggregation. This effect is further enhanced by increasing the length of a complementarity determining loop which, although expected to destabilize, contributes to nanobody stability. The effect of such variations depends on environmental conditions, however. Nanobodies with two disulfide bonds, for example, are prone to lose their functionality in the cytosol. Our study suggests strategies to engineer nanobodies that exhibit optimal performance parameters and gives insights into general mechanisms which evolved to prevent protein aggregation.
Highlights
The antibody repertoire of camelids contains heavy-chain antibodies (HCAbs), which represent a remarkable evolutionary exception: their structure comprises two heavy chains only, lacking the additional light chains of conventional antibodies
Its effect is pronounced in combination with a long CDR3 loop, further suggesting that an effective shielding of the former VH-VL interface is a prerequisite for nanobody thermoresistance and folding reversibility
To clarify the significance of nanobody aggregation besides reversible refolding upon heat-denaturation, we comprehensively characterized around 70 nanobodies in differential scanning fluorimetry (DSF) and turbidity measurements
Summary
The antibody repertoire of camelids contains heavy-chain antibodies (HCAbs), which represent a remarkable evolutionary exception: their structure comprises two heavy chains only, lacking the additional light chains of conventional antibodies. The derived antigen-binding domain – called nanobody or VHH (variable domain of the heavy chain of HCAbs) – is a natural single-domain antibody with several unique qualities Important is their tendency to bind structured, often cryptic epitopes that are frequently inaccessible to conventional antibodies. Soon after the discovery of HCAbs in 199314, several studies pointed at superior biophysical properties of the nanobody binder class[15,16] While their thermodynamic stability turned out to be comparable with conventional VH domains[17], several reports suggested that the reversibility of nanobody denaturation represents the most remarkable difference to conventional binders[15,17,18]. Its effect is pronounced in combination with a long CDR3 loop, further suggesting that an effective shielding of the former VH-VL interface is a prerequisite for nanobody thermoresistance and folding reversibility
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