The structural basis of nanobody unfolding reversibility and thermoresistance

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.

The Therapeutic Potential of Nanobodies

“…Moreover, nanobodies in general have unexpected physical properties: prolonged shelf life at + 4 °C and at − 20 °C, tolerance to increased temperature (60-80 °C, several weeks at 37 °C), resistance to proteolytic degradation, exposure to non-physiological pH (pH range 3.0-9.0), elevated pressure (500-750 MPa) and chemical denaturants (2-3 M guanidinium chloride, 6-8 M urea), all of which barely harm their antigen-binding capacity [54]. The nanobody robustness is mainly attributed to its efficient refolding capacity after chemical or thermal denaturation [55], although this reversible refolding upon thermal denaturation was recently questioned [56]. The monomeric structure of nanobodies and the lack of post-translational modifications allow for their expression in microbial systems including Escherichia coli, Saccharomyces cerevisiae and Pichia pastoris, which reduces the manufacturing costs [20,57].…”

An ultra-potent synthetic nanobody neutralizes SARS-CoV-2 by locking Spike into an inactive conformation

“…One of the most attractive properties that distinguishes nanobodies from traditional monoclonal antibodies is their extreme stability (21). We therefore tested Nb6, Nb6-tri, mNb6, and mNb6-tri for stability regarding temperature, lyophilization, and aerosolization.…”

Selective Engagement of FcγRIV by a M2e-Specific Single Domain Antibody Construct Protects Against Influenza A Virus Infection

“…Two clones (M2e-VHH-23m and M2e-VHH-66m) isolated with the first, and one clone (M2e-VHH-10m) isolated with the second panning strategy, were selected for further characterization. These VHHs had a cysteine residue at position 50 in the CDR2 and position 100b in the CDR3 (Kabat numbering), allowing the formation of an additional stabilizing disulfide bound, and were devoid of N-glycosylation sequons (Figure 1A) (51). The M2e-specific VHHs and an irrelevant control F-VHH-4 directed against the F protein of human respiratory syncytial virus, were subsequently expressed in Pichia pastoris in a secreted format (52).…”