Development and Humanization of a Camelid-Derived Neutralizing Nanobody Targeting Botulinum Neurotoxin Type A

Botulinum neurotoxin (BoNT), produced by Clostridium botulinum, is one of the most potent neurotoxins, causing botulism, a potentially fatal paralytic disease. Among different serotypes, BoNT/A is the most potent and the primary cause of botulism. The receptor-binding domain (Hc) of BoNT is crucial for targeting host cell receptors, making it a key therapeutic target. This study aims to develop neutralizing antibodies against BoNT/A by isolating camelid-derived nanobodies (Nbs) targeting BoNT/A-Hc (AHc).

A panel of Nbs specifically targeting AHc was isolated from a camelid immune phage display library. The VHH fragments were fused with human IgG Fc (hFc) to generate VHH-hFc fusion proteins. Neutralizing activity was evaluated using a murine lethality assay following pre-incubation of antibody and toxin. Potent Nbs were humanized through homology modeling, which predicts a theoretically reduced risk of immunogenicity. The affinity of the parent and humanized antibodies to AHc was measured using biolayer interferometry (BLI). Their biophysical properties, including thermal stability and aggregation propensity, were assessed by differential scanning fluorimetry (DSF), static light scattering (SLS), and dynamic light scattering (DLS).

Fifty-nine unique VHH clones were identified, four of which provided complete protection against a 20 LD₅₀ BoNT/A challenge in mice. The most potent antibodies, 1A1 and 1A3, exhibited ED₅₀ of 0.47 μg per mouse, corresponding to a neutralizing potency of 4.3 IU/mg. Upon humanization, the variants hA1 and hA3 showed strong binding affinities, with K_D ranging from 1 to 2 nM. The humanized variant hA1 demonstrated superior neutralizing activity against BoNT/A, exhibiting an ED₅₀ of 0.12 μg and a potency of 17.1 IU/mg, which represents an approximate four-fold increase compared to the parental antibody (1A1). Additionally, hA1 demonstrated enhanced thermal stability and minimal aggregation propensity.

Humanized Nbs could serve as an attractive platform for developing novel botulism therapeutics, with potential advantages over traditional serum antitoxins in safety profile, clinical tolerability and scalable manufacturing. These favorable traits render them viable candidates for next-generation antitoxins against BoNT/A poisoning. Humanized Nb hA1 displays potent neutralizing activity and optimized biophysical properties, implying its value as a preclinical lead for botulism intervention. Nevertheless, systematic follow-up studies covering post-exposure protective efficacy, direct immunogenicity testing and comprehensive safety profiling are required to fully validate its translational prospects.