A novel single-domain antibody multimer that potently neutralizes tetanus neurotoxin

Highlights • VHH were developed that cover 5 epitope bins on tetanus neurotoxinTeNT. • VHH monomers and multimers possessed up to picomolar affinity towards TeNT. • The in vivo mouse toxin-neutralizing test revealed very potent VHH multimers. • The VHH multimer production yields in yeast allow commercialization. • An innovative bivalent bispecific VHH multimer tetanus antitoxin was obtained.
Tetanus antitoxin, produced in animals, has been used for the prevention and treatment of tetanus for more than 100 years. The availability of antitoxins, ethical issues around production, and risks involved in the use of animal derived serum products are a concern. We therefore developed a llama derived single-domain antibody (VHH) multimer to potentially replace the conventional veterinary product. In total, 28 different tetanus neurotoxin (TeNT) binding VHHs were isolated, 14 of which were expressed in yeast for further characterization. Four VHH monomers (T2, T6, T15 and T16) binding TeNT with high affinity (KD