| Summary: | 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 < 1 nM), covering different antigenic domains as revealed by epitope binning, and including 3 monomers (T6, T15 and T16) that inhibited TeNT binding to neuron gangliosides, were chosen as building blocks to generate 11 VHH multimers. These multimers contained either 1 or 2 different TeNT binding VHHs fused to 1 VHH binding to either albumin (A12) or immunoglobulin (G13) to extend serum half-life in animals. Multimers consisting of 2 TeNT binding VHHs showed more than a 10-fold increase in affinity (KD of 4–23 pM) when compared to multimers containing only one TeNT binding VHH. The T6 and T16 VHHs showed synergistic in vivo TeNT neutralization and, when incorporated into a single VHH trimer (T6T16A12), they showed a very high TeNT neutralizing capacity (1,510 IU/mg).
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