A bispecific nanobody to provide full protection against lethal scorpion envenoming

Envenoming following scorpion sting is a common emergency in many parts of the world. Our aim was to ameliorate the current 100-kDa horse plasma antivenom serum (PAS)-derived Fab′₂ to more quickly reach the highly diffusible scorpion toxins (7 kDa). We immunized dromedaries with toxins from Androctonus australis hector (Aah) scorpions and cloned the single-domain antibody fragments or nanobodies (15 kDa) from their B cells. Nanobodies against AahI′ toxin (with AahII the most toxic compound of the venom) were retrieved from the libraries, and their AahI′-toxin neutralization was monitored in mice. Remarkably, the NbAahI′F12 fully protected mice against 100 LD₅₀ of AahI′ administered intracerebroventricularly. Moreover, where PAS failed completely to neutralize 2 LD₅₀ of crude venom injected subcutaneously, the designed bispecific NbF12-10 against AahI′/AahII toxins succeeded in neutralizing 5 LD₅₀. Finally, in a challenge assay in which mice were subcutaneously injected with a lethal dose of scorpion venom, the subsequent intravenous injection of 85 μg of NbF12-10 protected all mice, even if the whole procedure was repeated 3 times. Furthermore, the NbF12-10 remained fully protective when mice with severe signs of envenoming were treated a few minutes before the untreated mice died.