Generation of Multivalent Nanobody-Based Proteins with Improved Neutralization of Long α-Neurotoxins from Elapid Snakes

Recombinantly produced biotherapeutics hold promise for improving the current standard of care for snakebite envenoming over conventional serotherapy. Nanobodies have performed well in the clinic, and in the context of antivenom, they have shown the ability to neutralize long alpha-neurotoxins in vivo. Here, we showcase a protein engineering approach to increase the valence and hydrodynamic size of neutralizing nanobodies raised against a long alpha-neurotoxin (alpha-cobratoxin) from the venom of the monocled cobraNaja kaouthia. Based on the p53 tetramerization domain, a panel of anti-alpha-cobratoxin nanobody-p53 fusion proteins, termed Quads, were produced with different valences, inclusion or exclusion of Fc regions for endosomal recycling purposes, hydrodynamic sizes, and spatial arrangements, comprising up to 16 binding sites. Measurements of binding affinity and stoichiometry showed that the nanobody binding affinity was retained when incorporated into the Quad scaffold, and all nanobody domains were accessible for toxin binding, subsequently displaying increased blocking potency in vitro compared to the monomeric format. Moreover, functional assessment using automated patch-damp assays demonstrated that the nanobody and Quads displayed neutralizing effects against long alpha-neurotoxins from both N. kaouthia and the forest cobra N. melanoleuca. This engineering approach offers a means of altering the valence, endosomal recyclability, and hydrodynamic size of existing nanobody-based therapeutics in a simple plug-and-play fashion and can thus serve as a technology for researchers tailoring therapeutic properties for improved neutralization of soluble targets such as snake toxins.

Automated summary

This study presents a protein engineering approach to increase the valence of neutralizing nanobodies against snake toxins. The engineered nanobody fusion proteins, called Quads, showed improved blocking potency in vitro compared to the monomeric format. This technology offers a means of tailoring therapeutic properties for improved neutralization of soluble targets such as snake toxins.