Following transformation into theE. of toxin-specific broadly-neutralizing human monoclonal antibodies with established reactivity across related venom toxins from different STAT2 snake species and show efficacy in rodent models. == Introduction == Each year, snakebite envenoming exacts a high death toll and leaves hundreds of thousands of other victims maimed for life1. Antivenoms based on polyclonal antibodies isolated from the plasma of immunized animals are currently the only specific treatment option against severe envenomings2,3. While these medicines are essential and life-saving and will remain a cornerstone in snakebite therapy for years to come, an opportunity now exists to modernize treatment by exploiting the benefits of recombinant DNA and antibody technology4. Indeed, recombinant antibodies and antibody fragments have already been generated against a variety of snake venom toxins58, as well as multiple studies involving monoclonal antibodies derived using hybridoma technology have been reported912(see Laustsen et al.13for a comprehensive overview and Pucca et al.2for an overview of the historical context). Within this area of research, it has also been demonstrated that monoclonal antibodies targeting snake venom toxins can be developed using various platforms, such as phage display technology6, an in vitro methodology that can be used to actively select for antibodies with high-affinity and cross-reactivity14,15. In addition, the use of human antibody libraries in combination with phage display technology allows for the discovery of fully human antibodies that are likely to have high treatment tolerability in patients16. It has been speculated that monoclonal antibodies developed by these means could be used to formulate recombinant antivenoms that elicit fewer adverse reactions, are cost-competitive to existing therapy, and can be fine-tuned to have superior efficacy1620. Phage display technology could be particularly valuable Adenosine for discovering monoclonal antibodies against highly potent toxins with low immunogenicity that fail to elicit a strong antibody response in animals used for immunization21,22. This is the case for low molecular mass neurotoxins and cytotoxins of the three-finger toxin (3FTx) family, which are abundant in Elapidae venoms, such as cobra and mamba venoms2326. These elapid venoms, unlike Viperidae venoms, typically consist of neurotoxic and cytoxic components that elicit tissue damage as well as paralysis in bite victims1. However, antibodies derived directly from nave libraries Adenosine often lack sufficiently high affinity to enable toxin neutralization15. Affinity can be improved by further site-directed or random mutagenesis of the antibody paratopes, which can also lead to broadening of the neutralizing capacity of nave antibodies27. However, in addition to mutation of the antibody binding regions, retaining the heavy chains and exploring alternative light chains, a technique known as light chain-shuffling, has shown significant promise as well21,28. Here, a phage display library is generated by pairing a heavy or light Adenosine chain from a specific antibody with a nave repertoire of the partner chain and performing a new selection campaign15. Nevertheless, until now it remained unknown whether this technology could be used to generate antibodies that possess high affinity while simultaneously having Adenosine a broad neutralization capacity, i.e., are able to neutralize several related toxins from the venoms of different snake species. Previously, using a nave human scFv-based phage display library15, we described the discovery and characterization of the human monoclonal antibody, 368_01_C05, against -cobratoxin (P01391), a potent neurotoxin from the monocled cobra,Naja kaouthia. Notably, this antibody could prolong the survival of mice injected with lethal doses of -cobratoxin, although it failed to prevent lethality15. As a follow-up development, in the present study we constructed light-chain-shuffled antibody libraries based on this clone with the aim of using a phage display-based cross-panning campaign to simultaneously improve the affinity and expand the neutralizing capacity of the antibody against -neurotoxins from the venoms of several snake species. Cross-panning was carried out between -cobratoxin29and -elapitoxin30, a neurotoxin from the venom of the black mamba,Dendroaspis polylepis25. These two -neurotoxins share 70% sequence identity and both cause neuromuscular blockade by binding to the nicotinic acetylcholine receptor (nAChR) in muscle cells29,30. In this work, we cross-panned the chain-shuffled scFv library on these two toxins under stringent conditions to discover antibodies with improved affinity and cross-reactivity in comparison to the parent antibody. Using this strategy, we were able to generate an antibody that not only has improved affinity to -cobratoxin, but also significantly broadened cross-neutralization capacity against other -neurotoxins from the venoms of elapid snakes from the generaDendroaspis, Ophiophagus, Bungarus, andNaja. == Results == == Affinity maturation, cross-panning,.