Neutralizing snake toxins with monoclonal antibodies – Discovery, engineering, and new pharmacology

Snakebite envenoming is a devasting neglected tropical disease that is estimated to kill over 60,000 people annually and leaves many snakebite envenoming survivors with life-altering permanent disabilities such as amputations. The only specific treatment for snakebite envenoming is antivenom derived from the plasma of hyper-immunized animals, which has saved countless lives since its invention over 125 years ago. Unfortunately, this treatment is also related to several drawbacks, including adverse effects due to its animal origin, batch-to-batch variations, and a low therapeutic content. This has led researchers worldwide to propose novel snakebite envenoming treatments. In this PhD thesis, I focus on advancing the field of snakebite antivenoms, specifically focusing on recombinant antivenoms based on oligoclonal mixtures of human monoclonal antibodies (mAbs). These antivenoms are hypothesized to cause fewer adverse effects due to the human origin of the mAbs, have limited batch-to-batch variation as they can be manufactured by cell cultivation techniques, and have a higher therapeutic content due to tight control of the antivenom components.
In the work presented here, phage display technology has been employed to discover human mAbs specifically targeting snake toxins. One of the toxins I directed focus on was myotoxin II from the notorious pit viper Bothrops asper, where two discovered mAbs showed almost full neutralization of the myotoxic effects of myotoxin II in both in vitro and in vivo preincubation models. One of the human mAbs was added to a currently used antivenom to fortify its neutralizing capabilities, which resulted in an improvement of the antivenom’s ability to neutralize myotoxicity. However, when the antibody was tested in a model which closer mimics a real snakebite, known as a rescue model, contradicting results occurred as the mAb changed from being toxin-neutralizing to toxin-enhancing. While these results prevented a clear conclusion regarding the fortification of antivenoms, the results instead demonstrated the first reported case of antibody dependent enhancement (ADE) of the toxic effects exerted by a toxin from the animal kingdom. Follow-up experiments showed that antibody recycling using the neonatal Fc receptor (FcRn) likely contributed to the ADE. An important takeaway from the observed ADE is the contradicting results between the preincubation and the rescue in vivo models, since only the preincubation model is labeled as “essential” by WHO for testing antivenom efficacy. Thus, these results highlight the necessity of assessing even well-known antibody formats in representative preclinical models when evaluating their therapeutic utility against toxins or venoms, to avoid potential deleterious effects.
Finally, through a broad exploratory study, this PhD thesis has helped shed light on the determinants driving the discovery of cross-reactive mAbs using phage display technology. This study demonstrated that toxin cross-panning increased the fraction of cross-reactive antibodies in the discovery output. Further, it compared different antigen parameters as potential predictors for cross-reactive feasibility and found that antigen function might be a potential predictor, whereas the antigen sequence, structure, or surface homology did not appear particularly useful as standalone predictors.
It is my hope that the work presented here can help facilitate the future development of the next generation of antivenoms through an improved understanding of the pitfalls and the possibilities regarding the discovery and testing of human mAbs against snake venom toxins.