Black mamba (Dendroaspis polylepis)

Snakebite at first glance does not seem to be a problem, which should concern a modern human. Despite numerous achievements in the field of pharmacology, it is however among the most neglected diseases of our era.

Grim statistics provided by the WHO (World Health Organisation) show that, each year, 5 million bites occur, leading to 150,000 deaths and a striking 400,000 amputations. The lucky ones who manage to avoid a deadly outcome of snakebite envenoming are often mentally and physically scarred for life. Victims are mostly the children and young workers on the fields, left with mutilated bodies, disabling them from getting married, working, or otherwise providing for their families. Needless to say, snakebite is a significant socio-economic burden to rural areas and in tropical regions of the world, where most bites occur.

Watch this video from the Global Snakebite Initiative (GSI) about the global burden of snakebite envenoming:

Snake venom is a sophisticated product of evolution, and venom compositions differ greatly among different snake species. Variability is even observed between individual specimens because of varying environmental factors in the habitats where the specific specimens are collected. Venoms contain myriads of disparate proteins with bioactive (toxic) effects. A snake may express more than 50 toxin-encoding genes, which makes it exceptionally difficult to develop effective snakebite antivenoms.

As an example, the venom of the notorious black mamba consists of many different paralysing neurotoxins and a multitude of excitatory dendrotoxins (and many other venom components), which synergistically combine their effects and cause strong neurotoxic symptoms, such as flaccid paralysis and involuntary muscle contractions. Spitting cobra venoms, on the other hand, derive their toxic effects both from paralysing neurotoxins and “flesh-eating” cytotoxins. A third strategy can be exemplified by the venom of a rattlesnake, which typically forces the blood of its victims to coagulate, leading to internal bleeding, once all blood factors are exhausted.

Antiserum – The Traditional Treatment Against Snakebite

The current medical treatment against snakebite is based on administration of animal-derived antiserum containing toxin-targeting antibodies. Antiserum is produced by repetitive immunization of large animals (typically horses or sheep) with selected snake venoms to build up an antibody response against the different components from the venoms. After a prolonged immunization period, which can take up to 1.5 years, blood is drawn and the serum is isolated by removal of the blood cells. Antibodies present in the serum are then purified by precipitating other serum proteins, after which the antibody solution is formulated to ensure better stability of the antiserum for long-term storage. Finally, the antiserum is ready to be delivered to the patient.

Antiserum manufacture

Figure 1: Schematic overview of traditional animal-derived antiserum manufacture.

Although animal-derived antisera are effective in neutralizing snake venoms, they have a number of significant drawbacks, such as being time-consuming and costly to produce, having significant batch-to-batch variation, and being of heterologous (non-human) origin. Their heterologous nature may cause antisera to inflict adverse effects in patients due to their incompatibility with the human immune system. Another problem with existing antisera, is that only a small subset of the antibodies present in traditional antisera recognizes snake venom components. Traditional manufacturing processes generally do not include steps to separate therapeutically active antibodies from the rest of the antibodies that the production animal has raised against other pathogens and antigens during its lifetime. Consequently, up to 80% (for certain antisera) of patients experience side effects, including serum sickness and anaphylaxis, which in the worst-case scenarios may result in death of the patient.

Lack of appropriate technology does not prevent us from implementing alternative manufacturing strategies for snakebite antivenoms. In comparison, other protein-based therapies, such as insulin and human blood factors, are no longer derived from pigs or human donors respectively, but instead produced recombinantly (in the human form) by cellular expression in large fermentation/cultivation tanks. This switch from traditional manufacture of these biologics has had enormous impact on patient outcomes, but we are yet to see antivenoms enter the modern era of biopharmaceuticals and adapt the same biotechnological manufacturing strategies.

Recombinant Antivenom – A New Hope?

As opposed to traditional animal-derived antivenom, the Tropical Pharmacology Lab at the Technical University of Denmark see a novel solution for how to tackle snakebite. Researchers there are working on developing mixtures of human monoclonal antibodies that will be safer and more efficacious compared to the existing antisera. Eventually, they hope to replace conventional envenoming therapy with so-called recombinant antivenoms based on toxin-targeting human antibodies.

Once available to snakebite victims, recombinant antivenoms would have quite a few benefits compared to animal-derived antisera. As the recombinant antivenoms would be based on human antibodies, they would first of all be compatible with the human immune system. Also, a recombinant antivenom based on carefully selected antibodies can be designed to target only medically relevant toxins (and not irrelevant non-toxic venom components), bringing the content of therapeutically active antibodies up to 100%. Additionally, recombinant antivenoms could be produced by mammalian cell cultivation, making production less time-consuming and less expensive, which would pave the way for low cost therapies for impoverished victims living in rural parts of the tropics.

Manufacture using mammalian cells would also enable optimization and standardization of the process, which would lower batch-to-batch variation. Manufacturing an effective cocktail of human antibodies capable of neutralizing the medically most relevant toxins in selected snake venoms could be done through oligoclonal recombinant antibody expression in a single bioreactor containing different cell lines, expressing different monoclonal antibodies. Purification could then be performed on the mix of antibodies, providing the final oligoclonal recombinant antivenom capable of neutralizing a multitude of snake venom toxins.


Oligoclonal expression of human antibody mixtures in mammalian cells

Figure 2: Schematic representation of the oligoclonal expression of human antibody mixtures in mammalian cells.

The discovery approach they are following at the Tropical Pharmacology Lab combines the fields toxicovenomics (the study of venom proteomes in relation to toxicity) and antibody discovery by phage display selection. The first step involves snake venom fractionation by chromatographic means to isolate medically relevant toxins. Thereafter, these toxins are used as targets in our antibody discovery process to find antibodies that can target and neutralize these toxins. If these antibodies show promising results in different tests, they are advanced into development with the aim of using them as part of an oligoclonal recombinant snakebite antivenom.

“In our research, we are lucky to work closely together with international collaborators, including Instituto Clodomiro Picado in Costa Rica, where they are experts on toxinology and antivenom research, and IONTAS from the United Kingdom, where they are experts in phage display technology and antibody discovery”

Together with their close collaborators, the team´s goal is to harness biotechnology and pave the way for novel recombinant antivenoms that can save lives and limbs of snakebite victims worldwide.

By Urska Pus, stud.M.Sc and Andreas Hougaard Laustsen, M.Sc.Eng, PhD, Postdoctoral Fellow, Technical University of Denmark