Snaking Towards Synthetic Antivenoms

While walking through the woods, humans instinctively scan the forest floor for signs of danger. A rustling of leaves or an unexpected shape in the periphery causes the heart to race and sends shivers down the spine. Although humans and snakes share a mutual desire to avoid each other, snakes camouflaged in the underbrush can startle even the most vigilant hikers. While their attacks are defensive, encounters with certain snake species can be fatal. 

Snakebite envenoming kills more than 100,000 people each year and leaves many more with permanent disabilities.¹ Antivenoms based on animal-derived antibodies are the primary therapeutic counterattacks against envenoming, but these treatments have two major shortcomings: their nonhuman origin can trigger dangerous immune reactions, and they fail to efficiently target the wide variety of harmful snake venom toxins. 

“The strategy that is currently being used for treating snake bites is over 100 years old,” said Kartik Sunagar, an evolutionary geneticist at the Indian Institute of Science. In a paper published in Science Translational Medicine, Sunagar and a global team of researchers applied modern technologies to address this long-standing public health problem. They developed a synthetic human antibody that blocks the lethal effects of a key neurotoxin found across a number of deadly snake species.² The antibody, which broadly binds multiple variants of this neurotoxin, exemplifies how modernizing and streamlining the antivenom discovery pipeline may help usher in safer therapies for snake bite victims. 

“This is a state of the art approach,” said Christiane Berger-Schaffitzel, a biochemist at the University of Bristol who was not involved in the research. “It’s a fantastic example of how this can be successful and how this can really lead to a broadly neutralizing antibody, which is urgently needed for many of the toxins.”

Historically, scientists have generated antivenoms by immunizing large animals, such as horses, sheep, llamas, and camels, with sublethal doses of snake venoms, isolating the serum from the blood, and transforming the resulting amalgam of antibodies into a treatment.³ In some cases, however, patients’ immune systems flag these animal antibodies as foreign, triggering serum sickness and anaphylactic shock. 

Snake venoms are also incredibly diverse biochemical cocktails, both across and within species.^4,5 “That makes it so complicated to generate a universal antivenom,” said Berger-Schaffitzel. “You can’t just import antivenom from other regions of the world because it needs to be tailored to the snakes that are there locally.”

In their study, Sunagar and his team took a major step towards a broad-spectrum antivenom by targeting a key toxin subfamily: long-chain three-finger alpha-neurotoxins (3FTx-L).⁶ These lethal proteins, found in the venoms of many snake species, including cobras, kraits, and mambas, block nicotinic acetylcholine receptors (nAChR) at neuromuscular junctions, causing paralysis and death. 

The research team developed a fully synthetic platform to explore, at scale, the molecular recognition that occurs between the immune system and antigens. After engineering mammalian cells to produce several 3FTx-L variants, they used yeast display to screen a library of 100 billion synthetic human antibodies against the toxins to identify those with a strong and broad binding profile. “Animals may not even come up with some of these combinations,” said Sunagar. “Because it is a synthetic library, it contains much more diversity than what you would find in nature.”

After several rounds of selection, the researchers identified a few dozen promising candidates that bound all 3FTx-L variants. However, one antibody, which they called 95Mat5, stood out as being the most potent. To test their antibody’s performance in human cells, the researchers administered 95Mat5 alongside the 3FTx-L variant alpha-bungarotoxin. This lethal toxin is found in the venom of the banded krait, a highly venomous snake species found in Asia, including India where Sunagar and his team work.  Their antibody interfered with alpha-bungarotoxin’s ability to block nAChR, but they wanted to understand the dynamics of this interaction.

“It is a great antibody that they’ve discovered—no doubt about that—but what is novel and the strength of the study is that they went in, did crystal structures, and identified the epitope that is bound and could explain the mechanism of action, which is something that’s rarely done in the antivenom field,” said Andreas Laustsen-Kiel, a bioengineer at the Technical University of Denmark who was not involved in the study. 

By peering into the crystal structure, the researchers discovered that 95Mat5 binds to the same region of alpha-bungarotoxin that the toxin uses to attach to nAChR. Laustsen-Kiel said that structural information on toxin recognition can help scientists rationally design the best antibodies for a particular toxin. 

To put their antibody to the test in animals, the researchers preincubated 95Mat5 with alpha-bungarotoxin and injected the cocktail into mice, all of which survived. Although this is the gold-standard assay for testing antivenoms, many snakebite victims wait hours before receiving an antidote. This inspired the researchers to run rescue experiments in which they administered the antivenom 20 minutes—a long time for a mouse—after envenoming with either the full venom of the monocellate cobra or the black mamba. While untreated mice died within three hours, those that received the 95Mat5 antibody lived with no signs of neurotoxicity for at least 24 hours.

“This is one antibody that is neutralizing venomous snakes across continents, which is a very broad spectrum of neutralization which has not been seen before,” said Sunagar. However, he noted that the antibody cocktail still needs some tweaks. While the treatment completely neutralized some snake venoms, it was less effective against king cobra envenoming. The authors hypothesized that this could be due to other alpha-neurotoxins and non-3FTx components present in the king cobra venom.⁷  Sunagar noted that they still need to discover additional antibodies that can neutralize other lethal ingredients in snake venoms before they can get closer to creating a universal, or at least broad-spectrum, antivenom.

Snakebite envenoming, a neglected tropical disease, disproportionately affects people living in low- and middle-income countries.⁸ “There is an urgent need to treat this socioeconomic disease,” said Sunagar. Sunagar and his team hope that their framework provides a blueprint for generating other broadly neutralizing antivenom antibodies.

Laustsen-Kiel and his colleagues are using similar methods to develop human antibodies against snake venoms.^9,10 Noting the broader implications of these efforts towards antibody-based therapies, Laustsen-Kiel said, “Snakebite is a fantastic playing ground for scientists to help solve an important mission, but at the same time, develop new methods and knowledge that can be applied broadly in drug development and lead to better therapies for envenomings, poisonings, infectious diseases, and maybe even other things.”

References

1. Gutiérrez JM, et al. Snakebite envenoming. Nat Rev Dis Primers. 2017;3:17063. 
2. Khalek IS, et al. Synthetic development of a broadly neutralizing antibody against snake venom long-chain α-neurotoxins. Sci Transl Med. 2024;16(735):eadk1867.
3. León G, et al. Current technology for the industrial manufacture of snake antivenoms. Toxicon. 2018;151:63-73.
4. Casewell NR, et al. Causes and consequences of snake venom variation. Trends Pharmacol Sci. 2020;41(8):570-581.
5. Rashmi U, et al. Remarkable intrapopulation venom variability in the monocellate cobra (Naja kaouthia) unveils neglected aspects of India’s snakebite problem. J Proteomics. 2021;242:104256.
6. Nirthanan S. Snake three-finger α-neurotoxins and nicotinic acetylcholine receptors: Molecules, mechanisms and medicine. Biochem Pharmacol. 2020;181:114168. 
7. Jaglan A, et al. The royal armoury: Venomics and antivenomics of king cobra (Ophiophagus hannah) from the Indian Western Ghats. Int J Biol Macromol. 2023;252(Pt 2):126708.
8. Bhaumik S, et al. Mitigating the chronic burden of snakebite: Turning the tide for survivors. Lancet. 2021;398(10309):1389-1390.
9. Ledsgaard L, et al. Discovery and optimization of a broadly-neutralizing human monoclonal antibody against long-chain α-neurotoxins from snakes. Nat Commun. 2023;14(1):682.
10. Laustsen AH. Recombinant snake antivenoms get closer to the clinic. Trends Immunol. 2024;45(4):225-227.