An in vitro potency assay using nicotinic acetylcholine receptor binding works well with antivenoms against Bungarus candidus and Naja naja

Ratanabanangkoon, Kavi; Simsiriwong, Pavinee; Pruksaphon, Kritsada; Tan, Kae Yi; Chantrathonkul, Bunkuea; Eursakun, Sukanya; Tan, Choo Hock

doi:10.1038/s41598-018-27794-3

Cited by 11 publications

(5 citation statements)

References 37 publications

(47 reference statements)

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“…The receptor blocking assay was adapted from Ratanabanangkoon et al . 36 Black MaxiSorp plates (Nunc) were coated overnight with 100 µL of 5 µg/mL human α7-acetylcholine receptor chimera (adapted from Ref. 37 ) in PBS.…”

Section: Methodsmentioning

confidence: 99%

In vitro discovery of a human monoclonal antibody that neutralizes lethality of cobra snake venom

Ledsgaard

Laustsen

Pus

et al. 2022

mAbs

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The monocled cobra ( Naja kaouthia ) is among the most feared snakes in Southeast Asia due to its toxicity, which is predominantly derived from long-chain α-neurotoxins. The only specific treatment for snakebite envenoming is antivenom based on animal-derived polyclonal antibodies. Despite the lifesaving importance of these medicines, major limitations in safety, supply consistency, and efficacy create a need for improved treatments. Here, we describe the discovery and subsequent optimization of a recombinant human monoclonal immunoglobulin G antibody against α-cobratoxin using phage display technology. Affinity maturation by light chain-shuffling resulted in a significant increase in in vitro neutralization potency and in vivo efficacy. The optimized antibody prevented lethality when incubated with N. kaouthia whole venom prior to intravenous injection. This study is the first to demonstrate neutralization of whole snake venom by a single recombinant monoclonal antibody, thus providing a tantalizing prospect of bringing recombinant antivenoms based on human monoclonal or oligoclonal antibodies to the clinic.

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Section: Methodsmentioning

confidence: 99%

In vitro discovery of a human monoclonal antibody that neutralizes lethality of cobra snake venom

Ledsgaard

Laustsen

Pus

et al. 2022

mAbs

View full text Add to dashboard Cite

show abstract

“…The receptor blocking assay was adapted from Ratanabanangkoon et al (35) . Black Maxisorp plates (Nunc) were coated overnight with 100 µL of 5 µg/mL human α7-acetycholine receptor chimera (adapted from (36) ) in PBS.…”

Section: Methodsmentioning

confidence: 99%

In vitro discovery and optimization of a human monoclonal antibody that neutralizes neurotoxicity and lethality of cobra snake venom

Ledsgaard

Laustsen

Pus

et al. 2021

Preprint

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The monocled cobra (Naja kaouthia) is one of the most feared snakes in Southeast Asia. It is a highly dangerous species with a potent venom deriving its toxicity predominantly from abundant long-chain α-neurotoxins. The only specific treatment for snakebite envenoming is antivenom, which is based on animal-derived polyclonal antibodies. Despite the lifesaving importance of these medicines over the past 120 years, and their ongoing role in combating snakebite disease, major limitations in safety, supply consistency, and efficacy creates a need for a new generation of improved treatments based on modern biotechnological techniques. Here, we describe the initial discovery and subsequent optimization of a recombinant human monoclonal immunoglobin G (IgG) antibody against α-cobratoxin using phage display technology. Affinity maturation of the parental antibody by light chain-shuffling resulted in an 8-fold increase in affinity, translating to a significant increase in in vitro neutralization potency and in vivo efficacy. While the parental antibody prolonged survival of mice challenged with purified α-cobratoxin, the optimized antibody prevented lethality when incubated with N. kaouthia whole venom prior to intravenous injection. This study is the first to demonstrate neutralization of whole snake venom by a single recombinant monoclonal antibody. Importantly, this suggests that for venoms whose toxicity relies on a single predominant toxin group, such as that of N. kaouthia, as little as one monoclonal antibody may be sufficient to prevent lethality, thus providing a tantalizing prospect of bringing recombinant antivenoms based on human monoclonal or oligoclonal antibodies to the clinic.One Sentence SummaryA recombinant human monoclonal immunoglobulin G antibody, discovered and optimized using in vitro methods, was demonstrated to neutralize the lethal effect of whole venom from the monocled cobra in mice via abrogation of α-neurotoxin-mediated neurotoxicity.

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“…Other studies have reported the ability of a N. kaouthia PLA 2 to block acetylcholine currents in Lymnaea stagnalis neurons or to compete with α-bungarotoxin for binding to Torpedo or α7-type receptors (Vulfius et al, 2014). The purified Torpedo nAChR has also been employed in in vitro assays to assess antivenom efficacy against the venoms of N. kaouthia (Thai monocled cobra), N. naja (Indian cobra), and B. candidus (Malayan krait) (Ratanabanangkoon et al, 2017, Ratanabanangkoon et al, 2018).…”

Section: Introductionmentioning

confidence: 99%

A Decoy-Receptor Approach Using Nicotinic Acetylcholine Receptor Mimics Reveals Their Potential as Novel Therapeutics Against Neurotoxic Snakebite

et al. 2019

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Snakebite is a neglected tropical disease that causes 138,000 deaths each year. Neurotoxic snake venoms contain small neurotoxins, including three-finger toxins (3FTxs), which can cause rapid paralysis in snakebite victims by blocking postsynaptic transmission via nicotinic acetylcholine receptors (nAChRs). These toxins are typically weakly immunogenic and thus are often not effectively targeted by current polyclonal antivenom therapies. We investigated whether nAChR mimics, also known as acetylcholine binding proteins (AChBPs), could effectively capture 3FTxs and therefore be developed as a novel class of snake-generic therapeutics for combatting neurotoxic envenoming. First, we identified the binding specificities of 3FTx from various medically important elapid snake venoms to nAChR using two recombinant nAChR mimics: the AChBP from Lymnaea stagnalis and a humanized neuronal α7 version (α7-AChBP). We next characterized these AChBP-bound and unbound fractions using SDS-PAGE and mass spectrometry. Interestingly, both mimics effectively captured long-chain 3FTxs from multiple snake species but largely failed to capture the highly related short-chain 3FTxs, suggesting a high level of binding specificity. We next investigated whether nAChR mimics could be used as snakebite therapeutics. We showed that while α7-AChBP alone did not protect against Naja haje (Egyptian cobra) venom lethality in vivo , it significantly prolonged survival times when coadministered with a nonprotective dose of antivenom. Thus, nAChR mimics are capable of neutralizing specific venom toxins and may be useful adjunct therapeutics for improving the safety and affordability of existing snakebite treatments by reducing therapeutic doses. Our findings justify exploring the future development of AChBPs as potential snakebite treatments.

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An in vitro potency assay using nicotinic acetylcholine receptor binding works well with antivenoms against Bungarus candidus and Naja naja

Cited by 11 publications

References 37 publications

In vitro discovery of a human monoclonal antibody that neutralizes lethality of cobra snake venom

In vitro discovery of a human monoclonal antibody that neutralizes lethality of cobra snake venom

In vitro discovery and optimization of a human monoclonal antibody that neutralizes neurotoxicity and lethality of cobra snake venom

A Decoy-Receptor Approach Using Nicotinic Acetylcholine Receptor Mimics Reveals Their Potential as Novel Therapeutics Against Neurotoxic Snakebite

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