Synthetic antibodies block receptor binding and current‐inhibiting effects of α‐cobratoxin from Naja kaouthia

Abstract

Each year, thousands of people fall victim to envenomings caused by cobras.These incidents often result in death due to paralysis caused by α-neurotoxins from the three-finger toxin (3FTx) family, which are abundant in elapid venoms. Due to their small size, 3FTxs are among the snake toxins that are most poorly neutralized by current antivenoms, which are based on polyclonal antibodies of equine or ovine origin. While antivenoms have saved countless lives since their development in the late 18th century, an opportunity now exists to improve snakebite envenoming therapy via the application of new biotechnological methods, particularly by developing monoclonal antibodies against poorly neutralized α-neurotoxins. Here, we describe the use of phagedisplayed synthetic antibody libraries and the development and characterization of six synthetic antibodies built on a human IgG framework and developed against α-cobratoxinthe most abundant long-chain α-neurotoxin from Naja kaouthia venom. The synthetic antibodies exhibited sub-nanomolar affinities to α-cobratoxin and neutralized the curare-mimetic effect of the toxin in vitro. These results demonstrate that phage display technology based on synthetic repertoires can be used to rapidly develop human antibodies with druggrade potencies as inhibitors of venom toxins.

In vivo neutralization of coral snake venoms with an oligoclonal nanobody mixture in a murine challenge model

“…In this study, we developed a prototype mixture based on cross-neutralizing V H Hs and demonstrated its ability to neutralize lethality induced by two North American coral snake venoms in a mouse model. Previous studies have shown that a single monoclonal antibody can neutralize the lethal effects of snake venom when lethality is caused by one predominant toxin 39 , 40 , that such antibodies can be further developed to become broadly-neutralizing 19 , and that oligoclonal antibodies can be used to neutralize multiple different toxins 18 . However, here, we go beyond the state of the art by demonstrating that the concepts from these previous studies can be merged and applied to a simpler antibody format, namely the V H H format, thereby providing a proof of concept for a different type of antivenom 41 .…”

De novo designed proteins neutralize lethal snake venom toxins

“…As a representative long-chain α-neurotoxin, we chose α-cobratoxin from Naja kaouthia, one of the most extensively characterized toxins from the 3FTx family 43 (Figure 1). From 42 RFdiffusion designs against α-cobratoxin, one candidate had a binding affinity of 1.3 µM using BLI (Supplementary Figure S3).…”

Anin vitroassay to investigate venom neurotoxin activity on muscle-type nicotinic acetylcholine receptor activation and for the discovery of toxin-inhibitory molecules

“…The following method was adapted from Fitch et al [24] and Wang et al [25] and, as in section 2.2, all reagents were acquired from Gibco, Thermo Fisher Scientific, Paisley, UK, unless stated otherwise. One vial of FLIPR membrane potential dye (Component A, Explorer Kit Blue, R8042, Molecular Devices, San Jose, CA, USA) was dissolved in 36 mL assay buffer to create the dye solution.…”

“…Wang et al [25] previously demonstrated antagonism by α-BgTx, a Lc-α-NTx isolated from the venom of B. multicinctus, to the nicotine response of TE671 cells using membrane potential dye [32]. Consequently, α-BgTx was used along with a commercially available Sc-α-NTx, namely sNTx1 from the venom of N. pallida, which was used in previous studies to investigate Sc-α-NTx activity on nAChRs under the name 'toxin α' and originally thought to be isolated from venom of N. nigricollis [46].…”

“…However, these techniques are laborious, low-throughput, and resource-intensive, and are therefore barriers to identifying novel neurotoxin-inhibiting molecules (e.g., monoclonal antibodies, peptides, and/or small molecule drugs). More recently, automated patch-clamping has been introduced as a high-throughput method that allows for similar types of electrophysiological recordings [24][25][26]. However, this approach requires sophisticated equipment that is not available in most laboratories.…”