Discovery and optimization of a broadly-neutralizing human monoclonal antibody against long-chain α-neurotoxins from snakes

Abstract

Snakebite envenoming continues to claim many lives across the globe, necessitating the development of improved therapies. To this end, broadly-neutralizing human monoclonal antibodies may possess advantages over current plasma-derived antivenoms by offering superior safety and high neutralization capacity. Here, we report the establishment of a pipeline based on phage display technology for the discovery and optimization of high affinity broadly-neutralizing human monoclonal antibodies. This approach yielded a recombinant human antibody with superior broadly-neutralizing capacities in vitro and in vivo against different long-chain α-neurotoxins from elapid snakes. This antibody prevents lethality induced by Naja kaouthia whole venom at an unprecedented low molar ratio of one antibody per toxin and prolongs the survival of mice injected with Dendroaspis polylepis or Ophiophagus hannah whole venoms.

Discovery of broadly-neutralizing antibodies against brown recluse spider and Gadim scorpion sphingomyelinases using consensus toxins as antigens

“…The scFv genes from the third selection round were amplified using PCR and subcloned using NcoI and NotI restriction endonuclease sites into the pSANG10-3F vector for expression of soluble scFvs transformed into E. coli BL21(DE3) cells (New England Biolabs), as previously descrived [6,10]. The pSANG10-3F vector contains a FLAG-tag and a 6xHis tag in phase with the scFv.…”

“…Recently, it has been demonstrated that broadly-neutralizing mAbs (bnAbs) targeting animal toxins can be developed using cross-panning strategies in phage display selection campaigns 5,6 , by semi-rational design and directed evolution 7 , and by high-throughput screening of B-cells from immunized individuals 8 . Here, we demonstrate the utility of a fourth strategy that uses consensus toxins, which are artificial toxins based on the average sequence of related toxins 9 .…”

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

“…The Sc-α-NTx 'SHORT NEUROTOXIN alpha (NP)' (listed with the recommended name 'short neurotoxin 1' (sNTx1) on the UniProt database; P01426) isolated from Naja pallida venom was purchased from Latoxan (L8101, Valence, France), and the Lc-α-NTx, 'αbungarotoxin' (α-BgTx), isolated from Bungarus multicinctus venom was purchased from Biotium (0010-1, Fremont, CA, USA). Lymnaea stagnalis (Ls-AchBP) was prepared as previously described [17], as were the fully human monoclonal antibodies (mAbs) 2551_01_A12, 2554_01_D11 and 367_01_H01 in IgG1 format [21]. Samples of the various small molecule drugs used for screening were obtained by request from the Open Chemical Repository of the Developmental Therapeutics Program (https://dtp.cancer.gov) (Division of Cancer Treatment and Diagnosis, National Cancer Institute, Rockville, MD, USA), except for nicotine (see section 2.1.2) and varespladib (SML1100, Sigma-Aldrich, Gillingham, UK).…”

“…To explore the utility of our assay as a functional screen to detect novel toxin-inhibitory molecules, we selected representatives of these different therapeutic formats (antivenoms, small molecule drugs, nAChR-mimicking proteins, and monoclonal antibodies) and assessed their ability to inhibit the nAChR antagonism stimulated by representative neurotoxic snake venoms (from N. haje and D. polylepis) and α-NTxs (α-BgTx and sNTx1). In line with the WHO guidelines for preclinical testing of antivenoms [13] and many other in vitro and in vivo approaches to assess venom inhibition [17,21,28,52], we performed these experiments with an initial pre-incubation step, where inhibitor and venom/toxin were co-incubated at 37 °C for 30 min before assaying, to give the inhibitor maximal opportunity to exhibit neutralisation (Fig. 5).…”

“…In recent years, several new approaches to either improve, supplement, or replace existing antivenoms have been described [17][18][19][20][21]. Because neurotoxic envenoming can rapidly become life-threatening, toxins that act on the nAChR are priority targets for the discovery of novel therapeutics.…”