Development of Cys38 knock-out and humanized version of NbAahII10 nanobody with improved neutralization of AahII Scorpion toxin

Abstract

During scorpion envenoming, highly toxic small polypeptides of the venom diffuse rapidly within the victim, causing serious medical problems. Nanobodies (Nbs), the recombinant single-domain antigen-binding fragments of camel-specific heavy-chain only antibodies, offer special advantages in therapy over classic antibody fragments due to their robustness and smaller size, matching the size of the scorpion toxins. Recently, a potent AahII scorpion toxin-neutralizing Nb was identified. However, this NbAahII10 contains a single Cys in its first antigen-binding loop, leading to Nb dimerization upon prolonged storage. In this work, we first investigate the efficacy of NbAahII10 variants in which this Cys was substituted by Ala, Ser or Thr. Second, the NbAahII10 Cys/Ser mutant displaying the best functional properties is subsequently humanized. It is demonstrated that the maximally humanized version of NbAahII10 Cys/Ser maintains its high affinity for the antigen without conceding much on expression yield and stability. More importantly, its neutralizing capacity is preserved as all mice survive injections of seven LD(50) and 50% of mice survived nine LD(50) of the scorpion toxin. Thus, this humanized Nb is the best candidate to develop a therapy in human against the most toxic venom compound of one of the most dangerous scorpions.

Wheat germin vitrotranslation to produce one of the most toxic sodium channel specific toxins

“…The amino acid sequence analysis showed that NbAahII10 contains a single cysteine in its CDR1 (complementary-determining region 1) [17], which leads to dimerization of the Nb upon storage. To avoid this dimerization, the cysteine was substituted by serine and the resulting NbAahII10 C/S was shown to be identical to freshly prepared NbAahII10 in recognizing its cognate antigen.…”

“…Immunotherapy remains probably the most efficient treatment after envenomation, but the outcome depends on both accurate identification of the scorpion species involved and the timely anti-venom administration [16]. Because of their high affinity and specificity, small size and robust behaviour, the single-domain antibodies, referred to as Nbs (nanobodies), have been proposed to substitute the polyclonal Fab’ 2 to treat the scorpion envenoming [17,18]. Indeed, a bispecific Nb construct comprising an Nb neutralizing AahI’ toxin and an Nb neutralizing AahII toxin was proven to protect mice and rats that received a subcutaneous lethal dose of the scorpion venom [3].…”

“…We also studied the role of the oxidoreductases, h QSOX1b (human quiescin-sulphhydryl oxidase) and h PDI (human protein disulphide isomerase), as possible protein-folding catalysts. Finally, we investigated the toxicity of this rAahII (after removal of the GST (glutathione S-transferase) tag which leaves two extra amino acids at the N-terminal of the rAahII relative to the native AahII) and the potential of nanobody NbAahII10 ( Androctonus australis hector nanobody 10), raised against native AahII toxin [3,17,26], to neutralize the toxicity of the purified rAahII toxin. Our results demonstrate the successful expression of soluble and active rAahII toxin and the ability of NbAahII10 to neutralize its toxicity in mice.…”

Evolution of alternative methodologies of scorpion antivenoms production

“…The Fab' domain from equine IgG is 3 times larger than VHH domain from nanobodies, so its pharmacokinetics is more dynamic in nanobodies. For these reasons nanobodies have been reported to be very effective tools in addition to their ability to neutralize scorpions toxins rapidly (Hmila et al, 2008;Abderrazek et al, 2009Abderrazek et al, , 2011Hmila et al, 2010;Ezzine et al, 2012 Q5 ; Hmila et al, 2012). The advantage of using recombinant antibodies in general is directly linked to production yield and the ability to express antigen recognition domains only.…”

Evaluation of three different formats of a neutralizing single chain human antibody against toxin Cn2: Neutralization capacity versus thermodynamic stability

“…A variety of antibody fragments against scorpion toxins has been produced and their capacity to neutralize the toxins in vitro and in vivo has been extensively tested [18][19][20][21][22][23][24][25][26][27]. As observed in other antigen-antibody systems, the in vivo neutralizing capacity of the anti toxin recombinant antibodies is influenced by the structural format of the molecule.…”