Exploiting Cross-reactivity to Neutralize Two Different Scorpion Venoms with One Single Chain Antibody Fragment

Abstract

We report the optimization of a family of human single chain antibody fragments (scFv) for neutralizing two scorpion venoms. The parental scFv 3F recognizes the main toxins of Centruroides noxius Hoffmann (Cn2) and Centruroides suffusus suffusus (Css2), albeit with low affinity. This scFv was subjected to independent processes of directed evolution to improve its recognition toward Cn2 (Riaño-Umbarila, L., Juárez-González, V. R., Olamendi-Portugal, T., Ortíz-León, M., Possani, L. D., and Becerril, B. (2005) FEBS J. 272, 2591-2601) and Css2 (this work). Each evolved variant showed strong cross-reactivity against several toxins, and was capable of neutralizing Cn2 and Css2. Furthermore, each variant neutralized the whole venoms of the above species. As far as we know, this is the first report of antibodies with such characteristics. Maturation processes revealed key residue changes to attain expression, stability, and affinity improvements as compared with the parental scFv. Combination of these changes resulted in the scFv LR, which is capable of rescuing mice from severe envenomation by 3 LD 50 of freshly prepared whole venom of C. noxius (7.5 g/20 g of mouse) and C. suffusus (26.25 g/20 g of mouse), with surviving rates between 90 and 100%. Our research is leading to the formulation of an antivenom consisting of a discrete number of human scFvs endowed with strong cross-reactivity and low immunogenicity.

Effectiveness of the Androctonus Australis Hector Nanobody Nbf12-10 Antivenom to Neutralize Significantly the Toxic Effect and Tissue Damage Provoked by Fraction of Androctonus mauretanicus (Morocco) Scorpion Venom

“…Indeed, Am venom molecules (F3 fraction) could be detected easily by both, monospecific NbAahI'F12 and the bispecific NbF12-10 construct, which is suggestive for the presence of AahI related toxins or epitopes within the F3 fraction of Am venom. Similar results reported that human single chain fragment (scFv) recognized toxins of Centruroides noxius Hoffmann and Centruroides suffuses scorpion venom and rescue mice from severe envenomation by whole venom of Centruroides suffuses and Centruroides noxius [65]. Also, polyclonal anti-venom against Hadruroides lunatus displayed cross reactivity with venom from Tityus serrulatus, Centruroides sculpturatus and Androctonus australis hector scorpions [66].…”

Optimal Neutralization of Centruroides noxius Venom Is Understood through a Structural Complex between Two Antibody Fragments and the Cn2 Toxin

“…1A) that exclusively binds and modifies the gate functions of the human sodium channel Nav1.6 (11). We were able to obtain the scFv LR after three cycles of directed evolution of the parental scFv 3F and incorporating a V101F mutation into one of the derived variant scFvs (9004G) (12) (Fig. 1B).…”

“…The scFv C1 variants were generated using strategies described previously (12,13) and those described in this study. We learned that only a few changes were required to obtain scFvs that neutralize the Cn2 and Cll1 toxins.…”

“…In neutralization tests using 3 LD 50 of fresh whole venom in mice, scFv LR was capable of inhibiting nearly all symptoms caused by the C. suffusus suffusus venom. However, for the neutralization of C. noxius venom, some symptoms of intoxication were still observed, although survival rates between 90 and 100% were reached (rescue and preincubation assays) (12). These symptoms may be due to the presence of other toxins from the venom, which are less abundant but are not neutralized by the scFv LR.…”

Structural Insights into Antibody Sequestering and Neutralizing of Na+ Channel α-Type Modulator from Old World Scorpion Venom

“…The distinctive pharmacological properties are accompanied by distinctive immunological properties, as illustrated by the total absence of cross-reactivity between the ␣-and ␤-toxin classes (65). Moreover, unlike ␣-toxins, the ␤-toxins share greater structural homologies, as exemplified by significant cross-reactivity of a serum raised against a Centruroides immunogen with the main toxin from another Centruroides species (67,68). Hence, the availability of a crystal structure for a ␤-toxin bound to an antibody fragment is more likely to provide a canonical template for studying immunoreactivity of diverse members in this class of toxins than it is for ␣-toxins.…”