Using a recombinant bispecific antibody to block Na+-channel toxins protects against experimental scorpion envenoming

Abstract

In recent years, several molecular engineering methods of designing bispecific antibodies in various formats have been developed. Tandem-scFvs comprising two scFvs fused together via a peptide are 55-kDa molecules, and are one of the most promising and most straightforward approaches to bispecific antibody production. We report an attempt to design more effective antivenoms to the Androctonus australis scorpion using murine scFvs as building blocks to create a unique bispecific molecule that neutralizes the potent neurotoxins AahI and AahII. The tandem-scFv was produced in recombinant bacteria, purified by immobilized metal ion affinity chromatography, and analyzed by polyacrylamide gel electrophoresis, Western blot, gel filtration, mass spectrometry, and direct and competitive radioimmunoassay. In vivo, it neutralized the binding of the AahI and AahII toxins to their receptor, and protected mice against experimental envenomation. The findings reported here highlight the potential of recombinant antibody fragments for protecting against scorpion venom toxicity.

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

“…IgG4C1 binds AahII, whereas IgG9C2, generated against AahI, also binds AahIII (29) and AahIV (this work) with high affinities. The intraperitoneal injection of a bispecific tandem-scFv combining the variable domains of IgG4C1 and IgG9C2 or the concomitant intraperitoneal injections of singlechain homomeric diabodies also derived from these IgGs were found to protect experimentally envenomed mice against the overall toxicity of subcutaneous injection of up to 3 LD 50 of the Aah venom (11). 8 Hence, in conjunction with recent strategies involving selection of scFvs using phage-display technology (74), production of camelid antibodies because of their particular suitability for therapeutic use (10), prediction of potential epitopic regions through bioinformatics approaches (75), or the raising of antibodies against discontinuous epitopes (30,76), our structural data provide new templates for further enhancement of the binding affinity and neutralizing capacity of antiAah antivenoms to reach more efficient immunotherapy in humans.…”

VHH, bivalent domains and chimeric Heavy chain-only antibodies with high neutralizing efficacy for scorpion toxin AahI′

“…These constructs were welcomed for offering a promising protection against scorpion envenoming (Mousli et al, 1999;Devaux et al, 2001;Aubrey et al, 2003Aubrey et al, , 2004. Recent exercises introduced a bispecific tandem scFv from these constructs that recognize both, the AahI and the AahII toxin (Juste et al, 2007). However, the functional expression of this scFv tandem was rather low (100 g/L) and its stability remains questionable.…”

Recombinant therapeutic monoclonal antibodies: Mechanisms of action in relation to structural and functional duality

“…Several reports on mAbs directed to toxins have pointed out that tetanus toxin [11], botulinum neurotoxin (BoNT) [12], or ricin [13] can be very potently neutralized in vitro and in vivo by combining several mAbs binding non-overlapping epitopes. It has also been shown recently that a bispecific tandem-scFv neutralized the binding of both the AahI and AahII toxins of scorpion venom and protected mice against experimental envenomation [14]. Finally, a mAb that binds the BoNT/A1 subtype with high affinity and the BoNT/A2 subtype with low affinity has recently been engineered to increase the affinity for BoNT/A2 while maintaining that for BoNT/A1 [15].…”