Isolation, characterization and pentamerization of α-cobrotoxin specific single-domain antibodies from a naïve phage display library: Preliminary findings for antivenom development

Conventional antivenoms to snakebite generated from the serum of immunized animals, often elicit adverse reactions and have mismatched pharmacokinetic profiles with their target toxins due to antibody/toxin size discrepancies which results in poor neutralization. Furthermore, animal immunization protocols are often lengthy and have batch to batch variability. Recombinant VHH-based antivenoms may help overcome these problems. Three VHH fragments with specificity to α-cobrotoxin, a snake neurotoxin from Naja kaouthia venom, were isolated from a naïve llama VHH phage-display library. α-Cobrotoxin-binding specificity was determined using a phage-displayed VHH ELISA format. Sequence analysis shows two of the three clones differ by only two amino acid substitutions, while the third is unique. Surface plasmon resonance analysis determined the KD values of the interactions to be 2, 3 and 3 μM. These affinities are too low for α-cobrotoxin detection in a standard ELISA format, or for practical use as therapeutic agents. However, improved functional affinity was obtained via antibody pentamerization and α-cobrotoxin detection was possible using a pentabody-based ELISA. Development of antivenoms composed of a mixture of antibody fragments, such as VHHs and VHH multimers, may help match the pharmacokinetic profiles of complex venoms, improving antivenom biodistribution, and toxin neutralization while reducing adverse effects in humans.