In Vivo Neutralization of α-Cobratoxin with High-Affinity Llama Single-Domain Antibodies (V(H)Hs) and a V(H)H-Fc Antibody

Small recombinant antibody fragments (e.g. scFvs and V(H)Hs), which are highly tissue permeable, are being investigated for antivenom production as conventional antivenoms consisting of IgG or F(ab’)(2) antibody fragments do not effectively neutralize venom toxins located in deep tissues. However, antivenoms composed entirely of small antibody fragments may have poor therapeutic efficacy due to their short serum half-lives. To increase serum persistence and maintain tissue penetration, we prepared low and high molecular mass antivenom antibodies. Four llama V(H)Hs were isolated from an immune V(H)H-displayed phage library and were shown to have high affinity, in the low nM range, for α-cobratoxin (α–Cbtx), the most lethal component of Naja kaouthia venom. Subsequently, our highest affinity V(H)H (C2) was fused to a human Fc fragment to create a V(H)H2-Fc antibody that would offer prolonged serum persistence. After in planta (Nicotiana benthamiana) expression and purification, we show that our V(H)H2-Fc antibody retained high affinity binding to α–Cbtx. Mouse α–Cbtx challenge studies showed that our highest affinity V(H)Hs (C2 and C20) and the V(H)H2-Fc antibody effectively neutralized lethality induced by α–Cbtx at an antibody:toxin molar ratio as low as ca. 0.75×:1. Further research towards the development of an antivenom therapeutic involving these anti-α-Cbtx V(H)Hs and V(H)H2-Fc antibody molecules should involve testing them as a combination, to determine whether they maintain tissue penetration capability and low immunogenicity, and whether they exhibit improved serum persistence and therapeutic efficacy.