Bispecific single-domain antibody (VHH) fused with human IgG1 Fc with dual specificity effectively neutralize Naja Kaouthia venom

Naja kaouthia, or the monocled cobra, is one of the most medically important snakes in Thailand, responsible for approximately 17% of snakebite cases. Conventional horse-derived antivenoms are lifesaving, yet they may trigger severe allergic reactions and exhibit batch to batch variability. Nanobodies (VHH) are promising alternatives as recombinant antivenoms having demonstrated the ability to neutralize snake venom both in vitro and in vivo. However, a major challenge in developing them is the diverse and complex composition of snake venoms, which requires therapies capable of targeting multiple toxins. To address this, we developed a bispecific VHH that simultaneously targets the two main toxins in N. kaouthia venoms, α-neurotoxin (αNTX) and phospholipase A2 (PLA2), fused to a human IgG Fc domain (bispecific VHH-Fc), which was selected to prolong serum half-life and reduce the immunogenicity risks associated with animal-derived antivenoms. The bispecific VHH-Fc, along with two monospecific nanobodies (VHH-αNTX-Fc and VHH-PLA2-Fc), was expressed in Chinese hamster ovary (CHO) cells and purified from culture supernatant after 5-6 days. Immunoblotting confirmed the successful expression and Fc fusion of these constructs, as detected by anti-human IgG-Fc antibodies conjugated to horseradish peroxidase (HRP). Importantly, antigen-binding assays demonstrated that the bispecific VHH-Fc exhibited the the strongest binding signal to crude N. kaouthia venom compared to the monospecific nanobodies. In in vivo murine neutralization assays, the bispecific VHH-Fc showing higher survival than equine-derived antivenom (33%) and comparable efficacy to a VHH-Fc cocktail under the tested conditions. Complete protection was achieved at higher doses. These results demonstrate that the bispecific VHH-Fc can be efficiently produced in a mammalian expression system and possesses strong binding and neutralizing activity against N. kaouthia venom under the defined experimental conditions. Our findings support the bispecific VHH-Fc as a promising next-generation therapeutic candidate for the treatment of snakebite envenoming, while highlighting the importance of integrating binding and functional assays when evaluating antibody efficacy.