Thermal stabilization of anti-α-cobratoxin single domain antibodies

There is an unmet need for snake antivenoms that can be stored ready to use near the point of care. To address that need we have taken two anti-α-cobratoxin single domain antibodies and increased their thermal stability to improve their ambient temperature shelf-life. The anti-α-cobratoxin single domain antibodies C2 and C20 were first isolated, and demonstrated to be toxin neutralizing by Richard et al., 2013 (Richard, G., Meyers, A.J., McLean, M.D., Arbabi-Ghahroudi, M., MacKenzie, R., Hall, J.C., 2013. In vivo neutralization of alpha-cobratoxin with high-affinity llama single-domain antibodies (VHHs) and a VHH-Fc antibody. PLoS One 8, e69495). To thermal stabilize C2 and C20, we first made changes to their frame work 1 region that we had previously identified to be stabilizing, as well as reverted to the hallmark amino acids highly conserved in VHH domains; these changes improved their melting temperature (Tm) by 2 and 6 °C respectively. The further addition of a non-canonical disulfide bond raised the Tm an additional 13 and 9 °C respectively; giving final Tm values of 86 and 75 °C. Testing these mutants at 1 mg/mL at a range of elevated temperatures for an hour; we found that at 65 °C the wild type C2 and C20 had lost 35 and 95% of their binding activity respectively, while the mutants with the added disulfide bond retained nearly 100% of their initial binding activity. While significant work remains to formulate and field a shelf-stable antivenom, our results indicate such a product should be attainable in the near future.