Thermal stabilization of anti-α-cobratoxin single domain antibodies. (April 2017)

• Record Type: Journal Article
• Authors: Anderson, George P.; Liu, Jessica H.; Zabetakis, Dan; Liu, Jinny L.; Goldman, Ellen R.
• Is Part Of: Toxicon. Volume 129(2017)
• Page Start: 68
• Page End: 73
• Publication Date: 2017-04
• Subjects: Naja naja kaouthia -- Cobratoxin -- Antivenoms -- Snake venoms -- Single domain antibody -- Thermal stability; Toxins -- Periodicals; Venom -- Periodicals; 615.9
• DOI: 10.1016/j.toxicon.2017.02.008
• Languages: English
• ISSNs: 0041-0101

Abstract

Abstract: 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. Graphical abstract: Highlights: Two previously isolated anti- α-cobratoxin single domain antibodies were stabilized. Making point mutations and adding a disulfide bond raised the Tm between 9 and 13 °C. Both modified sdAb retained ∼100% of their binding activity after an hour at 65 °C.