title: Thermal stabilization of anti-α-cobratoxin single domain antibodies.
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.
authors.affiliation: US Naval Research Laboratory, Center for Biomolecular Science and Engineering, 4555 Overlook Ave SW, Washington, DC, 20375, USA. Electronic address: george.anderson@nrl.navy.mil.
authors.forename: George P
authors.initials: GP
authors.lastname: Anderson
authors.affiliation: Science and Engineering Apprenticeship Program, US Naval Research Laboratory, 4555, Overlook Ave SW, Washington, DC, USA.
authors.forename: Jessica H
authors.initials: JH
authors.lastname: Liu
authors.affiliation: US Naval Research Laboratory, Center for Biomolecular Science and Engineering, 4555 Overlook Ave SW, Washington, DC, 20375, USA.
authors.forename: Dan
authors.initials: D
authors.lastname: Zabetakis
authors.affiliation: US Naval Research Laboratory, Center for Biomolecular Science and Engineering, 4555 Overlook Ave SW, Washington, DC, 20375, USA.
authors.forename: Jinny L
authors.initials: JL
authors.lastname: Liu
authors.affiliation: US Naval Research Laboratory, Center for Biomolecular Science and Engineering, 4555 Overlook Ave SW, Washington, DC, 20375, USA.
authors.forename: Ellen R
authors.initials: ER
authors.lastname: Goldman
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000997:Antivenins / Q000737:chemistry / Q000494:pharmacology*; D002942:Circular Dichroism; D003039:Cobra Neurotoxin Proteins / Q000276:immunology*; D017815:Elapidae; D055550:Protein Stability; D061905:Single-Domain Antibodies / Q000737:chemistry / Q000494:pharmacology*; D020349:Surface Plasmon Resonance; D013696:Temperature
chemical_list: D000997:Antivenins; D003039:Cobra Neurotoxin Proteins; D061905:Single-Domain Antibodies; C025154:alpha-cobratoxin
keywords: Antivenoms; Cobratoxin; Naja naja kaouthia; Single domain antibody; Snake venoms; Thermal stability