Abstract

Excellent results regarding improved therapeutic properties have been often obtained through the conversion of a single-chain variable fragment (scFv) into a noncovalent dimeric antibody (diabody) via peptide linker shortening. We utilized this approach to obtain a dimeric version of the human scFv 6009F, which was originally engineered to neutralize the Cn2 toxin of Centruroides noxius scorpion venom. However, some envenoming symptoms remained with diabody 6009F. Diabody 6009F was subjected to directed evolution to obtain a variant capable of eliminating envenoming symptoms. After two rounds of biopanning, diabody D4 was isolated. It exhibited a single mutation (E43G) in framework 2 of the heavy-chain variable domain. Diabody D4 displayed an increase in T(m) (thermal transition midpoint temperature) of 6.3°C compared with its dimeric precursor. The importance of the E43G mutation was tested in the context of the human scFv LR, a highly efficient antibody against Cn2, which was previously generated by our group [Riaño-Umbarila, L., Contreras-Ferrat, G., Olamendi-Portugal, T., Morelos-Juárez, C., Corzo, G., Possani, L. D. and Becerril, B. (2011). J. Biol. Chem.286, 6143-6151]. The new variant, scFv LER, displayed an increase in T(m) of 3.4°C and was capable of neutralizing 2 LD(50) of Cn2 toxin with no detectable symptoms when injected into mice at a 1:1 toxin-to-antibody molar ratio. These results showed that the E43G mutation might increase the therapeutic properties of these antibody fragments. Molecular modeling and dynamics results suggest that the rearrangement of the hydrogen-bonding network near the E43G mutation could explain the improved functional stability and neutralization properties of both the diabody D4 and scFv LER.

Links

Publisher Full Text

Authors

Rodríguez-Rodríguez ER, Ledezma-Candanoza LM, Contreras-Ferrat LG, Olamendi-Portugal T, Possani LD, Becerril B, Riaño-Umbarila L

Institution

Departamento de Medicina Molecular y Bioprocesos, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Apartado Postal 510-3, Cuernavaca, Morelos 62250, Mexico. remi@ibt.unam.mx

Source

Journal of molecular biology 423:3 2012 Oct 26 pg 337-50

MeSH

Amino Acid Sequence
Animals
Antibodies, Neutralizing
Antibody Affinity
Humans
Immunoglobulin Fc Fragments
Immunoglobulin Heavy Chains
Mice
Molecular Dynamics Simulation
Neutralization Tests
Protein Binding
Protein Structure, Tertiary
Scorpion Venoms
Single-Chain Antibodies
Surface Plasmon Resonance

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

22835504