title: A single mutation in framework 2 of the heavy variable domain improves the properties of a diabody and a related single-chain antibody.
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.
authors: 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
authors: Everardo Remi
authors: ER
authors: Rodríguez-Rodríguez
authors: Luis M
authors: LM
authors: Ledezma-Candanoza
authors: Luis Gabriel
authors: LG
authors: Contreras-Ferrat
authors: Timoteo
authors: T
authors: Olamendi-Portugal
authors: Lourival D
authors: LD
authors: Possani
authors: Baltazar
authors: B
authors: Becerril
authors: Lidia
authors: L
authors: Riaño-Umbarila
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D057134:Antibodies, Neutralizing; D000915:Antibody Affinity; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D051379:Mice; D056004:Molecular Dynamics Simulation; D009500:Neutralization Tests; D011485:Protein Binding / Q000276:immunology; D017434:Protein Structure, Tertiary; D012604:Scorpion Venoms / Q000737:chemistry / Q000276:immunology*; D057127:Single-Chain Antibodies / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D020349:Surface Plasmon Resonance
chemical_list: D057134:Antibodies, Neutralizing; C035413:Centruroides toxin; D007141:Immunoglobulin Fc Fragments; D007143:Immunoglobulin Heavy Chains; D012604:Scorpion Venoms; D057127:Single-Chain Antibodies