Departamento de Medicina Molecular y Bioprocesos, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Apartado Postal 510-3, Cuernavaca, Morelos 62250, México.
J Biol Chem. 2011 Feb 25;286(8):6143-51. doi: 10.1074/jbc.M110.189175. Epub 2010 Dec 14.
We report the optimization of a family of human single chain antibody fragments (scFv) for neutralizing two scorpion venoms. The parental scFv 3F recognizes the main toxins of Centruroides noxius Hoffmann (Cn2) and Centruroides suffusus suffusus (Css2), albeit with low affinity. This scFv was subjected to independent processes of directed evolution to improve its recognition toward Cn2 (Riaño-Umbarila, L., Juárez-González, V. R., Olamendi-Portugal, T., Ortíz-León, M., Possani, L. D., and Becerril, B. (2005) FEBS J. 272, 2591-2601) and Css2 (this work). Each evolved variant showed strong cross-reactivity against several toxins, and was capable of neutralizing Cn2 and Css2. Furthermore, each variant neutralized the whole venoms of the above species. As far as we know, this is the first report of antibodies with such characteristics. Maturation processes revealed key residue changes to attain expression, stability, and affinity improvements as compared with the parental scFv. Combination of these changes resulted in the scFv LR, which is capable of rescuing mice from severe envenomation by 3 LD(50) of freshly prepared whole venom of C. noxius (7.5 μg/20 g of mouse) and C. suffusus (26.25 μg/20 g of mouse), with surviving rates between 90 and 100%. Our research is leading to the formulation of an antivenom consisting of a discrete number of human scFvs endowed with strong cross-reactivity and low immunogenicity.
我们报告了一系列人源单链抗体片段（scFv）的优化，以中和两种蝎子毒液。亲本 scFv 3F 识别 Centruroides noxius Hoffmann（Cn2）和 Centruroides suffusus suffusus（Css2）的主要毒素，但亲和力较低。该 scFv 经历了独立的定向进化过程，以提高其对 Cn2（Riaño-Umbarila，L.，Juárez-González，V. R.，Olamendi-Portugal，T.，Ortíz-León，M.，Possani，L. D.，和 Becerril，B.（2005）FEBS J. 272，2591-2601）和 Css2（本工作）的识别。每个进化变体对多种毒素均表现出强烈的交叉反应性，并能够中和 Cn2 和 Css2。此外，每个变体都能中和上述物种的整个毒液。据我们所知，这是首次报道具有这种特征的抗体。成熟过程揭示了关键残基的变化，与亲本 scFv 相比，实现了表达、稳定性和亲和力的提高。这些变化的组合导致了 scFv LR 的产生，它能够挽救 3LD（50）新鲜制备的 C. noxius（7.5μg/20g 小鼠）和 C. suffusus（26.25μg/20g 小鼠）全毒液引起的严重中毒的小鼠，存活率在 90%至 100%之间。我们的研究正在导致一种由具有强烈交叉反应性和低免疫原性的离散数量的人源 scFv 组成的抗毒液的配方。
