Exploiting cross-reactivity to neutralize two different scorpion venoms with one single chain antibody fragment

Abstract

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.

FIGURE 1.

Evolutionary conservation of the main toxins of C. noxius and C. suffusus. A multiple sequence alignment was generated with Clustal W2. The amino acid sequences of toxins Cll1, Cll2, Cn2, Css2, Css4, and Cn3 are shown. Symbols indicate: asterisks, absolute conservation; colons, conservation of identity and physicochemical properties; dots, conserved physicochemical properties; blank spaces, strikingly different residues.

FIGURE 2.

Cross-reactivity of scFv 6009F and scFv 9004G against closely related scorpion toxins. An ELISA was used to test recognition toward Cn2, Css2, Css4, Cn3, Cll1, and Cll2. Concentrations used were: toxin, 3 μg ml⁻¹; scFv, 5 μg ml⁻¹. Bars: white, scFv 6009F; black, scFv 9004G. Error bars of four experiments are shown.

FIGURE 3.

a, maturation process against toxins Cn2 and Css2. Antibody variants isolated during three cycles of directed evolution. Amino acid changes that appeared in each variant are shown indicating their structural location. Underlined changes were relevant for the improvement of antibody variants (see text for details). b, annealing of sequences of scFvs 6009F and 9004G.

FIGURE 4.

scFv recognition to Cn2 upon treatment under strong denaturing conditions. Antibody variants were incubated with increasing concentrations of guanidinium chloride before testing their recognition to toxin by ELISA. Bars: white, scFv 9004G; black, scFv 6009F; hatched, scFv LR. Error bars of four experiments are shown.