The venom of the scorpion Centruroides limpidus, which causes the highest number of stings in Mexico, is neutralized by two recombinant antibody fragments.

Phage display and directed evolution have made it possible to generate recombinant antibodies in the format of single chain variable fragments (scFvs) capable of neutralizing different toxins and venoms of Mexican scorpions. Despite having managed to neutralize a significant number of venoms, some others have not yet been completely neutralized, due to the diversity of the toxic components present in them. An example is the venom of the scorpion Centruroides limpidus, which contains three toxins of medical importance, called Cll1, Cll2 and Cl13. The first two are neutralized by scFv 10FG2, while Cl13, due to its sequence divergence, was not even recognized. For this reason, the aim of the present work was the generation of a new scFv capable of neutralizing Cl13 toxin and thereby helping to neutralize the whole venom of this scorpion. By hybridoma technology, a monoclonal antibody (mAb B7) was generated, which was able to recognize and partially neutralize Cl13 toxin. From mAb B7, its scFv format was obtained, named scFv B7 and subjected to three cycles of directed evolution. At the end of these processes, scFv 11F which neutralized Cl13 toxin was obtained. This scFv, administered in conjunction with scFv 10FG2, allowed to fully neutralize the whole venom of Centruroides limpidus scorpion.

Generation of a Broadly Cross-Neutralizing Antibody Fragment against Several Mexican Scorpion Venoms.

The recombinant antibody fragments generated against the toxic components of scorpion venoms are considered a promising alternative for obtaining new antivenoms for therapy. Using directed evolution and site-directed mutagenesis, it was possible to generate a human single-chain antibody fragment with a broad cross-reactivity that retained recognition for its original antigen. This variant is the first antibody fragment that neutralizes the effect of an estimated 13 neurotoxins present in the venom of nine species of Mexican scorpions. This single antibody fragment showed the properties of a polyvalent antivenom. These results represent a significant advance in the development of new antivenoms against scorpion stings, since the number of components would be minimized due to their broad cross-neutralization capacity, while at the same time bypassing animal immunization.

Broadening the neutralizing capacity of a family of antibody fragments against different toxins from Mexican scorpions.

New approaches aimed at neutralizing the primary toxic components present in scorpion venoms, represent a promising alternative to the use of antivenoms of equine origin in humans. New potential therapeutics developed by these approaches correspond to neutralizing antibody fragments obtained by selection and maturation processes from libraries of human origin. The high sequence identity shared among scorpion toxins is associated with an important level of cross reactivity exhibited by these antibody fragments. We have exploited the cross reactivity showed by single chain variable antibody fragments (scFvs) of human origin to re-direct the neutralizing capacity toward various other scorpion toxins. As expected, during these evolving processes several variants derived from a parental scFv exhibited the capacity to simultaneously recognize and neutralize different toxins from Centruroides scorpion venoms. A sequence analyses of the cross reacting scFvs revealed that specific mutations are responsible for broadening their neutralizing capacity. In this work, we generated a set of new scFvs that resulted from the combinatorial insertion of these point mutations. These scFvs are potential candidates to be part of a novel recombinant antivenom of human origin that could confer protection against scorpion stings. A remarkable property of one of these new scFvs (ER-5) is its capacity to neutralize at least three different toxins and its complementary capacity to neutralize the whole venom from Centruroides suffusus in combination with a second scFv (LR), which binds to a different epitope shared by Centruroides scorpion toxins.