FAMILY OF VARIABLE RECOMBINANT HUMAN ANTIBODIES FOR NEUTRALISING THE SCORPION TOXINS CN2 AND CSS2 AND THEIR RESPECTIVE VENOMS: CENTRUROIDES NOXIUS AND CENTRUROIDES SUFFUSUS SUFFUSUS.

摘要

The present invention reveals the method for obtaining a new family of single-chain fragment variable recombinant human antibodies (ScFV). The invention particularly describes a scFv family resulting from independent processes of directed evolution for remarkably improving its binding affinity for the Centruroides noxius Hoffman (Cn2) and Centruroides suffusus suffusus (Css2)toxins, compared to the binding affinity of the parenting antibody, for said toxins. This family presented an improved crossed reactivity against several toxins, particularly neutralising toxins Cn2 and Css2. In addition, each variable neutralising the total venoms of both species, thus being the first antibodies reported with said characteristics. Maturation processes were used to reveal changes in key residues for achieving expression levels, stability and an improved affinity compared to those presented by the parenting scFv. These changes were introduced in the scFv 9004G antibody and the obtained scFv was called, in the present invention, LR, this new variable being able to rescue mice from a severe poisoning caused by 3 LD50 from a total venom fresh preparation of C, noxius (7.5 µ g/ 20 g of mouse) and C. suffusus (26.25µ g/ 20 g of mouse), with success rates of from about 90% and 100%. Finally, the LER variable results from the change in the position 43 in the LR variable. This variable is able to neutralise toxin Cn2 up to a molecular ratio of 1:1. The invention is also related to the DNAs encoding for the members of the new family of scFv, with the molecular vectors that contain said DNAs, with the cells containing these vectors and the methods for producing the antibodies fragment variants, and with pharmaceutical compositions, the use of said antibody fragments for the manufacture of compositions, matrixes that comprise the antibody fragments and diagnosis methods based thereon.