Working Towards Snake Envenoming Treatment Improvement: Development of a Bothrops atrox SVMP-Targeting mAb

Bothrops atrox causes most snakebite envenomings in the Brazilian Amazon. Snake venom metalloproteinases (SVMPs) and phospholipases A₂ (PLA₂s) are two of the main toxin classes associated with local tissue damage. Although equine-derived antivenom is the gold-standard treatment in Brazil, part of the enzymatic activity of SVMPs and PLA₂s remains poorly neutralized by it. In an effort to improve envenoming treatment, and in alignment with the WHO 2030 agenda, monoclonal antibodies (mAbs) against the main toxins have been explored as complementary tools. In this context, this work aimed to obtain strategic fractions of B. atrox venom for mouse immunization and the generation of mAbs targeting SVMPs and PLA₂s by hybridoma technology. Venom was first fractionated by size-exclusion chromatography (SEC) and characterized by LC-MS/MS for relative abundance and by enzymatic assays for SVMP and PLA₂ activities, using azocasein, DQ-gelatin and NOBA. The pool selected to generate anti-PLA₂ mAbs contained 57% PLA₂s and intensely hydrolyzed NOBA, while the one used for anti-SVMP contained 73% metalloproteinases and showed robust gelatinolytic activity sensitive to 1,10-phenanthroline. Hybridomas were obtained by fusing spleen cells with SP2/0-Ag14 myeloma cells, and clones were isolated and screened by indirect ELISA against the respective pools. Although anti-PLA₂ hybridomas showed appropriate antigen recognition by ELISA, they did not sustain clonal expansion, and a new immunization is in progress. For anti-SVMP, two clones producing antibodies reactive to the antigen were identified. The mAb secreted by clone P1A2 was purified and tested in immunoprecipitation assays, which indicated that mAb-P1A2 interacted with 55 and 25 kDa venom toxins, consistent with PIII and PI SVMPs. IgG fragments of 17–60 kDa were also detected, indicating cleavage of the antibody by venom proteinases. Importantly, pre-incubation with mAb-P1A2 during the renaturation step of zymography reduced the gelatinolytic activity of the 55 kDa venom band. Our results show that activity-based screening of venom fractions is an important strategy for the development of new antivenoms. mAb-P1A2 is the first functional candidate obtained by this approach, and its neutralizing potential, together with the generation of anti-PLA₂ mAbs, is currently under investigation.