*Article* **The Deleterious Effects of Shiga Toxin Type 2 Are Neutralized In Vitro by FabF8:Stx2 Recombinant Monoclonal Antibody**

**Daniela Luz 1,†, Fernando D. Gómez 2,†, Raíssa L. Ferreira 1, Bruna S. Melo 1, Beatriz E. C. Guth 3, Wagner Quintilio 4, Ana Maria Moro 4, Agostina Presta 2, Flavia Sacerdoti 2, Cristina Ibarra 2, Gang Chen 5, Sachdev S. Sidhu 5, María Marta Amaral 2,\* and Roxane M. F. Piazza 1,\***


**Abstract:** Hemolytic Uremic Syndrome (HUS) associated with Shiga-toxigenic *Escherichia coli* (STEC) infections is the principal cause of acute renal injury in pediatric age groups. Shiga toxin type 2 (Stx2) has in vitro cytotoxic effects on kidney cells, including human glomerular endothelial (HGEC) and Vero cells. Neither a licensed vaccine nor effective therapy for HUS is available for humans. Recombinant antibodies against Stx2, produced in bacteria, appeared as the utmost tool to prevent HUS. Therefore, in this work, a recombinant FabF8:Stx2 was selected from a human Fab antibody library by phage display, characterized, and analyzed for its ability to neutralize the Stx activity from different STEC-Stx2 and Stx1/Stx2 producing strains in a gold standard Vero cell assay, and the Stx2 cytotoxic effects on primary cultures of HGEC. This recombinant Fab showed a dissociation constant of 13.8 nM and a half maximum effective concentration (EC50) of 160 ng/mL to Stx2. Additionally, FabF8:Stx2 neutralized, in different percentages, the cytotoxic effects of Stx2 and Stx1/2 from different STEC strains on Vero cells. Moreover, it significantly prevented the deleterious effects of Stx2 in a dose-dependent manner (up to 83%) in HGEC and protected this cell up to 90% from apoptosis and necrosis. Therefore, this novel and simple anti-Stx2 biomolecule will allow further investigation as a new therapeutic option that could improve STEC and HUS patient outcomes.

**Keywords:** STEC; Stx2; antibody fragment; monoclonal antibody

**Key Contribution:** Here we describe the generation, characterization, and efficacy of one Fab antibody fragment anti-Stx2 in protecting cells against Stx2 cytotoxic effects and its ability to neutralize Stx produced by STEC strains, which is demonstrated herein for the first time.
