Internal Toxin Neutralizer for Treating STEC-infection

AbstractThe Shiga toxin-producing E. coli (STEC) is the most common cause of bloody diarrhea and afflicts anestimated 73,000 people in the US annually, causing significant morbidity. The most recent and largest STECoutbreak occurred in Germany in 2011, affecting >3,800 people, including 54 deaths. Currently there is noeffective treatment for STEC infection. The pathology of STEC infection derives from two exotoxins ? Shigatoxin 1 (Stx1) and Shiga toxin 2 (Stx2) ? that are secreted by STEC in the gut. Although antibiotic treatment canreduce the load of STEC, it also augments Shiga toxin release, leading to increased risk of developing the moreserious hemolytic uremic syndrome (HUS) and kidney failure (up to 25%). Consequently, the CDC recommendsthat antibiotics not be used in STEC patients and that only supportive therapy (e.g. oral and i.v. fluid, paincontrol) be used. Although anti-toxin antibodies have been identified, the inability of antibodies to cross the cellmembrane renders them powerless against toxins already absorbed by the host cells, limiting their clinicalapplication. We hypothesize that a cytosol-accessible anti-toxin should be able to neutralize both extracellularand intracellular Shiga toxin, leading to a much-prolonged therapeutic window and better therapeutic efficacy.The overall goal of this study is to engineer a panel of intracellular toxin neutralizers (ITNs) against Shiga toxin2 (Stx2). As a scaffold for the proposed ITN, we will use a designed ankyrin repeat protein (DARPin). DARPinsrepresent a versatile class of binding proteins that have been engineered to bind diverse targets with up topicomolar affinity and possess low immunogenicity. In this project, we will first isolate DARPins that bind andneutralize Stx2 (Aim 1). Concurrently, we will screen a panel of cell-penetrating peptides (CPPs) for their abilityto transport ITNs into cells (Aim 2). In Aim 3, we will assemble anti-Stx2 ITNs using the best anti-Stx2 DARPinand CPP and evaluate the therapeutic potential of these anti-Stx2 ITNs in vitro and in vivo. The approach ofusing ITN to combat toxins in circulation offers a new paradigm for the treatment of both STEC and non-STECbacterial infections.