# Shiga Toxin Therapeutics: Beyond Neutralization
## 3 Current State of Shiga Toxin-Targeted Therapeutic Development  
The field of Shiga toxin therapeutic development has focused on inhibiting the toxins at various points in the pathway between entry into the cell and ribosomal depurination by activated toxin A subunits (Figure 1). The therapeutic targeting of the toxin is conceptually justified due to a direct causal link between Shiga toxin and the development of HUS [10; 12]. While this review will briefly summarize the current state of toxin-directed therapeutics, a more complete review was recently provided by [PERSON] and [PERSON] [24]. Toxin-neutralizing therapeutics and vaccines for other toxin-based diseases, such as tetanus and anthrax, have yielded excellent clinical outcomes, thus providing inspiration for Shiga-like toxin (STX)-neutralizing therapeutic strategies [47; 48] (Table 1). Trials using polyclonal anti-sera or monoclonal anti-Siga toxin antibodies in animal models of Shiga toxicosis successfully rescue from mortality if treatment is given within 48 h of toxin exposure [49; 50; 51; 52]. Monoclonal humanized murine anti-Shiga toxin antibodies have reached Phase II clinical trials within the U.S., but none have completed Phase III trials [53; 54]. Camelid heavy-chain-only antibody constructs containing heavy-chain oligomers specific for the toxin have been developed and are effective in murine and porcine models, but have not been used in humans to date [55]. The heavy-chain antibody constructs have been administered as a bolus injection or introduced via a replication-incompetent adenoviral construct, with both routes of administration conferring protection against STXs [56]. One of the challenges facing antibody-based Shiga toxin neutralizers is the variety of toxin subtypes present in various STEC strains. A monoclonal antibody must neutralize STX1 and multiple STX2 subtypes to be universally effective in STEC patients, since each STEC strain can secrete one or more subtypes. Figure 1: **Therapeutic targeting of Shiga toxin internalization and retrograde trafficking. ** Shiga toxins bind to cell membrane globotriaosylceramide (Gb3) to initiate internalization via endocytosis. The toxin then undergoes retrograde trafficking from endosomes through the Golgi apparatus and endoplasmic reticulum. The holotoxin is processed during trafficking to release active toxin A subunits into the cytosol to interact with 60S ribosomal components, resulting in ribotoxicity through depurination of rRNA adenine residues within the sacrin–ricin loop. Therapies inhibiting the interaction of Shiga toxin B subunits with Gb3 at the cell surface or inhibiting aspects of the retrograde trafficking system seek to ameliorate Shiga toxicity by preventing the toxin from reaching cytosolic ribosomes to initiate ribotoxicity. STX: Shiga-like toxin. Specific multivalent binding of the Shiga toxin B subunit to Gb3 in all STX subtypes relevant to the human disease has led to the generation of synthetic Gb3 analog constructs capable of inhibitory toxin binding. Synsorb-Pk(r) is a silicon dioxide backbone containing bound Gb3 capable of neutralizing Shiga toxins in vitro [57]. A Phase II trial in pediatric HUS patients failed to show any clinical improvement following treatment with Synsorb-Pk(r), possibly due to the inability of the orally administered drug to adsorb toxin produced at the mucosal surface or injected directly into epithelial cells by STEC [58]. The [PERSON] group has generated metallic backbone tetravalent peptides capable of binding to the Gb3 binding sites of STX1 and STX2 B subunits [59; 60]. Variations of the construct have been found to be protective when administered systemically in murine and non-human primate models of Shiga toxicosis, but no human trials have been performed [61; 62].