**1. Introduction**

The hemolytic uremic syndrome (HUS) in children is mostly caused by Shiga toxinproducing *Escherichia coli* (STEC) infection, which is also responsible for outbreaks in the United States, Europe, South America, and Japan [1–3]. In Argentina, where post-diarrheal HUS is endemic, around 300 new cases are reported each year [4]. Since the early 2000s,

**Citation:** Luz, D.; Gómez, F.D.; Ferreira, R.L.; Melo, B.S.; Guth, B.E.C.; Quintilio, W.; Moro, A.M.; Presta, A.; Sacerdoti, F.; Ibarra, C.; et al. The Deleterious Effects of Shiga Toxin Type 2 Are Neutralized In Vitro by FabF8:Stx2 Recombinant Monoclonal Antibody. *Toxins* **2021**, *13*, 825. https://doi.org/10.3390/ toxins13110825

Received: 14 October 2021 Accepted: 15 November 2021 Published: 22 November 2021

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epidemiologically, the emergence of the non-O157 STEC infection, replacing the traditionally predominant O157 serogroup occurrence [5]. The contamination by STEC strains is usually by contaminated food or water ingestion, person-to-person transmission, or contact with ruminants or its contaminated environment [6]. The primary infection symptom is diarrhea, which is an average incubation phase of three days that could turn bloody in about 60% of patients. However, Shiga toxins (Stx) released by STEC triggers thrombogenic and inflammatory microvascular endothelial cell alterations, leading to HUS in 5–15% of STEC infection cases. HUS is defined by hemolytic anemia, thrombocytopenia, and acute renal injury [7,8]. Besides death, this syndrome can lead to long-term consequences such as hypertension and renal disease because of the high sensitivity to the Stx of the microvascular endothelial cells in the kidney [9].

The Stx toxins produced by STEC are Stx1 and Stx2, they appear to differ significantly in their effectiveness to induce protein synthesis inhibition and cytotoxicity, with some subtypes of Stx2 more potent than Stx1, on the other hand, other subtypes have similar potency [10]. Stxs is AB5 type toxin, consisting of a homo-pentameric B subunit (7.7 kDa per monomer) which binds to the host receptor globotriaosylceramide (Gb3) and mediate the enzymatically active A subunit (~32 kDa) endocytosis. Once inside the cell, the A subunit depurinates the conserved adenine residue of 28S eukaryotic rRNA, stopping peptide elongation and leading to cell death [11–13]. No specific drug has proved effective as specific therapy for STEC-HUS, which remains as symptomatic care. The antibiotics administration in STEC infection and STEC-HUS remains controversial, with some bacteriostatic antibiotics having a beneficial effect while others can increase the Stx liberation by the bacteria [14]. Proofs of evidence of an advantage from complement blockade therapy in STEC-HUS are also lacking [15]. One alternative treatment for STEC infection and possibly for HUS is neutralizing anti-Stx antibody therapy.

Monoclonal antibodies (mAb) against Stx have been evaluated in animal models (reviewed in [16,17]). Moreover, few mAbs candidates have also been tested in healthy volunteers during phase I studies [18,19]. In addition, a chimeric anti-Stx1 and Stx2 mAb was challenged in a phase II study in South America, but definite evidence of its therapeutic efficacy remains vague [20,21].

In addition to conventional antibodies, recombinant antibodies can be an attractive replacement to avoid animal immunization and other limitations of hybridoma technology, a successful, but cumbersome and costly approach to generate monoclonal antibodies [22,23]. In this context, we may include a family of Stx2B-binding VHHs that neutralize Stx2 in vitro at a nanomolar to the subnanomolar range [24] and the FabC11:Stx2 generated by phage display technology and produced very efficiently using bacterial protein synthesis systems which were able to prevent Stx2 toxicity to human kidney cells and in mice [25,26]. Therefore, the generation of such molecules and studies concerning their applicability will provide new therapeutic options for treating STEC infections to prevent or ameliorate HUS outcomes.

Herein, also employing phage display antibody library F [27], a monovalent FabF8:Stx2 was generated, and efficiently produced in the bacterial system with neutralizing qualities against Stx. We introduce a novel and simple antitoxin agent as a new therapeutic option for STEC infections therapy.

#### **2. Results**

#### *2.1. Selection of FabF8:Stx2 from a Human Antibody Fragment Phage Display Library*

The FabF8:Stx2 was generated from the selection using purified Stx2a toxin and a human synthetic antibody phage display library (library F) developed by Persson et al. [27]. The cloning was confirmed by sequencing (Figure 1A). The 48 kDa fragment corresponds to the purified Fab fragment, however, a 25 kDa protein also appears, which corresponds to non-assembled variable chains (Figure 1B). As determined by surface plasmon resonance, the purified FabF8:Stx2 showed an affinity constant (K*D*) of 13.8 nM (Figure S1). The halfmaximum effective concentration (EC50) was determined as being 160 ng/mL (calculated

as described in the material and methods) as well as, specificity just for the selected toxin, with no significant cross-reactivity to Stx1 toxin (Figure 1C).

**Figure 1.** The FabF8:Stx2 generation. (**A**) FabF8:Stx2 gene cloning. Electrophoretic profile on 1.5% agarose gel stained with SYBR (1:1000) of restriction analyzes of FabF8:Stx2 clone. (1) 1Kb molecular weight marker (Invitrogen); (2) Clone F8 anti-Stx2 (FabF8:Stx2); (**B**) FabF8:Stx2 purification. Electrophoretic profile on 15% non-denaturing polyacrylamide gel stained with Coomassie blue of sample eluted from the purifications of Fab fragment. (1) Blueyed molecular weight marker (GE); (2) Clone F8 anti-Stx2. (**C**) ELISA assay to assess cross-reaction of ligands against Stx toxins (5 μg/mL) using EC50 concentration of FabF8:Stx2.
