A Single VHH-Based Toxin-Neutralizing Agent and an Effector Antibody Protect Mice against Challenge with Shiga Toxins 1 and 2

Abstract

b Shiga toxin-producing Escherichia coli (STEC) is a major cause of severe food-borne disease worldwide, and two Shiga toxins, Stx1 and Stx2, are primarily responsible for the serious disease consequence, hemolytic-uremic syndrome (HUS). Here we report identification of a panel of heavy-chain-only antibody (Ab) V H (VHH) domains that neutralize Stx1 and/or Stx2 in cell-based assays. VHH heterodimer toxin-neutralizing agents containing two linked Stx1-neutralizing VHHs or two Stx2-neutralizing VHHs were generally much more potent at Stx neutralization than a pool of the two-component monomers tested in cell-based assays and in vivo mouse models. We recently reported that clearance of toxins can be promoted by coadministering a VHHbased toxin-neutralizing agent with an antitag monoclonal antibody (MAb), called the "effector Ab," that indirectly decorates each toxin molecule with four Ab molecules. Decoration occurs because the Ab binds to a common epitopic tag present at two sites on each of the two VHH heterodimer molecules that bind to each toxin molecule. Here we show that coadministration of effector Ab substantially improved the efficacy of Stx toxin-neutralizing agents to prevent death or kidney damage in mice following challenge with Stx1 or Stx2. A single toxin-neutralizing agent consisting of a double-tagged VHH heterotrimer-one Stx1-specific VHH, one Stx2-specific VHH, and one Stx1/Stx2 cross-specific VHH-was effective in preventing all symptoms of intoxication from Stx1 and Stx2 when coadministered with effector Ab. Overall, the availability of simple, defined, recombinant proteins that provide cost-effective protection against HUS opens up new therapeutic approaches to managing disease.

A Heterodimer of a VHH (Variable Domains of Camelid Heavy Chain-only) Antibody That Inhibits Anthrax Toxin Cell Binding Linked to a VHH Antibody That Blocks Oligomer Formation Is Highly Protective in an Anthrax Spore Challenge Model

“…Heterodimeric VNAs Protect against Anthrax Toxin and Spore Infection in Mice-Linking toxin-neutralizing VHHs into heteromultimeric VNAs has been found to improve toxin affinity and, more importantly, to substantially improve in vivo antitoxin efficacy (11)(12)(13)16). A heterodimeric VNA (VNA2-PA) consisting of the two potent neutralizing VHHs, JIK-B8 and JKH-C7, separated by a short unstructured peptide, was expressed and purified (amino acid sequence shown in Fig.…”

“…32 and 6). We have found that VHH heteromultimer VNAs possess additional potential therapeutic advantages over conventional mAb products such as 1) the option to co-administer an anti-tag effector antibody that binds multiple tags on the VNAs (11,13) to promote antibody Fc effector functions such as serum clearance (33), 2) the ability to target multiple toxins with one biomolecule (12,16), and 3) the use of VNA gene therapy as an effective means to provide prolonged antitoxin protection (34,35). Simple addition of an albumin binding peptide (34,36) can dramatically improve the serum stability of VNAs (34).…”

“…In this work we hypothesized that a novel recombinant antitoxin agent consisting of a heterodimer of two distinct toxin-neutralizing, camelid anti-anthrax PA heavy chain VHH binding domains would serve as an effective therapeutic for anthrax exposure. This hypothesis was based on our earlier findings of unusually high in vivo antitoxin potency achieved for Clostridium botulinum neurotoxins (11), ricin (13), Shiga toxins (12), and Clostridium difficile (16) toxins using heteromultimers of toxinneutralizing VHHs called VNAs.…”

“…All VHHs panned on PA83 or PA63 recognized both PA83 and PA63. VHH coding sequences were determined for 24 clones displaying clearly unique fingerprints (12). Sequence alignments revealed 11 distinct homology groups.…”

“…VHHs often target active sites that may be inaccessible to larger conventional antibodies (7,8) and have proven to be effective as toxin neutralizing agents (9 -16). We have found that linking two or more neutralizing VHHs recognizing non-overlapping epitopes into heteromultimers (VHH-based neutralizing agents (VNAs)) often provides major improvements in the in vivo protection from toxin exposure as compared with the unlinked component VHHs (11)(12)(13)16).…”