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

Mahtab Moayeri,Clinton E Leysath,Jacqueline M Tremblay,Catherine Vrentas,Devorah Crown,Stephen H Leppla,Charles B Shoemaker

doi:10.1074/jbc.m114.627943

Abstract

Anthrax disease is caused by a toxin consisting of protective antigen (PA), lethal factor, and edema factor. Antibodies against PA have been shown to be protective against the disease. Variable domains of camelid heavy chain-only antibodies (VHHs) with affinity for PA were obtained from immunized alpacas and screened for anthrax neutralizing activity in macrophage toxicity assays. Two classes of neutralizing VHHs were identified recognizing distinct, non-overlapping epitopes. One class recognizes domain 4 of PA at a well characterized neutralizing site through which PA binds to its cellular receptor. A second neutralizing VHH (JKH-C7) recognizes a novel epitope. This antibody inhibits conversion of the PA oligomer from "pre-pore" to its SDS and heat-resistant "pore" conformation while not preventing cleavage of full-length 83-kDa PA (PA83) by cell surface proteases to its oligomer-competent 63-kDa form (PA63). The antibody prevents endocytosis of the cell surface-generated PA63 subunit but not preformed PA63 oligomers formed in solution. JKH-C7 and the receptor-blocking VHH class (JIK-B8) were expressed as a heterodimeric VHH-based neutralizing agent (VNA2-PA). This VNA displayed improved neutralizing potency in cell assays and protected mice from anthrax toxin challenge with much better efficacy than the separate component VHHs. The VNA protected virtually all mice when separately administered at a 1:1 ratio to toxin and protected mice against Bacillus anthracis spore infection. Thus, our studies show the potential of VNAs as anthrax therapeutics. Due to their simple and stable nature, VNAs should be amenable to genetic delivery or administration via respiratory routes.

Highlights

Anthrax toxin is the primary cause of pathology from exposure to anthrax spores
In this work we hypothesized that a novel recombinant antitoxin agent consisting of a heterodimer of two distinct toxin-neutralizing, camelid anti-anthrax protective antigen (PA) heavy chain Variable domains of camelid heavy chain-only antibodies (VHHs) 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

Summary

Background

Anthrax toxin is the primary cause of pathology from exposure to anthrax spores. Results: Two linked single domain antibodies (VHHs), each neutralizing anthrax toxicity by different mechanisms, potently protect mice from anthrax spore challenge. 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]. Linking JIK-B8 and JKH-C7 VHHs into a heterodimeric VNA resulted in an agent with an IC50 of ϳ100 pM that proved highly effective in protecting mice from anthrax toxin or spore challenges. This VNA should offer new and costeffective therapeutic options for treating anthrax exposures

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION