Delayed Toxicity Associated with Soluble Anthrax Toxin Receptor Decoy-Ig Fusion Protein Treatment

et al. (2012) Delayed Toxicity Associated with Soluble Anthrax Toxin Receptor Decoy-Ig Fusion Protein Treatment. PLoS ONE 7(4): e34611. doi:10.1371/journal.pone.0034611 Delayed Toxicity Associated with Soluble Anthrax Toxin Receptor Decoy-Ig Fusion Protein Treatment Diane Thomas 0 John Naughton 0 Christopher Cote 0 Susan Welkos 0 Marianne Manchester 0 John A. T. Young 0 Nicholas J. Mantis, Wadsworth Center, New York State Dept. Health, United States of America 0 1 Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, La Jolla, California, United States of America, 2 Nomis Center for Immunobiology and Microbial Pathogenesis, The Salk Institute for Biological Studies, La Jolla, California, United States of America, 3 Bacteriology Division, U.S. Army Medical Research Institute of Infectious Diseases , Fort Detrick, Frederick, Maryland , United States of America Soluble receptor decoy inhibitors, including receptor-immunogloubulin (Ig) fusion proteins, have shown promise as candidate anthrax toxin therapeutics. These agents act by binding to the receptor-interaction site on the protective antigen (PA) toxin subunit, thereby blocking toxin binding to cell surface receptors. Here we have made the surprising observation that co-administration of receptor decoy-Ig fusion proteins significantly delayed, but did not protect, rats challenged with anthrax lethal toxin. The delayed toxicity was associated with the in vivo assembly of a long-lived complex comprised of anthrax lethal toxin and the receptor decoy-Ig inhibitor. Intoxication in this system presumably results from the slow dissociation of the toxin complex from the inhibitor following their prolonged circulation. We conclude that while receptor decoy-Ig proteins represent promising candidates for the early treatment of B. anthracis infection, they may not be suitable for therapeutic use at later stages when fatal levels of toxin have already accumulated in the bloodstream. - Funding: This work was supported by National Institutes of Health grant AI 076852 (JATY and MM) and by the Nomis Foundation and the James B. Pendleton Charitable Trust (JATY). Work performed at USAMRIID was sponsored by the Defense Threat Reduction Agency JSTO-CBD as plan #CBM.VAXBT.03.10.RD.004/ Medical Research/Material Command Research Plan. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: JATY is a scientific founder of Pharmathene, Inc. MM and JATY hold stock in Pharmathene, Inc. Pharmathenes product portfolio related to anthrax includes * SparVaxTM - recombinant protective antigen (rPA) anthrax vaccine, and * ValortimH - fully human monoclonal antibody antitoxin treatment of anthrax. JATY and MM have a US patent Appl. No.: 12/070,384, Antitoxin and vaccine platform based on nodavirus VLPs. JATY has US patent application 11/ 340,424, Anthrax antitoxins. This does not alter the authors adherence to all the PLoS ONE policies on sharing data and materials. . These authors contributed equally to this work. Anthrax toxin is the major virulence factor of B. anthracis, the causative agent of anthrax. There are two forms of anthrax toxin, each of which contains the protective antigen (PA) toxin subunit. Edema toxin (EdTx) pairs PA with edema factor (EF), a calcium and calmodulin-dependent adenylate cyclase; and lethal toxin (LeTx) is comprised of PA and lethal factor (LF), a zinc-dependent metalloprotease that cleaves and inactivates MAP kinase kinase signaling pathways [1,2,3]. The first step of intoxication involves binding of an 83 kD form of PA (PA83) to cell surface receptors. Two different cellular receptors for PA83 have been identified, designated as ANTXR1 and ANTXR2 [4,5]. PA83 binds to an extracellular domain of each receptor that is related in structure to the integrin-like von Willebrand Factor type A (VWA) domain [4,5]. Following receptor-binding, PA83 is cleaved to a 63 kD form by a cell surface furin-like protease, and the resultant PA63 fragment spontaneously assembles into either heptameric or octameric prepore complexes [6,7,8,9]. Alternatively, these oligomeric PA complexes may assemble prior to receptor binding following PA83 to PA63 cleavage by a serum protease within the bloodstream of infected animals [10,11]. The toxin complexes are taken up into cells by receptor-mediated endocytosis [12,13,14,15]. Entry into an acidic endosomal compartment stimulates PA63 prepore-topore conversion and LF and EF translocation into the cytosol leading to toxicity [16]. There is a great deal of interest in developing anthrax antitoxins (reviewed in [17]). The rationale for developing these inhibitors is that they may complement existing vaccine and antibiotic-based therapies, and may be especially useful to treat disease caused by either vaccine-, or antibiotic-resistant bacterial strains. Several different anthrax anti-toxins are being developed including monoclonal antibodies, small molecule inhibitors, receptor decoys, substrate analogs, and dominant-negative toxin subunits. We first demonstrated the utility of a soluble receptor decoy as a candidate anthrax therapeutic [18]. That inhibitor, based upon the soluble VWA domain of ANTXR2, had several desirable features including a high binding affinity for PA (Kd = 170 pM) [19]. The receptor decoy inhibitor also efficiently neutralized both wild-type PA, as well as altered forms of PA that were engineered to be resistant to therapeutic monoclonal antibodies [20,21]. Several other groups have generated receptor decoy inhibitors by fusing the ANTXR2 VWA domain to the Fc portions of either human IgG1 or IgG2 [21,22]. These reagents have the additional benefit of having an increased circulation half-life in vivo, thereby increasing their antitoxin potencies. The receptor decoy-IgG2 protein protected rats against short-term intoxication by anthrax lethal toxin, and also protected mice against killing following intratracheal administration of attenuated B. anthracis Sterne spores [22]. The receptor decoy-IgG1 protein protected rabbits against killing following an inhalational challenge with fully virulent B. anthracis Ames spores [21]. In this study we also generated and tested several different receptor decoy-Ig fusion proteins in vitro and in vivo. In contrast to previous reports, we found that these inhibitors delayed killing, but did not protect, rats that were challenged with a lethal dose of anthrax lethal toxin. The mechanism of delayed toxicity was studied by following the fate of the toxin subunits following coadministration with anthrax lethal toxin. Our previous studies showed that a soluble, bacteriallyproduced form of the ANTXR2 VWA domain (termed receptor decoy inhibitor, or RDI) protected against LeTx challenge in rats, but only when it was administered concurrently or within 5 minutes of dosing with toxin [18]. That RDI protein was compr (...truncated)