Anthrax toxin is a three-part toxin secreted by Bacillus anthracis, consisting of three components: protective antigen (PA), lethal factor (LF), and edema factor (EF). Among the three components of the toxin, PA is the cellular binding moiety, which binds to its cellular receptors Tumor Endothelium Marker-8 (TEM8) and Capillary Morphogenesis Protein-2 (CMG2). Upon binding to target cell surface, PA is proteolytically activated by the ubiquitously expressed cell surface furin protease, resulting in the formation of active PA oligomer, which in turn binds and translocates the two enzymatic moieties LF and EF into the cytosol of cells. LF, which forms lethal toxin (LT) with PA, cleaves several mitogen-activated protein kinase kinases (MEKs) and rodent inflammasome sensor Nlrp1. EF, which forms edema toxin (ET) with PA, is an adenylate cyclase that generates abnormally high concentrations of cAMP. LT and ET are two of the major virulence factors of B. anthracis. Vaccines are important in anthrax treatment and prevention. In the year of 2015, by collaborating with Drs. Zhaochun Chen and Robert H. Purcell (Laboratory of Infectious Diseases, NIAID), we have evaluated the immunogenicity of the B. anthracis poly-gamma-D-glutamic acid capsular material (PGA) conjugated to recombinant protective antigen (rPA, the toxin cellular binding component) or to tetanus toxoid (TT) in two anthrax-naive juvenile chimpanzees. In a previous study of these conjugates, highly protective monoclonal antibodies (MAbs) against PGA were generated. This study examined the polyclonal antibody response of the same animals. Preimmune antibodies to PGA with titers of >1000 were detected in the chimpanzees. The maximal titer of anti-PGA was induced within 1 to 2 weeks following the 1st immunization, with no booster effects following the 2nd and 3rd immunizations. Thus, the anti-PGA response in the chimpanzees resembled a secondary immune response. Screening of sera from nine unimmunized chimpanzees and six humans revealed antibodies to PGA in all samples, with an average titer of 1000. An anti-PA response was also observed following immunization with PGA-rPA conjugate, similar to that seen following immunization with rPA alone. However, in contrast to anti-PGA, preimmune anti-PA antibody titers and those following the 1st immunization were <300, with the antibodies peaking above 10000 following the 2nd immunization. The polyclonal anti-PGA shared the MAb 11D epitope and, similar to the MAbs, exerted opsonophagocytic killing of B. anthracis. Most important, the PGA-TT-induced antibodies protected mice from a lethal challenge with virulent B. anthracis spores. Our data support the use of PGA conjugates, especially PGA-rPA targeting both toxin and capsule, as an expanded-spectrum anthrax vaccine. The absolute requirement for PAs proteolytic activation in the toxins action provided us the opportunity to reengineer PA to be selectively activated by tumor-associated proteases, generating tumor-targeting toxins. We have previously designed and characterized variants of anthrax lethal toxin that are selectively activated by either matrix-metalloproteinases (MMPs) or urokinase plasminogen activator (uPA) activities. In the year of 2015, by collaborating with Dr. Toni M. Antalis (University of Maryland School of Medicine), we have further extended this work by generating membrane-anchored serine protease-activated anthrax toxins for tumor targeting. The membrane-anchored serine proteases are a unique group of trypsin-like serine proteases that are tethered to the cell surface via transmembrane domains or glycosyl-phosphatidylinositol-anchors. Overexpressed in tumors, with pro-tumorigenic properties, they are attractive targets for protease-activated prodrug-like anti-tumor therapies. We mutated PAs native activation sequence to a sequence derived from protein C inhibitor (PCI) that can be cleaved by membrane-anchored serine proteases, to generate the mutant protein PA-PCIS. PA-PCIS was resistant to furin cleavage in vitro, yet cytotoxic to multiple human tumor cell lines when combined with FP59, a chimeric anthrax toxin lethal factor-Pseudomonas exotoxin fusion protein. Molecular analyses showed that PA-PCIS can be cleaved in vitro by several serine proteases including the membrane-anchored serine protease testisin, and mediates increased killing of testisin-expressing tumor cells. Treatment with PA-PCIS also potently attenuated the growth of testisin-expressing xenograft tumors in mice. The data expands the evidence that anthrax toxins can be engineered to target tumor cell-surface proteases so as to function as potent tumoricidal agents.
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