Infection Site Targeted Antitoxin Antibody (ISTAb) against Bacillus anthracis

Project Summary
Bacillus anthracis (Ba) is a Gram-positive spore forming bacterium that is listed as an agent of highest
concern (Category A) by NIAID and CDC. Ba is easy to grow, and its spores can be formulated into highly
stable powder form and disseminated as aerosol or used to contaminate food or water. In 2001, letters laced
with powdered anthrax spores were mailed to several US politicians. Twenty-two people, including 12 mail
handlers, were infected, and five of them died. B. anthracis virulence largely depends on two key toxins
generated by combination of the protective antigen (PA) associated with either lethal factor (LF) or edema
factor (EF). Although some oral antibiotics and a vaccine are available for use, in practice these treatments
cannot adequately address the adverse effects of bacterial toxins released post exposure. In our recently
completed R41 project, we developed and tested a novel approach to target neutralizing anti-PA antibodies
specifically to the site of infection in vitro and in vivo. The approach exploits the cell wall targeting domains
(CWT) of well characterized phage endolysins (PlyG, PlyL and PlyB) that bind with species-specificity and
high affinity to cell wall components of Ba. These CWTs are fused to specific antitoxin neutralizing
monoclonal antibodies (mAbs) to generate Infection Site Targeted Antitoxin antibodies (ISTAbs). ISTAb
technology provides two therapeutic advantages: immediate toxin neutralization at the site of infection
preventing toxemia, and opsonophagocytic killing by phagocytes to simultaneously clear both bacteria and
toxin. We compared nine ISTAb candidates (three CWTs and three mAbs) based on in vitro assays (cell
binding and toxin neutralization) and selected one ISTAb (AVP-21D9-PlyG) for pre- and post-challenge in
vivo studies in mice. This ISTAb exhibited significantly higher level of protection than the parental IgG. This
R42 is aimed to take this lead ISTAb molecule into the next level in therapeutic pipeline. In this proposal,
we will produce and extensively characterize next-generation AVP-21D9-PlyG ISTAbs, including stability
and in vivo efficacy studies in mice and nonhuman primates (NHP), and develop a stable formulation. In
Aim 1, we will use computer-aided optimizations to generate 3-5 ISTAb variants to remove potential
liabilities that may complicate downstream development. In Aim 2, two lead candidates will be tested in
mouse models. One lead molecule will be tested in an NHP model for PK and post-challenge efficacy. In
Aim 3: The final ISTAb will be subjected to accelerated stability and PK studies, formulation, and generation
of stable cell lines in CHO-S cells. The combination of immediate toxin clearance, phagocytic killing, and
concurrent use of antibiotics, is expected to create synergy and yield a treatment that is far superior to the
current standard of care. Furthermore, this technology can be applied to a variety of other bacterial
pathogens where toxins play a key role in pathogenesis. Overall, this approach has board application as a
platform technology across multiple pathogens.Bacillus anthracis (Ba), a spore-forming bacterium is listed as a select agent by CDC. It can
cause disease in animals and humans by three routes of infection: inhalational, gastrointestinal,
and skin. Ba is a Tier 1 agent that poses the greatest risk of misuse, with significant potential for
mass casualties. For instance, in 2001, powdered anthrax spores containing letter were
deliberately mailed to two U.S. Senators and several news media offices. Twenty-two people
contracted anthrax and five died as a result (http://www.cdc.gov/anthrax/bioterrorism/threat.html).
Protective antigen (PA), a binding protein is a crucial component of anthrax toxin. It can form a
toxic pair when combine either with: lethal factor (LF) or edema factor (EF). In this proposal, we
seek to use a novel technology called Infection site targeted antitoxin antibodies (ISTAbs), a
fusion protein fused with Ba bacteriophage CWT with anti-PA monoclonal antibody to target
antitoxins exactly to the site of infection. This technology can provide a novel approach for
treatment of Bacillus anthracis infections.