Infection Site Targeted Antitoxin Antibody (istab) Against Bacillus Anthracis

Project SummaryThe Gram-positive bacterium Bacillus anthracis is a very strong candidate for potential bio- weaponization, andbelieved to have actually been weaponized by the former Soviet Union. Anthrax spores are readily found innature or produced in the laboratory, are resistant to harsh conditions, and can survive for a long time in theenvironment. The microscopic spores could be formulated in powder form, sprays, food, or water. Two key toxinsgenerated by combination of the protective antigen (PA) with either lethal factor (LF) or edema factor (EF) playa critical role in B. anthracis virulence. Current CDC recommendations following potential exposure toaerosolized B. anthracis spores consist of a combination of oral antibiotics and PA-based anthrax vaccine.However, in practice, these treatments cannot adequately address the adverse effects of bacterial toxinsreleased post exposure. In this R41 proposal we intend to develop a novel approach to target neutralizing anti-toxin antibodies specifically to the site of infection. The approach exploits the cell wall targeting domains (CWT)of well characterized phage endolysins: PlyG, PlyL and PlyB which bind with species-specificity and high affinityto cell wall components of B. anthracis. Theses CWTs will be fused to specific antitoxin neutralizing monoclonalantibodies to generate Infection Site Targeted Antitoxin antibodies (ISTAbs). ISTAbs are expected toaccumulate at the site of infection where they are needed most, and capture and sequester the toxins, thusimmediately neutralizing the effects of the toxins and preventing their release into circulation. Bacterium-toxincomplex is then expected to be cleared by phagocytes. In this proposal, we will use three anthrax-PA neutralizingmonoclonal antibodies fused to high affinity phage endolysin CWTs to generate ISTAbs. In Aim 1 we will screenfor best binding CWTs from ten phage endolysins, including those from PlyG, PlyL, and PlyB. We willcharacterize them based on in vivo and in vitro binding. In Aim 2, based on Aim 1 results, we will select 3 CWTsfor generating up to nine ISTAbs by fusing the CWTs with three highly neutralizing anti-Anthrax monoclonalantibody as scaffold and characterize them for in vitro binding and toxin neutralizing activity. In Aim 3 we willfurther characterize the selected ISTAbs based on stability; bacterial cell binding specificity and affinity, andperformance in opsonophagocytic killing assays. In Aim 4, we will perform efficacy testing in pre-challenge andpost challenge treatment mouse models and also explore potential immunogenicity of the ISTAbs.Since ISTAb technology provides two therapeutic advantages: immediate toxin neutralization at the site ofinfection and opsonophagocytic killing by phagocyte, there is a high probability that these molecules willsynergize with existing antibiotics. The combination of immediate toxin clearance, phagocytic killing, andconcurrent use of antibiotics is expected to create synergy and yield a treatment that is far superior to the currentstandard of vaccine plus antibiotics. Furthermore, this technology can be applied to a variety of other bacterialpathogens where toxins play a key role in pathogenesis. Overall, this approach has board application as aplatform technology across multiple pathogens.