Rapid Discovery and Characterization of Synthetic Neutralizing Antibodies against Anthrax Edema Toxin

Abstract

Anthrax, a lethal, weaponizable disease caused by Bacillus anthracis, acts through exotoxins which are primary mediators of systemic toxicity and also targets for neutralization by passive immunotherapy. The ease of engineering Bacillus anthracis strains resistant to established therapy and the historic use of the microbe in bioterrorism present a compelling test case for platforms that permit the rapid and modular development of neutralizing agents. In vitro antigen-binding fragment (Fab) selection offers the advantages of speed, sequence level molecular control, and engineering flexibility compared to traditional monoclonal antibody pipelines. By screening an unbiased, chemically-synthetic phage Fab library and characterizing hits in cell-based assays, we identified two high-affinity neutralizing Fabs, A4 and B7, against anthrax edema factor (EF), a key mediator of anthrax pathogenesis. Engineered homodimers of these Fabs exhibited potency comparable to the best reported neutralizing monoclonal antibody against EF at preventing EF-induced cyclic AMP production. Using internalization assays in COS cells, B7 was found to block steps prior to EF internalization. This work demonstrates the efficacy of synthetic alternatives to traditional antibody therapeutics against anthrax, while also demonstrating a broadly generalizable, rapid, and modular screening pipeline for neutralizing antibody generation.

Anthrax outbreak: exploring its biological agents and public health implications

“…The cutaneous form is the most common and fatal anthrax form, which is associated with edema and necrosis and the consequential black eschar sign in infected individuals; it has a mortality rate of approximately 20% in untreated individuals (21). The gastrointestinal form is considered rare and is characterized by flu-like symptoms.…”

Protein Engineering: Advances in Phage Display for Basic Science and Medical Research

“…A rapid and efficient screening to tailor phage-display for the selection of neutralizing antibody was set up in the KossLab for the Anthrax model. The high-affinity neutralizing sABs selected against edema toxin, an adenylate cyclase and a major mediator of anthrax pathogenesis, has demonstrated efficacy of the synthetic alternatives to the traditional antibody therapeutics: its potency to inhibit edema-toxin catalyzed cAMP production in human cells is comparable with that reported for the best-performing monoclonal antibodies [ 126 ].…”

Bacillus anthracis as a biological warfare agent: infection, diagnosis and countermeasures

“…Comparing to the standard antibiotics, the therapy by antibodies against anthrax toxin can be employed for prophylactic purposes because patients can exert good tolerability to such therapy. The applied antibodies can be specifi c to edema and lethal factor of anthrax toxin and the neutralization of the whole toxin is principle of the therapy (63,64). Antigen binding synthetic fragment of an antibody with a high affi nity to edema factor appears promising (63).…”

“…The applied antibodies can be specifi c to edema and lethal factor of anthrax toxin and the neutralization of the whole toxin is principle of the therapy (63,64). Antigen binding synthetic fragment of an antibody with a high affi nity to edema factor appears promising (63). Combination of an antibiotics with an antitoxin (antibody specifi c to anthrax toxin) is recommended as a highly effective way how to protect from anthrax (65).…”