Ultrapotent neutralization of Bacillus anthracis toxin by a human antibody via blockade of PA oligomerization.

Anthrax lethal toxin is a key virulence factor of Bacillus anthracis. However, non-canonical epitopes of protective antigen (PA) and alternative neutralization mechanisms beyond receptor blockade or prevention of proteolytic cleavage remain underexplored. Here, we characterize a fully human monoclonal antibody, 22F1, with ultrapotent toxin neutralization in vitro (IC50 = 0.027 nM) and in vivo (molar ratio 1:7.5-15). Unlike known PA antibodies, 22F1 selectively binds the post-cleavage form PA63, not full-length PA83, due to shielding of the epitope by PA20. Cryo-EM reveals a novel conformational epitope spanning domains D1', D2, and D3. 22F1 does not block receptor engagement, PA proteolytic activation, or lethal factor binding. Instead, its epitope lies near the inter-protomer interface; binding of a single 22F1 Fab to any PA63 protomer exerts a dual steric hindrance effect, simultaneously preventing additional Fab binding to adjacent protomers and blocking PA63 oligomerization. This "single-hit" mechanism enables one antibody molecule to efficiently block prepore formation, achieving high potency. Our findings advance anthrax antitoxin research and provide a paradigm for targeting other proteins with similar activation-oligomerization-pore formation mechanisms.