Bispecific antibody engineered from two non-protective antibodies confers full protection against anthrax toxin through inhibition of PA83 cleavage

Anthrax is a highly contagious zoonotic disease, posing a great threat to human life and health. Protective antibodies against anthrax protective antigens (PA) can play a crucial role in the prevention and treatment of anthrax infections. However, the functions and mechanisms of the large number of non-protective antibodies elicited during the infection or vaccination of anthrax have not been clearly understood. Here, we found that two non-protective antibodies, 2A6 and 8A7, exerted an enhanced neutralizing effect both <i>in vitro</i> and <i>in vivo</i>. In the monoclonal antibodies (mAbs) combination, 8A7 increased the aggregation of PA, which might favor the inhibition of PA pore formation by 2A6. Structural analysis demonstrated distinct interaction patterns between the monoclonal antibodies, with 8A7 primarily interacting with domain 3 of the PA protein, while 2A6 establishes direct contact with domain 2. We further constructed bispecific antibodies in the form of DVD-Ig. The 2A6-8A7 conserved the parental antibody functions in promoting PA aggregation and inhibiting pore formation and simultaneously exhibited a distinct neutralizing mechanism through preventing PA83 cleavage. This study provides new insights into how the non-protective antibodies work in collaboration <i>in vivo</i> and opens new avenues for optimizing future anthrax prevention and control strategies.IMPORTANCEAnthrax remains a bioterrorism threat and public health challenge, where protective antibodies targeting protective antigen (PA) are critical for treatment. However, the role of non-protective antibodies-abundant yet poorly understood components of immune responses-has been unclear. This study reveals that two non-protective antibodies (2A6 and 8A7) unexpectedly block anthrax toxin activity in combination. By combining these antibodies, we observed enhanced PA aggregation and inhibition of toxin pore formation, with structural results showing their distinct binding sites on PA. Furthermore, engineered bispecific antibodies derived from this pair not only exhibited superior neutralization but also uncovered a novel mechanism: blocking the cleavage of PA, a critical step in toxin activation. These findings challenge the traditional view of non-protective antibodies as biologically inert, offering a transformative strategy to repurpose them into potent therapies. This work advances anthrax treatment design and deepens our understanding of how antibody combinations mimic natural immunity to combat pathogens.