Potent neutralization of anthrax edema toxin by a humanized monoclonal antibody that competes with calmodulin for edema factor binding

This study describes the isolation and characterization of a neutralizing monoclonal antibody (mAb) against anthrax edema factor, EF13D. EF13D neutralized edema toxin (ET)-mediated cyclic AMP (cAMP) responses in cells and protected mice from both ET-induced footpad edema and systemic ET-mediated lethality. The antibody epitope was mapped to domain IV of EF. The mAb was able to compete with calmodulin (CaM) for EF binding and displaced CaM from EF-CaM complexes. EF-mAb binding affinity (0.05–0.12 nM) was 50- to 130-fold higher than that reported for EF-CaM. This anti-EF neutralizing mAb could potentially be used alone or with an anti-PA mAb in the emergency prophylaxis and treatment of anthrax infection.Infection by inhalational anthrax is often fatal if treatment is delayed. Anthrax bacteria can be killed by vigorous treatment with antibiotics, but patients may still die because the lethality of anthrax is largely because of the action of toxins (1). Anti-toxin neutralizing monoclonal antibodies (mAbs) are the only viable choice for immediate neutralization of toxin and they could augment the effectiveness of antibiotics.Anthrax bacteria produce 3 toxin components: Protective antigen (PA), lethal factor (LF), and edema factor (EF) (2, 3). PA binds to cellular receptors and acts as a vehicle to deliver LF or EF into the cytosol where they exert their enzymatic activities (4–8). LF is a zinc-dependent protease that cleaves mitogen-activated protein kinase kinases and causes lysis of macrophages (9, 10). EF is a calcium-calmodulin (CaM)-dependent adenylate cyclase and causes local inflammation and edema (11). The combination of PA with LF results in lethal toxin (LT). LT can replicate symptoms of anthrax disease when injected into animals (12). PA together with EF forms edema toxin (ET) and ET can produce a range of toxic effects in the host (11, 13).PA has been regarded as the most important target for prophylaxis and therapy of anthrax, because PA is common to both LTs and ETs, initiates the toxic process via receptor binding, and is highly immunogenic. In fact, PA is the major component in the current anthrax vaccine and the target for most of the available human or human-like neutralizing mAbs that have been shown to be very effective in protection against anthrax toxin or spore challenge (14–19). However, there is evidence that LF and EF may play important roles in providing protective immunity (20–22). Furthermore, concerns that PA could potentially be manipulated, such that it would no longer be neutralized by current anti-PA neutralizing mAbs have led to interest in therapeutics against the other 2 toxin components. A mixture of mAbs that recognize distinct epitopes on multiple toxin components (PA, LF, or EF) would not only enhance the protective efficacy but also broaden the spectrum of protection. Thus, in recent years, several anti-LF mAbs have also been reported (23–27). However, no anti-EF neutralizing mAbs have been reported to date. A previous report had indicated that immunization with the PA-binding N-terminal domain of EF (amino acids 1–254) resulted in polyclonal sera containing both EF and LF neutralizing activities (28).The purpose of this study was to determine (i) if anti-EF neutralizing mAbs could be isolated; (ii) the effectiveness of such antibodies against anthrax ET effects; and (iii) the neutralization mechanism of these antibodies. We have made a Fab combinatorial phage display library from chimpanzees that were immunized with anthrax toxins (17). From the library, 4 EF-specific Fabs were recovered, and 1 of them had potent neutralizing activity independent of the homologous PA-binding N-terminal (1–254) domain of LF. In this report, we describe the detailed characterization of these anti-EF clones.