Novel Chimpanzee/Human Monoclonal Antibodies That Neutralize Anthrax Lethal Factor,and Evidence for Possible Synergy with Anti-Protective Antigen Antibody

Three chimpanzee Fabs reactive with lethal factor (LF) of anthrax toxin were isolated and converted into complete monoclonal antibodies (MAbs) with human γ1 heavy-chain constant regions. In a macrophage toxicity assay, two of the MAbs, LF10E and LF11H, neutralized lethal toxin (LT), a complex of LF and anthrax protective antigen (PA). LF10E has the highest reported affinity for a neutralizing MAb against LF (dissociation constant of 0.69 nM). This antibody also efficiently neutralized LT in vitro, with a 50% effective concentration (EC₅₀) of 0.1 nM, and provided 100% protection of rats against toxin challenge with a 0.5 submolar ratio relative to LT. LF11H, on the other hand, had a slightly lower binding affinity to LF (dissociation constant of 7.4 nM) and poor neutralization of LT in vitro (EC₅₀ of 400 nM) and offered complete protection in vivo only at an equimolar or higher ratio to toxin. Despite this, LF11H, but not LF10E, provided robust synergistic protection when combined with MAb W1, which neutralizes PA. Epitope mapping and binding assays indicated that both LF10E and LF11H recognize domain I of LF (amino acids 1 to 254). Although domain I is responsible for binding to PA, neither MAb prevented LF from binding to activated PA. Although two unique MAbs could protect against anthrax when used alone, even more efficient and broader protection should be gained by combining them with anti-PA MAbs.Anthrax is a highly lethal infectious disease caused by the spore-forming bacterium Bacillus anthracis. The deliberate distribution of anthrax spores through the U.S. mail system in 2001 resulted in five deaths among the 11 individuals who contracted inhalational anthrax (18). This incident highlighted the great threat posed by the potential use of anthrax in terrorism and warfare. The lethality of inhalational anthrax is primarily due to the action of anthrax toxins. The bacterium produces three toxin components; these are protective antigen (PA) (83 kDa), lethal factor (LF) (85 kDa), and edema factor (EF) (89 kDa) (13, 32). PA binds to host cell anthrax toxin receptors and is cleaved by cell surface furin to produce a 63-kDa peptide, PA63 (activated PA). Anthrax toxin receptor-bound PA63 oligomerizes to a heptamer and translocates up to three molecules of LF or EF from the cell surface via endosomes to the cytosol. Therefore, PA functions as a vehicle to mediate the cellular uptake of LF and EF (for a review, see reference 44). PA with LF forms lethal toxin (LT), and PA with EF forms edema toxin (ET). LF is a zinc-dependent endopeptidase that cleaves mitogen-activated protein kinase kinases and disrupts intracellular signaling (8, 30, 40). LT can replicate symptoms of anthrax disease when injected into animals (27). EF is a calcium-calmodulin-dependent adenylate cyclase that transforms ATP to cyclic AMP, and ET has a range of toxic effects in the host (12, 20). These toxins are the dominant virulence factors for anthrax disease, and vaccination against their common component, PA, is sufficient for protection against anthrax disease.Currently antibiotics are the only choice for clinical treatment of anthrax disease. Although effective, antibiotics have limitations. Exposure to the bacterium followed by bacterial division leads to production of large quantities of the anthrax toxins. Thus, unless exposure is diagnosed early enough for antibiotic treatment to prevent significant replication, patients will succumb to disease even after the killing of all bacteria. The current PA-based vaccine approved by the U.S. Food and Drug Administration is also not effective postexposure in protecting newly infected individuals, as it requires repeated administration and at least 4 weeks for development of anti-PA protective titers. Thus, in the absence of any small-molecule toxin inhibitors, monoclonal antibodies (MAbs) against toxin components are the only viable candidates for immediate neutralization of the effects of toxin. Although PA has been the primary target for passive protection (5, 25, 31, 35, 41, 43), it has been suggested that immunity to LF and EF can also play an important role in protection (14, 33, 34), and thus these proteins may represent alternative targets for antibody therapy against anthrax. In a previous study, the protective effects of anti-PA and anti-LF antibodies were greatly synergized by their combination (3). Furthermore, concerns that PA may be mutated within currently recognized neutralization epitopes such that anti-PA therapies would no longer be effective against this toxin warrant the further development of antibodies targeting the other toxin components. A cocktail of more than one MAb that could recognize distinct epitopes on multiple toxin proteins (PA, LF, and EF) could certainly broaden the spectrum of protection against anthrax. In recent years, several anti-LF neutralizing MAbs have been reported (1, 21, 24, 37, 46). However, only one of them was a human antibody; the others were rodent MAbs that would need further manipulation before use in humans.Chimpanzee immunoglobulins (Igs) are virtually identical to human Igs and may have clinically useful applications (9). As part of a larger study (5), we recovered chimpanzee MAbs specific for LF from a combinatorial cDNA library of antibody genes developed from chimpanzees that had been immunized with anthrax toxins. In this work we describe the detailed characterization of these anti-LF antibodies.