In this study, we used the cell-based anthrax toxin neutralization assay as the sole method to select a human anti-PA MAb with potential clinical utility, as opposed to other approaches based on antibody affinity or inhibition of receptor binding. As predicted from previous studies correlating potent toxin neutralization activity with protection in animals, MAb 1303 was effective in the prevention of anthrax lethality in rabbits and NHPs despite the aerosolized administration of greater than 100 times the lethal dose of virulent spores. To our knowledge, the lowest dose tested of MAb 1303 (1 mg/kg) is the lowest dose of an anti-PA MAb to show 100% protection in the NHP model. Our unexpected observation was the Fc receptor dependence of toxin neutralization for this anti-PA antibody.
Unlike previously described anti-PA MAbs, MAb 1303 activity was only partially mediated by the direct attachment of the antibody binding domains. Experiments using MAb 1303 F(ab′)2 fragments and Fc receptor-blocking antibodies, in two different assay systems, definitively demonstrated the requirement for Fc receptor binding by the Fc domain of MAb 1303 for full toxin-neutralizing potency. In the absence of Fc receptor interaction, MAb 1303 was 1 to 2 logs less effective than under conditions that promote Fc receptor interactions. Our results may suggest that immune complexes consisting of cell-bound PA, MAb 1303, and Fc receptors are formed at the cell surface, resulting in interference with the subsequent oligomerization of PA into viable toxin complexes and their translocation. Consistent with this hypothesis, MAb 1303 failed to neutralize preformed toxin complexes but could significantly prevent cell death up to 20 min after the addition of PA and LF subunits to J774 cells (data not shown).
Although an Fc receptor dependence of anthrax toxin neutralization was not demonstrated with prior antibodies, we examined whether this feature was unique to MAb 1303. Interestingly, a similar Fc-dependent characteristic was found with the second most potent human MAb generated in our studies as well as with the well-characterized, neutralizing anti-PA MAb 1G3 (data not shown) (15
). Furthermore, we demonstrated that the toxin-neutralizing activity of (pooled) human and animal polyclonal immune sera was also substantially abrogated in the presence of an anti-Fc receptor MAb. Fc receptor blockade had no inhibitory effect on the toxin neutralization of an anti-PA MAb that functions by blocking PA binding to cell receptors, consistent with the expectation that such activity would only be active on circulating PA, not after binding to the cell surface. Consistent with the TNA results, we were unable to demonstrate that the polyclonal immune sera contained significant blocking activity for PA binding to cell receptors, suggesting that the natural predominant mechanism of anthrax toxin neutralization by antibodies elicited by anthrax vaccines do not function by inhibiting initial PA binding but via other epitopes that permit an Fc receptor-mediated interaction on the cell surface.
The precise mechanism by which Fc receptor interaction with anti-PA MAbs results in enhanced activity remains to be established. MAb binding to receptor-bound PA and Fc receptors may result in immune complex formation at the cell surface that are endocytosed into degradation pathways mediated by the Fc receptor, or simply interfere with the proper toxin assembly. Recent studies by Abrami et al. demonstrated that the cellular internalization of the anthrax toxin involves a relatively slow clustering of the anthrax toxin receptor (ATR) into lipid rafts (2
). The investigators also showed that palmitoylation prevents ATR from association with lipid rafts and premature internalization (1
), and only heptameric PA63 (not monomeric PA83) can cluster the ATR into lipid rafts. Thus, endocytosis of the ATR-bound monomeric PA remains slow until the processing and oligomerization of PA63 occurs. Presumably, this would allow time for MAb 1303 to bind ATR-PA83 and ATR-PA63 (before heptamerization) complexes and simultaneously engage Fc receptors with the Fc portion of the MAb. Such interactions may result in premature internalization of the ATR-PA complex or prevent the appropriate migration to lipid rafts for toxin assembly and endocytosis. The fact that the in vitro toxin neutralization assay requires significantly less MAb 1303 than PA83 (Fig. ) suggests that one molecule of 1303 may be interacting with multiple PA molecules or that a single immune complex may be sufficient to interfere with heptamer formation/function. We are currently investigating how these mechanisms may be contributing to the neutralizing effect of MAb 1303 and other anti-PA antibodies.
Interestingly, Mohamed et al. found that certain anti-PA MAbs could enhance the cytotoxicity mediated by lethal toxin in an Fc receptor-dependent manner (19
). Since our selection was for antibodies that mediate potent neutralization of lethal toxin, it is unknown whether human MAbs may also mediate the enhancing effect described with some murine anti-PA MAbs. Alternatively, it may be the epitope specificity that determines the nature of the Fc receptor interaction and its outcome on toxin activity.
The absolute relevance of the Fc receptor interaction to the in vivo efficacy of anti-PA antibodies remains to be understood. MAb 1303 does protect against anthrax toxin cytotoxicity in the absence of Fc receptor interactions (albeit at significantly higher concentrations); thus, in vivo protection cannot be unambiguously attributed to the Fc-enhanced neutralization activity. Although some cells that are targets for the anthrax toxin in vivo do not express Fc receptors, many cells of the immune system which are critical to the innate response to pathogens have abundant expression of Fc receptors. In particular, dendritic cells have been implicated as a critical cell in the initial response to B. anthracis
). Therefore, it is reasonable to consider the Fc receptor interactions of anti-PA MAbs to be important to their efficacy in vivo.
In addition to defining a novel mechanism of action for anti-PA antibodies, our studies with delayed administration of MAb 1303 to infected and symptomatic animals suggests this MAb may be effective in a therapeutic setting. Since antibiotics have limited efficacy after exposure, development of additional therapeutic options for symptomatic individuals is of high importance. A recent report has suggested that passive immunity can be effectively combined with antibiotics resulting in greater therapeutic efficacy (10
). Therapeutic modeling in animals is challenging with anthrax because of the lack of consistent clinical symptoms prior to death, which makes it difficult to separate postexposure prophylaxis from therapeutic intervention. Further studies with this novel antibody in a therapeutic setting, alone and in combination with antibiotics, will be important to establish the utility of this MAb in the defense against anthrax.