Circulating polyclonal antibodies that recognize a disease-causing pathogen can elicit a therapeutic benefit through a variety of different effector mechanisms. One important mechanism is to attach multiple antibody Fc domains to a pathogen, and these are then bound by low-affinity Fc receptors in the liver and cleared from the circulation. Accelerated clearance in this way has the potential to improve disease when the target of the antibodies is a harmful biomolecule, such as a neurotoxin, that does not itself act on the liver cells as it is being removed from circulation and the body.
We show here that rapid serum clearance of the neurotoxin BoNT/A will occur if the toxin is bound by several different small binding agents that each contain a common epitope tag and if a single anti-tag monoclonal antibody is coadministered. A model for the proposed tagged-binding-agent clearance mechanism is shown in Fig. . Several antitoxin binding agents become associated with the toxin at different sites. Since each binding agent contains one or two copies of an epitope tag, coadministration of an anti-tag MAb leads to the toxin becoming decorated with antibodies. We suggest that the presence of multiple antibodies bound to the toxin through the binding agents triggers toxin clearance through the liver by the same Fc-dependent process. Most likely, the mechanism involves Fc receptor-mediated clearance similar to that responsible for clearance of multimeric antibodies or immune complexes (6
). Also possible are complement-mediated mechanisms of immune complex clearance (22
FIG. 6. Model for Fc decoration of toxin leading to rapid liver clearance through low-affinity Fc receptors. BoNT toxin, tagged binding agents, and anti-tag MAbs are indicated. Binding agents are represented with one or two epitope tags, indicated with triangles. (more ...)
Several lines of evidence indicate that the toxin is removed from serum by mechanisms dependent on the antibody Fc domain. First, clearance is dependent on the presence of both the tagged antitoxin binding agent and the anti-tag MAb. The use of tagged binding agents lacking specificity for the toxin has no antitoxin efficacy, even in the presence of anti-tag MAb. Second, a second epitope tag on one of the toxin binding agents improves the antitoxin efficacy of the pool about the same amount as does the addition of another, different single-tagged binding agent. This implies that the additional epitope tag on the toxin binding agent results in an additional MAb Fc domain becoming associated with the toxin, just as would occur if another single-tagged binding agent were present. Finally, we show that the accelerated clearance occurs mostly or entirely through the liver, which is strongly suggestive of Fc-mediated clearance.
The result showing that the use of a single binding agent with two epitope tags improves the efficacy of the binding agent pool implies that the therapeutic efficacy could be increased further by engineering each of the binding agents to contain two epitope tags. The need for fewer binding agents would further reduce development and production costs for products employing this therapeutic strategy. While it is apparent that engineering two or even more epitope tags onto each of the binding agents will provide improved clearance efficacy, these binding agents will themselves become bound to two or more MAbs, and this will likely lead to their rapid liver clearance even when they are not bound to their pathogenic targets and thus reduce or negate further therapeutic benefit.
The potential of a pool of small antitoxin binding agents for therapeutic applications was further demonstrated by showing that treatments were successful even when the agent was delivered 2 and 4 h after intoxication with a dose of 10 LD50 of BoNT/A. Current polyclonal antitoxins are used primarily after it is shown that a patient may have been exposed to toxin, but before major symptoms have developed. This test showed that the efficacy of these agents was not dependent on premixing toxin and antitoxin (which is the standard laboratory method). For the postintoxication testing, we compared the use of small binding agent antitoxins to the use of polyclonal anti-BoNT/A antisera. We used doses of both treatments that were previously shown to maximally protect mice from a dose of about 1,000 LD50 when premixed with toxin and then administered (higher doses of toxin were lethal). Although the tagged binding agents appeared to be more effective than the polyclonal sera administered postintoxication, this is an imperfect comparison and cannot yet be interpreted to suggest that the new therapy is superior in terms of efficacy. Nevertheless, the study does indicate that tagged binding agent antitoxins may be at least equivalent to conventional antitoxins when used in a postintoxication therapy.
Agents that promote rapid clearance of toxins or other pathogenic molecules would provide therapeutic benefit in many disease situations beyond botulinum toxin exposure (e.g., exposure to other toxins and venoms, cytokines, etc.). The results presented here suggest that pools of tagged agents recognizing different epitopes on a pathogenic molecule, together with anti-tag antibodies, have promise as therapeutic agents to promote clearance of the pathogenic molecule from patients. Such binding agents should be much more rapid to identify and more convenient and economical to commercialize than polyclonal antisera or a pool of different MAbs (17
). A wide variety of high-throughput strategies have been developed to rapidly identify small binding agents specific to defined targets (reviewed in references 11
). Many of these binding agents are non-antibody-based and consist of scaffolds with excellent commercial and therapeutic properties, such as low production costs, long shelf lives, serum stability, low toxicities, and low levels of immunogenicity. Although the therapeutic strategy suggested here currently requires coadministration with an anti-tag MAb, the same MAb could be used in all therapeutic applications, and therefore it could be selected for optimal commercial and therapeutic properties, produced, and stockpiled. Furthermore, the MAb isotype (IgG1 in this study) could be altered if different isotypes provided improved therapeutic benefits. Alternatively, the antibody might be engineered for improved Fc binding to the Fc receptor or replaced altogether by a bispecific binding agent recognizing both the epitope tag and Fc receptors. In some situations, prior immunization with an epitope tagged immunogen could produce preexisting anti-tag polyclonal antibodies to obviate an anti-tag MAb. As such, we believe that this general strategy may find therapeutic applications in both biodefense and general medicine.