Efficient serum clearance of botulinum neurotoxin achieved using a pool of small antitoxin binding agents

Abstract

Antitoxins for botulinum neurotoxins (BoNTs) and other toxins are needed that can be produced economically with improved safety and shelf-life properties compared to conventional therapeutics with large-animal antisera. Here we show that protection from BoNT lethality and rapid BoNT clearance through the liver can be elicited in mice by administration of a pool of epitope-tagged small protein binding agents together with a single anti-tag monoclonal antibody (MAb). The protein binding agents used in this study were single-chain Fv domains (scFvs) with high affinity for BoNT serotype A (BoNT/A). The addition of increasing numbers of differently tagged scFvs synergistically increased the level of protection against BoNT/A. It was not necessary that any of the BoNT/A binding agents possess toxin-neutralizing activity. Mice were protected from a dose equivalent to 1,000 to 10,000 50% lethal doses (LD(50)) of BoNT/A when given three or four different anti-BoNT scFvs, each fused to an E-tag peptide, and an anti-E-tag IgG1 MAb. Toxin protection was enhanced when an scFv contained two copies of the E tag. Pharmacokinetic studies demonstrated that BoNT/A was rapidly cleared from the sera of mice given a pool of anti-BoNT/A scFvs and an anti-tag MAb but not from the sera of mice given scFvs alone or anti-tag MAb alone. The scFv pool and anti-tag MAb protected mice from lethality when administered up to 2 h following exposure of mice to a dose equivalent to 10 LD(50) of BoNT/A. These results suggest that it will be possible to rapidly and economically develop and produce therapeutic antitoxins consisting of pools of tagged binding agents that are administered with a single, stockpiled anti-tag MAb.

FIG. 1.

BoNT/A-neutralizing activity of anti-BoNT/A scFvs in primary granule cerebellar neurons. (A) Neutralization of BoNT/A with 35 nM anti-BoNT/A scFvs. BoNT/A (0.1 nM) and scFvs were mixed and added to cultured neurons for 3 h. The extent of intoxication was monitored by SDS-PAGE and Western blotting of protein extracts to assess the extent of SNAP25 cleavage. Western blots were probed with anti-SNAP25 antibody. (B) Dose-response curve of BoNT/A neutralization by anti-BoNT/A scFv 2. BoNT/A (0.1 nM) and various dilutions of scFv 2, as indicated, were mixed and added to neuronal cell cultures for 3 h prior to Western blotting for SNAP25. Toxin neutralization was assessed by inhibition of SNAP25 cleavage by BoNT/A as described above.

FIG. 2.

Synergistic improvement in protection of mice from BoNT intoxication by administration of increasing numbers of different tagged anti-BoNT/A scFvs and an anti-tag MAb. The percent survival within groups of five mice is shown at various times postintoxication. The intoxicating dose (LD₅₀) is constant within each plot, as noted. Treatment groups varied in the content of anti-BoNT scFv and the presence or absence of anti-E-tag MAb, as indicated. All agents were administered intravenously. Control animals (Co) were given PBS alone. (A) Twenty micrograms of the indicated scFv was given to each mouse. Five micrograms of anti-E-tag MAb was given per mouse, where indicated (+M). (B and C) Each mouse received a total of 6 μg of the scFvs indicated. Where more than one scFv was included, as indicated, each was proportionally represented (e.g., two scFvs were present at 3 μg each). Five micrograms of anti-E-tag MAb was given per mouse, where indicated (+M). Two groups of mice were given scFv 2 and anti-E-tag MAb (2+M), one with the MAb administered i.v. and one with the MAb given intraperitoneally, with the same results.

FIG. 3.

Improved protection of mice from BoNT intoxication by administration of a pool of anti-BoNT/A scFvs and anti-tag MAb when one scFv contains a second epitope tag. The percent survival within groups of five mice is shown at various times postintoxication. The intoxicating dose (LD₅₀) is constant within each plot, as noted. Treatment groups varied in the content of anti-BoNT scFv and the presence of anti-E-tag MAb, as indicated. All agents were administered i.v. Controls (Co) received PBS alone. (A to C) Each mouse received a total of 2 μg of scFv, proportionally represented. 7E2, scFv 7 with two copies of the E tag. Ten micrograms of anti-E-tag MAb was given per mouse, where indicated (+M). In panel C, three other groups (3+7+M, 3+7E2+M, and 2+3+7+M) had the same survival as the control group.

FIG. 4.

Pharmacokinetics of BoNT/A clearance in mice coinfused with anti-BoNT/A scFvs, with or without anti-E-tag MAb. A series of experiments were performed to assess the tissue distribution and clearance of botulinum toxin following various treatment regimens. (A) ¹²⁵I-botulinum toxin type A (5 ng/mouse) was incubated for 1 h at room temperature with buffer (▪) or a mixture of scFvs (2, 3, 7-E2, and 21; 1.5 μg each/dose) and anti-E-tag MAb (10 μg/dose) (•). Groups of six mice were injected i.v. with the toxin treatments and sacrificed at the indicated times. The serum toxin concentration was determined by scintillation counting (error bars indicate standard errors of the means [SEM]). Animals were sacrificed, and the levels of toxin in blood were determined. (B) Iodinated toxin was incubated with buffer (open bars) or with the same scFv and MAb pool used in panel A (filled bars) and administered to groups of six mice. The mice were sacrificed 30 min later and perfused. The livers (columns A and B) and spleens (columns C and D) were excised, and the toxin concentrations were determined by scintillation counting. (C) Iodinated toxin was incubated with buffer or with 10 μl of BoNT/A antiserum, the scFv mixture alone, the anti-tag MAb alone, or the scFv and anti-tag MAb mixture used in panel A. The mice were sacrificed 30 min later, blood was collected, and the animals were perfused. Toxin concentrations in the blood, liver, and spleen were determined by scintillation counting.

FIG. 5.

Postintoxication administration of tagged antitoxin scFv pools protects mice from a dose of 10 LD₅₀ of BoNT/A within a therapeutic window. The percent survival within groups of five mice is shown at various times postintoxication. A dose of 10 LD₅₀ of BoNT/A was administered via intraperitoneal injection. The “scFv pool” given intravenously to each mouse consisted of 1.5 μg each of four scFvs (2, 3, 7, and 8) and 5 μg of anti-E-tag MAb. This dose of the scFv pool was previously shown to maximally protect mice from a dose of 1,000 LD₅₀ of BoNT/A when preincubated and coadministered with toxin (not shown). Mice receiving the scFv pool at 2 h postintoxication showed moderate signs of intoxication but survived. Lethality was delayed when the pool was given at 4 h postintoxication. As a positive control, groups of mice received a dose of polyclonal anti-BoNT/A sufficient to maximally protect mice from a dose of 1,000 LD₅₀ of BoNT/A when preincubated and coadministered with toxin (not shown). One of five mice that received CDC antitoxin at 2 h postintoxication survived. All five mice that received CDC antitoxin at 4 h postintoxication died within 24 h, as did the controls (Co) receiving no antitoxin treatment.

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. Anti-tag MAbs bind to epitope tags to decorate the toxin with multiple Fc domains, leading to accelerated clearance.