A Novel, Orally Delivered Antibody Therapy and Its Potential to Prevent Clostridioides difficile Infection in Pre-clinical Models

Abstract

Clostridioides difficile infection (CDI) is a toxin-mediated infection in the gut and a major burden on healthcare facilities worldwide. We rationalized that it would be beneficial to design an antibody therapy that is delivered to, and is active at the site of toxin production, rather than neutralizing the circulating and luminal toxins after significant damage of the layers of the intestines has occurred. Here we describe a highly potent therapeutic, OraCAb, with high antibody titers and a formulation that protects the antibodies from digestion/inactivation in the gastrointestinal tract. The potential of OraCAb to prevent CDI in an in vivo hamster model and an in vitro human colon model was assessed. In the hamster model we optimized the ratio of the antibodies against each of the toxins produced by C. difficile (Toxins A and B). The concentration of immunoglobulins that is effective in a hamster model of CDI was determined. A highly significant difference in animal survival for those given an optimized OraCAb formulation versus an untreated control group was observed. This is the first study testing the effect of oral antibodies for treatment of CDI in an in vitro gut model seeded with a human fecal inoculum. Treatment with OraCAb successfully neutralized toxin production and did not interfere with the colonic microbiota in this model. Also, treatment with a combination of vancomycin and OraCAb prevented simulated CDI recurrence, unlike vancomycin therapy alone. These data demonstrate the efficacy of OraCAb formulation for the treatment of CDI in pre-clinical models.

Keywords: Clostridioides difficile infection (CDI); formulation protecting antibodies from digestion/inactivation; immunotherapy of CDI; in vitro human gut model of CDI; in vivo hamster model of CDI; oral antibodies.

FIGURE 1

Treatment design of the in vivo hamster model of CDI. Each experiment contains sentinel group (x4) that receive clindamycin only and an untreated control group (x10) receiving clindamycin and challenge with C. difficile.

FIGURE 2

In vitro gut model experimental design. Timeline for gut models, including equilibration of microbial community, induction of CDI and addition of placebo, OraCAb antibodies or antibodies + vancomycin.

FIGURE 3

Inhibition of human trypsin with DEW and purified BBI determined by Nα-Benzoyl-DL-Arginine-p-Nitroanilide (BAPNA) colorimetric assay. Concentrations of DEW and BBI that gave half-maximal inhibition of human trypsin (EC₅₀) are given in the table.

FIGURE 4

Stability of IgG (anti-TxA4) in OraCAb formulation in SGF. OraCAb was incubated for 2.5 h in SGF at different v/v ratios and full protection of the IgG was found when the volume of OraCAb was 30% (3/10 ratio) or higher compared with the volume of the SGF. Around 50% of the IgG was found inactivated/degraded in SGF containing 10 or 20% OraCAb. Concentrations of IgG in OraCAb that give half maximum inhibition of TcdA in a cell-based toxin neutralization assay (EC₅₀) are given in the table. The calculations were based on the concentration of IgG prior to mixing the formulations with SGF.

FIGURE 5

Survival rates of hamsters after C. difficile challenge. Hamsters were challenged with C. difficile spores via the oral route on day 0, placed on one of the oral therapeutic regimes until the end of day 4 and monitored until day 15. An untreated control group, receiving challenge only, was included for each experiment. (A) Groups were treated with OraCAb containing different ratios of anti-TxB4: anti-TxA4 antibodies with either BBI or DEW as trypsin inhibitors. (B) Groups were treated using OraCAb formulated with antibody concentration of 150 mg/ml at a 3:1 anti-TxB4: anti-TxA4 ratio in the presence or absence of DEW. (C) Groups were treated with OraCAb containing different ratios of anti-TxB4: anti-TxA4 antibodies. The antibodies in OraCAb that contained anti-TxB4 IgG only were at the same concentration as anti-TxB4 antibodies in the 3:1 anti-TxB4: anti-TxA4 formulation. All OraCAb formulations contained DEW. (D) Groups were treated with OraCAb containing DEW and three different antibody concentrations (50, 100, or 150 mg/ml) at a fixed 3:1 anti-TxB4: anti-TxA4 ratio. The table shows the outcome of the statistical analyses of the data.

FIGURE 6

Mean C. difficile total viable counts (log₁₀ cfu/ml), spore counts (log₁₀ cfu/ml) and cytotoxin titer [relative units (RU)] in vessel three of the in vitro gut model. (A) – Model 1 (placebo); (B) – Model 2 (antibodies); (C) – Model 3 (placebo + antibodies); (D) – Model 4 (vancomycin + antibodies). The limit of detection is 1.22 log₁₀ cfu/ml for total viable counts and 1.52 log₁₀ cfu/ml for spore counts; horizontal dotted lines.

FIGURE 7

Mean viable counts of selected microbiota communities in vessel three of the in vitro gut model. (A) – Model 1 (placebo); (B) – Model 2 (antibodies). The limit of detection is 1.22 log₁₀ cfu/ml; horizontal dotted line.