Designed Ankyrin Repeat Protein (DARPin) Neutralizers of TcdB from Clostridium difficile Ribotype 027

(A and B) DARPins strongly exhibited the ability to neutralize (A) and bind (B) the different TcdB toxins. conc., concentration. (C) TcdB neutralization potency of different DARPins and bezlotoxumab. For neutralization assays, serially diluted immobilized-metal affinity chromatography (IMAC)-purified DARPins were mixed with the appropriate toxin and then added to Vero cells seeded the night before in 96-well plates. The cell viability was quantified by the CellTiterGlo assay 72 h later and normalized to naive Vero cells. For ELISAs, the MaxiSorp plates were coated with the appropriate toxin followed by treatment with serially diluted DARPins. The amounts of plate-bound DARPins (containing Myc tags) were quantified using an anti-c-Myc antibody. Data in panel A represent averages of results from at least 2 independent experiments. Data presented in panel B are representative of results from two independent experiments performed in duplicate.

Anti-TcdB_UK1 DARPins block the interaction between TcdB and its receptor CSPG4. ***, P < 0.001 (t test). The wells of an ELISA plate were coated with TcdB_UK1 followed by treatment with a 1 nM concentration of a CSPG4 extracellular domain-GFP fusion protein alone or in a mixture with a 250 nM concentration of the indicated DARPins. The amounts of plate-bound CSPG4 were detected using an anti-GFP antibody. The data are representative of results from two independent experiments and of averages of results from quadruplicate samples.

DARPin dimer U3D16 showed enhanced neutralization ability against TcdB_VPI (A) and TcdB_M68 (B). Serially diluted DARPins were mixed with the appropriate toxins and then added to Vero cells that had been seeded the night before. Cell viability was quantified by the CellTiterGlo assay 72 h later and normalized to naive Vero cells. The error bars represent mean deviations of results from two independent experiments. (C) U3 lacks the ability to bind to TcdB_UK1 as determined by ELISA. The ELISA plates were coated with the appropriate toxin and then blocked with BSA prior to the addition of serially diluted DARPin U3. The amounts of plate-bound DARPin were quantified using an anti-c-Myc antibody. The data are representative of results from two independent experiments performed in duplicate. OD₄₅₀, optical density at 450 nm.

TcdB_UK1 lacks significant ability to interact with FZD2. The wells of an ELISA plate were coated with TcdB_VPI or TcdB_UK1 followed by treatment with CSPG4-EC-GFP (1 nM) (A), FZD2-Fc (4 nM) (B), or PVRL3 (100 nM) (C). The amount of plate-bound CSPG4, FZD2, or PVRL3 was detected using each of the respective antibodies. Results are representative of at least two independent experiments. Error bars represent the mean deviations of results from duplicate samples.

Vero and Caco-2 cells exhibit different levels of sensitivity to TcdB_UK1 and TcdB_VPI. Vero cells (A) or Caco-2 cells (B) were incubated with serial dilutions of TcdB_UK1 or TcdB_VPI. The cell viability was quantified using CellTiter-Glo reagent 72 h later and normalized to naive cells. The error bars represent standard deviations of results from two independent experiments performed in triplicate.