Selection and characterization of ultrahigh potency designed ankyrin repeat protein inhibitors of C. difficile toxin B

Abstract

Clostridium difficile infection (CDI) is a major nosocomial disease associated with significant morbidity and mortality. The pathology of CDI stems primarily from the 2 C. difficile-secreted exotoxins-toxin A (TcdA) and toxin B (TcdB)-that disrupt the tight junctions between epithelial cells leading to the loss of colonic epithelial barrier function. Here, we report the engineering of a series of monomeric and dimeric designed ankyrin repeat proteins (DARPins) for the neutralization of TcdB. The best dimeric DARPin, DLD-4, inhibited TcdB with a half maximal effective concentration (EC50) of 4 pM in vitro, representing an approximately 330-fold higher potency than the Food and Drug Administration (FDA)-approved anti-TcdB monoclonal antibody bezlotoxumab in the same assay. DLD-4 also protected mice from a toxin challenge in vivo. Cryo-electron microscopy (cryo-EM) studies revealed that the 2 constituent DARPins of DLD-4-1.4E and U3-bind the central and C-terminal regions of the delivery domain of TcdB. Competitive enzyme-linked immunosorbent assay (ELISA) studies showed that the DARPins 1.4E and U3 interfere with the interaction between TcdB and its receptors chondroitin sulfate proteoglycan 4 (CSPG4) and frizzled class receptor 2 (FZD2), respectively. Our cryo-EM studies revealed a new conformation of TcdB (both apo- and DARPin-bound at pH 7.4) in which the combined repetitive oligopeptides (CROPS) domain points away from the delivery domain. This conformation of the CROPS domain is in stark contrast to that seen in the negative-stain electron microscopy (EM) structure of TcdA and TcdB at the same pH, in which the CROPS domain bends toward and "kisses" the delivery domain. The ultrapotent anti-TcdB molecules from this study serve as candidate starting points for CDI drug development and provide new biological tools for studying the pathogenicity of C. difficile. The structural insights regarding both the "native" conformation of TcdB and the putative sites of TcdB interaction with the FZD2 receptor, in particular, should help accelerate the development of next-generation anti-C. difficile toxin therapeutics.

Fig 1. Characterization of leading monomeric DARPin hits.

(A) Monomeric DARPins are able to protect Vero cells from TcdB-induced cytopathic effect at nanomolar concentrations. IMAC-purified DARPins were added to Vero cells (2 × 10³ cells/well) together with TcdB toxin (5 pg/mL). Cell viability was quantified 72 hours later by the CellTiterGlo assay and normalized to naïve Vero cells. Error bars represent the standard deviation of at least 2 independent experiments performed in duplicate. (B) Relative binding of DARPins to TcdB was determined using ELISA. Serially diluted DARPins were added to microtiter plates coated with 4 μg/mL of TcdB. Results are representative of 2 independent experiments. (C) DARPin monomer TcdB-neutralization potency. Data are the averages of at least 2 independent experiments. DARPin, designed ankyrin repeat protein; EC₅₀, half maximal effective concentration; ELISA, enzyme-linked immunosorbent assay; IMAC, Immobilized metal affinity chromatography; TcdB, C. difficile toxin B.

Fig 2. Characterization of dimeric DARPins.

(A) Dimeric DARPins protect Vero cells from the TcdB-induced cytopathic effect at picomolar concentrations. IMAC-purified DARPin dimers were added to Vero cells (1.5 × 10³ cells/well) together with TcdB toxin (5 pg/mL). Cell viability was quantified 72 hours later by the CellTiterGlo assay and normalized to naïve Vero cells. Error bars represent the standard deviation of 2 independent experiments performed in duplicate. (B) Relative binding of DARPin dimers to TcdB was determined using ELISA. Serially diluted DARPins were added to microtiter plates coated with 4 μg/mL of TcdB. Results are representative of 2 independent experiments, and the error bars represent mean deviation from duplicate samples. (C) TcdB-neutralization potency of DARPin dimers. Data are the averages of at least 2 independent experiments. Conc, concentration; DARPin, designed ankyrin repeat protein; EC₅₀, half maximal effective concentration; ELISA, enzyme-linked immunosorbent assay; IMAC, Immobilized metal affinity chromatography; TcdB, C. difficile toxin B.

Fig 3. DARPin dimers exhibit superior toxin-neutralization potency relative to the constituent monomers.

IMAC-purified DARPin dimers or monomers were added to Vero cells (1.5 × 10³ cells/well) together with TcdB (5 pg/mL). Cell viability was quantified 72 hours later by CellTiterGlo assay and normalized to naïve Vero cells. Error bars represent the standard deviation of at least 2 independent experiments performed in duplicate. Conc, concentration; DARPin, designed ankyrin repeat protein; IMAC, Immobilized metal affinity chromatography; TcdB, C. difficile toxin B.

Fig 4. DARPin dimers demonstrate avidity in TcdB binding.

Binding of selected DARPin monomers, pooled monomers, or dimers to TcdB was determined using ELISA. Combinations of serially diluted DARPins were added to microtiter plates coated with 4 μg/mL of TcdB. Results are representative of 2 independent experiments. Conc, concentration; DARPin, designed ankyrin repeat protein; ELISA, enzyme-linked immunosorbent assay; TcdB, C. difficile toxin B.

Fig 5. DARPin dimers offer superior protection to Vero cells against the toxicity of TcdB from C. difficile strains VPI 10463 (ribotype 087) and M68 (ribotype 017).

(A) IMAC-purified DARPins were added to Vero cells (1.5 × 10³ cells/well) together with TcdB toxin (2.5 pg/mL). Cell viability was quantified 72 hours later by the CellTiterGlo assay and normalized to naïve Vero cells. Error bars represent the standard deviation of at least 2 independent experiments done in duplicate. Bezlotoxumab is the FDA-approved monoclonal antibody for treating recurrent CDI [7]. a-GFP is a GFP-binding DARPin [15] and was used here as a negative control. (B) DARPin dimer TcdB-neutralization potency. Data are the averages of at least 2 independent experiments. CDI, Clostridium difficile infection; Conc, concentration; DARPin, designed ankyrin repeat protein; EC₅₀, half maximal effective concentration; FDA, Food and Drug Administration; IMAC, Immobilized metal affinity chromatography; TcdB, C. difficile toxin B.

Fig 6. In vivo studies.

(A) Mice were IP injected with TcdB (1.5 μg/kg) alone (PBS, n = 10) or together with DLD-4 (0.25 or 2.5 mg/kg, n = 10) or bezlotoxumab (10 mg/kg, n = 10). Mouse survival rate was monitored, and data were analyzed by Kaplan–Meier survival analysis with log-rank test of significance; p = 0.04 (DLD-4 versus PBS). (B) DLD-4 (5 mg/mouse) was mixed with TcdB (15 μg/mouse) in 100 μL PBS and injected immediately into the cecum of mice (n = 7). The control group received the same dose of TcdB alone (n = 10). Mouse survival was monitored for 3 days, and the data were analyzed by Kaplan–Meier survival analysis; p = 0.349 (TcdB alone versus TcdB + DLD-4). IP, intraperitoneally; TcdB, C. difficile toxin B.

Fig 7. Cryo-EM structure of the TcdB–DLD-4 complex.

(A) Domain organization of TcdB and its interaction with the 2 modules of the DLD-4 DARPin. The binding sites on TcdB for DLD-4 are indicated by the blue and orange triangles for the U3 and 1.4E modules, respectively. (B) Density map of the TcdB–DLD-4 complex with the functional domains and DARPin modules in different colors. (C) The same map but rotated 90° as indicated by the arrow. (D) The model fitted into the map as viewed from the eye cartoon labeled in Panel B. The density map is iso-surfaced at a threshold of 9 σ. (E) Zoom-in of the region labeled within the dashed black box in Panel C to show the linker between DARPins U3 and 1.4E. The density map is iso-surfaced at a threshold of 3.8 σ. APD, autoprocessing domain; CROPS, combined repetitive oligopeptides; cryo-EM, cryo-electron microscopy; DARPin, designed ankyrin repeat protein; GTD, glucosyltransferase domain; TcdB, C. difficile toxin B.

Fig 8. Interactions between TcdB and DLD-4.

(A) Overall model of the complex. (B) Zoom-in view to show the interactions between U3 DARPin and TcdB as viewed in the direction labeled by an eye cartoon in Panel A. (C) Zoom-in view to show the interactions between the DARPin 1.4E and TcdB at the region as labeled by red dashed box in Panel A. APD, autoprocessing domain; CROPS, combined repetitive oligopeptides; DARPin, designed ankyrin repeat protein; GTD, glucosyltransferase domain; TcdB, C. difficile toxin B.

Fig 9. Charge contribution of binding between U3 and TcdB_VPI.

Relative binding of U3 to different immobilized mutants of FBD (A) or different U3 mutants to immobilized TcdB_VPI (B) was determined using ELISA. In both cases, serially diluted WT or mutant U3 proteins were added to microtiter plates coated with 4 μg/mL of the TcdB_VPI (A) or FBD variants (B). Results are the average of 3 independent experiments, and the error bars represent the standard deviation. (C) Relative binding of FZD2-Fc to immobilized TcdB_VPI. The ELISA plates were coated with TcdB_VPI followed by treatment with 1 nM of FZD2-Fc alone or in combination with 250 nM of WT or different U3 mutants. The error bars represent mean deviation from 2 independent experiments. ELISA, enzyme-linked immunosorbent assay; FBD, frizzled binding domain; FZD2-Fc, Fc-tagged extracellular domain of FZD2; TcdB, C. difficile toxin B; WT, wild type.

Fig 10. DARPin DLD-4 blocks the interaction between TcdB and its cellular receptors.

ELISA plates were coated with TcdB_VPI followed by treatment with the 1 nM of the extracellular domains of CSPG4 (A) or FZD2 (B) alone or in mixture with 250 nM of the indicated DARPins. The amount of TcdB-bound CSPG4 and FZD2 was detected using the respective antibodies. Data were normalized to each receptor binding to TcdB in the absence of DARPins (“No DARPin”). Both 1.4E and DLD-4 blocked the interaction between CSPG4 and TcdB, whereas U3 had no significant effect on the TcdB–CSPG4 interaction. Similarly, U3 and DLD-4 significantly blocked the interaction between FZD2 and TcdB but not 1.4E. Error bars represent the standard deviation of 3 independent experiments done in duplicate. *p < 0.005, t test. CSPG4, chondroitin sulfate proteoglycan 4; DARPin, designed ankyrin repeat protein; ELISA, enzyme-linked immunosorbent assay; FZD2, frizzled class receptor 2; TcdB, C. difficile toxin B.