Mechanism of action and epitopes of Clostridium difficile toxin B-neutralizing antibody bezlotoxumab revealed by X-ray crystallography

Abstract

The symptoms of Clostridium difficile infections are caused by two exotoxins, TcdA and TcdB, which target host colonocytes by binding to unknown cell surface receptors, at least in part via their combined repetitive oligopeptide (CROP) domains. A combination of the anti-TcdA antibody actoxumab and the anti-TcdB antibody bezlotoxumab is currently under development for the prevention of recurrent C. difficile infections. We demonstrate here through various biophysical approaches that bezlotoxumab binds to specific regions within the N-terminal half of the TcdB CROP domain. Based on this information, we solved the x-ray structure of the N-terminal half of the TcdB CROP domain bound to Fab fragments of bezlotoxumab. The structure reveals that the TcdB CROP domain adopts a β-solenoid fold consisting of long and short repeats and that bezlotoxumab binds to two homologous sites within the CROP domain, partially occluding two of the four putative carbohydrate binding pockets located in TcdB. We also show that bezlotoxumab neutralizes TcdB by blocking binding of TcdB to mammalian cells. Overall, our data are consistent with a model wherein a single molecule of bezlotoxumab neutralizes TcdB by binding via its two Fab regions to two epitopes within the N-terminal half of the TcdB CROP domain, partially blocking the carbohydrate binding pockets of the toxin and preventing toxin binding to host cells.

Keywords: Antibody; Bacterial Toxin; Crystal Structure; Drug Action; Epitope Mapping.

FIGURE 1.

C. difficile toxins and toxin fragments. A, domain structures of TcdA and TcdB showing the putative receptor binding (CROP) domains at the C terminus. B, TcdB CROP domain showing SRs (green) and LRs (gray) and highlighting the four peptides used in this study, B1, B2, B3, and B4. The first of four CROP units, consisting of one LR flanked by three SRs on the N-terminal side and two SRs on the C-terminal side, is highlighted in red.

FIGURE 2.

Identification of the bezlotoxumab-binding regions within the TcdB CROP domain. A, Coomassie Blue-stained polyacrylamide gel and Western blot showing binding of bezlotoxumab to intact TcdB and to peptides B1, B2, and B3 but not to peptide B4 or to the TcdB catalytic domain (GTD). Molecular masses (kDa) of markers are indicated on the left. B, binding of bezlotoxumab to full-length TcdB, B1, B2, and B3, but not B4, as assessed by TdF and SPR. C, summary of HDX-MS experiments showing regions of the CROP domain that are protected from deuteration in the presence of bezlotoxumab and extent of protection. D, details of the HDX-MS analysis showing deuteration levels (color key at lower right) for peptide B1 alone (top, −B) and in the presence of bezlotoxumab (bottom, +B). Each box represents a distinct peptide identified by MS and is subdivided into four time points: 30, 100, 300, and 1000 s from top to bottom (see “Experimental Procedures”). The numbering used is that of full-length TcdB. The differences between deuteration levels in the presence versus the absence of bezlotoxumab were averaged over all time points, and significant differences are reported in Fig. 2C.

FIGURE 3.

Crystal structure of the N-terminal half of the TcdB CROP domain bound to two bezlotoxumab Fab fragments. A, side view showing parallel binding of the two Fab fragments (Fab1 and Fab2) to their respective epitopes, E1 and E2. LRs are shown in gray and SRs in green. Fab heavy chains are shown in yellow (Fab1) and peach (Fab2) and light chains in pink (Fab1) and purple (Fab2). B, bottom-up view showing the Fab fragments bound perpendicularly to the curvature of the CROP domain. Residues of the CROP domain that directly interact with the heavy chains (yellow and peach) or light chains (pink and purple) of the Fab fragments are highlighted on the CROP surface. C, partial sequence of the light chains (Lc) and heavy chains (Hc) of bezlotoxumab, showing the six complementarity-determining regions (identified using the Molecular Operating Environment software from Chemical Computing Group); residues that interact with peptide B2 are highlighted in gray. D, sequence alignment of the two bezlotoxumab epitope regions (E1 and E2) and the two non-binding homologous regions (E3 and E4) of the TcdB CROP domain. Bezlotoxumab-interacting residues in E1 and E2 (and corresponding residues in E3 and E4) are highlighted in gray, with conserved and non-conserved substitutions (compared with E1) shown in green and red, respectively. Regions protected by bezlotoxumab in the HDX-MS experiment (Fig. 2c) are underlined, and residues putatively involved in carbohydrate binding are identified by asterisks. SRs and LRs are shown as green and gray boxes, respectively.

FIGURE 4.

Bezlotoxumab epitopes and interactions with Fab residues. A, interacting residues within E1 (green) and Fab1 (yellow and pink for heavy and light chains, respectively). B, overlap of the Fab-interacting residues within E1 (green) and E2 (blue). C, structural conservation of a key salt bridge between Arg-100^Fab/hc Glu residues in E1 (green) and E2 (blue). D, role of Gly-1963^E1 (Ala-2095^E2) in enabling key interactions between Ser-1900^E1 (Asn-2031^E2) and Trp-33^Fab/hc, Tyr-52^Fab/hc, and Arg-99^Fab/hc.

FIGURE 5.

Surface representation of peptide B2 with bezlotoxumab epitopes and putative carbohydrate-interacting residues. Regions that interact with bezlotoxumab (as identified by x-ray crystallography) are shown in yellow on the surface of B2. Residues that are protected from deuteration in the HDX-MS analysis are shown in red. Residues putatively involved in carbohydrate binding are shown in blue. Overlapping regions are shown in orange (x-ray and HDX-MS), green (x-ray and carbohydrate binding), purple (HDX-MS and carbohydrate binding), or brown (x-ray, HDX-MS, and carbohydrate binding).

FIGURE 6.

Inhibitory effect of bezlotoxumab on activity and binding of TcdB in Vero cells. A, neutralization of the cytotoxic effects of purified TcdB by bezlotoxumab. The concentration of TcdB used (10 pg/ml) was previously determined to cause a ∼95% decrease in cell viability in this assay. B, Western blots of Vero cell membranes following incubation of cells with 50 ng/ml (lanes 3–5) or 100 ng/ml (lanes 6–8) TcdB, in the absence and presence of bezlotoxumab or actoxumab (200 μg/ml). Binding experiments were carried out at 37 °C in the presence of the endocytosis inhibitor chlorpromazine fixed at 14 μm. Lane 1, 1 ng of purified TcdB (no membranes); lane 2, membranes from cells incubated without TcdB; lanes 3 and 6, membranes from cells incubated with TcdB in the absence of bezlotoxumab; lanes 4 and 7, membranes from cells incubated with TcdB in the presence of bezlotoxumab; lanes 5 and 8, membranes from cells incubated with TcdB in the presence of actoxumab. Levels of TcdB are shown in the top panel, and levels of cadherin (loading control) are shown in the bottom panel. C, quantification of membrane-bound TcdB (normalized to the 1 ng of TcdB control lane and to cadherin band densities) following incubation of Vero cells in the presence of 50 or 100 ng/ml TcdB alone (black bars) or in the presence of bezlotoxumab (white bars) or actoxumab (hatched bars). Values are averages ± S.D. of two independent determinations.

FIGURE 7.

SEC-MALLS analysis of bezlotoxumab, B1, and bezlotoxumab-B1 immune complexes. A, light scattering and molecular mass distributions of complexes formed at various bezlotoxumab/B1 molar ratios (1:0 (magenta), 0:1 (olive green), 1:5 (bright green), 1:1 (red), and 5:1 (navy blue)) as a function of elution time. B, average detected molecular mass of each complex. The molecular masses of the B1-bezlotoxumab complexes are approximately equal to the sum of B1 alone and bezlotoxumab alone, indicating a 1:1 stoichiometry.

FIGURE 8.

Model of the TcdB CROP domain bound to a full-length molecule of bezlotoxumab. The C-terminal (left) half of the CROP domain was modeled based on the structure of the B2 peptide. The Fc region of bezlotoxumab is based on a published high-resolution structure of human IgG1. The four putative carbohydrate binding regions are shown in dark blue within the otherwise green CROP domain.

FIGURE 9.

Alignment of the structures of various CROP-antibody complexes. The backbones of the CROP domains of TcdA and TcdB were used for the alignment. Binding of nanobodies A20.1 (green) and B39 (white) as well as of Fab2 (yellow and cyan) and Fab1 (pink and purple) is shown, as is the TcdA carbohydrate ligand α-Gal-(1,3)-β-Gal-(1,4)-β-GlcNAcO(CH₂)₈CO₂CH₃.