Structure-guided design of a potent Clostridiodes difficile toxin A inhibitor

Abstract

Crystal structures of camelid heavy-chain antibody variable domains (V_HHs) bound to fragments of the combined repetitive oligopeptides domain of Clostridiodes difficile toxin A (TcdA) reveal that the C-terminus of V_HH A20 was located 30 Å away from the N-terminus of V_HH A26. Based on this observation, we generated a biparatopic fusion protein with A20 at the N-terminus, followed by a (GS)₆ linker and A26 at the C-terminus. This A20-A26 fusion protein shows an improvement in binding affinity and a dramatic increase in TcdA neutralization potency (>330-fold [IC ₅₀]; ≥2,700-fold [IC ₉₉]) when compared to the unfused A20 and A26 V_HHs. A20-A26 also shows much higher binding affinity and neutralization potency when compared to a series of control antibody constructs that include fusions of two A20 V_HHs, fusions of two A26 V_HHs, a biparatopic fusion with A26 at the N-terminus and A20 at the C-terminus (A26-A20), and actoxumab. In particular, A20-A26 displays a 310-fold (IC ₅₀) to 29,000-fold (IC ₉₉) higher neutralization potency than A26-A20. Size-exclusion chromatography-multiangle light scattering (SEC-MALS) analyses further reveal that A20-A26 binds to TcdA with 1:1 stoichiometry and simultaneous engagement of both A20 and A26 epitopes as expected based on the biparatopic design inspired by the crystal structures of TcdA bound to A20 and A26. In contrast, the control constructs show varied and heterogeneous binding modes. These results highlight the importance of molecular geometric constraints in generating highly potent antibody-based reagents capable of exploiting the simultaneous binding of more than one paratope to an antigen.

Keywords: Clostridiodes difficile; VHH; biparatopic; inhibitor; nanobody; single-domain antibody; toxin.

Figure 1

Model of the A20-A26 fusion protein bound to the CROPs domain of Clostridioides difficile TcdA. (A) The structure of full-length C. difficile TcdA was generated by superimposing the crystal structure of the N-terminal 1832 amino acid residues of TcdA (4R04) (Chumbler et al., 2016) onto the crystal structure of TcdB (6OQ5) (Chen et al., 2019). To model the CROP domain missing from the TcdA structure, we made the assumption that the orientation of the CROP domain relative to the N-terminal domains is similar in the two toxins. By superimposing the N-terminal short repeat of a model of the TcdA CROP domain (Ho et al., 2005; Pruitt et al., 2010; Pruitt and Lacy, 2012) onto the N-terminal short repeat in the CROP domain of TcdB (6OQ5), a model of full-length TcdA was generated. The resulting model shows similar features to the low resolution cryo-EM envelopes of full-length TcdA (Pruitt et al., 2010; Pruitt and Lacy, 2012). The structure of the complex formed between A20-A26 and TcdA was generated by modeling the structure of a (GS)₆ linker in an extended antiparallel β-strand conformation using PyMOL and positioning this linker between the C-terminus of the A20 V_HH and the N-terminus of the A26 V_HH observed in the crystal structure of the complex formed between A20, A26 and the TcdA-A2 fragment (4NC1) (Murase et al., 2014). TcdA is shown in cartoon ribbon and semi-transparent surface representations. A20, A26 and the A20-A26 V_HH domains are drawn in cartoon representation. Each polypeptide chain is colored according to the colors of the rainbow, starting from blue at the N-terminus to red at the C-terminus. RBD, receptor binding domain; GT, glucosyl transferase. (B) Schematic representation of the V_HH constructs generated in this work.

Figure 2

Biophysical characterization of anti-TcdA V_HH constructs. (A) Representative SDS-PAGE analysis of V_HH monomers and dimers is shown under reducing (+DTT [dithiothreitol]) and non-reducing (−DTT) conditions. M, protein molecular mass standards AU, absorbance unit. (B) SEC analysis of the antibodies with arrows indicating the elution positions of ovalbumin (44 kDa) and myoglobulin (17 kDa) protein standards. (C) Titration of the antibodies against full-length TcdA by ELISA. B39-B39 (a dimer of the anti-TcdB V_HH B39 with a (GS)₆ linker) is used as a negative control. Error bars indicate standard deviation (SD) of three technical replicates. (D) Representative SPR sensorgrams comparing off-rate of V_HHs, V_HH dimers and CDA1 mAb binding to immobilized TcdA-A2 fragment at 37°C with 5min or 60min dissociations. Black lines represent raw data points; red lines are fits to a separate k_d 1:1 binding model. RU, resonance unit.

Figure 3

In vitro neutralization of TcdA cytotoxicity. (A) The panel of antibodies at 100 nM were incubated with TcdA for 72 h and the percentage of live Vero cells were quantified by the colorimetric proliferation reagent WST-1. MDX1388 (anti-TcdB mAb) and B39-B39 (a dimer of the B39 V_HH with a (GS)₆ linker) were negative control antibodies. (B) The most potent antibodies were further titrated to evaluate their performance based on potency (IC₅₀ and IC₉₉) and efficacy (maximum inhibition) values. (C) Comparison of neutralizing potency of the structure-guided A20-A26 construct containing a 12-amino acid linker, to A20-A26 constructs containing 5, 10, and 15 amino acid linkers. In all assays the TcdA concentration was 260 pM and antibody concentrations as described. Error bars indicate standard deviation (SD) of three technical replicates.

Figure 4

Binding modes and UPLC-SEC chromatograms of TcdA-A2-V_HH complexes. (A) Binding modes for the interaction of TcdA-A2 and heterodimeric V_HHs in solution were determined based on molecular mass (M_obs), retention time (T_r) and TcdA neutralization data. (B) Chromatograms are shown for TcdA-A2 (red lines), the various antibodies (blue lines) and the 1:1 molar mixture of the two proteins (green lines). The M_obss of the peak in each chromatogram was estimated from MALS UV. Schematic representations of TcdA-A2, V_HH or V_HH-V_HH and complexes of the two proteins are shown for each peak. AU, absorbance unit.