The ternary complex of the TcdA-A2 fragment bound to A20 and A26 (PDB 4NC1) indicated that the N-terminus of A26 was located 30 Å away from the C-terminus of A20. Models of linkers containing a repeating Gly-Ser dipeptide motif with backbone geometry similar to that seen in antiparallel β-strands were generated using PyMOL (Version 2.0, Schrödinger LLC). A linker with six (GS) dipeptides appeared to be sufficient to bridge the gap observed in the 4NC1 crystal structure, but the presence of disordered residues at the N- and C-termini of the V_HH proteins introduced a modest degree of uncertainty into the design. As a result, a series of constructs with a small range of linker lengths were generated.

V_HHs A20, A26, and B39 [anti-TcdB control V_HH; (Hussack et al., 2018)] were expressed in Escherichia coli TG1 cells as described previously (Hussack et al., 2011). The optimized DNA sequences encoding A20-A20, A26-A26, A26-A20, A20-A26 (Hussack et al., 2011), and B39-B39 (Hussack et al., 2018) dimers were synthesized and subcloned into pTT5 (Durocher et al., 2002) expression plasmid (Thermo Fisher, Ottawa, Canada). The two monomer units in each dimer are separated by a (Gly-Ser)₆ linker sequence. Three additional A20-A26 constructs with Gly₄Ser, (Gly₄Ser)₂, and (Gly₄Ser)₃ amino acid linkers, hereafter referred to as A20-A26 (G₄S), A20-A26 (G₄S)₂, and A20-A26 (G₄S)₃, were also subcloned into pTT5. All V_HH dimers, the anti-TcdA IgG1 reference antibody actoxumab (CDA1) and the anti-TcdB IgG1 reference antibody bezlotoxumab (MDX1388) were expressed in HEK293-6E cells as described (Hussack et al., 2018). Five days after the transfection, supernatants were harvested by centrifugation at 3,000 × g for 10 min and filtered through 0.2-μm filter (Millipore, Etobicoke, Canada), dialyzed against phosphate-buffered saline (PBS) overnight at 4°C, and then the His₆-tagged dimers were purified by immobilized metal-ion affinity chromatography using a HisTrap column (Cytiva Life Sciences, Mississauga, Canada) and an AKTÄ™ FPLC (Cytiva Life Sciences). CDA1 and MDX1388 were purified by protein A affinity chromatography. The eluted proteins were buffer exchanged into PBS using Amicon devices, sterilized through 0.2-μm filtration and stored at −80°C. Antibody concentrations were determined from their respective molar extinction coefficients and A₂₈₀ absorbance, measured on a NanoDrop 3300 fluorospectrometer (Thermo Fisher). Protein purity was assessed by reducing and non-reducing sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE).

Purified proteins (400 μg in PBS) were loaded onto a Superdex™ 75 Increase 10/30 GL (Cytiva Life Sciences) column, at a flow rate of 0.5 mL/min, controlled by an AKTÄ™ FPLC (Cytiva Life Sciences). To determine the size of each construct, a standard curve was generated using the Bio-Rad™ Size-Exclusion Chromatography Standard (BioRad, Hercules, CA) and apparent molecular masses (M_apps) of antibodies were calculated by interpolation using their elution volumes (V_es). Size-exclusion chromatography (SEC) chromatograms were normalized as described (Kim et al., 2012).

Nunc-immuno microtitre plate wells with Maxisorp surface (Thermo Fisher) were coated in triplicates with 30 ng/well of full-length TcdA (List Biological Laboratories, Inc., Campbell, CA) in 0.05 M carbonate–bicarbonate buffer pH 9.6, and incubated overnight at 4°C. Wells were blocked with 1% (w/v) bovine serum albumin in PBS for 1 h at room temperature, washed with PBS/0.05% (v/v) Tween 20 and then incubated for 1 h with decreasing concentrations of His₆-tagged V_HH constructs in PBS containing 0.2% bovine serum albumin and 0.05% Tween 20 (PBS-TB). Following washing, the binding to TcdA was probed by incubating the wells for 1 h at room temperature with 0.2 ng/mL polyclonal rabbit anti-His₆ antibody conjugated to horseradish peroxidase (Bethyl Laboratories, Montgomery, TX) in PBS-TB. Wells were washed and incubated at room temperature for 15 min with 100 μL TMB peroxidase substrate solution (KPL, Gaithersburg, MD). Then, 50 μL of 1 M H₂SO₄ was added to stop enzymatic reactions and absorbance was read at 450 nm on a Multiskan Enzyme-linked immunosorbent assay (ELISA) plate reader (Thermo Fisher). Data analysis was performed using Prism software version 8.3 (GraphPad Software, Inc., La Jolla, CA).

All surface plasmon resonance (SPR) experiments were performed on a Biacore 3000 instrument (Cytiva Life Sciences) at 37°C in HBS-EP running buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.005% (v/v) surfactant P20). Recombinant TcdA-A2 fragment (Greco et al., 2006), encompassing the C-terminal CROPs domain of TcdA (aa 2,556–2,710) from C. difficile reference strain 10463, was amine coupled to a CM5 sensor chip at pH 4.5 in acetate buffer using standard methods recommended by the manufacturer (Cytiva Life Sciences) to create a high-density toxin A surface with ~8,500 resonance units immobilized. An ethanolamine-blocked flow cell served as a reference surface. The flow rate for all experiments was 40 μL/min and SEC-purified antibodies were injected for 120 s at a single concentration that varied depending on the antibody (CDA1: 50 nM; A26 and B39: 10 nM; A20: 5 nM; A20-A20, A26-A26, A20-A26 and A26-A20: 1 nM). Antibody dissociation was followed for 300 s for the monomeric antibodies (A20, A26, and B39) and 3,600 s for the bivalent antibodies (A20-A20, A26-A26, A20-A26, A26-A20 and CDA1). Complete regeneration of the TcdA-A2 surface was achieved with a 6 s pulse of 10 mM glycine, pH 2.0, for all antibodies except CDA1 (12 s pulse of 5 mM NaOH), all at a flow rate of 100 μL/min. Off-rates (k_ds, s⁻¹) were determined by fitting the dissociation phase (1,500–3,500 s for bivalent antibodies, 150–300 s for A20, 130–200 s for A26) of each sensorgram to a separate k_d 1:1 binding model using the BIAevaluation v4.1 software (Cytiva Life Sciences). With one exception (A20-A26), all antibodies achieved the minimum 5% dissociation required to accurately report an off-rate (Katsamba et al., 2006).

Vero cells (CCL-81) were obtained from ATCC (Manassas, VA) and cultured according to ATCC’s instructions in 96 well microtiter plates (Nunc) at 2 × 10⁴ cells/well in MEM media (Gibco) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (Gibco) at 37°C in 5% CO₂. The neutralization activity of antibodies was determined by co-incubation with 80 ng/mL (260 pM) of full-length TcdA, with antibody and toxin added simultaneously to the cells. After 72 h of incubation, the cell viability was quantified with the Cell Proliferation Assay Reagent WST-1 (Roche Diagnostics, Laval, Canada) according to the manufacturer’s instructions. Briefly, the media was replaced with 100 μL of MEM (without fetal bovine serum) containing 10% of WST-1, incubated for 40 min at 37°C in 5% CO₂ and finally the absorbance read at 450 nm. The neutralizing activity was calculated as % inhibition:

A450_test is the absorbance of cells incubated with TcdA and varying concentrations of antibodies;

A450_low is the absorbance of cells incubated only with TcdA (0% inhibition); and

A450_high is the absorbance of cells incubated only with media (100% inhibition).

Monomeric and dimeric V_HHs were mixed with TcdA-A2 at 1:1 molar ratios in PBS at final concentrations of 1.91 mg/mL and incubated at 4°C overnight. Control experiments which included monomeric V_HHs alone (0.91 mg/mL), dimeric V_HHs alone (0.91 mg/mL) and TcdA-A2 alone (1 mg/mL) were performed under the same conditions as the V_HH-TcdA-A2 mixtures. UPLC-SEC analysis of protein samples was performed on a BEH200 SEC column (4.6 × 150 mm, 1.7 μm particle column, Waters, Milford, MA) using a Waters H-Class Acquity UPLC system equipped with a diode array detector, a Wyatt MiniDawn multiangle light scattering (MALS) detector, and a Wyatt Optilab T-rEX refractive index detector (Wyatt Technology, Santa Barbara, CA). The mobile phase was PBS (HyClone SH30028.02, Cytiva Life Sciences) containing 0.02% (v/v) polysorbate 20 at a flow rate of 0.4 mL/min. The column temperature was 30°C. Molar masses were determined using Astra software version 6.1.7.17 (Wyatt Technology) using either absorbance at 280 nm (A₂₈₀) or refractive index (RI) as the concentration measure. Similar results were obtained using either A₂₈₀ or RI, but band broadening reduced resolution in the RI signal so masses determined using the A₂₈₀ signal are reported.

We previously solved the X-ray structures of two C. difficile TcdA-binding V_HHs, A20 (K_D = 2 nM) and A26 (K_D = 12 nM), in complex with fragments from the CROPs domain (formerly referred to as the RBD, receptor binding domain) of TcdA (Hussack et al., 2011; Murase et al., 2014). Because in the ternary complex of the TcdA-A2 fragment bound to a single molecule of A20 and a single molecule of A26 (PDB 4NC1) the N-terminus of A26 was observed to be located 30 Å away from the C-terminus of A20, we hypothesized that a short linker peptide could be used to fuse the two V_HH domains together in a recombinant fusion protein (Figure 1). Models of linkers containing a repeating Gly-Ser dipeptide motif were generated with backbone geometry similar to that seen in antiparallel β-strands. A linker with six Gly-Ser dipeptides (GS)₆ appeared to be sufficient to bridge the gap observed in the 4NC1 crystal structure, but the presence of disordered residues at the N- and C-termini of the V_HH proteins indicate that the ideal lengths and geometric details of linkers is challenging to model precisely. As a result, in addition to A20-A26 with a (GS)₆ linker, a series of constructs with a range of linker lengths were generated (Figure 1B). These included A20-A26 (G₄S), A20-A26 (G₄S)₂, and A20-A26 (G₄S)₃, which possessed linkers of shorter [(G₄S)], similar [(G₄S)₂] and longer [(G₄S)₃] distances than the (GS)₆ construct. The crystal structures also clearly indicated that the polarity of the arrangement of V_HH domains (i.e., whether the A20 V_HH domain or the A26 V_HH domain is N-terminal to the other V_HH domain in the fusion protein) would likely be critical, as the reverse orientation of V_HH domains would not be expected to allow for the simultaneous binding of both V_HH domains to the same molecule of TcdA. To test this prediction, the A26-A20 fusion protein was generated with the reverse polarity (i.e., A26 at the N-terminus and A20 at the C-terminus). To evaluate whether the tethering of two V_HH domains generates a non-specific multivalency effect, two other fusion proteins containing two A20 V_HH domains (A20-A20) and two A26 V_HH domains (A26-A26), each tethered by the (GS)₆ linker, were also generated.

While monomeric V_HHs were expressed in the periplasm of E. coli, the dimeric V_HHs were expressed in mammalian HEK293-6E cells to obtain higher expression yields. Nonetheless, in contrast to A20-A26 (G₄S) and A20-A26 (G₄S)₂, which could be expressed in high yields similar to the dimers with the (GS)₆ linker (Table 1), the A20-A26 (G₄S)₃ construct was surprisingly only expressed at very low yields despite repeated attempts, excluding its full analysis in the current study. The proteins were purified by immobilized metal-ion affinity chromatography and ran as single bands of the expected molecular masses on SDS-PAGE under reducing and non-reducing conditions (Figure 2A). Control proteins (V_HH B39, V_HH-V_HH B39-B39, mAbs CDA1 and MDX1388) were expressed in E. coli or HEK293-6E cells and purified by immobilized metal-ion affinity chromatography or protein A affinity chromatography (data not shown). Purified yields of A20 and A26 were in the range of 15–20 mg/L, and yields of the four dimeric V_HHs ranged from 80–114 mg/L (Table 1). The SEC profiles of all V_HHs and V_HH dimers produced single, monodispersed peaks devoid of aggregates (100% “monomeric”) with V_es consistent for a V_HH monomer to dimer transition and relative to protein standards (Figure 2B; Table 1).

Binding of V_HHs and V_HH-V_HHs to immobilized TcdA was demonstrated by ELISA (Figure 2C; Table 1). The four dimeric constructs, A20-A20, A26-A26, A20-A26 and A26-A20, showed the strongest binding to TcdA with similar apparent EC₅₀s of 32–47 pM. In contrast, monomeric A20 and A26 demonstrated significantly lower TcdA binding, with EC₅₀s of 194 pM (A20) and 14.6 nM (A26). The off-rates (k_ds, s⁻¹) of all constructs were determined by SPR by flowing V_HHs, V_HH-V_HHs or control CDA1 mAb over amine coupled TcdA for long dissociation times at 37°C (Figure 2D; Table 1). Consistent with the ELISA results, A20 and A26 dissociated rapidly with k_ds of 2.5 × 10⁻² s⁻¹ and 7.5 × 10⁻³ s⁻¹, respectively. All dimeric constructs clearly showed avid binding and demonstrated very slow dissociation rates as expected; however, subtle differences were evident. Of the dimeric proteins, A26-A26 dissociated the fastest followed by A20-A20 and A26-A20, which had similar k_ds to the CDA1 IgG benchmark. The dissociation of A20-A26 was very slow (6.9 × 10⁻⁶ s⁻¹), which is at the instrument limit of detection, achieving only 4% dissociation after an hour and corresponding to an estimated half-life of 28.1 h (Table 1).

We compared the TcdA neutralizing potency of the various V_HH constructs and controls in a 72 h TcdA inhibition assay using Vero cells. Initially, toxin neutralization was performed on monomers, dimers and the CDA1 benchmark mAb at a concentration of 100 nM (Figure 3A). The A20-A26 construct was a superior neutralizer in comparison to other monomers (A20, A26, and A20 + A26), dimers (A20-A20, A26-A26, and A26-A20) and CDA1, reaching nearly 100% TcdA neutralization. Moreover, while A26 demonstrated significant TcdA inhibition, A20 did not show any despite its higher affinity for TcdA, underlining the critical role epitope location plays in TcdA neutralization. Next, we performed neutralization experiments with various antibody concentrations (Figure 3B) to determine IC₅₀, IC₉₉ and maximum toxin inhibition values (Table 2). Antibody titration curves demonstrated a dramatic shift in neutralizing potency of A20-A26 relative to the other mono- and biparatopic constructs tested. The IC₅₀ of A20-A26 (0.16 nM) was far superior to A26-A20 (49.7 nM), A20-A20 (174.5 nM), A26-A26 (39 nM), and CDA1 (30 nM). The IC₉₉ of A20-A26 (0.27 nM) was even more dramatic in relation to comparators A26-A20 (7,866 nM), A20-A20 (2,123 nM), A26-A26 (1,189 nM) and CDA1 (3,342 nM). In particular, relative to the reverse orientation control A26-A20 and CDA1, A20-A26 outperformed these antibodies by 29,000- and 12,500-fold, respectively. Antibody efficacy, reported as the maximum level of TcdA inhibition achieved, was also superior for the A20-A26 construct reaching 94% compared to A26-A20 (85%), A20-A20 (85%), A26-A26 (89%) and CDA1 (78%).

Given the potency of A20-A26, three additional constructs were designed with varying linker lengths of (G₄S), (G₄S)₂, and (G₄S)₃ separating the two V_HHs. Neutralization experiments comparing the original A20-A26 construct containing the (GS)₆ linker with the various G₄S linkers revealed essentially identical neutralizing potency (Figure 3C). This is reflected in similar IC₅₀ (0.16 nM, 0.22 nM, 0.23 nM, and 0.29 nM) and IC₉₉ (0.27 nM, 0.54 nM, 0.42 nM, and 0.46 nM) values for A20-A26, A20-A26 (G₄S), A20-A26 (G₄S)₂, and A20-A26 (G₄S)₃, respectively (Table 2). The antibody efficacy of A20-A26 (94%) slightly exceeded constructs with shorter linkers A20-A26 (G₄S) (85%) and A20-A26 (G₄S)₂ (88%), and was essentially the same as the construct with a longer linker A20-A26 (G₄S)₃ (94%) (Table 2).

To gain further insight into the mechanism of action underlying the high neutralization potencies observed with A20-A26 dimers, several monomeric and dimeric V_HHs were incubated with TcdA-A2 in solution at 1:1 molar ratios for formation of antibody-TcdA-A2 complexes. The toxin/antibody mixtures were then subjected to SEC-MALS analysis to obtain observed molecular masses (M_obss) and retention times (T_rs) of free (TcdA-A2 [Ag], antibody [Ab]), and complexed (Ag.Ab) species. M_obs and T_r values, complemented with neutralization data, were used to determine the types of binding complexes (“species” and “modes”) formed (Figure 4; Table 3).

As expected, the individual unmixed TcdA-A2 and V_HH samples treated under the same conditions as the Ag-Ab mixes had M_obss very close to the theoretical M_r values, indicating the lack of significant homotypic interactions for each individual protein. The M_obss for A20:TcdA-A2 and A26:TcdA-A2 complexes were consistent with a 1:1 TcdA-A2-V_HH complex type (Ag.Ab) and the previous SEC data obtained for these complexes (Murase et al., 2014). The extent of complex formation was higher for A20 (80%) than for A26 (69%), consistent with its higher affinity for TcdA-A2 (Hussack et al., 2011). Homodimeric A20-A20 gave a major complex (92%) of (Ag)₂.Ab type and a minor complex (8%) of Ag.Ab type. For the A26-A26 homodimer, however, it was not clear from the M_obss, whether the complex was of Ag.Ab or (Ag)₂.Ab type for the major SEC species (76%) or of Ag.Ab or Ag type for the minor SEC species (24%) due to intermediary M_obs values relative to expected M_rs. Nonetheless, all possible complexes point to a lack of binding avidity. Thus, as in the case of A20 vs A26 V_HH, a higher % of complex formation for A20-A20 may be due to a higher intrinsic affinity of A20 for TcdA-A2. Moreover, the lack of binding avidity explains why A26 and A26-A26 have similar potencies, and in the case of A20-A20, it suggests the acquired neutralization capability of A20 upon homodimerization may be due to increased steric hindrance.

A20-A26 formed predominantly an Ag.Ab type complex (91%), and along with its ultra-potent neutralization capability, indicates that both V_HH domains in A20-A26 simultaneously engage with a single molecule of TcdA-A2 in a biparatopic fashion with 1:1 stoichiometry (Mode 1), as predicted from the original design based on the crystal structure (Figure 4). Conversely, A26-A20 did not appear to form any biparatopic complexes, a result that is also consistent with the crystal structure predictions. It formed two major complexes at 55% and 43% with significantly different T_rs but similar M_obss, indicative of (Ab)₂.(Ag)₂ tetrameric (Modes 2, 3, and 4) and (Ab)₂.Ag and/or Ab.(Ag)₂ trimeric complexes (Modes 5 and 6), respectively. Of the three tetrameric possibilities, one can adopt a cross-biparatopic binding arrangement (Mode 2), while the other two arrange in such a manner that excludes binding avidity (Modes 3 and 4, Figure 4). The far weaker neutralization potency of the A26-A20 construct relative to the A20-A26 biparatopic construct, on the one hand, and its similar neutralization capability to monomeric and homodimeric A20/A26 constructs, on the other, indicate that only one V_HH domain in A26-A20 is able to bind to a single epitope in TcdA-A2 within any one particular molecular complex, thus excluding the possibility of Mode 2 binding. Interestingly, A20-A26 (G₄S) and A20-A26 (G₄S)₂ which had the same V_HH-V_HH orientations as A20-A26, formed complexes with M_obss consistent with Mode 1 and Mode 2 biparatopic Ab.Ag engagement. Molecular models based on the crystal structures indicate that the much shorter linker found in A20-A26 (G₄S) when compared with A20-A26 would likely prevent the formation of complexes where both of the A20 and A26 V_HH domains are able to bind to a single molecule of TcdA-A2 simultaneously. This likely explains why only 53% of the complexes detected for A20-A26 (G₄S) show 1:1 stoichiometry (Ab.Ag; Mode 1), whereas 47% of the complexes show 2:2 stoichiometry ((Ab)₂.(Ag)₂; Mode 2) based on the M_obs and T_r parameters. In comparison, 91% of the complexes formed by A20-A26 and A20-A26 (G₄S)₂, which both have linkers with similar lengths that are expected to allow both V_HH domains to bind simultaneously to a single molecule of TcdA-A2, show 1:1 stoichiometry (Ab.Ag; Mode 1). Only up to 9% of the complexes have 2:2 stoichiometry ((Ab)₂.(Ag)₂; Mode 2). A20-A26 (G₄S)₃ was not analyzed by SEC-MALS, because it could not be expressed in sufficient quantities. However, it is expected to possess a similar binding mode to that observed for A20-A26 and A20-A26 (G₄S)₂, because the longer linker length would be expected to allow both V_HH domains to bind to a single molecule of TcdA-A2 simultaneously.