Development of Nanobodies as Theranostic Agents against CMY-2-Like Class C β-Lactamases

Abstract

Three soluble single-domain fragments derived from the unique variable region of camelid heavy-chain antibodies (VHHs) against the CMY-2 β-lactamase behaved as inhibitors. The structure of the complex VHH cAb_CMY-2(254)/CMY-2 showed that the epitope is close to the active site and that the CDR3 of the VHH protrudes into the catalytic site. The β-lactamase inhibition pattern followed a mixed profile with a predominant noncompetitive component. The three isolated VHHs recognized overlapping epitopes since they behaved as competitive binders. Our study identified a binding site that can be targeted by a new class of β-lactamase inhibitors designed on the sequence of the paratope. Furthermore, the use of mono- or bivalent VHH and rabbit polyclonal anti-CMY-2 antibodies enables the development of the first generation of enzyme-linked immunosorbent assay (ELISA) for the detection of CMY-2 produced by CMY-2-expressing bacteria, irrespective of resistotype.

Keywords: CMY-2 β-lactamase; antibiotic resistance; class C beta-lactamase; nanobodies; new antibiotic scaffold; paratopes; structural biology.

FIG 1

Sequence alignment of VHHs directed against CMY-2. FR, framework; CDR, complementarity-determining region.

FIG 2

Binding characterization of the three selected VHHs by biolayer interferometry. (A to C) Qualitative binding specificity of cAb_CMY-2(250) (A), cAb_CMY-2(254) (B), and cAb_CMY-2(272) (C). Names and classes (in parentheses) of the tested β-lactamases are indicated. (D to F) Quantitative binding measurements of cAb_CMY-2(250) (D), cAb_CMY-2(254) (E), and cAb_CMY-2(272) (F). The experimental data (Δλ) recorded with seven different concentrations were fitted using a 1:1 binding model. The negative control (CTRL −) corresponds to CMY-2 directly loaded onto the biosensor. Each experiment was carried out twice independently.

FIG 3

Competition binding assay between VHHs directed against CMY-2 monitored by BLI. The biotinylated VHH cAb_CMY-2(254) was loaded onto a streptavidin biosensor (SA sensor), while the analyte corresponds to complexes cAb_CMY-2(250)/CMY-2, cAb_CMY-2(254)/CMY-2, and cAb_CMY-2(272)/CMY-2 in different molar ratios. The negative control corresponds to the signal recorded when the complex cAb_CMY-2(254)/CMY-2 was directly loaded onto the nonfunctionalized biosensor. All the binding rates were calculated by fitting a simple exponential mathematic model to the first 120 s of the association phase. This experiment was performed twice independently.

FIG 4

Binding properties of rabbit pAbs directed against the β-lactamase CMY-2. (A) Indirect ELISA where the antigens were absorbed on a Maxisorp plate (except for the CTRL −) to investigate the specificity of pAbs. (B) Qualitative binding specificity assay of pAbs for CMY-2 monitored by BLI. (C) Quantitative binding measurements of pAbs for CMY-2 by BLI. The experimental data (Δλ) recorded with five different concentrations were fitted using a 1:1 binding model. The negative control corresponds to anti-CMY-2 pAbs directly loaded onto the biosensor. The BLI experiments were performed twice independently.

FIG 5

Sandwich ELISA on purified enzymes for CMY-2 detection. (A) LOD where cAb_CMY-2(254) was employed as antibody for capture and pAbs for detection (blue solid line) or inversely (blue dashed line). Curves were fitted with equation I in Text S1. The inset includes the Abs₄₅₀ for the CTRL −. The LOD was calculated from an average Abs₄₅₀ of the CTRL − plus three times the standard deviation. They are represented in red solid and dashed lines for VHH used as capture and detection antibody, respectively. (B) Specificity assessment using cAb_CMY-2(254) as capture agent and the pAbs for revelation (gray) or the inverse (gray, pattern). In both experiments, the CTRL − corresponds to the same ELISA without antigen. All averages and standard deviations are results from at least four measurements. CAP, capture; REV, revelation.

FIG 6

Quantitative binding measurements of the monovalent VHH cAb_CMY-2(254) (A) and the bivalent VHH cAb_CMY-2(254)_BIV (B) performed by BLI. The experimental data (Δλ) recorded with five different concentrations were fitted using a 1:1 binding model. The negative control corresponds to VHHs directly loaded onto the biosensor. Experiments were performed twice independently.

FIG 7

Sandwich ELISA for CMY-2 detection using the bivalent VHH cAb_CMY-2(254)_BIV. (A) Limits of detection where the VHH cAb_CMY-2(254)_BIV was used as antibody for capture and pAbs for detection (blue solid line) or the inverse (blue dashed line). The inset includes the Abs₄₅₀ for the negative control. The LOD was calculated from an average Abs₄₅₀ of the CTRL − plus three times the standard deviation. They are represented in red full and dashed lines for VHH used as capture and detection antibody, respectively. (B) Specificity measurement by use of cAb_CMY-2(254)_BIV as antibody for capture and the pAbs for the detection (gray) or inversely (gray, pattern). The negative control corresponds to the ELISA without antigen. All averages and standard deviations are results from at least four measurements.

FIG 8

Residual activity of CMY-2 in complex with cAb_CMY-2(254) for β-lactam ring substrates. Reporter substrates corresponded to 100 μM ampicillin (blue line), 100 μM cephalothin, 100 μM cephaloridin, and 40 μM nitrocefin. Concentrations of CMY-2 used for each reporter substrate were 5 nM, 1 nM, 0.2 nM, and 0.5 nM, respectively. All data were fitted on a one-phase exponential decay equation from the graph prism program with values resulted from two experiments performed independently.

FIG 9

Inhibitory model of CMY-2 activity for nitrocefin by the VHH cAb_CMY-2(254). (A) $K_m^{\mathit{app}}$ and $k_{\mathit{cat}}^{\mathit{app}}$ (parameters derived from the complete hydrolysis of 40 μM nitrocefin by CMY-2 in complex with the VHH cAb_CMY-2(254). Experiments were performed using CMY-2 at a concentration of 0.5 nM. $k_{\mathit{cat}}^{\mathit{app}}$ data were fitted using the one-phase exponential decay equation from graph prism. (B) Trend of 1/$k_{\mathit{cat}}^{\mathit{app}}$ values as a function of VHH cAb_CMY-2(254) concentration. All values resulted from three independent experiments.

FIG 10

Inhibitory model for CMY-2 activity for cephaloridin (A), cephalothin (B), and ampicillin (C) by cAb_CMY-2(254). $k_{\mathit{cat}}^{\mathit{app}}$ and 1/$k_{\mathit{cat}}^{\mathit{app}}$ parameters were obtained from the linear phase (equation III in Text S1) of hydrolysis of the corresponding substrate. Concentrations of CMY-2 were at 0.2 nM, 1 nM, and 5 nM for cephaloridin, cephalothin, and ampicillin, respectively.

FIG 11

Binding molecular characterization of the complex cAb_CMY-2(254)/CMY-2. (A) Cartoon representing the overall view of the complex cAb_CMY-2(254)/CMY-2. (B) Surface representation of CMY-2 in the complex cAb_CMY-2(254)/CMY-2. (C) Hydrophobic interactions between CDR1 and the N-terminal extremity of the VHH and CMY-2. (D) Hydrogen bonds between CDR3 of the VHH and CMY-2. CDR1, CDR2, and CDR3 of the VHH are colored in purple, green, and cyan, respectively, while frameworks are represented in yellow. CMY-2 is represented in gray, while residues constituting motif 1 (S₆₄XXK₆₇), motif 2 (Y₁₅₀XN₁₅₂), and motif 3 (K₃₁₅T₃₁₆G₃₁₇) of the CMY-2 active site are colored in orange. Hydrogen bonds are highlighted by a red dotted line. Residues G108 and D109 from CDR3 of cAb_CMY-2(254) are not illustrated in the model due to a lack of information in the electronic density.

FIG 12

Superposition of complex cAb_CMY-2(254)/CMY-2 and P99 (PDB code 1XX2) (A) and CMY-10 (PDB code 1ZKJ) (B). CMY-2, P99, and CMY-10 are colored in gray, red, and magenta. Only CDR3 of the VHH is illustrated in cyan, and active site residues are in orange. H-bonds are represented with a red dashed line.

FIG 13

Superposition of the complex cAb_CMY-2(254)/CMY-2 and the crystal structure of AmpC WT β-lactamase from E. coli in complex with a covalently bound cephalothin (PDB code 1KVM). CMY-2 is in gray, AmpC in blue, cephalothin in green, and CDR3 of the VHH in cyan.