Beta-lactamase inhibitors derived from single-domain antibody fragments elicited in the camelidae

Abstract

Small, soluble single-domain fragments derived from the unique variable region of dromedary heavy-chain antibodies (VHHs) against enzymes are known to be potent inhibitors. The immunization of dromedaries with the TEM-1 and BcII beta-lactamases has lead to the isolation of such single-domain antibody fragments specifically recognizing and inhibiting those beta-lactamases. Two VHHs were isolated that inhibit TEM-1 and one BcII inhibiting VHH was identified. All inhibitory VHHs were tight-binding inhibitors. The 50% inhibitory concentrations were determined for all inhibitors and they were all in the same range as the enzyme concentration used in the assay. Addition of the VHHs to the TEM-1 beta-lactamase, expressed on the surface of bacteria, leads to a higher ampicillin sensitivity of the bacteria. This innovative strategy could generate multiple potent inhibitors for all types of beta-lactamases.

FIG. 1

Analysis of antigen-specific antibodies. (A) Microtiter plates were coated with BcII at a concentration of 1 μg/ml. (B) TEM-1 was immobilized at a concentration of 2 μg/ml. The plates were incubated with serial dilutions of the immunoglobulin subclasses, isolated from serum after immunization. Bound IgG1 (▪), IgG2 (●), and IgG3 (▴) were subsequently detected with a rabbit anti-dromedary IgG antiserum and anti-rabbit IgG-alkaline phosphatase conjugate. OD₄₁₀ was measured after 10 min.

FIG. 2

Amino acid sequence of the isolated binders against the TEM-1 β-lactamase, named cAbTemxx, and against the BcII β-lactamase, named cAbBCIIxx. Numbering and deoxycytidine (CDR) designations are according to the methods of Kabat et al. (14).

FIG. 3

IC₅₀ determinations for the different inhibitors. The residual enzymatic activity was measured as a function of antibody fragment concentration. For all measurements, 100 μM nitrocefin was used as substrate. This was performed for cAbBCII10 (A), cAbTem02 (B), and cAbTem13 (C). For the experiment shown in panel A, 0.2 μM BcII was used, whereas 0.05 μM TEM-1 was used for the experiments shown in panels B and C.

FIG. 4

In vivo inhibition of TEM-1 when expressed as a fusion protein with OprI on the surface of E. coli. A total of 4 × 10⁷ cells were preincubated for 1 h with 0 to 3.5 μg of cAb in a final volume of 100 μl. The cells were plated on ampicillin-containing agar plates. Shown are the CFU (per milliliter) results obtained in the presence of increasing amounts of cAbTem13, ranging from 0 to 3.5 μg (▪), and the results obtained in the presence of increasing amounts of cAbBCII10, ranging from 0 to 3.5 μg (●).