Engineered pH-dependent recycling antibodies enhance elimination of Staphylococcal enterotoxin B superantigen in mice

Abstract

A new modality in antibody engineering has emerged in which the antigen affinity is designed to be pH dependent (PHD). In particular, combining high affinity binding at neutral pH with low affinity binding at acidic pH leads to a novel antibody that can more effectively neutralize the target antigen while avoiding antibody-mediated antigen accumulation. Here, we studied how the in vivo pharmacokinetics of the superantigen, Staphylococcal enterotoxin B (SEB), is affected by an engineered antibody with pH-dependent binding. PHD anti-SEB antibodies were engineered by introducing mutations into a high affinity anti-SEB antibody, 3E2, by rational design and directed evolution. Three antibody mutants engineered in the study have an affinity at pH 6.0 that is up to 68-fold weaker than the control antibody. The pH dependency of each mutant, measured as the pH-dependent affinity ratio (PAR - ratio of affinity at pH 7.4 and pH 6.0), ranged from 6.7-11.5 compared to 1.5 for the control antibody. The antibodies were characterized in mice by measuring their effects on the pharmacodynamics and pharmacokinetics (PK) of SEB after co-administration. All antibodies were effective in neutralizing the toxin and reducing the toxin-induced cytokine production. However, engineered PHD antibodies led to significantly faster elimination of the toxin from the circulation than wild type 3E2. The area under the curve computed from the SEB PK profile correlated well with the PAR value of antibody, indicating the importance of fine tuning the pH dependency of binding. These results suggest that a PHD recycling antibody may be useful to treat intoxication from a bacterial toxin by accelerating its clearance.

Keywords: Staphylococcal enterotoxin B; antigen pharmacokinetics; monoclonal antibody; pH-dependent antibody.

Figure 1.

Effect of antibody binding on the antigen PK. (a). A conventional high affinity antibody increases the serum stability of the antigen because the antigen remains bound to the antibody in the endosome and is recycled to the serum along with the antibody. (b). An engineered PHD antibody releases the bound antigen in the acidic environment of the endosome to allow endolysosomal degradation of the molecule while the antibody is returned to the serum. This creates a net flow of the antigen from the serum to the lysosome and increases the rate of antigen elimination compared to a high affinity antibody with pH independent binding.

Figure 2.

(a). SEB (pink) bound to wt anti-SEB 3E2 Fab (PDB: 3W2D). Heavy chain (VH) and light chain (VL) variable domains are colored cyan and orange, respectively. The antibody residues within 4.5 Å of SEB are shown as sticks. (b). 3E2 scFv mutants containing interfacial histidine mutations were displayed on the yeast surface and analyzed by flow cytometry for SEB binding. The amount of biotinylated SEB bound was measured using SAPE. The mean fluorescence intensity of the displaying population is plotted. The labeling was performed at pH 7.4 and 6.0. The binding at pH 6.0 is lower for the VH-Y124H mutant (m13). (c). The equilibrium binding affinity K_D of wild type 3E2 and VH-Y124H was measured by yeast surface display. The representative binding curves at pH 7.4 (black) and pH 6.0 (red) and their fitted values are shown.

Figure 3.

Fluorescence-activated cell sorting of m13 scFv library expressed on the yeast surface. (a). Cells were first incubated with unbiotinylated SEB, washed in PBST at pH 6.0, and finally incubated with biotinylated SEB at pH 7.4. Bound SEB was detected with SAPE. The cells were also labeled with anti-FLAG antibody and anti-mouse antibody-FITC to impose selection based on the scFv expression. The polygon represents the pool collected during each sorting. (b). The labeling and washing conditions during each round of sorting. The “percentage collected” refers to the number of cells collected divided by the number of cells sorted.

Figure 4.

(a). Mutations found in the clones selected from the yeast library are shown as yellow sticks. (b). The interaction between VH residue 72 and SEB(D99) was modeled. i. VH-Y72 (wild type) does not interact directly with SEB(D99), but ii. VH-Y72H, which is found in several selected mutants, may contribute to improved affinity by forming a hydrogen bond (dotted line). iii. VH-Y72R may stabilize the interaction further through electrostatic interaction. Minor adjustments of surface residues allow the formation of a network of salt bridges (dotted lines), involving VH-D50, VH-R72 and SEB(D99).

Figure 5.

Dissociation study of full-length anti-SEB antibody. Wt, L2, L6, or L6.R was immobilized on ELISA surface and then incubated with a saturating concentration of biotinylated SEB. After washing in PBST pH 6.0 or 7.4 for 15 min, bound SEB was detected using streptavidin-HRP.

Figure 6.

The concentrations of inflammatory cytokines IL-2, IL-6 and IFNγ in plasma were measured 3 hr after SEB injection, either alone (NT, no treatment) or together with various treatment antibodies. The untreated mice had the highest concentrations for all three cytokines (n = 3). The co-injection of SEB (i.p.) and antibodies (i.v.) reduced the cytokine production, indicating that the antibodies are all capable of neutralizing the toxicity of SEB. For each measured cytokine, pair-wise two-tailed comparisons were made between NT and each antibody treatment. The maximum p value is indicated. No statistical significance was observed among various antibody treatments.

Figure 7.

In vivo characterization of PHD antibodies. Mice were injected with 20 µg i.p. SEB immediately followed by 150 µg antibody i.v. (a). Total SEB concentration in plasma was determined from sandwich ELISA. The mean value is shown (n = 3 animals) for each time point. The standard deviations are represented by positive error bars. LOD, limit of detection. (b). Total antibody concentration in plasma was determined by ELISA at various time points. (c). A linear correlation between the SEB AUC (in units of nM●hr) and the PAR values of the antibodies is observed with a high Pearson correlation coefficient.