Amino terminal recognition by a CCR6 chemokine receptor antibody blocks CCL20 signaling and IL-17 expression via β-arrestin

Abstract

Background: CCR6 chemokine receptor is an important target in inflammatory diseases. Th17 cells express CCR6 and a number of inflammatory cytokines, including IL-17 and IL-22, which are involved in the propagation of inflammatory immune responses. CCR6 antagonist would be a potential treatment for inflammatory diseases such as psoriasis or rheumatoid arthritis. The aim of this study is to develop an antagonistic monoclonal antibody (mAb) against human CCR6 receptor (hCCR6).

Results: We generate monoclonal antibodies against hCCR6 immunizing Balb/c mice with hCCR6 overexpressing cells. The antibodies were tested by flow cytometry for specific binding to hCCR6, cloned by limiting dilution and resulted in the isolation and purification monoclonal antibody 1C6. By ELISA and flow cytometry, was determined that the antibody obtained binds to hCCR6 N-terminal domain. The ability of 1C6 to neutralize hCCR6 signaling was tested and we determined that 1C6 antibody were able to block response in β-arrestin recruitment assay with IC₅₀ 10.23 nM, but did not inhibit calcium mobilization. In addition, we found in a chemotaxis assay that 1C6 reduces the migration of hCCR6 cells to their ligand CCL20. Finally, we determined by RT-qPCR that the expression of IL-17A in Th17 cells treated with 1C6 was inhibited.

Conclusions: In the present study, we applied whole cell immunization for successfully obtain an antibody that is capable to neutralize hCCR6 signaling and to reduce hCCR6 cells migration and IL-17 expression. These results provide an efficient approach to obtain therapeutic potential antibodies in the treatment of CCR6-mediated inflammatory diseases.

Keywords: Bias signaling; CCR6; GPCR; Inflammation; Th17 cells; Therapeutic antibody.

Fig. 1

Establishment of anti-hCCR6 mAb. a Serum antibody titer of selected mouse after immunization with P815-c-myc-hCCR6 cells was tested in hCCR6-RBL and mock-RBL cell membrane ELISA. b SDS-PAGE analysis of 1C6 mAb purified from hybridoma supernatant using Protein G Sepharose. Positions of molecular size markers are shown on the right. **** p < 0.001

Fig. 2

Binding of anti-hCCR6 mAb to RBL transfected cells with hCCR6. a–d Comparison of the reactivity against mock cells and HA-hCCR6-expressing RBL cells between control isotype a, KM4703 b, 1C6 c and HA d antibodies at 20 μg/mL. e–h Representative flow cytometry analysis of RBL-hCCR6 and mock cells stained with different doses of mAb 1C6 (1–10 μg/mL). Blue histograms of hCCR6⁺ cells are superimposed over grey histograms from mock transfectans. One representative analysis from three independent experiments is shown. The histogram overlays were performed using BD CellQuest Pro Software

Fig. 3

Crossreactivity of 1C6 mAb with other GPCRs. Stably RBL transfectants expressing hCCR1 a, hCCR3 b, hCCR4 c, hCCR5 d, hCXCR1 e, hCXCR2 f, hCXCR3 g or hCXCR4 h were stained with 1 μg/mL of 1C6 (blue histograms) or anti HA antibodies (grey histograms) and analyzed by flow cytometry. One representative analysis from three independent experiments is shown. The histogram overlays were performed using BD CellQuest Pro Software

Fig. 4

Eppitope mapping. a–f Extracellular domains and N-terminal of hCCR6 were swapped with the homologous region of mCCR6. Flow cytometric analysis was performed using 1 μg/mL of mAb 1C6 (blue histograms) or anti-HA (grey histograms) for staining hCCR6 a, mCCR6 b or swapped hCCR6 mutants c–f expressing HA tagged RBL cells. One representative analysis from three independent experiments is shown. g Binding of 0.5 μg/mL of isotype, KM4703 and 1C6 antibodies to human CCR6 N-terminal region by ELISA. Means and SD of triplicates are plotted. **** p < 0.001

Fig. 5

Characterization of mAB 1C6 effect on ligand-induced signaling. a, b Antagonistic potency of 20 μg/mL 1C6, KM4703 and mouse IgG1 was assessed against 10 nM of CCL20 on calcium flux a and β-arrestin b assays. Data were expressed in terms of normalized percent inhibition (NPI). c Assessment of inhibition was carried out in the presence of increasing concentrations of purified 1C6 antibody. Data from β-arrestin assay were expressed in terms of relative luminescence units (RLU). Means and SD of triplicates are plotted. * p < 0.05; **** p < 0.001

Fig. 6

Inhibition of CCL20 mediated chemotaxis by 1C6 antibody. a Transwell chemotaxis assay on RBL-HA-CCR6 cells in the presence of increasing concentration of CCL20. The migrated cells were analyzed at 22 h of culture. b Assessment of hCCR6 cells chemotactic response to 125 nM CCL20 was carried out in presence or absence of 20 μg/mL 1C6. The chemotaxis index was calculated using the cells migrated in presence of CCL20 by the cells migrated in absence of CCL20 (control). All data are expressed as the mean ± SD of triplicated samples. * p < 0.05; **** p < 0.001

Fig. 7

Effect of 1C6 antibody on Th17 cells. Human Th17 cells or control cells without Th17 polarizing conditions were culture with anti-CCR6 antibodies for 5 days. a The concentration of CCL20 in the cell culture supernatants was measured by ELISA. Means and SD of triplicates are plotted. *** p < 0.01. b The mRNA expression of IL-17A was measured by real-time PCR. Data were normalized to the reference gene β-actin and the normalized expression ratio was calculated using 2^-ΔΔCt method. Means and SD of duplicates are plotted. **** p < 0.001