A Human C-C Chemokine Receptor 3 Monoclonal Antibody Inhibited Pulmonary Inflammation In Allergic Mice

IntroductionAsthma is an inflammatory disease in respiratory system, which is characterized by airway inflammations, hyperresponsiveness (AHR). Clinical study and mouse model of asthma showed leukocytes were involved in the pathophysiologic process of asthma that eosinophil are found to play key role in the disease, due to the toxic granular proteins they secrete and the membrane products. In response to the chemokines generated locally, eosinophils from the microcirculation accumulate in the airway and result in pulmonary inflammation.Many small molecular proteins (8-10 kDa) have been identified and characterized as chemokines by their actions on distinct subtypes of leukocytes. Chemokines regulate immune cells by binding to 7-transmenbrane G protein-coupled receptors including CCR1, CCR2 and CCR3 et al. The distribution and expression of these receptors vary in distinct cells.CCR3 is a major chemokine receptor expressed on the surface of eosinophil, which is responsible for the cells activation and chemotaxis. In addition to eosinophil, CCR3 also exist in basophil, mast cell, and Th2 cell. In vitro and vivo studies, several C-C types of chemokines are shown to be active on eosinophil in allergic inflammation. Chemokines that are selective for CCR3 include eotaxin, eotaxin-2, and eotaxin-3, whereas RANTES, MCP-3, and MCP-4 bind to other chemokine receptors other than CCR3^[11]. The chemokines that contribute to aberrant eosinophil recruitment to the airways in asthma are not certain. It may be that different chemokines, operate in the process of asthma by different immune mechanisms. It was reported that the binding of an oligodeoxynucleotide, so-called anti-sense, to thecomplementary sequence of a messenger RNA can prevent the synthesis of the encoded protein. So making the blockade of a common receptor is more appealing. CCR3 have been identified and characterized from other species, such as mice, rat, guinea pig and non-human primates, and antibody of CCR3 are generated to aim at this attractive biological target for therapeutic intervention.The treatment of asthma has been improved by the implementation of management guideline in recent years, with further development in the study for the mechanism of asthma. Many medicines such as corticosteroids, theophylline etc. have been used to control asthma as anti-inflammatory agents. However, the effects of those drugs are not satisfied in the clinical practice. Therefore, a new agent for asthma treatment is required anxiously. For these reasons, CCR3 has been more important as a significant anti-inflammatory pharmacological target, and CCR3 antagonists are currently being developed for the treatment of asthma and other allergic disorders. Administration of an anti-CCR3 monoclonal antibody (mAb) was capable of inducing a targeted reduction of eosinophils in peripheral blood. Inhibitors of CCR3 showed these agents were effective on inhibiting eosinophil recruitment in allergen models of asthma. We also reported anti-mouse CCR3 mAb inhibited airway inflammation in mice^[18]. However there is no report to evaluate the effects of anti-human CCR3 antibody on airway inflammation, and mucus secretion in the mice model of asthma.Objective The purpose of the study is to generated murine mAb against the humanCCR3, using the 30-amino acid synthetic peptide which is the NH2-terminal of CCR3 as the immunizing antigen. Furthermore, we want to investigate the therapeutic effects of this specific antibody study in an athmatic mice sensitized and challenged with ovalbumin.Methods A peptide was synthesized, corresponding to the predicted NH2 terminus of the human CCR3 amino acid sequence. The peptide conjugated to Keyhole limpethemocyanin (KLH) was injected into BALB/c mice at 6-day intervals. Mice sera were tested by ELISA and the spleen cells of mouse that produced the most potent serum titer larger than 1: 6250 were used for fusion with SP2/0 cells. Positive hybridoma clones were selected by enzyme-linked immunosorbent assay. Hybridoma clones producing anti-human CCR3 mAbs were injected into mice for ascites production. The IgG fraction from the ascites fluid was purified and dialyzed. Western blot analysis was performed to identify the specificity of Mouse Anti-human CCR3 mAb. In Vivo study to determine the inhibitory effect of the antibody, mice were sensitized on the d 0 and d 14 by ip injection of ovalbumin (Ova) mixed with aluminum. At d 24, 25, and 26, mice were challenged by aerosolized 1% Ova in saline for 40 min. 32 mice were divided into 4 groups (8 mice each group), Saline group, Ova group, Anti-CCR3 group, and IgG group. Antibody was administered by ip injection before OVA challenge at d 24, d 25, d 26 at the dose of 3 mg per kg, and the non-specific IgG were administered at the same time point in IgG group. The bronchoalveolar lavage fluid (BALF), histopathology, mucus secretion were examined.Results:Generation and identification of the anti-human CCR3 mAbEnzyme-linked immunosorbent assay was employed to detect the affinity of the generated antibody. Antibodies from three different hybridoma clones, which are PBND03, PBND04, and PBND05 show high affinity with the peptide. The affinities are PBND03>1.28×l0⁹, PBND04≥2×10⁷, PBND05≥3.2×10⁸ respectively. To further identify the antibody we produced, those three antibodies were chosen to perform western-blot as the primary antibody. Lysate of Daudi cell expressing CCR3 was used to determine the specificity of the generated antibody to CCR3. As shown in Fig.1, remarkable strips were observed. The antibody from PBND03 clone was employed for further study in vivo because of its highest affinity.The anti-human CCR3 mAb decreased leukocyte in the BALF of allergic miceTo examine the effect of anti-human CCR3 mAb on inflammatory cells accumulation responding to allergen challenge, BALF was recovered. Total cells of BALF were counted and differentiated using morphological criteria. The total number of leukocytes and eosinophils in the Ova group was significantly greater than that in the Saline group. A comparison of individual cell numbers per ml of BALF reveals that these increases were mainly due to increases in eosinophil populations. There are no differences in total number of leukocytes and eosinophil between Ova group and IgG group. In comparison to Ova group, total number of leukocytes and eosinophils in anti-human CCR3 mAb groups was decreased significantly. No significant differences in neutrophil and lymphocyte were observed between the groups.The anti-human CCR3 mAb inhibited pulmonary inflammation in allergic miceTo determine if anti-human CCR3 mAb inhibit the airway inflammatory infiltration,we observed the pulmonary pathology stained with H&E of mice in all groups. Compared with mice treated with saline, exposure to Ova resulted in the development of a characteristic peribronchial and perivascular airway tissue infiltration. In comparison to mice in Ova group, no obvious change of pulmonary inflammation was observed in the mice administered with non-specific IgG. However, the allergic mice administered with anti-human CCR3 mAb exhibited a remarkable reduced inflammation in the lung tissue.The anti-human CCR3 mAb reduced mucus overprodution of allergic miceTo evaluate the inhibitory effect of anti-human CCR3 mAb on hypersecretion of mucus in airway, PAS stain was performed. In allergic mice, morphometric analyses of the PAS stained lung sections revealed obviously increase in mucus cells hypertrophy and in the percentage of airway mucus, which is described by HR and MOR respectively. Anti-human CCR3 mAb significantly reduced the mucus secretion. Compared with mice in Ovagroup, mice treated with anti-human CCR3 mAbexhibited decreased HR and MOR. No reduction of mucus hypersecretion induced by allergen was observed in mice treated with non-specific IgG mice.The anti-human CCR3 mAb decreased IL-4 in BALF of allergic miceTo determine the effect of anti-human CCR3 mAb on the pulmonary immune response in allergic mice, the concentration of IL-4, IFN-γ in BALF were measured. The IL-4 level in the Ova group was significantly greater than that in the Saline group. The mice treated with anti-human CCR3 mAb showed significant decrease of IL-4 in comparison to the allergic mice. The IFN-γ levels in Ova group was less than that in the Saline group and there were no differences between the anti-human CCR3 mAb groups and Ova group in IFN-γ levels. It was showed non-specific IgG had no effect on these cytokine levels in allergic mice.ConclusionWe found the antibody we generated was specific to CCR3. The allergic mice treated with the anti-human CCR3 antibody exhibited significant reduction of pulmonary inflammation and mucus overproduction accompanied by the alteration of cytokine in BALF. It is suggested the blockade of CCR3 is an appealing therapeutical target for asthma. The present research may provide an experimental basis for further study of this agent.