Application of anti-CD47 antibody in preparation of medicine for treating chronic sinusitis with nasal polyp

Abstract

The invention provides application of an anti-CD47 antibody in preparation of a medicine for treating chronic sinusitis with nasal polyp, and relates to the technical field of biological medicine. The invention relates to an application of an anti-CD47 antibody in preparation of a medicine for treating chronic sinusitis with nasal polyp. The anti-CD47 antibody provided by the invention is used for treating important pathological characteristics, namely eosinophilic granulocytes, which influence the severity and recurrence of CRSwNP diseases, and has the advantage of directly solving the important pathological characteristics of the diseases.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to the use of anti-CD47 antibodies in the preparation of medicines for treating chronic sinusitis with nasal polyps. Background Art

[0002] Chronic rhinosinusitis with nasal polyps (CRSwNP) is a common chronic heterogeneous nasal disease characterized by persistent inflammation of the nasal mucosa, often accompanied by clinical symptoms such as nasal congestion, runny nose, and anosmia, which seriously affects the quality of life. The nasal mucosa of CRSwNP is often accompanied by different inflammatory cell infiltrations including eosinophils, lymphocytes, plasma cells, and neutrophils. Among them, CRSwNP with eosinophil infiltration as the main infiltration is defined as eosinophil-infiltrated CRSwNP (ECRSwNP), otherwise it is defined as non-eosinophil-infiltrated CRSwNP (nonECRSwNP). Studies in recent years have shown that ECRSwNP is the most difficult and important subtype of CRSwNP to cure, often with more severe symptoms, poor drug effects, and a postoperative recurrence rate of up to 98.5%. Eosinophilia is the key pathological feature of ECRSwNP. Therefore, controlling the number of eosinophils is the key to fundamentally treating ECRSwNP.

[0003] Despite treatment with emerging monoclonal antibody drugs, some patients still have poor efficacy or relapse after stopping treatment. Monoclonal antibodies are expensive, and ineffective treatment causes economic burden and delays the disease. Therefore, new therapeutic targets will help improve the overall efficacy of ECRSwNP. CD47 is a self-marking protein that is widely and lowly expressed on the surface of normal cells. Signal regulatory proteins α (SIRPα) is the main receptor of CD47 signals. It binds to CD47 and recruits protein tyrosine phosphatases (SHP1 / 2) through phosphorylation to transmit the "don't eat me" signal to protect normal cells from damage by the immune system. However, in some abnormal cells, it is often regulated by transcription factors such as NF-κB, resulting in its high expression. Studies in the field of cancer have shown that CD47 is abnormally highly expressed in cancer cells. Blocking this signal axis can reduce cancer cells and achieve the effect of treating cancer. However, the effect on nasal mucosal eosinophils is still unclear. Summary of the invention

[0004] In order to solve the technical problems existing in the prior art, the embodiment of the present invention provides the use of anti-CD47 antibodies in the preparation of a drug for treating chronic sinusitis with nasal polyps. The technical solution is as follows:

[0005] Use of anti-CD47 antibodies in the preparation of drugs for treating chronic sinusitis with nasal polyps.

[0006] Optionally, the chronic sinusitis with nasal polyps is eosinophil-infiltrated chronic sinusitis with nasal polyps.

[0007] Optionally, the anti-CD47 antibody reduces the number of eosinophils in the nasal mucosa of CRS patients.

[0008] A medicine or a medicine composition for treating chronic sinusitis with nasal polyps, characterized by comprising an anti-CD47 antibody.

[0009] Optionally, the chronic sinusitis with nasal polyps is eosinophil-infiltrated chronic sinusitis with nasal polyps.

[0010] Optionally, the anti-CD47 antibody reduces the number of eosinophils in the nasal mucosa of CRS patients.

[0011] Application of CD47 as a target in the preparation of drugs for the treatment of chronic sinusitis with nasal polyps.

[0012] Optionally, the chronic sinusitis with nasal polyps is eosinophil-infiltrated chronic sinusitis with nasal polyps.

[0013] The beneficial effects brought about by the technical solution provided by the embodiment of the present invention include at least:

[0014] First, the inventors used immunohistochemistry and Spearman correlation analysis to find that CD47 was significantly highly expressed, especially in the nasal polyp tissue of ECRSwNP, and was strongly positively correlated with the number of tissue eosinophils. In order to verify whether CD47 is a target for solving nasal mucosal eosinophilia, the inventors constructed two animal models, an acute eosinophilia mouse model and a CRSwNP mouse model, and confirmed from the in vivo level of the animal that anti-CD47 antibodies can significantly reduce the number of nasal mucosal eosinophils in the modeling group. In addition, the inventors further confirmed from the human tissue level that anti-CD47 antibodies can reduce the number of nasal mucosal eosinophils in CRSwNP by in vitro culture of nasal polyp tissues of CRSwNP patients. The anti-CD47 antibodies provided by the present invention have the following advantages in the preparation of drugs for treating chronic sinusitis with nasal polyps: the incidence of ECRSwNP in the West accounts for 80% of CRSwNP, and in recent years, its proportion in Asia has also been increasing. The continuous increase in the number of people with the disease, coupled with the limited efficacy of existing treatments, has led to an urgent need for new therapeutic targets. Anti-CD47 antibody therapy affects the severity and recurrence of CRSwNP, namely eosinophils, an important pathological feature of the disease, and has the advantage of directly addressing the important pathological features of the disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0016] Figure 1A and Figure 1B The figure shows that CD47 expression is significantly increased in CRSwNP nasal polyps and is significantly correlated with the number of tissue eosinophils; Figure 1A It is a representative picture of the CD47 levels in CRSwNP nasal polyp tissues and control nasal mucosal tissues detected by immunohistochemistry and a figure showing the statistical results of CD47 immunohistochemical scoring; Figure 1B is a graph of the Spearman correlation analysis of the correlation between the CD47 level in nasal polyp tissue and the counts of tissue eosinophils, lymphocytes, plasma cells, and neutrophils under high-power microscope (×400);

[0017] Figure 2A and Figure 2B The figure is a diagram showing that blocking CD47 reduces the number of eosinophils in the nasal mucosa of a mouse model of acute nasal eosinophilia; Figure 2A It is a schematic diagram of the mouse model of acute nasal mucosal eosinophilia and the experimental scheme of anti-CD47 antibody / isotype control IgG2a treatment; Figure 2B are representative images of hematoxylin-eosin (H&E) staining of the nasal mucosa and the results of eosinophil counts in the nasal mucosa;

[0018] Figure 3A and Figure 3B This is a diagram showing that blocking CD47 reduces the number of eosinophils in the nasal mucosa of the CRSwNP mouse model; Figure 3A This is a schematic diagram of the CRSwNP mouse model and the anti-CD47 antibody / isotype control IgG2a treatment experimental protocol; Figure 3B It is a representative picture of H&E staining of nasal mucosa and the result of eosinophil count of nasal mucosa;

[0019] Figure 4 The figure is a graph showing that blocking CD47 reduces the number of eosinophils in cultured human nasal polyp tissues. DETAILED DESCRIPTION

[0020] The technical solution of the present invention is described below in conjunction with the accompanying drawings.

[0021] the term

[0022] 1. Chronic rhinosinusitis with nasal polyps (CRSwNP)

[0023] 2. Signal regulatory proteins α (SIRPα)

[0024] 3. Immunohistochemical score (HIS)

[0025] 4. Hematoxylin-eosin staining (H&E)

[0026] 5. One-way analysis of variance analysis (One-way ANOVA)

[0027] 6. Papain

[0028] 7. Phosphate buffered saline (PBS)

[0029] 8. Aspergillus oryzae proteinase (AP)

[0030] 9. Ovalbumin (OVA)

[0031] First, the inventors collected nasal polyp tissues and normal nasal mucosal tissues from CRSwNP patients. Immunohistochemistry revealed that CD47 was significantly highly expressed in nasal polyp tissues of ECRSwNP, and was strongly positively correlated with the number of tissue eosinophils. In order to confirm the effect of CD47 on the number of eosinophils in the nasal mucosa of CRSwNP, the inventors constructed an acute nasal mucosal eosinophilia mouse model and a CRSwNP mouse model. After treatment with anti-CD47 antibodies, it was found that the number of eosinophils in the nasal mucosa was significantly reduced. Further, the inventors collected 6 fresh nasal polyp tissues from CRSwNP patients for in vitro culture, and the number of eosinophils in the tissue was also reduced after treatment with anti-CD47 antibodies. In summary, it is shown that anti-CD47 antibodies can reduce the number of eosinophils in the nasal mucosa of CRSwNP. Details are as follows:

[0032] Example 1

[0033] (1) Demographic and clinical characteristics of the population in this study.

[0034] According to the diagnostic criteria of the 2020 European Position Paper on Rhinosinusitis and Nasal Polyps (EPOS 2020), 27 CRSwNP patients and 5 controls were recruited from the Department of Otorhinolaryngology, Beijing Tongren Hospital, Capital Medical University. The definition of ECRSwNP and nonECRSwNP patients was based on hematoxylin-eosin staining (H&E staining) of nasal polyp tissues. The diagnosis was based on the counts of eosinophils, neutrophils, lymphocytes, and plasma cells in the histopathological sections under high-power microscopy (400 times). The specific diagnostic criteria for each subtype were based on the literature Lou et al. Rhinology. 2016; 54(2):150-9. The inclusion criteria for CRSwNP patients were as follows: ① Patients aged between 18 and 70 years old; ② Meet the diagnostic criteria of EPOS 2020. ECRSwNP was defined as patients with a percentage of eosinophils >27% or eosinophils >55 per high-power field under high-power microscope (400x), otherwise defined as nonECRSwNP. Inclusion criteria for controls: patients with nasal septum deviation or surgery requiring resection of the uncinate process or inferior turbinate to expose the field of view. Exclusion criteria for CRSwNP patients and controls: oral or nasal glucocorticoids, antibiotics, or immunomodulators within 4 weeks before surgery. Nasal polyp tissues of ECRSwNP and nonECRSwNP patients and nasal mucosal tissues of the uncinate process or turbinate of controls were obtained during surgery and used as samples for further research. This study was approved by the Ethics Committee of Beijing Tongren Hospital Affiliated to Capital Medical University, and all patients signed informed consent. The demographic and clinical characteristics of controls, nonECRSwNP, and ECRSwNP are shown in Table 1.

[0035] Table 1 Demographic and clinical characteristics

[0036]

[0037] Note: IQR, interquartile range.

[0038] (2) CD47 expression increases in the nasal mucosa of CRSwNP and is significantly correlated with the number of tissue eosinophils

[0039] Nasal polyp tissues of CRSwNP patients and nasal mucosal tissues of controls were fixed with 4% paraformaldehyde and then embedded in paraffin. After gradient dewaxing and dehydration, 4 μm tissue sections were incubated with anti-CD47 antibody (1:100 dilution, 63000S, CST) at 4°C overnight. The next day, enzyme-labeled goat anti-rabbit IgG polymer (PV-6001, Zhongshan Jinqiao) was incubated at room temperature for 1 hour and then DAB was used for color development. Continuous photography was performed under a pathological microscope at a 400x field of view. The immunohistochemical score (HIS) was obtained by combining the percentage of positive cells and the staining intensity of positive cells, ignoring the epithelium. The percentage of positive cells (no positive cells were scored as 0 points, 1% to 10% positive cells were scored as 1 point, 11% to 50% were scored as 2 points, 51% to 80% were scored as 3 points, and 81% to 100% were scored as 4 points); the positive cell staining intensity (negative was scored as 0 points, weak positive was scored as 1 point, moderate positive was scored as 2 points, and strong positive was scored as 3 points), and the product of the percentage of positive cells and the positive cell staining intensity was the IHS of the tissue in this case. Among them, the classification method of positive cell staining intensity is as follows: all stained tissues are divided into four categories according to the staining intensity: cells that are not stained are negative, cells with the strongest staining are strongly positive, weak positive refers to a category with staining but the lowest staining intensity, and moderate positive refers to a category with staining intensity between weak positive and strong positive. At least 5 non-repeated fields of view were selected for each section at a 400x field of view of subepithelial tissue (with the epithelium as the location, including the epithelium and the tissue below). SPSS20.0 was used for nonparametric Kruskal Wallis one-way ANOVA analysis and post hoc Dunn's multiple comparison test to analyze the differences among multiple groups. P < 0.05 was considered statistically significant. Figure 1A As shown in the figure, compared with the healthy control group, the expression of CD47 was significantly increased in ECRSwNP nasal polyp tissue (p < 0.001), and it showed an up-regulated trend in the nonECRSwNP group. Compared with the nonECRSwNP group, the expression of CD47 in the ECRSwNP group increased and the difference was statistically significant (p < 0.05).

[0040] The Spearman rank correlation method was used for correlation analysis (r > 0.6 for strong correlation; 0.4 < r < 0.6 for moderate correlation; r < 0.4 for weak correlation) to analyze the correlation between CD47 expression level and the number of eosinophils, lymphocytes, plasma cells, and neutrophils in nasal polyp tissues. P < 0.05 was considered statistically significant. Figure 1BAs shown in the results, CD47 was strongly positively correlated with the number of eosinophils (r = 0.63, p < 0.01), and had no significant correlation with lymphocytes, plasma cells, and neutrophils (p > 0.05). The results showed that CD47 was closely related to the number of eosinophils in nasal polyp tissue.

[0041] (3) Blocking CD47 reduces the number of eosinophils in the nasal mucosa of a mouse model of acute nasal eosinophilia

[0042] Construction of mouse model of acute nasal mucosal eosinophilia and treatment with anti-CD47 antibody or isotype control: 32 7-week-old wild-type Balb / c mice (Beijing Weitonglihua Laboratory Animal Technology Co., Ltd.). All mice were housed in an SPF-grade environment with 12 hours of light / 12 hours of darkness, 22±2℃, and a relative humidity of 50±5%, and had free access to food and drinking water. All animal experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals. After 1 week of adaptive feeding of mice, 10 μg of papain (papain, Sigma) was diluted in sterile phosphate buffered saline (PBS) to a total volume of 20 μL, and 20 μL was instilled intranasally into each mouse, after which the mouse head was kept in a downward position to prevent the reagent from flowing into the lungs. Once a day for 3 days. 200 μg of anti-CD47 antibody (BE0270, Bio X cell) or isotype control IgG2a (BE0089, Bio X cell) was diluted in sterile PBS to a total volume of 100 μL. Each mouse was injected intraperitoneally with 100 μL once a day for 5 days. On the third day, 20 μL of the above papain working solution was dripped into the nose once a day for 3 days. The above experimental protocol was as follows: Figure 2A As shown. 24 hours after the last intranasal instillation or intraperitoneal injection, the mice were anesthetized and perfused intracardially with normal saline. The skin and soft tissues of the head were removed by dissection, and the mandible was removed. The mouse skull was fixed with 4% paraformaldehyde for 24 hours and decalcified in 5% nitric acid for 1 week. The tissue was then paraffin-embedded according to standard procedures. 4μm tissue sections were subjected to H&E staining to evaluate the eosinophils in the nasal mucosa.

[0043] The results are as follows Figure 2B As shown in the figure, compared with the control group, the number of eosinophils in the nasal mucosa in the Papain modeling group increased significantly (p < 0.05). Compared with the Papain + isotype control IgG2a group, the number of eosinophils in the nasal mucosa in the papain + anti-CD47 antibody group decreased significantly (p < 0.001). The results show that anti-CD47 antibodies can reduce the number of eosinophils in the nasal mucosa of the mouse model of acute nasal mucosal eosinophilia.

[0044] (4) Blocking CD47 reduces the number of eosinophils in the nasal mucosa of the CRSwNP mouse model

[0045] Construction of CRSwNP mouse model and treatment with anti-CD47 antibody or isotype control: 24 7-week-old wild-type C57BL / 6 mice (Beijing Weitonglihua Laboratory Animal Technology Co., Ltd.). All mice were housed in an SPF-grade environment with 12 h light / 12 h dark, 22±2°C, and a relative humidity of 50±5%, and had free access to food and drinking water. All animal experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals. After 1 week of adaptive feeding of mice, 2 units of Aspergillus oryzae protease (AP, Sigma) and 75 μg of ovalbumin (OVA, Sigma) were mixed and diluted in sterile PBS to a total volume of 20 μL, and 20 μL was instilled intranasally into each mouse, after which the mouse head was kept in a downward position to prevent the reagent from flowing into the lungs. This was done 3 times a week for 12 weeks. Starting from the 6th week of AP and OVA intranasal administration, 200 μg of anti-CD47 antibody (BE0270, Bio X cell) or isotype control IgG2a (BE0089, Bio X cell) was diluted in sterile PBS to a total volume of 100 μL and injected intraperitoneally into each mouse with 100 μL once every 3 days for 6 weeks. Figure 3A As shown. 24 hours after the last intranasal instillation or intraperitoneal injection, the mice were anesthetized and perfused intracardially with normal saline. The skin and soft tissues of the head were removed by dissection, and the mandible was removed. The mouse skull was fixed with 4% paraformaldehyde for 24 hours and decalcified in 5% nitric acid for 1 week. The tissue was then paraffin-embedded according to standard procedures. 4μm tissue sections were subjected to H&E staining to evaluate the eosinophils in the nasal mucosa.

[0046] The results are as follows Figure 3B As shown in the figure, compared with the control group, the number of eosinophils in the nasal mucosa of the CRSwNP modeling group increased significantly (p < 0.001). Compared with the CRSwNP+isotype control IgG2a group, the number of eosinophils in the nasal mucosa of the CRSwNP+anti-CD47 antibody group decreased significantly (p < 0.001). The results showed that anti-CD47 antibodies can reduce the number of eosinophils in the nasal mucosa of the CRSwNP mouse model.

[0047] (5) Blocking CD47 reduces the number of eosinophils in human nasal polyp tissue cultured in vitro

[0048] Six fresh nasal polyp tissues from CRSwNP patients were cut into 0.1 g cubes. 5 μg of anti-CD47 antibody (MAB4670, R&D) or isotype control IgG1 (MAB002, R&D) was diluted to a total volume of 10 μL in sterile RIPM1640 medium containing 1% penicillin-streptomycin mixture and 10% fetal bovine serum. 10 μL of anti-CD47 antibody or isotype control was injected with a microsyringe and cultured for 24 hours. After fixation with 4% paraformaldehyde, the tissue was paraffin-embedded according to standard procedures. 4 μm tissue sections were subjected to H&E staining to evaluate the eosinophils in the nasal mucosa. The results are shown in Figure 2. Figure 4 As shown, the number of eosinophils in nasal polyp tissues was significantly reduced after anti-CD47 antibody treatment compared with the isotype control IgG1 group (p < 0.05).

[0049] The above is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art can easily think of changes or substitutions within the technical scope disclosed by the present invention, which should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims

1. Application of anti-CD47 antibodies in the preparation of drugs for the treatment of chronic sinusitis with nasal polyps.

2. The use according to claim 1, characterized in that: The chronic sinusitis with nasal polyps is eosinophil infiltration type chronic sinusitis with nasal polyps.

3. The use according to claim 1, characterized in that: The anti-CD47 antibody reduces the number of eosinophils in the nasal mucosa of patients with CRS.

4. A medicine or a pharmaceutical composition for treating chronic sinusitis with nasal polyps, characterized in that: Includes anti-CD47 antibodies.

5. The drug or pharmaceutical composition according to claim 4, characterized in that The chronic sinusitis with nasal polyps is eosinophil infiltration type chronic sinusitis with nasal polyps.

6. The drug or pharmaceutical composition according to claim 4, characterized in that The anti-CD47 antibody reduces the number of eosinophils in the nasal mucosa of patients with CRS.

7. Application of CD47 as a target in the preparation of drugs for the treatment of chronic sinusitis with nasal polyps.

8. The use according to claim 7, characterized in that: The chronic sinusitis with nasal polyps is eosinophil infiltration type chronic sinusitis with nasal polyps.

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