Application of anti-CD84 antibody in preparation of medicine for preventing and treating pulmonary fibrosis

Abstract

The invention provides application of an anti-CD84 antibody in preparation of a medicine for preventing and treating pulmonary fibrosis, and belongs to the technical field of biological medicine. The invention relates to an application of an anti-CD84 antibody in preparation of a medicine for preventing and treating pulmonary fibrosis. According to the application, the biological function of CD84 is blocked by using the anti-CD84 antibody, so that the pulmonary fibrosis degree of bleomycin-induced pulmonary fibrosis and a mouse disease model of pulmonary fibrosis adaptation diseases can be effectively reduced, and the activation of pro-inflammatory macrophages and / or pro-fibrosis macrophages can be effectively inhibited, thereby preventing further deterioration of pulmonary fibrosis. Therefore, the application provided by the invention provides a new means for preventing and treating pulmonary fibrosis.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and specifically relates to the application of an anti-CD84 antibody in the preparation of a medicine for preventing and treating pulmonary fibrosis. Background Art

[0002] Pulmonary fibrosis (PF) is a disease characterized by persistent damage to the alveoli, which leads to varying degrees of inflammation and fibrosis in the alveoli and interstitium, and then to destruction of the lung structure and respiratory failure. It is therefore also called interstitial lung disease (ILD). The causes of pulmonary fibrosis include known causes such as physical, chemical, and biological factors, as well as pulmonary fibrosis of unknown etiology. Pulmonary fibrosis with known causes, such as interstitial lung disease associated with connective tissue diseases, includes rheumatoid arthritis-associated interstitial lung disease (RA-ILD), systemic sclerosis-associated interstitial lung disease (SSc-ILD), and systemic lupus erythematosus-associated interstitial lung disease (SLE-ILD). The cause and mechanism of the disease are mainly due to the body's excessive immune response to self- or foreign antigens, which causes the activation of multiple immune cells such as lung lymphocytes, which then secrete a large number of cytokines, causing pulmonary fibrosis by mediating immune responses. Pulmonary fibrosis of unknown etiology includes idiopathic pulmonary fibrosis (IPF). IPF belongs to the idiopathic interstitial pneumonia (IIP) group in the interstitial lung disease (ILDs) family. It is the most common and most serious chronic interstitial lung disease of unknown etiology. IPF originates from abnormal repair after recurrent or persistent alveolar epithelial damage. Under multiple persistent injuries from known or unknown endogenous and exogenous damaging factors, damaged lung epithelial cells initiate the damage repair mechanism, epithelial regeneration and repair are insufficient, and the remaining cells undergo mesenchymal transformation, presenting a pro-fibrotic phenotype, secreting a large number of pro-fibrotic factors, causing abnormal activation and proliferation of fibroblasts, and excessive extracellular matrix deposition, leading to the formation of fibrous scars and honeycomb cysts, destroying the alveolar structure, and causing an irreversible and continuous decline in lung function, ultimately leading to respiratory failure and death. IPF is clinically manifested as progressive dyspnea accompanied by irritating dry cough, and the disease often continues to progress.

[0003] Currently, there are two pulmonary fibrosis treatment drugs on the market worldwide, namely pirfenidone and nintedanib, both of which can slow down the decline of forced vital capacity (FVC) of patients, but are not effective in improving acute exacerbation of IPF and prolonging survival. Therefore, it is of great significance to seek safe and effective new drugs for the prevention and / or treatment of pulmonary fibrosis and its indications.

[0004] Macrophages are the most abundant immune cells in lung tissue (about 70%), and play an important role in the pathogenesis of pulmonary fibrosis, and participate in the regulation and repair of tissue abnormalities in pulmonary fibrosis. Macrophages include M1 macrophages, M2 macrophages, and M2-like macrophages. Studies have shown that proinflammatory factors and oxygen free radicals are related to the occurrence and development of pulmonary fibrosis. In the bleomycin-induced pulmonary fibrosis model, inhibiting the expression of M1 macrophage FR-β in the validation stage can reduce pulmonary fibrosis. However, anti-infection treatment has no effect on patients with pulmonary fibrosis, and the role of inflammation in pulmonary fibrosis has been questioned. The researchers used a bleomycin-induced mouse pulmonary fibrosis model and found that the elimination of inherent M1 macrophages and circulating monocytes in the mouse lungs during the inflammatory stage did not affect the occurrence and development of pulmonary fibrosis. The number of M2 macrophages in the alveolar lavage fluid of IPF patients increased. At the same time, studies have shown that M2 macrophages may have anti-fibrotic effects, and M2 macrophage markers-mannose receptor and MFGE8 both have anti-fibrotic effects. M2-like macrophages are more profibrotic and can overexpress IL-10 to promote fibrosis. Glucocorticoids can induce M2-like macrophages, which explains why glucocorticoid treatment is not only ineffective in some fibrosis patients, but also has certain harms and aggravates the degree of fibrosis.

[0005] CD84 (SLAMF5) is a member of the signaling lymphocyte activation molecule (SLAM) family. It is mainly expressed in bone marrow-derived lymphocytes, monocytes, macrophages, etc., and plays an important role in immune cell communication, differentiation, and microenvironment regulation. Currently, CD84 is often used as a biomarker for some immune-related diseases in clinical practice. For example, in rheumatoid arthritis, CD84 + Patients are more likely to benefit from anti-TNF-α therapy; in patients with systemic lupus erythematosus, high expression of CD84 is significantly positively correlated with disease progression. Some in vitro studies have also demonstrated the potential clinical value of anti-CD84 therapy, such as CD84 neutralizing antibodies can significantly alleviate T cell differentiation in RA mouse models and alleviate the malignant progression of chronic lymphatic leukemia mouse models, indicating that CD84 may be a reliable target for the clinical treatment of lymphatic leukemia. However, there are currently no reports on the involvement of CD84 in the formation or treatment of pulmonary fibrosis. Summary of the invention

[0006] In view of this, the purpose of the present invention is to provide a use of a CD84 inhibitor in the preparation of a drug for preventing and treating pulmonary fibrosis, by inhibiting the expression of CD84 gene or protein or blocking the biological function of CD84 to inhibit the activation of macrophages and their transfer to pulmonary fibrosis, thereby achieving the purpose of preventing and / or treating pulmonary fibrosis.

[0007] The present invention provides an application of an anti-CD84 antibody in preparing a medicine for preventing and / or treating pulmonary fibrosis.

[0008] Preferably, the anti-CD84 antibody comprises an anti-CD84 monoclonal antibody and / or an anti-CD84 polyclonal antibody.

[0009] Preferably, the monoclonal antibodies against CD84 include CA2 monoclonal antibody and AD5 monoclonal antibody.

[0010] Preferably, the amino acid sequence of the heavy chain variable region sequence of the CA2 monoclonal antibody is shown as SEQ ID NO:1; and the amino acid sequence of the light chain variable region sequence of the CA2 monoclonal antibody is shown as SEQ ID NO:2.

[0011] Preferably, the amino acid sequence of the heavy chain variable region sequence of the AD5 monoclonal antibody is shown as SEQ ID NO:3; the amino acid sequence of the light chain variable region sequence of the AD5 monoclonal antibody is shown as SEQ ID NO:4.

[0012] Preferably, the pulmonary fibrosis includes idiopathic pulmonary fibrosis and / or pulmonary fibrosis indications.

[0013] Preferably, the pulmonary fibrosis indications include one or more of the following: systemic sclerosis-related interstitial lung disease, spontaneous systemic lupus erythematosus interstitial lung disease, rheumatoid arthritis-related interstitial lung disease, myositis / dermatomyositis-related interstitial lung disease and hypersensitivity pneumonitis-related interstitial lung disease.

[0014] Preferably, the drug is a drug that inhibits the activation of macrophages;

[0015] Preferably, the macrophages include pro-inflammatory macrophages and / or pro-fibrotic macrophages.

[0016] The present invention also provides a medicine for preventing and / or treating pulmonary fibrosis, comprising an anti-CD84 antibody and a medically acceptable adjuvant.

[0017] Preferably, the anti-CD84 antibody comprises an anti-CD84 monoclonal antibody and / or an anti-CD84 polyclonal antibody;

[0018] Preferably, the monoclonal antibodies against CD84 include CA2 monoclonal antibody and AD5 monoclonal antibody;

[0019] Further preferably, the amino acid sequence of the heavy chain variable region sequence of the CA2 monoclonal antibody is shown as SEQ ID NO:1; and the amino acid sequence of the light chain variable region sequence of the CA2 monoclonal antibody is shown as SEQ ID NO:2.

[0020] Further preferably, the amino acid sequence of the heavy chain variable region sequence of the AD5 monoclonal antibody is shown as SEQ ID NO:3; the amino acid sequence of the light chain variable region sequence of the AD5 monoclonal antibody is shown as SEQ ID NO:4.

[0021] The present invention provides an application of an anti-CD84 antibody in the preparation of a drug for preventing and / or treating pulmonary fibrosis. The present invention utilizes an anti-CD84 antibody to inhibit the biological function of the CD84 protein, which not only effectively reduces the degree of pulmonary fibrosis in bleomycin-induced pulmonary fibrosis and a mouse disease model of pulmonary fibrosis indications, but also effectively inhibits the activation of pro-inflammatory macrophages and / or pro-fibrotic macrophages, thereby preventing further deterioration of pulmonary fibrosis. It can be seen that the application provided by the present invention provides a new means for the prevention and treatment of pulmonary fibrosis.

[0022] Furthermore, the present invention specifically defines the types of pulmonary fibrosis, specifically including idiopathic pulmonary fibrosis and / or pulmonary fibrosis indications. The pulmonary fibrosis indications include one or more of the following: systemic sclerosis-related interstitial lung disease, spontaneous systemic lupus erythematosus interstitial lung disease and rheumatoid arthritis-related interstitial lung disease. In the embodiments of the present invention, by inhibiting the function of CD84 protein, it is possible to significantly inhibit skin thickening, reduce the area of ​​pulmonary fibrosis, reduce hydroxyproline content and collagen content, and thereby alleviate bleomycin-induced collagen deposition in mouse skin, thereby achieving the purpose of treating bleomycin-induced systemic sclerosis-related interstitial lung disease. At the same time, the embodiment of the present invention also verifies the therapeutic effect of inhibiting the function of CD84 protein on spontaneous systemic lupus erythematosus interstitial lung lesions. The results show that blocking CD84 can alleviate spontaneous skin thickening in MRL / Lpr mice, alleviate spontaneous splenomegaly in MRL / Lpr mice, alleviate concurrent fibrosis of lung tissue caused by SLE, and alleviate inflammatory cell infiltration of lung tissue caused by SLE. It can be seen that inhibiting the function of CD84 achieves the purpose of treating spontaneous systemic lupus erythematosus interstitial lung lesions. In addition, the present invention also verifies the role of CD84 in the occurrence and development of rheumatoid arthritis-related interstitial lung disease. By blocking CD84, the severity of arthritis caused by type II collagen immunity can be alleviated, the formation of lung fibrosis caused by RA can be alleviated, and the infiltration of inflammatory cells in lung tissue caused by RA can be alleviated. It can be seen that inhibiting the function of CD84 achieves the purpose of treating rheumatoid arthritis-related interstitial lung disease. In summary, CD84 can be used as a therapeutic target for systemic sclerosis-related interstitial lung disease, spontaneous systemic lupus erythematosus interstitial lung disease and rheumatoid arthritis-related interstitial lung disease, providing new ideas for the development, screening and preparation of related clinical drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 is the binding curve of CD84 monoclonal antibody CA2 with hCD84 and mCD84;

[0024] Figure 2 The results show that CD84 monoclonal antibody CA2 inhibits IL-4 / 13-induced activation of profibrotic macrophages;

[0025] Figure 3 The results are as follows: CD84 monoclonal antibody CA2 inhibits LPS / IFN-γ-induced pro-inflammatory macrophage activation; A is the expression result of IL-6; B is the expression result of TNF-α; C is IL-1β;

[0026] Figure 4 is the binding curve of CD84 monoclonal antibody AD5 with hCD84 and mCD84;

[0027] Figure 5 The results show that CD84 monoclonal antibody AD5 inhibits IL-4 / 13-induced activation of profibrotic macrophages;

[0028] Figure 6 These are the results of CD84 monoclonal antibody AD5 inhibiting LPS / IFN-γ-induced pro-inflammatory macrophage activation; A is the expression result of IL-6; B is the expression result of TNF-α; and C is IL-1β. DETAILED DESCRIPTION

[0029] The present invention provides an application of an anti-CD84 antibody in preparing a medicine for preventing and / or treating pulmonary fibrosis.

[0030] In the present invention, the CD84 preferably includes a macrophage-derived CD84 gene and / or a CD84 protein. The macrophage is a macrophage derived from lung tissue.

[0031] In the present invention, the anti-CD84 antibody preferably includes an anti-CD84 protein monoclonal antibody or an anti-CD84 protein polyclonal antibody. The monoclonal antibody of CD84 preferably includes CA2 monoclonal antibody and AD5 monoclonal antibody. The amino acid sequence of the heavy chain variable region sequence of the CA2 monoclonal antibody is preferably as shown in SEQ ID NO:1; the amino acid sequence of the light chain variable region sequence of the CA2 monoclonal antibody is preferably as shown in SEQ ID NO:2. The amino acid sequence of the heavy chain variable region sequence of the AD5 monoclonal antibody is preferably as shown in SEQ ID NO:3; the amino acid sequence of the light chain variable region sequence of the AD5 monoclonal antibody is preferably as shown in SEQ ID NO:4. The present invention has no special restrictions on the preparation method of the CD84 protein polyclonal antibody, and the preparation method of polyclonal antibodies well known in the art can be used, for example, using CD84 protein as an antigen to immunize an animal, blood collection and serum collection, and antibodies that can bind to CD84 protein are identified as polyclonal antibodies. The present invention has no special restrictions on the preparation method of the anti-CD84 protein monoclonal antibody, and the preparation method of monoclonal antibodies well known in the art can be used, such as in vitro recombinant expression.

[0032] In the present invention, the pulmonary fibrosis preferably includes idiopathic pulmonary fibrosis. The idiopathic pulmonary fibrosis (IPF) is manifested as abnormal repair after alveolar epithelial damage. Under multiple continuous damages of exogenous and exogenous damaging factors, the damage repair mechanism is activated, the epithelial regeneration and repair are insufficient, and the remaining cells undergo mesenchymal transformation, presenting a pro-fibrotic phenotype, secreting a large amount of pro-fibrotic factors, causing abnormal activation and proliferation of fibroblasts, and producing excessive extracellular matrix deposition, leading to the formation of fibrous scars and honeycomb capsules, and the alveolar structure is destroyed, causing the irreversible continuous decline of lung function, and ultimately leading to respiratory failure and death. IPF is clinically manifested as progressive dyspnea with irritating dry cough, and the condition often continues to progress. In an embodiment of the present invention, the pulmonary fibrosis is preferably induced by bleomycin.

[0033] In the present invention, the drug is preferably a drug that inhibits the activation of macrophages. The macrophages preferably include pro-inflammatory macrophages and / or pro-fibrotic macrophages.

[0034] In the present invention, the bleomycin-induced pulmonary fibrosis disease model was used as the research object. The expression of CD84 in the peripheral blood of patients with pulmonary fibrosis was detected. The results showed that compared with the healthy control group, the expression of CD84 in the peripheral blood of IPF patients increased significantly, with significant differences. +Macrophages infiltrate and promote the formation of fibrotic foci in the lung tissue of the pulmonary fibrosis model. This suggests that CD84 may be involved in the development of pulmonary fibrosis. The present invention further confirms the connection between the development of pulmonary fibrosis by regulating the expression level of CD84 or the function of the protein. Compared with the control group, the function of CD84 in the macrophages can reduce the migration of macrophages to the lungs in pulmonary fibrosis, and inhibit the activation of profibrotic macrophages and proinflammatory macrophages, thereby inhibiting the expression of profibrotic factors (TGF-β1 and IL-10) and proinflammatory factors (IL-6, TNF-α and IL-1β), and inhibiting the progress of pulmonary fibrosis to a certain extent. At the same time, compared with the control, the hydroxyproline content can be significantly reduced by missing CD84 protein in macrophages, thereby effectively reducing the collagen deposition in lung tissue, and the lack of CD84 in macrophages can significantly reduce the area of ​​bleomycin-induced pulmonary fibrosis, thereby inhibiting the formation of pulmonary fibrosis.

[0035] In the present invention, the pulmonary fibrosis preferably also includes pulmonary fibrosis indications. The pulmonary fibrosis indications preferably include one or more of the following: systemic sclerosis-related interstitial lung disease, spontaneous systemic lupus erythematosus interstitial lung disease, rheumatoid arthritis-related interstitial lung disease, myositis / dermatomyositis-related interstitial lung disease and hypersensitivity pneumonitis-related interstitial lung disease.

[0036] In the examples of the present invention, the thickness of the dermis, the degree of pulmonary fibrosis, and the hydroxyproline content of the skin and lung tissues were used as detection indicators to evaluate whether CD84 could be used as a therapeutic target for interstitial lung disease associated with systemic sclerosis. The results showed that blocking CD84 with a monoclonal antibody could alleviate the thickening of mouse skin caused by bleomycin, significantly reduce the collagen content in the skin, and thus reduce the collagen deposition in the mouse skin caused by bleomycin, significantly reduce the area of ​​pulmonary fibrosis caused by SSc, and ultimately achieve the purpose of alleviating the condition of interstitial lung disease associated with systemic sclerosis.

[0037] In the examples of the present invention, the dermis thickness, spleen coefficient, degree of pulmonary fibrosis, and total number of BALF cells were used as detection indicators to evaluate whether CD84 could be used as a therapeutic target for spontaneous interstitial lung lesions in systemic lupus erythematosus. The results showed that blocking CD84 with monoclonal antibodies could alleviate mouse skin thickening, alleviate spontaneous splenomegaly in MRL / Lpr mice, significantly reduce the area of ​​fibrosis, and significantly reduce the total number of BALF cells, thereby alleviating inflammatory cell infiltration in lung tissue caused by SLE. It can be seen that the therapeutic purpose of spontaneous interstitial lung lesions in systemic lupus erythematosus can be achieved by inhibiting the function of CD84.

[0038] In the embodiments of the present invention, RA score, degree of pulmonary fibrosis and total number of BALF cells are used as detection indicators to evaluate whether CD84 can be used as a therapeutic target for interstitial lung disease associated with rheumatoid arthritis. The present invention uses male DBA1 / J wild-type mice induced by type II collagen as a model. The results show that blocking CD84 by monoclonal antibodies can alleviate mouse skin thickening, alleviate spontaneous splenomegaly in MRL / Lpr mice, significantly reduce the area of ​​fibrosis, and significantly reduce the total number of BALF cells, thereby alleviating inflammatory cell infiltration of lung tissue caused by SLE. It can be seen that by inhibiting the function of CD84, the therapeutic purpose of spontaneous systemic lupus erythematosus interstitial lung lesions can be achieved.

[0039] The present invention also provides a medicine for preventing and / or treating pulmonary fibrosis, comprising an anti-CD84 antibody and a medically acceptable adjuvant.

[0040] In the present invention, the anti-CD84 antibody includes anti-CD84 monoclonal antibody and / or anti-CD84 polyclonal antibody; the CD84 monoclonal antibody includes CA2 monoclonal antibody and AD5 monoclonal antibody. The CA2 monoclonal antibody and AD5 monoclonal antibody are preferably obtained by recombinant expression. Experimental verification shows that the CA2 monoclonal antibody and AD5 monoclonal antibody have good affinity for CD84 antigen, ensuring the function of inhibiting CD84, thereby achieving the prevention and / or treatment of pulmonary fibrosis.

[0041] The present invention has no special restrictions on the types of the auxiliary materials, and any auxiliary materials known in the art can be used. The present invention has no special restrictions on the preparation method of the drug, and any preparation method of the drug known in the art can be used.

[0042] The application of an anti-CD84 antibody provided by the present invention in the preparation of a drug for preventing and treating pulmonary fibrosis is described in detail below in conjunction with the examples, but they should not be construed as limiting the scope of protection of the present invention.

[0043] Example 1

[0044] CD84 monoclonal antibody CA2 antigen affinity test

[0045] 1. Experimental Methods

[0046] 293T cells were inoculated into 6-well plates, and hCD84 (NM_001184879.2) and mCD84 (NM_013489.4) were overexpressed by transfecting plasmid pCDNA3.1. In each well of each group, 200 μL of OPTI-MEM was used as the dispersion system, and 2 μg of pCDNA3.1 and 6 μL of transfection reagent (Lipo2000) were added. After incubation for 5 minutes, the two were mixed, and the bottom of the tube was flicked to mix them thoroughly. After incubation for 10 minutes, they were added to the well plate. After 48 hours, the samples were collected and the binding of anti-humanCD84 antibody CA2 to hCD84 and mCD84 was detected by flow cytometry. The light chain variable region sequence Lc of CA2 antibody is as follows:

[0047] MRPSIQFLGLLLFWLHGAQCDIQMTQSPSSSLSASLGGKVTITCKASQDINKYIAWYQQKSGKGPRLLIHYTSTLQPGIPSRFSGSGSGRDYSFSISNLEPEDIATYYCLQYDNLYTFGGGTKLEIK(SEQ ID NO:1);

[0048] The heavy chain variable region sequence Hc of the CA2 antibody is as follows:

[0049] EVQLVESGGDLVRPGGSLKLSCAASGFTFTDYGMSWVRQTPDKRLEWVATISTASTYTFYPDSVKGRFTISRDNAKNTLYLQMSSLKSEDTAIFYCVRHGHYYGSRYTWLAYWGQGTLVTVSA (SEQ ID NO: 2).

[0050] The preparation of CA2 antibody is to clone the gene sequence encoding CA2 sequence into the eukaryotic expression vector pCDNA3.4, transfect into 293F cells, collect the culture supernatant after 7 days of culture, and obtain it by protein G gel purification. Among them, the gene sequence encoding CA2 light chain variable region is as follows:

[0051] atgagacccagcatccagttcctgggcctgctgctgttctggctgcacggcgcccagtgcgacatccagatgacccagagccccagcagcctgagcgccagcctgggcggcaaggtgaccatcacctgcaaggccagccaggacatcaacaagtacatcgcctggtaccagcagaagagcggcaagggccccagactgctgatccactacaccagcaccctgcagcccggcatccccagcagattcagcggcagcggcagcggcagagactacagcttcagcatcagcaacctggagcccgaggacatcgccacctactactgcctgcagtacgacaacctgtacaccttcggcggcggcaccaagctggagatcaag(SEQ ID NO:5). The gene sequence encoding the variable region of the CA2 heavy chain is as follows:

[0052] gaggtgcagctggtggagagcggcggcgacctggtgagacccggcggcagcctgaagctgagctgcgccgccagcggcttcaccttcaccgactacggcatgagctgggtgagacagacccccgacaagagactggagtgggtggccaccatcagcaccgccagcacctacaccttctaccccgacagcgtgaagggcagattcaccatcagcagagacaacgccaagaacaccctgtacctgcagatgagcagcctgaagagcgaggacaccgccatcttctactgcgtgagacacggccactactacggcagcagatacacctggctggcctactggggccagggcaccctggtgaccgtgagcgcc(SEQID NO:6).

[0053] 2. Experimental results

[0054] Digest 293T cells with trypsin and resuspend, add 800 μL PBS to wash, centrifuge at 800 rpm for 5 min, discard the supernatant, add 1 mL Cell Staining buffer to wash, centrifuge at 800 rpm for 5 min, discard the supernatant, resuspend with 100 μL Cell Staining buffer, add 1 μL blocking solution TruStain FCXTM, block on ice for 15 min, add 800 μL Cell Staining buffer to wash once, centrifuge at 800 rpm for 5 min, discard the supernatant, and resuspend with 100 μL Cell Staining buffer. Then, 0.1, 1, 3, 10 and 30 μg / mL of CA2 or control IgG antibody was added to each well and incubated on ice for 30 min. At the end of incubation, 800 μL of Cell Staining buffer was added and washed twice. Then, anti-mouse-FITC (1:200) was added and incubated on ice for 45 min. At the end of incubation, 800 μL of Cell Staining buffer was added and washed twice. After resuspending in 200 μL PBS, the binding of CA2 to hCD84 or mCD84 was detected by flow cytometry.

[0055] Depend on Figure 1 It can be seen that compared with the control IgG, CA2 has obvious binding curves with both hCD84 and mCD84, proving that CA2 can bind to human and mouse CD84 antigens.

[0056] Example 2

[0057] CD84 monoclonal antibody CA2 can inhibit the activation of profibrotic macrophages

[0058] 1. Experimental Methods

[0059] CA2 monoclonal antibody inhibits the activation of profibrotic macrophages

[0060] THP1 cells were inoculated in 6-well plates under sterile conditions, with 1×10 cells in each well. 6 , PMA was added 24 hours later to make its working solution 100 ng / mL, and control antibody (Ctl IgG) and anti-human CD84 monoclonal antibody CA2 were added 2.5 μg each after PMA stimulation for 24 hours. Interleukin 4 (IL-4) and interleukin 13 (IL-13) were added 6 hours later with a working concentration of 20 ng / mL to co-stimulate THP1 cells, and the cells were collected 24 hours later.

[0061] Add 1mL Trizol to each well, blow off the cells, transfer to a 1.5mL RNase free centrifuge tube, and let stand for 5-10min to fully lyse the cells. Add 200μL chloroform to each tube, vortex thoroughly for 15s, and let stand at room temperature for 2-3min. Centrifuge at 4℃12000rpm for 15min, transfer 400μL of the upper water layer to a new RNase free 1.5mL centrifuge tube, add 400μL isopropanol, mix well, and let stand at room temperature for 10min. Centrifuge at 4℃12000rpm for 10min to obtain flocculent precipitate as RNA, discard the supernatant, add 1mL 75% ethanol, and wash the precipitate. Centrifuge at 4℃7000rpm for 5min, discard the supernatant, repeat once, aspirate the excess solution, evaporate at room temperature, and the RNA becomes colorless, indicating that the ethanol has evaporated completely. Add RNase free water to dissolve, place in an ice box for 15min, and use a micro-spectrophotometer to detect and record the RNA concentration. Then, the RNA was reversed into cDNA, and the reverse program was: 42℃15min, 95℃3min. Then, the RT-PCR system was configured: 2×SYBR Green Mix 10μL per tube, 1μL of upstream and downstream primers per tube, 6μL of RNase free water, and 2μL of cDNA template. After completion, Roche was used to detect the expression of the target gene. The primer sequences involved are shown in Table 1:

[0062] Table 1 Primer sequences

[0063]

[0064] Depend on Figure 1 It can be seen that CA2 can significantly inhibit the activation of profibrotic macrophages induced by IL-4 / 13, as manifested by the significant inhibition of the expression of profibrotic factors TGF-β1 and IL-10.

[0065] Example 3

[0066] CD84 monoclonal antibody CA2 can inhibit the activation of pro-inflammatory macrophages

[0067] 1. Experimental Methods

[0068] THP1 cells were inoculated in 6-well plates under sterile conditions, with 1×10 cells in each well. 6 , PMA was added 24 hours later to make the working solution 100 ng / mL, 2.5 μg each of control antibody (Ctl IgG) and anti-human CD84 monoclonal antibody CA2 were added 24 hours after PMA stimulation, LPS / IFN-γ was added 6 hours later, the working solution concentration was 100 ng / mL, and the samples were collected 24 hours later.

[0069] Add 1mL Trizol to each well, blow off the cells, transfer to a 1.5mL RNase free centrifuge tube, and let stand for 5-10min to fully lyse the cells. Add 200μL chloroform to each tube, vortex thoroughly for 15s, and let stand at room temperature for 2-3min. Centrifuge at 4℃12000rpm for 15min, transfer 400μL of the upper water layer to a new RNase free 1.5mL centrifuge tube, add 400μL isopropanol, mix well, and let stand at room temperature for 10min. Centrifuge at 4℃12000rpm for 10min to obtain flocculent precipitate as RNA, discard the supernatant, add 1mL 75% ethanol, and wash the precipitate. Centrifuge at 4℃7000rpm for 5min, discard the supernatant, repeat once, aspirate the excess solution, evaporate at room temperature, and the RNA becomes colorless, indicating that the ethanol has evaporated completely. Add RNase free water to dissolve, place in an ice box for 15min, and use a micro-spectrophotometer to detect and record the RNA concentration. Then, RNA was reversed into cDNA, and the reverse program was: 42℃15min, 95℃3min. Then, the RT-PCR system was configured: 2×SYBR Green Mix 10μL per tube, 1μL of upstream and downstream primers per tube, 6μL of RNase free water, and 2μL of cDNA template. The primer sequences used are shown in Table 2:

[0070] Table 2 Primer sequences

[0071]

[0072] After completion, the eight rows of tubes were placed in a Roche LC96 instrument and the program was set as pre-denaturation: 95°C (300s); denaturation: 95°C (30s); annealing: 55°C (30s); extension: 72°C (30s), for a total of 40 cycles to detect the expression of the target gene.

[0073] Depend on Figure 2 It can be seen that CA2 monoclonal antibody can inhibit LPS / IFN-γ-induced macrophage activation, which is manifested by the significant inhibition of the expression of inflammatory factors IL-6, TNF-α and IL-1β.

[0074] Example 4

[0075] CD84 monoclonal antibody CA2 can inhibit bleomycin-induced pulmonary fibrosis

[0076] 1. Experimental Methods

[0077] C57BL6 / J male mice, 7-8 weeks old, 8 mice in each group. The pulmonary fibrosis model was established by intratracheal injection of bleomycin (2U / kg), and the control group was injected with NaCl. The day of modeling was day 0, and -CA2 (5mg / kg) was intraperitoneally injected on days 0-7 of modeling, once every 2 days, and the mice were killed on the 21st day.

[0078] 2. Experimental results

[0079] 2.1 Determination of Hydroxyproline Content: After the mice were dissected, the hydroxyproline (HYP) content of the mouse lung tissue was determined. Specifically, after the mice were killed, the right lung of the mouse was separated, placed in a 5mL ampoule, placed in a 120°C oven for drying, hydrolyzed under the action of hydrochloric acid, the pH value was adjusted to 6.5-8.0, the residue was filtered with a 0.5μm filter membrane, PBS (phosphate-balanced saline) was added to adjust the total volume to 10mL, 50μL of sample was taken, 350μL of deionized water was added, 200μL of chloramine T solution was added and incubated at room temperature for 20min, 200μL of perchloric acid was added and incubated at room temperature for 5min, 200μL of p-dimethylaminobenzaldehyde (P-DMAB) was added and incubated at 65°C for 20min. Take 200 μL to a 96-well plate to measure the absorbance of the sample at 570 nm, draw a standard curve using the standard reading, and then calculate the hydroxyproline concentration Cs of the measured sample according to the formula obtained from the standard curve. Convert it to the amount of hydroxyproline contained in the entire right lung according to the following formula I.

[0080] The amount of hydroxyproline contained in the entire right lung = Cs × 8 (dilution factor of the measured sample) × 10 (total sample volume) Formula I

[0081] The results are shown in Table 3. Compared with the control group, the HYP content in the right lung of the model group was significantly increased, and the HYP content in the CA2 group was significantly decreased compared with the BLM group, indicating that the loss of CD84 in macrophages can significantly reduce the collagen deposition in lung tissue caused by bleomycin.

[0082] Table 3 Effect of CA2 on hydroxyproline content in mouse lung tissue

[0083] Grouping NaCl BLM CA2 HYP (μg / right whole lung) 91.21±3.17 161.42±10.51 100.15±5.91

[0084] Note: Data are expressed as "mean ± standard error".

[0085] 2.2 Statistics of pulmonary fibrosis area: The lung tissues of mice were paraffin-embedded, and then H&E staining was performed on the lung tissue sections of mice. After collecting images, the pulmonary fibrosis area in the sections was calculated using Image-Pro Plus software. The percentage of pulmonary fibrosis area = pulmonary fibrosis area / total area of ​​lung sections × 100%. As shown in Table 4, the average pulmonary fibrosis area of ​​mice in the BLM group reached 28.5%, while the pulmonary fibrosis area of ​​the CA2 group decreased to 17.2%, indicating that CA2 monoclonal antibody can significantly reduce the formation of bleomycin-induced pulmonary fibrosis.

[0086] Table 4 Effect of CD84 deficiency on the area of ​​pulmonary fibrosis in mice

[0087] Grouping BLM CA2 Pulmonary fibrosis area percentage (%) 28.54±4.15 17.15±3.92

[0088] Note: Data are expressed as "mean ± standard error".

[0089] Example 5

[0090] CD84 monoclonal antibody CA2 can inhibit bleomycin-induced systemic sclerosis-related interstitial lung disease (SSc-ILD)

[0091] 1. Experimental Methods

[0092] Male C57BL / 6 mice were subcutaneously injected with bleomycin (0.2U / mouse / day) for 28 consecutive days to establish the SSc-ILD model. The day of modeling was D1, and the mice were weighed every day. Seven days after modeling, the mice were regrouped according to the weight loss rate, with 8 mice in each group. The specific groupings were: control group (NaCl), model group (BLM), CA2 (Ab-CD84, 5mg / kg) group. The mice were treated on the 7th day. The Ab-CD84 group was intraperitoneally injected once every 3 days according to the dose, and the NaCl group and BLM group were injected with an equal amount of saline as a control. The general state of the mice was observed once in the morning and afternoon every day. The mice were killed and analyzed after the drug was given on the 28th day.

[0093] 2. Index detection

[0094] After the experiment, the dermis thickness, degree of pulmonary fibrosis, and hydroxyproline content in skin and lung tissues of each group of mice were tested. The experimental results are as follows:

[0095] 2.1 Dermis thickness detection: The dermis fibrosis thickness detection was specifically to fix the skin samples with 10% formalin, dehydrate, embed in paraffin, cut into 5 μm thick sections, and perform H&E staining. Table 5 shows the statistical results of the dermis thickness of each group of mice. Compared with the control group, the skin of the BLM group mice was significantly thickened, while the skin thickness of the CA2 administration group mice was significantly reduced, indicating that blocking CD84 with CA2 can alleviate the thickening of mouse skin caused by bleomycin.

[0096] Table 5 Effect of CA2 on skin thickness in SSc-ILD mice

[0097] Grouping NaCl BLM CA2 Dermis thickness (μm) 203.5±13.5 372.2±17.1 229.4±27.9

[0098] Note: Data are expressed as "mean ± standard error".

[0099] 2.2 Determination of skin collagen content: Skin collagen content detection is hydroxyproline content detection. Specifically, after killing mice, separate mouse skin tissue, put it into a 5mL ampoule, place it in a 120℃ oven to dry, adjust the pH value to 6.5-8.0 after hydrolysis under the action of hydrochloric acid, filter the residue with a 0.5μm filter membrane, add PBS (phosphate-balanced saline) to adjust the total volume to 10mL, take 50μL of sample, add 350μL of deionized water, add 200μL of chloramine T solution and incubate at room temperature for 20min, add 200μL of perchloric acid and incubate at room temperature for 5min, add 200μL of p-dimethylaminobenzaldehyde (P-DMAB) and incubate at 65℃ for 20min. Take 200μL to a 96-well plate to measure the absorbance of the sample at 570nm, draw a standard curve using the standard reading, and then calculate the hydroxyproline concentration Cs of the measured sample according to the formula obtained from the standard curve. The hydroxyproline content per unit weight of skin is calculated according to the following formula III.

[0100] The amount of hydroxyproline contained in the skin = Cs × 8 (dilution factor of the sample) × 10 (total sample volume) / 0.01 (skin weight, g) Formula III

[0101] As shown in Table 6, compared with the control group, the HYP content per gram of skin in the BLM group mice was significantly increased, while the HYP content in the skin of the CA2-administered mice was significantly decreased, which proves that blocking CD84 with CA2 can effectively alleviate the collagen deposition in the mouse skin caused by bleomycin.

[0102] Table 6 Effect of CA2 on collagen content in the skin of SSc-ILD mice

[0103] Grouping NaCl BLM CA2 Skin HYP (μg / g) 173.6±10.8 231.6±16.1 192.5±16.2

[0104] Note: Data are expressed as "mean ± standard error".

[0105] 2.3 Statistics of pulmonary fibrosis degree: After drug administration, H&E staining was performed on the lung tissue sections of mice. After collecting images, the pulmonary fibrosis area in the sections was calculated using Image-Pro Plus software, and the percentage of pulmonary fibrosis area was calculated according to Formula II.

[0106] Pulmonary fibrosis area percentage = pulmonary fibrosis area / total lung section area × 100% Formula II

[0107] As shown in Table 7, SSc caused by continuous subcutaneous injection of BLM can further lead to fibrosis of lung tissue, while the fibrosis area of ​​lung tissue in mice in the CA2 administration group was significantly reduced, indicating that blocking CD84 with CA2 can alleviate the formation of pulmonary fibrosis caused by SSc.

[0108] Table 7 Effect of CA2 on the area of ​​pulmonary fibrosis in SSc-ILD mice

[0109] Grouping BLM CA2 Pulmonary fibrosis area percentage (%) 3.2±0.25 0.46±0.18

[0110] Note: Data are expressed as "mean ± standard error".

[0111] 2.4 Determination of lung hydroxyproline content: Lung collagen content detection is also known as hydroxyproline content detection. Specifically, after killing mice, the mouse skin tissue or right lung is separated and placed in a 5mL ampoule bottle, placed in a 120℃ oven for drying, hydrolyzed under the action of hydrochloric acid, the pH value is adjusted to 6.5-8.0, the residue is filtered with a 0.5μm filter membrane, PBS (phosphate-balanced saline) is added to adjust the total volume to 10mL, 50μL of sample is taken, 350μL of deionized water is added, 200μL of chloramine T solution is added and incubated at room temperature for 20min, 200μL of perchloric acid is added and incubated at room temperature for 5min, 200μL of p-dimethylaminobenzaldehyde (P-DMAB) is added and incubated at 65℃ for 20min. 200μL is taken to a 96-well plate to measure the absorbance of the sample at 570nm, and a standard curve is drawn using the standard readings, and then the hydroxyproline concentration Cs of the measured sample is calculated according to the formula obtained from the standard curve. The hydroxyproline content was obtained by converting the hydroxyproline content in the entire right lung into the amount of hydroxyproline contained in the entire right lung according to the following formula I.

[0112] The amount of hydroxyproline contained in the entire right lung = Cs × 8 (dilution factor of the sample tested) × 10 (total sample volume) Formula I

[0113] As shown in Table 8, compared with the control group, the HYP content in the right whole lung of the BLM group was significantly increased, while the HYP content in the right whole lung of the mice in the CA2 administration group was significantly decreased, which proves that blocking CD84 with CA2 can effectively alleviate the collagen deposition in the lungs of mice caused by SSc.

[0114] Table 8 Effect of CA2 on collagen content in the right lung of SSc-ILD mice

[0115] Grouping NaCl BLM CA2 Lung tissue HYP (μg / right whole lung) 118.1±2.5 146.9±1.9 125.3±4.3

[0116] Note: Data are expressed as "mean ± standard error".

[0117] Example 6

[0118] CD84 monoclonal antibody CA2 can inhibit spontaneous systemic lupus erythematosus interstitial lung disease (SLE-ILD) in MRL / Lpr mice

[0119] 1. Experimental Methods

[0120] MRL / Lpr male mice, 5 mice per group. Spontaneous SLE-ILD occurred when the mice were raised to 16 weeks of age, and drug administration began: control group (Ctl), model group (Model), CA2 (Ab-CD84, 5mg / kg) group. The mice were treated with drug administration from the 16th week. The Ab-CD84 group was intraperitoneally injected once every 3 days according to the dose, and the NaCl group and the Model group were injected with an equal amount of saline as a control. The general condition of the mice was observed once in the morning and afternoon every day, and the drug administration lasted until the 18th week.

[0121] 2. Index detection

[0122] 2.1 Dermis thickness detection: The dermis fibrosis thickness detection was specifically to fix the skin samples with 10% formalin, dehydrate, embed in paraffin, cut into 5 μm thick sections, and perform H&E staining. Table 9 shows the statistical results of the dermis thickness of each group of mice. Compared with the control group, the skin of the Model group mice was significantly thickened, while the skin thickness of the CA2 administration group mice was significantly reduced, indicating that the use of CA2 to block CD84 can alleviate the spontaneous skin thickening of MRL / Lpr mice.

[0123] Table 9 Effect of CA2 on skin thickness in SLE-ILD mice

[0124] Grouping NaCl Model CA2 Dermis thickness (μm) 363.4±18.1 447.3±19.6 382.9±20.7

[0125] Note: Data are expressed as "mean ± standard error".

[0126] 2.2 Spleen coefficient: The spleen coefficient was specifically measured by weighing the spleen weight of the mice after the mice were killed, and the spleen coefficient was obtained by spleen weight / body weight. The specific results are shown in Table 10. As shown in Table 10, compared with the control group, the spleen coefficient of the model group was significantly increased, while the spleen coefficient of the mice in the CA2 administration group was significantly decreased, indicating that blocking CD84 with -CA2 can alleviate the spontaneous splenomegaly of MRL / Lpr mice.

[0127] Table 10 Effect of CA2 on spleen coefficient of SLE-ILD mice

[0128] Grouping NaCl Model CA2 Spleen coefficient 0.00435±0.00026 0.015913±0.00051 0.007429±0.00037

[0129] Note: Data are expressed as "mean ± standard error".

[0130] 2.3 Statistics of pulmonary fibrosis degree: After drug administration, H&E staining was performed on the lung tissue sections of mice. After collecting images, Image-Pro Plus software was used to calculate the pulmonary fibrosis area in the sections and the percentage of pulmonary fibrosis area.

[0131] As shown in Table 11, obvious fibrosis occurred in the lung tissue of the model group, while the fibrosis area of ​​the mice in the CA2 administration group was significantly reduced, proving that blocking CD84 can alleviate the concurrent fibrosis of lung tissue caused by SLE.

[0132] Table 11 Effect of CA2 on the area of ​​pulmonary fibrosis in SLE-ILD mice

[0133] Grouping Model CA2 Pulmonary fibrosis area percentage (%) 12.83±2.19 4.93±1.04

[0134] Note: Data are expressed as "mean ± standard error".

[0135] 2.4 Total number of BALF cells: The number of inflammatory cells in alveolar lavage fluid was specifically determined by anesthetizing mice and fixing them on the operating table, inserting the indwelling needle cannula into the trachea, and then injecting 1mL PBS into the mouse lungs along the trachea with a syringe to completely lavage the lungs, repeating three times, collecting the lavage fluid, centrifuging the lavage fluid, adding red blood cell lysis solution to the cell sediment to resuspend the cells, lysing at room temperature for 10min, centrifuging to remove the lysis solution, resuspending with 100μL PBS, and taking 20μL of the cell suspension to detect the number of live cells in a live cell counter (three fields of view were randomly selected for each sample), thereby obtaining the number of inflammatory cells in the alveolar lavage fluid of the test object. As shown in Table 12, compared with the control group, the total number of BALF cells in the model group increased significantly, while the total number of BALF cells in the CA2 administration group mice decreased significantly, indicating that blocking CD84 with anti-mCD84CA2 can alleviate the infiltration of inflammatory cells in lung tissue caused by SLE.

[0136] Table 12 Effect of CA2 on the total number of inflammatory cells in BALF of SLE-ILD mice

[0137] Grouping NaCl Model CA2 <![CDATA[Total number of BALF cells (×10 5 )]]> 1.21±0.103 3.92±0.519 1.39±0.276

[0138] Note: Data are expressed as "mean ± standard error".

[0139] Example 7

[0140] CD84 monoclonal antibody CA2 can inhibit type II collagen-induced rheumatoid arthritis-related interstitial lung disease (RA-ILD)

[0141] 1. Experimental Methods

[0142] Male DBA1 / J wild-type mice (6-8 weeks old) were used as experimental animals. The model was established by intradermal injection of type II collagen (150 μg / mouse) emulsified with immune adjuvant at the base of the tail. The mice were immunized 5 times in total. After the initial immunization, booster immunization was performed every 3 weeks. The control group mice were injected with the same amount of adjuvant at the same time. The day of modeling was D0, and the mice were weighed every day. At the 12th week of modeling, the mice were regrouped according to the weight loss rate, with 6 mice in each group. The specific grouping was as follows: control group (Ctl), model group (Model), CA2 (Ab-CD84, 5 mg / kg) group, and drug treatment was started. The Ab-CD84 group was intraperitoneally injected once every 3 days according to the dose, and the NaCl group and Model group were injected with the same amount of saline as control. The general state of the mice was observed once in the morning and afternoon every day, and the drug was administered until the 20th week.

[0143] 2. Index detection

[0144] The degree of RA was scored throughout the experiment; after the experiment, the forced vital capacity, degree of pulmonary fibrosis and total number of BALF cells of each group of mice were tested. The experimental results are as follows:

[0145] 2.1 RA score: The mouse arthritis index test refers to the scoring of the mouse arthritis index starting from the second booster immunization on day 21, and scoring every three days until day 50. Each paw of each mouse was scored, and the sum of the scores of the four limbs was the arthritis index of a mouse. The scoring criteria are as follows: 0 = no erythema or swelling; 1 = mild erythema or swelling of one toe; 2 = erythema or swelling of more than one toe; 3 = erythema and swelling of the ankle or wrist; 4 = severe erythema and severe edema of the toes and ankles or fingers and wrists, and the ankle or wrist cannot be bent normally. The mouse arthritis index ranges from 0 to 16. The experimental results are shown in Table 13. Compared with the control group, the RA score of the model group was significantly increased; while the arthritis score of the mice in the CA2 administration group was significantly reduced, proving that blocking CD84 can alleviate the severity of arthritis caused by type II collagen immunity.

[0146] Table 13 Effect of CA2 on the severity of arthritis in RA-ILD mice

[0147] Grouping Model CA2 RA score 12.53±2.19 3.94±0.81

[0148] Note: Data are expressed as "mean ± standard error".

[0149] 2.2 Degree of pulmonary fibrosis: After administration, H&E staining was performed on the lung tissue sections of mice. After collecting images, Image-Pro Plus software was used to calculate the pulmonary fibrosis area in the sections and the percentage of pulmonary fibrosis area.

[0150] The results are shown in Table 14. Compared with the control group, the model group showed obvious fibrosis, while the degree of lung fibrosis in the CA2-administered group was significantly reduced, proving that blocking CD84 can alleviate the formation of lung fibrosis caused by RA.

[0151] Table 14 Effect of CA2 on the degree of pulmonary fibrosis in RA-ILD mice

[0152] Grouping Model CA2 Pulmonary fibrosis area percentage (%) 8.12±2.01 2.10±1.33

[0153] Note: Data are expressed as "mean ± standard error".

[0154] 2.3 Total number of BALF cells: The number of inflammatory cells in alveolar lavage fluid was specifically determined by anesthetizing mice and fixing them on the operating table, inserting the indwelling needle cannula into the trachea, and then injecting 1mL PBS into the mouse lungs along the trachea with a syringe to completely lavage the lungs, repeating three times, collecting the lavage fluid, centrifuging the lavage fluid, adding red blood cell lysis solution to the cell sediment to resuspend the cells, lysing at room temperature for 10min, centrifuging to remove the lysis solution, resuspending with 100μL PBS, and taking 20μL of the cell suspension to detect the number of live cells in a live cell counter (three fields of view were randomly selected for each sample), thereby obtaining the number of inflammatory cells in the alveolar lavage fluid of the test object. As shown in Table 15, compared with the control group, the total number of BALF cells in the model group increased significantly, while the total number of BALF cells in the CA2 administration group mice decreased significantly, indicating that blocking CD84 with anti-mCD84CA2 can alleviate the infiltration of inflammatory cells in lung tissue caused by RA.

[0155] Table 15 Effect of CA2 on the total number of inflammatory cells in BALF of RA-ILD mice

[0156] Grouping NaCl Model CA2 <![CDATA[Total number of BALF cells (×10 5 )]]> 0.821±0.113 6.124±1.017 1.512±0.391

[0157] Example 8

[0158] CD84 monoclonal antibody AD5 antigen affinity test

[0159] 1. Experimental Methods

[0160] 293T cells were inoculated into 6-well plates, and hCD84 (NM_001184879.2) and mCD84 (NM_013489.4) were overexpressed by transfecting plasmid pCDNA3.1. In each group, 200 μL of OPTI-MEM was used as the dispersion system, and 2 μg of siRNA and 6 μL of transfection reagent (Lipo2000) were added to each well. After incubation for 5 minutes, the two were mixed, and the bottom of the tube was flicked to mix them thoroughly. After incubation for 10 minutes, they were added to the well plate. After 48 hours, the samples were collected and the binding of anti-human CD84 antibody AD5 to hCD84 and mCD84 was detected by flow cytometry. The light chain variable region sequence Lc of the AD5 antibody is as follows:

[0161] MESQTQVFVYMLLWLSGVDGDIVMTQSQKFMSTSVGDRVSVTCKASQNVGTNVAWYQQKPGQSPKALIYSASYRYSGVPDRFTGSGSGTDFTLTIINVQSEDLAEYFCQQYNSYPYTFGGGTKLEIK(SEQ ID NO:3);

[0162] The heavy chain variable region sequence Hc of the AD5 antibody is as follows:

[0163] QIQLVQSGPELKKPGETVKISCKASGYTFTNYGMNWVKQAPGKGLKWMGWTNTYTGEPTYADDFKGRFAFSLETSASTAYLQINNLKNEDMATYFCARWGYMIFDYWGQGTTLTVSS (SEQ ID NO: 4).

[0164] The preparation of AD5 antibody is to clone the gene sequence encoding the AD5 sequence into the eukaryotic expression vector pCDNA3.4, transfect into 293F cells, collect the culture supernatant after 7 days, and purify it using protein G gel. Among them, the gene sequence encoding the variable region of the light chain of AD5 is as follows:

[0165] atggagagccagacccaggtgttcgtgtacatgctgctgtggctgagcggcgtggacggcgacatcgtgatgacccagagccagaagttcatgagcaccagcgtgggcgacagagtgagcgtgacctgcaaggccagccagaacgtgggcaccaacgtggcctggtaccagcagaagcccggccagagccccaaggccctgatctacagcgccagctacagatacagcggcgtgcccgacagattcaccggcagcggcagcggcaccgacttcaccctgaccatcatcaacgtgcagagcgaggacctggccgagtacttctgccagcagtacaacagctacccctacaccttcggcggcggcaccaagctggagatcaag(SEQ ID NO:7);

[0166] The gene sequence encoding the variable region of the AD5 heavy chain is as follows:

[0167] cagatccagctggtgcagagcggccccgagctgaagaagcccggcgagaccgtgaagatcagctgcaaggccagcggctacaccttcaccaactacggcatgaactgggtgaagcaggcccccggcaagggcctgaagtggatgggctggaccaacacctacaccggcgagcccacctacgccgacgacttcaagggcagattcgccttcagcctggagaccagcgccagcaccgcctacctgcagatcaacaacctgaagaacgaggacatggccacctacttctgcgccagatggggctacatgatcttcgactactggggccagggcaccaccctgaccgtgagcagc(SEQ ID NO:8).

[0168] 2. Experimental results

[0169] Digest 293T cells with trypsin and resuspend, add 800 μL PBS to wash, centrifuge at 800 rpm for 5 min, discard the supernatant, add 1 mL Cell Staining buffer to wash, centrifuge at 800 rpm for 5 min, discard the supernatant, resuspend with 100 μL Cell Staining buffer, add 1 μL blocking solution TruStain FCXTM, block on ice for 15 min, add 800 μL Cell Staining buffer to wash once, centrifuge at 800 rpm for 5 min, discard the supernatant, and resuspend with 100 μL Cell Staining buffer. Then, AD5 or control IgG antibody was added to each well at 0.1, 1, 3, 10, and 30 μg / mL, and incubated on ice for 30 min. After incubation, 800 μL Cell Staining buffer was added and washed twice. Then, anti-mouse-FITC (1:200) was added and incubated on ice for 45 min. After incubation, 800 μL Cell Staining buffer was added and washed twice. After resuspending in 200 μL PBS, the binding of AD5 to hCD84 or mCD84 was detected by flow cytometry.

[0170] Depend on Figure 4 It can be seen that compared with the control IgG, AD5 has obvious binding curves with both hCD84 and mCD84, proving that AD5 can bind to human and mouse CD84 antigens.

[0171] Example 9

[0172] CD84 monoclonal antibody AD5 can inhibit the activation of profibrotic macrophages

[0173] 1. Experimental Methods

[0174] AD5 monoclonal antibody inhibits the activation of profibrotic macrophages

[0175] THP1 cells were inoculated in 6-well plates under sterile conditions, with 1×10 cells in each well. 6 , PMA was added 24 hours later to make its working solution 100 ng / mL, and control antibody (Ctl IgG) and anti-human CD84 monoclonal antibody AD5 were added 2.5 μg each after PMA stimulation for 24 hours. Interleukin 4 (IL-4) and interleukin 13 (IL-13) were added 6 hours later with a working concentration of 20 ng / mL to co-stimulate THP1 cells, and the cells were collected 24 hours later.

[0176] Add 1mL Trizol to each well, blow off the cells, transfer to a 1.5mL RNase free centrifuge tube, and let stand for 5-10min to fully lyse the cells. Add 200μL chloroform to each tube, vortex thoroughly for 15s, and let stand at room temperature for 2-3min. Centrifuge at 4℃12000rpm for 15min, transfer 400μL of the upper water layer to a new RNase free 1.5mL centrifuge tube, add 400μL isopropanol, mix well, and let stand at room temperature for 10min. Centrifuge at 4℃12000rpm for 10min to obtain flocculent precipitate as RNA, discard the supernatant, add 1mL 75% ethanol, and wash the precipitate. Centrifuge at 4℃7000rpm for 5min, discard the supernatant, repeat once, aspirate the excess solution, evaporate at room temperature, and the RNA becomes colorless, indicating that the ethanol has evaporated completely. Add RNase free water to dissolve, place in an ice box for 15min, and use a micro-spectrophotometer to detect and record the RNA concentration. Then, the RNA was reversed into cDNA, and the reverse program was: 42℃15min, 95℃3min. Then, the RT-PCR system was configured: 2×SYBR Green Mix 10μL per tube, 1μL of upstream and downstream primers per tube, 6μL of RNase free water, and 2μL of cDNA template. After completion, Roche was used to detect the expression of the target gene. Among them, the primer sequences used are shown in Table 16:

[0177] Table 16 Primer sequences

[0178]

[0179] Depend on Figure 5 It can be seen that Ab-CD84 can significantly inhibit the activation of profibrotic macrophages induced by IL-4 / 13, as shown by the significant inhibition of the expression of profibrotic factors TGF-β1 and IL-10.

[0180] Example 10

[0181] CD84 monoclonal antibody AD5 (Ab-CD84 antibody) can inhibit the activation of pro-inflammatory macrophages

[0182] 1. Experimental Methods

[0183] THP1 cells were inoculated in 6-well plates under sterile conditions, with 1×10 cells in each well. 6 , PMA was added 24 hours later to make the working solution 100 ng / mL, 2.5 μg each of control antibody (Ctl IgG) and Ab-CD84 were added 24 hours after PMA stimulation, LPS / IFN-γ was added 6 hours later, and the working solution concentration was 100 ng / mL, and samples were collected 24 hours later.

[0184] Add 1mL Trizol to each well, blow off the cells, transfer to a 1.5mL RNase free centrifuge tube, and let stand for 5-10min to fully lyse the cells. Add 200μL chloroform to each tube, vortex thoroughly for 15s, and let stand at room temperature for 2-3min. Centrifuge at 4℃, 12000rpm for 15min, transfer 400μL of the upper water layer to a new RNase free 1.5mL centrifuge tube, add 400μL isopropanol, mix well, and let stand at room temperature for 10min. Centrifuge at 4℃, 12000rpm for 10min to obtain flocculent precipitate as RNA, discard the supernatant, add 1mL 75% ethanol, and wash the precipitate. Centrifuge at 4℃, 7000rpm for 5min, discard the supernatant, repeat once, aspirate the excess solution, evaporate at room temperature, and the RNA becomes colorless, indicating that the ethanol has evaporated completely. Add RNase free water to dissolve, place in an ice box for 15min, and use a micro-spectrophotometer to detect and record the RNA concentration. Then, RNA was reversed into cDNA, and the reverse program was: 42℃15min, 95℃3min. Then, the RT-PCR system was configured: 2×SYBR Green Mix 10μL per tube, upstream and downstream primers 1μL per tube, RNase free water 6μL, cDNA template 2μL. The primer sequences used are shown in Table 17:

[0185] Table 17 Primer sequences

[0186]

[0187] After completion, the eight rows of tubes were placed in a Roche LC96 instrument and the program was set as pre-denaturation: 95°C (300s); denaturation: 95°C (30s); annealing: 55°C (30s); extension: 72°C (30s), for a total of 40 cycles to detect the expression of the target gene.

[0188] Depend on Figure 6 It can be seen that Ab-CD84 antibody can inhibit LPS / IFN-γ-induced macrophage activation, which is manifested by the significant inhibition of the expression of inflammatory factors IL-6, TNF-α and IL-1β.

[0189] Embodiment 11

[0190] CD84 monoclonal antibody AD5 (Ab-CD84 antibody) can inhibit bleomycin-induced pulmonary fibrosis

[0191] 1. Experimental Methods

[0192] C57BL6 / J male mice, 7-8 weeks old, 8 mice per group. The pulmonary fibrosis model was established by intratracheal injection of bleomycin (2U / kg), and the control group was injected with NaCl. The day of modeling was day 0, and Ab-CD84 (5 mg / kg) was intraperitoneally injected on days 0-7 of modeling, once every 2 days, and the mice were killed on the 21st day.

[0193] 2. Experimental results

[0194] 2.1 Determination of Hydroxyproline Content: After the mice were dissected, the hydroxyproline (HYP) content of the mouse lung tissue was determined. Specifically, after the mice were killed, the right lung of the mouse was separated, placed in a 5mL ampoule, placed in a 120°C oven for drying, hydrolyzed under the action of hydrochloric acid, the pH value was adjusted to 6.5-8.0, the residue was filtered with a 0.5μm filter membrane, PBS (phosphate-balanced saline) was added to adjust the total volume to 10mL, 50μL of sample was taken, 350μL of deionized water was added, 200μL of chloramine T solution was added and incubated at room temperature for 20min, 200μL of perchloric acid was added and incubated at room temperature for 5min, 200μL of p-dimethylaminobenzaldehyde (P-DMAB) was added and incubated at 65°C for 20min. Take 200 μL to a 96-well plate to measure the absorbance of the sample at 570 nm, draw a standard curve using the standard reading, and then calculate the hydroxyproline concentration Cs of the measured sample according to the formula obtained from the standard curve. Convert it to the amount of hydroxyproline contained in the entire right lung according to the following formula I.

[0195] The amount of hydroxyproline contained in the entire right lung = Cs × 8 (dilution factor of the measured sample) × 10 (total sample volume) Formula I

[0196] The results are shown in Table 18. Compared with the control group, the HYP content in the right lung of the model group was significantly increased, and compared with the BLM group, the HYP content in the Ab-CD84 group was significantly decreased, indicating that the loss of CD84 in macrophages can significantly reduce the lung tissue collagen deposition caused by bleomycin.

[0197] Table 18 Effect of Ab-CD84 on hydroxyproline content in mouse lung tissue

[0198] Grouping NaCl BLM Ab-CD84 HYP (μg / right whole lung) 85.67±2.96 176.9±4.81 102.62±3.14

[0199] Note: Data are expressed as "mean ± standard error".

[0200] 2.2 Statistics of pulmonary fibrosis area: The lung tissues of mice were paraffin-embedded, and then H&E staining was performed on the lung tissue sections of mice. After collecting images, the pulmonary fibrosis area in the sections was calculated using Image-Pro Plus software. The percentage of pulmonary fibrosis area = pulmonary fibrosis area / total area of ​​lung sections × 100%. As shown in Table 19, the average pulmonary fibrosis area of ​​mice in the BLM group reached 31.5%, while the pulmonary fibrosis area of ​​the Ab-CD84 group decreased to 18.5%, indicating that blocking CD84 can significantly reduce the formation of bleomycin-induced pulmonary fibrosis.

[0201] Table 19 Effect of CD84 deficiency on the area of ​​pulmonary fibrosis in mice

[0202] Grouping BLM Ab-CD84 Pulmonary fibrosis area percentage (%) 31.45±2.76 18.5±1.23

[0203] Note: Data are expressed as "mean ± standard error".

[0204] Example 12

[0205] CD84 monoclonal antibody AD5 (Ab-CD84 antibody) can inhibit bleomycin-induced systemic sclerosis-related interstitial lung disease (SSc-ILD)

[0206] 1. Experimental Methods

[0207] Male C57BL / 6 mice were subcutaneously injected with bleomycin (0.2U / mouse / day) for 28 consecutive days to establish the SSc-ILD model. The day of modeling was D1, and the mice were weighed every day. Seven days after modeling, the mice were regrouped according to the weight loss rate, with 8 mice in each group. The specific groupings were: control group (NaCl), model group (BLM), and Ab-CD84 (5mg / kg) group. The mice were treated on the 7th day. The Ab-CD84 group was intraperitoneally injected once every 3 days according to the dose, and the NaCl group and BLM group were injected with an equal amount of saline as a control. The general state of the mice was observed once in the morning and afternoon every day. The mice were killed and analyzed after the drug was given to the 28th day.

[0208] 2. Index detection

[0209] After the experiment, the dermis thickness, degree of pulmonary fibrosis, and hydroxyproline content in skin and lung tissues of each group of mice were tested. The experimental results are as follows:

[0210] 2.1 Dermis thickness detection: The dermis fibrosis thickness detection is specifically to fix the skin sample with 10% formalin, dehydrate, embed in paraffin, cut into 5 μm thick sections, and perform H&E staining. Table 20 shows the statistical results of the dermis thickness of each group of mice. Compared with the control group, the skin of the BLM group mice was significantly thickened, while the skin thickness of the Ab-CD84 group mice was significantly reduced, indicating that blocking CD84 can alleviate the thickening of mouse skin caused by bleomycin.

[0211] Table 20 Effect of Ab-CD84 on skin thickness in SSc-ILD mice

[0212] Grouping NaCl BLM Ab-CD84 Dermis thickness (μm) 198.56±11.2 403.78±13.56 245±17.8

[0213] Note: Data are expressed as "mean ± standard error".

[0214] 2.2 Determination of skin collagen content: Skin collagen content detection is hydroxyproline content detection. Specifically, after killing mice, separate mouse skin tissue, put it into a 5mL ampoule, place it in a 120℃ oven to dry, adjust the pH value to 6.5-8.0 after hydrolysis under the action of hydrochloric acid, filter the residue with a 0.5μm filter membrane, add PBS (phosphate-balanced saline) to adjust the total volume to 10mL, take 50μL of sample, add 350μL of deionized water, add 200μL of chloramine T solution and incubate at room temperature for 20min, add 200μL of perchloric acid and incubate at room temperature for 5min, add 200μL of p-dimethylaminobenzaldehyde (P-DMAB) and incubate at 65℃ for 20min. Take 200μL to a 96-well plate to measure the absorbance of the sample at 570nm, draw a standard curve using the standard reading, and then calculate the hydroxyproline concentration Cs of the measured sample according to the formula obtained from the standard curve. The hydroxyproline content per unit weight of skin is calculated according to the following formula III.

[0215] The amount of hydroxyproline contained in the skin = Cs × 8 (dilution factor of the sample) × 10 (total sample volume) / 0.01 (skin weight, g) Formula III

[0216] As shown in Table 21, compared with the control group, the HYP content per gram of skin in the BLM group mice was significantly increased, while the HYP content in the skin of the Ab-CD84 administration group mice was significantly decreased, proving that the use of Ab-CD84 blocking antibody can effectively alleviate the collagen deposition in the mouse skin caused by bleomycin.

[0217] Table 21 Effect of Ab-CD84 on collagen content in the skin of SSc-ILD mice

[0218] Grouping NaCl BLM Ab-CD84 Skin HYP (μg / g) 157.23±8.19 259.53±10.67 189.3±17.1

[0219] Note: Data are expressed as "mean ± standard error".

[0220] 2.3 Statistics of pulmonary fibrosis degree: After drug administration, H&E staining was performed on the lung tissue sections of mice. After collecting images, the pulmonary fibrosis area in the sections was calculated using Image-Pro Plus software, and the percentage of pulmonary fibrosis area was calculated according to Formula II.

[0221] Pulmonary fibrosis area percentage = pulmonary fibrosis area / total lung section area × 100% Formula II

[0222] As shown in Table 22, SSc caused by continuous subcutaneous injection of BLM can further lead to fibrosis of lung tissue, while the fibrosis area of ​​lung tissue in the Ab-CD84 administration group of mice was significantly reduced, indicating that the use of CD84 blocking antibodies can alleviate the formation of pulmonary fibrosis caused by SSc.

[0223] Table 22 Effect of Ab-CD84 on the area of ​​pulmonary fibrosis in SSc-ILD mice

[0224]

[0225]

[0226] Note: Data are expressed as "mean ± standard error".

[0227] 2.4 Determination of lung hydroxyproline content: Lung collagen content detection is also known as hydroxyproline content detection. Specifically, after killing mice, the mouse skin tissue or right lung is separated and placed in a 5mL ampoule bottle, placed in a 120℃ oven for drying, hydrolyzed under the action of hydrochloric acid, the pH value is adjusted to 6.5-8.0, the residue is filtered with a 0.5μm filter membrane, PBS (phosphate-balanced saline) is added to adjust the total volume to 10mL, 50μL of sample is taken, 350μL of deionized water is added, 200μL of chloramine T solution is added and incubated at room temperature for 20min, 200μL of perchloric acid is added and incubated at room temperature for 5min, 200μL of p-dimethylaminobenzaldehyde (P-DMAB) is added and incubated at 65℃ for 20min. 200μL is taken to a 96-well plate to measure the absorbance of the sample at 570nm, and a standard curve is drawn using the standard readings, and then the hydroxyproline concentration Cs of the measured sample is calculated according to the formula obtained from the standard curve. The hydroxyproline content was obtained by converting the hydroxyproline content in the entire right lung into the amount of hydroxyproline contained in the entire right lung according to the following formula I.

[0228] The amount of hydroxyproline contained in the entire right lung = Cs × 8 (dilution factor of the sample tested) × 10 (total sample volume) Formula I

[0229] As shown in Table 23, compared with the control group, the HYP content in the right whole lung of the BLM group was significantly increased, while the HYP content in the right whole lung of the mice in the Ab-CD84 administration group was significantly decreased, proving that the use of CD84 blocking antibodies can effectively alleviate the collagen deposition in the lungs of mice caused by SSc.

[0230] Table 23 Effect of Ab-CD84 on collagen content in the right lung of SSc-ILD mice

[0231] Grouping NaCl BLM Ab-CD84 Lung tissue HYP (μg / right whole lung) 124.52±3.1 164.92±1.2 14.68±5.1

[0232] Note: Data are expressed as "mean ± standard error".

[0233] Embodiment 13

[0234] CD84 monoclonal antibody can inhibit spontaneous systemic lupus erythematosus interstitial lung disease (SLE-ILD) in MRL / Lpr mice

[0235] 1. Experimental Methods

[0236] MRL / Lpr male mice, 5 mice per group. Spontaneous SLE-ILD occurred when the mice were raised to 16 weeks of age, and drug administration began: control group (Ctl), model group (Model), Ab-CD84 (5 mg / kg) group. The mice were treated with drug administration from the 16th week, and the Ab-CD84 group was intraperitoneally injected once every 3 days according to the dose, and the NaCl group and the Model group were injected with an equal amount of normal saline as a control. The general condition of the mice was observed once in the morning and afternoon every day, and the drug administration lasted until the 18th week.

[0237] 2. Index detection

[0238] 2.1 Dermis thickness detection: The dermis fibrosis thickness detection is specifically to fix the skin sample with 10% formalin, dehydrate, embed in paraffin, cut into 5 μm thick sections, and perform H&E staining. Table 24 shows the statistical results of the dermis thickness of each group of mice. Compared with the control group, the skin of the Model group mice was significantly thickened, while the skin thickness of the Ab-CD84 administration group mice was significantly reduced, indicating that the use of CD84 blocking antibodies can alleviate the spontaneous skin thickening of MRL / Lpr mice.

[0239] Table 24 Effect of Ab-CD84 on skin thickness in SLE-ILD mice

[0240] Grouping NaCl Model Ab-CD84 Dermis thickness (μm) 328.92±16.8 489.34±18.6 378.6±15.7

[0241] Note: Data are expressed as "mean ± standard error".

[0242] 2.2 Spleen coefficient: The spleen coefficient test was specifically to weigh the spleen weight of the mice after the mice were killed, and the spleen coefficient was obtained by spleen weight / body weight. The specific results are shown in Table 25. As shown in Table 25, compared with the control group, the spleen coefficient of the model group was significantly increased, while the spleen coefficient of the Ab-CD84 administration group was significantly decreased, indicating that the use of CD84 blocking antibodies can alleviate the spontaneous splenomegaly of MRL / Lpr mice.

[0243] Table 25 Effect of Ab-CD84 on spleen coefficient of SLE-ILD mice

[0244] Grouping NaCl Model Ab-CD84 Spleen coefficient 0.00561±0.00014 0.019476±0.00038 0.006528±0.00042

[0245] Note: Data are expressed as "mean ± standard error".

[0246] 2.3 Statistics of pulmonary fibrosis degree: After drug administration, H&E staining was performed on the lung tissue sections of mice. After collecting images, Image-Pro Plus software was used to calculate the pulmonary fibrosis area in the sections and the percentage of pulmonary fibrosis area.

[0247] As shown in Table 26, obvious fibrosis occurred in the lung tissue of the model group, while the fibrosis area of ​​the mice in the Ab-CD84 administration group was significantly reduced, proving that blocking CD84 can alleviate the concurrent fibrosis of lung tissue caused by SLE.

[0248] Table 26 Effect of Ab-CD84 on the area of ​​pulmonary fibrosis in SLE-ILD mice

[0249] Grouping Model Ab-CD84 Pulmonary fibrosis area percentage (%) 14.02±1.87 5.02±0.97

[0250] Note: Data are expressed as "mean ± standard error".

[0251] 2.4 Total number of BALF cells: The number of inflammatory cells in alveolar lavage fluid was specifically determined by anesthetizing the mice and fixing them on the operating table, inserting the indwelling needle cannula into the trachea, and then injecting 1mL PBS into the mouse lungs along the trachea with a syringe to completely lavage the lungs. This was repeated three times, and the lavage fluid was collected. The lavage fluid was centrifuged, and the cell pellet was added with red blood cell lysis solution to resuspend the cells. The cells were lysed at room temperature for 10 minutes, and the lysis solution was centrifuged and aspirated. After resuspending with 100μL PBS, 20μL of the cell suspension was taken to detect the number of live cells in a live cell counter (three fields of view were randomly selected for each sample), and the number of inflammatory cells in the alveolar lavage fluid of the test object was obtained. As shown in Table 27, compared with the control group, the total number of BALF cells in the model group increased significantly, while the total number of BALF cells in the Ab-CD84 administration group mice decreased significantly, indicating that blocking CD84 with anti-mCD84 can alleviate the infiltration of inflammatory cells in lung tissue caused by SLE.

[0252] Table 27 Effect of Ab-CD84 on the total number of inflammatory cells in BALF of SLE-ILD mice

[0253] Grouping NaCl Model Ab-CD84 <![CDATA[Total number of BALF cells (×10 5 )]]> 1.34±0.302 4.09±0.318 1.52±0.324

[0254] Note: Data are expressed as "mean ± standard error".

[0255] Embodiment 14

[0256] CD84 monoclonal antibody can inhibit type II collagen-induced rheumatoid arthritis-associated interstitial lung disease (RA-ILD)

[0257] 1. Experimental Methods

[0258] The experimental animals used male DBA1 / J wild-type mice (6-8 weeks old) and were modeled by intradermal injection of type II collagen (150 μg / mouse) emulsified with immune adjuvant at the base of the tail. A total of 5 immunizations were performed. After the initial immunization, booster immunizations were performed every 3 weeks. The control group mice were injected with the same amount of adjuvant at the same time. The day of modeling was D0, and the mice were weighed every day. At the 12th week of modeling, they were regrouped according to the weight loss rate, with 6 mice in each group. The specific grouping was as follows: control group (Ctl), model group (Model), Ab-CD84 (5 mg / kg) group, and drug treatment was started. The Ab-CD84 group was intraperitoneally injected once every 3 days according to the dose, and the NaCl group and Model group were injected with the same amount of saline as control. The general state of the mice was observed once in the morning and afternoon every day, and the drug was administered until the 20th week.

[0259] 2. Index detection

[0260] The degree of RA was scored throughout the experiment; after the experiment, the forced vital capacity, degree of pulmonary fibrosis and total number of BALF cells of each group of mice were tested. The experimental results are as follows:

[0261] 1.1RA score: The mouse arthritis index test refers to the scoring of the mouse arthritis index starting from the second booster immunization on day 21, and scoring every three days until day 50. Each paw of each mouse was scored, and the sum of the scores of the four limbs was the arthritis index of a mouse. The scoring criteria are as follows: 0 = no erythema or swelling; 1 = mild erythema or swelling of one toe; 2 = erythema or swelling of more than one toe; 3 = erythema and swelling of the ankle or wrist; 4 = severe erythema and severe edema of the toes and ankles or fingers and wrists, and the ankle or wrist cannot be bent normally. The mouse arthritis index ranges from 0 to 16. The experimental results are shown in Table 28. Compared with the control group, the RA score of the model group was significantly increased; while the arthritis score of the mice in the Ab-CD84 administration group was significantly reduced, proving that blocking CD84 can alleviate the severity of arthritis caused by type II collagen immunity.

[0262] Table 28 Effect of Ab-CD84 on the severity of arthritis in RA-ILD mice

[0263] Grouping Model Ab-CD84 RA score 13.45±1.73 3.79±0.75

[0264] Note: Data are expressed as "mean ± standard error".

[0265] 1.2 Degree of pulmonary fibrosis: After administration, H&E staining was performed on the lung tissue sections of mice. After collecting images, Image-Pro Plus software was used to calculate the pulmonary fibrosis area in the sections and the percentage of pulmonary fibrosis area.

[0266] The results are shown in Table 29. Compared with the control group, the model group showed obvious fibrosis, while the degree of pulmonary fibrosis in the Ab-CD84 administration group was significantly reduced, proving that blocking CD84 can alleviate the formation of pulmonary fibrosis caused by RA.

[0267] Table 29 Effect of Ab-CD84 on the degree of pulmonary fibrosis in RA-ILD mice

[0268] Grouping Model Ab-CD84 Pulmonary fibrosis area percentage (%) 7.68±3.26 1.97±0.89

[0269] Note: Data are expressed as "mean ± standard error".

[0270] 2.3 Total number of BALF cells: The number of inflammatory cells in alveolar lavage fluid was specifically determined by anesthetizing mice and fixing them on the operating table, inserting the indwelling needle cannula into the trachea, and then injecting 1mL PBS into the mouse lungs along the trachea with a syringe to completely lavage the lungs, repeating three times, collecting the lavage fluid, centrifuging the lavage fluid, adding red blood cell lysis solution to the cell sediment to resuspend the cells, lysing at room temperature for 10min, centrifuging to remove the lysis solution, resuspending with 100μL PBS, and taking 20μL of the cell suspension to detect the number of live cells in a live cell counter (three fields of view were randomly selected for each sample), thereby obtaining the number of inflammatory cells in the alveolar lavage fluid of the test object. As shown in Table 30, compared with the control group, the total number of BALF cells in the model group increased significantly, while the total number of BALF cells in the Ab-CD84 administration group mice decreased significantly, indicating that blocking CD84 with anti-mCD84 can alleviate the infiltration of inflammatory cells in lung tissue caused by RA.

[0271] Table 30 Effect of Ab-CD84 on the total number of inflammatory cells in BALF of RA-ILD mice

[0272] Grouping NaCl Model Ab-CD84 <![CDATA[Total number of BALF cells (×10 5 )]]> 0.932±0.205 7.832±1.092 1.601±0.594

[0273] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Use of an anti-CD84 antibody in the preparation of a drug for preventing and / or treating pulmonary fibrosis.

2. The application according to claim 1, characterized in that: The anti-CD84 antibody includes an anti-CD84 monoclonal antibody and / or an anti-CD84 polyclonal antibody.

3. The application according to claim 2, characterized in that: The monoclonal antibodies against CD84 include CA2 monoclonal antibody and AD5 monoclonal antibody.

4. The use according to claim 3, characterized in that: The amino acid sequence of the heavy chain variable region sequence of the CA2 monoclonal antibody is shown in SEQ ID NO: 1; the amino acid sequence of the light chain variable region sequence of the CA2 monoclonal antibody is shown in SEQ ID NO:

2.

5. The application according to claim 3, characterized in that: The amino acid sequence of the heavy chain variable region sequence of the AD5 monoclonal antibody is shown in SEQ ID NO:3; the amino acid sequence of the light chain variable region sequence of the AD5 monoclonal antibody is shown in SEQ ID NO:

4.

6. The use according to claim 1, characterized in that: The pulmonary fibrosis includes idiopathic pulmonary fibrosis and / or pulmonary fibrosis indications.

7. The use according to claim 6, characterized in that: The pulmonary fibrosis indications include one or more of the following: systemic sclerosis-related interstitial lung disease, spontaneous systemic lupus erythematosus interstitial lung disease, rheumatoid arthritis-related interstitial lung disease, myositis / dermatomyositis-related interstitial lung disease and hypersensitivity pneumonitis-related interstitial lung disease.

8. The use according to any one of claims 1 to 7, characterized in that: The drug is a drug that inhibits the activation of macrophages; Preferably, the macrophages include pro-inflammatory macrophages and / or pro-fibrotic macrophages.

9. A drug for preventing and / or treating pulmonary fibrosis, characterized in that: The invention comprises an anti-CD84 antibody and medically acceptable excipients.

10. The drug according to claim 9, characterized in that: The anti-CD84 antibody includes an anti-CD84 monoclonal antibody and / or an anti-CD84 polyclonal antibody; Preferably, the monoclonal antibodies against CD84 include CA2 monoclonal antibody and AD5 monoclonal antibody; Further preferably, the amino acid sequence of the heavy chain variable region sequence of the CA2 monoclonal antibody is shown as SEQ ID NO:1; and the amino acid sequence of the light chain variable region sequence of the CA2 monoclonal antibody is shown as SEQ ID NO:

2. Further preferably, the amino acid sequence of the heavy chain variable region sequence of the AD5 monoclonal antibody is shown as SEQ ID NO:3; the amino acid sequence of the light chain variable region sequence of the AD5 monoclonal antibody is shown as SEQ ID NO:4.

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