Use of an anti-cd84 antibody in the preparation of a medicament for preventing or treating pulmonary fibrosis

Abstract

The application provides an application of an anti-CD84 antibody in preparation of a drug for preventing and treating pulmonary fibrosis, and belongs to the technical field of biological medicine. The application of the anti-CD84 antibody in preparation of the drug for preventing and treating pulmonary fibrosis. The biological function of CD84 is blocked by the anti-CD84 antibody, which can not only effectively reduce the pulmonary fibrosis degree of a bleomycin-induced pulmonary fibrosis and a pulmonary fibrosis indication mouse disease model, but also effectively inhibit the activation of pro-inflammatory macrophages and / or pro-fibrosis macrophages, so as to prevent further deterioration of the pulmonary fibrosis. It can be seen that the application provided by the application provides a new means for preventing and treating the pulmonary fibrosis.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of an anti-CD84 antibody in the preparation of drugs for the prevention and treatment of pulmonary fibrosis. Background Technology

[0002] Pulmonary fibrosis (PF) is a disease characterized by persistent damage to the alveoli, leading to varying degrees of inflammation and fibrosis in the alveoli and pulmonary interstitium, ultimately resulting in lung structural destruction and respiratory failure. It is also known as interstitial lung disease (ILD). The causes of pulmonary fibrosis include known factors such as physical, chemical, and biological factors, as well as pulmonary fibrosis of unknown etiology. Pulmonary fibrosis with known causes includes connective tissue disease-related interstitial lung diseases, such as 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 pathogenesis and mechanism are mainly due to an excessive immune response to self- or foreign antigens, activating various immune cells such as pulmonary lymphocytes, which then secrete large amounts of cytokines, mediating the immune response and causing pulmonary fibrosis. Pulmonary fibrosis of unknown etiology, including idiopathic pulmonary fibrosis (IPF), belongs to the idiopathic interstitial pneumonia (IIP) group within the interstitial lung disease (ILD) family. IPF is one of the most common and severe chronic interstitial lung diseases of unknown etiology. IPF originates from abnormal repair following recurrent or persistent alveolar epithelial damage. Under multiple persistent damage from known or unknown endogenous and exogenous damaging factors, damaged lung epithelial cells initiate damage repair mechanisms. Insufficient epithelial regeneration and repair leads to mesenchymal-like transformation of surviving cells, exhibiting a pro-fibrotic phenotype and secreting large amounts of pro-fibrotic factors. This causes abnormal activation and proliferation of fibroblasts, resulting in excessive extracellular matrix deposition, leading to fibrotic scarring and celluloid formation. Alveolar structure is destroyed, causing an irreversible and continuous decline in lung function, ultimately leading to respiratory failure and death. Clinically, IPF manifests as progressive dyspnea accompanied by an irritating dry cough, and the disease often continues to progress.

[0003] Currently, two drugs for treating pulmonary fibrosis are available worldwide: pirfenidone and nintedanib. Both can slow the decline in forced vital capacity (FVC), but they are not very effective in improving acute exacerbations of IPF or prolonging survival. Therefore, seeking safe and effective new drugs is of great significance for the prevention and / or treatment of pulmonary fibrosis and its indications.

[0004] Macrophages are the most abundant immune cells in lung tissue (approximately 70%) and play a crucial role in the pathogenesis of pulmonary fibrosis (PF), participating in the regulation and repair of tissue abnormalities during PF. Macrophages include M1 macrophages, M2 macrophages, and M2-like macrophages. Studies have shown that pro-inflammatory factors and oxygen free radicals are associated with the development and progression of PF. In a bleomycin-induced PF model, inhibiting FR-β expression in M1 macrophages alleviated PF during the validation phase. However, anti-infective therapy was ineffective in patients with PF, raising questions about the role of inflammation in PF. Researchers used a bleomycin-induced mouse PF model and found that the inflammatory phase cleared intrinsic M1 macrophages and circulating monocytes from the mouse lungs without affecting the occurrence and progression of PF. The number of M2 macrophages increased in the bronchoalveolar lavage fluid of IPF patients, and studies have shown that M2 macrophages may have anti-fibrotic effects, with M2 macrophage markers—mannose receptor and MFGE8—both exhibiting anti-fibrotic activity. M2-like macrophages are more pro-fibrotic and can overexpress IL-10, promoting fibrosis. Glucocorticoids can induce M2-like macrophages, which explains why glucocorticoid treatment is not only ineffective in some fibrosis patients but also harmful, exacerbating the degree of fibrosis.

[0005] CD84 (SLAMF5) is a member of the signaling lymphocyte activation molecule (SLAM) family, primarily expressed in bone marrow-derived lymphocytes, monocytes, and macrophages. It plays a crucial role in immune cell communication, differentiation, and microenvironment regulation. Currently, CD84 is frequently used clinically as a biomarker for some immune-related diseases, such as rheumatoid arthritis. + Patients are more likely to benefit from anti-TNF-α therapy; in patients with systemic lupus erythematosus (SLE), high CD84 expression is significantly positively correlated with disease progression. Some in vitro studies have also demonstrated the potential clinical value of anti-CD84 therapy; for example, CD84 neutralizing antibodies can significantly alleviate T cell differentiation in a mouse model of RA and slow the malignant progression of chronic lymphocytic leukemia in a mouse model, suggesting that CD84 may be a reliable target for clinical treatment of lymphocytic leukemia. However, there are currently no reports on CD84's involvement 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 an application of a CD84 inhibitor in the preparation of a drug for the prevention and treatment of pulmonary fibrosis, which inhibits the expression of the CD84 gene or protein or blocks the biological function of CD84 to inhibit the activation of macrophages and their metastasis to pulmonary fibrosis, thereby achieving the purpose of prevention and / or treatment of pulmonary fibrosis.

[0007] This invention provides the use of an anti-CD84 antibody in the preparation of a medicament for the prevention and / or treatment of pulmonary fibrosis.

[0008] Preferably, the anti-CD84 antibody includes a monoclonal antibody against CD84 and / or a polyclonal antibody against CD84.

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

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

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

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

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

[0014] Preferably, the drug is a drug that inhibits the activation of macrophages; Preferably, the macrophages include pro-inflammatory macrophages and / or pro-fibrotic macrophages.

[0015] The present invention also provides a medicament for the prevention and / or treatment of pulmonary fibrosis, comprising an anti-CD84 antibody and medically acceptable excipients.

[0016] Preferably, the anti-CD84 antibody includes a monoclonal antibody against CD84 and / or a polyclonal antibody against CD84; Preferably, the monoclonal antibody against CD84 includes CA2 monoclonal antibody and AD5 monoclonal antibody; More preferably, the amino acid sequence of the heavy chain variable region of the CA2 monoclonal antibody is shown in SEQ ID NO:1; and the amino acid sequence of the light chain variable region of the CA2 monoclonal antibody is shown in SEQ ID NO:2.

[0017] More preferably, the amino acid sequence of the heavy chain variable region of the AD5 monoclonal antibody is shown in SEQ ID NO:3; and the amino acid sequence of the light chain variable region of the AD5 monoclonal antibody is shown in SEQ ID NO:4.

[0018] This invention provides the application of an anti-CD84 antibody in the preparation of drugs for the prevention and / or treatment of pulmonary fibrosis. This invention utilizes the anti-CD84 antibody to inhibit the biological function of the CD84 protein, effectively reducing the degree of pulmonary fibrosis in bleomycin-induced pulmonary fibrosis and mouse models of pulmonary fibrosis, and also effectively inhibiting the activation of pro-inflammatory macrophages and / or pro-fibrotic macrophages, thereby preventing further deterioration of pulmonary fibrosis. Therefore, the application provided by this invention offers a new approach for the prevention and treatment of pulmonary fibrosis.

[0019] Furthermore, this invention specifically defines the types of pulmonary fibrosis, including idiopathic pulmonary fibrosis and / or indications for pulmonary fibrosis. The indications for pulmonary fibrosis include one or more of the following: systemic sclerosis-associated interstitial lung disease, spontaneous systemic lupus erythematosus interstitial lung disease, and rheumatoid arthritis-associated interstitial lung disease. In the embodiments of this invention, by inhibiting the function of CD84 protein, skin thickening can be significantly inhibited, the area of ​​pulmonary fibrosis can be reduced, and the content of hydroxyproline and collagen can be decreased, thereby alleviating bleomycin-induced collagen deposition in mouse skin, and thus achieving the purpose of treating bleomycin-induced systemic sclerosis-associated interstitial lung disease. The embodiments of this invention also verified the therapeutic effect of inhibiting CD84 protein function on spontaneous systemic lupus erythematosus (SLE) interstitial lung disease. Results showed that blocking CD84 could alleviate spontaneous skin thickening, spontaneous splenomegaly, SLE-induced pulmonary fibrosis, and SLE-induced inflammatory cell infiltration in the lungs. Therefore, inhibiting CD84 function achieves the therapeutic goal of spontaneous SLE interstitial lung disease. Furthermore, this invention also verified the role of CD84 in the development and progression of rheumatoid arthritis-related interstitial lung disease. Blocking CD84 could alleviate the severity of arthritis caused by type II collagen immunity, alleviate RA-induced pulmonary fibrosis, and alleviate RA-induced inflammatory cell infiltration in the lungs. Therefore, inhibiting CD84 function achieves the therapeutic goal of rheumatoid arthritis-related interstitial lung disease. In summary, CD84 can serve as a therapeutic target for systemic sclerosis-associated interstitial lung disease, spontaneous systemic lupus erythematosus interstitial lung disease, and rheumatoid arthritis-associated interstitial lung disease, providing new insights for the development, screening, and preparation of related clinical drugs. Attached Figure Description

[0020] Figure 1 The binding curves of the CD84 monoclonal antibody CA2 with hCD84 and mCD84 are shown. Figure 2 The results show that the CD84 monoclonal antibody CA2 inhibited IL-4 / 13-induced profibrotic macrophage activation; Figure 3 The results show the inhibition of LPS / IFN-γ-induced pro-inflammatory macrophage activation by the CD84 monoclonal antibody CA2; where A represents IL-6 expression, B represents TNF-α expression, and C represents IL-1β. Figure 4 The binding curves of the CD84 monoclonal antibody AD5 with hCD84 and mCD84 are shown. Figure 5 The results show that the CD84 monoclonal antibody AD5 inhibited IL-4 / 13-induced activation of profibrotic macrophages; Figure 6 The results show the effects of CD84 monoclonal antibody AD5 inhibiting LPS / IFN-γ-induced pro-inflammatory macrophage activation; where A represents IL-6 expression, B represents TNF-α expression, and C represents IL-1β. Detailed Implementation

[0021] This invention provides the use of an anti-CD84 antibody in the preparation of a medicament for the prevention and / or treatment of pulmonary fibrosis.

[0022] In this invention, the CD84 preferably includes the CD84 gene and / or CD84 protein derived from macrophages. The macrophages are macrophages derived from lung tissue.

[0023] In this invention, the anti-CD84 antibody preferably comprises an anti-CD84 protein monoclonal antibody or an anti-CD84 protein polyclonal antibody. The CD84 monoclonal antibody preferably comprises CA2 monoclonal antibody and AD5 monoclonal antibody. The amino acid sequence of the heavy chain variable region of the CA2 monoclonal antibody is preferably as shown in SEQ ID NO:2; the amino acid sequence of the light chain variable region of the CA2 monoclonal antibody is preferably as shown in SEQ ID NO:1. The amino acid sequence of the heavy chain variable region of the AD5 monoclonal antibody is preferably as shown in SEQ ID NO:4; the amino acid sequence of the light chain variable region of the AD5 monoclonal antibody is preferably as shown in SEQ ID NO:3. This invention does not impose any special limitations on the preparation method of the CD84 protein polyclonal antibody; any method well-known in the art for preparing polyclonal antibodies can be used, such as immunizing animals with CD84 protein as an antigen, collecting blood serum, and identifying antibodies that can bind to CD84 protein as polyclonal antibodies. This invention does not impose any special limitations on the preparation method of the anti-CD84 protein monoclonal antibody; any method well-known in the art for preparing monoclonal antibodies can be used, such as in vitro recombinant expression.

[0024] In this invention, the pulmonary fibrosis preferably includes idiopathic pulmonary fibrosis (IPF). IPF is characterized by abnormal repair following alveolar epithelial injury. Under the combined and persistent damage from multiple endogenous and exogenous damaging factors, the damage repair mechanism is initiated, but epithelial regeneration and repair are insufficient. Surviving cells undergo mesenchymal-like transformation, exhibiting a pro-fibrotic phenotype, secreting large amounts of pro-fibrotic factors, causing abnormal activation and proliferation of fibroblasts, resulting in excessive extracellular matrix deposition, leading to the formation of fibrotic scars and celluloid cysts, destruction of alveolar structure, irreversible and continuous decline in lung function, and ultimately respiratory failure and death. Clinically, IPF presents as progressive dyspnea accompanied by an irritating dry cough, and the condition often continues to progress. In this embodiment of the invention, the pulmonary fibrosis is preferably induced by bleomycin.

[0025] In this 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.

[0026] In this embodiment of the invention, a bleomycin-induced pulmonary fibrosis (IPF) model was used as the research subject. The expression of CD84 in the peripheral blood of IPF patients was detected. The results showed that compared with the healthy control group, the expression of CD84 in the peripheral blood of IPF patients was significantly increased, exhibiting a statistically significant difference. Meanwhile, CD84... + Macrophages infiltrate lung tissue in a pulmonary fibrosis model and promote the formation of fibrotic foci. This suggests that CD84 may be involved in the development and progression of pulmonary fibrosis. This invention further confirms the link between CD84 expression levels and protein function and the development and progression of pulmonary fibrosis. Compared with the control group, knocking out or blocking CD84 function in macrophages reduced macrophage migration to the lungs in pulmonary fibrosis and inhibited the activation of pro-fibrotic and pro-inflammatory macrophages, thereby inhibiting the expression of pro-fibrotic factors (TGF-β1 and IL-10) and pro-inflammatory factors (IL-6, TNF-α, and IL-1β), and inhibiting the progression of pulmonary fibrosis to some extent. Simultaneously, compared with the control, the deletion of CD84 protein in macrophages significantly reduced hydroxyproline content, thereby effectively reducing collagen deposition in lung tissue. Furthermore, the deletion of CD84 in macrophages significantly reduced the area of ​​bleomycin-induced pulmonary fibrosis, thus inhibiting the formation of pulmonary fibrosis.

[0027] In this invention, the pulmonary fibrosis preferably also includes indications for pulmonary fibrosis. The indications for pulmonary fibrosis preferably include one or more of the following: systemic sclerosis-associated interstitial lung disease, spontaneous systemic lupus erythematosus interstitial lung disease, rheumatoid arthritis-associated interstitial lung disease, myositis / dermatomyositis-associated interstitial lung disease, and allergic pneumonia-associated interstitial lung disease.

[0028] In this embodiment of the invention, dermal thickness, degree of pulmonary fibrosis, and hydroxyproline content in skin and lung tissue were used as detection indicators to evaluate whether CD84 could serve as a therapeutic target for systemic sclerosis-related interstitial lung disease. The results showed that blocking CD84 with monoclonal antibodies could alleviate bleomycin-induced skin thickening in mice, significantly reduce skin collagen content, thereby reducing bleomycin-induced skin collagen deposition in mice, significantly reducing the area of ​​pulmonary fibrosis caused by SSc, and ultimately achieving the goal of alleviating the condition of systemic sclerosis-related interstitial lung disease.

[0029] In this embodiment of the invention, dermal thickness, spleen coefficient, degree of pulmonary fibrosis, and total BALF cell count were used as detection indicators to evaluate whether CD84 could serve as a therapeutic target for spontaneous systemic lupus erythematosus (SLE) interstitial lung disease. The results showed that blocking CD84 with monoclonal antibodies could alleviate skin thickening in mice, alleviate spontaneous splenomegaly in MRL / Lpr mice, significantly reduce the fibrotic area, and significantly decrease the total BALF cell count, thereby alleviating SLE-induced inflammatory cell infiltration in lung tissue. Therefore, inhibiting CD84 function can achieve the therapeutic goal of spontaneous SLE interstitial lung disease.

[0030] In this embodiment of the invention, RA score, degree of pulmonary fibrosis, and total BALF cell count were used as detection indicators to evaluate whether CD84 could serve as a therapeutic target for rheumatoid arthritis-related interstitial lung disease. This invention used type II collagen-induced male DBA1 / J wild-type mice as a model. Results showed that blocking CD84 with monoclonal antibodies could alleviate skin thickening in mice, alleviate spontaneous splenomegaly in MRL / Lpr mice, significantly reduce the fibrotic area, and significantly decrease the total BALF cell count, thereby alleviating SLE-induced inflammatory cell infiltration in lung tissue. Therefore, inhibiting CD84 function can achieve the therapeutic goal of spontaneous systemic lupus erythematosus interstitial lung disease.

[0031] The present invention also provides a medicament for the prevention and / or treatment of pulmonary fibrosis, comprising an anti-CD84 antibody and medically acceptable excipients.

[0032] In this invention, the anti-CD84 antibody includes a monoclonal antibody against CD84 and / or a polyclonal antibody against CD84; the CD84 monoclonal antibody includes CA2 monoclonal antibody and AD5 monoclonal antibody. The CA2 and AD5 monoclonal antibodies are preferably obtained through recombinant expression. Experimental verification shows that the CA2 and AD5 monoclonal antibodies have good affinity for the CD84 antigen, ensuring the function of inhibiting CD84, thereby achieving the prevention and / or treatment of pulmonary fibrosis.

[0033] This invention does not impose any special restrictions on the type of excipients; any excipients well known in the art can be used. Similarly, this invention does not impose any special restrictions on the preparation method of the drug; any drug preparation method well known in the art can be used.

[0034] The following detailed description, in conjunction with embodiments, illustrates the application of an anti-CD84 antibody provided by the present invention in the preparation of drugs for the prevention and treatment of pulmonary fibrosis. However, these descriptions should not be construed as limiting the scope of protection of the present invention.

[0035] Example 1 CD84 monoclonal antibody CA2 antigen affinity assay 1. Experimental Methods 293T cells were seeded into 6-well plates and overexpressed hCD84 (NM_001184879.2) and mCD84 (NM_013489.4) by transfection with plasmid pCDNA3.1. In each well of each group, 200 μL of OPTI-MEM was used as a dispersion medium, along with 2 μg of pCDNA3.1 and 6 μL of transfection reagent (Lipo2000). After incubation for 5 min, the two were mixed thoroughly by gently tapping the bottom of the tube, and incubated for a total of 10 min before being added to the wells. Samples were collected after 48 h, and the binding of the anti-human CD84 antibody CA2 to hCD84 and mCD84 was detected by flow cytometry. The Lc sequence of the light chain variable region of the CA2 antibody is as follows: MRPSIQFLGLLLFWLHGAQCDIQMTQSPSSSLSASLGGKVTITCKASQDINKYIAWYQQKSGKGPRLLIHYTSTLQPGIPSRFSGSGSGRDYSFSISNLEPEDIATYYCLQYDNLYTFGGGTKLEIK (SEQ ID NO: 1); The heavy chain variable region sequence Hc of the CA2 antibody is as follows: EVQLVESGGDLVRPGGSLKLSCAASGFTFTDYGMSWVRQTPDKRLEWVATISTASTYTFYPDSVKGRFTISRDNAKNTLYLQMSSLKSEDTAIFYCVRHGHYYGSRYTWLAYWGQGTLVTVSA (SEQ ID NO: 2).

[0036] The CA2 antibody was prepared by cloning the gene sequence encoding CA2 into the eukaryotic expression vector pCDNA3.4, transfecting it into 293F cells, culturing for 7 days, collecting the culture supernatant, and purifying it using a protein G gel. The gene sequence encoding the CA2 light chain variable region is as follows: atgagacccagcatccagttcctgggcctgctgctgttctggctgcacggcgcccagtgcgacatccagatgacccagagccccagcagcctgagcgccagcctgggcggcaaggtgaccatcacctgcaaggccagccaggacatcaacaagtacatcgcctggtaccagcagaagagcggcaagggccccagactgctgatccactacaccagcaccctgcagcccggcatccccagcagattcagcggcagcggcagcggcagagactacagcttcagcatcagcaacctggagcccgaggacatcgccacctactactgcctgcagtacgacaacctgtacaccttcggcggcggcaccaagctggagatcaag (SEQ ID NO:5). The gene sequence encoding the variable region of the CA2 heavy chain is as follows: gaggtgcagctggtggagagcggcggcgacctggtgagacccggcggcagcctgaagctgagctgcgccgccagcggcttcaccttcaccgactacggcatgagctgggtgagacagacccccgacaagagactggagtgggtggccaccatcagcaccgccagcacctacaccttctaccccgacagcgtgaagggcagattcaccatcagcagagacaacgccaagaacaccctgtacctgcagatgagcagcctgaagagcgaggacaccgccatcttctactgcgtgagacacggccactactacggcagcagatacacctggctggcctactggggccagggcaccctggtgaccgtgagcgcc (SEQID NO:6).

[0037] 2. Experimental results 293T cells were digested and resuspended using trypsin, washed with 800 μL PBS, centrifuged at 800 rpm for 5 min and the supernatant was discarded. Cells were washed with 1 mL Cell Staining buffer, centrifuged at 800 rpm for 5 min and the supernatant was discarded. Cells were resuspended with 100 μL Cell Staining buffer, and 1 μL TruStain FCXTM blocking solution was added. Cells were blocked on ice for 15 min, washed once with 800 μL Cell Staining buffer, centrifuged at 800 rpm for 5 min and the supernatant was discarded. Cells were resuspended with 100 μL Cell Staining buffer. Afterwards, 0.1, 1, 3, 10, and 30 μg / mL of CA2 or control IgG antibody were added to each well, and the mixture was incubated on ice for 30 min. After incubation, the cells were washed twice with 800 μL of Cell Staining buffer, followed by the addition of anti-mouse-FITC (1:200) and incubation on ice for 45 min. After incubation, the cells were washed twice with 800 μL of Cell Staining buffer, resuspended in 200 μL of PBS, and the binding of CA2 to hCD84 or mCD84 was detected by flow cytometry.

[0038] 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.

[0039] Example 2 CD84 monoclonal antibody CA2 can inhibit the activation of pro-fibrotic macrophages. 1. Experimental Methods CA2 monoclonal antibody inhibition of pro-fibrotic macrophage activation assay THP1 cells were seeded aseptically into 6-well plates at a density of 1 × 10⁶ cells per well. 6 After 24 hours, PMA was added to make the working solution 100 ng / mL. After 24 hours of PMA stimulation, 2.5 μg each of control antibody (Ctl IgG) and anti-human CD84 monoclonal antibody CA2 were added. After 6 hours, interleukin-4 (IL-4) and interleukin-13 (IL-13) were added at a working concentration of 20 ng / mL to jointly stimulate THP1 cells. Cells were harvested after 24 hours.

[0040] Add 1 mL of Trizol to each well to lyse cells, then transfer to a 1.5 mL RNase-free centrifuge tube. Incubate for 5-10 min to allow for complete cell lysis. Add 200 μL of chloroform to each tube, vortex for 15 s, and incubate at room temperature for 2-3 min. Centrifuge at 12000 rpm for 15 min at 4°C. Transfer 400 μL of the upper aqueous layer to a new 1.5 mL RNase-free centrifuge tube, add 400 μL of isopropanol, mix well, and incubate at room temperature for 10 min. Centrifuge at 12000 rpm for 10 min at 4°C to obtain a flocculent precipitate of RNA. Discard the supernatant, add 1 mL of 75% ethanol, and wash the precipitate. Centrifuge at 7000 rpm for 5 min at 4°C, discard the supernatant, repeat once, aspirate excess solution, and allow to evaporate at room temperature. The ethanol should evaporate completely when the RNA becomes colorless. Dissolve the RNA in RNase-free water, incubate on ice for 15 min, and then use a micro-spectrophotometer to detect and record the RNA concentration. The RNA was then reverse-engineered into cDNA using the following reverse-engineering program: 42℃ for 15 min, then 95℃ for 3 min. The RT-PCR system was then prepared as follows: 10 μL of 2×SYBR Green Mix per tube, 1 μL each of forward and reverse primers per tube, 6 μL of RNase-free water, and 2 μL 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. Table 1 Primer Sequences

[0041] Depend on Figure 1 It can be seen that CA2 can significantly inhibit IL-4 / 13-induced activation of profibrotic macrophages, as evidenced by the significant inhibition of the expression of profibrotic factors TGF-β1 and IL-10.

[0042] Example 3 CD84 monoclonal antibody CA2 can inhibit the activation of pro-inflammatory macrophages. 1. Experimental Methods THP1 cells were seeded aseptically into 6-well plates at a density of 1 × 10⁶ cells per well. 6 PMA was added 24 h later to make the working solution 100 ng / mL. After 24 h of PMA stimulation, 2.5 μg each of control antibody (Ctl IgG) and anti-human CD84 monoclonal antibody CA2 were added. After 6 h, LPS / IFN-γ was added to make the working solution 100 ng / mL. The sample was collected 24 h later.

[0043] Add 1 mL of Trizol to each well to lyse cells, then transfer to a 1.5 mL RNase-free centrifuge tube. Incubate for 5-10 min to allow for complete cell lysis. Add 200 μL of chloroform to each tube, vortex for 15 s, and incubate at room temperature for 2-3 min. Centrifuge at 12000 rpm for 15 min at 4°C. Transfer 400 μL of the upper aqueous layer to a new 1.5 mL RNase-free centrifuge tube, add 400 μL of isopropanol, mix well, and incubate at room temperature for 10 min. Centrifuge at 12000 rpm for 10 min at 4°C to obtain a flocculent precipitate of RNA. Discard the supernatant, add 1 mL of 75% ethanol, and wash the precipitate. Centrifuge at 7000 rpm for 5 min at 4°C, discard the supernatant, repeat once, aspirate excess solution, and allow to evaporate at room temperature. The ethanol should evaporate completely when the RNA becomes colorless. Dissolve the RNA in RNase-free water, incubate on ice for 15 min, and then use a micro-spectrophotometer to detect and record the RNA concentration. The RNA was then reverse-engineered into cDNA using the following reverse-engineering program: 42℃ for 15 min, then 95℃ for 3 min. The RT-PCR system was then prepared as follows: 10 μL of 2×SYBR Green Mix per tube, 1 μL each of forward and reverse primers per tube, 6 μL of RNase-free water, and 2 μL of cDNA template. The primer sequences used are shown in Table 2. Table 2 Primer Sequences

[0044] After completion, the eight rows of tubes were placed into a Roche LC96 instrument, and the program was set as follows: pre-denaturation: 95℃ (300 s); denaturation: 95℃ (30 s); annealing: 55℃ (30 s); extension: 72℃ (30 s), for a total of 40 cycles, to detect the expression of the target gene.

[0045] Depend on Figure 2 It is known that CA2 monoclonal antibody can inhibit LPS / IFN-γ-induced macrophage activation, as evidenced by the significant inhibition of the expression of inflammatory factors IL-6, TNF-α and IL-1β.

[0046] Example 4 CD84 monoclonal antibody CA2 can inhibit bleomycin-induced pulmonary fibrosis. 1. Experimental Methods Male C57BL6 / J mice, 7-8 weeks old, 8 mice per group. A pulmonary fibrosis model was established by intratracheal injection of bleomycin (2 U / kg), while the control group was injected with NaCl. The day of model establishment was day 0. From day 0 to day 7 of model establishment, α-CA2 (5 mg / kg) was administered intraperitoneally every 2 days. The mice were euthanized on day 21.

[0047] 2. Experimental Results 2.1 Hydroxyproline content determination: After dissecting the mice, the hydroxyproline (HYP) content in the mouse lung tissue was determined. Specifically, after euthanizing the mice, the right lung was separated, placed in a 5mL ampoule, dried in an oven at 120℃, hydrolyzed in hydrochloric acid, and the pH was adjusted to 6.5-8.0. The residue was filtered through a 0.5μm filter membrane, and PBS (phosphate-balanced saline) was added to adjust the total volume to 10mL. 50μL of the sample was taken, 350μL of deionized water was added, 200μL of chloramine T solution was added, and the mixture was incubated at room temperature for 20min. 200μL of perchloric acid was added, and the mixture was incubated at room temperature for 5min. 200μL of p-dimethylaminobenzaldehyde (P-DMAB) was added, and the mixture was incubated at 65℃ for 20min. Measure the absorbance of the sample in a 96-well plate at 570 nm. Plot a standard curve using the standard readings, and then calculate the hydroxyproline concentration Cs of the sample using the formula derived from the standard curve. Convert the concentration to the total amount of hydroxyproline in the right lung using the following formula I.

[0048] The total amount of hydroxyproline in the right lung = Cs × 8 (dilution factor of the sample) × 10 (total sample volume) Formula I 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 absence of CD84 in macrophages can significantly reduce bleomycin-induced collagen deposition in lung tissue.

[0049] Table 3. Effects of CA2 on hydroxyproline content in mouse lung tissue.

[0050] Note: Data are expressed as mean ± standard error.

[0051] 2.2 Calculation of pulmonary fibrosis area: Mouse lung tissue was paraffin-embedded, and then H&E staining was performed on the lung tissue sections. After image acquisition, the pulmonary fibrosis area in the sections was calculated using Image-Pro Plus software. The percentage of pulmonary fibrosis area = pulmonary fibrosis area / total lung section area × 100%. As shown in Table 4, the average pulmonary fibrosis area in the BLM group reached 28.5%, while the pulmonary fibrosis area in the CA2 group decreased to 17.2%, indicating that CA2 monoclonal antibody can significantly reduce bleomycin-induced pulmonary fibrosis formation.

[0052] Table 4. Effects of CD84 deficiency on the area of ​​pulmonary fibrosis in mice.

[0053] Note: Data are expressed as mean ± standard error.

[0054] Example 5 CD84 monoclonal antibody CA2 can inhibit bleomycin-induced systemic sclerosis-associated interstitial lung disease (SSc-ILD). 1. Experimental Methods Male C57BL / 6 mice were used to establish an SSc-ILD model using bleomycin (0.2 U / mouse / day) administered subcutaneously for 28 consecutive days. Day 1 was the day of model establishment, and mice were weighed daily. Seven days after model establishment, mice were regrouped based on their weight loss rate, with eight mice in each group: control group (NaCl), model group (BLM), and CA2 (Ab-CD84, 5 mg / kg) group. Treatment began on day 7. The Ab-CD84 group received intraperitoneal injections every 3 days, while the NaCl and BLM groups received equal volumes of saline as controls. The general condition of the mice was observed twice daily, once in the morning and once in the afternoon. On day 28, the mice were euthanized for analysis.

[0055] 2. Indicator Testing After the experiment, the dermal thickness, degree of pulmonary fibrosis, and hydroxyproline content in the skin and lung tissue of each group of mice were measured. The results are as follows: 2.1 Dermal Thickness Measurement: The dermal fibrosis thickness was measured by fixing skin samples in 10% formalin, dehydrating, embedding in paraffin, cutting into 5µm thick sections, and performing H&E staining. Table 5 shows the statistical results of dermal thickness in each group of mice. Compared with the control group, the skin thickness of mice in the BLM group was significantly increased, while the skin thickness of mice in the CA2 administration group was significantly decreased, indicating that blocking CD84 with CA2 can alleviate bleomycin-induced skin thickening in mice.

[0056] Table 5. Effect of CA2 on skin thickness in SSc-ILD mice

[0057] Note: Data are expressed as mean ± standard error.

[0058] 2.2 Determination of Skin Collagen Content: The determination of skin collagen content, specifically hydroxyproline content, involved euthanizing mice, isolating mouse skin tissue, placing it in a 5mL ampoule, drying it in a 120℃ oven, hydrolyzing it with hydrochloric acid, adjusting the pH to 6.5-8.0, filtering the residue through a 0.5μm filter membrane, adding PBS (phosphate-balanced saline) to adjust the total volume to 10mL, taking 50μL of the sample, adding 350μL of deionized water, adding 200μL of chloramine T solution, incubating at room temperature for 20min, adding 200μL of perchloric acid, incubating at room temperature for 5min, adding 200μL of p-dimethylaminobenzaldehyde (P-DMAB), incubating at 65℃ for 20min. 200μL of the sample was transferred to a 96-well plate and the absorbance was measured at 570nm. A standard curve was plotted using the standard readings, and the hydroxyproline concentration Cs of the measured sample was then calculated using the formula derived from the standard curve. The hydroxyproline content per unit weight of skin can be calculated using Formula III below.

[0059] The amount of hydroxyproline in the skin = Cs × 8 (dilution factor of the sample) × 10 (total sample volume) / 0.01 (skin weight, g) Formula III As shown in Table 6, compared with the control group, the HYP content per gram of skin in mice in the BLM group was significantly increased, while the HYP content in the skin of mice in the CA2 administration group was significantly decreased, demonstrating that blocking CD84 with CA2 can effectively alleviate bleomycin-induced collagen deposition in mouse skin.

[0060] Table 6. Effects of CA2 on collagen content in the skin of SSc-ILD mice.

[0061] Note: Data are expressed as mean ± standard error.

[0062] 2.3 Statistics on the degree of pulmonary fibrosis: After the administration was completed, H&E staining was performed on the lung tissue sections of mice. After the images were acquired, 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.

[0063] Percentage of pulmonary fibrosis area = (Area of ​​pulmonary fibrosis / Total area of ​​lung sections) × 100% (Formula II) As shown in Table 7, continuous subcutaneous injection of BLM can lead to SSc and further cause pulmonary fibrosis. In contrast, the fibrotic area of ​​the lung tissue in mice treated with CA2 was significantly reduced, indicating that blocking CD84 with CA2 can alleviate the formation of pulmonary fibrosis caused by SSc.

[0064] Table 7. Effect of CA2 on the area of ​​pulmonary fibrosis in SSc-ILD mice.

[0065] Note: Data are expressed as mean ± standard error.

[0066] 2.4 Determination of Lung Hydroxyproline Content: The determination of lung collagen content, also known as hydroxyproline content, involves euthanizing mice, separating skin tissue or the right lung, placing it in a 5mL ampoule, drying it in a 120℃ oven, hydrolyzing it with hydrochloric acid, adjusting the pH to 6.5-8.0, filtering the residue through a 0.5μm filter membrane, adding PBS (phosphate-balanced saline) to adjust the total volume to 10mL, taking 50μL of sample, adding 350μL of deionized water, adding 200μL of chloramine T solution, incubating at room temperature for 20min, adding 200μL of perchloric acid, incubating at room temperature for 5min, adding 200μL of p-dimethylaminobenzaldehyde (P-DMAB), incubating at 65℃ for 20min. 200μL of the sample is then transferred to a 96-well plate, and the absorbance at 570nm is measured. A standard curve is plotted using the standard readings, and the hydroxyproline concentration Cs of the measured sample is calculated using the formula derived from the standard curve. The amount of hydroxyproline contained in the entire right lung can be converted using the following formula I, thus obtaining the hydroxyproline content.

[0067] The total amount of hydroxyproline in the right lung = Cs × 8 (dilution factor of the sample) × 10 (total sample volume) Formula I As shown in Table 8, compared with the control group, the HYP content in the right lung of mice in the BLM group was significantly increased, while the HYP content in the right lung of mice in the CA2 administration group was significantly decreased, demonstrating that blocking CD84 with CA2 can effectively alleviate SSc-induced collagen deposition in the lungs of mice.

[0068] Table 8. Effects of CA2 on collagen content in the right whole lung of SSc-ILD mice.

[0069] Note: Data are expressed as mean ± standard error.

[0070] Example 6 CD84 monoclonal antibody CA2 can inhibit spontaneous systemic lupus erythematosus interstitial lung disease (SLE-ILD) in MRL / Lpr mice. 1. Experimental Methods Male MRL / Lpr mice, five mice per group. At 16 weeks of age, spontaneous SLE-ILD was observed, and drug administration began: control group (Ctl), model group (Model), and CA2 (Ab-CD84, 5 mg / kg) group. Treatment began at week 16. The Ab-CD84 group received intraperitoneal injections every 3 days, while the NaCl group and Model group received equal volumes of saline as controls. The general condition of the mice was observed twice daily, in the morning and afternoon, until week 18.

[0071] 2. Indicator Testing 2.1 Dermal Thickness Measurement: Specifically, the dermal fibrosis thickness was measured by fixing skin samples in 10% formalin, dehydrating, embedding in paraffin, cutting into 5µm thick sections, and performing H&E staining. Table 9 shows the statistical results of dermal thickness in each group of mice. Compared with the control group, the skin thickness of mice in the Model group was significantly increased, while the skin thickness of mice in the CA2-treated group was significantly decreased, indicating that blocking CD84 with CA2 can alleviate spontaneous skin thickening in MRL / Lpr mice.

[0072] Table 9. Effect of CA2 on skin thickness in SLE-ILD mice

[0073] Note: Data are expressed as mean ± standard error.

[0074] 2.2 Spleen Coefficient: The spleen coefficient was determined by euthanizing the mice, weighing their spleens, and dividing the spleen weight by the body weight. The results are shown in Table 10. Table 10 shows that compared to the control group, the spleen coefficient in the model group was significantly increased, while the spleen coefficient in the CA2-treated group was significantly decreased, indicating that blocking CD84 with CA2 can alleviate spontaneous splenomegaly in MRL / Lpr mice.

[0075] Table 10 Effect of CA2 on spleen coefficient in SLE-ILD mice

[0076] Note: Data are expressed as mean ± standard error.

[0077] 2.3 Statistics on the degree of pulmonary fibrosis: After the administration was completed, H&E staining was performed on the lung tissue sections of mice. After the images were acquired, the pulmonary fibrosis area in the sections was calculated using Image-Pro Plus software, and the percentage of pulmonary fibrosis area was calculated.

[0078] 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 lung tissue fibrosis caused by SLE.

[0079] Table 11 Effect of CA2 on pulmonary fibrosis area in SLE-ILD mice

[0080] Note: Data are expressed as mean ± standard error.

[0081] 2.4 Total Cell Count in BALF: The detection of inflammatory cell count in bronchoalveolar lavage fluid (BALF) involved anesthetizing and fixing mice on a worktable, inserting an indwelling needle into the trachea, and then injecting 1 mL of PBS into the lungs via the trachea to completely lavage the lungs. This process was repeated three times. The lavage fluid was collected, centrifuged, and the cell pellet was resuspended in erythrocyte lysis buffer. The cells were lysed at room temperature for 10 min, centrifuged again, and the lysate was discarded. 20 μL of the cell suspension was then resuspended in 100 μL of PBS, and the number of viable cells was detected using a live cell counter (three fields of view were randomly selected for each sample). This yielded the inflammatory cell count in the BALF. Table 12 shows that compared to the control group, the total BALF cell count in the model group was significantly increased, while the total BALF cell count in the CA2-treated group was significantly decreased, indicating that blocking CD84 with anti-mCD84CA2 can alleviate inflammatory cell infiltration in lung tissue caused by SLE.

[0082] Table 12 Effect of CA2 on the total number of inflammatory cells in BALF of SLE-ILD mice

[0083] Note: Data are expressed as mean ± standard error.

[0084] Example 7 The CD84 monoclonal antibody CA2 can inhibit type II collagen-induced rheumatoid arthritis-associated interstitial lung disease (RA-ILD). 1. Experimental Methods Male DBA1 / J wild-type mice (6-8 weeks old) were used to establish the model by intradermal injection of type II collagen (150 μg / mouse) emulsified with adjuvant at the base of the tail. The mice were immunized five times, with booster immunizations every three weeks after the initial immunization. Control mice received the same amount of adjuvant at the same time. Day 0 was the day of model establishment, and mice were weighed daily. At week 12, mice were regrouped according to their weight loss rate, with six mice in each group: control group (Ctl), model group (Model), and CA2 (Ab-CD84, 5 mg / kg) group. Treatment began, with the Ab-CD84 group receiving intraperitoneal injections every three days at the prescribed dose. The NaCl group and Model group received equal volumes of saline as a control. The general condition of the mice was observed twice daily, in the morning and afternoon, until week 20 of treatment.

[0085] 2. Indicator Testing 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 in each group of mice were measured. The results are as follows: 2.1 RA Score: The mouse arthritis index was determined by scoring the mice every three days starting from day 21 after the second booster immunization, until day 50. Each paw of each mouse was scored, and the scores of all four limbs were summed to obtain the mouse's arthritis index. The scoring criteria were 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, with inability to bend the ankle or wrist normally. The mouse arthritis index ranged 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 CA2-treated group was significantly decreased, demonstrating that blocking CD84 can alleviate the severity of arthritis induced by type II collagen immunity.

[0086] Table 13 Effect of CA2 on the severity of arthritis in RA-ILD mice

[0087] Note: Data are expressed as mean ± standard error.

[0088] 2.2 Degree of pulmonary fibrosis: After drug administration, H&E staining was performed on mouse lung tissue sections. After image acquisition, the pulmonary fibrosis area in the sections was calculated using Image-Pro Plus software, and the percentage of pulmonary fibrosis area was calculated.

[0089] The results are shown in Table 14. Compared with the control group, the model group showed obvious fibrosis, while the CA2-treated group showed a significant reduction in the degree of pulmonary fibrosis, proving that blocking CD84 can alleviate RA-induced pulmonary fibrosis.

[0090] Table 14 Effects of CA2 on the degree of pulmonary fibrosis in RA-ILD mice

[0091] Note: Data are expressed as mean ± standard error.

[0092] 2.3 Total Cell Count in BALF: The detection of inflammatory cell count in bronchoalveolar lavage fluid (BALF) involved anesthetizing and fixing mice on a worktable, inserting an indwelling needle into the trachea, and then injecting 1 mL of PBS into the lungs via the trachea to completely lavage the lungs. This process was repeated three times. The lavage fluid was collected, centrifuged, and the cell pellet was resuspended in erythrocyte lysis buffer. The cells were lysed at room temperature for 10 min, centrifuged again, and the lysate was discarded. 20 μL of the cell suspension was then resuspended in 100 μL of PBS, and the number of viable cells was detected using a live cell counter (three fields of view were randomly selected for each sample). This yielded the inflammatory cell count in the BALF. Table 15 shows that compared to the control group, the total BALF cell count in the model group was significantly increased, while the total BALF cell count in the CA2-treated group was significantly decreased, indicating that blocking CD84 with anti-mCD84CA2 can alleviate RA-induced inflammatory cell infiltration in lung tissue.

[0093] Table 15 Effect of CA2 on the total number of inflammatory cells in BALF of RA-ILD mice

[0094] Example 8 CD84 monoclonal antibody AD5 antigen affinity assay 1. Experimental Methods 293T cells were seeded into 6-well plates and overexpressed hCD84 (NM_001184879.2) and mCD84 (NM_013489.4) by transfection with plasmid pCDNA3.1. In each well of each group, 200 μL of OPTI-MEM was used as a dispersion medium, along with 2 μg of siRNA and 6 μL of transfection reagent (Lipo2000). After incubation for 5 min, the two were mixed thoroughly by gently tapping the bottom of the tube, and incubated for a total of 10 min before being added to the wells. Samples were collected after 48 h, and the binding of the anti-human CD84 antibody AD5 to hCD84 and mCD84 was detected by flow cytometry. The Lc sequence of the light chain variable region of the AD5 antibody is as follows: MESQTQVFVYMLLWLSGVDGDIVMTQSQKFMSTSVGDRVSVTCKASQNVGTNVAWYQQKPGQSPKALIYSASYRYSGVPDRFTGSGSGTDFTLTIINVQSEDLAEYFCQQYNSYPYTFGGGTKLEIK (SEQ ID NO: 3); The heavy chain variable region sequence Hc of the AD5 antibody is as follows: QIQLVQSGPELKKPGETVKISCKASGYTFTNYGMNWVKQAPGKGLKWMGWTNTYTGEPTYADDFKGRFAFSLETSASTAYLQINNLKNEDMATYFCARWGYMIFDYWGQGTTLTVSS (SEQ ID NO: 4).

[0095] The AD5 antibody was prepared by cloning the gene sequence encoding AD5 into the eukaryotic expression vector pCDNA3.4, transfecting it into 293F cells, and collecting the culture supernatant after 7 days. The antibody was then purified using a protein G gel. The gene sequence encoding the AD5 light chain variable region is as follows: atggagagccagacccaggtgttcgtgtacatgctgctgtggctgagcggcgtggacggcgacatcgtgatgacccagagccagaagttcatgagc accagcgtgggcgacagagtgagcgtgacctgcaaggccagccagaacgtgggcaccaacgtggcctggtaccagcagaagcccggccagagcccc aaggccctgatctacagcgccagctacagatacagcggcgtgcccgacagattcaccggcagcggcagcggcaccgacttcaccctgaccatcatcaacgtgcagagcgaggacctggccgagtacttctgccagcagtacaacagctacccctacaccttcggcggcggcaccaagctggagatcaag (SEQ ID NO:7); The gene sequence encoding the AD5 heavy chain variable region is as follows: cagatccagctggtgcagagcggccccgagctgaagaagcccggcgagaccgtgaagatcagctgcaaggccagcggctacaccttcaccaactacggcatgaactgggtgaagcaggcccccggcaagggcctgaagtggatgggctggaccaacacctacaccggc gagcccacctacgccgacgacttcaagggcagattcgccttcagcctggagaccagcgccagcaccgcctacctgcagatcaacaacctgaagaacgaggacatggccacctacttctgcgccagatggggctacatgatcttcgactactggggccagggcaccaccctgaccgtgagcagc (SEQ ID NO:8).

[0096] 2. Experimental Results 293T cells were digested and resuspended using trypsin, washed with 800 μL PBS, centrifuged at 800 rpm for 5 min and the supernatant was discarded. Cells were washed with 1 mL Cell Staining buffer, centrifuged at 800 rpm for 5 min and the supernatant was discarded. Cells were resuspended with 100 μL Cell Staining buffer, and 1 μL TruStain FCXTM blocking solution was added. Cells were blocked on ice for 15 min, washed once with 800 μL Cell Staining buffer, centrifuged at 800 rpm for 5 min and the supernatant was discarded. Cells were resuspended with 100 μL Cell Staining buffer. Afterwards, add 0.1, 1, 3, 10, or 30 μg / mL of AD5 or control IgG antibody to each well and incubate on ice for 30 min. After incubation, wash twice with 800 μL of Cell Staining buffer, then add anti-mouse-FITC (1:200) and incubate on ice for 45 min. After incubation, wash twice with 800 μL of Cell Staining buffer, resuspend in 200 μL of PBS, and then use flow cytometry to detect the binding of AD5 to hCD84 or mCD84.

[0097] 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.

[0098] Example 9 CD84 monoclonal antibody AD5 can inhibit the activation of pro-fibrotic macrophages. 1. Experimental Methods AD5 monoclonal antibody inhibition of pro-fibrotic macrophage activation assay THP1 cells were seeded aseptically into 6-well plates at a density of 1 × 10⁶ cells per well. 6 PMA was added 24 h to make the working solution 100 ng / mL. After 24 h of PMA stimulation, 2.5 μg each of control antibody (Ctl IgG) and anti-human CD84 monoclonal antibody AD5 were added. After 6 h, interleukin-4 (IL-4) and interleukin-13 (IL-13) were added at a working concentration of 20 ng / mL to jointly stimulate THP1 cells. Cells were harvested 24 h later.

[0099] Add 1 mL of Trizol to each well to lyse cells, then transfer to a 1.5 mL RNase-free centrifuge tube. Incubate for 5-10 min to allow for complete cell lysis. Add 200 μL of chloroform to each tube, vortex for 15 s, and incubate at room temperature for 2-3 min. Centrifuge at 12000 rpm for 15 min at 4°C. Transfer 400 μL of the upper aqueous layer to a new 1.5 mL RNase-free centrifuge tube, add 400 μL of isopropanol, mix well, and incubate at room temperature for 10 min. Centrifuge at 12000 rpm for 10 min at 4°C to obtain a flocculent precipitate of RNA. Discard the supernatant, add 1 mL of 75% ethanol, and wash the precipitate. Centrifuge at 7000 rpm for 5 min at 4°C, discard the supernatant, repeat once, aspirate excess solution, and allow to evaporate at room temperature. The ethanol should evaporate completely when the RNA becomes colorless. Dissolve the RNA in RNase-free water, incubate on ice for 15 min, and then use a micro-spectrophotometer to detect and record the RNA concentration. The RNA was then reverse-engineered into cDNA using the following reverse-engineering program: 42℃ for 15 min, then 95℃ for 3 min. The RT-PCR system was then prepared as follows: 10 μL of 2×SYBR Green Mix per tube, 1 μL each of forward and reverse primers per tube, 6 μL of RNase-free water, and 2 μL of cDNA template. After completion, Roche assays were used to detect the expression of the target gene. The primer sequences used are shown in Table 16. Table 16 Primer Sequences

[0100] Depend on Figure 5 It can be seen that Ab-CD84 can significantly inhibit IL-4 / 13-induced activation of profibrotic macrophages, as evidenced by the significant inhibition of the expression of profibrotic factors TGF-β1 and IL-10.

[0101] Example 10 The CD84 monoclonal antibody AD5 (Ab-CD84 antibody) can inhibit the activation of pro-inflammatory macrophages. 1. Experimental Methods THP1 cells were seeded aseptically into 6-well plates at a density of 1 × 10⁶ cells per well. 6 PMA was added 24 h later to make the working solution 100 ng / mL. After 24 h of PMA stimulation, 2.5 μg each of control antibody (Ctl IgG) and Ab-CD84 were added. After 6 h, LPS / IFN-γ was added to make the working solution 100 ng / mL. The sample was collected 24 h later.

[0102] Add 1 mL of Trizol to each well to lyse cells, then transfer to a 1.5 mL RNase-free centrifuge tube. Incubate for 5-10 min to allow for complete cell lysis. Add 200 μL of chloroform to each tube, vortex for 15 s, and incubate at room temperature for 2-3 min. Centrifuge at 12000 rpm for 15 min at 4°C. Transfer 400 μL of the supernatant to a new 1.5 mL RNase-free centrifuge tube, add 400 μL of isopropanol, mix well, and incubate at room temperature for 10 min. Centrifuge at 12000 rpm for 10 min at 4°C to obtain a flocculent precipitate of RNA. Discard the supernatant and add 1 mL of 75% ethanol to wash the precipitate. Centrifuge at 7000 rpm for 5 min at 4°C, discard the supernatant, repeat once, aspirate excess solution, and allow to evaporate at room temperature. The ethanol should evaporate completely when the RNA becomes colorless. Dissolve the RNA in RNase-free water, incubate on ice for 15 min, and then use a micro-spectrophotometer to detect and record the RNA concentration. The RNA was then reverse-engineered into cDNA using the following reverse-engineering program: 42℃ for 15 min, then 95℃ for 3 min. The RT-PCR system was then prepared as follows: 10 μL of 2×SYBR Green Mix per tube, 1 μL each of forward and reverse primers per tube, 6 μL of RNase-free water, and 2 μL of cDNA template. The primer sequences used are shown in Table 17. Table 17 Primer Sequences

[0103] After completion, the eight rows of tubes were placed into a Roche LC96 instrument, and the program was set as follows: pre-denaturation: 95℃ (300 s); denaturation: 95℃ (30 s); annealing: 55℃ (30 s); extension: 72℃ (30 s), for a total of 40 cycles, to detect the expression of the target gene.

[0104] Depend on Figure 6It can be seen that the Ab-CD84 antibody can inhibit LPS / IFN-γ-induced macrophage activation, as evidenced by the significant inhibition of the expression of inflammatory factors IL-6, TNF-α and IL-1β.

[0105] Example 11 CD84 monoclonal antibody AD5 (Ab-CD84 antibody) can inhibit bleomycin-induced pulmonary fibrosis. 1. Experimental Methods Male C57BL6 / J mice, 7-8 weeks old, 8 mice per group. A pulmonary fibrosis model was established by intratracheal injection of bleomycin (2 U / kg), while the control group was injected with NaCl. The day of model establishment was day 0. From day 0 to day 7 of model establishment, β-Ab-CD84 (5 mg / kg) was administered intraperitoneally every 2 days. The mice were euthanized on day 21.

[0106] 2. Experimental Results 2.1 Hydroxyproline content determination: After dissecting the mice, the hydroxyproline (HYP) content in the mouse lung tissue was determined. Specifically, after euthanizing the mice, the right lung was separated, placed in a 5mL ampoule, dried in an oven at 120℃, hydrolyzed in hydrochloric acid, and the pH was adjusted to 6.5-8.0. The residue was filtered through a 0.5μm filter membrane, and PBS (phosphate-balanced saline) was added to adjust the total volume to 10mL. 50μL of the sample was taken, 350μL of deionized water was added, 200μL of chloramine T solution was added, and the mixture was incubated at room temperature for 20min. 200μL of perchloric acid was added, and the mixture was incubated at room temperature for 5min. 200μL of p-dimethylaminobenzaldehyde (P-DMAB) was added, and the mixture was incubated at 65℃ for 20min. Measure the absorbance of the sample in a 96-well plate at 570 nm. Plot a standard curve using the standard readings, and then calculate the hydroxyproline concentration Cs of the sample using the formula derived from the standard curve. Convert the concentration to the total amount of hydroxyproline in the right lung using the following formula I.

[0107] The total amount of hydroxyproline in the right lung = Cs × 8 (dilution factor of the sample) × 10 (total sample volume) Formula I 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 the HYP content in the Ab-CD84 group was significantly decreased compared with the BLM group, indicating that the absence of CD84 in macrophages can significantly reduce bleomycin-induced collagen deposition in lung tissue.

[0108] Table 18 Effects of Ab-CD84 on hydroxyproline content in mouse lung tissue

[0109] Note: Data are expressed as mean ± standard error.

[0110] 2.2 Calculation of pulmonary fibrosis area: Mouse lung tissue was paraffin-embedded, and then H&E staining was performed on the lung tissue sections. After image acquisition, the pulmonary fibrosis area in the sections was calculated using Image-Pro Plus software. The percentage of pulmonary fibrosis area = pulmonary fibrosis area / total lung section area × 100%. As shown in Table 19, the average pulmonary fibrosis area in the BLM group reached 31.5%, while the pulmonary fibrosis area in the Ab-CD84 group decreased to 18.5%, indicating that blocking CD84 can significantly reduce bleomycin-induced pulmonary fibrosis formation.

[0111] Table 19 Effect of CD84 deficiency on the area of ​​pulmonary fibrosis in mice

[0112] Note: Data are expressed as mean ± standard error.

[0113] Example 12 The CD84 monoclonal antibody AD5 (Ab-CD84 antibody) can inhibit bleomycin-induced systemic sclerosis-associated interstitial lung disease (SSc-ILD). 1. Experimental Methods Male C57BL / 6 mice were used to establish an SSc-ILD model by subcutaneous injection of bleomycin (0.2 U / mouse / day) for 28 consecutive days. Day 1 was the day of model establishment, and mice were weighed daily. Seven days after model establishment, mice were regrouped based on their weight loss rate, with eight mice in each group: control group (NaCl), model group (BLM), and Ab-CD84 (5 mg / kg) group. Treatment began on day 7. The Ab-CD84 group received intraperitoneal injections every three days, while the NaCl and BLM groups received equal volumes of saline as controls. The general condition of the mice was observed twice daily, once in the morning and once in the afternoon. The mice were euthanized and analyzed on day 28 after treatment.

[0114] 2. Indicator Testing After the experiment, the dermal thickness, degree of pulmonary fibrosis, and hydroxyproline content in the skin and lung tissue of each group of mice were measured. The results are as follows: 2.1 Dermal Thickness Measurement: Specifically, skin samples were fixed in 10% formalin, dehydrated, embedded in paraffin, cut into 5µm thick sections, and stained with H&E. Table 20 shows the statistical results of dermal thickness in each group of mice. Compared with the control group, the skin thickness of mice in the BLM group was significantly increased, while the skin thickness of mice in the Ab-CD84 administration group was significantly decreased, indicating that blocking CD84 can alleviate bleomycin-induced skin thickening in mice.

[0115] Table 20 Effect of Ab-CD84 on skin thickness in SSc-ILD mice

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

[0117] 2.2 Determination of Skin Collagen Content: The determination of skin collagen content, specifically hydroxyproline content, involved euthanizing mice, isolating mouse skin tissue, placing it in a 5mL ampoule, drying it in a 120℃ oven, hydrolyzing it with hydrochloric acid, adjusting the pH to 6.5-8.0, filtering the residue through a 0.5μm filter membrane, adding PBS (phosphate-balanced saline) to adjust the total volume to 10mL, taking 50μL of the sample, adding 350μL of deionized water, adding 200μL of chloramine T solution, incubating at room temperature for 20min, adding 200μL of perchloric acid, incubating at room temperature for 5min, adding 200μL of p-dimethylaminobenzaldehyde (P-DMAB), incubating at 65℃ for 20min. 200μL of the sample was transferred to a 96-well plate and the absorbance was measured at 570nm. A standard curve was plotted using the standard readings, and the hydroxyproline concentration Cs of the measured sample was then calculated using the formula derived from the standard curve. The hydroxyproline content per unit weight of skin can be calculated using Formula III below.

[0118] The amount of hydroxyproline in the skin = Cs × 8 (dilution factor of the sample) × 10 (total sample volume) / 0.01 (skin weight, g) Formula III As shown in Table 21, compared with the control group, the HYP content per gram of skin in mice in the BLM group was significantly increased, while the HYP content in the skin of mice in the Ab-CD84 administration group was significantly decreased, demonstrating that the Ab-CD84 blocking antibody can effectively alleviate bleomycin-induced collagen deposition in mouse skin.

[0119] Table 21 Effects of Ab-CD84 on skin collagen content in SSc-ILD mice

[0120] Note: Data are expressed as mean ± standard error.

[0121] 2.3 Statistics on the degree of pulmonary fibrosis: After the administration was completed, H&E staining was performed on the lung tissue sections of mice. After the images were acquired, 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.

[0122] Percentage of pulmonary fibrosis area = (Area of ​​pulmonary fibrosis / Total area of ​​lung sections) × 100% (Formula II) As shown in Table 22, continuous subcutaneous injection of BLM can lead to SSc and further cause pulmonary fibrosis. However, the fibrotic area of ​​the lung tissue in mice treated with Ab-CD84 was significantly reduced, indicating that CD84 blocking antibodies can alleviate the formation of pulmonary fibrosis caused by SSc.

[0123] Table 22 Effect of Ab-CD84 on pulmonary fibrosis area in SSc-ILD mice

[0124] Note: Data are expressed as mean ± standard error.

[0125] 2.4 Determination of Lung Hydroxyproline Content: The determination of lung collagen content, also known as hydroxyproline content, involves euthanizing mice, separating skin tissue or the right lung, placing it in a 5mL ampoule, drying it in a 120℃ oven, hydrolyzing it with hydrochloric acid, adjusting the pH to 6.5-8.0, filtering the residue through a 0.5μm filter membrane, adding PBS (phosphate-balanced saline) to adjust the total volume to 10mL, taking 50μL of sample, adding 350μL of deionized water, adding 200μL of chloramine T solution, incubating at room temperature for 20min, adding 200μL of perchloric acid, incubating at room temperature for 5min, adding 200μL of p-dimethylaminobenzaldehyde (P-DMAB), incubating at 65℃ for 20min. 200μL of the sample is then transferred to a 96-well plate, and the absorbance at 570nm is measured. A standard curve is plotted using the standard readings, and the hydroxyproline concentration Cs of the measured sample is calculated using the formula derived from the standard curve. The amount of hydroxyproline contained in the entire right lung can be converted using the following formula I, thus obtaining the hydroxyproline content.

[0126] The total amount of hydroxyproline in the right lung = Cs × 8 (dilution factor of the sample) × 10 (total sample volume) Formula I As shown in Table 23, compared with the control group, the HYP content in the right lung of mice in the BLM group was significantly increased, while the HYP content in the right lung of mice in the Ab-CD84 administration group was significantly decreased, demonstrating that CD84 blocking antibody can effectively alleviate SSc-induced collagen deposition in the lungs of mice.

[0127] Table 23 Effects of Ab-CD84 on collagen content in the right whole lung of SSc-ILD mice

[0128] Note: Data are expressed as mean ± standard error.

[0129] Example 13 CD84 monoclonal antibody can inhibit spontaneous systemic lupus erythematosus interstitial lung disease (SLE-ILD) in MRL / Lpr mice. 1. Experimental Methods Male MRL / Lpr mice, five mice per group. At 16 weeks of age, spontaneous SLE-ILD was observed, and drug administration began: control group (Ctl), model group (Model), and Ab-CD84 (5 mg / kg) group. Treatment began at week 16. The Ab-CD84 group received intraperitoneal injections every 3 days, while the NaCl group and Model group received equal volumes of saline as controls. The general condition of the mice was observed twice daily, in the morning and afternoon, until week 18.

[0130] 2. Indicator Testing 2.1 Dermal Thickness Detection: Specifically, skin samples were fixed in 10% formalin, dehydrated, embedded in paraffin, cut into 5µm thick sections, and stained with H&E. Table 24 shows the statistical results of dermal thickness in each group of mice. Compared with the control group, the skin of mice in the Model group was significantly thicker, while the skin thickness of mice in the Ab-CD84 administration group was significantly reduced, indicating that the CD84 blocking antibody can alleviate spontaneous skin thickening in MRL / Lpr mice.

[0131] Table 24 Effect of Ab-CD84 on skin thickness in SLE-ILD mice

[0132] Note: Data are expressed as mean ± standard error.

[0133] 2.2 Spleen Coefficient: The spleen coefficient was determined by euthanizing the mice, weighing their spleens, and dividing the spleen weight by the body weight. The results are shown in Table 25. Table 25 shows that the spleen coefficient in the model group was significantly higher than that in the control group, while the spleen coefficient in the Ab-CD84-treated group was significantly lower, indicating that the CD84-blocking antibody can alleviate spontaneous splenomegaly in MRL / Lpr mice.

[0134] Table 25 Effect of Ab-CD84 on spleen coefficient in SLE-ILD mice

[0135] Note: Data are expressed as mean ± standard error.

[0136] 2.3 Statistics on the degree of pulmonary fibrosis: After the administration was completed, H&E staining was performed on the lung tissue sections of mice. After the images were acquired, the pulmonary fibrosis area in the sections was calculated using Image-Pro Plus software, and the percentage of pulmonary fibrosis area was calculated.

[0137] As shown in Table 26, obvious fibrosis occurred in the lung tissue of the model group, while the fibrosis area of ​​mice in the Ab-CD84 administration group was significantly reduced, proving that blocking CD84 can alleviate lung tissue fibrosis caused by SLE.

[0138] Table 26 Effect of Ab-CD84 on pulmonary fibrosis area in SLE-ILD mice

[0139] Note: Data are expressed as mean ± standard error.

[0140] 2.4 Total Cell Count in BALF: The detection of inflammatory cell count in bronchoalveolar lavage fluid (BALF) involved anesthetizing and fixing mice on a control table, inserting an indwelling needle into the trachea, and then injecting 1 mL of PBS into the lungs via the trachea to completely lavage the lungs. This process was repeated three times. The lavage fluid was collected, centrifuged, and the cell pellet was resuspended in erythrocyte lysis buffer. The cells were lysed at room temperature for 10 min, centrifuged again, and the lysate was discarded. 20 μL of the cell suspension was then resuspended in 100 μL of PBS, and the number of viable cells was detected using a live cell counter (three fields of view were randomly selected for each sample). This yielded the inflammatory cell count in the BALF. Table 27 shows that compared to the control group, the total BALF cell count in the model group was significantly increased, while the total BALF cell count in the Ab-CD84 administration group was significantly decreased, indicating that blocking CD84 with anti-mCD84 can alleviate inflammatory cell infiltration in lung tissue caused by SLE.

[0141] Table 27 Effect of Ab-CD84 on the total number of inflammatory cells in BALF of SLE-ILD mice

[0142] Note: Data are expressed as mean ± standard error.

[0143] Example 14 CD84 monoclonal antibody can inhibit type II collagen-induced rheumatoid arthritis-associated interstitial lung disease (RA-ILD). 1. Experimental Methods Male DBA1 / J wild-type mice (6-8 weeks old) were used to establish the model by intradermal injection of type II collagen (150 μg / mouse) emulsified with adjuvant at the base of the tail. A total of 5 immunizations were administered, with booster immunizations every 3 weeks after the initial immunization. Control mice received the same amount of adjuvant at the same time. Day 0 was the day of model establishment, and mice were weighed daily. At week 12, mice were regrouped according to their weight loss rate, with 6 mice in each group: control group (Ctl), model group (Model), and Ab-CD84 (5 mg / kg) group. Treatment began, with the Ab-CD84 group receiving intraperitoneal injections every 3 days, and the NaCl group and Model group receiving equal volumes of saline as a control. The general condition of the mice was observed twice daily, in the morning and afternoon, until week 20 of treatment.

[0144] 2. Indicator Testing 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 in each group of mice were measured. The results are as follows: 1.1 RA Score: The mouse arthritis index was determined by scoring the mice every three days, starting from the second booster immunization on day 21, until day 50. Each paw of each mouse was scored, and the scores of all four limbs were summed to obtain the mouse's arthritis index. The scoring criteria were 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, with the ankle or wrist unable to bend normally. The mouse arthritis index ranged 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 Ab-CD84-treated group was significantly decreased, demonstrating that blocking CD84 can alleviate the severity of arthritis induced by type II collagen immunity.

[0145] Table 28 Effect of Ab-CD84 on the severity of arthritis in RA-ILD mice

[0146] Note: Data are expressed as mean ± standard error.

[0147] 1.2 Degree of pulmonary fibrosis: After drug administration, H&E staining was performed on mouse lung tissue sections. After image acquisition, the pulmonary fibrosis area in the sections was calculated using Image-Pro Plus software, and the percentage of pulmonary fibrosis area was calculated.

[0148] The results are shown in Table 29. Compared with the control group, the model group showed obvious fibrosis, while the Ab-CD84 administration group showed a significant reduction in the degree of pulmonary fibrosis, proving that blocking CD84 can alleviate pulmonary fibrosis caused by RA.

[0149] Table 29 Effects of Ab-CD84 on the degree of pulmonary fibrosis in RA-ILD mice

[0150] Note: Data are expressed as mean ± standard error.

[0151] 2.3 Total Cell Count in BALF: The detection of inflammatory cell count in bronchoalveolar lavage fluid (BALF) involved anesthetizing and fixing mice on a worktable, inserting an indwelling needle into the trachea, and then injecting 1 mL of PBS into the lungs via the trachea to completely lavage the lungs. This process was repeated three times. The lavage fluid was collected, centrifuged, and the cell pellet was resuspended in erythrocyte lysis buffer. The cells were lysed at room temperature for 10 min, centrifuged again, and the lysate was discarded. 20 μL of the cell suspension was then resuspended in 100 μL of PBS, and the number of viable cells was detected using a live cell counter (three fields of view were randomly selected for each sample). This yielded the inflammatory cell count in the BALF. Table 30 shows that compared to the control group, the total BALF cell count in the model group was significantly increased, while the total BALF cell count in the Ab-CD84 administration group was significantly decreased, indicating that blocking CD84 with anti-mCD84 can alleviate RA-induced inflammatory cell infiltration in lung tissue.

[0152] Table 30 Effect of Ab-CD84 on the total number of inflammatory cells in BALF of RA-ILD mice

[0153] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The use of an anti-CD84 antibody in the preparation of a medicament for the prevention and / or treatment of pulmonary fibrosis, wherein the anti-CD84 antibody is a CA2 monoclonal antibody or an AD5 monoclonal antibody; The amino acid sequence of the heavy chain variable region of the CA2 monoclonal antibody is shown in SEQ ID NO:2, and the amino acid sequence of the light chain variable region of the CA2 monoclonal antibody is shown in SEQ ID NO:

1. The amino acid sequence of the heavy chain variable region of the AD5 monoclonal antibody is shown in SEQ ID NO:4, and the amino acid sequence of the light chain variable region of the AD5 monoclonal antibody is shown in SEQ ID NO:

3. The pulmonary fibrosis is idiopathic pulmonary fibrosis and / or an indication for pulmonary fibrosis; the indication for pulmonary fibrosis includes one or more of the following: systemic sclerosis-associated interstitial lung disease, spontaneous systemic lupus erythematosus interstitial lung disease, and rheumatoid arthritis-associated interstitial lung disease.

2. Use according to claim 1, characterized in that, The drug is a drug that inhibits the activation of macrophages.

3. Use according to claim 2, characterized in that, The macrophages include pro-inflammatory macrophages and / or pro-fibrotic macrophages.

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