Use of an anti-cd84 antibody in the preparation of a medicament for preventing and / or treating inflammatory bowel disease

Abstract

The application provides application of an anti-CD84 antibody in preparation of a medicine for preventing and / or treating inflammatory bowel disease (IBD), and belongs to the technical field of biological medicines.The anti-CD84 antibody is used as a medicine to inhibit the biological function of CD84, and not only can the activation of proinflammatory macrophages be inhibited, but also the intestinal inflammation and pathological changes of a patient with inflammatory bowel disease can be relieved, and the level of an inflammatory factor in serum can be reduced, so that the purpose of preventing and treating inflammatory bowel disease is achieved.Therefore, the application provided by the application provides a new means for preventing and treating inflammatory bowel disease.

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 / or treatment of inflammatory bowel disease. Background Technology

[0002] Inflammatory bowel disease (IBD) consists of ulcerative colitis (UC) and Crohn's disease (CD). It is a chronic, progressive, and relapsing intestinal disease that severely impacts patients' quality of life and daily routines, and increases the burden of healthcare.

[0003] Traditional treatments for IBD include aminosalicylic acid derivatives, antibiotics, immunosuppressants, and glucocorticoids, but their efficacy is not always satisfactory.

[0004] Given the significant driving role of immune abnormalities in the progression of IBD, current IBD treatments primarily focus on anti-inflammatory or immunomodulatory therapies, especially some biologic antibodies such as the anti-TNF monoclonal antibody infliximab and the anti-interleukin (IL) 12-23 antibody ustekinumab. Additionally, tofacitinib, a small molecule drug targeting JAK, has been approved by the FDA for the treatment of mild to severe UC. However, immunomodulation often leads to adverse reactions such as diarrhea or lymphopenia, prolonging treatment duration, and high relapse rates limit their clinical application. Systemic administration of anti-inflammatory drugs is a major cause of adverse reactions. Therefore, the development of drugs specifically targeting colonic inflammation is of great importance for the treatment of IBD. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide an application of an anti-CD84 antibody in the preparation of a drug for the prevention and / or treatment of inflammatory bowel disease, which achieves the effects of alleviating the severity of enteritis, colorectal lesions and reducing inflammatory response by inhibiting the expression and function of CD84.

[0006] This invention provides the use of an anti-CD84 antibody in the preparation of a medicament for the prevention and / or treatment of inflammatory bowel disease.

[0007] Preferably, the CD84 antibody includes a monoclonal antibody against the CD84 protein and / or a polyclonal antibody against the CD84 protein.

[0008] Preferably, the monoclonal antibody against CD84 protein includes CA2 monoclonal antibody and AD5 monoclonal antibody;

[0009] The light chain variable region sequence of the CA2 monoclonal antibody is shown in SEQ ID NO:1;

[0010] The heavy chain variable region sequence of the CA2 monoclonal antibody is shown in SEQ ID NO:2;

[0011] The light chain variable region sequence of the AD5 monoclonal antibody is shown in SEQ ID NO:3;

[0012] The heavy chain variable region sequence of the AD5 monoclonal antibody is shown in SEQ ID NO:4.

[0013] Preferably, the CD84 is the CD84 of macrophages.

[0014] Preferably, the drug is a drug that inhibits the activation of pro-inflammatory macrophages.

[0015] Preferably, the treatment of inflammatory bowel disease includes alleviating intestinal epithelial damage caused by pro-inflammatory macrophage activation, reducing the severity of enteritis, alleviating colorectal lesions, and reducing inflammatory factor levels.

[0016] Preferably, the relief of colorectal lesions includes restoring colorectal length.

[0017] Preferably, the inflammatory factors include at least one of the following: TNF-α, IL-1β, and IL-6.

[0018] Preferably, the inflammatory bowel disease includes ulcerative colitis and Crohn's disease.

[0019] This invention provides a medicament for the prevention and / or treatment of inflammatory bowel disease, comprising an anti-CD84 antibody and medically acceptable excipients;

[0020] Preferably, the anti-CD84 antibody includes a monoclonal antibody against the CD84 protein and / or a polyclonal antibody against the CD84 protein;

[0021] More preferably, the monoclonal antibody against CD84 protein includes CA2 monoclonal antibody and AD5 monoclonal antibody;

[0022] The light chain variable region sequence of the CA2 monoclonal antibody is shown in SEQ ID NO:1;

[0023] The heavy chain variable region sequence of the CA2 monoclonal antibody is shown in SEQ ID NO:2;

[0024] The light chain variable region sequence of the AD5 monoclonal antibody is shown in SEQ ID NO:3;

[0025] The heavy chain variable region sequence of the AD5 monoclonal antibody is shown in SEQ ID NO:4.

[0026] This invention provides the application of an anti-CD84 antibody in the preparation of drugs for the prevention and / or treatment of inflammatory bowel disease (IBD). Using a classic animal model of IBD induced by dextran sulfate sodium (DSS) as the experimental subject, this invention uses an anti-CD84 antibody as a drug to inhibit the biological function of CD84. Results show that compared with the control group (NaCl), the Disease Activity Index (DAI) score of the model group was significantly increased, while the DAI score of the anti-CD84 antibody-treated group was significantly lower than that of the model group. This indicates that inhibiting the biological function of CD84 is beneficial in alleviating the severity of DSS-induced enteritis in mice. Furthermore, enteritis typically leads to shortening of the colorectal lesions. This invention also statistically analyzed the length of the colorectal lesions in the control group, model group, and treated group mice. Results show that compared with the control group, the colorectal lesions in the model group mice were significantly shortened, while the colorectal lesions in the treated group mice showed significant recovery in length. This demonstrates that inhibiting the biological function of CD84 is beneficial in significantly alleviating DSS-induced colorectal lesions. This invention also examined the secretion of inflammatory factors in the three treatment groups. The results showed that, compared to the control group, the secretion levels of key pro-inflammatory factors in the model group were significantly increased, while the secretion levels of serum TNF-α, IL-1β, and IL-6 in the treatment groups were significantly decreased. This demonstrates that inhibiting the biological function of CD84 is beneficial in significantly reducing the levels of serum pro-inflammatory factors in IBD mice, thereby reducing the inflammatory response in the mice. Therefore, the above experimental results indicate that anti-CD84 antibodies can effectively achieve the therapeutic and preventative effects of inflammatory bowel disease. Furthermore, since CD84 is not an inflammatory factor, blocking CD84 function does not affect immune surveillance, and CD84 blockade is often used as a therapeutic target for reversing tumor-induced immunosuppression, without posing other safety risks. Therefore, reagents that inhibit CD84 expression or function provide a new approach for preparing drugs to prevent and / or treat inflammatory bowel disease. Attached Figure Description

[0027] Figure 1 The binding curves of the CD84 monoclonal antibody CA2 with hCD84 and mCD84 are shown.

[0028] Figure 2 The results show the effectiveness of the CD84 monoclonal antibody CA2 in inhibiting LPS / IFN-γ-induced pro-inflammatory macrophage activation.

[0029] Figure 3 The results of the DAI score for IBD mice;

[0030] Figure 4 Results of colon and rectum length measurement in IBD mice;

[0031] Figure 5 The results of ELISA detection of serum inflammatory factors in IBD mice;

[0032] Figure 6Results of intestinal epithelial barrier damage factor protein detection;

[0033] Figure 7 The binding curves of the CD84 monoclonal antibody AD5 with hCD84 and mCD84 are shown.

[0034] Figure 8 The results show the effect of the CD84 monoclonal antibody AD5 on inhibiting LPS / IFN-γ-induced pro-inflammatory macrophage activation;

[0035] Figure 9 The results of the DAI score for IBD mice;

[0036] Figure 10 Results of colon and rectum length measurement in IBD mice;

[0037] Figure 11 The results of ELISA detection of serum inflammatory factors in IBD mice;

[0038] Figure 12 The results are from the detection of intestinal epithelial barrier damage factor proteins. Detailed Implementation

[0039] This invention provides the use of an anti-CD84 antibody in the preparation of a medicament for the prevention and / or treatment of inflammatory bowel disease.

[0040] In this invention, the inflammatory bowel disease preferably includes Crohn's disease and ulcerative colitis. Crohn's disease mainly occurs in the digestive tract, while ulcerative colitis mainly occurs near the rectum and anus. In this embodiment of the invention, a disease animal model induced by dextran sulfate sodium (DSS) is used as a classic animal model of inflammatory bowel disease. Compared with the control group, the disease activity index (DAI) score of the model group was significantly increased, and the colon and rectum of the model group mice were significantly shortened, indicating that the model mice had severe enteritis. At the same time, the secretion levels of key pro-inflammatory factors in the model group were significantly increased, indicating that the model mice had a severe inflammatory response in the intestine.

[0041] In this invention, the CD84 is preferably CD84 from macrophages. The macrophages preferably include pro-inflammatory macrophages. The anti-CD84 antibody preferably includes a monoclonal antibody against the CD84 protein and / or a polyclonal antibody against the CD84 protein. The monoclonal antibody against the CD84 protein includes CA2 monoclonal antibody and AD5 monoclonal antibody; the light chain variable region sequence of the CA2 monoclonal antibody is shown in SEQ ID NO:1; the heavy chain variable region sequence of the CA2 monoclonal antibody is shown in SEQ ID NO:2; the light chain variable region sequence of the AD5 monoclonal antibody is shown in SEQ ID NO:3; and the heavy chain variable region sequence of the AD5 monoclonal antibody is shown in SEQ ID NO:4. The source of the polyclonal antibody against the CD84 protein is not particularly limited in this invention; any method well-known in the art for preparing polyclonal antibodies can be used, such as using CD84 protein derived from macrophages as an immunogen, repeatedly immunizing animals, and collecting antiserum. The small molecule inhibitor of the CD84 protein has an inhibitory effect on the function of the CD84 protein.

[0042] In this invention, the drug is preferably a drug that inhibits the activation of pro-inflammatory macrophages. The therapeutic effects on inflammatory bowel disease (IBD) preferably include alleviating intestinal epithelial damage caused by pro-inflammatory macrophage activation, reducing the severity of enteritis, alleviating colorectal lesions, and reducing the levels of inflammatory factors. In an embodiment of this invention, the biological function of CD84 is blocked by a monoclonal antibody against CD84 protein. Results show that the monoclonal antibody against CD84 protein can alleviate the severity of DSS-induced enteritis in mice, restore the length of the colon and rectum, and reduce the secretion levels of serum inflammatory factors TNF-α, IL-1β, and IL-6, thereby achieving the purpose of preventing and / or treating IBD. Therefore, this invention uses CD84 as a therapeutic target and develops a drug for the prevention and treatment of IBD by using agents that inhibit CD84 expression or function. This invention does not impose any special limitations on the dosage form and preparation method of the drug; any drug preparation method well known in the art can be used.

[0043] This invention provides a medicament for the prevention and / or treatment of inflammatory bowel disease, comprising an anti-CD84 antibody and medically acceptable excipients.

[0044] In this invention, the anti-CD84 antibody preferably comprises a monoclonal antibody against the CD84 protein and / or a polyclonal antibody against the CD84 protein. The monoclonal antibody against the CD84 protein includes CA2 monoclonal antibody and AD5 monoclonal antibody. The light chain variable region sequence of the CA2 monoclonal antibody is shown in SEQ ID NO:1; the heavy chain variable region sequence of the CA2 monoclonal antibody is shown in SEQ ID NO:2. The light chain variable region sequence of the AD5 monoclonal antibody is shown in SEQ ID NO:3; the heavy chain variable region sequence of the AD5 monoclonal antibody is shown in SEQ ID NO:4. This invention uses a recombinant expression method to prepare CA2 and AD5 monoclonal antibodies. Experimental verification shows that the CA2 and AD5 monoclonal antibodies have good binding affinity to the CD84 antigen, thus providing a new approach for the prevention and treatment of IBD.

[0045] The following examples illustrate the application of a reagent for inhibiting CD84 expression or function provided by the present invention in the preparation of drugs for the prevention and treatment of inflammatory bowel disease. However, these examples should not be construed as limiting the scope of protection of the present invention.

[0046] Example 1

[0047] CD84 monoclonal antibody CA2 antigen affinity assay

[0048] 1. Experimental Methods

[0049] 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 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:

[0050] MRPSIQFLGLLLFWLHGAQCDIQMTQSPSSSLSASLGGKVTITCKASQ DINKYIAWYQQKSGKGPRLLIHYTSTLQPGIPSRFSGSGSGRDYSFSISNLE PEDIATYYCLQYDNLYTFGGGTKLEIK(SEQ ID NO:1);

[0051] The heavy chain variable region sequence Hc of the CA2 antibody is as follows:

[0052] EVQLVESGGDLVRPGGSLKLSCAASGFTFTDYGMSWVRQTPDKRLE WVATISTASTYTFYPDSVKGRFTISRDNAKNTLYLQMSSLKSEDTAIFYCV RHGHYYGSRYTWLAYWGQGTLVTVSA (SEQ ID NO: 2).

[0053] The CA2 antibody was prepared by cloning the gene sequence encoding CA2 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 CA2 light chain variable region is as follows:

[0054] atgagacccagcatccagttcctgggcctgctgctgttctggctgcacggcgcccagtgcgacatccagatg

[0055] acccagagccccagcagcctgagcgccagcctgggcggcaaggtgaccatcacctgcaaggccagccaggac

[0056] atcaacaagtacatcgcctggtaccagcagaagagcggcaagggccccagactgctgatccactacaccagcacc

[0057] ctgcagcccggcatccccagcagattcagcggcagcggcagcggcagagactacagcttcagcatcagcaacct

[0058] ggagcccgaggacatcgccacctactactgcctgcagtacgacaacctgtacaccttcggcggcggcaccaagct

[0059] ggagatcaag (SEQ ID NO:5). The gene sequence encoding the CA2 heavy chain variable region is as follows:

[0060] gaggtgcagctggtggagagcggcggcgacctggtgagacccggcggcagcctgaagctgagctgcgccgccagcggcttcaccttcaccgactacggcatgagctgggtgagacagacccccgacaagagactggagtgggtggccaccatcagcaccgccagcacctacaccttctaccccgaca gcgtgaagggcagattcaccatcagcagagacaacgccaagaacaccctgtacctgcagatgagcagcctgaagagcgaggacaccgccatcttctactgcgtgagacacggccactactacggcagcagcagatacacctggctggcctactggggccagggcaccctggtgaccgtgagcgcc(SEQID NO:6).

[0061] 2. Experimental Results

[0062] 293T cells were digested and resuspended using trypsin, washed with 800 μL of PBS, centrifuged at 800 rpm for 5 min and the supernatant was discarded. Cells were washed with 1 mL of Cell Staining buffer, centrifuged at 800 rpm for 5 min and the supernatant was discarded. Cells were resuspended with 100 μL of Cell Staining buffer, and 1 μL of TruStain FCXTM blocking solution was added. Cells were blocked on ice for 15 min, washed once with 800 μL of Cell Staining buffer, centrifuged at 800 rpm for 5 min and the supernatant was discarded. Cells were resuspended with 100 μL of Cell Staining buffer. Afterwards, 0.1, 1, 3, 10, or 30 μg / mL of CA2 or control IgG antibody was added to each well, and the mixture was incubated on ice for 30 min. After incubation, the cells were washed twice with 800 μL Cell Staining buffer, followed by the addition of anti-mouse-FITC (1:200), and incubated on ice for 45 min. After incubation, the cells were washed twice with 800 μL Cell Staining buffer, and the cells were resuspended in 200 μL PBS. The binding of CA2 to hCD84 or mCD84 was then detected by flow cytometry.

[0063] 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.

[0064] Example 2

[0065] CD84 monoclonal antibody CA2 can inhibit M1 macrophage polarization.

[0066] 1. Experimental Methods

[0067] 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, LPS / IFN-γ was added to make the working solution 100 ng / mL. The sample was collected after 24 hours.

[0068] 2. Experimental Results

[0069] 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, eight tubes were placed in 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.

[0070] Depend on Figure 2 It can be seen that CA2 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.

[0071] Example 3

[0072] CD84 monoclonal antibody CA2 can inhibit DSS-induced inflammatory bowel disease (IBD).

[0073] 1. Experimental Methods

[0074] Male C57BL / 6 mice (6-8 weeks old) were used as experimental animals. A 14-day IBD model was established using DSS induction. From day 1 to 7, 3% DSS solution was used for modeling. During this period, the control group received sterile drinking water, while the model group and the treatment group received 3% DSS solution. Day 0 was the day of modeling, and mice were weighed daily. On day 7, the 3% DSS solution was replaced with sterile drinking water, and mice were regrouped according to their weight loss rate, with 5 mice in each group: control group (NaCl), model group (DSS), and CA2 antibody (Ab-CD84, 5 mg / kg) group. Treatment began, with the Ab-CD84 group receiving intraperitoneal injections every 2 days, while the NaCl and Model groups received equal volumes of saline as controls. The general condition of the mice was observed twice daily, in the morning and afternoon, until day 14 of treatment.

[0075] 2. Experimental index detection

[0076] The Disease Activity Index (DAI) of mice was scored daily during the experiment. After the experiment, the colorectal length and serum inflammatory factor levels of mice in each group were measured. The specific steps are as follows:

[0077] 2.1 DAI Score: During the modeling period, various survival indicators of mice were observed and recorded daily. The disease activity index of mice was statistically analyzed based on the percentage of weight loss, fecal viscosity, and fecal blood. Detailed scoring rules are shown in Table 1.

[0078] Table 1. DAI Scoring Rules for IBD Mice

[0079]

[0080] Experimental results are as follows Figure 3 As shown, the DAI score of the model group began to increase from day 4 of modeling, while the DAI score of the mice gradually decreased after Ab-CD84 was administered on day 7, until it decreased significantly on day 14, proving that blocking CD84 can alleviate the severity of DSS-induced enteritis in mice.

[0081] 2.2 Colorectal Length: Enteritis often leads to shortening of the colorectal lesions; therefore, measuring colorectal length is used to assess the severity of IBD. The colorectal portion of mice was separated, and its length was calculated.

[0082] The results are as follows Figure 4As shown, the colorectal length of mice in the DSS model group was significantly shortened, while Ab-CD84 administration significantly restored the colorectal length. Further analysis of the specific length revealed that Ab-CD84 significantly restored colorectal length, demonstrating that blocking CD84 can significantly alleviate colorectal lesions caused by DSS.

[0083] 2.3 Serum inflammatory factor levels: Serum inflammatory factor detection refers to obtaining whole blood from mice by collecting blood from the eyeballs during necropsy, letting it stand for more than 30 minutes, centrifuging at 3000 rpm for 30 minutes to obtain serum, and then using the detection method of the ELISA kit (product source see Table 2) to quantitatively detect the protein content of key inflammatory factors TNF-α, IL-1β and IL-6 in the serum.

[0084] Table 2 Brands and catalog numbers of ELISA kits

[0085] IL-6 Shanghai Enzyme-Linked Biotechnology ml002293-2 TNF-α Shanghai Enzyme-Linked Biotechnology ml002095-2 IL-1β Shanghai Enzyme-Linked Biotechnology ml301814-2

[0086] The results are as follows Figure 5 As shown, the secretion levels of key inflammatory factors in the model group were significantly increased, and Ab-CD84 administration significantly reduced the protein levels of serum TNF-α, IL-1β, and IL-6, demonstrating that blocking CD84 can significantly reduce the level of serum inflammatory factors in IBD mice.

[0087] 2.4 Degree of Intestinal Barrier Damage: The degree of intestinal barrier damage was assessed by detecting the expression of tight junction proteins Occludin and ZO-1 in intestinal tissue homogenate. Proteins were extracted from intestinal tissue. Specifically, the intestinal tissue was homogenized, sonicated seven times, and centrifuged at 12000 rpm for 10 min at 4℃. The supernatant was used as the protein extraction solution. Protein concentration was quantified using a BCA kit, and absorbance was measured at 562 nm using a microplate reader. A standard curve was constructed based on absorbance and protein standard concentration, and the sample loading volume was calculated. 5× Loading buffer was added to the protein extraction solution, and the mixture was boiled at 100℃ for 7 min. The calculated sample volume was then loaded, and protein electrophoresis was performed sequentially using a stacking gel at 80V for 30 min and a separating gel at 120V for 1 h. After electrophoresis, the gel plates were removed, and a piece of gel near the target protein was cut and placed in a transfer clamp for transfer at 100V for 2 h. After transfer, the membrane was blocked in 5% skim milk prepared with TBST solution for 1 h. Next, the membrane site corresponding to the target protein was cut and placed in an incubation box containing the corresponding antibody, and incubated overnight at 4°C on a shaker. The next day, the incubation box was removed, the primary antibody was recovered, an appropriate amount of TBST solution was added for washing, the corresponding secondary antibody was added, and the mixture was incubated at room temperature on a shaker for 2 hours. After washing, the image was developed. Chemiluminescence imaging was used to photograph and analyze the band results to detect the expression levels of the two key factors.

[0088] The results are as follows Figure 6 As shown, the intestinal barrier of mice in the DSS group showed significant damage, but Ab-CD84 administration significantly restored the expression of Occludin and ZO-1, demonstrating that blocking CD84 can significantly alleviate intestinal barrier damage caused by DSS.

[0089] Example 4

[0090] CD84 monoclonal antibody AD5 antigen affinity assay

[0091] 1. Experimental Methods

[0092] 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:

[0093] MESQTQVFVYMLLWLSGVDGDIVMTQSQKFMSTSVGDRVSVTCKA SQNVGTNVAWYQQKPGQSPKALIYSASYRYSGVPDRFTGSGSGTDFTLTII NVQSEDLAEYFCQQYNSYPYTFGGGTKLEIK(SEQ ID NO:3);

[0094] The heavy chain variable region sequence Hc of the AD5 antibody is as follows:

[0095] QIQLVQSGPELKKPGETVKISCKASGYTFTNYGMNWVKQAPGKGLK WMGWTNTYTGEPTYADDFKGRFAFSLETSASTAYLQINNLKNEDMATYF CARWGYMIFDYWGQGTTLTVSS (SEQ ID NO: 4).

[0096] 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:

[0097] atggagagccagacccaggtgttcgtgtacatgctgctgtggctgagcggcgtggacggcgacatcgtgatgacccag

[0098] agccagaagttcatgagcaccagcgtgggcgacagagtgagcgtgacctgcaaggccagccagaacgtgggcacca

[0099] acgtggcctggtaccagcagaagcccggccagagccccaaggccctgatctacagcgccagctacagatacagcgg

[0100] cgtgcccgacagattcaccggcagcggcagcggcaccgacttcaccctgaccatcatcaacgtgcagagcgaggacc

[0101] tggccgagtacttctgccagcagtacaacagctacccctacaccttcggcggcggcaccaagctggagatcaag

[0102] (SEQ ID NO:7);

[0103] The gene sequence encoding the variable region of the AD5 heavy chain is as follows:

[0104] cagatccagctggtgcagagcggccccgagctgaagaagcccggcgagaccgtgaagatcagctgcaaggccagc

[0105] ggctacaccttcaccaactacggcatgaactgggtgaagcaggcccccggcaagggcctgaagtggatgggctggac

[0106] caacacctacaccggcgagcccacctacgccgacgacttcaagggcagattcgccttcagcctggagaccagcgcca

[0107] gcaccgcctacctgcagatcaacaacctgaagaacgaggacatggccacctacttctgcgccagatggggctacatgat

[0108] cttcgactactggggccagggcaccaccctgaccgtgagcagc (SEQ ID NO: 8).

[0109] 2. Experimental Results

[0110] 293T cells were digested and resuspended using trypsin, washed with 800 μL of PBS, centrifuged at 800 rpm for 5 min and the supernatant was discarded. Cells were washed with 1 mL of Cell Staining buffer, centrifuged at 800 rpm for 5 min and the supernatant was discarded. Cells were resuspended with 100 μL of Cell Staining buffer, and 1 μL of TruStain FCXTM blocking solution was added. Cells were blocked on ice for 15 min, washed once with 800 μL of Cell Staining buffer, centrifuged at 800 rpm for 5 min and the supernatant was discarded. Cells were resuspended with 100 μL of 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 Cell Staining buffer, then add anti-mouse-FITC (1:200) and incubate on ice for 45 min. After incubation, wash twice with 800 μL Cell Staining buffer, resuspend in 200 μL PBS, and then use flow cytometry to detect the binding of AD5 to hCD84 or mCD84.

[0111] Depend on Figure 7 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.

[0112] Example 5

[0113] CD84 monoclonal antibody AD5 can inhibit M1 macrophage polarization.

[0114] 1. Experimental Methods

[0115] 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 AD5 (Ab-CD84) were added. After 6 hours, LPS / IFN-γ was added to make the working solution 100 ng / mL. The sample was collected after 24 hours.

[0116] 2. Experimental Results

[0117] 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 sequences of the forward and reverse primers are shown in Table 3. After preparing the reaction system, eight tubes were placed in a Roche LC96 instrument. 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. The primers used are shown in the following table:

[0118] Table 3 Primer sequences

[0119]

[0120] Depend on Figure 8 It can be seen that Ab-CD84 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.

[0121] Example 6

[0122] CD84 monoclonal antibodies can inhibit DSS-induced inflammatory bowel disease (IBD).

[0123] 1. Experimental Methods

[0124] Male C57BL / 6 mice (6-8 weeks old) were used as experimental animals. A 14-day IBD model was established using DSS-induced mice. From day 1 to 7, 3% DSS solution was used for modeling. During this period, the control group received sterile drinking water, while the model group and the treatment group received 3% DSS solution. Day 0 was the day of modeling, and mice were weighed daily. On day 7, the 3% DSS solution was replaced with sterile drinking water, and mice were regrouped according to their weight loss rate, with 5 mice in each group: control group (NaCl), model group (DSS), and AD5 antibody (Ab-CD84, 5 mg / kg) group. Treatment began, with the Ab-CD84 group receiving intraperitoneal injections every 2 days, while the NaCl and Model groups received equal volumes of saline as controls. The general condition of the mice was observed twice daily, in the morning and afternoon, until day 14 of treatment.

[0125] 2. Experimental Results

[0126] The Disease Activity Index (DAI) of mice was scored daily during the experiment. After the experiment, the colorectal length and serum inflammatory factor levels of mice in each group were measured. The specific steps are as follows:

[0127] 2.1 DAI Score: During the modeling period, various survival indicators of mice were observed and recorded daily. The disease activity index of mice was statistically analyzed based on the percentage of weight loss, fecal viscosity, and fecal blood. Detailed scoring rules are shown in Table 4.

[0128] Table 4. DAI Scoring Rules for IBD Mice

[0129]

[0130] Experimental results are as follows Figure 9 As shown, the DAI score of the model group began to increase from day 4 of modeling, while the DAI score of the mice gradually decreased after Ab-CD84 was administered on day 7, until it decreased significantly on day 14, proving that blocking CD84 can alleviate the severity of DSS-induced enteritis in mice.

[0131] 2.2 Colorectal Length: Enteritis often leads to shortening of the colorectal lesions; therefore, measuring colorectal length is used to assess the severity of IBD. The colorectal portion of mice was separated, and its length was calculated.

[0132] The results are as follows Figure 10 As shown, the colorectal length of mice in the DSS model group was significantly shortened, while Ab-CD84 significantly restored the length of the colorectal length, proving that blocking CD84 can significantly alleviate colorectal lesions caused by DSS.

[0133] 2.3 Serum Inflammatory Factor Levels: Serum inflammatory factor detection involved obtaining whole blood from mice via ocular sampling during necropsy, allowing it to stand for more than 30 minutes, centrifuging at 3000 rpm for 30 minutes to obtain serum, and then using an ELISA kit to quantitatively detect the levels of key inflammatory factors TNF-α, IL-1β, and IL-6 proteins in the serum. The brands and catalog numbers of the ELISA kits are shown in Table 5.

[0134] Table 5 Sources of ELISA kits

[0135] IL-6 Shanghai Enzyme-Linked Biotechnology ml002293-2 TNF-α Shanghai Enzyme-Linked Biotechnology ml002095-2 IL-1β Shanghai Enzyme-Linked Biotechnology ml301814-2

[0136] The results are as follows Figure 11 As shown, the secretion levels of key inflammatory factors in the model group were significantly increased, and Ab-CD84 administration significantly reduced the protein levels of serum TNF-α, IL-1β, and IL-6, demonstrating that blocking CD84 can significantly reduce the level of serum inflammatory factors in IBD mice.

[0137] 2.4 Degree of Intestinal Barrier Damage: The degree of intestinal barrier damage was determined by detecting the expression of tight junction proteins Occludin and ZO-1 in intestinal tissue homogenate. RNA was extracted from intestinal tissue, which was homogenized with Trizol and transferred to 1.5 mL RNase-free centrifuge tubes. The cells were allowed to lyse completely for 5-10 min. 200 μL of chloroform was added to each tube, vortexed for 15 s, and allowed to stand at room temperature for 2-3 min. The tubes were then centrifuged at 12000 rpm for 15 min at 4°C. 400 μL of the supernatant was transferred to a new 1.5 mL RNase-free centrifuge tube, and 400 μL of isopropanol was added. The mixture was mixed and allowed to stand at room temperature for 10 min. Centrifugation at 12000 rpm for 10 min at 4°C yielded a flocculent precipitate of RNA. The supernatant was discarded, and 1 mL of 75% ethanol was added to wash the precipitate. The precipitate was then centrifuged at 7000 rpm for 5 min at 4°C, the supernatant was discarded, and this process was repeated once. Excess solution was aspirated, and the ethanol was allowed to evaporate at room temperature. The colorless RNA indicated complete ethanol evaporation. RNase-free water was added to dissolve the RNA, and the mixture was placed in an ice box for 15 minutes before the RNA concentration was measured and recorded using a micro-spectrophotometer. The RNA was then reverse-engineered into cDNA using the following reverse-engineering program: 42℃ for 15 minutes, then 95℃ for 3 minutes. 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, eight tubes were placed in 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. The primer sequences involved are shown in Table 6.

[0138] Table 6 Primer sequences

[0139]

[0140] like Figure 12 As shown, the intestinal barrier of mice in the DSS group showed significant damage, but Ab-CD84 administration significantly restored the expression of Occludin and ZO-1, demonstrating that blocking CD84 can significantly alleviate intestinal barrier damage caused by DSS.

[0141] 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 inflammatory bowel disease, wherein the anti-CD84 antibody is a CA2 monoclonal antibody or an AD5 monoclonal antibody; The light chain variable region sequence of the CA2 monoclonal antibody is shown in SEQ ID NO:1, and the heavy chain variable region sequence of the CA2 monoclonal antibody is shown in SEQ ID NO:

2. The light chain variable region sequence of the AD5 monoclonal antibody is shown in SEQ ID NO:3, and the heavy chain variable region sequence of the AD5 monoclonal antibody is shown in SEQ ID NO:

4. The inflammatory bowel disease mentioned is ulcerative colitis.

2. The application according to claim 1, characterized in that, The CD84 mentioned is the CD84 of macrophages.

3. The application according to claim 1, characterized in that, The drug is a drug that inhibits the activation of pro-inflammatory macrophages.

4. The application according to claim 1, characterized in that, The treatment for inflammatory bowel disease includes restoring colon and rectum length and reducing inflammatory factor levels.

5. The application according to claim 4, characterized in that, The inflammatory factors include at least one of the following: TNF-α, IL-1β, and IL-6.

6. A medicament for the prevention and / or treatment of ulcerative colitis, characterized in that, Includes anti-CD84 antibodies and medically acceptable excipients; The anti-CD84 antibody is either a CA2 monoclonal antibody or an AD5 monoclonal antibody. The light chain variable region sequence of the CA2 monoclonal antibody is shown in SEQ ID NO:1, and the heavy chain variable region sequence of the CA2 monoclonal antibody is shown in SEQ ID NO:

2. The light chain variable region sequence of the AD5 monoclonal antibody is shown in SEQ ID NO:3, and the heavy chain variable region sequence of the AD5 monoclonal antibody is shown in SEQ ID NO:4.

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