Application of anti-CD84 antibody in preparation of medicine for preventing and / or treating inflammatory bowel disease

Abstract

The invention 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 medicine. According to the application, a dextran sodium sulfate (DSS)-induced inflammatory bowel disease classical animal model is taken as an experimental subject, and an anti-CD84 antibody is taken as a drug to inhibit the biological function of CD84, so that not only can the activation of proinflammatory macrophages be inhibited, but also enteritis and lesion of patients with inflammatory bowel diseases can be relieved, and the level of inflammatory factors in serum can be reduced; therefore, the purpose of preventing and treating inflammatory bowel diseases is achieved. Therefore, the application provided by the invention provides a new means for preventing and treating the inflammatory bowel disease.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to the use of an anti-CD84 antibody in the preparation of a drug for preventing and / or treating inflammatory bowel disease. Background Art

[0002] Inflammatory bowel disease (IBD), consisting of ulcerative colitis (UC) and Crohn's disease (CD), is a chronic, progressive and recurrent intestinal disease. IBD seriously affects the quality of life and daily life of patients and increases the medical burden.

[0003] Traditional drugs for treating IBD include aminosalicylic acids, antibiotics, immunosuppressants and glucocorticoids, but the efficacy is not satisfactory.

[0004] Given the important driving role of immune abnormalities in the progression of IBD, current IBD treatment drugs are mainly anti-inflammatory or immunomodulatory, especially some biological drug antibodies, such as the anti-TNF monoclonal antibody infliximab and the anti-interleukin (IL) 12-23 antibody ustekinumab; in addition, there is also a small molecule drug tofacitinib targeting JAK approved by the FDA for the treatment of mild and severe UC. However, immunomodulation often leads to adverse reactions such as diarrhea or lymphopenia, which prolongs the treatment time, and the high recurrence rate also limits their clinical application. Systemic administration of anti-inflammatory drugs is the main cause of adverse reactions. Therefore, the development of drugs that specifically target colon inflammation is of great significance for the treatment of IBD. Summary of the invention

[0005] In view of this, the purpose of the present invention is to provide an anti-CD84 antibody for use in the preparation of a drug for preventing and / or treating inflammatory bowel disease, thereby achieving the effects of alleviating the severity of enteritis, colorectal lesions and reducing inflammatory responses by inhibiting the expression and function of CD84.

[0006] The present invention provides an application of an anti-CD84 antibody in preparing a medicine for preventing and / or treating inflammatory bowel disease.

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

[0008] Preferably, the anti-CD84 protein monoclonal antibodies include 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 macrophage CD84.

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

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

[0016] Preferably, the relief of colorectal lesions comprises 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] The present invention provides a drug for preventing and / or treating inflammatory bowel disease, comprising an anti-CD84 antibody and a medically acceptable excipient;

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

[0021] Further preferably, the anti-CD84 protein monoclonal antibodies include 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] The present invention provides an application of an anti-CD84 antibody in the preparation of a drug for preventing and / or treating inflammatory bowel disease. The present invention uses a classic animal model of inflammatory bowel disease induced by dextran sulfate sodium salt (DSS) as an experimental subject, and uses an anti-CD84 antibody as a drug to inhibit the biological function of CD84. The results show that compared with the control group (NaCl), the disease activity index (DAI) score of the model group was significantly increased, while the disease activity index score of the anti-CD84 antibody administration group was significantly lower than that of the model group, indicating that inhibiting the biological function of CD84 is beneficial to alleviating the severity of DSS-induced enteritis in mice. At the same time, enteritis usually leads to shortening of colorectal lesions. The present invention also statistically analyzes the length of the colorectum of mice in the control group, model group and administration group, and the results show that compared with the control group, the colorectum of mice in the model group was significantly shortened, while the length of the colorectum of mice in the administration group was significantly restored, which proves that inhibiting the biological function of CD84 is beneficial to significantly alleviating colorectal lesions caused by DSS. The present invention also detects the secretion of inflammatory factors in the above three treatment groups. The results show that compared with the control group, the secretion level of key pro-inflammatory factors in the model group was significantly increased, while the secretion levels of TNF-α, IL-1β, and IL-6 in the serum of the mice in the drug-treated group were significantly reduced, proving that inhibiting the biological function of CD84 is beneficial to significantly reduce the level of serum pro-inflammatory factors in IBD mice, thereby reducing the inflammatory response of mice. It can be seen that the above experimental results show that anti-CD84 antibodies can effectively achieve the efficacy of the treatment and prevention of inflammatory bowel disease. At the same time, since CD84 is not an inflammatory factor, blocking the function of CD84 will not affect immune surveillance, and CD84 blocking is often used as a therapeutic target for reversing tumor-induced immunosuppression, and will not bring other safety risks. Therefore, reagents that inhibit the expression or function of CD84 provide a new means for preparing drugs for the prevention and / or treatment of inflammatory bowel disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 is the binding curve of CD84 monoclonal antibody CA2 with hCD84 and mCD84;

[0028] Figure 2 The results show that CD84 monoclonal antibody CA2 can inhibit LPS / IFN-γ-induced pro-inflammatory macrophage activation.

[0029] Figure 3 DAI scoring results for IBD mice;

[0030] Figure 4 The colorectal length measurement results of IBD mice;

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

[0032] Figure 6It is the test result of intestinal epithelial barrier damage factor protein;

[0033] Figure 7 is the binding curve of CD84 monoclonal antibody AD5 with hCD84 and mCD84;

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

[0035] Fig. 9 DAI scoring results for IBD mice;

[0036] Fig.10 The colorectal length measurement results of IBD mice;

[0037] Fig.11 The results of ELISA test of serum inflammatory factors in IBD mice;

[0038] Fig.12 These are the results of intestinal epithelial barrier damage factor protein detection. DETAILED DESCRIPTION

[0039] The present invention provides an application of an anti-CD84 antibody in preparing a medicine for preventing and / or treating inflammatory bowel disease.

[0040] In the present invention, the inflammatory bowel disease preferably includes Crohn's disease and ulcerative colitis. Crohn's disease mostly occurs in the digestive tract, and ulcerative colitis mainly occurs near the rectum and anus. In an embodiment of the present invention, a disease animal model induced by dextran sulfate sodium salt (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 colorectum of the mice in the model group was significantly shortened, indicating that the model mice had severe enteritis, and the secretion level of key pro-inflammatory factors in the model group was significantly increased, indicating that the model mice had severe inflammatory reactions in the intestine.

[0041] In the present invention, the CD84 is preferably the CD84 of macrophages. The macrophages preferably include pro-inflammatory macrophages. The anti-CD84 antibody preferably includes a monoclonal antibody against CD84 protein and / or a polyclonal antibody against CD84 protein. The monoclonal antibodies against CD84 protein include 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. The source of the polyclonal antibody against CD84 protein of the present invention is not particularly limited, and the preparation method of polyclonal antibodies well known in the art can be used, for example, using CD84 protein derived from macrophages as an immune antigen, immunizing animals multiple times, and collecting antiserum. The small molecule inhibitor of CD84 protein has an inhibitory effect on the function of CD84 protein.

[0042] In the present invention, the drug is preferably a drug that inhibits the activation of pro-inflammatory macrophages. The manifestations of the treatment of inflammatory bowel disease preferably include alleviating intestinal epithelial damage caused by the activation of pro-inflammatory macrophages, alleviating the severity of enteritis, alleviating colorectal lesions and reducing the level of inflammatory factors. In an embodiment of the present invention, the biological function of CD84 is blocked by a monoclonal antibody against CD84 protein. The results show that the monoclonal antibody against CD84 protein can alleviate the severity of DSS-induced enteritis in mice, restore the length of the colorectum, and reduce the secretion level of inflammatory factors TNF-α, IL-1β, and IL-6 in the serum, thereby achieving the purpose of preventing and / or treating IBD. It can be seen that the present invention uses CD84 as a therapeutic target, and uses an agent that inhibits the expression or function of CD84 as an active agent to develop a drug for preventing and treating IBD. The present invention has no special restrictions on the dosage form and preparation method of the drug, and the preparation method of the drug known in the art can be used.

[0043] The present invention provides a medicine for preventing and / or treating inflammatory bowel disease, comprising an anti-CD84 antibody and a medically acceptable adjuvant.

[0044] In the present invention, the anti-CD84 antibody preferably includes a monoclonal antibody against CD84 protein and / or a polyclonal antibody against CD84 protein. The monoclonal antibodies against CD84 protein include 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. The present invention uses a recombinant expression method to prepare CA2 monoclonal antibody and AD5 monoclonal antibody. It has been experimentally verified that the CA2 monoclonal antibody and the AD5 monoclonal antibody have good binding properties to the CD84 antigen, thereby providing a new means for the prevention and treatment of IBD.

[0045] The following is a detailed description of the use of an agent for inhibiting CD84 expression or function provided by the present invention in the preparation of a drug for preventing and treating inflammatory bowel disease in conjunction with the examples, but they should not be construed as limiting the scope of protection of the present invention.

[0046] Example 1

[0047] CD84 monoclonal antibody CA2 antigen affinity test

[0048] 1. Experimental Methods

[0049] 293T cells were inoculated into 6-well plates, and hCD84 (NM_001184879.2) and mCD84 (NM_013489.4) were overexpressed by transfecting plasmid pCDNA3.1. In each group, 200 μL of OPTI-MEM was used as the dispersion system in each well, and 2 μg of siRNA and 6 μL of transfection reagent (Lipo2000) were added. After incubation for 5 minutes, the two were mixed, and the bottom of the tube was flicked to mix them thoroughly. After incubation for 10 minutes, they were added to the well plate. After 48 hours, the samples were collected and the binding of anti-human CD84 antibody CA2 to hCD84 and mCD84 was detected by flow cytometry. The light chain variable region sequence Lc of 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 preparation of CA2 antibody is to clone the gene sequence encoding CA2 sequence into the eukaryotic expression vector pCDNA3.4, transfect into 293F cells, collect the culture supernatant after 7 days, and purify it by protein G gel. Among them, the gene sequence encoding 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 gcgtgaagggcagattcaccatcagcagagacaacgccaagaacaccctgtacctgcagatgagcagcctgaagagcgaggacaccgccatcttctactgcgtgagacacggccactactacggcagcagcagtacacctggctggcctactggggccagggcaccctggtgaccgtgagcgcc(SEQID NO:6).

[0061] 2. Experimental results

[0062] Digest 293T cells with trypsin and resuspend, add 800 μL PBS to wash, centrifuge at 800 rpm for 5 min, discard the supernatant, add 1 mL Cell Staining buffer to wash, centrifuge at 800 rpm for 5 min, discard the supernatant, resuspend with 100 μL Cell Staining buffer, add 1 μL blocking solution TruStain FCXTM, block on ice for 15 min, add 800 μL Cell Staining buffer to wash once, centrifuge at 800 rpm for 5 min, discard the supernatant, and resuspend with 100 μL Cell Staining buffer. Then, 0.1, 1, 3, 10, and 30 μg / mL of CA2 or control IgG antibody was added to each well and incubated on ice for 30 min. At the end of incubation, 800 μL Cell Staining buffer was added and washed twice. Then, anti-mouse-FITC (1:200) was added and incubated on ice for 45 min. At the end of incubation, 800 μL Cell Staining buffer was added and washed twice. After resuspending in 200 μL PBS, the binding of CA2 to hCD84 or mCD84 was detected by flow cytometry.

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

[0068] 2. Experimental results

[0069] Add 1mL Trizol to each well, blow off the cells, transfer to a 1.5mL RNase free centrifuge tube, and let stand for 5-10min to fully lyse the cells. Add 200μL chloroform to each tube, vortex thoroughly for 15s, and let stand at room temperature for 2-3min. Centrifuge at 4℃12000rpm for 15min, transfer 400μL of the upper water layer to a new RNase free1.5mL centrifuge tube, add 400μL isopropanol, mix well, and let stand at room temperature for 10min. Centrifuge at 4℃12000rpm for 10min to obtain flocculent precipitate as RNA, discard the supernatant, add 1mL 75% ethanol, and wash the precipitate. Centrifuge at 4℃7000rpm for 5min, discard the supernatant, repeat once, aspirate the excess solution, evaporate at room temperature, and the RNA becomes colorless, indicating that the ethanol has evaporated completely. Add RNase free water to dissolve, place in an ice box for 15min, and use a micro-spectrophotometer to detect and record the RNA concentration. Afterwards, the RNA was reversed into cDNA, and the reverse program was: 42℃15min, 95℃3min. Then the RT-PCR system was configured: 2×SYBR Green Mix 10μL per tube, 1μL of upstream and downstream primers per tube, 6μL of RNase free water, and 2μL of cDNA template. After completion, the eight rows of tubes were placed in the Roche LC96 instrument, and the program was set as pre-denaturation: 95℃ (300s); denaturation: 95℃ (30s); annealing: 55℃ (30s); extension: 72℃ (30s), 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 (6-8 weeks old) were used as experimental animals. A DSS-induced 14-day IBD model was established. A 3% DSS solution was used to model the model from day 1 to day 7. During the modeling period, the control group used sterile drinking water, and the model group and the drug-treated group were given 3% DSS solution, respectively. The day of modeling was D0, and the mice were weighed every day. On the 7th day of modeling, the 3% DSS solution was replaced with sterile drinking water, and the mice were regrouped according to the weight loss rate, with 5 mice in each group. The specific grouping was as follows: control group (NaCl), model group (DSS), CA2 antibody (Ab-CD84, 5 mg / kg) group. Drug treatment was started. The Ab-CD84 group was intraperitoneally injected once every 2 days according to the dose, and the NaCl group and the Model group were injected with an equal amount of saline as a control. The general condition of the mice was observed once in the morning and afternoon every day, and the drug was administered until the 14th day.

[0075] 2. Experimental indicator detection

[0076] During the experiment, the disease activity index (DAI) of mice was scored every day. After the experiment, the colorectal length and serum inflammatory factor levels of each group of mice were tested. The specific steps are as follows:

[0077] 2.1DAI score: During the modeling period, the survival indicators of mice were observed and recorded every day, and the disease activity index of mice was statistically analyzed based on the weight loss ratio, feces viscosity and blood in stool. Detailed scoring rules are shown in Table 1.

[0078] Table 1 DAI scoring rules for IBD mice

[0079]

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

[0081] 2.2 Colorectal length: Enteritis usually leads to shortened colorectal lesions, so the severity of IBD can be determined by measuring the length of the colorectum. The colorectal sections of mice were separated and the lengths were counted.

[0082] The results are as follows Figure 4As shown in the figure, the colorectum of mice in the DSS model group was significantly shortened, while the administration of Ab-CD84 significantly restored the length of the colorectum of mice. Further statistics on the specific length showed that Ab-CD84 had a significant effect on the restoration of colorectal length, proving 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 the mouse eyeball when the mouse is killed. After standing for more than 30 minutes, the blood is centrifuged at 3000 rpm for 30 minutes to obtain serum. The ELISA kit (product source see Table 2) is then used to quantitatively detect the protein levels 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] Gene name ELISA kit brand Part Number IL-6 Shanghai ELISA ml002293-2 TNF-α Shanghai ELISA ml002095-2 IL-1β Shanghai ELISA ml301814-2

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

[0087] 2.4 Intestinal barrier damage: The degree of intestinal barrier damage was achieved by detecting the expression of tight junction proteins Occludin and ZO-1 in intestinal tissue homogenate. The protein of intestinal tissue was extracted by homogenizing the intestinal tissue, ultrasonically disrupting it 7 times, centrifuging it at 4°C and 12000rpm for 10min, taking the supernatant as the protein extract, quantifying the protein concentration using the BCA kit, measuring the absorbance at 562nm using an enzyme marker, making a standard curve based on the absorbance and protein standard concentration, and calculating the sample loading volume. Add 5× Loading buffer to the protein extract, boil it at 100°C for 7min, load the sample according to the calculated sample volume, and perform protein electrophoresis in accordance with the concentrated gel at 80V for 30min and the separation gel at 120V for 1h. After the protein electrophoresis, remove the gel plate, cut the gel block near the target protein and place it in the transfer clip, and transfer the membrane at 100V for 2h. After the transfer, block it in 5% skim milk prepared in TBST solution for 1h. After that, cut the membrane position corresponding to the target protein, put it into the incubation box containing the corresponding antibody, and incubate it on a shaker at 4°C overnight. The next day, take out the incubation box to recover the primary antibody, add an appropriate amount of TBST solution for washing, add the corresponding secondary antibody, incubate it on a shaker at room temperature for 2 hours, and then develop it after washing. Use a chemiluminescence imager to take pictures 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 obvious damage, but the administration of Ab-CD84 could significantly restore the expression of Occludin and ZO-1, proving that blocking CD84 can significantly alleviate the intestinal barrier damage caused by DSS.

[0089] Example 4

[0090] CD84 monoclonal antibody AD5 antigen affinity test

[0091] 1. Experimental Methods

[0092] 293T cells were inoculated into 6-well plates, and hCD84 (NM_001184879.2) and mCD84 (NM_013489.4) were overexpressed by transfecting plasmid pCDNA3.1. In each group, 200 μL of OPTI-MEM was used as the dispersion system, and 2 μg of siRNA and 6 μL of transfection reagent (Lipo2000) were added to each well. After incubation for 5 minutes, the two were mixed, and the bottom of the tube was flicked to mix them thoroughly. After incubation for 10 minutes, they were added to the well plate. After 48 hours, the samples were collected and the binding of anti-human CD84 antibody AD5 to hCD84 and mCD84 was detected by flow cytometry. The light chain variable region sequence Lc of the AD5 antibody is as follows:

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

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

[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 inoculated in 6-well plates under sterile conditions, with 1×10 cells in each well. 6 , PMA was added 24 hours later to make the working solution 100 ng / mL, 24 hours after PMA stimulation, 2.5 ug each of control antibody (Ctl IgG) and anti-human CD84 monoclonal antibody AD5 (Ab-CD84) were added, and LPS / IFN-γ was added 6 hours later with a working solution concentration of 100 ng / mL, and samples were collected 24 hours later.

[0116] 2. Experimental results

[0117] Add 1mL Trizol to each well, blow off the cells, transfer to a 1.5mL RNase free centrifuge tube, and let stand for 5-10min to fully lyse the cells. Add 200μL chloroform to each tube, vortex thoroughly for 15s, and let stand at room temperature for 2-3min. Centrifuge at 4℃12000rpm for 15min, transfer 400μL of the upper water layer to a new RNase free1.5mL centrifuge tube, add 400μL isopropanol, mix well, and let stand at room temperature for 10min. Centrifuge at 4℃12000rpm for 10min to obtain flocculent precipitate as RNA, discard the supernatant, add 1mL 75% ethanol, and wash the precipitate. Centrifuge at 4℃7000rpm for 5min, discard the supernatant, repeat once, aspirate the excess solution, evaporate at room temperature, and the RNA becomes colorless, indicating that the ethanol has evaporated completely. Add RNase free water to dissolve, place in an ice box for 15min, and use a micro-spectrophotometer to detect and record the RNA concentration. Afterwards, the RNA was reversed into cDNA, and the reversal program was: 42℃15min, 95℃3min. Then the RT-PCR system was configured: 2×SYBR Green Mix 10μL per tube, 1μL of upstream and downstream primers per tube, 6μL of RNase free water, and 2μL of cDNA template. The upstream and downstream primer sequences involved are shown in Table 3. After the reaction system was prepared, the eight rows of tubes were placed in the Roche LC96 instrument, and the program was set to pre-denaturation: 95℃ (300s); denaturation: 95℃ (30s); annealing: 55℃ (30s); extension: 72℃ (30s), a total of 40 cycles, to detect the expression of the target gene. Among them, 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, which is manifested by the significant inhibition of the expression of inflammatory factors IL-6, TNF-α and IL-1β.

[0121] Example 6

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

[0123] 1. Experimental Methods

[0124] Male C57BL / 6 (6-8 weeks old) were used as experimental animals. A 14-day IBD model of DSS-induced mice was established. A 3% DSS solution was used to model the model from day 1 to day 7. During the modeling period, the control group used sterile drinking water, and the model group and the drug-treated group were given 3% DSS solution respectively. The day of modeling was D0, and the mice were weighed every day. On the 7th day of modeling, the 3% DSS solution was replaced with sterile drinking water, and the mice were regrouped according to the weight loss rate, with 5 mice in each group. The specific grouping was as follows: control group (NaCl), model group (DSS), AD5 antibody (Ab-CD84, 5 mg / kg) group, and drug treatment was started. The Ab-CD84 group was intraperitoneally injected once every 2 days according to the dose, and the NaCl group and the Model group were injected with an equal amount of saline as a control. The general condition of the mice was observed once in the morning and afternoon every day, and the drug was administered until the 14th day.

[0125] 2. Experimental results

[0126] During the experiment, the disease activity index (DAI) of mice was scored every day. After the experiment, the colorectal length and serum inflammatory factor levels of each group of mice were tested. The specific steps are as follows:

[0127] 2.1DAI score: During the modeling period, the survival indicators of mice were observed and recorded every day, and the disease activity index of mice was statistically analyzed based on the weight loss ratio, feces viscosity and blood in stool. Detailed scoring rules are shown in Table 4.

[0128] Table 4 DAI scoring rules for IBD mice

[0129]

[0130] The experimental results are as follows Fig. 9 As shown, it can be seen that the DAI score of the model group began to increase from the 4th day of modeling, and after the administration of Ab-CD84 on the 7th day, the DAI score of the mice gradually decreased until it was significantly reduced on the 14th day, proving that blocking CD84 can alleviate the severity of DSS-induced enteritis in mice.

[0131] 2.2 Colorectal length: Enteritis usually leads to shortened colorectal lesions, so the severity of IBD can be determined by measuring the length of the colorectum. The colorectal sections of mice were separated and the lengths were counted.

[0132] The results are as follows Fig.10 As shown, the colorectum of mice in the DSS model group was significantly shortened, while Ab-CD84 had a significant effect on the restoration of 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 refers to obtaining whole blood from the eyeballs of mice when they are killed, centrifuging at 3000 rpm for 30 minutes to obtain serum, and then using ELISA test kits to quantitatively detect the key inflammatory factors TNF-α, IL-1β, and IL-6 protein content in the serum. The brands and product numbers of ELISA kits are shown in Table 5.

[0134] Table 5 Sources of ELISA kits

[0135] Gene name ELISA kit brand Part Number IL-6 Shanghai ELISA ml002293-2 TNF-α Shanghai ELISA ml002095-2 IL-1β Shanghai ELISA ml301814-2

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

[0137] 2.4 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 homogenates. RNA from intestinal tissue was extracted, and the intestinal tissue was homogenized with Trizol and transferred to a 1.5 mL RNase free centrifuge tube. The tube was allowed to stand for 5-10 min to allow the cells to fully lyse. 200 μL of chloroform was added to each tube, vortexed for 15 s, and allowed to stand at room temperature for 2-3 min. Centrifuge at 4°C 12000 rpm for 15 min, transfer 400 μL of the upper water layer to a new RNasefree 1.5 mL centrifuge tube, add 400 μL of isopropanol, mix well, and stand at room temperature for 10 min. Centrifuge at 4°C 12000 rpm for 10 min to obtain a flocculent precipitate as RNA, discard the supernatant, add 1 mL of 75% ethanol, and wash the precipitate. Centrifuge at 4°C 7000 rpm for 5 min, discard the supernatant, repeat once, aspirate the excess solution, evaporate at room temperature, and the RNA becomes colorless, indicating that the ethanol has evaporated cleanly. Add RNase free water to dissolve, place in an ice box for 15 minutes, and then use a micro-spectrophotometer to detect and record the RNA concentration. Then reverse the RNA into cDNA, and the reversal program is: 42℃15min, 95℃3min. Then perform RT-PCR system configuration: 2×SYBR Green Mix 10μL per tube, 1μL of upstream and downstream primers per tube, 6μL of RNase free water, and 2μL of cDNA template. After completion, then put eight rows of tubes into the Roche LC96 instrument, and set the program to pre-denaturation: 95℃ (300s); denaturation: 95℃ (30s); annealing: 55℃ (30s); extension: 72℃ (30s), a total of 40 cycles, to detect the expression of the target gene. Among them, the primer sequences involved are shown in Table 6.

[0138] Table 6 Primer sequences

[0139]

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

[0141] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

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

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

3. The application according to claim 2, characterized in that: The monoclonal antibodies against CD84 protein include 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.

4. The use according to claim 1, characterized in that: The CD84 is the CD84 of macrophages.

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

6. The use according to claim 1, characterized in that: The treatment of inflammatory bowel disease manifestations includes alleviating intestinal epithelial damage caused by activation of pro-inflammatory macrophages, alleviating the severity of enteritis, alleviating colorectal lesions and reducing the level of inflammatory factors.

7. The use according to claim 6, characterized in that: The relief of colorectal lesions includes restoration of colorectal length.

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

9. The use according to any one of claims 1 to 8, characterized in that: The inflammatory bowel diseases include ulcerative colitis and Crohn's disease.

10. A drug for preventing and / or treating inflammatory bowel disease, characterized in that: comprising an anti-CD84 antibody and a medically acceptable excipient; Preferably, the anti-CD84 antibody comprises a monoclonal antibody against CD84 protein and / or a polyclonal antibody against CD84 protein; Further preferably, the anti-CD84 protein monoclonal antibodies include 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.

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