Use of cadm3 in the preparation of a product for treating an autoimmune disease

Abstract

The application discloses application of CADM3 in preparation of products for treating autoimmune diseases, belongs to the technical field of biological medicine, and the amino acid sequence of the CADM3 is shown as SEQ ID NO.2.The application discloses that CADM3 is up-regulated on the surface of activated APC, in vitro cell intervention experiments are carried out by using prepared hCADM3-Ig fusion protein, it is confirmed that the fusion protein can effectively block the activation and proliferation of T cells, and further regulate the overactive autoimmune response.Through animal experiment verification, compared with the control group, the hCADM3-Ig fusion protein treatment group can significantly improve the neurological deficit symptoms of EAE model mice, obviously reduce the intestinal dysfunction of IBD model mice, effectively reduce the degree of inflammatory reaction in the two models, and significantly inhibit the activation and proliferation of pathological T cells.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biomedicine, in particular to the application of CADM3 in the preparation of products for treating autoimmune diseases. BACKGROUND

[0002] Autoimmune diseases are a group of clinical syndromes caused by abnormal recognition and attack of the immune system on self-tissues, with a prevalence rate of 5-10%, and a sustained upward trend. They include type I diabetes, multiple sclerosis (MS) and rheumatoid arthritis (RA), etc. The pathogenesis of autoimmune diseases is complex, but the abnormal activation of self-reactive T cells attacking self-tissue antigens is an important pathogenic mechanism.

[0003] The prevalence of autoimmune diseases is increasing year by year. The current commonly used treatment methods include: 1. Traditional immunosuppression and regulation drugs, mainly relying on the application of glucocorticoids and immunosuppressive agents (such as prednisone, azathioprine, cyclosporine, etc.). They can reduce the disability rate of patients with autoimmune diseases to a certain extent, improve the survival rate of patients, and delay the progression of the disease. However, this non-specific treatment method cannot fundamentally solve the problem of self-reactive T cells attacking self-antigens caused by impaired self-tolerance. At the same time, long-term use may increase the risk of infection, metabolic disorders and organ toxicity, and some patients are not sensitive to treatment or are prone to relapse after drug withdrawal; 2. Biological agents and targeted therapy biological agents (such as anti-TNF-α, anti-CD20 monoclonal antibodies), which play a role by specifically blocking inflammatory factors or immune cell functions. For example, the use of rituximab in patients with rheumatoid arthritis can quickly control inflammation and reduce bone destruction, but it is expensive (such as the cost of biological agents for half a year is 300,000-1,000,000 yuan), and only a limited number of patients have limited efficacy, and may increase the risk of infection or malignancy; 3. Cell therapy and gene editing technology, which targets pathogenic B cells or autoantibodies by gene editing to modify T cells, and is applied to neuromyelitis optica, myasthenia gravis and other nervous system autoimmune diseases. However, the current research sample size is small, the long-term safety is unknown, and the indications are mostly limited to B cell-mediated diseases.

[0004] CADM3, full name is Cell Adhesion Molecule 3, belongs to the CADM protein family. Its molecular structure contains three extracellular immunoglobulin-like domains (including two Ig-like C domains and one Ig-like V domain), a transmembrane domain and a short intracellular region. At present, the research on CADM3 mainly focuses on the development of the nervous system and its function as an adhesion molecule in cell adhesion and signal transduction related functions. In recent years, it has been found that CADM3 has a dual role in tumor development. For example, in non-small cell lung cancer and gastric cancer, CADM3 is often down-regulated due to high methylation in the promoter region, and restoring its expression can inhibit the activity of the EGFR / ERK pathway and induce tumor cell apoptosis; on the contrary, in breast cancer, overexpression of CADM3 can activate the integrin beta1 / FAK pathway to promote tumor cell migration and angiogenesis. There is no related report on CADM3 improving autoimmune diseases. SUMMARY

[0005] The purpose of the present application is to provide the application of CADM3 in the preparation of products for treating autoimmune diseases, to solve the problems existing in the prior art, and the CADM3 protein provided by the present application can effectively block the activation and proliferation of T cells, and further regulate the overactive autoimmune response.

[0006] To achieve the above purpose, the present application provides the following scheme:

[0007] The present application provides the application of CADM3 or CADM3-Ig fusion protein in the preparation of products for treating autoimmune diseases, and the amino acid sequence of the CADM3 is shown in SEQ ID NO. 2;

[0008] The CADM3-Ig fusion protein is prepared by the following method: the coding gene of the CADM3 is connected to the pCMV6-AC-FC-S plasmid, the cells are transfected, the cells are cultured, the supernatant is collected, and the CADM3-Ig fusion protein is obtained after concentration and purification;

[0009] The nucleotide sequence of the coding gene of the CADM3 is shown in SEQ ID NO. 1.

[0010] Optionally, the autoimmune diseases include autoimmune encephalomyelitis and inflammatory bowel disease.

[0011] Optionally, the CADM3 or CADM3-Ig fusion protein plays a role in treating autoimmune diseases by inhibiting the proliferation and activation of T cells.

[0012] Optionally, the CADM3 or CADM3-Ig fusion protein plays a role in treating autoimmune diseases by inhibiting T cells from secreting inflammatory factors.

[0013] The present application also provides a medicine for treating autoimmune diseases, characterized in that the medicine comprises CADM3 or CADM3-Ig fusion protein.

[0014] The amino acid sequence of the CADM3 is shown in SEQ ID NO. 2.

[0015] The CADM3-Ig fusion protein is prepared by connecting the coding gene of the CADM3 to pCMV6-AC-FC-S plasmid, transfecting cells, culturing the cells, collecting supernatant, and obtaining the CADM3-Ig fusion protein after concentration and purification.

[0016] The nucleotide sequence of the coding gene of the CADM3 is shown in SEQ ID NO. 1.

[0017] Optionally, the dosage form of the medicine comprises tablets, injections, inhalants, granules, pills, capsules or liniments.

[0018] Optionally, the medicine further comprises pharmaceutically acceptable excipients.

[0019] Optionally, the excipients comprise at least one of diluents, fillers, excipients, binders, humectants, disintegrants, absorption promoters, surfactants, adsorption carriers, lubricants and flavoring agents.

[0020] Optionally, the administration mode of the medicine comprises oral or non-gastrointestinal administration.

[0021] The present application discloses the following technical effects:

[0022] 1. Innovation of target

[0023] The present application first discloses the biological function of CADM3 molecule as a new B7 family related co-inhibitory molecule. The CADM3 protein is specifically expressed on the surface of antigen presenting cells, and significantly inhibits T cell immune response by combining with the receptor on the surface of activated T cells, thereby providing a new target for the treatment of autoimmune diseases.

[0024] 2. Immune regulation function of hCADM3-Ig fusion protein

[0025] The hCADM3-Ig fusion protein prepared by the application can significantly inhibit T cell activation and proliferation through in vitro experiments. In animal models, hCADM3-Ig fusion protein treatment can significantly improve the clinical symptoms of multiple sclerosis (EAE model) and inflammatory bowel disease (DSS-induced colitis model), and reduce histopathological damage.

[0026] 3. Wide treatment applicability

[0027] The technical solution of the application is not only applicable to multiple sclerosis and inflammatory bowel disease, but also can be extended to other autoimmune diseases, including but not limited to rheumatoid arthritis, type I diabetes, psoriasis, autoimmune thyroiditis, etc.

[0028] 4. Synergistic treatment potential

[0029] The hCADM3-Ig fusion protein of the application can be used in combination with existing therapies (such as immunosuppressants, biological agents) to enhance efficacy and reduce drug dosage, thereby reducing the risk of side effects and improving treatment safety. At the same time, the technical solution of the application can also be combined with other treatment methods, such as immunosuppressants, biological agents, etc., to achieve better treatment effect.

[0030] The application discloses that CADM3 is up-regulated on the surface of activated APC, and in vitro cell intervention experiments using the prepared hCADM3-Ig fusion protein confirm that the fusion protein can effectively block the activation and proliferation of T cells, and further regulate excessive autoimmune response. Through animal experiment verification, the hCADM3-Ig fusion protein treatment group has the following advantages over the control group: significantly improving the neurological deficit symptoms of EAE model mice, significantly reducing the intestinal dysfunction of IBD model mice, effectively reducing the degree of inflammatory response in the two models, and significantly inhibiting the activation and proliferation of pathological T cells.

[0031] The application develops an innovative treatment scheme based on CADM3 molecules, which has significant advantages in multiple dimensions: in the clinical application aspect, it can effectively meet the urgent treatment needs of patients with autoimmune diseases, improve treatment effect and improve the quality of life of patients; in the economic aspect, by optimizing the treatment scheme, the consumption of medical resources and the expenditure of treatment cost can be greatly reduced; in the business aspect, its technical innovation and sustainable development potential will build core competitive advantage for related enterprises and create considerable economic benefits. Combining these advantages, the application shows great commercialization prospects and broad market development space. BRIEF DESCRIPTION OF DRAWINGS

[0032] Figure 1Sequence alignment of CADM3 and B7 family related molecules; A: Multiple sequence alignment of mCADM3 ectodomain and some known B7 and BTN family members; B: Phylogenetic tree of multiple sequence alignment of mCADM3 ectodomain and some known B7 and BTN family members;

[0033] Figure 2 Homology and similarity analysis of CADM3 and B7 family related molecules; A: Protein sequence alignment of mCADM3 and hCADM3 ectodomain; B: Multiple sequence alignment similarity and homology statistics of mCADM3 ectodomain and some known B7 and BTN family members;

[0034] Figure 3 Expression analysis of CADM3 molecule on mouse immune cells; A: Typical profile of mCADM3 protein expression on resting and activated immune cells; B: Statistics of mCADM3 protein expression on immune cells;

[0035] Figure 4 Expression analysis of CADM3 putative receptor on immune cells; A: Typical profile of CADM3 putative receptor expression on resting and activated immune cells; B: Statistics of CADM3 putative receptor expression on resting and activated immune cells; C: Expression of CD28, CTLA-4, PD-1 and BTLA genes; D: Flow cytometry analysis of biotinylated hCADM3-Ig or control Ig binding to transfected cells;

[0036] Figure 5 In vitro experiment to detect the functional effect of commercial hCADM3 protein on T cells; A: Typical profile and statistics of CD69 expression in mouse CD4 + , CD8 + T cells treated with hCADM3 protein; B: Typical profile and statistics of Ki67 expression in mouse CD4 + , CD8 + T cells treated with hCADM3 protein;

[0037] Figure 6 Typical profile and statistics of CD44 + , CD62L + and CD44 lo , CD62L hi expression in mouse CD4 hi and CD8 lo T cells treated with hCADM3 and PD-L1 proteins;

[0038] Figure 7For the hCADM3 gene expression plasmid map and validation of the hCADM3-Ig fusion protein; A: hCADM3 gene expression plasmid map; B: Left lane 1 is MW markers; Left lane 2 is Coomassie Brilliant Blue stained SDS-PAGE; Left lane 3 is anti-IgG 2a antibody Western blot; Left lane 4 is anti-hCADM3 antibody Western blot;

[0039] Figure 8 To detect the effect of hCADM3-Ig fusion protein on T cell function in vitro; A: Effect of hCADM3-Ig fusion protein on mouse CD4 + CD8 + Typical and statistical graphs of CD69 expression in T cells; B: hCADM3-Ig fusion protein on mouse CD4 + CD8 + Typical and statistical graphs of Ki67 expression in T cells;

[0040] Figure 9 hCADM3-Ig fusion protein for mouse CD4 + and CD8 + CD44 in T cells lo CD62L hi and CD44 hi CD62L lo Typical and statistical graphs for expressing [the concept / object].

[0041] Figure 10 hCADM3-Ig fusion protein alleviates disease progression in EAE model mice; A: Mean clinical score of EAE model mice; B: H&E staining of mouse spinal cord tissue;

[0042] Figure 11 Analysis of immune cell expression in the spleen of EAE model mice; A: Mouse CD4 + CD8 + Typical and statistical graphs of CD69 expression in T cells; B: Mouse CD4 + CD8 + Typical and statistical graphs of Ki67 expression in T cells; C: mouse CD4 + CD8 + CD44 in T cells lo CD62L hi and CD44 hi CD62L lo Typical and statistical graphs of expression; D: mouse CD4 + Typical and statistical graphs showing the percentages of INF-γ and IL-17A in T cells; E: CD4 +CD25 + FoxP3 + The percentage of Tregs and its statistical results;

[0043] Figure 12 Analysis of mRNA expression of inflammatory factors in spinal cord tissue of EAE model mice;

[0044] Figure 13 To alleviate the progression of IBD in IBD model mice using hCADM3-Ig fusion protein; A: Percentage change in body weight in IBD model mice; B: DIA score in IBD model mice; C: H&E staining of colon tissue in mice; D: Analysis of mRNA expression of inflammatory factors in colon tissue;

[0045] Figure 14 Analysis of immune cell expression in the spleen of IBD model mice; A: Mouse CD4 + CD8 + Typical and statistical graphs of CD69 expression in T cells; B: Mouse CD4 + CD8 + Typical and statistical graphs of Ki67 expression in T cells; C: mouse CD4 + CD8 + CD44 in T cells lo CD62L hi and CD44 hi CD62L lo Typical and statistical graphs illustrating the situation;

[0046] Figure 15 Analysis of the expression of immune cells and polar cells in the spleen of IBD model mice; A: CD4 + CD25 + FoxP3 + Percentage of Tregs and their statistical results; B: Mouse CD4 + Typical and statistical graphs showing the percentages of INF-γ and IL-17A in T cells; C:F4 / 80 + CD206 in cells hi MHCII lo M1 macrophages and CD206 lo MHCII hi Typical and statistical diagrams of M2 macrophage expression. Detailed Implementation

[0047] SEQ ID NO.1 (nucleotide sequence encoding CADM3):

[0048]

[0049] SEQ ID NO.2: (Amino acid sequence of CADM3):

[0050] NLSQDGYWQEQDLELGTLAPLDEAISSTVWSSPDMLASQDSQPWTSDETVVAGGTVVLKCQVKDHEDSSLQWSNPAQQTLYFGEKRALRDNRIQLVTSTPHE LSISISNVALADEGEYTCSIFTMPVRTAKSLVTVLGIPQKPIITGYKSSLREKDTATLNCQSSGSKPAARLTWRKGDQELHGEPTRIQEDPNGKTFTVSSSV TFQVTREDDGASIVCSVNHESLKGADRSTSQRIEVLYTPTAMIRPDPPHPREGQKLLLHCEGRGNPVPQQYLWEKEGSVPPLKMTQESALIFPFLNKSDSGTYGCTATSNMGSYKAYYTLNVNDPSPVPSSSSTYHAIIGGIVAFIVFLLLIMLIFLGHYLIRHKGTYLTHEAKGSDDAPDADTAIINAEGGQSGGDDKKEYFI.

[0051] The CADM3 described in this invention is obtained by PCR amplification using the full-length extracellular cDNA of hCADM3 as a template and primer pair F / R. The nucleotide sequences of the primer pairs described in this invention are as follows: F: 5'-CACTTGTCACGAATTCGGCGAACCTCTCCCAGGACGGC-3' (SEQ ID NO.3), R: 5'-CTCGAGCGGCCGCGTACGCGTGATGAAATATTCCTTCTTGTCG-3' (SEQ ID NO.4).

[0052] Example 1

[0053] 1. Comparison of homology and similarity between CADM3 and related molecules of the B7 family

[0054] The amino acid sequences of CADM3, B7, and BTN family members were downloaded from the Uniprot website. Sequence alignment was performed using Multiple Sequence Alignment by CLUSTALW (Multiple Sequence Alignment-CLUSTALW(genome.jp)), followed by statistical analysis. A developmental tree was constructed using MEGA11. The results of the sequence alignment analysis and developmental tree construction are available in [link to documentation]. Figures 1-2 .

[0055] It can be seen from Figure 1 the results that the extracellular domain of mCADM3 has a high degree of similarity and homology with multiple B7 family members; the sequences of mCADM3 and hCADM3 have a high degree of similarity ( Figure 2 in A); the homology of the extracellular domain of mCADM3 with the extracellular domains of mouse B7-1, B7-DC (PD-L1), PD-L2, B7-H3, and B7-H4 is 20.69%, 27.93%, 30.56%, 31.58%, and 23.93%, respectively; the homology with the extended B7 family molecules BTN1A1, BTN2A2, and ERMAP is 24.39%, 23.58%, and 19.64%, respectively ( Figure 2 in B). Based on the above structural characteristics and sequence similarities, CADM3 may have functional properties similar to those of B7 molecules.

[0056] 2. Expression analysis of CADM3 molecule in mouse immune cells

[0057] Single-cell suspensions were prepared from mouse spleens and cultured for 3 days with stimulation using anti-CD3 (1 μg / mL) / CD28 (3 μg / mL) antibodies to obtain activated T cells; LPS (5 μg / mL) was co-cultured with splenocytes for 3 days to obtain activated B cells and APC cells (macrophages, dendritic cells, and monocytes). Static and activated immune cells were stained with anti-CD4, CD8, CD19, F4 / 80, CD11c, CD11b, and anti-CADM3 antibodies or isotype control antibodies, and the expression levels of CADM3 on different immune cells were detected by flow cytometry.

[0058] The specific detection steps are as follows:

[0059] Preparation of spleen immune cell suspension: SPF-grade C57BL / 6 mice (purchased from the Animal Experiment Center of Guizhou Medical University, production license number: SCXK (Guizhou) 2025-0001) were used. The mice were sacrificed by cervical dislocation after anesthesia. After disinfecting the body surface with 75% ethanol, the spleens were removed aseptically and placed in pre-cooled PBS for preservation. The spleens were transferred to a 200-mesh sieve in a sterile culture dish and gently ground with a syringe piston until the tissue was completely dispersed. The cell suspension was collected into a 15 mL centrifuge tube and centrifuged at 3000 rpm for 5 min, and the supernatant was discarded to remove tissue debris. 3 mL of pre-cooled 1×red blood cell lysis solution was added to resuspend the cell pellet, incubated at room temperature for 5 min, then 10 mL of PBS was added to terminate the lysis, and centrifuged at 3000 rpm for 5 min, and the supernatant was discarded. The cells were resuspended with PBS containing 2% FBS and filtered through a 200-mesh nylon sieve to remove cell clumps, and the cell concentration was counted with a cell counter and adjusted to 1×10 7 / mL.

[0060] Flow cytometry assay: The in vitro immune cell stimulation assay was performed using 96-well plates pre-coated with LPS (5 μg / mL) and anti-CD3 antibody (1 μg / mL). The procedure was as follows: after taking the pre-coated plates out from 4°C, 200 μL of normal C57BL / 6 mouse spleen single cell suspension was added to each well, and 3 replicates were set for each group. The plates were incubated in a 37°C, 5% CO2 incubator for 72 h. After incubation, the cells were collected by centrifugation at 3000 rpm for 5 min, and a resting immune cell suspension was prepared. Then, multi-color flow antibody labeling was performed: APC-CD4, PerCP / Cy5.5-CD8a, PE-CD19, PerCP / Cy5.5-F4 / 80, PE-CD11c, PE-CD11b, and FITC-CADM3 antibodies and isotype controls were added at a ratio of 1:100, and incubated at 4°C in the dark for 45 min. After washing with PBS, the cells were resuspended with 200 μL of PBS and transferred to flow tubes, and finally the expression of CADM3 on the surface of each immune cell was analyzed by flow cytometry.

[0061] As shown in Figure 3 , Fig. 2A shows that CADM3 is expressed in resting immune cells, and the expression level in activated T cells and antigen-presenting cells (APCs) is significantly increased. Figure 3 Fig. 2B shows results consistent with those of Fig. 2A. Figure 3 Figure 3

[0062] Example 2: Assay for the expression of CADM3 receptor molecules in mouse immune cells

[0063] Assay for the expression of CADM3 receptor in immune cells: A mouse spleen single cell suspension was prepared, and biotin-labeled CADM3 Ig protein was incubated with the cells, with the addition of CD16 / 32 blocking antibody to eliminate non-specific binding of Fc receptors. Staining was performed using mouse-specific surface molecule antibodies (anti-CD4, anti-CD8, anti-CD19, anti-CD11b, anti-F4 / 80, anti-CD11c, anti-CD14, anti-CD68), and FACS analysis was performed 72 h later to determine the expression of CADM3 putative receptors on the surface of T cells (CD4 + , CD8 + ), B cells (CD19 + ), and APCs (CD11b + monocytes, F4 / 80 + macrophages, CD11c + dendritic cells).

[0064] ​​To detect the binding of hCADM3-Ig fusion protein (see Example 4 for preparation method) and Control Ig (purchased from sinobiological, Cat: 51094-MNAH) to known B7 family receptor transfected cells: the purchased plasmids of CD28 (sino biological, Cat: HG11524-ACG), CTLA-4 (sino biological, Cat: HG11159-ACG), PD-1 (sino biological, Cat: HG10377-CH), BTLA (sino biological, Cat: HG11895-UT) genes were transfected into HEK-293F cells. The transfected gene positive HEK-293F cells were obtained according to the resistance gene screening, and were detected by staining with the corresponding CD28, CTLA-4, PD-1, BTLA protein antibodies, and a blank control was set up. The flow cytometry was used to detect the transfected gene positive rate. After obtaining the purified transfected gene positive HEK-293F cells, the hCADM3-Ig fusion protein or Control Ig was combined therewith, and PE-labeled streptavidin was used for staining, and the flow cytometry was used to detect the binding of hCADM3-Ig to the transfected cells.

[0065] As shown in Figure 4 , the expression levels of the putative CADM3 receptor in activated T cells and antigen presenting cells (APCs) were significantly increased (Fig. 1A-B). The expression of CD28, CTLA-4, PD-1, BTLA proteins transfected into 293F cells was significantly up-regulated, indicating that the plasmid transfection was successful (Fig. 1C-B). hCADM3-Ig did not bind to CD28, CTLA-4, PD-1, BTLA (Fig. 1D). It can be seen that the putative CADM3 receptor is significantly up-regulated in activated T cells, and does not bind to CD28, PD-1, CTLA-4, BTLA, etc. known B7 family receptors. Figure 4 Figure 4 Figure 4

[0066] Example 3 Effect of commercial hCADM3 protein on T cell function

[0067] 1. Effect of hCADM3 protein on T cell proliferation and activation function by flow cytometry

[0068] ​​​hCADM3 protein was purchased from Sino Biological (Cat: 11214-H08H) through commercial channels. Normal C57BL / 6 mouse spleen mononuclear cell suspension was prepared, and the culture plate was coated with anti-CD3 (1 μg / mL) / CD28 (3 μg / mL) antibody and gradient concentration of hCADM3 protein (1600 ng / mL, 3200 ng / mL, 6400 ng / mL) or equal molar mass Control Ig (purchased from Sino Biological, Cat: 51094-MNAH). After 16-18 h of stimulation and culture, the cells were collected, stained with anti-CD4, -CD8, -CD69 flow cytometry antibodies, and the expression level of T cell early activation marker CD69 was detected by flow cytometry. The culture time was further extended to 72 h, and the change of T cell proliferation marker CFSE expression was detected by FACS. The preparation of mouse spleen mononuclear cell suspension and the steps of flow cytometry antibody staining were as described above.

[0069] 2. Flow cytometry detection of the effect of hCADM3 protein on T cell differentiation

[0070] PD-L1 is a star molecule of the B7 family with clear co-inhibitory function. hPD-L1 Ig protein was purchased through commercial channels as a control. Anti-CD3 (1 μg / mL) / CD28 (3 μg / mL) antibody and gradient concentration of hCADM3 protein (1600 ng / mL, 3200 ng / mL, 6400 ng / mL) or equal molar mass PD-L1 Ig protein were used to coat the culture plate. The spleen mononuclear cell suspension of normal C57BL / 6 mice was plated into a 96-well plate, and after 72 h of culture, the cells were collected, stained with anti-CD4, -CD8, -CD44, -CD62L antibodies, and the expression of CD44 lo CD62L hi Naive T cells and CD44 hi CD62L lo Effector T cell proportion.

[0071] The results are as follows Figures 5-6As shown, by interfering mouse T cells with different concentrations (1600 ng / mL, 3200 ng / mL, 6400 ng / mL) of hCADM3 Ig, after 16-18 h of culture, the expression change of T cell activation related molecule CD69 was detected by FACS, and the results showed that different concentrations of hCADM3 Ig could significantly inhibit the activation of T cells. After 72 h of cell culture, the proliferation of T cells (Ki67) was detected by FACS, and the results showed that different concentrations of hCADM3 Ig could significantly inhibit the proliferation of T cells. FACS was used to detect the expression of CD62L, an initial T cell marker, and CD44, an effector T cell marker, and the results showed that different concentrations of hCADM3 Ig and hPD-L1 Ig could significantly inhibit the differentiation of T cells into effector T cells. Thus, it is shown that hCADM3 has the function of negatively regulating T cell response.

[0072] Example 4 Preparation of hCADM3-Ig fusion protein and verification of the effect on T cell function

[0073] 1. Preparation of hCADM3-Ig fusion protein

[0074] Obtain the CADM3 gene sequence: retrieve the full-length gene sequence of hCADM3 extracellular region (NM_021189.5) from NCBI, use hCADM3 full-length cDNA (purchased from Youbao Biology, product number: G106471) as a template, and amplify the hCADM3 full-length sequence by PCR with the primers shown in SEQ ID NO. 3 and SEQ ID NO. 4, and recover the hCADM3 target gene by gel cutting.

[0075] Construct the hCADM3 gene expression plasmid: use MIU1 and Sigf1 enzymes to cut the pCMV6-AC-FC-S expression vector into a linear fragment, then connect the hCADM3 target gene to the pCMV6-AC-FC-S plasmid by In-Fusion HD Enzyme Premix DNA ligase to obtain the hCADM3 gene expression plasmid pCMV6-AC-FC-S-CADM3-SS (the map is shown in Figure 7 A).

[0076] Transfection: transfect the constructed hCADM3 plasmid into HEK-293F cells, and select 200 μg / mL concentration of G418 geneticin to screen the stable expression cell strain.

[0077] Preparation and verification of hCADM3-Ig fusion protein: After the stable cell line was cultured for 2 weeks in FreeStyleTM293 expression medium, the supernatant was collected, concentrated, and then the protein was purified using a Protein A / G protein purification column. The hCADM3 was hung on the purification column through the Fc tag, and the hCADM3-Ig fusion protein was obtained by eluting the column. The results are shown in Figure 7 B, gel electrophoresis (SDS-PAGE) and Western Blot showed that a single band of protein that could bind to anti-Fc Ab and anti-hCADM3 Ab was obtained, which was the hCADM3-Ig fusion protein (hCADM3-Fc fusion protein) after purification.

[0078] The hCADM3-Ig fusion protein contains a FC tag protein because a tag protein (FC tag protein) needs to be introduced in the purification of the hCADM3-Ig fusion protein.

[0079] 2. In vitro verification of the effect of the prepared hCADM3-Ig fusion protein on T cells

[0080] Flow cytometry detection of the effect of hCADM3-Ig fusion protein on T cell proliferation and activation: In order to verify whether the prepared hCADM3-Ig fusion protein has a function on T cell response, mouse T cells were intervened by different concentrations (1600 ng / mL, 3200 ng / mL, 6400 ng / mL) of hCADM3-Ig fusion protein and equal molar mass Control Ig, and after 16-18 h of culture, the expression changes of T cell activation related molecules CD69 were detected by FACS. Further extending the culture time to 72 h, the expression changes of T cell proliferation marker Ki67 were detected by FACS lo CD62L hi CD44 hi CD62L lo Effector T cell proportion. The preparation of mouse spleen single cell suspension and flow cytometry antibody staining steps are as described above.

[0081] The results are shown in Figures 8-9As shown, different concentrations of hCADM3-Ig fusion protein can significantly inhibit the activation of T cells. After 72 h of cell culture, the proliferation of T cells (Ki67) was detected by FACS, and the results showed that different concentrations of hCADM3 can significantly inhibit the proliferation of T cells. FACS was used to detect the expression of CD62L, an initial T cell marker, and CD44, an effector T cell marker, and the results showed that different concentrations of hCADM3-Ig fusion protein can significantly inhibit the differentiation of T cells into effector T cells. Thus, it is shown that the hCADM3-Ig fusion protein prepared by the present application has the function of negatively regulating T cell response.

[0082] Example 5 Influence of hCADM3-Ig fusion protein on autoimmune disease model of encephalomyelitis EAE

[0083] 1. Construction of EAE mouse model

[0084] 200 μg MOG 35-55 Short peptide powder (Shanghai Jill Biochemical Co., Ltd., 51716) was dissolved in 100 μL PBS, mixed thoroughly to ensure complete dissolution of the short peptide. 100 μL of the above solution was mixed with 400 μg of complete Freund's adjuvant FCA (Sigma Company, F5881) of Mycobacterium tuberculosis H37Ra. The mixture was placed on ice and emulsified thoroughly using an ultrasonic emulsifier until a uniform white emulsion (MOG 35-55 / FCA) was formed. The temperature and time during emulsification were strictly controlled to ensure the stability and immunogenicity of the emulsion. 4-6 week old female mice were selected as experimental animals, and the prepared MOG 35-55 / CFA emulsion was injected subcutaneously at 4 points on the back of the mice, with a total injection volume of 0.2 mL per mouse (containing 200 μg MOG 35-55 short peptide and 400 μg of Mycobacterium tuberculosis H37Ra). To enhance the immune effect, 500 ng of inactivated pertussis toxin was injected intraperitoneally into each mouse on day 0 and day 2. Strict aseptic operation was performed during injection to avoid infection.

[0085] The constructed model mice were divided into a treatment group (hCADM3-Ig fusion protein) and a control group (Control Ig protein) (n = 20). From the 13th day, the mice in the treatment and control groups were injected intraperitoneally with 25 μg of hCADM3-Ig fusion protein and 25 μg of Control Ig protein, respectively, twice a week. The incidence was observed every day and the clinical scores were recorded. The experiment ended after 42 days, and subsequent related experiments were performed.

[0086] 2. H&E staining of mouse spinal cord tissue

[0087] The spinal cord tissues of the hCADM3-Ig and Control Ig groups of mice were taken, paraffin-embedded, sectioned, deparaffinated, stained with H&E, and observed under a microscope.

[0088] 3. Flow cytometry detection of mouse spleen immune cell phenotype analysis

[0089] (1) T cell activation and proliferation in the two groups of spleens: the spleens of the two groups of mice were taken, single-cell suspensions were prepared, and anti-CD4, -CD8, -CD69, and -Ki67 antibodies were used for staining, and flow cytometry was used to detect CD4 + T cells and CD8 + T cells CD69, Ki67 expression changes.

[0090] (2) Anti-CD4, -CD8, -CD44, and -CD62L antibody staining, flow cytometry detection of CD4 + T cells and CD8 + T cells CD44 lo CD62L hi Naive T cells and CD44 hi CD62L lo Effector T cell proportion.

[0091] (3) Flow cytometry detection of Tregs expression in the two groups of spleens: anti-CD4, -CD25, and -FOXP3 antibodies were used for staining, and flow cytometry was used to detect the expression of Tregs in the immune cells of the two groups of spleens.

[0092] (4) Flow cytometry detection of Th1 / Th17 cell expression in the two groups of spleens: anti-CD4, -INF-γ, and -IL-17A antibodies were used for staining, and flow cytometry was used to detect the expression of Th1 / Th17 in the two groups of CD4 + T cells. The preparation of mouse spleen single-cell suspensions and flow cytometry antibody staining steps are as described above.

[0093] 4. qPCR detection of spinal cord tissue

[0094] The spinal cord tissues of the hCADM3-Ig and Control Ig groups of mice were taken, total RNA was extracted, reverse-transcribed into cDNA, primers for IL-6, IL-17, TNF-α, TGF-β, IL-4, and IL-10 were designed (Table 1), and PCR amplification was performed on the machine, and the obtained data was statistically analyzed.

[0095] Table 1 Primer sequences

[0096]

[0097] According to MonAmp TM Green qPCR Mix kit instruction, Real-time qPCR reaction system was prepared as follows:

[0098] Table 2 Real-time qPCR reaction system

[0099]

[0100] Mix well to avoid foaming, put into StepOnePlus PCR instrument, set two-step PCR amplification standard program:

[0101] First step: pre-denaturation: 95℃, 30 sec. Second step: denaturation: 95℃, 10 sec; annealing & extension: 60℃, 35 sec; 40 cycles.

[0102] Results: Compared with the control group, the clinical score of the treatment group mice was significantly reduced after intraperitoneal injection of hCADM3-Ig fusion protein for 4 weeks, indicating that the severity of the disease was obviously relieved Figure 10 A). Further histopathological analysis showed that the degree of inflammatory cell infiltration in the spinal cord tissue of mice treated with hCADM3-Ig was significantly reduced compared with the Control Ig control group Figure 10 B).

[0103] Flow cytometry was used to detect immune cells in the spleen of each group. The results showed that compared with the control group, the proportion of CD4 + and CD8 + T cells in the spleen of EAE mice treated with hCADM3-Ig fusion protein was significantly reduced Figure 11 A), and the proportion of Ki67, a proliferation marker, in CD4 + and CD8 + T cells was also significantly reduced Figure 11 B). The proportion of CD44 lo CD62L hi initial T cells in the spleen of mice in the treatment group increased, while the proportion of CD44 hi CD62L lo effector T cells decreased Figure 11 C), indicating that hCADM3-Ig fusion protein can effectively inhibit the proliferation and activation of T cells in the spleen of EAE mice. Further analysis found that the proportion of Tregs in the spleen of mice treated with hCADM3-Ig fusion protein was significantly increased Figure 11 E), while the proportion of pro-inflammatory INF-γ and IL-17A cells showed a decreasing trend Figure 11Fig. 6h. Effect of CADM3-Ig fusion protein on inflammatory bowel disease (IBD). Fig. 6i. Effect of CADM3-Ig fusion protein on inflammatory bowel disease (IBD). Fig. 6j. Effect of CADM3-Ig fusion protein on inflammatory bowel disease (IBD). Figure 12

[0104] Example 6h. Effect of CADM3-Ig fusion protein on inflammatory bowel disease (IBD).

[0105] 1. Construction of IBD mouse model

[0106] C57BL / 6 mice were fed with 2.5% DSS, and from the 3rd day, the mice were injected intraperitoneally with 25 μg of hCADM3-Ig (experimental group) and 25 μg of Control Ig protein (control group), respectively, once every other day, for a total of 3 times. The development of IBD was monitored for 12 days. The body weight and DIA score of the mice were recorded.

[0107] 2. H&E staining of colon tissue

[0108] The colon tissues of the hCADM3-Ig and Control Ig groups of mice were paraffin-embedded, sectioned, deparaffinated, and stained with H&E. The cell nuclei were stained with hematoxylin, and the cytoplasm was stained with eosin. The sections were dehydrated and mounted for microscopic observation.

[0109] 3. qPCR detection of colon tissue

[0110] The colon tissues of the hCADM3-Ig and Control Ig groups of mice were extracted for total RNA, which was reverse-transcribed into cDNA. Primers for TNF-a and IL-1β were designed, and PCR amplification was performed. The obtained data were statistically analyzed.

[0111] 4. Flow cytometry detection of mouse spleen immune cell phenotype analysis

[0112] (1) T cell activation and proliferation in the spleen of the two groups: The spleens of the two groups of mice were taken, and single-cell suspensions were prepared. Anti-CD4, -CD8, -CD69, and -Ki67 antibodies were used for staining, and flow cytometry was used to detect the expression of CD4 + T cells and CD8 + T cells CD69, and Ki67.

[0113] (2) Anti-CD4, -CD8, -CD44, and -CD62L antibody staining, flow cytometry detection of CD4 + T cells and CD8 + T cells CD44 lo CD62L hi T cells and CD44 hi CD62L lo Effector T cell proportion.

[0114] ​(3) Flow cytometry was used to detect the expression of Tregs in the spleen of the two groups: anti-CD4, -CD25, and -FOXP3 antibodies were used for staining, and flow cytometry was used to detect the expression of Tregs in the immune cells of the spleen of the two groups.

[0115] (4) Flow cytometry analysis of Th1 / Th17 cell expression in spleen of the two groups: Anti-CD4, -INF-γ, and -IL-17A antibody staining, and flow cytometry analysis of CD4 in the two groups. + Expression of Th1 / Th17A in T cells. The preparation of mouse spleen single-cell suspension and flow cytometry antibody staining procedures are as described above.

[0116] (5) Flow cytometry was used to detect the polarization of macrophages in the two groups of spleens: -F4 / 80, -MHCII and -CD206 antibodies were added to each well for staining, and the expression of MHCII in M1 macrophages and CD206 in M2 macrophages was detected by flow cytometry.

[0117] Results: The rate of weight change in the treatment group was significantly lower than that in the control group, and the DIA score was significantly lower. Figure 13 AB staining and H&E staining results showed that, compared with the control group, the treatment group had more regular colonic epithelial cell structure, more intact intestinal mucosa, and reduced inflammatory cell infiltration. Figure 13 (C). qPCR results showed decreased expression of pro-inflammatory factors and increased expression of anti-inflammatory factors. Figure 13 (D).

[0118] Flow cytometry results showed that, compared with the control group, the hCADM3-Ig fusion protein significantly inhibited CD4. + T cells and CD8 + T cell activation and proliferation ( Figure 14 (AB), CD44 in the spleen of mice in the treatment group lo CD62L hi The proportion of naïve T cells increased, while CD44... hi CD62L lo The proportion of effector T cells decreased ( Figure 14 (C) Treatment with hCADM3-Ig fusion protein significantly increased the percentage of spleen Treg cells and decreased the proportion of INF-γ and IL-17A cells. Furthermore, it reduced M1 pro-inflammatory macrophages and increased M2 anti-inflammatory macrophages. Figure 15 This indicates that hCADM3-Ig can reduce the severity of IBD, an autoimmune disease in mice, by inhibiting T cell activation and proliferation, as well as the release of inflammatory factors.

[0119] Based on the above experimental results, this invention reveals that CADM3 is upregulated on the surface of activated APCs. In vitro cell intervention experiments using the prepared hCADM3-Ig fusion protein confirmed that the fusion protein can effectively block the activation and proliferation of T cells, thereby regulating the overactive autoimmune response.

[0120] This invention validates the therapeutic effect of hCADM3-Ig fusion protein on autoimmune diseases through animal experiments. Specific implementation methods include: In an experimental autoimmune encephalomyelitis (EAE) model: 1. assessing changes in neurological function scores; 2. analyzing pathological changes in spinal cord tissue; 3. quantitatively detecting the levels of inflammatory factors in the central nervous system; 4. detecting splenic T cell activation markers and proliferation capacity. In an inflammatory bowel disease (IBD) model: 1. continuously monitoring weight changes; 2. measuring the expression of inflammatory factors in colonic tissue; 3. observing the pathological characteristics of intestinal tissue; 4. analyzing the splenic T cell immune response status. Experimental results show that, compared with the control group, the hCADM3-Ig fusion protein treatment group significantly improved neurological deficit symptoms in EAE model mice, significantly alleviated intestinal dysfunction in IBD model mice, effectively reduced the degree of inflammatory response in both models, and significantly inhibited pathological T cell activation and proliferation.

[0121] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. Use of CADM3 or a CADM3-Ig fusion protein in the preparation of a product for the treatment of an autoimmune disease, characterized in that, The amino acid sequence of the CADM3 is shown as SEQ ID NO. 2; The CADM3-Ig fusion protein is prepared by the following method: the coding gene of the CADM3 is connected to the pCMV6-AC-FC-S plasmid, the cell is transfected, the cell is cultured, the supernatant is collected, and the CADM3-Ig fusion protein is obtained after concentration and purification; The nucleotide sequence of the coding gene of the CADM3 is shown as SEQ ID NO. 1; The autoimmune disease is multiple sclerosis and colitis.

2. Use according to claim 1, wherein The CADM3 or CADM3-Ig fusion protein plays a role in treating autoimmune diseases by inhibiting the proliferation and activation of T cells.

3. The use according to claim 1, wherein The CADM3 or CADM3-Ig fusion protein plays a role in treating autoimmune diseases by inhibiting the secretion of inflammatory factors by T cells.

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