Application of CADM3 in preparation of products for treating autoimmune diseases

Abstract

The invention discloses application of CADM3 in preparation of a product for treating autoimmune diseases, and belongs to the technical field of biological medicines, and the amino acid sequence of the CADM3 is shown as SEQ ID NO.2. The expression of CADM3 on the surface of activated APC is up-regulated, an in-vitro cell intervention experiment is carried out by using the prepared hCADM3-Ig fusion protein, and it is proved that the fusion protein can effectively block activation and proliferation of T cells, and then over-active autoimmune response is regulated and controlled. Animal experiments prove that compared with a control group, an hCADM3-Ig fusion protein treatment group can remarkably improve the neurological function defect symptom of an EAE model mouse, remarkably relieve intestinal dysfunction of an IBD model mouse, effectively reduce the inflammatory reaction degree in the two models and remarkably inhibit activation and proliferation of pathological T cells.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of CADM3 in the preparation of products for treating autoimmune diseases. Background Technology

[0002] Autoimmune diseases are a group of clinical syndromes caused by the immune system's abnormal recognition and attack of its own tissues. Their prevalence has reached 5-10% and is showing a continuous upward trend. These include type 1 diabetes, multiple sclerosis (MS), and rheumatoid arthritis (RA). The pathogenesis of autoimmune diseases is complex, but abnormal activation of autoreactive T cells, which attack their own tissue antigens and cause damage, is a crucial pathogenic mechanism.

[0003] The prevalence of autoimmune diseases is increasing year by year. Currently, commonly used treatment methods include: 1. Traditional immunosuppressive and modulatory drugs, mainly relying on the application of glucocorticoids and immunosuppressants (such as prednisone, azathioprine, cyclosporine, etc.). To a certain extent, these drugs reduce the disability rate of patients with autoimmune diseases, 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 autoreactive T cells attacking self-antigens due to impaired self-tolerance. At the same time, long-term use may lead to increased risk of infection, metabolic disorders and organ toxicity. Some patients are not sensitive to treatment or are prone to relapse after discontinuation of medication; 2. Biologics and targeted therapy. Biologics (such as anti-TNF-α and anti-CD20 monoclonal antibodies) work by specifically blocking inflammatory factors or immune cell function. For example, rituximab can quickly control inflammation and reduce bone destruction in patients with rheumatoid arthritis, but it is expensive (e.g., the cost of biologics can reach 30,000 to 100,000 yuan over six months), has limited efficacy in only some patients, and may increase the risk of infection or malignant tumors; 3. Cell therapy and gene editing technology, which modify T cells to target pathogenic B cells or autoantibodies through gene editing, can be applied to neuromyelitis optica, myasthenia gravis and other autoimmune diseases of the nervous system, but the current research sample size is small, the long-term safety is unclear, and the indications are mostly limited to B cell-mediated diseases.

[0004] CADM3, short for Cell Adhesion Molecule 3, belongs to the CADM protein family. Its molecular structure includes three extracellular immunoglobulin-like domains (including two Ig-like C domains and one Ig-like V domain), one transmembrane domain, and a short intracellular region. Current research on CADM3 mainly focuses on its role in nervous system development and its function as an adhesion molecule in cell adhesion and signal transduction. In recent years, CADM3 has been found to play a dual role in tumorigenesis and development. For example, in non-small cell lung cancer and gastric cancer, CADM3 expression is often downregulated due to promoter hypermethylation; restoring its expression can inhibit EGFR / ERK pathway activity and induce tumor cell apoptosis. Conversely, in breast cancer, overexpression of CADM3 can activate the integrin β1 / FAK pathway, promoting tumor cell migration and angiogenesis. Currently, there are no reports on CADM3 improving autoimmune diseases. Summary of the Invention

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

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] This invention provides the use of CADM3 or CADM3-Ig fusion protein in the preparation of products for treating autoimmune diseases, wherein the amino acid sequence of CADM3 is shown in SEQ ID NO.2;

[0008] The CADM3-Ig fusion protein was prepared by the following method: the coding gene of CADM3 was ligated into the pCMV6-AC-FC-S plasmid, cells were transfected, the cells were cultured, the supernatant was collected, and the CADM3-Ig fusion protein was obtained after concentration and purification.

[0009] The nucleotide sequence of the gene encoding 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 exerts a therapeutic effect on autoimmune diseases by inhibiting the proliferation and activation of T cells.

[0012] Optionally, the CADM3 or CADM3-Ig fusion protein can exert a therapeutic effect on autoimmune diseases by inhibiting the secretion of inflammatory factors by T cells.

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

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

[0015] The CADM3-Ig fusion protein was prepared by the following method: the coding gene of CADM3 was ligated into the pCMV6-AC-FC-S plasmid, cells were transfected, the cells were cultured, the supernatant was collected, and the CADM3-Ig fusion protein was obtained after concentration and purification.

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

[0017] Optionally, the dosage form of the drug includes tablets, injections, inhalers, granules, pills, capsules, or liniments.

[0018] Optionally, the drug may also include pharmaceutically acceptable excipients.

[0019] Optionally, the excipients include at least one of diluents, fillers, excipients, binders, humectants, disintegrants, absorption promoters, surfactants, adsorbent carriers, lubricants, and flavorings.

[0020] Optionally, the medication may be taken orally or via non-gastrointestinal routes.

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

[0022] 1. Target innovation

[0023] This invention reveals for the first time the biological function of CADM3 as a novel B7 family-associated co-inhibitory molecule. CADM3 protein is specifically expressed on the surface of antigen-presenting cells and significantly inhibits T cell immune responses by binding to receptors on the surface of activated T cells, providing a novel target for the treatment of autoimmune diseases.

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

[0025] The hCADM3-Ig fusion protein prepared in this invention has been shown in in vitro experiments to significantly inhibit T cell activation and proliferation. In animal models, treatment with the hCADM3-Ig fusion protein significantly improved the clinical symptoms of multiple sclerosis (EAE model) and inflammatory bowel disease (DSS-induced colitis model) and reduced histopathological damage.

[0026] 3. Wide range of therapeutic applications

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

[0028] 4. Potential for synergistic treatment

[0029] The hCADM3-Ig fusion protein of this invention can be used in combination with existing therapies (such as immunosuppressants and biologics) to enhance efficacy and reduce drug dosage, thereby reducing the risk of side effects and improving treatment safety. Furthermore, the technical solution of this invention can also be combined with other treatment methods, such as immunosuppressants and biologics, to achieve better therapeutic effects.

[0030] This invention reveals that CADM3 expression is upregulated on the surface of activated APCs. In vitro cell intervention experiments using the prepared hCADM3-Ig fusion protein confirmed that this fusion protein can effectively block T cell activation and proliferation, thereby regulating an overactive autoimmune response. Animal experiments verified 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.

[0031] This invention develops an innovative treatment based on CADM3 molecules, which exhibits significant advantages in multiple dimensions: In clinical application, it effectively meets the urgent treatment needs of patients with autoimmune diseases, improving treatment outcomes and enhancing patients' quality of life; economically, by optimizing the treatment plan, it can significantly reduce the consumption of medical resources and treatment costs; commercially, its technological innovation and sustainable development potential will enable related enterprises to build core competitive advantages and create considerable economic benefits. Considering these advantages, this invention demonstrates enormous commercialization prospects and broad market development potential. Attached Figure Description

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

[0033] Figure 2 Homology and similarity analysis of CADM3 and related molecules of the B7 family; A: Protein sequence comparison of the extracellular domains of mCADM3 and hCADM3; B: Multiple sequence alignment similarity and homology statistics of the extracellular domain of mCADM3 with some known members of the B7 and BTN families;

[0034] Figure 3 Analysis of CADM3 expression in mouse immune cells; A: Typical mCADM3 protein expression in resting and activated immune cells; B: Statistical results of mCADM3 protein expression in immune cells;

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

[0036] Figure 5 To investigate the effect of commercially available hCADM3 protein on T cell function in vitro; A: Effect of hCADM3 protein on mouse CD4 + CD8 + Typical and statistical graphs of CD69 expression in T cells; B: hCADM3 protein's effect on mouse CD4 + CD8 + Typical and statistical graphs of Ki67 expression in T cells;

[0037] Figure 6 hCADM3 and PD-L1 proteins on 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].

[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 members of the B7 family; 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, APC cells (macrophages, dendritic cells, and monocytes). Resting 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 storage. The spleens were transferred to a 200-mesh sieve in a sterile petri 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 lysate 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: In vitro immune cell stimulation experiments were performed using 96-well plates pre-coated with LPS (5 μg / mL) and anti-CD3 antibody (1 μg / mL). The specific procedure was as follows: After removing the pre-coated plate at 4℃, 200 μL of normal C57BL / 6 mouse spleen single-cell suspension was added to each well, with 3 replicates per group. The plates were incubated at 37℃ in a 5% CO2 incubator for 72 h. After incubation, cells were collected by centrifugation at 3000 rpm for 5 min, and a resting-state immune cell suspension was prepared. Subsequently, multicolor flow cytometry antibody labeling was performed: APC-CD4, PerCP / Cy5.5-CD8a, PE-CD19, PerCP / Cy5.5-F4 / 80, PE-CD11c, PE-CD11b, FITC-CADM3 antibody, and isotype controls were added at a 1:100 ratio, and the plates were incubated at 4℃ in the dark for 45 min. After washing with PBS, the cells were resuspended in 200 μL of PBS and transferred to flow cytometry tubes. Finally, the expression of CADM3 on the surface of each immune cell was analyzed by flow cytometry.

[0061] like Figure 3 As shown, Figure 3 The results showed that CADM3 was expressed in resting immune cells, and its expression level was significantly increased in activated T cells and antigen-presenting cells (APCs). Figure 3 The result displayed in B is the same as Figure 3 The results for A are consistent.

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

[0063] To detect the expression of CADM3 receptor in immune cells: Mouse spleen single-cell suspensions were prepared, and biotin-labeled CADM3Ig protein was co-incubated with the cells. CD16 / 32 blocking antibody was added simultaneously to eliminate non-specific binding of the Fc receptor. Staining was performed using mouse-specific surface molecular antibodies (anti-CD4, anti-CD8, anti-CD19, anti-CD11b, anti-F4 / 80, anti-CD11c, anti-CD14, anti-CD68). FACS analysis was performed on T cells (CD4+) after 72 hours. + CD8 + ), B cells (CD19) + ) and APCs (CD11b + Mononuclear cells, F4 / 80 + Macrophages, CD11c + Expression of CADM3 putative receptor on the surface of dendritic cells.

[0064] To detect the binding of hCADM3-Ig fusion protein (preparation method see Example 4) and Control Ig (purchased from sinobiological, Cat:51094-MNAH) to cells transfected with known B7 family receptors: Plasmids containing the purchased CD28 (sinobiological, Cat:HG11524-ACG), CTLA-4 (sinobiological, Cat:HG11159-ACG), PD-1 (sinobiological, Cat:HG10377-CH), and BTLA (sinobiological, Cat:HG11895-UT) genes were transfected into HEK-293F cells. HEK-293F cells with positive transfection genes were obtained through screening based on resistance genes, and staining was performed using antibodies against the corresponding CD28, CTLA-4, PD-1, and BTLA proteins. A blank control was also included. Flow cytometry was used to detect the gene positivity rate. After obtaining purified transfected gene-positive HEK-293F cells, hCADM3-Ig fusion protein or Control Ig was bound to them, and streptavidin was stained with PE. Flow cytometry was used to detect the binding of hCADM3-Ig to transfected cells.

[0065] like Figure 4 As shown, the putative CADM3 receptor expression levels were significantly increased in both activated T cells and antigen-presenting cells (APCs). Figure 4 (AB). The expression of CD28, CTLA-4, PD-1, and BTLA proteins was significantly upregulated in 293F cells after transfection, indicating successful plasmid transfection. Figure 4 (CB). hCADM3-Ig does not bind to CD28, CTLA-4, PD-1, or BTLA. Figure 4 (D). This suggests that the proposed CADM3 receptor is significantly upregulated on activated T cells and does not bind to known B7 family receptors such as CD28, PD-1, CTLA-4, and BTLA.

[0066] Example 3: Effects of commercially available hCADM3 protein on T cell function

[0067] 1. Flow cytometry detection of the effect of hCADM3 protein on T cell proliferation and activation function

[0068] hCADM3 protein (Sino Biological, Cat: 11214-H08H) was purchased commercially. Single-cell suspensions of normal C57BL / 6 mouse spleens were prepared using anti-CD3 (1 μg / mL) / CD28 (3 μg / mL) antibodies and gradient concentrations of hCADM3 protein (1600 ng / mL, 3200 ng / mL, 6400 ng / mL) or equimolar amounts of Control Ig (purchased from Sino Biological, Cat: 51094-MNAH) coated culture plates. After 16-18 hours of stimulation and culture, cells were collected and stained with anti-CD4, -CD8, and -CD69 flow cytometry antibodies. The expression level of CD69, an early T cell activation marker, was detected by flow cytometry. The culture time was further extended to 72 hours, and changes in the expression of the T cell proliferation marker CFSE were detected by FACS. The preparation of mouse spleen single-cell suspensions and flow cytometry antibody staining procedures were as described above.

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

[0070] PD-L1 is a star molecule in the B7 family with a clear co-inhibitory function. hPD-L1 Ig protein was purchased commercially as a control. Culture plates were coated with anti-CD3 (1 μg / mL) / CD28 (3 μg / mL) antibodies and gradient concentrations of hCADM3 protein (1600 ng / mL, 3200 ng / mL, 6400 ng / mL) or equimolar amounts of PD-L1 Ig protein. Single-cell suspensions of spleen cells from normal C57BL / 6 mice were seeded into 96-well plates and cultured for 72 h. Cells were then collected and stained with anti-CD4, -CD8, -CD44, and -CD62L antibodies. CD44+ and CD8T cells were detected by flow cytometry. lo CD62L hi Naïve T cells and CD44 hi CD62L lo The proportion of effector T cells.

[0071] The results are as follows Figures 5-6As shown, mouse T cells were treated with different concentrations (1600 ng / mL, 3200 ng / mL, and 6400 ng / mL). After culturing for 16-18 hours, the expression of CD69, a T cell activation-related molecule, was detected by FACS. The results showed that all concentrations of hCADM3 Ig significantly inhibited T cell activation. After 72 hours of cell culture, T cell proliferation (Ki67) was detected by FACS. The results showed that all concentrations of hCADM3 Ig significantly inhibited T cell proliferation. The expression of the naive T cell marker CD62L and the effector T cell marker CD44 was detected by FACS. The results showed that both hCADM3 Ig and hPD-L1 Ig significantly inhibited T cell differentiation into effector T cells. This indicates that hCADM3 has a negative regulatory function on T cell responses.

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

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

[0074] Obtaining the CADM3 gene sequence: The full-length extracellular region gene sequence of hCADM3 (NM_021189.5) was obtained by searching NCBI. Using the full-length hCADM3 cDNA (purchased from UBO Biotechnology, product number: G106471) as a template, the full-length hCADM3 sequence was amplified by PCR using the primer pairs shown in SEQ ID NO.3 and SEQ ID NO.4. The hCADM3 target gene was then recovered by gel excision.

[0075] Construction of hCADM3 gene expression plasmid: The pCMV6-AC-FC-S expression vector was cleaved into a linear fragment using MIU1 and Sigf1 enzymes. The hCADM3 target gene was then ligated into the pCMV6-AC-FC-S plasmid using In-Fusion HD Enzyme Premix DNA ligase to obtain the hCADM3 gene expression plasmid pCMV6-AC-FC-S-CADM3-SS (see diagram). Figure 7 (A)

[0076] Transfection: The constructed hCADM3 plasmid was transfected into HEK-293F cells, and cell lines that stably expressed the plasmid were selected using 200 μg / mL G418 genetic mycin.

[0077] Preparation and validation of hCADM3-Ig fusion protein: Stable cell lines expressing hCADM3 were cultured in FreeStyle™ 293 expression medium for 2 weeks. The supernatant was collected, concentrated, and purified using a Protein A / G protein purification column. hCADM3 was attached to the purification column via an Fc tag. Elution of the column yielded the hCADM3-Ig fusion protein. Results are as follows: Figure 7 In the B sample, gel electrophoresis (SDS-PAGE) and Western blotting showed that a single band of protein that could bind to anti-Fc Ab and anti-hCADM3 Ab was obtained, which is the purified hCADM3-Ig fusion protein (hCADM3-Fc fusion protein).

[0078] Because a tag protein (FC tag protein) needs to be introduced during the purification of the hCADM3-Ig fusion protein, the hCADM3-Ig fusion protein contains an FC tag protein.

[0079] 2. In vitro validation of the effect of the prepared hCADM3-Ig fusion protein on T cell function.

[0080] Flow cytometry analysis of the effects of hCADM3-Ig fusion protein on T cell proliferation and activation: To verify whether the prepared hCADM3-Ig fusion protein has a functional effect on T cell response, mouse T cells were treated with different concentrations (1600 ng / mL, 3200 ng / mL, 6400 ng / mL) of hCADM3-Ig fusion protein and equimolar amounts of Control Ig. After culturing for 16-18 h, the expression changes of CD69, a T cell activation-related molecule, were detected by FACS. The culture time was further extended to 72 h, and the expression changes of Ki67 and CD44, T cell proliferation markers, were detected by FACS. lo CD62L hi Naïve T cells and CD44 hi CD62L lo The proportion of effector T cells. The preparation of mouse spleen single-cell suspension and flow cytometry antibody staining procedures are as described above.

[0081] The results are as follows Figures 8-9As shown, different concentrations of hCADM3-Ig fusion protein significantly inhibited T cell activation. After 72 hours of cell culture, T cell proliferation was detected by FACS (Ki67), and the results showed that different concentrations of hCADM3 significantly inhibited T cell proliferation. FACS analysis of the expression of the naïve T cell marker CD62L and the effector T cell marker CD44 showed that different concentrations of hCADM3-Ig fusion protein significantly inhibited T cell differentiation into effector T cells. This indicates that the hCADM3-Ig fusion protein prepared in this invention has a function of negatively regulating T cell responses.

[0082] Example 5: Effect of hCADM3-Ig fusion protein on EAE (epileptic encephalitis) model of autoimmune disease

[0083] 1. Construction of EAE mouse model

[0084] 200 μg MOG 35-55 Short peptide powder (Shanghai Jier Biochemical Co., Ltd., 51716) was dissolved in 100 μL of PBS and thoroughly mixed to ensure complete dissolution. 100 μL of the above solution was then mixed with 400 μg of complete Freund's adjuvant FCA (Sigma, F5881) for Mycobacterium tuberculosis H37Ra. The mixture was placed on ice and emulsified thoroughly using an ultrasonic emulsifier until a homogeneous white emulsion (MOG) was formed. 35-55 / FCA). Temperature and time were strictly controlled during emulsification to ensure the stability and immunogenicity of the emulsion. Female mice aged 4-6 weeks were selected as experimental animals, and the prepared MOG was... 35-55 / CFA emulsion was administered subcutaneously at four points on the back of mice, with a total injection volume of 0.2 mL (containing 200 μg MOG) per mouse. 35-55 Short peptides and 400 μg of Mycobacterium tuberculosis H37Ra were administered. To enhance the immune response, each mouse was intraperitoneally injected with 500 ng of inactivated pertussis toxin on day 0 and day 2, respectively. Strict aseptic technique was maintained during the injection process 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). Starting from day 13, the mice in the treatment group and the control group were injected intraperitoneally with 25 μg of hCADM3-Ig fusion protein and 25 μg of Control Ig protein, respectively, twice a week. The disease status was observed and the clinical score was recorded daily. The experimental endpoint was reached after 42 days, and subsequent related experiments were carried out.

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

[0087] Spinal cord tissues from mice in the hCADM3-Ig and Control Ig groups were collected, embedded in paraffin, sectioned, dewaxed, and stained with hematoxylin and eosin for cell nuclei and eosin for cytoplasm. The sections were then dehydrated, mounted, and observed under a microscope.

[0088] 3. Phenotypic analysis of mouse spleen immune cells by flow cytometry

[0089] (1) T cell activation and proliferation in the spleen of the two groups: Spleens of mice from both groups were collected, and single-cell suspensions were prepared. Anti-CD4, -CD8, -CD69, and -Ki67 antibodies were used for staining, and CD4 was detected by flow cytometry. + T cells and CD8 + Changes in the expression of CD69 and Ki67 on T cells.

[0090] (2) Staining with anti-CD4, -CD8, -CD44 and -CD62L antibodies, and flow cytometry detection of CD4. + T cells and CD8 + CD44 in T cells lo CD62L hi Naïve T cells and CD44 hi CD62L lo The proportion of effector T cells.

[0091] (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.

[0092] (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. + The expression of Th1 / Th17 in T cells. The preparation of mouse spleen single-cell suspension and flow cytometry antibody staining procedures are as described above.

[0093] 4. Spinal cord tissue qPCR detection

[0094] Spinal cord tissues from hCADM3-Ig and Control Ig mice were collected, total RNA was extracted, and reverse transcribed into cDNA. Primers for IL-6, IL-17, TNF-α, TGF-β, IL-4 and IL-10 were designed (Table 1), and the samples were added to the PCR machine for PCR amplification. The obtained data were statistically analyzed.

[0095] Table 1 Primer sequences

[0096]

[0097] According to MonAmp TM The Green qPCR Mix kit instructions specify the following Real-time qPCR reaction mixture:

[0098] Table 2 Real-time qPCR reaction system

[0099]

[0100] Mix thoroughly to avoid foaming, place in a StepOnePlus PCR instrument, and set the standard two-step PCR amplification program:

[0101] Step 1: Pre-denaturation: 95℃, 30 sec. Step 2: Denaturation: 95℃, 10 sec; Annealing & Extension: 60℃, 35 sec; 40 cycles.

[0102] Results: Compared with the control group, mice in the treatment group showed significantly lower clinical scores after 4 weeks of intraperitoneal injection of hCADM3-Ig fusion protein, indicating a significant reduction in disease severity. 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 spleen of EAE mice treated with hCADM3-Ig fusion protein had significantly higher levels of CD4+ cells. + and CD8 + The expression rate of CD69, a marker of T cell activation, was significantly reduced. Figure 11 In CD4, the proliferation marker Ki67 is present in CD4+. + and CD8 + The proportion of expression in T cells was also significantly reduced. Figure 11 (Middle B). 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 11 The results showed that the hCADM3-Ig fusion protein effectively inhibited the proliferation and activation of T cells in the spleen of EAE mice. Further analysis revealed that the proportion of Tregs in the spleen of mice treated with the hCADM3-Ig fusion protein was significantly increased. Figure 11 In the middle E), the proportion of pro-inflammatory INF-γ and IL-17A cells showed a decreasing trend. Figure 11(D). qPCR analysis of changes in inflammatory factors in spinal cord tissue showed that CADM3 protein treatment decreased the expression of pro-inflammatory factors and increased the expression of anti-inflammatory factors. Figure 12 ).

[0104] Example 6: Effect of hCADM3-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. Starting from day 3, the mice were intraperitoneally injected with 25 μg hCADM3-Ig (experimental group) and 25 μg Control Ig protein (control group), respectively, once every other day for a total of 3 injections. IBD development was monitored for 12 days. Mouse body weight and DIA score were recorded.

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

[0108] Colon tissues from mice in the hCADM3-Ig and Control Ig groups were collected, paraffin-embedded, sectioned, dewaxed, and stained with hematoxylin and eosin for cell nuclei and eosin for cytoplasm. The sections were then dehydrated, mounted, and observed under a microscope.

[0109] 3. qPCR detection of colon tissue

[0110] Colon tissues from hCADM3-Ig and Control Ig mice were collected, total RNA was extracted, reverse transcribed into cDNA, primers TNF-α and IL-1β were designed, and the samples were loaded onto a PCR machine for amplification. The obtained data were statistically analyzed.

[0111] 4. Phenotypic analysis of mouse spleen immune cells by flow cytometry

[0112] (1) T cell activation and proliferation in the spleen of the two groups: Spleens of mice from both groups were collected, and single-cell suspensions were prepared. Anti-CD4, -CD8, -CD69, and -Ki67 antibodies were used for staining, and CD4 was detected by flow cytometry. + T cells and CD8 + Changes in the expression of CD69 and Ki67 on T cells.

[0113] (2) Staining with anti-CD4, -CD8, -CD44 and -CD62L antibodies, and flow cytometry detection of CD4. + T cells and CD8 + CD44 in T cells lo CD62L hi Naïve T cells and CD44 hi CD62L lo The proportion of effector T cells.

[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. The application of CADM3 or CADM3-Ig fusion protein in the preparation of products for treating autoimmune diseases, characterized in that, The amino acid sequence of CADM3 is shown in SEQ ID NO.2; The CADM3-Ig fusion protein was prepared by the following method: the coding gene of CADM3 was ligated into the pCMV6-AC-FC-S plasmid, cells were transfected, the cells were cultured, the supernatant was collected, and the CADM3-Ig fusion protein was obtained after concentration and purification. The nucleotide sequence of the gene encoding CADM3 is shown in SEQ ID NO.

1.

2. The application as described in claim 1, characterized in that, The autoimmune diseases mentioned include autoimmune encephalomyelitis and inflammatory bowel disease.

3. The application as described in claim 1, characterized in that, The CADM3 or CADM3-Ig fusion protein exerts its therapeutic effect on autoimmune diseases by inhibiting the proliferation and activation of T cells.

4. The application as described in claim 1, characterized in that, The CADM3 or CADM3-Ig fusion protein exerts its therapeutic effect on autoimmune diseases by inhibiting the secretion of inflammatory factors by T cells.

5. A drug for treating autoimmune diseases, characterized in that, The drug includes CADM3 or CADM3-Ig fusion protein; The amino acid sequence of CADM3 is shown in SEQ ID NO.2; The CADM3-Ig fusion protein was prepared by the following method: the coding gene of CADM3 was ligated into the pCMV6-AC-FC-S plasmid, cells were transfected, the cells were cultured, the supernatant was collected, and the CADM3-Ig fusion protein was obtained after concentration and purification. The nucleotide sequence of the gene encoding CADM3 is shown in SEQ ID NO.

1.

6. The drug as described in claim 5, characterized in that, The dosage forms of the drug include tablets, injections, inhalers, granules, pills, capsules, or liniments.

7. The drug as described in claim 5, characterized in that, The drug also includes pharmaceutically acceptable excipients.

8. The drug as described in claim 7, characterized in that, The excipients include at least one of the following: diluent, filler, excipient, binder, humectant, disintegrant, absorption promoter, surfactant, adsorbent carrier, lubricant, and flavoring agent.

9. The drug as described in claim 5, characterized in that, The medication can be taken orally or via non-gastrointestinal routes.

Images