Use of trem-2 in the preparation of a therapeutic drug and / or diagnostic reagent for multiple sclerosis

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to application of TREM-2 in preparation of a therapeutic drug and / or a diagnostic reagent for multiple sclerosis. The application research finds that TREM-2 is highly expressed in peripheral blood CD4+ T cells of multiple sclerosis patients, and positively correlated with the severity of the disease, indicating that TREM-2 can be used as a diagnostic marker for multiple sclerosis. The marker can be taken from peripheral blood, and has the characteristics of small trauma, low cost, rapidness and high sensitivity compared with other examinations such as cerebrospinal fluid examination, evoked potential and head CT, magnetic resonance imaging and the like. The application also finds that TREM-2 is involved in the transformation of pathogenic Th17 cells and the secretion of IL-6 and IL-17, indicating that intervention of the expression of TREM-2 can relieve and even hopefully treat multiple sclerosis.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to the application of TREM-2 in the preparation of therapeutic drugs or diagnostic reagents for multiple sclerosis. Background Technology

[0002] Multiple sclerosis (MS) is a common autoimmune disease of the nervous system, affecting approximately 2 million people worldwide. It is the most common cause of permanent disability in young people, excluding trauma, and is often referred to as the cancer of wheelchairs. It imposes a severe economic and psychological burden on families and society.

[0003] The pathogenesis of multiple sclerosis (MS) primarily involves the autoimmune system attacking myelin proteins, leading to impaired nerve conduction. Different nerves are affected, resulting in varying clinical symptoms. Previous research has suggested that the molecular mechanism involves peripheral APCs (autoimmune cells) activating Th cells, and these antigens share similarities with some molecules in the central nervous system (CNS). During this process, Th cells differentiate into Th1 / 17 cells and secrete VLA-4 to gain the ability to penetrate the blood-brain barrier (BBB). Once inside the CNS, these autoreactive T cells are reactivated, producing characteristic cytokines that activate nearby immune or neuronal cells and attract B cells into the CNS. Plasma cells produce antibodies that attack myelin proteins. Simultaneously, anti-inflammatory cytokines (such as IL-10) and other immunomodulatory mechanisms, such as regulatory T cells (Tregs) or NK cells, function, leading to the resolution of inflammation. Therefore, some patients experience relapses and remissions. Based on the pathogenesis, we are the first to propose that an imbalance of Th subtypes (Th1, Th17, Th9, Th2, and Tregs, etc.) is the basis for disease progression and transient remissions. This view has gained widespread acceptance in the scientific community. Although the regulation of Th subtype balance is an effective target for the treatment of MS, clinical treatments targeting Th cells have fallen far short of expectations.

[0004] Because the reactive inflammatory Th cell subset plays a crucial role in the pathogenesis of MS, most FDA-approved treatments for autoimmune diseases target T cells. First-line drugs include betaine (IFN-β), glatilmer acetate (GA), dimethyl fumarate (DMF), and fingolimod. IFN-β and GA are not very effective in slowing the progression of disability in progressive MS, and their effectiveness is correlated with serum IL-22 levels. However, due to their long-term efficacy and safety, they are the most common first-line treatments for relapsing-remitting MS (RRMS). DMF can lead to a decrease in peripheral T cells, resulting in an inflammatory state. Fingolimod can promote T cell retention in lymph nodes, leading to a high relapse rate after discontinuation. Previously approved monoclonal antibodies include natalizumab, alemtuzumab, daclizumab, and ocrelizumab. Natalizumab targets VLAN-4 (highly expressed on activated T cells and B cells), inhibiting the interaction between VLAN-4 and vascular cell adhesion molecule-1 (VCAM-1), thereby blocking T cell penetration into the BBB. However, discontinuation can lead to abnormal inflammatory activity, MS relapse, and progressive leukoencephalopathy. Alemtuzumab and daclizumab target CD52 (highly expressed on T cells and B cells), both inducing immune responses. Daclizumab, due to its high risk of causing hepatitis and inflammatory brain diseases such as encephalitis and meningoencephalitis, had its marketing authorization revoked in 2018. Ogrizumab is a humanized monoclonal antibody targeting CD20, and is also believed to target B cells. It effectively eliminates a subset of highly activated CD20+ T cells that produce inflammatory cytokines, and the absence of CD20+ inflammatory T cells may be the reason for the therapeutic effect of oligrinumab. However, this monoclonal antibody can cause immunodeficiency side effects, such as ulcerative colitis and leukoencephalopathy. Although drugs targeting Th cells are more effective, they often suffer from poor drug resistance, significant relapse, and numerous side effects due to the lack of targeted regulation and channeling of abnormally activated cells. Therefore, there is an urgent need to develop therapeutic approaches that target key regulatory targets of abnormally activated inflammatory Th cells.

[0005] Triggering receptors expressed on myeloid cells (TREMs), as pattern recognition receptors, have received widespread attention in the treatment of tumors and infections in recent years. These molecules are widely expressed in myeloid cells, such as macrophages, monocytes, and dendritic cells, in previous studies. Representative examples include TREM-1 and TREM-2. Both molecules can bind to DAP12 to perform their respective functions. TREM-1 amplifies the TLR signaling pathway and promotes host inflammation, while TREM-2 inhibits bacterial or fungal infections and the secretion of pro-inflammatory cytokines induced by them. In neurodegenerative diseases, TREM-2 can also promote microglial activation, playing a crucial role in inflammation regulation. Various molecules have been reported to bind to TREM-2, including certain cell surface molecules, bacterial or heat shock proteins, as well as lipids exposed during axonal injury and nucleic acids released by dying cells. Although substantial evidence suggests that TREM-2 has anti-inflammatory effects in innate immune cells, the regulatory role of TREM-2 in infectious and inflammatory diseases remains controversial in in vivo studies. On the one hand, TREM-2 fine-tuned the inflammatory response in a Gram-negative bacteria-induced sepsis mouse model. In a chemically induced liver injury model, TREM-2-deficient mice exhibited exacerbated liver damage and inflammation. On the other hand, TREM-2 deficiency reduced the inflammatory response and limited organ damage and death induced by Burkholderia pseudovilli infection.

[0006] The mechanism of action of TREM-2 on CD4+ T cells is not yet fully understood. Previous studies have shown that TREM-2 can promote Th1 cell responses through its unique CD3ζ / ZAP70 pathway, suggesting that TREM-2 may serve as a potential regulatory target for the transformation of T cells into pro-inflammatory Th cells.

[0007] However, current research on the expression changes and functional activities of TREM-2 in multiple sclerosis (MS) is scarce, especially regarding the function of TREM-2 in disease-specific pathogenic T lymphocytes, for which there are no reported studies. Currently, immunotherapy for MS has significant limitations and ineffectiveness. To improve early diagnosis and drug treatment outcomes for MS, and to improve patient survival, there is an urgent need to find new immunoregulatory molecules to enhance the diagnostic rate and efficacy of immunotherapy for MS. Summary of the Invention

[0008] In view of the current diagnosis and treatment methods for multiple sclerosis, which have low early diagnosis rates and poor long-term treatment effects, this invention provides the application of myeloid cell triggering receptor-2 (TREM-2) expression in the preparation of therapeutic drugs and / or diagnostic reagents for multiple sclerosis.

[0009] The present invention achieves the above objectives through the following technical solutions:

[0010] This invention provides the application of TREM-2 as a target in the preparation of diagnostic and / or prognostic reagents for multiple sclerosis.

[0011] Triggering receptor 2 (TREM2) expressed on myeloid cells has been widely expressed in myeloid cells, such as macrophages, monocytes, and dendritic cells, in previous studies. It has received considerable attention in the fields of cancer and infection treatment in recent years.

[0012] This study found that, using flow cytometry, TREM-2 expression in peripheral blood CD4+ lymphocytes of multiple sclerosis patients was significantly higher than in healthy controls. Further analysis using real-time quantitative PCR revealed that the expression of pro-inflammatory factors IFN-γ, IL-17, TNF-α, and GM-CSF all increased in tandem with TREM-2 expression. This suggests that TREM-2 is closely related to disease progression and immune status. Therefore, TREM-2 could serve as a biomarker for the diagnosis and prognosis of multiple sclerosis.

[0013] Preferably, TREM-2 is expressed on the surface of CD4+ T lymphocytes.

[0014] Preferably, the reagent is used to detect the expression level of TERM-2 on the surface of CD4+ T lymphocytes.

[0015] Preferably, the reagent is an antibody that can specifically bind to TREM-2.

[0016] Preferably, the antibody can be a monoclonal antibody carrying TREM-2 as a detection marker.

[0017] More preferably, the antibody is derived from CD4+ T cells.

[0018] The present invention also provides the above-mentioned diagnostic and / or prognostic reagents for multiple sclerosis prepared with TREM-2 as the target.

[0019] The expression abundance of TREM-2 in CD4+ T cells of patients with multiple sclerosis is significantly higher than that in healthy individuals. Furthermore, the disease progression is closely related to the body's immune status. Therefore, detecting TREM-2 expression in CD4+ T cells has high diagnostic accuracy and sensitivity. Multiple sclerosis can be diagnosed and its progression predicted by detecting the expression level of TREM-2 in peripheral blood CD4+ T cells.

[0020] Specifically, when used as a diagnostic reagent, the diagnostic process includes: detecting the expression level of TREM-2 in CD4+ T cells of a subject using an antibody that specifically binds to TREM-2, and comparing it with a reference level. When the expression level of TREM-2 is significantly increased, the diagnostic result is positive; when the expression level of TREM-2 does not change significantly, the diagnostic result is negative. The reference level is the expression level of TREM-2 in peripheral blood samples from healthy individuals.

[0021] Specifically, when used as a prognostic reagent, the prognostic process includes: using antibodies that specifically bind to TREM-2 to detect the expression level of TREM-2 in blood samples (or biopsy tissue samples) of the subject at different stages of the disease, comparing it with a reference level, plotting the expression level curve of TREM-2 at different stages of the disease, thereby determining the dynamic progression of the patient and guiding medication.

[0022] This invention also provides the use of TREM-2 antibody in the preparation of diagnostic and / or prognostic reagents for multiple sclerosis.

[0023] Preferably, the antibody may be conjugated to or have a detection marker.

[0024] This invention has found that using MOG 35-55 Under protein stimulation, TREM-2 promotes the secretion of cytokines, including interleukin-6 (IL-6) and interleukin-17 (IL-17), by CD4+ cells. Further knockout of TREM-2 reduced the differentiation of CD4+ T cells and the levels of these two cytokines secreted.

[0025] Therefore, by intervening in the expression of trigger receptor 2 targeting myeloid cells in vivo, the excessive inflammatory response in patients with multiple sclerosis can be reduced, which can then be used to treat multiple sclerosis.

[0026] Therefore, another objective of this invention is to provide the application of TREM-2 as a target in the preparation of functional products for the treatment of multiple sclerosis, wherein the functional products have the effect of inhibiting the progression of multiple sclerosis.

[0027] Preferably, the functional product has the function of downregulating the expression, transcription, or expression product of the TREM-2 gene.

[0028] Preferably, the functional product includes one or more of the following: TREM-2 protein inhibitors, TREM-2 gene-deficient or silenced immune-related cells, their differentiated cells, or gene recombinant constructs.

[0029] More preferably, the functional product includes:

[0030] (i) Activating antibodies, nucleotides, lentiviruses or adenoviruses that target the TREM-2 transcript and can inhibit the expression of the TREM-2 gene expression product or gene transcription;

[0031] (ii) Contains a TREM-2 competing sequence and is able to form a repressor molecule that inhibits the expression of TREM-2 gene expression products or gene transcription after being transfected into the body;

[0032] (iii) Immune-associated cells, their differentiated cells, or constructs after the TREM-2 gene sequence is suppressed.

[0033] Therefore, the functional products may include siRNA, fusion protein, specific antibody, and antisense RNA targeting TREM-2.

[0034] Compared with the prior art, the beneficial effects of the present invention are:

[0035] This invention reveals that TREM-2 is highly expressed in peripheral blood CD4+ T cells of patients with multiple sclerosis (MS) and is correlated with disease severity. This indicates that TREM-2 can serve as a diagnostic and prognostic biomarker for MS. This biomarker can be derived from peripheral blood, and direct blood sample testing reduces patient discomfort compared to complex procedures involving multiple indicators such as biochemistry, coagulation, and blood saturation. Furthermore, it is less invasive and highly sensitive.

[0036] This invention also investigated the intervention of TREM-2 knockout in mouse CD4+ T cells, and found that TREM-2 knockout significantly reduced the levels of cytokines IL-6 and IL-17, suggesting that intervention in TREM-2 expression can alleviate or even potentially treat multiple sclerosis.

[0037] The molecular markers for the diagnosis and prognosis of multiple sclerosis provided by this invention have the advantages of being simple, rapid, minimally invasive, and easy to repeat, and have broad application prospects. Attached Figure Description

[0038] Figure 1 The graph shows the expression level of TREM-2 on the surface of peripheral blood CD4+ T cells in healthy individuals and patients with multiple sclerosis. *** indicates P < 0.001.

[0039] Figure 2 Clinical scores and clinical manifestations of the disease model induced by TREM-2 knockout are shown in the figure. Figure 2 A represents the CFA control group, WT-EAE group, and CD4 group. cre TREM-2 flox Clinical scores of mice in the EAE group; Figure 2 B is a diagram and statistical chart showing the detection of blood-brain barrier damage in mice using the Evans blue assay. Figure 2 C is for WT-EAE and CD4 cre TREM-2 flox -Inflammatory infiltration in the brain of EAE mice; Figure 2 D represents the infiltration of Th cells in the brain and spinal cord; where * indicates P < 0.05; ** indicates P < 0.01; *** indicates P < 0.001.

[0040] Figure 3 Correlation analysis of TREM-2 proportion with IL-6 and IL-17, where, Figure 3 A represents the concentration of IL-17 in the peripheral blood of CFA and EAE mice; Figure 3 B shows the expression of IL-6 in the spleen of CFA and EAE mice; Figure 3 C represents the concentration of IL-17 and TREM-2. + CD4 + Correlation of T cell ratio; Figure 3 D represents IL-6 expression and TREM-2 + CD4 + The correlation of T cell proportion; where *** indicates P < 0.001.

[0041] Figure 4 This is a scale diagram of Th1, Th2, and Th17 after TREM-2 knockout, where... Figure 4 A represents the proportion of Th1; Figure 4 B represents the proportion of Th2; Figure 4 C represents the proportion of Th17; where ns indicates statistical difference (P>0.05); * indicates P<0.05; ** indicates P<0.01.

[0042] Figure 5 For MOG 35-55 TREM-2 in EAE model mice under protein stimulation conditions - / - -CD4 + T cells and WT-CD4 + A diagram showing the levels of IL-6 and IL-17 secreted by T cells; among them... Figure 5 A is a statistical graph of IL-17 concentration in the culture medium; Figure 5B is a statistical graph of IL-6 concentration in the culture medium; where ** indicates P < 0.01. Detailed Implementation

[0043] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0044] Unless otherwise specified, the experimental methods used in the embodiments of this invention are conventional methods; unless otherwise specified, the materials and reagents used are commercially available.

[0045] Example 1: Analysis of expression levels on the surface of CD4+ T cells in peripheral blood of patients with multiple sclerosis

[0046] (1) Collection of test samples:

[0047] Blood samples from the experimental group: patients with active, relapsing-remitting multiple sclerosis.

[0048] Blood samples from control group: Asymptomatic subjects undergoing physical examinations

[0049] (2) Detection methods and procedures:

[0050] a. Collect 5 mL of peripheral blood from the experimental group and the control group, centrifuge at 3000 rpm for 5 min, and aspirate the supernatant serum;

[0051] b. Isolate lymphocytes using lymphocyte separation medium (TMB);

[0052] c. Lyse red blood cells to prepare a single-cell suspension, and adjust the cell count to 1×10⁻⁶. 8 50 μL of the sample was collected and added to TREM-2 antibody (R&D Company, catalog number FAB1278P) and CD4 antibody (Biolegend).

[0053] e. Mix well and incubate at 4 degrees Celsius in the dark for 30 minutes. Wash twice with PBS and then detect using flow cytometry.

[0054] f. Analysis of the proportion of TREM-2 positive cells to CD4 positive cells under FSC and SSC gating.

[0055] Results analysis:

[0056] like Figure 1As shown, under FSC and SSC gating, TREM-2 was expressed in peripheral blood CD4 cells of both multiple sclerosis patients and healthy controls, but the expression level in the patient group was significantly higher than that in the healthy control group, and the difference was statistically significant. This indicates that TREM-2 is highly expressed in peripheral blood CD4 cells of multiple sclerosis patients.

[0057] Example 2: Clinical scoring and performance analysis of the disease model induced by TREM-2 knockout

[0058] (1) Sample collection:

[0059] The brains and spinal cords of the mice used for modeling were selected (6 mice in each group were observed for clinical scoring; 3 mice were dissected).

[0060] (2) Detection methods and procedures:

[0061] a.Use MOG 35-5 5. Construction of an animal model of induced multiple sclerosis—Experimental autoimmune encephalomyelitis (EAE). The subjects were C57BL / 6WT or TREM-2f / f-CD4-Cre mice;

[0062] b. Observe and record the mouse status according to the EAE clinical scoring criteria;

[0063] c. After the clinical score of the WT-EAE group mice reached 3 points, 3 mice from each group were selected and injected with Evans Blue (EB) via the tail vein. The mice were then euthanized by exsanguination shock 6-8 hours after injection.

[0064] d. Dissection of the central nervous system (cerebrum, cerebellum, brainstem, and spinal cord) after cardiac perfusion;

[0065] e. Soak in formamide solution and collect the supernatant;

[0066] f. Set up the EB standard curve and calculate the total EB content in lung tissue using a 620nm microplate reader;

[0067] e. Half of the brain was paraffin-embedded and stained with hematoxylin and eosin (HE), and the other half was ground up;

[0068] g. The ground suspension was centrifuged using a Percoll gradient centrifugation method to obtain immune cells;

[0069] h. cells were labeled with CD45 and detected by flow cytometry.

[0070] Results analysis:

[0071] like Figure 2As shown, TREM-2 knockout mice on CD4 cells exhibited significantly decreased clinical scores, and reduced levels of EB, inflammatory infiltration, and CD4+ in the central nervous system. + The T cell infiltration was reduced. This indicates that knocking out TREM-2 on CD4 cells inhibits the induction of EAE, mainly manifested as a reduction in clinical symptoms and a decrease in the degree of blood-brain barrier damage.

[0072] Example 3 TREM-2 + CD4 + Association analysis of T cell proportion with IL-6 and IL-17

[0073] (1) Sample collection:

[0074] Building MOG 35-55 -EAE model, to obtain brain tissue.

[0075] (2) Detection methods and procedures:

[0076] a.Use MOG 35-55 An EAE model was induced. The subjects were C57BL / 6WT mice. A negative control model, CFA, was also established.

[0077] b. Once the clinical score of the WT-EAE group mice reached 3 points, they were euthanized by exsanguination shock.

[0078] c. Remove the spleen and grind it;

[0079] d. Prepare as a single-cell suspension;

[0080] e. Cells were labeled with CD45, CD4, and TREM-2 and analyzed by flow cytometry;

[0081] f. Centrifuge blood to obtain plasma;

[0082] g. ELISA method for detecting IL-6 and IL-17 concentrations.

[0083] Results analysis:

[0084] like Figure 3 As shown, compared with the CFA control group, EAE mice had higher concentrations of IL-6 and IL-17 in their blood. TREM-2 in the spleen of EAE mice was also observed. + The proportion of CD4 cells was positively correlated. This indicates that the expression of TREM-2 on CD4 cells is related to the Th17-related pro-inflammatory factors IL-6 and IL-17.

[0085] Example 4: Analysis of Th1, Th2, and Th17 after TREM-2 knockout

[0086] (1) Sample collection:

[0087] Building MOG 35-55 -EAE model, to obtain spleen tissue.

[0088] (2) Detection methods and procedures:

[0089] a.Use MOG 35-55 An EAE model was induced. The subjects were C57BL / 6WT or TREM-2f / f-CD4-Cre mice. A negative control model, CFA, was also established.

[0090] b. Once the clinical score of the WT-EAE group mice reached 3 points, they were euthanized by exsanguination shock.

[0091] c. Dissection of the spleen;

[0092] d. Grind the broken red blood cells, filter and centrifuge to obtain a single-cell suspension;

[0093] e. Take 1×10 6 Cells were labeled with CD4, IL-17, IL-4 and IFN-γ and then analyzed by flow cytometry.

[0094] Results analysis:

[0095] like Figure 4 As shown, after constructing the EAE model, pro-inflammatory cells such as Th1 and Th17 cells significantly increased, while anti-inflammatory cells of Th2 cells decreased accordingly. However, in CD4 cell TREM-2 knockout mice, Th17 cell counts decreased after inducing the EAE model. This indicates that knocking out TREM-2 on CD4 cells inhibits the increase of pro-inflammatory CD4+ T cells—Th17 cells.

[0096] Example 5: After TREM-2 knockout, MOG 35-55 Analysis of IL-6 and IL-17 expression levels secreted by CD4+ T cells under protein stimulation conditions

[0097] (1) Sample collection:

[0098] Spleen tissue was collected from experimental mice, including 6 control mice, 6 WT-EAE modeling mice, and 6 WT-EAE modeling mice.

[0099] (2) The detection method includes the following steps:

[0100] a.Use MOG 35-55 An EAE model was induced. The subjects were C57BL / 6WT or TREM-2f / f-CD4-Cre mice. A negative control model, CFA, was also established.

[0101] b. Once the clinical score of the WT-EAE group mice reached 3 points, they were euthanized by exsanguination shock.

[0102] c. Dissection of the spleen;

[0103] d. Grind the broken red blood cells, filter and centrifuge to obtain a single-cell suspension;

[0104] e. Take 1×10 6 Each cell is 1×10 6 Incubation was performed at a concentration of [number] cells / mL. MOG was added during this period. 35-55 Provide specific stimulation;

[0105] f. After 48 hours, the supernatant was collected for ELISA to detect the expression of IL-6 and IL-17.

[0106] Results analysis:

[0107] like Figure 5 As shown, MOG 35-55 Under specific stimulation, spleen cells secrete more IL-6 and IL-17. However, the secretion levels of cells from mice with TREM-2 knockout on CD4 were significantly reduced. This indicates that TREM-2 knockout can effectively inhibit the expression of pro-inflammatory factors IL-6 and IL-17.

Claims

1. The application of TREM-2 in the preparation of functional products for treating multiple sclerosis, characterized in that, The functional product can inhibit the progression of multiple sclerosis; the functional product has the function of downregulating the expression, transcription, or expression product of the TREM-2 gene.

2. The application according to claim 1, characterized in that, The functional products include one or more of the following: TREM-2 protein inhibitors, TREM-2 gene-deficient or silenced immune-related cells, their differentiated cells, or gene recombinant constructs.

3. The application according to claim 1, characterized in that, The functional products include: (i) Activating antibodies, nucleotides, lentiviruses or adenoviruses that target the TREM-2 transcript and are able to inhibit the expression of the TREM-2 gene expression product or gene transcription; (ii) Constructs containing TREM-2 competing sequences and capable of forming repressor molecules that inhibit the expression of TREM-2 gene expression products or gene transcription after being transfected into the body; (iii) Immune-associated cells, their differentiated cells or constructs after the TREM-2 gene sequence is suppressed.

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