TREM2-targeted chimeric antigen receptor and application of TREM2-targeted chimeric antigen receptor in preparation of medicine for treating atherosclerosis

Abstract

The invention belongs to the field of biological medicine, and provides a TREM2-targeted chimeric antigen receptor and application thereof in preparation of medicine for treating atherosclerosis, and the chimeric antigen receptor comprises a single-chain variable region fragment specifically binding to TREM2, a hinge region, a transmembrane region, a costimulatory signal structural domain and a signal structural domain; wherein the single-chain variable region fragment is derived from an anti-TREM2 monoclonal antibody clone ab52. The invention further provides a novel therapy capable of precisely targeting and removing core pathogenic cells driving disease progression in atheromatous plaques. The TREM2 targeting chimeric antigen receptor can specifically target TREM2 and efficiently remove pathogenic macrophages in plaques, has good in-vivo safety and provides a new choice for treatment of atherosclerosis.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a chimeric antigen receptor targeting TREM2 and its application in the preparation of drugs for treating atherosclerosis. Background Technology

[0002] Atherosclerosis (AS) is a chronic inflammatory vascular disease and one of the leading causes of cardiovascular disease morbidity and mortality worldwide. Its pathological features include the accumulation of lipids, immune cells (especially macrophages), and necrotic debris in the arterial wall, forming atherosclerotic plaques. Macrophages within the plaque take up modified lipoproteins and transform into foam cells, becoming key factors driving inflammatory responses, expanding the necrotic core, and leading to plaque instability. Once an unstable plaque ruptures, it can trigger acute thrombotic events such as myocardial infarction and ischemic stroke, seriously threatening human health.

[0003] Currently, first-line clinical treatments primarily focus on intensive lipid-lowering therapies, such as statins and PCSK9 inhibitors. These therapies reduce the incidence of cardiovascular events to some extent by lowering circulating low-density lipoprotein cholesterol (LDL-C) levels. However, existing therapies have significant limitations: they often struggle to effectively reverse existing atherosclerotic plaques, lack sufficient ability to regulate the persistent malignant inflammatory microenvironment within the plaques, and cannot precisely eliminate pathogenic immune cell populations that drive disease progression. Therefore, there is an urgent need in this field for an innovative therapy that can directly target the inflammatory core of atherosclerosis and fundamentally alter the disease course.

[0004] In recent years, chimeric antigen receptor T cell (CAR-T) therapy has shown significant clinical efficacy in the field of cancer treatment. This technology involves genetically engineering a patient's own T cells to express chimeric receptors that specifically recognize tumor cell surface antigens, thereby achieving precise and efficient elimination of cancer cells. The success of this technology has prompted researchers to explore its application to non-tumor diseases, particularly chronic inflammatory diseases driven by specific pathological cell populations. Applying the CAR-T technology platform to the treatment of atherosclerosis could theoretically achieve precise intervention at the root cause of the disease by designing CAR-T cells that can specifically recognize and eliminate pathogenic macrophages within plaques. However, turning this idea into reality faces a core challenge: finding and validating an ideal target antigen. This antigen must be stably and highly expressed on diseased cells (such as foam cells / macrophages within plaques) while exhibiting low or absent expression levels in healthy vital organs and tissues, thereby minimizing off-target toxicity and ensuring treatment safety.

[0005] Triggering receptor expressed on myeloid cells 2 (TREM2) has become a promising target in atherosclerosis research in recent years. Multiple studies have shown that TREM2 is specifically highly expressed on macrophages / foam cells in atherosclerotic plaques in humans and mice, particularly enriched in a pathogenic subset known as "lipid-associated macrophages," while its expression level is very low in most normal tissues. TREM2 regulates lipid metabolism, cytotoxicity, and survival of macrophages in the plaque microenvironment, and its high expression is closely related to disease severity. This "disease-enriched, normal tissue-scarce" expression pattern makes TREM2 a highly attractive target for precision immunotherapy.

[0006] Although the potential of TREM2 as a therapeutic target has been preliminarily recognized, no related technologies or products utilizing TREM2-targeting CAR-T cells to treat atherosclerosis have been disclosed or launched to date. Current technologies lack CAR molecular design targeting this target, in vitro and in vivo functional validation, and systemic safety assessment protocols. Therefore, developing a CAR-T cell therapy that can specifically target TREM2, efficiently eliminate pathogenic macrophages within plaques, and possess good in vivo safety is of great significance for filling the gaps in current atherosclerosis treatment and advancing the development of immunotherapy for cardiovascular diseases. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention aims to provide a chimeric antigen receptor targeting TREM2 and its application in the preparation of drugs for treating atherosclerosis. This invention is the first to propose and validate myeloid cell trigger receptor 2 as an ideal target for CAR-T cell therapy in atherosclerosis. TREM2 is specifically highly expressed on lipid-associated macrophages / foam cells within atherosclerotic plaques, while its expression level is very low or undetectable in most healthy tissues. This "lesion-enriched, normal tissue-scarce" expression pattern is the biological basis for ensuring treatment specificity and avoiding off-target toxicity. The TREM2-CAR-T cells in this invention can specifically home to and accumulate in aortic atherosclerotic lesions, co-localizing with macrophages within the lesion, achieving precise local targeting. Treatment with the aforementioned TREM2-CAR-T cells not only significantly reduces plaque area and necrotic core but, more importantly, alters plaque composition, increases collagen content, and induces plaque stabilization.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] In a first aspect, the present invention provides a chimeric antigen receptor targeting TREM2, the chimeric antigen receptor comprising: a single-chain variable region fragment specifically binding to TREM2, a hinge region, a transmembrane region, a co-stimulatory signaling domain, and a signaling domain; wherein the single-chain variable region fragment is derived from an anti-TREM2 monoclonal antibody clone ab52.

[0010] Preferably, the hinge area is a CD28 hinge area.

[0011] Preferably, the transmembrane region and co-stimulatory signal domain are CD28 transmembrane and co-stimulatory domains.

[0012] Preferably, the signal structure domain is the CD3ζ signal structure domain.

[0013] Preferably, the amino acid sequence of the single-chain variable region fragment is shown in SEQ ID NO:2.

[0014] Preferably, the amino acid sequences of the hinge region, transmembrane region, and co-stimulatory signaling domain are as shown in SEQ ID NO:3.

[0015] Preferably, the amino acid sequence of the signal domain is shown in SEQ ID NO:4.

[0016] Optimized CAR Molecular Structure Design: This invention designs and constructs a specific CAR molecular structure targeting TREM2. Its key components include: Antigen Recognition Domain: Employing a single-chain variable region fragment derived from the anti-TREM2 monoclonal antibody clone ab52. This scFv has been shown to specifically recognize and bind to both human and murine TREM2 proteins simultaneously, ensuring the reliability of preclinical studies and its potential versatility for clinical translation. Signal Transduction Domain: Utilizing a combination of a CD28 co-stimulatory domain and a CD3ζ signal transduction domain. This combination aims to provide sufficient activation signals to T cells, not only eliciting potent immediate cytotoxicity but also potentially promoting T cell survival, proliferation, and memory formation in vivo, thereby potentially achieving long-term therapeutic effects with a single infusion.

[0017] Adaptability of treatment strategies to disease models: This invention is based on the mature ApoE... - / - In a mouse model of atherosclerosis, the efficacy of CAR-T cells at different disease stages (preventive and therapeutic interventions) was systematically evaluated. Through intravenous infusion, TREM2.CAR-T cells specifically homed and accumulated in atherosclerotic lesions of the aorta, co-localizing with macrophages within the lesions, achieving localized and specific clearance of the lesions. Treatment not only significantly reduced plaque area and necrotic core, but more importantly, altered plaque composition, increased collagen content, and induced plaque stabilization.

[0018] In-depth analysis of the mechanism of action and verification of safety: This invention goes beyond a simple cell clearance effect, utilizing technologies such as single-cell RNA sequencing to elucidate the multidimensional therapeutic mechanism of TREM2 CAR-T cells: specifically clearing inflammatory TREM2 cells. + Macrophage subsets (especially Slamf9) + (Subgroups); reduce chemotactic recruitment of monocytes; promote the infiltration and activation of immune cells with tissue protection functions (such as ILC2), thereby reshaping the immune microenvironment of the lesion and promoting inflammation resolution. Meanwhile, no significant systemic toxicity, weight changes, or major organ damage were detected during the observation period of several weeks, preliminarily validating the safety window of this strategy.

[0019] In a second aspect, the present invention provides a nucleic acid molecule that encodes the chimeric antigen receptor targeting TREM2 described in the first aspect.

[0020] Thirdly, the present invention provides a vector comprising at least one copy of the nucleic acid molecule described in the second aspect.

[0021] Preferably, the vector includes a cloning vector or an expression vector.

[0022] Preferably, the expression vector includes a viral expression vector, which includes a retroviral vector, an adenovirus vector, an adeno-associated virus vector, or a lentiviral vector.

[0023] Fourthly, the present invention provides a recombinant virus, which is prepared by a method comprising the following steps: co-transfecting packaging cells with the vector described in the third aspect and a packaging helper plasmid to obtain the recombinant virus.

[0024] Fifthly, the present invention provides an immune effector cell that expresses the chimeric antigen receptor targeting TREM2 described in the first aspect.

[0025] Preferably, the immune effector cells contain the nucleic acid molecule described in the second aspect, or the vector described in the third aspect, or the recombinant virus described in the fourth aspect.

[0026] Preferably, the immune effector cells are T cells or NK cells, with T cells being the most preferred.

[0027] In a sixth aspect, the present invention provides a pharmaceutical composition comprising the immune effector cells described in the fifth aspect.

[0028] In a seventh aspect, the present invention provides the use of any one or a combination of at least two of the following: the chimeric antigen receptor targeting TREM2 described in the first aspect, the nucleic acid molecule described in the second aspect, the carrier described in the third aspect, the immune effector cells described in the fifth aspect, or the pharmaceutical composition described in the sixth aspect, in the preparation of a medicament for the prevention and / or treatment of atherosclerosis and related cardiovascular diseases.

[0029] The present invention also provides a method for treating atherosclerosis, comprising administering to a subject in need a therapeutically effective amount of immune effector cells expressing the chimeric antigen receptor targeting TREM2 as described in the first aspect.

[0030] The present invention also provides the use of the TREM2 target for screening or developing cell therapy products for the treatment of atherosclerosis.

[0031] Eighthly, the present invention provides the use of TREM2 as a target in the preparation of a drug for treating atherosclerosis, wherein the drug specifically eliminates an inflammatory macrophage subset that highly expresses TREM2, and the inflammatory macrophage subset also highly expresses Slamf9.

[0032] Preferably, the drug reduces the chemotactic recruitment of monocytes.

[0033] Preferably, the drug promotes the infiltration and activation of immune cells with tissue-protective functions.

[0034] Preferably, the drug is a TREM2-targeting CAR-T cell.

[0035] In this invention, the TREM2-specific CAR-T cells, when used for treatment, selectively eliminate inflammatory macrophage subsets within plaques that highly express both TREM2 and Slamf9, while relatively preserving other macrophage subsets with lower TREM2 expression. After intravenous infusion, these CAR-T cells exhibit specific homing to atherosclerotic lesions, leading to increased plaque collagen deposition and reduced necrotic core.

[0036] The innovation and specificity of the target selection in this invention: This invention is the first to propose and validate myeloid trigger receptor 2 (TREM2) as an ideal target for CAR-T cell therapy in atherosclerosis. TREM2 is specifically highly expressed on lipid-associated macrophages / foam cells within atherosclerotic plaques, while its expression level is very low or undetectable in most healthy tissues. This "lesion-enriched, normal tissue-scarce" expression pattern is the biological basis for ensuring treatment specificity and avoiding off-target toxicity. This invention systematically confirms the feasibility of TREM2 as a therapeutic target through single-cell sequencing and histochemical analysis of clinical samples and mouse models.

[0037] Compared with the prior art, the present invention has the following beneficial effects:

[0038] This invention overcomes the limitations of existing therapies, such as their inability to precisely eliminate pathogenic cells, and solves the safety challenge of target selection when applying CAR-T technology to this disease. Specifically, while existing lipid-lowering therapies can regulate systemic lipid levels, they cannot precisely target and eliminate the core pathogenic cells driving disease progression within atherosclerotic plaques—namely, lipid-associated macrophages (LAMs) with high TREM2 expression. Furthermore, they are also unable to effectively reverse existing plaques or inhibit the malignant inflammatory microenvironment within them. Therefore, the primary task of this invention is to provide a novel therapy capable of actively identifying and efficiently eliminating these pathogenic cells. In addition, extending CAR-T technology to atherosclerosis, a non-tumor disease, faces the core challenge of finding safe and efficient targets. This invention also verifies the feasibility of TREM2 as a target in specific embodiments, ensuring that the CAR-T cells developed thereby can specifically act on the lesion site while avoiding off-target toxicity to vital organs such as the liver and brain, which have low TREM2 expression, thus effectively solving the key safety issues faced in the translation of this technology. Furthermore, the method developed in this invention not only achieves the fundamental goal of reducing plaque area but also fundamentally improves the composition and properties of plaques. By shrinking the necrotic core, increasing collagen deposition, and reducing matrix metalloproteinase expression, it induces plaques to transform from vulnerable to stable, thereby reducing their rupture risk. Simultaneously, this invention addresses the problem of how systemically infused therapeutic cells can accurately home to and accumulate in atherosclerotic lesions, co-localize with target cells, and effectively perform their functions, rather than being meaninglessly distributed or cleared in non-target organs. Ultimately, this invention provides an innovative treatment regimen that may require only a single infusion to produce long-term, durable therapeutic effects with manageable systemic side effects, thus offering a novel and groundbreaking immunotherapy strategy for the clinical management of atherosclerosis. Attached Figure Description

[0039] Figure 1 This is a visualization of UMAP based on single-cell RNA sequencing data from human coronary artery samples.

[0040] Figure 2 Violin diagrams showing the expression of lipid-related genes APOE, FABP5, and GPNMB in various macrophage clusters.

[0041] Figure 3 Stacked bar chart showing the proportions of different macrophage clusters in patients with mild and severe atherosclerosis.

[0042] Figure 4Violin plot showing the expression distribution of TREM2 and GPNMB in each cluster of mild and severe patients.

[0043] Figure 5 Multiple staining results of coronary artery tissue from patients with severe atherosclerosis.

[0044] Figure 6 The results are from flow cytometry analysis of TREM2 expression on the surface of target cells.

[0045] Figure 7 The activation status of TREM2.CAR-T cells after co-culturing with different target cells.

[0046] Figure 8 The results of quantitative analysis of the activation and proliferation functions of TREM2.CAR-T cells.

[0047] Figure 9 This is a schematic diagram of the experimental timeline.

[0048] Figure 10 This study aimed to quantify the average fluorescence intensity of the aorta in HFD-fed mice at week 8 using in vitro fluorescence imaging (pseudo-color, with red representing high signal) and the mean DiR fluorescence intensity in the aortic region.

[0049] Figure 11 Immunofluorescence staining (scale bar: 100 μm) of frozen sections of the aortic root and quantitative statistics.

[0050] Figure 12 For in vitro fluorescence imaging of major organs (heart, liver, spleen, lung, kidney) and quantification of the average DiR fluorescence intensity of each organ (vertical axis: ×10) 6 Photons per second; Horizontal axis: organ).

[0051] Figure 13 This is a schematic diagram of the experimental timeline.

[0052] Figure 14 To assess lipid deposition and quantitatively analyze the percentage of total aortic plaque area using Oil Red O (ORO) staining of the entire aorta.

[0053] Figure 15 The results show H&E staining of the aortic root (top row: 100 μm scale bar; bottom row: 20 μm magnification) and quantitative analysis of the proportion of necrotic core area.

[0054] Figure 16 Masson trichrome staining of the aortic root (top row: 100 μm scale bar; bottom row: 20 μm magnification) and quantitative analysis of collagen area ratio.

[0055] Figure 17 Immunohistochemical staining of CD68 in the aortic root (top row: scale bar 100 μm; bottom row: 20 μm) and CD68+ Cell density quantification (vertical axis: number of cells per high-power field; horizontal axis: group).

[0056] Figure 18 Immunofluorescence staining of CD206 (M2 marker) in the aortic root (scale bar as above) and CD206 + Cells account for CD68 + Cell percentage quantification (vertical axis: percentage %).

[0057] Figure 19 TREM2 immunofluorescence staining of the aortic root (scale bar as above) and TREM2 + Cells account for CD68 + Quantitative analysis of cell proportions.

[0058] Figure 20 Immunofluorescence staining of iNOS (M1 marker) in the aortic root (scale bar as above) and iNOS + Cells account for CD68 + Cell percentage quantification (vertical axis: percentage %). Detailed Implementation

[0059] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention in any way.

[0060] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0061] Example 1

[0062] This embodiment proposes and verifies that myeloid cell trigger receptor 2 can be used as an ideal target for CAR-T cell therapy for atherosclerosis, and constructs CAR-T cells targeting TREM2 based on this target.

[0063] 1. Histochemical analysis of human vascular samples.

[0064] Human coronary artery samples were obtained from patients who underwent heart transplantation. All collected human coronary artery samples were cardiac-protected using in situ cardiac anesthesia. Transthoracic echocardiography was performed prior to tissue collection for real-time assessment of in vivo structure and function. The clinical study was conducted in accordance with the principles of the Declaration of Helsinki and was approved by the Ethics Committee of Fuwai Hospital. All participants provided written informed consent before participating in the study. Tissue specimens were fixed overnight in 4% (v / v) formalin, embedded in paraffin, and cut into 5 µm thick sections. Sections were subjected to hematologic and epithelial processing (H&E), immunohistochemistry, and immunohistochemical fluorescence staining for histopathological evaluation. H&E and immunohistochemical images were examined using a digital microscope. Fluorescence images were observed and quantified under a stereomicroscope (Leica DMi8, Leica Microsystems, Germany).

[0065] 2. Construction and sample analysis of a mouse model of atherosclerosis.

[0066] The Animal Ethics and Welfare Committee of Fuwai Hospital, Shenzhen, Chinese Academy of Medical Sciences, approved all animal experiments involved in this invention. To validate the expression pattern of TREM2 (same as Trem2) at the in vivo level and evaluate the therapeutic potential of TREM2.CAR-T cells, we established a standardized mouse model of atherosclerosis and performed a systematic analysis.

[0067] Specifically, 8-week-old male ApoE were selected. - / - Mice (C57BL / 6J background) were randomly divided into two groups after a one-week acclimatization period: the experimental group was fed a Western-type high-fat diet (HFD, 45% fat for energy, 0.15% cholesterol) to induce atherosclerosis; the control group was fed a standard diet (CD). All mice were housed under specific pathogen conditions, and their body weight and food intake were monitored regularly. To simulate different stages of disease progression and assess the impact of intervention timing, we administered a single infusion of 1×10⁻⁶ mg / L via the tail vein at two key time points: week 4 (corresponding to early plaque formation) and week 8 (corresponding to late / established plaque stage) after the start of the high-fat diet. 6TREM2 CAR-T cells (dissolved in 200 μL sterile PBS) were collected. Control groups were established, including a high-fat diet + PBS injection, a high-fat diet + untransduced T cell injection, and a normal diet with no treatment. Sample size was pre-determined through power analysis to ensure statistical power of the results. At the pre-specified endpoint (week 13 of high-fat feeding), mice were euthanized by CO2 asphyxiation, followed by cardiac puncture and complete dissection of the aorta and major organs. A portion of the aortic tissue was immediately used for whole-aortic Oil Red O staining to assess plaque burden, while another portion was fixed in formalin, paraffin-embedded, and frozen sectioned for subsequent H&E staining, Masson's trichrome staining, and immunohistochemical / immunofluorescence analysis targeting markers such as CD68, TREM2, iNOS, and CD206. All histological assessments were performed by researchers unaware of the experimental groups to ensure data objectivity.

[0068] After euthanizing mice, the aorta (from the origin of the heart to the bifurcation of the iliac artery) was dissected and placed in PBS buffer at 4°C. Adhesive and connective tissue attached to the vessel wall were removed under a dissecting microscope. The aortic tissue was transferred to an enzymatic digestion solution containing collagenase IV (1.5 mg / mL), dispersin (1.5 mg / mL), and DNase I (20 U / mL), and digested with shaking at 37°C for 45 minutes. The mixture was agitated every 15 minutes during digestion. After digestion, the cells were filtered sequentially through 70 μm and 40 μm cell sieves, and the cell suspension was collected and resuspended in PBS containing 2% fetal bovine serum. Anti-mouse CD45 microbeads were used to enrich immune cells from the single-cell suspension using magnetic bead sorting. Cell viability was assessed by trypan blue staining (>85%) before sorting, and CD45 levels were detected by flow cytometry. + Cell purity (>90%). Adjust the cell suspension to approximately 700-1200 cells / μL for subsequent experiments. Single-cell RNA-seq libraries were constructed using the 10x Genomics Chromium Next GEM Single Cell 3' kit (v3.2). CD45 + Cells, along with gel beads and partitioning oil (used to separate individual cells), are loaded into the microfluidic channels of the chip to achieve single-cell isolation and barcode labeling. Subsequent steps include cell lysis, mRNA capture, reverse transcription to generate barcoded and UMI-labeled cDNA, and cDNA amplification via PCR. The amplified products are fragmented, end-repaired, A-tailed, and ligated with sequencing adapters to construct the final library. Library quality is assessed using an Agilent 2100 Bioanalyzer to evaluate fragment distribution, and quantitative PCR is used to determine library concentration.

[0069] Mouse aortic tissue digested with collagenase was used to enrich CD45 by flow cytometry. + Immune cells. Single-cell capture and library construction were performed using the 10x Genomics Chromium Next GEM Single Cell 3' kit (v3.2), followed by sequencing on the Illumina NovaSeq 6000 platform.

[0070] 3. Plasmid construction.

[0071] DNA sequences encoding mouse codon-optimized anti-Trem2 scFv, mouse CD28 hinge, transmembrane and signal domains, and mouse CD3z domain were cloned into an MSCV retroviral construct. A FLAG marker was introduced at the 5' end of the scFv (downstream of the signal peptide). A vector expressing the mouse Trem2 gene was constructed by cloning mTrem2 cDNA into the gamma retroviral vector SFG and introducing a double-helix IRES-DNGFR marker upstream of it.

[0072] The cDNA sequence is shown in SEQ ID NO:1.

[0073] The amino acid sequence of Trem2 scFv is shown in SEQ ID NO:2.

[0074] The amino acid sequences of the signal peptide and the FLAG label are shown in SEQ ID NO:5.

[0075] The amino acid sequences of the mouse CD28 hinge, transmembrane, and signaling domain are shown in SEQ ID NO:3.

[0076] Signaling domain: The amino acid sequence of the mouse CD3z domain is shown in SEQ ID NO:4.

[0077] The amino acid sequence of the chimeric antigen receptor targeting mouse TREM2 is shown in SEQ ID NO:6.

[0078] 4. Cell culture system.

[0079] Human Burkitt lymphoma cell line Daudi and mouse macrophage cell line RAW 264.7 were purchased from Procell. Daudi cells and their derivatives were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum, 2 mM GlutaMax, and 100 U / mL penicillin / streptomycin. RAW 264.7 cells were cultured in DMEM medium supplemented with 10% fetal bovine serum, 2 mM GlutaMax, and 100 U / mL penicillin / streptomycin. Mouse T cells were maintained in complete mouse T cell culture medium consisting of RPMI-1640 basal medium supplemented with 10% fetal bovine serum, 2 mM GlutaMax, 100 U / mL penicillin, 100 μg / mL streptomycin, 50 μM β-mercaptoethanol, 1×MEM non-essential amino acid solution, and 1 mM sodium pyruvate. To construct Daudi cells expressing mouse TREM2, Daudi cells were transduced using a retroviral vector encoding a GFP-puro bicistronic cassette and FLAG-tagged mouse Trem2 cDNA. After transduction, GFP-positive cells were selected using 2 μg / mL puromycin, and mouse TREM2-positive cells were obtained by flow cytometry sorting with the FLAG antibody. To induce RAW 264.7 cells to differentiate into foam cells, cells were co-incubated with 50 μg / mL oxidized low-density lipoprotein for 48 hours. Mouse bone marrow-derived macrophages were obtained from the femurs of 6-8 week old BALB / c mice and cultured for 6 days in 24-well plates without tissue culture treatment in complete RPMI-1640 medium supplemented with 10 ng / mL mouse M-CSF. The obtained BMDM remained in the unpolarized M0 state. All cell lines were validated by STR identification and mycoplasma contamination was regularly detected.

[0080] 5. Preparation of CAR constructs and retroviral supernatants

[0081] A single-chain variable region fragment of the monoclonal antibody ab52, which specifically recognizes mouse and human TREM2 protein, was cloned into an MSCV retroviral vector containing a CD28 hinge region, a transmembrane domain, and a CD28 / CD3ζ signaling domain. A FLAG tag was inserted between the scFv and the hinge region for flow cytometry detection. Retroviral supernatant was prepared by co-transfecting the transfer vector and pCL-Eco packaging vector (3:1 ratio) into HEK293T cells. Transfection was performed using... Transfection was performed according to the instructions. Culture supernatant containing virus particles was collected 48 hours and 72 hours after transfection.

[0082] 6. Preparation and functional verification of mouse TREM2.CAR-T cells.

[0083] Mouse CAR-T cells were prepared according to the method published by our team (patent number: ZL202411202471.0). Splenic cells from 6-8 week old BALB / c mice were collected and T cells were sorted using the MojoSort mouse CD3 T cell isolation kit. The sorted T cells were placed in mouse T cell culture medium and stimulated for 2 days with plate-coated anti-mouse CD3 / CD28 antibody. Activated T cells were transduced by co-incubation with retroviral supernatant in RetroNectin-coated culture plates. Two days after transduction (day 4), T cells were collected and expanded in mouse T cell culture medium containing recombinant human IL-7 (10 ng / mL) and recombinant human IL-15 (5 ng / mL). CAR transduction efficiency was assessed by detecting cell surface FLAG tags. All in vitro and in vivo experiments were performed on day 5 after transduction. To verify the specificity and cytotoxicity of TREM2.CAR-T cells, in vitro co-culture experiments were conducted. Different numbers (1-5) × 10 5 CAR-T cells or simulated transduced T cells, with different numbers (1-2) × 10 5 Target cells expressing or not expressing the TREM2 antigen were co-cultured. T cell activation and cytokine secretion were detected after 24 hours of co-culture; specific lysis of target cells and CAR-T cell proliferation were detected after 3 days of co-culture. Twenty-four hours before the proliferation assay, CAR-T cells were pre-stained with CellTraceViolet dye. Proliferation was assessed after co-culture based on dye dilution (with resting CAR-T cells as a control).

[0084] 7. Flow cytometry and sorting.

[0085] Cell surface staining was performed at room temperature for 15 minutes or at 4°C for 30 minutes in the presence of mouse Fc blockers. The flow cytometry antibodies used included: Biolegend's BV421-labeled anti-FLAG antibody, FITC-labeled anti-mouse CD69 antibody, PerCP-labeled anti-mouse CD25 antibody, Pacific Blue (blue fluorescent dye)-labeled anti-mouse CD3 antibody, and PerCP-Cy5.5-labeled anti-mouse CD11b antibody; and R&D Systems' APC-labeled anti-human / mouse TREM2 antibody. Flow cytometry data were acquired using a Novocyte Quanteon flow cytometer and analyzed using FlowJo software.

[0086] 8. Histological and immunohistochemical analysis.

[0087] Aortic tissue was fixed in formalin, embedded in paraffin, and sectioned for hematoxylin-eosin staining, Masson's trichrome staining, and immunohistochemical or immunofluorescence staining for proteins such as CD68, TREM2, iNOS, and CD206. Lipid deposition throughout the aorta was assessed using Oil Red O staining. All histological image analyses were performed by researchers unaware of their experimental group assignments.

[0088] 9. Statistical analysis.

[0089] Statistical analysis was performed using GraphPad Prism 9.5.0 software. The Mann-Whitney U test was used for comparisons between two groups, and the Kruskal-Wallis test, supplemented by Dunn's post-hoc test, was used for comparisons of three or more groups. Data are expressed as mean ± standard deviation, and P < 0.05 was considered statistically significant.

[0090] 10. Results.

[0091] (1) TREM2 + Lipid-associated macrophages are enriched in the arteries of patients with severe atherosclerosis.

[0092] Foam macrophages play a crucial role in the progression of atherosclerosis by initiating inflammation, assisting monocyte recruitment, and promoting fibrous plaque formation. Recent single-cell RNA sequencing studies have identified TREM2 as a specific marker for lipid-associated foam macrophages. TREM2, as a surface receptor for various lipoproteins, promotes lipid uptake by macrophages, a key step in foam cell formation. Simultaneously, TREM2 binds to DAP12, initiating inflammatory signaling and driving the inflammatory response after lipoprotein binding. In addition to foam macrophages, TREM2 is also expressed in pathologically affected liver macrophages and microglia. Therefore, these results suggest that TREM2 could serve as a potential target antigen for CAR-T cells, enabling them to precisely eliminate pathogenic foam macrophages in arteries, thereby promoting plaque regression.

[0093] Figures 1-5 TREM2 was displayed + Lipid-associated macrophages are enriched in the arteries of patients with severe atherosclerosis.

[0094] ( Figure 1 UMAP visualization based on single-cell RNA sequencing data from human coronary artery samples shows that monocytes cluster into six subpopulations (c1-c6). c5 is a cluster of TREM2+ foam macrophages (foamy Mφ, dark blue). The other subpopulations are indicated by arrow labels, including c1 monocytes (blue), c2 inflammatory macrophages (inflammatory Mφ, light blue), c3 HSPA1A...+ Macrophages (Stress Mφ, yellow), c4 CX3CR1 + Macrophages (red) and C6 resident macrophages (Resident Mφ, dark yellow). Figure 2 A violin diagram showing the expression of lipid-related genes APOE, FABP5, and GPNMB in various macrophage clusters. Color and ( Figure 1 The graph is consistent with the data, with the horizontal axis representing gene names and the vertical axis representing log-normalized counts. The c5 cluster showed the highest expression level, suggesting it belongs to lipid-associated macrophages. Figure 3 A stacked bar chart showing the proportion of each macrophage cluster in patients with mild and severe atherosclerosis. The horizontal axis represents disease severity, and the vertical axis represents the percentage of each cluster. The colors correspond to ( ). Figure 1 Clusters in ) . c5 (TREM2) in severely ill patients + The proportion of foam macrophages was significantly increased. Figure 4 Violin plots showing the expression distribution of TREM2 and GPNMB in each cluster of mild and severe patients. Each subplot represents a cluster on the horizontal axis and gene expression levels on the vertical axis; color indicates disease grouping. Severe patients, particularly in the c5 cluster, showed significantly upregulated expression of both markers, suggesting that a lipid-rich environment drives their expression program. Figure 5 Multiple staining of coronary artery tissue from a patient with severe atherosclerosis. Left: H&E staining showing plaque structure (necrotic core and fibrous cap); Middle: Masson trichrome staining showing collagen deposition (blue); Right: Immunofluorescence staining showing TREM2 (green), CD68 (red), and DAPI (blue). Yellow arrows indicate TREM2. + CD68 + Macrophages were observed to confirm their enrichment and localization in plaques. Scale bar: 50 μm. All data were derived from single-cell RNA sequencing and histological analysis of human coronary artery samples. Statistical significance was determined using the Wilcoxon rank-sum test; P < 0.05, P < 0.01, P < 0.001.

[0095] By performing single-cell RNA sequencing analysis on coronary artery samples from healthy donors and patients with atherosclerosis, we were able to detect CD68... + CSF1R + A unique TREM2 was confirmed within the mononuclear phagocyte population. + The existence of subgroup (c5) Figure 1 , Figure 2 and Figure 3 Consistent with previous studies, this c5 cluster also highly expresses lipid uptake-related genes (such as APOE, FABP5) and GPNMB (…). Figure 2Notably, the C5 cluster was highly enriched in patients with severe atherosclerosis compared to healthy donors or patients with milder lesions. Figure 3 and Figure 4 Importantly, we observed an overall upregulation of TREM2 and other foam cell markers (such as GPNMB) expression in patients with severe atherosclerosis, even in non-foam cell clusters. Figure 4 These data suggest that a lipid-rich and inflammatory environment drives the expression program of these lipid-related genes, thereby promoting foam cell formation and eventual plaque development. TREM2 in disease lesions + The presence of macrophages was also confirmed by tissue Masson staining and immunofluorescence staining. Figure 5 In summary, our clinical cohort and overlapping observations with others confirm that TREM2 is a marker of foam macrophages in advanced atherosclerosis.

[0096] (2) Development and validation of TREM2-specific CAR-T cells.

[0097] We hypothesized that eliminating TREM2-positive lipid-associated macrophages could halt the progression of atherosclerosis and restore aortic function. To this end, we developed a chimeric antigen receptor targeting the TREM2 protein, whose single-chain variable region was derived from the monoclonal antibody clone ab52 and linked to the mouse CD28 co-stimulatory domain and CD3ζ intracellular signaling domain. This receptor was transduced into mouse spleen T cells; T cells transduced with an empty vector served as a control. The TREM2.CAR molecule was detected at high levels by an anti-FLAG-tagged antibody, and the resulting CAR-T cells exhibited typical cytotoxic T cell functional characteristics. We tested the effector function of this TREM2.CAR-T cell by co-culturing it with different cell lines, including Daudi cells ectopically expressing the mouse Trem2 gene, the RAW 264.7 macrophage line naturally expressing TREM2, and bone marrow-derived M0 macrophages. When co-cultured with TREM2… + During co-culture, TREM2 CAR-T cells exhibited strong activation and proliferation capabilities, as evidenced by the expression of activation markers CD25 / CD69 and the dilution of CellTrace dye. CAR-T cells showed significant cytotoxicity against Daudi-mTREM2 and RAW 264.7 cell lines, accompanied by the production of cytokines (IFN-γ, TNF-α, and IL-2) and an increase in T cell numbers, while no such effect was observed against Daudi cells that do not express TREM2.

[0098] Figures 6-8 The construction and in vitro functional validation of TREM2-specific CAR-T cells were demonstrated.

[0099] ( Figure 6 Flow cytometry detection of TREM2 expression on the surface of target cells. Left image: Daudi (wild type, TREM2) - Right image: RAW 264.7 macrophage cell line (naturally expressing TREM2) and its foam cells induced by oxLDL (RAW 264.7 + oxLDL). Red in the image represents anti-TREM2-APC staining, and gray filler represents isotype controls. oxLDL treatment significantly upregulated TREM2 expression, confirming its role in foam cell formation. The TREM2 positivity rate in all cell lines was >80%. Figure 7 TREM2. Activation of CAR-T cells after co-culturing with different target cells. Flow cytometry scatter plot: vertical axis is CD25 (PerCP), horizontal axis is FSC-A (cell size). Top row: with TREM2 - Daudi or TREM2 + After co-culturing Daudi-TREM2 for 24 hours, CD3 + CAR + CD25 in T cells + The proportions were 17.3% and 86.7%, respectively. Bottom row: After co-culturing with RAW 264.7 or BMDM-M0 expressing TREM2 naturally, CD25... + The proportions were 80.9% and 97.4%, respectively, indicating that CAR-T cells can specifically recognize and activate TREM2. + Target cells. Figure 8 Quantitative analysis of activation and proliferation function of TREM2 CAR-T cells. Right: Bar chart showing CD25 after 24 hours of co-culture. + CD69 + Percentage of double-positive T cells (vertical axis), target cell types (horizontal axis). Compared with TREM2. + The activation rate of target cells co-cultured was significantly higher than that of TREM2. - The Daudi control group (data are expressed as mean ± SD, n=3, Kruskal-Wallis test was used) Left: CellTrace Violet dye dilution assay to assess T cell proliferation after 72 hours of co-culture. The vertical axis represents dye intensity (log scale), and the horizontal axis represents cell count. (Compared to TREM2) + The target cell co-culture group showed significant dye dilution (proliferation index >5-fold), while the Daudi co-culture group showed weak proliferation, confirming antigen-dependent proliferation.

[0100] The experiment used mouse spleen-derived TREM2 CAR-T cells (transduction efficiency >70%, with CD8+).+ The subsets were predominantly subpopulations, with an effector-to-target ratio of 5:1. Flow cytometry data were collected using Novocyte Quanteon and analyzed using FlowJo. Statistical significance was assessed using the Mann-Whitney U test ( ). ).

[0101] To further verify in vivo whether TREM2.CAR-T cells can effectively home to atherosclerotic plaques, we infused Dil-labeled TREM2.CAR-T cells or transduced T cells to ApoE patients who had been fed a high-fat diet for 12 weeks and had severe atherosclerotic lesions in their aorta. - / - In mice, we observed extensive infiltration of labeled CAR-T cells into the aorta within 48 hours post-infusion, a phenomenon not observed with simulated T cells. The distribution of CAR-T cells in the spleen and liver was comparable to that of simulated T cells, suggesting specificity for aortic infiltration. Furthermore, immunofluorescence staining confirmed that CAR-T cells localized near CD68-positive macrophages in the aorta. In summary, our data demonstrate that TREM2 CAR-T cells exhibit specific and potent cytotoxicity against TREM2-positive cells in vitro and effectively home to atherosclerotic aortas in vivo.

[0102] Figures 9-12 This demonstrates the role of TREM2.CAR-T cells in ApoE. - / - Homing characteristics and safety assessment in a mouse model of atherosclerosis.

[0103] ( Figure 9 Schematic diagram of the experimental timeline. 8-week-old male ApoE - / - Mice were given a high-fat diet (HFD, green box) to induce plaque formation. At week 8, mice were injected via tail vein with: (i) saline; (ii) DiR-labeled T-cell mimics (DiR@Ctrl-T); (iii) DiR-labeled TREM2.CAR-T cells (DiR@TREM2.CAR-T); (iv) free DiR dye (DIR); and (v) unlabeled TREM2.CAR-T cells (TREM2.CAR-T). Mice were sacrificed 48 hours after injection, and major organs were collected for in vitro fluorescence imaging (n=3 / group). Arrows indicate intervention and observation time points. Figure 10 (a) In vitro fluorescence imaging of the aorta of HFD-fed mice at week 8 (pseudo-color, red represents high signal). (iv) Corresponding to ( Figure 9 In each group of the TREM2.CAR-T treatment group (iii, v), the aortic fluorescence signal was significantly enhanced, indicating that cells specifically homed to the lesion site; the signal was weak in the control group (i, ii). Images were acquired using an IVIS optical imaging system. Figure 10 Quantitative analysis of the mean fluorescence intensity of DiR in the aortic region (B). The signal intensity of the TREM2.CAR-T group (iii, v) was significantly higher than that of the Saline and DIR@Ctrl-T groups (B). One-way ANOVA (Tukey post-hoc test) confirmed the specific accumulation of CAR-T cells within plaques. Data are expressed as mean ± SD (n=3). Figure 11 (Center A) Immunofluorescence staining of frozen sections of the aortic root (scale bar: 100 μm). Left: Saline group; Right: DiR@TREM2.CAR-T group. Staining shows DAPI (blue, nucleus), CD68 (cyan, macrophages), and DiR (green, labeled CAR-T cells). In the TREM2.CAR-T group, green DiR signal was enriched in CD68. + The regions (indicated by yellow arrows for co-localization) show the CAR-T cell infiltration into the macrophage-rich plaque microenvironment. Images were acquired using a Leica confocal microscope, with a co-localization factor >0.7. Figure 11 (B) Quantitative statistics. Figure 12 (a) In vitro fluorescence imaging of major organs (heart, liver, spleen, lungs, kidneys) to assess systemic distribution. (iv) Treatment is the same as ( Figure 9 The TREM2.CAR-T group showed significantly enhanced cardiac (including aortic) signals, while the signals in the liver, spleen, lungs, and kidneys were similar to those in the control group, suggesting no significant off-target accumulation. Figure 12 (B) Quantitative analysis of average DiR fluorescence intensity in each organ (vertical axis: ×10) 6 Photons / second; Horizontal axis: organ). TREM2. The cardiac signal in the CAR-T group was significantly higher than that in the Saline group ( There were no statistically significant differences among the remaining organ groups (P>0.05), indicating that the treatment strategy has a good safety window. Data are expressed as mean ± SD (n=3).

[0104] (3) The anti-atherosclerotic effect of TREM2.CAR-T cells.

[0105] To evaluate the preventive and therapeutic effects of TREM2.CAR-T cells on atherosclerosis, we conducted an ApoE study. - / - CAR-T cells were infused into mice at week 4 or 8 after starting a high-fat diet. We demonstrated that even after 4 weeks of high-fat diet, ApoE cells... - / -Atherosclerotic lesions were present in the mouse aorta, and the necrotic core and collagen area progressively worsened with continued high-fat diet. A single infusion of TREM2 CAR-T cells significantly reduced the area stained with Oil Red O throughout the aorta, with prophylactic treatment showing a more significant therapeutic effect in the aortic arch region. Furthermore, the necrotic core area (H&E staining) and collagen area (Masson staining) of atherosclerotic lesions in the aortic root and brachiocephalic artery were also significantly reduced, especially with early CAR-T cell application. Along with the reduction in lesion area, we observed a simultaneous decrease in the aortic macrophage population after CAR-T cell treatment. Highly enriched CD68 and TREM2-positive cells in atherosclerotic lesions were significantly cleared by CAR-T cells. Interestingly, staining for M1 and M2 macrophage markers iNOS and CD206 was significantly reduced, suggesting that macrophage clearance was not targeted at a specific polarization state.

[0106] Despite exhibiting potent anti-atherosclerotic activity, CAR-T cell therapy did not affect mouse body weight, and the results were consistent across different genetic backgrounds and dietary conditions. Furthermore, in previously reported cases with TREM2... + No significant tissue damage was observed in the liver and brain of macrophages, and the levels of tissue damage biomarkers in serum were unaffected, indicating that TREM2.CAR-T cells were well-tolerated and did not cause any significant adverse reactions. In conclusion, these data demonstrate that TREM2.CAR-T cells have a potent anti-atherosclerotic effect, whether used for prophylactic infusion or for the treatment of existing plaques.

[0107] Figures 13-16 This demonstrates the role of TREM2.CAR-T cells in ApoE. - / - Evaluation of the efficacy of preventive and therapeutic interventions in a mouse model of atherosclerosis.

[0108] ( Figure 13 Schematic diagram of the experimental timeline. 8-week-old male ApoE - / - Mice were given a high-fat diet (HFD, green box) from week 1 to induce atherosclerosis. Group G1 (blue box) received a single injection of 4 × 10⁴ g of acetic acid via the tail vein at week 4 (early plaque stage). 6 One TREM2 CAR-T cell was administered to the G2 group (green box) for 5 weeks; the G2 group (late plaque stage) received the same dose of cells at week 8 (intervention period) for 5 weeks. Control groups included a normal diet (Ctrl) and a no-intervention HFD group. All animals were sacrificed at week 13 for endpoint analysis (n=6 / group). Arrows indicate treatment and sampling time points. Figure 14(A) Oil Red O (ORO) staining of the entire aorta to assess lipid deposition. The overall staining from the ascending aorta to the iliac bifurcation is shown in the Ctrl, HFD, CAR-T G1, and CAR-T G2 groups. Extensive red lipid plaques were visible in the HFD group; plaque area was significantly reduced in the CAR-T G1 and G2 groups, especially in the G1 group (early intervention). Images were acquired using an anatomical microscope and quantitatively analyzed using ImageJ. Figure 14 (B) Quantitative statistics on the percentage of total aortic plaque area. The vertical axis of the bar chart represents the percentage (%) of plaque area to total aortic area. The plaque proportion in the HFD group was significantly higher than that in the Ctrl group. ); The CAR-T G1 group had a reduction of approximately 60% compared to HFD ( Group G2 decreased by approximately 45% ( There was no significant difference between groups G1 and G2 (ns). Data are expressed as mean ± SD (n=6), and one-way ANOVA and Tukey post-hoc test were used. Figure 15 (A) H&E staining of the aortic root (top row: 100 μm scale bar; bottom row: 20 μm magnification). The Ctrl group showed normal vessel wall structure; the HFD group showed a thick fibrous cap, lipid pools, and necrotic core (arrows); the CAR-T G1 and G2 groups showed reduced plaque thickness, thinner fibrous caps, and decreased cell density, suggesting reduced inflammation and tissue remodeling. Figure 15 Quantitative analysis of the proportion of necrotic core area in the HFD group (B group) showed that the proportion of necrotic core area was significantly larger than that in the Ctrl group. ); Both CAR-T G1 and G2 groups showed significantly smaller reductions compared to the HFD group ( ), and the effect of group G1 was better than that of group G2 ( Data are expressed as mean ± SD (n=6), and Kruskal-Wallis test and Dunn post-hoc test were used. Figure 16 (A) Masson's trichrome staining of the aortic root (top row: 100 μm scale bar; bottom row: 20 μm magnification). Blue indicates collagen deposition. The Ctrl group showed sparse collagen distribution; the HFD group showed reduced collagen and expanded necrotic areas; the CAR-T G1 and G2 groups showed enhanced collagen signal, especially in the fibrous cap region, suggesting plaque stabilization. Figure 16 Quantitative analysis of collagen area ratio in the HFD group (B group) showed that the collagen ratio was significantly lower than that in the Ctrl group. Both CAR-TG1 and G2 groups showed significantly higher levels than the HFD group ( Group G1 had the highest collagen content (ns, compared with G2). Data are expressed as mean ± SD (n=6), and one-way ANOVA and Tukey post-hoc test were used.

[0109] Figures 17-20The results showed that TREM2.CAR-T cell therapy reduced pro-inflammatory macrophages and increased the relative proportion of M2-like macrophages.

[0110] ( Figure 17 (A) Immunohistochemical staining of CD68 in the aortic root (top row: scale bar 100 μm; bottom row: 20 μm). Macrophages were sparse in the Ctrl group; dense brown CD68 was observed in the HFD group. + The signal was mainly distributed in the plaque shoulder and necrotic core; CD68 in CAR-TG1 and G2 groups + The cell count was significantly reduced, especially in the G1 group, indicating that pathogenic macrophages were effectively eliminated. Figure 17 (B) CD68 + Cell density quantification (vertical axis: cells per high-power field; horizontal axis: group). The HFD group was significantly higher than the Ctrl group. ); Both CAR-T G1 and G2 groups showed significantly lower levels compared to the HFD group ( There was no significant difference between the two groups (ns). Data are expressed as mean ± SD (n=6), and one-way ANOVA and Tukey post-hoc test were used. Figure 18 (A) Immunofluorescence staining of CD206 (M2 marker) in the aortic root (scale bar as above). The Ctrl group showed weak CD206 signal; the HFD group showed only scattered CD206. + Cells; CAR-T G1 and G2 groups showed enhanced green CD206 signaling, and cells were branched, distributed in the fibrous cap and neoendothelial subepithelial region, suggesting enhanced M2 polarization. Figure 18 (B) CD206 + Cells account for CD68 + Cell percentage quantification (vertical axis: percentage %). The HFD group had the lowest percentage (P<0.0001 vs. Ctrl); both CAR-T G1 and G2 groups were significantly higher than the HFD group (P<0.0001), with the G1 group slightly higher than the G2 group (ns). Data are expressed as mean ± SD (n=6), and Kruskal-Wallis test and Dunn post-hoc test were used. Figure 19 (A) Immunofluorescence staining of TREM2 in the aortic root (scale bar as above). The Ctrl group showed almost no TREM2 signal; the HFD group showed dense green TREM2. + Clusters were found in foam cell regions; TREM2 signaling was significantly weakened in the CAR-T G1 and G2 groups, with residual signaling mainly present in scattered cells with low expression, confirming TREM2. hi Macrophages were targeted and eliminated. Figure 19 (B) TREM2 + Cells account for CD68 + Cell proportion quantification. The HFD group had the highest proportion ( ); Both CAR-T G1 and G2 groups showed significantly lower levels compared to the HFD group ( ), and the effect of group G1 was better than that of group G2 ( Data are expressed as mean ± SD (n=6), and one-way ANOVA and Tukey post-hoc test were used. Figure 20 (A) Immunofluorescence staining of iNOS (M1 marker) in the aortic root (scale bar as above). The Ctrl group showed weak iNOS signal; the HFD group showed extensive red iNOS. + Signals were distributed in areas of active inflammation; red signals were significantly reduced in the CAR-T G1 and G2 groups, with residual iNOS. + The cells were rare, suggesting that M1-type pro-inflammatory polarization was suppressed. Figure 20 (B) iNOS + Cells account for CD68 + Cell percentage quantification (vertical axis: percentage %). The HFD group had the highest percentage ( ); Both CAR-T G1 and G2 groups showed significantly lower levels compared to the HFD group ( There was no significant difference between the two groups (ns). Data are presented as mean ± SD (n=6) and analyzed using the Mann-Whitney U test. All IHC / IF slides were acquired using a Leica DMi8 confocal microscope with secondary antibody labeled with horseradish peroxidase or Alexa Fluor. Blinded counting was performed using ImageJ (≥5 high-power fields per slide). Antibody sources: CD68 (Abcam, ab125212), CD206 (R&D, AF2535), TREM2 (BioLegend, 145412), iNOS (Abcam, ab15323). Experiments were performed under SPF conditions, with approval from the animal ethics committee, and in accordance with ARRIVE guidelines. .

[0111] The applicant declares that the above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.

Claims

1. A chimeric antigen receptor targeting TREM2, characterized in that, The chimeric antigen receptor comprises: a single-chain variable region fragment that specifically binds to TREM2, a hinge region, a transmembrane region, a co-stimulatory signaling domain, and a signaling domain; wherein the single-chain variable region fragment is derived from the anti-TREM2 monoclonal antibody clone ab52.

2. The chimeric antigen receptor targeting TREM2 according to claim 1, characterized in that, The hinge area is a CD28 hinge area; Preferably, the transmembrane region and the co-stimulatory signal domain are CD28 transmembrane and co-stimulatory domains; Preferably, the signal structure domain is a CD3ζ signal structure domain; Preferably, the amino acid sequence of the single-chain variable region fragment is shown in SEQ ID NO:2; Preferably, the amino acid sequences of the hinge region, transmembrane region, and co-stimulatory signaling domain are as shown in SEQ ID NO:3; Preferably, the amino acid sequence of the signal domain is shown in SEQ ID NO:

4.

3. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the chimeric antigen receptor targeting TREM2 as described in claim 1 or 2.

4. A carrier, characterized in that, The vector comprises at least one copy of the nucleic acid molecule of claim 3; Preferably, the vector comprises: a cloning vector or an expression vector; Preferably, the expression vector includes a viral expression vector, which includes a retroviral vector, an adenovirus vector, an adeno-associated virus vector, or a lentiviral vector.

5. A recombinant virus, characterized in that, The recombinant virus is prepared by a method comprising the following steps: co-transfecting packaging cells with the vector of claim 4 and a packaging helper plasmid to obtain the recombinant virus.

6. An immune effector cell, characterized in that, The immune effector cells express the chimeric antigen receptor targeting TREM2 as described in claim 1 or 2; Preferably, the immune effector cells contain the nucleic acid molecule of claim 3, or the vector of claim 4, or the recombinant virus of claim 5; Preferably, the immune effector cells are T cells or NK cells, with T cells being the most preferred.

7. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the immune effector cells of claim 6.

8. Use of any one or a combination of at least two of the following: the chimeric antigen receptor targeting TREM2 as described in claim 1 or 2; the nucleic acid molecule as described in claim 3; the carrier as described in claim 4; the immune effector cell as described in claim 6; or the pharmaceutical composition as described in claim 7, in the preparation of a medicament for the prevention and / or treatment of atherosclerosis and related cardiovascular diseases.

9. The application of TREM2 as a target in the preparation of drugs for treating atherosclerosis, characterized in that, The drug specifically eliminates an inflammatory macrophage subset that highly expresses TREM2, and the inflammatory macrophage subset also highly expresses Slamf9; Preferably, the drug reduces the chemotactic recruitment of monocytes; Preferably, the drug promotes the infiltration and activation of immune cells with tissue-protective functions.

10. The application according to claim 9, characterized in that, The drug is a CAR-T cell that targets TREM2.

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