Immunosuppressive dendritic cell, and preparation method therefor and use thereof

Abstract

A chimeric antigen receptor dendritic cell overexpressing CTLA4-Ig, PD-L1 and IL-10, and a preparation method therefor, wherein the amino acid sequences of CTLA4-Ig, PD-L1, and IL -10 are set forth in SEQ ID NOs: 17-19, respectively, and the amino acid sequence of the chimeric antigen receptor targeting fibroblast activation protein α-positive cells is set forth in SEQ ID NO: 20. The chimeric antigen receptor dendritic cell is used for preventing and / or treating cardiac fibrosis.

Description

An immunosuppressive dendritic cell, its preparation method and application

[0001] This application claims priority to Chinese Patent Application No. 202510285966.2, filed on March 11, 2025, entitled "An Immunosuppressive Dendritic Cell and Its Preparation Method and Application", the entire contents of which are incorporated herein by reference. Technical Field

[0002] This application belongs to the field of cell drug technology, specifically relating to an immunosuppressive dendritic cell, its preparation method, and its application. Background Technology

[0003] Cardiac fibrosis is considered a major driver of heart failure progression. Following cardiac injury (including ischemic and non-ischemic), fibroblasts proliferate, leading to the accumulation of large amounts of extracellular matrix accompanied by inflammatory damage and tissue structure disruption, resulting in poor cardiac remodeling and thus affecting normal cardiac function. However, due to the lack of strategies with clinical translational potential, there are currently no specific antifibrotic drugs in clinical practice to slow or reverse cardiac fibrosis.

[0004] In the pathogenesis of heart failure, the activation of the innate immune system is the first rapid response to myocardial injury, activating subsequent adaptive immunity. Considering that immune cells integrate multiple physiological mechanisms, such as cell signaling and intercellular interactions, making them excellent mediators of immune responses, the delivery of immunomodulatory cells has been considered a feasible approach. Dendritic cells (DCs) are the body's most potent professional antigen-presenting cells (APCs), capable of efficiently uptake, processing, and presenting antigens. Mature DCs effectively activate naive T-cell-level immune responses, while tolerant DCs inhibit T-cell activation and immune responses. Therefore, DCs are central to initiating, regulating, and maintaining immune responses. Currently, there are no reports on the application of DCs in the treatment of cardiac fibrosis. Summary of the Invention

[0005] In view of this, the purpose of this application is to provide dendritic cells that overexpress CP-IL10 fusion protein or CP-IL10-CAR fusion protein, which can reduce the clearance of DCs by immune cells in the body, inhibit cardiac inflammation and fibrosis, and promote cardiac repair by overexpressing CTLA4-Ig, PD-L1 and IL-10; and to prepare targeted and inhibitory DCs by fusing FAP CAR with CP-IL10, which further enhances the repair effect of DCs on cardiac fibrosis.

[0006] This application provides a dendritic cell or CAR dendritic cell overexpressing CP-IL10, wherein the dendritic cell or CAR dendritic cell overexpresses CTLA4-Ig, PD-L1 and IL-10;

[0007] The surface of the CAR dendritic cells also expresses a chimeric antigen receptor that targets fibroblast activation protein α-positive cells.

[0008] Preferably, the amino acid sequence of the CTLA4-Ig is shown in SEQ ID NO: 17;

[0009] The amino acid sequence of the PD-L1 is shown in SEQ ID NO: 18;

[0010] The amino acid sequence of the IL-10 is shown in SEQ ID NO: 19;

[0011] The amino acid sequence of the chimeric antigen receptor targeting fibroblast activation protein α-positive cells is shown in SEQ ID NO: 20.

[0012] Preferably, the dendritic cells or CAR dendritic cells include autologous dendritic cells or allogeneic dendritic cells.

[0013] Preferably, the dendritic cells or dendritic cells in the CAR dendritic cells are derived from at least one of the following: peripheral blood cells, bone marrow cells, hematopoietic stem cells, embryonic stem cells, and induced pluripotent stem cells.

[0014] This application provides a method for constructing dendritic cells or CAR dendritic cells overexpressing CP-IL10, comprising the following steps:

[0015] Dendritic cells were introduced into dendritic cells by introducing CTLA4-Ig gene fragments, PD-L1 gene fragments and IL-10 gene fragments, or CTLA4-Ig gene fragments, PD-L1 gene fragments and IL-10 gene fragments and the coding sequence of a chimeric antigen receptor targeting fibroblast activation protein α positive cells, to obtain dendritic cells overexpressing CP-IL-10 or CAR dendritic cells.

[0016] Preferably, the method of introducing dendritic cells includes at least one of the following: virus-mediated, lipid nanoparticle-mediated, and electroporation.

[0017] This application provides a drug for preventing and treating cardiac fibrosis, wherein the active pharmaceutical ingredient includes dendritic cells or CAR dendritic cells overexpressing CP-IL10 or prepared by the preparation method.

[0018] Preferably, the dosage form of the drug includes an injection solution and / or an injection powder;

[0019] In the drug, the density of dendritic cells or CAR dendritic cells overexpressing CP-IL10 is 1.2 × 10⁻⁶. 4 per μL.

[0020] This application provides the use of the CP-IL10 overexpressing dendritic cells or CAR dendritic cells, or the CP-IL10 overexpressing dendritic cells or CAR dendritic cells prepared by the preparation method, in the preparation of drugs for the prevention and / or treatment of cardiac fibrosis.

[0021] Preferably, the cardiac fibrosis includes at least one of the following diseases: myocardial infarction, aortic arch coarctation, and myocardial ischemia-reperfusion.

[0022] This application provides dendritic cells overexpressing CP-IL10 (CP-IL10-DSs) or CAR dendritic cells (CP-IL10-CAR-DSs), wherein the dendritic cells overexpress CTLA4-Ig, PD-L1, and IL-10, and the surface of the CAR dendritic cells also expresses a chimeric antigen receptor targeting fibroblast activation protein α-positive cells. In this invention, by overexpressing CP and IL-10 proteins in dendritic cells, it is beneficial to reduce the clearance of exogenous DCs by immune cells and inhibit cardiac immune and inflammatory responses; simultaneously, by preparing fibroblast activation protein α-positive cells by expressing these proteins on the surface of DCs, it is beneficial to target fibroblasts, achieve precise drug delivery, and further enhance the inhibitory effect of immunosuppressive DCs on cardiac inflammation and fibrosis. In this embodiment, mice with permanent cardiac fibrosis caused by myocardial ischemia-reperfusion injury, acute myocardial infarction, or aortic arch coarctation were used as subjects. After tail vein injection of CP-IL10-CAR-DCs or CTL-DCs (empty viral vector transfected DC control), the cardiac fibrosis in mice was significantly improved. Moreover, the drug effect of CP-IL10-CAR-DCs was better than that of CTL-DCs combination, downregulating the expression level of cardiac fibrosis-related proteins, alleviating fibrosis and improving cardiac function. Attached Figure Description

[0023] Figure 1 shows the results of flow cytometry detection of CAR and PD-L1 co-expression in CP-IL10-CAR-DCs;

[0024] Figure 2 shows the echocardiographic results of cardiac function in a model of ischemia-reperfusion-induced cardiac fibrosis induced by CP-IL10-CAR-DCs.

[0025] Figure 3 shows the staining results of cardiac tissue fibrosis in a model of ischemia-reperfusion-induced cardiac fibrosis induced by CP-IL10-CAR-DCs.

[0026] Figure 4 shows the effect of the CP-IL10-CAR-DCs composition on the expression of cardiac fibrosis-related proteins in a model of ischemia-reperfusion-induced cardiac fibrosis.

[0027] Figure 5 shows the effect of the CP-IL10-CAR-DCs composition on inflammation-related genes in cardiac tissue in a model of ischemia-reperfusion-induced cardiac fibrosis.

[0028] Figure 6 is a heatmap of next-generation sequencing results of the CP-IL10-CAR-DCs composition on cardiac tissue inflammation-related genes in an ischemia-reperfusion-induced cardiac fibrosis model;

[0029] Figure 7 shows the echocardiographic results of cardiac function in an acute myocardial infarction-induced cardiac fibrosis model using CP-IL10-CAR-DCs.

[0030] Figure 8 shows the staining results of cardiac tissue fibrosis in an acute myocardial infarction-induced cardiac fibrosis model using CP-IL10-CAR-DCs.

[0031] Figure 9 shows the effects of the CP-IL10-CAR-DCs composition on inflammation-related genes in cardiac tissue in an acute myocardial infarction-induced cardiac fibrosis model.

[0032] Figure 10 shows the echocardiographic results of cardiac function in a model of cardiac fibrosis induced by aortic arch constriction using CP-IL10-CAR-DCs.

[0033] Figure 11 shows the staining results of cardiac function in a model of cardiac fibrosis induced by aortic arch constriction using CP-IL10-CAR-DCs.

[0034] Figure 12 shows the effect of CP-IL10-CAR-DCs on the expression of cardiac tissue fibrosis marker proteins in a model of cardiac fibrosis caused by aortic arch constriction.

[0035] Figure 13 shows the therapeutic effect of allogeneic cell therapy on cardiac function;

[0036] Figure 14 shows the results of Sirius red staining of cardiac tissue;

[0037] Figure 15 shows the expression levels of fibrotic proteins in the overall cardiac tissue of mice.

[0038] Figure 16 shows the CBA results of serum samples from mice on day 28. Detailed Implementation

[0039] This application provides a CP-IL10-CAR-dendritic cell, wherein the dendritic cell overexpresses CTLA4-Ig, PD-L1 and IL-10, and the surface of the dendritic cell also expresses a chimeric antigen receptor that targets fibroblast activation protein α positive cells.

[0040] In this application, CP is an abbreviation for CTLA4-Ig fusion protein and PD-L1. The amino acid sequence of CTLA4-Ig is as shown in SEQ ID NO: 17.

[0041] (MACLGLRRYKAQLQLPSRTWPFVALLTLLFIPVFSEAIQVTQPSVVLASSHGVASFPCEYSPSHNTDEVRVTVLRQTNDQMTEVCATTFTEKNTVGFLDYPFCSGTFNESRVNLTIQGLRAVDTGLYLCKVELMYPPPYFVGMGNGTQIYVISRVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDI SKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLT CMITDFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK*); the amino acid sequence of PD-L1 is as SEQ ID NO:18(MRIFAGIIFTACCHLLRAFTITAPKDLYVVEYGSNVTMECRFPVERELDLLALVVYWEKEDEQVIQFVAGEEDLKPQHSNFRGRASLPKDQLLKGNAALQITDVKLQDAGVYCCIISYGGADYKRITLKVNAPYRKINQRISVDPATSEHE LICQAEGYPEAEVIWTNSDHQPVSGKRSVTTSRTEGMLLNVTSSLRVNATANDVFYCTFWRSQPGQNHTAELIIPELPATHPPQNRTHWVLLGSILLFLIVVSTVLLFLRKQVRMLDVEKCGVEDTSSKNRNDTQFEET); the amino acid sequence of IL-10 is such as SEQ ID NO: 19

[0042] (MPGSALLCCLLLLTGMRISRGQYSREDNNCTHFPVGQSHMLLELRTAFSQVKTFFQTKDQLDNILLTDSLMQDFKGYLGCQALSEMIQFYLVEVMPQAEKHGPEIKEHLNSLGEKLKTLRMRLRRCHRFLPCENKSKAVEQVKSDFNKLQDQGVYKAMNEFDIFINCIEAYMMIKMKS*). The amino acid sequence of the chimeric antigen receptor targeting fibroblast activation protein α-positive cells is as shown in SEQ ID NO: 20.

[0043]

[0044] In this application, PD-L1 is Programmed cell death ligand 1, also known as cluster of differentiation 274 (CD274) or B7 homolog 1 (B7-H1). PD-L1, by binding to programmed cell death receptor-1 (PD-1), can transmit inhibitory signals, reducing CD8+ in lymph nodes. +Proliferation of T cells. The nucleotide sequence of the coding sequence of said PD-L1 is shown as SEQ ID NO: 22 (ATGAGGATATTTGCTGGCATTATATTCACAGCCTGCTGTCACTTGCTACGGGCGTTTACTATCACGGCTCCAAAGGACTTGTACGTGGTGGAGTATGGCAGCAACGTCACGATGGAGTGCAGATTCCCTGTAGAACGGGAGCTGGACCTGCTTGCGTTAGTGGTGTACTGGGAAAAGGAAGATGAGCAAGTGATTCAGTTTGTGGCAGGAGAGGAGGACCTTAAGCCTCAGCACAGCAACTTCAGGGGGAGAGCCTCGCTGCCAAAGGACCAGCTTTTGAAGGGAAATGCTGCCCTTCAGATCACAGACGTCAAGCTGCAGGACGCAGGCGTTTACTGCTGCATAATCAGCTACGGTGGTGCGGACTACAAGCGAATCACGCTGAAAGTCAATGCCCCATACCGCAAAATCAACCAGAGAATTTCCGTGGATCCAGCCACTTCTGAGCATGAACTAATATGTCAGGCCGAGGGTTATCCAGAAGCTGAGGTAATCTGGACAAACAGTGACCACCAACCCGTGAGTGGGAAGAGAAGTGTCACCACTTCCCGGACAGAGGGGATGCTTCTCAATGTGACCAGCAGTCTGAGGGTCAACGCCACAGCGAATGATGTTTTCTACTGTACGTTTTGGAGATCACAGCCAGGGCAAAACCACACAGCGGAGCTGATCATCCCAGAACTGCCTGCAACACATCCTCCACAGAACAGGACTCACTGGGTGCTTCTGGGATCCATCCTGTTGTTCCTCATTGTAGTGTCCACGGTCCTCCTCTTCTTGAGAAAACAAGTGAGAATGCTAGATGTGGAGAAATGTGGCGTTGAAGATACAAGCTCAAAAAACCGAAATGATACACAATTCGAGGAGACG).

[0045] In this application, the IL-10 is interleukin-10, also known as cytokine synthesis inhibitory factor (CSIF), a pleiotropic cytokine that can exert immunosuppressive or immunostimulatory effects in various cell types. IL-10 reduces antigen presentation by downregulating the expression of major histocompatibility antigen II on the surface of monocytes, downregulates T lymphocyte activity, and inhibits the activation, migration, and adhesion of inflammatory cells. Simultaneously, IL-10 also inhibits the synthesis and release of inflammatory factors. Exogenous IL-10 strongly inhibits the synthesis of IL-1, IL-6, IL-8, TNF-α, GM-CSF, and G-CSF at the transcriptional level, thereby exerting an anti-inflammatory effect. The nucleotide sequence encoding IL-10 is shown in SEQ ID NO:23.

[0046] In this application, the nucleotide sequence of the chimeric antigen receptor targeting fibroblast activation protein α-positive cells is as shown in SEQ ID NO: 24. Fibroblast activation protein (FAP) is a protein whose expression is significantly increased during fibrosis, but expressed at low levels in normal tissues and organs. Therefore, FAP is a naturally excellent target for activated fibroblasts. This application, by expressing a CAR targeting FAP on the surface of dendritic cells, can promote the precise localization of dendritic cells to fibrotic cells, thereby enabling dendritic cells to exert their repair function in cardiac fibrosis.

[0047] In this application, the dendritic cells preferably include, according to species, human-derived dendritic cells and / or animal-derived dendritic cells. The dendritic cells preferably include, according to origin, autologous dendritic cells and / or allogeneic dendritic cells. The autologous dendritic cells preferably refer to dendritic cells derived from patients with cardiac fibrosis to be treated, and the prepared CP-IL10-CAR dendritic cells are used for the treatment of specific patients with cardiac fibrosis derived from dendritic cells. The allogeneic dendritic cells preferably refer to dendritic cells derived from a non-self individual, and the dendritic cells or CAR dendritic cells prepared based on this, overexpressing the CP fusion protein, are used for the treatment of non-specific patients.

[0048] This application provides a method for constructing the CP-IL10-CAR-dendritic cells, comprising the following steps:

[0049] The CTLA4-Ig gene fragment, PD-L1 gene fragment, and IL-10 gene fragment, or the CTLA4-Ig gene fragment, PD-L1 gene fragment, IL-10 gene fragment, and the coding sequence of the chimeric antigen receptor targeting fibroblast activation protein α positive cells, are introduced into dendritic cells to obtain CP-IL10-DCs or CP-IL10-CAR-DCs.

[0050] In this application, the method of introducing dendritic cells preferably includes at least one of the following: virus-mediated, lipid nanoparticle-mediated, and electroporation. In the virus-mediated method, the virus preferably includes lentivirus and / or adenovirus.

[0051] In this application, the nucleotide sequence of the CTLA4-Ig gene fragment is shown in SEQ ID NO:21. The nucleotide sequence of the PD-L1 gene fragment is shown in SEQ ID NO:22. The nucleotide sequence of the IL-10 gene fragment is shown in SEQ ID NO:23. The nucleotide sequence of the coding sequence for the chimeric antigen receptor targeting fibroblast activation protein α-positive cells is shown in SEQ ID NO:24. The scFv nucleotide sequence of the FAP containing the CD8LS and Hinge+TM transmembrane regions is shown in SEQ ID NO:25.

[0052] In this application, the coding sequences for the CTLA4-Ig gene fragment and the chimeric antigen receptor targeting fibroblast activation protein α-positive cells were synthesized by Guangzhou Aiji Biotechnology Co., Ltd. The PD-L1 and IL-10 gene fragments were obtained by PCR amplification. The primers for amplifying the PD-L1 gene included a forward primer with the nucleotide sequence shown in SEQ ID NO:1 and a reverse primer with the nucleotide sequence shown in SEQ ID NO:2. The primers for amplifying the IL-10 gene included a forward primer with the nucleotide sequence shown in SEQ ID NO:3 and a reverse primer with the nucleotide sequence shown in SEQ ID NO:4. The template for amplifying the PD-L1 and IL-10 gene fragments was a cDNA library obtained by reverse transcription of total RNA from mouse spleen. This application does not impose any special limitations on the homologous recombination technology; homologous recombination technology well known in the art can be used.

[0053] In this application, the recombinant viral vector also includes an IRES region. The IRES region is located between the coding sequence of the chimeric antigen receptor targeting fibroblast activation protein α-positive cells and CTLA4-Ig, between CTLA4-Ig and PD-L1, and between PD-L1 and IL-10. The nucleotide sequence of the IRES region is shown in SEQ ID NO:26. The IRES region can recruit ribosomes to translate the mRNA.

[0054] In this embodiment, CP-IL10-CAR-dendritic cells were prepared using a lentiviral vector, specifically the pLenti CMV GFP Pmlmlmluro (Addgene, Plasmid#17448) vector. During cloning, the cloning sites for the exogenous gene preferably include AgeI and AscI.

[0055] In this application, the recombinant viral vector preferably further includes sequencing verification to obtain a second recombinant viral vector containing the expected target fragment for subsequent viral packaging.

[0056] This application does not impose any particular limitation on the method of viral packaging; any viral packaging method well known in the art can be used. In the embodiments of this application, the recombinant viral vector, PMD2, and PSPAX2 are mixed, and after adding transfection reagent, host cells are treated. After the cells develop pathopathic effects, the supernatant is collected, and the virus is concentrated to obtain recombinant lentivirus. The mass ratio of the recombinant viral vector, PMD2, and PSPAX2 is 3:2:3. This application does not impose any particular limitation on the type of host cell; any host cell well known in the art can be used, such as 293FT cells.

[0057] After obtaining the recombinant lentivirus, this application infects dendritic cells with the recombinant virus to obtain CP-IL10-CAR-dendritic cells.

[0058] In this application, the density of the dendritic cells is preferably (1-10) × 10⁻⁶. 6 / well. The infection titer of the recombinant virus is preferably 10. During infection, 10 μg / mL of polybrene transfection reagent is added. The transfection time is preferably 22–26 h, but can be 24 h. After infection, the culture medium is replaced with fresh medium and cultured for 45–50 h, but can be 48 h. Flow cytometry was used to detect DC efficiency, CTLA4-Ig and PD-L1 expression, and CAR efficiency. The results showed that compared with CTL-DCs prepared from empty plasmid virus, PD-L1 expression was significantly increased in CP-IL10-CAR-DCs, and the co-expression efficiency of CAR and PD-L1 was approximately 40% or higher.

[0059] This application provides a drug for preventing and treating cardiac fibrosis, wherein the active pharmaceutical ingredient includes the CP-IL10-CAR-dendritic cells or the CP-IL10-CAR-dendritic cells prepared by the preparation method.

[0060] In this application, the dosage form of the drug preferably includes an injection solution and / or an injection powder. The injection solution further includes a 0.9% (w / w) sodium chloride aqueous solution. In the drug, the density of the CP-IL10-CAR-dendritic cells is not less than 1.0 × 10⁻⁶. 4 The number of cells / μL can be 1.2 × 10⁻⁶. 4 cells / μL ~ 2.0 × 10⁻⁶ 4 per μL.

[0061] This application provides the use of the CP-IL10-CAR-dendritic cells or the CP-IL10-CAR-dendritic cells prepared by the preparation method in the preparation of drugs for the prevention and / or treatment of cardiac fibrosis.

[0062] In this application, the cardiac fibrosis preferably includes at least one of the following disease inductions: myocardial infarction, aortic arch coarctation, and myocardial ischemia-reperfusion. In this application, the treatment of cardiac fibrosis preferably includes at least one of the following aspects: downregulating the expression levels of cardiac fibrosis-related proteins, regulating the expression levels of cardiac inflammatory factors, reducing the degree of cardiac fibrosis, and improving cardiac function. In this application, the cardiac fibrosis-related proteins include at least one of the following: α-SMA, Collagen-I, and Preiostin. Regulating the expression levels of cardiac inflammatory factors includes downregulating the expression levels of pro-inflammatory factors and / or increasing the expression levels of anti-inflammatory factors. The pro-inflammatory factors include at least one of the following: IL-6, MCP-1, TNF-α, SNRPE, CXCR2, IL2RB1, CXCL9, and NIRC5. The anti-inflammatory factors include at least one of the following: IL-10, FOXP3, CAR8, TFRC, Slc41a3, and CD274.

[0063] The following detailed description, in conjunction with embodiments, illustrates an immunosuppressive dendritic cell, its preparation method, and its application provided in this application. However, these descriptions should not be construed as limiting the scope of protection of this application.

[0064] Example 1

[0065] A method for preparing CP-IL10-CAR-DCs

[0066] 1. Construction methods of mouse-derived CTLA4-Ig gene, PD-L1 gene, IL-10 gene and FAP CAR overexpression vector

[0067] 1. RNA was extracted from the spleen of normal mice after grinding, and then reverse transcribed to obtain a cDNA library. This cDNA library was used as a PCR template to design PCR amplification primers for the mouse PD-L1 and IL-10 genes. The specific sequences are as follows:

[0068] PD-L1 F:

[0069] PD-L1R:

[0070] IL-10F:

[0071] IL-10R:

[0072] The mouse-derived CTLA4-Ig sequence and the scFv sequence of FAP were synthesized by Guangzhou Aiji Biotechnology Co., Ltd. The nucleotide sequence encoding CTLA4-Ig is shown in SEQ ID NO:21, and the nucleotide sequence of the scFv sequence of FAP is as follows:

[0073]

[0074] 2. Preparation method and cell infection of murine lentivirus overexpressing CTLA4-Ig, PD-L1, and IL-10 genes.

[0075] The pLenti CMV FAP CAR IRES CTLA4-Ig IRES PD-L1 IRES IL-10Puro plasmids prepared in step 1 above were extracted using a plasmid extraction kit and then packaged as lentiviruses. Specifically, 293FT culture medium was placed in a 15cm culture dish. When the density reached 80%–90%, the medium was replaced with fresh culture medium in an incubator 2 hours before lentivirus packaging. For virus packaging, 1mL of serum-free opti-MEM (Thermo Fisher) medium was placed in a 1.5mL EP tube, and 10μg PMD2, 15μg PSPAX2, and 15μg pLenti CMV FAP CAR IRES CTLA4-Ig IRES PD-L1 IRES IL-10Puro or pLenti CMV IRES Puro plasmid (control plasmid) were added and mixed thoroughly by inverting the tube. Then add 58 μL of 1 mg / mL PEI transfection reagent, shake rapidly on a shaker for 10 s, and incubate at room temperature in the dark for 15 min. Then add to the above 293FT cells, and replace with fresh culture medium after 8 h. Collect the supernatant 72 h after medium replacement, centrifuge, add virus concentrate, concentrate overnight at 4℃, centrifuge to collect the precipitate, and obtain lentivirus overexpressing CP-IL10-CAR or empty vector lentivirus (control lentivirus), which is then frozen at -80℃ for later use. For viral infection, DCs are prepared according to a 10... 6 / wells were cultured in a low-adsorption six-well plate, and 150 μl of 1×10⁻⁶ solution was added. 7 Cells were infected with PFU / mL virus solution and 10μg / mL polybrene for 24 hours, then replaced with fresh culture medium. Cells were collected for use 48 hours after the medium change.

[0076] 3. Flow cytometry analysis of DC efficiency, CP expression, and CAR efficiency

[0077] After 48 hours of viral infection, the CP-IL10-CAR-DCs obtained above were collected in 1.5 mL EP tubes, with CTL-DCs used as a control. The cells were stained with flow cytometry antibodies, washed twice with pre-cooled PBS, resuspended in PBS, and transferred to flow cytometry tubes for flow cytometry analysis. The results were analyzed using Flowjo software.

[0078] 4. Detection results of PD-L1 and CAR expression in CP-IL10-CAR-DCs drugs

[0079] The results are shown in Figure 1. Flow cytometry analysis showed that the co-expression of CAR and PD-L1 was significantly increased in CP-IL10-CAR-DCs compared to CTL-DCs. Flow cytometry analysis using the G4S linker, a special structure in the CAR structure, revealed that the CAR expression efficiency was over 40%, while the CAR expression efficiency in CTL-DCs was only about 7.5%.

[0080] Example 2

[0081] Preparation methods of CTL-DCs or CP-IL10-CAR-DCs drugs

[0082] 1. Ingredient ratio: The concentration of CTL-DCs or CP-IL10-CAR-DCs prepared in Example 1 was 1.5 × 10⁻⁶ cells. 6 125 μL, sodium chloride injection (0.9%).

[0083] 2. Preparation Method: Male C57 / B6 mice were purchased and euthanized under anesthesia. Bone marrow from the tibia and femur was collected and cleaved. The marrow was then cultured for one week in RMPI-1640 complete medium containing recombinant mouse GM-CSF (20 ng / mL) and IL-4 (10 ng / mL) to obtain mouse dendritic cells (DCs). The efficiency of DCs was assessed using an anti-mouse CD11c flow cytometry antibody. Cells were infected with an empty vector lentivirus, and after 48 hours, cells were collected to obtain CTL-DCs. Alternatively, mouse DCs were infected with an overexpressing CP-IL10-CAR lentivirus, and after 48 hours, cells were collected to obtain CP-IL10-CAR-DCs. The collected CTL-DCs or CP-IL10-CAR-DCs were counted and suspended in 0.9% sodium chloride solution, then aliquoted and ready for use.

[0084] Example 3

[0085] The therapeutic effect of CP-IL10-CAR-DCs on ischemia-reperfusion-induced cardiac fibrosis

[0086] 1. Experimental Materials

[0087] 1.1 Laboratory Animals

[0088] C57BL / 6 mice, SPF grade, 6–8 weeks old, male; purchased from Shanghai Slack Laboratory Animal Co., Ltd.

[0089] 1.2. Cell therapy drugs

[0090] Take the CTL-DCs or CP-IL10-CAR-DCs drugs obtained in Example 2.

[0091] 1.3 Main Reagents

[0092] 0.9% sodium chloride solution (Sigma-Aldrich, USA); phosphate buffered saline (PBS, Gibco, USA); dimethyl sulfoxide (Gibco, USA); anti-α-SMA antibody, Collagen-1 antibody, Periodin antibody, Fibronectin antibody (Abcam, UK); anti-β-actin antibody (CST Biotech, USA); RIPA (Thermo Fisher Scientific, USA); BCA protein quantification kit (Beyotime Biotechnology Co., Ltd., China); anti-PD-L1 antibody, G4S-linker antibody (BD Biotech, USA); secondary antibody (goat anti-rabbit, CST Biotech, USA); PrimerScript TM RT reagent kit with gDNA Eraser reverse transcription kit (Dalian Takara Bio Co., Ltd.); Premix Ex Taq TM II (Dalian Takara Bio Inc.); all other reagents were domestically produced analytical grade.

[0093] 1.4 Main Instruments

[0094] Clean bench, CO2 incubator (Thermo Scientific, USA); inverted microscope (Chongqing Aote Optical Instrument Co., Ltd.); low-speed centrifuge, high-speed centrifuge (Eppendorf, Germany); ChemiDoc™ Touch imaging system, electrophoresis apparatus (Bio-Rad, USA); real-time PCR instrument (Roche).

[0095] 2. Methods

[0096] 2.1 Establishment, grouping, and intervention of a model of ischemia-reperfusion-induced cardiac fibrosis

[0097] Forty male C57BL / 6 mice aged 6–8 weeks were used. Thirty of these mice were anesthetized with atropine and sodium pentobarbital before surgery, and connected to a ventilator for stabilization before undergoing thoracotomy. After locating the left anterior descending coronary artery, a 7-0 atraumatic suture needle was inserted through the myocardial surface about 2 mm from the lower edge of the left atrial appendage and exited near the conus medullaris. After stabilization for 15 minutes, a polyethylene tube about 2 mm long was placed at the ligation site on the heart surface and ligated with a slipknot. After 45 minutes of ligation, the polyethylene tube was removed, and the left anterior descending coronary artery was reopened, and myocardial tissue reperfusion was restored, thus establishing a mouse model of myocardial ischemia-reperfusion. The other 10 mice served as a control group and underwent only thoracotomy.

[0098] Thirty model mice were randomly divided into a model group, a CTL-DCs group, and a CP-IL10-CAR-DCs group, with 10 mice in each group. The CTL-DCs group and the CP-IL10-CAR-DCs group received a tail vein injection of 125 μl of a 1.2 × 10⁻⁶ CTL-DCs solution on the third day after modeling. 4 The drug obtained in Example 2 was administered via tail vein injection to both the control and model groups. Echocardiography was performed on mice on day 1 before modeling and on days 3, 7, 14, and 28 after modeling to assess changes in cardiac function. Mice were euthanized by cervical dislocation on day 28 after modeling, and their tissues were used for the following experiments.

[0099] 2.2 Protein Immunoblotting

[0100] 2.2.1 Protein extraction and quantification

[0101] Heart tissue was collected and lysed with an appropriate amount of RIPA (containing 1% PMSF) lysis buffer to extract total protein. The tissue was incubated on ice for 30 min. The tissue was then centrifuged at 12000 rpm for 30 min, and the supernatant was transferred to a new centrifuge tube. 2 μL of the supernatant was used for protein quantification. The remaining protein was added to an appropriate amount of loading buffer, mixed well, and then heated at 100℃ for denaturation for 5 min, and stored at -80℃. The protein quantification process was as follows: The sample was diluted 50-fold with lysis buffer, and 100 μL was added to a 96-well cell culture plate. Following the instructions of the BCA protein concentration assay kit, solutions A and B (volume ratio of 1:50) were added to centrifuge tubes and gently pipetted to prepare a mixture. 100 μL of the mixture was added to each well. The plate was covered with a film and incubated at 37℃ for 30 min. The absorbance was measured at 490 nm using a microplate reader. A standard curve was plotted based on the absorbance values, and the protein concentration of each sample was calculated.

[0102] 2.2.2 Protein electrophoresis and Western blotting

[0103] Prepare a 10% separating gel. After the separating gel solidifies, prepare a stacking gel and add it to the top layer of the separating gel. Insert a comb and gently remove the comb after the gel solidifies. Add protein markers and samples to each well of a 10% SDS-PAGE membrane. Electrophore at 80V for 30 min, then at 120V for 1.5 h. Transfer the membrane (nitrocellulose membrane) at constant voltage (100V) and room temperature for 90 min. Remove the membrane, label both sides, and block with 5% BSA at room temperature for 2 h. Wash three times with 1×TBS-T for 5 min each time. Dilute the antibody to the working concentration with 5% BSA according to the antibody instructions and hybridize with the nitrocellulose membrane overnight at 4℃. Wash three times with 1×TBS-T for 5 min each time. Dilute the secondary antibody 1:1000 and hybridize at room temperature for 1 h. Wash three times with 1×TBS-T for 5 min each time. Expose the membrane. Scan the membrane and calculate the gray values ​​of each protein band using Image J (v 1.45) software.

[0104] 2.3 Real-time quantitative PCR

[0105] 2.3.1 Total RNA extraction and reverse transcription

[0106] Take a small amount of tissue, grind it, and add an appropriate amount of trizol for lysis. Incubate at room temperature for 3 minutes until liquefaction, then transfer the lysate to a clean, enzyme-free microcentrifuge tube. Add 0.2 mL of pre-chilled chloroform, quickly and vigorously invert to mix, and incubate on ice for 5 minutes to ensure complete lysis of the protein complex. Centrifuge (4℃, 13000g, 15 minutes). Aspirate the upper aqueous phase and transfer it to another enzyme-free microcentrifuge tube. Add 400 μL of isopropanol, quickly and vigorously invert to mix, and incubate at -20℃ for 30 minutes to precipitate RNA. Centrifuge (4℃, 13000g, 10 minutes). A small amount of white precipitate will be visible at the bottom of the tube. Discard the supernatant, add 1 mL of 70% ethanol, invert to mix, wash to remove impurities, and incubate on ice for 5 minutes. Centrifuge (4℃, 7600g, 5min), remove as much ethanol as possible, and dry at room temperature for 5min until the residual ethanol has completely evaporated and the RNA precipitate is clear. Finally, add 30μL of nuclease-free water to dissolve the RNA. Take 1μL of RNA to determine the RNA concentration and purity, OD. 260 / 280 The ratio is between

[0107] A value between 1.8 and 2.0 indicates good RNA quality. Then, cDNA was synthesized according to the procedure in the reverse transcription kit instructions and used as a template for real-time quantitative PCR.

[0108] 2.3.2 Real-time quantitative PCR (qPCR detection)

[0109] The 15 μL real-time reaction system contained: 7.8 μL SYBR Green (5×), 0.6 μL each of forward and reverse primers (10 μmol / L), 1.5 μL cDNA diluted 1:15, 4.5 μL H2O, and 0.3 μL ROX. The reaction program on the real-time PCR instrument was set to 95℃ for 30 s; 60℃ for 30 s, 40 cycles; and 72℃ for 90 s. Each sample was measured three times. The relative amount of mRNA was expressed as Ct value, and the average relative expression level was determined by 2... －△△Ct Analysis of calculation methods.

[0110] The primer sequences used are shown in Table 1.

[0111] Table 1 Primer sequences for qPCR detection

[0112] 2.3.3 Next-generation sequencing

[0113] After anesthesia, the infarcted area of ​​the mouse heart was harvested, preserved in liquid nitrogen, and sent to Novogene for second-generation BulkRNAseq. Bioinformatics analysis was performed on the raw data, using software tools including FastQC (raw data quality assessment) and Trimmomatic (removal of low-quality bases and adapter sequences). Sequence alignment and variant detection were performed using STAR (transcriptomics), with DESeq2 used for differential expression analysis, and differential expression thresholds (e.g., |log2FC|>1 and FDR<0.05).

[0114] 2.4 Staining tissue with Sirius red, the specific experimental steps are as follows:

[0115] 1) Tissue fixation: The heart was immersed in a 4% paraformaldehyde solution and then subjected to routine dehydration and embedding.

[0116] 2) Slice to 3-6 μm, bake the slices and then dewax to water as usual.

[0117] 3) Use a histochemical pen to outline the tissue contour. Then stain with Sirius red staining solution for 30-60 minutes, and rinse slightly with running water.

[0118] 4) Starting with 75% ethanol, after a series of dehydration steps, the film is finally cleared with xylene and sealed with neutral resin.

[0119] 2.5 Mouse cardiac ultrasound

[0120] 1) Echocardiography was performed on mice using the Vevo3100 small animal ultrasound system before ischemia-reperfusion surgery and on days 3, 7, 14 and 28 after surgery.

[0121] 2) Remove hair from the mouse's chest with depilatory cream at least one day in advance to expose the skin on the left side of the heart.

[0122] 3) Place the mice on a 37°C constant temperature plate and administer gas anesthesia, then record their physiological state.

[0123] 4) Determine the position of the apex and left ventricular outflow tract by observing the imaging images, and measure on the long axis of the left ventricle to ensure alignment of the apex and the outflow tract. Next, rotate the probe 90° counter-clockwise to locate the papillary muscles and acquire short-axis ultrasound images. The acquired images should include at least five cardiac cycles. Measure the required data using the accompanying instrument analysis software.

[0124] 2.6 Statistical Methods

[0125] Image data were processed and analyzed using ImageJ software, while other data were statistically processed and analyzed using SPSS 17.0 software. The difference was considered statistically significant if p < 0.05.

[0126] 3. Results

[0127] 3.1 Effects of CP-IL10-CAR-DCs on Cardiac Function

[0128] The results are shown in Figure 2. Echocardiographic results indicated that, compared with the CTL-DCs and the model group, the cardiac function of mice injected with CP-IL10-CAR-DCs was significantly improved after modeling, with an ejection fraction (LVEF) improvement of approximately 12% compared to the model group and approximately 8% compared to the CTL-DCs group.

[0129] 3.2 Effects of CP-IL10-CAR-DCs on cardiac tissue fibrosis

[0130] The results are shown in Figure 3. Sirius red staining results showed that the cardiac tissue structure of the untreated group mice was clear. The cardiac tissue of the model group and the CTL-DCs group mice showed sheet-like collagen fibers, indicating diffuse cardiac fibrosis. The cardiac tissue of the CP-IL-10-CAR-DCs group mice showed fibrosis, but the degree of fibrosis was significantly reduced compared to the model group and the CTL-DCs group. No diffuse fibrosis was observed in the CP-IL10-CAR-DCs group; the lesions were focally distributed, and the degree of fibrosis was less than that in the CTL-DCs group.

[0131] 3.3 Effects of CP-IL10-CAR-DCs on the expression of fibrosis-related proteins in cardiac tissue

[0132] The results are shown in Figure 4. Compared with the normal control group, the expression levels of cardiac fibrosis marker proteins in the model group mice were significantly increased, indicating that the mouse cardiac fibrosis model was successfully established. Compared with the model group and the CTL-DCs group, the overall cardiac fibrosis protein expression levels in the CP-IL10-CAR-DCs group mice were significantly decreased, indicating that the CP-IL10-CAR-DCs drug can effectively alleviate cardiac fibrosis.

[0133] 3.4 Effects of CP-IL10-CAR-DCs on Inflammation-Related Genes in Cardiac Tissue

[0134] As shown in Figure 5, compared with the control group, the mRNA expression levels of various inflammatory chemokines in the cardiac tissue of mice in the model group and CTL-DCs group were significantly increased, indicating that low-dose CTL-DCs had a limited effect on alleviating cardiac inflammation. Compared with the model group and CTL-DCs group, the expression of IL-6, MCP-1, and TNF-α in the cardiac tissue of mice in the CP-IL-10-CAR-DCs group was significantly downregulated, while the expression of inhibitory factors IL-10 and FOXP3 was increased (*p<0.05, **p<0.01, ***p<0.001), indicating that CP-CAR-DCs play an important role in the treatment of cardiac fibrosis and inflammation, enhancing the therapeutic effect.

[0135] As shown in Figure 6, compared with the control group, the expression of most pro-inflammatory genes in the heart, such as TNF, Il12rb1, H2-Q6, and Cxcr2, decreased after treatment in the CP-IL-10-CAR-DCs group, while the expression of inhibitory factors IL-10, FOXP3, and CD274 increased. This indicates that CP-CAR-DCs play an important role in the treatment of cardiac fibrosis and inflammation, enhancing the therapeutic effect.

[0136] 5. Conclusion

[0137] In a mouse model of ischemia-reperfusion-induced cardiac fibrosis, low-dose CP-IL10-CAR-DCs significantly improved cardiac function and symptoms of cardiac fibrosis, and downregulated the expression of fibrotic proteins in cardiac tissue. Furthermore, CP-IL10-CAR-DCs played a crucial role in treating the inflammatory process of cardiac fibrosis, specifically by downregulating the expression of IL-6, MCP-1, and TNF-α genes in cardiac tissue and upregulating the expression of genes such as IL10 and FOXP3. In contrast, equal doses of CTL-DCs showed no effect on improving cardiac fibrosis. Therefore, CP-IL10-CAR-DCs can reduce the dosage of DCs and enhance the therapeutic effect in the treatment of cardiac fibrosis.

[0138] Example 4

[0139] The therapeutic effect of CP-IL10-CAR-DCs on cardiac fibrosis caused by acute myocardial infarction

[0140] 1. Experimental Materials

[0141] 1.1 Laboratory Animals

[0142] C57BL / 6 mice, SPF grade, 6–8 weeks old, male; purchased from Shanghai Slack Laboratory Animal Co., Ltd.

[0143] 1.2. Cell therapy drugs

[0144] Take the CTL-DCs or CP-IL-10-CAR-DCs drugs obtained in Example 1.

[0145] 1.3 Main reagents: Same as in Example 3;

[0146] 1.4 Main instruments: Same as in Example 3;

[0147] 2. Methods

[0148] 2.1 Establishment, grouping, and intervention of a cardiac fibrosis model

[0149] Forty male C57BL / 6 mice aged 6-8 weeks were used. Thirty of these mice were anesthetized preoperatively with atropine and sodium pentobarbital, along with muscle relaxants such as toluidine. After stabilization via mechanical ventilation, thoracotomy was performed. The left anterior descending coronary artery was located, and a 7-0 atraumatic suture needle was used to puncture the myocardium approximately 2 mm below the lower edge of the left atrial appendage, exiting near the conus medullaris for ligation. The chest was closed, the skin was sutured, and the mice were placed on a heated blanket to await resuscitation, thus establishing an acute myocardial infarction mouse model. The remaining 10 mice served as a control group and underwent only thoracotomy. The 30 model mice were randomly divided into a model group, a CTL-DCs group, and a CP-IL10-CAR-DCs group, with 10 mice in each group. On the third day after modeling, the CTL-DCs and CP-IL10-CAR-DCs groups received a tail vein injection of 125 μl of a 1.2 × 10⁻⁶ concentration. 4 The drug obtained in Example 2 was administered via tail vein injection to both the control and model groups. Echocardiography was performed on mice on day 1 before modeling and on days 3, 7, 14, and 28 after modeling to assess changes in cardiac function. Mice were euthanized by cervical dislocation on day 28 after modeling and their tissues were collected for the experiment. Other experimental methods are described in Example 3.

[0150] 3. Results

[0151] 3.1 Effects of CP-IL10-CAR-DCs on Cardiac Function

[0152] The results are shown in Figure 7. Echocardiography results indicated that, compared with the CTL-DCs and the model group, the cardiac function of mice injected with CP-IL10-CAR-DCs was significantly improved after modeling, with an ejection fraction (LVEF) improvement of approximately 11% compared with the model group and approximately 9% compared with the CTL-DCs group.

[0153] 3.2 Effects of CP-IL10-CAR-DCs on cardiac tissue fibrosis

[0154] The results are shown in Figure 8. Sirius red staining results showed that the cardiac tissue structure of the untreated group mice was clear. The cardiac tissue of the model group and the CTL-DCs group mice showed sheet-like collagen fibers, indicating diffuse cardiac fibrosis. The cardiac tissue of the CP-IL10-CAR-DCs group mice showed fibrosis, but the degree of fibrosis was significantly reduced compared to the model group and the CTL-DCs group. No diffuse fibrosis was observed in the CP-IL10-CAR-DCs group; the lesions were focally distributed, and the degree of fibrosis was less than that in the CTL-DCs group.

[0155] 3.3 Effects of CP-IL10-CAR-DCs on the expression of fibrosis-related proteins in cardiac tissue

[0156] The results are shown in Figure 9. Compared with the normal control group, the expression levels of cardiac fibrosis marker proteins in the model group mice were significantly increased, indicating that the mouse cardiac fibrosis model was successfully established. Compared with the model group and the CTL-DCs group, the overall cardiac fibrosis protein expression levels in the CP-CAR-DCs group mice were significantly decreased (***, p < 0.001, **, p < 0.01), indicating that CP-IL10-CAR-DCs drugs can effectively alleviate cardiac fibrosis.

[0157] 4. Conclusion

[0158] Acute myocardial infarction (AMI) is a common and fatal cardiovascular disease, with approximately 7 million new cases worldwide each year. Following AMI, due to impaired cardiac contractile function caused by cardiac fibrosis, about 20.4% of patients develop heart failure. In a mouse model of AMI-induced cardiac fibrosis, intervention with CP-IL10-CAR-DCs significantly improved cardiac function and reduced symptoms of cardiac fibrosis, downregulating the expression of fibrotic proteins in cardiac tissue. However, equal doses of CTL-DCs showed no effect on improving cardiac fibrosis. Therefore, CP-IL10-CAR-DCs can reduce the dosage of DCs and enhance therapeutic efficacy in the treatment of cardiac fibrosis.

[0159] Example 5

[0160] The therapeutic effect of CP-IL10-CAR-DCs on cardiac fibrosis caused by aortic arch coarctation

[0161] 1. Experimental Materials

[0162] 1.1 Laboratory Animals

[0163] C57BL / 6 mice, SPF grade, 6–8 weeks old, male; purchased from Shanghai Slack Laboratory Animal Co., Ltd.

[0164] 1.2. Cell therapy drugs

[0165] Take the CP-CAR-DCs and CTL-DCs obtained in Example 1.

[0166] 1.3 Main reagents: Same as in Example 3;

[0167] 1.4 Main instruments: Same as in Example 3;

[0168] 2. Methods

[0169] 2.1 Establishment, grouping, and intervention of a cardiac fibrosis model

[0170] Forty male C57BL / 6 mice aged 6-8 weeks were used. Thirty of these mice were anesthetized preoperatively with atropine and sodium pentobarbital, along with a muscle relaxant such as toluidine. After anesthesia, the mice's chest was shaved and the skin prepared. The mice were fixed supine on the operating table with medical tape (head facing right, laterally towards the operator). A transverse incision was made parallel to the midpoint of the forearm. The sternum was transversely severed between the first and second ribs, with the incision just wide enough to interrupt the sternum; a larger incision could easily sever large blood vessels. After opening the thoracic cavity with a chest expander, the thymus was exposed by separating the muscle layers. The aortic arch and its branches beneath the thymus tissue could be clearly observed through blunt dissection. A suture was threaded through a small opening in the fascia between the aortic arch, brachiocephalic trunk, and left common carotid artery. A 27G needle was used, and after ligation, the needle was withdrawn to narrow the aortic arch. The chest was closed, the skin was sutured, and the mice were placed on a heated blanket to await recovery. Thirty mice with modeling were randomly divided into a model group, a CTL-DCs group, and a CP-IL10-CAR-DCs group, with 10 mice in each group. The CTL-DCs group and the CP-IL10-CAR-DCs group received a tail vein injection of 125 μl of a 1.2 × 10⁻⁶ CTL-DCs solution at a concentration of 1.2 × 10⁻⁶ CTL-DCs at week four after modeling. 4 The drug obtained in Example 2 was administered via tail vein injection to both the control and model groups. Echocardiography was performed on mice before TAC and at 2, 4, 6, 8, and 12 weeks post-operation using a Vevo2000 small animal ultrasound system. Mice were euthanized by cervical dislocation at week 12 post-modeling for experimental use. Other experimental methods are described in Example 3.

[0171] 3. Results

[0172] 3.1 Effects of CP-IL10-CAR-DCs on Cardiac Function

[0173] The results are shown in Figure 10. Echocardiographic results indicated that, compared with the CTL-DCs and the model group, the cardiac function of mice injected with CP-IL10-CAR-DCs was significantly improved after modeling. At week 12 of modeling, the left ventricular ejection fraction (LVEF) was about 23% better than that of the model group and about 13% better than that of the CTL-DCs group.

[0174] 3.2 Effects of CP-IL10-CAR-DCs on cardiac tissue fibrosis

[0175] The results are shown in Figure 11. Sirius red staining results showed that the cardiac tissue structure of the untreated group mice was clear. The cardiac tissue of the model group and the CTL-DCs group mice showed sheet-like collagen fibers, indicating diffuse cardiac fibrosis. The cardiac tissue of the CP-IL10-CAR-DCs group mice showed fibrosis, but the degree of fibrosis was significantly reduced compared to the model group and the CTL-DCs group. No diffuse fibrosis was observed in the CP-IL10-CAR-DCs group; the lesions were focally distributed, and the degree of fibrosis was less than that in the CTL-DCs group.

[0176] 3.3 Effects of CP-IL10-CAR-DCs on the expression of fibrosis-related proteins in cardiac tissue

[0177] The results are shown in Figure 12. Compared with the normal control group, the expression levels of cardiac fibrosis marker proteins in the model group mice were significantly increased, indicating that the mouse cardiac fibrosis model was successfully established. Compared with the model group and the CTL-DCs group, the overall cardiac fibrosis protein expression levels in the CP-CAR-DCs group mice were significantly decreased (***, p < 0.001, **, p < 0.01), indicating that CP-IL10-CAR-DCs drugs can effectively alleviate cardiac fibrosis.

[0178] 4. Conclusion

[0179] Dilated cardiomyopathy (DC) is a myocardial disease characterized by ventricular dilation and decreased systolic function. Epidemiological studies show that it can occur at any age, but is more common in young adults. Recent studies have found a close relationship between DC and myocardial fibrosis, which can lead to further deterioration of cardiac structure and function. The formation of fibrosis is usually associated with inflammation, ischemia, or genetic factors, which not only exacerbate ventricular dilation but may also cause electrophysiological abnormalities, increasing the risk of heart failure and arrhythmias. In animal models, aortic arch coarctation surgery can effectively simulate this pathophysiological process. Here, we demonstrate that intervention with a CP-CAR-DC combination significantly improves cardiac function symptoms caused by aortic arch coarctation and downregulates the expression level of fibrosis proteins in cardiac tissue, thereby effectively delaying the progression of DC and its cardiac fibrosis.

[0180] Example 6

[0181] The therapeutic effect of allogeneic CP-IL10-CAR-DCs on ischemia-reperfusion-induced cardiac fibrosis

[0182] 1. Experimental Materials

[0183] 1.1 Laboratory Animals

[0184] C57BL / 6 mice, SPF grade, 6–8 weeks old, male; purchased from Shanghai Slack Laboratory Animal Co., Ltd.

[0185] 1.2. Cell therapy drugs

[0186] The CP-IL10-CAR-DCs drug obtained from the Balb / c example was used.

[0187] 1.3 Main reagents: Same as in Example 3; (Reagent: BD) TM Cytometric Bead Array(CBA)Mouse Enhanced Sensitivity Master Buffer Kit 562248

[0188] 2. The microsphere-based cytokine detection CBA method is as follows:

[0189] 2.1 Open all the standard samples (lyophilized microspheres) and transfer all the standard microspheres to the same 15mL conical centrifuge tube;

[0190] 2.2 Add 4 mL of Assay Diluent to the centrifuge tube;

[0191] 2.3 It needs to be allowed to stand at room temperature for at least 15 minutes to reach equilibrium;

[0192] 2.4 Gently mix the standard with a pipette; do not vortex or shake violently.

[0193] 2.5 Mark several 12×75mm centrifuge tubes with “Top, 1:3, 1:9, 1:27, 1:81, 1:243 and 1:729”;

[0194] 2.6 Use a pipette to transfer 460 μL of Assay Diluent into a Top tube;

[0195] 2.7 Use a pipette to transfer 400 μL of Assay Diluent into all remaining 12 × 75 mm centrifuge tubes;

[0196] 2.8 Pipette 40 μL of the standard solution into a Top tube. Gently vortex to mix for no more than 3 seconds;

[0197] 2.9 Transfer 200 μl of diluent from the Top tube to a 1:3 tube; transfer 200 μl of standard diluent from the 1:3 tube to a 1:9 tube... and so on up to the 1:729 tube;

[0198] 2.10 Prepare another tube containing Assay Diluent as a negative control at 0 fg / mL;

[0199] 2.11 Determine the number of samples to be tested and the types of freely assortable cytokines (to avoid insufficient liquid due to multiple sample additions during the experiment, it is recommended to mix the amounts of 2-3 samples).

[0200] 2.12 Vortex each Capture Bead tube for at least 15 seconds to thoroughly resuspend the beads;

[0201] 2.13 Determine the total amount of capture microspheres required for dilution in the experiment, based on 20 μl / sample;

[0202] 2.14 Determine the volume of each type of capture microsphere at a rate of 1 μl / sample;

[0203] 2.15 Determine the volume of the Capture Bead Diluent: Total number of captured microspheres - Factor number × Volume of captured microspheres = Volume of Capture Bead Diluent;

[0204] 2.16 Mix various microspheres and microsphere dilution solutions in the same test tube and label it "Mixed Microspheres" (prepare fresh and do not store after mixing);

[0205] 2.17 Dilute the sample with Assay Diluent at an appropriate ratio (e.g., 1:10 or 1:100). Serum samples should generally be diluted at least 1:3.

[0206] 2.18 Mix the sample diluent thoroughly before transferring the sample to a flow cytometer containing the trapping microspheres;

[0207] 2.19 To facilitate analysis in FCAP Array software, it is recommended to collect consecutive standards from highest to lowest concentration or from lowest to highest concentration.

[0208] Note: You can use the same numbers calculated as for "Hybrid Capture Microspheres";

[0209] 2.20 Determine the number of samples to be tested, identify the types of freely combinable cytokines, and prepare extra antibodies to ensure that each sample has sufficient antibodies;

[0210] 2.21 Determine the total volume of diluted mouse detection antibody (Part A) required for the experiment. Each sample requires 20 μl of diluted mouse detection antibody.

[0211] 2.22 Determine the required volume of each mouse detection antibody (Part A), with 1 μl of high-concentration detection antibody required for each sample;

[0212] 2.23 Determine the amount of Detection Reagent Diluent A required to detect antibody A;

[0213] 2.24 After mixing all antibody A and antibody dilution solution A, store at 4°C protected from light until use;

[0214] 2.25 Open a small vial of lyophilized Detection Reagent (Part B) (50 Tests);

[0215] 2.26 Add 0.55 mL (550 μL) of antibody dilution buffer B;

[0216] 2.27 Incubate in the dark at room temperature for 15 minutes; then gently mix by blowing air up and down.

[0217] 2.28 Add 4.5 mL of antibody dilution solution B to a 15 mL centrifuge tube;

[0218] 2.29 Add 0.5 mL of the dissolved Detection Reagent (Part B) to the above 15 mL centrifuge tube, vortex gently, and store at 4°C, protected from light, until use;

[0219] 2.30 Add 50 μL of standard diluent to the first eight flow cytometry tubes. Add 50 μL of each unknown sample to the remaining flow cytometry tubes;

[0220] 2.31 Vortex the mixed trapping microspheres for at least 5 seconds; add 20 μL of the mixed trapping beads to each of the above flow cytometry tubes and mix gently;

[0221] 2.32 Incubate at room temperature in the dark for 2 hours;

[0222] 2.33 Add 20 μL of the mixed detection antibody A to each flow cytometry tube;

[0223] 2.34 Incubate at room temperature in the dark for 2 hours;

[0224] 2.35 Add 1 mL of wash buffer to each flow cytometer tube and centrifuge at 200 g for 5 minutes, then discard the supernatant;

[0225] 2.36 Add 100 μL of sensitivity-enhancing antibody B to each flow cytometry tube and mix gently;

[0226] 2.37°C, incubate at room temperature in the dark for 1 hour;

[0227] 2.38 Add 1 mL of wash buffer to each flow cytometer tube and centrifuge at 200 g for 5 minutes, then discard the supernatant;

[0228] 2.39 Add 300 μL of wash buffer to each flow cytometer tube and resuspend it before running.

[0229] 3. Treatment methods

[0230] The ischemia-reperfusion-induced cardiac fibrosis establishment and other experimental methods are the same as in Example 3;

[0231] 4. Results

[0232] 4.1 Allogeneic CP-IL10-CAR-DCs improve cardiac function

[0233] As shown in Figure 13, this trial evaluated the therapeutic effect of allogeneic cell therapy on cardiac function. The results were similar to those of autologous cell therapy. Compared with untreated myocardial ischemia-reperfusion model mice, the cardiac function of the CP-IL10-CAR-DCs treatment group was significantly improved by about 10% after modeling.

[0234] 4.2 Effects of allogeneic CP-IL10-CAR-DCs on cardiac tissue fibrosis

[0235] The results are shown in Figure 14. Sirius red staining results showed that the heart tissue of mice in the model group contained sheet-like collagen fibers, indicating diffuse cardiac fibrosis. The heart tissue of mice in the allogeneic CP-IL-10-CAR-DCs group also showed fibrosis, but the degree of fibrosis was significantly reduced compared to the model group. No diffuse fibrosis was observed in the CP-IL10-CAR-DCs group; the lesions were focally distributed, and the degree of fibrosis was less than that in the model group.

[0236] 4.3 Effects of allogeneic CP-IL10-CAR-DCs on the expression of fibrosis-related proteins in cardiac tissue

[0237] The results are shown in Figure 15. Compared with the model group, the expression level of fibrotic proteins in the overall cardiac tissue of mice in the allogeneic CP-IL10-CAR-DCs group was significantly reduced, indicating that CP-IL10-CAR-DCs can effectively alleviate cardiac fibrosis.

[0238] 4.4 Treatment with allogeneic CP-IL10-CAR-DCs did not induce a cytokine storm in mice.

[0239] The results are shown in Figure 16. The CBA results of serum samples from mice on day 28 showed that CP-IL10-CAR-DCs did not cause an increase in serum levels such as IFN-γ, IL-6, IL-17a, IL-10, IL-1β, and TNF, demonstrating the safety of CP-IL10-CAR-DCs.

[0240] 5. Conclusion

[0241] Allogeneic CP-IL10-CAR-DCs treatment significantly improved symptoms of myocardial ischemia-reperfusion fibrosis, downregulated the expression level of fibrotic proteins in cardiac tissue, and did not cause a cytokine storm, demonstrating good safety. The effectiveness of allogeneic cell therapy expands the clinical application value of this drug and provides new ideas and potential strategies for cell therapy of cardiac fibrosis.

[0242] The above description is only a preferred embodiment of this application. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of this application, and these improvements and modifications should also be considered within the scope of protection of this application.

Claims

1. A dendritic cell or CAR dendritic cell overexpressing CP-IL10, characterized in that, The dendritic cells or CAR dendritic cells overexpress CTLA4-Ig, PD-L1, and IL-10; The surface of the CAR dendritic cells also expresses a chimeric antigen receptor that targets fibroblast activation protein α-positive cells.

2. The dendritic cells or CAR dendritic cells overexpressing CP-IL10 according to claim 1, characterized in that, The amino acid sequence of the CTLA4-Ig is shown in SEQ ID NO: 17; The amino acid sequence of the PD-L1 is shown in SEQ ID NO: 18; The amino acid sequence of the IL-10 is shown in SEQ ID NO: 19; The amino acid sequence of the chimeric antigen receptor targeting fibroblast activation protein α-positive cells is shown in SEQ ID NO:

20.

3. The dendritic cells or CAR dendritic cells overexpressing CP-IL10 according to claim 1, characterized in that, The dendritic cells or CAR dendritic cells include autologous dendritic cells or allogeneic dendritic cells.

4. Dendritic cells overexpressing CP or CAR dendritic cells according to any one of claims 1 to 3, characterized in that, The dendritic cells or dendritic cells in the CAR dendritic cells may be derived from at least one of the following sources: peripheral blood cells, bone marrow cells, hematopoietic stem cells, embryonic stem cells, and induced pluripotent stem cells.

5. A method for constructing dendritic cells or CAR dendritic cells overexpressing CP-IL10 as described in any one of claims 1 to 4, characterized in that, The method for constructing dendritic cells overexpressing CP-IL10 includes the following steps: introducing CTLA4-Ig gene fragments, PD-L1 gene fragments and IL-10 gene fragments into dendritic cells to obtain dendritic cells overexpressing CP-IL10. The method for constructing CAR dendritic cells includes the following steps: introducing the coding sequences of CTLA4-Ig gene fragment, PD-L1 gene fragment, IL-10 gene fragment and chimeric antigen receptor targeting fibroblast activation protein α positive cells into dendritic cells to obtain CAR dendritic cells.

6. The construction method according to claim 5, characterized in that, The methods for introducing dendritic cells include at least one of the following: virus-mediated, lipid nanoparticle-mediated, and electroporation.

7. The construction method according to claim 6, characterized in that, The viruses used in the virus-mediated transmission include lentiviruses and / or adenoviruses.

8. A drug for preventing and treating cardiac fibrosis, characterized in that, The active pharmaceutical ingredient includes dendritic cells or CAR dendritic cells overexpressing CP-IL10 as described in any one of claims 1 to 4, or dendritic cells or CAR dendritic cells overexpressing CP-IL10 prepared by the preparation method described in claim 5 or 6.

9. The drug according to claim 8, characterized in that, The dosage forms of the drug include injection solutions and / or injection powders; In the drug, the density of dendritic cells or CAR dendritic cells overexpressing CP-IL10 is 1.2 × 10⁻⁶. 4 per μL.

10. The use of dendritic cells or CAR dendritic cells overexpressing CP-IL10 as described in any one of claims 1 to 4, or dendritic cells or CAR dendritic cells overexpressing CP-IL10 prepared by the preparation method described in claim 5 or 6, in the preparation of medicaments for the prevention and / or treatment of cardiac fibrosis.

11. The application according to claim 10, characterized in that, The cardiac fibrosis includes at least one of the following diseases: myocardial infarction, aortic arch coarctation, and myocardial ischemia-reperfusion.

12. A method for preventing and / or treating cardiac fibrosis, characterized in that, Dendritic cells or CAR dendritic cells overexpressing CP-IL10 as described in any one of claims 1 to 4, or dendritic cells or CAR dendritic cells overexpressing CP-IL10 prepared by the preparation method described in claim 5 or 6, are injected into patients with cardiac fibrosis.

13. The method according to claim 12, characterized in that, The cardiac fibrosis includes at least one of the following diseases: myocardial infarction, aortic arch coarctation, and myocardial ischemia-reperfusion.

14. The method according to claim 12, characterized in that, The treatment of cardiac fibrosis includes at least one of the following aspects: downregulating the expression level of cardiac fibrosis-related proteins, regulating the expression level of cardiac inflammatory factors, reducing the degree of cardiac fibrosis, and improving cardiac function.

15. The method according to claim 14, characterized in that, The cardiac tissue fibrosis-related proteins include at least one of the following: α-SMA, Collagen-I, and Preiostin.

16. The method according to claim 14, characterized in that, The regulation of cardiac tissue inflammatory factor expression levels includes downregulating pro-inflammatory factor expression levels and / or increasing anti-inflammatory factor expression levels.

17. The method according to claim 16, characterized in that, The pro-inflammatory factors include at least one of the following: IL-6, MCP-1, TNF-α, SNRPE, CXCR2, IL2RB1, CXCL9, and NIRC5.

18. The method according to claim 16, characterized in that, The anti-inflammatory factors include at least one of the following: IL-10, FOXP3, CAR8, TFRC, Slc41a3, and CD274.

Images