Medicine for treating myocardial ischemia-reperfusion injury or myocardial infarction and recombinant cell for expressing chimeric antigen receptor used by medicine

Abstract

The invention discloses a medicine for treating myocardial ischemia-reperfusion injury or myocardial infarction and a recombinant cell for expressing a chimeric antigen receptor. The invention provides an application of a recombinant macrophage for expressing a chimeric antigen receptor or a substance loaded with a nucleic acid molecule for coding the chimeric antigen receptor in preparation of drugs. The medicine is a medicine for treating myocardial ischemia reperfusion injury or / and myocardial infarction; the chimeric antigen receptor comprises an extracellular domain, a transmembrane domain and an intracellular domain. The invention provides a first preclinical evidence for treating the possibility of myocardial ischemia-reperfusion injury or / and myocardial infarction based on adoptive cell therapy of the chimeric antigen receptor macrophage.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine and relates to a drug for treating myocardial ischemia-reperfusion injury or myocardial infarction and a recombinant cell expressing a chimeric antigen receptor used therein. Background Art

[0002] Cardiovascular disease (CVD) is one of the leading causes of death worldwide and includes a variety of conditions, including myocardial infarction, heart failure, and arrhythmias. With the aging of society and changes in lifestyle, the incidence of CVD is increasing year by year. While traditional treatments for CVD, such as medication and interventional surgery, have improved patient outcomes to some extent, they still have numerous drawbacks, such as significant side effects and high recurrence rates.

[0003] In recent years, with the advancement of immunology and molecular biology, cell therapy has gradually become a new strategy for the treatment of cardiovascular diseases. The emergence of chimeric antigen receptor (CAR) technology, in particular, has provided new insights into the treatment of cardiovascular diseases. CAR technology uses genetic engineering to fuse specific antigen recognition domains with signaling domains to construct chimeric antigen receptors. These receptors are then introduced into immune cells, enabling them to specifically recognize and kill target cells.

[0004] Macrophages, key cells in the immune system, possess powerful phagocytic and antigen-presenting abilities and play a crucial role in the development and progression of cardiovascular disease. Introducing chimeric antigen receptors into macrophages, enabling them to specifically recognize and eliminate pathological cells, holds promise as a new therapeutic approach. However, the application of chimeric antigen receptor macrophages (CAR-Ms) in cardiovascular disease remains exploratory, and their preparation methods and mechanisms of action require further research and optimization.

[0005] Lipid nanoparticles (LNPs) are nanoscale particles composed of lipids and are widely used in drug delivery systems, especially for the delivery of nucleic acid (e.g., mRNA, siRNA) drugs and vaccines. They have attracted considerable attention in gene-based therapies due to their ease of preparation, high biocompatibility, payload capacity, and ability to support repeated dosing. Summary of the Invention

[0006] The purpose of the present invention is to provide a drug for treating myocardial ischemia-reperfusion injury or myocardial infarction and a recombinant cell expressing a chimeric antigen receptor used therein.

[0007] The present invention provides the use of recombinant macrophages expressing chimeric antigen receptors in the preparation of a drug; the drug is a drug for treating myocardial ischemia-reperfusion injury and / or myocardial infarction; the chimeric antigen receptor comprises an extracellular domain, a transmembrane domain and an intracellular domain.

[0008] The extracellular domain has a FAP antigen binding region, and the FAP antigen binding region is scFv that binds to FAP and / or Fab that binds to FAP and / or scFab that binds to FAP and / or scIgG that binds to FAP.

[0009] The transmembrane domain comprises the following (a) and / or (b) and / or (c) and / or (d): (a) the hinge region and transmembrane region of CD8, (b) the hinge region and transmembrane region of CD4, (c) the hinge region and transmembrane region of CD28, and (d) the hinge region and transmembrane region of CD3ζ.

[0010] The intracellular domain comprises a CD147 intracellular signaling domain and / or a CD3 zeta intracellular signaling domain and / or an FcεRIγ intracellular signaling domain and / or a CD27 intracellular signaling domain and / or a CD28 intracellular signaling domain and / or a CD137 intracellular signaling domain and / or a CD134 intracellular signaling domain and / or a CD80 intracellular signaling domain and / or a CD86 intracellular signaling domain and / or an FcRγ intracellular signaling domain.

[0011] As a specific form, the extracellular domain is a scFv that binds to FAP, the transmembrane domain is the hinge region and transmembrane region of CD8, and the intracellular domain is the CD147 intracellular signaling domain.

[0012] FAP: Fibroblast Activation Protein. Specifically, the FAP is human FAP or mammalian FAP. Exemplarily, the mammalian FAP may be a mouse.

[0013] scFv: single-chain antibody fragment. Fab: Antigen-binding fragment. scFab: single-chain Fab fragment. scIgG: single-chain immunoglobulin.

[0014] CD8: CD8 differentiation antigen. CD4: CD4 differentiation antigen. CD28: CD28 differentiation antigen. CD3ζ: ζ chain of CD3 differentiation antigen. CD147: CD147 differentiation antigen. FcεRIγ: γ chain of FcεRI, a member of the antigen receptor superfamily. CD27: CD27 differentiation antigen. CD137: CD137 differentiation antigen. CD134: CD134 differentiation antigen. CD80: CD80 differentiation antigen. CD86: CD86 differentiation antigen. FcRγ: Fc receptor γ chain.

[0015] The chimeric antigen receptor may further comprise a marker domain.

[0016] For example, the marker domain may be EGFP protein.

[0017] Exemplarily, the scFv that specifically binds to FAP is shown as positions 22-265 of SEQ ID NO: 1. Exemplarily, the hinge region and transmembrane region of CD8 are shown as positions 266-332 of SEQ ID NO: 1. Exemplarily, the intracellular signaling domain of CD147 is shown as positions 333-372 of SEQ ID NO: 1. Exemplarily, the EGFP protein is shown as positions 393-631 of SEQ ID NO: 1.

[0018] As a specific example, the chimeric antigen receptor is as follows (e1) or (e2) or (e3) or (e4) or (e5):

[0019] (e1) a protein whose amino acid sequence includes positions 22-372 of SEQ ID NO: 1;

[0020] (e2) a protein whose amino acid sequence includes positions 22-631 of SEQ ID NO: 1;

[0021] (e3) a protein whose amino acid sequence includes positions 1-372 of SEQ ID NO: 1;

[0022] (e4) a protein whose amino acid sequence includes SEQ ID NO: 1;

[0023] (e5) A protein identical to (e1) or (e2) or (e3) or (e4); the identity is 60%, 70%, 80%, 90% or 95% or more.

[0024] In one embodiment, (e1) is a protein having an amino acid sequence of positions 22 to 372 of SEQ ID NO: 1. In one embodiment, (e2) is a protein having an amino acid sequence of positions 22 to 631 of SEQ ID NO: 1. In one embodiment, (e3) is a protein having an amino acid sequence of positions 1 to 372 of SEQ ID NO: 1. In one embodiment, (e4) is a protein having an amino acid sequence of SEQ ID NO: 1.

[0025] The present invention also provides a complex, characterized in that the complex is a substance loaded with a nucleic acid molecule encoding any of the above-mentioned chimeric antigen receptors.

[0026] In one embodiment of the present invention, the nucleic acid molecule is mRNA.

[0027] As a specific form, the mRNA is the RNA shown in SEQ ID NO: 3 (in SEQ ID NO: 3, A represents adenine nucleotide, G represents guanine nucleotide, C represents cytosine nucleotide, and T represents N1-methyl pseudouracil nucleotide). As a specific form, the mRNA is mRNA-FAP CAR. mRNA-FAP CAR is an RNA shown in SEQ ID NO: 3 (in SEQ ID NO: 3, A represents adenine nucleotide, G represents guanine nucleotide, C represents cytosine nucleotide, and T represents N1-methyl pseudouracil nucleotide) with a cap structure (Cap1-GAG) added to the 5' end and a ployA structure added to the 3' end.

[0028] Specifically, the complex can be one or more of lipid nanoparticles (LNP-FAP CAR) loaded with the nucleic acid molecule, polymer nanoparticles loaded with the nucleic acid molecule, exosomes loaded with the nucleic acid molecule, dendrimers loaded with the nucleic acid molecule, cell membrane-coated nanoparticles loaded with the nucleic acid molecule, metal organic frameworks loaded with the nucleic acid molecule, carbon nanotubes loaded with the nucleic acid molecule, and microvesicles loaded with the nucleic acid molecule. Specifically, the complex has the ability to target immune cells. The complex contains DOPS. The complex targets and binds to immune cells through DOPS. Specifically, the immune cells are macrophages and / or monocytes.

[0029] As an example, the preparation method of the complex (LNP-FAP CAR) includes the following steps:

[0030] (1) The RNA shown in SEQ ID NO: 3 was prepared by in vitro transcription (the nucleotide raw materials used in in vitro transcription were: ATP, GTP, CTP and N1-Me-pUTP; in SEQ ID NO: 3, A represents adenine nucleotide, G represents guanine nucleotide, C represents cytosine nucleotide, and T represents N1-methyl pseudouracil nucleotide), and then a cap structure (Cap1-GAG) was added to the 5' end and a ployA structure was added to the 3' end. The resulting RNA was named mRNA-FAP CAR;

[0031] (2) Mixing five raw materials in ethanol to obtain solution A; the five raw materials are ALC-0315, DSPC, cholesterol, DMG-PEG 2000 and DOPS sodium salt; the molar ratios of ALC-0315, DSPC, cholesterol, DMG-PEG 2000 and DOPS sodium salt are 39.4:8:36.3:1.4:15, respectively;

[0032] (3) dissolving the RNA prepared in step (1) in citrate buffer (pH 3.0, 10 mM) to obtain solution B;

[0033] (4) 1 volume of solution A and 3 volumes of solution B were mixed (N / P ratio of 10:1), and then incubated at room temperature for 10 minutes.

[0034] The method further comprises a purification step.

[0035] Specifically, the purification step is step (5).

[0036] Step (5): After completing step (4), the entire system is transferred to a dialysis bag, dialyzed in PBS buffer, and then the liquid in the dialysis bag is collected, which is the LNP-FAP CAR solution (containing lipid nanoparticles loaded with mRNA-FAP CAR).

[0037] The present invention also provides the use of any of the above-mentioned complexes in the preparation of a medicine; the medicine is a medicine for treating myocardial ischemia-reperfusion injury and / or myocardial infarction.

[0038] The present invention also provides a drug for treating myocardial ischemia-reperfusion injury and / or myocardial infarction, which contains any of the above-mentioned complexes.

[0039] The present invention also provides a protein, which is any of the above chimeric antigen receptors.

[0040] The present invention also provides a biomaterial, which is as follows (f1) or (f2) or (f3) or (f4):

[0041] (f1) a nucleic acid molecule encoding the protein;

[0042] (f2) a recombinant vector containing the nucleic acid molecule described in (f1);

[0043] (f3) a recombinant cell containing the nucleic acid molecule described in (f1);

[0044] (f4) A recombinant cell containing the recombinant vector described in (f2).

[0045] In the above biological material, the nucleic acid molecule encoding the protein is a DNA molecule (such as a single-stranded DNA molecule or a double-stranded DNA molecule) or an RNA molecule (such as a single-stranded RNA molecule or a double-stranded RNA molecule).

[0046] Specifically, the nucleic acid molecule is as follows (g1) or (g2) or (g3) or (g4) or (g5) or (g6) or (g7) or (g8) or (g9) or (g10):

[0047] (g1) the DNA molecule shown at positions 64-1116 of SEQ ID NO: 2;

[0048] (g2) the DNA molecule shown in SEQ ID NO: 2, 64-1893;

[0049] (g3) the DNA molecule shown in positions 1-1116 of SEQ ID NO: 2;

[0050] (g4) the DNA molecule shown in SEQ ID NO: 2;

[0051] (g5) a DNA molecule identical to (g1) or (g2) or (g3) or (g4); the identity is 60%, 70%, 80%, 90% or 95% or greater;

[0052] (g6) the RNA molecule shown at positions 64-1116 of SEQ ID NO: 3;

[0053] (g7) RNA molecules shown in SEQ ID NO: 3, numbers 64-1893;

[0054] (g8) the RNA molecule shown in positions 1-1116 of SEQ ID NO: 3;

[0055] (g9) RNA molecule represented by SEQ ID NO: 3;

[0056] (g10) An RNA molecule that is identical to (g6) or (g7) or (g8) or (g9); the identity is 60%, 70%, 80%, 90% or greater than 95%.

[0057] In the above-mentioned biological material, the recombinant vector may be a recombinant plasmid having the nucleic acid molecule. Specifically, the recombinant vector is a recombinant virus (such as a recombinant lentivirus) having the nucleic acid molecule. Specifically, the recombinant plasmid is a recombinant plasmid obtained by inserting the nucleic acid molecule into an expression plasmid. Specifically, the expression plasmid may be a lentiviral expression plasmid. Specifically, the recombinant virus is obtained by introducing the recombinant plasmid into a host cell and culturing it. Specifically, the recombinant virus is obtained by introducing the recombinant plasmid and a lentiviral helper plasmid into a host cell and culturing it. Specifically, the lentiviral helper plasmid is a psPAX2 plasmid and a pCMV-VSV-G plasmid. The host cell is a cell used for virus packaging. Specifically, the host cell is a HEK293T cell.

[0058] As an example, the recombinant plasmid is as follows: the small fragment between the EcoRI and XbaI restriction sites of the Lenti-EF1A-T2A-EGFP-PGK-Puro vector is replaced with the double-stranded DNA molecule shown in SEQ ID NO: 2, while keeping other sequences unchanged to obtain a recombinant plasmid.

[0059] In the above-mentioned biomaterial, the recombinant cell may be a recombinant immune cell. In a specific embodiment of the present invention, the recombinant immune cell is a recombinant macrophage. In a specific embodiment of the present invention, the recombinant cell may be an M1 type recombinant macrophage. Specifically, the recombinant cell is a recombinant macrophage obtained by introducing the nucleic acid molecule into a macrophage. Specifically, the recombinant cell is a recombinant macrophage obtained by introducing the recombinant vector into a macrophage. Specifically, the recombinant cell is a recombinant macrophage obtained by introducing the recombinant vector into a macrophage and then polarizing it to the M1 type. In a specific embodiment of the present invention, the recombinant immune cell is a recombinant monocyte.

[0060] As an example, the method for preparing the recombinant cell includes the following steps:

[0061] (1) Mice were taken, and bone marrow was collected from the femur, tibia, and hip bones. The bone marrow was treated with red blood cell lysis buffer, then centrifuged and the supernatant was discarded. The precipitate was resuspended in sterile water and filtered through a 40 μm pore size cell filter. The filtrate was collected as the cell suspension.

[0062] (2) Add the cell suspension prepared in step (1) to complete culture medium containing 50 ng / mL M-CSF to a cell content of 1×10 6 / mL, and then cultured for 5 days;

[0063] (3) After completing step (2), the recombinant virus solution and polybrene (MOI = 50, the concentration of polybrene in the system is 8 μg / mL) are added to the culture system, and then cultured for 5 days;

[0064] (4) After completing step (3), LPS was added to the culture system to a concentration of 200 ng / mL, and then cultured for 3 hours. The cells were then collected to obtain the recombinant macrophages (FAP CAR-Ms).

[0065] Exemplarily, the mouse may be a C57BL / 6J mouse.

[0066] The present invention also protects the use of the protein or the biomaterial in preparing a drug; the drug is a drug for treating myocardial ischemia-reperfusion injury and / or myocardial infarction.

[0067] The medicine for treating myocardial ischemia-reperfusion injury and / or myocardial infarction is a medicine for treating myocardial ischemia-reperfusion injury and / or myocardial infarction in animals.

[0068] In a specific embodiment of the present invention, the myocardial ischemia-reperfusion injury is acute myocardial ischemia-reperfusion injury.

[0069] In a specific embodiment of the present invention, the myocardial infarction is acute myocardial infarction.

[0070] The animal is a mammal, such as a human or a mouse.

[0071] In one embodiment of the present invention, the animal is a non-aging animal.

[0072] In one embodiment of the present invention, the animal is a juvenile, young adult or middle-aged animal.

[0073] Beneficial Effects of the Invention: The present invention is the first to utilize recombinant macrophages expressing chimeric antigen receptors (which can be recombinant macrophages prepared in vitro) or monocytes / macrophages with chimeric antigen receptors (cells formed by a substance loaded with the nucleic acid molecule targeting and binding to monocytes / macrophages in vivo) to treat myocardial ischemia-reperfusion injury and myocardial infarction, providing the first preclinical evidence for the feasibility of adoptive cell therapy based on chimeric antigen receptor macrophages for the treatment of myocardial ischemia-reperfusion injury and myocardial infarction. In summary, the research of the present invention provides a new immunotherapy strategy for patients with cardiovascular diseases, including myocardial ischemia-reperfusion injury and myocardial infarction. BRIEF DESCRIPTION OF THE DRAWINGS

[0074] Figure 1 This is the result of qRT-PCR detection of the relative expression level of the Fap gene in Example 1.

[0075] Figure 2 These are exemplary photographs and quantitative statistical results of multiple immunofluorescence staining in Example 1; in Panel A, the hearts of the I / R group are divided into the myocardial infarction area (center) and the border area (border).

[0076] Figure 3 This is the result of qRT-PCR detection of the relative expression level of the target gene in step 3 of Example 2.

[0077] Figure 4 This is a photo of the FAP CAR-Ms in step 3 of Example 2 under a fluorescence microscope.

[0078] Figure 5 This is the result of detecting the expression of FAP in primary cardiac fibroblasts in step 2 of Example 3.

[0079] Figure 6 This is the result of testing the phagocytic ability of phagocytes on primary cardiac fibroblasts in step 3 of Example 3.

[0080] Figure 7 This is the result of evaluating cardiac function by echocardiography in Example 4.

[0081] Figure 8 These are the results of evaluating myocardial fibrosis by Sirius red-stained pathological sections in Example 4.

[0082] Figure 9 These are the results of evaluating myocardial fibrosis using Masson-stained pathological sections in Example 4.

[0083] Figure 10 This is the result of immunofluorescence staining of the I / R+CAR-Ms group in step 4 of Example 4.

[0084] Figure 11 These are the results of multiple immunofluorescence staining of each group in step 4 of Example 4.

[0085] Figure 12 The figures are the results of in vitro staining (upper panel) and flow cytometry (lower panel) evaluation of the phagocytic ability of FAP CAR-monocytes / macrophages in Example 7.

[0086] Figure 13 These are the results of flow cytometry evaluation of eGFP expression in monocytes, T cells, B cells, NK cells, and neutrophils in the blood in Example 8.

[0087] Figure 14 This is the result of immunofluorescence staining to detect the biodistribution of FAP CAR-monocytes / macrophages in Example 9.

[0088] Figure 15These are the results of myocardial fibrosis detection using Masson trichrome staining and Sirius red staining in Example 9.

[0089] Figure 16 This is the result of immunofluorescence staining to detect myocardial fibrosis in Example 9.

[0090] Figure 17 These are the results of the echocardiographic assessment of cardiac function 14 days after I / R in Example 9.

[0091] Figure 18 This is the result of qRT-PCR analysis of cardiotoxicity-related genes in Example 9.

[0092] Figure 19 These are the results of the echocardiographic assessment of cardiac function 2 months after I / R in Example 9.

[0093] Figure 20 This is the result of the echocardiographic assessment of cardiac function in step 1 of Example 10.

[0094] Figure 21 This is the result of the echocardiographic assessment of cardiac function in step 2 of Example 10.

[0095] Figure 22 Schematic diagram of the elements of the recombinant plasmid in Example 2. DETAILED DESCRIPTION

[0096] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0097] Unless otherwise noted, the experimental methods in the following examples are conventional methods and were performed according to the techniques and conditions described in literature in the field or according to product specifications. Materials and reagents used in the following examples are commercially available unless otherwise noted. Unless otherwise noted, the quantitative experiments in the following examples were performed in triplicate, and the results were averaged. Unless otherwise noted, cell culture conditions were 37°C and 5% CO2.

[0098] Complete culture medium: DMEM medium containing 10% FBS and 1% penicillin-streptomycin. Red blood cell lysate: TBD Science, product catalog number NH4CL2009. M-CSF (Human Recombinant M-CSF): STEMCELL Technologies, product catalog number 78057. Polybrene: Source Leaf Biotechnology, product catalog number S24797. LPS (lipopolysaccharide): Sigma-Aldrich, product catalog number L2880. Collagenase 2: Worthington, product catalog number LS004176. Collagenase 4: Worthington, product catalog number LS004188. DiD (cell membrane far-red fluorescent probe): Beyotime, product catalog number C1039, operate according to the instructions. DiI (cell membrane red fluorescent probe): Beyotime, product catalog number C1036, operate according to the instructions. CMFDA (CellTracker TM Fluorescent Probes) were purchased from Invitrogen, product catalog number A66434, and the procedure was performed according to the manufacturer's instructions. FAP rabbit mAb was purchased from Abclonal, product catalog number A23789. Goat anti-rabbit IgG (H+L) cross-adsorbed secondary antibody, Alexa Fluor TM 488: Thermo Fisher Scientific, catalog number A11008. Hoechst: Beyotime, catalog number C1022. Anti-GFP: Abcam, catalog number ab6662. Lipofectamine 2000: Invitrogen, catalog number 11668019. psPAX2 plasmid: Addgene, catalog number #12260. pCMV-VSV-G plasmid: Addgene, catalog number #8454. N1-Me-pUTP stands for N1-Methyl-Pseudouridine-5'-Triphosphate.

[0099] EDTA buffer: contains 130mmol / L NaCl, 5mmol / L KCl, 0.5mmol / L NaH2PO4, 10mmol / L HEPES, 10mmol / L Glucose, 10mmol / L BDM, 10mmol / L Taurine and 5mmol / L EDTA, with the balance being pure water.

[0100] Perfusion buffer: contains 130mmol / L NaCl, 5mmol / L KCl, 0.5mmol / L NaH2PO4, 10mmol / L HEPES, 10mmol / L Glucose, 10mmol / L BDM, 10mmol / L Taurine and 1mmol / L MgCl2, and the balance is pure water.

[0101] Collagenase buffer: contains 0.5 mg / ml collagenase 2 and 0.5 mg / ml collagenase 4, and the balance is pure water.

[0102] Stop buffer: perfusion buffer containing 5% FBS.

[0103] The Lenti-EF1A-T2A-EGFP-PGK-Puro vector (i.e., "Lenti-EF1A-T2A-EGFP-PGK-Puro vector" in the literature) is described in the following literature: Lei, A., Yu, H., Lu, S. et al. A second-generation M1-polarized CAR macrophage with antitumor efficacy. Nat Immunol 25, 102–116 (2024). https: / / doi.org / 10.1038 / s41590-023-01687-8.

[0104] MIRI: Myocardial ischemia-reperfusion injury. Myocardial ischemia refers to a pathological condition in which reduced blood perfusion to the heart leads to decreased oxygen supply, abnormal myocardial energy metabolism, and an inability to support normal cardiac function. When blood perfusion is restored to the ischemic tissue, significant pathophysiological changes occur in the myocardial cells and local vascular network in the reperfused area. These changes, in combination, can promote further tissue damage.

[0105] I / R: Ischemia / Reperfusion.

[0106] BMDM: bone marrow-derived macrophages.

[0107] CAR: chimeric antigen receptor.

[0108] FAP CAR-Ms: macrophages expressing FAP chimeric antigen receptor.

[0109] FAP CAR-monocytes / macrophages: Macrophages exist in the form of monocytes in peripheral blood. Therefore, macrophages expressing FAP chimeric antigen receptors in peripheral blood are called FAP CAR-monocytes / macrophages.

[0110] LNP: lipid nanoparticle.

[0111] Method of I / R surgery: The experimental animals were anesthetized by inhalation of 2% isoflurane, and a 1.2 cm incision was made on the left side of the chest to expose the fourth intercostal space; a small hole was made in the fourth intercostal space, the heart was squeezed out, a silk thread was placed below the course of the left coronary artery, and the left anterior descending coronary artery (LAD) was ligated. When the anterior wall of the left ventricle turned white, it indicated that the ligation was successful. After 40 minutes of ischemia, the ligature was loosened and the ischemic area was reperfused. After reperfusion, the heart was immediately returned to the chest cavity, the air was evacuated, and the muscles and skin were sutured.

[0112] Sham surgery method: The experimental animals were anesthetized by inhalation of 2% isoflurane, and a 1.2 cm incision was made on the left side of the chest to expose the fourth intercostal space. A small hole was made in the fourth intercostal space, the heart was squeezed out, and a silk thread was placed below the course of the left coronary artery. After 40 minutes, the silk thread was withdrawn, and the heart was immediately returned to the chest cavity. The air was evacuated and the muscles and skin were sutured.

[0113] Example 1: Identification of FAP as a target for MIRI therapy

[0114] 1. Group processing

[0115] The experimental animals were 6-8 week old C57BL / 6J mice.

[0116] I / R group: received I / R surgery; Sham group: received sham surgery.

[0117] 2. qRT-PCR analysis showed that the expression level of Fap gene was significantly upregulated after I / R injury

[0118] On days 1, 3, and 7 after surgery, n mice were sampled from each group. The mice were sacrificed and heart tissue was obtained. Total RNA was extracted and reverse-transcribed to obtain cDNA. Real-time fluorescence quantitative PCR was performed using the cDNA as a template. The data were normalized to the internal reference gene (18S gene) to obtain the relative expression level of the Fap gene.

[0119] The primers used for detecting the Fap gene are as follows:

[0120] FapFw: 5′-TAAGGAGGGGATAGTCATTGCC-3′;

[0121] Fap Re: 5′-TGCCACATTTGAAAAGACCAGT-3′.

[0122] The primers used to detect the 18S gene are as follows:

[0123] 18S Fw: 5′-GCTTAATTTGACTCAACACGGGA-3′;

[0124] 18S Re: 5′-AGCTATCAATCTGTCAATCCTGTC-3′.

[0125] qRT-PCR results are shown in Figure 1 (n=8) Compared with the Sham group, the Fap mRNA levels in the hearts of the I / R group mice were significantly upregulated starting from the 3rd day after I / R surgery and lasted until the 7th day.

[0126] 3. Multiple immunofluorescence staining revealed that FAP expression levels in cardiac fibroblasts were significantly increased after I / R injury

[0127] On days 3 and 7 after surgery, n mice were sampled from each group. The mice were sacrificed and their hearts were removed. Frozen sections (10 μm thick) were prepared and then subjected to multiple immunofluorescence staining. FAP was the target: the primary antibody was FAP rabbit mAb (Abclonal, A23789); the secondary antibody was Goat anti-rabbit IgG (H+L) cross-adsorbed secondary antibody, Alexa Fluor TM 488 (Thermo Fisher Scientific, A11008). PDGFRα as the target: the primary antibody was PDGFR alpha rabbit pAb (Abclonal, A2103); the secondary antibody was HRP-labeled goat anti-rabbit IgG (Servicebio, GB23303). α-SMA as the target: the primary antibody was Smooth Muscle Actin (SMA) mouse mAb (Immunoway, YM6566); the secondary antibody was 647-conjugated goat anti-mouse IgG (H+L) (Abclonal, AS059) was used. Cell nuclei were stained with Hoechst (Beyotime, C1022). Images were taken using a fluorescence microscope and quantified using Image J software.

[0128] The results of multiple immunofluorescence staining are shown in Figure 2A (n=6). Starting from the third day after I / R surgery, the FAP positive signals in the marginal and central areas of the hearts of mice in the I / R group increased significantly, and PDGFRα + / α-SMA + / FAP + The proportion of triple-positive cells increased significantly, suggesting that FAP co-localized with cardiac fibroblasts, and its expression increased over time and maintained a high level on the 7th day.

[0129] The results of the study showed that FAP is a cell surface protein whose expression level is significantly increased in activated cardiac fibroblasts after I / R injury and can serve as a potential target for MIRI treatment.

[0130] 4. Multiple immunofluorescence staining revealed no significant changes in FAP expression levels in cardiac fibroblasts of aged animals

[0131] Male C57BL / 6J mice were housed normally. Mice were randomly sacrificed at 30 and 70 weeks of age, and heart tissue was collected. Heart tissue was then subjected to multiple immunofluorescence staining. FAP was the target: the primary antibody was a FAP rabbit mAb (Abclonal, A23789); the secondary antibody was a Goat anti-rabbit IgG (H+L) cross-adsorbed secondary antibody, Alexa Fluor TM 488 (Thermo Fisher Scientific, A11008). Vimentin was the target: the primary antibody was Vimentin Antibody (Abmart, T55134S); the secondary antibody was HRP-labeled goat anti-rabbit IgG (Servicebio, GB23303). Hoechst was used to stain the cell nuclei. The results are shown in Figure 2 B (n=5). There were no significant differences in the FAP expression ratio or FAP / Vimentin ratio between 30- and 70-week-old mouse hearts. These results indicate that FAP expression levels in cardiac fibroblasts from aged and young mice do not differ significantly, suggesting that FAP is not a potential target for cardiac fibrosis in aged animals.

[0132] Example 2: Preparation of FAP CAR-Ms

[0133] 1. Preparation of recombinant lentivirus

[0134] 1. Preparation of recombinant plasmid

[0135] The double-stranded DNA molecule shown in SEQ ID NO: 2 was used to replace the small fragment between the EcoRI and XbaI restriction sites of the Lenti-EF1A-T2A-EGFP-PGK-Puro vector, while keeping other sequences unchanged, to obtain a recombinant plasmid (the recombinant plasmid has been sequenced and verified. The schematic diagram of the recombinant plasmid is shown in Figure 22 The DNA molecule represented by SEQ ID NO:2 encodes the protein represented by SEQ ID NO:1. In SEQ ID NO:1, amino acid residues 1-21 constitute the signal peptide, amino acid residues 22-265 constitute the anti-mouse FAP single-chain variable fragment, amino acid residues 266-308 constitute the CD8 hinge region, amino acid residues 309-332 constitute the CD8 transmembrane region, amino acid residues 333-372 constitute the CD147 intracellular signaling domain, amino acid residues 375-392 constitute the T2A peptide, and amino acid residues 393-631 constitute the EGFP protein. The signal peptide is removed from SEQ ID NO:1 to form the mature protein, which is represented by residues 22-631 in SEQ ID NO:1.

[0136] 2. Prepare recombinant lentivirus.

[0137] Preparation method for transfection complex for one culture dish: Take a 1.5 ml EP tube, add 1 ml serum-free DMEM medium, 80 μl Lipofectamine 2000, 6 μg psPAX2 plasmid, 4 μg pCMV-VSV-G plasmid, and 10 μg recombinant plasmid prepared in step 1, mix well, and then let it stand at room temperature for 5 minutes.

[0138] HEK293T cells were cultured in a 10 cm diameter dish to a confluence of 90%-95%. The transfection complex was added and cultured for 48 hours (the supernatant was collected and then an equal volume of new complete medium was added). The cells were then cultured for an additional 24 hours (the supernatant was collected). The two supernatants were combined and filtered through a 0.45 μm filter to collect the filtrate. The filtrate was centrifuged at 19400 g for 135 minutes at 4°C. The supernatant was discarded and the pellet was resuspended in 1 ml of complete medium to obtain the viral fluid containing the recombinant lentivirus (referred to as the viral fluid). The fluid was aliquoted and stored at -80°C.

[0139] 2. Preparation of FAP CAR-Ms and BMDM wt

[0140] 1. Obtain BMDMs

[0141] Bone marrow was collected from the femur, tibia, and hip bones of adult male C57BL / 6J mice using a 25-gauge needle. The bone marrow was first treated with red blood cell lysis buffer for 1 minute, then centrifuged (4°C, 1700 rpm, 5 min) and the supernatant discarded. The precipitate was resuspended in sterile water and filtered through a 40 μm pore cell strainer. The filtrate was collected as the cell suspension.

[0142] 2. Add the cell suspension prepared in step 1 to complete culture medium containing 50 ng / mL M-CSF to a cell content of 1×10 6 cells / mL and then cultured for 5 days.

[0143] 3. After completing step 2, add the virus solution prepared in step 1 and polybrene (MOI = 50, the concentration of polybrene in the system is 8 μg / mL) to the culture system, and then culture for 5 days.

[0144] 4. After completing step 3, LPS was added to the culture system and its concentration in the system was 200 ng / mL (the function of LPS is to induce M1 polarization of macrophages), and then cultured for 3 hours, and then the cells were collected, namely FAP CAR-Ms.

[0145] 5. After completing step 2, add polybrene to the culture system to a concentration of 8 μg / mL, and then culture for 5 days.

[0146] 6. After completing step 5, add LPS to the culture system and make its concentration in the system 200ng / mL, then culture for 3 hours, and then collect the cells, which are BMDM wt (expressed in BMDMs).

[0147] 3. Detecting the expression of target genes

[0148] The test cells are: FAP CAR-Ms or BMDM prepared in step 2 wt .

[0149] 1. qRT-PCR

[0150] The test cells were taken, total RNA was extracted and reverse transcribed to obtain cDNA, and real-time fluorescence quantitative PCR was performed using cDNA as a template. The data were normalized according to the internal reference gene (18S gene) to obtain the relative expression level of the target gene.

[0151] PCR was performed using primer pair A (consisting of mFap scFv Fw and mFap scFv Re, the target sequence is located in the scFv coding region of SEQ ID NO: 2) and primer pair B (consisting of CD8 TM +CD147 IC Fw and CD8 TM+CD147 IC Re, and the target sequence is located in the CD8 transmembrane region coding region and the CD147 intracellular signaling domain coding region).

[0152] mFap scFv Fw: 5′-CAGAGACAACGCCAAGAACA-3′;

[0153] mFap scFv Re: 5′-CCATGGCGTAGTAGCCGTAT-3′.

[0154] CD8 TM +CD147 IC Fw::5′-TTTACATCTGGGCACCCTTG-3′;

[0155] CD8 TM +CD147 IC Re: 5′-GAGTTCCACTGCCCTTCAGT-3′.

[0156] The primers used to detect the 18S gene were the same as those in step 2 of Example 1.

[0157] See the results Figure 3 (n=3), the results using primer pair A are shown in the left panel, and the results using primer pair B are shown in the right panel. The target gene was successfully expressed in FAPCAR-Ms.

[0158] 2. GFP fluorescence observation

[0159] The test cells were taken and EGFP fluorescence was observed under a fluorescence microscope.

[0160] See the results Figure 4 The target gene was successfully expressed in FAP CAR-Ms.

[0161] Example 3: Verification of the phagocytic ability of macrophages

[0162] 1. Obtaining Primary Cardiac Fibroblasts

[0163] The experimental animals were 8-12 week old male C57BL / 6J mice.

[0164] I / R group: received I / R surgery; Sham group: received sham surgery.

[0165] On the third day after surgery, the animals were anesthetized with 1.5%-2% isoflurane. The chest cavity was opened to expose the heart. After the descending aorta was severed, 3 mL of EDTA buffer was immediately injected into the right ventricle to flush the heart. The ascending aorta was clamped with a hemostat, and the heart was transferred to a culture dish containing fresh EDTA buffer. Next, 10 mL of EDTA buffer, 3 mL of perfusion buffer, and 30-50 mL of collagenase buffer were sequentially injected into the left ventricle for digestion. The heart was then gently pulled into fragments approximately 1 mm in diameter using forceps, and 5 mL of stop buffer was added to the culture dish. The contents of the culture dish were then filtered through a 100 μm pore size cell strainer, and the filtrate was collected. The filtrate was allowed to stand for 20 minutes (at which time the myocardial cells would sink), and the supernatant (containing cardiac fibroblasts) was collected and centrifuged at 300 g at 4°C for 5 minutes. The supernatant was discarded, and the precipitate was collected to obtain primary cardiac fibroblasts.

[0166] 2. Detection of FAP expression in primary cardiac fibroblasts

[0167] Primary cardiac fibroblasts prepared from mice in the I / R group and mice in the Sham group in step 1 were respectively taken for immunofluorescence staining. The primary antibody used was FAP rabbit mAb; the secondary antibody used was Goat anti-rabbit IgG (H+L) cross-adsorbed secondary antibody, Alexa Fluor TM 488. Cell nuclei were stained with Hoechst and observed under a fluorescence microscope (Andor Dragonfly 505).

[0168] See the results Figure 5 The expression of FAP was observed in primary cardiac fibroblasts of mice in the I / R group.

[0169] 3. Phagocytosis of target cells by macrophages

[0170] Target cells: Primary cardiac fibroblasts prepared from I / R group mice in step 1.

[0171] Macrophages: FAP CAR-Ms or BMDM prepared in step 2 of Example 2 wt .

[0172] Target cells were labeled with DiD. Macrophages were labeled with CMFDA. DiD-labeled target cells and CMFDA-labeled macrophages were co-cultured in complete culture medium containing 50 ng / mL M-CSF for two hours at 37°C (cell ratio 1:1) and then observed using an Andor Dragonfly 505 microscope.

[0173] See photos Figure 6 FAP CAR-Ms have phagocytic effects on cardiac fibroblasts and BMDM wt It has no phagocytic effect on cardiac fibroblasts.

[0174] Example 4: FAP CAR-Ms improve cardiac function and reduce myocardial fibrosis in mice after I / R

[0175] 1. Group processing

[0176] Experimental animals: 8-12 week old male C57BL / 6J mice.

[0177] I / R+CAR-M group: I / R surgery was performed, and FAP CAR-Ms suspension was injected into the tail vein 3 days after surgery (the injection volume for each mouse was 200 μl, containing 4×10 6 The FAP CAR-Ms suspension was obtained by suspending the FAP CAR-Ms prepared in step 2 of Example 2 in PBS buffer.

[0178] I / R+BMDM group: I / R surgery was performed and BMDM was injected into the tail vein 3 days after surgery. wt The injection volume for a single mouse was 200 μl, containing 4×10 6 BMDM wt ). BMDM wt The suspension is prepared by mixing the BMDM prepared in step 2 of Example 2 wt The obtained protein was resuspended in PBS buffer.

[0179] I / R+PBS group: I / R surgery was performed, and PBS buffer was injected through the tail vein on day 3 after the surgery (the injection volume for each mouse was 200 μl).

[0180] Sham group: sham operation was performed.

[0181] 2. Evaluate cardiac function through echocardiography

[0182] Two weeks after surgery, mice in each group underwent echocardiography, with the output data including left ventricular fractional shortening (LVFS, %), left ventricular ejection fraction (LVEF, %), left ventricular end-diastolic dimension (LVEDD, mm), and left ventricular end-systolic dimension (LVESD, mm). The results of three consecutive cardiac cycles were analyzed using ImageJ software.

[0183] See the results Figure 7(n=8). Compared with the sham group, the LVEF and LVFS of the I / R+PBS group were significantly decreased, and the LVEDD and LVESD were significantly increased, indicating that cardiac function was significantly impaired after I / R. Compared with the I / R+PBS group, there were no significant differences in the above four indicators of the I / R+BMDM group, that is, there was no significant improvement in cardiac function. Compared with the I / R+PBS group, the LVEF and LVFS of the I / R+CAR-M group were significantly increased, and the LVEDD and LVESD were significantly decreased, that is, cardiac function was significantly improved.

[0184] 3. Assessment of cardiac function through histopathological examination

[0185] Two weeks after the operation, the mice were sacrificed, and the hearts were removed for paraffin section preparation (5 μm thickness). The sections were stained with Sirius red and Masson staining, and the fibrosis area was measured using Image J.

[0186] The results of Sirius red staining are shown in Figure 8 (n=5). The results of Masson staining are shown in Figure 9 (n=5). Compared with the sham group, the cardiac scar area of mice in the I / R+PBS group was significantly larger, and the proportion of fibrosis area was significantly increased. Compared with the I / R+PBS group, the proportion of fibrosis area in the I / R+BMDM group was not significantly different, indicating no significant improvement in myocardial fibrosis. Compared with the I / R+PBS group, the proportion of fibrosis area in the I / R+CAR-M group was significantly reduced, indicating reduced myocardial fibrosis.

[0187] 4. Positioning in the body

[0188] Five mice were sampled from each of the I / R+CAR-M groups on days 4, 7, and 14 after surgery. The mice were sacrificed, their hearts were removed, paraffin sections were prepared, and immunofluorescence staining was performed. The primary antibody used was FAP rabbit mAb (Abclonal, A23789); the secondary antibody used was Goat anti-rabbit IgG (H+L) cross-adsorbed secondary antibody, Alexa Fluor TM 488 (Thermo Fisher Scientific, A11008). Cell nuclei were stained with Hoechst (Beyotime, C1022). Observation was performed under a fluorescence microscope (Andor Dragonfly 505). Figure 10 Immunofluorescence staining showed that FAP CAR-Ms could migrate to the myocardium one day after injection into mice and target FAP after I / R. +Cardiac fibroblasts are expressed and remain in the heart for up to 2 weeks.

[0189] The sampling time points were 4 days, 7 days and 14 days after surgery. Five mice were sampled in each group at each sampling time point. The mice were sacrificed and the hearts were removed. Paraffin sections were made and then subjected to multiple immunofluorescence staining. PDGFRα was the target: the primary antibody was PDGFR alpha rabbit pAb (Abclonal, A2103); the secondary antibody was HRP-labeled goat anti rabbit IgG (Servicebio, GB23303). α-SMA was the target: the primary antibody was Smooth Muscle Actin (SMA) mouse mAb (Immunoway, YM6566); the secondary antibody was 647-conjugated goat anti-mouse IgG (H+L) (Abclonal, AS059). Cell nuclei were stained with Hoechst (Beyotime, C1022). Images were taken using a fluorescence microscope and quantified using Image J software. Figure 11 Compared with the I / R+PBS group and the I / R+BMDM group, the α-SMA expression in the I / R+CAR-M group was significantly higher than that in the I / R+PBS group and the I / R+BMDM group. + / PDGFRα + The number of fibroblasts in double-positive patients was significantly reduced, and this difference was first seen on day 4 after I / R and was more obvious on days 7 and 14 after I / R. These results indicate that FAP CAR-Ms can target activated cardiac fibroblasts, reduce their number, and thus improve myocardial fibrosis after I / R.

[0190] Example 5: FAP CAR-Ms have a protective effect and are non-toxic in mouse hearts

[0191] In Example 4, RNA sequencing (RNA-seq) was performed on mouse heart tissue two weeks after surgery.

[0192] Principal component analysis (PCA) revealed significant gene expression differences between the I / R+CAR-M and I / R+PBS groups. A total of 1,193 upregulated and 519 downregulated genes were identified in the I / R+PBS group. Of these, 475 I / R-upregulated genes were downregulated in the FAP CAR-M group, and 230 I / R-downregulated genes were upregulated in the FAP CAR-M group. Subsequently, gene ontology (GO) enrichment analysis identified the major upregulated and downregulated biological processes in the FAP CAR-M group. Among the top 10 significantly upregulated pathways, 7 were related to fatty acid catabolism, indicating that FAP CAR-Ms attenuated I / R-induced adverse metabolic reprogramming from fatty acid metabolism to glycolysis. Conversely, among the 10 pathways primarily downregulated in the FAP CAR-M group, 8 were related to extracellular matrix organization and 2 were related to inflammatory responses, suggesting that FAP CAR-Ms possess anti-fibrotic and anti-inflammatory cardioprotective effects.

[0193] Furthermore, the expression of 74 genes associated with cardiotoxicity was analyzed. GSEA showed that cardiotoxicity was upregulated after I / R and rescued by FAP CAR-Ms. Specifically, proinflammatory genes (such as Serpine1, Pla2g4a, and Gpm6a) were upregulated after I / R but could be suppressed by FAP CAR-Ms treatment. Similarly, profibrotic genes including Bgn, Tgfb2, Postn, Col3a1, Vcan, and Timp1 were significantly upregulated after I / R but could be suppressed by FAP CAR-Ms treatment. Furthermore, Ckm, which has been reported to exert cardioprotective effects by enhancing bioenergetics and improving contractility during reperfusion, was significantly upregulated by FAP CAR-Ms. These data suggest that FAP CAR-Ms did not cause cardiotoxicity.

[0194] Example 6: Preparation of RNA-loaded LNPs

[0195] Phosphatidylserine (PS) is a lipid that is externalized during apoptosis. Monocytes and macrophages can target and recognize PS through scavenger receptors (such as SR-A1 and SR-B1) that are highly expressed on their surfaces, thereby eliminating apoptotic cells. Dioleoylphosphatidylserine (DOPS), a derivative of PS, can mimic the process by which PS is recognized, thus enhancing macrophage targeting by adding DOPS.

[0196] 1. Preparation of LNP-FAP CAR

[0197] 1. The RNA set forth in SEQ ID NO: 3 was prepared by in vitro transcription (the nucleotide raw materials used in the in vitro transcription were: ATP, GTP, CTP, and N1-Me-pUTP; in SEQ ID NO: 3, A represents adenine nucleotide, G represents guanine nucleotide, C represents cytosine nucleotide, and T represents N1-methylpseudouracil nucleotide). A cap structure (Cap1-GAG) was then added to the 5' end and a ployA structure was added to the 3' end. The resulting RNA was designated mRNA-FAP CAR. The RNA molecule set forth in SEQ ID NO: 3 encodes the protein set forth in SEQ ID NO: 1.

[0198] 2. Mix five raw materials in ethanol (ethanol is used as the reaction solvent) to obtain Solution A. The five raw materials are ALC-0315, DSPC, cholesterol, DMG-PEG 2000, and DOPS sodium salt; the molar ratios of ALC-0315, DSPC, cholesterol, DMG-PEG 2000, and DOPS sodium salt are 39.4:8:36.3:1.4:15, respectively. DOPS is dissolved in a small amount of tetrahydrofuran and added to the ethanol, while the other four raw materials are added directly to the ethanol.

[0199] ALC-0315 (CAS No.: 2036272-55-4), DSPC (CAS No.: 816-94-4), and DMG-PEG 2000 (CAS No.: 160743-62-4) are all products of SINOPEG. Cholesterol (CAS No.: 57-88-5) is a product of Innochem. DOPS sodium salt (CAS No.: 90693-88-2) is a product of Aladdin.

[0200] 3. Dissolve the RNA prepared in step 1 in citrate buffer (pH 3.0, 10 mM) to obtain solution B.

[0201] 4. Mix 1 volume of solution A and 3 volumes of solution B (N / P ratio of 10:1), incubate at room temperature for 10 minutes, then transfer to a dialysis bag (molecular weight cutoff of 10 kDa or 20 kDa) and dialyze in PBS buffer for 2 hours. The liquid in the dialysis bag is then collected to obtain the LNP-FAP CAR solution (containing lipid nanoparticles loaded with mRNA-FAP CAR) and stored at 4°C. The average particle size, polydispersity index (PDI), and zeta potential of the LNPs were measured by dynamic light scattering (DLS, Zetasizer Nano ZS instrument). The mRNA encapsulation efficiency was determined by the RiboGreen assay.

[0202] 2. Preparation of LNP-FAP CARtrun

[0203] 1. RNA lacking the CD147 intracellular signaling domain nucleotide sequence compared to SEQ ID NO: 3 was prepared by in vitro transcription (the nucleotide raw materials used in in vitro transcription were: ATP, GTP, CTP and N1-Me-pUTP; A represents adenine nucleotide, G represents guanine nucleotide, C represents cytosine nucleotide, and T represents N1-methylpseudouracil nucleotide). Then, a cap structure (Cap1-GAG) was added to the 5' end and a ployA structure was added to the 3' end. The resulting RNA was named mRNA-FAPCARtrun.

[0204] 2. Same as step 2 in step 1.

[0205] 3. Same as step 3 in step 1.

[0206] 4. Same as step 1, step 4.

[0207] The LNP-FAP CARtrun solution (containing lipid nanoparticles loaded with mRNA-FAP CARtrun) was obtained and stored at 4°C. The average particle size, polydispersity index (PDI), and zeta potential of the LNPs were measured by dynamic light scattering (DLS, Zetasizer Nano ZS instrument). The mRNA encapsulation efficiency was determined by RiboGreen assay.

[0208] 3. Preparation of LNP-Luc

[0209] 1. The RNA represented by SEQ ID NO: 4 was prepared by in vitro transcription (the nucleotide raw materials used in the in vitro transcription were: ATP, GTP, CTP, and N1-Me-pUTP; A represents adenine nucleotide, G represents guanine nucleotide, C represents cytosine nucleotide, and T represents N1-methylpseudouracil nucleotide). Then, a cap structure (Cap1-GAG) was added to the 5' end and a ployA structure was added to the 3' end. The resulting RNA was named mRNA-Luc.

[0210] 2. Same as step 2 in step 1.

[0211] 3. Same as step 3 in step 1.

[0212] 4. Same as step 1, step 4.

[0213] The resulting LNP-Luc solution (containing mRNA-Luc-loaded lipid nanoparticles) was stored at 4°C. The average particle size, polydispersity index (PDI), and zeta potential of the LNPs were measured by dynamic light scattering (DLS, Zetasizer Nano ZS instrument). The mRNA encapsulation efficiency was determined by the RiboGreen assay.

[0214] Example 7. Preparation of FAP CAR-macrophages in vitro and evaluation of phagocytosis

[0215] 1. Preparation of NIH3T3 cells overexpressing FAP

[0216] 1. Replace the small fragment between the EcoRI and XbaI restriction sites of the Lenti-EF1A-T2A-EGFP-PGK-Puro vector with the mouse Fap gene (NCBI Reference Sequence: NM_007986.3, 10-DEC-2024), keeping other sequences unchanged, to obtain a recombinant plasmid (the recombinant plasmid has been sequenced and verified).

[0217] 2. Preparation of recombinant lentivirus

[0218] Preparation method for transfection complex for one culture dish: Take a 1.5 ml EP tube, add 1 ml serum-free DMEM medium, 80 μl Lipofectamine 2000, 6 μg psPAX2 plasmid, 4 μg pCMV-VSV-G plasmid, and 10 μg recombinant plasmid prepared in step 1, mix well, and then let it stand at room temperature for 5 minutes.

[0219] HEK293T cells were cultured in a 10 cm diameter dish to a confluence of 90%-95%, and then the transfection complex was added. The cells were then cultured for 48 hours (the supernatant was collected, and then an equal volume of new complete medium was added). The cells were then cultured for an additional 24 hours (the supernatant was collected). The two supernatants were combined, filtered through a 0.45 μm filter, and the filtrate was collected. The filtrate was concentrated and purified using the PEG-8000 concentration method.

[0220] 3. The recombinant lentivirus prepared in step 2 was used to infect NIH3T3 cells (RRID:CVCL_0594) to obtain recombinant cells, namely, NIH3T3 cells overexpressing FAP.

[0221] 2. Evaluation of FAP CAR-macrophage phagocytosis

[0222] 1. Preparation of Cardiac Macrophages

[0223] Six- to eight-week-old C57BL / 6J mice were anesthetized with 1.5% isoflurane. The chest cavity was opened to expose the heart. After the descending aorta was cut, 3 mL of EDTA buffer was immediately injected into the right ventricle to flush the heart. The ascending aorta was clamped with a hemostat, and the heart was transferred to a culture dish containing fresh EDTA buffer. Next, 10 mL of EDTA buffer, 3 mL of perfusion buffer, and 30-50 mL of collagenase buffer were sequentially injected into the left ventricle for digestion. The heart was then gently pulled into fragments approximately 1 mm in diameter using forceps, and 5 mL of stop buffer was added to the culture dish. The contents of the culture dish were then filtered through a 100 μm cell strainer, and the filtrate was collected. Four cycles of gravity sedimentation (15-20 min each) were then performed using the perfusion buffer. The supernatants from each cycle were collected, combined, and centrifuged at 300 g for 5 min. The supernatant was discarded, and the pellet was collected to obtain cardiac macrophages.

[0224] 2. Preparation of FAP CAR-macrophages

[0225] Cardiac macrophages were cultured in DMEM medium containing 10% FBS, 1% penicillin / streptomycin, 20 ng / mL M-CSF, and 2 μg / mL of the LNP-FAP CAR (based on mRNA) prepared in step 1 of Example 6 for 24 hours. The cells were then collected to obtain FAP CAR-macrophages.

[0226] 3. Preparation of FAP CARtrun-macrophages

[0227] Cardiac macrophages were cultured for 24 hours in DMEM medium containing 10% FBS, 1% penicillin / streptomycin, 20 ng / mL M-CSF, and 2 μg / mL of LNP-FAP CARtrun (based on mRNA) prepared in step 2 of Example 6. The cells were then harvested to obtain FAP CARtrun-macrophages.

[0228] 4. Evaluation of the Phagocytosis of FAP CAR-macrophages and FAP CARtrun-macrophages

[0229] Target cells: NIH3T3 cells (using 3T3 wt ) or NIH3T3 cells overexpressing FAP prepared in step 1 (using 3T3 FAP-OE (indicated by ). Macrophages include: FAP CAR-macrophages or FAP CARtrun-macrophages.

[0230] Target cells were labeled with DiD, and macrophages were labeled with DiI. DiD-labeled target cells and DiI-labeled macrophages were co-cultured (1:1 cell ratio) in complete culture medium containing 50 ng / mL M-CSF at 37°C for two hours. The cells were then observed using an Andor Dragonfly 505 microscope and analyzed using a BD Fortessa flow cytometer.

[0231] See the sample photos Figure 12 The results of flow cytometry analysis and phagocytosis ratio statistics are shown in the figure above. Figure 12 Figure 2 (n=3). FAP CAR-macrophages showed targeted phagocytosis of FAP-overexpressing NIH3T3 cells, but not wild-type NIH3T3 cells. FAP CARtrun-macrophages had no significant phagocytic effect on either NIH3T3 cells or NIH3T3 cells overexpressing FAP.

[0232] Example 8: Assessing the ability of test substances to target and bind to monocytes / macrophages in vivo

[0233] The test substances were: PBS buffer, LNP-FAP CAR prepared in step 1 of Example 6, LNP-FAP CARtrun prepared in step 2 of Example 6, or LNP-Luc prepared in step 3 of Example 6.

[0234] Eight-week-old male C57BL / 6J mice underwent I / R surgery. Mice were divided into four groups and injected with the test substance on the third day after surgery. The I / R+LNP-mock group received LNP-Luc, the I / R+LNP-FAP CARtrun group received LNP-FAP CARtrun, and the I / R+LNP-FAP CAR group received LNP-FAP CAR. The injection volume was 200 μL per mouse, and the dose was 2 mg / kg body weight (based on mRNA). The I / R+PBS group received PBS buffer in a 200 μL injection volume per mouse. Peripheral blood was collected 6 hours after injection for FACS analysis.

[0235] FACS test results are shown in Figure 13 In the I / R+LNP-FAP CAR group, more than 20% of peripheral blood monocytes were GFP-positive, and only a very low proportion of T cells, B cells, NK cells, and neutrophils were GFP-positive, indicating that FAP CAR-monocytes were generated in vivo.

[0236] Example 9: LNP-FAP CAR treatment alleviates myocardial injury after I / R in mice

[0237] 1. Group processing

[0238] The experimental animals were 8-week-old male C57BL / 6J mice.

[0239] I / R group: received I / R surgery; Sham group: received sham surgery.

[0240] The experimental animals after I / R surgery were divided into 4 groups and injected with the test substance once on the 1st, 3rd, 5th, 7th, 9th and 11th day after surgery. The test substance injected into the I / R+LNP-Luc group was LNP-Luc prepared in step 3 of Example 6, the test substance injected into the I / R+LNP-FAP CARtrun group was LNP-FAPCARtrun prepared in step 2 of Example 6, and the test substance injected into the I / R+LNP-FAP CAR group was LNP-FAP CAR prepared in step 1 of Example 6. The single injection volume was 200 μL / animal, and the single dose was 2 mg / kg body weight (based on the amount of mRNA). The test substance injected into the PBS group was PBS buffer, and the single injection volume was 200 μL / animal.

[0241] The experimental animals that underwent sham surgery did not receive any special treatment after surgery.

[0242] 2. FAP CAR-monocyte / macrophage biodistribution

[0243] On day 4 after surgery, five mice were randomly selected from each of the sham group and the I / R + LNP-FAP CAR group. Hearts were harvested, paraffin sections were prepared, and immunofluorescence staining was performed. GFP was used as the target, and the primary antibody was anti-GFP. FAP was used as the target, and the primary antibody was FAP rabbit mAb. Hoechst (Beyotime, C1022) was used to stain cell nuclei.

[0244] See the results Figure 14 GFP-positive cells infiltrated the myocardium and were adjacent to FAP-expressing cardiac fibroblasts. These results suggest that LNP-FAP CAR targets and binds to monocytes / macrophages in vivo, generating FAP CAR-monocytes / macrophages that can infiltrate the myocardium after I / R.

[0245] LNP-FAP CAR therapy reduces myocardial fibrosis

[0246] On the 4th and 14th days after surgery, 5 mice were randomly selected from each group. The mice were sacrificed and the hearts were removed. Frozen sections were prepared and then subjected to immunofluorescence staining. The primary antibody used was FAP rabbit mAb. PDGFRα was the target: the primary antibody was PDGFR alpha rabbit pAb (Abclonal, A2103). CollagenⅢ was the target: the primary antibody was Collagen Type III (N-terminal) Rabbit pAb (Proteintech, 22734-1-AP). α-SMA was the target: the primary antibody was Smooth Muscle Actin (SMA) mouse mAb. Hoechst was used to stain the cell nuclei. The results are shown in Figure 2. Figure 16 Compared with the I / R+PBS group, the FAP in the I / R+LNP-FAPCAR group was + Fibroblasts, α-SMA + PDGFRα + The areas of fibroblasts and CollagenⅢ were significantly reduced. Compared with the I / R+PBS group, there was no significant difference in the I / R+LNP-Luc or I / R+LNP-FAP CARtrun groups.

[0247] Two weeks after the operation, the mice were sacrificed and the hearts were removed to prepare paraffin sections (5 μm thickness). The sections were stained with Sirius red and Masson staining, and the fibrosis area was measured using Image J. Figure 15 Compared with the I / R+PBS group, the I / R+LNP-FAP CAR group showed a significant decrease in extracellular matrix by both Masson trichrome staining and Sirius red staining on day 14 after I / R. There were no significant differences in either the I / R+NNP-Luc or I / R+LNP-FAP CARtrun groups compared with the I / R+PBS group.

[0248] The results showed that LNP-FAP CAR treatment effectively reduced activated fibroblasts and alleviated myocardial fibrosis after I / R.

[0249] LNP-FAP CAR therapy improves cardiac function

[0250] Two weeks after surgery, mice in each group underwent echocardiography, and the output data included left ventricular ejection fraction (LVEF, %), fractional shortening (LVFS, %), left ventricular end-diastolic diameter (LVEDD, mm), and left ventricular end-diastolic internal diameter (LVESD, mm). The results of three consecutive cardiac cycles were analyzed using ImageJ software.

[0251] See the results Figure 17. Compared with the Sham group, the LVEF and LVFS in the I / R+PBS group were significantly decreased, and the LVEDD and LVESD were significantly increased on the 14th day after I / R, indicating impaired cardiac function. Compared with the I / R+PBS group, there were no significant differences in these parameters between the mice in the I / R+LNP-Luc group or the I / R+LNP-FAP CARtrun group. Compared with the I / R+PBS group, the I / R+LNP-FAP CAR group showed significant improvements in LVEF, LVFS, LVEDD and LVESD. The results showed that LNP-FAP CAR treatment enhanced cardiac function after I / R.

[0252] 5. Detection of potential toxicity of LNP-FAP CAR therapy

[0253] From surgery to day 14 after surgery, mice in the I / R+LNP-FAP CAR- group showed no signs of illness, weight change, or body temperature change. On day 14 after surgery, various organs were harvested for histopathological evaluation, and no toxic effects on major non-cardiac organs were found. On day 14 after surgery, cardiac tissue RNA was extracted and converted to cDNA, and qRT-PCR analysis of cardiotoxicity-related genes was performed. The results are shown in Figure 18 Compared with the I / R+PBS group, the I / R+LNP-FAP CAR group did not show significant upregulation, indicating lower cardiotoxicity.

[0254] VI. LNP-FAP CAR therapy improves cardiac function in the long term

[0255] Two months after surgery, mice in each group underwent echocardiography, and the output data included left ventricular ejection fraction (LVEF, %), fractional shortening (LVFS, %), left ventricular end-diastolic diameter (LVEDD, mm), and left ventricular end-diastolic internal diameter (LVESD, mm). The results of three consecutive cardiac cycles were analyzed using ImageJ software.

[0256] See the results Figure 19 . Compared with the Sham group, the LVEF and LVFS in the I / R+PBS group were significantly decreased, and the LVEDD and LVESD were significantly increased 2 months after I / R, indicating impaired cardiac function. Compared with the I / R+PBS group, there were no significant differences in these parameters between the mice in the I / R+LNP-Luc group or the I / R+LNP-FAP CARtrun group. Compared with the I / R+PBS group, the I / R+LNP-FAP CAR group showed significant improvements in LVEF, LVFS, LVEDD and LVESD. The results showed that LNP-FAP CAR treatment improved the cardiac function of mice 2 months after I / R.

[0257] Example 10: Delayed LNP-FAP CAR treatment can improve cardiac function

[0258] The experimental animals were 8-week-old male C57BL / 6J mice.

[0259] 1. The first method of delayed administration

[0260] The experimental animals underwent I / R surgery and were then divided into two groups. The test substance was injected once on the 7th, 9th, 11th, 13th, 15th, and 17th days after surgery. The test substance injected into the I / R+LNP-FAP CAR group was the LNP-FAP CAR prepared in step 1 of Example 6, with a single injection volume of 200 μL / animal and a single dose of 2 mg / kg body weight (based on the amount of mRNA). The test substance injected into the PBS group was PBS buffer, with a single injection volume of 200 μL / animal.

[0261] Echocardiography was performed 28 days after surgery, and the output data included left ventricular ejection fraction (LVEF, %) and fractional shortening (LVFS, %). The results of three consecutive cardiac cycles were analyzed using ImageJ software.

[0262] See the results Figure 20 (n=5) Compared with the I / R+PBS group, the I / R+LNP-FAP CAR group showed significant improvement in LVEF and LVFS.

[0263] 2. The second delayed drug administration method

[0264] The experimental animals underwent I / R surgery and were then divided into two groups. The test substance was injected once on the 14th, 16th, 18th, 20th, 22nd, and 24th days after surgery. The I / R+LNP-FAP CAR group was injected with the LNP-FAP CAR prepared in step 1 of Example 6, with a single injection volume of 200 μL per animal and a single dose of 2 mg / kg body weight (based on mRNA amount). The PBS group was injected with PBS buffer, with a single injection volume of 200 μL per animal.

[0265] Echocardiography was performed 28 days after surgery, and the output data included left ventricular ejection fraction (LVEF, %) and fractional shortening (LVFS, %). The results of three consecutive cardiac cycles were analyzed using ImageJ software.

[0266] See the results Figure 21 (n=5) Compared with the I / R+PBS group, the I / R+LNP-FAP CAR group showed significant improvements in LVEF, LVFS, LVEDD, and LVESD.

[0267] The results of this example show that delaying LNP-FAP CAR treatment can also improve cardiac function after I / R.

[0268] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be practiced over a wide range of parameters, concentrations, and conditions without departing from the spirit and scope of the present invention and without unnecessary experimentation. Although specific embodiments have been given herein, it should be understood that further modifications may be made to the present invention. In summary, this application is intended to encompass any variations, uses, or improvements to the present invention, including those made by conventional techniques known in the art that depart from the scope of the present invention. Applications of the essential features may be made within the scope of the following claims.

Claims

1. Use of recombinant macrophages expressing a chimeric antigen receptor in the preparation of a drug; the drug is a drug for treating myocardial ischemia-reperfusion injury and / or myocardial infarction; the chimeric antigen receptor comprises an extracellular domain, a transmembrane domain, and an intracellular domain.

2. The use according to claim 1, characterized in that: The extracellular domain has a FAP antigen binding region, and the FAP antigen binding region is a scFv that binds to FAP and / or a Fab that binds to FAP and / or a scFab that binds to FAP and / or a scIgG that binds to FAP; The transmembrane domain comprises the following (a) and / or (b) and / or (c) and / or (d): (a) the hinge region and transmembrane region of CD8; (b) the hinge region and transmembrane region of CD4; (c) the hinge region and transmembrane region of CD28; (d) the hinge region and transmembrane region of CD3ζ; The intracellular domain comprises a CD147 intracellular signaling domain and / or a CD3 zeta intracellular signaling domain and / or an FcεRIγ intracellular signaling domain and / or a CD27 intracellular signaling domain and / or a CD28 intracellular signaling domain and / or a CD137 intracellular signaling domain and / or a CD134 intracellular signaling domain and / or a CD80 intracellular signaling domain and / or a CD86 intracellular signaling domain and / or an FcRγ intracellular signaling domain.

3. The use according to claim 1 or 2, characterized in that: The extracellular domain is a scFv that binds to FAP, the transmembrane domain is the hinge region and transmembrane region of CD8, and the intracellular domain is the CD147 intracellular signaling domain.

4. The use according to claim 1, 2 or 3, characterized in that: The chimeric antigen receptor is as follows (e1) or (e2) or (e3) or (e4) or (e5): (e1) a protein whose amino acid sequence includes positions 22-372 of SEQ ID NO: 1; (e2) a protein whose amino acid sequence includes positions 22-631 of SEQ ID NO: 1; (e3) a protein whose amino acid sequence includes positions 1-372 of SEQ ID NO: 1; (e4) a protein whose amino acid sequence includes SEQ ID NO: 1; (e5) A protein identical to (e1) or (e2) or (e3) or (e4); the identity is 60%, 70%, 80%, 90% or 95% or more.

5. Use of the complex in the preparation of a drug; the drug is a drug for treating myocardial ischemia-reperfusion injury and / or myocardial infarction; the complex is a substance loaded with a nucleic acid molecule encoding the chimeric antigen receptor according to any one of claims 1 to 4.

6. A composite, characterized in that: The complex is the complex described in claim 5.

7. A drug for treating myocardial ischemia-reperfusion injury and / or myocardial infarction, comprising the complex according to claim 6.

8. A protein which is the chimeric antigen receptor according to any one of claims 1 to 4.

9. Biological material, which is (f1) or (f2) or (f3) or (f4) as follows: (f1) a nucleic acid molecule encoding the protein of claim 8; (f2) a recombinant vector containing the nucleic acid molecule described in (f1); (f3) a recombinant cell containing the nucleic acid molecule described in (f1); (f4) A recombinant cell containing the recombinant vector described in (f2).

10. Use of the protein according to claim 8 or the biomaterial according to claim 9 in the preparation of a medicament; the medicament is a medicament for treating myocardial ischemia-reperfusion injury and / or myocardial infarction.

Images