FAP-targeted anti-inflammatory CAR-macrophage, preparation method thereof and application of macrophage in preparation of drugs for treating fibrosis diseases

Abstract

The invention discloses an anti-inflammatory CAR-macrophage targeting FAP, a preparation method thereof and application of the anti-inflammatory CAR-macrophage in preparation of drugs for treating fibrosis diseases, and belongs to the technical field of biological medicines. The invention provides an FAP-specific chimeric antigen receptor, an mRNA expression vector of the FAP-specific chimeric antigen receptor, a His-labeled STING signal channel inhibitor and an mRNA expression vector of the His-labeled STING signal channel inhibitor. In-vivo and in-vitro macrophage editing can be effectively realized by delivering CAR mRNA and TRIM13mNRA through LNP, the ability of targeted phagocytosis of FAP positive cells is endowed, meanwhile, anti-inflammatory phenotypic polarization of macrophages is promoted, local inflammation is improved, and fibrosis progress driven by inflammation is synergistically restrained.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technologies, and particularly relates to an anti-inflammatory CAR-macrophage targeting FAP, a preparation method thereof, and an application thereof in the preparation of a medicament for treating fibrotic diseases. Background Art

[0002] Fibrotic diseases are pathological reactions caused by abnormal wound healing after chronic injury of organs or tissues, characterized by excessive deposition and structural remodeling of the extracellular matrix (ECM), ultimately leading to progressive loss of organ function. Fibrotic diseases can occur in multiple organ systems such as the liver (e.g., viral hepatitis, metabolic dysfunction-related steatohepatitis), lung (idiopathic pulmonary fibrosis), heart (myocardial fibrosis), kidney (renal interstitial fibrosis), etc. Their common pathological features include ECM metabolic imbalance (increased synthesis and inhibited degradation), reduction of functional parenchymal cells, and formation of pathological scars. In severe cases, they can progress to organ sclerosis or failure.

[0003] Chimeric Antigen Receptor T Cell (CAR-T) therapy is a treatment strategy that transforms autologous or healthy donor peripheral blood T cells of patients into immune cells capable of specifically recognizing target antigens through gene editing technology in vitro, and then transfuses these edited T cells back into the patient's body to eliminate target cells. The FDA approved two CAR-T drugs, Novartis' Kymriah and Gilead's Yescarta, for the treatment of malignant hematological tumors in 2017. However, CAR-T therapy has shown limited efficacy in the treatment of malignant solid tumors. Solid tumors are often accompanied by pathological features such as extracellular matrix deposition and vascular abnormalities, which significantly hinder the tumor infiltration of T cells and affect their ability to eliminate tumor cells. Chimeric antigen receptor macrophages (CAR-Ms) therapy is a new type of cell therapy that emerged after CAR-T. Macrophages themselves have strong invasiveness and can present antigens to activate the adaptive immune response, showing unique advantages in the treatment of solid tumors. On the other hand, CAR-macrophage therapy shows potential prospects in the treatment of fibrotic diseases. Fibroblast activation protein (FAP) is a type II transmembrane glycoprotein that is highly specifically expressed in fibrotic diseases. Under normal physiological conditions, FAP is hardly expressed; while in the pathological progression of fibrotic diseases (such as pulmonary fibrosis, myocardial fibrosis, and renal fibrosis), FAP is significantly upregulated in myofibroblasts, and this spatio-temporal specific expression pattern makes it the core antigen for targeted therapy. Fibroblast activation protein (FAP) is a type II transmembrane glycoprotein that is specifically expressed in aHSCs, and its expression difference between quiescent and activated HSCs can serve as a target antigen for CAR-Ms to play a recognition and elimination role. In addition, chronic inflammation is an important factor promoting the development of fibrosis. The limited anti-inflammatory ability of CAR-Ms in fibrotic foci reduces their therapeutic effect on fibrosis. Activation of the macrophage STING pathway induces downstream pro-inflammatory responses to promote myofibroblast differentiation. Inhibiting the STING pathway is a potential way to relieve inflammation and assist CAR-Ms in treating fibrotic diseases. TRIM13 is a ubiquitin ligase that regulates STING homeostasis and can promote its degradation by catalyzing STING ubiquitination, playing a dynamic regulatory role in the macrophage response to pathogenic DNA. Currently, there is no publicly available fibrotic treatment drug based on the above principles. Summary of the Invention

[0004] In view of the deficiencies of the prior art, the present invention provides an anti-inflammatory CAR-macrophage targeting FAP, its preparation method, and its application in the preparation of drugs for treating fibrotic diseases. The anti-inflammatory CAR-macrophage is a CAR-macrophage targeting FAP and having an anti-inflammatory effect. This CAR-macrophage can specifically target the FAP target, and at the same time co-express TRIM13 to inhibit the polarization of the pro-inflammatory phenotype of macrophages, reduce the inflammation of fibrotic lesions, and contribute to the co-treatment of fibrotic diseases.

[0005] In the first aspect of the present invention, there is provided an anti-inflammatory CAR-macrophage targeting FAP clearance, and the anti-inflammatory CAR-macrophage contains a FAP-specific CAR targeting and a pro-phenotype polarization protein;

[0006] In the embodiment of the present invention, a FAP-specific CAR is constructed, including an extracellular domain, a transmembrane region, and an intracellular domain;

[0007] The front signal peptide segment is the front signal peptide of CD8α; the single-chain antibody fragment is a monoclonal antibody specific for FAP; the hinge region is selected from the hinge regions of IghG1, CD8α, or CD28; the transmembrane region is selected from the transmembrane regions of CD3ζ, CD28, CD8, or CD4; the intracellular domain contains an activation signal domain or an activation signal domain and a co-stimulatory domain; the activation signal domain is selected from the intracellular regions of FcεRIγ or CD3ζ; the co-stimulatory domain is selected from the intracellular regions of CD28, 4-1BB, or ICOS;

[0008] Preferably, the hinge region is the hinge region of CD8α; the transmembrane region is the transmembrane region of CD8; the intracellular region is the intracellular region of CD3ζ; a representative amino acid sequence example of the FAP-specific CAR is shown as SEQ ID NO.1.

[0009] The pro-phenotype polarization protein includes, but is not limited to, IL-10, IL-4, or STING signaling pathway inhibitors, etc.;

[0010] The STING signaling pathway inhibitor is a TRIM13 protein containing a his tag, and its representative amino acid sequence is shown as SEQ ID NO.2.

[0011] In the second aspect of the present invention, an expression vector is provided. The recombinant expression vector contains a gene sequence encoding a FAP-specific CAR, including but not limited to plasmids, linear mRNAs, and circular mRNAs; the mRNA includes a 5' cap, a 5' untranslated region, a CAR sequence, a 3' untranslated region, and a 3' polyA tail, and its representative nucleotide sequence is as shown in SEQ ID NO.3; preferably, the mRNA encoding anti-FAP CAR is prepared by the DNA template T7 transcriptase method.

[0012] The mRNA is circular RNA or linear RNA.

[0013] In the third aspect of the present invention, an expression vector is provided. The recombinant expression vector contains a gene sequence encoding a STING signaling pathway inhibitor, including but not limited to plasmids, linear mRNAs, and circular mRNAs; the STING signaling pathway inhibitor is a TRIM13 protein containing a his tag, and the mRNA includes a 5' cap, a 5' untranslated region, a CAR sequence, a 3' untranslated region, and a 3' polyA tail, and its representative nucleotide sequence is as shown in SEQ ID NO.4.

[0014] Preferably, the mRNA encoding TRIM13 is prepared by the DNA template T7 transcriptase method.

[0015] More preferably, the mRNA is circular RNA or linear RNA.

[0016] In the fifth aspect of the present invention, a method for preparing the anti-inflammatory CAR-macrophage targeting FAP is provided: it is obtained by introducing the mRNA encoding CAR and the mRNA encoding His-tagged TRIM13 into macrophages.

[0017] Preferably, the introduction methods include but are not limited to viral vectors (adenoviral vectors, lentiviral vectors, etc.), electroporation, and non-viral vectors (lipid nanoparticles, polymer nanoparticles, etc.) for delivery.

[0018] More preferably, the introduction method is to use non-viral vector lipid nanoparticles.

[0019] In the sixth aspect of the present invention, a lipid nanoparticle is provided, which contains mRNA and a delivery vector; the mRNA is selected from one of the mRNA encoding FAP-specific CAR or the mRNA encoding the STING signaling pathway inhibitor TRIM13, or is a lipid nanoparticle targeting co-loading the mRNA encoding FAP-specific CAR and the mRNA encoding the STING signaling pathway inhibitor TRIM13.

[0020] A lipid nanoparticle that co-targets mRNA encoding FAP-specific CAR and mRNA encoding STING signaling pathway inhibitor TRIM13 provided by the present invention is denoted as CAR / TRIM13@LNPs or CT@LNPs. The lipid nanoparticle includes mRNA encoding FAP-specific CAR, mRNA encoding STING signaling pathway inhibitor TRIM13, and a delivery vector.

[0021] The preparation method of the lipid nanoparticle that co-targets mRNA encoding FAP-specific CAR and mRNA encoding STING signaling pathway inhibitor TRIM13 includes the following steps:

[0022] (1) Dissolve mRNA encoding FAP-specific CAR and mRNA encoding STING signaling pathway inhibitor TRIM13 in potassium hydrogen phthalate-sodium hydroxide buffer to obtain an mRNA mixture;

[0023] (2) Dissolve ionizable lipid P3T14, cholesterol, DMG-PEG 2000 and DOPE in ethanol to obtain a lipid nanoparticle solution, called a mixed lipid solution;

[0024] (3) Add the mRNA mixture and the mixed lipid solution to a microfluidic device, and prepare a lipid nanoparticle delivery system that co-targets mRNA encoding FAP-specific CAR and mRNA encoding STING signaling pathway inhibitor TRIM13 by the microfluidic method.

[0025] Preferably according to the present invention, in step (1), the mass ratio of CAR mRNA to TRIM13 is 1:1.

[0026] Preferably according to the present invention, in step (2), the structure of the ionizable lipid P3T14 is shown in the following formula:

[0027]

[0028] Preferably, in step (2), the molar ratio of the ionizable lipid P3T14, DOPE, cholesterol, and DMG-PEG2000 is 25:35:5:40; the solvent of the above lipids is ethanol; wherein, the concentration of the ionizable lipid P3T14 is 10 mg / mL, and the concentrations of DOPE, cholesterol, and DMG-PEG 2000 are 5 mg / mL.

[0029] Preferably according to the present invention, in step (3), the mass ratio of the mRNA in the mRNA mixture to the ionizable lipid P3T14 in the mixed lipid solution (lipid nanoparticle solution) is 1:1 - 1:30, more preferably 1:10.

[0030] The seventh aspect of the present invention further provides the use of the above-mentioned FAP-targeting specific CAR, mRNA encoding FAP3-specific CAR, STING signaling pathway inhibitor TRIM13, mRNA encoding STING signaling pathway inhibitor TRIM13, FAP-targeting anti-inflammatory CAR-macrophages, or lipid nanoparticles co-loaded with mRNA encoding FAP-specific CAR and mRNA encoding STING signaling pathway inhibitor TRIM13 in the preparation of a medicament for treating fibrotic diseases.

[0031] The eighth aspect of the present invention provides a medicament for treating fibrosis, which contains mRNA encoding FAP-targeting specific CAR and mRNA encoding STING signaling pathway inhibitor TRIM13, or a recombinant expression vector inserted with mRNA encoding FAP-targeting specific CAR and mRNA encoding STING signaling pathway inhibitor TRIM13, or contains FAP-targeting anti-inflammatory CAR-macrophages, or contains lipid nanoparticles (CT@LNPs) co-loaded with mRNA encoding FAP-targeting specific CAR and mRNA encoding STING signaling pathway inhibitor TRIM13, or contains lipid nanoparticles (C@LNPs) encoding FAP-targeting specific CAR.

[0032] Preferably, the medicament for treating fibrosis is CT@LNPs.

[0033] Furthermore, the CAR-macrophage medicament can be edited and infused in vitro or generated in vivo.

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

[0035] 1. The present invention provides FAP-targeting and anti-inflammatory CAR-macrophages, which can specifically target the FAP target, and at the same time can inhibit the STING signaling pathway of macrophages by co-expressing TRIM13, thereby achieving the synergistic effect of CAR-macrophages in clearing FAP-positive myofibroblasts and regulating local inflammation, and thus obtaining a highly efficient anti-fibrotic effect.

[0036] 2. The present invention provides lipid nanoparticles (CT@LNPs) co-loaded with mRNA encoding FAP-targeting specific CAR and mRNA encoding STING signaling pathway inhibitor TRIM13, which can successfully deliver mRNA encoding FAP-targeting specific CAR and mRNA encoding STING signaling pathway inhibitor TRIM13, and can be used for in vitro editing of macrophages and in vivo editing of macrophages, simplifying the process of engineering immune cell therapy, effectively avoiding off-target risks and systemic toxic side effects.

[0037] 3. After treatment with the lipid nanoparticles (CT@LNPs) co-loaded with the mRNA encoding the FAP-specific CAR and the STING signaling pathway inhibitor TRIM13 provided by the present invention, liver fibrosis in the mouse liver fibrosis animal model was significantly improved, and the levels of TNFα and IFNγ decreased significantly; in the mouse pulmonary fibrosis animal model, the pulmonary function of the mice was significantly improved after administration, indicating that the preparation has the function of anti-pulmonary fibrosis; at the same time, the in vivo safety evaluation test proved that the lipid nanoparticles have good biosafety and have good application prospects in fibrotic diseases. Description of the Drawings

[0038] The specification drawings forming a part of the present invention are used to provide a further understanding of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation to the present invention.

[0039] Figure 1 It is a schematic diagram of the structure of the chimeric antigen receptor in Example 1 of the present application.

[0040] Figure 2 It is a synthetic route diagram of the ionizable lipid P3T14 in Example 3 of the present application.

[0041] Figure 3 It is the nuclear magnetic characterization result of the ionizable lipid P3T14 in Example 3 of the present application.

[0042] Figure 4 It is the physicochemical property characterization of CT@LNPs in Example 4 of the present application. Among them, the left figure is the particle size, the middle figure is the Zeta potential, and the right figure is the encapsulation efficiency.

[0043] Figure 5 It is the measurement result of the influence of CT@LNPs on macrophage viability in Example 5 of the present application.

[0044] Figure 6 It is the measured result of the transfection efficiency of CT@LNPs on macrophages in Example 6 of the present application.

[0045] Figure 7 It is the measured result of the phagocytic ability of macrophages on FAP-positive cells in Example 7 of the present application.

[0046] Figure 8 It is the in vivo pharmacodynamic result of CT@LNPs in fibrotic mice in Example 8 of the present application. Among them, A is the hydroxyproline content in each group after treatment, B is the IFNγ content in each group after treatment, C is the TNFα content in each group after treatment, and D is the liver fibrosis-related staining sections (including Masson staining and Sirius red staining).

[0047] Figure 9This is the in vivo pharmacodynamic results in the mouse model of pulmonary fibrosis in Example 8 of this application. Among them, A is the hydroxyproline content in each group after treatment, and B - G are pulmonary resistance, functional residual capacity, dynamic compliance, forced vital capacity, tidal volume, and inspiratory capacity in sequence.

[0048] Figure 10 This is the in vivo safety characterization result diagram of CT@LNPs in Example 9 of this application. Among them, the left figure is the ALT level after treatment, and the right figure is the AST level after treatment. Detailed implementation manners

[0049] It should be noted that the following detailed descriptions are all exemplary and are intended to provide further illustration of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present invention belongs.

[0050] Example 1: Construction of a specific CAR targeting FAP

[0051] Using the myofibroblast marker FAP as a specific antigen to design the extracellular domain of CAR. As Figure 1 shown, the chimeric antigen receptor includes a CD8 front signal peptide, a specific anti - FAP monoclonal antibody (anti - FAP - scFv), a myc tag gene, a CD8α hinge region, a CD8 transmembrane region, and a CD3ζ intracellular domain.

[0052] The specific construction process is as follows: Search for the amino acid sequences of each component through NCBI, optimize the sequences, and then concatenate the sequences. The concatenation order is the CD8 front signal peptide, anti - FAP - scFv, myc tag gene, CD8α hinge region, CD8 transmembrane region, and CD3ζ intracellular domain, to construct a specific CAR targeting FAP. Its amino acid sequence is shown in SEQ ID NO.1, and the nucleotide sequence is shown in SEQ ID NO.3.

[0053] Example 2: Construction of the STING signaling pathway inhibitor TRIM13

[0054] According to the TRIM13 sequence shown in the NCBI database (NCBI ID: NM_001164220), insert the His tag gene. Its amino acid sequence is shown in SEQ ID NO.2, and the nucleotide sequence is shown in SEQ ID NO.4.

[0055] Example 3: Preparation of the ionizable lipid P3T14

[0056] As Figure 2As shown, 5 mL of anhydrous DMF was added to a mixture of 6.715 g of tert-butyldimethylchlorosilane, 1.54 g of tris(hydroxymethyl)aminomethane, and 6.304 g of the catalyst imidazole, and the reaction was carried out at 25 °C for 12 h to obtain Tris-TBS. 3.000 g of Tris-TBS was mixed with 1.000 g of 4-methyl-1-piperazinebutyric acid in 10 mL of DCM. In the presence of 1.800 g of EDCI and 1.260 g of HOBT as catalysts, 7.200 mL of TEA was used as an acid-binding agent, and the reaction was carried out at ice bath temperature and then at room temperature for 24 h to obtain P3TBS. 0.500 g of P3TBS and 0.8652 g of TBAF·3H2O were dissolved in 5 mL of tetrahydrofuran. The solution of TBAF·3H2O in tetrahydrofuran was added dropwise to the P3TBS solution under ice bath stirring, and the reaction was carried out for 3 h. The reaction solution was dried by rotary evaporation; 5 mL of anhydrous DCM and 380 μL of triethylamine were added under ice bath stirring and in the presence of nitrogen, and then 0.635 g of myristoyl chloride was slowly added dropwise; the reaction was carried out at room temperature for 24 h to obtain the target product P3T14.

[0057] The P3T14 prepared in this example was characterized by NMR, and the NMR results are as Figure 3 shown.

[0058] From Figure 3 it can be seen that the ionizable lipid P3T14 was successfully synthesized, and its structure is as shown in the following formula:

[0059]

[0060] Example 4, Preparation and Characterization of CAR / TRIM13@LNPs

[0061] 4.1, A method for preparing a lipid nanoparticle (CT@LNPs) co-loaded with mRNA encoding a FAP-specific CAR and mRNA encoding a STING signaling pathway inhibitor TRIM13, comprising the following steps:

[0062] (1) 40 μL each of CAR mRNA (1 mg / mL) and TRIM13 mRNA (1 mg / mL) were mixed evenly and diluted to 240 μL with a potassium hydrogen phthalate-sodium hydroxide buffer solution with pH = 4 to obtain an mRNA mixture;

[0063] (2) 10 mg of ionizable lipid P3T14, 5.88 mg of cholesterol, 5.45 mg of DMG-PEG 2000 and 12.93 mg of DOPE were dissolved in 1 mL of ethanol to obtain a mixed lipid solution, or a lipid nanoparticle solution;

[0064] (3) Add 240 μL of the mRNA mixture and 80 μL of the mixed lipid solution to the microfluidic device. The mass ratio of mRNA in the mRNA mixture to the ionizable lipid P3T14 in the mixed lipid solution is 1:10. Lipid nanoparticles (CT@LNPs) for co-loading mRNA encoding FAP-specific CAR and mRNA encoding the STING signaling pathway inhibitor TRIM13 are prepared by the microfluidic method.

[0065] 4.2, A method for preparing lipid nanoparticles (C@LNPs / T@LNPs) co-loading mRNA encoding a FAP-specific CAR or mRNA encoding the STING signaling pathway inhibitor TRIM13, comprising the following steps:

[0066] (1) Take 80 μL of CAR mRNA (1 mg / mL) or TRIM13 mRNA (1 mg / mL), and dilute it to 240 μL with a potassium hydrogen phthalate-sodium hydroxide buffer solution with pH = 4 to obtain an mRNA solution.

[0067] (2) Dissolve 10 mg of the ionizable lipid P3T14, 5.88 mg of cholesterol, 5.45 mg of DMG-PEG 2000 and 12.93 mg of DOPE in 1 mL of ethanol to obtain a mixed lipid solution.

[0068] (3) Add 240 μL of the mRNA solution and 80 μL of the mixed lipid solution to the microfluidic device. The mass ratio of mRNA in the mRNA mixture to the ionizable lipid P3T14 in the mixed lipid solution is 1:10. Lipid nanoparticles (C@LNPs / T@LNPs) for co-loading mRNA encoding a FAP-specific CAR or mRNA encoding the STING signaling pathway inhibitor TRIM13 are prepared by the microfluidic method.

[0069] The encapsulation efficiency of CT@LNPs was measured using a RiboGreen kit, and its hydrated particle size and surface potential were measured by a Malvern particle size analyzer. The results are as Figure 4 shown.

[0070] As Figure 4 known, CT@LNPs can efficiently encapsulate mRNA, and the encapsulation efficiency is 82 ± 3.03%; the hydrated particle size of CT@LNPs is 163.97 ± 2.39, PDI < 0.3, and the particle size is uniform.

[0071] Example 5. Cytotoxicity investigation of CAR / TRIM13@LNPs

[0072] (1) Extraction, isolation and culture of bone marrow-derived macrophages (BMDMs)

[0073] After euthanizing C57BL / 6 mice, the femurs and tibias were taken, the bone marrow was flushed, and the cells were collected by centrifugation after passing through a 70-μm cell strainer. The cells were cultured in DMEM medium containing 10 ng / mL M-CSF for seven days to induce their differentiation into macrophages.

[0074] (2) Macrophages were seeded in 96-well plates (5×10 3 cells / well), cultured overnight to allow them to adhere, and then different gradient concentrations of Lgmn / CAR-LNPs (gradient concentrations were 100, 250, 500, 1000, 1250 ng / ml, calculated based on mRNA concentration) in medium solution were added, and co-incubated for 24 hours to detect the viability of BMDMs cells. The results are as Figure 5 shown.

[0075] As Figure 5 can be seen, CT@LNPs had no obvious cytotoxicity and had good safety.

[0076] Example 6. In vitro macrophage transfection experiment of CAR / TRIM13@LNPs

[0077] Macrophages were seeded in 24-well plates (6×10 4 cells / well). After 24 hours, they were divided into two groups, and CT@LNPs (60 ng / well, calculated based on mRNA) and an equal volume of PBS solution (CT@LNPs group and Control group) were added respectively. After co-incubating for 24 hours, the cells were collected to detect the transfection efficiency. The results are as Figure 6 shown, where the positive control was the commercially available transfection reagent LipoSmartmRNATransfection Reagent. At the same time, the transfection efficiencies of ionizable lipids P1T14 and P2T14 with similar structures were also tested. The structures of P1T14 and P2T14 are shown as follows:

[0078]

[0079]

[0080] As Figure 6 can be seen, the proportion of BMDMs that could simultaneously express anti-FAP CAR protein and TRIM13 protein in BMDMs treated with CT@LNPs provided by the present invention reached 27.67±2.80%, indicating that CT@LNPs could successfully deliver two kinds of mRNAs, and CT@LNPs could be used to construct anti-inflammatory CAR-macrophages specifically targeting FAP. And the transfection efficiency was higher than that of CT@LNPs prepared with ionizable lipids P1T14 and P2T14 with similar structures.

[0081] Example 7. Investigation of the phagocytic ability of CAR / TRIM13@LNPs on FAP-positive cells in vitro

[0082] Lipid nanoparticles loaded with mRNA encoding a FAP-specific CAR (C@LNPs), lipid nanoparticles loaded with mRNA encoding a STING signaling pathway inhibitor TRIM13 (T@LNPs), and lipid nanoparticles loaded with mRNA encoding a FAP-specific CAR and mRNA encoding TRI1M3 (CT@LNPs) were prepared respectively according to the method described in Example 4.

[0083] Lipid nanoparticles loaded with mRNA encoding a FAP-specific CAR (C@LNPs) were prepared respectively according to the method described in Example 2. Mouse FAP plasmid was transfected into HEK293T cells using Lipofectamine TM 3000 to prepare FAP-293T target cells. FAP-293T cells were stained with 10 μM Cell-Tracker Red (37 °C for 30 minutes), washed three times with PBS, digested with trypsin, and then counted for standby. BMDMs were seeded at 3×10 5 / well. After 24 hours, the medium was replaced with the medium containing each preparation and treated for 12 hours. Subsequently, the cells were co-incubated with the stained FAP-293T (1×10 5 / well) for 12 hours. After collecting the cells, BMDMs were labeled with FITC anti-CD11b, and the proportion of Cell-Tracker Red+ and CD11b+ cells was detected by flow cytometry as the phagocytosis rate to evaluate the targeted phagocytic ability. The results are as Figure 7 shown.

[0084] As shown in Figure 7 A, BMDMs in the C@LNPs and CT@LNPs treatment groups showed relatively high phagocytosis efficiency, and there was no statistical difference. It can be seen that both of them can effectively clear 293T cells expressing FAP and have a strong anti-fibrotic effect. In addition, beads with surface-anchored FAP were co-incubated with CAR-macrophages. The results of confocal imaging are as Figure 7 shown in B. The red staining is the beads with anchored FAP, and the green staining is the macrophages or CAR-macrophages stained with Cell-Tracker Green CMFPD. Representative pictures show that macrophages increase the uptake of anchored FAP beads after expressing the FAP-specific CAR.

[0085] Example 8. In vivo pharmacodynamic evaluation of CAR / TRIM13@LNPs

[0086] A mouse liver fibrosis animal model was constructed by intraperitoneal injection of a carbon tetrachloride olive oil solution (volume ratio 1 / 4, twice a week, 2 μL / g) for four consecutive weeks, and intraperitoneal injection of the olive oil solution was used as the control group. After the modeling, the mice were randomly divided into 2 groups, including a control group (n = 3), and PBS, PBS, and CT@LNPs (1 mg / kg, calculated based on mRNA) were injected via the tail vein once every three days for a total of four administrations. After treatment, the liver fibrosis levels of the mice in each group were detected by measuring the hydroxyproline content in the liver; after the treatment, ELISA was used to detect the TNFα and IFNγ indexes in the serum to evaluate the therapeutic effect of chronic liver inflammation. The results are as Figure 8 shown.

[0087] As can be seen from Figure 8 A in, after treatment with CT@LNPs, the hydroxyproline content in the liver decreased significantly. As can be seen from Figure 8 B and C in, after treatment with CT@LNPs, the levels of TNFα and IFNγ decreased significantly, indicating that the inflammation was alleviated. As can be seen from Figure 8 D in, the fibrosis-related staining area decreased significantly, further proving that the fibrosis was effectively improved.

[0088] A mouse pulmonary fibrosis model was constructed by intratracheal instillation of a bleomycin saline solution (3 U / kg). After the modeling, the mice were randomly divided into 2 groups, including a control group (n = 3), and PBS, PBS, and CT@LNPs (1 mg / kg, calculated based on mRNA) were administered by aerosol inhalation once every three days for a total of four administrations. After treatment, the therapeutic effects of the mice in each group were evaluated by measuring the hydroxyproline content in the lung tissue and lung function-related parameters (lung resistance (RL), functional residual capacity (FRC), dynamic compliance (Cdyn), forced vital capacity (FVC), tidal volume (TV), and inspiratory capacity (IC)).

[0089] As can be seen from Figure 9 A in, after treatment with CT@LNPs, the hydroxyproline content in the lung tissue decreased significantly, suggesting the regression of fibrotic lesions. As can be seen from Figure 9 B-G in, after treatment, the lung resistance and functional residual capacity decreased, while the dynamic compliance, forced vital capacity, tidal volume, and inspiratory capacity increased, indicating obvious improvement in lung function.

[0090] Example 9. In vivo safety evaluation of CAR / TRIM13@LNPs

[0091] Male C57BL / 6 mice were randomly divided into four groups and injected with PBS, C@LNPs, T@LNPs, and CT@LNPs (1 mg / kg, calculated based on mRNA) via the tail vein. The drugs were administered once every three days for three times. Peripheral blood of the mice was collected on the day after the third administration, and the levels of ALT and AST were measured. The results are as Figure 10 shown.

[0092] As Figure 10 can be seen, compared with the PBS group, there was no significant difference in the levels of ALT and AST in the CT@LNPs group, and they were within the normal range, indicating good biosafety.

[0093] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention can have various modifications and changes. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A FAP-targeting anti-inflammatory CAR-macrophage, characterized in that, The anti-inflammatory CAR-macrophages described above contain a FAP-specific CAR targeting and a protein for promoting phenotypic polarization; The FAP-specific CAR targeting fibrosis contains an extracellular domain, a transmembrane region, and an intracellular domain; the extracellular domain includes a front signal peptide segment, a single-chain antibody fragment, a myc tag gene, and a hinge region; or only includes a front signal peptide segment, a single-chain antibody fragment, and a hinge region; The front signal peptide segment is the front signal peptide of CD8α; the single-chain antibody fragment is a monoclonal antibody specific for FAP; the hinge region is selected from the hinge regions of IghG1, CD8α, or CD28; the transmembrane region is selected from the transmembrane regions of CD3ζ, CD28, CD8, or CD4; the intracellular domain contains an activation signal domain or an activation signal domain and a co-stimulatory domain; the activation signal domain is selected from the intracellular regions of FcεRIγ or CD3ζ; the co-stimulatory domain is selected from the intracellular regions of CD28, 4-1BB, or ICOS; The proteins for promoting phenotypic polarization include, but are not limited to, IL-10, IL-4, or STING signaling pathway inhibitors.

2. The anti-inflammatory CAR-macrophages targeting FAP according to claim 1, characterized in that, The CAR contains a hinge region, a transmembrane region, and an intracellular region; the hinge region is the hinge region of CD8α; the transmembrane region is the transmembrane region of CD8; the intracellular region is the intracellular region of CD3ζ; the representative amino acid sequence of the FAP-specific CAR is shown in SEQ ID NO.

1.

3. The anti-inflammatory CAR-macrophage targeting FAP according to claim 1, characterized in that, The protein for promoting phenotypic polarization is a STING signaling pathway inhibitor; the STING signaling pathway inhibitor is a TRIM13 protein containing a his tag, and its amino acid sequence is shown in SEQ ID NO.

2.

4. A recombinant expression vector, characterized in that, The recombinant expression vector contains a gene sequence encoding a FAP-specific CAR, including but not limited to plasmids, linear mRNAs, or circular mRNAs; The nucleotide sequence of the linear mRNA is shown in SEQ ID NO.3; Alternatively, the recombinant expression vector encodes a TRIM13 protein containing a his tag, including but not limited to plasmids, linear mRNAs, or circular mRNAs; The nucleotide sequence of the linear mRNA is shown in SEQ ID NO.

4.

5. Method for preparing anti-inflammatory CAR-macrophages targeting FAP, characterized in that, The recombinant expression vector described in claim 4 is introduced into macrophages; The introduction methods include but are not limited to electroporation, viral transfection, or non-viral vector delivery; Preferably, the non-viral vector delivery introduction method uses a non-viral vector lipid nanoparticle.

6. A lipid nanoparticle, characterized in that, Contains mRNA and a delivery vector; The mRNA is selected from at least one of the mRNA encoding a FAP-specific CAR or the mRNA encoding a STING signaling pathway inhibitor TRIM13; the nucleotide sequence of the mRNA encoding a FAP-specific CAR is shown in SEQ ID NO.3; the nucleotide sequence of the mRNA encoding a STING signaling pathway inhibitor TRIM13 is shown in SEQ ID NO.

4.

7. The method for preparing the lipid nanoparticles according to claim 6, characterized in that, The steps are as follows: Step 1: Dissolve the mRNA encoding FAP-specific CAR or the mRNA encoding the STING signaling pathway inhibitor TRIM13 in a potassium hydrogen phthalate-sodium hydroxide buffer to obtain an mRNA mixture; Step 2: Dissolve the ionizable lipid P3T14, cholesterol, DMG-PEG 2000 and DOPE in ethanol to obtain a mixed lipid solution; Among them, the structure of the ionizable lipid P3T14 is shown in the following formula: The mass ratio of the mRNA in the mRNA mixture to the ionizable lipid P3T14 in the mixed lipid solution is 1:1 - 1:30; Step 3: Add the mRNA mixture and the mixed lipid solution into a microfluidic device, and prepare a lipid nanoparticle delivery system encoding FAP-specific CAR mRNA or encoding the STING signaling pathway inhibitor TRIM13 by microfluidics.

8. The method for preparing the lipid nanoparticles according to claim 6, characterized in that, The steps are as follows: Step 1: Dissolve the mRNA encoding FAP-specific CAR and the mRNA encoding the STING signaling pathway inhibitor TRIM13 in a potassium hydrogen phthalate-sodium hydroxide buffer to obtain an mRNA mixture; Step 2, dissolve the ionizable lipid P3T14, cholesterol, DMG-PEG 2000 and DOPE in ethanol to obtain a mixed lipid solution; Among them, the structure of the ionizable lipid P3T14 is shown in the following formula: The mass ratio of the mRNA in the mRNA mixture to the ionizable lipid P3T14 in the mixed lipid solution is 1:1 - 1:30; Step 3: Add the mRNA mixture and the mixed lipid solution into a microfluidic device, and prepare a lipid nanoparticle delivery system co-loaded with the mRNA encoding FAP-specific CAR and the mRNA encoding the STING signaling pathway inhibitor TRIM13 by microfluidics.

9. Use of the anti-inflammatory CAR-macrophage targeting FAP according to any one of claims 1-2, the recombinant expression vector according to claim 4, or the lipid nanoparticle according to claim 6 in the preparation of a drug for fibrotic diseases.

10. A drug for treating fibrotic diseases, characterized in that, The drug contains at least one of the anti-inflammatory CAR-macrophage targeting FAP according to claim 1, the recombinant expression vector according to claim 4, or the lipid nanoparticle according to claim 6.

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