A CAR-macrophage targeting FAP with enhanced efferocytosis, and its preparation method and application

Abstract

The present invention relates to a CAR-macrophage targeting FAP and enhancing efferocytosis, as well as its preparation method and application. The CAR-macrophage contains a FAP-specific CAR targeting fibrosis and an efferocytosis enhancer Lgmn. The amino acid sequence of the FAP-specific CAR targeting fibrosis is shown in SEQ ID NO.1, and the nucleotide sequence is shown in SEQ ID NO.2; the amino acid sequence of the efferocytosis enhancer is shown in SEQ ID NO.3, and the nucleotide sequence is shown in SEQ ID NO.4. The CAR-macrophage can specifically target the FAP target and enhance the efferocytosis signaling pathway, thereby achieving a combination of CAR-macrophage therapy and enhancing intracellular degradation capacity. By expressing Lgmn protein, the rapid degradation and clearance of engulfed activated fibroblasts is enhanced, and the phagocytic effect of CAR-macrophages on activated fibroblasts is exerted and amplified, thereby achieving an efficient anti-fibrosis effect.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a CAR-macrophage targeting FAP with enhanced endocytosis, as well as a preparation method and application thereof. Background Art

[0002] Ischemic heart disease and heart failure (HF) caused by myocardial infarction (MI) remain the leading causes of death worldwide. After MI, adverse cardiac remodeling manifests as excessive fibrosis, which significantly impairs cardiac function, leading to heart failure and increased cardiovascular risk. Cardiac fibroblasts (CFs) are overactivated in response to various cardiac insults and play a key role in cardiac fibrosis and extracellular matrix (ECM) deposition. Inhibiting or eliminating activated CFs is crucial for alleviating fibrosis and improving cardiac function after MI, but no effective treatment has yet been found.

[0003] Chimeric antigen receptor (CAR) cell therapy offers a promising precision medicine approach by reprogramming immune cells to eliminate specific target cells. CAR-T cells have shown potential therapeutic efficacy in targeted removal of activated CFs, reducing cardiac fibrosis and restoring cardiac function. However, CAR-T cells may face several limitations during their application, including poor tissue penetration and severe side effects such as cachexia, anemia, and fatal bone toxicity. These issues limit the widespread clinical application of CAR-T cells. Compared with CAR-T cell therapy, chimeric antigen receptor-macrophage (CAR-MΦ) cells offer significant advantages. First, CAR-MΦs can more effectively penetrate infarcted tissue, resulting in better clinical outcomes. Second, CAR-MΦs do not self-replicate, significantly reducing the risk of cytokine release syndrome. Furthermore, CAR-MΦs have a shorter circulation time and exhibit less off-target toxicity than CAR-T cells, thereby improving treatment safety.

[0004] CAR-MΦ therapy has shown great potential in the treatment of fibrotic diseases. For example, systemic injection of CAR-MΦ targeting uPAR has been shown to reduce liver fibrosis and cirrhosis. In addition, transplantation of FAP-targeted +Bone marrow-derived macrophages (BMDMs) derived from myofibroblasts can reduce cardiac fibrosis and improve cardiac function in an AngII / PE-induced cardiac injury model. However, despite the impressive results achieved in these studies, the complex process and high cost of producing large quantities of fibrosis-specific macrophages in vitro remain major obstacles to the widespread application of CAR-MΦ therapy. In addition, the large number of dead cells after MI leads to an excessive burden of efferocytosis for CAR-MΦs, which is manifested in the lack of internalization and degradation of CAR-MΦs, thereby limiting the continuous clearance of cargo within the phagolysosome, triggering secondary necrosis, hindering the resolution of inflammation, and limiting the anti-fibrotic efficacy of CAR-MΦs.

[0005] Therefore, developing a CAR-MΦ that can deliver precise treatment while also enhancing the ability of macrophages to clear dead cells is crucial for inflammation resolution and immunotherapy of cardiac fibrosis. Summary of the Invention

[0006] In response to the deficiencies of the prior art, the present invention provides a CAR-macrophage targeting FAP with enhanced efferocytosis, as well as a preparation method and application thereof.

[0007] The technical solutions of the present invention are as follows:

[0008] The first aspect of the present invention provides a FAP-specific CAR targeting fibrosis, comprising an extracellular domain, a transmembrane region, and an intracellular domain;

[0009] The extracellular domain includes a front-end signal peptide segment (Leader), an antigen recognition domain (scFv), a flg tag gene and a hinge region (Hinge);

[0010] Wherein, the front-end signal peptide segment is the CD8α front-end signal peptide, the antigen recognition domain is a specific anti-fibrosis FAP monoclonal antibody (anti-FAP-scFv); the hinge region is selected from the hinge region of IghG1, CD8α or CD28; the transmembrane region is selected from the transmembrane region of CD4, CD8α, CD28 or CD3ζ; the intracellular domain is a signal transduction domain, selected from the intracellular domain of FcεRIγ or CD3ζ.

[0011] Preferably according to the present invention, the hinge region is the hinge region of CD8α; the transmembrane region is the transmembrane region of CD8α; and the intracellular domain is the intracellular domain of CD3ζ.

[0012] Further preferably, the amino acid sequence of the FAP-specific CAR targeting fibrosis is shown in SEQ ID NO.1.

[0013] The second aspect of the present invention provides an mRNA encoding the above-mentioned FAP-specific CAR targeting fibrosis, wherein the mRNA comprises a 5' cap, a 5' non-coding region, a CAR sequence, a 3' non-coding region and a 3' polyA tail, and its nucleotide sequence is shown in SEQ ID NO.2.

[0014] Preferably, according to the present invention, the mRNA encoding the FAP-specific CAR targeting fibrosis is prepared according to the DNA template T7 transcriptase method.

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

[0016] A recombinant expression vector containing mRNA encoding a FAP-specific CAR targeting fibrosis.

[0017] A third aspect of the present invention provides a macrophage efferocytosis enhancer, wherein the macrophage efferocytosis enhancer is Lgmn protein, LC3 protein, MERTK protein, NRP1 protein or CD68 protein.

[0018] Further preferably, the efferocytosis enhancer is the Lgmn protein containing a his-tagged gene, the amino acid sequence of which is shown in SEQ ID NO. 3. This protein confers efficient degradation of phagolysosomal cargo, thereby enhancing the clearance ability of CAR-macrophages on activated fibroblasts.

[0019] In a fourth aspect, the present invention provides an mRNA encoding the above-mentioned efferocytosis enhancer Lgmn. The mRNA comprises a 5' cap, a 5' non-coding region, an Lgmn sequence, a 3' non-coding region and a 3' polyA tail, and its nucleotide sequence is shown in SEQ ID NO.4.

[0020] Preferably, according to the present invention, the mRNA encoding the efferocytosis enhancer Lgmn is prepared according to the DNA template T7 transcriptase method.

[0021] Further preferably, the mRNA is circular RNA or linear RNA.

[0022] A recombinant expression vector containing mRNA encoding an efferocytosis enhancer Lgmn.

[0023] The fifth aspect of the present invention provides a CAR-macrophage targeting FAP and having enhanced efferocytosis, wherein the CAR-macrophage contains the above-mentioned FAP-specific CAR targeting fibrosis and the efferocytosis enhancer Lgmn.

[0024] Preferably, according to the present invention, the CAR-macrophages simultaneously express FAP-specific CAR targeting fibrosis and the efferocytosis enhancer Lgmn.

[0025] Preferably, according to the present invention, the CAR-macrophages targeting FAP and having enhanced efferocytosis are prepared according to the following method: mRNA encoding a FAP-specific CAR targeting fibrosis and mRNA containing an efferocytosis enhancer Lgmn are introduced into macrophages.

[0026] Further preferably, the introduction method includes but is not limited to electroporation, viral transfection, and non-viral vector delivery.

[0027] A recombinant expression vector contains an mRNA encoding a FAP-specific mRNA targeting fibrosis and an mRNA encoding an efferocytosis enhancer Lgmn.

[0028] The sixth aspect of the present invention provides a lipid nanoparticle (Lgmn / CAR-LNPs) that co-loads mRNA encoding a FAP-specific CAR targeting fibrosis and mRNA encoding an efferocytosis enhancer Lgmn, comprising mRNA encoding a FAP-specific CAR targeting fibrosis, mRNA encoding an efferocytosis enhancer Lgmn, and a delivery vector.

[0029] According to a preferred embodiment of the present invention, the method for preparing lipid nanoparticles (Lgmn / CAR-LNPs) co-loading mRNA encoding FAP-specific CAR targeting fibrosis and mRNA encoding Lgmn, an enhancer of efferocytosis, comprises the following steps:

[0030] (1) Dissolve CAR mRNA and Lgmn mRNA in 50 mM citric acid buffer (pH = 4) to obtain an mRNA mixture;

[0031] (2) dissolving ionizable lipid D-2, cholesterol, PEG-lipid, and DOPE in ethanol to obtain a mixed lipid solution;

[0032] (3) The mRNA mixture and the mixed lipid solution were added to the microfluidic device, and lipid nanoparticles (Lgmn / CAR-LNPs) co-loaded with mRNA encoding FAP-specific CAR targeting fibrosis and mRNA encoding Lgmn, an enhancer of endocytosis, were prepared by microfluidic method.

[0033] Preferably, according to the present invention, in step (1), the mass ratio of the CAR mRNA to the Lgmn mRNA is 1:1; the concentration of the CAR mRNA in the mRNA mixture is 0.11-0.12 mg / mL, and the concentration of the Lgmn mRNA is 0.11-0.12 mg / mL.

[0034] Preferably, according to the present invention, in step (2), the structure of the ionizable lipid D-2 is shown below:

[0035]

[0036] Further preferably, the preparation method of the ionizable lipid D2 is as follows:

[0037] N-Boc-ethylenediamine is coupled with 1-bromononane to generate an intermediate, and then the Boc group of the intermediate is removed using trifluoroacetic acid, and then coupled with a cyclic dicarboxylic acid derivative of pyroglutamic acid to obtain the ionizable lipid D2.

[0038] According to the preferred embodiment of the present invention, in step (2), the molar ratio of the ionizable lipid D-2, cholesterol, PEG-lipid and DOPE is 15:20:10:0.5; the concentration of the ionizable lipid D2 in the plasmid nanoparticle solution is 10 mg / mL;

[0039] The PEG-lipid is a mixture of DMG-PEG, DSPE-PEG-MPEP and DSPE-PEG-S2P, and the molar ratio of the three is 1:1:1.

[0040] Preferably, according to the present invention, in step (3), the mass ratio of the total mRNA in the mRNA mixture to the ionizable lipid D-2 in the mixed lipid solution is 1:10.

[0041] In a seventh aspect, the present invention provides the use of the above-mentioned FAP-specific CAR targeting fibrosis, a recombinant expression vector containing mRNA encoding the FAP-specific CAR targeting fibrosis, an efferocytosis enhancer Lgmn, a recombinant expression vector containing mRNA encoding the efferocytosis enhancer Lgmn, CAR-macrophages with enhanced efferocytosis targeting FAP, a recombinant expression vector containing mRNA encoding the FAP-specific mRNA targeting fibrosis and mRNA encoding the efferocytosis enhancer Lgmn, or lipid nanoparticles (Lgmn / CAR-LNPs) co-loaded with mRNA encoding the FAP-specific CAR targeting fibrosis and mRNA encoding the efferocytosis enhancer Lgmn in the preparation of drugs for the treatment of fibrotic diseases.

[0042] Preferably according to the present invention, the fibrotic disease is cardiac fibrosis.

[0043] The eighth aspect of the present invention also provides a drug for treating fibrosis, which contains an mRNA encoding FAP-specific mRNA targeting fibrosis and an mRNA encoding an efferocytosis enhancer Lgmn, or a recombinant expression vector encoding an mRNA encoding FAP-specific mRNA targeting fibrosis and an mRNA encoding an efferocytosis enhancer Lgmn has been inserted, or a CAR-macrophage targeting FAP efferocytosis enhancer has been inserted, or a recombinant expression vector encoding an mRNA encoding FAP-specific mRNA targeting fibrosis and an mRNA encoding an efferocytosis enhancer Lgmn has been inserted, or lipid nanoparticles (Lgmn / CAR-LNPs) co-carrying an mRNA encoding a FAP-specific CAR targeting fibrosis and an mRNA encoding an efferocytosis enhancer Lgmn.

[0044] Preferably according to the present invention, the fibrotic disease is cardiac fibrosis.

[0045] The technical features and advantages of the present invention are as follows:

[0046] 1. The present invention provides a CAR-macrophage targeting FAP with enhanced efferocytosis. The CAR-macrophage can specifically target the FAP target and enhance the efferocytosis signaling pathway, thereby combining CAR-macrophage therapy with enhanced intracellular degradation capacity. By expressing the Lgmn protein, the rapid intracellular degradation and clearance of engulfed activated fibroblasts is enhanced, thereby exerting and amplifying the phagocytic effect of CAR-macrophages on activated fibroblasts, thereby achieving a highly effective anti-fibrosis effect.

[0047] 2. The present invention provides a lipid nanoparticle (Lgmn / CAR-LNPs) that co-loads mRNA encoding FAP-specific CAR targeting fibrosis and mRNA encoding Lgmn, an enhancer of cell efferocytosis. The lipid nanoparticle successfully delivers mRNA encoding FAP-specific CAR targeting fibrosis and mRNA encoding Lgmn, an enhancer of cell efferocytosis, through a highly biocompatible LNP delivery system. It can be used for editing macrophages in vitro as well as in vivo, simplifying the process of engineered immune cell therapy and effectively avoiding off-target risks and systemic toxic side effects.

[0048] 3. The lipid nanoparticles (Lgmn / CAR-LNPs) provided by the present invention, which co-load mRNA encoding FAP-specific CAR targeting fibrosis and mRNA encoding Lgmn, an enhancer of efferocytosis, can directly generate FAP-targeted CAR-macrophages with enhanced efferocytosis in situ within infarcted cardiac tissue, greatly simplifying the delivery and generation process of CAR-macrophages and reducing systemic adverse reactions associated with macrophage reprogramming. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1Schematic diagram of the structure of the chimeric antigen receptor (CAR) in Example 1 of the present application.

[0050] Figure 2 This is the synthetic route of the ionizable lipid D-2 in Example 2 of the present application.

[0051] Figure 3 This is the NMR characterization result of the ionizable lipid D-2 in Example 2 of the present application.

[0052] Figure 4 This is the characterization of the physical and chemical properties of Lgmn / CAR-LNPs in Example 3 of this application.

[0053] Figure 5 These are the results of measuring the effect of Lgmn / CAR-LNPs on macrophage viability in Example 4 of this application.

[0054] Figure 6 These are the results of measuring the transfection efficiency of Lgmn / CAR-LNPs on macrophages in Example 5 of this application.

[0055] Figure 7 These are the results of measuring the phagocytic ability of the engineered macrophages on FAP-expressing target cells in Example 6 of the present application.

[0056] Figure 8 The effect of Lgmn / CAR-LNPs on infarct size in mice with myocardial infarction in Example 7 of the present application;

[0057] In the figure, Masson staining represents the myocardial infarction area and the statistical results of infarction area. **** indicates that after statistical analysis, there is a significant difference compared with the control group, P < 0.0001.

[0058] Figure 9 These are M-mode ultrasound images and statistical graphs of cardiac function indicators (ejection fraction EF, contraction fraction FS, end-systolic volume (s), end-diastolic volume (d), left ventricular end-systolic diameter (D), and end-diastolic diameter (D)) of mice in each group on days 0, 7, 14, 21, and 28 after treatment with Lgmn / CAR-LNPs in Example 7 of the present application;

[0059] In the figure, **** indicates that after statistical analysis, there is a significant difference compared with the control group, P < 0.0001.

[0060] Figure 10 This is a diagram showing the in vivo safety characterization results of Lgmn / CAR-LNPs in Example 8 of the present application. DETAILED DESCRIPTION

[0061] The present invention is further described below with reference to the following examples, which are only used to illustrate the present invention and are not intended to limit the scope of protection of the present invention.

[0062] Unless otherwise specified, the drugs and reagents involved in the examples are all common commercially available products; unless otherwise specified, the experimental operations involved in the examples are all performed according to routine operations in the art.

[0063] Example 1. FAP-specific CAR targeting fibrosis and efferocytosis enhancer

[0064] 1. The extracellular domain of the chimeric antigen receptor (CAR) was designed using the fibrosis-specific marker fibroblast activation protein (FAP) as the specific antigen.

[0065] like Figure 1 As shown, the extracellular to intracellular segment of the chimeric antigen receptor includes, in sequence, a CD8 front-end signal peptide, a specific anti-fibrosis FAP monoclonal antibody (anti-FAP-scFv), a CD8α hinge region, a CD8α transmembrane region, a CD3ζ signal transduction domain, and a flg-tag marker gene.

[0066] The specific construction process is as follows: the amino acid sequence of each element is searched through the NCBI database, the sequence is optimized, and then the sequence is concatenated. The concatenation order is CD8 front-end signal peptide, anti-FAP-scFv, flag tag gene, CD8α hinge region, CD8 transmembrane region, and CD3ζ intracellular domain. A FAP-specific CAR targeting cardiac fibrosis is constructed. Its amino acid sequence is shown in SEQ ID NO.1, and its nucleotide sequence is shown in SEQ ID NO.2. The mRNA of the CAR is synthesized by Yunzhou Biotechnology (Guangzhou) Co., Ltd.

[0067] 2. Provide a macrophage efferocytosis enhancer, the efferocytosis enhancer is an Lgmn protein containing a his-tagged gene, and its amino acid sequence is shown in SEQ ID NO.3. This protein confers efficient degradation of phagolysosomal cargo, thereby enhancing the clearance ability of CAR-macrophages on activated fibroblasts. The mRNA includes a 5' cap, a 5' non-coding region, an Lgmn sequence, a 3' non-coding region, and a 3' polyA tail, and the nucleotide sequence is shown in SEQ ID NO.4. The Lgmn mRNA is synthesized by Yunzhou Biotechnology (Guangzhou) Co., Ltd.

[0068] Example 2: Preparation of ionizable lipid D-2

[0069] like Figure 2As shown, N-boc-ethylenediamine (5 mmol) was dissolved in 20 mL of anhydrous acetonitrile, potassium carbonate (10 mmol) and 1-bromononane (12 mmol) were added, and the reaction was stirred at 80°C for 72 h. The mixture was filtered, and the filtrate was concentrated under reduced pressure and purified by silica gel column chromatography (petroleum ether:ethyl acetate volume ratio = 5:1) to obtain intermediate 1. Intermediate 1 (2 mmol) was dissolved in 2 mL of dichloromethane, and 2 mL of trifluoroacetic acid was added under ice bath. The reaction was stirred at room temperature for 4 h, and the reaction solution was spin-dried to obtain intermediate 2, which was directly used for the next step without purification. Intermediate 2 (2 mmol), a cyclic diisopropylpyrrolidinoglutamic acid derivative (1 mmol), benzotriazole-1-yl-oxytripyrrolidinophosphine hexafluorophosphate (PyBOP, 2.5 mmol) and N-ethyldiisopropylamine (DIPEA, 6 mmol) were dissolved in 6 mL of N,N-dimethylformamide (DMF) and stirred at room temperature for 48 h. The product was washed and extracted and purified by silica gel column chromatography (dichloromethane: methanol volume ratio = 8:1) to obtain ionizable lipid D-2.

[0070] The ionizable lipid D-2 prepared in this example was characterized by nuclear magnetic resonance, and the nuclear magnetic resonance results are as follows: Figure 3 shown.

[0071] Depend on Figure 3 It can be seen that the ionizable lipid D-2 was successfully synthesized, and its structure is shown in the following formula:

[0072]

[0073] Example 3. Preparation and Characterization of Lgmn / CAR-LNPs

[0074] A method for preparing lipid nanoparticles (Lgmn / CAR-LNPs) co-loaded with mRNA encoding FAP-specific CAR targeting fibrosis and mRNA encoding Lgmn, an enhancer of efferocytosis, comprises the following steps:

[0075] (1) Dissolve 20 μL of 1 mg / mL CAR mRNA and 20 μL of 1 mg / mL Lgmn mRNA in 50 mM citric acid buffer (pH = 4) to obtain 900 μL of mRNA mixture;

[0076] (2) 40 μL of 10 mg / mL ionizable lipid D-2, 48.67 μL of 5 mg / mL cholesterol, 46.8 μL of 5 mg / mL DOPE, and 7.9 μL of 5 mg / mL PEG-lipid were dissolved in ethanol to obtain 300 μL of a mixed lipid solution;

[0077] The PEG-lipid is a mixture of DMG-PEG, DSPE-PEG-MPEP and DSPE-PEG-S2P, and the molar ratio of the three is 1:1:1;

[0078] (3) 900 μL of the mRNA mixture was rapidly mixed with 300 μL of the mixed lipid solution at a flow rate ratio of 3:1 in a microfluidic device to prepare lipid nanoparticles (Lgmn / CAR-LNPs) that co-loaded mRNA encoding FAP-specific CAR targeting fibrosis and mRNA encoding Lgmn, an enhancer of endocytosis. The mass ratio of the mRNA in the mRNA mixture to the ionizable lipid D2 in the mixed lipid solution was 1:10.

[0079] The encapsulation efficiency of Lgmn / CAR-LNPs was determined using a RiboGreen kit, and the hydrated particle size and surface potential were measured using a Malvern particle size analyzer. Figure 4 shown.

[0080] Depend on Figure 4 It can be seen that Lgmn / CAR-LNPs can efficiently encapsulate mRNA, with an encapsulation efficiency of up to 93.5±2.08%; the hydrated particle size of Lgmn / CAR-LNPs is 161.49±5.5nm, the particle size is uniform, and the Zeta potential is close to neutrality.

[0081] Example 4: Cytotoxicity Study of Lgmn / CAR-LNPs

[0082] 1. Extraction, isolation and culture of bone marrow-derived macrophages (BMDMs)

[0083] Femurs and tibias were removed from euthanized C57BL / 6 mice, and the bone marrow was flushed and collected by centrifugation through a 70μm cell strainer. The cells were cultured for seven days in DMEM supplemented with 10ng / mL M-CSF to induce differentiation into macrophages.

[0084] 2. Macrophages were seeded into 96-well plates (5×10 3 After culturing overnight to allow attachment, different gradient concentrations of Lgmn / CAR-LNPs (gradient concentrations of 100, 250, 500, 1000, and 1250 ng / ml, calculated based on mRNA concentration) were added to the culture medium solution and incubated for 24 hours to detect BMDMs cell viability. The results are shown in the figure. Figure 5 shown.

[0085] Depend on Figure 5 It can be seen that Lgmn / CAR-LNPs have no obvious cytotoxicity and good safety.

[0086] Example 5: In vitro macrophage transfection experiment of Lgmn / CAR-LNPs

[0087] Macrophages were seeded in 24-well plates (6 × 10 4 cells / well), and 24 hours later, the cells were divided into two groups, and Lgmn / CAR-LNPs (60 ng / well, calculated as mRNA) and an equal volume of PBS solution were added, namely the LNPs group and the Control group. After incubation for 24 hours, the cells were collected and the transfection efficiency was detected. The results are shown in Figure 2. Figure 6 shown.

[0088] Depend on Figure 6 It can be seen that the simultaneous expression of CAR protein in macrophages treated with Lgmn / CAR-LNPs can reach 54.37%, and Lgmn expression is also significantly upregulated, indicating that Lgmn / CAR-LNPs can successfully co-deliver two mRNAs. Lgmn / CAR-LNPs can be used to construct CAR-macrophages with enhanced FAP-targeting efferocytosis.

[0089] Example 6: Investigation of the anti-fibrotic ability of Lgmn / CAR-LNPs in vitro

[0090] 1. According to the method described in Example 3, lipid nanoparticles (CAR-LNPs) loaded with mRNA encoding FAP-specific CAR targeting fibrosis and lipid nanoparticles (Lgmn / CAR-LNPs) loaded with mRNA encoding FAP-specific CAR targeting fibrosis and mRNA encoding the enhancer Lgmn for enhancing endocytosis were prepared respectively.

[0091] 2. The mouse FAP-EGFP plasmid was transfected into HEK293T cells (ATCC CRL-3216) using LipofectamineTM 3000 (Invitrogen L3000015) to prepare EGFP-FAP-HERK293T target cells.

[0092] 3. Macrophages were seeded into 24-well plates (6×10 4 After the cells adhered to the wall, they were divided into three groups and added with PBS, CAR-LNPs, and Lgmn / CAR-LNPs (60 ng / well, calculated as mRNA), namely the Control group, CAR group, and Lgmn / CAR group. After incubation for 24 hours, EGFP-FAP-HERK293T target cells (6×10 4 After 12 hours of continuous culture, the cells were collected and the phagocytosis of EGFP-FAP-HERK293T target cells by the engineered macrophages in each group was determined by flow cytometry. Figure 7 shown.

[0093] Depend on Figure 7 The Lgmn / CAR group exhibited the highest phagocytic efficiency, indicating that Lgmn / CAR-LNPs further induced macrophage clearance of FAP-positive cells, demonstrating a potent anti-fibrotic effect. Furthermore, compared to the CAR group, the Lgmn / CAR group, through the expression of Lgmn protein, enhanced the rapid intracellular degradation and clearance of activated fibroblasts, amplifying the phagocytic effect of CAR-macrophages on activated fibroblasts, resulting in a more effective anti-fibrotic effect.

[0094] Example 7. In vivo pharmacodynamic evaluation of Lgmn / CAR-LNPs

[0095] According to the method described in Example 3, lipid nanoparticles loaded with mRNA encoding FAP-specific CAR targeting fibrosis (CAR-LNPs), lipid nanoparticles loaded with mRNA encoding Lgmn, an enhancer of cell efferocytosis (Lgmn-LNPs), and lipid nanoparticles loaded with mRNA encoding FAP-specific CAR targeting fibrosis and mRNA encoding Lgmn, an enhancer of cell efferocytosis (Lgmn / CAR-LNPs) were prepared respectively.

[0096] C57BL / 6 male mice were used to establish an acute myocardial infarction model by ligating the left anterior descending coronary artery. The specific construction method was as follows:

[0097] (1) After the mouse is deeply anesthetized with isoflurane, it is placed on a surgical anesthesia mouse mask, the precordial area is depilated, and the mouse's limbs are fixed;

[0098] (2) A surgical opening is made in the third and fourth intercostal spaces in the precordial area, and the muscle tissue is bluntly separated to expose the apex of the heart;

[0099] (3) Use the needle holder in the right hand to break through the chest wall between the third and fourth intercostal spaces, and use the left hand to compress the chest cavity until the heart is completely exposed outside the chest wall. Ligate the left anterior descending coronary artery of the mouse heart using 8-0 surgical sutures while looking directly at the mouse heart.

[0100] (4) Release your left hand and use the needle holder to guide the heart back into the chest cavity, squeezing the chest cavity to expel any air that may be present to prevent pneumothorax;

[0101] (5) Use 4-0 surgical sutures to close the surgical opening in the chest wall, and use the mouse electrocardiogram to observe whether the myocardial infarction model is successful. If the model is successful, the mouse lead electrocardiogram will show obvious ST segment elevation.

[0102] Mice with acute myocardial infarction were randomly divided into four groups (n=6-8) and injected with PBS, CAR-LNPs, Lgmn-LNPs, and Lgmn / CAR-LNPs (0.3 mg / kg, calculated as mRNA) via tail vein, namely the MI group, CAR group, Lgmn group, and Lgmn / CAR group. The drugs were administered once every three days for four times. At the same time, normal C57BL / 6 male mice (n=8) were sham-operated as a control, namely the Sham group. The myocardial tissues of the mice in each group were collected for Masson staining 28 days after modeling to detect the myocardial infarction area of ​​the mice in each group. The results are shown in Figure 3. Figure 8 shown.

[0103] Depend on Figure 8 It can be seen that the injection of CAR-LNPs, Lgmn-LNPs, and Lgmn / CAR-LNPs can significantly reduce the myocardial infarction area in mice 28 days after myocardial infarction, among which Lgmn / CAR-LNPs has the best effect and the smallest myocardial infarction area.

[0104] The cardiac ultrasound function of mice in each group was detected on the 0th, 7th, 14th, 21st and 28th day after injection. Figure 9 shown.

[0105] Depend on Figure 9 It can be seen that compared with the MI group, the ejection fraction and short-axis shortening rate of MI mice in the CAR-LNPs, Lgmn-LNPs, and Lgmn / CAR-LNPs injection groups were significantly increased, and the left ventricular end-diastolic volume and left ventricular inner diameter were significantly decreased, all indicating improved cardiac function, among which Lgmn / CAR-LNPs had the best effect.

[0106] Example 8. In vivo safety evaluation of Lgmn / CAR-LNPs

[0107] According to the method described in Example 3, lipid nanoparticles loaded with mRNA encoding FAP-specific CAR targeting fibrosis (CAR-LNPs), lipid nanoparticles loaded with mRNA encoding Lgmn, an enhancer of cell efferocytosis (Lgmn-LNPs), and lipid nanoparticles loaded with mRNA encoding FAP-specific CAR targeting fibrosis and mRNA encoding Lgmn, an enhancer of cell efferocytosis (Lgmn / CAR-LNPs) were prepared respectively.

[0108] An acute myocardial infarction model mouse was constructed according to the method described in Example 7.

[0109] Mice with acute myocardial infarction were randomly divided into four groups (n=4) and injected with PBS, CAR-LNPs, Lgmn-LNPs, and Lgmn / CAR-LNPs (0.3 mg / kg, calculated as mRNA) via tail vein, namely the MI group, CAR group, Lgmn group, and Lgmn / CAR group. The drugs were administered once every three days for four times. At the same time, normal C57BL / 6 male mice (n=4) were sham-operated as a control, namely the Sham group. Peripheral blood was collected from the mice the day after the third administration to determine the hepato-renal toxicity. The results are shown in Table 1. Figure 10 shown.

[0110] Depend on Figure 10 It can be seen that compared with the MI group, there was no significant difference in the indicators of the CAR group, Lgmn group and Lgmn / CAR group and they were all within the normal range, proving their good biosafety.

[0111] The above are only preferred embodiments of the present invention. The scope of protection of the present invention is not limited to the above embodiments. All technical solutions based on the concept of the present invention are within the scope of protection of the present invention. It should be pointed out that for those skilled in the art, any modification, equivalent replacement or improvement without departing from the principle of the present invention should be considered as within the scope of protection of the present invention.

Claims

1. A CAR-macrophage targeting FAP with enhanced efferocytosis, characterized in that: The CAR-macrophages contain FAP-specific CAR targeting fibrosis and an efferocytosis enhancer Lgmn; The amino acid sequence of the FAP-specific CAR targeting fibrosis is shown in SEQ ID NO.1, and the nucleotide sequence of the mRNA encoding the FAP-specific CAR targeting fibrosis is shown in SEQ ID NO.2; The efferocytosis enhancer is the Lgmn protein containing a his-tagged gene, the amino acid sequence of which is shown in SEQ ID NO.3, and the nucleotide sequence of which is shown in SEQ ID NO.

4.

2. The FAP-targeting CAR-macrophage with enhanced efferocytosis according to claim 1, characterized in that: The preparation was performed as follows: mRNA encoding a FAP-specific CAR targeting fibrosis and mRNA containing the efferocytosis enhancer Lgmn were introduced into macrophages.

3. A recombinant expression vector, characterized in that: The recombinant expression vector contains the FAP-specific mRNA encoding the fibrosis-targeting mRNA according to claim 1 and the mRNA encoding the efferocytosis enhancer Lgmn.

4. A lipid nanoparticle co-loaded with mRNA encoding a FAP-specific CAR targeting fibrosis and mRNA encoding an efferocytosis enhancer Lgmn, characterized in that: It comprises the mRNA encoding the FAP-specific CAR targeting fibrosis according to claim 1, the mRNA encoding the efferocytosis enhancer Lgmn, and a delivery vector.

5. The method for preparing lipid nanoparticles co-loaded with mRNA encoding FAP-specific CAR targeting fibrosis and mRNA encoding Lgmn, an enhancer of efferocytosis, according to claim 4, characterized in that: The steps include: (1) Dissolve CAR mRNA and Lgmn mRNA in 50 mM, pH 4 citric acid buffer to obtain an mRNA mixture; The mass ratio of the CAR mRNA to the Lgmn mRNA is 1:1; the concentration of the CAR mRNA in the mRNA mixture is 0.11-0.12 mg / mL, and the concentration of the Lgmn mRNA is 0.11-0.12 mg / mL; (2) dissolving ionizable lipid D-2, cholesterol, PEG-lipid, and DOPE in ethanol to obtain a mixed lipid solution; Wherein, the structure of the ionizable lipid D-2 is shown below: ； The molar ratio of the ionizable lipid D-2, cholesterol, PEG-lipid and DOPE is 15:20:10:0.5; the concentration of the ionizable lipid D2 in the nanoparticle solution is 10 mg / mL; The PEG-lipid is a mixture of DMG-PEG, DSPE-PEG-MPEP and DSPE-PEG-S2P, with a molar ratio of 1:1:1; (3) Adding the mRNA mixture and the mixed lipid solution to the microfluidic device, and preparing lipid nanoparticles co-loaded with mRNA encoding FAP-specific CAR targeting fibrosis and mRNA encoding Lgmn, an enhancer of endocytosis, by microfluidic method; The mass ratio of the total mRNA in the mRNA mixture to the ionizable lipid D-2 in the mixed lipid solution is 1:

10.

6. Use of the FAP-targeting CAR-macrophage with enhanced efferocytosis according to claim 1, the recombinant expression vector according to claim 3, or the lipid nanoparticles co-loaded with mRNA encoding the FAP-specific CAR targeting fibrosis and mRNA encoding the efferocytosis enhancer Lgmn according to claim 4 in the preparation of a drug for treating fibrotic diseases; The fibrotic disease is cardiac fibrosis.

7. A drug for treating fibrosis, characterized in that: The drug contains the CAR-macrophages with enhanced FAP efferocytosis targeting according to claim 1, the recombinant expression vector according to claim 3, or the lipid nanoparticles according to claim 4 that co-load mRNA encoding the FAP-specific CAR targeting fibrosis and mRNA encoding the efferocytosis enhancer Lgmn; the fibrotic disease is cardiac fibrosis.

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