FAP-targeted CAR-macrophage with enhanced intercellular burial effect and preparation method and application of CAR-macrophage

Abstract

The invention relates to a FAP-targeted CAR-macrophage with an enhanced intercellular effect as well as a preparation method and application of the FAP-targeted CAR-macrophage. The CAR-macrophage contains a FAP specific CAR for targeting fibrosis and a burial effect enhancer Lgmn, the amino acid sequence of the FAP specific CAR for targeting fibrosis is as shown in SEQ ID NO. 1, and the nucleotide sequence of the FAP specific CAR for targeting fibrosis is as shown in SEQ ID NO. 2; the amino acid sequence of the cell burial effect enhancer is as shown in SEQ ID NO.3, and the nucleotide sequence of the cell burial effect enhancer is as shown in SEQ ID NO.4. The CAR-macrophage can specifically target an FAP target spot, can also enhance a cellular burial effect signal channel, realizes combination of CAR-macrophage therapy and intracellular degradation capability improvement, enhances intracellular rapid degradation and removal of phagocytized activated fibroblasts by expressing Lgmn protein, exerts and amplifies the phagocytic effect of the CAR-macrophage on the activated fibroblasts, and improves the activity of the CAR-macrophage on the activated fibroblasts. Therefore, an efficient anti-fibrosis effect is obtained.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a CAR-macrophage targeting FAP and having enhanced endocytosis, and 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 increasing cardiovascular risk. Cardiac fibroblasts (CFs) are overactivated in response to various cardiac injuries and play a key role in cardiac fibrosis and extracellular matrix (ECM) deposition. Inhibiting or eliminating activated CFs is crucial to reduce fibrosis and improve cardiac function after MI, and no effective treatment has yet been found.

[0003] Chimeric antigen receptor (CAR) cell therapy offers a promising precision therapy by reprogramming immune cells to eliminate specific target cells. CAR-T targeted removal of activated CFs has shown potential therapeutic effects, reducing cardiac fibrosis and restoring cardiac function. However, CAR-T cells may face some limitations during application, including poor tissue penetration and severe side effects such as cachexia, anemia, and fatal bone toxicity. These problems limit the widespread application of CAR-T cells in actual clinical practice. Compared with CAR-T cell therapy, chimeric antigen receptor-macrophages (CAR-MΦ) have obvious advantages. First, CAR-MΦs can penetrate infarcted tissue more effectively, resulting in better clinical effects. Second, CAR-MΦs do not self-proliferate, thus significantly reducing the risk of cytokine release syndrome. In addition, CAR-MΦs have a shorter circulation time and lower off-target toxicity than CAR-T cells, thereby improving the safety of treatment.

[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 targeting FAP +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 mass production of fibrosis-specific macrophages in vitro remain the main obstacles to the widespread application of CAR-MΦ therapy. In addition, the large number of dead cells after MI causes CAR-MΦs to face an excessive cell burial burden, which is specifically manifested in the lack of CAR-MΦs internalization and degradation function, thereby limiting the continuous clearance of cargo in phagolysosomes, thereby 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 view of the deficiencies in the prior art, the present invention provides a CAR-macrophage targeting FAP and having enhanced efferocytosis, as well as a preparation method and application thereof.

[0007] The technical solution of the present invention is 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] Among them, 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 as 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] The third aspect of the present invention provides a macrophage efferocytosis enhancer, wherein the efferocytosis enhancer is Lgmn protein, LC3 protein, MERTK protein, NRP1 protein or CD68 protein.

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

[0019] The fourth aspect of the present invention provides mRNA encoding the above-mentioned efferocytosis enhancer Lgmn, wherein 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 the nucleotide sequence thereof 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 contains 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 to obtain the obtained cells.

[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] Preferably, according to 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 endocytosis, comprises the following steps:

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

[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 as shown below:

[0035]

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

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

[0038] Preferably, 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 CAR targeting fibrosis and mRNA encoding the efferocytosis enhancer Lgmn, or lipid nanoparticles (Lgmn / CAR-LNPs) co-loading mRNA encoding the FAP-specific CAR targeting fibrosis and mRNA encoding the efferocytosis enhancer Lgmn in the preparation of drugs for treating 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 mRNA encoding FAP-specific targeting fibrosis and mRNA encoding Lgmn, a recombinant expression vector having inserted mRNA encoding FAP-specific targeting fibrosis and mRNA encoding Lgmn, a CAR-macrophage targeting FAP efferocytosis enhancement, or a recombinant expression vector having inserted mRNA encoding FAP-specific targeting fibrosis and mRNA encoding Lgmn, a fibrosis enhancer, or lipid nanoparticles (Lgmn / CAR-LNPs) co-loading mRNA encoding FAP-specific CAR targeting fibrosis and mRNA encoding Lgmn, a fibrosis enhancer.

[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 and having enhanced efferocytosis. The CAR-macrophage can specifically target the FAP target and enhance the efferocytosis signal pathway, thereby realizing the combination of CAR-macrophage therapy and improving the intracellular degradation ability. By expressing Lgmn protein, the rapid intracellular degradation and clearance of engulfed activated fibroblasts is enhanced, and the phagocytic effect of CAR-macrophages on activated fibroblasts is exerted and amplified, thereby obtaining a highly efficient anti-fibrosis effect.

[0047] 2. 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 enhancer of efferocytosis, Lgmn. The lipid nanoparticle successfully delivers mRNA encoding a FAP-specific CAR targeting fibrosis and mRNA encoding an enhancer of efferocytosis, Lgmn, through a highly biocompatible LNP delivery system. The lipid nanoparticle can be used for editing macrophages in vitro or in vivo, thereby 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 in the 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 a synthetic route for 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 a characterization of the physical and chemical properties of Lgmn / CAR-LNPs in Example 3 of the present application.

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

[0054] Figure 6 These are the results of measuring the transfection efficiency of Lgmn / CAR-LNPs on macrophages in Example 5 of the present 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 the infarction area 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] Fig. 9 The M-mode ultrasound images and cardiac function indexes (ejection fraction EF, contraction fraction FS, end-systolic volume Volume; s, end-diastolic volume Volume; d, left ventricular end-systolic diameter Diameter; s and end-diastolic diameter Diameter; d) of each group of mice after 0, 7, 14, 21 and 28 days after Lgmn / CAR-LNPs treatment in Example 7 of the present application are statistical graphs;

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

[0060] Fig.10 This is a graph 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 in conjunction with examples, which are only used to illustrate the present invention but not 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; the experimental operations involved in the examples are all carried out according to routine operations in the art unless otherwise specified.

[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 a specific antigen.

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

[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 in the order of CD8 front-end signal peptide, anti-FAP-scFv, flag tag gene, CD8α hinge region, CD8 transmembrane region, CD3ζ intracellular domain, and 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 burial enhancer, the burial enhancer is a Lgmn protein containing a his tag gene, and its amino acid sequence is shown in SEQ ID NO.3. The protein confers efficient degradation of phagolysosomal cargo, thereby enhancing the clearance ability of CAR-macrophages for 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 (5mmol) was dissolved in 20mL of anhydrous acetonitrile, potassium carbonate (10mmol) and 1-bromononane (12mmol) were added, the mixture was stirred at 80°C for 72h, and the mixture was filtered. The filtrate was concentrated under reduced pressure and purified by silica gel column chromatography (volume ratio of petroleum ether: ethyl acetate = 5:1) to obtain intermediate 1. Intermediate 1 (2mmol) was dissolved in 2mL of dichloromethane, 2mL of trifluoroacetic acid was added under ice bath, the mixture was stirred at room temperature for 4h, and the reaction solution was spin-dried to obtain intermediate 2, which was directly subjected to the next step of reaction without purification. Intermediate 2 (2 mmol), pyroglutamic acid cyclic di-derivative (1 mmol), benzotriazol-1-yl-oxytripyrrolidinophosphine hexafluorophosphate (PyBOP, 2.5 mmol) and N-ethyldiisopropylamine (DIPEA, 6 mmol) were dissolved in 6 mL of N,N-dimethylformamide (DMF), stirred and reacted 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 NMR. The NMR 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-loading 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 mixed solution;

[0076] (2) dissolving 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 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) co-loaded with mRNA encoding FAP-specific CAR targeting fibrosis and mRNA encoding Lgmn, an enhancer of endocytosis. The mass ratio of mRNA in the mRNA mixture to ionizable lipid D2 in the mixed lipid solution was 1:10.

[0079] The encapsulation efficiency of Lgmn / CAR-LNPs was determined using the RiboGreen kit, and the hydrated particle size and surface potential were determined 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 rate 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] After euthanasia, C57BL / 6 mice were harvested from the femur and tibia, and the bone marrow was washed and centrifuged through a 70 μm cell strainer to collect the cells. The cells were cultured in DMEM medium containing 10 ng / mL M-CSF for seven days to induce differentiation into macrophages.

[0084] 2. Macrophages were seeded in 96-well plates (5×10 3 Cells / well), cultured overnight to allow attachment, then added with different gradient concentrations of Lgmn / CAR-LNPs (gradient concentrations of 100, 250, 500, 1000, 1250 ng / ml, calculated based on mRNA concentration) culture medium solution, incubated for 24 hours to detect BMDMs cell viability. The results are shown in 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 After 24 hours, 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 24 hours of co-incubation, the cells were collected and the transfection efficiency was detected. The results are shown in 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 the expression of Lgmn is also significantly upregulated, indicating that Lgmn / CAR-LNPs can successfully co-deliver two mRNAs, and Lgmn / CAR-LNPs can be used to construct CAR-macrophages with enhanced FAP targeting efferocytosis.

[0089] Example 6: Investigation of the anti-fibrosis 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 Lgmn, an enhancer of cell thrombosis, were prepared respectively.

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

[0092] 3. Inoculate macrophages in 24-well plates (6×10 4 After the cells adhered to the wall, they were divided into three groups, and PBS, CAR-LNPs, and Lgmn / CAR-LNPs (60 ng / well, calculated as mRNA) were added, 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 engineered macrophages in each group was measured by flow cytometry. Figure 7 shown.

[0093] Depend on Figure 7 It can be seen that the Lgmn / CAR group showed the highest phagocytic efficiency, indicating that Lgmn / CAR-LNPs further induced macrophages to clear FAP-positive cells and had a strong anti-fibrotic effect. Compared with the CAR group, the Lgmn / CAR group enhanced the rapid intracellular degradation and clearance of engulfed activated fibroblasts by expressing Lgmn protein, exerted and amplified the phagocytic effect of CAR-macrophages on activated fibroblasts, and thus obtained a more efficient 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 efflux (Lgmn-LNPs), and lipid nanoparticles loaded with mRNA encoding FAP-specific CAR targeting fibrosis and mRNA encoding Lgmn, an enhancer of cell efflux (Lgmn / CAR-LNPs) were prepared respectively.

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

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

[0098] (2) A surgical opening is made at 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. Use 8-0 surgical sutures to ligate the left anterior descending coronary artery of the mouse 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] The mice with acute myocardial infarction model were randomly divided into four groups (n=6-8), and PBS, CAR-LNPs, Lgmn-LNPs, Lgmn / CAR-LNPs (0.3 mg / kg, calculated as mRNA) were injected into the tail vein, namely the MI group, CAR group, Lgmn group and Lgmn / CAR group, respectively. The drugs were administered once every three days for 4 times. At the same time, normal C57BL / 6 male mice (n=8) were subjected to sham surgery as the control, namely the Sham group. The myocardial tissues of the mice in each group were taken for Masson staining 28 days after the modeling, and the myocardial infarction area of ​​the mice in each group was detected. The results are shown in Fig. 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. Fig. 9 shown.

[0105] Depend on Fig. 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 efflux (Lgmn-LNPs), and lipid nanoparticles loaded with mRNA encoding FAP-specific CAR targeting fibrosis and mRNA encoding Lgmn, an enhancer of cell efflux (Lgmn / CAR-LNPs) were prepared respectively.

[0108] The acute myocardial infarction model mice were constructed according to the method described in Example 7.

[0109] Acute myocardial infarction model mice were randomly divided into four groups (n=4), and PBS, CAR-LNPs, Lgmn-LNPs, Lgmn / CAR-LNPs (0.3 mg / kg, calculated as mRNA) were injected into the tail vein, namely the MI group, CAR group, Lgmn group and Lgmn / CAR group, respectively. The drugs were administered once every three days for 4 times. At the same time, normal C57BL / 6 male mice (n=4) were subjected to sham surgery as a control, namely the Sham group. The peripheral blood of mice was collected the day after the third administration to determine the liver and kidney toxicity. The results are shown in Fig.10 shown.

[0110] Depend on Fig.10 It can be seen that compared with the MI group, there were no significant differences 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, and the protection scope of the present invention is not limited to the above embodiments. All technical solutions under the concept of the present invention belong to the protection scope of the present invention. It should be pointed out that for ordinary technicians in this technical field, any modification, equivalent replacement or improvement without departing from the principle of the present invention should be regarded as the protection scope of the present invention.

Claims

1. A FAP-specific CAR targeting fibrosis, characterized in that: Includes extracellular domain, transmembrane region and intracellular domain; The extracellular domain includes a front-end signal peptide segment, an antigen recognition domain, a flg tag gene and a hinge region; 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; 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; Preferably, 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; Further preferably, the amino acid sequence of the FAP-specific CAR targeting fibrosis is as shown in SEQ ID NO.

1.

2. An mRNA encoding the FAP-specific CAR targeting fibrosis according to claim 1, characterized in that: The nucleotide sequence is shown in SEQ ID NO.

2.

3. A macrophage efferocytosis enhancer, characterized in that: The efferocytosis enhancer is Lgmn protein containing his tag gene, and its amino acid sequence is shown in SEQ ID NO.

3.

4. An mRNA encoding the efferocytosis enhancer Lgmn according to claim 3, characterized in that: The nucleotide sequence is shown in SEQ ID NO.

4.

5. A CAR-macrophage targeting FAP and having enhanced efferocytosis, characterized in that: The CAR-macrophage contains the FAP-specific CAR targeting fibrosis according to claim 2 and the efferocytosis enhancer Lgmn according to claim 4; Preferably, 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 the efferocytosis enhancer Lgmn are introduced into macrophages.

6. A recombinant expression vector, comprising the mRNA encoding the FAP-specific targeted fibrosis according to claim 2 and the mRNA encoding the efferocytosis enhancer Lgmn according to claim 4.

7. 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 includes mRNA encoding FAP-specific CAR targeting fibrosis, mRNA encoding Lgmn, an enhancer of efferocytosis, and a delivery vector.

8. 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 7, characterized in that: The steps include: (1) dissolving CAR mRNA and Lgmn mRNA in potassium hydrogen phthalate-sodium hydroxide buffer to obtain an mRNA mixed solution; 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 in the following formula: 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; 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; (3) adding the mRNA mixture and the mixed lipid solution to the microfluidic device, and preparing lipid nanoparticles co-carrying mRNA encoding FAP-specific CAR targeting fibrosis and mRNA encoding Lgmn, an enhancer of endocytosis, by a 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.

9. Use of the FAP-specific CAR targeting fibrosis according to claim 1, the efferocytosis enhancer Lgmn according to claim 3, the CAR-macrophage targeting FAP efferocytosis-enhanced according to claim 5, the recombinant expression vector according to claim 6, 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 7 in the preparation of drugs for treating fibrotic diseases; The fibrotic disease is cardiac fibrosis.

10. A drug for treating fibrosis, characterized in that: The drug contains the FAP-specific CAR targeting fibrosis according to claim 1, the efferocytosis enhancer Lgmn according to claim 3, the CAR-macrophage targeting FAP efferocytosis enhancer according to claim 5, the recombinant expression vector according to claim 6, 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 7; The fibrotic disease is cardiac fibrosis.

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