A nanoliposome delivery system for constructing FAP-targeted CAR-T in situ, a preparation method and application thereof

Abstract

This invention discloses an in-situ constructed FAP-targeting CAR-T nanoliposome delivery system, its preparation method, and its applications, belonging to the field of biomedical technology. This invention provides a biocompatible nanoliposome delivery system whose surface is modified with a CD3 antibody that can capture T cells through circulation, delivering plasmid DNA encoding FAP-CAR into the T cells, thus in-situ transforming T cells into CAR-T cells in vivo. The nanoliposome delivery system provided by this invention can significantly alleviate myocardial fibrosis caused by myocardial infarction and restore cardiac function, providing a research basis for the clinical application of CAR-T cell therapy based on in-situ nanoengineering in myocardial infarction.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to an in-situ constructed FAP-targeting CAR-T nanoliposome delivery system, its preparation method, and its application. Background Technology

[0002] Myocardial infarction (MI) is a critical event leading to severe cardiac damage. The incidence of adverse cardiac remodeling after MI remains high, and long-term prognosis is generally poor. Over the past two decades, numerous studies have been dedicated to improving cardiac outcomes after MI. Initially, treatment focused primarily on reversing mechanical changes, such as reducing preload, afterload, and volume overload. Pharmacological therapies, including renin-angiotensin system blockers (e.g., angiotensin-converting enzyme inhibitors, ACEIs) or beta-blockers, were recommended to reduce morbidity and mortality in MI patients. Despite these advances, clinical outcomes for MI patients remain unsatisfactory, with a 5-year survival rate of less than 50%. Therefore, new therapies are needed to prevent further deterioration of cardiac function, and innovative drugs are urgently required to treat MI.

[0003] Myocardial fibrosis is a core pathological feature of cardiac injury following myocardial infarction. After myocardial infarction, ischemia and necrosis of cardiomyocytes stimulate the activation and proliferation of fibroblasts, leading to excessive extracellular matrix deposition and the progression of myocardial fibrosis. This condition increases cardiac stiffness, reduces ventricular compliance, and adversely affects systolic and diastolic function, thereby exacerbating cardiac dysfunction. Targeting fibrosis has become a key therapeutic strategy for limiting post-myocardial infarction injury. While some drugs, such as ACE inhibitors and beta-blockers, are used to relieve ischemia and inhibit the release of fibrosis-related factors, they primarily delay the progression of myocardial fibrosis through indirect mechanisms rather than directly targeting its core pathological process. Currently, there are no drugs specifically approved by the U.S. Food and Drug Administration (FDA) for the treatment of cardiac fibrosis.

[0004] Immunotherapy targeting cardiac fibroblasts has recently made encouraging progress. Fibroblast activating protein (FAP) is a cell surface glycoprotein highly expressed by cardiac fibroblasts in damaged heart tissue. In a mouse model of hypertensive cardiac injury induced by angiotensin II and norepinephrine, adoptive transfer of engineered chimeric antigen receptors (CARs) expressing FAP significantly reduced cardiac fibrosis and improved cardiac function.

[0005] Nevertheless, this CAR The clinical application of T-technology is still constrained by many factors, such as CAR. CARs require complex in vitro preparation processes and strict quality control. Unlike traditional drugs, CARs require... T is an autologous T-cell therapy based on the integration of the CAR gene, meaning CAR CAR cells must complete a complex process of blood sample collection, separation, activation, transfection, amplification, detection, formulation, transportation, and administration in the shortest possible time. Currently, clinically used CAR cells mainly rely on viral vectors and autologous immune cells for in vitro preparation, which requires a relatively long processing time (3-6 weeks). The in vitro preparation of T cells requires strict GMP cleanliness requirements and significant investment. Each patient's CAR... T-cell therapy requires personalized treatment and cannot be mass-produced, complicating timely prevention and treatment for myocardial infarction patients. Therefore, it can currently only be performed in a few research centers or specific laboratories worldwide. Thus, how to develop CAR-T therapy remains a key question. A new T-cell preparation technology avoids the complex in vitro preparation process and strict quality control, reducing CAR-T risk. The technological barriers of T-therapy are a major challenge that researchers in cell immunotherapy need to overcome.

[0006] Emerging research is exploring the use of targeted nanomaterials combined with mRNA transfection to generate CAR-T cells in situ, bypassing complex in vitro preparation processes. While transient mRNA CAR-T systems based on lipid nanoparticles (LNPs) avoid preparation delays, their clinical application remains limited by cold chain transportation requirements, high production costs, and uncertain efficacy in acute myocardial injury. In contrast, plasmid DNA offers significant advantages: room temperature stability, low-cost production, and transient expression kinetics matching the upregulation time window of fibroblast activation protein (FAP) after myocardial infarction. However, existing DNA delivery methods, such as electroporation and viral vector systems, still face key challenges: low transfection efficiency due to innate immune recognition, the inability to simultaneously achieve T cell-specific targeting and controllable transient expression in vivo, and the potential immunogenicity risks posed by viral vectors. Although these strategies have been applied in preclinical studies of diseases such as cancer, their therapeutic effects on myocardial fibrosis remain unclear. Therefore, developing a safe and efficient in situ CAR therapy that overcomes these obstacles is crucial for treating myocardial fibrosis and improving cardiac function. Summary of the Invention

[0007] In view of this, the present invention provides a novel CAR-T cell therapy based on in-situ nanoengineering. This therapy uses a biocompatible liposome nanodelivery system to deliver plasmid DNA encoding FAP-CAR into target cells, which can be T cells, NK cells, or macrophages. In a specific embodiment of the present invention, the liposome nanodelivery system, due to its surface modification with CD3 antibodies, can capture T cells through circulation, thereby transforming T cells into CAR-T cells in vivo. Furthermore, the present invention adds chloroquine to the lipid membrane, which, by inhibiting the interferon (IFN) reaction, can effectively enhance the transfection of plasmids encoding FAP-CAR into T cells, thereby improving transfection efficiency. Moreover, the present invention uses protamine sulfate to further improve the encapsulation efficiency of the plasmid. The technical solution provided by this invention aims to solve the following technical problems: (1) There is a lack of treatment methods that directly target myocardial fibrosis, that is, current drugs mainly slow down the progression of fibrosis through indirect mechanisms and cannot directly intervene in the core pathological process of fibrosis; (2) Traditional CAR-T cell therapy requires in vitro cell manipulation, which takes a long time, is difficult to exert its effects quickly, and has low clinical feasibility; (3) Lipid nanoparticles that encapsulate DNA will further reduce the preparation cost and improve the feasibility of treatment methods.

[0008] This invention includes the following technical solutions: In a first aspect, the present invention provides an in situ CAR-T nanoliposome delivery system, characterized in that the nanoliposome delivery system comprises a lipid membrane and a plasmid carrying a CAR gene encapsulated by the lipid membrane, and the surface of the nanoliposome delivery system is modified with a CD3 antibody.

[0009] Those skilled in the art can modify CD3 antibodies using conventional techniques. In one specific embodiment of the present invention, the CD3 antibody is coupled with DSPE-PEG2000-Mal and then modified onto the surface of nanoliposomes.

[0010] Furthermore, the nanoliposome delivery system also includes protamine, which is co-encapsulated with the plasmid in a lipid membrane. Those skilled in the art have found that protamine can promote plasmid aggregation, improve the plasmid encapsulation efficiency of the liposomes, and unexpectedly, it has been discovered that protamine protects the plasmid from DNase degradation, thereby enhancing plasmid bioavailability. In some embodiments of the present invention, the mass ratio of protamine to plasmid is (1-2):1, such as 1:1, 1.5:1, or 2:1. In a specific embodiment of the present invention, the mass ratio of protamine to plasmid is 1:1.

[0011] The lipid membrane is selected from any membrane material known to those skilled in the art that can be used to form liposomes. Preferably, the lipid membrane comprises cholesterol, 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), and distearate phosphatidylethanolamine. Polyethylene glycol (DSPE-PEG2000), distearate phosphatidylethanolamine One or more of polyethylene glycol-maleimide (DSPE-PEG2000-Mal).

[0012] In a specific embodiment of the present invention, the lipid membrane comprises cholesterol and 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), with a molar mass ratio of cholesterol to DOTAP of 1:(1-1.2).

[0013] Preferably, the lipid membrane further contains chloroquine. It has been unexpectedly discovered by those skilled in the art that chloroquine in the lipid membrane can significantly enhance the transfection rate of plasmid-transfected T cells and enhance lysosomal escape.

[0014] In a preferred embodiment of the present invention, the mass ratio of chloroquine to plasmid DNA is (4-5):1.

[0015] In this invention, the plasmid carrying the CAR gene encodes a CAR (chimeric antigen receptor) targeting FAP, and the structure of the CAR is shown in Formula I: X1-scFv-H-TM-CS-X2(Ⅰ) In the formula, "-" represents a linking peptide or peptide bond; X1 does not exist, or X1 is a signal peptide sequence; scFv is a humanized or murine single-chain antibody targeting FAP; H represents the hinge area; TM stands for transmembrane region; CS is a co-stimulatory signaling molecule; X2 is an intracellular signal transduction region originating from CD3ζ.

[0016] In a specific embodiment of the present invention, X1 is a CD8 leader.

[0017] The H is selected from the hinge region of the following proteins: CD4, CD7, CD8, CD28, 4-1BB, or a combination of two or more of them.

[0018] In a preferred embodiment of the present invention, H is the hinge region from which CD8 originates.

[0019] The TM is selected from one or more of the transmembrane regions derived from the following proteins: CD3ε, CD4, CD8, CD9, CD16, CD22, CD28, CD33, CD80, CD86, 4-1BB, CTLA-4, PD-1, and LAG-3.

[0020] In a preferred embodiment of the present invention, the TM is a transmembrane region derived from CD8.

[0021] The CS is selected from one or more of the following protein-derived co-stimulatory signaling molecules: CD2, CD3, CD7, CD27, CD28, CD30, CD40, CD70, CD83, CD86, CD127, CD134, 4-1BB, OX40, ICOS, GITR, PD-1, PD1L, B7-H3, DAP10, CDS, ICAM-1, LFA-1, and NKG2C.

[0022] In a preferred embodiment of the present invention, the CS is a co-stimulatory signaling molecule derived from 4-1BB.

[0023] In a specific embodiment of the present invention, the structure of the CAR is shown in Formula II: CD8 leader-scFv-CD8H-CD8TM-41BB-CD3ζ (Ⅱ) The scFv fragment is a single-chain antibody targeting FAP, and the antibody clone number is selected from 4G5 or MO36.

[0024] In a specific embodiment of the present invention, the plasmid carrying the CAR gene encodes a CAR targeting FAP, which is either a 4G5 CAR or a MO36 CAR, and the structure of the 4G5 CAR or MO36 CAR is as follows: Figure 3 As shown.

[0025] Specifically, the nucleotide sequence of the 4G5 CAR is shown in SEQ ID NO.1; the nucleotide sequence of the MO36 CAR is shown in SEQ ID NO.2. Since the inflammatory cytokine secretion levels of the in vitro constructed 4G5 CAR-T cells are higher than those of the MO36 CAR-T cells, this invention preferably uses the CAR constructed based on clone number 4G5.

[0026] The plasmid vector carrying the 4G5 CAR gene can be selected from any plasmid vector conventionally used in the art. In a specific embodiment of the present invention, the plasmid vector carrying the CAR gene is selected from pCDH-T2A-copGFP, and the plasmid structure carrying the 4G5 CAR gene is as follows. Figure 4 As shown.

[0027] The nanoliposome delivery system provided by this invention can deliver a target gene (CAR gene) into cells via plasmid transfection, wherein the cells are selected from T cells, NK cells, or macrophages. In this invention, it is preferable to deliver the CAR gene into T cells to generate CAR-T cells targeting FAP in situ in vivo.

[0028] In a second aspect, the present invention provides a method for preparing the in-situ constructed CAR-T nanoliposome delivery system, characterized in that the method comprises the following steps: S1) Construct a plasmid carrying the CAR gene, wherein the CAR is selected from 4G5 CAR or MO36 CAR; S2) Formation of liposome membrane: Cholesterol and 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) are dissolved in an organic solvent (chloroform) to form an oil phase solution, and the lipid membrane is obtained by rotary evaporation; S3) Dissolve the plasmid carrying the CAR gene in an aqueous solvent (buffer solution with pH 4-5, such as sodium acetate solution, sodium citrate solution, etc.), then add DSPE-PEG2000 and DSPE-PEG2000-Mal in sequence, add the formed aqueous solution to the lipid membrane, and form liposomes loaded with plasmid through the hydrated lipid membrane. S4) The liposomes loaded with plasmids are subjected to graded extrusion to achieve nanoscale size, and then CD3 antibody is modified on the surface of the nanoliposomes to obtain a nanoliposome delivery system.

[0029] Preferably, the components forming the lipid membrane in step S2 also include chloroquine. Specifically, the molar mass ratio of cholesterol to DOTAP is 1:(1-1.2), and the mass ratio of chloroquine to plasmid DNA is (4-5):1.

[0030] Preferably, the aqueous solution in step S3 further contains protamine, which forms a complex with the plasmid, wherein the mass ratio of protamine to plasmid is 1:1.

[0031] Preferably, in step S3, the mass ratio of lipid membrane to plasmid is (20-50):1, specifically 20:1, 27:1, 30:1, 35:1, 40:1, or 50:1. More preferably, the mass ratio of lipid membrane to plasmid is 20:1.

[0032] Preferably, the graded extrusion in step S4 includes using a micro extruder to sequentially extrude the liposomes through 800nm, 400nm, and 100nm nuclear pore membranes to form a nanoscale liposome suspension.

[0033] Preferably, the method for modifying the surface of the nanoliposomes with CD3 antibody in step S4 is as follows: reducing the disulfide bonds in the CD3 antibody, adding the extruded nanoliposomes, and carrying out a maleimide-thiol Michael addition reaction in a reaction system at 25°C and pH 7.0 to obtain nanoliposomes with CD3 antibody modified on the surface.

[0034] In a third aspect, the present invention provides the application of the in-situ constructed CAR-T nanoliposome delivery system in the preparation of a drug for treating myocardial infarction.

[0035] The drug has at least one of the following effects: 1) Improves cardiac function, including systolic and diastolic function; 2) Reduce the area of ​​myocardial cell fibrosis; 3) Alleviate the progression of heart failure.

[0036] In specific embodiments of the present invention, the application includes administering a therapeutically effective amount of the nanoliposome delivery system to a subject in need, either alone or in combination with other agents or other treatment methods.

[0037] Specifically, the other agents are selected from drugs commonly used in the art for the treatment of myocardial infarction, including but not limited to ACEIs and beta-blockers.

[0038] In this invention, "single administration" and "combined administration" refer to administering one, two, or more reagents to a subject so that the reagent and / or its metabolites are simultaneously present in the subject. "Combined administration" includes administering two or more active ingredients, each in a separate formulation, simultaneously, or at different times, or administering two or more active ingredients in the same formulation.

[0039] In a fourth aspect of the invention, the invention provides a pharmaceutical composition, characterized in that the pharmaceutical composition comprises a therapeutically effective amount of the in-situ constructed CAR-T nanoliposome delivery system of the first aspect of the invention.

[0040] The term "therapeutic effective amount" refers to the amount of active substance sufficient to treat the disease. This effective amount may vary depending on the intended application or the subject and disease condition, such as weight and age, severity of the disease, method of administration, etc., and can be readily determined by those skilled in the art.

[0041] Furthermore, the pharmaceutical composition also includes pharmaceutically acceptable excipients.

[0042] In a preferred embodiment of the invention, the pharmaceutical composition is suitable for non-gastrointestinal administration, such as via intravenous, intramuscular, intradermal, and subcutaneous routes. Therefore, depending on the applicable dosage form, the pharmaceutically acceptable excipient is selected from antioxidants, buffers, antibacterial agents, suspending agents, solubilizers, or solutes that make the formulation isotonic with the blood of the subject.

[0043] In one specific embodiment of the present invention, the pharmaceutical composition is an injection administered to the subject via intravenous injection.

[0044] The technical solution provided by this invention has the following advantages: 1) The nanoliposome delivery system provided by the present invention has good biocompatibility and carries a plasmid that encodes a CAR gene that can target FAP protein. Due to the modification of the surface of the nanoliposome with CD3 antibody, the nanoliposome delivery system can target T cells, enabling the nanoliposome to capture T cells through circulation and realize the transfection of plasmid to T cells. In addition, the present invention found that adding chloroquine to the lipid membrane can effectively inhibit the interferon (IFN) reaction and enhance the transfection efficiency of plasmid to T cells.

[0045] 2) The present invention unexpectedly discovered that protamine has the effect of promoting plasmid aggregation. The present invention preferably encapsulates a mixture of plasmid and protamine in a lipid membrane, which can significantly improve the plasmid encapsulation efficiency.

[0046] 3) In a mouse model of myocardial infarction, the nanoliposome delivery system for in situ construction of CAR-T cells provided by this invention can significantly reduce myocardial fibrosis and preserve cardiac function. During the experiment, no mice died due to excessive clearance of fibroblasts by CAR-T cells. On the contrary, these cells protected the heart from damage after myocardial infarction.

[0047] 4) The nanoliposome delivery system provided by this invention can generate transient CAR-T cells targeting FAP-overexpressing fibroblasts in situ in vivo, effectively solving the problem of in vitro preparation of CAR-T cells in clinical applications, and providing a research basis for the clinical application of CAR-T cell therapy based on in situ nanoengineering in myocardial infarction. Attached Figure Description

[0048] Figure 1 A schematic diagram of the CAR structure shown in Equation I.

[0049] Figure 2 A schematic diagram of the CAR structure shown in Formula II.

[0050] Figure 3 Structural diagrams of 4G5 CAR and MO36 CAR.

[0051] Figure 4 A schematic diagram of a plasmid carrying the CAR gene.

[0052] Figure 5 Evaluation of FAP expression and FAP CAR generation in a mouse model of cardiac fibrosis; (a) M-mode echocardiography of mice after left anterior descending artery ligation. (b) Masson trichrome staining of the heart in fibrotic mice. (cd) Detection of FAP and α-SMA expression in cardiac fibrosis by flow cytometry and Western blot. (ef) Schematic diagram and in vitro experiments of CAR-T cell generation via lentivirus (gj). (g) Effector cells (FAP CAR-Jurkat T) and target cells (NIH / 3T3). FAP After co-culturing for 1 hour, flow cytometry was used to analyze the recognition of target cells by CAR molecules. (hi) Target cells (NIH / 3T3) FAP (j) After co-culturing with effector cells (CAR-T4G5 and CAR-TMO36) for 24 hours, target cell death was assessed. (j) Flow cytometry was used to determine the cell death of cells derived from NIH / 3T3. FAP Cytokine secretion levels of activated CAR-T cells. Statistical significance: ns (p>0.05); *p<0.05; **p<0.01; ****p<0.0001.

[0053] Figure 6 Synthesis and characterization of FAP CAR-LNP. (a) Synthetic route of FAP CAR-LNP; (b) Dynamic light scattering analysis of FAP CAR-LNP and Fake-LNP, and corresponding measurements of (c) mean diameter and (d) polydispersity index (PDI). DNA presence in FAP CAR-LNP was demonstrated by (e) zeta potential analysis and (f) Picogreen fluorescence flow cytometry. (g) DNA loading capacity of FAP CAR-LNP at different LNP-to-DNA weight ratios was determined by gel electrophoresis. (h) The protective effect of Protamine on DNA loading during fractionation extrusion was characterized by gel electrophoresis. (ij) Nanoparticle stability of FAP CAR-LNP in (i) plasma and (j) water was measured by DLS. (k) Chloroquine-promoted endosome escape in primary human T cells was observed by confocal microscopy. Primary human T cells were treated with Cy5-labeled FAPCAR-LNPs (with and without chloroquine, red) for 3 hours, followed by lysosomal labeling (green) and fluorescence microscopy analysis. Arrows indicate colocalization of endosomes / lysosomes with untreated chloroquine FAPCAR-LNPs (yellow). Scale bar is 20 μm.

[0054] Figure 7Encapsulation analysis of protamine and chloroquine in FAP CAR-LNP; ab are the UV-Vis spectra of (a) protamine and (b) chloroquine in FAP CAR-LNP (25.6 μg) dissolved in 1 mL chloroform after ultrasonic disruption.

[0055] Figure 8 In vitro toxicity assessment of FAP CAR-LNP; ab shows the cell viability of (a) human T cells and (b) mouse T cells after 24 hours of treatment with different concentrations of FAP CAR-LNP, as determined by the CCK-8 assay.

[0056] Figure 9 Verification results of the DNA cleavage protective effect of protamine.

[0057] Figure 10 FAP CAR-LNP achieved T cell targeting and CAR T cell generation in vitro and in vivo; (ad) Primary human T cells were treated with Cy5-labeled FAP CAR-LNP for 2 or 4 days by flow cytometry. Targeting efficiency was characterized by Cy5-positive cell population and mean fluorescence intensity (MFI) of Cy5. Transfection efficiency and FAP expression were determined by GFP-positive cell population and MFI of GFP. Saline, cell-free DNA, Lipo 3000, chloroquine-free FAP CAR-LNP, and FAP CAR-LNP (isotype) treatment groups served as negative controls. (e) The in vitro cytotoxic effects of CAR T cells derived from primary human T cells transduced with FAP CAR-LNP were verified. T cells were treated with FAP CAR-LNP and its control for 24 hours, followed by interaction with NIH / 3T3. FAP (left) or NIH / 3T3 (right) cells were co-cultured for 24 hours at different effector cell to target cell ratios. Target cell death was determined by luciferase assay. (f) Biodistribution of FAP CAR-LNP in healthy C57BL / 6 mice, assessed by Cy5 fluorescence 2 hours after intravenous injection (iv). (g) Flow cytometry analysis of FAP CAR-LNP-mediated T cell targeting in the spleen 2 hours after intravenous injection, indicated by Cy5 fluorescence in CD3+ T cells. (h) Flow cytometry analysis of in situ CAR T cell generation in mice 2 days after intravenous injection of FAP CAR-LNP and its control group, determined by GFP expression in CD3+ spleen cells. Statistical significance: ns. p>0.05; *p<0.05; **p<0.01; ****p<0.0001.

[0058] Figure 11 Re-edited T cells improve cardiac function and prevent the progression of heart failure after myocardial infarction; (a) Schematic diagram of in vivo treatment and characterization using a C57BL / 6 mouse model of myocardial infarction. (b) Comparison of left ventricular ejection fraction (LVEF) over time between the FAP CAR-LNP treatment group and the control group. (c) M-mode echocardiography of mice in different treatment groups on day 1 and day 21. (de) Measurements of end-diastolic (d) and end-systolic (e) volumes (in μL) on day 21. (f) Masson trichrome staining of cardiac tissue collected on day 21, showing the fibrotic region (blue) located below the papillary muscles, at four consecutive sites spaced 350 μm apart. Scale bar: 500 μm. (g) Quantitative analysis of ventricular fibrosis (percentage). Statistical significance: ns. p>0.05; *p<0.05; **p<0.01; ****p<0.0001. Detailed Implementation

[0059] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0060] Evaluation of FAP expression and FAP CAR generation in a MI mouse model Experimental animal models are valuable for accurately examining the spatiotemporal dynamics of myocardial fibrosis (CF) during cardiac healing. Notably, both myocardial infarction and surgical ligation of the left anterior descending artery (LAD) lead to rapid cardiomyocyte death in the ischemic left ventricle. The infarcted region (the area directly affected by ischemic injury) primarily undergoes alternative fibrosis, a response to cardiomyocyte loss and accompanying functional tissue loss. Therefore, this invention employs a myocardial infarction (MI) mouse model to investigate the expression of protein targets within the fibrotic region.

[0061] The inventors of this invention manufactured a MI mouse model (C57BL / 6 mice) according to existing technology, with a sham-operated group (Sham) as a control. Compared with the sham-operated group, the left ventricular diameter of the MI group mice was significantly increased, indicating impaired cardiac function. Figure 5 a), and cardiac fibrosis peaked within the first week after injury, with significant and widespread fibrosis observed throughout the myocardium. Figure 5 b).

[0062] FAP is a cell surface protein that is widely upregulated in the context of myocardial fibrosis following hypertension. FAP is also considered a promising target for the treatment of myocardial fibrosis. In the MI mouse model created in this invention, compared with sham-operated controls, FAP expression was increased in cardiac fibrosis (…). Figure 5 c, d). As a positive control, the cardiac fibroblast marker α-smooth muscle actin (α-SMA) was also detected. Figure 5 d). It has been reported that FAP is undetectable in other cardiomyocyte types; therefore, this invention develops a CAR strategy for FAP fibroblasts.

[0063] The single-chain antibody variable fragment (scFv) sequence is derived from antibodies with clone numbers 4G5 and MO36. This embodiment designs two CAR molecular constructs (CAR4G5 and CARMO36, structures as shown below). Figure 3 As shown, it includes an extracellular single-chain antibody variable fragment (scFv) that recognizes human and mouse FAP, as well as the CD8 transmembrane domain and cytoplasmic domain.

[0064] To evaluate the feasibility of CAR, this invention used lentiviral constructs to generate Jurkat T cells expressing FAP CAR (referred to as CAR-J cells) and evaluated their ability to recognize FAP antigens in vitro. Figure 5 e, f). CAR-J cells were mixed with NIH / 3T3 cells overexpressing FAP (referred to as NIH / 3T3). FAP The co-culture experiment showed that CAR molecules can effectively recognize and bind to FAP antigen ( Figure 5 g).

[0065] Subsequently, this invention utilized primary human T cells to generate CARs. 4G5 -T and CAR MO36 -T cells to evaluate CAR-T cell response to NIH / 3T3 FAP Cell-specific killing ability. Target cells (NIH / 3T3) were used at effector cell to target cell ratios of 1:1 and 4:1 (E:T). FAP Co-cultured with CAR-T cells and untransduced control T cells. Compared with untransduced control T cells, CAR-T cells showed increased activity against NIH / 3T3 antibodies. FAP The cells exhibited significantly enhanced cytotoxicity. Figure 5 In contrast, CAR-T cells showed very limited killing effect on NIH / 3T3 cells that do not express FAP (h). Figure 5 i). In addition, CAR 4G5-T and CAR MO36 -T cells secreted significantly higher levels of activating and cytotoxic factors, including interleukin-2 (IL-2), interleukin-4 (IL-4), interferon-γ (IFN-γ), and tumor necrosis factor-α (TNF-α). Figure 5 j). Given CAR 4G5 -T's inflammatory cytokine secretion levels were higher than CAR's. MO36 -T, and the CAR4G5 construct was used in subsequent experiments. Overall, this invention designed a CAR molecule capable of recognizing FAP antigens and effectively activating T cells in vitro.

[0066] Preparation Example 1: Construction of a plasmid carrying the CAR gene The complete coding sequence (CDS) of the FAP gene was searched using Genebank (NCBI). Antibodies with clone numbers 4G5 and MO36 were obtained. By analyzing the hypervariable region (complementarity-determining region, CDR) sequences of these antibodies, corresponding single-chain antibody variable region fragments (scFv) were designed and constructed. Subsequently, based on the scFv as the basic structural domain, the complete gene sequence of the chimeric antigen receptor (CAR) molecule was designed and synthesized. The nucleic acid sequences of 4G5 CAR and MO36 CAR were obtained using a whole-genome synthesis method (the sequences are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively). The synthesized CAR gene and the vector plasmid pCDH-T2A-copGFP were double-digested with EcoRI and NotI restriction endonucleases. Finally, the digested 4G5CAR and MO36 CAR gene fragments were inserted into the pCDH-T2A-copGFP vector backbone, which had been digested with the same double enzymes, through a ligation reaction to construct the recombinant plasmid. Figure 4 ).

[0067] Since previous in vitro FAP CAR-T experiments have confirmed that CAR4G5 is superior to CARMO36, in subsequent experiments, unless otherwise specified, all CARs used will refer to the CAR4G5 construct.

[0068] Preparation Example 2: Synthesis of FAP-CAR-LNP The FAP CAR-LNP described in this invention is prepared via a modified water-in-oil emulsion method. First, a lipid membrane composed of cholesterol, 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), and chloroquine is formed by rotary evaporation. Liposomes are then formed by hydrating the lipid membrane, during which pre-mixed plasmids and protamine are encapsulated within the liposomes. Subsequently, the liposomes are subjected to fractional extrusion to achieve nanoscale size, and finally, an antibody targeting CD3 is externally modified. Figure 6a). The specific operating steps are as follows: 1) Preparation of lipid thin films Accurately weigh the following components: • Cholesterol: 9.02 mg • 1,2-Dioleoyl-3-trimethylammonium propane (DOTAP): 14.67 mg • Chloroquine diphosphate: 3.2 mg Each component was dissolved separately in 1 mL of chloroform. 28.8 μL of cholesterol solution, 30 μL of DOTAP solution, and 47.6 μL of chloroquine solution were transferred to a 50 mL round-bottom flask. 1 μL of CY5 fluorescent dye (10 mM) was added, and the mixture was sonicated (100 W, 5 min) until completely dissolved. The solution was then evaporated under reduced pressure in a rotary evaporator (40°C, 30 rpm, 0.09 MPa) until a uniform blue lipid film formed on the flask wall. The film was then dried in a vacuum drying oven (25°C, 0.01 MPa) for 12 hours to remove residual solvent.

[0069] 2) Preparation of plasmid-protamine complex Add 47 μg of plasmid DNA and 4.7 μL of protamine solution (10 mg / mL) to a 1.5 mL centrifuge tube, vortex for 1 min (2000 rpm), and incubate at 4°C for 12 hours to form a complex.

[0070] 3) Liposome hydration and extrusion Add the following to the above complex in sequence: •5.1 μL DSPE-PEG2000 (10 mg / mL) •1.7 μL DSPE-PEG2000-Mal (10 mg / mL) Vortex to mix (10 s), and let stand at room temperature for 15 min. Add preheated 200 mM sodium acetate buffer (pH 5.2) to a final volume of 350 μL, and transfer to a round-bottom flask containing the lipid membrane. Vortex (2 min, 2500 rpm) to hydrate the lipid membrane, and then extrude it five times sequentially through a polycarbonate membrane (800 nm → 400 nm → 100 nm) in a liposome extruder (60°C, 100 bar) to obtain a monodisperse liposome suspension.

[0071] 4) Buffer replacement Transfer the liposomes to a pretreated dialysis bag (MWCO 14 kDa), place them in 1 L PBS buffer (pH 7.4), and dialyze magnetically (500 rpm) in the dark for 2 hours (4°C). Collect the concentrated liposomes from the dialysis bag.

[0072] 5) Antibody conjugation 10 μL of TCEP (10 mM) and 1 μL of anti-mouse CD3 antibody (10 mg / mL) were incubated at room temperature for 20 min to reduce disulfide bonds. 100 μL of liposomes were added, and the pH was adjusted to 7.0 with 100 mM NaOH. The mixture was reacted at 25°C for 3 hours in a rotary mixer (15 rpm) to achieve covalent coupling of DSPE-PEG2000-Mal with the antibody via maleimide-thiol Michael addition.

[0073] Comparative Example 1: Preparation of Fake LNP The preparation method is the same as in Preparation Example 2, except that step 2) does not contain plasmid DNA. That is, compared with the liposomes obtained in Preparation Example 2, the liposomes prepared this time do not contain plasmid DNA, but only protamine, forming the Fake LNP.

[0074] Comparative Example 2: Preparation of FAP CAR-LNP (w / o protamine) The preparation method is the same as in Preparation Example 2, except that step 2) does not contain protamine. That is, compared with the liposomes obtained in Preparation Example 2, the liposomes prepared this time do not contain protamine, but only plasmid DNA.

[0075] Preparation of FAP CAR-LNP (w / o chloroquine) in Comparative Example 3 The preparation method is the same as in Preparation Example 2, except that in step 1), chloroquine diphosphate is not added during the formation of the lipid film. That is, compared with the lipid particles obtained in Preparation Example 2, the lipid body shell formed this time does not contain chloroquine.

[0076] Comparative Example 4: Preparation of FAP CAR-LNP isotype / FAP CAR-LNP (isotype) The preparation method is the same as in Preparation Example 2, except that in step 5), when performing antibody conjugation, the CD3 antibody is replaced with isotype to obtain FAP CAR-LNP modified with anti-CD3 isotype antibody (isotype).

[0077] Characterization of FAP CAR-LNP Dynamic light scattering analysis (DLS) showed that the average hydrated particle size of FAP CAR-LNP was 135 nm, and the polydispersity index (PDI) was 0.1 ± 0.01. Figure 6 The bd indicates a uniform particle size distribution. The zeta potential of FAP CAR-LNP was measured to be -25.1 ± 1.16 mV, while its DNA-free control group (referred to as Fake LNP) was positively charged (bd). Figure 6 e). Compared to the FakeLNP group, the mean fluorescence intensity (MFI) of Picogreen (an ultrasensitive nucleic acid probe) in the FAP CAR-LNP group was significantly increased. Figure 6 f). These results indicate that the plasmid DNA was successfully loaded into the FAP CAR-LNP.

[0078] The amount of plasmid DNA loaded was determined by gel electrophoresis. Figure 6 (g) Liposomes were prepared with LNP to plasmid DNA mass ratios of 20:1, 27:1, 30:1, 35:1, 40:1, and 50:1, where LNP refers to the lipid film component that does not contain plasmid DNA. It can be seen that when the LNP to DNA mass ratio is as low as 20, the DNA can still be completely encapsulated within the LNP due to the aggregation effect of protamine on the plasmid. Figure 2h further shows that plasmids not pre-mixed with protamine (referred to as w / o protamine) partially unpacked within the FAP CAR-LNP during the fractionation extrusion process. Based on this, the preferred LNP to DNA mass ratio of this invention is 20:1.

[0079] The encapsulation of protamine and chloroquine was analyzed by UV-Vis spectroscopy. In the FAPCAR-LNP prepared in this invention, the loading capacity and efficiency of protamine were 50 μg / mg and 100%, respectively, while the loading capacity and efficiency of chloroquine were 95 μg / mg and 51.4%, respectively. Figure 7 a, b). The stability of FAP CAR-LNP in plasma and water was evaluated by DLS analysis. Figure 6 (i, j). The results showed that the particle size and dispersibility of FAP CAR-LNP in water remained essentially unchanged for at least 11 days. In plasma, the particle size increased slightly, but the PDI remained below 0.7 for 120 minutes, indicating that the FAP CAR-LNP prepared in this invention is not prone to aggregation during circulation.

[0080] The cytotoxicity of FAPCAR-LNP to primary human and mouse T cells was assessed after 24 hours of treatment (co-incubation of FAPCAR-LNP with primary human and mouse T cells), and the results showed that it was non-toxic. Figure 8 a, b). We observed that in primary human T cells, chloroquine-free FAP CAR-LNP (referred to as FAP CAR-LNP w / o chloroquine) co-localized with lysosomes (yellow portion) ( Figure 6 In contrast, when chloroquine was present, FAP CAR-LNP did not colocalize with lysosomes, indicating that chloroquine facilitated the escape of LNPs from lysosomes.

[0081] To evaluate the condensation and protection effects of protamine on plasmids, this invention compared the encapsulation performance of lipid nanoparticles (LNPs) prepared by gradient extrusion in systems with and without protamine. Three systems were set up: free DNA, Lipofectamine 3000-DNA complex, and DNA-LNP with and without protamine. After treatment with DNase for 15 minutes, the protective effect was evaluated by agarose gel electrophoresis (lanes 5-8, with lanes 1-4 representing the control group without DNase treatment). The experimental results demonstrate that protamine can effectively protect plasmids from DNase degradation and significantly improve plasmid bioavailability. Figure 9 ).

[0082] Evaluation of the targeting effect of FAP CAR-LNP on T cells and the generation of CAR T cells To determine the targeting effect of FAP CAR-LNP and its ability to generate CAR T cells, this invention treated freshly isolated and activated primary human T cells with Cy5-labeled FAP CAR-LNP containing a plasmid encoding FAP CAR and green fluorescent protein (GFP). Figure 10 a). FAP CAR-LNP modified with anti-CD3 isotype antibody (referred to as FAP CAR-LNP isotype, i.e., FAP CAR-LNP (isotype)), chloroquine-free FAP CAR-LNP (FAP CAR-LNP (w / oCQ)), cell-free plasmid DNA, lipofectamine 3000 (Lipo 3000), and saline were used as controls. Compared with the isotype control, CD3-targeted FAP CAR-LNP showed higher efficiency in capturing T cells. Figure 10b). Although both FAP CAR-LNP and its isotype control successfully captured the majority of T cells (99.3% and 87.3%, respectively, two days after treatment), the mean Cy5 fluorescence intensity (MFI) of the former was significantly higher than that of the latter. Figure 10 c). This observation indicates that CD3 targeting significantly increased the number of LNPs per T cell, thereby greatly improving LNP utilization. Four days later, the MFI of Cy5 decreased sharply, indicating that FAP CAR-LNPs were degraded within T cells.

[0083] Consistent with this finding, the transfection efficiency of FAP CAR-LNP, as determined by GFP expression, was significantly higher than that of all control groups two days after treatment and continued to increase over four days. Figure 10 d). In contrast, the commercially available lipid reagent Lipofectamine 3000, commonly used for nucleic acid delivery and transfection, failed to transfect these primary T cells (i.e., the liposomes prepared using the commercially available reagent Lipofectamine 3000 to encapsulate plasmid DNA were used to transfect primary T cells). This inefficiency may stem from the fact that plasmids adhere to positively charged lipid surfaces, making them more easily cleared by T cells during delivery. The transfection efficiency of chloroquine-free FAPCAR-LNP was significantly lower than that of chloroquine-containing FAPCAR-LNP, because chloroquine enhances the ability of lysosomes to escape ( Figure 6 (k) and effectively inhibit pattern recognition receptor-induced interferon response, thereby synergistically improving transfection efficiency by reducing the clearance of exogenous plasmids from T cells. These results highlight the importance of protamine-mediated plasmid aggregation and encapsulation, and chloroquine in enhancing plasmid transfection.

[0084] Subsequently, we validated that CAR T cells generated using FAP CAR-LNP responded to NIH / 3T3 antibodies in vitro in a dose-dependent manner. FAP Cellular cytotoxicity. Results showed that FAP CAR-T cells induced NIH / 3T3... FAP Complete cell lysis, but failure to kill NIH / 3T3 cells ( Figure 10 e). The performance of FAP CAR-LNP in primary mouse T cells was further validated, with similar results.

[0085] We conducted a preliminary study on the performance of in situ CAR T cell generation via intravenous injection of Cy5-labeled FAP CAR-LNP loaded with the FAP CAR-GFP plasmid. As expected, FAP CAR-LNP was mainly distributed in the T cell-rich spleen two hours after injection, due to CD3 targeting (…). Figure 10 f). Furthermore, some FAP CAR-LNPs accumulate in the liver, likely due to the high concentration of mononuclear phagocytes (especially Kupffer cells) in the liver, which are known for capturing nanoparticles. Flow cytometry analysis of spleen cells two hours after injection confirmed that FAP CAR-LNPs effectively captured T cells (f). Figure 10 g). FAP CAR-LNP transfected T cells significantly increased GFP expression after two days. Figure 10 This demonstrated the generation of T cells in vivo. Pathological analysis showed that intravenous injection of FAP CAR-LNP did not cause organ damage in mice.

[0086] The effectiveness of in situ nanoengineered CAR T cells in alleviating myocardial fibrosis and restoring cardiac function after myocardial infarction (MI). This invention investigates the effect of FAP CAR-LNP engineered CAR T cells on reducing myocardial fibrosis and cardiac function in a mouse model of myocardial infarction (MI). Figure 11 a). Cardiac injury was induced in mice via LAD ligation to simulate acute myocardial infarction (MI). Our dosing regimen was based on FAP expression patterns, with significantly increased FAP expression in the infarcted area compared to the non-infarcted area, peaking at 7 days post-infarction and gradually returning to baseline at 28 days. One day post-surgery, the left ventricular ejection fraction (LVEF) decreased ( Figure 11 b). We administered FAPCAR-LNP twice intravenously at a dose of 12.8 mg / kg on the second and fifth days postoperatively. The Fake LNP treatment group served as the control group, and the saline treatment group served as the blank control group. Cardiac function was monitored by echocardiography from days 7 to 21 postoperatively. Figure 11 c). We observed a significant improvement in cardiac function in impaired mice treated with FAP CAR-LNP. Left ventricular systolic function (measured by ejection fraction (EF)) was preserved in MI-impaired mice treated with FAP CAR-LNP. In contrast, LVEF in the saline-treated and fake LNP-treated groups decreased continuously over a period of 7 to 14 days. Furthermore, both left ventricular diastolic and systolic function were significantly improved in the FAP CAR-LNP group compared to the saline and fake LNP control groups. Figure 11d, e). Histological analysis using Masson's trichrome staining showed a significant reduction in cardiac fibrosis and a mitigation of heart failure progression in mice. Figure 11 (f, g). Furthermore, we did not document any cases of cardiac rupture due to a lack of scar tissue, a potential outcome of CAR-T cell therapy.

[0087] To further verify whether the improvement in cardiac function was attributed to the effect of in situ generated FAP CAR T cells, we established a myocardial infarction (MI) model using immunodeficient BALB / c-nude mice and treated them with FAP CAR-LNP. The results showed that the nude mice exhibited weakness on day 14. FAP CAR-LNP treatment failed to alleviate cardiac function and fibrosis, demonstrating the crucial role of FAP CAR-T cells in post-MI treatment.

[0088] The above specific embodiments are merely illustrative of the invention and do not represent a limitation thereof. Those skilled in the art will recognize that other variations of the specific structure of this invention are possible.

Claims

1. A nanoliposome delivery system for in-situ construction of CAR-T, characterized in that, The nanoliposome delivery system comprises a lipid membrane and a plasmid carrying a CAR gene encapsulated by the lipid membrane, and the surface of the nanoliposome delivery system is modified with a CD3 antibody. The plasmid carrying the CAR gene encodes a CAR that targets FAP, and the structure of the CAR is shown in Formula II: CD8 leader-scFv-CD8H-CD8TM-41BB-CD3ζ (Ⅱ) The scFv fragment is a single-chain antibody targeting FAP, and the antibody clone number is selected from 4G5; the CAR targeting FAP is 4G5 CAR, and the nucleotide sequence of the 4G5 CAR is shown in SEQ ID NO.

1.

2. The nanoliposome delivery system according to claim 1, characterized in that, The nanoliposome delivery system also includes protamine, which is co-encapsulated with the plasmid in a lipid membrane, and the mass ratio of protamine to plasmid is (1-2):

1.

3. The nanoliposome delivery system according to claim 1, characterized in that, The lipid membrane material is selected from a combination of cholesterol, DOTAP, and chloroquine.

4. The method for preparing the in-situ constructed CAR-T nanoliposome delivery system according to claim 1, characterized in that, The method includes the following steps: S1) Construct a plasmid carrying the CAR gene, wherein the CAR is selected from 4G5 CAR; S2) Cholesterol and DOTAP are dissolved in an organic solvent to form an oil phase solution, and the lipid membrane is obtained by rotary evaporation; S3) Dissolve the plasmid carrying the CAR gene in an aqueous solvent, add DSPE-PEG2000 and DSPE-PEG2000-Mal in sequence, add the resulting aqueous solution to the lipid membrane, and form liposomes loaded with plasmid through the hydrated lipid membrane. S4) The liposomes loaded with plasmids are subjected to graded extrusion to achieve nanoscale size, and the surface is modified with CD3 antibody to obtain a nanoliposome delivery system.

5. The method according to claim 4, characterized in that, The components forming the lipid membrane in step S2 also include chloroquine, the molar mass ratio of cholesterol to DOTAP is 1:(1-1.2), and the mass ratio of chloroquine to plasmid DNA is (4-5):

1.

6. The method according to claim 4, characterized in that, The aqueous solution in step S3 also contains protamine, which forms a complex with the plasmid, wherein the mass ratio of protamine to plasmid is (1-2):

1.

7. The method according to claim 4, characterized in that, The method for modifying the surface of the nanoliposomes with CD3 antibody in step S4 is as follows: reduce the disulfide bonds in the CD3 antibody, add the extruded nanoliposomes, and carry out a maleimide-thiol Michael addition reaction in a reaction system at 25°C and pH 7.0 to obtain nanoliposomes with CD3 antibody modified on the surface.

8. The use of the in-situ constructed CAR-T nanoliposome delivery system according to any one of claims 1-3 in the preparation of a drug for treating myocardial infarction.

9. The application according to claim 8, characterized in that, The drug has at least one of the following effects: 1) Improves cardiac function, including systolic and diastolic function; 2) Reduce the area of ​​myocardial cell fibrosis; 3) Alleviate the progression of heart failure.

10. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises a therapeutically effective amount of the in-situ constructed CAR-T nanoliposome delivery system according to any one of claims 1-3, and pharmaceutically acceptable excipients.

Images