Chimeric genetically-engineered NANO vesicle used for removing pathogenic antibodies, preparation method therefor, and use thereof

Abstract

Provided are a chimeric genetically-engineered nano vesicle used for removing pathogenic antibodies, a preparation method therefor, and the use thereof, belonging to the technical field of biology. By means of lentivirus particle transfection, a stably transfected cell line of a protein A / G gene is constructed, so as to stably express protein A and protein G. A fusion protein A / G can overcome the defect of poor IgG3 binding capacity of pure protein A, thereby improving the effect of antibody IgG adsorption. In addition, using PLGA nanoparticles having relatively uniform particle sizes as the core of the target vesicle and mixing same with engineered cell membranes that are ruptured by compression can promote self-assembly of cell membranes, so as to form extracellular vesicle mimetics, thereby improving the stability of the target nano vesicle. The prepared and separated nano vesicle can be applied to in-vivo adsorption and removal of antibodies, has good biological safety, and avoids the risk of dialysis catheterization operation during using processes and the risks such as bleeding and allergy during treatment processes of immunoadsorption columns.

Description

Chimeric genetically engineered nanovesicle for clearing pathogenic antibodies and its preparation method and application Technical Field

[0001] The present invention belongs to the field of biotechnology, and specifically relates to a chimeric genetically engineered nanovesicle for clearing pathogenic antibodies, and a preparation method and application thereof. Background Art

[0002] As an important component of the immune system, antibodies play a key role in defending against the invasion of foreign pathogens and protecting the body's health. However, in certain circumstances (such as autoimmune diseases and organ transplantation), antibodies can be harmful to the body. In autoimmune diseases, immune disorders cause autoantibodies to recognize and attack the body's own cells, tissues, and organs, inducing immune inflammatory responses and tissue damage. Currently, there are over 100 known autoimmune diseases, including common ones such as rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, inflammatory bowel disease, myasthenia gravis, and Goodpasture's syndrome. Autoimmune diseases are not uncommon in the human population and can cause significant disability and mortality rates. In organ transplantation, recipients' antibodies specific to donor antigens can induce antibody-mediated rejection, leading to graft dysfunction or even graft loss. In the treatment of autoimmune diseases and organ transplantation, eliminating the patient's pathogenic antibodies can effectively alleviate symptoms and improve poor prognosis.

[0003] Immunoadsorption is an in vitro therapeutic approach that utilizes a high-affinity adsorbent (antigen, antibody, or certain specific substances with physicochemical affinity) immobilized on a carrier to form an immunoadsorption column, selectively or specifically removing pathogenic immune complexes and antibodies from a patient's blood. Compared to plasma exchange, it is relatively selective in removing pathogenic antibodies, with advantages such as not losing plasma components such as albumin, not requiring additional fresh plasma, and preventing blood-borne diseases. Currently, the most commonly used immunoadsorption column for pathogenic antibody removal in clinical practice is the Staphylococcal Protein A immunoadsorption column. Other immunoadsorption columns include those containing Protein A-like polypeptides, tryptophan or phenylalanine, and anti-IgG polyadsorbents, each with its own unique characteristics in terms of antibody adsorption strength and specificity. Furthermore, new immunoadsorbents are also being developed. For example, CN107138139A discloses an immunoadsorbent that uses covalent coupling to graft small molecule functional ligands onto an adsorption carrier material. This immunoadsorbent utilizes high-density activation, and the use of a coupling spacer arm to enable flexible attachment of the small molecule functional ligand improves functional ligand stability, adsorption capacity, and selectivity. However, the antibody adsorption treatment method still has some defects, such as the need to establish additional vascular access, and the risks associated with allergies and bleeding during the treatment process.

[0004] Extracellular vesicles are phospholipid bilayer vesicles secreted by cells that can carry mRNA, microRNA, cytokines, and membrane molecules. Extracellular vesicles have the advantages of biocompatibility, biodegradability, low toxicity, and non-immunogenicity, and can be used as an effective delivery carrier for antibody adsorbents. After genetic engineering, the target protein can be packaged into extracellular vesicles, and combined with the targeting properties of the extracellular vesicles themselves, it can be used for the efficient treatment of diseases. At present, the preparation method of engineered nanovesicles has been applied in research fields such as red blood cells, macrophages, and platelets. However, the top-down strategy for preparing engineered nanovesicles still has certain limitations, such as uneven size of the prepared particles and encapsulation problems.

[0005] Therefore, in order to solve the defects of the existing technology, it is necessary to propose a new method for preparing nanovesicles that can effectively eliminate pathogenic antibodies.

[0006] Summary of the Invention

[0007] In view of the problems existing in the prior art, the purpose of the present invention is to design and provide a chimeric genetically engineered nanovesicle for clearing pathogenic antibodies and a technical solution for its preparation method and application.

[0008] The present invention is specifically implemented by the following technical solutions:

[0009] The first aspect of the present invention provides a method for preparing chimeric genetically engineered macrophages, comprising the following steps:

[0010] S1: Construct a lentiviral vector and use engineered cells to package the lentivirus to obtain lentiviral particles containing the fusion coding genes of protein A and protein G;

[0011] S2: The virus obtained in S1 is used to infect macrophages, and a stable cell line expressing the protein A and protein G fusion genes with high purity is obtained through culture screening.

[0012] Furthermore, the engineered cells in S1 are HEK-293T cells.

[0013] Furthermore, the amino acid sequence of the protein A in S1 is shown as SEQ ID NO.1, and the amino acid sequence of the protein B is shown as SEQ ID NO.2.

[0014] Furthermore, the step S2 specifically includes the following steps:

[0015] Culture the target macrophage cells to a density of 60-70%, add the lentiviral particles obtained in S1, shake well, and then culture in an incubator for 48-72 hours;

[0016] The cells were infected for the second time after they reached the logarithmic growth phase;

[0017] Add 2-4 μg / mL of puromycin for screening for 48-72 hours; then, reduce the puromycin maintenance concentration to 0.8-1.2 μg / mL, and continue screening and amplifying the infected cells for 24-48 hours to obtain a stable cell line expressing a specific gene with high purity.

[0018] The second aspect of the present invention provides a method for preparing antibody-adsorbed nanovesicles using the chimeric genetically engineered macrophages prepared by the above method, which comprises the following steps:

[0019] 1) Preparation of PLGA nanospheres by emulsification reagent evaporation method;

[0020] 2) Collect high-purity stable cells expressing a specific gene, repeatedly pass through a polycarbonate filter, and centrifuge and wash to obtain cell membranes;

[0021] 3) The PLGA nanospheres described in step 1) and the cell membranes described in step 2) are mixed uniformly in proportion, and the mixtures are sequentially passed through 400 nm, 200 nm, and 100 nm polycarbonate membranes, extruded back and forth to obtain nanovesicles with a chimeric engineered membrane encapsulating a PLGA core;

[0022] 4) The nanovesicles described in step 3) are mixed with a phosphatidylserine solution for surface modification to obtain nanovesicles for antibody adsorption.

[0023] Further, the step 1) is specifically as follows:

[0024] The DCM solution containing PLGA was added dropwise to a 2-4% w / v PVA aqueous solution and sonicated to form an oil-in-water emulsion;

[0025] The resulting emulsion was stirred on a magnetic stirrer at room temperature overnight;

[0026] The solution was evaporated using a rotary evaporator at 40-60 °C to remove the DCM solvent residue;

[0027] The emulsion was transferred to a centrifuge tube, ultracentrifuged, and washed with deionized water. The washed microspheres were placed in a cryovial, vacuum-lyophilized, and stored at -20°C for later use.

[0028] Furthermore, in step 2), the repeated passage through the polycarbonate filter is specifically as follows: first passing through a polycarbonate membrane with a pore size of 10 μm 3-5 times, and then passing through a membrane with a pore size of 5 μm 3-5 times; the centrifugation parameters are 4° C., 2000 g, and 15-20 minutes.

[0029] Furthermore, in step 3), the PLGA nanospheres and the cell membrane are mixed at a mass ratio of 10-20:1.

[0030] Furthermore, in step 4), the concentration of the phosphatidylserine solution is 10%-15%.

[0031] The third aspect of the present invention provides antibody-adsorbed nanovesicles obtained by the above-mentioned preparation method.

[0032] A fourth aspect of the present invention provides the use of the above-mentioned antibody-adsorbed nanovesicles in the preparation of a drug, wherein the use of the drug is any one of the following (a1) to (a6):

[0033] (a1) adsorption and removal of antibodies;

[0034] (a2) blocking activation of the complement system;

[0035] (a3) Reduce inflammatory accumulation and protect the microvascular endothelium of transplanted kidneys;

[0036] (a4) inhibiting the proliferation and activation of B cells in secondary lymphoid tissues;

[0037] (a5) Alleviate alloreactive antibody-mediated renal allograft damage;

[0038] (a6) Improve transplant kidney function and long-term prognosis of kidney transplant recipients.

[0039] The beneficial effects of the present invention are specifically as follows:

[0040] 1. A chimeric genetically engineered stable cell line was constructed, which can absorb all IgG subtypes;

[0041] The present invention provides a method for preparing chimeric genetically engineered macrophages. Through lentiviral particle transfection, a stably transfected cell line expressing both Protein A and Protein G is constructed. The fusion protein A / G can compensate for the weak binding ability of simple Protein A to IgG3, thereby enhancing the adsorption of IgG antibodies.

[0042] 2. The constructed nanovesicles are stable, uniform in size, and have guaranteed integrity;

[0043] By using PLGA nanoparticles with relatively uniform particle size as the core of the target vesicles and mixing them with the extruded and broken engineered cell membranes, the cell membrane self-assembly is promoted to form extracellular vesicle mimics, thereby improving the stability of the target nanovesicles.

[0044] 3. Nanovesicles can adsorb and remove antibodies in the body, reducing invasive procedures and bleeding risks;

[0045] The nanovesicles prepared and separated in the present invention can be used to adsorb and remove antibodies in vivo, have good biosafety, and avoid the risks of dialysis catheterization during the use of current immunoadsorption columns and the risks of bleeding, allergies, etc. during treatment.

[0046] 4. After being phagocytosed by macrophages, nanovesicles can exert an immunomodulatory effect and reduce the use of immunosuppressants.

[0047] The method for preparing and isolating nanovesicles described in the present invention can induce phagocytosis of macrophages, promote the effective degradation and clearance of antigens and antibodies, and at the same time, can upregulate the immunosuppressive function of macrophages after phagocytosis, which helps to improve the effect of treating antibody-mediated rejection reactions in organ transplantation. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] FIG1 shows the expression of fusion protein A / G on the cell membrane of chimeric genetically engineered macrophages;

[0049] FIG2 is a characterization of chimeric genetically engineered antibodies adsorbed onto nanovesicles;

[0050] Figure 3 shows the biocompatibility characterization of chimeric genetically engineered antibody-adsorbed nanovesicles. The groups in the bar graphs of Figures 3B and 3C on Day 1, Day 5, and Day 7 are specifically ranked as follows: from left to right, they are control group, Vector-trapper vesicle group, chimeric vesicles group, and DSA-trapper vesicle group, respectively.

[0051] FIG4 shows the antibody adsorption effect of chimeric genetically engineered nanovesicles in vitro and in vivo;

[0052] Figure 5 shows that chimeric genetically engineered antibody-adsorbed nanovesicles inhibit the proliferation and activation of B cells and plasma cells in secondary lymphoid tissues. The groups in the bar graphs 1week and 3week3 of Figure 5 are ranked as follows: from left to right: Isograft group, Allograft group, DSA-trapper vesicle group, Chimeric-vesicles group, and Vector-trapper vesicle group;

[0053] Figure 6 shows that chimeric genetically engineered antibodies adsorbed onto nanovesicles inhibit complement system activation and inflammation around microvascular endothelium, thereby alleviating transplant kidney damage;

[0054] Figure 7 shows that chimeric genetically engineered antibody-adsorbed nanovesicles alleviate chronic fibrosis of transplanted kidneys, improve renal function and significantly prolong the survival of allogeneic renal transplant recipients. DETAILED DESCRIPTION

[0055] The present invention will be further described below with reference to specific embodiments. It should be emphasized that the embodiments are intended to illustrate the present invention and are not intended to limit the scope of application of the technology used in the present invention. The scope of the present invention is specifically defined by the appended claims.

[0056] If specific techniques and conditions are not specified in the protocol, the techniques or conditions described in the literature in this field will be followed. The manufacturers of the test kits and instruments used are not specified and are all commercially available through official channels.

[0057] Case Study 1: Preparation of Chimeric Genetically Engineered Macrophages

[0058] S1 constructs a lentiviral vector and uses engineered cells to package the lentivirus to obtain lentiviral particles containing the fusion coding genes of protein A and protein G. Specifically:

[0059] When HEK293T cells reached 70-80% confluency in a culture dish, mix Opti-MEM, pCMV delta R8.2, pCMV-VSV-G, pCDH-CMV-Protein A / G-EGFP-Puro plasmid, and Xtreme Gene 9 in appropriate proportions and incubate the mixture at room temperature for 20-30 minutes. Aspirate the culture medium from the HEK293T cells using a pipette and add approximately 8-10 mL of fresh DMEM. Add the transfection mixture to the HEK293T cells using a 200 μL micropipette and incubate in a 37°C cell culture incubator for approximately 48 hours. On day 3, collect the supernatant from the HEK293T dish using a 10 mL disposable sterile pipette. Transfer the collected supernatant to a 10 mL syringe and filter through a 0.45 μm PVDF filter in a laminar flow hood. Quickly freeze the viral supernatant using liquid nitrogen and store at -80°C until needed.

[0060] S2 uses the virus obtained in S1 to infect macrophages, and through culture screening, obtains a stable cell line expressing the protein A and protein G fusion genes with high purity. Specifically:

[0061] Raw264.7 cells were cultured in a 6-well plate to a density of approximately 60-70%. The virus solution was taken out of the -80°C refrigerator and thawed. Polybrene was added to the virus solution to a final concentration of 8 μg / mL. The virus solution was added to the Raw264.7 cell culture dish, shaken well, and placed in an incubator for culture for two days. A secondary infection can be performed when the cells reach the logarithmic growth phase. 2-4 μg / mL puromycin was added to the cell line culture dish for 48-72 hours to screen for cells expressing the fusion gene; then, the puromycin concentration was reduced to maintain the concentration at 0.8-1.2 μg / mL, and the cells infected with the lentivirus were continued to screen and amplify the cells expressing the fusion gene for 24-48 hours, thereby obtaining a stable cell line expressing the protein A and protein G fusion coding genes. The amino acid sequence of the protein A is shown in SEQ ID NO.1, and the amino acid sequence of the protein B is shown in SEQ ID NO.2.

[0062] The protein A has a 516 amino acid sequence (SEQ ID NO. 1):

[0063] The protein B has a 448 amino acid sequence (SEQ ID NO. 2):

[0064] Using DAPI to label the nucleus and F-actin to label intracellular actin, confocal fluorescence microscopy revealed that the expression rate of fusion protein A / G in engineered macrophages exceeded 95% (Figure 1A). Furthermore, chimeric genetically engineered macrophages were isolated by flow cytometry and centrifuged at 1000-1200 rpm for 5 minutes before being resuspended in RPMI 1640 medium containing 10% FBS, thereby obtaining a stable cell line expressing fusion protein A / G with a purity of up to 96.28% (Figures 1B and C).

[0065] Implementation Case 2: Method for preparing antibody-adsorbed nanovesicles by chimeric genetically engineered macrophages.

[0066] 1) PLGA nanospheres were prepared by an emulsification reagent volatilization method, specifically:

[0067] Prepare a 2.5% PVA solution. Stir the solution overnight at room temperature using a magnetic stir bar and filter through a 0.2 μm vacuum filter. Add 75 mg of PLGA to a 10 mL glass vial and add 5 mL of DCM solution to dissolve the PLGA. Transfer 25 mL of the 2-4% w / v PVA aqueous solution to a 250 mL flask in an ice bath and perform a primary emulsification using a probe sonicator. Use a glass pipette to add the PLGA-containing DCM solution dropwise to the PVA solution and sonicate to form an oil-in-water emulsion. Stir the resulting emulsion overnight at 500 rpm on a magnetic stirrer in a fume hood to reduce the DCM solvent. Evaporate the solution using a rotary evaporator at 40°C for 2 hours to remove any residual DCM solvent. Transfer the emulsion to a centrifuge tube and centrifuge at 10,000 rpm for 5 minutes in an ultracentrifuge. Wash the washed microspheres three times with deionized water. Place the washed microspheres in cryovials and freeze-dry them under vacuum. Store at -20°C until use.

[0068] 2) Extraction of chimeric genetically engineered macrophage cell membranes, specifically:

[0069] The chimeric genetically engineered macrophages obtained in Example 1 were cultured in a 150 mm culture dish until the growth density exceeded 90%. The cells were separated using 2 mM ethylenediaminetetraacetic acid (EDTA) / PBS to preserve their surface proteins. The cells were pelleted and 1 × 10 8Cells / mL were resuspended in PBS. The cell suspension was added to pre-chilled Tris-magnesium buffer and hypotonic incubated overnight with 1% phenylmethylsulfonyl fluoride (PMSF). The suspension was then repeatedly passed through a polycarbonate filter mounted in a microextruder (Avanti). The cells were first passed through a 10 μm pore polycarbonate membrane 3-5 times, and then through a 5 μm pore membrane in a newly assembled device 3-5 times. Mechanical extrusion disrupted the cell structure to produce a homogenate. A 1 M sucrose solution was then added to the homogenate to a final concentration of 0.25 M. The resulting extrudate was centrifuged at 2000 g for 15-20 minutes at 4°C. The pellet was discarded and the supernatant removed. This centrifugation was repeated 2-3 times to remove the nuclei and organelles. The collected supernatant was centrifuged at 3000 g at 4°C to collect the pellet, washed once with ice-cold Tris-magnesium buffer, and then centrifuged to obtain the purified chimeric engineered cell membrane.

[0070] 3) Nanovesicles with chimeric engineered membranes encapsulating PLGA cores

[0071] The PLGA nanospheres prepared in step 1) and the chimeric engineered cell membrane extracted in step 2) were mixed uniformly at a mass ratio of 10-20:1 and sonicated in a water bath for 20-30 minutes. Using a microextruder, the nanospheres were passed through 400 nm, 200 nm, and 100 nm polycarbonate membranes, extruding back and forth 15-20 times to obtain nanovesicles containing a chimeric engineered membrane encapsulating the PLGA core.

[0072] 4) A 10% phosphatidylserine solution was mixed with the chimeric engineered membrane-PLGA nanovesicles for surface modification to prepare nanovesicles for antibody adsorption.

[0073] Case Study 3: Characterization of Chimeric Genetically Engineered Antibody-Adsorbed Nanovesicles (DSA-trapper)

[0074] Transmission electron microscopy revealed that the chimeric genetically engineered antibody-adsorbed nanovesicles had a PLGA core, a round, bilayer-like membrane structure, a high electron density region within the vesicles, and a low electron density region outside the vesicles (Figure 2A). Malvern particle size analyzer analysis revealed that the average diameter of the chimeric genetically engineered antibody-adsorbed nanovesicles was 122.58±9.79nm, and the average zeta potential was -41.34±3.16mV (Figure 2B).

[0075] Western blot analysis (Figure 2C) revealed that the chimeric antibody-adsorbed nanovesicles and chimeric vesicles were rich in protein A and protein G, whereas the PLGA particle control group and the empty vesicle group contained no protein A / G. The successful expression of the fusion protein and the establishment of a stable cell line were further verified at the molecular level. Compared with PLGA nanoparticles, proteins on the macrophage membrane (including TLR4 and IL-1RII) were highly inherited by the chimeric antibody-adsorbed nanovesicles. This facilitates the induction of macrophage target molecules by the chimeric antibody-adsorbed nanovesicles, neutralizing proinflammatory cytokines and directly inhibiting allogeneic immune responses.

[0076] Case Study 4: Biocompatibility Testing of Chimeric Genetically Engineered Antibody-Adsorbed Nanovesicles (DSA-trapper)

[0077] To determine the biocompatibility of chimeric genetically engineered antibody-adsorbed nanovesicles, we conducted in vitro biocompatibility studies by co-incubating human renal tubular epithelial cells with DSA-trapper vesicles, Vector-trapper vesicles (unengineered macrophage membrane vesicles), or Chimeric vesicles (chimeric engineered cell membrane vesicles without phosphatidylserine) for 7 days. Cell viability was assessed by live / dead staining. The results showed that cells in all four groups were able to maintain good viability and proliferation capacity, with a mortality rate of less than 5% (Figure 3A). Notably, quantitative statistics further supported the good biocompatibility of the DSA-trapper vesicle group compared with the control group (Figure 3B). Cell proliferation and toxicity assays are important parameters for evaluating clinical application in the preclinical experimental stage. Therefore, we performed CCK-8 assays to better understand the effects of DSA-trapper vesicles on cell proliferation and toxicity. Over time, cells in all four groups showed similar growth trends, and there were no significant differences in cell proliferation and cytotoxic effects among the control group, Vector-trapper vesicle group, Chimeric vesicle group, and DSA-trapper vesicle group (Figure 3C).

[0078] To verify the biosafety of DSA-trapper vesicles in mice, we injected DSA-trapper vesicles, chimeric vesicles, and Vector-trapper vesicles into mice via the tail vein and analyzed changes in blood routine, liver function, and renal function on days 7 and 21. The results showed no significant differences in blood routine (red blood cell, white blood cell, and platelet counts), liver function (alanine aminotransferase, aspartate aminotransferase, and albumin), and renal function (glomerular filtration rate and urine albumin-to-creatinine ratio) between the DSA-trapper vesicle group and the control group on days 7 and 21.

[0079] Case Study 5: In vitro and in vivo antibody adsorption experiments using chimeric genetically engineered nanovesicles (DSA-trapper)

[0080] In vitro antibody adsorption assay: The adsorption efficiency of DSA-trapper vesicles for IgG3 and IgM was tested using a concentration gradient. As shown in Figure 4, the adsorption curves demonstrate that DSA-trapper vesicles exhibited over 95% adsorption efficiency for IgG3 and over 80% for IgM, with IC50 values ​​of 281 μg / mL and 916 μg / mL, respectively. Vector-trapper exhibited no significant adsorption efficiency (Figure 4A).

[0081] In vivo antibody adsorption experiment: First, we established a mouse model of antibody-mediated rejection injury after allogeneic renal transplantation. Five groups were included in the experiment: isograft, allograft, DSA-trapper vesicles, vector-trapper vesicles, and chimeric vesicles. Group details are as follows: a) Isograft group: renal transplantation was performed using C57BL / 6 mice as donors and C57BL / 6 mice as recipients, with an equal volume of sterile saline injected postoperatively; b) Allograft group: renal transplantation was performed using BALB / C mice as donors and C57BL / 6 mice as recipients, with an equal volume of sterile saline injected postoperatively; c) DSA-trapper vesicle group: renal transplantation was performed using BALB / C mice as donors and C57BL / 6 mice as recipients, with intravenous administration of DSA-trapper vesicles. d) Vector-trapper vesicle group: BALB / C mice served as donors and C57BL / 6 mice served as recipients underwent kidney transplantation surgery, and Vector-trapper vesicles were administered intravenously. e) Chimeric vesicle group: BALB / C mice served as donors and C57BL / 6 mice served as recipients underwent kidney transplantation surgery, and chimeric vesicles were administered intravenously. Serum DSA levels in the transplanted mice were subsequently monitored after renal transplantation. In the Allograft group, serum DSA levels gradually increased with postoperative time, with the DSA IgG immunofluorescence intensity increasing from less than 1,000 at the time of surgery to over 10,000 at 8 weeks postoperatively. Compared with the Allograft, Chimeric vesicle, and Vector-trapper vesicle groups, the serum DSA immunofluorescence intensity in the DSA-trapper vesicle group significantly decreased by 92.74% at 8 weeks postoperatively and remained close to baseline levels (Figure 4B). In addition, similar trends were observed in the adsorption and clearance effects of DSA-trapper vesicles on DSA in IgM, IgG1, IgG2b, IgG2c, and IG3 subtypes of DSA.

[0082] Case Study 6: Chimeric Genetically Engineered Antibody-Adsorbed Nanovesicles (DSA-trapper) Inhibits the Proliferation and Activation of B Cells and Plasma Cells in Secondary Lymphoid Tissues

[0083] The induction of DSA production by memory B cells and long-lived plasma cells upon re-stimulation with donor alloantigens is a hallmark immune process of antibody-mediated rejection. The therapeutic effect of DSA-trapper vesicles on antibody-mediated immune activation was further evaluated by quantitative analysis of germinal center B cells (GCs), spleen-derived plasma cells (SPPCs), and spleen-derived memory B cells (SPMCs). In the Allograft transplantation group, the ratios of GCs, SPPCs, and SPMCs were significantly elevated one week after transplantation, approximately three times that of the isograft group. These ratios further increased three weeks after transplantation, reaching 2.97%, 0.94%, and 2.92%, respectively. Changes in the chimeric vesicle and vector-trapper groups were similar to those in the Allograft group. In the group treated with DSA-trapper vesicles, the ratios of GCs, SPPCs, and SPMCs remained low at 0.83%, 0.40%, and 0.62%, respectively, three weeks after transplantation, showing no significant difference from the isograft group (Figure 5).

[0084] Implementation Case 7: Chimeric genetically engineered antibody-adsorbed nanovesicles (DSA-trapper) inhibit complement system activation and microvascular endothelial inflammation, alleviate renal injury and chronic fibrosis, improve renal function and significantly prolong the survival of allogeneic transplant recipients.

[0085] Circulating DSA binds to alloantigens on the surface of vascular endothelial cells, activating the complement system to form a membrane attack complex or directly mediating renal graft endothelial injury through antibody-dependent cell-mediated cytotoxicity. Our study found that immunofluorescence analysis of allograft-treated kidney tissue revealed extensive deposition of complement C3 and immunoglobulin G (IgG) in the glomerular and peritubular capillaries at both one and three weeks after transplantation. In contrast, the mean immunofluorescence intensities of complement C3 and IgG in the DSA-trapper vesicle-treated group remained low after surgery, showing no significant difference from those in the isograft group (Figure 6A,B). C4d deposition in each group was assessed by quantitative analysis of the mean positive staining area by C4d immunohistochemistry. Compared to approximately 30–40% of the C4d deposited area in the Allograft, Chimeric vesicle, and Vector-trapper groups, the C4d-positive area in the DSA-trapper vesicle-treated group remained low at both one and three weeks after surgery, similar to that in the isograft group (Figure 6C,E,F). This suggests that DSA-trapper vesicles effectively inhibit the complement system.

[0086] Furthermore, we found that platelets (labeled by CD41) and neutrophils (labeled by Ly6G) were aggregated and distributed in the interstitial tissue surrounding microvessels (labeled by CD31) in the Allograft, Chimeric vesicle, and Vector-trapper groups at 1 and 3 weeks after surgery. Treatment with DSA-trapper vesicles significantly improved the inflammatory accumulation of platelets and neutrophils around microvessels at 1 and 3 weeks after surgery (Figure 6D,G,H). Transmission electron microscopy further observed adhesion of immune inflammatory cells to the microvascular endothelium, infiltration of the interstitial tissue around the microvessels, and endothelial damage in the Allograft, Chimeric vesicle, and Vector-trapper groups. However, no significant inflammatory cell infiltration was observed in the DSA-trapper vesicle-treated group (Figure 6I). This suggests that DSA-trapper vesicles can effectively protect the microvascular endothelium of transplanted kidneys and alleviate inflammatory cell infiltration in the graft.

[0087] Immunofluorescence analysis revealed that 8 weeks after surgery, the transplanted kidneys in the Allograft group showed glomerular sclerosis and tubular atrophy, with a significant decrease in the podocyte function protein PODXL and the tubular epithelial function protein AQP-1 (Figure 7A). Neither the chimeric vesicle-treated nor the vector-trapper-treated groups showed significant improvement, similar to the pathological findings in the Allograft group. In contrast, the transplanted kidneys in the DSA-trapper vesicle-treated group showed well-patented glomeruli and normal tubular morphology. The relative fluorescence intensity of podocyte and tubular function markers was more than twice that of the positive control groups and showed no significant difference compared to the isograft-treated mice (Figure 7B).

[0088] Eight weeks after renal transplantation, PAS and MASSON staining revealed that DSA-trapper vesicle treatment significantly improved glomerular and tubular morphology. However, the Allograft, Chimeric vesicle, and Vector-trapper groups primarily demonstrated glomerular sclerosis, tubular atrophy, interstitial fibrosis, and tubulointerstitial lymphocytic infiltration. Furthermore, peritubular capillary stratification, podocyte loss, and glomerular basement membrane thickening were observed in the Allograft group, while DSA-trapper vesicle treatment significantly improved renal ultrastructure (Figure 7F,G). Quantification of glomerular sclerosis and tubular atrophy / interstitial fibrosis (IFTA) scores in histopathological sections of transplanted kidneys revealed significantly higher glomerular sclerosis and IFTA scores in the Allograft, Chimeric vesicle, and Vector-trapper groups compared with the DSA-trapper and isograft groups (Figure 7C,D).

[0089] Post-transplant renal function testing in mice revealed a gradual increase in serum creatinine levels in the Allograft, Chimeric vesicle, and Vector-trapper groups. Compared to the Allograft group, the DSA-trapper group significantly improved allograft function, with serum creatinine levels significantly reduced by 81.28±7.44% and 87.36±6.91% at four and eight weeks post-transplant, respectively (Figure 7E). Furthermore, urine albumin-to-creatinine ratio (UACR) was measured in mice. While UACR remained low in the DSA-trapper and isograft groups at four and eight weeks post-transplant, UACR levels increased significantly by more than 10-fold in the Allograft, Chimeric vesicle, and Vector-trapper groups. Similar trends in glomerular filtration rate were observed at four and eight weeks post-transplant (Figures 7H,I). These results suggest that DSA-trapper vesicles can effectively protect renal function. Finally, by observing the survival rate of kidney-transplanted mice, DSA-trapper vesicle treatment significantly improved the survival rate of kidney-transplanted mice compared with the Allograft group, Chimeric vesicles group, and Vector-trapper group (Figure 7J).

[0090] In summary, chimeric genetically engineered antibody-adsorbed nanovesicles (DSA-trapper) therapy can effectively adsorb DSA antibodies, inhibit complement system activation and inflammation around the microvascular endothelium, reverse renal transplant fibrosis, significantly improve renal transplant function, and prolong the survival of renal transplant recipients, providing a promising treatment method for the prevention and treatment of antibody-mediated renal transplant rejection.

Claims

1. A method for preparing chimeric genetically engineered macrophages, characterized in that The following steps are involved: S1: construct a lentiviral vector, use engineered cells to package the lentivirus, and obtain lentiviral particles containing protein A and protein G fusion coding genes; S2: The virus obtained in S1 is used to infect macrophages, and a stable cell line expressing the fusion gene of protein A and protein G with high purity is obtained through culture screening.

2. A method for preparing a chimeric genetically engineered macrophage according to claim 1, characterized in that The amino acid sequence of the protein A in S1 is shown in SEQ ID NO.1, and the amino acid sequence of the protein B is shown in SEQ ID NO.

2.

3. The method for preparing a chimeric genetically engineered macrophage according to claim 1, characterized in that The S2 specifically includes the following steps: The target cell macrophages were cultured to a density of 60-70%, and the lentiviral particles obtained in S1 were added, shaken well, and then placed in an incubator for 48-72 hours; When the cells reach the logarithmic growth phase, they are infected again; Puromycin 2-4 μg / mL was added for screening for 48-72 hours; then, the puromycin maintenance concentration was reduced to 0.8-1.2 μg / mL, and the infected cells were screened and amplified for 24-48 hours to obtain a stable cell line expressing a specific gene with high purity.

4. A method for preparing antibody-adsorbed nanovesicles using the chimeric genetically engineered macrophages prepared according to any one of claims 1 to 3, characterized in that The following steps are involved: 1) PLGA nanoparticles were prepared by using an emulsified reagent volatilization method; 2) Collect high-purity stable cells expressing specific genes, repeatedly pass through polycarbonate filters, and centrifuge and wash to obtain cell membranes; 3) The PLGA nanospheres described in step 1) and the cell membranes described in step 2) are mixed uniformly according to a certain proportion, and are sequentially passed through 400 nm, 200 nm, and 100 nm polycarbonate membranes, and squeezed back and forth to obtain nanovesicles with a chimeric engineered membrane encapsulating a PLGA core; 4) The nanovesicles in step 3) are mixed with a phosphatidylserine solution for surface modification to obtain nanovesicles for antibody adsorption.

5. The preparation method according to claim 4, characterized in that The step 1) is specifically as follows: The DCM solution containing PLGA was added dropwise to a 2-4% w / v PVA aqueous solution and sonicated to form an oil-in-water emulsion; The resulting emulsion was stirred on a magnetic stirrer at room temperature overnight; The solution was evaporated using a rotary evaporator at 40-60 °C to remove the DCM solvent residue; The emulsion was transferred to a centrifuge tube, ultracentrifuged, washed with ionized water, and the washed microspheres were placed in a cryotube for vacuum freeze-drying and stored at -20°C for later use.

6. The preparation method according to claim 4, characterized in that In the step 2), the repeated passing through the polycarbonate filter is specifically: first passing through a polycarbonate membrane with a pore size of 10 μm for 3-5 times, and then passing through a membrane with a pore size of 5 μm for 3-5 times; the centrifugal treatment parameters are 4° C., 2000 g, and 15-20 minutes.

7. The preparation method according to claim 4, characterized in that In the step 3), the PLGA nanospheres and the cell membrane are mixed at a mass ratio of 10-20:

1.

8. The preparation method according to claim 4, characterized in that The concentration of the phosphatidylserine solution in step 4) is 10%-15%.

9. Antibody-adsorbed nanovesicles obtained by the preparation method of claims 4-8.

10. Use of the antibody-adsorbed nanovesicles according to claim 9 in the preparation of a drug, wherein the drug is used for any one of the following (a1) to (a6): (a1) adsorption and removal of antibodies; (a2) blocking activation of the complement system; (a3) Reduce inflammatory accumulation and protect the microvascular endothelium of transplanted kidneys; (a4) Inhibit the proliferation and activation of B cells in secondary lymphoid tissues; (a5) Alleviate alloreactive antibody-mediated renal allograft injury; (a6) Improve transplanted kidney function and long-term prognosis of kidney transplant recipients.

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