Chimeric gene engineering nano vesicle for removing pathogenic antibody as well as preparation method and application of chimeric gene engineering nano vesicle

Abstract

The invention discloses a chimeric gene engineering nano vesicle for removing pathogenic antibodies as well as a preparation method and application of the chimeric gene engineering nano vesicle, and belongs to the technical field of biology. According to the invention, a stable transfection cell line of a protein A / G gene is constructed through lentiviral particle transfection, so that the protein A and the protein G can be stably expressed. The fusion protein A / G can overcome the defect of weak binding capacity of pure protein A and IgG3, and the adsorption effect of the antibody IgG is improved. The PLGA nanoparticles with relatively uniform particle size are adopted as the core of the target vesicle and are mixed with the engineered cell membrane broken by extrusion to promote self-assembly of the cell membrane and form an extracellular vesicle simulant, so that the stability of the target nano vesicle is improved. The prepared and separated nano vesicles can be applied to in-vivo adsorption and antibody removal, the biological safety is good, and the dialysis indwelling catheter operation risk in the use process of an existing immunoadsorption column and the risks of bleeding, allergy and the like in the treatment process are avoided.

Description

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 part of the immune system, antibodies play a key role in resisting the invasion of foreign pathogenic microorganisms and protecting the health of the body. However, in some cases (autoimmune diseases, organ transplantation, etc.), antibodies are harmful to the body. In autoimmune diseases, due to immune disorders, autoantibodies recognize and attack the body's own cells, tissues and organs, inducing immune inflammatory reactions and tissue damage. There are more than 100 known autoimmune diseases, including rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, inflammatory bowel disease, myasthenia gravis, and goodpasture-nephritis syndrome. Autoimmune diseases are not uncommon in the population and can cause a considerable disability and mortality rate. In organ transplantation, antibodies specific to donor antigens in the recipient can induce antibody-mediated rejection damage reactions, leading to graft dysfunction or graft loss. In the treatment of autoimmune diseases and organ transplantation, clearing the patient's pathogenic antibodies can effectively relieve symptoms and improve poor prognosis.

[0003] Immunoadsorption is an in vitro treatment method that uses an adsorbent with high affinity (antigen, antibody or certain specific substances with physicochemical affinity) to be fixed on a carrier to make an immunoadsorption column, selectively or specifically remove pathogenic immune complexes and antibodies in the patient's blood. Compared with plasma exchange, it relatively selectively removes pathogenic antibodies, has the advantages of not losing plasma components such as albumin, not needing to supplement fresh plasma, and avoiding blood-borne diseases. At present, the commonly used immunoadsorption column for pathogenic antibody removal in clinical practice is the Staphylococcus protein A immunoadsorption column. Other immunoadsorption columns also include immunoadsorption columns with different adsorbents such as protein A polypeptides, tryptophan or phenylalanine, and anti-IgG multi-antibody adsorbents, each with its own characteristics in the intensity and specificity of antibody adsorption. In addition, new immunoadsorbents are also being developed. For example, CN107138139A discloses an immunoadsorbent that uses a covalent coupling method to graft small molecule functional ligands on an adsorption carrier material. Its activation method is high-density activation, and the coupling spacer arm is used to achieve flexible connection of small molecule functional ligands, which can improve the stability, adsorption capacity and selectivity of functional ligands. However, the antibody adsorption treatment method still has some defects, such as the need to establish additional vascular access, and the risks of allergies, bleeding, etc. 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 engineered nanovesicles prepared by the top-down strategy still have 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. Summary of the invention

[0006] 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 preparation method and application technical solution thereof.

[0007] The present invention is specifically implemented by the following technical solutions:

[0008] The first aspect of the present invention provides a method for preparing chimeric genetically engineered macrophages, which comprises the following steps:

[0009] 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;

[0010] 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.

[0011] Furthermore, the engineered cells in S1 are HEK-293T cells.

[0012] 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.

[0013] Further, the S2 specifically includes the following steps:

[0014] 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;

[0015] When the cells reach the logarithmic growth phase, they are infected again;

[0016] 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.

[0017] 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:

[0018] 1) PLGA nanoparticles were prepared by using an emulsified reagent volatilization method;

[0019] 2) Collect high-purity stable cells expressing specific genes, repeatedly pass through polycarbonate filters, and centrifuge and wash to obtain cell membranes;

[0020] 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;

[0021] 4) The nanovesicles in step 3) are mixed with a phosphatidylserine solution for surface modification to obtain nanovesicles for antibody adsorption.

[0022] Further, the step 1) is specifically as follows:

[0023] 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;

[0024] The resulting emulsion was stirred on a magnetic stirrer at room temperature overnight;

[0025] The solution was evaporated using a rotary evaporator at 40-60 °C to remove the DCM solvent residue;

[0026] 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.

[0027] Furthermore, in 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.

[0028] Furthermore, in step 3), the PLGA nanospheres and the cell membrane are mixed at a mass ratio of 10-20:1.

[0029] Furthermore, in step 4), the concentration of the phosphatidylserine solution is 10%-15%.

[0030] The third aspect of the present invention provides antibody-adsorbed nanovesicles obtained by the above preparation method.

[0031] The 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)-(a6):

[0032] (a1) adsorption and removal of antibodies;

[0033] (a2) blocking activation of the complement system;

[0034] (a3) Reduce inflammatory accumulation and protect the microvascular endothelium of transplanted kidneys;

[0035] (a4) Inhibit the proliferation and activation of B cells in secondary lymphoid tissues;

[0036] (a5) Alleviate alloreactive antibody-mediated renal allograft injury;

[0037] (a6) Improve transplanted kidney function and long-term prognosis of kidney transplant recipients.

[0038] The beneficial effects of the present invention are specifically as follows:

[0039] 1. A chimeric genetically engineered stable cell line was constructed to absorb all IgG subtypes;

[0040] The present invention provides a method for preparing chimeric genetically engineered macrophages, which constructs a stable cell line of protein A / G genes by transfection with lentiviral particles, thereby stably expressing protein A and protein G. Fusion protein A / G can make up for the disadvantage of weak binding ability of simple protein A to IgG3 and improve the adsorption effect of antibody IgG.

[0041] 2. The constructed nanovesicles are stable, uniform in size, and have guaranteed integrity;

[0042] 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.

[0043] 3. Nanovesicles can adsorb and remove antibodies in the body, reducing invasive procedures and bleeding risks;

[0044] The nanovesicles prepared and separated in the present invention can be used for adsorbing and removing antibodies in vivo, have good biosafety, and avoid the risks of dialysis catheterization operation during the use of current immunoadsorption columns and the risks of bleeding, allergies, etc. during treatment.

[0045] 4. After being phagocytosed by macrophages, nanovesicles can exert an immunomodulatory effect and reduce the use of immunosuppressants.

[0046] The method for preparing and isolating nanovesicles of 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

[0047] Figure 1 The fusion protein A / G is expressed on the cell membrane of chimeric genetically engineered macrophages;

[0048] Figure 2 Characterization of chimeric genetically engineered antibody-adsorbed nanovesicles;

[0049] Figure 3 To characterize the biocompatibility of chimeric genetically engineered antibody-adsorbed nanovesicles, Figure 3 The order of the groups on Day 1, Day 5, and Day 7 in the bar graphs of B and 3C is as follows: from left to right, they are the control group, the Vector-trapper vesicle group, the chimeric vesicles group, and the DSA-trapper vesicle group;

[0050] Figure 4 The in vitro and in vivo antibody adsorption effects of chimeric genetically engineered nanovesicles;

[0051] Figure 5 Chimeric genetically engineered antibodies adsorbed into nanovesicles inhibit the proliferation and activation of B cells and plasma cells in secondary lymphoid tissues. Figure 5 Column Figure 1 The specific order of groups in week and 3weeks is: from left to right: Isograft group, Allograft group, DSA-trapper vesicle group, Chimeric-vesicles group and Vector-trapper vesicle group;

[0052] Figure 6 Chimeric genetically engineered antibodies adsorb nanovesicles to inhibit complement system activation and inflammation around microvascular endothelium, thereby alleviating transplant kidney damage;

[0053] Figure 7 Chimeric genetically engineered antibody-adsorbed nanovesicles can alleviate chronic fibrosis of transplanted kidneys, improve renal function and significantly prolong the survival of allogeneic renal transplant recipients. DETAILED DESCRIPTION

[0054] The present invention is further described below in conjunction with 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.

[0055] If the specific technology and conditions are not specified in the protocol, the technology or conditions described in the literature in this field shall be followed. The manufacturers of the kits and instruments used are not specified, and all are products that can be purchased through formal channels.

[0056] Implementation Case 1: Preparation Method of Chimeric Genetically Engineered Macrophages

[0057] S1 constructs a lentiviral vector, uses engineered cells to package the lentivirus, and obtains lentiviral particles containing protein A and protein G fusion coding genes. Specifically:

[0058] When the HEK293T cells in the culture dish reached 70-80% confluency, Opti-MEM, pCMV delta R8.2, pCMV-VSV-G, pCDH-CMV-Protein A / G-EGFP-Puro plasmid, and Xtreme gene 9 were mixed in proportion and the mixture was incubated at room temperature for 20-30 minutes; the medium was aspirated from the HEK293T cells using a pipette and about 8-10 mL of fresh DMEM medium was added. The transfection mixture was added to the HEK293T cells using a 200 μL micropipette and incubated in a 37°C cell culture incubator for about 48 hours. On the third day, the supernatant was collected from the HEK293T culture dish using a 10 mL disposable sterile pipette. The collected supernatant was transferred to a 10 mL syringe and then filtered through a 0.45 μm PVDF filter in a laminar flow hood. The viral supernatant was quickly frozen using liquid nitrogen and then stored in a -80°C refrigerator for later use.

[0059] S2 uses the virus obtained in S1 to infect macrophages, and obtains a stable cell line expressing protein A and protein G fusion genes with high purity through culture screening. Specifically:

[0060] Raw264.7 cells were cultured in a 6-well plate to a density of about 60-70%, the virus solution was taken out from the -80°C refrigerator and thawed, and 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 grow to the logarithmic growth phase. 2-4 μg / mL puromycin was added to the cell line culture dish for 48-72 hours to screen cells expressing fusion genes; 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 protein A and protein G fusion encoding 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.

[0061] The protein A has a 516 amino acid sequence (SEQ ID NO.1):

[0062]

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

[0064]

[0065] Using DAPI to label the cell nucleus and F-actin to label intracellular actin, we observed using confocal fluorescence microscopy and found that the expression rate of fusion protein A / G in engineered macrophages exceeded 95% ( Figure 1 A). Further, chimeric genetically engineered macrophages were separated by flow cytometry, and resuspended in RPMI1640 medium containing 10% FBS after centrifugation at 1000-1200 r / min for 5 minutes, thereby obtaining a stable cell line expressing fusion protein A / G with a purity of up to 96.28% ( Figure 1 B and C).

[0066] Implementation Case 2: Method for preparing antibody-adsorbed nanovesicles by chimeric genetically engineered macrophages.

[0067] 1) PLGA nanospheres were prepared by an emulsified reagent volatilization method, specifically:

[0068] Prepare 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 bottle and add 5 mL of DCM solution to dissolve the PLGA. Transfer 25 mL of 2-4% w / v PVA aqueous solution to a 250 mL flask in an ice bath and use a probe sonicator for initial emulsification. Use a glass pipette to add the DCM solution containing PLGA dropwise to the PVA solution and then sonicate to form an oil-in-water emulsion. Stir the resulting emulsion at 500 rpm on a magnetic stirrer at room temperature in a fume hood overnight to reduce the DCM solvent. Evaporate the solution using a rotary evaporator at 40°C for 2 hours to remove the DCM solvent residue. Transfer the emulsion to a centrifuge tube and centrifuge it at 10,000 rpm for 5 minutes using an ultracentrifuge. Wash it three times with deionized water, place the washed microspheres in a cryovial, vacuum freeze-dry, and store at -20°C for later use.

[0069] 2) Extraction of chimeric genetically engineered macrophage cell membrane, specifically:

[0070] 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 precipitated and 1×10 8 Cells / mL were resuspended in PBS. The cell suspension was added to pre-cooled Tris-magnesium buffer and 1% volume of phenylmethylsulfonyl fluoride (PMSF) was added for hypotonic incubation overnight. Repeatedly passed through a polycarbonate filter installed in a micro-extruder (Avanti). The cells first passed through a 10μm pore size polycarbonate membrane 3-5 times, and then passed through a 5μm pore size membrane in a newly assembled device 3-5 times. The cell structure was destroyed by mechanical extrusion to obtain a cell homogenate, and 1M sucrose solution was added to the cell homogenate to a final concentration of 0.25M. The extruded solution was centrifuged at 2000g for 15-20 minutes at 4°C, the precipitate was discarded and the supernatant was removed, and the above centrifugation was repeated 2-3 times to remove the cell nucleus and organelles. The collected supernatant was centrifuged at 4°C and 3000g to collect the precipitate, washed once with ice Tris-magnesium buffer and centrifuged to collect the purified chimeric engineered cell membrane.

[0071] 3) Nanovesicles with chimeric engineered membranes encapsulating PLGA core

[0072] The PLGA nanospheres prepared in step 1) and the chimeric engineered cell membrane extracted in step 2) are mixed evenly at a mass ratio of 10-20:1, and then placed in a water bath in an ultrasonicator for 20-30 minutes. A micro extruder is used to sequentially pass through 400nm, 200nm and 100nm polycarbonate membranes, and squeeze back and forth 15-20 times to obtain nanovesicles with a chimeric engineered membrane encapsulating a PLGA core.

[0073] 4) A 10% concentration of phosphatidylserine solution was mixed with the chimeric engineered membrane-PLGA nanovesicles for surface modification to prepare nanovesicles for antibody adsorption.

[0074] Case Study 3: Characterization of Chimeric Genetically Engineered Antibody-Adsorbed Nanovesicles (DSA-trapper)

[0075] Through transmission electron microscopy observation, the chimeric genetically engineered antibody adsorbed nanovesicles have a PLGA core, a round, double phospholipid bilayer membrane structure, a high electron density area inside the vesicle, and a low electron density area outside the vesicle ( Figure 2 A). Malvern particle size analyzer test results showed 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 2 B).

[0076] Western blot analysis showed that ( Figure 2 C), the chimeric genetically engineered antibody adsorbed nanovesicles and the protein A and protein G content in the chimeric vesicles were rich, while the PLGA particle control group and the empty vesicle group did not contain protein A / G. The successful expression of the fusion protein and the successful construction of the stable cell line were further successfully verified at the molecular level. Compared with PLGA nanoparticles, proteins on the macrophage membrane (including TLR4 and IL-1RII) were highly inherited to the chimeric genetically engineered antibody adsorbed nanovesicles. This is conducive to the chimeric genetically engineered antibody adsorbed nanovesicles to induce macrophage target molecules, neutralize proinflammatory cytokines and directly inhibit allogeneic immune responses.

[0077] Case study 4: Biocompatibility testing of chimeric genetically engineered antibody-adsorbed nanovesicles (DSA-trapper)

[0078] To determine the biocompatibility of chimeric genetically engineered antibody-adsorbed nanovesicles, we performed in vitro biocompatibility studies by incubating DSA-trapper vesicles, Vector-trapper vesicles (unengineered macrophage membrane vesicles), or Chimericvesicles (chimeric engineered cell membrane vesicles without phosphatidylserine) with human renal tubular epithelial cells for 7 days. Cell viability was assessed by live / dead staining. The results showed that the cells in the four groups were able to maintain good viability and proliferation ability, with a mortality rate of less than 5% ( Figure 3 A). It is noteworthy that compared with the control group, quantitative statistics further support the good biocompatibility of the DSA-trapper vesicle group ( Figure 3 B). Cell proliferation and toxicity assays are important parameters for evaluating clinical applications in the preclinical experimental stage. We therefore 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 were cultured with similar growth trends, and there was no significant difference in cell proliferation and toxicity effects among the control group, Vector-trapper vesicle group, Chimeric vesicles group, and DSA-trapper vesicle group ( Figure 3 C).

[0079] In order to verify the biosafety of DSA-trapper vesicles in mice, we injected DSA-trapper vesicles, chimeric vesicles, and Vector-trapper vesicles into mice through the tail vein and analyzed the changes in blood routine, liver function, and kidney function on days 7 and 21. The results showed that there were no significant differences in blood routine (red blood cell, white blood cell, platelet count), liver function (alanine aminotransferase, aspartate aminotransferase, albumin index) and kidney function (glomerular filtration rate, urine albumin creatinine ratio) analysis between DSA-trapper vesicles and the control groups on days 7 and 21.

[0080] Case study 5: In vitro and in vivo antibody adsorption experiments of chimeric genetically engineered nanovesicles (DSA-trapper)

[0081] In vitro antibody adsorption experiment: The adsorption effect of DSA-trapper vesicles on IgG3 and IgM was detected by concentration gradient. Figure 4 As shown in the figure, the adsorption curves showed that the adsorption effect of DSA-trapper vesicles on IgG3 was more than 95%, and the adsorption effect on IgM was more than 80%, with IC50 values ​​of 281μg / mL and 916μg / mL, respectively, while Vector-trapper did not show obvious adsorption effect ( Figure 4 A).

[0082] In vivo antibody adsorption experiment: First, we constructed a mouse allogeneic renal transplantation antibody-involved rejection injury model. Five groups were set up in the experiment: Isograft group, Allograft group, DSA-trapper vesicle group, Vector-trapper vesicle group and chimeric vesicle group. The specific information of each group is as follows: a) Isograft group: C57BL / 6 mice were used as donors and C57BL / 6 mice were used as recipients for renal transplantation, and an equal amount of sterile saline was injected after surgery; b) Allograft group: BALB / C mice were used as donors and C57BL / 6 mice were used as recipients for renal transplantation, and an equal amount of sterile saline was injected after surgery; c) DSA-trapper vesicle group: BALB / C mice were used as donors and C57BL / 6 mice were used as recipients for renal transplantation, and DSA-trapper vesicles were given intravenous injection for treatment. d) Vector-trapper vesicle group: BALB / C mice were used as donors and C57BL / 6 mice were used as recipients for renal transplantation, and Vector-trapper vesicles were given intravenous injection for treatment. e) Chimericvesicles group: BALB / C mice were used as donors and C57BL / 6 mice were used as recipients for renal transplantation, and chimericvesicles were given intravenous injection for treatment. Subsequently, the serum DSA levels of transplanted mice in each group were monitored after renal transplantation. In the Allograft group, the serum DSA level gradually increased with the extension of postoperative time, and the IgG immunofluorescence intensity of DSA increased from less than 1000 at the time of surgery to more than 10000 at 8 weeks after surgery. Compared with the Allograft group, the Chimeric vesicles group and the Vector-trapper vesicle group, the serum DSA immunofluorescence intensity of the DSA-trapper vesicle group was significantly reduced by 92.74% at 8 weeks after surgery and maintained at close to the baseline level ( Figure 4 B). 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.

[0083] Case study 6: Experiment on inhibition of proliferation and activation of B cells and plasma cells in secondary lymphoid tissue by chimeric genetically engineered antibody-adsorbed nanovesicles (DSA-trapper)

[0084] The induction of DSA production by memory B cells and long-lived plasma cells after 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 increased significantly at 1 week after surgery, which was about 3 times that of the Isograft group, and further increased at 3 weeks after surgery, reaching 2.97%, 0.94% and 2.92%, respectively. The chimeric vesicles group and the vector-trapper group had similar levels of change as the Allograft group. In the group treated with DSA-trapper vesicles, the cell ratios of GCs, SPPCs and SPMCs remained at a low level of 0.83%, 0.40% and 0.62%, respectively, 3 weeks after transplantation, which was not significantly different from the Isograft group ( Figure 5 ).

[0085] 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.

[0086] 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 endothelial injury of the transplanted kidney through antibody-dependent cell-mediated cytotoxicity. Our study found that immunofluorescence of transplanted kidney tissues in the Allograft group showed a large amount of deposition of complement C3 and immunoglobulin IgG in the glomerular and peritubular capillary regions 1 week and 3 weeks after renal transplantation. The average immunofluorescence intensity of complement C3 and IgG in the DSA-trapper vesicle treatment group remained at a low level after surgery, with no significant difference from the Isograft group ( Figure 6 A, B). The C4d deposition in each group was evaluated by quantitative analysis of the average positive staining area of ​​C4d immunohistochemistry. Compared with the C4d deposition area of ​​about 30-40% in the Allograft group, Chimeric vesicles group, and Vector-trapper group, the C4d positive area in the DSA-trapper vesicle treatment group continued to remain at a lower level at 1 and 3 weeks after surgery, which was similar to the Isograft group ( Figure 6 C, E, F). These results suggest that DSA-trapper vesicles can effectively inhibit the complement system.

[0087] Furthermore, we found that platelets (CD41 marker) and neutrophils (Ly6G marker) were observed to aggregate and distribute in the interstitial tissue around microvessels (CD31 marker) in the Allograft group, Chimeric vesicles group, and Vector-trapper group at 1 week and 3 weeks after surgery. DSA-trapper vesicle treatment significantly improved the inflammatory aggregation of platelets and neutrophils around microvessels at 1 week and 3 weeks after surgery ( Figure 6 D, G, H). Through transmission electron microscopy, we further observed the adhesion of immune inflammatory cells to the microvascular endothelium, infiltration of the interstitial tissue around the microvascular endothelium, and endothelial damage in the Allograft group, Chimeric vesicles group, and Vector-trapper group, while no obvious inflammatory cell infiltration was observed in the DSA-trappers vesicle treatment group ( Figure 6 I). These results suggest that DSA-trapper vesicles can effectively protect the microvascular endothelium of transplanted kidneys and alleviate the infiltration of inflammatory cells in transplanted kidneys.

[0088] By immunofluorescence, 8 weeks after surgery, the transplanted kidney tissues in the Allograft group showed glomerular sclerosis, tubular atrophy, and a significant decrease in podocyte function protein PODXL and tubular epithelial function protein AQP-1 ( Figure 7 A). There was no significant improvement in the Chimeric vesicles treatment group and the Vector-trapper treatment group, and the pathological results were similar to those in the Allograft group. However, the DSA-trapper vesicle treatment group showed good glomerular patency and normal tubular morphology in the transplanted kidneys. The relative fluorescence intensity of podocyte and tubular function markers was more than twice that of the positive control groups, and there was no significant difference with the isograft group ( Figure 7 B).

[0089] Eight weeks after renal transplantation, PAS staining and MASSON staining showed that DSA-trapper vesicle treatment significantly improved the morphology of glomeruli and tubules. However, the Allograft group, Chimeric vesicles treatment group, and Vector-trapper treatment group mainly showed glomerular sclerosis, tubular atrophy, interstitial fibrosis, and tubulointerstitial lymphocyte infiltration. In addition, peritubular capillary stratification, podocyte shedding, and glomerular basement membrane thickening were shown in the Allograft group, while the renal ultrastructure was significantly improved in the DSA-trapper vesicle treatment group ( Figure 7F, G). Quantitative evaluation of glomerular sclerosis and tubular atrophy / interstitial fibrosis (IFTA) scores in the transplanted kidney tissue pathological sections showed that the glomerular sclerosis scores and IFTA scores in the Allograft group, Chimeric vesicles group, and Vector-trapper group were significantly higher than those in the DSA-trapper vesicle group and isograft group ( Figure 7 C,D).

[0090] The results of renal function tests after mouse kidney transplantation showed that the serum creatinine in the Allograft group, Chimeric vesicles treatment group and Vector-trapper treatment group showed a gradual increase. Compared with the Allograft group, the DSA-trapper vesicle treatment group significantly improved transplanted kidney function, with serum creatinine levels significantly reduced by 81.28±7.44% and 87.36±6.91% at 4 and 8 weeks after transplantation, respectively. Figure 7 E). At the same time, we detected the urine albumin-to-creatinine ratio (UACR) of mice. At 4 and 8 weeks after surgery, the UACR of the DSA-trapper vesicle-treated group and the isograft-treated group remained at a low level, while the UACR levels of the Allograft-treated group, the Chimeric vesicle-treated group, and the Vector-trapper-treated group increased significantly by more than 10 times. We found that the glomerular filtration rate levels at 4 and 8 weeks after transplantation showed a similar trend of change ( Figure 7 H, I). The above results suggest that DSA-trapper vesicles can effectively protect renal function. Finally, by observing the survival rate of mice with transplanted kidneys, DSA-trapper vesicle treatment significantly improved the survival rate of mice with transplanted kidneys compared with the Allograft group, Chimeric vesicles group and Vector-trapper group ( Figure 7 J).

[0091] In summary, chimeric genetically engineered antibody-adsorbed nanovesicles (DSA-trapper) therapy can effectively adsorb DSA antibodies, inhibit complement system activation and microvascular endothelial inflammation, reverse kidney fibrosis, significantly improve kidney function, and prolong the survival of kidney transplant recipients, providing a promising treatment method for the prevention and treatment of antibody-mediated kidney transplant rejection.

Claims

1. A method for preparing chimeric genetically engineered macrophages, Features 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, Features 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. A method for preparing a chimeric genetically engineered macrophage according to claim 1, Features 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, Features 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, Features 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, Features 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, Features 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, Features 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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