Fusion protein, cell membrane particle containing fusion protein and application of fusion protein

Abstract

The invention discloses a fusion protein, cell membrane particles containing the fusion protein and application of the fusion protein. The fusion protein comprises a CD8 antibody and an IL-2 mutant, the CD8 antibody and the IL-2 mutant are connected through a cleavable connecting assembly, and the IL-2 mutant can stimulate Treg proliferation in a biased mode. The prepared cell membrane particle for expressing the aCD8-IL2 fusion protein can enhance retention and proliferation of Foxp3 + T cells in spleen and pancreas islet in a mouse body and reduce infiltration of CD8 + T cells, so that the cell membrane particle can be applied to immunotherapy of new type I diabetes mellitus.

Description

[0001] This application claims the priority of a Chinese patent application with the application number 202311822814.9, titled "A Fusion Protein, Cell Membrane Particles Containing the Same, and Their Applications", filed on December 27, 2023. Technical Field

[0002] The present invention relates to the field of immunotherapy drug delivery, and particularly to a fusion protein, cell membrane particles containing the same, and their applications. Background Art

[0003] Diabetes is one of the most major chronic diseases currently. At present, the incidence of diabetes still shows an increasing trend year by year, seriously threatening the healthy life of humans. Under the combined action of genetic and environmental factors, the body has an absolute lack of insulin secretion or insulin resistance, leading to disorders in glucose metabolism in the body, and then obvious hyperglycemia. Long-term disorders in glucose metabolism in the body often cause damage to multiple organs, triggering a series of complications, resulting in chronic progressive lesions or functional decline and failure of organs and tissues such as the heart, eyes, kidneys, nerves, and blood vessels.

[0004] Type I diabetes is caused by the abnormal attack of autoreactive immune cells on β-cells, especially the destruction of β-cells in the pancreas mediated by T-cells. Among them, CD4 + T-cells can assist B-cells and T-cells, promoting the activation of macrophages and islet-specific CD8 + T-cells, and CD8 + T-cells are the "direct killers", directly inducing the death of β-cells. However, regulatory Treg cells help protect β-cells and inhibit the destruction of β-cells. Based on the above disease mechanism of type I diabetes, various types of drugs have been developed, including small molecule immunosuppressants, targeted T-cell antibodies (such as teplizumab, thymoglobulin antibody, abatacept), targeted B-cell (such as rituximab), cytokines (such as interleukin-2 (IL2)), etc. to prevent autoimmune attacks and delay or reverse the development of diabetes. Some drugs are undergoing clinical studies and have very significant effects on delaying the progression of the disease, but these effects are not continuously effective for all patients, and new drugs need to be further developed. Bispecific drugs are expected to improve the immune tolerance of patients and increase the treatment efficiency. Summary of the Invention

[0005] To solve the above problems, on the one hand, the present invention provides a fusion protein, which comprises a CD8 antibody and an IL-2 mutant, wherein the CD8 antibody and the IL-2 mutant are connected by a cleavable linker component, and the IL-2 mutant can preferentially stimulate the proliferation of Tregs.

[0006] In some embodiments, the fusion protein further comprises a transmembrane region.

[0007] In some specific embodiments, the transmembrane region is the CD28 transmembrane region or the CD8 receptor transmembrane region.

[0008] In some more specific embodiments, the CD28 transmembrane region has the amino acid sequence shown in SEQ ID NO:16.

[0009] In some embodiments, the fusion protein further comprises a signal peptide.

[0010] In some specific embodiments, the signal peptide is the CD8 receptor expression signal peptide, the platelet-derived growth factor receptor PDGFR signal peptide, or the antibody secretion signal peptide.

[0011] In some more specific embodiments, the signal peptide has the amino acid sequence shown in SEQ ID NO:2.

[0012] In some embodiments, the IL-2 mutant is linked to the transmembrane region through a hinge region.

[0013] In some specific embodiments, the hinge region is the IgG4 hinge region.

[0014] In some more specific embodiments, the hinge region has the amino acid sequence shown in SEQ ID NO:14.

[0015] In some embodiments, the CD8 antibody is a single-chain antibody.

[0016] In some specific embodiments, the CD8 antibody comprises: a heavy chain variable region (VH) having the amino acid sequence shown in SEQ ID NO:4, and a light chain variable region (VL) having the amino acid sequence shown in SEQ ID NO:8.

[0017] In some embodiments, the cleavable linker component is an extracellular granzyme B-responsive peptide.

[0018] In some specific embodiments, the cleavable linker component has the amino acid sequence shown in SEQ ID NO:10.

[0019] In some embodiments, the heavy chain variable region (VH) and the light chain variable region (VL) are linked by a linker.

[0020] In some specific embodiments, the linker has the amino acid sequence shown in SEQ ID NO:6.

[0021] On the other hand, the present invention provides a polynucleotide encoding the above-mentioned fusion protein.

[0022] On the other hand, the present invention provides an expression vector comprising the above-mentioned polynucleotide.

[0023] On the other hand, the present invention provides a transformant comprising the above-mentioned expression vector.

[0024] On the other hand, the present invention provides a transformant comprising the above-mentioned polynucleotide or the above-mentioned expression vector.

[0025] On the other hand, the present invention provides a cell membrane particle obtained by collecting after disrupting the above-mentioned transformant.

[0026] In some embodiments, the cell membrane particles are prepared by the following steps:

[0027] 1) Collect cells;

[0028] 2) Disperse the collected cells in a buffer to obtain a cell suspension;

[0029] 3) Disrupt the cell suspension with a cell ultrasonic disruptor;

[0030] 4) Centrifuge the ultrasonicated cell suspension to obtain a supernatant;

[0031] 5) Centrifuge the collected supernatant to collect cell membranes;

[0032] 6) Resuspend the cell membrane precipitate with a buffer and then centrifuge to collect cell membranes;

[0033] 7) Resuspend the cell membrane precipitate with a buffer again and extrude it through polycarbonate membranes with different pore sizes for multiple times to obtain cell membrane particles with uniform size.

[0034] In some embodiments, the centrifugation conditions in step 4) are 3000 - 5000 rpm / min for 5 - 20 min.

[0035] In some embodiments, the centrifugation conditions in step 6) are 15000 - 20000 rpm / min for 30 - 120 min.

[0036] In some embodiments, the "multiple times" in step 7) is 10 - 20 times, for example 15 times; the "different".

[0037] In some embodiments, the different pore sizes in step 7) are, for example, 800 nm, 400 nm, and 200 nm.

[0038] On the other hand, the present invention provides a pharmaceutical composition comprising the above-mentioned fusion protein or the above-mentioned cell membrane particles.

[0039] On the other hand, the present invention provides a kit, which comprises the above-mentioned fusion protein, the above-mentioned polynucleotide, the above-mentioned transformant or the above-mentioned cell membrane particles.

[0040] On the other hand, the present invention provides a method for preparing a fusion protein, which comprises the step of culturing the above-mentioned transformant.

[0041] On the other hand, the present invention provides the use of the above-mentioned fusion protein in the preparation of a medicament for treating and / or preventing autoimmune diseases.

[0042] In some embodiments, the autoimmune disease is diabetes.

[0043] In some specific embodiments, the autoimmune disease is type I diabetes.

[0044] Based on common general knowledge in the art, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred examples of the present invention.

[0045] The reagents and raw materials used in the present invention are all commercially available.

[0046] The positive and progressive effects of the present invention are as follows:

[0047] The fusion protein of aCD8-IL2 provided by the present invention (for example, existing in the form of cell membrane particles) significantly reduces the blood glucose level of mice compared with the fusion protein of IL-2 (for example, existing in the form of cell membrane particles) and the fusion protein of aCD8 (for example, existing in the form of cell membrane particles). The data are as follows: in the aCD8-IL2 group, 7 out of 9 diabetic mice restored to the normal blood glucose state; in the IL-2 group, 5 out of 9 diabetic mice restored to the normal blood glucose state; in the aCD8 group, 4 out of 9 diabetic mice restored to the normal blood glucose state. And the fusion protein of aCD8-IL2 can significantly reduce the number of dead cells of OVA-B16. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 It is a schematic diagram of the gene sequence of the aCD8-IL2 fusion protein.

[0049] Figure 2 It is a schematic diagram of the gene sequence of the IL2 fusion protein.

[0050] Figure 3 It is a schematic diagram of the gene sequence of the aCD8 fusion protein.

[0051] Figure 4 It is a flow cytometry diagram of 8ILGM cells.

[0052] Figure 5 It is a flow cytometry diagram of ILGM cells.

[0053] Figure 6 Confocal fluorescence imaging map after incubation of 8ILGM with CD8 protein.

[0054] Figure 7 Confocal fluorescence imaging map after incubation of ILGM with CD25 protein.

[0055] Figure 8 Confocal fluorescence imaging map after incubation of 8GM with CD8 protein.

[0056] Figure 9 DLS particle size distribution map of CHO-L1, ILGM, 8ILGM, 8GM cell membrane particles ( Figure 9 part a), Zeta potential statistical chart ( Figure 9 part b).

[0057] Figure 10 Scanning electron microscopy image of CHO-K1 cell membrane particles ( Figure 10 part a), cryo-transmission electron microscopy image ( Figure 10 part b) and transmission electron microscopy image ( Figure 10 part c).

[0058] Figure 11 Number of dead OVA-B16 cells after incubation of OT-1 CD8+ T cells and OVA-B16 cells with CHO-L1, ILGM, 8ILGM, 8GM cell membrane particles ( Figure 11 part a), mortality rate of OVA-B16 cells ( Figure 11 part b)

[0059] Figure 12 Blood glucose level curve graph of NOD mice under different treatment conditions ( Figure 12 part a) and body weight change graph ( Figure 12 part b).

[0060] Figure 13 Statistical chart of diabetes incidence in NOD mice in different treatment groups.

[0061] Figure 14 Immunofluorescence staining map of mouse pancreas after different treatments (glucagon and insulin, Figure 14 part a) and fluorescence area statistical chart ( Figure 14 part b).

[0062] Figure 15 Immunofluorescence staining map of mouse pancreas after different treatments (glucagon and Foxp3, Figure 15 part a) and fluorescence area statistical chart ( Figure 15 part b).

[0063] Figure 16 Immunofluorescence staining images of mouse spleens after different treatments (Foxp3, Figure 16 part a), and statistical graphs of fluorescence areas ( Figure 16 part b).

[0064] Figure 17 Flow cytometry diagrams of lymphocytes in the peripheral blood of mice after different treatments ( Figure 17 parts a - c), and statistical graphs of the proportion of CD3 + T cells in CD45 + cells, the proportion of CD4 + T cells in CD3 + T cells, the proportion of CD8 + T cells in CD3 + T cells, and the proportion of Foxp + T cells in CD4 + T cells ( Figure 17 parts d - e).

[0065] Figure 18 H&E staining images of mouse pancreas after different treatments.

[0066] Figure 19 H&E staining images of the major organs of mice after different treatments, including the heart, liver, spleen, lung, and kidney. Detailed implementation methods

[0067] The present invention will be further illustrated by the following examples, but the present invention is not limited to the scope of the described examples. For the experimental methods without specific conditions in the following examples, they are carried out according to conventional methods and conditions, or selected according to the product instructions.

[0068] "Host cell" refers to a cell that has been transformed with a nucleic acid sequence or is capable of being transformed and thereby expressing a target gene. This term includes the progeny of the parental cell, regardless of whether the progeny are identical to the original parental cell in morphology or genetic composition, as long as the target gene is present.

[0069] "Operably linked" refers to the arrangement of elements, where the components so described are configured to perform their normal functions. Thus, a given signal peptide operably linked to a polypeptide directs the secretion of the polypeptide from the cell. In the case of a promoter, a promoter operably linked to a coding sequence will direct the expression of the coding sequence. The promoter or other control elements do not have to be contiguous with the coding sequence as long as they function to direct its expression. For example, there can be intervening untranslated but transcribed sequences between the promoter sequence and the coding sequence, and the promoter sequence can still be considered to be "operably linked" to the coding sequence.

[0070] Plasmid vectors are artificially constructed on the basis of natural plasmids to adapt to laboratory operations. Compared with natural plasmids, plasmid vectors usually carry one or more selectable marker genes (such as antibiotic resistance genes) and a synthetic multiple cloning site sequence containing multiple restriction endonuclease recognition sites, and most of the non-essential sequences are removed to minimize the molecular weight for convenient genetic engineering operations. The commonly used plasmid vectors are generally between 1 kb and 10 kb in size, such as pBR322, PUC series, pGEM series, pET series, and pBluescript (abbreviated as pBS), etc.

[0071] Type 1 diabetes is a type of diabetes in which there is not enough insulin production, leading to elevated blood sugar levels in the body. The symptoms of type 1 diabetes include frequent urination, increased thirst, increased hunger, weight loss, blurred vision, feeling tired, and poor wound healing. Although the cause of type 1 diabetes is not clear, its underlying mechanism involves the autoimmune destruction of insulin-producing β cells in the pancreas.

[0072] In the following examples and effect examples, all reagents are of reagent grade or other grades better than reagent grade. The specific sources are as follows:

[0073] Sucrose was purchased from Macklin.

[0074] HEPES (1 M, pH = 7.2 - 7.4) was purchased from Solarbio.

[0075] EDTA was purchased from Macklin.

[0076] Protease inhibitor (product number: P8340) was purchased from Sigma.

[0077] Polycarbonate membranes (800 nm, 400 nm, 200 nm) were purchased from Whatman.

[0078] Lipofectamine TM 3000 transfection reagent (product number: L3000075) was purchased from Thermofisher.

[0079] CD8 protein (AB276832) for flow cytometry analysis and confocal fluorescence imaging was purchased from Abcam, and CD25 protein (Z03400) was purchased from GenScript.

[0080] Glucagon antibody (AB92517), insulin antibody (AB6995), and anti-Foxp3 antibody (AB253297) for immunofluorescence staining were purchased from Abcam.

[0081] The secondary antibodies (Anti-rabbit IgG(H+L), Anti-rat IgG(H+L)) were purchased from Cell Signaling Technology.

[0082] NOD / LTJ mice were purchased from Beijing HFK Bioscience.

[0083] All mouse studies were conducted according to the protocols approved by the Laboratory Animal Management Committee of Zhejiang University (Approval number for animal ethics and welfare: ZJU20220116).

[0084] Example 1 Construction of Engineered aCD8-IL2 Fusion Protein Cells (8ILGM Cells)

[0085] Experimental materials: transfection reagent Lipofectamine TM 3000; flow cytometer Beckman moflo Astrios EQ. Experimental steps:

[0086] 1. Construction of the engineered aCD8-IL2 fusion protein gene sequence

[0087] As Figure 1 shown, the fusion protein gene aCD8-IL2 containing the targeting CD8 and human IL2 mutant protein includes, in sequence: signal peptide, single-chain antibody against CD8 (anti-CD8 scFv), extracellular granzyme B-responsive peptide, human IL2 mutant protein, IgG4 hinge region (IgG4 Hinge), CD28 transmembrane region (CD28 TM), EGFP fluorescent protein.

[0088] The nucleotide sequence of the signal peptide is as shown in SEQ ID NO:1, and the amino acid sequence is as shown in SEQ ID NO:2;

[0089] The nucleotide sequence of the heavy chain VH of the single-chain antibody against CD8 is as shown in SEQ ID NO:3, and the amino acid sequence is as shown in SEQ ID NO:4;

[0090] The nucleotide sequence of the hinge linker (Linker) of the single-chain antibody against CD8 is as shown in SEQ ID NO:5, and the amino acid sequence is as shown in SEQ ID NO:6;

[0091] The nucleotide sequence of the light chain VL of the single-chain antibody against CD8 is as shown in SEQ ID NO:7, and the amino acid sequence is as shown in SEQ ID NO:8;

[0092] The nucleotide sequence of the extracellular granzyme B-responsive peptide is shown in SEQ ID NO:9, and the amino acid sequence is shown in SEQ ID NO:10;

[0093] The nucleotide sequence of the human IL2 mutant protein is shown in SEQ ID NO:11, and the amino acid sequence is shown in SEQ ID NO:12;

[0094] The nucleotide sequence of the IgG4 hinge region is shown in SEQ ID NO:13, and the amino acid sequence is shown in SEQ ID NO:14;

[0095] The nucleotide sequence of the CD28 transmembrane region is shown in SEQ ID NO:15, and the amino acid sequence is shown in SEQ ID NO:16;

[0096] The nucleotide sequence of the intracellular EGFP fluorescent protein is shown in SEQ ID NO:17, and the amino acid sequence is shown in SEQ ID NO:18;

[0097] The nucleotide sequence of the fusion protein aCD8-IL2 is shown in SEQ ID NO:19, and the amino acid sequence is shown in SEQ ID NO:20.

[0098] 2. Construction of stable transfected cell lines by transfection with the plasmid of aCD8-IL2 fusion protein gene

[0099] Step 1: Take CHO-K1 cells in the logarithmic growth phase. 24 hours before transfection, seed the cells in a 6-well plate at 2.5×10 5 cells per well. When the cell confluence reaches 70%-90% within 24 hours, transfection can be carried out;

[0100] Step 2: Use LipofectamineTM3000 reagent. Dilute 3.75 μL of Lipofectamine TM 3000 reagent with 250 μL of Opti-MEM TM medium and mix well to obtain solution I;

[0101] Step 3: Dilute DNA with Opti-MEM TM medium to prepare a DNA premix. Add 2.5 μg of the plasmid carrying the aCD8-IL2 fusion protein and the neomycin resistance gene to 250 μL of Opti-MEM TM medium, and then add P3000 TM reagent (2 μL / μg DNA) and mix well to obtain solution II;

[0102] Step 4: Thoroughly mix Solution I and II, transfer the DNA-lipid complex to the wells of a 6-well plate after incubating at room temperature for 15 min, and place the transfected cells in an incubator for routine culture.

[0103] Step 5: Change the medium 24 h after plasmid transfection. Transfer the cells at a ratio of 1:10 to a 24-well plate 48 h later, and simultaneously transfer untransfected cells at the same density as a control; after the cells adhere to the wall, add G418, and change the medium in a timely manner according to the cell death situation and the nutritional status of the medium; when all the cells in the control group die and there are viable cells in the drug-administered group, obtain preliminary resistant clones.

[0104] Step 6: Use a flow cytometer Beckman moflo Astrios EQ to sort the cells for a stable transfected cell line of resistant monoclonal cells, collect the positive population cells into a 96-well plate for monoclonal culture, and freeze them in liquid nitrogen after culturing and amplifying.

[0105] Example 2 Identification of Cells Engineered with aCD8-IL2 Fusion Protein

[0106] Step 1: Incubate the 8ILGM cells constructed in Example 1 with 2% BSA on ice for 15 min for blocking.

[0107] Step 2: Incubate the above cells with CD8 protein and CD25 protein on ice for 30 min respectively, and wash twice with PBS.

[0108] Step 3: Incubate the above cells with APC-labeled anti-Fc protein on ice for 30 min respectively, and wash twice with PBS.

[0109] Step 4: Analyze the cells using a flow cytometer. As Figure 4 shown, the green fluorescence shift is the eGFP protein expressed by 8ILGM cells, and the red fluorescence shift indicates that the csFv of the CD8 antibody expressed by the cells can bind to the CD8 protein, and the IL2 protein can bind to the CD25 protein (IL2Rα).

[0110] Example 3 Preparation of Cell Membrane Particles Expressing aCD8-IL2 Fusion Protein

[0111] Step 1: Collect the 8ILGM cells cultured in a 15-cm culture dish, wash twice with PBS (800 rpm / min, 4 °C, centrifuge for 5 min), and collect the cells.

[0112] Step 2: Disperse the collected cells in 2 mL of homogenization buffer, where the homogenization solution is an aqueous solution containing 0.25 M sucrose, 1 mM EDTA, 20 mM Hepes-NaOH, and protease inhibitors (pH = 7.4).

[0113] Step 3: The cell suspension was disrupted by a cell ultrasonic disruptor for 5 minutes. The instrument parameters were set to 150 W, 3 seconds on, 7 seconds off, for 5 minutes.

[0114] Step 4: The ultrasonicated cell suspension was centrifuged under the conditions of 4000 rpm / min, 10 min, and 4 °C. After centrifugation, the supernatant was collected and the cell debris precipitate at the bottom was removed.

[0115] Step 5: The collected supernatant was centrifuged to collect the cell membrane. The centrifugation conditions were 18000 rpm / min, 60 min, and 4 °C. After centrifugation, the supernatant was removed and the cell membrane precipitate at the bottom of the centrifuge tube was collected.

[0116] Step 6: The cell membrane precipitate was resuspended in 2 mL of PBS buffer (pH = 7.2) and centrifuged again under the conditions of 18000 rpm / min, 60 min, and 4 °C. After centrifugation, the supernatant was removed and the cell membrane precipitate at the bottom of the centrifuge tube was collected.

[0117] Step 7: The cell membrane precipitate was resuspended in 2 mL of PBS buffer (pH = 7.2). The above cell membrane suspension was extruded through a polycarbonate membrane with a pore size of 800 nm using an avestin liposome extruder, at least 15 times back and forth; then extruded through a polycarbonate membrane with a pore size of 400 nm again, at least 15 times back and forth; and then extruded through a polycarbonate membrane with a pore size of 200 nm again, at least 15 times back and forth; to obtain uniformly sized cell membrane particles. The hydrated particle size of the cell membrane particles was 186.2 ± 2.4 nm.

[0118] Construction of Engineered IL2 Protein-expressing Cells (ILGM Cells) in Example 4

[0119] Experimental Materials: Transfection Reagent Lipofectamine TM 3000; Flow Cytometer Beckman mofloAstrios EQ Experimental Steps:

[0120] 1. Construction of the Engineered ILGM Fusion Protein Gene Sequence

[0121] As Figure 2 shown, the fusion protein gene ILGM containing the human-derived IL2 mutant protein includes, in sequence: IL-2 signal peptide (IL-2 signal peptide), human-derived IL2 mutant protein, IgG4 hinge region (IgG4 Hinge), CD28 transmembrane region (CD28 TM), and EGFP fluorescent protein.

[0122] The nucleotide sequence of the IL-2 signal peptide is as shown in SEQ ID NO:21, and the amino acid sequence is as shown in SEQ ID NO:22: MYRMQLLSCIALSLALVTNS;

[0123] The nucleotide sequence of the human IL2 mutant protein is shown in SEQ ID NO: 11; the nucleotide sequence of the IgG4 hinge region is shown in SEQ ID NO: 13; the nucleotide sequence of the CD28 transmembrane region is shown in SEQ ID NO: 15; the nucleotide sequence of the intracellular EGFP fluorescent protein is shown in SEQ ID NO: 17; the nucleotide sequence of the fusion protein ILGM is shown in SEQ ID NO: 23, and the amino acid sequence is shown in SEQ ID NO: 24.

[0124] 2. Construction of stable transfected cell lines by transfection of ILGM fusion protein gene plasmid

[0125] Step 1: Take CHO-K1 cells in the logarithmic growth phase. Inoculate the cells into a 6-well plate 24 h before transfection, with 2.5×10 5 cells per well. When the cell confluence reaches 70%-90% within 24 h, transfection can be carried out;

[0126] Step 2: Use Lipofectamine TM 3000 reagent. Dilute 3.75 μL of Lipofectamine TM 3000 reagent with 250 μL of Opti-MEM TM medium and mix well to obtain solution I;

[0127] Step 3: Dilute DNA with Opti-MEM TM medium to prepare a DNA premix. Add 2.5 μg of the plasmid carrying the ILGM fusion protein and the neomycin resistance gene to 250 μL of Opti-MEM TM medium, and then add P3000 TM reagent (2 μL / μg DNA) and mix well to obtain solution II;

[0128] Step 4: Mix solutions I and II well. After incubating at room temperature for 15 min, transfer the DNA-lipid complex to the wells of the 6-well plate. After transfection, the cells are placed in an incubator for routine culture;

[0129] Step 5: Change the medium 24 h after plasmid transfection. After 48 h, passage the cells at a ratio of 1:10 to a 24-well plate, and at the same time passage the untransfected cells at the same density as a control; after the cells adhere to the wall, add G418, and change the medium in a timely manner according to the cell death situation and the nutritional status of the medium; when all the cells in the control group die and there are living cells in the drug-administered group, obtain the preliminary resistant clones.

[0130] Step 6: Use the Beckman moflo Astrios EQ flow cytometer to sort the cells into resistant monoclonal stable transfected cell lines, collect the positive population of cells into a 96-well plate for monoclonal culture, and cryopreserve them in liquid nitrogen after culture and amplification.

[0131] Identification of Cells Expressing Engineered IL2 Protein in Example 5

[0132] Step 1: Incubate the ILGM cells constructed in Example 5 with 2% BSA on ice for 15 min for blocking.

[0133] Step 2: Incubate the above cells with CD25 protein on ice for 30 min, and wash twice with PBS.

[0134] Step 3: Incubate the above cells with APC-labeled anti-Fc antibody on ice for 30 min, and wash twice with PBS.

[0135] Step 4: Analyze the cells using a flow cytometer. As Figure 5 shown, the green fluorescence shift is the eGFP protein expressed by ILGM cells, and the red fluorescence shift indicates that the IL2 protein expressed by ILGM cells can bind to CD25 protein (IL2Rα).

[0136] Preparation of Cell Membrane Particles Expressing IL2 Protein in Example 6

[0137] Step 1: Collect the ILGM cells cultured in a 15-cm culture dish, wash twice with PBS (800 rpm / min, 4 °C, centrifuge for 5 min), and collect the cells.

[0138] Steps 2-7 are the same as those in Example 3 above.

[0139] Construction of Engineered aCD8 Fusion Protein Cells (8GM Cells) in Example 7

[0140] Experimental Materials: Transfection Reagent Lipofectamine TM 3000; Beckman moflo Astrios EQ flow cytometer. Experimental Steps:

[0141] 1. Construct the gene sequence of engineered aCD8 fusion protein

[0142] As Figure 3 shown, it contains the fusion protein gene aCD8 targeting CD8, including in sequence: signal peptide, single-chain antibody targeting CD8 (anti-CD8 scFv), extracellular granzyme B-responsive peptide, IgG4 hinge region (IgG4 Hinge), CD28 transmembrane region (CD28 TM), EGFP fluorescent protein.

[0143] The nucleotide sequence of the signal peptide is shown in SEQ ID NO:1, and the amino acid sequence is shown in SEQ ID NO:2;

[0144] The nucleotide sequence of the VH of the single-chain antibody targeting CD8 is shown in SEQ ID NO:3, and the amino acid sequence is shown in SEQ ID NO:4;

[0145] The nucleotide sequence of the linker of the single-chain antibody targeting CD8 is shown in SEQ ID NO:5, and the amino acid sequence is shown in SEQ ID NO:6;

[0146] The nucleotide sequence of the VL of the single-chain antibody targeting CD8 is shown in SEQ ID NO:7, and the amino acid sequence is shown in SEQ ID NO:8;

[0147] The nucleotide sequence of the extracellular granzyme B-responsive peptide is shown in SEQ ID NO:9, and the amino acid sequence is shown in SEQ ID NO:10;

[0148] The nucleotide sequence of the IgG4 hinge region is shown in SEQ ID NO:13, and the amino acid sequence is shown in SEQ ID NO:14;

[0149] The nucleotide sequence of the CD28 transmembrane region is shown in SEQ ID NO:15, and the amino acid sequence is shown in SEQ ID NO:16;

[0150] The nucleotide sequence of the intracellular EGFP fluorescent protein is shown in SEQ ID NO:17, and the amino acid sequence is shown in SEQ ID NO:18;

[0151] The nucleotide sequence of the fusion protein aCD8 is shown in SEQ ID NO:25, and the amino acid sequence is shown in SEQ ID NO:26.

[0152] 2. Construction of stable transfected cell lines by transfection of aCD8 fusion protein gene plasmid

[0153] Step 1: Take CHO-K1 cells in the logarithmic growth phase. 24 hours before transfection, inoculate the cells in a 6-well plate, with 2.5×10 5 cells per well. When the cell confluence reaches 70%-90% within 24 hours, transfection can be carried out;

[0154] Step 2: For transfection, use LipofectamineTM3000 reagent. Dilute 3.75 μL of Lipofectamine TM 3000 reagent with 250 μL of Opti-MEM TM medium and mix well to obtain Solution I;

[0155] Step 3: Use Opti-MEM TM Dilute DNA with the medium to prepare a DNA premix, 250 μL of Opti-MEM TM Add 2.5 μg of the plasmid carrying the aCD8 fusion protein and the neomycin resistance gene to the medium, and then add P3000 TM reagent (2 μL / μg DNA), and mix well to form Solution II;

[0156] Step 4: Mix Solutions I and II well, transfer the DNA-lipid complex to the wells of a 6-well plate after incubating at room temperature for 15 min, and culture the transfected cells in an incubator routinely after transfection.

[0157] Step 5: Change the medium 24 h after plasmid transfection. Passage the cells 1:10 into a 24-well plate 48 h later, and passage the untransfected cells at the same density as a control at the same time; after the cells adhere to the wall, add G418, and change the medium in a timely manner according to the cell death situation and the nutritional status of the medium; when all the cells in the control group die and there are living cells in the drug-administered group, obtain the preliminary resistant clones.

[0158] Step 6: Use the flow cytometer Beckman moflo Astrios EQ to sort the cells for the resistant monoclonal stable transfected cell line, collect the positive population cells into a 96-well plate for monoclonal culture, and freeze them in liquid nitrogen after culture and amplification.

[0159] Example 8 Preparation of 8GM Cell Membrane Particles

[0160] Step 1: Collect CHO-K1 cells cultured in a 15-cm culture dish, wash them twice with PBS (800 rpm / min, 4 °C, centrifuge for 5 min), and collect the cells.

[0161] Steps 2-7 are the same as those in Example 3 above.

[0162] Example 9 Preparation of CHO-K1 Cell Membrane Particles

[0163] Step 1: Collect CHO-K1 cells cultured in a 15-cm culture dish, wash them twice with PBS (800 rpm / min, 4 °C, centrifuge for 5 min), and collect the cells.

[0164] Steps 2-7 are the same as those in Example 3 above.

[0165] Effect Example

[0166] (1) Binding of 8ILGM Cells and CD8 Protein

[0167] Test Object: 8ILGM cells constructed according to Example 1

[0168] Test equipment: Laser confocal microscope

[0169] Test procedure: The 8ILGM cells constructed in Example 1 were seeded on a 3.5-cm confocal cell culture dish at 200,000 cells / dish. Block with 2% BSA and incubate on ice for 15 min. Incubate the above cells with CD8 protein with an Fc tag on ice for 30 min, and wash twice with PBS. Incubate with APC-labeled anti-Fc IgG on ice for 30 min, and wash twice with PBS. Incubate with cell membrane dye DiI and nuclear dye DAPI for 20 min and 10 min respectively, and wash twice with PBS. Place the confocal culture dish on the confocal microscope for imaging.

[0170] Test results: As Figure 6 shown, the details are as follows:

[0171] As Figure 6 shown, the 8ILGM cells constructed in Example 1 had green fluorescence, indicating the successful expression of eGFP protein. The red fluorescence of APC was co-localized with the fluorescence of cell membrane dye DiI, indicating the successful expression of aCD8-IL2 fusion protein on the cell membrane. At the same time, the conformation of the csFv region of aCD8 antibody was accurately expressed and could interact with CD8 protein.

[0172] (2) Binding of ILGM cells to CD25 protein

[0173] Test subject: ILGM cells constructed according to Example 5

[0174] Test equipment: Laser confocal microscope

[0175] Test procedure: The ILGM cells constructed in Example 5 were seeded on a 3.5-cm confocal cell culture dish at 200,000 cells / dish. Block with 2% BSA and incubate on ice for 15 min. Incubate the above cells with CD25 protein on ice for 30 min, and wash twice with PBS. Incubate with APC-labeled anti-Fc IgG on ice for 30 min, and wash twice with PBS. Incubate with cell membrane dye DiI and nuclear dye DAPI for 20 min and 10 min respectively, and wash twice with PBS. Place the confocal culture dish on the confocal microscope for imaging.

[0176] Test results: As Figure 7 shown, the details are as follows:

[0177] As Figure 7 shown, the ILGM cells constructed in Example 5 had green fluorescence, indicating the successful expression of eGFP protein. The red fluorescence of APC was co-localized with the fluorescence of cell membrane dye DiI, indicating the successful expression of IL2 protein on the cell membrane. At the same time, the conformation of IL2 protein was accurately expressed and could interact with CD25 protein.

[0178] (3) Binding of 8GM cells and CD8 protein

[0179] Test subject: 8GM cells constructed according to Example 7

[0180] Test equipment: Laser confocal microscope

[0181] Test procedure: The 8GM cells constructed in Example 7 were seeded on a 3.5 cm confocal cell culture dish, 200,000 cells / dish. Block with 2% BSA and incubate on ice for 15 min. Incubate the above cells with CD8 protein with an Fc tag on ice for 30 min, and wash twice with PBS. Incubate with APC-labeled anti-Fc IgG on ice for 30 min, and wash twice with PBS. Incubate with the nuclear dye DAPI for 20 min, 10 min, and wash twice with PBS. Place the confocal culture dish on the confocal microscope for imaging.

[0182] Test results: As Figure 8 shown, the details are as follows:

[0183] As Figure 8 shown, the 8GM cells constructed in Example 7 have green fluorescence, indicating successful expression of the eGFP protein. The red fluorescence of APC indicates that the conformation of the csFv region of the fusion protein aCD8 antibody on the 8GM cells is accurately expressed and can interact with the CD8 protein.

[0184] (4) Measurement of the hydrated particle size and Zeta potential of CHO-K1, ILGM, 8ILGM, and 8GM cell membrane particles

[0185] Test subject: 8ILGM cell membrane particles, ILGM cell membrane particles, 8GM cell membrane particles, and CHO-K1 cell membrane particles prepared according to Example 3, Example 6, Example 8, and Example 9;

[0186] Test equipment: Litesizer 500 laser particle size analyzer;

[0187] Test results: As Figure 9 shown, the details are as follows:

[0188] As Figure 9 shown, the cell membrane particles prepared in Example 3, Example 6, Example 8, and Example 9 are uniform in size, with an average hydrated particle size in the range of 180 - 210 nm, maintaining the negative charge of the cell membrane, and the Zeta potential in the range of -10 mV to -26 mV.

[0189] (5) Microscopic morphology test of CHO-K1 cell membrane particles

[0190] Test subject: CHO-K1 cell membrane particles prepared according to the protocol of Example 9;

[0191] Test equipment: Field emission scanning electron microscope (Thermo PEI, Nova Nano 450), cryo-transmission electron microscope (Thermo PEI, Talos F200C 200kv), 120kV cryo-transmission electron microscope (Thermo Scientific TalosL120C);

[0192] Test results: As Figure 10 shown, the details are as follows:

[0193] As Figure 10 shown, the cell membrane particles prepared according to the scheme of Example 9 are mainly single-layer membrane particles. The transmission electron microscope images show bowl-shaped nanoparticles with double-sided concavity and uniform particle size. Figure 10 Part a of Figure 10 is the scanning electron microscope image of the membrane particles, Figure 10 part b of

[0194] (6) Tests on the killing ability of 8ILGM, ILGM, 8GM, and CHO-K1 cell membrane particles against CD8 + T cells

[0195] Test subjects: 8ILGM cell membrane particles, ILGM cell membrane particles, 8GM cell membrane particles, and CHO-K1 cell membrane particles prepared according to Example 3, Example 6, Example 8, and Example 9;

[0196] Test equipment: Flow cytometer (BD, FACSCelesta), multifunctional microplate reader (Biotek, SynergyH1);

[0197] Test content 1: Collect single-cell suspensions of OT-1 mouse spleens, stimulate with OVA antigen, IL2, and IL7 factors for 3 days to activate OVA antigen-specific CD8 + T cells, and use a mouse CD8 + T cell isolation kit to isolate OT-1 CD8 + T cells in the spleens of OT-1 mice. Incubate OVA-specific CD8 + T cells with OVA-B16 tumor cells and different cell membrane particles for 48h, collect all cells, and label CD8 with anti-mouse Brilliant Violet 650 TM CD45 antibody +T cells, APC-Annexin V / 7-AAD kit was used to label cell death, and flow cytometry was used to detect and evaluate the killing of OVA-B16 cells by OT-1 CD8 + T cells in the presence of different cell membrane particles.

[0198] Test content 2: OVA-specific CD8 + T cells were co-incubated with OVA-B16 tumor cells expressing firefly luciferase (Luci-OVA-B16) and different cell membrane particles for 48 h. The supernatant medium was removed, and potassium luciferin solution was added. The bioluminescence of the cells was immediately detected using a microplate reader to evaluate the killing of Luci-OVA-B16 cells by OT-1 CD8 + T cells in the presence of different cell membrane particles.

[0199] Test results: As shown in part a of Figure 11 , the 8ILGM group could reduce the number of dead OVA-B16 cells and increase the proportion of live OVA-B16 cells; as shown in part b of Figure 11 , compared with the OT-1 CD8 + T cell group, the 8ILGM and 8GM groups could reduce the death of OVA-B16 tumor cells, indicating that the addition of 8ILGM and 8GM cell membrane particles could inhibit the killing ability of OT-1 CD8 + T cells against OVA-B16 cells.

[0200] (5) Diabetes treatment tests of 8ILGM, ILGM, 8GM, and CHO-K1 cell membrane particles

[0201] Test subjects: Three kinds of cell membrane particles prepared according to the schemes of Example 3, Example 6, Example 8, and Example 9.

[0202] Test content: Female NOD mice were used as a spontaneous diabetes animal model. When the blood glucose level of NOD mice was higher than 250 mg / dL for two consecutive days, they were regarded as newly diagnosed diabetic mice. The newly diagnosed diabetic mice were randomly divided into 3 groups, denoted as groups I-III. Group I was the CHO-K1 group (n = 8), group II was the ILGM group (n = 9), and group III was the 8ILGM group (n = 9). The three groups of newly diagnosed diabetic mice were injected with CHO-K1 cell membrane particles, ILGM cell membrane particles, and 8ILGM cell membrane particles via the tail vein, respectively, at a dose of 30 mg / kg (calculated according to the amount of membrane protein). Injection was performed once every 2 days, and treatment was stopped on the 40th day. During the treatment period, the blood glucose level and body weight of the mice were monitored using a Roche blood glucose meter and a weighing scale. During the treatment process, if the body weight of the mice decreased by 15%-20%, the mice were immediately euthanized. After the treatment was completed, the mice were euthanized for further analysis.

[0203] Test results: As shown in part a of Figure 12 , compared with control group I, the ILGM cell membrane particles and 8ILGM cell membrane particles in groups II and III can effectively reverse the blood glucose level of newly diagnosed diabetic NOD mice and restore it to the normal range. The treatment effect of group III is better than that of group II. Within 40 days of treatment, in group III, the blood glucose of 7 out of 9 NOD mice returned to the normal level; in group II, the blood glucose of 5 out of 9 NOD mice returned to the normal level; in group I, the blood glucose of 8 newly diagnosed diabetic NOD mice was higher than 250 mg / dL. As shown in Figure 13 the diabetes incidence statistics, 8ILGM cell membrane particles can significantly reverse newly diagnosed diabetes, and the lowest incidence is about 78%.

[0204] As shown in Figure 12 part b of, the tail vein treatment of cell membrane particles does not cause a significant decrease in the body weight of mice, indicating that the treatment with cell membrane particles does not cause obvious biological toxicity. However, the progression of diabetes will cause a decrease in the body weight of mice. In the experiment, when the body weight of diabetic mice decreased by 15%-20%, the mice were immediately euthanized.

[0205] (6) Tissue immunofluorescence assay

[0206] Test subjects: The pancreatic tissue and spleen tissue of mice after the above treatment;

[0207] Test content: After 40 days of treatment of female NOD mice in groups I-III above, the female NOD mice and untreated NOD mice after onset were euthanized, and the pancreas and spleen of the mice were collected and fixed with paraformaldehyde for 24 hours to be embedded in paraffin. The pancreas and spleen were cut with a microtome and mounted on glass slides. The sections were dehydrated with xylene, absolute ethanol, 95% ethanol, 85% ethanol, 75% ethanol, incubated in 3% H2O2 for 10-20 min, heat repaired with EDTA for 15-20 min, and sealed in 3% BSA with 0.2% Triton-X 100 for 30 minutes. Then stained with different primary antibodies: glucagon antibody (Abcam, CAT. Ab92517), insulin antibody (Abcam, CAT. Ab6995), anti-Foxp3 antibody (Abcam, CAT. Ab253297) were incubated overnight at 4°C according to the manufacturer's instructions. With fluorescently labeled secondary antibody Anti-rabbit IgG(H+L), F(ab')2 fragment (Alexa 488 Conjugate) (CST, CAT. No. 4412); Anti-rat IgG(H+L), (Alexa After incubation with 647Conjugate (CST, catalog number 4418) for 30 min, the sections were stained with DAPI and analyzed using a digital slide scanner (Olympus VS200).

[0208] Test results: As Figure 14 shown in the immunofluorescence staining of the pancreas, where the green fluorescence represents glucagon after being stained and labeled, which also represents the islet part, and the red fluorescence staining area represents insulin after being stained and labeled. The red fluorescence staining area in the untreated group and group I after the onset of diabetes in NOD mice was significantly reduced, indicating that the islet part had developed lesions at this time and very little insulin was produced. The red fluorescence staining area in group II and group III was significantly larger, indicating that the islets could still produce sufficient insulin normally at this time. It can be seen that the treatment of group II and group III can relieve islet lesions and reverse the development of type I diabetes.

[0209] As Figure 15 shown in the immunofluorescence staining of the pancreas, where the green fluorescence represents glucagon after being stained and labeled, which also represents the islet part, and the red fluorescence staining area represents the Foxp3 molecule after being stained and labeled, which also represents Treg cells. The red fluorescence staining area in the untreated group and group I after the onset of diabetes in NOD mice was very small, almost none, indicating that the infiltration of Treg cells in the islet part was extremely low at this time. The red fluorescence staining area in group II and group III was significantly larger, indicating an increase in the infiltration of Treg cells in the islets. It can be seen that in group II and group III, the ILGM cell membrane particles and 8ILGM cell membrane particle treatment particles promote the infiltration and expansion of Treg cells in the pancreas, which is helpful for the treatment of diabetes.

[0210] As Figure 16 shown in the immunofluorescence staining of the spleen, the red fluorescence staining area represents the Foxp3 molecule after being stained and labeled, which also represents Treg cells. The second row of pictures is an enlargement of the first row of pictures. Compared with the untreated group and group I, the red fluorescence staining area in group II and group III was significantly larger, indicating an increase in the number of Treg cells in the spleen. The ILGM cell membrane particles and 8ILGM cell membrane particle treatment particles promote the expansion of Treg cells, which is helpful for the treatment of diabetes.

[0211] (6) Detection of lymphocytes in peripheral blood

[0212] Test subjects: Peripheral blood cells of NOD mice 20 days after the above treatment;

[0213] Test content: After 20 days of treatment of the female NOD mice in groups I-III above, blood was obtained through the orbital venous plexus of the mice. Healthy female NOD mice with normal blood glucose were used as the control group, and blood was obtained through the orbital venous plexus of the mice. Peripheral blood cells were subjected to erythrocyte lysis, staining and other operations, and finally flow cytometry analysis was performed;

[0214] Test results: As Figure 17 shown, intravenous injection of CHO-K1, ILGM and 8ILGM cell membrane particles did not cause significant changes in T cells in peripheral blood, indicating that cell membrane particle treatment did not affect systemic T cell changes. GranB in pancreatic lymph nodes + CD8 + The infiltration diagram of T cells showed that treatment with 8ILGM cell membrane particles significantly reduced the infiltration ratio of GranB + CD8 + T cells, indicating that the effect of aCD8-IL2 was better than that of IL2.

[0215] (7) Hematoxylin-eosin staining (H&E staining) of tissues

[0216] Test content: After 40 days of treatment of the female NOD mice in groups I-III above, the mice were euthanized and the main organs, including the pancreas, heart, liver, spleen, lung and kidney, were collected, fixed with paraformaldehyde for 24 hours for embedding in paraffin, sectioned and then subjected to H&E staining. And analysis was performed using a digital slide scanner (Olympus VS200). Untreated healthy female NOD mice were used as the control group and the same operations were performed;

[0217] Test results: As Figure 18 shown, compared with the untreated group, there were no obvious changes in the pancreatic tissues of the mice treated in groups I-III. The islets in group I were significantly reduced or difficult to observe, and islets could still be observed in the pancreases of the mice in the untreated group, group II and group III. It shows that ILGM cell membrane particles and 8ILGM cell membrane particles are helpful for the retention of islets and the treatment of diabetes.

[0218] As Figure 19 shown, there were no obvious differences in the main organs (including the pancreas, heart, liver, spleen, lung and kidney) of the mice in the untreated group and groups I-III, indicating that CHO-K1 cell membrane particles, ILGM cell membrane particles and 8ILGM cell membrane particles have good biocompatibility and will not cause obvious organ damage.

[0219] The sequences involved in the present invention are as follows:

[0220] The nucleotide sequence of the signal peptide is as shown in SEQ ID NO:1:

[0221] ATGGGCTGGTCCTGCATCATTCTTTTCTTGGTCGCTACTGCAACTGGAGTTCACTCC;

[0222] The amino acid sequence is shown in SEQ ID NO:2:

[0223] MGWSCIILFLVATATGVHS;

[0224] The VH nucleotide sequence of the single-chain antibody targeting CD8 is shown in SEQ ID NO:3:

[0225] CAGGTGCAGCTGAAAGAAAGTGGCCCAGGCCTGGTCCAGCCATCCCAAACACTGTCTCTCACCTGTACTGTTTCTGGGTTTAGCCTCACCAGTAATTCTGTGCACTGGGTGAGGCAGCCTCCTGGGAAAGGTTTAGAGTGGATGGGAGGGATTTGGGGCGATGGAGACACCGACTACAACTCTGCTCTCAAGTCTCGGTTATCCATAAGTCGAGACACGTCCAAAAATCAAGTATTTCTGAAGATGAACTCACTGCAGACTGATGACACAGCTATTTACTTCTGCACACCCCTGATTGGCTCTTGGTATTTTGACTTCTGGGGACCAGGTACCATGGTGACTGCCAGTTCC;

[0226] The amino acid sequence is shown in SEQ ID NO:4:

[0227] QVQLKESGPGLVQPSQTLSLTCTVSGFSLTSNSVHWVRQPPGKGLEWMGGIWGDGDTDYNSALKSRLSISRDTSKNQVFLKMNSLQTDDTAIYFCTPLIGSWYFDFWGPGTMVTASS;

[0228] The nucleotide sequence of the single-chain antibody hinge Linker targeting CD8 is shown in SEQ ID NO:5:

[0229] GGGGGCGGTGGCAGTGGGGGCGGTGGCTCGGGAGGCGGGGGCTCT;

[0230] The amino acid sequence is shown in SEQ ID NO:6:

[0231] GGGGSGGGGSGGGGS;

[0232] The nucleotide sequence of the single-chain antibody light chain VL targeting CD8 is shown in SEQ ID NO:7:

[0233] GACATTGTCATGACACAATCACCCAGCTCATTGGCAGTGTCTGCAGGAGAAAGAGTCACTCTCAACTGCAAAGCGTCGCAGAATGTGAGAAACAACATAGCCTGGTACCAGCAGAAACCAGGACAATCGCCGAAACTTCTGATCTACTATGCCAGCTACAGGTATACTGGCGTCCCTGATCGTTTTACGGGAGATGGCTTTGGCACAGACTTCACCCTGGCCATCAATAGCGTACAAGCTGACGATGCAGCCTTTTACTACTGCCAGCGCATCTATAATTCCCCTTATACTTTTGGAGCTGGCACTAAGTTGGAGTTAATCCGT;

[0234] The amino acid sequence is shown in SEQ ID NO:8:

[0235] DIVMTQSPSSLAVSAGERVTLNCKASQNVRNNIAWYQQKPGQSPKLLIYYASYRYTGVPDRFTGDGFGTDFTLAINSVQADDAAFYYCQRIYNSPYTFGAGTKLELIR;

[0236] The nucleotide sequence of the extracellular granzyme B-responsive peptide is shown in SEQ ID NO:9:

[0237] GGGGGGGGGGGAAGCATTGAGTTTGATGGCGGAGGCGGCAGCATTGAATTTGATGGGGGTGGTGGCAGT;

[0238] The amino acid sequence is shown in SEQ ID NO:10:

[0239] GGGGSIEFDGGGGSIEFDGGGGS;

[0240] The nucleotide sequence of the human IL2 mutant protein is shown in SEQ ID NO:11:

[0241] GCGCCTACAAGCAGCTCTACAAAAAAGACGCAACTCCAGCTCGAGCACCTGCTTCTGGATCTGCAGATGATCTTGAATGGCATCAATAACTACAAAAACCCAAAACTAACCAGGATGCTGACCTTCAAGTTTTACATGCCCAAGAAGGCCACAGAGCTGAAACATCTTCAGTGCTTGGAAGAAGAGCTAAAGCCGTTGGAAGAGGTGCTGAACTTGGCCCAGAGCAAGAACTTCCATCTTCGGCCTCGAGATCTCATTAGTAGAATCAACGTCATAGTTCTGGAGCTCAAAGGAAGTGAGACCACTTTCATGTGTGAGTATGCTGATGAGACAGCCACGATTGTGGAGTTCCTGAATCGCTGGATCACCTTTTCTCAGTCAATCATAAGCACTCTGACA;

[0242] The amino acid sequence is shown in SEQ ID NO: 12:

[0243] APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISRINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT;

[0244] The nucleotide sequence of the IgG4 hinge region is shown in SEQ ID NO: 13:

[0245] GAGTCTAAATATGGCCCTCCCTGTCCACCATGCCCTGGACAGCCCAGAGAGCCTCAAGTCTACACCTTACCTCCTTCCCAAGAAGAGATGACAAAGAATCAGGTGTCCCTAACCTGCCTGGTGAAGGGCTTCTACCCTTCAGACATTGCTGTGGAGTGGGAGAGCAATGGCCAGCCAGAAAACAACTACAAGACAACACCGCCAGTTCTGGACAGTGATGGCTCTTTCTTCCTTTATTCCCGTCTCACTGTAGACAAGAGCCGCTGGCAGGAGGGCAATGTTTTTTCGTGTTCTGTGATGCATGAGGCTCTTCACAACCACTACACTCAGAAGTCCCTTTCTCTGAGTCTCGGGAAG;

[0246] The amino acid sequence is shown in SEQ ID NO:14:

[0247] ESKYGPPCPPCPGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK;

[0248] The nucleotide sequence of the CD28 transmembrane region is shown in SEQ ID NO:15:

[0249] TTCTGGGCCCTGGTCGTTGTAGCTGGCGTTCTCTTCTGCTATGGGCTACTAGTGACCGTGGCCCTCTGTGTCATCTGGACT;

[0250] The amino acid sequence is shown in SEQ ID NO:16:

[0251] FWALVVVAGVLFCYGLLVTVALCVIWT;

[0252] The nucleotide sequence of the intracellular EGFP fluorescent protein is shown in SEQ ID NO:17:

[0253] ATGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCACGGCATGGACGAGCTGTACAAGTAA;

[0254] The amino acid sequence is shown in SEQ ID NO: 18:

[0255] MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITHGMDELYK

[0256] The nucleotide sequence of the fusion protein aCD8-IL2 is shown in SEQ ID NO:19:

[0257]

[0258] The amino acid sequence is shown in SEQ ID NO: 20:

[0259] MGWSCIILFLVATATGVHSDYKDDDDKQVQLKESGPGLVQPSQTLSLTCTVSGFSLTSNSVHWVRQPPGKGLEWMGGIWGDGDTDYNSALKSRLSISRDTSKNQVFLKMNSLQTDDTAIYFCTPLIGSWYFDFWGPGTMVTASSGGGGSGGGGSGGGGSDIVMTQSPSSLAVSAGERVTLNCKASQNVRNNIAWYQQKPGQSPKLLIYYASYRYTGVPDRFTGDGFGTDFTLAINSVQADDAAFYYCQRIYNSPYTFGAGTKLELIRGGGGSIEFDGGGGSIEFDGGGGSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISRINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLTESKYGPPCPPCPGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKFWALVVVAGVLFCYGLLVTVALCVIWTGGGGSGGGGSGGGGSDIQHSGGRMSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITHGMDELYK

[0260] The nucleotide sequence of the IL-2 signal peptide is shown in SEQ ID NO: 21:

[0261] ATGTATAGGATGCAGCTGCTGAGTTGCATTGCATTGTCTCTGGCCTTGGTGACGAACAGC;

[0262] The amino acid sequence is as shown in SEQ ID NO: 22:

[0263] MYRMQLLSCIALSLALVTNS;

[0264]

[0265] The amino acid sequence is as shown in SEQ ID NO: 24:

[0266] MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISRINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLTESKYGPPCPPCPGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKFWALVVVAGVLFCYGLLVTVALCVIWTGGGGSGGGGSGGGGSDIQHSGGRMSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITHGMDELYK

[0267] The nucleotide sequence of the fusion protein 8GM is as shown in SEQ ID NO: 25:

[0268]

[0269] The amino acid sequence is shown in SEQ ID NO: 26:

[0270] MGWSCIILFLVATATGVHSDYKDDDDKQVQLKESGPGLVQPSQTLSLTCTVSGFSLTSNSVHWVRQPPGKGLEWMGGIWGDGDTDYNSALKSRLSISRDTSKNQVFLKMNSLQTDDTAIYFCTPLIGSWYFDFWGPGTMVTASSGGGGSGGGGSGGGGSDIVMTQSPSSLAVSAGERVTLNCKASQNVRNNIAWYQQKPGQSPKLLIYYASYRYTGVPDRFTGDGFGTDFTLAINSVQADDAAFYYCQRIYNSPYTFGAGTKLELIRGGGGSIEFDGGGGSIEFDGGGGSESKYGPPCPPCPGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKFWALVVVAGVLFCYGLLVTVALCVIWTGGGGSGGGGSGGGGSDIQHSGGRMSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITHGMDELYK

[0271] Although the specific embodiments of the present invention have been described above, those skilled in the art should understand that this is only for illustration purposes. The protection scope of the present invention is defined by the appended claims. Without departing from the principles and essence of the present invention, those skilled in the art can make various changes or modifications to these embodiments, but these changes and modifications all fall within the protection scope of the present invention.

Claims

1. A fusion protein, characterized in that The fusion protein comprises a CD8 antibody and an IL-2 mutant, wherein the CD8 antibody and the IL-2 mutant are connected via a cleavable connection component, and the IL-2 mutant can preferentially stimulate Treg proliferation.

2. The fusion protein according to claim 1, characterized in that The fusion protein further comprises a transmembrane region; preferably, the transmembrane region is a CD28 transmembrane region or a CD8 receptor transmembrane region; more preferably, the CD28 transmembrane region has an amino acid sequence as shown in SEQ ID NO: 16; And / or, the fusion protein further comprises a signal peptide; preferably, the signal peptide is a CD8 receptor expression signal peptide, a platelet-derived growth factor receptor PDGFR signal peptide or an antibody secretion signal peptide; more preferably, the signal peptide has an amino acid sequence as shown in SEQ ID NO:

2.

3. The fusion protein according to claim 2, characterized in that The IL-2 mutant is connected to the transmembrane region via a hinge region; preferably, the hinge region is an IgG4 hinge region, and the hinge region has an amino acid sequence as shown in SEQ ID NO:

14.

4. The fusion protein according to any one of claims 1 to 3, characterized in that The CD8 antibody is a single-chain antibody; preferably, the CD8 antibody comprises: a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 4, and a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 8; And / or, the cleavable linking component is an extracellular granzyme B response peptide; preferably, the cleavable linking component has an amino acid sequence as shown in SEQ ID NO:

10.

5. The fusion protein according to claim 4, characterized in that The heavy chain variable region and the light chain variable region are connected by a linker; Preferably, the linker has an amino acid sequence as shown in SEQ ID NO:

6.

6. A polynucleotide, characterized in that The polynucleotide encodes the fusion protein according to any one of claims 1 to 5.

7. An expression vector, characterized in that: The expression vector comprises the polynucleotide according to claim 6.

8. A transformant, characterized in that The transformant comprises the expression vector according to claim 7.

9. A cell membrane particle, characterized in that: The cell membrane particles are obtained by collecting the transformant according to claim 8 after being crushed; preferably, they are prepared by the following steps: 1) Collect cells; 2) dispersing the collected cells in a buffer to obtain a cell suspension; 3) The cell suspension is disrupted using a cell ultrasonic disruptor; 4) centrifuging the cell suspension after ultrasound to obtain a supernatant; the preferred centrifugation conditions are 3000-5000 rpm / min, 5-20 min; 5) Centrifuging the collected supernatant to collect the cell membrane; 6) After the cell membrane precipitate is resuspended in a buffer solution, the cell membrane is collected by centrifugation; the preferred centrifugation condition is 15000-20000 rpm / min, 30-120 min; 7) After the cell membrane precipitate is resuspended in a buffer solution, it is squeezed through polycarbonate membranes of different pore sizes multiple times to obtain cell membrane particles of uniform size.

10. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the fusion protein according to any one of claims 1 to 5 or the cell membrane particles according to claim 9.

11. A kit, characterized in that: The kit comprises the fusion protein according to any one of claims 1 to 5, the polynucleotide according to claim 6 or the cell membrane particles according to claim 9.

12. A method for preparing a fusion protein, characterized in that: The method comprises the step of culturing the transformant according to claim 8.

13. Use of the fusion protein according to any one of claims 1 to 5 in the preparation of a medicament for treating and / or preventing an autoimmune disease, wherein the autoimmune disease is preferably diabetes, more preferably type 1 diabetes.

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