Immune cell-derived extracellular vesicle and application thereof

Abstract

The invention belongs to the field of immunology, and relates to an immune cell-derived extracellular vesicle and application thereof, the targeted extracellular vesicle is generated by immune cells, and the membrane of the targeted extracellular vesicle comprises at least one protein with a membrane fusion function and at least one targeting part. A nucleic acid sequence containing a targeting antibody and a protein with a membrane fusion function is used as an exogenous gene, immune cells are used as production cells to prepare the EV, and the EV which expresses the targeting antibody on the membrane surface and is high in membrane fusion efficiency is prepared. The EV internally contains granzyme and other toxic molecules, the targeting molecules are combined with tumor cell surface antigens, membrane fusion protein promotes the membrane fusion rate, the EV can directly deliver the granzyme and other toxic molecules to tumor cell cytoplasm, and the toxic molecules play an excellent tumor killing effect. The result shows that the apoptosis rate of the immune EV in the experimental group for remarkably inducing the apoptosis of the SK-BK-3 cells in vitro can reach 85-95%.

Description

Immune cell-derived extracellular vesicle and its use Technical Field

[0001] The present invention relates to the field of disease treatment drugs, and more particularly, to targeted extracellular vesicles derived from immune cells and uses thereof. Background Art

[0002] Cell-derived extracellular vesicles (EVs) are secreted by various cell types and carry important biomolecules. Based on their biogenesis, EVs can be divided into three subgroups: exosomes (30-150 nm in diameter), microvesicles (150-1000 nm), and apoptotic bodies (50-2000 nm). Recent evidence suggests that membrane vesicles may play a crucial role as mediators of intercellular communication. Exosomes have received considerable attention and have been extensively characterized in recent years.

[0003] EVs not only have low immunogenicity but can also fuse with receptor cell membranes, bypassing lysosomal engulfment and delivering drugs directly into the cytoplasm, significantly improving drug delivery efficiency. These advantages have led to their widespread application in delivering siRNA, miRNA, and small molecule drugs. These molecules can be loaded into EVs through various methods, including transfection, incubation, sonication, freeze-thaw cycles, and electroporation.

[0004] In addition to their potential as drug delivery molecules, immune cell-derived EVs also offer advantages in the treatment of tumor cells. Immune cell-derived EVs can modulate the tumor microenvironment by presenting antigens, regulating activation, or promoting immune cell proliferation. Furthermore, CTL-derived EVs can induce tumor cell apoptosis through the release of cytotoxic molecules. Recent studies have shown that EVs released by CAR-T cells can also effectively inhibit tumor growth. Furthermore, research and analysis have shown that CAR exosomes do not express PD1, so recombinant PD-L1 therapy does not weaken their anti-tumor effects. Furthermore, compared to CAR-T therapy, the use of CAR exosomes is relatively safe. Therefore, the widespread application of these immune cell-derived EVs is expected to become an effective strategy for treating cancer.

[0005] Summary of the Invention

[0006] In a first aspect, the present invention provides a targeted extracellular vesicle derived from an immune cell, which is produced by an immune cell and contains at least one protein with membrane fusion function and at least one targeting moiety on the membrane of the targeted extracellular vesicle. The targeting moiety can target target molecules of tumor cells and tumor stromal cells. The targeting moiety binds to the target molecule (the relevant tumor antigen carried on the tumor cell), and the targeted extracellular vesicle fuses with the tumor cell carrying the relevant tumor antigen. The protein with membrane fusion function promotes the fusion of the extracellular vesicle membrane and the tumor cell membrane. Tumor cell killing is carried out by releasing toxins in the targeted extracellular vesicle, thereby promoting tumor cell apoptosis.

[0007] As a preferred embodiment, the genes of the at least one protein having membrane fusion function and the at least one targeting portion are constructed into a vector and then transferred into immune cells.

[0008] As a preferred embodiment, the protein with membrane fusion function is derived from a viral protein, and the targeting moiety includes a receptor ligand, an antibody or a functional fragment thereof, a single domain antibody VHH or a DARPin.

[0009] As a preferred embodiment, the antibody is a single domain antibody.

[0010] As a preferred embodiment, the antibody is a VHH antibody.

[0011] As a preferred embodiment, the antibody is a humanized antibody.

[0012] As a preferred embodiment, the protein having membrane fusion function is a modified protein of a virus that can achieve membrane fusion function.

[0013] As a preferred embodiment, the amino acid sequence of the protein having membrane fusion function is shown in SEQ ID NO: 1.

[0014] As a preferred embodiment, the immune cells include T cells, CAR-T cells, B cells, natural killer cells, dendritic cells, macrophages or neutrophils.

[0015] As a preferred embodiment, the targeted extracellular vesicles (targeted EVs) are exosomes, microvesicles, extracellular granules, and apoptotic bodies.

[0016] As a preferred embodiment, the at least one target cell comprises a mammalian cell.

[0017] As a preferred embodiment, the mammalian cells are human cells.

[0018] As a preferred embodiment, the at least one target cell is a tumor cell, a tumor stromal cell or an immune cell.

[0019] As a preferred embodiment, the at least one target molecule is a cell surface marker or a cell surface receptor.

[0020] As a preferred embodiment, the cell surface marker or cell surface receptor is a tumor-associated antigen.

[0021] As a preferred embodiment, the targeting portion targets tumor-associated antigens, which include but are not limited to Her2, DEC205, CD33, CD30, CD22, CD79b, CLEC9A, Nectin-4, TROP2, TF, CEACAM5, CTLA4, CD3, CD7, CD11c, CD19, CD20, CD22, CD40, CD44, CD206, EGFR, EGFRvIII, fibroblast activation protein (FAP), CA9, MMP-2, PD-L1, SIRPa, Trop2, GPC1, GPC3, cMET, BCMA, VEGFR or Cladin18.2.

[0022] As a preferred embodiment, the targeting portion is a single domain antibody, and the amino acid sequence is shown in SEQ ID NO: 3.

[0023] As a preferred embodiment, the at least one targeting moiety comprises an anti-Her2 VHH antibody, an anti-DEC205 VHH antibody, an anti-CLEC9A VHH antibody, an anti-CEACAM5 VHH antibody, an anti-CTLA4 VHH antibody, an anti-CD3 VHH antibody, an anti-CD7 VHH antibody, an anti-CD11c VHH antibody, an anti-CD19 VHH antibody, an anti-CD20 VHH antibody, an anti-CD22 VHH antibody, an anti-CD40 VHH antibody, an anti-CD44 VHH antibody, an anti-CD206 VHH antibody, and an anti-CD40 VHH antibody. H antibody, anti-EGFR VHH antibody, anti-EGFRvIII VHH antibody, anti-fibroblast activation protein (FAP) VHH antibody, anti-CA9 VHH antibody, anti-MMP-2 VHH antibody, anti-PD-L1 VHH antibody, anti-SIRPa VHH antibody, anti-Trop2 VHH antibody, anti-GPC1 VHH antibody, anti-GPC3 VHH antibody, anti-cMET VHH antibody, anti-BCMA VHH antibody, anti-VEGFR VHH antibody, anti-Cladin18.2 VHH antibody.

[0024] In a second aspect, the present invention provides a composition comprising any one of the aforementioned immune cell-derived targeted extracellular vesicles; and a pharmaceutically acceptable excipient, diluent, or carrier.

[0025] The third aspect of the present invention provides a method for preparing any of the aforementioned immune cell-derived targeted extracellular vesicles, comprising the following steps:

[0026] (1) Constructing a recombinant expression vector containing an exogenous target protein gene fragment;

[0027] (2) transfecting the recombinant expression vector into immune cells for expression, culturing the transfected immune cells, and stimulating the immune cells to secrete targeted extracellular vesicles carrying the exogenous target protein.

[0028] As a preferred embodiment, the cells are T cells, NK cells and CAR-T cells.

[0029] A fourth aspect of the present invention provides use of any of the aforementioned targeted extracellular vesicles and the aforementioned composition in the preparation of anticancer drugs.

[0030] A fifth aspect of the present invention provides use of any of the aforementioned targeted extracellular vesicles and the aforementioned composition in cancer treatment. Beneficial effects

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] 1) The present invention uses nucleic acid sequences containing targeting molecules and membrane fusion proteins as exogenous genes and immune cells as producer cells to produce EVs. These EVs express the targeting molecules on their membrane surfaces and fuse efficiently with cell membranes. The EVs also contain toxic molecules such as granzymes. The targeting molecules bind to tumor cell surface antigens, and the membrane fusion proteins promote membrane fusion. This allows the EVs in the experimental group to deliver toxic molecules such as granzymes directly to the tumor cell cytoplasm. Results showed that the EVs in the experimental group significantly induced apoptosis in SK-BK-3 cells in vitro, with an apoptosis rate of 85-95%. In control group 1, EVs immunized with EVs that did not express modified Sindbis virus on their membranes induced an apoptosis rate of 25%-35% in SK-BK-3 cells in vitro, while EVs in control group 2 (which did not contain modified viral proteins or anti-Her2 VHH) induced an apoptosis rate of 8%-13%. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] In order to more clearly illustrate the technical solution of the present application, the following is a brief introduction to the drawings required for use in the embodiments. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0034] Figure 1 is a schematic diagram of the extracellular vesicles derived from immune cells of the present invention; wherein: 1. Anti-Her2 VHH; 2. Polypeptide A; 3. EV. DETAILED DESCRIPTION

[0035] The present invention is further described in detail below with reference to the embodiments so that those skilled in the art can implement the invention with reference to the description.

[0036] Targeting extracellular vesicles

[0037] In a first aspect, the present invention provides a targeted extracellular vesicle (EV) derived from an immune cell, which is produced by an immune cell, and the targeted extracellular vesicle membrane contains at least one protein with membrane fusion function and at least one targeting portion. The targeting portion can target target molecules of tumor cells and tumor stromal cells. The targeting portion binds to at least one target molecule (the relevant tumor antigen carried on the tumor cell), and the targeted extracellular vesicle fuses with the tumor cell carrying the relevant tumor antigen, and the protein with membrane fusion function promotes the fusion of the extracellular vesicle membrane and the tumor cell membrane. Tumor cell killing is carried out by releasing toxins in the targeted extracellular vesicle, promoting tumor cell apoptosis.

[0038] In one embodiment, the genes of the at least one protein having membrane fusion function and the at least one targeting portion are constructed into a vector and then transferred into immune cells.

[0039] As a preferred embodiment, the protein with membrane fusion function is derived from a viral protein, and the targeting moiety includes a receptor ligand, an antibody or a functional fragment thereof, a single domain antibody VHH or a DARPin.

[0040] In one embodiment, the EV is an exosome, a microvesicle, an extracellular granule, or an apoptotic body.

[0041] In one embodiment, the targeting moiety is a single domain antibody.

[0042] In one embodiment, wherein the targeting moiety is a VHH antibody.

[0043] In one embodiment, the targeting moiety is a humanized antibody.

[0044] In one embodiment, the amino acid sequence of the protein having membrane fusion function is shown in SEQ ID NO: 1.

[0045] In one embodiment, the immune cells include T cells, CAR-T cells, B cells, natural killer cells, dendritic cells, macrophages or neutrophils.

[0046] In one embodiment, the targeted extracellular vesicles (targeted EVs) are exosomes, microvesicles, ectosomes, or apoptotic bodies.

[0047] In one embodiment, the at least one target cell comprises a mammalian cell.

[0048] In one embodiment, the mammalian cell is a human cell.

[0049] In one embodiment, the at least one target cell is a tumor cell, a tumor stromal cell, or an immune cell.

[0050] In one embodiment, the at least one target molecule is a cell surface marker or a cell surface receptor.

[0051] In one embodiment, the cell surface marker or cell surface receptor is a tumor associated antigen.

[0052] In one embodiment, the targeting moiety targets a tumor-associated antigen, which includes but is not limited to Her2, DEC205, CD33, CD30, CD22, CD79b, CLEC9A, Nectin-4, TROP2, TF, CEACAM5, CTLA4, CD3, CD7, CD11c, CD19, CD20, CD22, CD40, CD44, CD206, EGFR, EGFRvIII, fibroblast activation protein (FAP), CA9, MMP-2, PD-L1, SIRPa, Trop2, GPC1, GPC3, cMET, BCMA, VEGFR or Cladin18.2.

[0053] In one embodiment, the targeting moiety is a single domain antibody, and the amino acid sequence is shown in SEQ ID NO: 3.

[0054] In one embodiment, the at least one targeting moiety comprises an anti-Her2 VHH antibody, an anti-DEC205 VHH antibody, an anti-CLEC9A VHH antibody, an anti-CEACAM5 VHH antibody, an anti-CTLA4 VHH antibody, an anti-CD3 VHH antibody, an anti-CD7 VHH antibody, an anti-CD11c VHH antibody, an anti-CD19 VHH antibody, an anti-CD20 VHH antibody, an anti-CD22 VHH antibody, an anti-CD40 VHH antibody, an anti-CD44 VHH antibody, an anti-CD206 VHH antibody, an anti-CD30 VHH antibody, an anti-CD31 VHH antibody, an anti-CD32 VHH antibody, an anti-CD33 VHH antibody, an anti-CD34 VHH antibody, an anti-CD36 VHH antibody, an anti-CD37 VHH antibody, an anti-CD38 VHH antibody, an anti-CD39 VHH antibody, an anti-CD40 VHH antibody, an anti-CD44 VHH antibody, an anti-CD37 VHH antibody, an anti-CD38 VHH antibody, an anti-CD39 VHH antibody, an anti-CD39 VHH antibody, an anti-CD31 VHH antibody, an anti-CD32 VHH antibody, an anti-CD33 VHH antibody, an anti-CD34 VHH antibody, an anti-CD36 VHH antibody, an anti-CD37 VHH antibody, an anti-CD38 VHH antibody, an anti-CD39 ... Antibodies, anti-EGFR VHH antibodies, anti-EGFRvIII VHH antibodies, anti-fibroblast activation protein (FAP) VHH antibodies, anti-CA9 VHH antibodies, anti-MMP-2 VHH antibodies, anti-PD-L1 VHH antibodies, anti-SIRPa VHH antibodies, anti-Trop2 VHH antibodies, anti-GPC1 VHH antibodies, anti-GPC3 VHH antibodies, anti-cMET VHH antibodies, anti-BCMA VHH antibodies, anti-VEGFR VHH antibodies, anti-Cladin18.2 VHH antibodies.

[0055] Composition

[0056] In a second aspect, the present invention provides a composition comprising any one of the aforementioned immune cell-derived targeted extracellular vesicles; and a pharmaceutically acceptable excipient, diluent, or carrier.

[0057] Preparation method of targeted extracellular vesicles

[0058] The third aspect of the present invention provides a method for preparing any of the aforementioned immune cell-derived targeted extracellular vesicles, comprising the following steps:

[0059] (1) Constructing a recombinant expression vector containing an exogenous target protein gene fragment;

[0060] (2) transfecting the recombinant expression vector into immune cells for expression, culturing the transfected immune cells, stimulating the immune cells to secrete targeted extracellular vesicles carrying the exogenous target protein, and then collecting and purifying the targeted extracellular vesicles.

[0061] Methods for collecting and purifying targeted extracellular vesicles include ultracentrifugation, filtration centrifugation, density gradient centrifugation, immunomagnetic bead method, PS affinity method, and chromatography. Preferably, the collection method uses ultracentrifugation combined with immunomagnetic bead method for purification.

[0062] In one embodiment, the cells are T cells, NK cells and CAR-T cells.

[0063] A fourth aspect of the present invention provides use of any of the aforementioned targeted extracellular vesicles and the aforementioned composition in the preparation of anticancer drugs.

[0064] A fifth aspect of the present invention provides use of any of the aforementioned targeted extracellular vesicles and the aforementioned composition in cancer treatment.

[0065] Definitions and Examples

[0066] The following definitions are provided to facilitate understanding of the terms used herein.

[0067] "Extracellular vesicles" (EVs): are cell-derived membrane structures, including exosomes, microvesicles, virus-like particles, large vesicles, oncosomes, nanovesicles and apoptotic bodies. These extracellular vesicles are generally classified based on their size, specific markers, cell origin and biogenesis process. Exosomes are 30-160 nm vesicles of endosomal origin released from cells when the multivesicular body (MVB) membrane fuses with the plasma membrane. Exosomes are produced by a variety of cell types, and their release can be induced by a variety of stimuli, including stress, hypoxia, cell death and viral infection. Classical microvesicles (also called microparticles) are 100 nm-1 μm vesicles released from cells by shedding of the plasma membrane. Cancer cells can also secrete larger microvesicles (>1 μm) called oncosomes, which differ from classical microvesicles only in size. Like exosomes, microvesicle release can be induced by stress and viral infection, and their contents are heterogeneous. Apoptotic bodies are large EVs that are released from apoptotic cells by blebbing and range in size from 200 nm to 5 μm. These phosphatidylserine and annexin V-coated EVs contain cytoplasmic contents from dying cells. Traditionally, EVs that precipitate at 100,000 g are referred to as exosomes, but in fact, this precipitate contains a combination of microvesicles and exosomes. Although their biogenesis pathways are different, exosomes and microvesicles have many similarities and are difficult to distinguish from each other once released from cells. Recently, the International Society for Extracellular Vesicles recommended that the term small EV (sEV) should be used for particles smaller than 200 nm, while the term large EV (lEV) should be used for particles larger than 200 nm. Among EVs, there is a type of nanosized vesicle (cell-derived nanovesicles, CNV) that resembles nEVs in appearance and structure.

[0068] "Targeted EV" is used synonymously herein to refer to an EV comprising a recombinant polypeptide as defined herein and thus having an engineered affinity for a target molecule (provided by the targeting moiety).

[0069] "Targeting moiety" means a molecule that is capable of binding to a target molecule with sufficient affinity and specificity to enable targeting of EVs to target cells expressing the target molecule. Non-limiting examples of targeting moieties include antibodies, functional fragments thereof, engineered fragments thereof, ligands (which target receptors), designed ankyrin repeat proteins (DARPins) (which bind to target proteins), and domains that mediate specific protein-protein interactions.

[0070] In one embodiment, tumor-associated antigens include but are not limited to the following: Her2, DEC205, CLEC9A, CEACAM5, CTLA4, CD3, CD7, CD11c, CD19, CD20, CD22, CD40, CD44, CD206, EGFR, EGFRvIII, fibroblast activation protein (FAP), CA9, MMP-2, PD-L1, SIRPa, Trop2, GPC1, GPC3, cMET, BCMA, VEGFR, Cladin18.2.

[0071] Table 1: Exemplary targeting moieties

[0072] "Single-domain antibodies" (sdAbs), also known as nanobodies, like VHHs, are antibody fragments composed of a single monomeric variable antibody domain. Like intact antibodies, they are able to selectively bind to specific antigens. With a molecular weight of only 12-15 kDa, single-domain antibodies are much smaller than ordinary antibodies composed of two heavy chains and two light chains. sdAbs are produced by immunizing dromedary camels, llamas, alpacas, or sharks, or can be engineered from ordinary IgGs with four chains.

[0073] The term "functional fragment" means a portion of an antibody that retains the paratope (comprising the complementarity determining regions or CDRs) and is capable of binding to the same target molecule as the parent antibody from which it is derived. Examples include Fab and F(ab')2 fragments.

[0074] The term "engineered fragment" refers to a recombinant polypeptide that is derived from a parent antibody and retains the paratope, and is therefore able to bind to the same target molecule as the parent antibody. An example is a single-chain variable fragment (VHH), which is a fusion protein of the variable regions of the heavy chain (VH) and light chain (VL) of an immunoglobulin, connected by a short linker peptide of typically 10 to about 25 amino acids.

[0075] The term "DARPin" refers to a repeating protein containing several repeating domains (usually 4 to 6 repeats) of typically 33 amino acids. DARPins can be selected and used as alternative scaffolds for specific targeting because they can bind to their target antigens with high affinity and specificity. A key advantage of using DARPins compared to monoclonal antibodies is that DARPins typically have a low molecular weight, containing 40 to 100 amino acid residues. For example, HER2 is frequently overexpressed in breast cancer cells. DARPins that bind to the extracellular domain of HER2 can be selected and used to direct therapeutic EVs to malignant cells expressing HER2.

[0076] The term "target molecule" refers to a molecule to which a targeting moiety binds. Such a molecule may be a cell surface molecule, for example, a polypeptide, lipid or polysaccharide that can be specifically bound by a targeting moiety.

[0077] The term "target cell" means a cell that expresses a target molecule to which a targeting moiety binds and to which a payload (if applicable) and / or cargo (if applicable) is directed.

[0078] "Tumor-associated antigen" (TAA) means any immunogen that is associated with tumor cells and that is absent or less abundant in healthy cells or corresponding healthy cells (depending on the application and requirements). For example, in the context of an organism, a tumor-associated antigen can be unique to a tumor cell. A TAA can be, for example, a tumor-specific mutation, an aberrantly spliced ​​protein, an oncofetal antigen, or an endogenous retroviral protein. A TAA can be a neoantigen comprising a neoepitope. A neoantigen is an antigen that has not been previously discovered.

[0079] Example 1: Construction of targeted EVs

[0080] Construct a plasmid (use genetic engineering to synthesize the nucleic acid sequence encoding polypeptide A and the nucleic acid sequence encoding anti-Her2 VHH into a universal plasmid template, or synthesize the nucleic acid sequence encoding the recombinant protein containing polypeptide A and anti-Her2 VHH (nanobody) into a universal plasmid vector).

[0081] The amino acid sequence number of the modified membrane fusion protein from the sindbis virus is shown in SEQ ID NO: 1 (named as polypeptide A).

[0082] The nucleic acid sequence encoding the anti-Her2 VHH antibody is numbered as shown in SEQ ID NO: 2. The amino acid sequence encoding the anti-Her2 VHH antibody is numbered as shown in SEQ ID NO: 3.

[0083] Table 2 Amino acid sequence of modified virus

[0084] Table 3 Nucleic acid sequences encoding anti-Her2 VHH antibodies

[0085] Table 4 Amino acid sequences encoding VHH antibodies

[0086] Example 2

[0087] EVs produced using HEK293 cells as donors and validated for membrane fusion function against SK-BK-3 cell membranes

[0088] HEK293 cells were used as donors. The plasmid or lentivirus containing the nucleic acid sequence encoding polypeptide A and the recombinant protein of anti-Her2 VHH in Example 1 was transfected into HEK293 cells to express membrane-bound anti-Her2 VHH and membrane fusion protein polypeptide A, and targeted EVs were produced and collected. The final constructed EV had targeting and membrane fusion functions, with targeting provided by the anti-Her2 VHH antibody and membrane fusion function provided by the membrane fusion protein polypeptide A. The final EV was named B-EV (i.e., targeted EV).

[0089] EV and A-EV were constructed as controls. EV was an unmodified vesicle produced by wild-type HEK293 cells, and A-EV was an HEK293-produced A-EV that lacked anti-Her2 VHH. (The A-EV was prepared by genetically engineering the nucleic acid sequence encoding polypeptide A into a universal plasmid template and transfecting HEK293 cells.) EV, A-EV, and modified B-EV all demonstrated the presence of classic EV markers, including the membrane-bound protein CD81 and the cytoplasmic protein TSG101.

[0090] This step investigated whether the modified B-EV could fuse to the membrane of SK-BK-3 cells expressing Her2. The procedure was as follows: SK-BK-3 cells were co-cultured with B-EV for 30 minutes, sufficient time for Her2 / anti-Her2 VHH interaction. Data showed that the average fusion efficiency of membrane fusion protein peptide A molecules fused to SK-BK-3 cells in each group was 62%. These results confirmed that the modified B-EV could effectively bind to and fuse to the plasma membrane of SK-BK-3 cells via anti-Her2 VHH antibodies. Characterization confirmed that B-EV could attach and fuse to the target cell plasma membrane within 20 minutes. EVs showed little attachment to the SK-BK-3 cell membrane; A-EVs adhered to the SK-BK-3 cell membrane via anti-Her2 VHHs but did not trigger membrane fusion. Experiments confirmed that only the modified B-EVs bound to and fused to the SK-BK-3 cell plasma membrane.

[0091] Example 3

[0092] Verification of the cell membrane fusion function of EVs prepared in Example 2 against BT-474 cells

[0093] BT-474 cells expressing Her2 were incubated for 30 minutes. Similarly, EVs showed little attachment to the BT-474 cell membrane. A-EVs adhered to the BT-474 cell membrane via anti-Her2 VHH but did not trigger membrane fusion. Only modified B-EVs bound to and fused with the BT-474 cell plasma membrane. Fluorescence resonance energy transfer assays confirmed fusion between B-EVs and BT-474 cell membranes, with an average fusion efficiency of 68% within 30 minutes at pH 5-6.

[0094] Example 4

[0095] Preparation of immune EVs using stably expressed cytotoxic T cells

[0096] Primary T cells were transfected with a plasmid or lentivirus containing the nucleic acid sequences encoding polypeptide A (modified viral protein) and the recombinant protein of anti-Her2 VHH in Example 1. The primary T cells were cultured in EV-free medium and stimulated to produce immune EVs in the experimental group. The stimulation method was as follows: During the first stimulation phase, primary T cells were co-cultured with Dynabeads human T activator CD3 / CD28. During the second stimulation phase, IL-2 was added every two days to produce extracellular vesicles, which were then isolated and purified from the cell culture supernatant.

[0097] At the same time, control group 1 immune EV and control group 2 immune EV were set up. In control group 1, the plasmid or lentivirus containing the nucleic acid sequence of anti-Her2 VHH was transfected into primary T cells, and the primary T cells were cultured in a culture medium without EV. After stimulation, control group 1 immune EV (without modified viral proteins) was produced. Control group 2 immune EV was EV produced by primary T cells (without modified viral proteins and anti-Her2 VHH).

[0098] 1) As shown in Figure 1, both polypeptide A and anti-Her2 VHH were expressed on the membrane of the experimental group immune EV. Only anti-Her2 VHH was expressed on the membrane of the control group 1 immune EV, while anti-Her2 VHH and polypeptide A were not detected on the membrane of the control group 2 immune EV. GZmB (granzyme), Fasl, and EV markers (CD63, CD9, HSP70, TSG101, and ANXA1) were detected in the experimental group immune EV, the control group 1 immune EV, and the control group 2 immune EV.

[0099] 2) Treatment of target cells to test apoptosis rate: SK-BK-3 cells expressing Her2 were used as target cells. EVs from the experimental group, control group 1, and control group 2 were incubated with these cells in vitro. The EVs from the experimental group, control group 1, and control group 2 were detected 3-6 hours later. Similarly, the three groups (EVs from the experimental group, control group 1, and control group 2) were treated with Her2-negative Mdamb231 cells.

[0100] After testing, the immune EVs in the experimental group strongly bound to SK-BK-3 cells, while the immune EVs in control group 1 and control group 2 had relatively poor binding ability to SK-BK-3 cells.

[0101] 3) The effects of experimental group immune EVs on SK-BK-3 cells after binding to them were investigated. SK-BK-3 cells were incubated with different concentrations of experimental group immune EVs and control group immune EVs for 3-6 hours, respectively, to examine the apoptosis rate of SK-BK-3 cells induced by different concentrations of EVs. The apoptosis rate of cells treated with PBS was used as the background value. Statistics showed that at EV concentrations of 200-300 μg / ml, experimental group immune EVs significantly induced apoptosis in SK-BK-3 cells in vitro, with an apoptosis rate of 85-95%. At the same concentration, control group 1 immune EVs induced an apoptosis rate of 25%-35% in SK-BK-3 cells in vitro, while control group 2 immune EVs induced an apoptosis rate of 8%-13% in SK-BK-3 cells.

[0102] 4) The results of EV treatment of MDA-MB-231 cells (Her2-negative) in each group are as follows: EV-induced apoptosis in the experimental group was 2-2.2%. However, EV-induced apoptosis in control group 1 was 1-1.2%, and in control group 2 was 0.9-1.1%.

[0103] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. A targeted extracellular vesicle derived from an immune cell, characterized in that: The targeted extracellular vesicle is produced by immune cells, and the membrane of the targeted extracellular vesicle comprises at least one protein with membrane fusion function and at least one targeting part.

2. The immune cell-derived targeted extracellular vesicle according to claim 1, characterized in that The at least one protein with membrane fusion function and at least one targeting part gene construct vector and then are transferred into immune cells.

3. The immune cell-derived targeted extracellular vesicle according to claim 1 or 2, characterized in that: The protein with membrane fusion function is derived from a viral protein, and the targeting part includes a receptor ligand, an antibody or a functional fragment thereof, a single domain antibody VHH or a DARPin.

4. The immune cell-derived targeted extracellular vesicle according to claim 3, characterized in that: The amino acid sequence of the protein having membrane fusion function is shown in SEQ ID NO:

1.

5. The immune cell-derived targeted extracellular vesicle according to claim 1, characterized in that: The immune cells include T cells, CAR-T cells, B cells, natural killer cells, dendritic cells, macrophages or neutrophils.

6. The immune cell-derived targeted extracellular vesicle according to claim 1 or 2, characterized in that: The targeting portion targets tumor-associated antigens, which include but are not limited to Her2, DEC205, CD33, CD30, CD22, CD79b, CLEC9A, Nectin-4, TROP2, TF, CEACAM5, CTLA4, CD3, CD7, CD11c, CD19, CD20, CD22, CD40, CD44, CD206, EGFR, EGFRvIII, fibroblast activation protein (FAP), CA9, MMP-2, PD-L1, SIRPa, Trop2, GPC1, GPC3, cMET, BCMA, VEGFR or Cladin18.

2.

7. The immune cell-derived targeted extracellular vesicle according to claim 3, characterized in that: The targeting portion is a single domain antibody, and the amino acid sequence is shown in SEQ ID NO:

3.

8. A composition comprising the immune cell-derived targeted extracellular vesicles according to any one of claims 1 to 7, and a pharmaceutically acceptable excipient, diluent or carrier.

9. The method for preparing the immune cell-derived targeted extracellular vesicles according to any one of claims 1 to 7, characterized in that: The steps include: (1) constructing a recombinant expression vector containing an exogenous target protein gene fragment; (2) transfecting the recombinant expression vector into immune cells for expression, culturing the transfected immune cells, and stimulating the immune cells to secrete targeted extracellular vesicles carrying the exogenous target protein.

10. Use of the targeted extracellular vesicle according to any one of claims 1 to 7 and the composition according to claim 8 in the preparation of anticancer drugs.

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