Construction and application of engineered extracellular vesicles

Abstract

The present invention provides engineered extracellular vesicles, pharmaceutical compositions containing the engineered extracellular vesicles, and applications thereof, as well as methods for constructing the engineered extracellular vesicles, which can be used for drug delivery, enrichment of target proteins in extracellular vesicles, or presentation of target proteins.

Description

[Technical Field]

[0001] [Cross reference] This application claims priority from a Chinese patent application filed with the State Intellectual Property Office of the People's Republic of China on September 30, 2022, bearing application number 202211213321.0 and entitled "Construction of Engineered Extracellular Vesicles and Their Applications," the entire contents of which are incorporated herein by reference.

[0002] The present invention belongs to the field of biomedicine, and in particular relates to engineered extracellular vesicles, their preparation methods and their applications. [Background technology]

[0003] Extracellular vesicles (EVs) are membrane vesicles secreted by cells and taken up by receptor cells. They are approximately 30–1000 nm in diameter. EVs function as carriers for intercellular sorting of biological macromolecules, such as proteins, RNA, and lipids, and are an important mediator of intercellular communication. EVs are widely present in various body fluids, are used as sample sources for liquid biopsies, and are also considered to be naturally adsorbed drug carriers.

[0004] EVs can be used as drug carriers to load and deliver drugs, such as therapeutic polypeptides and proteins. To ensure the efficacy of target proteins in EVs, it is necessary to ensure sufficient amounts of target proteins within the EVs. However, heterologously expressed non-exosomal target proteins within cells are not actively sorted into extracellular vesicles.

[0005] According to previous reports, there are currently approximately 20 types of proteins used for loading target proteins into EVs (Theranostics, 2019;9(4):1015-1028). Patent applications such as WO2013084000A2, WO2014168548A2, WO2018015535A1, and WO2019040920A1 also describe methods for loading proteins into EVs. Summary of the Invention [Means for solving the problem]

[0006] As a result of extensive research, the present inventors surprisingly discovered that the plexin family has the function of transporting and concentrating target proteins fused to itself into extracellular vesicles. Based on this finding, the present inventors have completed the present invention.

[0007] In a first aspect, the present invention provides engineered extracellular vesicles comprising a fusion protein, wherein the fusion protein comprises a fusion partner that is a protein localized on the extracellular vesicle membrane or a variant thereof, and preferably the fusion protein further comprises a target protein.

[0008] As used herein, the term "extracellular vesicles (EVs)" refers to double-membrane vesicles (EVs) secreted from cells or shed from the cell membrane. Their diameters range from 40 to 1000 nm, and their main forms include microvesicles (MVs) and exosomes (Exs). Extracellular vesicles are widely present in cell culture supernatants and various bodily fluids (blood, lymph, saliva, urine, semen, and breast milk). They contain various cellular proteins, lipids, DNA, mRNA, miRNA, and other proteins, and are involved in processes such as intercellular communication, cell migration, angiogenesis, and immune regulation. The term "engineered extracellular vesicles" refers to artificially synthesized EVs, EVs produced from cells after artificial intervention, or EVs produced from genetically engineered cells. The term "unengineered extracellular vesicles" refers to extracellular vesicles secreted from cells naturally and unmodified under normal conditions (e.g., physiological conditions).

[0009] As used herein, proteins include variants thereof. A "variant" of a protein refers to a protein that has a different amino acid sequence from the amino acid sequence of a naturally occurring protein (sometimes referred to as the wild type), but has the same or similar function as the wild type protein. A variant of a protein is a naturally occurring protein in which amino acids have been deleted, added, and / or substituted. The variant may also be a truncated form of the naturally occurring protein (sometimes referred to as a fragment thereof).

[0010] As used herein, the term "fusion partner" refers to a polypeptide segment fused with a target protein, which can be positioned on the membrane of an extracellular vesicle and deliver the target protein fused to itself into the extracellular vesicle.

[0011] In some embodiments of the invention, the fusion partner has the following characteristics: 1) the fusion partner is a type I transmembrane protein; 2) the transmembrane region and the adjacent intracellular region of the transmembrane protein comprise an α-helix having a length of 20 to 200 amino acids, preferably 30 to 90 amino acids, more preferably 40 to 80 amino acids, and a part or all of the transmembrane region is located in the α-helix; 3) The extracellular region contains 1 to 5 IPT domains.

[0012] As used herein, the term "type I transmembrane protein" refers to a single-pass transmembrane protein whose N-terminus is located outside the membrane and whose C-terminus is located inside the membrane. The "adjacent intracellular region" in the "transmembrane region and adjacent intracellular region" refers to an intracellular segment that is in close contact with the transmembrane region. As used herein, the term "α-helix" may refer to a single α-helix or multiple α-helices, for example, a coiled-coil domain formed from multiple α-helices.

[0013] As used herein, the term "IPT (Immunoglobulin-like fold Plexins Transcription factors) domain" is also called a TIG (Transcription factor ImmunoGlobin) domain and refers to a domain that has an immunoglobulin fold.

[0014] In some embodiments, the fusion partner is selected from the group consisting of plexin family members, such as PLXNA, PLXNB, PLXNC, and PLXND family members, e.g., selected from the group consisting of PLXNA1, PLXNA2, PLXNA3, PLXNA4, PLXNB1, PLXNB2, PLXNC1, and PLXND1.

[0015] Currently known plexin family members include PLXNA1 (Plexin A1), PLXNA2, PLXNA3, PLXNA4, PLXNB1, PLXNB2, PLXNC1, and PLXND1. Plexins are transmembrane protein receptors for semaphorins and are involved in many cellular activities related to cell proliferation, adhesion, basement membranes, cell motility, and invasion. These proteins are frequently expressed in brain tissue, the endocrine system, the gastrointestinal system, the respiratory system, the bone marrow, and the lymphatic system.

[0016] Taking PLXNA1 as an example, PLXNA1 can mediate cytoskeletal reorganization leading to cell migration and axon repulsion by directly binding to the transmembrane semaphorin 6D or indirectly binding to type 3 secreted semaphorins via neuropilin-1 and neuropilin-2. As shown in Figure 1A, the PLXNA1 protein contains multiple domains. Full-length PLXNA1 exists in two states: an activated state (Figure 1B) and an inactivated state (Figure 1C). Because full-length PLXNA1 has biological activity, it is not suitable as a fusion partner for transporting proteins of interest. Surprisingly, the inventors discovered that truncated extracellular and / or intracellular domains of PLXNA1, when fused to proteins of interest, can transport and concentrate the proteins in extracellular vesicles.

[0017] In particular, the fusion partner is selected from the group consisting of PLXNA1, PLXNA2 and fragments thereof. The fragment may be a fragment comprising an amino acid sequence selected from SEQ ID NOs: 2 to 6. In particular, the amino acid sequence of the fusion partner is selected from the group consisting of SEQ ID NOs: 2 to 6.

[0018] In some embodiments, the fusion protein is an engineered transmembrane protein expressed by an exogenous sequence, wherein the transmembrane protein comprises a fragment of formula (I): Xaa1…Xaa n Xaa (n+1) Xaa (n+2) Xaa (n+3) Xaa(n+4) [Formula I] (SEQ ID NO: 15) [In the formula, Xaa1 is selected from the group consisting of S, T, C, N, Y, and Q; n is any amino acid, where n is an integer from 1 to 20, and Xaa (n+1) , Xaa (n+2) and Xaa (n+4) are each independently selected from the group consisting of L, I, G, A and V; (n+3) is selected from the group consisting of T, S, C, N, Y and Q. Preferably, the fragment represented by formula (I) is located upstream of the transmembrane region.

[0019] In some embodiments, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, and more preferably 1, 2, 3, 4, or 5. In some other embodiments, Xaa1 is selected from the group consisting of S, T, C, and N, and Xaa (n+1) , Xaa (n+2) and Xaa (n+4) are each independently selected from the group consisting of L and I, (n+3) is selected from the group consisting of T, S, C, and N. In some further embodiments, Xaa1 is S and Xaa (n+1) , Xaa (n+2) and Xaa (n+4) is L and Xaa (n+3) is T. In one specific embodiment, the fragment of formula (I) is SDSLLTL (SEQ ID NO: 14).

[0020] In some embodiments, the fusion protein comprises a sequence selected from SEQ ID NOs: 2-6.

[0021] As used herein, the term "exogenous sequence" refers to a sequence that is not naturally occurring in a cell, but is instead an artificially constructed sequence. As used herein, amino acids may be natural or unnatural amino acids, and may be represented by three-letter or single-letter symbols known in the art, such as alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartic acid (Asp or D), cysteine ​​(Cys or C), glutamine (Gln or Q), glutamic acid (Glu or E), glycine (Gly or G), histidine (His or H), isoleucine (Ile or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y), and valine (Val or V). "Similar amino acids" are several amino acids that have similar structures and properties, and can be used interchangeably without significantly affecting the structure or properties of proteins when substituted for one another.

[0022] In some embodiments, the target protein may be located upstream of the fusion partner or downstream of the fusion partner. As used herein, "upstream" of a protein refers to the N-terminus or a portion adjacent to the N-terminus of a polypeptide, and "downstream" of a protein refers to the C-terminus or a portion adjacent to the C-terminus of a polypeptide. Depending on actual needs, the target protein may be encapsulated inside the extracellular vesicles or displayed on the outer surface of the extracellular vesicles.

[0023] In some embodiments of the invention, the protein of interest is a therapeutic peptide, a targeting peptide, an affinity tag, or a linker for attaching a therapeutic compound.

[0024] As used herein, the term "therapeutic peptide" refers to a protein or variant thereof having therapeutic activity, including, but not limited to, an antibody or antigen-binding fragment thereof, a receptor, a ligand, a cytokine, a hormone, etc. In some embodiments of the present invention, the therapeutic peptide is a human interleukin family member (e.g., IL-2, IL-7, IL-10, IL-11, IL-12, IL-15, and IL-23), a tumor necrosis factor family member (e.g., TNF, LTA, LTB, FASLG, TNFSF8, TNFSF9, TNFSF10, TNFSF11, TNFSF12, TNFSF13, TNFSF14, TNFSF15, TNFSF18, and EDA), an interferon family member (e.g., IL-2, IL-7, IL-10, IL-11, IL-12, IL-15, and IL-23), a leukemia family member (e.g., TNFSF ... These cytokines may include cytokines (IFN-α, IFN-β, and IFN-γ), T cell engagers (e.g., 4-1BB, OX40, CD28, CD40, CD40L, CD47, CD27, CD70, CD80, CD86, GITRL, ICOSL, CD155, CD112, TIM-3, BTLA), and other cytokines (e.g., G-CSF, EPO, TPO, GM-CSF, EGF, bFGF, FVIIa, ATIII, TNK, α-glucosidase, BMP-2, hirudin).

[0025] As used herein, the term "targeting peptide" refers to a polypeptide that is recognized and bound by a specific cell, i.e., a polypeptide that can target a specific cell. In some embodiments of the present invention, the targeting peptide is a polypeptide that targets a cell. The targeting peptide may be an antibody or an antigen-binding fragment thereof, a cell surface receptor, or a ligand.

[0026] As used herein, the term "affinity tag" refers to a short polypeptide chain capable of binding to a corresponding affinity agent, typically used for protein isolation and purification. In some embodiments of the present invention, the affinity tag is selected from the group consisting of His-tag, glutathione-S-transferase (GST), S-peptide, ZZ domain, albumin-binding domain (ABD), HA, Myc, FLAG™, maltose-binding protein (MBP), calmodulin-binding peptide (CBP), SUMO, streptococcal protein G (protein G), and Staphylococcus aureus protein A (protein A).

[0027] As used herein, the term "therapeutic compound linker" refers to a substance for attaching a therapeutic compound to a fusion partner.

[0028] In some embodiments of the present invention, the extracellular vesicles comprise two or more fusion proteins, for example, each of the two or more fusion proteins independently comprising a different protein of interest. The different proteins of interest can form fusion proteins with the same or different fusion partners, but the different fusion proteins may be carried in the same extracellular vesicle.

[0029] As used herein, the term "therapeutic agent" refers to a substance that has a therapeutic effect, such as a nucleotide, an amino acid, a lipid, a carbohydrate, a small molecule, an antibody, an enzyme, a ligand, a receptor, or a polypeptide. In some embodiments of the present invention, a therapeutic agent is encapsulated in the extracellular vesicles. The therapeutic agent may be a therapeutic peptide, a polynucleotide, or a small molecule compound.

[0030] In a second aspect, the present invention provides a pharmaceutical composition comprising an extracellular vesicle as described in the first aspect and a pharmaceutically acceptable carrier.

[0031] The term "pharmaceutically acceptable carrier" as used herein may be selected from the group consisting of water, buffer solution, isotonic salt solution such as PBS (phosphate buffer), glucose, mannitol, dextrose, lactose, starch, magnesium stearate, cellulose, magnesium carbonate, 3% glycerin, hyaluronic acid, ethanol, polyalkylene glycols such as polypropylene glycol, and triglycerides. The type of pharmaceutically acceptable carrier used depends particularly on whether the composition of the present invention is prepared for oral, nasal, intradermal, subcutaneous, intramuscular, or intravenous administration. The composition of the present invention may contain additives such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorants, flavoring agents, and / or fragrances.

[0032] In a third aspect, the present invention provides an engineered cell used to produce the extracellular vesicles described in the first aspect, the cell comprising a polynucleotide encoding the fusion protein.

[0033] In some embodiments of the present invention, the polynucleotide encoding the fusion protein is present in the cell in a free form and is introduced into the cell, for example, by instantaneous transfection. In other embodiments of the present invention, the polynucleotide is integrated into the genome of the cell. In other embodiments of the present invention, the polynucleotide is one or more types and encodes one or more fusion proteins.

[0034] In a fourth aspect, the present invention provides a method for treating a disease comprising administering to a subject in need thereof an extracellular vesicle as described in the first aspect, a pharmaceutical composition as described in the second aspect, or a cell as described in the third aspect.

[0035] As used herein, the term "subject" refers to a mammal, such as a human being, but may also refer to other animals, such as wild animals (e.g., herons, storks, cranes, etc.), domestic animals (e.g., ducks, geese, etc.), or laboratory animals (e.g., orangutans, monkeys, rats, mice, rabbits, guinea pigs, groundhogs, ground squirrels, etc.).

[0036] The compositions of the present invention can be administered in a variety of ways known in the art. It will be understood by those skilled in the art that the route and / or pattern of administration will vary depending on the desired results. To administer the compounds of the present invention by a specific route of administration, it may be necessary to coat the compound with or administer the compound together with a material to prevent inactivation. For example, the compound can be administered to a subject in an appropriate carrier, such as a fat pad or diluent. Pharmaceutically acceptable diluents include saline and aqueous buffer solutions. Pharmaceutical carriers include sterile aqueous solutions or dispersions, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. The use of such media and reagents for pharmaceutically active substances is known in the art.

[0037] According to the present invention there is provided the use of an extracellular vesicle as described in the first aspect, a pharmaceutical composition as described in the second aspect or a cell as described in the third aspect in the manufacture of a medicament for the treatment of a disease.

[0038] In a fifth aspect, the present invention provides a method for producing extracellular vesicles as described in the first aspect, comprising: 1) providing a cell containing a polynucleotide encoding the fusion protein; 2) culturing the cells under conditions suitable for expression of the fusion protein; and 2) isolating the extracellular vesicles.

[0039] In some embodiments of the present invention, the introduction is transient, for example, by lipofection, co-transfection, etc., where a polynucleotide encoding the fusion protein is introduced into a host cell. In other embodiments, the introduction is stable, for example, by genome editing techniques, where a polynucleotide encoding the fusion protein is inserted into the genome of a host cell, and the engineered extracellular vesicles are stably expressed in the host cell.

[0040] Any cells that can be used for protein expression and that are well known to those skilled in the art can be used in the present invention, and eukaryotic cells such as expi293, HEK293 and HEK293F are preferred.

[0041] In a sixth aspect, the present invention provides a method for delivering a protein of interest to a target cell, the method comprising: 1) providing an extracellular vesicle as described in the first aspect, wherein the fusion protein comprises a protein of interest to be delivered; and 2) contacting the extracellular vesicles with the target cells.

[0042] In a seventh aspect, the present invention provides a method for isolating extracellular vesicles as described in the first aspect, comprising: 1) expressing the fusion protein containing an affinity tag in a cell; 2) contacting the extracellular vesicles with a binding agent capable of binding to the affinity tag; and 3) isolating the extracellular vesicles by binding of the affinity tag to the binding agent.

[0043] As used herein, the term "binding agent" refers to a substance that can bind to an affinity tag. The binding agent binds to the affinity tag with high affinity, which is advantageous for the isolation of the fusion protein linked to the affinity tag by the binding agent.

[0044] In an eighth aspect, the invention provides a fusion protein in which an engineered transmembrane protein is expressed with an exogenous sequence. The transmembrane protein comprises a fragment represented by the following formula (I): Xaa1…Xaa n Xaa (n+1) Xaa (n+2) Xaa (n+3) Xaa (n+4) [Formula I] (SEQ ID NO: 15) [In the formula, Xaa1 is selected from the group consisting of S, T, C, N, Y, and Q; n is any amino acid, where n is an integer from 1 to 20, and Xaa (n+1) , Xaa (n+2) and Xaa (n+4) are each independently selected from the group consisting of L, I, G, A and V; (n+3) is selected from the group consisting of T, S, C, N, Y and Q. Preferably, the fragment represented by formula (I) is located upstream of the transmembrane region.

[0045] In some embodiments, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, and more preferably 1, 2, 3, 4, or 5. In some other embodiments, Xaa1 is selected from the group consisting of S, T, C, and N, and Xaa (n+1) , Xaa (n+2) and Xaa (n+4) are each independently selected from the group consisting of L and I, (n+3) is selected from the group consisting of T, S, C, and N. In some further embodiments, Xaa1 is S and Xaa (n+1) , Xaa (n+2) and Xaa (n+4) is L and Xaa (n+3) is T. In one specific embodiment, the fragment of formula (I) is SDSLLTL (SEQ ID NO: 14).

[0046] In some embodiments of the invention, the fusion protein may comprise a fragment selected from SEQ ID NOs:2-6.

[0047] In a ninth aspect, the present invention provides the use of the fusion protein in the production of engineered extracellular vesicles.

[0048] In a tenth aspect, the present invention provides a method for loading a molecule of interest into an extracellular vesicle of the present invention, comprising contacting the molecule of interest with the extracellular vesicle, wherein the molecule of interest and the fusion partner are linked to moiety A and moiety B, respectively, which binds to moiety A by affinity, wherein the binding is non-covalent and reversible. Any molecular pairing capable of affinity binding known in the art can be used in the present invention.

[0049] Traditional methods for producing engineered extracellular vesicles (EVs) involve first designing a plasmid to conjugate a scaffold protein with a molecule of interest, then transfecting the plasmid into engineered cells. After culturing the cells under specific conditions, the EVs carrying the molecule of interest are secreted into the supernatant, followed by purification, characterization, and identification. However, this production method is time-consuming, cost-intensive, and has a long cycle time, making it unsuitable for personalized loading of different molecules of interest. In response to the drawbacks of traditional methods, such as time, cost, and cycle time, and the need for personalized loading of different molecules of interest, our method provides a more versatile, simple, and efficient method for loading target proteins into EVs. By producing a single type of engineered EV and co-incubating it in vitro with a specific tagged protein of interest, the target protein can be rapidly loaded.

[0050] In some embodiments, the portion A and the portion B are selected from the group consisting of NbALFA / ALFA, biotin / avidin, strepII / streptactin, InteinN / C, SpyCatcher / Spy, and ProteinA / Fc. The molecule of interest and the fusion partner are linked to the portion A and the portion B, respectively, such that upon contact, the portions A and B bind with affinity, and the molecule of interest is loaded onto the EV via the fusion partner and displayed on the surface of the EV.

[0051] In some embodiments, the molecule of interest is a therapeutic peptide, a targeting peptide, an affinity tag, or a linker for attaching a therapeutic compound. When the molecule of interest is a polypeptide, part A or part B can be linked to the N-terminus, C-terminus, or internal part of the polypeptide, as needed. As used herein, the molecule of interest may be a protein of interest (POI).

[0052] In some embodiments, different target molecules can be loaded onto the same EV. For example, if different target molecules each carry a portion A and the EV carries a portion B, simultaneous contact of the different target molecules with the EV allows the different target molecules to be simultaneously loaded onto the EV. The ratio of the different target molecules can be adjusted according to actual needs. Furthermore, if different target molecules carry different types of portion A (A1, A2, A3, etc.) and the EV carries different types of portion B (B1, B2, B3, etc.), the different target molecules can also be loaded onto the same EV. [Brief explanation of the drawings]

[0053] [Figure 1] The structure of PLXNA1 and its different states are shown. A) Schematic diagram of PLXNA1 structure. B) Inactive state of PLXNA1, showing the Sema domain binding to the MRS overlap. C) Active state of PLXNA1, showing the Sema domain binding to a ligand. [Figure 2]1 shows a series of PLXNA1 truncations constructed in Example 3, in which mIL12 and eGFP are fused to the N- and C-termini of the truncations, respectively. [Figure 3] This shows the WB results of concentrating the target protein in EVs using the PLXNA1 truncation construct constructed in Example 3. [Figure 4] Example 3 shows the results of detecting the number of eGFP on EVs induced by PLXNA1 truncations using ELISA. [Figure 5] This shows a comparison of the enrichment effects of PLXNA1 with other EV proteins PTGFRN and Lamp2b in EVs in Example 4. [Figure 6] Shows the expression and concentration of PLXNA2, PLXNB1, PLXNB2, PLXNC1 and PLXND1 in EVs in Example 5. [Figure 7] The copy number of eGFP on each EV in three samples, P240, P241, and P242, is shown. [Figure 8] Sequence alignment of the transmembrane regions of PLXNA1 and PLXNA2 is shown, where the italicized sequence is the last IPT domain near the transmembrane region, the bolded sequence is a key sequence consisting of 7 amino acids, and the underlined sequence is the transmembrane region. [Figure 9] The effect of carrying key sequences on the enrichment of fusion proteins into EVs is shown in Figure 1. A) Schematic diagram of the structure of the P166 and P167 fusion proteins, B) WB detection results, and C) statistical results. [Figure 10A] The effect of mutations in key sequences on the enrichment of fusion proteins into EVs is shown. (A) shows the results of point mutations at each site. [Figure 10B] The effect of key sequence mutations on the enrichment of fusion proteins into EVs is shown. Here, B) shows the results of synonymous mutations. [Figure 10C] The effect of key sequence mutations on the enrichment of fusion proteins into EVs is shown. Here, C) indicates the result of an insertion mutation. [Figure 11]This shows that all of the different target proteins carried by the PLXNA1 truncations were sorted into EVs. [Figure 12] This shows that mIL12 expressed as fused to a truncated PLXNA1 still retains activity. [Figure 13] The correlation between luciferase activity and the number of EV particles is shown. [Figure 14] FIG. 1 is a schematic diagram showing loading of a protein of interest via molecular interactions based on the EV fusion partner of the present invention. [Figure 15] Example 11 shows the results of detecting the expression of NbALFA-Flag-PLXΔ in EVs by WB. [Figure 16] 1 shows the results of detecting NbALFA-PLXΔ-EV by nanoflow cytometry in Example 11. [Figure 17] 10 shows the results of detecting GFP loaded into NbALFA-PLXΔ-EVs by nanoflow cytometry in Example 11. [Figure 18] 11 shows the results of nanoflow cytometry detection of RVG polypeptide loaded onto NbALFA-PLXΔ-EV in Example 11. [Figure 19] 1 shows the results of detecting the affinity of RVG-labeled endoplasmic reticulum for the recipient cell N2a in Example 11. [Figure 20] 10 shows the results of nanoflow cytometry detection of trastuzumab scFv fragments loaded onto NbALFA-PLXΔ-EV in Example 11. [Figure 21] 1 shows the results of detecting the affinity of endoplasmic reticulum labeled with trastuzumab scFv fragments for recipient cells SK-BR-3. [Figure 22] 1 shows the measurement results of freeze-thaw cycles of EV in Example 11. [Figure 23] 10 shows the results of detecting the mean fluorescence intensity of GFP by nanoflow cytometry at 2 and 4 weeks after GFP-labeled NbALFA-PLXΔ-EVs were stored at 4°C or -80°C in Example 11. [Figure 24] 10 shows the WB detection results of different tags in Example 12. [Figure 25] 13 shows that the expression of mSA-Flag-PLXΔ in EVs in Example 13 was detected by WB. [Figure 26] 1 shows the results of detecting mSA-PLXΔ-EV by nanoflow cytometry in Example 13. [Figure 27] 13 shows the results of detecting mSA-PLXΔ-EV labeled with biotin-488 by nanoflow cytometry in Example 13. [Figure 28] 1 shows the results of detecting EVs labeled with FITC-HA by nanoflow cytometry in Example 13. [Figure 29] 10 shows the results of detecting Strep II-flag-PLXΔ-EV by nanoflow cytometry in Example 14. [Figure 30] 1 shows the results of detecting FITC-streptactin-labeled Strep II-flag-PLXΔ-EVs by nanoflow cytometry in Example 14. DETAILED DESCRIPTION OF THE INVENTION

[0054] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as understood by one of ordinary skill in the art.

[0055] Although the numerical ranges and approximations of parameters are given broad ranges in the present invention, the numerical values ​​set forth in the specific examples are described as precisely as possible. However, any numerical value necessarily contains some error due to the standard deviation present in each measurement. Furthermore, all ranges disclosed herein should be understood to include any and all subranges contained therein. For example, a range stated as "1 to 10" should be understood to include any and all subranges between the minimum value of 1 and the maximum value of 10 (including the endpoints), i.e., all subranges beginning with a minimum value of 1 or greater, such as 1 to 6.1, and all subranges ending with a maximum value of 10 or less, such as 5.5 to 10. Furthermore, any reference referred to as "incorporated herein" is understood to be incorporated in its entirety.

[0056] Also, it should be noted that as used herein, the singular forms include the plural forms of that object unless clearly and unambiguously limited to one object.

[0057] As used herein, terms such as "comprise," "include," "contain," and "included" are not limiting. Further, unless otherwise specified, "or" means "and / or."

[0058] Any patents, patent applications, publications cited herein, or discussions herein are hereby incorporated by reference in their entirety. [Example]

[0059] Some preferred embodiments and aspects of the present invention will now be described in more detail with reference to specific examples, which should not be construed as limiting the scope of the invention. Example 1: Materials and Methods The materials used in the examples of the present invention and their manufacturers are listed in Table 1 below.

[0060] [Table 1]

[0061] [Experimental Method] 1. Cell Transfection 1) Preparation of cells On the day of transfection, count the suspension-cultured Expi293F cells and determine the cell density at 2-5 x 10 6 The cells were then cultured in pre-warmed medium at a concentration of 2 × 10 6 / ml to a final volume of 30 ml.

[0062] 2) Transfection Preparation of solution A: 30 μg of plasmid was pipetted into a 15 ml centrifuge tube containing 1 ml of Opti-MEM, mixed well by gently blowing, and then allowed to stand for 5 minutes.

[0063] Preparation of solution B: 60 μl of PEI (working solution concentration 1 μg / μl) was pipetted and added to a 15 ml centrifuge tube containing 1 ml of OPTI-MEM, mixed well by slow blowing, and then allowed to stand for 5 minutes.

[0064] 1 ml of solution B was drawn up with a pipette and added to solution A, and after gentle mixing, the mixture was allowed to stand at room temperature for 20 minutes.

[0065] The mixed transfection reagent was added dropwise to the Expi293F cells while gently shaking to mix well.

[0066] 3) Addition of liquid after transfection Four to six hours after transfection, the cell culture bottles were sterilized with 75% alcohol and transferred to a biological safety cabinet.

[0067] 30 μl of anti-clumping agent was drawn up with a 100 μl pipette and added to the shake flask.

[0068] The suspension culture was carried out in an incubator at 37°C, 5% or 8% CO2, and 110 rpm.

[0069] 4) Addition of KT feed 22 to 24 hours after transfection, the cell culture bottles were sterilized with 75% ethanol and transferred to a biological safety cabinet, and 0.6 ml of KT feed and 2 g / L glucose were added to the cell culture medium.

[0070] In accordance with the "Standard Operating Procedure for Use, Cleaning, and Maintenance of 00-ZCZY-AS8V Shaking Incubators" (SOP-EQ-082), the incubators were set at 37°C, 5% or 8% CO2, and 110 rpm.

[0071] 5) Cell harvesting Cells were harvested after 4 days of culture.

[0072] The cell suspension was transferred to a 50 ml sterile centrifuge tube and centrifuged at 800 g and 4° C. for 10 minutes, after which the cells and supernatant were separated.

[0073] The cell supernatant was transferred to a separate 50 ml sterile centrifuge tube and centrifuged at 4000 g and 4° C. for 10 minutes.

[0074] The supernatant was aspirated, sterilized by filtering through a 0.22 μm filter, and then transferred to a 50 ml sterile centrifuge tube for subsequent use.

[0075] 2. EV Extraction The cell supernatant was added to an ultracentrifuge tube and centrifuged at 16,000 xg for 2 hours at 4°C, after which the supernatant was collected.

[0076] The supernatant obtained in the previous step was transferred to a clean ultracentrifuge tube and centrifuged at 100,000×g at 4° C. for 2 hours, after which the supernatant was discarded.

[0077] After centrifugation, the EVs pellet was resuspended in PBS.

[0078] The resuspended EVs were added to a 100 kD ultrafiltration tube and centrifuged at 4000 × g for 10 minutes. The EVs were then collected in a 1.5 mL centrifuge tube, and the total protein concentration was detected by BCA.

[0079] 3. Western blotting (WB) EVs containing 10 μg of total protein or cell lysates containing 20 μg of total protein were collected, and an appropriate amount of 5x loading buffer was added to a final concentration of 1x loading buffer. The mixture was then incubated at 95°C for 10 minutes. Running buffer (1 L) was prepared using the following formula: 100 mL of 10x running buffer (Cowin Biotech) + 900 mL of ddH2O. After gentle mixing, the mixture was ready for subsequent use. A precast gel (Beyotime) of the appropriate concentration was attached to the gel tank. After confirming there was no leakage from the assembled gel tank, the prepared running buffer was poured into the gel tank, ensuring that the running buffer exceeded the gel surface. The heat-denatured sample was pipetted and carefully added to each well of the SDS-PAGE gel (care should be taken not to apply too much force when adding the sample to avoid contaminating other wells with the sample in one well). The gel tank was powered on properly, and gel electrophoresis was performed at a voltage of 80 V. After the sample entered the separating gel, gel electrophoresis was continued at a voltage of 120 V. After the target protein was separated, gel electrophoresis was stopped.

[0080] An appropriately sized nitrocellulose (NC) membrane was collected and immersed in methanol for at least 20 seconds, then washed with an appropriate amount of pre-chilled transfer buffer for subsequent use. [The transfer buffer was prepared according to the following formula: 100 mL of 10x transfer buffer solution (Cowin Biotech) + 200 mL of methanol + 700 mL of ddH2O, pre-chilled after preparation.] After gel electrophoresis was completed, the gel was removed and immersed in pre-chilled transfer buffer. A sandwich model was then assembled in the following order: black side - sponge - filter paper - gel - NC membrane - filter paper - sponge - white side. The sandwich model was then placed in the transfer tank in the following order: black side - black side, white side - white side. Two iceboxes frozen overnight at -80°C were placed in the transfer tank, and the entire sandwich model was immersed in transfer buffer. The transfer tank was then attached and the membrane was transferred at a constant current of 350 mA for 1 hour. Blocking: 5% BSA blocking buffer (dissolved in 1x TBST) was prepared. After transfer, the membrane was removed and placed in blocking buffer and blocked for 1-2 hours on a horizontal shaker at room temperature. Primary antibody incubation: After blocking was complete, the membrane was washed three times with 1x TBST. A primary antibody solution was prepared in TBST at the specified ratio and incubated for the appropriate time. The preparation ratio and incubation time are recorded in Table 1 above. Secondary antibody incubation: After blocking was complete, the membrane was washed three times with 1x TBST. A secondary antibody solution was prepared in TBST at the specified ratio and incubated for the appropriate time. ECL chemiluminescence development: After secondary antibody incubation was complete, the membrane was washed three times with 1x TBST. Before development, ECL chemiluminescence solution A and solution B were mixed in a 1:1 ratio to prepare the membrane, and the membrane was developed using the Tanon-5200 Multi chemiluminescence gel imaging system.

[0081] 4. Quantitative detection of target proteins EVs containing 1.2 μg of total protein were collected and added to 250 μl of RIPA lysis buffer (containing 100x protease inhibitors), mixed thoroughly, and then lysed at 4°C for 1 hour. Then, the mixture was centrifuged at low speed to remove insoluble material. Thirty minutes before the ELISA experiment, the kit was removed and left at room temperature to achieve equilibration. Standards of different concentrations were prepared according to the manufacturer's instructions, and samples were diluted with the kit's diluent. The samples were incubated at 37°C for 90 minutes and washed four times in a plate washer. Next, 100 μl of antibody was added, incubated at 37°C for 60 minutes, and washed four times in a plate washer. Next, 100 μl of enzyme conjugate was added, incubated at 37°C for 30 minutes, washed four times in a plate washer, and allowed to develop for 15 minutes. Stop solution was then added, and the plate was read using a microplate reader.

[0082] 5. Measurement of EV Concentration The concentration and particle size of EVs were measured using a flow nanoanalyzer (NanoFCM, Xiamen, China) equipped with a 488 nm laser. First, the analyzer was used to calibrate mass-controlled microspheres (250 nm SiNPs) and identify the mixed microspheres (68-155S16-Exo). Next, EVs were diluted to 1 × 10 in PBS. 8 ~6×10 8 The EVs were diluted to a concentration range of 2,000–12,000 particles / mL (total particle detection count: 2,000–12,000 particles / min). Sample data were collected for 60 seconds, and the EV concentration and particle size distribution were analyzed using Flow NanoAnalyzer software (NF Professional 2.0 version).

[0083] 6. In Vitro IL-12 Activity Measurement A 70 μm cell strainer was placed in a culture dish, and 3 mL of mouse lymphocyte isolation solution was added.

[0084] Spleens from C57BL / 6 mice were collected under sterile conditions, placed in the culture dish prepared in the previous step, and polished by gently pressing with the end of a 5 mL rubber push rod until no red clumps remained.

[0085] The cell strainer was washed with 1 mL of mouse lymphocyte separation solution, and the cell suspension was collected in a 15 mL centrifuge tube.

[0086] 1 mL of RPMI-1640 was taken and slowly added to the above 15 mL centrifuge tube along the inner wall of the centrifuge tube, and the acceleration and deceleration speeds of the centrifuge were set to 3, after which the centrifuge tube was centrifuged at 800 g for 30 minutes.

[0087] The supernatant was discarded and the cells were resuspended in 10 mL of RPMI 1640 and centrifuged at 250 g for 10 min to collect and count the cells. 6 Adjusted to cells / mL.

[0088] After adding mouse CD3 antibody (0.5 μg / mL) and mixing well, the cells were cultured at 37°C and 5% CO for 2 days, during which the cell state (aggregation) was carefully observed.

[0089] On day 3, stimulated mouse spleen cells were collected and centrifuged at 300 g for 5 min.

[0090] The supernatant was discarded and the cells were resuspended in mouse spleen cell medium and centrifuged at 300 g for 5 minutes.

[0091] The supernatant was discarded and the cells were resuspended in mouse spleen cell medium and counted (cell viability >80%).

[0092] 1.5×10 5 Cells were plated in 96-well plates according to the plate layout at 90 μL / well and placed in an incubator set at 37° C. and 5% CO 2 .

[0093] Serially diluted samples were seeded into a 96-well plate containing pre-plated cells at 10 μL per well according to the plate layout, mixed gently, and 200 μL of sterile water was added to the surrounding wells to prevent evaporation. The plate was then incubated at 37°C in a 5% CO2 atmosphere for 40 hours.

[0094] The next day, the well plates containing the spleen cells and samples were centrifuged at 1500 rpm for 5 minutes, and 60 μL of the supernatant was collected and used to detect mouse IFNγ using an ELISA kit.

[0095] [Example 2] Expression and concentration of plexin family members in EVs Through extensive research, the inventors surprisingly discovered that Plexin family members are highly expressed in EVs, suggesting potential applications for loading and enriching target proteins in EVs. We constructed a PLXNA1 eukaryotic expression plasmid and overexpressed PLXNA1 protein in cells. EVs were collected according to the method described in Example 1, and PLXNA1 expression in EV-free cell lysates and EVs was detected by WB. The results showed that PLXNA1 protein was sorted into EVs and expressed at high levels, indicating enrichment in EVs.

[0096] The full-length sequence of PLXNA1 used in the experiment is as follows:

[0097] [Example 3] Construction and expression of PLXNA1 truncations PLXNA1 contains an extracellular domain, a transmembrane domain, and an intracellular domain. Because the extracellular and intracellular domains are involved in its biological function, full-length PLXNA1 is not suitable as a fusion partner for transporting target proteins. The inventors unexpectedly discovered that PLXNA1 lacking a portion of the extracellular domain and / or the intracellular domain can also transport target proteins into EVs. The inventors constructed a series of PLXNA1 truncations and fused two different polypeptides, mIL12 and eGFP (as a marker), to the N- and C-termini of the PLXNA1 truncations, respectively. A schematic diagram of the structures is shown in Figure 2. The fusion proteins also contained a Flag tag.

[0098] The amino acid sequence contained in the plasmid constructed in this example (P in the plasmid is an abbreviation for plasmid) is as follows:

[0099] P292(863-1316): A truncated form of PLXNA1 containing four IPT domains, a transmembrane region, and part of the adjacent sequence DPKILKLSPETGPRQGGTRLTITGENLGLRFEDVRLGVRVGKVLCSPVESEYISAEQIVCEIGDASSVRAHDALVEVCVRDCSPHYRALSPKRFTFVTPTFYRVSPSRGPLSGGT WIGIEGSHLNAGSDVAVSVGGRPCSFSWRNSREIRCLTPPGQSPGSAPIIININRAQLTNPEVKYNYTEDPTILRIDPEWSINSGGTLLTVTGTNLATVREPRIRAKYGGIEREN GCLVYNDTTMVCRAPSVANPVRSPPELGERPDELGFVMDNVRSLLVLNSTSFLYYPDPVLEPLSPTGLLELKPSSPLILKGRNLLPPAPGNSRLNYTVLIGSTPCTLTVSETQLLCEAPNLTGQHKVTVRAGGFEFSPGTLQVYSDSLLTLPAIVGIGGGGGLLLLVIVAVLIAYKRKSRDADRTLKRLQLQMDNLESRVALECKEAFAELQTDIHELTNDLDG (SEQ ID NO: 2)

[0100] P235(863-1300): a truncated form of PLXNA1 containing four IPT domains and transmembrane regions DPKILKLSPETGPRQGGTRLTITGENLGLRFEDVRLGVRVGKVLCSPVESEYISAEQIVCEIGDASSVRAHDALVEVCVRDCSPHYRALSPKRFTFVTPTFYRVSPSRGPLSGGTWIGIEGSHLNAGSDVAVSVGGRPCSFSWRNSREIRCLTPPGQSPGSAPIIININRAQLTNPEVKYNYTEDPTILRIDPEWSINSGGTLLTVTGTNLATVREPRIRAKYGGIERENGCLVYNDTTMVCRAPSVANPVRSPPELGERPDELGFVMDNVRSLLVLNSTSFLYYPDPVLEPLSPTGLLELKPSSPLILKGRNLLPPAPGNSRLNYTVLIGSTPCTLTVSETQLLCEAPNLTGQHKVTVRAGGFEFSPGTLQVYSDSLLTLPAIVGIGGGGGLLLLVIVAVLIAYKRKSRDADRTLKRLQLQMDNLESRVALECKEAF (SEQ ID NO: 3)

[0101] P236(960-1300): a truncated form of PLXNA1 containing three IPT domains and a transmembrane region TPTFYRVSPSRGPLSGGTWIGIEGSHLNAGSDVAVSVGGRPCSFSWRNSREIRCLTPPGQSPGSAPIIININRAQLTNPEVKYNYTEDPTILRIDPEWSINSGGTLLTVTGTNLATVREPRIRAKYGGIERENGCLVYNDTTMVCRAPSVANPVRSPPELGERPDELGFVMDNVRSLLVLNSTSFLYYPDPVLEPLSPTGLLELKPSSPLILKGRNLLPPAPGNSRLNYTVLIGSTPCTLTVSETQLLCEAPNLTGQHKVTVRAGGFEFSPGTLQVYSDSLLTLPAIVGIGGGGGLLLLVIVAVLIAYKRKSRDADRTLKRLQLQMDNLESRVALECKEAF (SEQ ID NO: 4)

[0102] P237(1046-1300): a truncated form of PLXNA1 containing two IPT domains and a transmembrane region EDPTILRIDPEWSINSGGTLLTVTGTNLATVREPRIRAKYGGIERENGCLVYNDTTMVCRAPSVANPVRSPPELGERPDELGFVMDNVRSLLVLNSTSFLYYPDPVLEPLSPTGLLELKPSSPLILKGRNLLPPAPGNSRLNYTVLIGSTPCTLTVSETQLLCEAPNLTGQHKVTVRAGGFEFSPGTLQVYSDSLLTLPAIVGIGGGGGLLLLVIVAVLIAYKRKSRDADRTLKRLQLQMDNLESRVALECKEAF (SEQ ID NO: 5)

[0103] P238(1143-1300): A truncated form of PLXNA1 containing one IPT domain and one transmembrane region SFLYYPDPVLEPLSPTGLLELKPSSPLILKGRNLLPPAPGNSRLNYTVLIGSTPCTLTVSETQLLCEAPNLTGQHKVTVRAGGFEFSPGTLQVYSDSLLTLPAIVGIGGGGGLLLLVIVAVLIAYKRKSRDADRTLKRLQLQMDNLESRVALECKEAF (SEQ ID NO: 6)

[0104] The plasmid was constructed according to the experimental method described in Example 1. The plasmid was transfected into 293 suspension cells and cultured until harvest. The supernatant was harvested and separated by low-speed centrifugation. The cells obtained from centrifugation were lysed, and the total protein content of the cell lysate was quantified using BCA. Exosomes were isolated from the supernatant of the harvested cells by ultra-high-speed centrifugation, and the total protein content of the EVs was quantified using BCA. Cell lysates and EVs containing the same total protein content were analyzed by Western blotting, and the band intensities were compared to determine whether the target protein was enriched in EVs. The results in Figure 3 demonstrate that different PLXNA1 truncations can enrich the target protein in EVs.

[0105] Detection of eGFP counts on EVs by ELISA 1) Concentration of exosome particles obtained (CONC) 粒子 (particles / mL) was detected by a nanoflow cytometer. 2) eGFP concentration CONC eGFP (ng / mL) was detected by ELISA kit. 3)Official copy / p=(CONC eGFP *N A ) / (CONC particle *M eGFP ) was used to calculate the average copy number of eGFP on a single EV particle (where N A is Avogadro's constant (approximately 6.02 × 10 23 ), and M eGFP represents the molar mass of eGFP).

[0106] The results are shown in Figure 4. The figure indicates that the copy numbers of P292 and P235 on a single EV particle were high. Based on this result, in subsequent examples, a truncated P235, i.e., PLXNA1(863-1300), was used as a fusion partner for constructing fusion proteins.

[0107] [Example 4] Comparison with PTGFRN and Lamp2b PTGFRN and Lamp2b are EV biomarker proteins reported in previous studies, and target proteins can be delivered to EVs (Delivery of siRNA to the mouse brain by systemic injection of targeted exosomes, Nature Biotechnology, volume 29, pp. 341–345 (2011)). Using the same carrier, we constructed expression plasmids for PLXNA1, PTGFRN, and Lamp2b fusion proteins.

[0108] P235:mIL12-PLXNA1(863-1300)-Flag-eGFP.

[0109] P320: mIL12-PTGFRN(FL)-Flag-eGFP; the sequence of the fusion protein is as follows:

[0110] P321: mIL12-Lamp2b(FL)-Flag-eGFP; the sequence of the fusion protein is as follows:

[0111] The three plasmids were each introduced into HEK293 cells, cultured, and the cell supernatant was collected and EVs were extracted. Cell lysates, supernatants, and EVs were added in protein-equivalent amounts, and the expression of the fusion proteins (anti-Flag antibody and anti-eGFP antibody) was detected by WB. The specific experimental procedures were the same as in Example 3, and the results are shown in Figure 5.

[0112] As shown in Figure 5, compared with the reported EV proteins PTGFRN and Lamp2b, the PLXNA1 truncation fusion protein showed a higher fold enrichment in EVs.

[0113] [Example 5] Other proteins of the plexin family To confirm whether other members of the same plexin family have similar functions, other members PLXNA2, PLXNB1, PLXNB2, PLXNC1, and PLXND1 were selected to construct plasmids.

[0114] P240:mIL12-PLXNB2(802-1217)-Flag-eGFP P241:mIL12-PLXNA2(857-1306)-Flag-eGFP P242:mIL12-PLXNB1(1068-1558)-Flag-eGFP P433:mIL12-PLXNC1(661-1003)-Flag-eGFP P434:mIL12-PLXND1(886-1341)-Flag-eGFP

[0115] The amino acid sequence of PLXNB2(802-1217) is as follows: PPVITRIQPETGPLGGGIRITILGSNLGVQAGDIQRISVAGRNCSFQPERYSVSTRIVCVIEAAETPFTGGVEVDVFGKLGRSPPNVQFTFQQPKPLSVEPQQGPQAGGTTLTIHGTH LDTGSQEDVRVTLNGVPCKVTKFGAQLQCVTGPQATRGQMLLEVSYGGSPVPNPGIFFTYRENPVLRAFEPLRSFASGGRSINVTGQGFSLIQRFAMVVIAEPLQSWQPPREAESLQP MTVVGTDYVFHNDTKVVFLSPAVPEEPEAYNLTVLIEMDGHRALLRTEAGAFEYVPDPTFENFTGGVKKQVNKLIHARGTNLNKAMTLQEAEAFVGAERCTMKTLTETDLYCEPPEVQPPPKRRQKRDTTHNLPEFIVKFGSREWVLGRVEYDTRVSDVPLSLILPLVIVPMVVVIAVSVYCYWRKSQQAEREYEKIKSQLEGLEESVRDRCKKEFTDLMIEMEDQTNDV (SEQ ID NO: 9)

[0116] The amino acid sequence of PLXNA2(857-1306) is as follows: NPQITEILTVSGPPEGGTRVTIHGVNLGLDFSEIAHHVQVAGVPCTPLPGEYIIAEQIVCEMGHALVGTTSGPVRLCIGECKPEFMTKSHQQYTFVNPSVLSLNPIRGPESGGT MVTITGHYLGAGSSVAVYLGNQTCEFYGRSMSEIVCVSPPSSNGLGPVPVSVSVDRAHVDSNLQFEYIDDPRVQRIEPEWSIASGHTPLTITGFNLDVIQEPRIRVKFNGKESVN VCKVVNTTTLTCLAPSLTTDYRPGLDTVERPDEFGFVFNNVQSLLIYNDTKFIYYPNPTFELLSPTGVLDQKPGSPIILKGKNLCPPASGGAKLNYTVLIGETPCAVTVSETQLLCEPPNLTGQHKVMVHVGGMVFSPGSVSVISDSLLTLPAIVSIAAGGSLLLIIVIIVLIAYKRKSRENDLTLKRLQMQMDNLESRVALECKEAFAELQTDINELTSD (SEQ ID NO: 10)

[0117] The amino acid sequence of PLXNB1(1068-1558) is as follows: PAPLIHSVEPLTGPVDGGTRVTIRGSNLGQHVQDVLGMVTVAGVPCAVDAQEYEVSSSLVCITGASGEEVAGATAVEVPGRGRGVSEHDFAYQDPKVHSIFPARGPRAGGTRLTLNGSKLLTGR LEDIRVVVGDQPCHLLPEQQSEQLRCETSPRPTPATLPVAVWFGATERRLQRGQFKYTLDPNITSAGPTKSFLSGGREICVRGQNLDVVQTPRIRVTVVSRMLQPSQGLGRRRRVVPETACSLGP SCSSQQFEEPCHVNSSQLITCRTPALPGLPEDPWVRVEFILDNLVFDFATLNPTPFSYEADPTLQPLNPEDPTMPFRHKPGSVFSVEGENLDLAMSKEEVVAMIGDGPCVVKTLTRHHLYCEPPVEQPLPRHHALREAPDSLPEFTVQMGNLRFSLGHVQYDGESPGAFPVAAQVGLGVGTSLLALGVIIIVLMYRRKSKQALRDYKKVQIQLENLESSVRDRCKKEFTDLMTEMTDLTSDL (SEQ ID NO: 11)

[0118] The amino acid sequence of PLXNC1(661-1003) is as follows: VFYIKSIEPQKVSTLGKSNVIVTGANFTRASNITMILKGTSTCDKDVIQVSHVLNDTHMKFSLPSSRKEMKDVCIQFDGGNCSSVGSLSYIALPHCSLIFPATTWISGGQNITMMGRNFDVIDNLIISHELKGNINVSEYCVATYCGFLAPSLKSSKVRTNVTVKLRVQDTYLDCGTLQYREDPRFTGYRVESEVDTELEVKIQKENDNFNISKKDIEITLFHGENGQLNCSFENITRNQDLTTILCKIKGIKTASTIANSSKKVRVKLGNLELYVEQESVPSTWYFLIVLPVLLVIVIFAAVGVTRHKSKELSRKQSQQLELLESELRKEIRDGFAELQMDK (SEQ ID NO: 12)

[0119] The amino acid sequence of PLXND1(886-1341) is as follows: GTCPAPEIHAIEPLSGPLDGGTLLTIRGRNLGRRLSDVAHGVWIGGVACEPLPDRYTVSEEIVCVTGPAPGPLSGVVTVNASKEGKSRDRFSYVLPLVHSLEPTMGPKAGGTRITI HGNDLHVGSELQVLVNDTDPCTELMRTDTSIACTMPEGALPAPVPVCVRFERRGCVHGNLTFWYMQNPVITAISPRSPVSGGRTITVAGERFHMVQNVSMAVHHIGREPTLCKVL NSTLITCPSPGALSNASAPVDFFINGRAYADEVAVAEELLDPEEAQRGSRFRLDYLPNPQFSTAKREKWIKHHPGEPLTLVIHKEQDSLGLQSHEYRVKIGQVSCDIQIVSDRIIHCSVNESLGAAVGQLPITIQVGNFNQTIATLQLGGSETAIIVSIVICSVLLLLSVVALFVFCTKSRRAERYWQKTLLQMEEMESQIREEIRKGFAELQTDMTDLTKELN (SEQ ID NO: 13)

[0120] Fusion protein expression, EV collection, and WB detection were performed as in Example 3, and the anti-Flag WB results are shown in Figure 6. The experimental results show that all five fusion proteins mentioned above were expressed in EVs, and among them, PLXNA2 was significantly enriched in EVs.

[0121] The particle concentration and eGFP content of the three samples, P240, P241, and P242, were detected, and the copy number of eGFP on each EV was calculated. The results are shown in Figure 7. It was found that the EVs of the three samples, P240, P241, and P242, all carried different amounts of the fusion protein.

[0122] [Example 6] Identification of polypeptide sequences important for EV enrichment Example 3 demonstrated that the truncated PLXNA1 still plays a significant role in enriching target proteins in EVs. Sequence alignment revealed a relatively conserved seven-amino acid sequence, SDSLLTL (SEQ ID NO: 14), between the transmembrane domain and IPT domain of PLXNA1 and PLXNA2 (see Figure 8). This sequence is presumed to play a special role in the active selection of EVs.

[0123] Therefore, plasmids P166 and P167 were constructed (Figure 9A). Compared to P166, P167 lacked the conserved 8-amino acid sequence. WB results shown in Figure 9B indicated that the expression levels of the fusion protein in cell supernatants and EVs were significantly reduced after cleavage of the SDSLLTL (SEQ ID NO: 14) sequence (positions 1237-1243 of the full-length sequence).

[0124] Furthermore, total IL-12 in the supernatant and IL-12 in EVs were detected by ELISA. The percentage of IL-12 in the supernatant and EVs was significantly higher in the P166 sample than in the P167 sample. This suggests that this portion of PLXNA1 (1237-1243) is a key sequence and plays an important role in actively sorting the fusion protein onto EVs.

[0125] [Example 7] Effect of key sequence mutations on protein selection Mutation studies were carried out on the key sequence SDSLLTL (SEQ ID NO: 14) to examine how the amino acids at each position in this sequence affect the selection efficiency of the fusion protein.

[0126] First, each amino acid in this sequence was mutated to alanine, one by one, resulting in the following sequence: ADSLLTL (SEQ ID NO: 21) SASLLTL (SEQ ID NO: 22) SDALLTL (SEQ ID NO: 23) SDSALTL (SEQ ID NO: 24) SDSLATL (SEQ ID NO: 25) SDSLLAL (SEQ ID NO: 26) SDSLLTA (SEQ ID NO: 27)

[0127] Next, we transiently transfected HEK293 cells with the constructed plasmid, extracted EVs from the supernatant, and performed WB experiments (anti-Flag) on ​​EVs and cell lysates at equivalent protein amounts. The results showed that mutations at P304 (S1237A), P307 (L1240A), P308 (L1241A), P309 (T1242A), and P310 (L1243A) significantly affected the sorting of the fusion protein into EVs, significantly reducing the sorting efficiency. On the other hand, mutations at P305 (D1238A) and P306 (S1239A) did not significantly affect the sorting efficiency (Figure 10A).

[0128] Similar amino acid mutations were also made at different positions in the key sequence. The resulting sequences are as follows: SESLLTL (SEQ ID NO: 28) SDSLITL (SEQ ID NO: 29)

[0129] The results showed that P311 (D1238E) and P312 (L1241I) hardly changed the selection efficiency of the fusion protein (FIG. 10B).

[0130] One, two, or three alanines were inserted between the amino acid S at position 1239 and the amino acid L at position 1240. The sequences are as follows: SDSALLTL (SEQ ID NO: 30) SDSAALLTL (SEQ ID NO: 31) SDSAAALLTL (SEQ ID NO: 32)

[0131] The results showed that inserting different lengths of amino acids into this position altered the selection efficiency of the fusion protein (Fig. 10C).

[0132] [Example 8] Different target proteins fused to PLXNA1 truncations can all be sorted into EVs To verify whether different proteins or polypeptides fused to POIs could be sorted into EVs, we constructed plasmids fused to different polypeptides and transiently transfected them into HEK293 cells. Cell lysates and EVs were collected, and the target proteins were detected by WB. Here, S-RBD represents the COVID-19 virus spike protein, eGFP represents green fluorescent protein, nanoluc represents a novel luciferase approximately 171 amino acids in size, and mCD3scFab represents a single-chain antigen-binding fragment of mouse CD3. The sequences of the fusion proteins carried by the plasmids are as follows:

[0133] P322: S-RBD-PLXNA1(863-1300)-Flag-eGFP; the sequence of the fusion protein is as follows: (SEQ ID NO: 16)

[0134] P323: mCD3scFab-PLXNA1(863-1300)-Flag-eGFP; the sequence of the fusion protein is as follows:

[0135] P324: eGFP-PLXNA1(863-1300)-Flag-nanoluc; the sequence of the fusion protein is as follows: (SEQ ID NO: 18)

[0136] As shown in Figure 11, the WB detection results showed that all of the fusion proteins were successfully detected as components of EVs.

[0137] [Example 9] Detection of biological activity of target protein in fusion protein To verify whether the fusion-expressed extracellular protein (POI) retained biological activity, mononuclear cells (PBMCs) were collected from the spleens of C57BL / 6 mice. HEK293 cells were transiently transfected with the P235 plasmid, and EVs were extracted from the supernatant. The content of the EV product, IL-12, was quantified by ELISA. The in vitro activity of P235EVs was determined using recombinant mIL-12 protein as a control. The results, shown in Figure 12, demonstrate that mIL-12 fused with the PLXNA1 truncated construct retained its activity.

[0138] [Example 10] Detection of biological activity of target protein in intracellular domain of fusion protein To verify whether the fusion-expressed intracellular protein retained biological activity, P324 EVs were extracted (see Example 8, P324:eGFP-PLXNA1(863-1300)-Flag-nanoluc) and luciferase activity was detected in EVs with different particle numbers. 10 μl of exosome sample was added to 10 μl of fluorescent substrate working solution, in which the substrate (Nano-Glo® Luciferase Assay Substrate) was diluted 1:50 with the corresponding buffer (Nano-Glo® Luciferase Assay Buffer). After 5 minutes of incubation, luminescence readings were measured using a microplate reader. As shown in Figure 13, there was a good linear relationship between luciferase activity and EV particle number.

[0139] Example 11: Loading of protein of interest (POI) via NbALFA / ALFA interaction based on EV fusion partners of the present invention The above examples demonstrate that the EV fusion partner of the present invention can load EVs with a target protein fused to it. The target protein can be an affinity tag, and the target molecule of interest can be loaded onto the EV surface by linking the affinity tag-binding moiety to the target molecule. In other words, by producing only one type of engineered EV and co-incubating it in vitro with target molecules labeled with different specific tags, rapid loading of the target molecule can be achieved, resulting in the production of EVs loaded with different proteins. See the schematic diagram in Figure 14. In the figure, A and B are molecules that can be non-covalently linked to each other.

[0140] I. Construction of NbALFA-PLXΔ-engineered extracellular vesicles (NbALFA-PLXΔ-EVs) The NbALFA / ALFA tag is an affinity tag combination known in the art (Hansjorg Götzke et al., 2019, Nat. Commun.; EP3849996A1). The PLXΔ sequence is the same as the sequence of the P292(863-1316) fragment (SEQ ID NO: 2) described in Example 3.

[0141] Plasmid construction: A plasmid was designed to express the fusion protein NbALFA-Flag-PLXΔ.

[0142] EV generation: The plasmids were introduced into HEK293 to construct mixed clone cells, which were then cultured. The cell supernatants were collected to extract EVs (also referred to herein as NbALFA-PLXΔ-EVs or NbALFA EVs), and cell lysates were also collected.

[0143] WB detection: NbALFA EVs and wild-type 293 control EVs were subjected to a WB experiment using equivalent protein amounts to analyze flag expression. The results are shown in Figure 15.

[0144] Nanoflow cytometry: NbALFA EVs were labeled with an FITC-conjugated anti-Flag antibody, and the Flag-positive rate was measured by nanoflow cytometry. The results are shown in Figure 16. To exclude nonspecific effects of the antibody, a homologous isotype FITC-conjugated antibody that does not specifically bind to Flag was used as the NbALFA EV isotype control. Specifically, an isotype antibody mouse IgG2b type: FITC plus mouse IgG2b isotype control (Proteintech / FITC-65128) was used.

[0145] As shown in Figure 15, no flag signal was detected in wild-type 293 (WT) cell lysates or EVs, but flag signals were detected in cell lysates and EVs transfected with NbALFA-Flag-PLXΔ, indicating that PLXΔ was enriched in EVs. As shown in Figure 16, the positive rate of NbALFA-EVs was over 70%.

[0146] II. Loading of GFP into NbALFA-PLXΔ-EVs Construction of GFP-ALFA: A plasmid was constructed, and the ALFA-conjugated GFP recombinant protein was expressed in prokaryotic cells and quantified.

[0147] EV generation: Mixed-type clonal cells expressing NbALFA-PLXΔ were cultured, and the cell supernatant was collected to extract EVs. Wild-type 293EVs were used as a negative control.

[0148] Nanoflow cytometry: EV particle concentrations were measured by nanoflow cytometry.

[0149] In vitro labeling: 1E+9 NbALFA-PLXΔ-EVs, 1% EV-free serum (FBS), and GFP-ALFA protein molecules (molecule-to-EV particle ratios of 100:1, 1000:1, and 10000:1, respectively) were added to a 100 μL PBS reaction system. The mixture was then thoroughly mixed and incubated at 25°C for 30 minutes. Excess molecules were removed by size exclusion.

[0150] Nano-flow cytometry: GFP mean fluorescence intensity was measured by nano-flow cytometry.

[0151] As a result, as shown in Figure 17, GFP signals were detected on the surface of EVs, demonstrating that GFP-ALFA was effectively loaded onto NbALFA-PLXΔ-EVs, and the fluorescence intensity increased as the number of GFP molecules increased.

[0152] III. Loading of RVG polypeptides into NbALFA-PLXΔ-EVs Preparation of RVG-HA-ALFA polypeptide: RVG-HA-ALFA was synthesized and quantified.

[0153] EV generation: Mixed-type clonal cells expressing NbALFA-PLXΔ were cultured, and the cell supernatant was collected to extract EVs. Wild-type 293EVs were used as a negative control.

[0154] Nanoflow cytometry: EV particle concentrations were measured by nanoflow cytometry.

[0155] In vitro labeling: 1E+9 NbALFA-PLXΔ-EVs, 1% EV-free serum (FBS), and RVG-HA-ALFA polypeptide molecules (molecule-to-EV particle ratios of 10:1, 100:1, 5000:1, 10000:1, 5000:1, 10000:1, and 50000:1, respectively) were added to a 100 μL PBS reaction system. The mixture was then thoroughly mixed and incubated at 25°C for 30 minutes. Excess molecules were removed by size exclusion.

[0156] Nanoflow cytometry: RVGs were labeled with FITC-conjugated anti-HA antibody and loaded into EVs, and the HA mean fluorescence intensity was measured by nanoflow cytometry.

[0157] As a result, as shown in Figure 18, compared with the negative control, RVG-ALFA was effectively labeled onto NbALFA-PLXΔ-EV, and labeling tended to reach saturation when the input ratio reached 5000.

[0158] Target activity detection: RVG-labeled vesicles were stained with the lipophilic green fluorescent reagent CMG (CellMask™ Green Plasma Membrane Stain, Invitrogen / C37608) and then co-incubated with mouse neuroblastoma cell line (N2a). The input ratios of extracellular vesicle particles to cells were set to 0, 500, 1000, 2500, 5000, 7500, and 10000, respectively, and the cells were incubated in an incubator for 2 hours. Afterwards, excess vesicles were washed with PBS, and CMG fluorescence intensity was measured by nanoflow cytometry. As shown in Figure 19, RVG-labeled vesicles had a stronger affinity for the receptor N2a cells compared to the wild-type control.

[0159] IV. Loading of trastuzumab scFv fragments into NbALFA-PLXΔ-EVs Synthesis of scFv-HA-ALFA polypeptide: A plasmid was constructed, and the ALFA-binding scFv recombinant protein (ALFA was located at the C-terminus of the scFv) was expressed in eukaryotic cells and quantified.

[0160] EV generation: Mixed-type clonal cells expressing NbALFA-PLXΔ were cultured, and the cell supernatant was collected to extract EVs. Wild-type 293EVs were used as a negative control.

[0161] Nanoflow cytometry: EV particle concentrations were measured by nanoflow cytometry.

[0162] In vitro labeling: 1E+11 NbALFA-PLXΔ-EVs, 1% EV-free serum (FBS), and scFv-HA-ALFA protein molecules (molecule to EV particle ratio of 10,000:1) were added to a 100 μL PBS reaction system. The mixture was then thoroughly mixed and incubated at 25°C for 30 minutes. Excess molecules were removed by size exclusion.

[0163] Nanoflow cytometry: scFv was labeled with FITC-conjugated anti-HA antibody and loaded into EVs, and the HA mean fluorescence intensity was measured by nanoflow cytometry.

[0164] As a result, as shown in Figure 20, scFv-ALFA was effectively labeled onto NbALFA-PLXΔ-EV, compared to the negative control and the isotype control (mouse IgG1 isotype control monoclonal antibody, Proteintech / 66360-1-Ig).

[0165] Target activity detection: scFv-labeled vesicles were stained with the lipophilic green fluorescent reagent CMG and then incubated with a breast cancer cell line (SK-BR-3). The input ratios of extracellular vesicle particles to cells were set at 0, 2500:1, 5000:1, 7500:1, 10000:1, 12500:1, 15000:1, 17500:1, 20000:1, 22500:1, and 25000:1, respectively, and the cells were incubated in an incubator for 2 hours. After washing the excess vesicles with PBS, the CMG fluorescence intensity was measured by nanoflow cytometry. As shown in Figure 21, the scFv-labeled vesicles showed stronger affinity for the receptor SK-BR-3 compared to the wild-type control.

[0166] V. NbALFA-PLXΔ-engineered extracellular vesicles labeled with target molecules have good stability Labeling NbALFA-PLXΔ-EVs with GFP-ALFA in vitro: In vitro labeling experiments were performed according to the method described in Section I, and GFP mean fluorescence intensity was measured by nanoflow cytometry.

[0167] Freeze-thaw cycle test: GFP-labeled NbALFA-PLXΔ-EVs were completely frozen at -80°C (approximately 15 minutes), then removed and allowed to thaw at room temperature. This was recorded as one freeze-thaw cycle. Eight freeze-thaw cycles were performed in this manner. The mean GFP fluorescence intensity after the first, third, fifth, and eighth freeze-thaw cycles was measured by nanoflow cytometry.

[0168] As a result, as shown in Figure 22, the labeled molecules remained stably bound before the eighth freeze-thaw cycle, but the fluorescence of the labeled molecules slightly decreased after the eighth freeze-thaw cycle.

[0169] Measurement of storage conditions: GFP-labeled NbALFA-PLXΔ-EVs were stored at 4°C or -80°C, and were removed at 2 and 4 weeks, respectively, and left at room temperature for 1 hour. The mean fluorescence intensity of GFP was then measured by nanoflow cytometry.

[0170] As a result, as shown in FIG. 23, the labeled molecule was stably bound within a storage period of 4 weeks at 4° C. or −80° C.

[0171] Example 12: Labeling of EVs with different protein combinations In vitro simultaneous labeling of NbALFA-PLXΔ-EVs with nanoluc-HA-ALFA and nanoluc-V5-ALFA: nanoluc-HA-ALFA and nanoluc-V5-ALFA were mixed in equal molecular weights and subjected to in vitro labeling according to the method described above. WB detection was performed using HA-tag and V5-tag antibodies, respectively. When in vitro labeling was performed using multiple proteins, the ratios of the various proteins were adjusted as necessary. As shown in Figure 24, WB detection revealed that differently tagged target molecules were simultaneously labeled into the endoplasmic reticulum.

[0172] Example 13: Loading of Protein of Interest (POI) via Biotin / Avidin Interaction Based on EV Fusion Partners of the Invention I. Construction of mSA-PLXΔ engineered extracellular vesicles (mSA-PLXΔ-EVs) Plasmid construction: A plasmid was designed to express the fusion protein mSA-Flag-PLXΔ (where mSA is the abbreviation for monomeric strepavidin).

[0173] EV generation: The plasmids were introduced into HEK293 to construct mixed clone cells, which were then cultured. The cell supernatant was collected to extract EVs (also referred to herein as mSA-PLXΔ-EVs or mSA EVs), and cell lysates were also collected.

[0174] WB detection: mSA EVs and wild-type 293 control EVs were subjected to WB experiments using equivalent amounts of protein to analyze flag expression.

[0175] Nanoflow cytometry: mSA EVs were labeled with FITC-conjugated anti-flag antibody, and the flag-positive rate was measured by nanoflow cytometry.

[0176] As shown in Figure 25, WB detection results showed that no flag signal was detected in wild-type 293 (WT) cell lysates or EVs, but flag signal was detected in cell lysates and EVs transfected with mSA-Flag-PLXΔ, indicating that PLXΔ was enriched in EVs. As shown in Figure 26, nanoflow cytometry measurements showed that the positive rate of mSA-EVs exceeded 70%.

[0177] II. Loading of biotin-modified molecules into mSA-PLXΔ engineered extracellular vesicles EV generation: Mixed clone cells expressing mSA-PLXΔ were cultured, and the cell supernatant was collected to extract EVs. Wild-type 293EVs were used as a negative control.

[0178] Nanoflow cytometry: EV particle concentrations were measured by nanoflow cytometry.

[0179] In vitro labeling: 1E+9 mSA-PLXΔ-EVs, 1% EV-free serum (FBS), and Alexa Fluor 488-PEG3K-biotin molecules (molecule to EV particle ratio of 10,000:1) were added to a 100 μL PBS reaction system. The mixture was then thoroughly mixed and incubated at 25°C for 30 minutes. Excess molecules were removed by size exclusion.

[0180] Nano-flow cytometry: The Alexa Fluor 488 fluorescence positive rate was measured by nano-flow cytometry.

[0181] As a result, as shown in Figure 27, biotin-488 was labeled in mSA-PLXΔ-EV compared to the negative control.

[0182] III. Loading of biotin-modified HA antibody into mSA-PLXΔ-EVs EV generation: Mixed clone cells expressing mSA-PLXΔ were cultured, and the cell supernatant was collected to extract EVs. Wild-type 293EVs were used as a negative control.

[0183] Nanoflow cytometry: EV particle concentrations were measured by nanoflow cytometry.

[0184] In vitro labeling: 1E+9 mSA-PLXΔ-EVs, 1% EV-free serum (FBS), and biotin-modified HA antibody (Invitrogen / 26183-BTIN) were added to a 100 μL PBS reaction mixture (molecule-to-EV particle ratio: 10,000:1). The mixture was then thoroughly mixed and incubated at 25°C for 30 minutes. Excess molecules were removed by size exclusion.

[0185] Binding to FITC-conjugated HA polypeptide (Ruixibio / R-YAF-010): ER labeled with the above-mentioned HA antibody was mixed with FITC-conjugated HA polypeptide (molecule to EV particle ratio of 10,000:1) and incubated at 25°C for 30 minutes. Excess molecules were removed by size exclusion. FITC fluorescence positivity was measured by nanoflow cytometry.

[0186] As a result, as shown in Figure 28, endoplasmic reticulum labeled with anti-HA antibody was labeled with FITC-HA due to the affinity of mSA-biotin.

[0187] Example 14: Loading of protein of interest (POI) via Strep II / Streptactin interaction based on the EV fusion partner of the present invention Expression of FITC-streptactin: A plasmid was constructed to express streptactin in prokaryotes and label it with FITC fluorescent molecules.

[0188] EV generation: A Strep II-flag-PLXΔ plasmid was engineered and transfected into HEK293 cells to generate mixed-type clones. The supernatants were collected and EVs were extracted from the cells. Wild-type 293EVs were used as a negative control.

[0189] Nanoflow cytometry: EV granule concentration and flag-positive rate were measured by nanoflow cytometry. As shown in Figure 29, the positive rate of Strep II-flag-PLXΔ-EV exceeded 80%.

[0190] In vitro labeling: 1E+9 mSA-PLXΔ vesicles, 1% EV-free serum (FBS), and FITC-streptactin molecules (molecule-to-EV particle ratio: 10,000:1) were added to a 100 μL PBS reaction mixture, which was then mixed thoroughly and incubated at 25°C for 30 minutes.

[0191] FITC detection: GFP was detected by nanoflow cytometry. As shown in Figure 30, FITC-streptactin effectively labeled EVs, with a positive rate of 73%.

[0192] All publications and patents mentioned in this application are incorporated herein by reference. Various modifications and variations made to the methods and compositions described herein without departing from the scope and spirit of the invention will be apparent to those skilled in the art. Although the invention has been described in terms of specific preferred embodiments, it should be understood that the invention sought to be protected should not be unduly limited to these specific embodiments. Indeed, various variations for carrying out the described modes of the invention that are obvious to those skilled in the art are intended to be within the scope of the claims.

Claims

1. 1. An engineered extracellular vesicle comprising a fusion protein, The fusion protein comprises a fusion partner that is a protein or a variant thereof that is located on the extracellular vesicle membrane, and preferably the fusion protein further comprises a target protein.

2. The fusion partner may be 1) It is a type I transmembrane protein; 2) the transmembrane region and the adjacent intracellular region of the transmembrane protein comprise an α-helix having a length of 20 to 200 amino acids, preferably 30 to 90 amino acids, more preferably 40 to 80 amino acids, and a part or all of the transmembrane region is located in the α-helix; and 3) The extracellular vesicle according to claim 1, characterized in that the extracellular region contains 1 to 5 IPT domains.

3. The extracellular vesicle of claim 1 or 2, wherein the fusion partner is selected from the group consisting of PLXNA, PLXNB, PLXNC, PLXND family members and variants thereof.

4. The extracellular vesicle of claim 3, wherein the fusion partner is selected from the group consisting of PLXNA1, PLXNA2, PLXNA3, PLXNA4, PLXNB1, PLXNB2, PLXNC1, PLXND1 and variants thereof, preferably selected from the group consisting of PLXNA1, PLXNA2 and variants thereof.

5. The extracellular vesicle of claim 4, wherein the fusion partner comprises a sequence selected from SEQ ID NOs: 2 to 6.

6. The extracellular vesicle according to any one of claims 1 to 5, wherein the target protein is located upstream or downstream of the fusion partner.

7. The extracellular vesicle according to any one of claims 1 to 6, wherein the target protein is a therapeutic peptide, a targeting peptide, an affinity tag, or a linker for linking a therapeutic compound.

8. the therapeutic peptide is an antibody and / or a cytokine; For example, the cytokine may be a human interleukin family member (e.g., IL-2, IL-7, IL-10, IL-11, IL-12, IL-15, and IL-23), a tumor necrosis factor family member (e.g., TNF, LTA, LTB, FASLG, TNFSF8, TNFSF9, TNFSF10, TNFSF11, TNFSF12, TNFSF13, TNFSF14, TNFSF15, TNFSF18, and EDA), an interferon (IFN-α, INF-β, and INF-β), or an IFN-α family member (e.g., IFN-α, INF-β, and INF-β).

8. The extracellular vesicle of claim 7, wherein the extracellular vesicle is selected from the group consisting of T cell activators (e.g., 4-1BB, OX40, CD28, CD40, CD40L, CD47, CD27, CD70, CD80, CD86, GITRL, ICOSL, CD155, CD112, TIM-3, BTLA), and other cytokines (e.g., G-CSF, EPO, TPO, GM-CSF, EGF, bFGF, FVIIa, ATIII, TNK, α-glucosidase, BMP-2, hirudin).

9. The extracellular vesicle of claim 7, wherein the targeting peptide is a polypeptide fragment that targets a cell selected from the group consisting of an antibody or an antigen-binding fragment thereof, a cell surface receptor and its ligand.

10. The extracellular vesicle of claim 7, wherein the affinity tag is selected from the group consisting of His tag, glutathione-S-transferase (GST), S-peptide, ZZ domain, albumin binding domain (ABD), HA, Myc, FLAG™, maltose binding protein (MBP), calmodulin-binding peptide (CBP), SUMO, streptococcal protein G (Protein G), and Staphylococcus aureus protein A (Protein A).

11. For example, the extracellular vesicle according to any one of claims 1 to 10, comprising two or more fusion proteins each independently containing a different target protein.

12. The extracellular vesicle according to any one of claims 1 to 11, wherein the extracellular vesicle encapsulates a therapeutic agent selected from the group consisting of therapeutic peptides, polynucleotides, and small molecule compounds.

13. A pharmaceutical composition comprising the extracellular vesicles of any one of claims 1 to 12 and a pharmaceutically acceptable carrier.

14. An engineered cell for producing extracellular vesicles according to any one of claims 1 to 12.

15. 15. The cell of claim 14, comprising a polynucleotide encoding said fusion protein, preferably wherein said polynucleotide is integrated into the genome.

16. A method for treating a disease, comprising administering the extracellular vesicle according to any one of claims 1 to 12, the pharmaceutical composition according to claim 13, or the cell according to claim 14 to a subject in need thereof.

17. A method for producing extracellular vesicles according to any one of claims 1 to 12, comprising: 1) providing a cell containing a polynucleotide encoding the fusion protein; 2) culturing the cells under conditions suitable for expression of the fusion protein; and 2) isolating the extracellular vesicles.

18. A method for delivering a protein of interest to a target cell, comprising: 1) providing the extracellular vesicles according to any one of claims 1 to 12, wherein the fusion protein comprises a target protein to be delivered; and 2) contacting the extracellular vesicles with the target cells.

19. A method for isolating extracellular vesicles according to any one of claims 1 to 12, comprising: 1) secreting the extracellular vesicles from cells capable of expressing the fusion protein containing an affinity tag; 2) contacting the extracellular vesicles with a binding agent capable of binding to the affinity tag; and 3) isolating the extracellular vesicles by binding of the affinity tag to the binding agent.

20. 1. An engineered transmembrane protein expressed by an exogenous sequence, comprising: The transmembrane protein comprises a fragment represented by the following formula (I): Xaa １ …Xaa ｎ Xaa （ｎ＋１） Xaa （ｎ＋２） Xaa （ｎ＋３） Xaa （ｎ＋４） [Formula I] (Sequence Number 15) [In the formula, Xaa １ is selected from the group consisting of S, T, C, N, Y and Q, and Xaa ２ ~Xaa ｎ is any amino acid, where n is an integer from 1 to 20; （ｎ＋１） , Xaa （ｎ＋２） and Xaa （ｎ＋４） are each independently selected from the group consisting of L, I, G, A and V; （ｎ＋３） is selected from the group consisting of T, S, C, N, Y and Q. Preferably, the fusion protein wherein the fragment represented by formula (I) is located upstream of the transmembrane region.

21. 21. The fusion protein of claim 20, wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, more preferably 1, 2, 3, 4 or 5.

22. Xaa １ is selected from the group consisting of S, T, C, and N; Xaa （ｎ＋１） , Xaa （ｎ＋２） and Xaa （ｎ＋４） are each independently selected from the group consisting of L and I; Xaa （ｎ＋３） is selected from the group consisting of T, S, C and N.

23. Xaa １ is S, Xaa （ｎ＋１） , Xaa （ｎ＋２） and Xaa （ｎ＋４） is L, Xaa （ｎ＋３） is T.

24. The fusion protein of claim 23, wherein the fragment represented by formula (I) is SDSLLTL (SEQ ID NO: 14).

25. The fusion protein of any one of claims 20 to 24, wherein the fusion protein comprises a sequence selected from SEQ ID NOs: 2 to 6.

26. A polynucleotide encoding the fusion protein according to any one of claims 20 to 25.

27. An engineered extracellular vesicle comprising the fusion protein of any one of claims 20 to 25.

28. A method for loading a molecule of interest into the extracellular vesicles according to any one of claims 1 to 12, comprising: contacting the target molecule with the extracellular vesicles, wherein said molecule of interest and said fusion partner are each linked to a moiety A and a moiety B that binds to moiety A by affinity.

29. 29. The method of claim 28, wherein said portion A and said portion B are selected from the group consisting of NbALFA / ALFA, biotin / avidin, strep II / streptactin, intein N / C, SpyCatcher / Spy, and protein A / Fc.

30. 30. The method of claim 28 or 29, wherein the molecule of interest is a therapeutic peptide, a targeting peptide, an affinity tag, or a linker for attaching a therapeutic compound.

Images