Engineered extracellular vesicles and uses thereof

Abstract

This invention provides engineered extracellular vesicles, pharmaceutical compositions comprising said engineered extracellular vesicles, methods for their preparation, and applications. The engineered extracellular vesicles can be used for drug delivery, expression of target proteins in extracellular vesicles, or purification of extracellular vesicles.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to an engineered extracellular vesicle, its preparation method, and its application. Background Technology

[0002] Extracellular vesicles (EVs) are membranous vesicles secreted by cells and capable of being taken up by recipient cells, with a diameter of approximately 30-1000 nm. EVs serve as carriers for the intercellular transport of biomolecules such as proteins, RNA, and lipids, and are important mediators of cell-to-cell communication. Because EVs are widely present in various bodily fluids, they can be used as a sample source for liquid biopsies and are also considered a naturally occurring drug carrier.

[0003] In the research and application of EVs, the preparation of EVs with specific functions is a key technical aspect. Taking drug delivery systems as an example, to improve the tissue targeting of EVs, it is necessary to highly express targeting molecules with high affinity for target cells on their surface. Alternatively, it may be necessary to deliver therapeutic peptides or proteins into EVs. For the development of EV in vitro diagnostic kits, it is necessary to obtain EVs loaded with specific analyte molecules as standards.

[0004] According to existing reports, approximately 20 proteins have been reported for use in 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 protein components into EVs. Summary of the Invention

[0005] The inventors of this application unexpectedly discovered a class of extracellular vesicle (EV) scaffold proteins during their research. These scaffold proteins are expressed at very low background levels in naturally secreted extracellular vesicles, but they exhibit unexpectedly excellent effects in EV construction, target protein display, and drug loading. Based on the above findings, the inventors completed this invention.

[0006] In a first aspect, the present invention provides an engineered extracellular vesicle (EV) comprising a transmembrane protein as a scaffold protein, the transmembrane protein being a member of the immunoglobulin superfamily, the transmembrane protein having a low basal expression level in unengineered extracellular vesicles.

[0007] In some embodiments of the present invention, the background expression level of the transmembrane protein in unengineered extracellular vesicles is lower than the background expression level of CD81. For example, the lower than the background expression level of CD81 means that in the mass spectrometry detection of total protein in the unengineered extracellular vesicles, the iBAQ value of CD81 is set to 1, and the ratio of the iBAQ value of the transmembrane protein to the iBAQ value of CD81 is less than 0.5, 0.4, 0.3, 0.2, 0.1, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02, or 0.01.

[0008] In some embodiments of the present invention, the transmembrane protein satisfies at least one of the following conditions:

[0009] (A) The transmembrane protein is similar to BCAM, meaning that when BCAM is used as a standard, the E value of the transmembrane protein compared to BCAM is <= 1.0E-4. The E value is calculated using formula (I), which is derived from the NCBI blast program.

[0010] E=K*m*n*exp(-lambda*S) (I)

[0011] Where K and lambda are constants related to the database and algorithm, m represents the length of the target sequence, n represents the size of the database, and the S value represents the homology between the two sequences. The higher the S score, the greater the homology between them.

[0012] (B) The transmembrane protein is a single-pass transmembrane protein, and the extracellular region of the single-pass transmembrane protein contains 1, 2, 3, 4, 5, 6, 7, 8, or 9 immunoglobulin domains (Ig domains); and

[0013] (C) The extracellular region of the transmembrane protein contains both an IgV domain and an IgC domain.

[0014] In some embodiments of the invention, the transmembrane protein satisfies a combination selected from the following: A and B, A and C, B and C, and A and B and C.

[0015] In some embodiments of the present invention, the transmembrane protein is selected from BCAM, MCAM, ICAM5, ALCAM, CD276, CADM1, VCAM1, CADM2, NECTIN4, CADM4, CADM3, AGER, CD96, and variants thereof.

[0016] In some embodiments of the present invention, the extracellular region of the transmembrane protein comprises 3, 4, or 5 immunoglobulin domains.

[0017] Specifically, the extracellular region of the transmembrane protein contains both an IgV domain and an IgC domain.

[0018] More specifically, the IgC domain is an IgC2 domain.

[0019] More specifically, the extracellular region of the transmembrane protein contains five immunoglobulin domains, which are two IgV domains and three IgC domains from the N-terminus to the C-terminus.

[0020] In some embodiments of the present invention, the transmembrane protein is selected from BCAM, MCAM, ALCAM, CD276, CADM1, CADM2, NECTIN4, CADM4, CADM3, AGER, CD96, and variants thereof;

[0021] Specifically, the transmembrane protein is selected from BCAM, MCAM, ALCAM, CADM1, CADM2, CADM4, CADM3, AGER, and variants thereof;

[0022] More specifically, the transmembrane protein is selected from BCAM, MCAM, ALCAM, and their variants.

[0023] In some embodiments of the present invention, the single transmembrane protein is a fusion protein containing a target polypeptide segment; preferably, the target polypeptide segment is located at the N-terminus or C-terminus of the fusion protein; preferably, the target polypeptide segment enables the fusion protein to have therapeutic, targeting, and / or affinity tagging functions.

[0024] In some embodiments of the present invention, the target polypeptide segment is a therapeutic polypeptide segment, for example, a member of the interleukin family (e.g., IL-2, IL-7, IL-10, IL-11, IL-12, IL-15, and IL-23), a member of the tumor necrosis factor family (e.g., TNF, LTA, LTB, FASLG, TNFSF8, TNFSF9, TNFSF10, TNFSF11, TNFSF12, TNFSF13, TNFSF14, TNFSF15, TNFSF18, and EDA), interferon (INF-α, INF-β, and INF-γ), or 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] In some embodiments of the present invention, the target polypeptide segment is a cell-targeting polypeptide segment; specifically, the cell-targeting polypeptide segment is selected from antibodies or their antigen-binding fragments, cell surface receptors, or ligands.

[0026] In some embodiments of the present invention, the target polypeptide segment is an affinity tag, specifically selected from His tag, glutathione thiotransferase (GST), S-peptide, ZZ domain, albumin-binding domain (ABD), HA, Myc, FLAG™, maltose-binding protein (MBP), calmodulin-binding peptide (CBP), SUMO, Streptococcus protein G (Protein G), and Staphylococcus aureus protein A (Protein A).

[0027] In some embodiments of the invention, the extracellular vesicles contain a therapeutic agent; in particular, the therapeutic agent is selected from therapeutic peptides, polynucleotides, and small molecule compounds.

[0028] In a second aspect, the present invention provides a pharmaceutical composition comprising the aforementioned extracellular vesicles and a pharmaceutically acceptable carrier.

[0029] In a third aspect, the present invention provides an engineered cell for producing the aforementioned extracellular vesicles.

[0030] In a fourth aspect, the present invention provides a method for treating a disease, comprising administering the extracellular vesicles, the pharmaceutical composition, or the cells to a subject in need of treatment.

[0031] In a fifth aspect, the present invention provides a method for preparing said extracellular vesicles, comprising:

[0032] 1) Express the single transmembrane protein in cells, and

[0033] 2) Isolate the extracellular vesicles.

[0034] In a sixth aspect, the present invention provides a method for delivering a therapeutic agent to target cells, comprising:

[0035] 1) Prepare the extracellular vesicles, wherein the transmembrane protein is a fusion protein containing an affinity tag, and

[0036] 2) Make the extracellular vesicles come into contact with the target cells.

[0037] In a seventh aspect, the present invention provides a method for isolating said extracellular vesicles, comprising:

[0038] 1) Express the transmembrane protein in cells, the transmembrane protein containing an affinity tag,

[0039] 2) Contact the extracellular vesicles with a binding agent capable of binding the affinity tag, and

[0040] 3) The extracellular vesicles are separated based on the binding of the affinity tag to the binding agent.

[0041] Unlike engineered extracellular vesicles reported in existing technologies, which preferentially select membrane proteins with high expression levels in EVs, this invention selects membrane proteins with very low or no basal expression in EVs. When using these proteins to construct EVs, there is less competition from endogenous proteins (e.g., non-fusion proteins of the EV itself), which helps these proteins (e.g., fusion proteins carrying the target protein) to be actively sorted into the EV, increasing their relative content in the EV and thus greatly improving the content of the active ingredient carried by the EV. Attached Figure Description

[0042] Figure 1 The expression of the EGFP-BCAM fusion protein in EVs is shown in Example 2, with PTGFRN and LAMP2B as controls.

[0043] Figure 2 The results of Western blot analysis of wild-type protein and fusion protein in total cellular protein and EV in Example 2 are shown. Figure 2 A) and the percentage of wild-type protein and fusion protein in the total of the two ( Figure 2 B).

[0044] Figure 3 The fluorescence detection results of EVs carrying the eGFP fusion protein in Example 3 are shown.

[0045] Figure 4 The results are from an ELISA assay, showing the expression level of the fusion protein carrying IL12 in EVs in Example 4.

[0046] Figure 5 The expression of different BCAM fusion proteins in EVs is shown.

[0047] Figure 6 The EC50 results of PBMCs stimulated by IL12 from different sources to produce IFN-γ are shown. Figure 6 A represents recombinant IL12 protein. Figure 6 B indicates EVs expressing PTGFRN-IL12. Figure 6 C represents the EV expressing BCAM-IL12.

[0048] Figure 7 The structures of some scaffold proteins of this invention are shown, derived from the Uniprot database.

[0049] Figure 8 The expression of the MCAM and EGFP fusion protein in EVs is shown in Example 8.

[0050] Figure 9 The detection results of IL12 carried by the captured EV are shown in Example 8.

[0051] Figure 10 The EC50 results of PBMCs stimulated by IL12 from different sources to produce IFN-γ are shown. Figure 10 A represents recombinant IL12 protein. Figure 10 B indicates the EV expressing BCAM-IL12. Figure 10 C indicates the EV expressing MCAM-IL12.

[0052] Figure 11 The results showed that fusion with BCAM and MCAM does not affect luciferase activity.

[0053] Figure 12 The structure of the BCAM truncated body is shown. Figure 12 A), the expression results of the fusion protein in cells and EVs ( Figure 12 B), and the activity determination of the full-length and truncated Δ4.

[0054] Figure 13 Example 11 shows the use of EV to carry DOX.

[0055] Figure 14 The DOX packet gradient experiment of Example 11 is shown.

[0056] Figure 15 The purification results of Example 12 are shown.

[0057] Figure 16 The experimental results of Example 13 are shown. Detailed Implementation

[0058] the term

[0059] Unless otherwise defined, all technical terms used herein have the same meaning as understood by one of ordinary skill in the art.

[0060] The term "extracellular vesicles (EVs)" as used in this article refers to vesicle-like bodies with a double-membrane structure that detach from the cell membrane or are secreted by cells. Their diameter ranges from 40 nm to 1000 nm, and they are mainly in the form of microvesicles (MVs) and exosomes (Exs). EVs are widely distributed in cell culture supernatants and various body fluids (blood, lymph, saliva, urine, semen, and breast milk), carrying a variety of cell-derived proteins, lipids, DNA, mRNA, miRNA, etc., and participating in processes such as intercellular communication, cell migration, angiogenesis, and immune regulation. The term "engineered extracellular vesicles" refers to artificially synthesized extracellular vesicles, extracellular vesicles produced by cells after artificial intervention, or extracellular vesicles produced by genetically engineered cells. The term "unengineered extracellular vesicles" refers to natural, unmodified extracellular vesicles secreted by cells under normal conditions (e.g., physiological conditions).

[0061] The term "scaffold protein" as used in this article refers to a protein that exists in its natural form in unengineered extracellular vesicles and can carry other proteins into the extracellular vesicles when forming fusion proteins with other proteins (target proteins / peptides).

[0062] The term "single transmembrane protein" as used herein refers to a protein that contains only one transmembrane segment, wherein the membrane is a lipid bilayer, which may be a cell membrane, organelle membrane, vesicle membrane, artificial lipid bilayer, etc.

[0063] The term "immunoglobulin domain" (Ig domain), sometimes also called an immunoglobulin-like domain, is an Ig domain named after immunoglobulin molecules. It contains approximately 70-110 amino acids and is classified according to size and function. Ig domains can be divided into IgV, IgC1, IgC2, or IgI. Most Ig domains are either variable Ig domains (IgV) or constant Ig domains (IgC). Some members of the IgSF (immunoglobulin superfamily) have Ig domains that are similar in amino acid sequence to the IgV domain but similar in size to the IgC domain; these are called IgC2 domains, while the standard IgC domain is called the IgC1 domain.

[0064] Extracellular vesicle proteins or transmembrane proteins as referred to herein include their variants. The term "variant" as used herein refers to a protein whose amino acid sequence differs from that of its naturally occurring counterpart but has the same or similar function. These protein variants are capable of expression in extracellular vesicles and of carrying target proteins / peptides into extracellular vesicles secreted by cells. Protein variants can arise from naturally occurring protein amino acids through deletion, addition, and / or substitution, and can also be truncated forms of the natural protein.

[0065] BCAM refers to basal cell adhesion molecule. MCAM refers to melanoma cell adhesion molecule, also known as cell surface glycoprotein MUC18 or CD146. ALCAM refers to activated leukocyte cell adhesion molecule, also known as CD166. CD276 is a cell surface antigen, also known as B7-H3. CADM1 refers to cell adhesion molecule 1. CADM2 refers to cell adhesion molecule 2. CADM3 refers to cell adhesion molecule 3. CADM4 refers to cell adhesion molecule 4. NECTIN4 refers to nectin cell adhesion molecule 4. AGER refers to advanced glycosylation endproduct-specific receptor. CD96 is a cell surface antigen.

[0066] As used herein, the term "therapeutic peptide fragment" refers to a protein or fragment or variant thereof with therapeutic activity, including but not limited to members of the human interleukin family (e.g., IL-2, IL-7, IL-10, IL-11, IL-12, IL-15, and IL-23), members of the tumor necrosis factor family (e.g., TNF, LTA, LTB, FASLG, TNFSF8, TNFSF9, TNFSF10, TNFSF11, TNFSF12, TNFSF13, TNFSF14, TNFSF15, TNFSF18, and EDA), interferons (INF-α, INF-β, and INF-γ), T cells, and other therapeutically active proteins. 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).

[0067] As used herein, the term "cell-targeting polypeptide segment" refers to a protein or fragment thereof, or a variant thereof, that has a cell-targeting effect. In some embodiments, the cell-targeting polypeptide segment is an antibody or an antigen-binding fragment thereof. In other embodiments, the cell-targeting polypeptide segment is a cell surface receptor or ligand.

[0068] As used in this article, the term "affinity tag" refers to a polypeptide segment capable of binding to a corresponding binding agent, including but not limited to His tag, glutathione thiotransferase (GST), S-peptide, ZZ domain, albumin-binding domain (ABD), HA, Myc, and FLAG. TM Maltose-binding protein (MBP), calmodulin-binding peptide (CBP), SUMO, Streptococcal protein G, and Staphylococcus aureus protein A. The term "binding agent" refers to a substance capable of specifically binding to an affinity tag.

[0069] The term "therapeutic agent" as used in this article refers to a substance with therapeutic effects, which may include nucleotides, amino acids, lipids, carbohydrates, small molecules, antibodies, enzymes, ligands, receptors, polypeptides, etc.

[0070] Although the numerical ranges and parameter approximations shown in the broad scope of this invention are intended to be approximated, the values ​​described in the specific embodiments are recorded as accurately as possible. However, any numerical value inherently contains a certain degree of error due to the standard deviation present in their respective measurements. Furthermore, all ranges disclosed herein should be understood to encompass any and all subranges contained therein. For example, the stated range “1 to 10” should be considered to include any and all subranges between the minimum value 1 and the maximum value 10 (inclusive); that is, all subranges beginning with a minimum value of 1 or greater, such as 1 to 6.1, and subranges ending with a maximum value of 10 or less, such as 5.5 to 10. Additionally, any references marked “incorporated herein” should be understood to be incorporated herein in their entirety.

[0071] It should also be noted that, as used herein, the singular form includes the plural form of the object it refers to, unless it is clearly and explicitly limited to a single object. The term "or" may be used interchangeably with the term "and / or" unless the context clearly indicates otherwise.

[0072] The term "object" as used in this article refers to mammals, such as humans, but can also be other animals, such as wild animals (e.g., herons, storks, cranes, etc.), livestock (e.g., ducks, geese, etc.) or laboratory animals (e.g., chimpanzees, monkeys, rats, mice, rabbits, guinea pigs, marmots, ground squirrels, etc.).

[0073] The compositions of the present invention can be administered by a variety of methods known in the art. Those skilled in the art will understand that the route and / or mode of administration will vary depending on the desired outcome. To administer the compounds of the present invention via a specific route of administration, it may be necessary to cover the compound with a material to prevent its inactivation, or to administer the compound co-with said material. For example, the compound may be administered to a subject in a suitable carrier, such as a liposome or diluent. Pharmaceutically acceptable diluents include saline solutions and aqueous buffers. Drug carriers include sterile aqueous solutions or dispersions and sterile powders for the provisional preparation of sterile, injectable solutions or dispersions. The use of such media and reagents for pharmaceutically active substances is known in the art.

[0074] The compositions of the present invention may also contain excipients, such as preservatives, humectants, emulsifiers, and dispersants. The presence of microorganisms can be prevented by the sterilization process described above and by including a variety of antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, etc. It may also be desirable to include isotonic agents in the compositions, such as sugars, sodium chloride, etc. Furthermore, prolonged absorption of injectable drug forms can be achieved by including agents that delay absorption, such as aluminum monostearate and gelatin.

[0075] Example

[0076] The following detailed embodiments further illustrate some preferred implementations and aspects of the present invention. These embodiments should not be construed as limiting the scope of the invention.

[0077] Example 1: Materials and Methods

[0078] The materials used in the embodiments of the present invention and their sources are shown in Table 1.

[0079] Table 1

[0080]

[0081]

[0082] Experimental methods

[0083] 1. Cell transfection

[0084] The progenitor cell line used in this embodiment of the invention is Expi293F cells (A14527CN, Thermo Fisher, America). One day before transfection, cells were trypsinized and counted, passaged, and plated to a total cell count of 1.5 × 10⁶ cells in 5 mL plates. The next day, transfection solutions were prepared: Solution A contained 1 μg sgAAVS1, 4 μg pDonor, and 125 μl Opti-MEM medium; after mixing, the mixture was allowed to stand for 5 min. Solution B contained 12.5 μl PEI (working solution concentration 1 μg / μl) and 125 μl Optipro-MEM medium; after mixing, the mixture was allowed to stand for 5 min. One hour before transfection, the cell culture medium was replaced with Opti-MEM, and the cells were placed in a cell culture incubator. Solution B was added dropwise to Solution A, gently mixed, and allowed to stand for 20 minutes. The prepared transfection reagent was added dropwise to T25 Flasks. The cells were cultured at 37°C and 5% CO₂ for 48 h.

[0085] Prepare fresh KOP293-Ex medium containing a final concentration of 2 μg / mL puromycin. Change the medium every 2-3 days. If the cells are overconcentrated, digest them and passage them 1:1, continuing to add puromycin for selection until the control group cells are mostly killed. After the control group cells are killed, transfer the experimental group cells to KOP293-Ex + 5% FBS (containing 2 μg / mL puromycin) medium. Incubate at 37°C with 5% CO2. Change the medium every 2-3 days until the cell confluence reaches 60-80%. Passage at a 1:1 ratio.

[0086] Gradually reduce serum levels and then culture in suspension after acclimatization.

[0087] 2. EV extraction

[0088] Remove the Expi293F cell culture flask from the incubator, shake well, and pipette a small amount of cells into a 1.5 mL EP tube. Pipette 20 μL of the cell suspension into a new EP tube, add 20 μL of tolphenol blue, mix thoroughly, and add 10 μL of the cell suspension to a cell counting chamber. Count the cells using a BIO-RAD cell counter. When the cell density is 4–6 × 10⁶ cells / mL and the viability is above 95%, dilute the cell suspension with KOP293-EX medium (Zhuhai Kairui Biotechnology Co., Ltd., K03256) to adjust the cell density to 2 × 10⁶ cells / mL. Transfer 300 mL of the cell suspension to a 1000 mL Erlenmeyer flask (Corning) for suspension culture. Culture conditions: 37℃, 5% CO₂, rotation speed 110 rpm, humidity above 75%. Record cell density and viability after 7 days. When the cell density is 1 × 10⁷ cells / mL and the viability is >90%, collect the cell supernatant and extract EVs.

[0089] After thoroughly mixing 300 mL of cell supernatant, aliquot it into 50 mL centrifuge tubes and centrifuge at 800 g for 10 min. Transfer the supernatant to a new 50 mL centrifuge tube and centrifuge at 4000 g for 10 min. Transfer the supernatant and filter it through a 0.22 μm filter membrane for sterilization. Concentrate the supernatant using a tangential flow membrane pack. The tangential flow parameters are as follows: cut-off molecular weight 10–30 kJ; tangential flow pore size 10–30 nm; tangential flow concentration factor: 10-fold concentration, i.e., 1000 mL of cell supernatant concentrated to 100 mL. Add the concentrated supernatant to an ultrafiltration tube and centrifuge at 10 wt% at 4 °C for 2 h. After centrifugation, resuspend the EV pellet in 1 mL of PBS in each tube, then rinse the ultrafiltration tube with 1 mL of PBS. Add the resuspended EVs to a 100 kDa ultrafiltration tube and centrifuge at 4000 g for 10 min. Adjust the centrifugation time according to different samples. Centrifuge to a final volume of 1 / 1000 of the original supernatant (e.g., concentrate 1000 mL of cell supernatant to 1 mL of EVs). Collect the EVs into a 1.5 mL centrifuge tube and perform BCA assay to determine the total protein concentration.

[0090] 3. Western blotting (WB)

[0091] Take 10 μg of total protein in EV or 20 μg of total protein in cell lysate, add an appropriate amount of 5x loading buffer to a final concentration of 1x loading buffer, and incubate at 95℃ for 10 min. Prepare running buffer (1L) according to the following formula: 100 mL 10x running buffer (Kangwei Century) + 900 mL ddH2O. Mix gently and set aside. Install the appropriate concentration of pre-cast gel (Beyotime) in the running gel tank. After confirming that the assembled running gel tank is leak-proof, pour the prepared running buffer into the running gel tank, ensuring that the running buffer covers the gel surface. Use a pipette to take an appropriate amount of heat-denatured sample and carefully add it to the well of the SDS-PAGE gel (be careful not to apply too much force when loading the sample to avoid contaminating the sample in another well). After correctly connecting the power supply of the running gel tank, run the gel at 80V. After the sample enters the separating gel, adjust the voltage to 120V and continue running until the target protein is separated.

[0092] Take an NC membrane of appropriate size, immerse it in methanol for at least 20 seconds, then wash it with a suitable amount of pre-cooled trans buffer and set aside. (Prepare the trans buffer as follows: 100mL 10x trans buffer (Kangwei Century) + 200mL methanol + 700mL ddH2O. Pre-cool after preparation.) Take out the runnated gel and immerse it in the pre-cooled trans buffer. Assemble the sandwich model in the following order: black side - cotton - filter paper - gel - NC membrane - filter paper - cotton - white side. Then place the sandwich model into the transfer tank in the order of "black side - black side, white side - white side". Add two ice packs that have been frozen overnight at -80°C to the transfer box, pour in the transfer solution to immerse the entire sandwich model, install the transfer box, and transfer at a constant current of 350mA for 1 hour. Blocking: Prepare a 5% BSA blocking solution (dissolved in 1X TBST), take out the transferred membrane and place it in the blocking solution, and block on a horizontal shaker at room temperature for 1-2 hours. Primary antibody incubation: After blocking, wash three times with 1X TBST, prepare the primary antibody solution in TBST according to the specified ratio, and incubate for an appropriate time. Record the preparation ratio and incubation time in Table 1. Secondary antibody incubation: After blocking, wash three times with 1X TBST, prepare the secondary antibody solution in TBST according to the specified ratio, and incubate for an appropriate time. ECL chemiluminescence color development: After secondary antibody incubation, wash three times with 1X TBST. Before color development, mix the ECL chemiluminescence AB solution at a 1:1 ratio and then perform color development on the Tanon-5200Multi chemiluminescence gel imaging system.

[0093] 4. Quantitative detection of the target protein (also known as POI, protein of interest).

[0094] Add 1.2 μg of total protein EV to 250 μl of RIPA lysis buffer (containing 100x protease inhibitor), mix thoroughly, and lyse at 4°C for 1 h. Centrifuge at low speed to remove insoluble matter. 30 min before the ELISA experiment, remove the kit and allow it to equilibrate at room temperature. Prepare standards of different concentrations according to the instructions, and dilute the samples using the diluent provided with the kit. Incubate the samples at 37°C for 90 min, then wash 4 times with a plate washer. Add 100 μl of antibody and incubate at 37°C for 60 min. Wash 4 times with a plate washer, then add 100 μl of enzyme conjugate and incubate at 37°C for 30 min. After washing 4 times with a plate washer, develop the color for 15 min. Add stop solution and read the values ​​using a microplate reader.

[0095] 5. EV Concentration Measurement

[0096] EV concentration was detected using a ZetaView PMX 110 (Particle Metrix, Meerbusch, Germany) equipped with a 405nm laser. The EVs were first diluted with PBS to approximately 1x10⁻⁶. 7 ~1x10 9 The concentration range of particles / mL was determined by capturing a 60-second video at a frame rate of 30 frames per second, and the particle size and concentration were analyzed using NTA software (ZetaView 8.02.28).

[0097] 6. Immunoprecipitation

[0098] Extracellular vesicles are rich in proteins and have many specific marker receptors on their surface, such as CD9, CD81, CD63, CD82, and TSG101. Therefore, extracellular vesicles can be captured by co-incubating them with magnetic beads coated with anti-labeling antibodies. Immunoaffinity techniques have advantages such as high specificity, high purity of extracellular vesicles, and no impact on the morphological integrity of extracellular vesicles, making them a preferred method for enriching and characterizing unique extracellular vesicles. After selectively capturing extracellular vesicles using magnetic beads modified with CD81 antibodies, and then labeling them with fluorescent antibodies specific to the extracellular fragment of the fusion protein, the fluorescence intensity of the magnetic beads can be statistically analyzed using flow cytometry to confirm that the fusion protein is indeed expressed extracellularly in the extracellular vesicles.

[0099] Extracellular vesicles were mixed in PBS to prepare Protein A agarose. The beads were washed twice with PBS, and then prepared to a 50% concentration with PBS. The tip of the pipette was trimmed to avoid damaging the agarose beads during handling. 100 μl of Protein A agarose beads (50%) were added to each 1 mL of extracellular vesicles, and the mixture was shaken at 4°C for 10 min (EP tube placed on ice on a horizontal shaker) to remove nonspecific proteins and reduce background. Afterward, the mixture was centrifuged at 14000g for 15 min at 4°C, and the supernatant was transferred to a new centrifuge tube to remove the Protein A beads. 10 μl of Flag primary antibody was then added, and the antigen-antibody mixture was incubated overnight at 4°C with gentle shaking or at room temperature for 2 h. 100 μL of Protein A agarose beads were added to capture the antigen-antibody complex, and the mixture was incubated overnight at 4°C with gentle shaking or at room temperature for 1 h. Centrifuge briefly at 14000 rpm for 5 seconds, collect the agarose bead-antigen-antibody complex, discard the supernatant, and wash 3 times with pre-cooled PBS, 800 μl each time. Resuspend the agarose bead-antigen-antibody complex in 60 μl of 2× loading buffer, mix gently, boil the loaded sample for 5 min to free the antigen, antibody, and beads, centrifuge, and electrophoresis the supernatant.

[0100] 7. In vitro activity assay of IL-12

[0101] Immediately after removing the PBMC culture medium from the liquid nitrogen tank, place it in a 37°C water bath and gently shake. Once the cells have mostly thawed, transfer them to a clean bench. Add the PBMC to a clean 15ml centrifuge tube, along with 5ml of PBMC culture medium. Centrifuge at 1300rpm for 5 minutes, then discard the supernatant. Resuspend the cells in 5ml of PBMC and place in a 25ml culture flask. Incubate overnight at 37°C with 5% CO2.

[0102] The following day, transfer PBMCs to a 15 mL centrifuge tube and centrifuge at 1300 rpm. After 5 minutes, discard the supernatant. Resuspend in 5 mL of PBMC activation medium and place in a 25 mL culture flask. Incubate at 37°C and 5% CO2 for 72 hours. After 72 hours, transfer the stimulated PBMCs to a 15 mL centrifuge tube and centrifuge at 1300 rpm. After 5 minutes, discard the supernatant and resuspend in 5 mL of PBMC medium. Repeat once. After resuspending in an appropriate volume of PBMC medium, centrifuge at 1.5 x 10⁻⁶. 5 Cells / well / 90μL were seeded in 96-well plates. Recombinant IL-12 protein or EV expressing IL-12 was diluted to nine experimental concentrations. 10μL of recombinant protein or EV was added to each well, mixed with PBMCs, and incubated at 37°C, 5% CO2 for 24 hours.

[0103] The following day, PBMCs and supernatant samples were transferred to new EP tubes or 96-well plates, centrifuged at 3000 rpm, and the supernatant was collected for human IFNγ detection.

[0104] 8. Mass spectrometry detection of extracellular vesicle proteins

[0105] LC-MS / MS Sample Preparation

[0106] Extracellular vesicle samples (200 μL, 500 μg / mL) were removed from a -80°C freezer and transferred to a 1.5 mL centrifuge tube. An appropriate amount of SDT protein lysis buffer (4% SDS, 10 mM DTT, 100 mM TEAB) was added to dissolve the vesicles. The mixture was vortexed and sonicated in an ice-water bath for 5 min to achieve complete lysis. The mixture was centrifuged at 12000 g for 15 min at 4°C. The supernatant was collected and added to a final concentration of 10 mM DTT (Sigma / D9163-25G), and reacted at 56°C for 1 h. Then, sufficient IAM (Sigma / I6125-25G) was added, and the mixture was reacted at room temperature in the dark for 1 h. Four volumes of pre-chilled -20°C acetone were added, and the mixture was precipitated at -20°C for at least 2 h. The precipitate was then collected by centrifugation at 12000 g for 15 min at 4°C. Then, 1 mL of pre-cooled acetone (Beijing Chemical Plant / 11241203810051) at -20℃ was added to resuspend and wash the precipitate. The precipitate was centrifuged at 12000g for 15 min at 4℃, collected, air-dried, and a suitable amount of protein lysis buffer (8M urea, 100mM TEAB, pH=8.5) was added to dissolve the protein precipitate.

[0107] Using the Bradford Protein Quantitative Kit (Beyotime), BSA standard protein solutions were prepared according to the manufacturer's instructions, with concentration gradients ranging from 0 to 0.5 μg / μL. Different concentration gradients of BSA standard protein solutions and different dilutions of the test sample solutions were added to 96-well plates, with the volume brought to 20 μL. Each gradient was repeated three times. 180 μL of G250 staining solution was added immediately, and the plates were incubated at room temperature for 5 min. The absorbance at 595 nm was measured. A standard curve was plotted using the absorbance of the standard protein solutions, and the protein concentration of the test samples was calculated. 20 μg of each protein sample was subjected to 12% SDS-PAGE gel electrophoresis. The stacking gel electrophoresis conditions were 80 V for 20 min, and the separating gel electrophoresis conditions were 120 V for 90 min. After electrophoresis, Coomassie Brilliant Blue R-250 staining was performed until the bands were clear.

[0108] Take the protein sample and add DB protein lysis buffer (8M urea, 100mM TEAB, pH=8.5) to a final volume of 100μL. Add trypsin (Promega / V5280) and 100mM TEAB (Sigma / T7408-500mL) buffer, mix well, and digest at 37℃ for 4h. Then add trypsin and CaCl2 and digest overnight. Adjust the pH to less than 3 with formic acid, mix well, and centrifuge at 12000g for 5min at room temperature. Slowly pass the supernatant through a C18 desalting column, then wash three times consecutively with washing buffer (0.1% formic acid, 3% acetonitrile). Add an appropriate amount of elution buffer (0.1% formic acid, 70% acetonitrile), collect the filtrate, and lyophilize.

[0109] Mobile phases A (100% water, 0.1% formic acid) and B (80% acetonitrile, 0.1% formic acid) were prepared. The lyophilized powder was dissolved in 10 μL of mobile phase A, centrifuged at 14000g for 20 min at 4°C, and 1 μg of the supernatant was injected for LC-MS analysis. An EASY-nLC™ 1200 nano-scale UHPLC system (Thermo Fisher / LC140) was used. The pre-column was a self-made pre-column (4.5cm × 75μm, 3μm), and the analytical column was a self-made analytical column (15cm × 150μm, 1.9μm). The elution conditions are shown in Table 1. A Q Exactive™ HF-X mass spectrometer (Thermo) with Nanospray Flex was used. TM The ESI ion source was set to 2.1 kV, the ion spray voltage to 320 °C, and the mass spectrometry was performed in data-dependent acquisition mode. The full scan range of the mass spectrometry was 350-1500 m / z, the first-order mass spectrometry resolution was set to 60000 (200 m / z), and the maximum C-trap capacity was 3 × 10⁻⁶ m / z. 6 The maximum C-trap injection time was 20 ms. The top 40 precursor ions by ion intensity in the full scan were fragmented using high-energy collisional fragmentation (HCD) and detected by secondary mass spectrometry. The resolution of the secondary mass spectrometry was set to 15000 (200 m / z), and the maximum C-trap capacity was 1 × 10⁻⁶. 5 The maximum C-trap injection time was 45 ms, the peptide fragmentation collision energy was set to 27%, and the threshold intensity was set to 2.2 × 10⁻⁶. 4 The dynamic exclusion range is set to 20s to generate raw mass spectrometry detection data.

[0110] Table 2 Elution gradient for liquid chromatography

[0111]

[0112] Data processing

[0113] All resulting spectra were searched using Proteome Discoverer 2.2 (PD2.2, Thermo) based on the Uniprot protein database. Search parameters were set as follows: precursor ion mass tolerance was 10 ppm, and fragment ion mass tolerance was 0.02 Da. Immobilization modification was cysteine ​​alkylation, variable modification was methionine oxidation, and N-terminal acetylation was allowed, with a maximum of two missed cleavage sites permitted. To improve the quality of the analytical results, PD2.2 further filtered the search results: peptide spectrum matches (PSMs) with a confidence level of 99% or higher were considered reliable PSMs; proteins containing at least one unique peptide were considered reliable proteins. Only reliable peptides and proteins were retained, and FDR (Fragmented Detail Recognition) verification was performed, removing peptides and proteins with an FDR greater than 1%.

[0114] Example 2: Expression of BCAM in EV

[0115] A stable transfected cell line of BCAM was constructed, and the expression of BCAM in EVs was detected. PTGFRN and LAMP2B were used as controls.

[0116] In this embodiment, the expression vector used is AAVS1_Puro_Tet3G_3xFLAG_Twin_Strep (Addgene, catalog number 92099). The CMV promoter and multiple cloning site fragment are derived from the pcDNA3.1(+) vector (Thermo, catalog number V790-20). Homologous recombination of the two yields the pAAVS1-puro-CMV-MCS plasmid, which is the backbone vector used subsequently. All sequences are inserted into the multiple cloning site of this backbone vector.

[0117] Plasmids were constructed by fusing EGFP (the target protein, POI) with BCAM and two other known EV proteins (PTGFRN and LAMP2B), respectively. The sequences of the inserted vectors are shown below:

[0118] pAAVS1-Puro-CMV-eGFP-BCAM

[0119] MEPPDAPAQARGAPRLLLLAVLLAAHPDAQAMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKSAGGGGSGGGGSEVRLSVPPLVEVMRGKSVILDCTPTGTHDHYMLEWFLTDRSGARPRLASAEMQGSELQVTMHDTRGRSPPYQLDSQGRLVLAEAQVGDERDYVCVVRAGAAGTAEATARLNVFAKPEATEVSPNKGTLSVMEDSAQEIATCNSRNGNPAPKITWYRNGQRLEVPVEMNPEGYMTSRTVREASGLLSLTSTLYLRLRKDDRDASFHCAAHYSLPEGRHGRLDSPTFHLTLHYPTEHVQFWVGSPSTPAGWVREGDTVQLLCRGDGSPSPEYTLFRLQDEQEEVLNVNLEGNLTLEGVTRGQSGTYGCRVEDYDAADDVQLSKTLELRVAYLDPLELSEGKVLSLPLNSSAVVNCSVHGLPTPALRWTKDSTPLGDGPMLSLSSITFDSNGTYVCEASLPTVPVLSRTQNFTLLVQGSPELKTAEIEPKADGSWREGDEVTLICSARGHPDPKLSWSQLGGSPAEPIPGRQGWVSSSLTLKVTSALSRDGISCEASNPHGNKRHVFHFGTVSPQTSQAGVAVMAVAVSVGLLLLVVAVFYCVRRKGGPCCRQRREKGAPPPGEPGLSHSGSEQPEQTGLLMGGASGGARGGSGGFGDEC(SEQ ID NO:1).

[0120] Among them, the BCAM signal peptide sequence is: MEPPDAPAQARGAPRLLLLAVLLAAHPDAQA (SEQ ID NO: 2);

[0121] The eGFP sequence is as follows:

[0122] MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK (SEQ ID NO:3);

[0123] The linker sequence is: GGGGSGGGGS (SEQ ID NO:4);

[0124] The BCAM sequence is as follows:

[0125] EVRLSVPPLVEVMRGKSVILDCTPTGTHDHYMLEWFLTDRSGARPRLASAEMQGSELQVTMHDTRGRSPPYQLDSQGRLVLAEAQVGDERDYVCVVRAGAAGTAEATARLNVFAKPEATEVSPNKGTLSVMEDSAQEIATCNSRNGNPAPKITWYRNGQRLEVPVEMNPEGYMTSRTVREASGLLSLTSTLYLRLRKDDRDASFHCAAHYSLPEGRHGRLDSPTFHLTLHYPTEHVQFWVGSPSTPAGWVREGDTVQLLCRGDGSPSPEYTLFRLQDEQEEVLNVNLEGNLTLEGVTRGQSGTYGCRVEDYDAADDVQLSKTLELRVAYLDPLELSEGKVLSLPLNSSAVVNCSVHGLPTPALRWTKDSTPLGDGPMLSLSSITFDSNGTYVCEASLPTVPVLSRTQNFTLLVQGSPELKTAEIEPKADGSWREGDEVTLICSARGHPDPKLSWSQLGGSPAEPIPGRQGWVSSSLTLKVTSALSRDGISCEASNPHGNKRHVFHFGTVSPQTSQAGVAVMAVAVSVGLLLLVVAVFYCVRRKGGPCCRQRREKGAPPPGEPGLSHSGSEQPEQTGLLMGGASGGARGGSGGFGDEC(SEQID NO:5)。

[0126] pAAVS1-Puro-CMV-eGFP-PTGFRN

[0127]

[0128] The PTGFRN signal peptide sequence is: MGRLASRPLLLALLSLALCRG (SEQ ID NO:7);

[0129] The PTGFRN sequence is as follows:

[0130] RVVRVPTATLVRVVGTELVIPCNVSDYDGPSEQNFDWSFSSLGSSFVELASTWEVGFPAQLYQERLQRGEILLRRTANDAVELHIKNVQPSDQGHYKCSTPSTDATVQGNYEDTVQVKVLADSLHVGPSARPPPSLSLREGEPFELRCTAASASPLHTHLALLWEVHRGPARRSVLALTHEGRFHPGLGYEQRYHSGDVRLDTVGSDAYRLSVSRALSADQGSYRCIVSEWIAEQGNWQEIQEKAVEVATVVIQPSVLRAAVPKNVSVAEGKELDLTCNITTDRADDVRPEVTWSFSRMPDSTLPGSRVLARLDRDSLVHSSPHVALSHVDARSYHLLVRDVSKENSGYYYCHVSLWAPGHNRSWHKVAEAVSSPAGVGVTWLEPDYQVYLNASKVPGFADDPTELACRVVDTKSGEANVRFTVSWYYRMNRRSDNVVTSELLAVMDGDWTLKYGERSKQRAQDGDFIFSKEHTDTFNFRIQRTTEEDRGNYYCVVSAWTKQRNNSWVKSKDVFSKPVNIFWALEDSVLVVKARQPKPFFAAGNTFEMTCKVSSKNIKSPRYSVLIMAEKPVGDLSSPNETKYIISLDQDSVVKLENWTDASRVDGVVLEKVQEDEFRYRMYQTQVSDAGLYRCMVTAWSPVRGSLWREAATSLSNPIEIDFQTSGPIFNASVHSDTPSVIRGDLIKLFCIITVEGAALDPDDMAFDVSWFAVHSFGLDKAPVLLSSLDRKGIVTTSRRDWKSDLSLERVSVLEFLLQVHGSEDQDFGNYYCSVTPWVKSPTGSWQKEAEIHSKPVFITVKMDVLNAFKYPLLIGVGLSTVIGLLSCLIGYCSSHWCCKKEVQETRRERRRLMSMEMD(SEQ ID NO:8)。

[0131] pAAVS1-Puro-CMV-eGFP-LAMP2B

[0132] MVCFRLFPVPGSGLVLVCLVLGAVRSYAMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKSAGGGGSGGGGSLELNLTDSENATCLYAKWQMNFTVRYETTNKTYKTVTISDHGTVTYNGSICGDDQNGPKIAVQFGPGFSWIANFTKAASTYSIDSVSFSYNTGDNTTFPDAEDKGILTVDELLAIRIPLNDLFRCNSLSTLEKNDVVQHYWDVLVQAFVQNGTVSTNEFLCDKDKTSTVAPTIHTTVPSPTTTPTPKEKPEAGTYSVNNGNDTCLLATMGLQLNITQDKVASVININPNTTHSTGSCRSHTALLRLNSSTIKYLDFVFAVKNENRFYLKEVNISMYLVNGSVFSIANNNLSYWDAPLGSSYMCNKEQTVSVSGAFQINTFDLRVQPFNVTQGKYSTAQECSLDDDTILIPIIVGAGLSGLIIVIVIAYVIGRRKSYAGYQTL(SEQ ID NO:9).

[0133] Among them, the LAMP2B signal peptide sequence is: MVCFRLFPVPGSGLVLVCLVLGAVRSYA (SEQ ID NO: 10);

[0134] The LAMP2B sequence is:

[0135] LELNLTDSENATCLYAKWQMNFTVRYETTNKTYKTVTISDHGTVTYNGSICGDDQNGPKIAVQFGPGFSWIANFTKAASTYSIDSVSFSYNTGDNTTFPDAEDKGILTVDELLAIRIPLNDLFRCSLSTLEKNDVVQHYWDVLVQAFVQNGTVSTNEFLCDKDKTSTVAPTIHTTVPSPTTTPTPKEKPEAG TYSVNNGNDTCLLATMGLQLNITQDKVASVININPNTTHSTGSCRSHTALLRLNSSTIKYLDFVFAVKNENRFYLKEVNISMYLVNGSVFSIANNNLSYWDAPLGSSYMCNKEQTVSVSGAFQINTFDLRVQPFNVTQGKYSTAQECSLDDDTILIPIIVGAGLSGLIIVIVIAYVIGRRKSYAGYQTL(SEQ ID NO:11).

[0136] Using a site-selective insertion method, the target plasmid was site-directedly integrated into the AAVS1 site of expi HEK293F (Thermo Fisher Scientific, catalog number A14527) to obtain a stably transfected cell line. EVs were extracted from the culture supernatant of the stably transfected cell line. Western blot analysis was used to detect eGFP and the EV protein marker CD9 in the EV products. The results showed that, similar to known EV proteins PTGFRN and LAMP2B, BCAM can also load its fused protein fragment (eGFP) into cellularly secreted EVs.

[0137] To further confirm that the target band in the EVs was indeed the introduced fusion protein, lysates from three stable transfected cell lines, EVs secreted by the cells, and EVs secreted by wild-type expi HEK293F cells were compared. Western blot results showed that bands with different molecular weights were observed in the stable transfected cell lines and their secreted EVs, allowing for the identification of wild-type protein and POI fusion protein based on molecular weight. In contrast, wild-type BCAM, due to its lower background expression level, actually helped increase the proportion of eGFP-BCAM fusion protein in the EVs relative to wild-type BCAM; that is, the amount of BCAM fusion protein in the obtained EVs was significantly higher than the background BCAM. (See [link to relevant documentation]). Figure 2 The wild-type protein band and fusion protein band in A were also significantly higher than the ratio of PTGFRN and LAMP2B fusion proteins to the background level. Figure 2B represents the percentage of wild-type protein and fusion protein in the total.

[0138] Example 3: Detection of eGFP-BCAM fusion protein expression in EVs

[0139] To evaluate the ability of BCAM as a protein to carry target protein POIs, three EVs fused to express eGFP (eGFP-LAMP2B, eGFP-PTGFRN, and eGFP-BCAM) were first collected. Particle concentration was determined using NTA (Nanoparticle Tracking Analysis). NTA) was used to dilute the samples to 1x10e9 particles / mL, and the fluorescence intensity was quantified using a multi-functional microplate reader. The results showed that the fluorescence intensity of eGFP-BCAM was higher than that of eGFP-PTGFRN, and significantly higher than that of eGFP-LAMP2B (NTA). Figure 3 This indicates that BCAM has a higher carrier efficiency compared to LAMP2B and PTGFRN.

[0140] Example 4: Detection of IL12-BCAM fusion protein expression in EVs

[0141] To further evaluate BCAM's ability to carry other effective components as a protein carrier, IL12 (approximately 60 kDa) was fused with BCAM, and a fusion protein of IL12 and PTGFRN was constructed as a control. The sequence of the insertion vector is as follows:

[0142] pAAVS1-Puro-CMV-hIL12-BCAM

[0143]

[0144] Among them, the hIL12 sequence is as follows:

[0145] MCHQQLVISWFSLVFLASPLVAIWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCSGGSGGGSGGGGSGGGGSGGGSGGRNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNAS (SEQ ID NO:13).

[0146] pAAVS1-Puro-CMV-hIL12-PTGFRN

[0147]

[0148] Stable transfected cell lines were constructed, and EVs were collected. After lysing EVs with an equal total protein content (0.5 μg), the abundance of their active ingredient (IL12) was detected using ELISA. The results showed that the expression level of IL12-BCAM in EVs was significantly higher than that of IL12-PTGFRN (…). Figure 4 The above experiments demonstrate that BCAM is a preferred EV scaffold protein, capable of effectively carrying the target protein fused with it into the EV.

[0149] Example 5: Detection of expression of other BCAM fusion proteins in EVs

[0150] To test BCAM's ability as a scaffold protein to carry and sort different active ingredients into EVs, fusion proteins (containing flag tags) of BCAM with POIs of different molecular weights were constructed. The inserted sequences in the vectors are as follows:

[0151] pAAVS1_Puro_CMV_SP_RVG_linker Flag-BCAM

[0152] MEPPDAPAQARGAPRLLLLAVLLAAHPDAQAYTIWMPENPRPGTPCDIFTNSRGKRASNGSAGGGGSDYKDDDDKGGGGSEVRLSVPPLVEVMRGKSVILDCTPTGTHDHYMLEWFLTDRSGARPRLASAEMQGSELQVTMHDTRGRSPPYQLDSQGRLVLAEAQVGDERDYVCVVRAGAAGTAEATARLNVFAKPEATEVSPNKGTLSVMEDSAQEIATCNSRNGNPAPKITWYRNGQRLEVPVEMNPEGYMTSRTVREASGLLSLTSTLYLRLRKDDRDASFHCAAHYSLPEGRHGRLDSPTFHLTLHYPTEHVQFWVGSPSTPAGWVREGDTVQLLCRGDGSPSPEYTLFRLQDEQEEVLNVNLEGNLTLEGVTRGQSGTYGCRVEDYDAADDVQLSKTLELRVAYLDPLELSEGKVLSLPLNSSAVVNCSVHGLPTPALRWTKDSTPLGDGPMLSLSSITFDSNGTYVCEASLPTVPVLSRTQNFTLLVQGSPELKTAEIEPKADGSWREGDEVTLICSARGHPDPKLSWSQLGGSPAEPIPGRQGWVSSSLTLKVTSALSRDGISCEASNPHGNKRHVFHFGTVSPQTSQAGVAVMAVAVSVGLLLLVVAVFYCVRRKGGPCCRQRREKGAPPPGEPGLSHSGSEQPEQTGLLMGGASGGARGGSGGFGDEC(SEQ ID NO:15).

[0153] Among them, the RVG sequence is: YTIWMPENPRPGTPCDIFTNSRGKRASNG(SEQ ID NO:16);

[0154] The Linker flag sequence is: GGGGS DYKDDDDK GGGGGS(SEQ ID NO:17).

[0155] pAAVS1_Puro_CMV_FLT3L_linker Flag-BCAM

[0156] MTVLAPAWSPTTYLLLLLLLSSGLSGTQDCSFQHSPISSDFAVKIRELSDYLLQDYPVTVASNLQDEELCGGLWRLVLAQRWMERLKTVAGSKMQGLLERVNTEIHFVTKCAFQPPPSCLRFVQTNISRLLQETSEQLVALKPWITRQNFSRCLELQCQPDSSTLPPPWSPRPLEATAPTAPQPPSAGGGGSDYKDDDDKGGGGSEVRLSVPPLVEVMRGKSVILDCTPTGTHDHYMLEWFLTDRSGARPRLASAEMQGSELQVTMHDTRGRSPPYQLDSQGRLVLAEAQVGDERDYVCVVRAGAAGTAEATARLNVFAKPEATEVSPNKGTLSVMEDSAQEIATCNSRNGNPAPKITWYRNGQRLEVPVEMNPEGYMTSRTVREASGLLSLTSTLYLRLRKDDRDASFHCAAHYSLPEGRHGRLDSPTFHLTLHYPTEHVQFWVGSPSTPAGWVREGDTVQLLCRGDGSPSPEYTLFRLQDEQEEVLNVNLEGNLTLEGVTRGQSGTYGCRVEDYDAADDVQLSKTLELRVAYLDPLELSEGKVLSLPLNSSAVVNCSVHGLPTPALRWTKDSTPLGDGPMLSLSSITFDSNGTYVCEASLPTVPVLSRTQNFTLLVQGSPELKTAEIEPKADGSWREGDEVTLICSARGHPDPKLSWSQLGGSPAEPIPGRQGWVSSSLTLKVTSALSRDGISCEASNPHGNKRHVFHFGTVSPQTSQAGVAVMAVAVSVGLLLLVVAVFYCVRRKGGPCCRQRREKGAPPPGEPGLSHSGSEQPEQTGLLMGGASGGARGGSGGFGDEC(SEQ ID NO:18)

[0157] Among them, the FLT3L sequence is as follows:

[0158] MTVLAPAWSPTTYLLLLLLLSSGLSGTQDCSFQHSPISSDFAVKIRELSDYLLQDYPVTVASNLQDEELCGGLWRLVLAQRWMERLKTVAGSKMQGLLERVNTEIHFVTKCAFQPPPSCLRFVQTNISRLLQETSEQLVALKPWITRQNFSRCLELQCQPDSSTLPPPWSPRPLEATAPTAPQPP(SEQ ID NO:19)。

[0159] pAAVS1-puro-CMV-SP-linker Flag-BCAM

[0160] MEPPDAPAQARGAPRLLLLAVLLAAHPDAQASAGGGGSDYKDDDDKGGGGSEVRLSVPPLVEVMRGKSVILDCTPTGTHDHYMLEWFLTDRSGARPRLASAEMQGSELQVTMHDTRGRSPPYQLDSQGRLVLAEAQVGDERDYVCVVRAGAAGTAEATARLNVFAKPEATEVSPNKGTLSVMEDSAQEIATCNSRNGNPAPKITWYRNGQRLEVPVEMNPEGYMTSRTVREASGLLSLTSTLYLRLRKDDRDASFHCAAHYSLPEGRHGRLDSPTFHLTLHYPTEHVQFWVGSPSTPAGWVREGDTVQLLCRGDGSPSPEYTLFRLQDEQEEVLNVNLEGNLTLEGVTRGQSGTYGCRVEDYDAADDVQLSKTLELRVAYLDPLELSEGKVLSLPLNSSAVVNCSVHGLPTPALRWTKDSTPLGDGPMLSLSSITFDSNGTYVCEASLPTVPVLSRTQNFTLLVQGSPELKTAEIEPKADGSWREGDEVTLICSARGHPDPKLSWSQLGGSPAEPIPGRQGWVSSSLTLKVTSALSRDGISCEASNPHGNKRHVFHFGTVSPQTSQAGVAVMAVAVSVGLLLLVVAVFYCVRRKGGPCCRQRREKGAPPPGEPGLSHSGSEQPEQTGLLMGGASGGARGGSGGFGDEC(SEQ ID NO:20)。

[0161] pAAVS1-puro-CMV-hCD24(1-57)-linker Flag-BCAM

[0162] MGRAMVARLGLGLLLLALLLPTQIYSSETTTGTSSNSSQSTSNSGLAPNPTNATTKASAGGGGSDYKDDDDKGGGGSEVRLSVPPLVEVMRGKSVILDCTPTGTHDHYMLEWFLTDRSGARPRLASAEMQGSELQVTMHDTRGRSPPYQLDSQGRLVLAEAQVGDERDYVCVVRAGAAGTAEATARLNVFAKPEATEVSPNKGTLSVMEDSAQEIATCNSRNGNPAPKITWYRNGQRLEVPVEMNPEGYMTSRTVREASGLLSLTSTLYLRLRKDDRDASFHCAAHYSLPEGRHGRLDSPTFHLTLHYPTEHVQFWVGSPSTPAGWVREGDTVQLLCRGDGSPSPEYTLFRLQDEQEEVLNVNLEGNLTLEGVTRGQSGTYGCRVEDYDAADDVQLSKTLELRVAYLDPLELSEGKVLSLPLNSSAVVNCSVHGLPTPALRWTKDSTPLGDGPMLSLSSITFDSNGTYVCEASLPTVPVLSRTQNFTLLVQGSPELKTAEIEPKADGSWREGDEVTLICSARGHPDPKLSWSQLGGSPAEPIPGRQGWVSSSLTLKVTSALSRDGISCEASNPHGNKRHVFHFGTVSPQTSQAGVAVMAVAVSVGLLLLVVAVFYCVRRKGGPCCRQRREKGAPPPGEPGLSHSGSEQPEQTGLLMGGASGGARGGSGGFGDEC(SEQ ID NO:21).

[0163] Among them, the hCD24(1-57) sequence is:

[0164] MGRAMVARLGLGLLLLALLLPTQIYSSETTTGTSSNSSQSTSNSGLAPNPTNATTKA(SEQID NO:22).

[0165] pAAVS1-puro-CMV-SP-hCD24(27-57)-linker Flag-BCAM

[0166] MEPPDAPAQARGAPRLLLLAVLLAAHPDAQASETTTGTSSNSSQSTSNSGLAPNPTNATTKASAGGGGSDYKDDDDKGGGGSEVRLSVPPLVEVMRGKSVILDCTPTGTHDHYMLEWFLTDRSGARPRLASAEMQGSELQVTMHDTRGRSPPYQLDSQGRLVLAEAQVGDERDYVCVVRAGAAGTAEATARLNVFAKPEATEVSPNKGTLSVMEDSAQEIATCNSRNGNPAPKITWYRNGQRLEVPVEMNPEGYMTSRTVREASGLLSLTSTLYLRLRKDDRDASFHCAAHYSLPEGRHGRLDSPTFHLTLHYPTEHVQFWVGSPSTPAGWVREGDTVQLLCRGDGSPSPEYTLFRLQDEQEEVLNVNLEGNLTLEGVTRGQSGTYGCRVEDYDAADDVQLSKTLELRVAYLDPLELSEGKVLSLPLNSSAVVNCSVHGLPTPALRWTKDSTPLGDGPMLSLSSITFDSNGTYVCEASLPTVPVLSRTQNFTLLVQGSPELKTAEIEPKADGSWREGDEVTLICSARGHPDPKLSWSQLGGSPAEPIPGRQGWVSSSLTLKVTSALSRDGISCEASNPHGNKRHVFHFGTVSPQTSQAGVAVMAVAVSVGLLLLVVAVFYCVRRKGGPCCRQRREKGAPPPGEPGLSHSGSEQPEQTGLLMGGASGGARGGSGGFGDEC(SEQ ID NO:23)

[0167] Among them, the hCD24(27-57) sequence is: SETTTGTSSNSSQSTSNSGLAPNPTNATTKA (SEQ ID NO:24).

[0168] pAAVS1-puro-CMV-CD3 scFab-linker Flag-BCAM

[0169]

[0170] The CD3 scFab sequence is as follows:

[0171] (SEQ ID NO:26).

[0172] Western blot results showed that POIs of different molecular weights could be expressed in EVs, and the molecular weights were consistent with expectations.

[0173] Example 6: Detection of the biological activity of the target protein in the BCAM fusion protein

[0174] To test whether the IL12 fusion protein on EVs has biological activity, recombinant IL12 protein (…) was used. Figure 6 A) EVs expressing IL12-PTGFRN Figure 6 B), and EVs expressing IL12-BCAM ( Figure 6 C) Stimulate PBMCs and detect IFN-γ secretion levels. EC50 results showed that fusion with BCAM and PTGFRN did not affect the biological activity of IL12.

[0175] Example 7: Identification of membrane proteins with similar functions to BCAM

[0176] Based on the discovery of BCAM, the inventors screened and identified membrane proteins with similar functions to BCAM. These proteins all belong to the immunoglobulin superfamily, have high similarity to BCAM, and exhibit low basal expression levels in EVs.

[0177] Using NCBI's BLAST software (Basic local alignment search tool, J MolBiol. 1990 Oct 5; 215(3):403-10.), the inventors screened a group of EV proteins with high similarity to BCAM in the immunoglobulin superfamily, as shown in Table 3.

[0178] Table 3

[0179]

[0180]

[0181] Query cover: The percentage of the query fragment (BCAM) length contained in the aligned fragment (POI). A higher value indicates a higher proportion of the query fragment length being included in the alignment (e.g., 100% when BCAM is the POI).

[0182] E Value: Expected value. The smaller the value, the higher the similarity between the two proteins (e.g., when BCAM is a POI, this value is 0). When the E value <= 1.0E-4, the protein is considered to have a high similarity to BCAM, as listed in Table 1. The E value is calculated by the NCBI blastp program, and the calculation method for the E value is as follows:

[0183] E=K*m*n*exp(-lambda*S) (I);

[0184] Where K and lambda are constants related to the database and algorithm, m represents the length of the target sequence, n represents the size of the database, the S value represents the homology between the two sequences, the higher the S score, the greater the homology between them, and the lower the calculated E value, the higher the similarity.

[0185] Total score: The higher the total score, the higher the consistency between the POI and the query fragment.

[0186] For some parameter settings, please refer to [link / reference].

[0187] https: / / blast.ncbi.nlm.nih.gov / Blast.cgi?PROGRAM=blastp&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome.

[0188] Furthermore, the inventors investigated the abundance of the proteins listed in Table 3 in EVs. The relative abundance of different proteins in extracellular vesicles and cells was detected using mass spectrometry, with the CD81 content (denoted as iBAQ) set to 1. The results are shown in Table 4.

[0189] Table 4

[0190]

[0191]

[0192] The MaxQuant program calculates protein intensity as the sum of the intensities of all identified peptides, divided by the theoretically observable number of peptides, to obtain the iBAQ (intensity-based absolute quantification) value. This is an approximately absolute quantification method that can effectively compare the expression abundance of different proteins in a sample (Global quantification of mammalian gene expression control, Nature. 2013 Mar 7; 495(7439):126-7). When the protein content in EVs is less than 5% of the CD81 content, i.e., the ratio of the iBAQ value of a protein to CD81 in Table 2 is <= 0.05, it is judged in this paper as having a low background expression level in EVs. CD81 is one of the common tetramembrane proteins in EVs and is set as a reference in this embodiment.

[0193] The structures of some of these proteins are as follows Figure 7 As shown, the structural analysis is derived from the Uniprot database.

[0194] Example 8: Detection of MCAM fusion protein expression in EVs

[0195] To verify the feasibility of constructing EVs using MCAM, an expression plasmid for the eGFP-MCAM fusion protein was constructed, with the following inserted sequence:

[0196] pAAVS1-Puro-CMV-eGFP-MCAM

[0197] MGLPRLVCAFLLAACCCCPRVAGMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKSAGGGGSGGGGSVPGEAEQPAPELVEVEVGSTALLKCGLSQSQGNLSHVDWFSVHKEKRTLIFRVRQGQGQSEPGEYEQRLSLQDRGATLALTQVTPQDERIFLCQGKRPRSQEYRIQLRVYKAPEEPNIQVNPLGIPVNSKEPEEVATCVGRNGYPIPQVIWYKNGRPLKEEKNRVHIQSSQTVESSGLYTLQSILKAQLVKEDKDAQFYCELNYRLPSGNHMKESREVTVPVFYPTEKVWLEVEPVGMLKEGDRVEIRCLADGNPPPHFSISKQNPSTREAEEETTNDNGVLVLEPARKEHSGRYECQGLDLDTMISLLSEPQELLVNYVSDVRVSPAAPERQEGSSLTLTCEAESSQDLEFQWLREETGQVLERGPVLQLHDLKREAGGGYRCVASVPSIPGLNRTQLVNVAIFGPPWMAFKERKVWVKENMVLNLSCEASGHPRPTISWNVNGTASEQDQDPQRVLSTLNVLVTPELLETGVECTASNDLGKNTSILFLELVNLTTLTPDSNTTTGLSTSTASPHTRANSTSTERKLPEPESRGVVIVAVIVCILVLAVLGAVLYFLYKKGKLPCRRSGKQEITLPPSRKSELVVEVKSDKLPEEMGLLQGSSGDKRAPGDQGEKYIDLRH

[0198] Among them, the MCAM signal peptide sequence is: MGLPRLVCAFLLAACCCCPRVAG;

[0199] The MCAM sequence is: VPGEAEQPAPELVEVEVGSTALLKCG

[0200] .

[0201] Using a site-selective insertion method, the target plasmid was site-specifically integrated into the AAVS1 site of expi HEK293F, resulting in a stably transfected cell line. EVs were extracted from the culture supernatant of the stably transfected cell line. Western blot analysis was used to detect eGFP and the EV protein marker CD9 in the EV products. The results showed that, similar to BCAM, MCAM can also load its fused eGFP into EVs secreted by cells.

[0202] To rule out the possibility that the active ingredient exists in a non-EV form, immunoprecipitation was used to capture purified EVs using magnetic beads modified with CD81 and CD9, respectively. First, the EVs were extracted from the supernatant using the purification method described in the examples; this was the sample before capture. Then, immunomagnetic beads were used to specifically capture EVs expressing four-transmembrane proteins in these samples; the samples on the magnetic beads were the captured samples. The EVs before and after capture were lysed, and IL12 was analyzed by Western blot. The results showed that IL12 was indeed present in the immunoprecipitated samples, indicating that the IL12 in the samples was located within the EVs, and not a free recombinant IL12 protein separated from the EVs during purification for some unknown reason. IL12, after fusion with PTGFRN, MCAM, and BCAM, was loaded into the EVs.

[0203] Example 9: Detection of the biological activity of the target protein in the MCAM fusion protein

[0204] 1. IL12 activity detection

[0205] To verify that fusion with MCAM does not alter the protein's biological activity, engineered cells expressing IL12-MCAM were constructed, and the EV (active cell count) of cells expressing IL12-MCAM was compared. Figure 10 C) and recombinant IL12 ( Figure 10 A) and EVs expressing IL12-BCAM ( Figure 10 B) In vitro activity. The results of the IFN-γ secretion activity test of PBMC showed that IL12-MCAM- has similar biological activity to IL12-BCAM, indicating that fusion with MCAM does not affect the biological activity of IL-12.

[0206] 2. Luciferase activity detection

[0207] Luciferase (molecular weight approximately 70 kDa) was fused with BCAM, MCAM, and PTGFRN, respectively, and stable transfected cell lines were constructed. The vector insertion sequences are as follows:

[0208] pAAVS1_Puro_CMV_SP_luciferase_linker Flag-BCAM

[0209]

[0210] The luciferase sequence is as follows:

[0211] (SEQ ID NO:28).

[0212] Each EV (extra molecule) was mixed at 20 μg with 60 μL of substrate, and its in vitro activity was measured. Chemiluminescence results showed that the luciferase activities of MCAM and BCAM were superior to those of the blank control, and the activity of the MCAM luciferase fusion protein was significantly higher than that of the PTGFRN luciferase fusion protein.

[0213] Example 10: The truncated BCAM can still carry the target protein in EV expression

[0214] 1. Expression of hIL-12 in stably transfected cells and EVs by two truncated variants of BCAM

[0215] Expression plasmids for the fusion protein of two truncated BCAM variants and hIL-12 were constructed. The vector insertion sequences are as follows:

[0216] pAAVS1-Puro-CMV-hIL12-BCAM(N32-628), ( Figure 12 (marked as FL)

[0217]

[0218] Among them, the BCAM (N32-628) sequence is as follows:

[0219] EVRLSVPPLVEVMRGKSVILDCTPTGTHDHYMLEWFLTDRSGARPRLASAEMQGSELQVTMHDTRGRSPPYQLDSQGRLVLAEAQVGDERDYVCVVRAGAAGTAEATARLNVFAKPEATEVSPNKGTLSVMEDSAQEIATCNSRNGNPAPKITWYRNGQRLEVPVEMNPEGYMTSRTVREASGLLSLTSTLYLRLRKDDRDASFHCAAHYSLPEGRHGRLDSPTFHLTLHYPTEHVQFWVGSPSTPAGWVREGDTVQLLCRGDGSPSPEYTLFRLQDEQEEVLNVNLEGNLTLEGVTRGQSGTYGCRVEDYDAADDVQLSKTLELRVAYLDPLELSEGKVLSLPLNSSAVVNCSVHGLPTPALRWTKDSTPLGDGPMLSLSSITFDSNGTYVCEASLPTVPVLSRTQNFTLLVQGSPELKTAEIEPKADGSWREGDEVTLICSARGHPDPKLSWSQLGGSPAEPIPGRQGWVSSSLTLKVTSALSRDGISCEASNPHGNKRHVFHFGTVSPQTSQAGVAVMAVAVSVGLLLLVVAVFYCVRRKGGPCCRQRREKGAPPPGEPGLSHSGSEQPEQTGLLMGGASGGARGGSGGFGDEC (SEQ ID NO: 30).

[0220] pAAVS1-Puro-CMV-hIL12-BCAM (N264-628), ( Figure 12 marked as Δ2 in)

[0221] MGMCHQQLVISWFSLVFLASPLVAIWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCSGGGGSGGGGSGGGGSGGGGSRNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNASGGGGSGGGGSGGGGSGGGGSTEHVQFWVGSPSTPAGWVREGDTVQLLCRGDGSPSPEYTLFRLQDEQEEVLNVNLEGNLTLEGVTRGQSGTYGCRVEDYDAADDVQLSKTLELRVAYLDPLELSEGKVLSLPLNSSAVVNCSVHGLPTPALRWTKDSTPLGDGPMLSLSSITFDSNGTYVCEASLPTVPVLSRTQNFTLLVQGSPELKTAEIEPKADGSWREGDEVTLICSARGHPDPKLSWSQLGGSPAEPIPGRQGWVSSSLTLKVTSALSRDGISCEASNPHGNKRHVFHFGTVSPQTSQAGVAVMAVAVSVGLLLLVVAVFYCVRRKGGPCCRQRREKGAPPPGEPGLSHSGSEQPEQTGLLMGGASGGARGGSGGFGDEC(SEQ ID NO:31)

[0222] Among them, the BCAM (N264-628) sequence is:

[0223] TEHVQFWVGSPSTPAGWVREGDTVQLLCRGDGSPSPEYTLFRLQDEQEEVLNVNLEGNLTLEGVTRGQSGTYGCRVEDYDAADDVQLSKTLELRVAYLDPLELSEGKVLSLPLNSSAVVNCSVHGLPTPALRWTKDSTPLGDGPMLSLSSITFDSNGTYVCEASLPTVPVLSRTQNFTLLVQGSPELKTAEIEPKADGSWREGDEVTLICSARGHPDPKLSWSQLGGSPAEPIPGRQGWVSSSLTLKVTSALSRDGISCEASNPHGNKRHVFHFGTVSPQTSQAGVAVMAVAVSVGLLLLVVAVFYCVRRKGGPCCRQRREKGAPPPGEPGLSHSGSEQPEQTGLLMGGASGGARGGSGGFGDEC(SEQ ID NO:32).

[0224] pAAVS1-Puro-CMV-hIL12-BCAM(N448-628),( Figure 12 labeled as Δ4 in)

[0225] MGMCHQQLVISWFSLVFLASPLVAIWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCSGGGGSGGGGSGGGGSGGGGSRNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNASGGGGSGGGGSGGGGSGGGGSPELKTAEIEPKADGSWREGDEVTLICSARGHPDPKLSWSQLGGSPAEPIPGRQGWVSSSLTLKVTSALSRDGISCEASNPHGNKRHVFHFGTVSPQTSQAGVAVMAVAVSVGLLLLVVAVFYCVRRKGGPCCRQRREKGAPPPGEPGLSHSGSEQPEQTGLLMGGASGGARGGSGGFGDEC(SEQ ID NO:33).

[0226] Among them, the BCAM (N448 - 628) sequence is:

[0227] PELKTAEIEEPKAADGSWREGDEVTLICSARGHPDPKLSWSQLGGSPAEPIPGRQGWVSSSLTLKVTSALSRDGISCEASNPHGNKRHVFHFGTVSPQTSQAGVAVMAVAVSVGLLLLVVAVFYCVRRKGGPCCRQRREKGAPPPGEPGLSGSEQPEQTGLLMGGASGGARGGSGGFGDEC (SEQ ID NO: 34).

[0228] The structures of the two truncated bodies are as follows: Figure 12 As shown in Figure A, the expression results of the fusion protein are as follows: Figure 12 As shown in B. The results show that the truncated BCAM still has the ability to carry the target protein to the EV. The activity of the fusion protein was detected using different concentration gradients, and the results showed that the fusion protein constructed with the truncated form also had activity ( Figure 12 C).

[0229] Example 11: Engineered EVs for drug delivery

[0230] Doxorubicin hydrochloride (DOX) is a cyclic nonspecific anticancer chemotherapy drug, mainly used clinically to treat acute lymphoblastic leukemia, acute myeloid leukemia, breast cancer, lung cancer, soft tissue sarcoma, and liver cancer. However, serious adverse reactions often occur during chemotherapy, such as cardiotoxicity, bone marrow suppression, nausea, and vomiting. Furthermore, DOX has a low molecular weight, easily diffuses in the body, and is relatively evenly distributed in tissues, producing toxic side effects on normal tissues and affecting antitumor activity. To reduce the toxic side effects of antitumor drugs and improve efficacy, EVs were used to encapsulate it, and the drug loading was measured. Doxorubicin hydrochloride is readily soluble in aqueous solution. 1 ml of DOX-HCl (850 μg / ml) was prepared using PBS solution. 1 ml of fresh EVs (12,000 ng / μl protein) was extracted from 293 cells. 200 μL (10E12 EVS) was taken from 1 mL of EVs solution and 50 μL of DOX-HCl (850 μg / mL) solution was added, containing 42.5 μg of DOX-HCl. The EVS and DOX-HCl were then mixed, and PBS was added to 1 mL. The mixture was incubated overnight (16 h) at 37 °C. The incubated solution was subjected to size exclusion testing using CL-2B packing material, and the first four fractions (500 μL / tube) were collected. 2 mL of the collected solution was added to a 100 KD ultrafiltration tube and centrifuged at 4000 g and 4 °C for 5 min to concentrate the solution. The concentrated solution yielded the DOX-loaded EVs drug solution. The pink DOX-HCl-loaded EVs solution was then subjected to Triton X-ray filtration to release the drug. The mixture was centrifuged at 150,000 g for 2 h, and the Triton X-ray supernatant was extracted. The absorbance was measured at 485 nm for concentration calculation.

[0231] The efficacy of DOX-loaded extracellular vesicles was evaluated. DOX was diluted to the following concentration gradients: 10000 nM, 3333.34 nM, 1111.11 nM, 370.37 nM, 123.46 nM, 41.15 nM, 13.71 nM, 4.58 nM, 1.53 nM, 0 nM; DOX-loaded extracellular vesicles were diluted to the following concentration gradients: 2500 nM, 833.34 nM, 277.78 nM, 92.59 nM, 30.86 nM, 10.28 nM, 3.43 nM, 1.14 nM, 0.38 nM, 0 nM. MCF7 cells were seeded in 96-well plates and cultured at 37°C and 5% CO2 for at least one day. One day later, the culture medium was replaced with either of the two gradient concentrations of DOX solution mentioned above, or with extracellular vesicle solution containing DOX. After culturing for another 72 hours, the cells were removed, and 10 μl of CCK8 solution was added to each well for an additional 2 hours of culturing. The OD450 absorbance was then measured. Figure 13 ).

[0232] Example 12: Engineered EVs for purification

[0233] By displaying affinity chromatography-friendly groups, such as flag tags, at the extracellular ends of scaffold proteins, EVs can be directly separated from the liquid phase using affinity chromatography / immunocapture methods.

[0234] Thoroughly resuspend the Anti-Flag Affinity Gel to form a homogeneous solution. Transfer 40 μL of the mixture (20 μL gel) to a new centrifuge tube. Centrifuge at 5,000–8,200 × g for 30 seconds to allow the gel to settle at the bottom of the tube. Let it stand for 1–2 minutes before adding the sample. Remove the supernatant, handling carefully to avoid aspirating the gel. Add 500 μL of TBS and gently resuspend the Anti-Flag Affinity Gel. Centrifuge at 10,000 rpm for 30 seconds, discard the supernatant, and repeat the above steps once. To remove trace amounts of unbound antibody from the gel, add 500 μL of 0.1 M glycine HCl to pH 3.5 to wash the gel. Centrifuge to remove the supernatant, add 3 volumes of TBS, gently shake for 2–3 minutes, centrifuge at 5,000–8,200 × g for 30 seconds, discard the supernatant, and repeat washing until the supernatant pH is neutral. Do not exceed 20 minutes for the Glycine HCl treatment. Add 200-1000 μL of extracellular vesicles, adjusting to a final volume of 1 mL if necessary. For the positive control, add 1 mL of TBS and 4 μL of 50 ng / μL Flag-BAP fusion protein (approximately 200 ng); for the negative control, add only 1 mL of PBS (protein-free). Incubate slowly at 4°C for 2 hours. To improve binding efficiency, incubation can be extended overnight. Centrifuge at 5,000-8,200 × g for 30 seconds and remove the supernatant. Mix the precipitate with 0.5 mL of TBS, gently shake, centrifuge at 5,000-8,200 × g for 30 seconds to remove the supernatant, and repeat 3 times.

[0235] Flag fusion extracellular vesicle elution (choose one of the following 3 methods): 1) Non-denaturing elution (3×Flag peptide). Prepare 3×Flag peptide elution buffer: Dissolve 3×Flag peptide in 0.5M Tris-HCl solution, pH 7.5 (containing 1M NaCl), to a final concentration of 25 μg / μL. Dilute to 5 μg / μL with ddH2O, add 3 μL of 5 μg / μL 3×Flag peptide, and add to 100 μL TBS (final concentration 150 ng / μL). Add 100 μL of 3×Flag peptide elution buffer, incubate at 4℃ for 30 min (with gentle shaking), centrifuge at 5,000-8,200×g for 30 s, and transfer the supernatant to a new tube (do not aspirate the gel). If used immediately, the supernatant can be stored at 4℃ to -20℃ for long-term storage. 2) Acidic elution (0.1M Gly-HCl, pH 3.5). Add 100 μL of 0.1 M Gly-HCl (pH 3.5), incubate at room temperature for 5 min (with gentle shaking), centrifuge at 5,000–8,200 × g for 30 s, and transfer the supernatant to a new tube (containing 10 μL of 0.5 M Tris-HCl (pH 7.5), 1.5 M NaCl). Avoid aspirating the gel. If used immediately, the supernatant can be stored at 4°C to -20°C for long-term storage. 3) Elute with SDS-PAGE loading buffer. Add 20 μL of 2× loading buffer (125 mM Tris-HCl, pH 6.8, 4% SDS, 20% glycerol, 0.004% bromophenol blue), boil for 3 min, and centrifuge at 5,000–8,200 × g for 30 s. The supernatant can be directly used for SDS-PAGE electrophoresis or Western blotting.

[0236] Take an appropriate size NC membrane, soak it in methanol for at least 20 seconds, then wash it with a suitable amount of pre-cooled trans buffer and set aside. Prepare the trans buffer according to the following formula: 100ml 10x trans buffer (Kangwei Century) + 200ml methanol + 700ml ddH2O. (Pre-cool after preparation) Take out the runnated gel and soak it in the pre-cooled trans buffer. Assemble the sandwich model in the following order: black side - cotton - filter paper - gel - NC membrane - filter paper - cotton - white side. Then place the sandwich model into the transfer tank in the order of "black side - black side, white side - white side". Add two ice packs that have been frozen overnight at -80°C to the transfer box, pour in the transfer solution to soak the entire sandwich model, install the transfer box, and transfer at a constant current of 350mA for 1 hour. Blocking: Prepare a 5% BSA blocking solution (dissolved in 1X TBST), take out the transferred membrane and place it in the blocking solution, and block on a horizontal shaker at room temperature for 1-2 hours. Primary antibody incubation (primary antibodies are the corresponding antibodies for each target protein): After blocking, wash three times with 1X TBST, prepare the primary antibody solution in TBST according to the specified ratio, and incubate for an appropriate time. Record the preparation ratio and incubation time in Table 1. Secondary antibody incubation: After blocking, wash three times with 1X TBST, prepare the secondary antibody solution in TBST according to the specified ratio, and incubate for an appropriate time. ECL chemiluminescence staining: After secondary antibody incubation, wash three times with 1X TBST. Before staining, mix the ECL chemiluminescence AB solution at a 1:1 ratio and then perform staining on the membrane using the Tanon-5200Multi chemiluminescence gel imaging system. Figure 15 Example 13: Validation of mouse T cell targeting of mCD3 scFab exosomes.

[0237] EVs mark

[0238] The mCD3 scFab expression and control exosomes used in this embodiment of the invention are the same as in Example X. During exosome extraction, the lipophilic orange-red fluorescent anthocyanin dye DiL (D3911, Invitrogen) was used for labeling. Specifically, for the cell supernatant from suspension culture, the recommended pretreatment steps are: aliquot into 50ml centrifuge tubes, centrifuge at 800g for 5 min to transfer the supernatant, centrifuge at 3000g for 20 min to transfer the supernatant, and filter the supernatant through a 0.22µm filter. Add the cell supernatant to a 100kd ultrafiltration tube. Centrifuge at 4,000g for 10 min. Concentrate the supernatant to a total volume of less than 200mL or a concentration factor of approximately 10 times. Add DiL at a concentration of 5µM and stain at 37°C for 30 min. After staining, filter through a 0.22µm filter. Centrifuge at 10,000g for 20 min to transfer the supernatant. Add the concentrated supernatant from the staining process to an ultrafiltration tube, centrifuge at 10,000g at 4°C for 2 hours, and resuspend the stained EVs in approximately 2 mL of PBS per 20 mL of supernatant. Add the stained EVs to a 100 kDa ultrafiltration tube, centrifuge at 4,000g for 10 minutes, and centrifuge until the final volume corresponds to approximately 1,000 times the original supernatant concentration. Store the stained EVs at 4°C protected from light.

[0239] Quantification of stained EVs: The stained EVs were diluted to a final concentration of approximately 1E+7-1E+8 particles / mL, and the number of exosome particles was recorded using a nanoflow cytometer (N30E, Fuliu Biotechnology).

[0240] Cellular active targeting

[0241] 24 hours before the formal experiment, spleen cells from C57BL / 6 mice were harvested and lysed with erythrocyte lysis buffer (Solarbio, R1010) to prepare mPBMCs. The prepared mPBMCs were seeded in 96-well plates at a density of 2E+5 cells / well. During the formal experiment, control exosomes and experimental exosomes were co-incubated with mPBMCs at concentrations of 1E+7, 1E+8, 1E+9, 2.5E+9, and 5E+9 particles / mL in a cell culture incubator (CLM-170B-8-NF, ESCO) at 37°C and 5% CO2 for 2 hours. Cells were then harvested for flow cytometry analysis.

[0242] Before flow cytometry, mPBMCs were washed once with sterile PBS, then incubated with APC-labeled anti-mCD3 antibody (Invitrogen, 17-0032-82) at 4°C for 1 hour. After incubation, they were washed again with PBS and resuspended for analysis. For the assay, mPBMCs without added stained exosomes and APC-mCD3 antibody were used as a gate control. Data analysis was performed using an NxT acoustic focusing flow cytometer (A24860, Thermo Fisher) and its accompanying software.

[0243] Figure 16 The experimental results are shown. Figure 16 A shows the DiL-labeled cell population (green cells) using the BL2-A (excitation: 488nm, emission filter: 569nm-601nm; area) parameter and the APC-labeled cell population (red cells) using the RL1-A (excitation: 637nm, emission filter: 656nm-684nm; area) parameter. Figure 16 B showed that exosomes expressing mCD3 scFab exhibited a significantly enhanced tendency for endocytosis in mouse T cells under co-incubation conditions of 2 h.

[0244] All disclosures and patents mentioned in this application are incorporated herein by reference. Various modifications and variations of the methods and compositions described herein will be apparent to those skilled in the art without departing from the scope and spirit of the invention. While the invention has been described through specific preferred embodiments, it should be understood that the claimed invention should not be unduly limited to these specific embodiments. In fact, various variations of the described modes of carrying out the invention that will be apparent to those skilled in the art are intended to be included within the scope of the appended claims.

Claims

1. An engineered extracellular vesicle comprising a transmembrane protein as a scaffold protein, said transmembrane protein being BCAM or a truncated form thereof, said BCAM having an amino acid sequence as shown in SEQ ID NO: 5, and said truncated form having an amino acid sequence as shown in SEQ ID NO: 32 or 34.

2. The extracellular vesicle according to claim 1, wherein the transmembrane protein is a fusion protein containing the target polypeptide segment. The target polypeptide segment is located at the N-terminus or C-terminus of the fusion protein. The target polypeptide segment enables the fusion protein to have therapeutic, targeting, and / or affinity tagging functions.

3. The extracellular vesicle according to claim 2, wherein the target polypeptide segment is a therapeutic polypeptide segment, and the therapeutic polypeptide segment is selected from members of the human interleukin family, members of the tumor necrosis factor family, interferons, and other cytokines.

4. The extracellular vesicle according to claim 2, wherein the target polypeptide segment is a polypeptide segment that targets cells.

5. The extracellular vesicle according to claim 4, wherein the target cell polypeptide segment is an antibody or its antigen-binding fragment, a cell surface receptor or ligand.

6. The extracellular vesicle according to claim 2, wherein the target polypeptide segment is an affinity tag. The affinity tag is selected from His tag, glutathione thiotransferase, S-peptide, ZZ domain, albumin-binding domain, HA, Myc, FLAG™, maltose-binding protein, calmodulin-binding peptide, SUMO, streptococcal protein G, and Staphylococcus aureus protein A.

7. The extracellular vesicle of claim 1, wherein the extracellular vesicle contains a therapeutic agent.

8. The extracellular vesicle of claim 7, wherein the therapeutic agent is selected from therapeutic peptides, polynucleotides, and small molecule compounds.

9. A pharmaceutical composition comprising an extracellular vesicle according to any one of claims 1 to 8 and a pharmaceutically acceptable carrier.

10. An engineered cell for producing extracellular vesicles according to any one of claims 1 to 8.

11. A method for preparing extracellular vesicles according to any one of claims 1 to 8, comprising: 1) Express the transmembrane protein in cells, and 2) Isolate the extracellular vesicles.

12. A method for delivering a therapeutic agent to target cells, said method not for disease diagnosis and treatment, comprising: 1) Preparation of extracellular vesicles according to any one of claims 1 to 8, wherein the transmembrane protein is a fusion protein containing an affinity tag, and 2) Make the extracellular vesicles come into contact with the target cells.

13. A method for isolating extracellular vesicles according to any one of claims 1 to 8, comprising: 1) Express the transmembrane protein in cells, the transmembrane protein containing an affinity tag, 2) Contact the extracellular vesicles with a binding agent capable of binding the affinity tag, and 3) The extracellular vesicles are separated based on the binding of the affinity tag to the binding agent.

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