CN116103338A

CN116103338A - Engineered extracellular vesicles and uses thereof

Info

Publication number: CN116103338A
Application number: CN202111320152.6A
Authority: CN
Inventors: 赵立波; 孔关义; 樊凤辉; 程立; 李鸿艳; 任亚芳; 吴洁
Original assignee: Beijing Echo Biotech Co ltd
Current assignee: Beijing Echo Biotech Co ltd
Priority date: 2021-11-09
Filing date: 2021-11-09
Publication date: 2023-05-12

Abstract

The invention provides an engineering extracellular vesicle, a pharmaceutical composition containing the engineering extracellular vesicle, a preparation method and application thereof. The engineered extracellular vesicles can be used for drug delivery, expression of a protein of interest in an extracellular vesicle, or purification of an extracellular vesicle.

Description

Engineered extracellular vesicles and uses thereof

Technical Field

The invention relates to the field of biological medicine, in particular to an engineering extracellular vesicle, a preparation method and application thereof.

Background

Extracellular vesicles (extracellular vesicle, EV) are membranous vesicle small bodies secreted by cells and capable of uptake by recipient cells, approximately 30-1000nm in diameter. EV is an important medium for cell-to-cell communication as a carrier for transporting biomacromolecules such as proteins, RNA, lipids and the like among cells. Because EV is widely present in various body fluids of the human body, it can be used as a sample source for liquid biopsies and is also considered as a natural domesticated drug carrier.

In research and application of EV, how to prepare EV with specific functions is a key technical link. For example, in order to develop a drug carrier and to improve tissue targeting of EV, it is necessary to express a targeting molecule having high affinity with a target cell on the surface of the target molecule. It is also desirable to carry a polypeptide or protein having therapeutic effects into the EV. For development of an EV in vitro diagnostic kit, it is necessary to obtain an EV loaded with a specific analyte molecule as a standard.

According to the existing reports, about 20 proteins are reported to be available for the loading of the protein of interest in EV (Theranostics, 2019;9 (4): 1015-1028). Patent applications WO2013084000A2, WO2014168548A2, WO2018015535A1, WO2019040920A1 and the like also describe methods for loading protein components into EVs.

Disclosure of Invention

The inventor of the application unexpectedly discovers a class of Extracellular Vesicle (EV) scaffold proteins in research, wherein the scaffold proteins have low background expression in the naturally secreted extracellular vesicles, but the proteins have unexpected excellent effects in the aspects of EV construction, target protein display, drug loading and the like. Based on the above findings, the inventors completed the present invention.

In a first aspect, the invention provides an engineered extracellular vesicle (extracellular vesicle, EV) comprising as scaffold protein a transmembrane protein which is a member of the immunoglobulin superfamily, the transmembrane protein having a low background expression level in the unengineered extracellular vesicle.

In some embodiments of the invention, the background expression level of the transmembrane protein in the unengineered extracellular vesicles is lower than the background expression level of CD81, e.g., the lower background expression level of CD81 means that in mass spectrometry detection of the unengineered extracellular vesicles total protein the iBAQ value of CD81 is set to 1, 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.

In some embodiments of the invention, wherein the transmembrane protein meets at least one of the following conditions:

(A) The transmembrane protein has similarity with BCAM, which means that when BCAM protein is used as a standard, the transmembrane protein has an E value < = 1.0E-4 compared with BCAM, the E value is calculated by formula (I), which is derived from NCBI blast program,

E＝K*m*n*exp(-lambda*S) (I)

wherein K and lambda are constants related to the database and the algorithm, m represents the length of the target sequence, n represents the size of the database, S value represents the homology of the two sequences, and the higher the S score is, the greater the homology between the two sequences is;

(B) The transmembrane protein is a single transmembrane protein whose extracellular region comprises 1, 2, 3, 4, 5, 6, 7, 8, or 9 immunoglobulin domains (immunoglobulin domain, ig domain); and

(C) The extracellular region of the transmembrane protein comprises both an IgV domain and an IgC domain.

In some embodiments of the invention, the transmembrane protein satisfies a combination selected from the group consisting of: a and B, A and C, B and C, and a and B and C.

In some embodiments of the invention, the transmembrane protein is selected from BCAM, MCAM, ICAM, ALCAM, CD276, CADM1, VCAM1, CADM2, necin 4, CADM3, agrer, CD96, and variants thereof.

In some embodiments of the invention, the extracellular region of the transmembrane protein comprises 3, 4 or 5 immunoglobulin domains,

in particular, the extracellular region of the transmembrane protein comprises both an IgV domain and an IgC domain,

more particularly, the IgC domain is an IgC2 domain,

more particularly, the extracellular region of the transmembrane protein comprises 5 immunoglobulin domains, the 5 immunoglobulin domains being 2 IgV domains and 3 IgC domains in order from the N-terminus to the C-terminus.

In some embodiments of the invention, the transmembrane protein is selected from BCAM, MCAM, ALCAM, CD, CADM1, CADM2, necin 4, CADM3, agrer, CD96, and variants thereof;

in particular, the transmembrane protein is selected from BCAM, MCAM, ALCAM, CADM, CADM2, CADM4, CADM3, agr, and variants thereof;

more particularly, the transmembrane protein is selected from BCAM, MCAM, ALCAM, and variants thereof.

In some embodiments of the invention, the single transmembrane protein is a fusion protein comprising a polypeptide segment of interest; preferably, the polypeptide segment of interest is located at the N-terminus or C-terminus of the fusion protein; preferably, the polypeptide segment of interest confers therapeutic, targeting and/or affinity tag function to the fusion protein.

In some embodiments of the invention, the polypeptide segment of interest is a therapeutic polypeptide segment, e.g., the therapeutic polypeptide segment is a human interleukin family member (e.g., IL-2, IL-7, IL-10, IL-11, IL-12, IL-15, and IL-23), tumor necrosis factor family member (e.g., TNF, LTA, LTB, FASLG, TNFSF, TNFSF9, TNFSF10, TNFSF11, TNFSF12, TNFSF13, TNFSF14, TNFSF15, TNFSF18, and EDA), interferon (INF- α, INF- β, and INF- γ), T cell engager (e.g., 4-1BB, OX40, CD28, CD40L, CD47, CD27, CD70, CD80, CD86, GITRL, ICOSL, CD155, CD112, TIM-3, BTLA), other cytokines (e.g., G-CSF, EPO, TPO, GM-CSF, EGF, bFGF, FVIIa, AT III, K, α -glucolase, BMP-2, BMP-2, and so forth).

In some embodiments of the invention, the polypeptide segment of interest is a cell-targeting polypeptide segment, in particular, the cell-targeting polypeptide segment is selected from an antibody or antigen binding fragment thereof, a cell surface receptor, or a ligand

In some embodiments of the invention, the polypeptide segment of interest is an affinity tag, in particular, the affinity tag is selected from the group consisting of His tag, glutathione thiol transferase (GST), S-peptide, ZZ domain, albumin Binding Domain (ABD), HA, myc, FLAGTM, maltose Binding Protein (MBP), calmodulin Binding Peptide (CBP), SUMO, streptococcal Protein G (Protein G), and xyloglucan Protein a (Protein a).

In some embodiments of the invention, the extracellular vesicles contain a therapeutic agent therein; in particular, the therapeutic agent is selected from the group consisting of therapeutic polypeptides, polynucleotides and small molecule compounds.

In a second aspect, the invention provides a pharmaceutical composition comprising said extracellular vesicles and a pharmaceutically acceptable carrier.

In a third aspect, the invention provides an engineered cell for use in the production of said extracellular vesicles.

In a fourth aspect, the invention provides a method of treating a disease comprising administering to a subject in need thereof said extracellular vesicles, said pharmaceutical composition or said cells.

In a fifth aspect, the present invention provides a method of preparing the extracellular vesicles, comprising

1) Expressing the single transmembrane protein in a cell, and

2) Isolating the extracellular vesicles.

In a sixth aspect, the invention provides a method of delivering a therapeutic agent to a target cell comprising

1) Preparing the extracellular vesicles, wherein the transmembrane protein is a fusion protein comprising an affinity tag, and

2) Contacting the extracellular vesicles with the target cells.

In a seventh aspect, the present invention provides a method of isolating the extracellular vesicles, comprising

1) Expressing the transmembrane protein in a cell, the transmembrane protein comprising an affinity tag,

2) Contacting the extracellular vesicles with a binding agent capable of binding the affinity tag, and

3) Isolating the extracellular vesicles based on the binding of the affinity tag to the binding agent.

Unlike the engineered extracellular vesicles reported in the prior art, the proteins used in the construction of extracellular vesicles in the prior art are all membrane proteins with high EV expression levels, whereas the present invention selects membrane proteins with low or no background expression on EV. When such proteins are used to construct EVs, less competition from endogenous proteins (e.g., non-fusion proteins of the EV itself) facilitates active sorting of such proteins (e.g., fusion proteins carrying the protein of interest) into the EV, increasing their relative content in the EV, and thereby greatly increasing the content of active ingredient carried by the EV.

Drawings

FIG. 1 shows the expression of EGFP-BCAM fusion protein in EV in example 2, PTGFRN and LAMP2B as controls.

FIG. 2 shows the Western blot detection results of wild-type and fusion proteins in total cellular proteins and EV (FIG. 2A) and the percentage of wild-type and fusion proteins in the sum of both (FIG. 2B) in example 2.

FIG. 3 shows the results of fluorescence detection of EV carrying eGFP fusion protein in example 3.

FIG. 4 shows ELISA detection results, showing the expression level of IL 12-carrying fusion protein in example 4 at EV.

FIG. 5 shows the expression of different BCAM fusion proteins in EV.

FIG. 6 shows the results of EC50 assays for stimulation of PBMC by IL12 of different origins to produce IFN-y; FIG. 6A shows IL12 recombinant protein, FIG. 6B shows EV expressing PTGFRN-IL12, and FIG. 6C shows EV expressing BCAM-IL 12.

FIG. 7 shows the structure of some scaffold proteins of the invention, derived from the Uniprot database.

FIG. 8 shows the expression of the MCAM and EGFP fusion protein of example 8 at EV.

FIG. 9 shows the results of detection of captured EV-carried IL12 in example 8.

FIG. 10 shows the results of EC50 assays for stimulation of PBMC by IL12 of different origins to produce IFN-y; FIG. 10A shows the recombinant protein for IL12, FIG. 10B shows the EV expressing BCAM-IL12, and FIG. 10C shows the EV expressing MCAM-IL 12.

FIG. 11 shows that fusion with BCAM and MCAM did not affect luciferase activity.

FIG. 12 shows the structure of the BCAM truncations (FIG. 12A), the results of the expression of the fusion proteins in cells and EVs (FIG. 12B), and the activity assays of full length and truncations Δ4.

Fig. 13 shows encapsulating DOX with EV in example 11.

Fig. 14 shows a DOX-entrapped gradient experiment of example 11.

FIG. 15 shows the purification results of example 12.

Fig. 16 shows the experimental results of example 13.

Detailed Description

Terminology

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

The term "extracellular vesicles (extracellular vesicles, EVs)" as used herein refers to vesicles of double membrane structure, which are detached from or secreted by cell membranes, and vary in diameter from 40nm to 1000nm, mainly in the form of Microvesicles (MVs) and Exosomes (Exosomes, exs). Extracellular vesicles are widely present in cell culture supernatants and various body fluids (blood, lymph, saliva, urine, semen, milk), and carry various proteins, lipids, DNA, mRNA, miRNA, etc. related to cell sources, and are involved in intercellular communication, cell migration, angiogenesis, and immunomodulation. The term "engineered extracellular vesicles" refers to extracellular vesicles that are synthesized artificially, or that are produced by cells after human intervention, or that are produced by cells after genetic engineering. The term "non-engineered extracellular vesicles" refers to extracellular vesicles that are secreted by natural, non-engineered cells under normal conditions (e.g., physiological conditions).

The term "scaffold protein" as used herein means that its native form is present in an unengineered extracellular vesicle, and is capable of carrying other proteins into the extracellular vesicle when forming a fusion protein with other proteins (proteins/polypeptides of interest).

The term "single transmembrane protein" as used herein refers to a protein comprising only one transmembrane segment, which membrane is a lipid bilayer membrane, which may be a cell membrane, an organelle membrane, a vesicle membrane, an artificial lipid bilayer, etc.

The term "immunoglobulin domain (immunoglobulin domain, ig domain), sometimes also referred to as immunoglobulin-like domain (Ig-like domain), as used herein, is an Ig domain named immunoglobulin molecule comprising about 70-110 amino acids and is classified according to size and function. Ig domains can be classified as IgV, igC1, igC2 or IgI, with most Ig domains belonging to either variable Ig domains (IgV) or constant Ig domains (IgC). The Ig domains of certain members of IgSF (immunoglobulin superfamily) are similar in amino acid sequence to IgV domains, but similar in size to IgC domains, these being referred to as IgC2 domains, while the standard IgC domains are referred to as IgC1 domains.

The term "variant" of a protein, as used herein, refers to a protein whose amino acid sequence differs from the naturally occurring corresponding protein amino acid sequence but has the same or similar function, and which is capable of being expressed in an extracellular vesicle, as well as of carrying the protein/polypeptide of interest into an extracellular vesicle secreted by the cell. Variants of a protein may be derived from naturally occurring protein amino acids via deletions, additions and/or substitutions, and may also be truncated forms of the native protein.

BCAM refers to the basal cell adhesion molecule (Basal cell adhesion molecule). MCAM refers to melanoma cell adhesion molecule (Melanoma Cell Adhesion Molecule), also known as the cell surface glycoprotein MUC18, CD146.ALCAM refers to activated leukocyte adhesion molecules (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 (Cell adhesion molecule 1). CADM2 refers to cell adhesion molecule 2 (Cell adhesion molecule 2). CADM3 refers to cell adhesion molecule 3 (Cell adhesion molecule 3). CADM4 refers to cell adhesion molecule 4 (Cell adhesion molecule 4). NECTIN4 refers to connexin cell adhesion molecule 4 (Nectin cell adhesion molecule 4). AGER refers to the late glycosylation end product specific receptor (Advanced glycosylation end product-specific receptor). CD96 is a cell surface antigen.

The term "therapeutic polypeptide segment" as used herein refers to a therapeutically active protein or fragment or variant thereof, including but not limited to human interleukin family members (e.g., IL-2, IL-7, IL-10, IL-11, IL-12, IL-15, and IL-23), tumor necrosis factor family members (e.g., TNF, LTA, LTB, FASLG, TNFSF, TNFSF9, TNFSF10, TNFSF11, TNFSF12, TNFSF13, TNFSF14, TNFSF15, TNFSF18, and EDA), interferons (INF- α, INF- β, and INF- γ), T cell engager (e.g., 4-1BB, OX40, CD28, CD40L, CD47, CD27, CD70, CD80, CD86, GITRL, ICOSL, CD155, CD112, ICOSL-3, BTLA), and other cytokines (e.g., G-CSF, EPO, TPO, GM-CSF, EGF, bFGF, FVIIa, AT, TNK, α -glucolase, TIM-2, and Tdin-y).

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

The term "affinity tag" as used herein refers to a polypeptide segment capable of binding to a corresponding binding agent, including but not limited to His tag, glutathione thiol transferase (GST), S-peptide, ZZ domain, albumin Binding Domain (ABD), HA, myc, FLAG ^TM Maltose Binding Protein (MBP), calmodulin Binding Peptide (CBP), SUMO, streptococcal Protein G (Protein G), and Staphylococcus aureus Protein A (Protein A). The term "binding agent" refers to a substance capable of specifically binding an affinity tag.

The term "therapeutic agent" as used herein refers to a substance having a therapeutic effect and may be a nucleotide, amino acid, lipid, carbohydrate, small molecule, antibody, enzyme, ligand, receptor, polypeptide, or the like.

Notwithstanding that the numerical ranges and approximations of the parameters set forth in the broad scope of the invention, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements. In addition, all ranges disclosed herein are to be understood to encompass any and all subranges subsumed therein. For example, a range recited as "1 to 10" should be considered to include any and all subranges between (inclusive of) the minimum value of 1 and the maximum value of 10; that is, all subranges beginning with a minimum value of 1 or more, e.g., 1 to 6.1, and ending with a maximum value of 10 or less, e.g., 5.5 to 10. In addition, any reference referred to as "incorporated herein" should be understood as being incorporated in its entirety.

It should be further noted that, as used in this specification, the singular forms include the plural of what is meant by them unless clearly and explicitly limited to one what is meant by them. The term "or" may be used interchangeably with the term "and/or" unless the context clearly indicates otherwise.

The term "subject" as used herein refers to a mammal, such as a human, but may also be other animals, such as wild animals (e.g., aigrette, geranium, crane, etc.), domestic animals (e.g., duck, goose, etc.), or laboratory animals (e.g., gorilla, monkey, rat, mouse, rabbit, guinea pig, woodchuck, ground squirrel, etc.).

The compositions of the present invention may be applied by a variety of methods known in the art. The skilled artisan will appreciate that the route and/or mode of administration will vary depending on the desired outcome. In order to administer the compounds of the present invention by a particular route of administration, it may be desirable to cover the compound with, or co-administer the compound with, a material that avoids its inactivation. For example, the compound may be administered to the subject in a suitable carrier, such as a liposome or diluent. Pharmaceutically acceptable diluents include saline and aqueous buffers. The pharmaceutical carrier includes sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. The use of such media and agents for pharmaceutically active substances is known in the art.

The compositions of the present invention may also contain adjuvants such as preserving, wetting, emulsifying and dispersing agents. The avoidance of the presence of microorganisms can be ensured both by the sterilization procedure hereinabove and by the inclusion of various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to include isotonic agents, for example, sugars, sodium chloride, and the like in the compositions. In addition, prolonged absorption of the injectable pharmaceutical form can be brought about by the inclusion of agents which delay absorption, for example, aluminum monostearate and gelatin.

Examples

Some preferred embodiments and aspects of the invention are further described below in conjunction with specific examples. These examples should not be construed as limiting the scope of the invention.

Example 1: materials and methods

The materials and sources used in the examples of the present invention are shown in Table 1.

TABLE 1

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Experimental method

1. Cell transfection

The mother cell line used in the examples of the present invention was an Expi293F cell (A14527 CN, thermo Fisher, america). Cells were trypsinized and counted one day prior to transfection, and cells were passaged and plated in total 1.5X106 cells, 5mL volume. The next day, a transfection solution is prepared, wherein solution A contains sgAAVS1 1 mug, pDONOR 4 mug and Opti-MEM culture medium 125 mug, which are evenly mixed and then left for 5min; the solution B contained PEI (working solution concentration 1. Mu.g/. Mu.l) 12.5. Mu.l and Optipro-MEM medium 125. Mu.l, which were mixed and allowed to stand for 5min. 1 hour prior to transfection, the cell culture medium was replaced with Opti-MEM and placed in a cell incubator. And (3) dripping the solution B into the solution A, mixing gently, and standing for 20 dishes. The prepared transfection reagent was added dropwise to the T25 flash. The cells were incubated at 37℃under 5% CO2 for 48h.

Fresh KOP293-Ex medium containing puromycin at a final concentration of 2 μg/mL was prepared, the medium was changed 1 time every 2-3 days, 1:1 passage after digestion if the cells were overfilled, and puromycin selection was continued until the control cells were essentially killed. After the control cells were killed, the experimental cells were grown in KOP293-Ex+5% FBS medium supplemented with 2. Mu.g/mL puromycin. Placing in an incubator at 37 ℃ and culturing with 5% CO 2. Fresh medium was changed 1 time every 2-3 days until the cell confluency reached 60-80%. Passaging was performed at 1:1.

Gradually reducing serum, domesticating and performing suspension culture.

EV extraction

The Expi293F cell culture flask was removed from the incubator, shaken well, and then a small amount of cells were pipetted into a 1.5mLEP tube, 20. Mu.l of the cell suspension was pipetted into a new EP tube, 20. Mu.l of phenol blue was added, thoroughly mixed, 10. Mu.l of the cell suspension was added to a cell counting plate, and counted by a cell counter (BIO-RAD). When the cell density was 4 to 6X 10.6 cells/mL and the activity was 95% or more, the cell suspension was diluted with KOP293-EX medium (KOD , R.Biotech., co., ltd., K03256) to adjust the cell density to 2X 10.6 cells/mL. 300mL of the cell suspension was transferred to a 1000mL Erlenmeyer flask (Corning) for suspension culture. Culture conditions: 37 ℃,5% CO2, the rotating speed is 110rpm, and the humidity is more than 75%. Cell density and viability were recorded after 7 days, and when cell density was 1×10 7 cells/mL and viability > 90%, cell supernatant was collected and EV was extracted.

300mL of cell supernatant was mixed well and then packed into 50mL centrifuge tubes, and centrifuged at 800g for 10min. The supernatant was transferred to a new 50mL centrifuge tube and centrifuged at 4000g for 10min at RT. The supernatant was transferred and the whole supernatant was filter sterilized through a 0.22um filter. The supernatant was concentrated using a tangential flow membrane pack. Tangential flow parameters are as follows: the cut off molecular weight is 10-30 k; tangential flow aperture is 10-30 nm; tangential flow concentration factor: concentrated 10-fold, i.e., 1000mL of cell supernatant was concentrated to 100mL. The concentrated supernatant was added to a super-separation tube and centrifuged at 10wg at 4℃for 2h. After centrifugation, 1mL of PBS was added to each tube to resuspend EVs pellet, and then 1mL of PBS was added to wash the super-tube. The resuspended EVs were added to a 100kd ultrafiltration tube, centrifuged for 4000g,10min and the centrifugation time adjusted according to different samples. Centrifuge to a final volume of 1/1000 of the original supernatant (e.g., 1000mL cell supernatant concentrated to 1mL EVs). EVs were collected into 1.5mL centrifuge tubes and BCA assay was performed for total protein concentration.

3. Western Blotting (WB)

An appropriate amount of 5x loading buffer was added to a final concentration of 1 x loading buffer,95 ℃for 10min, either EV 10 μg total protein or cell lysate 20 μg total protein. The running buffer (1L) was prepared as follows: 100mL 10x running buffer (well known as century) +900mL ddH2O. Mixing lightly for use. And installing the prefabricated glue (Biyun) with proper concentration in the glue running groove, and pouring the prepared running buffer into the glue running groove after confirming that the assembled glue running groove is not leaked, so that the running buffer is not over the glue surface. A proper amount of heat-denatured sample was taken with a pipette and carefully added to the wells of SDS-PAGE gels (note that loading was not performed too hard, avoiding contamination of one well with the sample from the other). After the gel running groove power supply is correctly connected, the gel is run at 80v voltage, after the sample enters the separation gel, the voltage is adjusted to 120v to continue the gel running, and the gel can be stopped after the target protein is separated.

And (3) taking NC films with proper sizes, soaking the NC films in methanol for at least 20 seconds, and then taking a proper amount of pre-cooled trans buffers for cleaning for later use. (Trans buffer is prepared according to the following formula: 100mL 10x trans buffer (well known as century) +200mL methanol+700 mL ddH2O. Precooling after preparation), the running glue is taken out, soaked in the precooled trans buffer, and a sandwich model is assembled according to the following sequence: black-cotton-filter-gum-NC membrane-filter-cotton-white. And putting the sandwich model into a film transfer groove according to the sequence of black surface-black surface and white surface-white surface. Two ice boxes frozen overnight in a refrigerator with the temperature of-80 degrees are added into the rotary film box, the whole sandwich model is soaked by pouring film transferring liquid, the rotary film box is installed, and the 350mA constant flow film is arranged for 1h. Closing: a5% BSA blocking solution (1 XTBST was used for dissolution) was prepared, and the transferred membrane was taken out and placed in a blocking solution, and blocked for 1-2 hours on a horizontal shaker at room temperature. Incubation resistance: after blocking, the cells were washed 3 times with 1 XTBE, and a primary antibody solution was prepared in TBST in proportion and incubated for a suitable period of time. And the ratio of the configuration and the incubation time are recorded in table 1. Secondary antibody incubation: after blocking, the mixture was washed 3 times with 1 XTBE, and a secondary antibody solution was prepared in TBST in proportion and incubated for a suitable period of time. ECL chemiluminescent color development: after the secondary antibody incubation was completed, the cells were washed 3 times with 1 XTBST. After ECL chemiluminescent AB solution is mixed and configured according to a ratio of 1:1 before color development, the film is developed on a solar chemiluminescent gel imaging system Tanon-5200 Multi.

4. Quantitative detection of proteins of interest (also known as POIs, protein of interest)

EV was added to 250. Mu.l of RIPA lysate (containing 100x protease inhibitor) at 1.2. Mu.g of total protein, and after thoroughly mixing, the mixture was lysed at 4℃for 1h. Centrifuging at low speed to remove insoluble substances. 30min before ELISA experiments, the kit was removed and allowed to stand at room temperature for equilibration. Standards of different concentrations were prepared according to the instructions and samples were diluted using the dilutions of the kit itself. The samples were incubated at 37℃for 90min and then washed 4 times with a plate washer. Mu.l of antibody was added and incubated at 37℃for 60 min. The plate washer was washed 4 times and 100. Mu.l of enzyme conjugate was added thereto and incubated at 37℃for 30 minutes. After washing 4 times with a plate washer, the color development is carried out for 15min. Stop solution was added and read using a microplate reader.

EV concentration measurement

EV concentration was detected using a ZetaView PMX 110 (Particle Metrix, meerbusch, germany) equipped with a laser having a wavelength of 405 nm. EV was first diluted to about 1X10 with PBS ⁷ ～1x10 ⁹ The concentration range of particles/mL was photographed at a frame rate of 30 frames/sec for a duration of 60 seconds and analyzed for particle size and concentration using NTA software (ZetaView 8.02.28).

6. Co-immunoprecipitation

Since extracellular vesicles are rich in proteins and have many specific marker receptors on their surface, such as CD9, CD81, CD63, CD82, and TSG101, extracellular vesicles can be captured after incubation with extracellular vesicles using anti-marker antibody coated magnetic beads. The immunoaffinity technology has the advantages of high specificity, capability of obtaining high-purity extracellular vesicles, no influence on the integrity of the extracellular vesicles, and the like, and is a preferred method for enriching and characterizing unique extracellular vesicles. After the extracellular vesicles are selectively captured by using the magnetic beads modified with the CD81 antibodies, the fluorescent intensity of the magnetic beads can be counted by using a cell flow type by matching with the markers of the fusion protein extracellular section specific fluorescent antibodies, so that the fact that the fusion protein is expressed outside the extracellular vesicles is confirmed.

Extracellular vesicles were mixed in PBS, prepared Protein A agarose, washed twice with PBS, then formulated with PBS to a 50% concentration, and the gun tip portion was subtracted to avoid damage to the agarose beads during procedures involving the agarose beads. Mu.l Protein A agarose beads (50%) were added to each 1mL extracellular vesicle and shaken at 4℃for 10min (EP tube on ice and on horizontal shaker) to remove non-specific heteropolyproteins and reduce background. After that, the supernatant was centrifuged at 14000g at 4℃for 15 min and transferred to a new centrifuge tube to remove the Protein A beads. After that, 10. Mu.l of Flag primary antibody was added, and the antigen-antibody mixture was slowly shaken at 4℃overnight or at room temperature for 2 hours. mu.L of Protein A agarose beads were added to capture the antigen-antibody complex, and the antigen-antibody mixture was slowly shaken overnight at 4℃or at room temperature for 1h. The agarose bead-antigen antibody complex was collected by instantaneous centrifugation at 14000rpm for 5s, the supernatant was removed, and washed 3 times with pre-chilled PBS, 800. Mu.l/time. Agarose bead-antigen antibody complexes were suspended with 60 μl of 2 Xloading buffer, gently mixed, the loaded samples were boiled for 5min to free antigen, antibody, beads, centrifuged, and the supernatant was electrophoresed.

IL-12 in vitro Activity assay

Immediately after the PBMC media was removed from the liquid nitrogen tank, it was placed in a 37℃water bath with gentle shaking. After the cells are substantially thawed, they are transferred to an ultra clean bench. PBMCs were added to clean 15ml centrifuge tubes and 5ml PBMC medium was added. After centrifugation at 1300rpm for 5 minutes, the supernatant was discarded. 5mL PBMC were resuspended in 25mL culture flask, 37℃and 5% CO ₂ And (5) culturing overnight.

The next day, transfer PBMCs to a 15mL centrifuge tube and centrifuge at 1300rpm. After 5 minutes, the supernatant was discarded. 5mL PBMC activation medium was resuspended in 25mL flask, 37℃and 5% CO ₂ Culturing for 72h. After 72h, stimulated PBMC were transferred to a 15mL centrifuge tube and centrifuged at 1300rpm. After 5 minutes, the supernatant, 5mL PBMC, was discardedThe medium was resuspended. Repeating once. After appropriate volume of PBMC media was resuspended at 1.5x10 ⁵ cells/well/90. Mu.L were seeded in 96-well plates. IL-12 recombinant protein, or expression of IL-12 EV diluted to 9 experimental concentration. 10 mu L of recombinant protein or EV was added to each well plate, and the mixture was mixed with PBMC and left at 37℃with 5% CO ₂ Culturing for 24 hours.

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

8. Mass spectrometry detection of extracellular vesicle proteins

LC-MS/MS sample preparation

Taking out extracellular vesicle sample (200 mu L,500 mu g/mL) from a refrigerator at-80 ℃, transferring to a 1.5mL centrifuge tube, adding a proper amount of SDT protein lysate (4%SDS,10mM DTT,100mM TEAB) for dissolution, shaking and mixing, and performing ice water bath ultrasonic treatment for 5min for full lysis. Centrifugation was performed at 12000G for 15min at 4℃and 10mM DTT (Sigma/D9163-25G) was added to the supernatant and reacted at 56℃for 1h, followed by addition of IAM (Sigma/I6125-25G) in an amount sufficient to react at room temperature in the absence of light for 1h. Adding 4 times of pre-cooled acetone at-20deg.C, precipitating at-20deg.C for at least 2 hr, centrifuging at 4deg.C and 12000g for 15min, and collecting precipitate. Then adding 1 mL-20deg.C pre-cooled acetone (Beijing chemical plant/11241203810051), re-suspending and washing the precipitate, centrifuging at 4deg.C and 12000g for 15min, collecting the precipitate, air drying, adding appropriate amount of protein solution (8M urea, 100mM TEAB, pH=8.5), and dissolving protein precipitate.

The BSA standard protein solution was prepared using the Bradford protein quantification kit (Biyun day) according to the instructions with a concentration gradient ranging from 0 to 0.5. Mu.g/. Mu.L. BSA standard protein solutions with different concentration gradients and sample solutions to be tested with different dilution factors are respectively taken and added into a 96-well plate, the volume is complemented to 20 mu L, and each gradient is repeated for 3 times. 180 mu L G of the staining solution was rapidly added, and the solution was left at room temperature for 5min, and the absorbance at 595nm was measured. And drawing a standard curve by using the absorbance of the standard protein liquid and calculating the protein concentration of the sample to be detected. 20 mug protein samples to be tested are respectively taken for 12% SDS-PAGE gel electrophoresis, wherein the electrophoresis conditions of the concentrated gel are 80V and 20min, and the electrophoresis conditions of the separating gel are 120V and 90min. After electrophoresis, coomassie brilliant blue R-250 is used for dyeing, and the color is decolorized until the band is clear.

Protein samples were taken, added to DB protein lysate (8M urea, 100mM TEAB, pH=8.5) to make up to 100. Mu.L, pancreatin (Promega/V5280) and 100mM TEAB (Sigma/T7408-500 mL) buffer were added, mixed well, digested for 4h at 37℃and then added with pancreatin and CaCl2, and digested overnight. Adding formic acid to adjust pH to less than 3, mixing, centrifuging at room temperature and 12000g for 5min, collecting supernatant, slowly passing through C18 desalting column, continuously cleaning with cleaning solution (0.1% formic acid and 3% acetonitrile) for 3 times, adding appropriate amount of eluent (0.1% formic acid and 70% acetonitrile), collecting filtrate, and lyophilizing.

Mobile phase a (100% water, 0.1% formic acid) and B (80% acetonitrile, 0.1% formic acid) were prepared. The lyophilized powder was dissolved in 10. Mu.LA solution, centrifuged at 14000g for 20min at 4℃and 1. Mu.g of the supernatant was sampled and assayed as liquid. Using an EASY-nLCTM 1200 nanoliter UHPLC system (Thermo Fisher/LC 140), the pre-column was a homemade pre-column (4.5 cm. Times.75 μm,3 μm) and the analytical column was a homemade analytical column (15 cm. Times.150 μm,1.9 μm) and the liquid chromatography elution conditions are shown in Table 1. Using a Q exact HF-X mass spectrometer (Thermo), nanospray Flex ^TM (ESI) ion source, ion spray voltage is set to be 2.1kV, ion transmission tube temperature is set to be 320 ℃, mass spectrum adopts a data dependent acquisition mode, mass spectrum full-scan range is set to be 350-1500 m/z, primary mass spectrum resolution is set to be 60000 (200 m/z), and maximum capacity of C-trap is 3 multiplied by 10 ⁶ The maximum injection time of C-trap is 20ms; selecting parent ion with ion intensity TOP 40 in full scan, and performing secondary mass spectrum detection by high energy collision fragmentation (HCD) method with the resolution of 15000 (200 m/z) and maximum capacity of C-trap of 1×10 ⁵ The maximum injection time of C-trap was 45ms, the fragmentation collision energy of the peptide fragment was set to 27%, and the threshold intensity was set to 2.2X10 ⁴ The dynamic exclusion range was set to 20s, generating mass spectrometry detection raw data.

TABLE 2 liquid chromatography elution gradient table

Data processing

All result spectra were searched using search software Proteome Discoverer 2.2.2 (PD 2.2, thermo) according to Uniprot protein database. The search parameters were set as follows: the mass tolerance of the precursor ions was 10ppm and the mass tolerance of the fragment ions was 0.02Da. The immobilization modification is alkylation modification of cysteine, the variable modification is methionine oxidation modification, the N end is acetylation modification, and the maximum number of the missed cleavage sites is allowed. To improve the quality of the analysis results, the PD2.2 software further filters the search results: the spectral peptide (Peptide Spectrum Matches, PSMs for short) with the credibility of more than 99 percent is credible PSMs, the protein containing at least one unique peptide segment is credible protein, only credible spectral peptide and protein are reserved, FDR verification is carried out, and the peptide segment and protein with the FDR more than 1 percent are removed.

Example 2: expression of BCAM in EV

Stably transfected cell lines of BCAM were constructed, and BCAM expression in EV was detected, with PTGFRN and LAMP2B as controls.

The expression vector used in this example was AAVS1_Puro_Tet3G_3xFLAG_Twin_strep (Addgene, cat. No. 92099), the CMV promoter and the multiple cloning site fragment were derived from pcDNA3.1 (+) vector (Thermo, cat. No. V790-20), and the two were homologously recombined to obtain pAAVS1-Puro-CMV-MCS plasmid, which was the backbone vector used in the following, and all sequences were inserted into the multiple cloning sites of the backbone vector.

Plasmids were constructed, and EGFP as a target Protein (POI) was fused with BCAM and two other known EV proteins (PTGFRN and LAMP 2B), respectively. The sequence of the insert vector is as follows:

pAAVS1-Puro-CMV-eGFP-BCAM

MEPPDAPAQARGAPRLLLLAVLLAAHPDAQAMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKSAGGGGSGGGGSEVRLSVPPLVEVMRGKSVILDCTPTGTHDHYMLEWFLTDRSGARPRLASAEMQGSELQVTMHDTRGRSPPYQLDSQGRLVLAEAQVGDERDYVCVVRAGAAGTAEATARLNVFAKPEATEVSPNKGTLSVMEDSAQEIATCNSRNGNPAPKITWYRNGQRLEVPVEMNPEGYMTSRTVREASGLLSLTSTLYLRLRKDDRDASFHCAAHYSLPEGRHGRLDSPTFHLTLHYPTEHVQFWVGSPSTPAGWVREGDTVQLLCRGDGSPSPEYTLFRLQDEQEEVLNVNLEGNLTLEGVTRGQSGTYGCRVEDYDAADDVQLSKTLELRVAYLDPLELSEGKVLSLPLNSSAVVNCSVHGLPTPALRWTKDSTPLGDGPMLSLSSITFDSNGTYVCEASLPTVPVLSRTQNFTLLVQGSPELKTAEIEPKADGSWREGDEVTLICSARGHPDPKLSWSQLGGSPAEPIPGRQGWVSSSLTLKVTSALSRDGISCEASNPHGNKRHVFHFGTVSPQTSQAGVAVMAVAVSVGLLLLVVAVFYCVRRKGGPCCRQRREKGAPPPGEPGLSHSGSEQPEQTGLLMGGASGGARGGSGGFGDEC(SEQ ID NO:1)。

wherein the BCAM signal peptide sequence is: MEPPDAPAQARGAPRLLLLAVLLAAHPDAQA (SEQ ID NO: 2);

the eGFP sequence is:

MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK(SEQ ID NO:3)；

the connection sequence (linker) is: GGGGSGGGGS (SEQ ID NO: 4);

the BCAM sequence is:

EVRLSVPPLVEVMRGKSVILDCTPTGTHDHYMLEWFLTDRSGARPRLASAEMQGSELQVTMHDTRGRSPPYQLDSQGRLVLAEAQVGDERDYVCVVRAGAAGTAEATARLNVFAKPEATEVSPNKGTLSVMEDSAQEIATCNSRNGNPAPKITWYRNGQRLEVPVEMNPEGYMTSRTVREASGLLSLTSTLYLRLRKDDRDASFHCAAHYSLPEGRHGRLDSPTFHLTLHYPTEHVQFWVGSPSTPAGWVREGDTVQLLCRGDGSPSPEYTLFRLQDEQEEVLNVNLEGNLTLEGVTRGQSGTYGCRVEDYDAADDVQLSKTLELRVAYLDPLELSEGKVLSLPLNSSAVVNCSVHGLPTPALRWTKDSTPLGDGPMLSLSSITFDSNGTYVCEASLPTVPVLSRTQNFTLLVQGSPELKTAEIEPKADGSWREGDEVTLICSARGHPDPKLSWSQLGGSPAEPIPGRQGWVSSSLTLKVTSALSRDGISCEASNPHGNKRHVFHFGTVSPQTSQAGVAVMAVAVSVGLLLLVVAVFYCVRRKGGPCCRQRREKGAPPPGEPGLSHSGSEQPEQTGLLMGGASGGARGGSGGFGDEC(SEQ ID NO:5)。

pAAVS1-Puro-CMV-eGFP-PTGFRN

MGRLASRPLLLALLSLALCRGMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKSAGGGGSGGGGSRVVRVPTATLVRVVGTELVIPCNVSDYDGPSEQNFDWSFSSLGSSFVELASTWEVGFPAQLYQERLQRGEILLRRTANDAVELHIKNVQPSDQGHYKCSTPSTDATVQGNYEDTVQVKVLADSLHVGPSARPPPSLSLREGEPFELRCTAASASPLHTHLALLWEVHRGPARRSVLALTHEGRFHPGLGYEQRYHSGDVRLDTVGSDAYRLSVSRALSADQGSYRCIVSEWIAEQGNWQEIQEKAVEVATVVIQPSVLRAAVPKNVSVAEGKELDLTCNITTDRADDVRPEVTWSFSRMPDSTLPGSRVLARLDRDSLVHSSPHVALSHVDARSYHLLVRDVSKENSGYYYCHVSLWAPGHNRSWHKVAEAVSSPAGVGVTWLEPDYQVYLNASKVPGFADDPTELACRVVDTKSGEANVRFTVSWYYRMNRRSDNVVTSELLAVMDGDWTLKYGERSKQRAQDGDFIFSKEHTDTFNFRIQRTTEEDRGNYYCVVSAWTKQRNNSWVKSKDVFSKPVNIFWALEDSVLVVKARQPKPFFAAGNTFEMTCKVSSKNIKSPRYSVLIMAEKPVGDLSSPNETKYIISLDQDSVVKLENWTDASRVDGVVLEKVQEDEFRYRMYQTQVSDAGLYRCMVTAWSPVRGSLWREAATSLSNPIEIDFQTSGPIFNASVHSDTPSVIRGDLIKLFCIITVEGAALDPDDMAFDVSWFAVHSFGLDKAPVLLSSLDRKGIVTTSRRDWKSDLSLERVSVLEFLLQVHGSEDQDFGNYYCSVTPWVKSPTGSWQKEAEIHSKPVFITVKMDVLNAFKYPLLIGVGLSTVIGLLSCLIGYCSSHWCCKKEVQETRRERRRLMSMEMD(SEQ ID NO:6)。

wherein, PTGFRN signal peptide sequence is: MGRLASRPLLLALLSLALCRG (SEQ ID NO: 7);

the PTGFRN sequence is:

RVVRVPTATLVRVVGTELVIPCNVSDYDGPSEQNFDWSFSSLGSSFVELASTWEVGFPAQLYQERLQRGEILLRRTANDAVELHIKNVQPSDQGHYKCSTPSTDATVQGNYEDTVQVKVLADSLHVGPSARPPPSLSLREGEPFELRCTAASASPLHTHLALLWEVHRGPARRSVLALTHEGRFHPGLGYEQRYHSGDVRLDTVGSDAYRLSVSRALSADQGSYRCIVSEWIAEQGNWQEIQEKAVEVATVVIQPSVLRAAVPKNVSVAEGKELDLTCNITTDRADDVRPEVTWSFSRMPDSTLPGSRVLARLDRDSLVHSSPHVALSHVDARSYHLLVRDVSKENSGYYYCHVSLWAPGHNRSWHKVAEAVSSPAGVGVTWLEPDYQVYLNASKVPGFADDPTELACRVVDTKSGEANVRFTVSWYYRMNRRSDNVVTSELLAVMDGDWTLKYGERSKQRAQDGDFIFSKEHTDTFNFRIQRTTEEDRGNYYCVVSAWTKQRNNSWVKSKDVFSKPVNIFWALEDSVLVVKARQPKPFFAAGNTFEMTCKVSSKNIKSPRYSVLIMAEKPVGDLSSPNETKYIISLDQDSVVKLENWTDASRVDGVVLEKVQEDEFRYRMYQTQVSDAGLYRCMVTAWSPVRGSLWREAATSLSNPIEIDFQTSGPIFNASVHSDTPSVIRGDLIKLFCIITVEGAALDPDDMAFDVSWFAVHSFGLDKAPVLLSSLDRKGIVTTSRRDWKSDLSLERVSVLEFLLQVHGSEDQDFGNYYCSVTPWVKSPTGSWQKEAEIHSKPVFITVKMDVLNAFKYPLLIGVGLSTVIGLLSCLIGYCSSHWCCKKEVQETRRERRRLMSMEMD(SEQ ID NO:8)。

pAAVS1-Puro-CMV-eGFP-LAMP2B

MVCFRLFPVPGSGLVLVCLVLGAVRSYAMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKSAGGGGSGGGGSLELNLTDSENATCLYAKWQMNFTVRYETTNKTYKTVTISDHGTVTYNGSICGDDQNGPKIAVQFGPGFSWIANFTKAASTYSIDSVSFSYNTGDNTTFPDAEDKGILTVDELLAIRIPLNDLFRCNSLSTLEKNDVVQHYWDVLVQAFVQNGTVSTNEFLCDKDKTSTVAPTIHTTVPSPTTTPTPKEKPEAGTYSVNNGNDTCLLATMGLQLNITQDKVASVININPNTTHSTGSCRSHTALLRLNSSTIKYLDFVFAVKNENRFYLKEVNISMYLVNGSVFSIANNNLSYWDAPLGSSYMCNKEQTVSVSGAFQINTFDLRVQPFNVTQGKYSTAQECSLDDDTILIPIIVGAGLSGLIIVIVIAYVIGRRKSYAGYQTL(SEQID NO:9)。

wherein, the LAMP2B signal peptide sequence is: MVCFRLFPVPGSGLVLVCLVLGAVRSYA (SEQ ID NO: 10);

the LAMP2B sequence is:

LELNLTDSENATCLYAKWQMNFTVRYETTNKTYKTVTISDHGTVTYNGSICGDDQNGPKIAVQFGPGFSWIANFTKAASTYSIDSVSFSYNTGDNTTFPDAEDKGILTVDELLAIRIPLNDLFRCNSLSTLEKNDVVQHYWDVLVQAFVQNGTVSTNEFLCDKDKTSTVAPTIHTTVPSPTTTPTPKEKPEAGTYSVNNGNDTCLLATMGLQLNITQDKVASVININPNTTHSTGSCRSHTALLRLNSSTIKYLDFVFAVKNENRFYLKEVNISMYLVNGSVFSIANNNLSYWDAPLGSSYMCNKEQTVSVSGAFQINTFDLRVQPFNVTQGKYSTAQECSLDDDTILIPIIVGAGLSGLIIVIVIAYVIGRRKSYAGYQTL(SEQ ID NO:11)。

the plasmid of interest was site-directed integrated into the AAVS1 site of expi HEK293F (Thermo FisherScientific, cat# a 14527) using a site-selective insertion method, resulting in a stably transfected cell line. The EVs in the culture supernatant of the stably transfected cell line were extracted. And detecting eGFP and EV protein marker CD9 in the product EVs by using a Western blot method. The results show that, like the known EV proteins PTGFRN and LAMP2B, BCAM is also able to load the protein fragment fused thereto (eGFP) into the EV secreted by the cells.

To further demonstrate that the band of interest in the above EVs was indeed a transferred fusion protein, three stably transfected cell line lysates, cell secreted EVs and wild type expi HEK293F secreted EVs were compared. The Western blot results show that bands with different molecular weights can be observed in the stably transfected cell line and the EV secreted by the stably transfected cell line, and that fusion proteins of wild-type proteins and POIs can be judged according to the molecular weights. In contrast, wild-type BCAM, because of having a lower background expression level, instead helps to increase the proportion of eGFP-BCAM fusion protein relative to wild-type BCAM in the EV, i.e., the amount of BCAM fusion protein in the EV obtained is significantly higher than background BCAM, see wild-type protein bands and fusion protein bands in fig. 2A; the ratio was also significantly higher than the ratio of PTGFRN and LAMP2B fusion proteins to background, and fig. 2B shows the percentage of wild-type protein and fusion protein in the sum of the two.

Example 3: detection of eGFP-BCAM fusion protein expression at EV

To assess the ability of BCAM as a protein to carry a protein of interest POI. Three EVs were first collected that fusion expressed eGFP (eGFP-LAMP 2B, eGFP-PTGFRN and eGFP-BCAM). Particle concentration was determined using NTA (Nanoparticle trackinganalysis) (Particle metric

NTA), the samples were diluted to 1x10e9 Particles/mL and their fluorescence intensity was quantified with a multifunctional microplate reader. The results showed that eGFP-BCAM has a higher fluorescence intensity than eGFP-PTGFRN and significantly higher than eGFP-LAMP2B (fig. 3), indicating that BCAM has higher carryover efficiency compared to LAMP2B and PTGFRN.

Example 4: detection of expression of IL12-BCAM fusion proteins at EV

To further evaluate the ability of BCAM as a protein carrier with other active ingredients, IL12 with a molecular weight of about 60kDa was fused to BCAM, while a fusion protein of IL12 and PTGFRN was constructed as a control. The sequence of the insert vector is as follows:

pAAVS1-Puro-CMV-hIL12-BCAM

MCHQQLVISWFSLVFLASPLVAIWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCSGGSGGGSGGGGSGGGGSGGGSGGRNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNASSAGGGGSGGGGSEVRLSVPPLVEVMRGKSVILDCTPTGTHDHYMLEWFLTDRSGARPRLASAEMQGSELQVTMHDTRGRSPPYQLDSQGRLVLAEAQVGDERDYVCVVRAGAAGTAEATARLNVFAKPEATEVSPNKGTLSVMEDSAQEIATCNSRNGNPAPKITWYRNGQRLEVPVEMNPEGYMTSRTVREASGLLSLTSTLYLRLRKDDRDASFHCAAHYSLPEGRHGRLDSPTFHLTLHYPTEHVQFWVGSPSTPAGWVREGDTVQLLCRGDGSPSPEYTLFRLQDEQEEVLNVNLEGNLTLEGVTRGQSGTYGCRVEDYDAADDVQLSKTLELRVAYLDPLELSEGKVLSLPLNSSAVVNCSVHGLPTPALRWTKDSTPLGDGPMLSLSSITFDSNGTYVCEASLPTVPVLSRTQNFTLLVQGSPELKTAEIEPKADGSWREGDEVTLICSARGHPDPKLSWSQLGGSPAEPIPGRQGWVSSSLTLKVTSALSRDGISCEASNPHGNKRHVFHFGTVSPQTSQAGVAVMAVAVSVGLLLLVVAVFYCVRRKGGPCCRQRREKGAPPPGEPGLSHSGSEQPEQTGLLMGGASGGARGGSGGFGDEC(SEQ ID NO:12)。

wherein, hIL12 sequence is:

MCHQQLVISWFSLVFLASPLVAIWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCSGGSGGGSGGGGSGGGGSGGGSGGRNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNAS(SEQ ID NO:13)。

pAAVS1-Puro-CMV-hIL12-PTGFRN

MCHQQLVISWFSLVFLASPLVAIWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCSGGSGGGSGGGGSGGGGSGGGSGGRNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNASSAGGGGSGGGGSRVVRVPTATLVRVVGTELVIPCNVSDYDGPSEQNFDWSFSSLGSSFVELASTWEVGFPAQLYQERLQRGEILLRRTANDAVELHIKNVQPSDQGHYKCSTPSTDATVQGNYEDTVQVKVLADSLHVGPSARPPPSLSLREGEPFELRCTAASASPLHTHLALLWEVHRGPARRSVLALTHEGRFHPGLGYEQRYHSGDVRLDTVGSDAYRLSVSRALSADQGSYRCIVSEWIAEQGNWQEIQEKAVEVATVVIQPSVLRAAVPKNVSVAEGKELDLTCNITTDRADDVRPEVTWSFSRMPDSTLPGSRVLARLDRDSLVHSSPHVALSHVDARSYHLLVRDVSKENSGYYYCHVSLWAPGHNRSWHKVAEAVSSPAGVGVTWLEPDYQVYLNASKVPGFADDPTELACRVVDTKSGEANVRFTVSWYYRMNRRSDNVVTSELLAVMDGDWTLKYGERSKQRAQDGDFIFSKEHTDTFNFRIQRTTEEDRGNYYCVVSAWTKQRNNSWVKSKDVFSKPVNIFWALEDSVLVVKARQPKPFFAAGNTFEMTCKVSSKNIKSPRYSVLIMAEKPVGDLSSPNETKYIISLDQDSVVKLENWTDASRVDGVVLEKVQEDEFRYRMYQTQVSDAGLYRCMVTAWSPVRGSLWREAATSLSNPIEIDFQTSGPIFNASVHSDTPSVIRGDLIKLFCIITVEGAALDPDDMAFDVSWFAVHSFGLDKAPVLLSSLDRKGIVTTSRRDWKSDLSLERVSVLEFLLQVHGSEDQDFGNYYCSVTPWVKSPTGSWQKEAEIHSKPVFITVKMDVLNAFKYPLLIGVGLSTVIGLLSCLIGYCSSHWCCKKEVQETRRERRRLMSMEMD(SEQ ID NO:14)。

stably transfected cell lines were constructed and EVs were collected. After cleavage of EV from the total amount of iso-protein (0.5. Mu.g), the abundance of its active ingredient (IL 12) was detected using ELISA, which showed that the expression level of IL12-BCAM in EV was significantly higher than that of IL12-PTGFRN (FIG. 4). The above experiments demonstrate that BCAM is a preferred EV scaffold protein and is effective in carrying the target protein fused thereto into the EV.

Example 5: detection of expression of other BCAM fusion proteins at EV

To test the ability of BCAM as a scaffold protein, carrying different active ingredients and sorting into EV, fusion proteins (Flag-containing tags) of BCAM and POI of different molecular weights were respectively constructed. The insert sequences in the vector are as follows:

pAAVS1_Puro_CMV_SP_RVG_linker Flag-BCAM

MEPPDAPAQARGAPRLLLLAVLLAAHPDAQAYTIWMPENPRPGTPCDIFTNSRGKRASNGSAGGGGSDYKDDDDKGGGGSEVRLSVPPLVEVMRGKSVILDCTPTGTHDHYMLEWFLTDRSGARPRLASAEMQGSELQVTMHDTRGRSPPYQLDSQGRLVLAEAQVGDERDYVCVVRAGAAGTAEATARLNVFAKPEATEVSPNKGTLSVMEDSAQEIATCNSRNGNPAPKITWYRNGQRLEVPVEMNPEGYMTSRTVREASGLLSLTSTLYLRLRKDDRDASFHCAAHYSLPEGRHGRLDSPTFHLTLHYPTEHVQFWVGSPSTPAGWVREGDTVQLLCRGDGSPSPEYTLFRLQDEQEEVLNVNLEGNLTLEGVTRGQSGTYGCRVEDYDAADDVQLSKTLELRVAYLDPLELSEGKVLSLPLNSSAVVNCSVHGLPTPALRWTKDSTPLGDGPMLSLSSITFDSNGTYVCEASLPTVPVLSRTQNFTLLVQGSPELKTAEIEPKADGSWREGDEVTLICSARGHPDPKLSWSQLGGSPAEPIPGRQGWVSSSLTLKVTSALSRDGISCEASNPHGNKRHVFHFGTVSPQTSQAGVAVMAVAVSVGLLLLVVAVFYCVRRKGGPCCRQRREKGAPPPGEPGLSHSGSEQPEQTGLLMGGASGGARGGSGGFGDEC(SEQ ID NO:15)。

wherein, RVG sequence is: YTIWMPENPRPGTPCDIFTNSRGKRASNG (SEQ ID NO: 16);

the Linker flag sequence is: GGGGSDYKDDDDKGGGGS (SEQ ID NO: 17).

pAAVS1_Puro_CMV_FLT3L_linker Flag-BCAM

MTVLAPAWSPTTYLLLLLLLSSGLSGTQDCSFQHSPISSDFAVKIRELSDYLLQDYPVTVASNLQDEELCGGLWRLVLAQRWMERLKTVAGSKMQGLLERVNTEIHFVTKCAFQPPPSCLRFVQTNISRLLQETSEQLVALKPWITRQNFSRCLELQCQPDSSTLPPPWSPRPLEATAPTAPQPPSAGGGGSDYKDDDDKGGGGSEVRLSVPPLVEVMRGKSVILDCTPTGTHDHYMLEWFLTDRSGARPRLASAEMQGSELQVTMHDTRGRSPPYQLDSQGRLVLAEAQVGDERDYVCVVRAGAAGTAEATARLNVFAKPEATEVSPNKGTLSVMEDSAQEIATCNSRNGNPAPKITWYRNGQRLEVPVEMNPEGYMTSRTVREASGLLSLTSTLYLRLRKDDRDASFHCAAHYSLPEGRHGRLDSPTFHLTLHYPTEHVQFWVGSPSTPAGWVREGDTVQLLCRGDGSPSPEYTLFRLQDEQEEVLNVNLEGNLTLEGVTRGQSGTYGCRVEDYDAADDVQLSKTLELRVAYLDPLELSEGKVLSLPLNSSAVVNCSVHGLPTPALRWTKDSTPLGDGPMLSLSSITFDSNGTYVCEASLPTVPVLSRTQNFTLLVQGSPELKTAEIEPKADGSWREGDEVTLICSARGHPDPKLSWSQLGGSPAEPIPGRQGWVSSSLTLKVTSALSRDGISCEASNPHGNKRHVFHFGTVSPQTSQAGVAVMAVAVSVGLLLLVVAVFYCVRRKGGPCCRQRREKGAPPPGEPGLSHSGSEQPEQTGLLMGGASGGARGGSGGFGDEC(SEQ ID NO:18)

Wherein, the FLT3L sequence is:

MTVLAPAWSPTTYLLLLLLLSSGLSGTQDCSFQHSPISSDFAVKIRELSDYLLQDYPVTVASNLQDEELCGGLWRLVLAQRWMERLKTVAGSKMQGLLERVNTEIHFVTKCAFQPPPSCLRFVQTNISRLLQETSEQLVALKPWITRQNFSRCLELQCQPDSSTLPPPWSPRPLEATAPTAPQPP(SEQ ID NO:19)。

pAAVS1-puro-CMV-SP-linker Flag-BCAM

MEPPDAPAQARGAPRLLLLAVLLAAHPDAQASAGGGGSDYKDDDDKGGGGSEVRLSVPPLVEVMRGKSVILDCTPTGTHDHYMLEWFLTDRSGARPRLASAEMQGSELQVTMHDTRGRSPPYQLDSQGRLVLAEAQVGDERDYVCVVRAGAAGTAEATARLNVFAKPEATEVSPNKGTLSVMEDSAQEIATCNSRNGNPAPKITWYRNGQRLEVPVEMNPEGYMTSRTVREASGLLSLTSTLYLRLRKDDRDASFHCAAHYSLPEGRHGRLDSPTFHLTLHYPTEHVQFWVGSPSTPAGWVREGDTVQLLCRGDGSPSPEYTLFRLQDEQEEVLNVNLEGNLTLEGVTRGQSGTYGCRVEDYDAADDVQLSKTLELRVAYLDPLELSEGKVLSLPLNSSAVVNCSVHGLPTPALRWTKDSTPLGDGPMLSLSSITFDSNGTYVCEASLPTVPVLSRTQNFTLLVQGSPELKTAEIEPKADGSWREGDEVTLICSARGHPDPKLSWSQLGGSPAEPIPGRQGWVSSSLTLKVTSALSRDGISCEASNPHGNKRHVFHFGTVSPQTSQAGVAVMAVAVSVGLLLLVVAVFYCVRRKGGPCCRQRREKGAPPPGEPGLSHSGSEQPEQTGLLMGGASGGARGGSGGFGDEC(SEQ ID NO:20)。

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

MGRAMVARLGLGLLLLALLLPTQIYSSETTTGTSSNSSQSTSNSGLAPNPTNATTKASAGGGGSDYKDDDDKGGGGSEVRLSVPPLVEVMRGKSVILDCTPTGTHDHYMLEWFLTDRSGARPRLASAEMQGSELQVTMHDTRGRSPPYQLDSQGRLVLAEAQVGDERDYVCVVRAGAAGTAEATARLNVFAKPEATEVSPNKGTLSVMEDSAQEIATCNSRNGNPAPKITWYRNGQRLEVPVEMNPEGYMTSRTVREASGLLSLTSTLYLRLRKDDRDASFHCAAHYSLPEGRHGRLDSPTFHLTLHYPTEHVQFWVGSPSTPAGWVREGDTVQLLCRGDGSPSPEYTLFRLQDEQEEVLNVNLEGNLTLEGVTRGQSGTYGCRVEDYDAADDVQLSKTLELRVAYLDPLELSEGKVLSLPLNSSAVVNCSVHGLPTPALRWTKDSTPLGDGPMLSLSSITFDSNGTYVCEASLPTVPVLSRTQNFTLLVQGSPELKTAEIEPKADGSWREGDEVTLICSARGHPDPKLSWSQLGGSPAEPIPGRQGWVSSSLTLKVTSALSRDGISCEASNPHGNKRHVFHFGTVSPQTSQAGVAVMAVAVSVGLLLLVVAVFYCVRRKGGPCCRQRREKGAPPPGEPGLSHSGSEQPEQTGLLMGGASGGARGGSGGFGDEC(SEQ ID NO:21)。

wherein the hCD24 (1-57) sequence is as follows:

MGRAMVARLGLGLLLLALLLPTQIYSSETTTGTSSNSSQSTSNSGLAPNPTNATTKA(SEQID NO:22)。

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

MEPPDAPAQARGAPRLLLLAVLLAAHPDAQASETTTGTSSNSSQSTSNSGLAPNPTNATTKASAGGGGSDYKDDDDKGGGGSEVRLSVPPLVEVMRGKSVILDCTPTGTHDHYMLEWFLTDRSGARPRLASAEMQGSELQVTMHDTRGRSPPYQLDSQGRLVLAEAQVGDERDYVCVVRAGAAGTAEATARLNVFAKPEATEVSPNKGTLSVMEDSAQEIATCNSRNGNPAPKITWYRNGQRLEVPVEMNPEGYMTSRTVREASGLLSLTSTLYLRLRKDDRDASFHCAAHYSLPEGRHGRLDSPTFHLTLHYPTEHVQFWVGSPSTPAGWVREGDTVQLLCRGDGSPSPEYTLFRLQDEQEEVLNVNLEGNLTLEGVTRGQSGTYGCRVEDYDAADDVQLSKTLELRVAYLDPLELSEGKVLSLPLNSSAVVNCSVHGLPTPALRWTKDSTPLGDGPMLSLSSITFDSNGTYVCEASLPTVPVLSRTQNFTLLVQGSPELKTAEIEPKADGSWREGDEVTLICSARGHPDPKLSWSQLGGSPAEPIPGRQGWVSSSLTLKVTSALSRDGISCEASNPHGNKRHVFHFGTVSPQTSQAGVAVMAVAVSVGLLLLVVAVFYCVRRKGGPCCRQRREKGAPPPGEPGLSHSGSEQPEQTGLLMGGASGGARGGSGGFGDEC(SEQ ID NO:23)

wherein the hCD24 (27-57) sequence is as follows: SETTTGTSSNSSQSTSNSGLAPNPTNATTKA (SEQ ID NO: 24).

pAAVS1-puro-CMV-CD3 scFab-linker Flag-BCAM

MGRLASRPLLLALLSLALCRGRMKIICLALVALLLTAQPAMAEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGSSGSGSGSTGTSSSGTGTSAGTTGTSASTSGSGSGGGGGSGGGGSAGGTATAGASSGSQVQLVESGGGVVQPGRSLRLSCAASGFKFSGYGMHWVRQAPGKGLEWVAVIWYDGSKKYYVDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARQMGYWHFDLWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTSAGGGGSDYKDDDDKGGGGSEVRLSVPPLVEVMRGKSVILDCTPTGTHDHYMLEWFLTDRSGARPRLASAEMQGSELQVTMHDTRGRSPPYQLDSQGRLVLAEAQVGDERDYVCVVRAGAAGTAEATARLNVFAKPEATEVSPNKGTLSVMEDSAQEIATCNSRNGNPAPKITWYRNGQRLEVPVEMNPEGYMTSRTVREASGLLSLTSTLYLRLRKDDRDASFHCAAHYSLPEGRHGRLDSPTFHLTLHYPTEHVQFWVGSPSTPAGWVREGDTVQLLCRGDGSPSPEYTLFRLQDEQEEVLNVNLEGNLTLEGVTRGQSGTYGCRVEDYDAADDVQLSKTLELRVAYLDPLELSEGKVLSLPLNSSAVVNCSVHGLPTPALRWTKDSTPLGDGPMLSLSSITFDSNGTYVCEASLPTVPVLSRTQNFTLLVQGSPELKTAEIEPKADGSWREGDEVTLICSARGHPDPKLSWSQLGGSPAEPIPGRQGWVSSSLTLKVTSALSRDGISCEASNPHGNKRHVFHFGTVSPQTSQAGVAVMAVAVSVGLLLLVVAVFYCVRRKGGPCCRQRREKGAPPPGEPGLSHSGSEQPEQTGLLMGGASGGARGGSGGFGDEC(SEQ ID NO:25)

Wherein the CD3 scFab sequence is:

MGRLASRPLLLALLSLALCRGRMKIICLALVALLLTAQPAMAEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGSSGSGSGSTGTSSSGTGTSAGTTGTSASTSGSGSGGGGGSGGGGSAGGTATAGASSGSQVQLVESGGGVVQPGRSLRLSCAASGFKFSGYGMHWVRQAPGKGLEWVAVIWYDGSKKYYVDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARQMGYWHFDLWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT(SEQ ID NO:26)。

the Western blot results show that POIs with different molecular weights can be expressed in EV, and the molecular weights are consistent with expectations.

Example 6: detection of biological Activity of target protein in BCAM fusion protein

To test whether the IL12 fusion protein on EV was biologically active, PBMC were stimulated with IL12 recombinant protein (FIG. 6A), EV expressing IL12-PTGFRN (FIG. 6B), and EV expressing IL12-BCAM (FIG. 6C), and secretion levels of IFN-gamma were detected. The results of EC50 showed that fusion with BCAM and pdgfr did not affect the biological activity of IL 12.

Example 7: identification of Membrane proteins having similar Functions to BCAM

Based on the discovery of BCAM, the inventors screened for membrane proteins with similar functions as BCAM. These proteins all belong to the immunoglobulin superfamily, have high similarity to BCAM, and have low background expression levels at EV.

Analysis was performed using BLAST software from NCBI (Basic local alignment search tool, J Mol biol.1990 Oct 5;215 (3): 403-10.), and the inventors screened a panel of EV proteins with higher similarity to BCAM in the immunoglobulin superfamily, as shown in Table 3.

TABLE 3 Table 3

Query cover: the inclusion of a percentage of the query fragment (BCAM) length in the pair Ji Pianduan (POI) is a larger number indicating that the length of the sequence being included in the alignment is a higher proportion of the query fragment (e.g., 100% when BCAM is the POI)

E Value: the smaller the value, the higher the similarity of the two (e.g., when BCAM is used as POI, the value is 0). When E value < = 1.0E-4, the protein was considered to be highly similar to BCAM, as listed in table 1. The E value is calculated by NCBI blastp program as follows:

E＝K*m*n*exp(-lambda*S) (I)；

where K and lambda are constants associated with the database and algorithm, m represents the length of the target sequence, n represents the size of the database, S value represents the homology of the two sequences, higher S score indicates greater homology between them, and lower calculated E value indicates higher similarity.

Total score: the greater the total score, the greater the POI follow-up query segment consistency.

The setting of some parameters can be seen in

https://blast.ncbi.nlm.nih.gov/Blast.cgiPROGRAM＝blastp&PAGE_TYPE＝BlastSearch&LINK_LOC＝blasthome.

Further, the inventors studied the abundance of the protein in table 3 in EV. The relative amounts of the different proteins in the extracellular vesicles and cells were measured by mass spectrometry, and the CD81 content (expressed as iBAQ) was set to 1, and the results are shown in table 4.

TABLE 4 Table 4

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The MaxQuant program calculates the protein intensity as the sum of all the identified peptide intensities divided by the number of peptides that are theoretically observable, resulting in a iBAQ (intensity based absolute quantification) value, which is an approximately absolute quantitative method that can effectively compare the abundance of expression between different proteins in a sample (Global quantification of mammalian gene expression control, nature.2013Mar 7;495 (7439): 126-7). When the content of the protein in EV is lower than 5% of the content of CD81, i.e. the ratio of iBAQ value to CD81 of one protein in table 2 < = 0.05, it is herein judged that the background expression level of the protein in EV is low. CD81 is one of the four transmembrane proteins commonly found in EVs, and is set as a reference in this example.

Some of these proteins were structurally analyzed from Uniprot database, as shown in fig. 7.

Example 8: detection of expression of MCAM fusion proteins at EV

To verify the feasibility of MCAM to construct EVs, an eGFP and MCAM fusion protein expression plasmid was constructed with the following insert sequences:

pAAVS1-Puro-CMV-eGFP-MCAM

MGLPRLVCAFLLAACCCCPRVAGMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKSAGGGGSGGGGSVPGEAEQPAPELVEVEVGSTALLKCGLSQSQGNLSHVDWFSVHKEKRTLIFRVRQGQGQSEPGEYEQRLSLQDRGATLALTQVTPQDERIFLCQGKRPRSQEYRIQLRVYKAPEEPNIQVNPLGIPVNSKEPEEVATCVGRNGYPIPQVIWYKNGRPLKEEKNRVHIQSSQTVESSGLYTLQSILKAQLVKEDKDAQFYCELNYRLPSGNHMKESREVTVPVFYPTEKVWLEVEPVGMLKEGDRVEIRCLADGNPPPHFSISKQNPSTREAEEETTNDNGVLVLEPARKEHSGRYECQGLDLDTMISLLSEPQELLVNYVSDVRVSPAAPERQEGSSLTLTCEAESSQDLEFQWLREETGQVLERGPVLQLHDLKREAGGGYRCVASVPSIPGLNRTQLVNVAIFGPPWMAFKERKVWVKENMVLNLSCEASGHPRPTISWNVNGTASEQDQDPQRVLSTLNVLVTPELLETGVECTASNDLGKNTSILFLELVNLTTLTPDSNTTTGLSTSTASPHTRANSTSTERKLPEPESRGVVIVAVIVCILVLAVLGAVLYFLYKKGKLPCRRSGKQEITLPPSRKSELVVEVKSDKLPEEMGLLQGSSGDKRAPGDQGEKYIDLRH

wherein, the MCAM signal peptide sequence is as follows: MGLPRLVCAFLLAACCCCPRVAG;

the MCAM sequence is: VPGEAEQPAPELVEVEVGSTALLKCG

LSQSQGNLSHVDWFSVHKEKRTLIFRVRQGQGQSEPGEYEQRLSLQDRGATLALTQVTPQDERIFLCQGKRPRSQEYRIQLRVYKAPEEPNIQVNPLGIPVNSKEPEEVATCVGRNGYPIPQVIWYKNGRPLKEEKNRVHIQSSQTVESSGLYTLQSILKAQLVKEDKDAQFYCELNYRLPSGNHMKESREVTVPVFYPTEKVWLEVEPVGMLKEGDRVEIRCLADGNPPPHFSISKQNPSTREAEEETTNDNGVLVLEPARKEHSGRYECQGLDLDTMISLLSEPQELLVNYVSDVRVSPAAPERQEGSSLTLTCEAESSQDLEFQWLREETGQVLERGPVLQLHDLKREAGGGYRCVASVPSIPGLNRTQLVNVAIFGPPWMAFKERKVWVKENMVLNLSCEASGHPRPTISWNVNGTASEQDQDPQRVLSTLNVLVTPELLETGVECTASNDLGKNTSILFLELVNLTTLTPDSNTTTGLSTSTASPHTRANSTSTERKLPEPESRGVVIVAVIVCILVLAVLGAVLYFLYKKGKLPCRRSGKQEITLPPSRKSELVVEVKSDKLPEEMGLLQGSSGDKRAPGDQGEKYIDLRH。

The plasmid of interest was site-directed integrated into the AAVS1 site of expi HEK293F using site-selective insertion to give a stably transfected cell line. The EVs in the culture supernatant of the stably transfected cell line were extracted. And detecting eGFP and EV protein marker CD9 in the product EVs by using a Western blot method. The results show that, like BCAM, MCAM is also able to load eGFP fused to it into EV secreted by cells.

To exclude the possibility that the active ingredient is present in a non-EV form, immunocapture was performed on the purified EV using magnetic beads modified with CD81 and CD9, respectively, by immunoprecipitation. The EVs were first extracted from the supernatant using the purification method of the examples, which is a pre-capture sample. Immune magnetic beads are then used to specifically capture EVs expressing four transmembrane proteins in these samples, and the captured samples are on the magnetic beads. Analysis of IL12 in the EV before and after capture by Western blot shows that IL12 is indeed present in the immunoprecipitated sample, indicating that IL12 in the sample is located in the EV, rather than free IL12 recombinant protein that was separated along with EVs during purification for some unknown reason. IL12 was loaded into EV after fusion with PTGFRN, MCAM and BCAM.

Example 9: detection of biological Activity of target protein in MCAM fusion protein

IL12 Activity assay

To verify that fusion with MCAM does not alter the biological activity of the protein, engineered cells expressing IL12-MCAM were constructed and the in vitro activity of EVs expressing IL12-MCAM (fig. 10C) and recombinant IL12 (fig. 10A) and EVs expressing IL12-BCAM (fig. 10B) were compared. The results of the assay for IFN-gamma secretion by PBMC showed that IL 12-MCAM-has similar biological activity as IL12-BCAM, indicating that fusion with MCAM does not affect the biological activity of IL-12.

2. Luciferase activity assay

Luciferase (molecular weight about 70 kDa) was fused to BCAM, MCAM and PTGFRN, respectively, and stable transfected cell lines were constructed. The vector insertion sequence is as follows:

pAAVS1_Puro_CMV_SP_luciferase_linker Flag-BCAM

MEPPDAPAQARGAPRLLLLAVLLAAHPDAQAMADAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDAHIEVDITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAPANDIYNERELLNSMGISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQSMYTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVRFSHARDPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYKIQSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGYGLTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMSGYVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESILLQHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVVFVDEVPKGLTGKLDARKIREILIKAKKGGKIAVSAGGGGSDYKDDDDKGGGGSEVRLSVPPLVEVMRGKSVILDCTPTGTHDHYMLEWFLTDRSGARPRLASAEMQGSELQVTMHDTRGRSPPYQLDSQGRLVLAEAQVGDERDYVCVVRAGAAGTAEATARLNVFAKPEATEVSPNKGTLSVMEDSAQEIATCNSRNGNPAPKITWYRNGQRLEVPVEMNPEGYMTSRTVREASGLLSLTSTLYLRLRKDDRDASFHCAAHYSLPEGRHGRLDSPTFHLTLHYPTEHVQFWVGSPSTPAGWVREGDTVQLLCRGDGSPSPEYTLFRLQDEQEEVLNVNLEGNLTLEGVTRGQSGTYGCRVEDYDAADDVQLSKTLELRVAYLDPLELSEGKVLSLPLNSSAVVNCSVHGLPTPALRWTKDSTPLGDGPMLSLSSITFDSNGTYVCEASLPTVPVLSRTQNFTLLVQGSPELKTAEIEPKADGSWREGDEVTLICSARGHPDPKLSWSQLGGSPAEPIPGRQGWVSSSLTLKVTSALSRDGISCEASNPHGNKRHVFHFGTVSPQTSQAGVAVMAVAVSVGLLLLVVAVFYCVRRKGGPCCRQRREKGAPPPGEPGLSHSGSEQPEQTGLLMGGASGGARGGSGGFGDEC(SEQ ID NO:27)。

wherein, the sequence of luciferase (luciferase) is:

MADAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDAHIEVDITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAPANDIYNERELLNSMGISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQSMYTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVRFSHARDPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYKIQSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGYGLTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMSGYVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESILLQHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVVFVDEVPKGLTGKLDARKIREILIKAKKGGKIAV(SEQ ID NO:28)。

after mixing 20. Mu.g of each EV with 60. Mu.L of substrate, the in vitro activity was examined. Chemiluminescence results show that both MCAM and BCAM have better luciferase activity than the blank control, and MCAM luciferase fusion protein activity is significantly higher than PTGFRN luciferase fusion protein.

Example 10: truncated BCAMs are still capable of carrying the protein of interest for EV expression

Expression of BCAM 2 truncations fused hIL-12 in stably transfected cells and EVs

An expression plasmid of fusion protein of 2 truncations of BCAM and hIL-12 is constructed. The vector insertion sequence is as follows:

pAAVS1-Puro-CMV-hIL12-BCAM (N32-628), (labeled FL in FIG. 12)

MGMCHQQLVISWFSLVFLASPLVAIWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCSGGGGSGGGGSGGGGSGGGGSRNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNASGGGGSGGGGSGGGGSGGGGSEVRLSVPPLVEVMRGKSVILDCTPTGTHDHYMLEWFLTDRSGARPRLASAEMQGSELQVTMHDTRGRSPPYQLDSQGRLVLAEAQVGDERDYVCVVRAGAAGTAEATARLNVFAKPEATEVSPNKGTLSVMEDSAQEIATCNSRNGNPAPKITWYRNGQRLEVPVEMNPEGYMTSRTVREASGLLSLTSTLYLRLRKDDRDASFHCAAHYSLPEGRHGRLDSPTFHLTLHYPTEHVQFWVGSPSTPAGWVREGDTVQLLCRGDGSPSPEYTLFRLQDEQEEVLNVNLEGNLTLEGVTRGQSGTYGCRVEDYDAADDVQLSKTLELRVAYLDPLELSEGKVLSLPLNSSAVVNCSVHGLPTPALRWTKDSTPLGDGPMLSLSSITFDSNGTYVCEASLPTVPVLSRTQNFTLLVQGSPELKTAEIEPKADGSWREGDEVTLICSARGHPDPKLSWSQLGGSPAEPIPGRQGWVSSSLTLKVTSALSRDGISCEASNPHGNKRHVFHFGTVSPQTSQAGVAVMAVAVSVGLLLLVVAVFYCVRRKGGPCCRQRREKGAPPPGEPGLSHSGSEQPEQTGLLMGGASGGARGGSGGFGDEC(SEQ ID NO:29)。

Wherein the BCAM (N32-628) sequence is:

EVRLSVPPLVEVMRGKSVILDCTPTGTHDHYMLEWFLTDRSGARPRLASAEMQGSELQVTMHDTRGRSPPYQLDSQGRLVLAEAQVGDERDYVCVVRAGAAGTAEATARLNVFAKPEATEVSPNKGTLSVMEDSAQEIATCNSRNGNPAPKITWYRNGQRLEVPVEMNPEGYMTSRTVREASGLLSLTSTLYLRLRKDDRDASFHCAAHYSLPEGRHGRLDSPTFHLTLHYPTEHVQFWVGSPSTPAGWVREGDTVQLLCRGDGSPSPEYTLFRLQDEQEEVLNVNLEGNLTLEGVTRGQSGTYGCRVEDYDAADDVQLSKTLELRVAYLDPLELSEGKVLSLPLNSSAVVNCSVHGLPTPALRWTKDSTPLGDGPMLSLSSITFDSNGTYVCEASLPTVPVLSRTQNFTLLVQGSPELKTAEIEPKADGSWREGDEVTLICSARGHPDPKLSWSQLGGSPAEPIPGRQGWVSSSLTLKVTSALSRDGISCEASNPHGNKRHVFHFGTVSPQTSQAGVAVMAVAVSVGLLLLVVAVFYCVRRKGGPCCRQRREKGAPPPGEPGLSHSGSEQPEQTGLLMGGASGGARGGSGGFGDEC(SEQ ID NO:30)。

pAAVS1-Puro-CMV-hIL12-BCAM (N264-628), (labeled Δ2 in FIG. 12)

MGMCHQQLVISWFSLVFLASPLVAIWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCSGGGGSGGGGSGGGGSGGGGSRNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNASGGGGSGGGGSGGGGSGGGGSTEHVQFWVGSPSTPAGWVREGDTVQLLCRGDGSPSPEYTLFRLQDEQEEVLNVNLEGNLTLEGVTRGQSGTYGCRVEDYDAADDVQLSKTLELRVAYLDPLELSEGKVLSLPLNSSAVVNCSVHGLPTPALRWTKDSTPLGDGPMLSLSSITFDSNGTYVCEASLPTVPVLSRTQNFTLLVQGSPELKTAEIEPKADGSWREGDEVTLICSARGHPDPKLSWSQLGGSPAEPIPGRQGWVSSSLTLKVTSALSRDGISCEASNPHGNKRHVFHFGTVSPQTSQAGVAVMAVAVSVGLLLLVVAVFYCVRRKGGPCCRQRREKGAPPPGEPGLSHSGSEQPEQTGLLMGGASGGARGGSGGFGDEC(SEQ ID NO:31)

Wherein the BCAM (N264-628) sequence is:

TEHVQFWVGSPSTPAGWVREGDTVQLLCRGDGSPSPEYTLFRLQDEQEEVLNVNLEGNLTLEGVTRGQSGTYGCRVEDYDAADDVQLSKTLELRVAYLDPLELSEGKVLSLPLNSSAVVNCSVHGLPTPALRWTKDSTPLGDGPMLSLSSITFDSNGTYVCEASLPTVPVLSRTQNFTLLVQGSPELKTAEIEPKADGSWREGDEVTLICSARGHPDPKLSWSQLGGSPAEPIPGRQGWVSSSLTLKVTSALSRDGISCEASNPHGNKRHVFHFGTVSPQTSQAGVAVMAVAVSVGLLLLVVAVFYCVRRKGGPCCRQRREKGAPPPGEPGLSHSGSEQPEQTGLLMGGASGGARGGSGGFGDEC(SEQ ID NO:32)。

pAAVS1-Puro-CMV-hIL12-BCAM (N448-628), (labeled as Δ4 in FIG. 12)

MGMCHQQLVISWFSLVFLASPLVAIWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCSGGGGSGGGGSGGGGSGGGGSRNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNASGGGGSGGGGSGGGGSGGGGSPELKTAEIEPKADGSWREGDEVTLICSARGHPDPKLSWSQLGGSPAEPIPGRQGWVSSSLTLKVTSALSRDGISCEASNPHGNKRHVFHFGTVSPQTSQAGVAVMAVAVSVGLLLLVVAVFYCVRRKGGPCCRQRREKGAPPPGEPGLSHSGSEQPEQTGLLMGGASGGARGGSGGFGDEC(SEQ ID NO:33)。

Wherein the BCAM (N448-628) sequence is:

PELKTAEIEPKADGSWREGDEVTLICSARGHPDPKLSWSQLGGSPAEPIPGRQGWVSSSLTLKVTSALSRDGISCEASNPHGNKRHVFHFGTVSPQTSQAGVAVMAVAVSVGLLLLVVAVFYCVRRKGGPCCRQRREKGAPPPGEPGLSHSGSEQPEQTGLLMGGASGGARGGSGGFGDEC(SEQ ID NO:34)。

the structure of the 2 truncations constructed is shown in FIG. 12A, and the fusion protein expression results are shown in FIG. 12B. The results show that truncated BCAMs still have the ability to carry the protein of interest to EV. The activity of the fusion protein was examined using different concentration gradients, and the results showed that fusion proteins constructed with truncations were also active (FIG. 12C).

Example 11: engineering EV for drug delivery

Doxorubicin hydrochloride (DOX) is a periodic nonspecific anticancer chemotherapeutic drug and is clinically used for treating acute lymphoblastic leukemia, acute granulocytic leukemia, breast cancer, lung cancer, soft tissue sarcoma, liver cancer, etc. However, serious adverse reactions such as cardiotoxicity, bone marrow suppression, nausea and vomiting, etc. often occur during chemotherapy; and DOX has low molecular weight, is easy to diffuse in vivo, is relatively evenly distributed in medium tissues, has toxic and side effects on normal tissues, and simultaneously has an anti-tumor effect. In order to reduce the toxic and side effects of the antitumor drug and improve the curative effect of the drug, EVs are used for carrying the antitumor drug and the drug carrying amount is detected. Doxorubicin hydrochloride is readily soluble in aqueous solutions. 1ml DOX-HCL (850. Mu.g/ml) was prepared using PBS solution. Fresh 1ml Evs (1.2 wang/. Mu.l protein) was extracted from 293 cells. 200ul (10E 12 Evs) was pipetted from 1ml of EVs solution and 50. Mu.l DOX-HCL (850. Mu.g/ml) solution was added, wherein DOX-HCL contained 42.5. Mu.g; evs were co-mixed with DOX-HCL, PBS was added to 1ml and incubated overnight (16 h) at 37 ℃. The co-incubated mixed solution was drained using CL-2B packing and the first four tube fractions (500 ul/tube) were collected. The collected 2ml solution was added to a 100KD ultrafilter tube, centrifuged at 4000g at 4℃for 5min, and concentrated. After concentration, we obtained the DOX-entrapped EVs drug solution. Adding the pink EVs solution coated with DOX-HCL into the solution, rupture the membrane, releasing the medicine, centrifuging for 2 hours at 150000g, extracting the solution of the supernatant of the triton, detecting the absorbance at 485nm, and converting the concentration.

The extracellular vesicles having DOX entrapped therein were evaluated for drug efficacy. DOX was diluted to the following concentration gradient: 10000nM,3333.34nM,1111.11nM,370.37nM,123.46nM,41.15nM,13.71nM,4.58nM,1.53nM,0nM; the DOX-entrapped extracellular vesicles were diluted to the following concentration gradient: 2500nM,833.34nM,277.78nM,92.59nM,30.86nM,10.28nM,3.43nM,1.14nM,0.38nM,0nM. MCF7 cells were seeded in 96-well plates and cultured at 37℃in 5% CO ₂ At least one day in the environment. One day later, the culture medium is replaced by the DOX solution with the two gradient concentrations or the extracellular vesicle solution with the DOX, and the mixture is taken out after being cultured for 72 hours, and added into each holeThe incubation was continued for 2 hours with 10. Mu.l of CCK8 solution. OD450 absorbance was measured after removal (fig. 13).

Example 12: engineered EV for purification

By displaying some groups, such as Flag tags, at the extracellular end of the scaffold protein that facilitate affinity chromatography. The EVs can be separated directly from the liquid phase using affinity chromatography/immunocapture methods.

The Anti-Flag affinity purified gel was resuspended thoroughly to form as homogeneous a solution as possible. 40. Mu.L of the mixture (20. Mu.L gel) was taken into a new centrifuge tube. 5,000-8,200Xg was centrifuged for 30s to precipitate the gel at the bottom of the centrifuge tube and allowed to stand for 1-2min before adding the sample. The supernatant was removed, at which time care was taken to avoid blotting into the gel. Add 500. Mu.L TBS, gently resuspend Anti-Flag Affinity Gel, centrifuge at 10,000rpm for 30s, discard supernatant and repeat the above steps 1 time. To remove traces of unbound antibody from the gel, the gel may be washed by adding 500 μl 0.1M glycine HCl,pH 3.5, centrifuging to remove the supernatant, adding 3 volumes of TBS, shaking gently for 2-3 minutes, centrifuging for 30s at 5,000-8,200xg, discarding the supernatant, and washing repeatedly until the pH of the supernatant is neutral. Glycine HCl treatment time is not more than 20min. ) 200-1000. Mu.L of extracellular vesicles were added and adjusted to a final volume of 1mL if necessary. Positive control, requiring the addition of 1mL TBS and 4. Mu.L of 50 ng/. Mu.L Flag-BAP fusion protein (about 200 ng); the negative control was added with 1mL PBS (without protein). Incubation was slow at 4℃for 2 hours. If the binding efficiency is improved, the binding efficiency can be prolonged to be overnight. Centrifugation was performed for 30s at 5,000-8,200Xg, and the supernatant was removed. The above precipitate was centrifuged for 30s with 0.5mL TBS, gently mixed, 5,000-8,200Xg to remove supernatant, and repeated 3 times.

Flag fusion extracellular vesicles eluted (one of the following 3 methods is optional). 1) Non-denaturing elution (3×flag polypeptide). Preparing a 3×flag polypeptide eluent: 3 Xflag polypeptide was dissolved in 0.5M Tris-HCl solution, pH 7.5 (containing 1M NaCl) to a final concentration of 25. Mu.g/. Mu.L. Diluted to 5. Mu.g/. Mu.L with ddH2O, 3. Mu.L of 5. Mu.g/. Mu.L of 3 XFlag polypeptide was added to 100. Mu.L of TBS (final concentration 150 ng/. Mu.L). mu.L of 3 XFlag polypeptide eluate was added separately, incubated at 4℃for 30min (shaking slowly), centrifuged at 5,000-8,200 Xg for 30s, and the supernatant was transferred to a new tube (without blotting into the gel). If used immediately, the supernatant can be stored at 4℃and-20℃for a long period of time. 2) Acidic elution (0.1M Gly-HCl, pH 3.5). 100. Mu.L of 0.1M Gly-HCl, pH 3.5, incubation at room temperature for 5min (shaking slowly), centrifugation at 5,000-8,200Xg for 30s, and transfer of supernatant to new tube (10. Mu.L, 0.5M Tris-HCl, pH 7.5,1.5M NaCl). Care should be taken not to suck the gel. If used immediately, the supernatant can be stored at 4℃and-20℃for a long period of time. 3) 20. Mu.L of 2 Xloading buffer (125 mM Tris-HCl, pH 6.8,4% SDS,20% glycerol, 0.004% bromophenol blue) was added to the SDS-PAGE loading buffer, boiled for 3min,5,000-8,200 Xg, and centrifuged for 30s. The supernatant can be directly subjected to SDS-PAGE or WB detection.

And (3) taking NC films with proper sizes, soaking the NC films in methanol for at least 20 seconds, and then taking a proper amount of pre-cooled trans buffers for cleaning for later use. The trans buffer is prepared according to the following formula: 100ml 10x trans buffer (well known as century) +200ml methanol+700 mlrdH2O. Pre-cooling after preparation), taking out the running glue, soaking in a pre-cooled trans buffer, and assembling a sandwich model according to the following sequence: black-cotton-filter-gum-NC membrane-filter-cotton-white. And putting the sandwich model into a film transfer groove according to the sequence of black surface-black surface and white surface-white surface. Two ice boxes frozen overnight in a refrigerator with the temperature of-80 degrees are added into the rotary film box, the whole sandwich model is soaked by pouring film transferring liquid, the rotary film box is installed, and the 350mA constant flow film is arranged for 1h. Closing: a5% BSA blocking solution (1 XTBST was used for dissolution) was prepared, and the transferred membrane was taken out and placed in a blocking solution, and blocked for 1-2 hours on a horizontal shaker at room temperature. Primary antibody incubation (primary antibody is the corresponding antibody to each protein of interest): after blocking, the cells were washed 3 times with 1 XTBE, and a primary antibody solution was prepared in TBST in proportion and incubated for a suitable period of time. And the ratio of the configuration and the incubation time are recorded in table 1. Secondary antibody incubation: after blocking, the mixture was washed 3 times with 1 XTBE, and a secondary antibody solution was prepared in TBST in proportion and incubated for a suitable period of time. ECL chemiluminescent color development: after the secondary antibody incubation was completed, the cells were washed 3 times with 1 XTBST. After ECL chemiluminescent AB solution was mixed at a ratio of 1:1 prior to development, the film was developed on a solar chemiluminescent gel imaging system Tanon-5200Multi (FIG. 15). Example 13: mouse T cell targeting validation of mCD3 scFab exosomes

EVs markers

The present examples used the mCD3 scFab expressed and the control exosomes were identical to example X. During exosome extraction, labeling was performed with lipophilic orange-red fluorescent cyanine dye DiL (D3911, invitrogen). The specific operation is as follows: for cell supernatants of suspension cultures, the recommended pretreatment step is sub-packaging into 50ml centrifuge tubes, centrifuging at RT 800g for 5min to transfer supernatant, centrifuging at 3000g for 20min to transfer supernatant, and filtering the supernatant through 0.22um filter. Cell supernatants were added to 100kd ultrafiltration tubes. Centrifuge 4,000g for 10min. The supernatant was concentrated to within 200mL total volume or about 10 times the concentration. DiL 37℃was added at a concentration of 5. Mu.M for 30min, and 0.22. Mu.m was filtered after completion of the staining. The supernatant was transferred by centrifugation at RT 10,000g for 20 min. The stained concentrated supernatant was added to a super-isolation tube, centrifuged at 10,000g at 4 degrees for 2h, and the stained EVs resuspended with about 2mLPBS per 20 mL. To a 100kd ultrafiltration tube, add dyed EVs, centrifuge 4,000g,10min, centrifuge to a final volume corresponding to about 1,000 times the original supernatant. The dyed EVs were stored in 4℃in the dark.

Staining EVs quantification: the stained EVs were diluted to a final concentration of about 1E+7-1E+8 parts per mL of particle count, and the exosome particle count was recorded using a nanofluidic detector (N30E, fuluidic).

Active targeting of cells

24 hours prior to the formal experiments, the spleen cells of C57BL/6 mice were taken and lysed with red blood cell lysate (Solarbio, R1010) to prepare mPBMCs, which were seeded at 2E+5cells/well in 96-well plates. In the formal experiments, control exosomes and experimental group exosomes were incubated with mpbccs at 1e+7, 1e+8, 1e+9, 2.5e+9, 5e+9particles/mL concentrations in 37 degrees, 5% co2 concentration cell incubators (CLM-170B-8-NF, ESCO) and harvested cells were incubated for 2h, respectively, for flow assays.

The mPBMCs were washed once with sterile PBS before flow assay, incubated at 4℃for 1h with APC-labeled anti-mCD 3 antibody (Invitrogen, 17-0032-82), washed once with PBS after incubation, and resuspended for assay. In the detection, mPBMCs cells without addition of chromosomal exosomes and APC-mCD3 antibody markers were used as loop gate controls, using

NxT soundWave focusing flow cytometry (a 24860, thermo Fisher) and its associated software for data analysis.

Fig. 16 shows the experimental results. FIG. 16A shows a population of DiL-labeled cells (green cell population) using BL2-A (excitation: 488nm, emission filter: 569nm-601nm; area) parameters, and a population of APC-labeled cells (red cell population) using RL1-A (excitation: 637nm, emission filter: 656nm-684nm, area) parameters. Figure 16B shows that the exosomes expressing mCD3 scFab have a significantly enhanced tendency to endocytosis of mouse T cells under co-incubation for 2 h.

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

Claims

1. An engineered extracellular vesicle comprising a transmembrane protein as a scaffold protein, the transmembrane protein being a member of the immunoglobulin superfamily, the transmembrane protein having a low background expression level in the unengineered extracellular vesicle.

2. The extracellular vesicle of claim 1, wherein the background expression level of the transmembrane protein in an unengineered extracellular vesicle is lower than the background expression level of CD81, e.g., the lower background expression level of CD81 means that in mass spectrometry detection of the unengineered extracellular vesicle total protein, the ratio of the iBAQ value of CD81 to the iBAQ value of CD81 is set to 1, which 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.

3. The extracellular vesicle of claim 1 or 2, wherein the transmembrane protein satisfies at least one of the following conditions:

E＝K*m*n*exp(-lambda*S) (I)

(B) The transmembrane protein is a single transmembrane protein, the extracellular region of the single transmembrane protein comprising 1, 2, 3, 4, 5, 6, 7, 8, or 9 immunoglobulin domains; and

4. The extracellular vesicle of claim 3, wherein the transmembrane protein satisfies a combination selected from the group consisting of: a and B, A and C, B and C, and a and B and C.

5. The extracellular vesicle of any one of claims 1-4, wherein the transmembrane protein is selected from BCAM, MCAM, ICAM, ALCAM, CD276, CADM1, VCAM1, CADM2, necin 4, CADM3, agrer, CD96, and variants thereof.

6. The extracellular vesicle of any one of claims 3-5, wherein the extracellular region of the transmembrane protein comprises 3, 4, or 5 immunoglobulin domains;

in particular, the extracellular region of the transmembrane protein comprises both an IgV domain and an IgC domain;

more particularly, the IgC domain is an IgC2 domain;

7. The extracellular vesicle of claim 6, wherein the transmembrane protein is selected from BCAM, MCAM, ALCAM, CD, CADM1, CADM2, necin 4, CADM3, agrer, CD96, and variants thereof;

8. The extracellular vesicle according to any one of claim 1 to 7, wherein the transmembrane protein is a fusion protein comprising a polypeptide segment of interest,

in particular, the polypeptide segment of interest is located at the N-terminus or C-terminus of the fusion protein,

In particular, the polypeptide segment of interest confers therapeutic, targeting and/or affinity tag functions to the fusion protein.

9. The extracellular vesicle of claim 8, wherein the polypeptide segment of interest is a therapeutic polypeptide segment selected from the group consisting of human interleukin family members (e.g., IL-2, IL-7, IL-10, IL-11, IL-12, IL-15, and IL-23), tumor necrosis factor family members (e.g., TNF, LTA, LTB, FASLG, TNFSF, TNFSF9, TNFSF10, TNFSF11, TNFSF12, TNFSF13, TNFSF14, TNFSF15, TNFSF18, and EDA), interferons (INF- α, INF- β, and INF- γ), T cellengager (e.g., 4-1bb, ox40, cd28, 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, AT, TNK, α -glucose, tim-hibmp).

10. The extracellular vesicle according to claim 8, wherein the polypeptide segment of interest is a cell-targeting polypeptide segment,

in particular, the cell-targeting polypeptide segment is an antibody or antigen-binding fragment thereof, a cell surface receptor or a ligand.

11. The extracellular vesicle according to claim 8, wherein the polypeptide segment of interest is an affinity tag,

In particular, the affinity tag is selected from the group consisting of His tag, glutathione thiol transferase (GST), S-peptide, ZZ domain, albumin Binding Domain (ABD), HA, myc, FLAGTM, maltose Binding Protein (MBP), calmodulin Binding Peptide (CBP), SUMO, streptococcal Protein G (Protein G), and gold staphylococcal Protein a (Protein a).

12. The extracellular vesicle according to any one of claims 1 to 11, wherein the extracellular vesicle comprises a therapeutic agent, in particular, the therapeutic agent is selected from the group consisting of a therapeutic polypeptide, a polynucleotide and a small molecule compound.

13. A pharmaceutical composition comprising an extracellular vesicle according to any one of claims 1 to 12 and a pharmaceutically acceptable carrier.

14. An engineered cell for the production of an extracellular vesicle according to any one of claims 1 to 12.

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

16. A method of preparing an extracellular vesicle according to any one of claims 1 to 12, comprising

1) Expressing the transmembrane protein in a cell, and

2) Isolating the extracellular vesicles.

17. A method of delivering a therapeutic agent to a target cell comprising

1) Preparing an extracellular vesicle according to any one of claims 1 to 12, wherein the transmembrane protein is a fusion protein comprising an affinity tag, and

2) Contacting the extracellular vesicles with the target cells.

18. A method of isolating an extracellular vesicle according to any one of claims 1 to 12, comprising