Chimeric adapter protein comprising o6-benzylguanine binding protein, and extracellular vesicle comprising same

Abstract

The present invention relates to a novel chimeric adaptor protein and an extracellular vesicle comprising same. The chimeric adaptor protein according to one aspect is abundantly expressed on the surface of the extracellular vesicle, has excellent conjugation efficiency, and has excellent bioavailability and modularity, such as preserving the integrity of the extracellular vesicle without interacting with other components on the surface of the extracellular vesicle. Extracellular vesicles, when loaded with a drug, can be advantageously used as an effective nanocarrier.

Description

Chimeric adaptor protein comprising O6-benzylguanine binding protein and extracellular vesicles comprising the same

[0001] It relates to a novel chimeric adaptor protein and an extracellular vesicle containing the same.

[0002] Extracellular vesicles (EVs) are nano-sized membrane-bound structures that have lower toxicity and reduced immunological activity compared to other nanoparticles or small molecules, and their potential as drug carriers is being actively studied. Furthermore, surface engineering, which modifies the activity of surface proteins, can improve target delivery efficiency and interaction with specific cell tissues.

[0003] Surface engineering of extracellular vesicles can be broadly categorized into genetic and chemical techniques. Genetic techniques require repetitive genetic manipulation of cells, which can be time-consuming and reduce engineering efficiency. Furthermore, the range of molecules that can be engineered onto the surface is limited to proteins. Among chemical techniques, noncovalent bond-mediated chemical techniques are prone to transient and unstable bonds, which may result in surface-engineered molecules that may not remain stable in certain environments or for extended periods of time. In contrast, covalent bond-mediated chemical techniques directly bind the engineered molecule to residues on the surface protein molecule of the extracellular vesicle, thereby inducing changes in surface protein activity. However, this technique is also challenging, as it requires balancing the need to minimize diversity and steric hindrance, while simultaneously modifying the surface of the extracellular vesicle without disrupting its integrity.

[0004] These researchers have been working to develop novel surface engineering technologies, resulting in the development of a chimeric adaptor protein (CAP) with a novel structure capable of conjugating with various functional molecules. The extracellular vesicles expressing the CAP of the present invention can serve as a universal platform for drug delivery.

[0005] One aspect provides a chimeric adaptor protein comprising an O6-benzyl guanine binding protein; a transmembrane domain; and a signaling domain.

[0006] Another aspect provides a construct comprising a gene encoding the chimeric adapter protein; a vector comprising the construct; or a recombinant cell comprising the construct or vector.

[0007] Another aspect is to provide extracellular vesicles comprising the chimeric adaptor protein or derived from the recombinant cell.

[0008] Another aspect is to provide extracellular vesicles loaded with a drug in the extracellular vesicles.

[0009] One aspect provides a chimeric adaptor protein comprising an O6-benzylguanine binding protein; a transmembrane domain; and a signaling domain.

[0010] As used herein, “chimeric adaptor protein (CAP)” refers to a synthetic complex protein designed to perform a new or improved function by combining domains of different proteins. A chimeric adaptor protein may include a specific protein binding domain, a functional domain that influences the activity of a pathway, and a linker region connecting each domain. The chimeric adaptor protein of the present invention may be expressed on the surface of an extracellular vesicle by binding to the extracellular vesicle. That is, the chimeric adaptor protein of the present invention may include a specific protein binding domain, a transmembrane domain, and a signal transduction domain. The chimeric adaptor protein of the present invention may be expressed on the surface of an extracellular vesicle by binding to an extracellular vesicle and by binding to a specific protein, thereby inducing signal transduction within the extracellular vesicle and altering the activity of the extracellular vesicle.

[0011] In this specification, “O6-benzyl guanine” is a compound in which a benzyl group is bonded to guanine, and is used as a substrate specifically recognized by a self-labeling enzyme such as SNAP-tag. SNAP-tag can label a molecule by binding to a benzyl guanine derivative. The O6-benzyl guanine is used interchangeably with 6-benzylguanine, O6-benzylguanine, 6-(benzyloxy)-7H-purin-2-amine, O6-Benzyl, NSC 637037, 6-O-benzylguanine, 6-benzyloxy guanine, 6-oxo-benzylguanine, or 4 6-Benzyloxyguanine.

[0012] As used herein, the term "self-labeling enzyme" or "self-labeling protein" refers to a protein that can attach a specific molecular tag or marker to a target protein on its own. Self-labeling proteins are extremely useful tools in the fields of cell biology, molecular biology, and biochemistry, as they can attach specific molecular tags or markers to target proteins without complex chemical reactions or modifications. The term "self-labeling protein" may be used interchangeably with "self-labeling protein" or "self-labeling protein." Unless otherwise specified, the term "self-labeling protein" used without modification is used to encompass, without limitation, any protein capable of attaching a molecular tag or marker to extracellular vesicles.

[0013] In one specific example, the O6-benzylguanine binding protein may comprise a self-labeling protein.

[0014] In one specific example, the O6-benzylguanine binding protein may comprise a SNAP-tag. The SNAP-tag has high affinity for O6-benzylguanine.

[0015] In one specific example, the O6-benzylguanine binding protein may be expressed on the surface of extracellular vesicles.

[0016] In one specific example, the O6-benzylguanine binding protein may comprise the amino acid sequence of SEQ ID NO: 2, a portion thereof, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0017] The chimeric adapter protein of the present invention can form site-specific conjugations with various functional molecules via the O6-benzylguanine binding protein. For example, the chimeric adapter protein can form a covalent bond with O6-benzylguanine via the O6-benzylguanine binding protein, and can bind to various functional molecules via O6-benzylguanine.

[0018] As used herein, the term "transmembrane domain" refers to a domain located in the cell membrane, connecting the extracellular domain and the intracellular domain. The transmembrane domain of the present invention may play a role in anchoring, displaying, or binding an O6-benzylguanine binding protein to the cell membrane of an extracellular vesicle.

[0019] In one specific example, the transmembrane domain is any one selected from the group consisting of platelet-derived growth factor receptor (PDGFR), epidermal growth factor receptor (EGFR), fibroblast growth factor receptor (FGFR), vascular endothelial growth factor receptor (VEGFR), hepatocyte growth factor receptor (HGFR), tropomyosin receptor kinase (Trk), insulin receptor (IR), Leukocyte receptor tyrosine kinase (LTK), angiopoietin receptor, cholecystokinin (CCK) receptor, neurotrophic factor (NGF) receptor, receptor tyrosine kinase-like orphan receptors (ROR), discoidin domain receptor (DDR), rearranged during transfection receptor (RETR), tyrosine-protein kinase-like (PTK), related to receptor tyrosine kinase (RYK), muscle-specific kinase (MuSK), CD63, CD9, and CD81. It may comprise the transmembrane domain above. Preferably, the transmembrane domain may comprise the transmembrane domain of platelet-derived growth factor receptor (PDGFR).

[0020] In one embodiment, the transmembrane domain may comprise the amino acid sequence of SEQ ID NO: 4, a portion thereof, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0021] In this specification, the term “signaling domain” refers to a domain that is located inside the cell membrane, i.e., inside the extracellular vesicle, and transmits a signal transmitted by combining an extracellular domain with a ligand, etc.

[0022] In one specific example, the signal transduction domain may include at least one signal transduction domain selected from the group consisting of CD9, CD63, C-terminus of CD63, CD81, LAMP-1, LAMP-2, Syntaxin-3, Syntenin-1, Syntenin-2, Syndecan-1, Syndecan-4, and Prostaglandin F2 Receptor Negative Regulator. Preferably, the signal transduction domain may include an N-terminal sorting domain (NTD) of syntenin. The N-terminal sorting domain is a signal sequence required for a protein to move to a specific organelle or location within a cell, and is involved in protein localization and transport.

[0023] In the chimeric adaptor protein of the present invention, the signal transduction domain can play a role in enriching the chimeric adaptor protein on the surface during the biosynthesis of extracellular vesicles.

[0024] In one specific example, the signal transduction domain may comprise the amino acid sequence of SEQ ID NO: 6, a portion thereof, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0025] In one specific example, the O6-benzylguanine binding protein and the transmembrane domain may be connected directly or via a linker. That is, the linker domain may be located between the O6-benzylguanine binding protein and the transmembrane domain.

[0026] Each domain of the chimeric adapter protein of the present invention may optionally be linked by a short oligopeptide or polypeptide linker. The linker may be a rigid linker or a flexible linker, and is not particularly limited in length or type, and any linker known in the art may be applied without limitation.

[0027] In the present invention, the linker may preferably include a rigid linker. More specifically, the linker may be (EAAAK)n, and the copy number "n" may be adjusted to optimize the linker. For example, the copy number n may be an integer from 5 to 10, and preferably n may be 8.

[0028] In one specific example, the linker may comprise an amino acid sequence of SEQ ID NO: 3, a portion thereof, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0029] In the chimeric adapter protein of the present invention, the linker can improve the conjugation efficiency of the O6-benzylguanine binding protein and O6-benzylguanine.

[0030] In one specific embodiment, the chimeric adapter protein may additionally comprise a reporter.

[0031] In this specification, the term “reporter” is used to monitor the introduction or expression efficiency of the self-labeling protein of the present invention, and any protein that can be monitored without damage can be used without limitation.

[0032] In one embodiment, the reporter can be luciferase, green fluorescent protein (GFP), modified green fluorescent protein (mGFP), enhanced green fluorescent protein (EGFP), red fluorescent protein (RFP), modified red fluorescent protein (mRFP), enhanced red fluorescent protein (ERFP), blue fluorescent protein (BFP), enhanced blue fluorescent protein (EBFP), yellow fluorescent protein (YFP), enhanced yellow fluorescent protein (EYFP), cyan fluorescent protein (CFP), or enhanced cyan fluorescent protein (ECFP).

[0033] In one specific example, the reporter may be positioned between the transmembrane domain and the signal transduction domain.

[0034] In one specific example, the reporter may comprise an amino acid sequence of SEQ ID NO: 5, a portion thereof, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0035] As described above, the chimeric adapter protein of the present invention is abundantly expressed on the surface of extracellular vesicles, and not only exhibits excellent conjugation efficiency with O6-benzylguanine, but also minimizes interference by not interacting with other components of the cell membrane, such as neighboring proteins. The chimeric adapter protein of the present invention strategically integrates a long, rigid linker and a syntenic NTD to minimize steric interference in the local environment on the surface of the extracellular vesicle, thereby enabling efficient binding to a target molecule and providing enhanced functionality. In addition, the extracellular vesicle into which the chimeric adapter protein of the present invention has been introduced preserves its integrity as a separate, distinct entity. In other words, the chimeric adapter protein of the present invention can impart modularity without disturbing the surrounding environment of the surface of the extracellular vesicle.

[0036] In one embodiment, the chimeric adapter protein may comprise the amino acid sequence of SEQ ID NO: 1, a portion thereof, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0037] As used herein, “homology” and “identity” refer to the degree of relationship between two given sequences, which may be expressed as a percentage. The terms homology and identity are often used interchangeably. Whether any two sequences are homologous or identical can be determined using a well-known computer algorithm, such as the “FASTA” program, using default parameters, as in, for example, Pearson et al (1988) [Proc. Natl. Acad. Sci. USA 85]: 2444. Alternatively, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needleman program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277) (version 5.0.0 or later) can be determined using the GCG program package (Devereux, J., et al, Nucleic Acids Research 12: 387 (1984)), BLASTP, BLASTN, FASTA (Atschul, [S.] [F.,] [ET AL, J MOLEC BIOL 215]: 403 (1990); Guide to Huge Computers, Martin J. Bishop, [ED.,] Academic Press, San Diego, 1994, and [CARILLO ETA / .](1988) SIAM J Applied Math 48: 1073). For example, sequence homology or identity can be determined using BLAST or ClustalW of the National Center for Biotechnology Information database in the United States.

[0038] Each domain of the chimeric adapter protein described herein may include not only the aforementioned domains, but also modified forms of each of the aforementioned domains. In this case, the modification may be performed by substituting, deleting, or adding one or more amino acids from the amino acid sequence of the wild-type domain without altering the function of the domains. Typically, the substitution may be performed by a conservative amino acid substitution that does not affect the charge, polarity, or hydrophobicity of the entire protein.

[0039]

[0040] Another aspect provides a construct comprising a gene encoding the chimeric adapter protein.

[0041] Another aspect provides a vector comprising the above construct.

[0042] As used herein, “construct” refers to a macromolecule or molecular complex comprising a polynucleotide that is delivered to a host cell either in vitro, in vivo, or ex vivo. The construct may additionally comprise a promoter sequence.

[0043] The term "coding" herein refers to the inherent property of a specific sequence of nucleotides in a polynucleotide, such as a gene, cDNA, or mRNA, to serve as a template for the synthesis of other polymers and macromolecules in a biological process, either a defined sequence of nucleotides (i.e., rRNA, tRNA, and mRNA) or a defined sequence of amino acids, and the biological properties resulting therefrom. Thus, a gene encodes a protein when transcription and translation of the mRNA corresponding to that gene in a cell or other biological system produces the protein. Both the coding strand, which is identical to the mRNA sequence and is the nucleotide sequence usually provided in the sequence listing, and the non-coding strand used as a template for transcription of a gene or cDNA, may be said to encode a protein or other product of that gene or cDNA.

[0044] As used herein, the term "vector" refers to any nucleic acid construct capable of delivering or directing the movement of foreign genetic material into a target cell where the polynucleotide can be replicated and / or expressed. The vector comprises the construct to be delivered. The vector may be a linear molecule or a circular molecule. The vector may be integrating or non-integrating.

[0045] In one specific example, the vector may be any one selected from the group consisting of a plasmid, a cosmid, a virus, a phage, a recombinant expression cassette, and a transposon, but is not limited thereto, and it is obvious that any vector that is suitable for the purpose of the present invention among vectors that can be commonly used may be used.

[0046] It is obvious that a gene sequence having some sequences deleted, modified, substituted or added from the construct or vector of the present invention can also be used in the present application, as long as it has the same or corresponding function as the gene of the present invention.

[0047] In one specific example, the construct may comprise a gene sequence of SEQ ID NO: 7, a portion thereof, or a gene sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0048] In one specific example, the construct may comprise one or more of the gene sequences of SEQ ID NOs: 8 to 12, a portion thereof, or a gene sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0049]

[0050] Another aspect is to provide a recombinant cell comprising the above construct or the above vector.

[0051] The recombinant cell may comprise a single vector containing a single gene encoding the chimeric adaptor protein. The recombinant cell may be introduced with a single vector containing each gene encoding the chimeric adaptor protein domain; or may be introduced with multiple vectors containing each gene. Each gene or vector may be introduced into the recombinant cell independently, simultaneously, sequentially, or in reverse order.

[0052] In one specific example, the recombinant cell may be an archaeal cell, a bacterial cell, a eukaryotic cell, a eukaryotic unicellular organism, a somatic cell, a germ cell, a hepatocyte, a plant cell, an animal cell, a mammalian cell, a mouse cell, a non-human primate cell, or a human cell.

[0053] Another aspect is to provide extracellular vesicles comprising the chimeric adaptor protein or derived from the recombinant cell.

[0054] As used herein, “extracellular vesicle (EV)” refers to any type of vesicle produced outside of a cell, which is a nano-sized membrane-bound structure produced in the endosomal compartment of most eukaryotic cells. “Extracellular vesicle,” “extracellular vesicle,” “vesicle or vesicle released outside of a cell,” etc. are all used interchangeably, and extracellular vesicles may include many different types, such as exosomes, ectosomes, microvesicles, microparticles, and exosome-like vesicles. Small extracellular vesicles (sEV) generally refer to extracellular vesicles having a diameter of 50 to 200 nm, and the extracellular vesicles of the present invention may be, but are not limited to, small extracellular vesicles.

[0055] Extracellular vesicles engineered with the chimeric adapter protein of the present invention can be used as powerful vehicles for targeted drug delivery based on the excellent bioavailability and pharmacokinetic properties of the extracellular vesicles themselves.

[0056] In one specific example, the extracellular vesicles may have a diameter of 0.1 to 1000 nm. Specifically, they may have a diameter of 50 to 200 nm.

[0057] In one specific example, the extracellular vesicles may further comprise O6-benzylguanine; or O6-benzylguanine and a functional molecule linked to the chimeric adaptor protein.

[0058] As used herein, the term "functional molecule" refers, without limitation, to any molecule capable of binding to the extracellular vesicles of the present invention by binding to O6-benzylguanine. Extracellular vesicles engineered with the chimeric adaptor protein of the present invention exhibit excellent engineering efficiency with the functional molecule.

[0059] Specifically, the functional molecule may be, but is not limited to, an organic fluorescent agent, an inorganic substance, an antibody or an antigen-binding fragment thereof, or a cytokine.

[0060] In one specific example, the organic fluorescence may be, but is not limited to, SNAP 549, Alexa Fluor 488, Alexa Fluor 546, Alexa Fluor 647, Cy3, Cy5, Cy7, FITC, Rhodamine B, Texas Red, DAPI, Fluorescein, Atto 488, Atto 550, or Atto 647.

[0061] In one specific example, the inorganic material may be a quantum dot (QD) or a QD conjugate, other nanoparticle (NP) or NPs conjugate. For example, it may be, but is not limited to, Streptavidin-QD, CdSe / ZnS-QD, CdTe-QD, PbS-QD, InP-QD, ZnS-QD, ZnO NP, BaTiO3NP, Fe3O4NP, CuO NP, Cu2O NP, Au NP, Streptavidin-Au NP or Ag NP.

[0062] In one specific example, the antibody or antigen-binding fragment thereof may include at least one selected from the group consisting of a monoclonal antibody, a domain antibody (dAb), a single chain antibody (scAb), a Fab fragment, a Fab' fragment, an F(ab')2 fragment, a scFab fragment, an Fv fragment, a dsFv fragment, a single chain variable fragment (scFv), a scFv-Fc fragment, a single domain heavy chain antibody, a single domain light chain antibody, an antibody variant, a multimeric antibody, a minibody, a diabody, a bispecific antibody, and a multispecific antibody.

[0063] In one specific example, the antibody or antigen-binding fragment thereof may be an anti-EGFR antibody. More specifically, it may be, but is not limited to, cetuximab, panitumumab, or necitumumab.

[0064] In one specific example, the cytokine may be, but is not limited to, IL-2, IL-7, IL-12, IL-15, IL-21, IFN-α, IFN-γ, TNF-α, TGF-β, IL-10, IL-1β, IL-6, IL-17 or GM-CSF.

[0065] In one specific example, the extracellular vesicles may be loaded with a drug. That is, the extracellular vesicles of the present invention can function as drug delivery vehicles and, depending on the type of drug, can have disease-preventing, ameliorating, or therapeutic effects. For example, applying a tumor-targeting therapeutic agent to the extracellular vesicles can have preventative, ameliorating, or therapeutic effects on various tumors.

[0066] The above drug may be included without limitation as long as it can be contained within an extracellular vesicle. Examples include, but are not limited to, RNA-based drugs (mRNA, siRNA, miRNA, etc.), therapeutic proteins, anticancer agents, antibodies, cytokines, gene editing tools, or lipid-based drugs.

[0067] Specifically, the anticancer agent may include a drug such as doxorubicin, cyclophosphamide, paclitaxel, carboplatin, cisplatin, methotrexate, ifosfamide, topotecan, vincristine, or tamoxifen. Preferably, the anticancer agent may be doxorubicin.

[0068] Also, examples of the above antibodies include alemtuzumab, apolizumab, acelizumab, atlizumab, bapineuzumab, bevacizumab, vivatuzumab mertansine, cantuzumab mertansine, cedelizumab, certolizumab pegol, cidfusituzumab, cidtuzumab, daclizumab, eculizumab, efalizumab, epratuzumab, erlizumab, felvizumab, fontolizumab, gemtuzumab ozogamicin, inotuzumab ozogamicin, ipilimumab, labetuzumab, lintuzumab, matuzumab, mepolizumab, motavizumab, motovizumab, natalizumab, nimotuzumab, nolovizumab, numavizumab, ocrelizumab, omalizumab, palivizumab, May include pascolizumab, pekfucituzumab, pectuzumab, pertuzumab, pexelizumab, ralivizumab, ranibizumab, reslivizumab, reslizumab, resaivizumab, rovelizumab, ruplizumab, sibrotuzumab, siplizumab, sontuzumab, tacatuzumab tetraxetan, tadoximusab, talizumab, tefibazumab, tocilizumab, toralizumab, trastuzumab, tucotuzumab celmoleukin, tucusituzumab, umavizumab, urtoxazumab, or visilizumab.

[0069]

[0070] The present invention is susceptible to various modifications and embodiments. Specific embodiments are illustrated in the drawings and described in detail in the following detailed description. However, this is not intended to limit the present invention to specific embodiments, but rather to encompass all modifications, equivalents, and alternatives falling within the spirit and technical scope of the present invention. In describing the present invention, detailed descriptions of related known technologies will be omitted if they are deemed to obscure the gist of the present invention.

[0071] Chimeric adaptor proteins, depending on the specificity, are abundantly expressed on the surface of extracellular vesicles, exhibit excellent conjugation efficiency, and exhibit excellent bioavailability and modularity, including the preservation of extracellular vesicle integrity without interacting with other components of the extracellular vesicle surface. They can also be useful as effective nanocarriers for drug loading within extracellular vesicles.

[0072] Figure 1 is a structural schematic diagram of an extracellular vesicle (sEV-CAP) expressing a chimeric adapter protein according to one specific example.

[0073] Figure 2 is a graph showing a TEM image of sEV-CAP according to one specific example and its diameter and size distribution.

[0074] Figure 3 is a graph comparing the production yield of sEV-CAP according to one specific example.

[0075] Figure 4 is an image showing the Western blotting results of sEV-CAP according to one specific example.

[0076] Figure 5 is an image showing the level of CAP expression in HEK293FT cells depending on the presence or absence of syntenin NTD, and is the result of comparative analysis of the intensity of EGFP bound to the intracellular domain of the chimeric adapter protein.

[0077] Figure 6 is an image quantifying the amount of CAP expression in a single extracellular vesicle with or without syntenin NTD based on the number of EGFP.

[0078] Figure 7 is a graph showing TIRF images and quantification of QD labeling yield showing EGFP and labeled QDs in cells expressing CAP according to one specific example.

[0079] Figure 8 is an image showing the trajectory of CAP-conjugated QDs in HEK293FT cells expressing CAP.

[0080] Figure 9 is a schematic diagram of sEV-CAP modular engineering combining various functional molecules.

[0081] FIG. 10 is a TIRF image and correlation coefficient showing O6-benzylguanine (BG)-dependent colocalization between sEV-CAP and various functional molecules according to one embodiment.

[0082] Figure 11 is an image showing the number distribution of each CAP and functional molecule in sEV-CAP to which functional molecules are bound.

[0083] Figure 12 is a TEM image of sEV-CAP (sEV-QD) bound to QD.

[0084] Figure 13 is a graph comparing the average number of QDs bound to sEV-CAP as analyzed by TEM with the results of TEM and optical microscopy (OM)-based stoichiometric analysis of sEV-QDs.

[0085] Figure 14 is a graph showing the results of linear regression analysis of each CAP and functional molecule in sEV-CAP combined with functional molecules.

[0086] Figure 15 is a schematic diagram of the surface engineering and activity testing of sEV-CAP.

[0087] Figure 16 is a graph showing SEAP activity induced by sEV-IL2 and a dose-response curve for sEV.

[0088] Figure 17 is a time-lapse EGFP fluorescence image in a single cell after sEV-Y treatment.

[0089] Figure 18 is a graph showing EGFP fluorescence intensity in a single cell over time after sEV-Y treatment (left) and EGFP fluorescence intensity at the single cell level 1 hour after sEV-Y treatment (right).

[0090] Figure 19 is a graph showing the results of mean square displacement (MSD) analysis on trajectories showing directional movement 2 hours after sEV-Y treatment.

[0091] Figure 20 is a schematic diagram showing drug delivery of sEV(drug)-Y according to one specific example.

[0092] Figure 21 is a TIRF image showing the loading of sEV-Y with doxorubicin (Dox) to generate sEV(Dox)-Y, and the co-localization of sEV-Y and Dox fluorescence to confirm the loading.

[0093] Figure 22 is a fluorescence image confirming intracellular delivery after sEV(Dox)-Y treatment and a graph quantifying the same.

[0094] Figure 23 is a graph showing cell survival rate after sEV(Dox)-Y treatment.

[0095] Figure 24 is a schematic diagram of a mouse drug administration schedule in a tumor targeting assay experiment of sEV-Y according to one specific example.

[0096] Figure 25 is an ex vivo fluorescence image of tumors and major organs after administration of wt sEV, sEV, and sEV-Y.

[0097] Figure 26 is a graph quantifying DiR intensity in tumors after administration of wt sEV, sEV, and sEV-Y.

[0098] Figure 27 is an image showing the accumulation of DiR-labeled sEV in tumor cryosections after administration of wt sEV, sEV, and sEV-Y.

[0099] Figure 28 is a schematic diagram of a mouse drug administration schedule in a drug delivery capability verification test of sEV-Y according to one specific example.

[0100] Figure 29 is a graph quantifying tumor bioluminescence measured in mice on the day of euthanasia.

[0101] Figure 30 is a graph showing tumor tissue photographs and tumor weights obtained from mice on the day of euthanasia.

[0102] Figure 31 is a tumor volume curve from the first day of treatment to 19 days in mice.

[0103] Figure 32 is a curve of body weight change over 19 days from the first day of treatment in mice.

[0104] Figure 33 is a graph showing the immunostaining image and the fold change of the quantified TUNEL signal for tumor tissue obtained after drug treatment in mice.

[0105] Figure 34 is a histological image of tumor tissue obtained after drug treatment in mice, stained with hematoxylin and eosin.

[0106] Hereinafter, preferred embodiments are presented to aid understanding of the present invention. However, the following embodiments are provided solely to facilitate a better understanding of the present invention and are not intended to limit the scope of the present invention. The embodiments are susceptible to various modifications, and thus the embodiments are not limited to the embodiments disclosed below and may be implemented in various forms.

[0107]

[0108] Example 1. Preparation of sEV-CAP

[0109] 1.1 Introduction of CAP

[0110] Extracellular vesicles (sEV-CAP) expressing a chimeric adaptor protein (CAP) according to one specific example were prepared as follows.

[0111] The vector encoding CAP was constructed by synthesizing the CAP gene and introducing it into the pLV2 vector. The vector encoding CAP was transfected into HEK293FT cells using Lipofectamine 2000, and the cells were cultured in culture medium supplemented with the antibiotic Hygromycin (300 μg ml-1) for 21 days.

[0112] The gene and amino acid sequences of CAP are shown in Tables 1 and 2 below.

[0113]

[0114] Type Amino acid sequence CAP sequence Full length (SEQ ID NO: 1) - tag (SEQ ID NO: 2) MDKDCEMKRTTLDSPLGKLELSGCEQGLHRIIFLGKGTSAADAVEVPAPAAVLGGPEPLMQATAWLNAYFHQPEAIEEFPVPALHHPVFQQESFTRQVLWKLLKVVKFGEVISYSHLAALAGNPAATAAVKTALSGNPVPILIPCHRVVQGDLDVGGYEGGLAVKEWLLAHEGHRLGKPGLGRigid linker (SEQ ID NO: 3) EAAAKEAAAKEAAAKEAAAKEAAAKEAAAKEAAAKPDGFR beta TM (SEQ ID NO: 4) AVGQDTQEVIVVPHSLPFKVVVISAILALVVLTIISLIILIMLWQKKPREGFP (SEQ ID NO:5)MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICCTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKSyntenin NTD(서열번버 6)SLYPSLEDLKVDKVIQAQTAFSANPANPAILSEASAPIPHDGNLYPRLYPELSQYMGLSLEGG

[0115]

[0116] 종류유전자 서열CAP 서열전장(서열번호 7)tgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccctgacctacggcgtgcagtgcttcagccgctaccccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagcacccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaagggcggtggcggttcctctctctatccatctctcgaagacttgaaggtagacaaagtaattcaggctcaaactgctttttctgcaaaccctgccaatccagcaattttgtcagaagcttctgctcctatccctcacgatggaaatctctatcccagactgtatccagagctctctcaatacatggggctgagtttagaaggcggtSNAP-tag(서열번호8)atggacaaagactgcgaaatgaagcgcaccaccctggatagccctctgggcaagctggaactgtctgggtgcgaacagggcctgcaccgtatcatcttcctgggcaaaggaacatctgccgccgacgccgtggaagtgcctgccccagccgccgtgctgggcggaccagagccactgatgcaggccaccgcctggctcaacgcctactttcaccagcctgaggccatcgaggagttccctgtgccagccctgcaccacccagtgttccagcaggagagctttacccgccaggtgctgtggaaactgctgaaagtggtgaagttcggagaggtcatcagctacagccacctggccgccctggccggcaatcccgccgccaccgccgccgtgaaaaccgccctgagcggaaatcccgtgcccattctgatcccctgccaccgggtggtgcagggcgacctggacgtggggggctacgagggcgggctcgccgtgaaagagtggctgctggcccacgagggccacagactgggcaagcctgggctgggtRigid linker(서열번호 9)gaagccgctgctaaggaagccgctgctaaggaagccgctgctaaggaagccgctgctaaggaagccgctgctaaggaagccgctgctaaggaagccgctgctaaggaagccgctgctaagPDGFR beta TM(서열번호 10)gctgtgggccaggacacgcaggaggtcatcgtggtgccacactccttgccctttaaggtggtggtgatctcagccatcctggccctggtggtgctcaccatcatctcccttatcatcctcatcatgctttggcagaagaagccacgtEGFP(서열번호11)atggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccctgacctacggcgtgcagtgcttcagccgctaccccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagcacccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaagSyntenin NTD(서열번호 12)tctctctatccatctctcgaagacttgaaggtagacaaagtaattcaggctcaaactgctttttctgcaaaccctgccaatccagcaattttgtcagaagcttctgctcctatccctcacgatggaaatctctatcccagactgtatccagagctctctcaatacatggggctgagtttagaaggcggt

[0117]

[0118] 1.2 sEV-CAP의 분리

[0119] sEVs were isolated from the culture supernatant of cells stably expressing the chimeric adapter protein (CAP) as in Example 1.

[0120] First, cells were stabilized for 16 hours after seeding. Cells were then washed three times with phosphate-buffered saline (PBS) and cultured for 24 hours in serum-free DMEM supplemented with 1% A / A, insulin (1 mg ml-1), transferrin (550 μg ml-1), and sodium selenium (670 ng ml-1). The culture supernatant was collected and centrifuged sequentially at 300 g for 5 minutes and at 2,500 g for 20 minutes, and then filtered through a 0.22 μm filter. SEVs were purified through a tangential flow filtration (TFF) system using a hollow fiber filter (0.05 μm pore size polysulfone MidiKros TFF device; Spectrum Laboratories). The filtered supernatant was continuously circulated while maintaining a pressure of less than 20 psi, which was monitored by an in-line analog pressure gauge. SEVs were then concentrated by diafiltration against PBS. Staining of sEV using DiR was performed according to the manufacturer's instructions, and free DiR was removed using PBS via a TFF system.

[0121]

[0122] Example 2. Surface engineering of sEV-CAP

[0123] Surface engineering of extracellular vesicles (sEV-CAP) expressing the chimeric adapter protein manufactured according to Example 1 with various functional molecules was performed as follows.

[0124] Specifically, sEV-CAP was mixed with BG-linker-conjugated molecules and reacted overnight in PBS at room temperature with rotation. For organic dye (Dye) conjugation, 1.0 μM SNAP 549 was reacted with sEV-CAP. For inorganic nanomaterial (QD, Au NP) conjugation, sEV-CAP was incubated with 1 μM BG-biotin, washed, and then reacted with 1.0 nM streptavidin-QD or 0.1 nM streptavidin-Au NP. For protein (antibody) conjugation, cetuximab was conjugated to RPE at the four N-glycan domains of the Fc region using the SiteClick™ Antibody Labeling Kit (Invitrogen) according to the manufacturer's instructions. Then, 20.0 μM RPE-conjugated cetuximab (Cet-RPE) was mixed with a 20-fold molar excess of BG-GLA-NHS and reacted. Simultaneously, 20 μM IL2 was pre-incubated with a 5-fold molar excess of BG-GLA-NHS at room temperature for 30 min. Each mixture was then subjected to a 7 K MWCO zeba spin desalting column (Thermo Fisher) to remove unconjugated BG-GLA-NHS. Finally, 1.0 nM BG-conjugated Cet-RPE or 1.0 nM BG-conjugated IL2 was reacted with sEV-CAP. After the reaction, the sEV-CAP conjugated to each molecule was washed using a TFF system to remove unconjugated molecules except for sEV-Au NPs. For control experiments, NH2-Tex, streptavidin-QD, and Cet-RPE without BG-linker were also reacted with sEV-CAP in PBS overnight at room temperature with rotation. After incubation, sEV-CAP and the control samples were washed using a TFF system to remove unconjugated functional molecules.

[0125]

[0126] Experimental Example 1. Evaluation of the Extracellular Vesicle Properties of sEV-CAP

[0127] The characteristics of extracellular vesicles (sEV-CAP) expressing a chimeric adapter protein according to one specific example were evaluated as follows.

[0128] First, we investigated whether sEV-CAP affects the intrinsic properties of extracellular vesicles. Specifically, sEVs were harvested and purified from HEK293FT cells by tangential flow filtration (TFF), and then sEVs expressing CAP (sEV-CAP), sEVs expressing CAP without the syntenin NTD (sEV-CAP) were isolated. w / o NTD ) and wild-type sEV (wt sEV) were compared and analyzed. The size and shape were compared through transmission electron microscopy (TEM) and shown in Fig. 2, the biosynthetic efficiency was compared through nanoparticle tracking analysis (NTA) and shown in Fig. 3, and the sEV marker was confirmed through Western blotting and shown in Fig. 4.

[0129] As shown in Figure 2, the diameter and size distribution between extracellular vesicles were similar.

[0130] As shown in Fig. 3, the production yield was wt sEV (710 ± 73 cell -1 day -1 ), sEV-CAP w / o NTD (668 ± 145 cells -1 day -1 ), sEV-CAP (743 ± 177 cells -1 day -1 ) appeared.

[0131] As shown in Figure 4, the presence of typical sEV markers (ALIX, Hsp70, TSG101, and CD9) and the absence of the non-sEV marker, the Golgi marker GM130, and co-expression of the SNAP tag were confirmed in all extracellular vesicles.

[0132] That is, sEV-CAP, sEV-CAP according to one specific example w / p NTD , and there were no significant differences in size, morphology, and biosynthetic efficiency between wt sEV.

[0133]

[0134] Experimental Example 2. Evaluation of the efficacy of sEV-CAP

[0135] The CAP structure of extracellular vesicles (sEV-CAP) expressing a chimeric adaptor protein according to one specific example was evaluated through the following experiments to have biophysical properties that contribute to efficient surface engineering of extracellular vesicles.

[0136]

[0137] 2.1 Verify CAP localization

[0138] The EGFP fluorescence intensity of CAP was measured in HEK293FT cells with or without syntenin NTD expression using total internal reflection fluorescence (TIRF) microscopy, and the results of comparative analysis of CAP expression at the cellular level are shown in Fig. 5. In addition, sEV expressing CAP (sEV-CAP) and sEV expressing CAP without syntenin NTD (sEV-CAP) w / o NTD ) fluorescence intensity of EGFP (I EGFP ) was measured at the single molecule level, and the content of individual sEVs was then I EGFP The results of quantification by dividing by are shown in Figure 6.

[0139] As shown in Fig. 5, syntenin NTD did not alter CAP expression in cells. On the other hand, as shown in Fig. 6, syntenin NTD significantly enhanced CAP localization in sEVs, increasing the number of constructs per sEV to 8±6 (sEV-CAP w / o NTD ) was confirmed to increase from 59±24 (sEV-CAP).

[0140]

[0141] 2.2 Measurement of macromolecular labeling efficiency

[0142] Additionally, the labeling efficiency of QDs was measured in HEK293FT cells to evaluate the binding efficiency of CAPs to macromolecules such as antibodies. Specifically, BG-conjugated quantum dots (QDs) were used in sequential treatments including BG-biotin, followed by streptavidin-QDs. QDs enable quantitative analysis through optical signals and, having a similar size to antibodies (approximately 15 nm), are suitable for evaluating potential steric constraints during protein functionalization. CAPs without CAPs or syntenin NTDs (CAPs) w / o NTD ) and wild-type HEK293FT cells are shown in Figure 7.

[0143] As shown in Figure 7, in the case of wild-type (wt) HEK293FT cells lacking the rigid linker and syntenin NTD, binding to BG was significantly reduced and indicated as non-reactive (NR). Each construct (CAP or CAP w / o NTD ) showed increased binding to BG upon rigid linker binding, and the addition of syntenin NTD did not impair binding efficiency, as shown in the TIRF images and graphs quantifying labeling yields showing labeled QDs and EGFP in HEK293FT cells expressing .

[0144]

[0145] 2.3 Measuring interactions with neighboring proteins

[0146] Because membrane receptors can be activated or inhibited through interactions, we tested whether CAP physically interacts (protein-protein interactions; PPI) with other proteins or organelles to assess potential functional disruption of neighboring proteins.

[0147] Specifically, we performed single-molecule tracking experiments in living cells and analyzed the diffusivity (D) changes of individual CAP trajectories using a hidden Markov model (HMM). The temporal variation of D , consistent with the Saffman-Delbruck equation, indicates interactions with surrounding objects. For comparison, SNAP-tagged fusion epidermal growth factor receptor (SNAP-EGFR) was used as a control, and the results are shown in Figure 8.

[0148] As shown in Figure 8, SNAP-EGFR showed reversible transitions between monomeric and dimeric states under similar conditions, whereas CAP and its partial constructs (lacking the syntenin NTD or linker) did not show detectable interactions with the surrounding environment. This suggests that within the temporal and spatial resolution (20 ms, 200 nm), CAP according to one embodiment does not engage in homo- or hetero-interactions between its individual constructs (coiled-coil domain of TMPDGFR, SNAP, syntenin NTD, or EGFP).

[0149]

[0150] In summary, the above results show that the CAP according to one specific example is localized to the extracellular vesicles that express it, increases the binding efficiency with the extracellular vesicles and the desired functional molecule, and has high stability as it does not physically interact with neighboring proteins.

[0151]

[0152] Experimental Example 3. Modular Engineering of sEV-CAP

[0153] The extracellular vesicle (sEV-CAP) platform expressing a chimeric adaptor protein according to one embodiment can be easily functionalized by simply combining it with a functional molecule (hereinafter, BG-X) conjugated to BG. To demonstrate this, experiments were performed on extracellular vesicles (sEV-X) introduced with various functional molecules (X). As in Example 2 described above, (i) an organic molecule expressed as a dye, (ii) an inorganic substance expressed as a QD, and (iii) a protein therapeutic expressed as an antibody (cetuximab conjugated to R-Phycoerythrin (RPE) and modified with BG via BG-GLA-NHS, hereinafter, Y) were introduced. A schematic diagram of the sEV-CAP modular engineering is shown in Fig. 9.

[0154] sEV-CAP was incubated with each BG-X, and unbound BG-X was removed, and the binding efficiency and presence of each post-synthetic product were evaluated.

[0155]

[0156] 3.1 Co-localization Analysis

[0157] First, co-localization analysis using TIRF microscopy was performed, and the results are shown in Fig. 10.

[0158] As shown in Figure 10, high co-localization was observed between sEV-CAP and all conjugated functional molecules, as indicated by the Pearson correlation coefficient (r (EGFP,X) ) showed a strong linear relationship of 0.67 for Dye, 0.65 for QD, and 0.65 for Y.

[0159]

[0160] 3.2 Calculating the stoichiometry of CAP and conjugated functional molecules in sEV-X

[0161] The stoichiometry of each functional molecule was calculated.

[0162] First, to quantify the number of functional molecules in each sEV-X, the fluorescence intensity of a single functional molecule was measured using TIRF. The dye or QD was used to measure single-molecule fluorescence intensity measurements (I Dye , I QD ) was obtained. For Y, site-specific click chemistry between the Fc region of cetuximab and fluorescent RPE was used, and the binding stoichiometry ranged from 1:1 to 1:4. The average intensity of RPE from individual Y molecules was assumed to represent the intensity of a single Y. Then, the stoichiometry of the functional molecule was calculated by dividing the fluorescence intensity of the functional molecule bound to sEV-CAP measured using TIRF by each single molecule intensity, and the results are shown in Fig. 11.

[0163] As shown in Figure 11, the stoichiometry for single sEV was determined to be 20 ± 12 for Dye, 5 ± 4 for QD, and 5 ± 4 for Y.

[0164]

[0165] Next, sEV-QDs were imaged using TEM. The QDs with the CdSe / ZnS core / shell structure appeared darker than the carbon-based sEVs, and d-spacing measurements confirmed that the molecules bound to the sEVs were QDs, and the results are shown in Figure 12. In addition, the number of QDs bound to a single sEV was quantified using TEM analysis, and an average of 4 ± 3 QDs were bound, and the results are shown in Figure 13.

[0166] As shown in Fig. 13, the number of QDs bound to sEV obtained through TEM analysis showed a similar trend to the results of the optical microscope-based stoichiometric analysis of sEV-QDs in Fig. 11.

[0167]

[0168] 3.3 Linear Regression Analysis

[0169] Additionally, linear regression analysis was performed to confirm the Pearson correlation between CAP and X in sEV-X, and the results are shown in Fig. 14.

[0170] As shown in Figure 14, the correlation coefficient r (CAP,X) is r for Dye (CAP,Dye) 0.69, r for QD (CAP,QD) 0.71, and r for Y (CAP,Y) 0.70, a consistent linear relationship was observed between functionalization reactivity and CAP amount. In addition, linear regression analysis allowed estimation of the minimum CAP amount required for effective X binding: 3.6 for Dye (intDye=0), 12.0 for QD (intQD=0), and 14.1 for Y (intY=0).

[0171]

[0172] These results support the validity and modularity of sEV-CAP functionalization according to one specific example.

[0173]

[0174] Experimental Example 4. Evaluation of the therapeutic effect of sEV-IL2 conjugates

[0175] To evaluate the functional enhancement of sEVs through the chimeric adaptor protein (CAP) platform, interleukin 2 (IL2; ~16 kDa), a small-molecule cytokine with therapeutic potential, was conjugated to sEV-CAP, and the effects of the engineered sEVs were evaluated as follows. A schematic diagram of the surface engineering and activity testing of sEV-CAP is shown in Figure 15.

[0176] To assess IL2-mediated signaling, a reporter system of target cells expressing the IL2 receptor (IL2R) was used. Specifically, the activity of the secretory embryonic alkaline phosphatase (SEAP) reporter gene induced by IL2R activation was measured, and the results are shown in Figure 16.

[0177] As shown in Figure 16, neither wt sEVs nor sEV-CAP lacking IL2 induced SEAP expression due to the absence of IL2R activating molecules, whereas sEV-IL2 induced SEAP activity by inducing expression in a dose-dependent manner. These results imply that sEV-CAP acquired additional IL2 functions through surface engineering, suggesting that such immune-activating agents, when applied to sEV-CAP, can be readily utilized for the treatment of various immune-related diseases.

[0178]

[0179] Experimental Example 5. Evaluation of the therapeutic effect of sEV-Y conjugate

[0180] 5.1 Confirmation of cell penetration and internalization of sEV-Y complexes

[0181] To evaluate the functional enhancement of sEV through the chimeric adaptor protein (CAP) platform, cetuximab (ScFv-Fc format; ~110 kDa, hereinafter Y) with therapeutic potential was conjugated to sEV-CAP (sEV-Y), and the effects of the engineered sEV were evaluated as follows. A schematic diagram of the surface engineering and activity testing of sEV-CAP is shown in Fig. 15.

[0182] First, the cellular uptake efficiency of sEV-Y targeting a specific antigen, EGFR, in A549 human lung cancer cells was investigated using time-lapse fluorescence images and EGFP fluorescence intensity, and is shown in Figs. 17 and 18.

[0183] Additionally, representative mean square displacement (MSD) analyses were performed on trajectories exhibiting directional motion 2 hours after treatment. Diffusion types were classified as superdiffusion (active transport), normal diffusion (random motion), and confined diffusion (restricted motion). The results are shown in Figure 19.

[0184] As shown in Figures 17 and 18, compared to sEV-CAP without Y, the amount of sEV-Y on the cell surface increased over time, resulting in approximately 3.8-fold improvement in specific targeting.

[0185] As shown in Figure 19, sEV-Y was internalized and exhibited hyperdiffusion indicating active transport along cellular microtubules, which is characteristic of receptor-mediated endocytosis.

[0186] That is, these results imply that sEV-CAP efficiently performed cell penetration and internalization using Y conjugated through surface engineering.

[0187]

[0188] 5.2 Confirmation of the efficacy of sEV-Y as a drug carrier

[0189] Based on the results of Experimental Example 5.1, the potential of sEV-CAP as a drug carrier was confirmed through the following experiments. Figure 20 is a schematic diagram showing drug delivery of sEV(drug)-Y according to one specific example.

[0190] Specifically, doxorubicin (Dox), a chemotherapeutic drug, was encapsulated within sEV-CAP via electroporation and then conjugated to Y to generate sEV(Dox)-Y. To confirm the internal loading of Dox, co-localization of EGFP and Dox fluorescence of sEV-CAP was confirmed via TIRF imaging, and the results are shown in Fig. 21. To evaluate whether sEV(Dox)-Y shows enhanced cellular uptake, EGFP fluorescence images were taken 2 hours after treating A549 cells with sEV(Dox)-Y, and the intensity was quantified, and is shown in Fig. 22. In addition, cytotoxicity induced by sEV(Dox)-Y was evaluated 48 hours after treating A549 cells with sEV(Dox)-Y, and is shown in Fig. 23.

[0191] As shown in Figure 21, co-localization of EGFP and Dox fluorescence of sEV-CAP was confirmed.

[0192] As shown in Figure 22, the presence of Y in sEV (Dox) improved drug delivery efficiency to target A549 cells.

[0193] As shown in Figure 23, it was confirmed that sEV(Dox)-Y significantly increased cytotoxicity.

[0194] These results imply that the cytotoxicity of sEV(Dox)-Y was observed due to enhanced drug delivery efficiency caused by increased cellular uptake and internalization of sEV mediated by Y, and that no co-localization was observed between EGFP and Dox after internalization, suggesting that Dox disperses within cells through free diffusion, enabling cytoplasmic drug delivery.

[0195]

[0196] Experimental Example 6. Confirmation of the in vivo efficacy of sEV-Y conjugates.

[0197] The targeting and drug delivery capabilities of sEVs engineered via the chimeric adaptor protein (CAP) platform were tested in a mouse model bearing subcutaneously implanted tumor cells.

[0198]

[0199] 6.1 Targeting Confirmation

[0200] A schematic diagram of the mouse drug administration schedule in a tumor targeting assay experiment of sEV-CAP and sEV-Y conjugated with cetuximab (ScFv-Fc format) is shown in Fig. 24. Specifically, fluorescence images were observed after administration of wt sEV, sEV, and sEV-Y in tumors and major organs, and the DiR fluorescence intensity was quantified, which is shown in Figs. 25 and 26. In addition, DiR fluorescence images in tumor cryosections were observed, which is shown in Fig. 27.

[0201] As shown in Figures 25 to 27, sEV-Y showed significant accumulation at the tumor site compared to non-targeted sEV (wt sEV and sEV-CAP without Y).

[0202]

[0203] 6.2 Confirming drug delivery capability

[0204] A schematic diagram of the mouse drug administration schedule in the drug delivery ability confirmation test of sEV-CAP and sEV-Y conjugated with cetuximab (ScFv-Fc format) is shown in Figure 28. Mice with tumors were administered an equivalent dose (1 mg kg -1 ) were treated with Dox, administered in an available form or encapsulated within non-targeted and targeted sEVs (sEV(Dox) and sEV(Dox)-Y, respectively).

[0205] First, tumor bioluminescence was measured on the day of euthanasia after administration of PBS, DOX, sEV(Dox), or sEV(Dox)-Y, and the quantified results are shown in Fig. 29. Tumor tissue photographs and tumor weights obtained on the day of euthanasia are shown in Fig. 30, and the tumor volume curve for 19 days from the first day of treatment is shown in Fig. 31.

[0206] As shown in Figures 29 and 30, available Dox and sEV(Dox) reduced tumor bioluminescence by approximately 40%, whereas sEV(Dox)-Y significantly reduced it by 90% compared to the untreated control. Tumor volume and tumor weight after euthanasia also showed that sEV(Dox)-Y inhibited tumor growth more effectively.

[0207] Meanwhile, as shown in Figure 32, the administered Dox dose was 1 mg kg -1 , total 6mg kg -1 This was well tolerated, with no significant differences in body weight changes observed between groups, suggesting negligible toxicity associated with the drug delivery system.

[0208]

[0209] To further evaluate the enhanced cell death by sEV(Dox)-Y, a TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) assay was performed, and the results are shown in Fig. 33, and a tumor histopathological analysis was performed, and the results are shown in Fig. 34.

[0210] As shown in Figure 33, the level of GFP bound to fragmented DNA, a marker of apoptosis, increased 2.5-fold upon Dox and sEV (Dox) treatment, but increased 4.8-fold upon sEV (Dox)-Y treatment compared to the control group. As shown in Figure 34, this correlated with severe tissue structural destruction and extensive necrosis within the tumor. This contrasts with the effects observed when Dox and sEV (Dox) alone were administered.

[0211]

[0212] In summary, sEV-CAP can be conjugated to functional molecules of various types and sizes. Furthermore, when applied to tumor-targeting therapeutics, sEV-CAP is effective and safe, with no cytotoxicity, making it suitable for the treatment of various tumors. Depending on the type of agent loaded, sEV-CAP is a functional technology for developing sEV-based therapeutics for various diseases, particularly cancer, and as a versatile bio-nanocarrier, it holds significant potential for clinical translation.

[0213]

[0214]

[0215]

[0216] Reference example

[0217]

[0218] Reference Example 1. Cell Culture

[0219] HEK293FT cells were cultured in the presence of penicillin (100 U -1 ), streptomycin (100 μg ml -1 ), amphotericin B (250 ng ml -1) were cultured in DMEM containing 10% FBS and 1% A / A. A549-Luc2 cells were cultured in RPMI-1640 medium containing 10% FBS and 1% A / A. Cells were cultured at 37°C in a humidified atmosphere with 5% CO2. HEK-Blue™ IL-2 cells (Invivogen) and Expi293F™ cells (Gibco) were cultured according to the manufacturer's instructions.

[0220]

[0221] Reference Example 2. Optical Microscope

[0222] Total internal reflection fluorescence (TIRF) and epi-fluorescence microscopy were performed using a stereomicroscope (Nikon, ECLPSE Ti2-E) equipped with a perfect focus system (PFS, TI2-N-ND-P), a motorized stage (TI2-S-SE-E), a stage-top incubator with temperature and CO2 control (Okolab, UNO-TH-PREMIXED), and an electron multiplication charge-coupled device (EM-CCD, Andor, iXorn Ultra 897). A laser (Nikon, LU-N4 Laser Unit, 488 / 561 wavelength) and a mercury lamp (Nikon, C-HGFIE Intensilight) were used as light sources for TIRF and Epi-microscopy, respectively. Imaging was performed using a 100× objective (Nikon, 1.49 NA, oil-immersion, CFI SR HP Apochromat TIRF). DAPI, DIR, and TUNEL staining of tissue sections were imaged using a confocal microscope (Olympus, FV1200) equipped with 405 nm, 488 nm, and 635 nm lasers. Histological images were imaged using a fluorescence microscope (Nikon, Eclipse Ni-E) equipped with 4× and 20× objectives. All imaging data from the light microscopy were processed and analyzed using ImageJ software.

[0223] For microscopy experiments, cells were plated and stabilized on fibronectin-coated glass-bottom dishes (MatTek) for 24 hours. If transfection was required, cells were transfected with vectors and cultured for an additional 24 hours. If using a designated label, cells were incubated with the label for 15 minutes at 37°C and then washed three times with PBS to remove unbound label. For sEV or functional molecules (dyes, QDs, antibodies), sEV or each molecule was fixed on a glass-bottom dish for 15 minutes. The dish was then washed three times with PBS to remove excess material.

[0224]

[0225] Reference Example 3. Quantitative analysis of EGFP expression at the single cell and single sEV levels.

[0226] Stable cell lines and sEVs expressing the EGFP fusion engineered protein (CAP) were imaged using a TIRF microscope with a 488 nm laser. Snapshots were taken with an exposure time of 100 ms. The number of EGFP cells in a single sEV was quantified based on the single fluorescence intensity of EGFP.

[0227]

[0228] Reference Example 4. Evaluation of expression level and binding efficiency for BG in cell membranes.

[0229] Stable cell lines expressing individual SNAP-tagged constructs (CAPs) were observed using total internal reflection fluorescence (TIRF) microscopy using a 488 nm laser to assess expression levels. To assess binding efficiency to BG, the same stable cell lines were incubated with 1.0 μM SNAP 549 or 1.0 nM streptavidin-QD. Cells treated with streptavidin-QD were pre-labeled with 1.0 μM BG-biotin and washed three times with PBS. Cells were then imaged at the same location using TIRF microscopy to detect SNAP 549 or streptavidin-QD using a 561 nm laser and EGFP using a 488 nm laser. The expression level of each construct was assessed by measuring the fluorescence intensity of EGFP. Binding efficiency was quantified as the ratio of SNAP 549 or QD fluorescence intensity to EGFP fluorescence intensity. All imaging was performed with an exposure time of 100 ms.

[0230]

[0231] Reference Example 5. Single molecule tracking.

[0232] HEK293FT cells transfected with vectors encoding the stable cell line (CAP-expressing cells) and the SNAP-tagged fusion epidermal growth factor receptor (SNAP-EGFR; control) were treated with 1.0 μM BG-biotin and then incubated with 1.0 nM streptavidin-QD. Fluorescence trajectories of each QD were acquired using TIRF microscopy at 561 nm. Time-lapse imaging was performed for 3 min with an exposure time of 20 ms per frame and no gaps.

[0233]

[0234] Reference Example 6. Size and concentration analysis of sEVs

[0235] The size distribution and absolute number of sEVs were analyzed using Nanoparticle Tracking Analysis (NTA; Malvern Panalytical, NanoSight LM10). sEVs were diluted in PBS until approximately 50–150 particles per frame were obtained. SEVs were captured for 30 s at a frame rate of 30 frames s-1 using NTA software version 2.3 (NanoSight). The sEV production yield was calculated by dividing the total number of sEVs harvested from the culture supernatant by the number of cells seeded.

[0236]

[0237] Reference Example 7. Western blot analysis

[0238] Purified sEVs or cells were lysed and quantified using a micro BCA protein assay kit. The lysates were boiled at 95°C for 5 minutes, and then sample buffer was added. Each sample (15 μg) was loaded onto a Bolt 4-12% Bis-Tris plus gel (Invitrogen) operating at 100 V and transferred to a nitrocellulose membrane using an iBlot system (Invitrogen). The membrane was cut to enable blotting with multiple antibodies. After blocking with 5% bovine serum albumin in Tris-buffered saline containing 0.05% Tween 20 (TBST), each membrane was incubated with primary antibodies overnight at 4°C. After washing with TBST, the blots were stained with secondary antibodies and imaged using a Chemidoc imaging system (Bio-Rad, ChemicDox XRS+ system).

[0239]

[0240] Reference Example 8. Quantitative analysis of CAP and conjugated functional molecule (X) in a single sEV-X.

[0241] CAP (EGFP) and each molecule (streptavidin-QD, SNAP 549, Cet-RPE) were immobilized on a plate for microscopic observation. For QD, SNAP 549, and Cet-RPE, 100 μl of a 10.0 pM solution in PBS buffer was incubated for 15 min. EGFP was prepared by immobilizing sEV-CAP for 15 min.

[0242] Fluorescence trajectories of QDs, SNAP 549, and EGFP were acquired using TIRF microscopy, using a 561 nm laser for QDs and SNAP 549, and a 488 nm laser for EGFP. Time-lapse imaging was performed for 3 min with 100 ms exposure per frame and no gaps. Fluorescence intensity histograms were constructed from the collected trajectories to identify major peaks corresponding to single QDs, SNAP 549, or EGFP molecules. Cet-RPE was imaged using TIRF microscopy using a 561 nm laser, snapshots were taken with 100 ms exposures, and the fluorescence intensity histograms were analyzed to determine the average intensity of single Cet-RPE. The average fluorescence intensity values ​​of individual QDs, SNAP 549, Cet-RPE, and EGFP were used to quantify functional molecules in single sEV-X. To ensure accurate fluorescence analysis of EGFP in single sEV-CAPs, QDs and Cet-RPE were imaged under 488 nm and 561 nm lasers. Fluorescence intensity under the 488 nm laser was quantified relative to that under the 561 nm laser, and this value was excluded from subsequent quantification experiments using EGFP.

[0243] After functionalizing sEV-CAP, EGFP was imaged using a 488 nm laser, and the conjugated molecule X was imaged using a 561 nm laser in a TIRF microscope. The fluorescence intensity of individual EGFP spots (sEV-X) was measured, and the corresponding fluorescence intensity of X at the same location was measured. The total fluorescence intensity of EGFP and X in a single sEV-X was divided by the intensity of each single molecule for quantitative analysis.

[0244]

[0245] Reference Example 9. Linear Regression Analysis of CAP with Functional Molecule (X) in Single sEV-X

[0246] The number of CAPs and Xs in a single sEV-X was obtained from the quantitative analysis of Reference Example 8. Linear regression analysis was performed to indicate the best-fit line and derive the X-intercept. In addition, 95% confidence intervals were calculated for both X and Y-intercepts to evaluate the correlation between CAPs and functional molecules.

[0247]

[0248] Reference Example 10. Pearson correlation between CAP and functional molecule (X) in a single sEV-X.

[0249] To assess the linear correlation between the fluorescence intensity of CAP (EGFP) and X or the number of CAP and X, the Pearson correlation coefficient (r) was calculated. This coefficient quantifies the degree of linear relationship between two variables from -1 (perfect negative correlation) to 1 (perfect positive correlation), with 0 indicating no linear relationship. In the colocalization analysis, x and y represent the fluorescence intensities of EGFP and X, respectively. In the quantitative analysis for single sEV-X, x and y represent the number of CAP and X molecules, respectively. The correlation was calculated using the following formula.

[0250]

[0251] Here x i Wow y irepresents individual sample points, and bar{x} and bar{y} are the means of x and y samples, respectively.

[0252]

[0253] Reference Example 11. sEV and Dox cell uptake assay

[0254] A549-Luc2 cells were seeded at 5 × 10 per well on fibronectin-coated plates. 3 Cells were seeded at a density of 10 × 10 ng / mL. After 24 h, cells were cultured with different treatments depending on the experimental focus. To prepare cetuximab-conjugated sEVs, cetuximab was first reacted with a 20-fold molar excess of BG-GLA-NHS at room temperature for 30 min. Then, the mixture was purified using a 7 K MWCO zeba spin desalting column to remove unconjugated BG-GLA-NHS. sEVs were then reacted with a 100-fold molar excess of BG-conjugated cetuximab (BG-Cet) overnight at room temperature. For sEV uptake experiments, cells were cultured with sEVs (3.7 × 10 9 particle ml -1 ) were treated. To analyze the cellular uptake of Dox by sEV, cells were treated with Dox (1 μg ml -1 ), sEV(Dox)(1.1 × 10 10 particle ml -1 ) or sEV(Dox)-Y(0.9 × 10 10 particle ml -1 ) was treated with an equivalent dose of Dox.

[0255] The green fluorescence of sEVs and the red fluorescence of Dox were observed using an epifluorescence microscope. For sEV delivery experiments, time-lapse imaging was performed at 10-minute intervals for 1 hour after treatment. Afterwards, cells were washed with 1 ml of PBS to remove extracellular sEVs, and intracellular sEVs were imaged to quantify delivery efficiency. Additionally, fluorescence trajectories of individual sEVs in the cells were obtained. Time-lapse imaging was performed continuously for 3 minutes with an exposure time of 100 ms per frame and no gaps. For Dox delivery experiments, cells were washed with 1 ml of PBS for 2 hours after treatment to remove extracellular sEVs and Dox, and internalized sEVs and Dox were imaged. The dishes were placed in a preheated microscope stage-top incubator maintained at 37°C, 5% CO2, and 80% humidity during imaging.

[0256]

[0257] Reference Example 12. Reporter Cell Assay

[0258] Reporter assays using HEK-Blue™ IL2 cells were performed according to the manufacturer's instructions. Cells were seeded at 5 × 10 per well in a 96-well plate. 4 Cells were seeded at a density of 10 cells / well and incubated with IL2 or sEV. After 24 h, 20 μl of the supernatant per well was incubated with 180 μl of Quanti-Blue™ solution in a humidified atmosphere (37°C, 5% CO2). After 1 h, secreted alkaline phosphatase was quantified by measuring the absorbance at 630 nm.

[0259]

[0260] Reference Example 13. MTS Test

[0261] A549-Luc2 cells were seeded at 1 × 10 per well in a 96-well plate. 4The cells were seeded at a density of 10 μg / mL. After 24 h, the cells were incubated with Dox or Dox-loaded sEVs. Cell viability was analyzed using the CellTiter 96® AQueous one solution reagent. After adding 20 μl of the reagent, the microplate was incubated in a humidified atmosphere (37°C, 5% CO2) for 1 h, and the absorbance was measured at 490 nm.

[0262]

[0263] Reference Example 14. Animal Testing

[0264] All animal experiments were performed in accordance with the animal experiment guidelines approved by the Institutional Animal Care and Use Committee (IACUC) of DGIST (Approval No. 23032701-0000). Six- to eight-week-old male BALB / c-nu mice (Orient Bio) were used in the study. Mice were injected with A549-Luc2 cells (1 × 10 in 100 μl of PBS). 7 (dog cells) were mixed with the same volume of Matrigel (Corning) and injected subcutaneously. Tumor volume (V) was calculated as V = length × width 2 / 2 was calculated.

[0265] To analyze the biodistribution of sEVs, tumor volumes were approximately 600 mm 3 When reached, mice were randomly grouped. PBS or DiR-labeled sEVs (2 × 10 per mouse) 11 (Dog particles) were injected intravenously through the tail vein. Mice were euthanized 4 hours after injection, and major organs (tumor, heart, lung, liver, spleen, kidney) were collected and fluorescence signals were quantified using an in vivo imaging system (Perkin Elmer, IVIS spectrum). To evaluate the therapeutic efficacy of Dox-loaded sEVs, tumor volumes of approximately 150–170 mm were used. 3 When reached, mice were randomly divided into four groups and administered an equivalent dose of Dox (1 mg kg) in soluble form or encapsulated in sEVs. -1) was intravenously injected six times every three days. Before euthanasia, bioluminescent signals were detected using an in vivo imaging system, and tumor tissues were excised, weighed, and histological analysis was performed.

[0266]

[0267] Reference Example 15. Immunofluorescence and histological analysis

[0268] In Reference Example 14, tissues harvested from rats were fixed in 4% paraformaldehyde, dehydrated in 30% sucrose in PBS, and embedded in OCT compound. Frozen tissues were sectioned at 10 μm thickness, and slides were washed with PBS. Apoptosis was detected using a TUNEL assay kit according to the manufacturer's instructions. Nuclei were stained with DAPI for 5 minutes at room temperature. After washing with PBS, the samples were mounted using antifade mounting medium. For hematoxylin and eosin staining, the slides were stained with hematoxylin for 3 minutes and washed with sterile distilled water. The slides were then stained with eosin for 1 minute and washed with water. After dehydration in 95% and absolute ethanol, the samples were mounted using mounting medium.

[0269]

[0270] Reference Example 16. Statistical Analysis

[0271] All data are expressed as the mean ± standard deviation of the indicated replicates. Statistical analyses were performed using GraphPad Prism 8.0 software with a two-tailed Student's t test and a one-way ANOVA with a post-hoc test. Quantitative and linear regression analyses and Pearson correlation coefficient calculations for the number of CAPs and Xs in single sEV-Xs were performed using GraphPad Prism 8.0. ImageJ software was used to calculate Pearson correlation coefficients for the fluorescence intensities of CAPs (EGFP) and Xs, and to measure the diameter and TUNEL signal of sEVs. Data for Pearson correlation coefficients were analyzed from 10 individual images. Data for TUNEL signals from each mouse were expressed as the mean value of five alternate sections.

[0272]

[0273] Reference Example 17. Experimental Materials

[0274] 1) For cell culture and transfection

[0275] HEK293FT (Thermo Fisher), Expi293F TM (Gibco), HEK-Blue TM IL-2 (Invivogen), A549-Luc2 (ATCC), fetal bovine serum (FBS; Hyclone), dulbecco's modified eagle medium (DMEM; Hyclone), roswell park memorial institute (RPMI) 1640 (Hyclone), Expi293F TM Expression medium (Gibco), antibiotic-antimycotic solution (A / A; GenDEPOT), insulin-transferrin-selenium (Gibco), hygromycin B (Gibco), puromycin (Invivogen), nomocin (Invivogen), HEK-Blue TM CLR Selection (Invivogen), lipofectamine® 2000 (Invitrogen), ExpiFectamine TM 293 Transfection Kit (Gibco).

[0276]

[0277] 2) For general experiments

[0278] SNAP-surface 549 (SNAP 549; BioLabs), QD 605 streptavidin conjugate (streptavidin-QD; Thermo Fisher), SNAP-biotin® (BG-biotin; BioLabs), phosphate-buffered saline (PBS; pH 7.4; Biowest), bovine fibronectin (fibronectin; Sigma-Aldrich), ethanol (99%; Alfa Aesar).

[0279]

[0280] 3) For surface modification of sEV-CAP

[0281] Recombinant human interleukin 2 (IL2; Genscript), streptavidin-gold nanoparticle conjugate (streptavidin-Au NP; abcam), texas red-(ACTG)5-NH2 (Cosmogentech), BG-GLA-NHS (NEB), dimethyl sulfoxide (DMSO; 99%, Sigma-Aldrich).

[0282]

[0283] 4) For in vitro experiments

[0284] Cloning restriction enzymes; BsmB I, EcoR I, Sfi I (Enzynomcis); T4 ligase (NEB); XL1-blue electrocompetent E. coli (Agilent); carbenicillin (Invitrogen); zeocin (Gibco); sodium phosphate monobasic dihydrate (≥99%; Sigma-Aldrich); sodium phosphate dibasic dihydrate (≥98.5%; Sigma-Aldrich); glycine (>99%; TCI America); hydrochloric acid (HCl; aqueous solution, 37%, Sigma-Aldrich); tris-aminomethane (Tris; ≥99.8%, Sigma-Aldrich); doxorubicin (Dox; Biosynth); CellTiter 96® AQueous one solution reagent (Promega); Quanti-Blue TM solution (Invivogen), electrolytic buffer (Invitrogen), Neon TM Resuspension buffer R (Invitrogen), formalin solution (10%, neutral buffer; Sigma-Aldrich), glutaraldehyde (50% in H2O, Sigma-Aldrich), sodium cacodylate trihydrate (SC; 98%, Sigma-Aldrich), SNAP-tag® purified protein (NEB), 5X SDS-PAGE loading buffer (Biosesang), coomassie brilliant blue R 250 (Sigma-Aldrich), methanol (99%, Alfa-Aesar).

[0285]

[0286] 5) Antibodies

[0287] Anti-SNAP-tag polyclonal rabbit antibody (NEB, P9310), anti-GM130 monoclonal mouse antibody (BD Biosciences, 610822), anti-β-actin polyclonal rabbit antibody (Cell Signaling, 4967), anti-ALIX monoclonal mouse antibody (Abcam, ab117600), anti-Hsp70 monoclonal mouse antibody (Abcam, ab2787), anti-TSG101 polyclonal rabbit antibody (Abcam, ab30871), or anti-CD9 polyclonal rabbit antibody (Abcam, ab223052), HRP-conjugated anti-mouse IgG antibody (Cell Signaling, 7076), or HRP-conjugated anti-rabbit IgG antibody (Cell Signaling, 7074).

[0288]

[0289] 6) For in vivo experiments

[0290] 1,1'-Dioctadecyl-3,3,3',3'-tetramethylindotricarbocyanine iodide (DiR; Invitrogen), Matrigel® basement membrane matrix (Corning), FSC22 frozen section compound (Leica biosystem), paraformaldehyde (4%, Biosesang), terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay kit 488 nm (Cell Signaling), 4',6-diamidino-2-phenylindole, dihydrochloride (DAPI; Thermo Scientific), hematoxylin (Dako), eosin (Dako), ProLongTM gold antifade mounting reagent (Invitrogen), Eukitt® quick-hardening mounting reagent (Sigma-Aldrich).

[0291]

[0292] The foregoing description of the present invention is provided for illustrative purposes only. Those skilled in the art will readily appreciate that the present invention can be readily modified into other specific forms without altering the technical spirit or essential characteristics of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.

Claims

1. A chimeric adaptor protein comprising an O6-benzyl guanine binding protein; a transmembrane domain; and a signal transduction domain.

2. A chimeric adaptor protein according to claim 1, wherein the O6-benzylguanine binding protein comprises a self-labeling protein (SLP).

3. A chimeric adapter protein according to claim 1, wherein the O6-benzylguanine binding protein comprises a SNAP-tag.

4. In claim 1, the transmembrane domain is selected from the group consisting of platelet-derived growth factor receptor (PDGFR), epidermal growth factor receptor (EGFR), fibroblast growth factor receptor (FGFR), vascular endothelial growth factor receptor (VEGFR), hepatocyte growth factor receptor (HGFR), tropomyosin receptor kinase (Trk), insulin receptor (IR), leukocyte receptor tyrosine kinase (LTK), angiopoietin receptor, cholecystokinin (CCK) receptor, neurotrophic factor (NGF) receptor, receptor tyrosine kinase-like orphan receptors (ROR), discoidin domain receptor (DDR), rearranged during transfection receptor (RETR), tyrosine-protein kinase-like (PTK), related to receptor tyrosine kinase (RYK), muscle-specific kinase (MuSK), CD63, CD9, and CD81. A chimeric adaptor protein comprising one or more transmembrane domains.

5. A chimeric adaptor protein according to claim 1, wherein the signal transduction domain comprises at least one signal transduction domain selected from the group consisting of CD9, CD63, the C-terminus of CD63, CD81, LAMP-1, LAMP-2, Syntaxin-3, Syntenin-1, Syntenin-2, Syndecan-1, Syndecan-4, and Prostaglandin F2 Receptor Negative Regulator.

6. A chimeric adapter protein according to claim 1, wherein the signal transduction domain comprises an N-terminal sorting domain of syntenin.

7. A chimeric adaptor protein according to claim 1, wherein the O6-benzylguanine binding protein and the transmembrane domain are connected directly or via a linker.

8. A chimeric adaptor protein according to claim 7, wherein the linker comprises a rigid linker.

9. A chimeric adapter protein according to claim 1, further comprising a reporter.

10. A chimeric adapter protein comprising an amino acid sequence represented by sequence number 1 according to claim 1.

11. A construct comprising a gene encoding a chimeric adapter protein of any one of claims 1 to 10.

12. A vector comprising the construct of claim 11.

13. A recombinant cell comprising the construct of claim 11; or a vector comprising the construct.

14. An extracellular vesicle comprising a chimeric adaptor protein according to any one of claims 1 to 10; or derived from a recombinant cell according to claim 13.

15. An extracellular vesicle according to claim 14, wherein O6-benzylguanine or O6-benzylguanine and a functional molecule are additionally linked to the chimeric adaptor protein.

16. An extracellular vesicle according to claim 15, wherein the functional molecule is an organic fluorescent substance, an inorganic substance, an antibody or an antigen-binding fragment thereof, or a cytokine.

17. An extracellular vesicle according to claim 14, wherein a drug is loaded inside the extracellular vesicle.

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