Chimeric adapter proteins comprising O6-benzylguanine binding proteins and extracellular vesicles comprising such proteins

Abstract

The present invention relates to a novel chimeric adapter protein and an extracellular vesicle containing the same, and more particularly, to a novel chimeric adapter protein and an extracellular vesicle containing the same, according to one aspect, the chimeric adapter protein can be abundantly expressed on the surface of the extracellular vesicle, has excellent binding efficiency, and does not interact with other components on the surface of the extracellular vesicle, thereby maintaining the integrity of the extracellular vesicle, etc. Good bioavailability and modularization are realized. When the medicine is loaded in the extracellular vesicles, the extracellular vesicles can also be used as an effective nano-carrier.

Description

Technical Field

[0001] This relates to a novel chimeric adaptor protein and extracellular vesicles containing the protein. Background Technology

[0002] Extracellular vesicles (EVs) are nanoscale membrane structures that exhibit low toxicity and reduced immunomodulatory activity compared to other nanoparticles or small molecules. Therefore, their potential as drug carriers is being actively studied. Surface engineering, which alters the activity of surface proteins, can improve targeted delivery efficiency and interactions with specific cells and tissues.

[0003] Extracellular vesicle surface engineering can be broadly categorized into genetic and chemical techniques. Genetic techniques require repeated cellular gene manipulation, are time-consuming, and may reduce engineering efficiency; furthermore, the range of engineerable molecules is limited to proteins. In chemical techniques, non-covalently mediated methods, due to the temporary and unstable nature of the bonding itself, may result in surface-engineered molecules failing to maintain stability under specific conditions or over long periods. In contrast, covalently mediated methods can directly bind the molecule to be engineered to the residues of extracellular vesicle surface proteins, thereby inducing changes in surface protein activity. However, this technique still presents significant challenges, as it requires balancing the need for diversity and minimizing steric hindrance, while simultaneously modifying the extracellular vesicle surface without compromising its integrity.

[0004] Our research team has been dedicated to developing a novel surface engineering technology and has developed a novel chimeric adaptor protein (CAP) that can be coupled to a variety of functional molecules. The extracellular vesicles expressing CAP in this invention can serve as drug carriers, acting as a universal platform. Summary of the Invention Technical issues

[0005] On the one hand, a chimeric adaptor protein is provided, comprising an O6-benzylguanine binding protein; a transmembrane domain; and a signal transduction domain.

[0006] On the other hand, a construct comprising a gene encoding the chimeric adaptor protein is provided; a vector comprising the construct; or a recombinant cell comprising the construct or the vector.

[0007] On the other hand, an extracellular vesicle is provided containing the chimeric adaptor protein; or derived from the recombinant cell.

[0008] On the other hand, extracellular vesicles containing drugs are provided. Technical solution

[0009] On the one hand, a chimeric adaptor protein is provided, comprising an O6-benzylguanine binding protein; a transmembrane domain; and a signal transduction domain.

[0010] In this specification, "chimeric adaptor protein (CAP)" refers to a synthetic complex protein designed to perform novel or enhanced functions by binding to the structural domains of different proteins. Chimeric adaptor proteins may include specific protein-binding domains, functional domains affecting pathway activity, and linker regions connecting the various domains. The chimeric adaptor protein of this invention can bind to and be expressed on the surface of extracellular vesicles. That is, the chimeric adaptor protein of this invention may include specific protein-binding domains, transmembrane domains, and signal transduction domains. By binding to extracellular vesicles, expressing on the surface of extracellular vesicles, and binding to specific proteins, the chimeric adaptor protein of this invention induces signal transduction within the extracellular vesicles and alters the activity of the extracellular vesicles.

[0011] In this specification, "O6-benzylguanine" is a compound in which guanine is bound to a benzyl group, used as a substrate for specific recognition by self-labeled enzymes such as SNAP-tag. SNAP-tag can bind to benzylguanine derivatives to label molecules. The O6-benzylguanine can be used interchangeably with 6-benzylguanine, O6-benzylguanine, 6-(benzyloxy)-7H-purine-2-amine, O6-benzyl, NSC 637037, 6-O-benzylguanine, 6-benzyloxyguanine, 6-oxobenzylguanine, or 4,6-benzyloxyguanine.

[0012] In this specification, "self-labeled enzyme" or "self-labeled protein" refers to a protein capable of attaching a specific molecular tag or marker to a target protein on its own. Self-labeled proteins are highly useful tools in cell biology, molecular biology, and biochemistry because they can attach specific molecular tags or markers to target proteins without complex chemical reactions or modification steps. The terms "self-labeled protein" and "self-tagged protein" are used interchangeably. Unless otherwise stated, an unmodified "self-labeled protein" refers to any protein that can attach a molecular tag or marker to extracellular vesicles without limitation.

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

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

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

[0016] In one specific example, the O6-benzylguanine binding protein may be an amino acid sequence comprising 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 with it.

[0017] The chimeric adaptor protein of the present invention can be site-specifically coupled to a variety of functional molecules through the O6-benzylguanine binding protein. For example, the O6-benzylguanine binding protein can form a covalent bond with O6-benzylguanine, and O6-benzylguanine can bind to a variety of functional molecules.

[0018] In this specification, a "transmembrane domain" refers to a domain that connects extracellular and intracellular domains and is located on the cell membrane. The transmembrane domain of this invention can be used to immobilize, display, or bind O6-benzylguanine-binding protein to the cell membrane of extracellular vesicles.

[0019] In one specific example, the transmembrane domain comprises any one or more transmembrane domains selected from the group consisting of: PDGFR (platelet-derived growth factor receptor), EGFR (epidermal growth factor receptor), FGFR (fibroblast growth factor receptor), VEGFR (vascular endothelial growth factor receptor), HGFR (hepatocyte growth factor receptor), Trk (tropomyosin receptor kinase), IR (insulin receptor), LTK (leukocyte receptor tyrosine kinase), angiopoietin receptor, CCK (cholecystokinin) receptor, neurotrophic factor (NGF) receptor, ROR (receptor tyrosine kinase-like orphan receptor), DDR (discoidovertebrate domain receptor), RETR (reverse rearrangement receptor), PTK (tyrosine protein kinase-like), RYK (receptor tyrosine kinase-associated), MuSK (muscle-specific kinase), CD63, CD9, and CD81. Preferably, the transmembrane domain may comprise the transmembrane domain of the platelet-derived growth factor receptor (PDGFR).

[0020] In one specific example, the transmembrane domain may be an amino acid sequence comprising 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 with it.

[0021] In this specification, "signaling domain" refers to the part located inside the cell membrane, that is, inside the extracellular vesicle, which delivers the signal after binding with extracellular domains and ligands.

[0022] In one specific example, the signal transduction domain comprises one or more signal transduction domains selected from the group consisting of: CD9, CD63, the C-terminus of CD63, CD81, LAMP-1, LAMP-2, Syntaxin-3, Syntenin-1, Syndecan-2, Syndecan-1, Syndecan-4, and Prostaglandin F2 Receptor Negative Regulator. Preferably, the signal transduction domain may comprise the N-terminal sorting domain (NTD) of a Syndecan-binding protein. The N-terminal sorting domain is a signal sequence required for protein movement to specific organelles or locations within the cell, and is associated with protein localization and transport.

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

[0024] In one specific example, the signal transduction domain may be an amino acid sequence comprising 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 with it.

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

[0026] The various domains of the chimeric adaptor protein of the present invention can be selectively linked by short oligopeptides or polypeptide linkers. The linkers can be rigid linkers or flexible linkers, and their length or type is not particularly limited; any linker known in the art can be used without restriction.

[0027] In this invention, the connector preferably comprises a rigid connector. More specifically, the connector can be (EAAAK)n, and the copy number "n" can be adjusted by considering connector optimization. For example, the copy number n can be an integer from 5 to 10, and preferably n can be 8.

[0028] In one specific example, the linker may be an amino acid sequence comprising 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 with it.

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

[0030] In one specific example, the chimeric adaptor protein may further include a reporter gene.

[0031] In this specification, "reporter gene" is used to monitor the introduction or expression efficiency of the self-labeled protein of the present invention. Any protein that can be monitored without causing damage may be used without restriction.

[0032] In one specific example, the reporter gene may 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 gene may be located between the transmembrane domain and the signal transduction domain.

[0034] In one specific example, the reporter gene may be an amino acid sequence containing 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 with it.

[0035] As described above, the chimeric adaptor protein of the present invention is abundantly expressed on the surface of extracellular vesicles, exhibits excellent binding efficiency to O6-benzylguanine, and is in a form with minimal interference, such as not interacting with neighboring proteins or other cell membrane components. The chimeric adaptor protein of the present invention, through strategic integration of long and robust linkers and multi-ligand glycan-binding proteins (NTDs), minimizes spatial interference in the local environment on the surface of extracellular vesicles, thereby achieving effective binding to target molecules and enhancing function. Furthermore, extracellular vesicles incorporating the chimeric adaptor protein of the present invention maintain their integrity as separate, independent entities. In other words, the chimeric adaptor protein of the present invention can impart modularity without interfering with the environment surrounding the surface of extracellular vesicles.

[0036] In one specific example, the chimeric adaptor protein may be an amino acid sequence comprising 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 with it.

[0037] In this specification, “homology” and “identity” refer to the degree of correlation between two given sequences, expressed as a percentage. The terms homology and identity are generally used interchangeably. Whether any two sequences are homologous or identical is determined using known computer algorithms such as the “FASTA” program, with the same default parameters as described, for example, in Pearson et al. (1988) [Proc. Natl. Acad. Sci. USA 85]:2444. Alternatively, the Needleman program in the EMBOSS software 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 executed using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) to determine (including the GCG package (Devereux, J. et al., Nucleic Acids Research 12: 387 (1984)), BLASTP, BLASTN, FASTA (Atschul, [S.][F.,] [ET AL, JMOLEC 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, BLAST or ClustalW from the National Biotechnology Information Database Center can be used to determine sequence homology or identity.

[0038] The various domains of the chimeric adaptor protein described above in this specification include not only the aforementioned domains but also modified forms of each of the domains. In this case, the modification can be performed by substituting, deleting, or adding one or more amino acids in the amino acid sequence of the wild-type domain without altering the function of the domain. Typically, the substitution can be performed using conservative amino acid substitutions that do not affect the overall charge, polarity, or hydrophobicity of the protein.

[0040] On the other hand, a construct is provided that includes a gene encoding the chimeric adaptor protein.

[0041] On the other hand, a carrier is provided that contains the aforementioned construct.

[0042] In this specification, "construct" refers to a macromolecule or molecular complex containing a polynucleotide delivered to a host cell via any means, including in vitro, in vivo, or in vitro delivery. A construct may further include a promoter sequence.

[0043] In this specification, the term "encoding" refers to the unique characteristics of a specific sequence of nucleotides in a polynucleotide such as a gene, cDNA, or mRNA that serves as a template for the synthesis of other polymers and macromolecules in a biological process, having a defined sequence of nucleotides (i.e., rRNA, tRNA, and mRNA) or an amino acid-defined sequence. Thus, a protein is encoded when a gene is transcribed and translated from its corresponding mRNA in a cell or other biological system to produce a protein. The coding strand of the nucleotide sequence, identical to the mRNA sequence and usually provided in the sequence listing, and the non-coding strand that serves as a template for gene or cDNA transcription, are both referred to as the protein or other product encoding that gene or cDNA.

[0044] In this specification, "vector" refers to any nucleic acid construct capable of delivering or directing the transfer of exogenous genetic material into target cells capable of replicating and / or expressing the polynucleotide. The vector comprises the delivered construct. The vector may be a linear or circular molecule. The vector may be integrated or non-integrated.

[0045] In one specific example, the vector may be any one selected from the group consisting of, but not limited to: plasmids, granules, viruses, bacteriophages, recombinant expression cassettes, and transposons; obviously, any vector that is in line with the purpose of this invention may be used among commonly available vectors.

[0046] Obviously, gene sequences that have the same or corresponding functions as the genes of this invention, and that are missing, modified, replaced, or added in a portion of the sequence of the construct or vector of this invention, can all be used in this application.

[0047] In one specific example, the construct may be a gene sequence containing 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 with it.

[0048] In one specific example, the construct may be a gene sequence containing any one or more of the gene sequences in SEQ ID NO: 8-12, a portion thereof, or a gene sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with them.

[0050] On the other hand, a recombinant cell is provided that comprises the construct or the vector.

[0051] The recombinant cell may contain a single vector encoding a single gene of the chimeric adaptor protein. The recombinant cell may contain a single vector with genes encoding the domains of the chimeric adaptor protein; or it may contain multiple vectors with the genes. 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 archaea cell, bacterial cell, eukaryotic cell, eukaryotic unicellular organism, somatic cell, germ cell, hepatocyte, plant cell, animal cell, mammalian cell, mouse cell, non-human primate cell, or human cell.

[0053] On the other hand, an extracellular vesicle is provided that contains the chimeric adaptor protein; or is derived from the recombinant cell.

[0054] In this specification, "extracellular vesicle (EV)" refers to nanoscale membrane structures surrounded by membranes generated in the endosomal compartments of most eukaryotic cells, thus representing all types of vesicles produced extracellularly. The terms "extracellular vesicle," "extracellular vesicle," and "vesicles or microvesicles released extracellularly" are used interchangeably. Extracellular vesicles can encompass various types, such as exosomes, ectosomes, microvesicles, microparticles, and exosome-like vesicles. Small extracellular vesicles (sEVs) typically refer to extracellular vesicles with a diameter of 50-200 nm. The extracellular vesicles of this invention can be, but are not limited to, small extracellular vesicles.

[0055] Extracellular vesicles engineered using the chimeric adaptor protein of this invention can be used as a powerful medium for targeted drug delivery due to their excellent bioavailability and pharmacokinetic properties.

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

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

[0058] In this specification, "functional molecule" refers to all molecules capable of binding to O6-benzylguanine and to the extracellular vesicles of the present invention, but is not limited thereto. Extracellular vesicles engineered with the chimeric adaptor protein of the present invention exhibit excellent engineering efficiency with the aforementioned functional molecule.

[0059] Specifically, the functional molecule may be, but is not limited to, organic fluorescence, inorganic substances, antibodies or their antigen-binding fragments, or cytokines.

[0060] In one specific example, the organic fluorescent agent 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, but is not limited to, quantum dots (QDs) or QD conjugates, other nanoparticles (NPs) or NP conjugates. For example, it may be streptavidin-QD, CdSe / ZnS-QD, CdTe-QD, PbS-QD, InP-QD, ZnS-QD, ZnO NP, BaTiO3 NP, Fe3O4 NP, CuO NP, Cu2O NP, Au NP, streptavidin-Au NP, or Ag NP.

[0062] In one specific example, the antibody or its antigen-binding fragment may be selected from any one or more of the following groups: monoclonal antibody, domain antibody (dAb), single chain antibody (scAb), Fab fragment, Fab' fragment, F(ab')2 fragment, scFab fragment, Fv fragment, dsFv fragment, single chain variable fragment (scFv), scFv-Fc fragment, single domain heavy chain antibody, single domain light chain antibody, variant antibody, multimeric antibody, mini antibody, biantibody, bispecific antibody, and multispecific antibody.

[0063] In one specific example, the antibody or its antigen-binding fragment 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 cytokines may be, but are 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 carry a drug. That is, the extracellular vesicles of the present invention can function as drug carriers and, depending on the type of drug, can have preventative, ameliorative, or therapeutic effects on diseases. For example, applying tumor-targeting therapeutic agents to the extracellular vesicles can have preventative, ameliorative, or therapeutic effects on various tumors.

[0066] The drug can be any drug that can be loaded inside an extracellular vesicle, and there are no limitations. For example, it can be, but is 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 drugs such as doxorubicin, cyclophosphamide, paclitaxel, carboplatin, cisplatin, methotrexate, ifosfamide, topotecan, vincristine, or tamoxifen. Preferably, the anticancer agent may be doxorubicin.

[0068] Additionally, examples of the antibodies may include alemtuzumab, apolizumab, aselizumab, atlizumab, bapineuzumab, bevacizumab, bivatuzumab-mertansine, cantuzumab-mertansine, cedelizumab, and cetelolizumab injection. pegol, Cidfusituzumab, Cidtuzumab, Daclizumab, Eculizumab, Efalizumab, Epratuzumab, Erlizumab, Felvizumab, Fontolizumab, Gemtuzumabozogamicin, Inotuzumab ozogamicin, ipilimumab, labetuzumab, lintuzumab, matuzumab, mepolizumab, motavizumab, motovizumab, natalizumab, nimotuzumab, nolovizumab, numavitizumab, ozogamicin Ocrelizumab, Omalizumab, Palivizumab, Pascolizumab, Pecfusituzumab, Pectuzumab, Pectuzumab, Pexelizumab, Ralivizumab, Ranibizumab, Reslivizumab, ReslizumabResyvizumab, rovelizumab, ruplizumab, sibrotuzumab, siplizumab, Sontuzumab, Tacatuzumab tetraxetan, Tadocizumab, Talizumab, Tefibazumab, Tocilizumab, Toralizumab, Trastuzumab, Tucotuzumab Celmoleukin, Tucusituzumab, Umavizumab, Urtoxazumab, or Visilizumab.

[0070] This invention can be modified in various ways and has multiple embodiments. Specific embodiments will be shown in the accompanying drawings and described in detail below. However, this does not mean that the invention is limited to specific implementations, but should be understood to cover all modifications, equivalents, and even substitutions falling within the scope of the invention's spirit and technology. In the description of this invention, detailed descriptions of related known technologies will be omitted if it is believed that they would obscure the key points of the invention. The effects of the invention

[0071] The chimeric adaptor protein described in one aspect is abundantly expressed on the surface of extracellular vesicles, exhibiting excellent binding efficiency and not interacting with other components on the surface of extracellular vesicles, thus maintaining the integrity of the extracellular vesicles and possessing good bioavailability and modularity. When extracellular vesicles carry drugs, they can also be utilized as an effective nanocarrier. Attached Figure Description

[0072] Figure 1 This is a structural design diagram of an extracellular vesicle (sEV-CAP) expressing a chimeric adaptor protein, based on a specific example.

[0073] Figure 2 The image shows a TEM image of sEV-CAP and its diameter and size distribution based on a specific example.

[0074] Figure 3 This is a graph comparing production yields of sEV-CAP based on a specific example.

[0075] Figure 4This is an image of the protein blotting results of sEV-CAP as shown in a specific example.

[0076] Figure 5 The image shown illustrates the expression level of CAP in HEK293FT cells based on the presence or absence of multiligand glycan-binding protein NTDs. It is a result of a comparative analysis of the binding intensity of EGFP to the intracellular domain of chimeric adaptor proteins.

[0077] Figure 6 An imaging image used to quantify CAP expression levels in a single extracellular vesicle based on the presence or absence of multiligand-binding protein NTDs.

[0078] Figure 7 The image shows a TIRF imaging plot of EGFP and QD-labeled cells expressing CAP, along with a plot of quantitative QD labeling yield, based on a specific example.

[0079] Figure 8 An imaging diagram of the CAP-coupled QD trajectory in HEK293FT cells expressing CAP.

[0080] Figure 9 A schematic diagram of sEV-CAP modular engineering that combines multiple functional molecules.

[0081] Figure 10 TIRF imaging plots and correlation coefficients for O6-benzylguanine (BG)-dependent co-occurrence between sEV-CAP and various functional molecules, as shown in a specific example.

[0082] Figure 11 An imaging diagram showing the number distribution of each CAP and functional molecule in the sEV-CAP that binds functional molecules.

[0083] Figure 12 This is a TEM image of sEV-CAP (sEV-QD) combined with QD.

[0084] Figure 13 This figure compares the average number of QDs bound on sEV-CAP obtained by TEM analysis with the sEV-QD stoichiometric analysis results based on TEM and optical microscopy (OM).

[0085] Figure 14 A graph showing the results of linear regression analysis between each CAP and the functional molecule in the sEV-CAP that incorporates the functional molecule.

[0086] Figure 15 This is a schematic diagram related to sEV-CAP surface engineering and activity testing.

[0087] Figure 16This is a graph showing sEV-IL2-induced SEAP activity and sEV dose-response curves.

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

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

[0090] Figure 19 A graph showing the mean square displacement (MSD) analysis results in the trajectory of the directional motion displayed after 2 hours of sEV-Y processing.

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

[0092] Figure 21 After loading doxorubicin (Dox) onto sEV-Y to generate sEV(Dox)-Y, the co-localization of sEV-Y and Dox fluorescence is used to determine whether loading has occurred, as shown in the TIRF imaging.

[0093] Figure 22 Fluorescent imaging images confirming intracellular delivery of sEV(Dox)-Y after treatment, and images showing its quantification.

[0094] Figure 23 A graph confirming cell viability after sEV(Dox)-Y treatment.

[0095] Figure 24 This is a schematic diagram of the dosing regimen for mice in a tumor-targeting analysis experiment based on a specific example of sEV-Y.

[0096] Figure 25 In vitro fluorescence imaging images of tumors and major organs after administration of wt sEV, sEV, and sEV-Y.

[0097] Figure 26 The graph shows the quantification of DiR intensity in the tumor after administration of wt sEV, sEV, and sEV-Y.

[0098] Figure 27 Imaging images of DiR-labeled sEV accumulation in frozen sections of tumors after administration of wt sEV, sEV, and sEV-Y.

[0099] Figure 28 A schematic diagram of the dosing regimen for mice in an experiment to confirm the drug delivery capability of sEV-Y based on a specific example.

[0100] Figure 29 A graph showing the bioluminescence of mouse tumors measured and quantified on the day of euthanasia.

[0101] Figure 30 The image shows a photograph of tumor tissue and a graph of tumor weight obtained from the mouse on the day of euthanasia.

[0102] Figure 31 The tumor volume curve is shown from day 1 to day 19 of treatment in mice.

[0103] Figure 32 The curve shows the weight change of mice from day 1 to day 19 of treatment.

[0104] Figure 33 Immunostaining imaging of tumor tissue obtained from mice after drug treatment and a graph showing the fold change in quantitative TUNEL signal.

[0105] Figure 34 Histological imaging of tumor tissue obtained in mice after administration of the drug, stained with hematoxylin and eosin. Detailed Implementation

[0106] The following preferred embodiments are presented to aid in understanding the present invention. However, the embodiments provided below are merely for the purpose of facilitating understanding of the invention and are not intended to limit the scope of the invention. Various changes can be made to the embodiments, and the embodiments are not limited to those disclosed below, but can be implemented in various forms.

[0108] Example 1: Preparation of sEV-CAP

[0109] 1.1 Introduction of CAP

[0110] The preparation of an extracellular vesicle (sEV-CAP) expressing a chimeric adaptor protein (CAP) according to a specific example is as follows.

[0111] The vector encoding CAP was prepared by synthesizing the CAP gene and then introducing it into the pLV2 vector. After transfecting the CAP-encoding vector into HEK293FT cells using Lipofectamine 2000, the cells were then treated with the antibiotic hygromycin (300 μg / ml). -1 Cultured in the culture medium for 21 days.

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

[0114] Table 1

[0116] Table 2

[0118] 1.2sEV-CAP separation

[0119] As shown in Example 1, sEVs were isolated from the culture supernatant of cells that stably express chimeric adaptor protein (CAP).

[0120] First, the cells were stabilized 16 hours after seeding. They were then washed three times with PBS and supplemented with 1% A / A and insulin (1 mg / ml). -1 ), transferrin (550 μg ml) -1 Sodium selenide (670 ng / ml) -1 The sEVs were cultured in serum-free DMEM for 24 hours. The culture supernatant was collected and centrifuged sequentially at 300g for 5 minutes and 2,500g for 20 minutes, followed by filtration through a 0.22 μm filter. The sEVs were purified using a tangential flow filtration (TFF) system with a hollow fiber filter (0.05 μm pore size polysulfone MidiKross TFF device, Spectrum Laboratories). The supernatant was continuously circulated while maintaining a pressure below 20 psi using an in-line analog pressure gauge. The sEVs were then concentrated after dialysis with PBS. SEVs were stained with DiR according to the manufacturer's instructions, and free DiR was removed using PBS via the TFF system.

[0122] Example 2: Surface Engineering of sEV-CAP

[0123] The surface engineering of extracellular vesicles (sEV-CAPs) expressing chimeric adaptor proteins prepared according to Example 1 with various functional molecules is described below.

[0124] Specifically, sEV-CAP was mixed with BG-linker-conjugated molecules and reacted at room temperature while being incubated overnight in PBS. 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 co-cultured with 1 μM BG-biotin and washed, then reacted with 1.0 nM streptavidin-QD or 0.1 nM streptavidin-Au NP. For protein conjugation (antibody), cetuximab was conjugated to RPE at the four N-glycosylation domains in 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 reacted with a 20-fold molar excess of BG-GLA-NHS. Simultaneously, 20 μM IL2 was pre-reacted with a 5-fold molar excess of BG-GLA-NHS at room temperature for 30 minutes. Subsequently, uncoupled BG-GLA-NHS in each mixture was removed using a 7 K MWCO zeba spin desalting column (Thermo Fisher). Finally, 1.0 nM MBG-coupled Cet-RPE or 1.0 nM BG-coupled IL2 was reacted with sEV-CAP. After the reaction, sEV-CAPs coupled to each molecule were washed with a TFF system to remove uncoupled molecules other than sEV-Au NP. In the control experiment, Cet-RPE without NH2-Tex, streptavidin-QD, and the BG linker was also reacted with sEV-CAP overnight in PBS at room temperature while spinning. After incubation, sEV-CAP and control samples were washed with a TFF system to remove uncoupled functional molecules.

[0126] Example 1: Evaluation of extracellular vesicle characteristics of sEV-CAP

[0127] The characteristics of an extracellular vesicle expressing a chimeric adaptor protein (sEV-CAP) are evaluated based on a specific example as follows.

[0128] First, we investigated whether sEV-CAP affects the intrinsic properties of extracellular vesicles. Specifically, after harvesting and purifying sEVs from HEK293FT cells via tangential flow filtration (TFF), we compared sEVs expressing CAP (sEV-CAP) and sEVs expressing CAP without the multiligand-binding protein NTD (sEV-CAP). w / o NTDA comparative analysis was conducted between the sEV and wild-type sEV (wt sEV). Size and morphology were compared using transmission electron microscopy (TEM), and the results are as follows: Figure 2 As shown; biosynthetic efficiency was compared using nanoparticle tracking analysis (NTA), and the results are as follows. Figure 3 As shown; the sEV marker was confirmed by Western blotting, and the results are as follows. Figure 4 As shown.

[0129] like Figure 2 As shown, the diameter and size distribution of extracellular vesicles are similar.

[0130] like Figure 3 As shown, the production yield was wt sEV (710 ± 73 cells). -1 sky -1 ), sEV-CAP w / o NTD (668 ± 145 cells) -1 sky -1 ), sEV-CAP (743 ± 177 cells) -1 sky -1 ).

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

[0132] In other words, based on a specific example, sEV-CAP, sEV-CAP w / p NTD Furthermore, wt sEVs showed no significant differences in size, morphology, or biosynthetic efficiency.

[0134] Experimental Example 2: Efficacy Evaluation of sEV-CAP

[0135] The following experiments evaluated the biophysical properties of the CAP structure of extracellular vesicles (sEV-CAP) expressed according to a specific example of chimeric adaptor protein, which contribute to efficient surface engineering of extracellular vesicles.

[0137] 2.1 CAP Positioning Confirmation

[0138] Total internal reflection fluorescence (TIRF) microscopy was used to measure the EGFP fluorescence intensity of CAP (capital protein) based on the expression of multiligand-binding glycan (NTD) proteins in HEK293FT cells. CAP expression levels were compared and analyzed at the cellular level. The results are as follows: Figure 5 As shown. Furthermore, sEVs expressing CAP (sEV-CAP) and sEVs expressing CAP without the multiligand-binding protein NTD (sEV-CAP) are also shown. w / o NTD In this study, the fluorescence intensity (Ig) of EGFP was measured at the single-molecule level. EGFP ), and divide the content of a single sEV by I EGFP Quantification was performed, and the results are as follows: Figure 6 As shown.

[0139] like Figure 5 As shown, the multi-ligand glycan-binding protein NTD did not alter CAP expression in cells. On the one hand, as... Figure 6 As shown, the multiligand glycan-binding protein NTD significantly enhanced the localization of CAP in sEVs, resulting in an increase in the number of constructs per sEV from 8 ± 6 (sEV-CAP). w / o NTD The value increased to 59±24 (sEV-CAP).

[0141] 2.2 Determination of giant molecule labeling efficiency

[0142] Furthermore, to evaluate the binding efficiency of large molecules such as antibodies to CAP, the labeling efficiency of QDs in HEK293FT cells was determined. Specifically, BG-conjugated quantum dots (QDs) were used in a sequential treatment containing BG-biotin, followed by streptavidin-QDs. QDs can be quantitatively analyzed via optical signals and have a size similar to antibodies (approximately 15 nm), making them suitable for assessing potential stereoconfinement during protein functionalization. CAP expressing CAP without CAP or multiligand glycan-binding protein NTDs (CAP...) w / o NTD The results of the comparative analysis of HEK293FT cells and wild-type HEK293FT cells are as follows: Figure 7 As shown.

[0143] like Figure 7 As shown, wild-type (wt) HEK293FT cells lacking rigid linkers and the multiligand-binding glycan-binding protein NTD exhibited significantly reduced binding to BG and were marked as unresponsive (NR). The expression of each construct (CAP or CAP) is shown in the diagram. w / o NTDTIRF imaging and quantitative labeling yield plots of QD and EGFP labeled in HEK293FT cells show that binding to BG increases upon rigid linker binding, confirming that binding efficiency is not impaired even with the addition of multiligand glycan-binding protein NTD.

[0145] 2.3 Interaction determination with neighboring proteins

[0146] Since membrane receptors can be activated or inhibited through interactions, this study verified whether CAPs engage in physical interactions (protein-protein interactions; PPIs) with other proteins or organelles to assess potential functional disorders of neighboring proteins.

[0147] Specifically, single-molecule tracking experiments were performed in living cells, and the diffusivity (D) of individual CAP trajectories was analyzed using a hidden Markov model (HMM). The change in D, consistent with the Saffman-Delbruck equation over time, reflects the interaction with surrounding objects. As a comparison, a SNAP-tagged fusion with epidermal growth factor receptor (SNAP-EGFR) was used as a control group, and the results are as follows... Figure 8 As shown.

[0148] like Figure 8 As shown, under similar conditions, SNAP-EGFR exhibits a reversible transition between monomeric and dimer states, while CAP and some of its constructs (excluding the multiligand-binding protein NTD or linker) do not show detectable interactions with their surroundings. This implies that, within a temporal and spatial resolution (20 ms, 200 nm), CAP, according to a specific example, does not participate in homologous or heterologous interactions between its constituent parts (the coiled-coil domain of TMPDGFR, SNAP, multiligand-binding protein NTD, or EGFP).

[0150] Based on the above results, it can be concluded that, according to a specific example, CAP is located in the extracellular vesicles that express it, which improves the binding efficiency with extracellular vesicles and target functional molecules, and has high stability by not physically interacting with neighboring proteins.

[0152] Experiment Example 3: Modular Engineering of sEV-CAP

[0153] According to a specific example, the extracellular vesicle platform expressing a chimeric adaptor protein (sEV-CAP) can be easily functionalized by simply binding to a functional molecule conjugated to BG (hereinafter referred to as BG-X). To verify this, experiments were conducted on extracellular vesicles (sEV-X) incorporating various functional molecules (X). As described in Example 2 above, the following were introduced: (i) organic molecules, represented by dyes; (ii) inorganic substances, represented by QDs; and (iii) protein therapeutic agents, represented by antibodies (cetuximab conjugated to R-phycoerythrin (RPE) and modified to BG by BG-GLA-NHS, hereinafter referred to as Y). A schematic diagram of the modular engineering of sEV-CAP is shown below. Figure 9 As shown.

[0154] sEV-CAP was cultured together with each BG-X, and after removing unbound BG-X, the binding efficiency and presence of each synthesized product were evaluated.

[0156] 3.1 Colocation Analysis

[0157] First, colocalization analysis was performed using TIRF microscopy, and the results are as follows: Figure 10 As shown.

[0158] like Figure 10 As shown, sEV-CAP exhibits high colocalization with all coupled functional molecules, with a Pearson correlation coefficient (r) of [missing value]. （EGFP,X） The coefficient of performance (COP) is 0.67 for dyes, 0.65 for QD, and 0.65 for Y, showing a strong linear relationship.

[0160] 3.2 Stoichiometric calculations of CAP and coupled 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 individual functional molecules was measured using TIRF. Single-molecule fluorescence intensity measurements were obtained using dyes or QD. 染料 I QDFor Y, site-specific click chemistry was used between the Fc region of cetuximab and the fluorescent RPE, with a binding stoichiometry range of 1:1 to 1:4. It was assumed that the average intensity of the RPE in a single Y molecule represented the intensity of a single Y molecule. 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 the following single-molecule intensities, as shown in the figure. Figure 11 As shown.

[0163] like Figure 11 As shown, for the results of a single sEV, the stoichiometry was determined to be 20±12 for dye, 5±4 for QD, and 5±4 for Y.

[0165] Then, TEM was used to image the sEV-QD. Compared with carbon-based sEVs, the CdSe / ZnS core / shell structured QD appeared darker. d-interval measurements confirmed that the molecule bound to the sEV was a QD, as shown in the results. Figure 12 As shown in the figure. Furthermore, the number of QDs bound to a single sEV was quantified using TEM analysis, with an average of 4 ± 3 QDs bound, as shown in the figure. Figure 13 As shown.

[0166] like Figure 13 As shown, the number of QDs bound to sEVs obtained through TEM analysis is... Figure 11 The sEV-QD chemometric analysis results based on optical microscopy showed a similar trend.

[0168] 3.3 Linear Regression Analysis

[0169] Furthermore, by performing linear regression analysis, the Pearson correlation between CAP and X in sEV-X was confirmed, and the results are as follows: Figure 14 As shown.

[0170] like Figure 14 As shown, the correlation coefficient r （CAP,X） When using dyes, the value is r. （CAP,染料） 0.69, r when QD （CAP,QD） 0.71, when Y is r （CAP,Y） The value of 0.70 indicates a consistent linear relationship between functionalization reactivity and CAP amount. Furthermore, linear regression analysis can estimate the minimum CAP amount required for effective X binding: 3.6 for dye (int dye = 0), 12.0 for QD (int QD = 0), and 14.1 for Y (int Y = 0).

[0172] The above results support the feasibility and modularity of sEV-CAP functionalization based on a specific example.

[0174] Experimental Example 4: Evaluation of the therapeutic effect of sEV-IL2 binding agents

[0175] To evaluate the functional enhancement of sEVs via the chimeric adaptor protein (CAP) platform, the therapeutically promising low molecular weight cytokine interleukin-2 (IL2; ~16 kDa) was conjugated to sEV-CAP, and the effect on engineered sEVs was evaluated as follows. A schematic diagram related to sEV-CAP surface engineering and activity testing is shown below. Figure 15 As shown.

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

[0177] like Figure 16 As shown, sEV-CAP, lacking wt sEVs or IL2, did not induce SEAP expression due to the absence of IL2R activating molecules. However, sEV-IL2 was induced in a dose-dependent manner, thereby triggering SEAP activity. These results indicate that sEV-CAP acquires the additional function of IL2 through surface engineering, suggesting that such immune activators, when applied to sEV-CAP, can be readily used to treat a variety of immune-related diseases.

[0179] Experimental Example 5: Evaluation of the therapeutic effect of sEV-Y binding agents

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

[0181] To evaluate the enhancement of sEV function via the chimeric adaptor (CAP) platform, the therapeutically promising, relatively high molecular weight cetuximab (ScFv-Fc form; ~110 kDa, hereinafter referred to as Y) was conjugated to sEV-CAP (sEV-Y). The effect on engineered sEVs was evaluated as follows. A schematic diagram related to sEV-CAP surface engineering and activity testing is shown below. Figure 15 As shown.

[0182] First, the cellular uptake efficiency of sEV-Y targeting the specific antigen EGFR in A549 human lung cancer cells was investigated using time-lapse fluorescence imaging and EGFP fluorescence intensity. The results are as follows: Figure 17 and Figure 18 As shown.

[0183] Furthermore, after 2 hours of processing, representative mean square displacement (MSD) analysis was performed on the trajectories showing directional motion. Diffusion types were categorized as superdiffusion (active transport), normal diffusion (random motion), and confined diffusion (confined motion). Results are as follows... Figure 19 As shown.

[0184] like Figure 17 and Figure 18 As shown, compared with sEV-CAP without Y, the amount of sEV-Y on the cell surface increases over time, which improves specific targeting by about 3.8 times.

[0185] like Figure 19 As shown, sEV-Y is internalized and exhibits hyperdiffusion of active transport along cellular microtubules, which corresponds to the characteristics of receptor-mediated endocytosis.

[0186] In other words, these results demonstrate that sEV-CAP effectively achieves cell penetration and internalization through surface engineering using coupled Y.

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

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

[0190] Specifically, the chemotherapy drug doxorubicin (Dox) was encapsulated within sEV-CAP via electroporation, and subsequently conjugated with Y to generate sEV(Dox)-Y. To confirm the internal loading of Dox, the colocalization of EGFP and Dox fluorescence in sEV-CAP was observed using TIRF imaging, and the results are as follows: Figure 21 As shown in the figure. To assess whether sEV(Dox)-Y exhibits enhanced cellular uptake capacity, EGFP fluorescence images were captured 2 hours after A549 cells were treated with sEV(Dox)-Y, and the intensity was quantified. The results are shown in the figure. Figure 22 As shown. Furthermore, after treating A549 cells with sEV(Dox)-Y for 48 hours, the sEV(Dox)-Y-induced cytotoxicity was assessed, and the results are as follows. Figure 23 As shown.

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

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

[0193] like Figure 23 As shown, sEV(Dox)-Y significantly enhanced cytotoxicity.

[0194] The above results indicate that the cytotoxicity of sEV(Dox)-Y was enhanced by increased cellular uptake and internalization of sEV mediated by Y, which improved drug delivery efficiency. Furthermore, no colocalization was observed between EGFP and Dox after internalization, suggesting that Dox disperses within the cell via free diffusion, thereby enabling cytoplasmic drug delivery.

[0196] Experimental Example 6: In vivo analysis of sEV-Y conjugates ( in-vivo Efficacy verification

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

[0199] 6.1 Target confirmation

[0200] In the tumor-targeting analysis experiment of cetuximab (ScFv-Fc form) conjugated with sEV-CAP and sEV-Y, the schematic diagram of the mouse dosing regimen is as follows: Figure 24 As shown. Specifically, fluorescence imaging was observed in tumors and major organs after administration of wt sEV, sEV, and sEV-Y, and their DiR fluorescence intensity was quantified. The results are shown below. Figure 25 and Figure 26 As shown. Furthermore, by observing DiR fluorescence imaging in frozen tumor sections, the results are as follows... Figure 27 As shown.

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

[0203] 6.2 Validation of drug delivery capability

[0204] In the drug delivery capability validation experiments of cetuximab (ScFv-Fc form) conjugated with sEV-CAP and sEV-Y, the schematic diagram of the mouse dosing regimen is shown below. Figure 28 As shown. Using an equal dose (1 mg kg) -1 Dox was used to treat mice with tumors, either in a soluble form or encapsulated in non-targeted and targeted sEVs (sEV(Dox) and sEV(Dox)-Y, respectively).

[0205] First, tumor bioluminescence was measured on the day of euthanasia following administration of PBS, DOX, sEV(Dox), or sEV(Dox)-Y, and the quantitative results are as follows: Figure 29 As shown; photos of the tumor tissue obtained on the day of euthanasia and the tumor weight are as follows. Figure 30 As shown; the tumor volume curve on day 19 from the first day of treatment is as follows. Figure 31 As shown.

[0206] like Figure 29 and Figure 30 As shown, soluble Dox and sEV(Dox) reduced tumor bioluminescence by approximately 40%, while sEV(Dox)-Y significantly reduced it by 90% compared to the untreated control group. Post-euthanasia tumor volume and weight also indicated that sEV(Dox)-Y more effectively inhibited tumor growth.

[0207] On the one hand, such as Figure 32 As shown, the dosage of Dox is 1 mg / kg. -1 The total dose is 6 mg / kg. -1 The drug was well tolerated, and no significant differences in weight change were observed between groups. In other words, toxicity related to the drug delivery system was negligible.

[0209] To further evaluate the enhanced apoptosis of sEV(Dox)-Y, TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) analysis was performed, and the results are as follows: Figure 33 As shown, tumor tissue pathological analysis was performed, and the results are as follows. Figure 34 As shown.

[0210] like Figure 33 As shown, the GFP level binding to fragmented DNA, a marker of apoptosis, increased 2.5-fold in Dox and sEV(Dox) treatments, and 4.8-fold in sEV(Dox)-Y treatment compared to the control group. Figure 34 As shown, this is correlated with severe tissue damage and extensive necrosis within the tumor. This contrasts with the effects observed when only Dox and sEV (Dox) were applied.

[0212] In summary, sEV-CAPs can be conjugated to functional molecules of various types and sizes. Furthermore, applying tumor-targeting therapeutic agents to sEV-CAPs allows for the easy treatment of a variety of tumors, demonstrating effectiveness and safety without cytotoxicity. Depending on the type of formulation loaded, developing functional technologies and multifunctional bionanocarriers for sEV-based therapeutics targeting multiple diseases, particularly cancer, holds immense potential for clinical translation.

[0216] Reference Example

[0218] Refer to Example 1. Cell Culture

[0219] HEK293FT cells were subjected to a solution containing penicillin (100 U). -1 Streptomycin (100 μg / ml) -1 Amphotericin B (250 ng / ml) -1 Cells were cultured in 10% FBS and DMEM containing 1% A / A. A549-Luc2 cells were cultured in RPMI-1640 medium containing 10% FBS and 1% A / A. Cells were cultured at 5% CO2 and 37°C under humid conditions. HEK-Blue™ IL-2 cells (Invivogen) and Expi293F™ cells (Gibco) were cultured according to the manufacturer's instructions.

[0221] Refer to 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 focusing 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). The TIRF and Epi-fluorescence microscopes used laser (Nikon LU-N4 laser unit, 488 / 561 nm wavelength) and mercury lamp (Nikon C-HGFIE Intensilight), respectively. Imaging was performed using a 100× objective lens (Nikon, 1.49 NA, oil immersion, CFI SR HP apochromatic TIRF). DAPI, DIR, and TUNEL imaging of tissue sections was performed using a confocal microscope (Olympus, FV1200) equipped with 405nm, 488nm, and 635nm lasers. Histological imaging was performed using a fluorescence microscope (Nikon, Eclipse Ni-E) equipped with 4× and 20× objectives. All imaging data from the optical microscopes were processed and analyzed using ImageJ software.

[0223] For samples used in microscopy, taking cells as an example, smear the cells and stabilize them in fibronectin-coated glass-bottomed culture dishes (MatTek) for 24 hours. If transfection is required, transfect the cells with the vector and culture for another 24 hours. When using a specified tag, incubate the cells and tag together at 37°C for 15 minutes, then wash three times with PBS to remove unbound tags. For sEVs or functional molecules (dyes, QDs, antibodies), fix the sEVs or individual molecules on a glass-bottomed culture dish for 15 minutes. Then wash the dish three times with PBS to remove excess material.

[0225] Refer to Example 3. Quantitative analysis of EGFP expression at the single cell and single sEV level.

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

[0228] Refer to Example 4. Evaluate the expression level and binding efficiency of BG in the cell membrane.

[0229] Stable cell lines expressing a single SNAP-tag construct (CAP) were observed using total internal reflection fluorescence (TIRF) microscopy with a 488 nm laser to assess their expression levels. To evaluate BG binding efficiency, the same stable cell lines were co-cultured with either 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. SNAP 549 or streptavidin-QD was then detected using a 561 nm laser, and EGFP was detected using a 488 nm laser via TIRF microscopy at the same locations. Expression levels of each construct were assessed by measuring EGFP fluorescence intensity. Binding efficiency was quantified by the ratio of SNAP 549 or QD fluorescence intensity to EGFP fluorescence intensity. All imaging was performed with an exposure time of 100 ms.

[0231] Refer to Example 5. Single-molecule tracking

[0232] HEK293FT cells transfected with a vector encoding a stable cell line (cells expressing CAP) and a SNAP-tagged fusion epidermal growth factor receptor (SNAP-EGFR; control group) were treated with 1.0 μM BG-biotin and then co-cultured with 1.0 nM streptavidin-QD. Fluorescence trajectories of each QD were obtained using a 561 nm TIRF microscope. Time-lapse imaging was performed with each frame exposed for 20 ms for 3 minutes without intervals.

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

[0235] 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 to 150 particles were contained per frame. sEVs were captured for 30 seconds at a rate of 30 frames per second using NTA software version 2.3 (NanoSight). sEV production yield was calculated by dividing the total number of sEVs harvested from the culture supernatant by the number of seeded cells.

[0237] Refer to Example 7. Western blot analysis

[0238] Purified sEVs or cells were broken down and quantified using a micro-BCA protein assay kit. The solution was 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) running at 100V and transferred to a nitrocellulose membrane using the iBlot system (Invitrogen). The membrane was cut to allow for blot analysis with various antibodies. After blocking with 5% bovine serum albumin in Tris-buffered saline containing 0.05% Tween 20 (TBST), the membranes were incubated overnight at 4°C with the primary antibody. After washing with TBST, the blots were stained with secondary antibody and imaged using a chemidoc imaging system (Bio-Rad, ChemicDox XRS+ system).

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

[0241] CAP (EGFP) and its constituent molecules (streptavidin-QD, SNAP 549, and Cet-RPE) were immobilized in culture dishes for microscopic observation. For QD, SNAP 549, and Cet-RPE, 100 μl of a 10.0 pM solution was incubated in PBS buffer for 15 minutes. EGFP was prepared by immobilizing sEV-CAP for 15 minutes.

[0242] Fluorescence trajectories of QD, SNAP 549, and EGFP were obtained using TIRF microscopy, with QD and SNAP 549 imaged using a 561 nm laser and EGFP imaged using a 488 nm laser. Time-lapse imaging was performed for 3 minutes with 100 ms exposures per frame, without intervals. Fluorescence intensity histograms were constructed from the collected trajectories to identify the major peaks corresponding to individual QD, SNAP 549, or EGFP molecules. Cet-RPE was imaged using a 561 nm laser and TIRF microscopy with snapshots taken at 100 ms exposures, and the average intensity of individual Cet-RPE molecules was determined by analyzing the fluorescence intensity histograms. Functional molecules on individual sEV-X molecules were quantified using the average fluorescence intensity values ​​of individual QD, SNAP 549, Cet-RPE, and EGFP. To ensure accurate fluorescence analysis of EGFP in individual sEV-CAPs, QD and Cet-RPE were imaged using 488 nm and 561 nm lasers. The fluorescence intensity under 488 nm laser was quantified relative to that under 561 nm laser, and this value was excluded in subsequent quantification experiments using EGFP.

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

[0245] Refer to Example 9. Linear regression analysis of CAP with functional molecule (X) in a single sEV-X.

[0246] In a single sEV-X, the number of CAPs and Xs was obtained from the quantitative analysis of Reference Example 8. Linear regression analysis was performed to plot the optimal line and derive the X slice. Furthermore, to assess the correlation between CAPs and functional molecules, 95% confidence intervals were calculated for both X and Y slices.

[0248] Refer to 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 molecules, the Pearson correlation coefficient (r) was calculated. This coefficient quantifies the degree of linearity between the two variables, ranging from -1 (perfectly negative correlation) to 1 (perfectly positive correlation), where 0 indicates no linear relationship. In colocalization analysis, x and y represent the fluorescence intensity of EGFP and X, respectively. In the quantitative analysis of a single sEV-X, x and y represent the number of CAP and X molecules, respectively. The correlation was calculated using the following formula.

[0250]

Mathematical Formula 1

[0251] Where x i With y i Representing a single sample point, the horizontal bars {x} and {y} are the average values ​​of the x and y samples, respectively.

[0253] Refer to Example 11. sEV and Dox cell uptake test

[0254] A549-Luc2 cells were aliquoted into fibronectin-coated culture dishes at a density of 5 × 10⁶ cells per well. 3Cell density. After 24 hours, cells were cultured with other treatments according to 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 minutes. Then, unconjugated BG-GLA-NHS was removed from the mixture using a 7 K MWCOzeba spin desalting column. The sEVs were then reacted overnight at room temperature with a 100-fold molar excess of BG-conjugated cetuximab (BG-Cet). For the sEV uptake assay, cells were treated with sEVs (3.7 × 10⁻⁶). 9 Particles (ml) -1 The cells were treated with sEVs. To analyze sEV-mediated Dox cell uptake, Dox (1 μg / ml) was used. -1 ), sEV (Dox) (1.1×10 10 Particles (ml) -1 ) or sEV(Dox)-Y(0.9×10 10 Particles (ml) -1 This treatment of cells is equivalent to a dose of Dox.

[0255] The green fluorescence of sEVs and the red fluorescence of Dox were observed using Epi-fluorescence microscopy. For sEV delivery experiments, time-lapse imaging was performed every 10 minutes for 1 hour after treatment. Subsequently, cells were washed with 1 ml PBS to remove extracellular sEVs, and intracellular sEVs were imaged to quantify delivery efficiency. The fluorescence trajectories of individual sEVs in the cells were also obtained. Time-lapse imaging was performed with each frame exposed for 100 ms for 3 minutes without intervals. In the Dox delivery experiment, cells were washed with 1 ml PBS for 2 hours after treatment to remove extracellular sEVs and Dox, and internalized sEVs and Dox were imaged. During imaging, the culture dishes were placed in an incubator on top of a preheated microscope stage, maintained at 37°C, 5% CO2, and 80% humidity.

[0257] Refer to Example 12. Reporter gene cell testing

[0258] Reporter gene assays were performed using HEK-Blue™ IL2 cells according to the manufacturer's instructions. Cells were cultured at 5 × 10⁶ cells per well. 4 The cells were seeded at a density of 100 μL in 96-well plates and incubated with IL-2 or sEV. After 24 hours, 20 μL of supernatant from each well was incubated with 180 μL of Quanti-Blue™ solution under high humidity conditions (37°C, 5% CO2). After 1 hour, the secreted alkaline phosphatase was quantified by measuring the absorbance at 630 nm.

[0260] Refer to Example 13. MTS test

[0261] A549-Luc2 cells were dispensed into 96-well plates at a density of 1 × 10⁶ cells per well. 4 Cell density was determined. After 24 hours, cells were cultured with sEVs loaded with Dox or Dox. Cell viability was analyzed using CellTiter 96® AQueous single-solution reagent. After adding 20 μl of reagent, the microplates were incubated under humidified conditions (37°C, 5% CO2) for 1 hour, and the absorbance was measured at 490 nm.

[0263] Refer to Example 14. Animal Experiments

[0264] All animal experiments were conducted in accordance with the Animal Experimentation Guidelines approved by the Institutional Animal Management and Use Committee (IACUC) of DGIST (Approval No. 23032701-0000). Six- to eight-week-old male BALB / c-nu mice (OrientBio) were used in this study. A549-Luc2 cells (1×10⁻⁶ cells in 100 μl PBS) were added. 7 A mixture of one cell and an equal volume of matrix gel (Corning) was injected subcutaneously into mice. Tumor volume (V) was expressed as V = length × width. 2 / 2 calculation.

[0265] To analyze the biodistribution of sEVs, when the tumor volume reached approximately 600 mm², 3 Mice were randomly divided into groups. sEVs labeled with PBS or DiR were injected intravenously via the tail vein (2 × 10⁻⁶ per mouse). 11 (Particles). Four hours after injection, mice were euthanized, and major organs (tumor, heart, lung, liver, spleen, kidney) were collected. Fluorescence signals were quantified using an in vivo imaging system (PerkinElmer, IVIS spectrum). To evaluate the therapeutic effect of sEVs loaded with Dox, tumors were collected when the tumor volume reached approximately 150-170 mm². 3 Mice were randomly divided into four groups and administered either a soluble form of Dox or an equivalent dose (1 mg / kg) encapsulated in sEVs. -1 The tumor was administered intravenously every three days for a total of six times. Before euthanasia, an in vivo imaging system was used to detect bioluminescent signals, and the tumor tissue was removed for weighing and tissue analysis.

[0267] Refer to Example 15. Immunofluorescence and Histological Analysis

[0268] In Example 14, tissues collected from mice were fixed with 4% paraformaldehyde, dehydrated in PBS with 30% sucrose, and then embedded in an OCT compound. Frozen tissues were sections 10 μm thick, and slides were washed with PBS. Apoptosis was detected using the TUNEL assay kit, following the manufacturer's instructions. Cell nuclei were stained with DAPI for 5 minutes at room temperature. After washing with PBS, samples were mounted using an antifluorescence quenching mounting medium. For hematoxylin-eosin staining, slides were stained with hematoxylin for 3 minutes and washed with sterile distilled water. Slides were then stained with eosin for 1 minute and washed with water. After dehydration in 95% and absolute ethanol, samples were mounted using the mounting medium.

[0270] Refer to Example 16. Statistical Analysis

[0271] All data are expressed as mean ± standard deviation of the indicated replicates. Statistical analyses were performed using GraphPad Prism 8.0 software, with two-tailed Student's t-tests and one-way ANOVA with post-hoc tests. Quantitative and linear regression analyses and Pearson correlation coefficients for the number of CAP and X values ​​for individual sEV-X were calculated using GraphPad Prism 8.0. ImageJ software was used to calculate the Pearson correlation coefficient between the fluorescence intensity of CAP (EGFP) and X, and to determine the diameter and TUNEL signal of sEVs. Data on Pearson correlation coefficients were analyzed from 10 individual images. TUNEL signal data for each mouse are expressed as the mean of five alternative portions.

[0273] Refer to Example 17. Experimental Materials

[0274] 1) Used 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 medium (Hyclone), Expi 293F TMExpression medium (Gibco), antibiotic-antifungal solution (A / A; GenDEPOT), insulin-transferrin-selenium (Gibco), hygromycin B (Gibco), puromycin (Invivogen), and Normocin (Invivogen), HEK-Blue. TM CLR screening reagent (Invivogen), lipofectamine® 2000 (Invitrogen), ExpiFectamine TM 293 Transfection Kit (Gibco).

[0277] 2) Used 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 plasma fibronectin (Sigma-Aldrich), ethanol (99%, Alfa Aesar).

[0280] 3) Used for sEV-CAP surface modification

[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), and dimethyl sulfoxide (DMSO; 99%, Sigma-Aldrich).

[0283] 4) Used for in vitro experiments

[0284] Cloning restriction endonucleases: BsmB I, EcoR I, Sfi I (Enzynomcis). T4 ligase (NEB). XL1-blue electroporation of competent Escherichia coli (Agilent), carbenicillin (Invitrogen), bleomycin (Zeocin) (Gibco). Sodium dihydrogen phosphate dihydrate (≥99%; Sigma-Aldrich), disodium hydrogen phosphate dihydrate (≥98.5%; Sigma-Aldrich), glycine (>99%; TCI America), hydrochloric acid (HCl; aqueous solution, 37%, Sigma-Aldrich), tris(hydroxymethyl)aminomethane (Tris; ≥99.8%, Sigma-Aldrich), doxorubicin (Dox; Biosynth), CellTiter 96® AQueous single-solution reagent (Promega), Quanti-Blue. TM Solution (Invivogen), Electrolysis buffer (Invitrogen), Neon TM Resuspension buffer R (Invitrogen), formalin solution (10%, neutral buffer; Sigma-Aldrich), glutaraldehyde (50% in H2O, Sigma-Aldrich), sodium dimethylarsenate 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).

[0286] 5) Antibodies

[0287] Anti-SNAP-tagged 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).

[0289] 6) Used in vivo ( in vivo )experiment

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

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

Claims

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

2. The chimeric adaptor protein according to claim 1, wherein, The O6-benzylguanine binding protein contains a self-labeled protein.

3. The chimeric adaptor protein according to claim 1, wherein, The O6-benzylguanine binding protein contains a SNAP-tag.

4. The chimeric adaptor protein according to claim 1, wherein, The transmembrane domain comprises any one or more transmembrane domains selected from the group consisting of: platelet-derived growth factor receptor, epidermal growth factor receptor, fibroblast growth factor receptor, vascular endothelial growth factor receptor, hepatocyte growth factor receptor, tropomyosin receptor kinase, insulin receptor, leukocyte receptor tyrosine kinase, angiopoietin receptor, cholecystokinin receptor, neurotrophic factor receptor, receptor tyrosine kinase-like orphan receptor, discoid domain receptor, transfection rearrangement receptor, tyrosine protein kinase-like receptor, receptor tyrosine kinase-associated receptor, muscle-specific kinase, CD63, CD9, and CD81.

5. The chimeric adaptor protein according to claim 1, wherein, The signal transduction domain includes any one or more signal transduction domains selected from the group consisting of: CD9, CD63, the C-terminus of CD63, CD81, LAMP-1, LAMP-2, synaptic fusion protein-3, polyligand-binding protein-1, polyligand-binding protein-2, polyligand-protein-1, polyligand-protein-4, and prostaglandin F2 receptor negative regulator.

6. The chimeric adaptor protein according to claim 1, wherein, The signal transduction domain includes the N-terminal sorting domain of a multiligand glycan-binding protein.

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

8. The chimeric adaptor protein according to claim 7, wherein, The connector includes a rigid connector.

9. The chimeric adaptor protein according to claim 1, further comprising a reporter gene.

10. The chimeric adaptor protein according to claim 1, comprising the amino acid sequence shown in SEQ ID NO:

1.

11. A construct comprising a gene encoding a chimeric adaptor protein as described in any one of claims 1 to 10.

12. A carrier comprising the construct of claim 11.

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

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

15. The extracellular vesicle according to claim 14, wherein, The chimeric adaptor protein is further linked with O6-benzylguanine; or further linked with O6-benzylguanine and a functional molecule.

16. The extracellular vesicle according to claim 15, wherein, The functional molecules are organic fluorescent substances, inorganic substances, antibodies or their antigen-binding fragments, or cytokines.

17. The extracellular vesicle according to claim 14, wherein, The extracellular vesicles contain drugs.

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