Interferon-induced transmembrane protein scaffold for engineering extracellular vesicles

Abstract

The present invention relates to an IFITM scaffold enabling high-efficiency engineering of extracellular vesicles. In the present invention, active substances to be expressed in extracellular vesicles are linked to the IFITM scaffold so that the active substances can be expressed in the vesicles at a high level. In particular, the active substance linked to the C-terminus of the IFITM scaffold is present on the surface of the vesicles to exhibit the intrinsic activity. In addition, the present invention provides a U-shaped IFITM scaffold, which enables two types of active substances linked to the N-terminus and the C-terminus of the IFITM scaffold to be present on the surface of extracellular vesicles at a 1:1 ratio.

Description

Interferon-induced transmembrane protein scaffold for extracellular vesicle engineering

[0001] The present invention relates to an interferon-induced transmembrane protein (IFITM) scaffold and the like, which can engineer extracellular vesicles with high efficiency.

[0002] Drug delivery systems (DDS) are used to ensure that therapeutic agents administered to the human body work effectively and to reduce side effects.

[0003] For example, antibodies, nanobodies, proteins, or polypeptides may lose efficacy or fail to be delivered to the target site when exposed to the in vivo environment. For instance, while targeted anticancer drugs are developed to act specifically on cancer cells, their therapeutic effect can be maximized if they can act more selectively on the cancerous tissue to be treated when administered into the human body.

[0004] Liposomes or micelles developed to date as drug delivery systems have increased drug retention time in vivo and improved pharmacokinetics, but they lack the ability to selectively target specific cells, such as immune cells or cancer cells. Furthermore, these existing drug delivery systems suffer from low biocompatibility and may cause problems due to unexpected side effects.

[0005] Meanwhile, recent studies have reported that the secretome contains various bioactive factors that regulate cell behavior, and in particular, since the secretome contains 'exosomes' or 'extracellular vesicles' that have intercellular signaling functions, research on their components and functions is actively underway.

[0006] Cells release various types of membrane vesicles into the extracellular environment, and these released vesicles are commonly referred to as extracellular vesicles (EVs). Extracellular vesicles are also called extracellular particles (EPs), small extracellular vesicles, large extracellular vesicles, artificial cell-derived vesicles (ACDVs), synthetic vesicles (SVs), cell membrane-derived vesicles, ectosomes, shedding vesicles, microvesicles, microparticles, and exosomes, and in some cases, they are used to distinguish them from exosomes.

[0007] Exosomes are vesicles ranging in size from tens to hundreds of nanometers that possess a double phospholipid membrane identical in structure to the cell membrane. They contain proteins, nucleic acids (DNA, mRNA, miRNA, etc.), and other materials referred to as exosome cargo. Exosome cargo contains a wide range of signaling factors, which are known to be specific to cell types and regulated differently depending on the environment of the secreting cell. As intercellular signaling mediators secreted by cells, exosomes are known to regulate cellular behaviors, including activation, proliferation, migration, differentiation, dedifferentiation, apoptosis, and necrosis of target cells, through which various cellular signals are transmitted. Exosomes contain genetic material and bioactive factors specific to the properties and state of the cell from which they originated. Stem cell-derived exosomes regulate cell behaviors such as migration, proliferation, and differentiation, and reflect the characteristics of stem cells related to tissue regeneration (Nature Review Immunology 2002 (2) 569-579).

[0008] In other words, exosomes, which are called avatars of cells, contain bioactive factors such as growth factors similar to cells and act as carriers that transport these bioactive factors between cells, that is, they serve as a means of communication between cells. Exosomes are known to be released not only from animal cells such as stem cells, immune cells, fibroblasts, and cancer cells, but also from the cells of various organisms such as plants, bacteria, fungi, and algae.

[0009] When such exosomes are used as drug carriers, they have the advantages of being biocompatible, allowing for repeated administration with low immunogenicity, and improving drug stability and absorption within the body. However, the method of passively loading drug cargoes into naturally occurring exosomes using hydrophobic properties has limitations, such as low manufacturing efficiency and inability to load sufficient drug cargoes into the exosomes.

[0010] Recently, a method has been proposed in which a target protein or peptide is fused to a transmembrane protein of an exosome during the gene construct fabrication stage, expressed within a cell, and the exosomes secreted or released into the cell culture medium are isolated to obtain exosomes loaded with the target protein or peptide (exosomes in which the target protein or peptide is fused to a transmembrane protein of an exosome) (see WO 2013 / 084000 A2; WO 2014 / 168548 A2).

[0011] However, just like other technical fields, the technical field to which this invention belongs also requires the continuous development of new technologies for effectively loading target proteins or peptides into exosomes and effectively delivering them to target sites for treatment.

[0012] The technical problem to be solved by the present invention is to provide an IFITM scaffold with increased engineering efficiency in an extracellular vesicle, a gene expression cassette encoding said IFITM scaffold, an IFITM expression vector comprising said expression cassette, a cell transformed with said vector, and a vesicle derived from said transformed cell.

[0013] In addition, the present invention aims to provide an IFITM scaffold capable of loading two types of active substances into an extracellular vesicle in a 1:1 ratio by constructing a U-shaped IFITM scaffold and connecting two types of active substances to the N-terminus and C-terminus of the scaffold, respectively, a gene expression cassette encoding the IFITM scaffold, an IFITM expression vector comprising the expression cassette, a cell transformed with the vector, and a vesicle derived from the transformed cell.

[0014] However, the technical problems that the present invention aims to solve are not limited to those mentioned above, and other unmentioned problems will be clearly understood by a person skilled in the art from the description below.

[0015] By providing the present invention, an active substance to be engineered into an extracellular vesicle can be connected to the IFITM backbone to load the active substance into the vesicle at a high level, and in particular, a type 2 transmembrane protein can be loaded into the extracellular vesicle to maintain its activity. Furthermore, the present invention can provide an extracellular vesicle that displays the active substance on the surface of the extracellular vesicle by connecting the active substance to the C-terminus of the IFITM backbone and exerts its inherent activity. Meanwhile, when an extracellular vesicle is engineered by connecting active substances exhibiting cell targeting ability and pharmacological activity to the C-terminus and N-terminus of the IFITM backbone of the present invention, respectively, it can be utilized as a target-specific drug delivery system. Moreover, the U-shaped IFITM backbone of the present invention can display two types of active substances connected to its N-terminus and C-terminus in a 1:1 ratio on the surface of the extracellular vesicle.

[0016] Figure 1 is an immunoblot result confirming the level of IFITM1 in extracellular vesicles derived from cells transformed with IFITM1 and its variant expression vectors.

[0017] Figure 2 is an immunoblot result confirming the level of IFITM3 in extracellular vesicles derived from cells transformed with an expression vector of an IFITM3 variant (IFITM3-mEGF) fused with IFITM3 and mature epidermal growth factor (mEGF).

[0018] Figure 3a is a schematic diagram of the structure of truncated EGF (tEGF) designed for comparison with a variant of IFITM3 (IFITM3-mEGF) fused with IFITM3 and mEGF.

[0019] Figure 3b is the result of an immunoblot confirming the levels of EGF in extracellular vesicles derived from cells transformed with the IFITM3-mEGF (lane 1) and tEGF (lane 2) expression vectors of Figure 3a.

[0020] Figure 3c is a graph showing the cell proliferation rate over time of 293FT cells treated with extracellular vesicles derived from cells transformed with IFITM3, its variant (IFITM3-mEGF), and tEGF expression vectors.

[0021] Figure 4a is a Western blot result confirming the level of IFITM3 in cells transiently transformed with an IFITM3 backbone expression vector fused with two types of LIGHT, Apelin, CAR-Apelin, or mEGF.

[0022] Figure 4b is a Western blot result confirming the levels of IFITM3 or EGF in cells stably transformed with an IFITM3 backbone expression vector fused with two types of LIGHT and Apelin, and in extracellular vesicles derived from said cells.

[0023] Figure 5 is a Western blot result confirming the expression levels of IFITM3 in cells and cell-derived extracellular vesicles transformed with IFITM3 and its variant (IFITM3-D1) expression vectors using a FLAG tag fused to each vector.

[0024] Figure 6 is a Western blot result confirming the level of IFITM3 in cells transformed with an expression vector of IFITM3 and its variant (IFITM3-mEGF) linked to an active molecule, and in extracellular vesicles derived from said transformed cells.

[0025] Figure 7 is a Western blot result confirming the level of IFITM3 in extracellular vesicles derived from cells transformed with CAR-Apelin-linked IFITM3 variant (IFITM-E2 and IFITM-E3) expression vectors and compared with IFITM3-mEGF.

[0026] Figure 8 is a Western blot result confirming the expression levels of mEGF and FLAG tags fused to the N-terminus and C-terminus, respectively, of the U-shaped IFITM backbone in cells transformed with a U-shaped IFITM backbone (mEGF-V6-sIFITM3) expression vector and extracellular vesicles derived from said cells.

[0027] Figure 9 is a Western blot result confirming the expression levels of mutant SIRPα (mSIRPα) and FLAG tag fused to the N-terminus and C-terminus, respectively, of the U-shaped IFITM backbone in cells transformed with a U-shaped IFITM backbone (mEGF-V9-sIFITM3) expression vector and extracellular vesicles derived from said cells.

[0028] It has been reported that IFITM (Interferon-Inducible Transmembrane Protein) increases cell permeability (Lou et al., Science 378, 1097-1104 (2022)). This suggests that it is possible to develop drugs that promote intracellular absorption by linking drugs to IFITM.

[0029] When using extracellular vesicles as drug delivery vehicles, IFITM present on the extracellular vesicle membrane can enhance the effect of endocytosis by modifying the characteristics of the cell membrane. Additionally, IFITM is a transmembrane protein that expresses its C-terminus on the surface of the extracellular vesicle, and has the advantage of presenting the C-terminus of the active molecule bound to the C-terminus of IFITM in a free state when the active molecule is a peptide or protein. Accordingly, when using IFITM in the engineering of extracellular vesicles, an active molecule having a cell-targeting function is attached to its C-terminus and an active molecule that performs function upon endocytosis is attached to its N-terminus, thereby providing an extracellular vesicle as a drug delivery vehicle with enhanced target specificity and endocytosis effects.

[0030] The inventors design IFITM and various variants of IFITM for efficient engineering of extracellular vesicles (EVs), and cells transformed with a vector for expressing said IFITM variants express said IFITM variants and, in particular, identify high levels of said IFITM variants in intracellular vesicles derived from said cells, thereby providing IFITM and said IFITM variants as scaffolds for engineering biologically active substances in extracellular vesicles.

[0031] Furthermore, the present invention designs a fusion protein in which IFITM3 is linked with EGF or an EGF variant, and cells transformed with a vector for expressing the fusion protein express the fusion protein, and confirmed high levels of expression of a biologically active substance fused with the fusion protein in vesicles derived from said cells. It was found that by fusing EGF or an EGF variant with the IFITM scaffold, higher expression in extracellular vesicles is possible compared to the existing IFITM scaffold. Accordingly, the present invention provides a fusion protein in which IFITM is linked with EGF or an EGF variant as a scaffold for effectively engineering a biologically active substance, and in said fusion protein, IFITM may be IFITM1, IFITM2, IFITM3, IFITM5, or IFITM10.

[0032] In addition, the present invention provides a U-shaped IFITM scaffold designed and fabricated using IFITM to enable the 1:1 initiation of two types of proteins intended for display on the surface of an extracellular vesicle.

[0033] The present invention provides a scaffold comprising the IFITM variant and a fusion protein utilizing IFITM, which enables the surface disclosure of a target protein in an extracellular vesicle, wherein the term "scaffold" is used interchangeably with "IFITM scaffold" in this specification. Furthermore, the present invention aims to provide an extracellular vesicle that discloses a biologically active substance on its surface by means of the scaffold.

[0034] In this specification, the biologically active substance is a biologically active molecule disclosed on the surface of an extracellular vesicle and is not limited to proteins, but may be a cargo such as a peptide, compound, DNA, or RNA that can be linked to the IFITM backbone. In the present invention, the biologically active substance is not limited to exhibiting pharmacological efficacy, provided that it exhibits activities such as targeting specific tissues or cells or increasing the efficiency of extracellular vesicle endocytosis. In this specification, the term biologically active substance may be used interchangeably with the terms target protein, target peptide, payload, active molecule, and active substance.

[0035] In the present invention, extracellular vesicles that disclose an active substance on their surface are referred to as surface-engineered extracellular vesicles.

[0036] In this specification, "extracellular vesicle" means a cell-derived vesicle comprising a membrane surrounding an internal space. Extracellular vesicles include all membrane-bound vesicles having a diameter smaller than that of the cell from which they originated. Generally, extracellular vesicles have a diameter in the range of 20 nm to 1000 nm and may contain various macromolecular cargoes in an internal space displayed on the outer surface of the extracellular vesicle and / or across the membrane. The cargoes may include small molecules, nucleic acids, proteins, carbohydrates, lipids, small molecules, and / or combinations thereof. For example, extracellular vesicles include, but are not limited to, apoptotic bodies, cell fragments, vesicles derived from cells by direct or indirect manipulation (e.g., continuous extrusion or alkaline solution treatment), vesiculated organelles, and vesicles generated from living cells (e.g., by direct plasma membrane budding or the fusion of late endosomes and plasma membranes). Extracellular vesicles may be derived from living or dead organisms, transplanted tissues or organs, and / or cultured cells.

[0037] In this specification, "exosome" means a cell-derived nanovesicle comprising a lipid bilayer membrane surrounding an internal space, which is generated from said cell by direct plasma membrane germination or the fusion of a late endosome with a plasma membrane. The exosome comprises lipids or fatty acids and polypeptides, and may optionally comprise a therapeutic active payload, a receiver (e.g., target moiety), a polynucleotide (e.g., RNA or DNA), a sugar (e.g., monosaccharide, polysaccharide, or glycan), or other molecules. The exosome may be derived from a producer cell and may be isolated from the producer cell based on its size, density, biochemical parameters, or a combination thereof. An exosome is a type of extracellular vesicle.

[0038] In this specification, “Surface-Engineered Extracellular Vesicle” means an extracellular vesicle having a membrane whose composition is modified. For example, the surface-engineered extracellular vesicle may have scaffold proteins or peptides on the surface of the extracellular vesicle at a higher (or lower) density than naturally occurring extracellular vesicles.

[0039] The surface-modified extracellular vesicles of the present invention may be produced from cells transformed or transfected with an exogenous sequence (e.g., a DNA structure encoding a scaffold protein or peptide).

[0040] In the present invention, the transformed cells for producing extracellular vesicles may be loaded with a payload having therapeutic efficacy. In this case, the loading of the payload within the extracellular vesicle may be initiated on the surface of the extracellular vesicle in a form in which the payload is connected to the C-terminus of the IFITM backbone of the present invention, loaded inside the extracellular vesicle by being connected to the N-terminus of the IFITM backbone, or loaded inside the extracellular vesicle separately from the IFITM backbone. To this end, the payload may be transduced into the cell as a gene encoding the payload within a gene cassette expressing the IFITM backbone of the present invention, or may be transduced into the cell as a gene encoding the payload within a gene cassette operating separately from the gene cassette expressing the IFITM backbone.

[0041] In the present invention, the IFITM backbone and the therapeutically active payload may be fusion proteins.

[0042] The surface-modified extracellular vesicles of the present invention may further comprise a targeting moiety that can be used to target a desired organ, tissue, or cell. Non-limiting embodiments of said targeting moiety include an antibody, an antigen-binding fragment of an antibody, an antigen-binding variant of an antibody, an antigen-binding fragment of an antigen-binding variant of an antibody, and an antigen-binding variant of an antigen-binding fragment of an antibody. said targeting moiety may be disclosed on the surface of the extracellular vesicle in a form connected to the C-terminus of the IFITM backbone, or disclosed on the surface of the extracellular vesicle separately from the IFITM.

[0043] In this specification, the term “variant” of a protein, peptide, or nucleic acid means a protein, peptide, or nucleic acid having at least one different amino acid or nucleotide compared to a wild-type protein, peptide, or nucleic acid. Variants of a protein, peptide, or nucleic acid include, but are not limited to, substitutions, deletions, frame shifts, or rearrangements of a protein, peptide, or nucleic acid. In this specification, the term variant may be used interchangeably with “mutant.”

[0044] In the present invention, transformation refers to the introduction of foreign DNA into a cell by transfection or transduction. Transduction may be performed by various methods known in the field, such as calcium phosphate-DNA co-precipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, electroshock, microinjection, liposome fusion, lipofectamine, and protoplast fusion. Additionally, transfection refers to the delivery of a gene into a cell using a virus or viral vector particle by means of infection. In this specification, transfection and transduction may be used interchangeably, and it is preferable to interpret both in a broad sense as transformation involving the delivery of a foreign gene to a host cell; a cell into which a foreign gene has been introduced by transfection or transduction is referred to as a transformant.

[0045] Meanwhile, transformation can be classified as stable transformation if the injected foreign gene is inserted into the host cell's chromosomal DNA or is capable of independent replication, and as transient transformation otherwise. In this invention, the term transformation is interpreted to encompass both stable transformation and transient transformation.

[0046] The present invention is capable of various modifications and may have various embodiments. Specific embodiments are illustrated in the drawings and described in detail in the detailed description below. However, this is not intended to limit the present invention to specific embodiments, and it should be understood that it includes all modifications, equivalents, and substitutions that fall within the spirit and scope of the present invention. In describing the present invention, detailed descriptions of related prior art are omitted if it is determined that such detailed descriptions may obscure the essence of the present invention.

[0047]

[0048] [Experimental Method]

[0049] 1. Cell culture and isolation of extracellular vesicles

[0050] 293FT cells were cultured in a CO2 incubator (37°C, 5% CO2) using Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS). To induce the expression of specific genes, the cells were subjected to transient transfection using a plasmid vector and a transforming agent, or stable cell lines were produced by infecting them with a recombinant retrovirus.

[0051] For transient transfection, cells were transfected using a transfection agent such as Lipofectamine 2000, Lipofectamine 3000, or polyethyleneimine (PEI). The cell medium was replaced with DMEM, and a mixture of DNA and infection agent was added to the cells. Then, the cells were cultured in a CO2 incubator (37°C, 5% CO2) for 24 hours. After 24 hours, the medium containing the infection agent and plasmid was removed and replaced with DMEM supplemented with 10% FBS, and the cells were cultured in a CO2 incubator (37°C, 5% CO2) for 24 hours. After 24 hours, the medium was replaced with DMEM medium supplemented with insulin-transferrin-selenium (Gibco) without serum, and the cells were cultured in a CO2 incubator (37°C, 5% CO2) for 48 hours.

[0052] To generate stable expression cells, Plat-E cells were used to produce retroviruses containing a retroviral vector that packages the DNA sequence of interest and the DNA sequence of the puromycin resistance gene. Specifically, Plat-E cells (2 x 10⁶ 6) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% FBS in a CO2 incubator (37°C, 5% CO2). When the cells reached 80–90% concentration, the cells were transformed using Lipofectamine 2000 with a retroviral vector encoding the DNA sequence of interest. After 24 hours, the culture medium was replaced with DMEM supplemented with 10% FBS and cultured for an additional 24 hours. Subsequently, the culture medium containing virus particles was collected, centrifuged at 3,000 rpm, and the supernatant was filtered through a 0.45 μm filter for use in infecting 293FT cells. After culturing 293FT cells in the virus-containing medium for 24 hours, they were cultured in a medium supplemented with puromycin and 10% FBS for at least 2 weeks to obtain cells that stably express a specific gene.

[0053] To isolate extracellular vesicles, the culture medium was collected after culturing the transformed cells in a serum-free medium for 48 hours. The culture medium was then centrifuged at 300 g for 10 minutes, 2000 g for 10 minutes, and 10,000 g for 30 minutes to obtain the supernatant. The supernatant was then filtered and centrifuged at 150,000 g for 3 hours. The extracellular vesicle pellet obtained from the centrifugation was resuspended in a phosphate-buffered saline (PBS) containing a protease inhibitor cocktail and stored at -20 ℃.

[0054]

[0055] 2. Design and Fabrication of the IFITM Framework

[0056] The amino acid sequences of wild-type IFITM1 and wild-type IFITM3 are as shown in Table 1 below.

[0057] Amino Acid Sequence (N-terminus → C-terminus) Sequence Number Wt IFITM1MHKEEHEVAV LGPPPSTILP RSTVINIHSE TSVPDHVVWS LFNTLFLNWC CLGFIAFAYS VKSRDRKMVG DVTGAQAYAS TAKCLNIWAL ILGILMTIGF ILLLVFGSVT VYHIMLQIIQ EKRGY1Wt IFITM3MNHTVQTFFS PVNSGQPPNY EMLKEEHEVA VLGAPHNPAP PTSTVIHIRS ETSVPDHVVW SLFNTLFMNP CCLGFIAFAY SVKSRDRKMV GDVTGAQAYA STAKCLNIWA LILGILMTIL LIVIPVLIFQ AYG2

[0058] 2-1. IFITM1 variant

[0059] The IFITM1 variant was constructed based on IFITM1-M0, in which the 13th amino acid of wt IFITM1 was substituted from P to A. IFITM1-M1 was constructed by deleting the “KRGY” at the C-terminus of IFITM1-M0. IFITM1-M2 was constructed by deleting the “H” at the N-terminus and the “KRGY” at the C-terminus of IFITM1-M0, and inserting “YEML” at the N-terminus. The specific amino acid sequences of the IFITM1 variants are shown in Table 2 below.

[0060] Amino Acid Sequence (N-terminus → C-terminus) Sequence Number IFITM1-M0MHKEEHEVAV LGAPPSTILP RSTVINIHSE TSVPDHVVWS LFNTLFLNWC CLGFIAFAYS VKSRDRKMVG DVTGAQAYAS TAKCLNIWAL ILGILMTIGF ILLLVFGSVT VYHIMLQIIQ EKRGY3IFITM1-M1MHKEEHEVAV LGAPPSTILP RSTVINIHSE TSVPDHVVWS LFNTLFLNWC CLGFIAFAYS VKSRDRKMVG DVTGAQAYAS TAKCLNIWAL ILGILMTIGF ILLLVFGSVT VYHIMLQIIQ E4IFITM1-M2MYEML KEEHEVAV LGAPPSTILP RSTVINIHSE TSVPDHVVWS LFNTLFLNWC CLGFIAFAYS VKSRDRKMVG DVTGAQAYAS TAKCLNIWAL ILGILMTIGF ILLLVFGSVT VYHIMLQIIQ E5

[0061]

[0062] 2-2. IFITM3 variant

[0063] A fusion protein (hereinafter referred to as 'IFITM3-mEGF') was designed by attaching mature EGF (mEGF) to the C-terminus of IFITM3. Truncated EGF (tEGF) was used to compare the efficacy of IFITM3-mEGF (Fig. 3a).

[0064] Meanwhile, a variant of IFITM3 was designed with the "MNHTVQTFFS PVNSGQPPNY E" sequence deleted at the N-terminal end of IFITM3, and this was named IFITM3-D1.

[0065] In addition, to produce variants of IFITM3-mEGF that were confirmed to improve engineering efficiency in extracellular vesicles, IFITM3-E2 and IFITM3-E3 were designed as variants by fusing IFITM3 with mEGF having a portion of the C-terminus deleted.

[0066] 아미노산 서열 (N말단→C말단)서열번호mEGF (E1)NSDSECPLSH DGYCLHDGVC MYIEALDKYA CNCVVGYIGE RCQYRDLKWW ELR6mEGF2 (E2)NSDSECPLSH DGYCLHDGVC MYIEALDKYA CN7mEGF3 (E3)NSDSECPLSH DGYCLHDGVC MYIEA8tEGFNSDSECPLSH DGYCLHDGVC MYIEALDKYA CNCVVGYIGE RCQYRDLKWW ELRVIVVAVC VVVLVMLLLL SLWGAHYYRT QKLLSKNPKN PYEESSRDVR SRRPADTEDG MSSCPQPWFV VIKEHQDLKN GGQPVAGEDG QAADGSMQPT SWRQEPQLCG MGTEQGCWIP VSSDKGSCPQ VMERSFHMPS YGTQTLEGGV EKPHSLLSAN PLWQQRALDP PHQMELTQ9IFITM3-D1MLKEEHEVA VLGAPHNPAP PTSTVIHIRS ETSVPDHVVW SLFNTLFMNP CCLGFIAFAY SVKSRDRKMV GDVTGAQAYA STAKCLNIWA LILGILMTIL LIVIPVLIFQ AYG10IFITM3-E2MNHTVQTFFS PVNSGQPPNY EMLKEEHEVA VLGAPHNPAP PTSTVIHIRS ETSVPDHVVW SLFNTLFMNP CCLGFIAFAY SVKSRDRKMV GDVTGAQAYA STAKCLNIWA LILGILMTIL LIVIPVLIFQ AYGEFNSDSE CPLSHDGYCL HDGVCMYIEA LDKYACN11IFITM3-E3MNHTVQTFFS PVNSGQPPNY EMLKEEHEVA VLGAPHNPAP PTSTVIHIRS ETSVPDHVVW SLFNTLFMNP CCLGFIAFAY SVKSRDRKMV GDVTGAQAYA STAKCLNIWA LILGILMTIL LIVIPVLIFQ AYGEFNSDSE CPLSHDGYCL HDGVCMYIEA12

[0067] The underlined parts in the sequence of tEGF in Table 3 above are the amino acid sequences for the transmembrane domain and cytosolic domain of EGF.

[0068] In IFITM3-E2 and IFITM3-E3 of Table 3 above, the underlined amino acids are those produced by the insertion of the restriction enzyme recognition site, and the parts in bold are mEGF2 (E2) and mEGF3 (E3).

[0069]

[0070] 2-3. IFITM framework fused with active molecules

[0071] To verify the activity of IFITM as a scaffold for engineering high levels of biologically active substances into extracellular vesicles, fusion proteins were constructed by linking biologically active molecules with wild-type IFITM3. As biologically active molecules, TNFSF14 (Tumor Necrosis Factor Superfamily Member 14, LIGHT), known to form tertial lymphoid structures (TLS) within the tumor microenvironment, Apelin, known for its therapeutic efficacy in pulmonary arterial hypertension, and EGF, which promotes cell growth, were used. Additionally, to detect the expression levels of the IFITM scaffold, a FLAG peptide was inserted after the first M (methionine) of IFITM. Each fusion protein is designated as "IFITM3-biologically active molecule".

[0072] Amino Acid Sequence (N-terminus → C-terminus) Sequence Number ApelinQRPRLSHKGP MPF13LIGHTv1HQRLG DIVAHLPDGG KGSWEKLIQD QRSHQANPAA HLTGANASLI GIGGPLLWET RLGLAFLRGL TYHDGALVTM EPGYYYVYSK VQLSGVGCPQ GLANGLPITH GLYKRTSRYP KELELLVSRR SPCGRANSSR VWWDSSFLGG VVHLEAGEEV VVRVPGNRLV RPRDGTRSYF GAFMV14LIGHTv2QRSHQANPAA HLTGANASLI GIGGPLLWET RLGLAFLRGL TYHDGALVTM EPGYYYVYSK VQLSGVGCPQ GLANGLPITH GLYKRTSRYP KELELLVSRR SPCGRANSSR VWWDSSFLGG VVHLEAGEEV VVRVPGNRLV RPRDGTRSYF GAFMV15CARCARSKNKDC16FLAGDYKDHDGDYK DHDIDYKDDD DK17

[0073]

[0074] Meanwhile, it is confirmed that IFITM3-mEGF functions as an IFITM scaffold capable of being highly engineered into extracellular vesicles. Accordingly, wild-type IFITM3 was replaced with IFITM3-mEGF as the scaffold, and a fusion protein was constructed in which a biologically active molecule was fused to the said scaffold.

[0075]

[0076] 2-5. U-shaped IFITM skeleton

[0077] To initiate two active substances into extracellular vesicles, a U-shaped IFITM scaffold was designed by fusing a portion of the IFITM3 sequence with a Type 1 transmembrane domain, ESM (V6 or V9). The U-shaped IFITM scaffold can initiate two active substances into the surface of extracellular vesicles in a 1:1 ratio.

[0078] Amino Acid Sequence (N-terminus → C-terminus) Sequence Number V6FKY PLLIGIGLST VIGLLSCLIG YCSS18V9DVLNAFKYPL LIGIGLSAVI GLLSCLIGYC SSHWC19sIFITM3SVKS RDRKMVGDVT GAQAYASTAK CLNIWALILG ILMTILLIVI PVLIFQAYG20mSIRPαEEELQIIQPD KSVLVAAGET ATLRCTITSL FPVGPIQWFR GAGPGRVLIY NQRQGPFPRV TTVSDTTKRN NMDFSIRIGN ITPADAGTYY CIKFRKGSPD DVEFKSGAGT ELSVRAKP21

[0079]

[0080] To construct a U-shaped IFITM scaffold, the sequence "MNHTVQTFFS PVNSGQPPNY EMLKEEHEVA VLGAPHNPAP PTSTVIHIRS ETSVPDHVVW SLFNTLFMNP CCLGFIAFAY" was deleted in the N-terminal direction of IFITM3 and named small IFITM3 (sIFITM3).

[0081] V6-sIFITM3v1, as a U-shaped IFITM backbone, was designed by attaching sIFITM3, which is a wild-type IFITM3 with some sequences deleted, to the C-terminus of the V6 or V9 domains indicated in bold in Table 5 above. The underlined amino acids are amino acids generated by the restriction enzyme recognition site in the expression vector.

[0082] To verify the efficiency of a U-shaped IFITM scaffold for engineering two types of biologically active substances into extracellular vesicles at high levels, a fusion protein with biologically active molecules linked to this scaffold was constructed. As for the biologically active molecules, Mutant signal-regulatory protein alpha (mSIRPα), which can enhance the phagocytic ability of macrophages in pathological cells, or EGF, which acts on cell proliferation, were used at the N-terminus, and a FLAG peptide was linked to the C-terminus to confirm extracellular exposure of the C-terminus.

[0083]

[0084] 2-6. Production of a Vector for IFITM Skeleton Expression

[0085] A plasmid vector for the expression of an active substance was constructed using the IFITM framework and framework described in experimental methods 2-1 to 2-5 above.

[0086] In the IFITM variant, the nucleotide sequences encoding each domain, namely the IFITM domain, EGF domain, V6 domain, V9 domain, and active molecule domain, are linked to restriction enzyme recognition site sequences, and the restriction enzyme recognition site sequences used were GGATCC, GAATTC, AGCGCT, or CTCGAG, which encode amino acids GS, EF, SA, or LE, respectively.

[0087] The genes inserted into the IFITM skeleton expression vector described above are shown in Table 6 below.

[0088] 염기서열 (5'→3')서열번호FLAG-IFITM1-M1ATGGACTACA AAGACCATGA CGGTGATTAT AAAGATCATG ACATCGATTA CAAGGATGAC GATGACAAGG GATCCCACAA GGAGGAACGAT GAGGCTGGGGGGG ACCCCCCAGC ACCATCCTTC CAAGGTCCAC CGTGATCAAC ATCCACAGCG AGACCTCCGT GCCCGACCAT GTCGTCTGGT CCCTGTTCAA CACCCTCTTC TTGAACTGGT GCTGTCTGGG CTTCATAGCA TTCGCCTACT CCGTGAAGTC TAGGGACAGG AAGGGGGCCGGCCG GCCTATGCCT CCACCGCCAA GTGCCTGAAC ATCTGGGCCC TGATTCTGGG CATCCTCATG ACCATTGGAT TCATCCTGTT ACTGGTATTC GGCTCTGTGA CAGTCTACCA TATTATGTTA CAGATAATAC AGGAATAG22FLAG-IFITM1-M2ATGGACTGAGACCATGATTGATT AAAGATCATG ACATCGATTA CAAGGATGAC GATGACAAGG GATCCTATGA GATGCTCAAG GAGGAACATG AGGTGGCTGT GCTGGGGGCA CCCCCCAGCA CCATCCTTCC AAGGTCCACC GTGATCAACA TCCACAGCGA GACCTCCGTG CCCGACCATCCTTGTCCCTTCCTT TGAACTGGTG CTGTCTGGGC TTCATAGCAT TCGCCTACTC CGTGAAGTCT AGGGACAGGA AGATGGTTGG CGACGTGACC GGGGCCCAGG CCTATGCCTC CACCGCCAAG TGCCTGAACA TCTGGGCCCT GATTCTGGGC ATCCTCATGA CCATTGATTGATTGTTGTTGTTGTT GCTCTGTGAC AGTCTACCAT ATTATGTTACAGTATACA GGAATAG23IFITM3-mEGFATGAATCACA CTGTCCAAAC CTTCTTCTCT CCTGTCAACA GTGGCCAGCC CCCCAACTAT GAGAGCTCA CGAGGTGGCT GTGCTGGGGG CGCCCCACAA CCCTGCTCCC CCGACGTCCA CCGTGATCCA CATCCGCAGC GAGACCTCCG TGCCCGACCA TGTCGTCTGG TCCCTGTTCA ACACCCTCTT CATGAACCCC TGCTGCCTGG GCTTCATAGC ATTCGCCTAC TCCGTGAAGT CTAGGGACAG GAAGATGGTT GGCGACGTGA CCGGGGCCCA GGCCTATGCC TCCACCGCCA AGTGCCTGAA CATCTGGGCC CTGATTCTGG GCATCCTCAT GACCATTCTG CTCATCGTCA TCCCAGTGCT GATCTTCCAG GCCTATGGAG AATTCAATAG TGACTCTGAA TGTCCCCTGT CCCACGATGG GTACTGCCTC CATGATGGTG TGTGCATGTA TATTGAAGCA TTGGACAAGT ATGCATGCAA CTGTGTTGTT GGCTACATCG GGGAGCGATG TCAGTACCGA GACCTGAAGT GGTGGGAACT GCGCTGA24IFITM3-LIGHTv1ATGAATCACA CTGTCCAAAC CTTCTTCTCT CCTGTCAACA GTGGCCAGCC CCCCAACTAT GAGATGCTCA AGGAGGACCA CGAGGTGGCT GTGCTGGGGG CGCCCCACAA CCCTGCTCCC CCGACGTCCA CCGTGATCCA CATCCGCAGC GAGACCTCCG TGCCCGACCA TGTCGTCTGG TCCCTGTTCA ACACCCTCTT CATGAACCCC TGCTGCCTGG GCTTCATAGC ATTCGCCTAC TCCGTGAAGT CTAGGGACAG GAAGATGGTT GGCGACGTGA CCGGGGCCCA GGCCTATGCCTCCACCGCCA AGTGCCTGAA CATCTGGGCC CTGATTCTGG GCATCCTCAT GACCATTCTG CTCATCGTCA TCCCAGTGCT GATCTTCCAG GCCTATGGAG AATTCCATCA ACGTCTTGGA GACATAGTAG CTCATCTGCC AGATGGAGGC AAAGGCTCCT GGGAGAAGCT GATACAAGAT CAACGATCTC ACCAGGCCAA CCCAGCAGCA CATCTTACAG GAGCCAACGC CAGCTTGATA GGTATTGGTG GACCTCTGTT ATGGGAGACA CGACTTGGCC TGGCCTTCTT GAGGGGCTTG ACGTATCATG ATGGGGCCCT GGTGACCATG GAGCCCGGTT ACTACTATGT GTACTCCAAA GTGCAGCTGA GCGGCGTGGG CTGCCCCCAG GGGCTGGCCA ATGGCCTCCC CATCACCCAT GGACTATACA AGCGCACATC CCGCTACCCG AAGGAGTTAG AACTGCTGGT CAGTCGGCGG TCACCCTGTG GCCGGGCCAA CAGCTCCCGA GTCTGGTGGG ACAGCAGCTT CCTGGGCGGC GTGGTACATC TGGAGGCTGG GGAAGAGGTG GTGGTCCGCG TGCCTGGAAA CCGCCTGGTC AGACCACGTG ACGGCACCAG GTCCTATTTC GGAGCTTTCA TGGTCTGA25IFITM3-LIGHTv2ATGAATCACA CTGTCCAAAC CTTCTTCTCT CCTGTCAACA GTGGCCAGCC CCCCAACTAT GAGATGCTCA AGGAGGAGCA CGAGGTGGCT GTGCTGGGGG CGCCCCACAA CCCTGCTCCC CCGACGTCCA CCGTGATCCA CATCCGCAGC GAGACCTCCG TGCCCGACCA TGTCGTCTGG TCCCTGTTCA ACACCCTCTT CATGAACCCC TGCTGCCTGG GCTTCATAGC ATTCGCCTACTCCGTGAAGT CTAGGGACAG GAAGATGGTT GGCGACGTGA CCGGGGCCCA GGCCTATGCC TCCACCGCCA AGTGCCTGAA CATCTGGGCC CTGATTCTGG GCATCCTCAT GACCATTCTG CTCATCGTCA TCCCAGTGCT GATCTTCCAG GCCTATGGAG AATTCCAACG ATCTCACCAG GCCAACCCAG CAGCACATCT TACAGGAGCC AACGCCAGCT TGATAGGTAT TGGTGGACCT CTGTTATGGG AGACACGACT TGGCCTGGCC TTCTTGAGGG GCTTGACGTA TCATGATGGG GCCCTGGTGA CCATGGAGCC CGGTTACTAC TATGTGTACT CCAAAGTGCA GCTGAGCGGC GTGGGCTGCC CCCAGGGGCT GGCCAATGGC CTCCCCATCA CCCATGGACT ATACAAGCGC ACATCCCGCT ACCCGAAGGA GTTAGAACTG CTGGTCAGTC GGCGGTCACC CTGTGGCCGG GCCAACAGCT CCCGAGTCTG GTGGGACAGC AGCTTCCTGG GCGGCGTGGT ACATCTGGAG GCTGGGGAAG AGGTGGTGGT CCGCGTGCCT GGAAACCGCC TGGTCAGACC ACGTGACGGC ACCAGGTCCT ATTTCGGAGC TTTCATGGTC TGA26IFITM3-CAR-Apelin13ATGAATCACA CTGTCCAAAC CTTCTTCTCT CCTGTCAACA GTGGCCAGCC CCCCAACTAT GAGATGCTCA AGGAGGAGCA CGAGGTGGCT GTGCTGGGGG CGCCCCACAA CCCTGCTCCC CCGACGTCCA CCGTGATCCA CATCCGCAGC GAGACCTCCG TGCCCGACCA TGTCGTCTGG TCCCTGTTCA ACACCCTCTT CATGAACCCC TGCTGCCTGG GCTTCATAGC ATTCGCCTAC TCCGTGAAGTCTAGGGACAG GAAGATGGTT GGCGACGTGA CCGGGGCCCA GGCCTATGCC TCCACCGCCA AGTGCCTGAA CATCTGGGCC CTGATTCTGG GCATCCTCAT GACCATTCTG CTCATCGGTCA TCCCAGTGCT GATCTTCCAG GCCTATGGAG AATTCTGTGC TAGAAGTAAG AACAAGGATT GTGAATTCCA GCGGCCCCGC CTCTCCCATA AGGGACCCAT GCCTTTCTGA27IFITM3-Apelin13ATGAATCACA CTGTCCAAAC CTTCTTCTCT CCTGTCAACA GTGGCCAGCC CCCCAACTAT GAGATGCTCA AGGAGGAGCA CGAGGTGGCT GTGCTGGGGG CGCCCCACAA CCCTGCTCCC CCGACGTCCA CCGTGATCCA CATCCGCAGC GAGACCTCCG TGCCCGACCA TGTCGTCTGG TCCCTGTTCA ACACCCTCTT CATGAACCCC TGCTGCCTGG GCTTCATAGC ATTCGCCTAC TCCGTGAAGT CTAGGGACAG GAAGATGGTT GGCGACGTGA CCGGGGCCCA GGCCTATGCC TCCACCGCCA AGTGCCTGAA CATCTGGGCC CTGATTCTGG GCATCCTCAT GACCATTCTG CTCATCGTCA TCCCAGTGCT GATCTTCCAG GCCTATGGAG AATTCCAGCG GCCCCGCCTC TCCCATAAGG GACCCATGCC TTTCTGA28FLAG-IFITM3-D1ATGGACTACA AAGACCATGA CGGTGATTAT AAAGATCATG ACATCGATTA CAAGGATGAC GATGACAAGG GATCCCTCAA GGAGGAGCAC GAGGTGGCTG TGCTGGGGGC GCCCCACAAC CCTGCTCCCC CGACGTCCAC CGTGATCCAC ATCCGCAGCG AGACCTCCGT GCCCGACCAT GTCGTCTGGT CCCTGTTCAACACCCTCTTC ATGAACCCCT GCTGCCTGGG CTTCATAGCA TTCGCCTACT CCGTGAAGTC TAGGGACAGG AAGATGGTTG GCGACGTGAC CGGGGCCCAG GCCTATGCCT CCACCGCCAA GTGCCTGAAC ATCTGGGCCC TGATTCTGGG CATCCTCATG ACCATTCTGC TCATCGTCAT CCCAGTGCTG ATCTTCCAGG CCTATGGATA G29IFITM3-mEGF-CAR-Apelin13ATGAATCACA CTGTCCAAAC CTTCTTCTCT CCTGTCAACA GTGGCCAGCC CCCCAACTAT GAGATGCTCA AGGAGGAGCA CGAGGTGGCT GTGCTGGGGG CGCCCCACAA CCCTGCTCCC CCGACGTCCA CCGTGATCCA CATCCGCAGC GAGACCTCCG TGCCCGACCA TGTCGTCTGG TCCCTGTTCA ACACCCTCTT CATGAACCCC TGCTGCCTGG GCTTCATAGC ATTCGCCTAC TCCGTGAAGT CTAGGGACAG GAAGATGGTT GGCGACGTGA CCGGGGCCCA GGCCTATGCC TCCACCGCCA AGTGCCTGAA CATCTGGGCC CTGATTCTGG GCATCCTCAT GACCATTCTG CTCATCGTCA TCCCAGTGCT GATCTTCCAG GCCTATGGAG AATTCAATAG TGACTCTGAA TGTCCCCTGT CCCACGATGG GTACTGCCTC CATGATGGTG TGTGCATGTA TATTGAAGCA TTGGACAAGT ATGCATGCAA CTGTGTTGTT GGCTACATCG GGGAGCGATG TCAGTACCGA GACCTGAAGT GGTGGGAACT GCGCCTCGAG TGTGCTAGAA GTAAGAACAA GGATTGTCAG CGGCCCCGCC TCTCCCATAA GGGACCCATG CCTTTCTGA30IFITM3-mEGF-LIGHTv3ATGAATCACACTGTCCAAAC CTTCTTCTCT CCTGTCAACA GTGGCCAGCC CCCCAACTAT GAGATGCTCA AGGAGGACCA CGAGGTGGCT GTGCTGGGGG CGCCCCACAA CCCTGCTCCC CCGACGTCCA CCGTGATCCA CATCCGCAGC GAGACCTCCG TGCCCGACCA TGTCGTCTGG TCCCTGTTCA ACACCCTCTT CATGAACCCC TGCTGCCTGG GCTTCATAGC ATTCGCCTAC TCCGTGAAGT CTAGGGACAG GAAGATGGTT GGCGACGTGA CCGGGGCCCA GGCCTATGCC TCCACCGCCA AGTGCCTGAA CATCTGGGCC CTGATTCTGG GCATCCTCAT GACCATTCTG CTCATCGTCA TCCCAGTGCT GATCTTCCAG GCCTATGGAG AATTCAATAG TGACTCTGAA TGTCCCCTGT CCCACGATGG GTACTGCCTC CATGATGGTG TGTGCATGTA TATTGAAGCA TTGGACAAGT ATGCATGCAA CTGTGTTGTT GGCTACATCG GGGAGGATG TCAGTACCGA GACCTGAAGT GGTGGGAACT GCGCCTCGAG CCAGCAGCAC ATCTTACAGG AGCCAACGCC AGCTTGATAG GTATTGGTGG ACCTCTGTTA TGGAGACAC GACTTGGCCT GGCCTTCTTG AGGGCTTGA CGTATCATGA TGGGGCCCTG GTGACCATGG AGCCCGGTTA CTACTATGTG TACTCCAAAG TGCAGCTGAG CGGCGTGGGC TGCCCCCAGG GGCTGGCCAA TGGCCTCCCC ATCACCCATG GACTATACAA GCGCACATCC CGCTACCCGA TAKE ACTGCTGGTC AGTCGGCGGT CACCCTGTGG CCGGGCCAAC AGCTCCCGAG TCTGGTGGGA CAGCAGCTTC CTGGGCGGCG TGGTACATCTGGAGGCTGGG GAAGAGGTGG TGGTCCGCGT GCCTGGAAAC CGCCTGGTCA GACCACGTGA CGGCACCAGG TCCTATTTCG GAGCTTTCAT GGTCTGA31IFITM3-mEGF-Apelin13ATGAATCACA CTGTCCAAAC CTTCTTCTCT CCTGTCAACA GTGGCCAGCC CCCCAACTAT GAGATGCTCA AGGAGGACCA CGAGGTGGCT GTGCTGGGGG CGCCCCACAA CCCTGCTCCC CCGACGTCCA CCGTGATCCA CATCCGCAGC GAGACCTCCG TGCCCGACCA TGTCGTCTGG TCCCTGTTCA ACACCCTCTT CATGAACCCC TGCTGCCTGG GCTTCATAGC ATTCGCCTAC TCCGTGAAGT CTAGGGACAG GAAGATGGTT GGCGACGTGA CCGGGGCCCA GGCCTATGCC TCCACCGCCA AGTGCCTGAA CATCTGGGCC CTGATTCTGG GCATCCTCAT GACCATTCTG CTCATCGTCA TCCCAGTGCT GATCTTCCAG GCCTATGGAG AATTCAATAG TGACTCTGAA TGTCCCCTGT CCCACGATGG GTACTGCCTC CATGATGGTG TGTGCATGTA TATTGAAGCA TTGGACAAGT ATGCATGCAA CTGTGTTGTT GGCTACATCG GGGAGCGATG TCAGTACCGA GACCTGAAGT GGTGGGAACT GCGCCTCGAG CAGCGGCCCC GCCTCTCCCA TAAGGGACCC ATGCCTTTCT GA32IFITM3-E2-CAR-Apelin13ATGAATCACA CTGTCCAAAC CTTCTTCTCT CCTGTCAACA GTGGCCAGCC CCCCAACTAT GAGATGCTCA AGGAGGTGGCT GTGCTGGGGG CGCCCCACAA CCCTGCTCCC CCGACGTCCA CCGTGATCCA CATCCGCAGC GAGACCTCCGTGCCCGACCA TGTCGTCTGG TCCCTGTTCA ACACCCTCTT CATGAACCCC TGCTGCCTGG GCTTCATAGC ATTCGCCTAC TCCGTGAAGT CTAGGGACAG GAAGATGGTT GGCGACGTGA CCGGGGCCCA GGCCTATGCC TCCACCGCCA AGTGCCTGAA CATCTGGGCC CTGATTCTGG GCATCCTCAT GACCATTCTG CTCATCGTCA TCCCAGTGCT GATCTTCCAG GCCTATGGAG AATTCAATAG TGACTCTGAA TGTCCCCTGT CCCACGATGG GTACTGCCTC CATGATGGTG TGTGCATGTA TATTGAAGCA TTGGACAAGT ATGCATGCAA CCTCGAGTGT GCTAGAAGTA AGAACAAGGA TTGTCAGCGG CCCCGCCTCT CCCATAAGGG ACCCATGCCT TTCTGA33IFITM3-E3-CAR-Apelin13ATGAATCACA CTGTCCAAAC CTTCTTCTCT CCTGTCAACA GTGGCCAGCC CCCCAACTAT GAGATGCTCA AGGAGGAGCA CGAGGTGGCT GTGCTGGGGG CGCCCCACAA CCCTGCTCCC CCGACGTCCA CCGTGATCCA CATCCGCAGC GAGACCTCCG TGCCCGACCA TGTCGTCTGG TCCCTGTTCA ACACCCTCTT CATGAACCCC TGCTGCCTGG GCTTCATAGC ATTCGCCTAC TCCGTGAAGT CTAGGGACAG GAAGATGGTT GGCGACGTGA CCGGGGCCCA GGCCTATGCC TCCACCGCCA AGTGCCTGAA CATCTGGGCC CTGATTCTGG GCATCCTCAT GACCATTCTG CTCATCGTCA TCCCAGTGCT GATCTTCCAG GCCTATGGAG AATTCAATAG TGACTCTGAA TGTCCCCTGT CCCACGATGG GTACTGCCTC CATGATGGTG TGTGCATGTATATTGAAGCA CTCGAGTGTG CTAGAAGTAA GAACAAGGAT TGTCAGCGGC CCCGCCTCTC CCATAAGGGA CCCATGCCTT TCTGA34mEGF-V6-sIFITM3-FLAGATGCTGCTCA CTCTTATCAT TCTGTTGCCA GTAGTTTCAA AATTTAGTTT TGTTAGTCTC TCAGCAAATA GTGACTCTGA ATGTCCCCTG TCCCACGATG GGTACTGCCT CCATGATGGT GTGTGCATGT ATATTGAAGC ATTGGACAAG TATGCATGCA ACTGTGTTGT TGGCTACATC GGGGAGCGAT GTCAGTACCG AGACCTGAAG TGGTGGGAAC TGCGCAGCGC TTTCAAGTAT CCCTTGCTGA TCGGCATCGG TCTGTCCACG GTCATCGGGC TCCTGTCCTG TCTCATCGGG TACTGCAGCT CCGAATTCTC CGTGAAGTCT AGGGACAGGA AGATGGTTGG CGACGTGACC GGGGCCCAGG CCTATGCCTC CACCGCCAAG TGCCTGAACA TCTGGGCCCT GATTCTGGGC ATCCTCATGA CCATTCTGCT CATCGTCATC CCAGTGCTGA TCTTCCAGGC CTATGGACTC GAGGACTACA AAGACCATGA CGGTGATTAT AAAGATCATG ACATCGATTA CAAGGATGAC GATGACAAGT GA35mSIRPA-V9-sIFITM3-FLAGATGGAGCCTG CCGGCCCCGC CCCCGGTAGA CTGGGCCCAC TGCTATGTCT GCTGCTGGCC GCTAGCTGCG CATGGTCCGG CGTAGCCGGC GAGGAGGAGC TGCAGATCAT TCAGCCCGAC AAGAGCGTGC TGGTGGCCGC CGGCGAGACC GCCACCCTGA GATGCACCAT CACAAGCCTG TTCCCCGTGG GCCCCATTCA GTGGTTCAGA GGCGCCGGCC CCGGCAGAGTGCTGATCTAC AATCAGAGAC AAGGCCCCTT CCCTAGAGTG ACCACCGTGA GCGACACCAC CAAGAGAAAC AACATGGACT TCAGCATCAG AATCGGCAAC ATCACCCCCG CCGACGCCGG CACCTACTAC TGCATCAAGT TCAGAAAGGG CAGCCCCGAC GACGTGGAGT TCAAGAGCGG CGCCGGCACC GAGCTGAGCG TGAGAGCCAA GCCCGGATCC GATGTACTCA ACGCCTTCAA GTATCCCCTC CTTATCGGCA TAGGGCTGAG CGCCGTGATT GGTTTGCTCT CATGCCTGAT AGGCTACTGT TCAAGTCACT GGTGTGAATT CTCCGTGAAG TCTAGGGACA GGAAGATGGT TGGCGACGTG ACCGGGGGCCC AGGCCTATGC CTCCACCGCC AAGTGCCTGA ACATCTGGGC CCTGATTCTG GGCATCCTCA TGACCATTCT GCTCATCGTC ATCCCAGTGC TGATCTTCCA GGCCTATGGA CTCGAGGACT ACAAAGACCA TGACGGTGAT TATAAAAGATC ATGACATCGA TTACAAGGAT GACGATGACA AGTGA36

[0089]

[0090] 3. Measurement of extracellular protein levels

[0091] The total amount of protein present in extracellular vesicles was measured using the bicinchoninic acid (BCA) protein assay or the Bradford protein assay.

[0092] For the analysis of vicinic acid protein, standard solutions were prepared, and 5 μl of bovine serum albumin at each concentration was applied to 96-well plates (2, 1, 0.5, 0.25, 0.125, and 0 mg / ml). Extracellular vesicle samples were diluted with PBS, and 5 μl was applied to the 96-well plates. Reagents A (500113, Bio-Rad) and S (500114, Bio-Rad) were mixed in a ratio of 50 to 1, and 25 μl of the reagent mixture was applied to the 96-well plates. 200 μl of reagent B (500115, Bio-Rad) was applied to the 96-well plates, and the plates were gently tapped. The samples were incubated in the dark for 15 minutes. Protein levels were measured using a microplate reader at a wavelength of 750 nm.

[0093] For Bradford protein analysis, standard solutions were prepared, and bovine serum albumin of each concentration was applied to E-tubes (10, 8, 6, 4, 2, and 0 μg / ml). 10 μl of the extracellular vesicle sample was mixed with 150 μl of PBS, and then 40 μl of Bradford reagent (500113, Bio-Rad) was added and mixed. Protein levels were measured using a microplate reader at a wavelength of 595 nm.

[0094]

[0095] 4. SDS-Polyacrylamide (SDS-PAGE) Electrophoresis and Western Blot

[0096] Transfused cells and purified extracellular vesicles were lysed by adding them to a lysis buffer containing a protease inhibitor cocktail (Calbiochem), and then mixed with SDS-PAGE sample buffer. SDS-PAGE was performed with a predetermined amount of cell and extracellular vesicle proteins for each experiment. After electrophoresis, the proteins present on the gel were transferred to a nitrocellulose membrane or a methanol-activated polyvinylidene difluoride (PVDF) membrane. Subsequently, the membranes were blocked for 90 minutes at room temperature with 5% skim milk dissolved in Tris-Buffered Saline (TBST) containing Tween-20, and then the membranes were incubated with the primary antibody at 4°C for 16 hours. To detect protein expression, CD81 (Santa Cruz Biotechnology, sc-166029), IFITM1 (Cell signaling, 13126S), IFITM3 (Abcam, ab15592; Sigma-Aldrich, HPA004337), EGF (Abcam, ab9695), Apelin (Abcam, ab125213), Actin (Sigma-Aldrich, A2066), and SIRPα (R&D System, MAB4546) antibodies were used as primary antibodies. After incubating with the primary antibodies, the membrane was washed at least three times with PBS, and then incubated for 2 hours with a secondary antibody conjugated with horseradish peroxidase (HRP) (DAKO, P0447; DAKO, P0448). For the FLAG antibody, an antibody conjugated with HRP was used (Sigma-Aldrich, A8592). Afterward, the membrane was washed at least three times using PBS, and then the blot was analyzed using an ECL solution in a Chemi-Doc imaging system (Bio-Rad).

[0097]

[0098] [Experimental Results]

[0099] Example 1. Derivation of an IFITM1 variant with excellent extracellular vesicle engineering efficiency

[0100] Cells were transformed and cultured with IFITM1 (P13A), IFITM1-M1, or IFITM1-M2 expression vectors. Protein immunoblotting was performed after SDS-PAGE using 10 μg and 0.5 μg of protein, respectively, of the cells and vesicles derived from the cells.

[0101] As a result, very low levels of IFITM1 were observed in vesicles derived from cells transformed with an IFITM1 (P13A) expression vector compared to vesicles derived from cells transformed with IFITM1-M1 or IFITM1-M2 expression vectors. From the above, it can be seen that the intracellularly engineered IFITM1-M1 and IFITM1-M2 variants are effectively expressed in extracellular vesicles. Furthermore, among the IFITM1 variants, it was confirmed that IFITM1-M1 can engineer extracellular vesicles to a higher level compared to IFITM1-M2 (Fig. 1).

[0102]

[0103] Example 2. Confirmation of IFITM3-mEGF expression levels in extracellular vesicles

[0104] A vector capable of expressing IFITM3-mEGF, in which mEGF is fused to the C-terminus of IFITM3, was transformed into cells through viral infection. SDS-PAGE and Western blot were performed on vesicles derived from the above cells and virus-transfected cells at amounts of 10 μg and 1 μg, respectively, based on protein content.

[0105] Figure 2 shows the immunoblot results. Lane 1 in Figure 2 shows the results regarding EGF expression in cells virally transduced with the IFITM3-mEGF expression vector. Thus, it was found that successful expression of IFITM3-mEGF was achieved within the cells. Meanwhile, lane 2 in Figure 2 shows the isolation of extracellular vesicles derived from IFITM3-mEGF-expressing cells and the confirmation of EGF levels, indicating that IFITM3-mEGF is expressed not only within the cells but also at high levels in extracellular vesicles.

[0106]

[0107] Example 3. Expression of a fusion protein in which an active molecule is linked to IFITM3 in an extracellular vesicle

[0108] 3-1. Measurement of the activity of biologically active substances loaded in extracellular vesicles

[0109] In Example 2, it was confirmed that the IFITM3-mEGF scaffold was loaded into extracellular vesicles at a high level. At this time, to confirm whether the loaded IFITM3-mEGF exhibited biological activity of EGF, 20 μg / ml of extracellular vesicles derived from cells transformed with an IFITM3-mEGF expression vector were added to 293FT cells cultured in DMEM medium (4.5 g / L glucose, FBS 5%), and cell proliferation was measured using a proliferation assay kit (Promega) after 3 days. For comparison, vesicles derived from cells transformed with IFITM3 or tEGF expression vectors were used.

[0110] As a result, it was confirmed that the IFITM3-mEGF fusion protein is loaded into extracellular vesicles at a high level, and the simultaneously loaded EGF effectively exerts its intrinsic activity (Figs. 3a, 3b, and 3c).

[0111]

[0112] 3-2. Confirmation of the Potential of the IFITM3 Backbone for Type 2 Membrane-Penetrating Protein Engineering

[0113] LIGHT and Apelin are both type 2 transmembrane proteins. In the case of Apelin, the C-terminus must be structurally free to exhibit intrinsic activity.

[0114] Accordingly, vectors expressing IFITM3-LIGHT (two types of LIGHT fragments; LIGHTv1, LIGHTv2), IFITM3-Apelin (two types of Apelin; Apelin, CAR-Apelin), and IFITM3-mEGF were constructed and transiently transformed into cells or virally transformed, and Western blot was performed using SDS-PAGE based on the protein amounts indicated in the drawing to confirm the expression levels in the cells and the levels in vesicles derived from the transformed cells.

[0115] As a result, expression of IFITM3-bound Cargo was confirmed in cells transiently transformed with an IFITM3-based active molecule expression vector, but expression was at very low levels except for IFITM3-mEGF and IFITM3-Apelin (Fig. 4a). When the expression of IFITM3-based fusion proteins was confirmed in virus-transformed cells and vesicles derived from said transformed cells, the expression of IFITM3-LIGHT and IFITM3-Apelin in vesicles derived from said transformed cells was at very low levels compared to IFITM3-mEGF (Fig. 2) (Fig. 4b).

[0116]

[0117] Example 4. IFITM3 variant scaffold with superior extracellular vesicle engineering efficiency compared to wild-type IFITM3

[0118] In Example 3, a variant of IFITM3 was designed as an IFITM scaffold to load active molecules into extracellular vesicles, and the level of its expression in extracellular vesicles was to be verified. Accordingly, a vector expressing the IFITM3 variant was constructed using the same method as in the previous examples, and after transforming it into cells, the level of the IFITM3 variant in extracellular vesicles was verified. The IFITM3 variant is an IFITM3 variant in which the antibody binding site of the IFITM3 antibody is deleted from wild-type IFITM3, and therefore cannot be detected by the IFITM3 antibody. Accordingly, the expression of the IFITM3 variant was verified by inserting a Flag tag sequence into the IFITM3 variant. As a result, it was confirmed that IFITM3-D1, in which a portion of the wild-type IFITM3 sequence was cut, was effectively expressed in extracellular vesicles (Fig. 5).

[0119]

[0120] Example 5. IFITM3-mEGF scaffold with superior extracellular vesicle engineering efficiency compared to wild-type IFITM3

[0121] In Example 3, it was confirmed that the active molecules (LIGHT, Apelin) fused to wild-type IFITM3 were not effectively expressed in extracellular vesicles. Accordingly, a vector for expressing a fusion protein was constructed in which an active molecule was linked to an IFITM3-based fusion protein (IFITM3-mEGF) with improved loading efficiency into extracellular vesicles as in Example 2, and transformed cells were produced to check the levels of each fusion protein in the transformed cells and in vesicles derived from the transformed cells. As a result, it was confirmed that IFITM3-mEGF functions as a scaffold capable of expressing the active molecule in extracellular vesicles at a higher level compared to wild-type IFITM3 (Fig. 6).

[0122]

[0123] Example 6. IFITM3-E2 / E3 scaffold with superior extracellular vesicle engineering efficiency compared to wild-type IFITM3

[0124] IFITM3-mEGF, which was confirmed to have an effect of improving the engineering efficiency of extracellular vesicles, is a form in which mEGF with EGF activity is fused with IFITM3. To eliminate the growth factor efficacy of EGF, IFITM3-mEGF variants (IFITM3-E2 and IFITM3-E3) were designed by fusing only a portion of mEGF with IFITM3. Vectors expressing the above IFITM3-mEGF, IFITM3-E2, and IFITM3-E3 were constructed and transformed into cells, and the levels of each variant were checked in the cells and vesicles derived from the transformed cells. As a result, it was found that IFITM3-E2 and IFITM3-E3 could engineer extracellular vesicles to a level similar to that of IFITM3-mEGF (Fig. 7).

[0125]

[0126] Example 7. IFITM-based U-shaped scaffold for loading two active substances into extracellular vesicles

[0127] Meanwhile, to load two active substances onto the surface of extracellular vesicles, a U-shaped IFITM scaffold was designed by fusing an IFITM3 variant with an ESM (V6 or V9), a Type 1 transmembrane domain with excellent extracellular vesicle engineering efficiency. To verify whether the U-shaped IFITM scaffold could express the two active substances on the surface of extracellular vesicles, fusion proteins were designed in which mEGF and FLAG peptides, or mSIRPα and FLAG peptides, were attached to the N-terminus or C-terminus of the U-shaped IFITM scaffold, respectively. A vector for expressing the fusion proteins was constructed and transformed into cells, and the levels of the fusion proteins in the cells and their derived extracellular vesicles were checked. As a result, it was confirmed that the U-shaped IFITM scaffold could effectively express the two proteins in extracellular vesicles (Figs. 8 and 9).

[0128]

[0129] Although the embodiments have been described above with reference to the limited drawings, those skilled in the art can apply various technical modifications and variations based on the above. For example, suitable results may be achieved even if the described techniques are performed in a different order than described, and / or if the components of the described system, structure, device, circuit, etc. are combined or assembled in a form different from described, or replaced or substituted by other components or equivalents.

[0130] Therefore, other implementations, other embodiments, and equivalents to the claims also fall within the scope of the claims set forth below.

Claims

1. As an IFITM backbone comprising the amino acid sequence of an IFITM1 variant, The above IFITM1 variant is an IFITM backbone in which the 13th amino acid of the wild-type IFITM, composed of the amino acids represented by SEQ ID NO. 1, is substituted with alanine.

2. In Paragraph 1, The above IFITM1 variant is an IFITM backbone consisting of the amino acid sequence of SEQ ID NO.

3.

3. In Paragraph 2, The above IFITM backbone is an IFITM backbone in which 1 to 4 amino acids are deleted from the N-terminus and / or C-terminus of the above IFITM1 variant.

4. In Paragraph 2, The above IFITM backbone is an IFITM backbone in which 1 to 4 amino acids are inserted at the N-terminus of the above IFITM1 variant.

5. In Paragraph 1, The above IFITM backbone is an IFITM backbone in which EGF or an EGF fragment is fused to the C-terminus of an IFITM1 variant.

6. In Paragraph 5, The IFITM backbone, wherein the EGF fragment comprises an amino acid sequence represented by one selected from the group consisting of SEQ ID NOs 6 to 8.

7. In Paragraph 1, The above IFITM backbone is an IFITM backbone in which biologically active substances are connected to the N-terminus and C-terminus of an IFITM1 variant.

8. IFITM backbone containing the structure [N-terminus - IFITM3 domain - EGF domain - C-terminus].

9. In Paragraph 8, The IFITM backbone, wherein the above IFITM3 domain comprises the amino acid sequence represented by SEQ ID NO.

2.

10. In Paragraph 8, The IFITM backbone comprises an amino acid sequence in which 1 to 21 amino acids are deleted from the N-direction of SEQ ID NO. 2, wherein the above IFITM3 domain comprises an amino acid sequence.

11. In Paragraph 8, IFITM backbone, wherein the EGF domain comprises an amino acid sequence represented by one selected from the group consisting of SEQ ID NOs 6 to 8.

12. U-shaped IFITM skeleton with a structure of [N-terminus - V6 or V9 domain - IFITM domain derived from IFITM1 or IFITM3 - C-terminus].

13. In Paragraph 12, A U-shaped IFITM skeleton in which the IFITM domain derived from IFITM1 or IFITM3 is the IFITM skeleton of claim 1 or 8.

14. In Paragraph 12, A U-shaped IFITM backbone in which the IFITM domain derived from the above IFITM3 comprises the amino acid sequence represented by SEQ ID NO.

20.

15. In Paragraph 12, A U-shaped IFITM backbone in which the IFITM domain derived from IFITM1 or IFITM3 comprises an amino acid sequence in which 1 to 80 amino acids are deleted from the N-terminal or C-terminal direction of the amino acid sequence represented by SEQ ID NO. 1 or SEQ ID NO.

2.

16. In Paragraph 12, A U-shaped IFITM backbone in which the above V6 or V9 domain comprises the amino acid sequence of SEQ ID NO. 18 or 19.

17. A polynucleotide encoding the IFITM backbone of claim 1, 8, or 12.

18. IFITM expression cassette containing the polynucleotide of claim 16.

19. An IFITM expression vector comprising the IFITM expression cassette of claim 17.

20. Transformed cells transformed with the vector of paragraph 18.

21. Extracellular vesicles derived from the transformed cells of paragraph 19.

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