Complex for being joined with extracellular vesicle and used, and use of complex

Abstract

The purpose of the present invention is to provide novel technology that enables more efficient joining of a useful component to an extracellular vesicle. Provided is a complex for being joined with extracellular vesicle and used, the complex: (1) containing a first binding part that joins directly or indirectly with a useful component; (2) containing a second binding part that specifically binds with the first binding part; and (3) binding together the first binding part and the second binding part.

Description

Complex for use in conjunction with extracellular vesicles and use thereof

[0001] The present invention relates to a complex for use in conjunction with extracellular vesicles and the use thereof.

[0002] Extracellular vesicles are vesicles secreted from cells, have a lipid bilayer membrane structure, and are known to encapsulate substances such as proteins, sugars, and nucleic acids. In recent years, various studies using extracellular vesicles have been conducted. For example, because extracellular vesicles are contained in body fluids such as blood, urine, and saliva, they are expected to be used as biomarkers in the diagnosis and treatment of diseases. In addition, extracellular vesicles are expected to be used as carriers in drug delivery systems (DDS) (Non-Patent Document 1).

[0003] Known examples of such research include a technique in which pores are formed in the membrane of extracellular vesicles by applying a stimulus such as electroporation or ultrasonic treatment to the vesicles, and then a substance is introduced into the vesicles through the pores. Another known technique involves preparing a fusion protein of CD63 (Cluster Differentiation 63) protein and a protein of interest, and then introducing the fusion protein into the extracellular vesicles by utilizing the migration ability of the CD63 protein into extracellular vesicles.

[0004] Tsukasa Kadota et al., The forefront of extracellular vesicles and exosomes research: Aiming for clinical application, Japanese Pharmacological Journal, 149(3), 119-122, 2017, Japanese Pharmacological Society

[0005] The present inventors have uniquely focused on the fact that the aforementioned technology for fusing CD63 protein with a target protein can only introduce one molecule of the target protein into extracellular vesicles per CD63 protein molecule. An object of the present disclosure is to provide a new technology that can more efficiently link useful components to extracellular vesicles.

[0006] As a result of extensive research, the present inventors have found that a useful component can be efficiently linked to extracellular vesicles by using a complex formed by linking a biotin protein ligase linked to a useful component via biotin with a biotin carboxyl carrier protein. Furthermore, since useful components can be efficiently linked to extracellular vesicles by utilizing the principle of specific binding between biotin protein ligase and biotin carboxyl carrier protein via biotin, the present inventors have found that, regardless of the combination of biotin protein ligase and biotin carboxyl carrier protein, a useful component can be efficiently linked to extracellular vesicles by using a complex formed by specifically binding a first binding moiety (e.g., biotin protein ligase, SpyCatcher, etc.) directly or indirectly linked to a useful component with a second binding moiety (e.g., biotin carboxyl carrier protein, SpyTag, etc.) that specifically binds to the first binding moiety. The present invention was completed through further research based on this finding, and the present disclosure encompasses, for example, the following representative inventions. Item 1. Item 2. A complex for use by linking to extracellular vesicles, comprising the following components: (1) comprising a first binding part directly or indirectly linked to a useful component, (2) comprising a second binding part that specifically binds to the first binding part, (3) the first binding part and the second binding part are linked together. Item 2. The complex according to Item 1, wherein the first binding part, the second binding part, and the specific bond are (i) or (ii) below: (i) the first binding part is biotin protein ligase, the second binding part is biotin carboxyl carrier protein, and the specific bond is a bond via biotin, or (ii) the first binding part is SpyCatcher, the second binding part is SpyTag, and the specific bond is an isopeptide bond. Item 3. The complex according to Item 1 or 2, wherein the useful component is at least one selected from the group consisting of proteins, peptides, amino acids, and nucleic acids. Item 4. Item 4. The complex according to any one of Items 1 to 3, further comprising an extracellular vesicle-targeting protein, wherein the extracellular vesicle-targeting protein and the second binding portion are directly or indirectly linked.Item 5. The complex according to Item 4, comprising two or more molecules of the second binding part per molecule of the extracellular vesicle-targeting protein. Item 6. Extracellular vesicles linked to the complex according to any one of Items 1 to 5. Item 7. A cell that secretes extracellular vesicles, comprising at least one vector selected from the group consisting of a vector capable of expressing a first binding part linked directly or indirectly to a useful ingredient and a vector capable of expressing a second binding part. Item 8. The cell that secretes extracellular vesicles according to Item 7, wherein the vector capable of expressing the second binding part is a vector capable of expressing a second binding part linked directly or indirectly to an extracellular vesicle-targeting protein. Item 9. Item 10. A method for producing the complex described in Item 4, comprising the following steps (a1) and (a2): (a1) mixing a first binding part linked directly or indirectly to a useful component with a second binding part linked directly or indirectly to an extracellular vesicle-targeting protein, (a2) binding the first binding part with the second binding part, thereby indirectly linking the useful component to the extracellular vesicle-targeting protein via the bond. Item 10. A method for producing extracellular vesicles carrying the complex described in any one of claims 1 to 5, comprising the following steps (b1) and (b2): (b1) mixing a first binding part linked directly or indirectly to a useful component with extracellular vesicles carrying a second binding part linked directly or indirectly to an extracellular vesicle-targeting protein, (b2) binding the first binding part with the second binding part, thereby indirectly linking the useful component to the extracellular vesicles via the bond.

[0007] According to the present disclosure, useful components can be efficiently linked to extracellular vesicles. Extracellular vesicles equipped with the complexes of the present disclosure can also be used as carriers in DDS and the like, and are useful for delivering useful components in the diagnosis, treatment, and the like of diseases.

[0008] FIG. 1 shows a model diagram of a biotinylation reaction using BPL, BCCP, and biotin. FIG. 2 shows a model diagram of a complex of the present disclosure in which two or more molecules of a second binding moiety (BCCP) are linked. FIG. 3 shows a model diagram of a complex of the present disclosure in which two or more molecules of a second binding moiety (SpyTag) are linked. FIG. 4 shows a model diagram of a complex of the present disclosure in which seven molecules of a second binding moiety (BCCP or SpyTag) are linked per molecule of extracellular vesicle-targeting protein. FIG. 5 shows the Gag-FLAG-5BCCP polynucleotide used in Test Example 1. FIG. 6 shows the gLuc-BPL polynucleotide used in Test Example 1. FIG. 7 shows an example of loading a useful ingredient into extracellular vesicles using a BCCP-BPL biotinylation reaction. FIG. 8 shows the results of Test Example 1. FIG. 8 demonstrates that active ingredients can be successfully linked to extracellular vesicles using the complex of the present disclosure. FIG. 9 shows the results of Test Example 2. FIG. 9 shows that the active ingredient was successfully linked to extracellular vesicles using the conjugate of the present disclosure. FIG. 10 shows the results of Test Example 2. FIG. 10 shows that the active ingredient was successfully linked to extracellular vesicles using the conjugate of the present disclosure. FIG. 11 shows the binding mode between the useful ingredient (PKI) and the first binding moiety, the binding mode between the extracellular vesicle-targeting protein (Gag) and the second binding moiety in Test Example 3, and the protocol of Test Example 3. FIG. 12 shows the results of Test Example 3. FIG. 12 shows that even when a PKI inhibitor (PKI) was used as the active ingredient, the active ingredient was successfully linked to extracellular vesicles, and further shows that the useful ingredient was successfully delivered to the target subject. FIG. 13 shows the results of Test Example 3. FIG. 13 shows that even when a PKI inhibitor (PKI) was used as the active ingredient, the active ingredient was successfully linked to extracellular vesicles, and further shows that the useful ingredient was successfully delivered to the target subject. Figure 14 shows the results of Test Example 3. Figure 14 shows that even when PKI was used as the active ingredient, the active ingredient was successfully linked to extracellular vesicles, and furthermore, it shows that the useful ingredient was successfully delivered to the target subject. Figure 15 shows the results of Test Example 5. Figure 15 shows that even in vivo, the active ingredient (PKI, STAT3i) was successfully linked to extracellular vesicles, and furthermore, it shows that the useful ingredient was successfully delivered to the target subject. Figure 16 shows the results of Test Example 5.Figure 15 shows that the active ingredient (PKI) was successfully linked to extracellular vesicles in vivo, and further shows that the useful ingredient was successfully delivered to the target subject.

[0009] Hereinafter, embodiments of the present disclosure will be described in more detail. In the present disclosure, the terms "contain" and "comprise" also mean "consist essentially of" and "consist of," respectively.

[0010] The present disclosure encompasses a complex for use in conjunction with extracellular vesicles, comprising the following components: (1) a first binding moiety directly or indirectly linked to a useful component, (2) a second binding moiety that specifically binds to the first binding moiety, and (3) the first binding moiety and the second binding moiety are bound to each other.

[0011] In the present disclosure, the first binding portion and the second binding portion are not limited as long as they can bind, but preferred examples include first binding portions and second binding portions that can specifically bind to each other.

[0012] Examples of the first binding moiety and the second binding moiety include biotin protein ligase (BPL) and biotin carboxyl carrier protein (BCCP), and preferably the first binding moiety is BPL and the second binding moiety is BCCP. Although this disclosure is not limited to this, it is known that BPL and BCCP can specifically bind via biotin, and this binding via a specific bond is known as a biotinylation reaction between BPL and BCCP.

[0013] Examples of the first binding moiety and the second binding moiety include SpyCatcher and SpyTag, and preferably the first binding moiety is SpyCatcher and the second binding moiety is SpyTag. Although not limiting the present disclosure, it is known that SpyCatcher and SpyTag can specifically bind via an isopeptide bond, and this binding via a specific bond is known as the SpyCatcher / SpyTag system.

[0014] Without limiting the present disclosure, examples of the first and second binding portions using BPL and BCCP, and SpyCatcher and SpyTag are described in more detail below.

[0015] Example using BPL and BCCP

[0016] A case will be described where the first binding moiety is BPL and the second binding moiety is BCCP. In the present disclosure, the BCCP is not limited, and examples of the origin of BCCP include archaea (Archaea). Examples of archaea include Sulfolobus archaea (Sulfolobus) such as Sulfolobus tokodaii. Examples of BCCP include preferably archaeal BCCP, more preferably BCCP of the genus Sulfolobus, and even more preferably BCCP of Sulfolobus tokodaii.

[0017] Although not limiting the present disclosure, examples of BCCP include the BCCP described in the Uniprot database under accession number F9VN46. As an example, the amino acid sequence encoding the BCCP of Sulfolobus tokodaii is the amino acid sequence represented by SEQ ID NO: 1. The amino acid sequence represented by SEQ ID NO: 1 corresponds to the amino acid sequence encoding the BCCP described in the Uniprot database under accession number F9VN46. Thus, the amino acid sequence encoding BCCP can be easily determined based on various databases related to proteins, etc. Furthermore, the molecular weight of BCCP is known to be approximately 7000 (approximately 7 kDa). Although not limiting the present disclosure, examples of the molecular weight of BCCP include preferably approximately 6000 to 8000 (approximately 6 to 8 kDa), and more preferably approximately 6500 to 7500 (approximately 6.5 to 7.5 kDa).

[0018] The amino acid sequence encoding BCCP may have modifications to the amino acid sequence, as long as BCCP and BPL can be linked via biotin.

[0019] Although not limiting the present disclosure, examples of such modifications include amino acid sequences that have a sequence identity of 90% or more, preferably 95% or more, more preferably 98% or more, and even more preferably 99% or more, or 99.5% or more to the amino acid sequence represented by SEQ ID NO: 1.

[0020] Furthermore, examples of such modifications include, but are not limited to, an amino acid sequence in which one or more amino acids have been deleted, substituted, or added in the amino acid sequence represented by SEQ ID NO: 1. Here, examples of the range of one or more amino acids include 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, and 1 or 2 amino acids.

[0021] In the present disclosure, amino acid sequence identity refers to the degree of amino acid sequence agreement between two or more comparable amino acid sequences. The level of amino acid sequence identity can be determined, for example, using the sequence analysis tool FASTA with default parameters. It can also be determined based on the BLAST algorithm by Karlin and Altschul (Karlin S, Altschul SF. "Methods for assessing the statistical significance of molecular sequence features by using general scoring schemes" Proc Natl Acad Sci USA. 87:2264-2268 1990), Karlin S, Altschul SF. "Applications and statistics for multiple high-scoring segments in molecular sequences" Proc Natl Acad Sci USA. 90:5873-7 (1993)). A program called BLASTX has been developed based on this BLAST algorithm. Specific techniques for these analysis methods are publicly known, and can be found on the website of the National Center of Biotechnology Information (NCBI) (http: / / www.ncbi.nlm.nih.gov / ).

[0022] The modified amino acid may be either a natural amino acid or an artificial amino acid. Examples of amino acids include hydrophobic amino acids, hydrophilic amino acids, basic amino acids, acidic amino acids, branched-chain amino acids, aromatic amino acids, and sulfur-containing amino acids. More specific examples of amino acids include valine, leucine, isoleucine, alanine, arginine, glutamine, lysine, aspartic acid, glutamic acid, proline, cysteine, threonine, methionine, histidine, phenylalanine, tyrosine, tryptophan, asparagine, glycine, and serine. Techniques for deleting, substituting, and / or adding one or more amino acids in a specific amino acid sequence are known.

[0023] Furthermore, while not limiting the present disclosure, conservative substitutions are a preferred example of substitutions. In the present disclosure, conservative substitution refers to the substitution of an amino acid residue with an amino acid residue having a side chain with similar properties. For example, substitution between amino acid residues having basic side chains such as lysine, arginine, and histidine constitutes a conservative substitution. Other examples of conservative substitutions include substitution between amino acid residues having acidic side chains such as aspartic acid and glutamic acid; amino acid residues having uncharged polar side chains such as glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine; amino acid residues having nonpolar side chains such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan; amino acid residues having β-branched side chains such as threonine, valine, and isoleucine; and amino acid residues having aromatic side chains such as tyrosine, phenylalanine, tryptophan, and histidine.

[0024] The complex of the present disclosure may contain two or more molecules of BCCP, and the BCCP may be used alone or in combination of two or more types.

[0025] In the present disclosure, BPL is not limited, and examples of the origin of BPL include Archaea. Examples of archaea include Sulfolobus archaea such as Sulfolobus tokodaii. Examples of BPL include preferably BPL from archaea, more preferably BPL from the genus Sulfolobus, and even more preferably BPL from Sulfolobus tokodaii. The origin of BPL may be the same as or different from that of BCCP, and is preferably the same.

[0026] Although not limiting the present disclosure, examples of BPL include BPL described in the Uniprot database under accession number F9VNG7, etc. As an example, an amino acid sequence encoding BPL from Sulfolobus tokodaii is the amino acid sequence represented by SEQ ID NO: 2. The amino acid sequence represented by SEQ ID NO: 2 corresponds to the amino acid sequence encoding BPL described in the Uniprot database under accession number F9VNG7 (Mass (Da) 26317). Thus, the amino acid sequence encoding BPL can be easily determined using various databases related to proteins, etc. The molecular weight of BPL is known to be approximately 26,000 (approximately 26 kDa). Although not limiting the present disclosure, examples of the molecular weight of BPL include, but are not limited to, approximately 25,000 to 28,000 (approximately 25 to 28 kDa), preferably approximately 26,000 to 27,000 (approximately 26 to 27 kDa).

[0027] The amino acid sequence encoding BPL may have modifications to the amino acid sequence, as long as BCCP and BPL can be linked via biotin.

[0028] Although not limiting the present disclosure, examples of such modifications include amino acid sequences that have a sequence identity of 90% or more, preferably 95% or more, more preferably 98% or more, and even more preferably 99% or more, or 99.5% or more to the amino acid sequence represented by SEQ ID NO: 2.

[0029] Furthermore, examples of such modifications include, but are not limited to, an amino acid sequence in which one or more amino acids have been deleted, substituted, or added in the amino acid sequence represented by SEQ ID NO: 2. Here, examples of the range of one or more amino acids include 1 to 20, preferably 1 to 15, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 or 2 amino acids.

[0030] Sequence identity is as described above. The amino acid after modification may be either a natural amino acid or an artificial amino acid, and the amino acid is as described above. Furthermore, although not limiting the present disclosure, a preferred example of substitution is conservative substitution, and conservative substitution is as described above.

[0031] The complex of the present disclosure may contain two or more molecules of BPL, and one type of BPL may be used alone, or two or more types may be used in combination.

[0032] Biotin is a known substance designated by CAS registration number 58-85-5, and is also known as one of the B vitamins.

[0033] The complex of the present disclosure includes a useful component directly or indirectly linked to BPL. Examples of useful components include proteins, peptides, amino acids, and nucleic acids. While not limiting the present disclosure, proteins may be reporter proteins, and examples thereof include fluorescent proteins such as Gaussia luciferase (gLuc) and green fluorescent protein (GFP). Examples of peptides include enzyme activity inhibitory peptides such as protein kinase A (PKA) inhibitory peptides and signal transducer and activator of transcription 3 (STAT3) inhibitory peptides. Examples of nucleic acids include DNA and RNA (small interfering RNA (siRNA), microRNA (miRNA), etc.). The useful components may be used alone or in combination of two or more. The complex of the present disclosure can be used as a protein drug, a nucleic acid drug, etc., depending on the useful component to be linked.

[0034] The useful component may be linked to the BPL by any linking means known in the art. Examples of indirect linking means include, but are not limited to, linking via a linker, linking via a nucleic acid-binding protein such as an RNA-binding protein (e.g., L7Ae), or linking via the SpyCatcher / SpyTag system.

[0035] Furthermore, as long as BCCP and BPL can be linked via biotin, the useful component may be linked to BCCP directly or indirectly. Linking of the useful component to BCCP may be carried out according to linking means known in the art, as described above.

[0036] The BPL linked to a useful component and BCCP are linked via biotin. Conventionally, biotinylation reactions using BCCP as a substrate and BPL as a catalytic enzyme have been known. For example, biotinylation reactions using BCCP and BPL from archaea, and biotinylation reactions using BCCP and BPL from Escherichia coli are known. From this perspective, in the complex of the present disclosure, the BPL linked to a useful component and BCCP are linked via biotin, preferably utilizing the biotinylation reaction mechanism. A model diagram of a biotinylation reaction using BCCP as a substrate and BPL as a catalytic enzyme is shown in Figure 1. Furthermore, for this reason, preferred examples of modifications to the amino acid sequences of BCCP and BPL are modifications that allow the biotinylation reaction mechanism to be utilized, and more preferred examples are modifications that allow the biotinylation reaction mechanism known in archaea to be utilized. Furthermore, without being limited thereto, whether or not the biotinylation reaction mechanism can be utilized can be determined according to the below-described Test Example 1. As shown in Test Example 1, if the amount of useful component carried (loading amount) in a complex (extracellular endoplasmic reticulum carrying the complex) prepared under biotin-added conditions is greater than the amount of useful component carried (loading amount) in a complex (extracellular endoplasmic reticulum carrying the complex) prepared under the same conditions except that biotin was not added, it is determined that the biotinylation reaction mechanism can be utilized, and if the amounts are the same or less, it is determined that the biotinylation reaction mechanism cannot be utilized.

[0037] The number of BCCP molecules in the complex of the present disclosure is not limited, and the complex contains one or more BCCP molecules. Here, the range of one or more is exemplified by one or more, preferably two or more, and in principle, there is no upper limit. The complex of the present disclosure preferably contains two or more BCCP molecules, more preferably 2 to 100, 2 to 80, 2 to 50, 2 to 30, 2 to 20, 2 to 10, 2 to 8, 2 to 5, etc. As mentioned above, the molecular weight of one BCCP molecule is approximately 7,000. As shown in Figure 2, a preferred example of the complex of the present disclosure is one in which multiple BCCP molecules are connected together.

[0038] The BCCPs are linked directly or indirectly, preferably indirectly via a linker or the like. Examples of linkers include a GS linker, a linker consisting only of glycine (G), a linker consisting only of serine (S), an EAAAK linker, and the like. The length of the linker sequence is not limited as long as the effects of the present disclosure can be obtained, and examples of the number of amino acid residues in the linker sequence are preferably 2 to 100, more preferably 3 to 50, and even more preferably 4 to 40, etc. As the GS linker, a linker consisting only of glycine (G), a linker consisting only of serine (S), an EAAAK linker, and the like are preferably used. ４ Examples of the linker include (S)n, where n is an integer of 1 to 6, preferably 2 to 5, and more preferably 3 or 4. Examples of the EAAAK linker include (EAAAK)n, where n is an integer of 1 to 6, preferably 2 to 5, and more preferably 3 or 4 (EAAAK is shown in SEQ ID NO: 3). One type of linker may be used alone, or two or more types may be used in combination.

[0039] In this complex, as shown in Figure 2, BCCP and BPL are linked via biotin. The content ratio is not limited, and examples thereof include a ratio of 0.5 or more, preferably 0.8 or more, and more preferably 1, of BPL molecules directly or indirectly linked to a useful component per BCCP molecule. The number of BPL molecules linked to a useful component per BCCP molecule is determined by dividing the number of BPL molecules directly or indirectly linked to the useful component by the number of BCCP molecules. More specifically, this is determined as described in Test Example 1 below. Note that in this complex, it is not necessary for a useful component to be linked to all BPLs, and it is not necessary for BPLs to be linked to all BCCPs. Figure 2 (E) shows an example in which a useful component is indirectly linked to BPL via SpyTag and SpyCatcher. BPL and SpyTag can be linked directly or indirectly, preferably indirectly via a linker or the like. The linker and the like are explained in the same manner as above.

[0040] Example Using SpyCatcher and SpyTag: A more detailed description will be given of the case where the first binding moiety is SpyCatcher and the second binding moiety is SpyTag. Specific binding via a covalent bond (specifically, an isopeptide bond) between SpyCatcher and SpyTag is conventionally known as the SpyCatcher / SpyTag system. The SpyCatcher / SpyTag system involves binding SpyTag, a short peptide of 14 amino acid residues with a molecular weight of 1.9 (1.9 kDa), to SpyCatcher, a small protein with a molecular weight of 12 (12 kDa). Upon mixing, SpyTag and SpyCatcher bind, spontaneously forming an isopeptide bond between them. This binding reaction is known to occur reproducibly over a wide range of pH, temperature, and buffer conditions. Therefore, by coexisting SpyTag and SpyCatcher in the medium shown in the test example below, SpyTag and SpyCatcher can specifically bind via an isopeptide bond.

[0041] Although not limited thereto, an example of an amino acid sequence encoding SpyTag is the amino acid sequence shown in SEQ ID NO: 4, and an example of an amino acid sequence encoding SpyCatcher is the amino acid sequence shown in SEQ ID NO: 5.

[0042] The amino acid sequence encoding SpyTag may have modifications to the amino acid sequence, provided that SpyTag and SpyCatche can be linked by an isopeptide bond.

[0043] Although not limiting the present disclosure, examples of such modifications include amino acid sequences that have a sequence identity of 90% or more, preferably 95% or more, more preferably 98% or more, and even more preferably 99% or more, or 99.5% or more to the amino acid sequence represented by SEQ ID NO: 4.

[0044] Furthermore, examples of such modifications include, but are not limited to, an amino acid sequence in which one or more amino acids have been deleted, substituted, or added in the amino acid sequence represented by SEQ ID NO: 4. Here, examples of the range of one or more amino acids include 1 to 5, 1 to 4, 1 to 3, and 1 or 2 amino acids.

[0045] The amino acid sequence encoding SpyCatcher may have modifications to the amino acid sequence, as long as the SpyTag and SpyCatcher can be linked by an isopeptide bond.

[0046] Although not limiting the present disclosure, examples of such modifications include amino acid sequences that have a sequence identity of 90% or more, preferably 95% or more, more preferably 98% or more, and even more preferably 99% or more, or 99.5% or more to the amino acid sequence represented by SEQ ID NO: 5.

[0047] Furthermore, examples of such modifications include, but are not limited to, an amino acid sequence in which one or more amino acids have been deleted, substituted, or added in the amino acid sequence represented by SEQ ID NO: 5. Here, examples of the range of one or more amino acids include 1 to 20, 1 to 15, 1 to 10, 1 to 8, 1 to 5, 1 to 4, 1 to 3, and 1 or 2 amino acids.

[0048] The sequence identity is as described above. The amino acid after modification may be either a natural amino acid or an artificial amino acid, and the amino acid is as described above. Furthermore, although not limiting the present disclosure, a preferred example of substitution is conservative substitution, and the conservative substitution is as described above.

[0049] The complex of the present disclosure may contain two or more SpyTags, and one or more SpyTags may be used alone or in combination. The complex of the present disclosure may contain two or more SpyCatchers, and one or more SpyCatchers may be used alone or in combination.

[0050] The complex of the present disclosure includes a useful component linked directly or indirectly to SpyCatcher. The useful component is as described above. The useful component may be used alone or in combination with two or more components. The complex of the present disclosure can be used as a protein drug, a nucleic acid drug, or the like, depending on the useful component to be linked.

[0051] The useful component may be linked to SpyCatcher by any linking method known in the art. Examples of indirect linking methods include, but are not limited to, linking via a linker, linking via a nucleic acid-binding protein such as an RNA-binding protein (e.g., L7Ae), and linking using the linkage between BCCP and BPL via the biotinylation reaction described above.

[0052] Furthermore, as long as SpyCatcher and SpyTag can be linked, the useful component may be linked to SpyTag. Linking of the useful component to SpyTag may be performed according to linking means known in the art, as described above.

[0053] SpyCatcher linked to a useful component and SpyTag are linked via an isopeptide bond. A model diagram of linkage using the SpyCatcher / SpyTag system is shown in Figure 3. The aforementioned amino acid sequence modifications in SpyTag and SpyCatcher / are modifications within the scope of enabling the system to be used. The usability of the system can be determined according to the procedures in the test examples described below. When SpyCatcher and SpyTag are coexisted in phosphate-buffered saline (PBS (137 mM NaCl, 10 mM NaHPO, 2.7 mM KCl, 1.8 mM KHPO, pH 7.4)) for 2 hours at room temperature (30°C), the complex is prepared. If the amount of useful component loaded (loaded) is greater than the amount of useful component loaded (loaded) in a complex prepared under the same conditions except for the absence of SpyCatcher and SpyTag, the system is considered usable. If the amounts are the same or less than the amounts loaded, the system is considered unusable.

[0054] The number of SpyTag molecules in the complex of the present disclosure is not limited, and the complex contains one or more SpyTag molecules. Here, the range of one or more is exemplified by one or more, preferably two or more, and in principle, there is no upper limit. The complex of the present disclosure preferably contains two or more SpyTag molecules, more preferably 2 to 100, 2 to 80, 2 to 50, 2 to 30, 2 to 20, 2 to 10, 2 to 8, 2 to 5, etc. As mentioned above, the molecular weight of one SpyTag molecule is approximately 1900. As shown in Figure 3, a preferred example of the complex of the present disclosure is one in which multiple SpyTags are connected together.

[0055] SpyTags are linked directly or indirectly, preferably indirectly via a linker or the like. The linker or the like is as described above. The linker may be used alone or in combination of two or more types.

[0056] In this complex, as shown in Figure 3, SpyTag and SpyCatcher are linked by an isopeptide bond. The content ratio is not limited, and examples thereof include a ratio of 0.5 or more, preferably 0.8 or more, and more preferably 1, of SpyCatcher molecules directly or indirectly linked to a useful component per SpyTag molecule. The number of SpyCatcher molecules linked to a useful component per SpyTag molecule is determined by dividing the number of SpyCatcher molecules linked to a useful component by the number of SpyTag molecules. More specifically, this is determined in accordance with Test Example 1 described below. In this complex, it is not necessary for a useful component to be linked to all SpyCachers, and it is not necessary for SpyCatcher to be linked to all SpyTags. Figure 3(B) shows an example in which a useful component is indirectly linked to SpyCatcher via the linkage between BCCP and BPL via the biotinylation reaction. SpyCatcher and BCCP can be linked directly or indirectly, and are preferably linked indirectly via a linker, etc. The linker, etc. is explained in the same manner as above.

[0057] The complex of the present disclosure, which includes a first binding portion and a second binding portion, may further include an extracellular vesicle-targeting protein. The extracellular vesicle-targeting protein may be either a protein directed toward the inside of extracellular vesicles or a protein directed toward the outside of extracellular vesicles. Examples of extracellular vesicle-targeting proteins include Gag protein, Lamp2 (liposome-associated membrane protein 2b), lactadherin, ExoSignal, and the like. Although not limiting the present disclosure, an example of a Gag protein is the amino acid sequence encoding the Gag protein described in Uniprot accession number P03332. These may be used alone or in combination of two or more. The complex of the present disclosure is used in conjunction with extracellular vesicles. For this reason, it is preferable that the complex of the present disclosure includes an extracellular vesicle-targeting protein.

[0058] Although not limiting the present disclosure, when the complex of the present disclosure further comprises an extracellular vesicle-targeting protein, the extracellular vesicle-targeting protein is usually linked directly or indirectly to the second binding moiety. Furthermore, the second binding moiety may be linked to either the N-terminal or C-terminal side of the extracellular vesicle-targeting protein, but is preferably linked to the C-terminal side. When linked indirectly, it is preferably linked via a FLAG tag, a linker, or the like. Examples of linkers include a GS linker, a linker consisting only of glycine (G), a linker consisting only of serine (S), and the like. As the GS linker, preferably (G ４Examples of such a linker include S)n, where n is an integer of 1 to 6, preferably 2 to 5, and more preferably 3 or 4. These may be used alone or in combination of two or more. The linkage may be performed according to procedures such as genetic engineering techniques known in the art. Furthermore, without limiting the present disclosure, the number of amino acid residues between the extracellular vesicle-targeting protein and the second binding part (i.e., in the example of FIG. 4, the number of amino acid residues between the second binding part (first BCCP or first SpyTag) closest to the extracellular vesicle-targeting protein and the extracellular vesicle-targeting protein) is preferably 0 to 80, more preferably 0 to 50, and even more preferably 0 to 30, for example.

[0059] When the complex contains an extracellular vesicle-targeting protein, the complex preferably contains about 1 to 100, more preferably about 2 to 80, and even more preferably about 3 to 50, about 3 to 30, about 3 to 20, about 3 to 10, about 3 to 8, or about 3 to 5 molecules of second binding moieties per molecule of extracellular vesicle-targeting protein. When an extracellular vesicle-targeting protein is contained, the complex can be linked to extracellular vesicles via the extracellular vesicle-targeting protein. Therefore, by using two or more molecules of second binding moieties per molecule of extracellular vesicle-targeting protein, useful components can be linked to extracellular vesicles more efficiently. Although not limiting the present disclosure, a model diagram of a complex of the present disclosure in which seven molecules of second binding moieties are linked per molecule of extracellular vesicle-targeting protein is shown in Figure 4.

[0060] In the present disclosure, the number of molecules of the second binding moiety per molecule of extracellular vesicle-targeting protein is determined by dividing the number of molecules of the second binding moiety by the number of molecules of the extracellular vesicle-targeting protein in a complex containing the extracellular vesicle-targeting protein. Test Example 1 described below is an example in which BCCP was used as the second binding moiety and a complex containing 5 molecules of BCCP per molecule of the extracellular vesicle-targeting protein was obtained.

[0061] The complex of the present disclosure is used by linking it to extracellular vesicles. The means for linking the complex to extracellular vesicles is not limited as long as the linkage is possible. A preferred example is linking the complex to extracellular vesicles via an extracellular vesicle-targeting protein. From this perspective, the complex preferably contains an extracellular vesicle-targeting protein. Linking the extracellular vesicle-targeting protein to extracellular vesicles may be performed according to known procedures such as genetic engineering techniques.

[0062] Extracellular vesicles are vesicles secreted from cells and have a lipid bilayer membrane structure. Based on differences in their production mechanisms, extracellular vesicles have traditionally been classified into exosomes (50-150 nm in diameter), microvesicles (100-1000 nm in diameter), and apoptotic vesicles (1000-5000 nm in diameter). More recently, they have been classified into small EVs (sEVs) (50-200 nm in diameter) and medium / large EVs (m / lEVs) (200-1000 nm in diameter) based on their size. In the present disclosure, the extracellular vesicles may be any of these, with preferred examples including exosomes and microvesicles.

[0063] In the complex of the present disclosure, as described above, a first binding part to which a useful component is fused can be linked to a second binding part. Thus, for example, as shown in Figure 4, by linking two or more molecules of the second binding part per molecule of the extracellular vesicle-targeting protein, it becomes possible to efficiently incorporate the useful component fused to the first binding part into the complex, and ultimately to more efficiently deliver the useful component to extracellular vesicles.

[0064] The complex of the present disclosure allows useful components to be efficiently loaded into extracellular vesicles. Extracellular vesicles equipped with the complex of the present disclosure can also be used as carriers in DDS and the like, and are useful for delivering useful components in the diagnosis, treatment, and the like of diseases.

[0065] The conjugate of the present disclosure can be produced according to the above-described procedures known in the art. Without limiting the present disclosure, the conjugate of the present disclosure can be produced by binding a first binding moiety, which is directly or indirectly linked to a useful ingredient, to a second binding moiety via the first binding moiety and the second binding moiety.

[0066] For example, the first binding moiety and the second binding moiety linked directly or indirectly to the useful component can be produced according to conventionally known protein expression methods. While not limiting the present disclosure, the first binding moiety can be obtained by introducing a vector capable of expressing the first binding moiety linked directly or indirectly to the useful component into a host cell to obtain a transformant, and culturing the transformant to express the first binding moiety linked directly or indirectly to the useful component. Alternatively, the second binding moiety can be obtained by introducing a vector capable of expressing the second binding moiety into a host cell to obtain a transformant, and culturing the transformant to express the second binding moiety. The vector capable of expressing the first binding moiety and the vector capable of expressing the second binding moiety may be introduced into the same host cell or into different host cells. When introduced into the same host cell, the first binding moiety and the second binding moiety are expressed in one cell. When introduced into different host cells, the first binding moiety or the second binding moiety is expressed in each cell.

[0067] A vector capable of expressing a second binding portion is preferably constructed so that two or more molecules of the second binding portion are linked together to enable expression of the second binding portion. Although not limiting the present disclosure, in the vector, a base sequence encoding a linker sequence or the like is preferably linked between the base sequence encoding the second binding portion and the base sequence encoding the second binding portion. The linker or the like is as described above.

[0068] A vector capable of expressing a first binding moiety directly or indirectly linked to a useful ingredient incorporates at least a base sequence encoding the useful substance and a base sequence encoding the first binding moiety. These base sequences are preferably linked via a base sequence encoding a linker sequence or the like. The linker sequence or the like is as described above.

[0069] The base sequence encoding the first binding portion and the base sequence encoding the second binding portion may be integrated together into one vector, or may be integrated into separate vectors.

[0070] A polynucleotide having a desired nucleotide sequence is constructed according to conventional procedures known in the art, and then inserted into an expression vector to obtain a desired expression vector.

[0071] Examples of expression vectors include those commonly used in the field of genetic engineering, and examples of their origin include bacteria such as Escherichia coli, yeast, viruses, mammals, etc., and may also be any vector, such as a plasmid, phage, or cosmid. The host cell is also not limited and may be determined appropriately depending on the type of expression vector used, with mammalian-derived cells being a preferred example. A promoter may be attached to the expression vector as needed, and for example, a promoter suitable for the host cell may be selected. In addition, the expression vector may also be attached with base sequences such as an enhancer, a splicing signal, a polyA addition signal, a drug resistance gene, or a marker gene such as green fluorescent protein (GFP). These may be attached to any position in the expression vector depending on the purpose.

[0072] The constructed vector is transformed into a host cell, and the resulting transformant is cultured to obtain the first binding moiety and the second binding moiety. By allowing the first binding moiety and the second binding moiety to coexist under conditions that allow binding between the first binding moiety and the second binding moiety, a useful component can be indirectly linked to the second binding moiety via the bond between the first binding moiety and the second binding moiety. If necessary, the culture thus obtained may be harvested, disrupted, and the complex may be purified from the resulting supernatant. The purification procedure may be carried out according to conventionally known protein purification procedures. Any further treatment may be carried out as long as the complex is obtained.

[0073] The medium for culturing the transformant is not limited as long as it can culture the host cells, and may be either a liquid medium or a solid medium. From the viewpoint of efficient culture, a liquid medium is preferred. Various liquid media are known, and examples thereof include LB medium (Luria-Bertani medium).

[0074] While not limiting the present disclosure, when the first binding moiety is BPL, the second binding moiety is BCCP, and the specific binding is via biotin, the solution or medium used during the binding reaction between the first binding moiety and the second binding moiety is not limited as long as specific binding is possible. Preferred examples of the medium include liquid media such as DMEM (Dulbecco's Modified Eagle's Medium) and Opti-MEM (registered trademark) medium (Thermo Fisher Scientific Inc.). The first binding moiety and the second binding moiety are contacted in the solution or medium at about 37°C for about 2 to 24 hours. The content of biotin in the solution or medium used during the binding reaction is not limited as long as the biotinylation reaction occurs. Preferred examples of the biotin content in the solution or medium are about 0.1 to 200 nM, more preferably about 0.1 to 150 nM, and even more preferably about 0.1 to 100 nM. Furthermore, the solution or medium may contain any component, as needed, such as antibiotics such as ampicillin, kanamycin, or chloramphenicol, serum such as FBS (fetal bovine serum), serum substitutes, galactose, or lactose, as long as the effect of the present disclosure is not impaired.

[0075] Although not limiting the present disclosure, when the first binding moiety is SpyCatcher and the second binding moiety is SpyTag, the solution or medium used during the binding reaction between the first binding moiety and the second binding moiety is not limited as long as specific binding (isopeptide bond) between them is possible, preferred examples include phosphate buffered saline (PBS) and the medium, and examples of contact in these include contact at about 30 to 37° C. for about 2 to 24 hours. Any component such as an antibiotic may be added to the solution or medium as long as it does not interfere with the effects of the present disclosure.

[0076] In this manner, the composite of the present disclosure can be produced.

[0077] As described above, the complex of the present disclosure may further contain an extracellular vesicle-targeting protein. When the complex contains an extracellular vesicle-targeting protein, the extracellular vesicle-targeting protein is preferably linked directly or indirectly to the second binding portion.

[0078] The linkage of the extracellular vesicle-targeting protein to the second binding moiety may also be carried out according to conventionally known procedures. For example, in a vector capable of expressing the second binding moiety, the base sequence encoding the extracellular vesicle-targeting protein may be linked directly or indirectly to either the 5'-end or 3'-end of the base sequence encoding the second binding moiety, preferably to the 5'-end of the base sequence encoding the second binding moiety. The base sequence encoding the extracellular vesicle-targeting protein is linked directly or indirectly to the base sequence encoding the second binding moiety, preferably via the tag sequence or the like. By using this vector, a second binding moiety to which the extracellular vesicle-targeting protein is directly or indirectly linked can be obtained. The linkage of the second binding moiety to the first binding moiety may be carried out in the same manner as described above.

[0079] The complex of the present disclosure is used by being linked to an extracellular vesicle. Although not limiting the present disclosure, in order to easily load the complex of the present disclosure onto an extracellular vesicle, the complex of the present disclosure is preferably linked to the extracellular vesicle via an extracellular vesicle-targeting protein.

[0080] The linking of the complex of the present disclosure to extracellular vesicles is not limited as long as the linking is possible, and may be performed according to procedures conventionally known in the art. For example, the linking procedure of the complex of the present disclosure to extracellular vesicles via an extracellular vesicle-targeting protein may be performed by mixing a complex having a first binding moiety directly or indirectly linked to a useful ingredient and a second binding moiety directly or indirectly linked to an extracellular vesicle-targeting protein with extracellular vesicles, thereby linking the extracellular vesicle-targeting protein to the extracellular vesicles, thereby allowing the complex of the present disclosure to be carried on the extracellular vesicles.

[0081] Alternatively, for example, a second binding moiety directly or indirectly linked to an extracellular vesicle-targeting protein may be mixed with extracellular vesicles to link the extracellular vesicles to the extracellular vesicles. The second binding moiety bound to the extracellular vesicles via the extracellular vesicle-targeting protein obtained in this manner may be mixed with a first binding moiety directly or indirectly linked to a useful component, and the second binding moiety and the first binding moiety may be linked as described above to obtain extracellular vesicles carrying the complex of the present disclosure. In these procedures, the procedure for mixing and linking the extracellular vesicle-targeting protein and extracellular vesicles is known, and linking may be performed according to a conventionally known procedure, for example, by mixing in PBS at about 4°C for about 2 hours. Furthermore, any solution or medium may be used as long as linking is possible, and the temperature and time may also be set appropriately.

[0082] Alternatively, for example, a second binding part directly or indirectly linked to an extracellular vesicle-targeting protein may be expressed in the presence of extracellular vesicles to obtain a second binding part bound to extracellular vesicles via the extracellular vesicle-targeting protein, and the second binding part may be mixed with a first binding part directly or indirectly linked to a useful ingredient, followed by binding the second binding part to the first binding part as described above to obtain extracellular vesicles carrying the complex of the present disclosure. From this perspective, a preferred example of the host cell into which the vector is introduced is a cell capable of secreting extracellular vesicles. Examples of host cells capable of secreting extracellular vesicles include various cancer cells such as HEK293 cells, dendritic cells, and B16 cells. These may be used alone or in combination of two or more.

[0083] In this way, extracellular vesicles carrying a first binding portion to which a useful component is linked and a second binding portion can be produced via an extracellular vesicle-targeting protein.

[0084] Therefore, it can be said that the present disclosure includes providing extracellular vesicles to which the complex is linked. Furthermore, it can be said that the present disclosure includes providing a cell that secretes extracellular vesicles, the cell comprising at least one vector selected from the group consisting of a vector capable of expressing a first binding part directly or indirectly linked to a useful ingredient and a vector capable of expressing a second binding part directly or indirectly linked to an extracellular vesicle-targeting protein. For example, the cell preferably comprises a vector capable of expressing a second binding part directly or indirectly linked to an extracellular vesicle-targeting protein.

[0085] Therefore, it can be said that the present disclosure also includes providing a method for producing the complex of the present disclosure, which comprises the following steps (a1) and (a2): (a1) mixing a first binding part directly or indirectly linked to a useful component with a second binding part directly or indirectly linked to an extracellular vesicle-targeting protein, and (a2) binding the first binding part with the second binding part, thereby indirectly linking the useful component to the extracellular vesicle-targeting protein via the bond.

[0086] Therefore, it can be said that the present disclosure also includes providing a method for producing extracellular vesicles carrying the complex of the present disclosure, which comprises the following steps (b1) and (b2): (b1) mixing extracellular vesicles carrying a first binding part directly or indirectly linked to a useful component and a second binding part directly or indirectly linked to an extracellular vesicle-targeting protein, and (b2) binding the first binding part to the second binding part, thereby indirectly linking the useful component to the extracellular vesicles via the bond.

[0087] According to the present disclosure, useful components can be loaded onto extracellular vesicles. In particular, according to the present disclosure, since multiple second binding moieties can be linked, useful components can be efficiently linked. Furthermore, according to the present disclosure, it is also possible to link two or more molecules of second binding moieties to one molecule of extracellular vesicle-targeting protein, so the complex can be easily linked to extracellular vesicles via the extracellular vesicle-targeting protein, and thus useful components can be efficiently loaded onto extracellular vesicles. Furthermore, as shown in Figures 2 to 4, according to the present disclosure, useful components can be linked in multiple stages of two or more, so useful components can be multiply loaded onto extracellular vesicles.

[0088] Furthermore, it has been confirmed that the conjugate of the present disclosure can deliver useful components to targets such as tumor macrophages. The conjugate of the present disclosure, and even extracellular vesicles to which the conjugate is linked, are also useful as carriers in DDS and the like, and are useful for delivering useful components in disease diagnosis, treatment, and the like. Furthermore, since the conjugate of the present disclosure can be used to deliver useful components, it is also useful in various fields other than the medical field.

[0089] Hereinafter, the embodiments of the present disclosure will be described more specifically with reference to examples, but the embodiments of the present disclosure are not limited to the following examples.

[0090] Test Example 11-1. Preparation of Complexes Supported on Extracellular Vesicles (Examples 1 and 2) Complexes were prepared according to the following procedure. Cell Culture: A commercially available murine macrophage-like cell line, RAW264.7, was purchased from ATCC (American Type Culture Collection) and cultured at 37°C in RPMI 1640 (Nissui Co. Ltd.) containing 10% FBS (fetal bovine serum). A commercially available human embryonic kidney cell line, HEK293 (Human Embryonic Kidney 293, CRL-1573), was purchased from ATCC and cultured at 37°C in Dulbecco's modified Eagle's medium (Nissui Co. Ltd.) containing 10% FBS. Mouse breast cancer cell line 4T1 was purchased from ATCC and cultured in RPMI 1640 containing 10% FBS at 37°C and 5% CO2.

[0091] Vector Construction The polynucleotide shown in Figure 5 was constructed according to the following procedure. The sequence of the polynucleotide is shown in SEQ ID NO: 6. Using a standard two-step PCR procedure, a nucleotide sequence encoding a FLAG tag (dotted line in Figure 5) was ligated via a linker sequence to the 3'-end of the nucleotide sequence encoding Gag protein (underlined line in Figure 5). Next, using a standard two-step PCR procedure, five repeats of a BCCP sequence (double underlined line in Figure 5) were ligated via a linker sequence ((G4S)3 linker) to the 3'-end of the nucleotide sequence encoding FLAG. The nucleotide sequences encoding BCCP were each linked via a (G4S)3 linker. This resulted in a polynucleotide (hereinafter sometimes referred to as Gag-FLAG-5BCCP polynucleotide) in which the nucleotide sequences encoding Gag protein, FLAG, and BCCP (five repeats) were linked in order from the 5'-end.

[0092] In this test example, Gag protein was used as an example of an extracellular vesicle-targeting protein. The nucleotide sequence encoding Gag protein is a sequence encoding the Gag protein described in Uniprot accession number P03332, and was designed by the inventors for optimized expression in mammalian cells. The nucleotide sequence encoding the FLAG tag encodes the amino acid sequence (DYKDDDDK) represented by SEQ ID NO: 7. The nucleotide sequence encoding BCCP encodes the amino acid sequence represented by SEQ ID NO: 1.

[0093] Separately, the polynucleotide shown in Figure 6 was constructed according to the following procedure. The sequence of this polynucleotide is shown in SEQ ID NO: 8. Using a standard two-step PCR procedure, the nucleotide sequence encoding BPL (double underlined in Figure 6) was linked via a linker sequence to the 3' end of the nucleotide sequence encoding the gLuc protein (Gaussia luciferase) (underlined in Figure 6). The gLuc protein was used as a model for a useful component to be linked to BPL. This resulted in a polynucleotide (hereinafter sometimes referred to as a gLuc-BPL polynucleotide) in which the nucleotide sequences encoding the gLuc protein and BPL were linked in order from the 5' end. The nucleotide sequence encoding BPL encodes the amino acid sequence shown in SEQ ID NO: 2.

[0094] Next, a Gag-FLAG-5BCCP polynucleotide was inserted into the multicloning site of the pcDNA3.1 vector (Thermo Fisher Scientific, Inc.) according to a conventional recombination procedure to obtain pCMV construct 1-1. Separately, a gLuc-BPL polynucleotide was inserted into the multicloning site of the pcDNA3.1 vector to obtain pCMV construct 1-2.

[0095] The resulting pCMV constructs 1-1 and 1-2 were introduced into HEK293 cells, which are extracellular vesicle (EV)-producing cells, at an arbitrary ratio, and then cultured for 24 or 48 hours according to known procedures for expression and recovery, followed by further culture in the presence of biotin for 24 hours. EVs were then collected from the resulting culture supernatant by centrifugation.

[0096] Specifically, construct 1-1 and construct 1-2 were introduced into EK293 cells and cultured for 24 or 48 hours. The medium was then replaced with Opti-MEM® medium (Thermo Fisher Scientific Inc., containing 0.4 μM biotin) for an additional 24 hours in the presence of biotin. The resulting cultures were then centrifuged (300 g, 10 minutes, 2000 g, 20 minutes, both at 4°C). The culture supernatants were then centrifuged (10,000 g, 30 minutes, 4°C) to separate the precipitate and supernatant fractions. Extracellular vesicles (medium / large EVs, hereafter referred to as MVs) were collected from the precipitate fraction. The complexes linked to extracellular vesicles obtained in this manner were designated Complex 1 (Example 1). Of Complex 1 (Example 1), the complex obtained by changing the medium to Opti-MEM medium (containing biotin) after the 24-hour culture was designated Complex 1-1 (Example 1-1), and the complex obtained by changing the medium to Opti-MEM medium (containing biotin) after the 48-hour culture was designated Complex 1-2 (Example 1-2).

[0097] The supernatant fraction was passed through a syringe filter with a pore size of 0.2 μm and subjected to ultracentrifugation (100,000 g, 1 hour, 4°C), from which the precipitate fraction was obtained. Extracellular vesicles (small EVs, hereinafter referred to as sEVs) were recovered from the resulting precipitate fraction. The complex linked to the extracellular vesicles thus obtained was designated Complex 2 (Example 2). Of Complex 2 (Example 2), the complex obtained by changing the medium to Opti-MEM (containing biotin) after the 24-hour culture was designated Complex 2-1 (Example 2-1), and the complex obtained by changing the medium to Opti-MEM (containing biotin) after the 48-hour culture was designated Complex 2-2 (Example 2-2).

[0098] As a reference example, a polynucleotide in which the nucleotide sequence encoding the Gag protein and the nucleotide sequence encoding the gLuc protein were linked via a nucleotide sequence encoding a FLAG tag (hereinafter sometimes referred to as the Gag-gLuc polynucleotide) was used instead of the Gag-FLAG-5BCCP polynucleotide and the gLuc-BPL polynucleotide. The polynucleotide was inserted into a vector in the same manner as above to obtain Reference Construct 1. Reference Construct 1 was introduced into HEK293 cells in the same manner as above, followed by culture and centrifugation to separate the precipitate and supernatant fractions. Extracellular vesicles (medium / large EVs, small EVs) were similarly recovered from each of the precipitate and supernatant fractions. The complexes linked to extracellular vesicles thus obtained were designated Reference Complex 1 (Reference Example 1) and Reference Complex 2 (Reference Example 2), respectively.

[0099] Furthermore, of Reference Complex 1 (Reference Example 1), the complex obtained by changing the medium to Opti-MEM medium (containing biotin) after the 24-hour culture was designated Reference Complex 1-1 (Reference Example 1-1), and the complex obtained by changing the medium to Opti-MEM medium (containing biotin) after the 48-hour culture was designated Reference Complex 1-2 (Reference Example 1-2). Furthermore, of Reference Complex 2 (Reference Example 2), the complex obtained by changing the medium to Opti-MEM medium (containing biotin) after the 24-hour culture was designated Reference Example 2-1, and the complex obtained by changing the medium to Opti-MEM medium (containing biotin) after the 48-hour culture was designated Reference Example 2-2.

[0100] 7 is a model diagram of an example of loading useful components into extracellular vesicles using the BCCP-BPL biotinylation reaction used in Test Example 1 (Examples 1 and 2). For each complex obtained as described above (Table 1), the expression level (loading amount) of gLuc in the extracellular vesicles was measured and calculated.

[0101]

[0102] To explain the measurement and calculation of the expression level of gLuc specifically, taking Example 1-1 as an example, MV contained in the culture supernatant of cells transfected with the vector was collected by stepwise centrifugation and suspended in PBS, and then luciferase activity was measured using a Picagene Dual Kit (Toyo Ink Co., Ltd., Picagene (registered trademark) from Toyo B-Net Co., Ltd.). That is, according to the kit's instruction manual, the MV membrane was dissolved in the lysis solution provided with the kit, and then a luciferase activity measurement reagent was mixed, and the luminescence level was measured with a luminometer. Measurements and calculations were performed in the same manner for Examples and Reference Examples other than Example 1-1.

[0103] 1-2. Results The results are shown in Figure 8. As shown in Figure 8, it was found that the useful components were efficiently loaded onto the extracellular vesicles in both the complexes of Example 1-1 and Example 1-2 (loaded onto extracellular vesicles MV (medium / large EV)). In particular, about six times the amount of gLuc was detected in Example 1-2 compared to Reference Example 1-2, demonstrating that the useful components were loaded onto the extracellular vesicles very efficiently.

[0104] Furthermore, although not shown in Figure 8, it was confirmed that the useful components were successfully loaded onto extracellular vesicles in the complexes of Examples 2-1 and 2-2 (loaded onto small endoplasmic reticulum sEVs (small EVs)). This demonstrates that the complexes of Examples 1 and 2 using the BCCP-BPL conjugation system are useful as a means of loading useful components onto extracellular vesicles.

[0105] In this test example, the molecular weight of BCCP per molecule was 7K, the molecular weight of BPL was 27K, and the number of BCCP molecules per molecule of extracellular vesicle-targeting protein was 5. The number of BPL molecules per molecule of BCCP was 1.

[0106] From these findings, it was confirmed that useful substances can be efficiently loaded onto extracellular vesicles by using a complex in which BCCP and BPL linked to a useful substance are linked via biotin.

[0107] Test Example 22-1 Preparation of Complexes Supported in Extracellular Vesicles (Examples 3 to 14) Complexes supported in extracellular vesicles were prepared in the same manner as in Test Example 1, except that the contact concentration of biotin during culture was changed.

[0108] Specifically, the constructs were introduced into EK293 cells as described in Test Example 1 and cultured for 24 hours. The medium was then replaced with DMEM (Dulbecco's Modified Eagle's Medium, Nissui Co. Ltd.) and cultured for another 24 hours. The medium was then replaced with biotin-containing or biotin-free medium and cultured for another 24 hours. The resulting culture medium was then centrifuged as described above to obtain various complexes. The biotin-containing medium used was Opti-MEM medium, FBS-containing DMEM medium (biotin content in the medium: approximately 0.2 nM [Table 9: DMEM + biotin (biotin present, approximately 0.2 nM)], or biotin content in the medium: 0.2 nM + 100 nM [Table 9: DMEM + biotin (biotin present, approximately 0.2 nM + 100 nM)]. FBS contains biotin, and the biotin content in the FBS-containing DMEM medium was adjusted by adjusting the amount of FBS and the amount of biotin solution added. DMEM medium was used as a biotin-free medium.

[0109] In addition, complexes were obtained in the same manner as described above, except that the introduction ratio of pCMV construct 1-1 and pCMV construct 1-2 was changed in Test Example 1. In Test Example 1, the introduction ratio (mass ratio of plasmid DNA) of CMV construct 1-1 and pCMV construct 1-2 was 1:1, while in this test, molecular weight ratios were 1:1, 1:2, and 2:1. In this test, these constructs were introduced into cells, and then the cells were cultured for 24 hours. The medium was replaced with DMEM medium, and the cells were further cultured for 24 hours. Subsequently, the cells were cultured in FBS-containing DMEM medium (biotin concentration in the medium: approximately 0.2 nM).

[0110] 2-2. Results The results are shown in Figures 9 and 10. Figure 9 shows the results of MVs derived from precipitate fractions contacted with biotin after 48 hours of culture following introduction of Construct 1-1 and Construct 1-2 into cells (Examples 3 to 5, Comparative Example 1), and the results of MVs derived from precipitate fractions contacted with biotin using Gag-gLuc polynucleotide instead of Construct 1-1 and Construct 1-2 (FBS-containing DMEM medium) (Reference Example 3). As shown in Figure 9, almost no useful components were loaded onto extracellular vesicles under biotin-free conditions (Comparative Example 1), whereas useful components were loaded onto extracellular vesicles under biotin-added conditions (Examples 3 to 5). This confirmed that the biotin-mediated BCCP-BPL linkage system was functioning.

[0111] Although Figure 9 shows the results for MV, similar measurements and calculations were performed on sEV derived from the supernatant fraction. When biotin was not added (Comparative Example 2), almost no useful components were loaded onto the extracellular vesicles, whereas when biotin was added (Examples 6 to 8), useful components were loaded onto the extracellular vesicles. This also confirmed that the biotin-mediated BCCP-BPL linkage system was functioning.

[0112] Furthermore, as shown in Figure 10, it was confirmed that useful components could be efficiently loaded into extracellular vesicles even when the ratio of BCCP to BPL was changed.

[0113] Test Example 33-1. Preparation of Complexes Supported on Extracellular Vesicles Complexes linked to extracellular vesicles were prepared using the same procedure as in Test Example 1, except that PKA inhibitor peptides (PKI) were used instead of gLuc protein as the useful component. Specifically, five repeats of a PKI-encoding sequence were linked to the 3'-end of a MYC tag-encoding sequence, and a BPL-encoding sequence was further linked to the 3'-end of the repeats. The PKI-encoding sequences were linked via a (G4S)3 linker. The MYC tag-encoding sequence and the PKI-encoding sequence, and the PKI-encoding sequence and the BPL-encoding sequence were also linked via linkers. This resulted in a polynucleotide in which the MYC tag, PKI (five repeats), and BPL-encoding sequences were linked in order from the 5'-end (hereinafter, sometimes referred to as the Myc tag-5PKI-BPL polynucleotide). The MYC tag-encoding sequence used in this test example encodes the amino acid sequence (EQKLISEEDL) set forth in SEQ ID NO:9. The base sequence encoding PKI used in this test example encodes the amino acid sequence (TYADFIASGRTGRRNAI) shown in SEQ ID NO: 10. Gag-FLAG-5BCCP polynucleotide was obtained in the same manner as in Test Example 1.

[0114] Next, the Gag-FLAG-5BCCP polynucleotide was inserted into the multicloning site of the pcDNA3.1 vector to obtain pCMV construct 2-1 in the same manner as in Test Example 1. Similarly, the Myc tag-5PKI-BPL polynucleotide was inserted into the multicloning site of the pcDNA3.1 vector to obtain pCMV construct 2-2.

[0115] The resulting pCMV constructs 2-1 and 2-2 were used in the same procedure as in Test Example 1, as shown in Figure 11. After gene transfer, the cells were cultured for 48 hours. The medium was then replaced with FBS-containing DMEM or Opti-MEM medium, and the cells were cultured for an additional 24 hours. Extracellular vesicle MVs were then recovered from the precipitated fraction. Western blotting was then performed according to procedures known in the art to confirm whether the useful component, the PKA inhibitor peptide, was incorporated into the extracellular vesicle MVs. Anti-myc and anti-rabbit IgG antibodies were used for detection.

[0116] 3-2. Results In this test example, a complex was produced in the same manner as in Test Example 1, except that PKI was used as the useful component. It was also confirmed that PKI and Gag coexist in extracellular vesicles. This confirmed that useful components can be loaded into extracellular vesicles even when PKI, a PKA inhibitor peptide, is used as the useful component instead of gLuc protein.

[0117] It has been known that tumor-associated macrophages (TAMs) present in the tumor microenvironment are involved in cancer progression and have the ability to inhibit the immune system against cancer. Therefore, in this test example, we investigated whether the function of TAMs can be inhibited using the complex. Specifically, PKA is known to be involved in the immune inhibitory action of TAMs, and increased PKA is known to be one of the causes of the immune inhibitory action of TAMs. Therefore, we used the complex (using Opti-MEM medium, Example 15) to act on TAM-like macrophages with endoplasmic reticulum loaded with PKI, a known PKA inhibitor, as an active ingredient.

[0118] The results are shown in Figures 12 to 14. Specifically, macrophages were differentiated into TAM-like cells by adding cancer cell culture supernatant, and then various samples, such as endoplasmic reticulum, were added. The mRNA expression of TAM marker molecules was measured by real-time RT-PCR. The results are shown in Figure 12. The same procedure was repeated to determine the degree of differentiation into M1 macrophages, a macrophage phenotype opposite to TAM. The mRNA expression of M1 marker molecules was measured by real-time RT-PCR. The results are shown in Figure 13. Furthermore, Western blotting was used to confirm whether PKI actually inhibited activation of the PKA pathway. In the figure, Mφ refers to control macrophages (no sample added).

[0119] As shown in Figures 12 and 13, when PKI was applied to extracellular vesicles prepared using the complex (Example 15) as described above (TAM + PKI-MV (1, 10, 30, 50 μg / mL) in both figures), the M2 marker level decreased and the M1 marker level increased. This means that the expression of M2 type (immunosuppressive type) macrophages decreased and the expression of M1 type (immunostimulatory type) macrophages increased, which means that the immunosuppressive function of macrophages was reduced. From this, it was understood that by using the complex in combination with extracellular vesicles, useful components can be efficiently delivered to target macrophages and even cancer cells, etc.

[0120] Furthermore, as shown in Figure 14, when PKI was applied to the endoplasmic reticulum (PKI-MVs in the figure), the rate of CREB phosphorylation decreased. This confirmed that the expected signal pathway was changed by adding extracellular vesicles carrying a PKA inhibitory peptide to TAM. This also confirmed that the use of the complex in combination with extracellular vesicles can efficiently deliver useful components to target cells such as macrophages.

[0121] From these findings, it was confirmed that the complex of the present disclosure can effectively deliver useful components to target subjects when used in combination with extracellular vesicles.

[0122] Test Example 44-1. Preparation of Complex Supported in Extracellular Vesicles (Example 16) A complex supported in extracellular vesicles was prepared in the same manner as in Test Example 3, except that a STAT3 inhibitor protein (STAT3i) was used instead of gLuc protein as the useful component. Specifically, five repeats of a STAT3i-encoding nucleotide sequence were linked to the 3'-end of a nucleotide sequence encoding a MYC tag, and a BPL-encoding nucleotide sequence was further linked to the 3'-end of the repeats. The STAT3i-encoding nucleotide sequences were linked via a (G4S)3 linker. The MYC tag-encoding nucleotide sequence and the STAT3i-encoding nucleotide sequence, and the STAT3i-encoding nucleotide sequence and the BPL-encoding nucleotide sequence were also linked via linkers. This resulted in a polynucleotide in which the MYC tag, STAT3i (five repeats), and BPL-encoding nucleotide sequences were linked in order from the 5'-end (hereinafter, sometimes referred to as a Myc tag-5STAT3i-BPL polynucleotide). The base sequence of STAT3 used in this test example encodes the amino acid sequence (VRHSALHMAVGPLSWPARVS) shown in SEQ ID NO: 11. Gag-FLAG-5BCCP polynucleotide was obtained in the same manner as in Test Example 3.

[0123] Next, the Gag-FLAG-5BCCP polynucleotide was inserted into the multicloning site of the pcDNA3.1 vector to obtain pCMV construct 3-1 in the same manner as in Test Example 3. Similarly, the Myc tag-5STAT3i-BPL polynucleotide was inserted into the multicloning site of the pcDNA3.1 vector to obtain pCMV construct 3-2.

[0124] Following the same procedure as in Test Example 1, the cells were cultured for 48 hours after gene transfer, then the medium was replaced with FBS-containing DMEM medium and further cultured for 24 hours. MVs were then recovered from the precipitated fraction. The mixing ratio (mass ratio of plasmid DNA) of pCMV construct 3-1 and pCMV construct 3-2 was 1:1 or 1:2. Extracellular vesicles loaded with the active ingredient STAT3i using a biotinylation reaction were designated Example 16 (mixing ratio 1:1) and Example 17 (mixing ratio 1:2). Next, as in Test Example 3, Western blotting and real-time RT-PCR were used to confirm whether the useful ingredient, STAT3 inhibitory protein, was incorporated into the extracellular vesicle MVs, and their immunosuppressive function was also confirmed.

[0125] 4-2. Results As a result, similar to Test Example 3, it was confirmed that the useful component STAT3i was efficiently loaded onto extracellular vesicles in both Examples 16 and 17.

[0126] Furthermore, as in Test Example 3, when STAT3i was administered using the extracellular vesicles of Example 16 (TAM + rS3-MV (10, 50 μg / mL)), M2 marker levels were reduced and M1 marker levels were increased. A similar trend was observed when the extracellular vesicles of Example 17 were used. From this, it was understood that in this test example, the expression of M2 type macrophages was reduced and the expression of M1 type macrophages was increased, thereby reducing the immune inhibitory function of macrophages. Furthermore, it was confirmed that the expected signal pathway was changed by adding extracellular vesicles carrying a STAT3 inhibitory protein to TAM.

[0127] This also confirms that the complex of the present disclosure, when used in combination with extracellular vesicles, can efficiently deliver useful components to target macrophages and even cancer cells.

[0128] Furthermore, these findings confirm that the complex of the present disclosure allows multiple BCCPs to be linked and used, thereby enabling efficient linkage of useful components to BCCPs. Furthermore, since two or more BCCP molecules could be linked to one molecule of extracellular vesicle-targeting protein, it was found that useful components could be efficiently loaded onto extracellular vesicles via BCCP. This confirms that useful components can be efficiently delivered to target subjects.

[0129] Test Example 55-1. Test Procedure The extracellular vesicles prepared in Test Example 3 (hereinafter referred to as PKI-MVs) or the extracellular vesicles of Example 16 prepared in Test Example 4 (hereinafter referred to as STAT3i-MVs) were administered into the tumor tissue of mice, and the change in intratumoral macrophages from M2 to M1 type, as well as the number and activation state of T cells in the tumor tissue, were evaluated. In addition, the extracellular vesicles (PKI-MVs) prepared in Test Example 3 were administered into the tumor tissue of tumor-bearing mice, and tests were conducted to examine their use in combination with conventional immune checkpoint therapy (administration of anti-PD-1 antibody).

[0130] Specifically, tumor-bearing mice were generated by intradermal transplantation of 4T1 cells, and the extracellular vesicles (PKI-MVs, STAT3i-MVs) were administered into the tumor tissue. The tumor tissue was then harvested, frozen, and immunostained to assess the differentiation state of macrophages and the number and activation state of T cells within the tumor tissue (Figure 15). Separately, tumor-bearing mice were similarly generated and administered the extracellular vesicles into the tumor tissue, along with intraperitoneal administration of an anti-PD-1 antibody. The tumor growth inhibitory effect was assessed by measuring tumor volume over time (Figure 16).

[0131] 5-2. Results The results are shown in Figures 15 and 16. Figure 15 shows the change in intratumoral macrophages from M2 to M1 (a) and the number and activation state of T cells in tumor tissue (b). In (a), the expression of M2 was higher than that of M1 in the group administered with PBS alone, whereas the expression of M2 was reduced and the expression of M1 was increased when either the PKI-MVs of Test Example 3 or the STAT3i-MVs of Test Example 4 was administered. Furthermore, the results in (b) showed that the number of T cells increased when either the PKI-MVs of Test Example 3 or the STAT3i-MVs of Test Example 4 was administered. These results demonstrate that the use of a complex formed by linking BPL and BCCP linked to a useful ingredient via biotin in combination with extracellular vesicles in vivo enables efficient delivery of useful ingredients.

[0132] Furthermore, as shown in Figure 16, administration of the PKI-MVs of Test Example 3 reduced tumor size in mice. Furthermore, when an anti-PD-1 antibody, known as a type of tumor treatment, was administered in combination with the PKI-MVs of Test Example 3, tumor size was reduced even more than when either antibody was administered alone. These findings also demonstrate that the conjugate of the present disclosure is useful for delivering active ingredients.

[0133] The test example uses BPL as the first binding moiety and BCCP as the second binding moiety, and by utilizing the principle of specific binding between BPL and BCCP, the complex of the test example was able to successfully load useful components onto extracellular vesicles and deliver the useful components to the target. This confirms that even when a means such as the SpyCatcher / SpyTag system, which utilizes specific binding similar to that between BPL and BCCP, is used instead of the combination of BPL and BCCP, useful components can be successfully loaded onto extracellular vesicles and delivered to the target.

Claims

1. A complex for use in linkage with extracellular vesicles, comprising: (1) a first binding part directly or indirectly linked to a useful ingredient; (2) a second binding part that specifically binds to the first binding part; and (3) the first binding part and the second binding part are linked to each other.

2. The complex of claim 1, wherein the first binding portion, the second binding portion and the specific bond are either (i) or (ii) below: (i) the first binding portion is biotin protein ligase, the second binding portion is biotin carboxyl carrier protein, and the specific bond is a bond via biotin; or (ii) the first binding portion is SpyCatcher, the second binding portion is SpyTag, and the specific bond is an isopeptide bond.

3. The complex according to claim 1, wherein the useful component is at least one selected from the group consisting of proteins, peptides, amino acids and nucleic acids.

4. The complex described in claim 1, further comprising an extracellular vesicle-targeting protein, wherein the extracellular vesicle-targeting protein is directly or indirectly linked to the second binding portion.

5. The complex described in claim 4, which contains two or more molecules of the second binding portion per molecule of the extracellular vesicle-targeting protein.

6. An extracellular vesicle to which the complex described in any one of claims 1 to 5 is linked.

7. A cell that secretes extracellular vesicles, comprising at least one vector selected from the group consisting of a vector capable of expressing a first binding part directly or indirectly linked to a useful ingredient and a vector capable of expressing a second binding part.

8. A cell secreting extracellular vesicles as described in claim 7, wherein the vector capable of expressing the second binding portion is a vector capable of expressing a second binding portion that is directly or indirectly linked to an extracellular vesicle-targeting protein.

9. A method for producing the complex described in claim 4, comprising the following steps (a1) and (a2): (a1) mixing a first binding part directly or indirectly linked to a useful component with a second binding part directly or indirectly linked to an extracellular vesicle-targeting protein; and (a2) binding the first binding part to the second binding part, thereby indirectly linking the useful component to the extracellular vesicle-targeting protein via the bond.

10. A method for producing extracellular vesicles carrying the complex described in claim 1, comprising the following steps (b1) and (b2): (b1) mixing extracellular vesicles carrying a first binding part directly or indirectly linked to a useful component and a second binding part directly or indirectly linked to an extracellular vesicle-targeting protein; and (b2) binding the first binding part to the second binding part, thereby indirectly linking the useful component to the extracellular vesicles via the bond.

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