Intracellular delivery of target substance by CD47-mediated endocytosis

Abstract

The present invention relates to a method for intracellular delivery of a target substance by CD47-mediated endocytosis and, more specifically, to an improved method for intracellular delivery of a target substance by CD47-mediated endocytosis initiated / induced by interaction between a ligand for CD47 and CD47 expressed on a target cell.

Description

Method for intracellular delivery of target substances by CD47-mediated endocytosis

[0001] This application claims the benefit of Korean Patent Application No. 10-2024-0050923, filed April 16, 2024, and Korean Patent Application No. 10-2025-0042865, filed April 2, 2025, the entire contents of which are incorporated herein by reference.

[0002] The present invention relates to a method for intracellular delivery of a target substance by CD47-mediated endocytosis, and more particularly, to an improved method for intracellular delivery of a target substance by CD47-mediated endocytosis initiated / induced by the interaction of a ligand for CD47 and CD47 expressed on a target cell.

[0003]

[0004] The mammalian cell membrane represents a largely impermeable barrier to extracellular substances, posing a major obstacle to drug development. Therefore, numerous cell delivery systems have been developed for intracellular delivery, including electroporation, physical methods like microinjection, and the use of cationic lipid constructs, protein transduction agents, and cell-penetrating peptides. Despite the development of various technologies, intracellular delivery remains one of the most challenging technical challenges in drug development.

[0005] Although clathrin-mediated endocytosis and micropinocytosis, the classical endocytic pathways that are major routes of intracellular transport, exhibit limitations in their ability to transport extracellular materials, clathrin-independent endocytosis offers the exception of being relatively free from endosomal entrapment and subsequent degradation. While avoiding endosomal entrapment and lysosomal degradation is a crucial step in the transport of extracellular materials, designing cell-penetrating and useful internalizing peptides that meet these goals remains challenging, despite serious efforts to design novel strategies to prevent degradation.

[0006] Meanwhile, extracellular particles (ECPs) are highly biocompatible particles released from cells. They can be divided into EVPs, which are spherical particles with a lipid bilayer, and non-vesicular EPPs, which are spherical particles without a lipid bilayer. EPPs are present in most body fluids, including blood and urine, and are known to mediate signaling between cells through physiologically active substances such as proteins, mRNA, and miRNA. Furthermore, EPPs are secreted by fusing with the cell membrane, protecting the loaded substances from plasma and immune components in the body and enabling material transfer to recipient cells through endocytosis. In particular, they can load a wide range of biological substances, such as proteins, membrane receptors, and nucleic acids, and are emerging as a new concept of therapeutic carriers for drug delivery systems.

[0007] However, since the delivery of extracellular substances using extracellular particles also involves clathrin-mediated endocytosis and macropinocytosis to a certain extent, it is necessary to avoid endosomal capture and lysosomal degradation processes for effective delivery of extracellular substances.

[0008]

[0009] Accordingly, the inventor of the present invention has conducted repeated research to develop surface-engineered extracellular particles that can bypass the clathrin-mediated endocytosis and macropinocytosis pathways in order to efficiently deliver a target substance into a cell using extracellular particles. As a result, the inventor has discovered that when a target substance is loaded into an extracellular particle containing a ligand capable of interacting with CD47 and delivered to a target cell, the rate of delivery into the cell by endocytosis of the clathrin-independent pathway is significantly improved, thereby completing the present invention.

[0010] Accordingly, the purpose of the present invention is to provide a method for delivering a target substance to a target cell, comprising a step of contacting extracellular particles loaded with a target substance, the extracellular particles comprising a ligand for CD47, a fragment thereof, a variant thereof, a variant of a fragment thereof, or a fragment of a variant thereof, with the target cell.

[0011] Another object of the present invention is to provide a composition for delivering a target substance to a target cell, comprising a ligand for CD47, a fragment thereof, a variant thereof, a variant of a fragment thereof, or a fragment of a variant thereof, and comprising extracellular particles loaded with a target substance.

[0012] Another object of the present invention is to provide a method for improving the delivery efficiency of a target substance into a target cell, comprising the following steps:

[0013] (a) a step of loading a target substance into an extracellular particle comprising a ligand for CD47, a fragment thereof, a variant thereof, a variant of a fragment thereof, or a fragment of a variant thereof; and

[0014] (b) A step of bringing the extracellular particles into contact with a target cell.

[0015] Another object of the present invention is to provide a use of a ligand for CD47, a fragment thereof, a variant thereof, a variant of a fragment thereof, or a fragment of a variant thereof for preparing a composition for delivering a target substance to a target cell.

[0016] Another object of the present invention is to provide a use of extracellular particles loaded with a target substance for preparing a composition for delivering a target substance to a target cell.

[0017]

[0018] In order to achieve the above-described object of the present invention, the present invention provides a method for delivering a target substance to a target cell, comprising a step of contacting an extracellular particle loaded with a target substance, the extracellular particle comprising a ligand for CD47, a fragment thereof, a variant thereof, a variant of a fragment thereof, or a fragment of a variant thereof, with the target cell.

[0019] In order to achieve another object of the present invention, the present invention provides a composition for delivering a target substance to a target cell, the composition comprising a ligand for CD47, a fragment thereof, a variant thereof, a variant of a fragment thereof, or a fragment of a variant thereof, and an extracellular particle loaded with a target substance.

[0020] In order to achieve another object of the present invention, the present invention provides the use of a ligand for CD47, a fragment thereof, a variant thereof, a variant of a fragment thereof, or a fragment of a variant thereof for preparing a composition for delivery of a target substance to a target cell.

[0021] In order to achieve another object of the present invention, the present invention provides the use of extracellular particles loaded with a target substance for preparing a composition for delivering a target substance to a target cell.

[0022]

[0023] Unless otherwise defined, all technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains. The following terms used herein have the meanings assigned to them below.

[0024] The terms "comprising" and "having" (and all forms of "having" and "having") as used herein are to be interpreted as inclusive or open-ended and do not exclude additional configurations, unrecited configurations or methods, etc.

[0025] As used herein, the terms "a" or "an" when used in conjunction with the term "comprising" in the claims and / or the specification may mean "one," but may also be interpreted as "one or more" and "at least one." The use of the term "or" in the claims is intended to mean "and / or" unless the context explicitly states that only alternatives are mentioned or that the alternatives are mutually exclusive. The use of the term "at least one" is understood to include any quantity of one or more, including but not limited to one, as well as 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 100, or any integer included therein. The term "at least one" may extend up to 100 or 1000 or more, depending on the term to which it is appended. Furthermore, the quantity of 100 / 1000 should not be considered limiting. Additionally, the use of the terms “at least one of X, Y and Z”, “selected from the group consisting of X, Y and Z” is understood to include X alone, Y alone and Z alone, as well as any combination of X, Y and Z.

[0026] As used herein, the terms "combinations thereof" or "their combinations" mean all permutations and combinations of the items listed above. For example, "A, B, C, or any combination thereof" is intended to include at least one of A, B, C, AB, AC, BC, or ABC. Where order is important in a particular context, BA, CA, CB, CBA, BCA, ACB, BAC, or CAB are also included. Combinations that include repetitions of one or more items or terms are also expressly included, such as BB, AAA, AAB, BBC, AAABCCCC, CBBAAA, CABABB, and the like. Those skilled in the art will appreciate that there is generally no limit to the number of items or terms in any combination, unless the context clearly dictates otherwise.

[0027] The term "about" as used herein is used to indicate that the values ​​include inherent error variations for the composition, the method used to administer the composition, or variations that exist between study subjects.

[0028] The term "substantially," as used herein, means that the subsequently described event or circumstance occurs completely, or that the subsequently described event or circumstance occurs to a significant degree or extent. For example, the term "substantially" means that the subsequently described event or circumstance occurs at least 90% of the time, at least 95% of the time, or at least 98% of the time.

[0029] The term "CD47 (cluster of differentiation 47)" in the present invention refers to a widely expressed transmembrane glycoprotein with a single Ig-like domain and five transmembrane domains, which functions as a cellular ligand or receptor for SIRPα, with binding mediated through the NH2-terminal V-like domain of SIRPα. CD47 is also constitutively upregulated in numerous cancers, such as leukemia. Furthermore, CD47 has been found to be overexpressed in inflammatory necroptotic cells that exacerbate inflammatory responses and in pathological fibroblasts, which are the root cause of fibrosis. Overexpression of CD47 enhances the pathogenicity of these pathological cells by allowing them to avoid phagocytosis by immune cells in the body. While targeting CD47 represents a unique mechanism of action for the treatment of rare and intractable diseases, the widespread expression of CD47 presents a therapeutic challenge. Furthermore, the treatment of leukemia and related hematologic disorders presents special challenges, such as the adverse effects of cancer on bone marrow and hematologic functions and the complications of tumor lysis syndrome. CD47 is upregulated on diseased cells, but is also ubiquitously expressed at relatively high levels on all normal cells, including natural killer (NK) cells, red blood cells (RBCs), and platelets. CD47 is a high-affinity receptor for thrombospondin-1 (TSP-1), a secreted glycoprotein that plays a role in vascular development and angiogenesis. In this latter capacity, TSP1-CD47 interaction inhibits nitric oxide signaling at multiple levels in vascular cells. The binding of TSP-1 to CD47 influences several fundamental cellular functions, including cell migration and adhesion, cell proliferation, or apoptosis, and plays a role in the regulation of angiogenesis and inflammation. CD47 also interacts with signal-regulating protein alpha (SIRPα), an inhibitory transmembrane receptor present on myeloid cells, including macrophages.The CD47 / SIRPα interaction induces bidirectional signaling, leading to various intercellular responses, including inhibition of phagocytosis, stimulation of cell-to-cell fusion, and T cell activation. CD47 also interacts with signal regulatory protein beta (SIRPβ), expressed on the surface of immune cells. The CD47 / SIRPβ interaction is primarily involved in signal transduction related to immune cell activation, and may particularly facilitate the activation of cells such as NK cells. CD47 also interacts with signal regulatory protein gamma (SIRPγ), expressed on the surface of immune cells. The CD47 / SIRPγ interaction is crucial for T cell activation and the regulation of immune responses. Furthermore, CD47 interacts with various membrane integrins, most commonly integrin αVβ3. These interactions result in the formation of CD47 / integrin complexes, which influence various cellular functions, including cell adhesion, proliferation, and migration. In the present invention, CD47 preferably refers to the human CD47 protein. The amino acid sequence and related information for human CD47 are known, for example, as NP_001768, NP_942088, NP_001369235, etc., and the gene sequence and related information are known, for example, as NM_001025079, NM_001025080, NM_001777, NM_198793, NM_001382306, etc.

[0030] As used herein, the term "receptor" refers to a biological molecule or molecular group capable of binding to a ligand. A receptor may be provided to transmit information within a cell, cell type, or organism. A receptor comprises at least one receptor unit, and each receptor unit may, for example, be composed of a protein molecule. A receptor can form a complex with a ligand as a binding partner. Various signaling information is transmitted, particularly through changes in the conformation of the receptor, following complexation with the ligand on the cell surface. In the present invention, the receptor may include CD47.

[0031] As used herein, the term "ligand" refers to a molecule or binding moiety that has a complementary structure to a receptor or can interact with a receptor (i.e., CD47) to form a complex. For example, the ligand may be used to refer to a molecule that can reversibly and / or irreversibly bind to a polypeptide or protein with high affinity. In some embodiments, the ligand may be understood to mean a protein, peptide, or fragment thereof that has a binding motif suitable for its amino acid sequence and is capable of forming a complex with the receptor. The terms "binds specifically" or "has selective affinity" as used herein with respect to a ligand mean that the ligand reacts or binds more frequently, more rapidly, for a longer period of time, with greater affinity, or a combination thereof, to a specific epitope compared to alternative substances, including unrelated proteins. "Specific binding" does not necessarily require exclusive binding, i.e., binding to a single target. Thus, in certain embodiments, a ligand may bind to more than one receptor.

[0032] The term "loading" in the present invention refers to the introduction, association, encapsulation, encasing, and / or encapsulation of a target substance into an extracellular particle (e.g., an extracellular vesicle). In one embodiment, the target substance may be completely or partially loaded into the interior of the extracellular particle. In another embodiment, the target substance may be loaded by being completely or partially exposed to the exterior of the extracellular particle.

[0033] As used herein, the term "association" refers to two or more substances being physically "associated" with each other when they directly or indirectly interact with each other, thereby bringing them into physical proximity and / or maintaining them in physical proximity. In some embodiments, two or more substances that are physically associated with each other are covalently linked to each other; in some embodiments, two or more substances that are physically associated with each other are not covalently linked to each other, but are non-covalently linked to each other, for example, by hydrogen bonding, van der Waals interactions, hydrophobic interactions, magnetism, and combinations thereof.

[0034] As used herein, the term "lipid bilayer particle" refers to a particle formed by a thin polar membrane composed of two layers of lipid molecules. The biological lipid bilayer particle may comprise an amphipathic phospholipid having a hydrophilic phosphate head and a hydrophobic tail composed of two fatty acid chains. In one embodiment, the lipid bilayer particle may be a cell membrane particle or a cell-derived membrane particle. The term "cell membrane particle" or "cell-derived membrane particle" as used herein should be interpreted to include all membrane-derived vesicles or particles that can be produced by blebbing or budding, and may also include hybrid vesicles produced by mixing cell-generated vesicles and synthetic vesicles, and vesicles or particles produced by mechanically treating cells. Thus, "cell-derived membrane particles" may include, but are not limited to, extracellular vesicles, virus particles, virus-like particles, and platelet-like particles. In another aspect, the lipid bilayer particles may be non-cell derived (e.g., liposomes).

[0035] In the present invention, the term "target cell" refers to any cell that will receive a target substance loaded into an extracellular particle, including a lipid bilayer particle.

[0036] As used herein, the term "contacting" refers to establishing a physical connection between two or more entities. In some embodiments, contacting a cell with an extracellular particle refers to manipulating the cell and the extracellular particle so that they can be physically connected. Methods for contacting cells with external entities, both in vivo and ex vivo, are well known in the biological arts. In some embodiments, contact between an extracellular particle and a mammalian cell present in a mammal can be accomplished by various routes of administration (e.g., intravenous, intramuscular, intradermal, and subcutaneous), and various amounts of the extracellular particle can be involved in contacting the cell.

[0037] As used herein, the term "extracellular vesicle" refers to a cell-derived vesicle comprising a membrane surrounding an internal space. Extracellular vesicles are composed of all membrane-bound vesicles smaller in diameter than the cell from which they are derived. Typically, extracellular vesicles range in diameter from 20 nm to 1000 nm and may contain various macromolecular cargoes within their internal space, displayed on the outer surface of the extracellular vesicle, and / or spanning the membrane. The cargoes may include small molecules, nucleic acids, proteins, carbohydrates, lipids, small molecules, and / or combinations thereof. By way of example and without limitation, extracellular vesicles include apoptotic bodies, cell fragments, vesicles derived from cells by direct or indirect manipulation (e.g., continuous extrusion or treatment with alkaline solutions), vesicular organelles, and vesicles generated by living cells (e.g., by direct plasma membrane budding or fusion of late endosomes with the plasma membrane). In one aspect, the extracellular vesicles may be selected from the group consisting of exosomes, microvesicles, and apoptotic bodies. The extracellular vesicles may be derived from living or dead organisms, transplanted tissues or organs, and / or cultured cells.

[0038] The term "exosome" as used herein refers to a cell-derived vesicle comprising a membrane surrounding an internal space, which is generated from the cell by direct plasma membrane budding or by fusion of late endosomes with the plasma membrane. Exosomes may contain lipids or fatty acids and polypeptides, and may optionally contain cargo as described above. Exosomes may be derived from producer cells and may be isolated from producer cells based on size, density, biochemical parameters, or a combination thereof. Exosomes are a type of extracellular vesicle.

[0039] The term "non-vesicular extracellular particle" in the present invention refers to a particle secreted from a cell and not surrounded by a cell membrane, and includes particles such as exomeres and supermeres. Exomeres are very small, less than 50 nm in size, and can transport proteins, lipids, nucleic acids, etc. Supermeres are even smaller than exomeres and are known to play a major role in RNA transfer in the body. These particles play an important role in the transmission and regulation of information between cells.

[0040] The term "endocytosis" as used herein refers to the process by which a eukaryotic cell internalizes components of the plasma membrane, cell surface receptors, and extracellular fluid. Endocytosis includes (1) phagocytosis and (2) pinocytosis, which itself includes (2a) micropinocytosis, which does not require receptor binding, (2b) clathrin-mediated endocytosis, (2c) caveolae-mediated endocytosis, and (2d) clathrin- / caveolae-independent endocytosis.

[0041] In the present invention, the term "CD47-mediated endocytosis" means endocytosis mediated and / or initiated by the binding of CD47 and its ligand, endocytosis mediated and / or initiated by the CD47 signaling pathway, and endocytosis in which a target substance of endocytosis enters the cell together with CD47.

[0042] The terms "extracellular vesicle protein", "extracellular vesicle (EV) protein", "exosomal protein", "exosomal polypeptide", "EV polypeptide" and "EV protein" are used interchangeably herein and should be understood to refer to any protein or polypeptide that can be utilized to transport a polypeptide structure, such as a CD47 ligand, into an extracellular vesicle. More specifically, the term "extracellular vesicle protein" should be understood to include any protein or polypeptide that can transport, move or shuttle a polypeptide structure into an extracellular vesicle, such as an exosome. Examples of such exosomal proteins include, for example, CD9, CD53, CD63, CD81, CD54, CD50, FLOT1, FLOT2, CD71, CD133, CD138, CD235a, syntenin-1, syntenin-2, Lamp2b, TSPAN8, TSPAN14, CD37, CD82, CD151, CD231, CD102, NOTCH1, NOTCH2, NOTCH3, NOTCH4, DLL1, DLL4, JAG1, JAG2, CD49d / ITGA4, ITGB5, ITGB6, ITGB7, CD11a, CD11b, CD11c, CD18 / ITGB2, CD41, CD49b, CD49c, CD49e, CD51, CD61, CD104, tetraspanin, Fc receptor, interleukin receptor, Immunoglobulin, MHC-I component, MHC-II component, CD2, CD3 epsilon, CD3 zeta, CD13, CD19, CD30, CD34, CD36, CD40, CD40L, CD44, CD45, CD45RA, CD47, CD86, CD110, CD111, CD115, CD117, CD125, CD135, CD184, CD200, CD279, CD273, CD274, CD362, COL6A1, AGRN, EGFR, GAPDH, GLUR2, GLUR3, HLA-DM, HSPG2, L1CAM,LAMB1, LAMC1, LFA-1, LGALS3BP, Mac-1 alpha, Mac-1 beta, MFGE8, SLIT2, STX3, TCRA, TCRB, TCRD, TCRG, VTI1A, VTI1B, PDGFR, GPI anchor protein, lactadherin, syndecan, synaptotagmin, ALIX (apoptosis-linked gene 2-interacting protein X), PTGFRN (prostaglandin F2 receptor inhibitor), fragments thereof, variants thereof, variants of fragments thereof, fragments of variants thereof, and any combination thereof, but numerous any polypeptides capable of transporting a polypeptide structure into an extracellular vesicle are included within the scope of the present invention. Extracellular vesicle proteins are generally of human origin and can be found in various publicly available databases such as Uniprot, RCSB, etc.

[0043] The term non-vesicular extracellular particle protein, as used herein, is used interchangeably herein and should be understood to relate to any protein or polypeptide that can be utilized to transport a polypeptide structure, such as one comprising a CD47 ligand, into a non-vesicular extracellular particle. More specifically, the term "non-vesicular extracellular particle protein" should be understood to include any protein or polypeptide that can transport, move, or shuttle a polypeptide structure into a non-vesicular extracellular particle. Examples of such non-vesicular extracellular particle proteins include, but are not limited to, HSP90AA / B, TGFβ-induced (TGFBI), HSPA13, LDHA / B, ENO1, ENO2, ALDOA, GPI, ACTN4, AGO1, AGO2, HEXOKINASE I, fragments thereof, variants thereof, variants of fragments thereof, fragments of variants thereof, and any combination thereof.

[0044] In the present invention, the term "SIRP" refers to a regulatory membrane glycoprotein expressed primarily in myeloid cells, but also in stem cells or neurons. Four types of SIRP have been reported: SIRPα, SIRPβ, SIRPγ, and SIRPδ. Among them, SIRPα and SIRPγ are known as inhibitory receptors and interact with CD47, a transmembrane protein widely expressed on various diseased cells. The interaction between SIRP and CD47 is referred to as the "don't eat me" signal. This interaction negatively regulates the effector functions of innate immune cells, such as phagocytosis of diseased cells. This is similar to the self-signaling provided by MHC I family molecules through Ig-like or Ly49 receptors. Diseased cells that overexpress CD47 activate SIRPα or SIRPγ, inhibiting macrophage-mediated destruction. Recent studies have shown that high-affinity mutations in SIRPα mask CD47 in cancer cells, thereby increasing their phagocytosis.

[0045] The term "SIRPα" as used herein refers to a cell surface type I transmembrane protein expressed in macrophages and a member of the SIRP / SHPS (CD172) family within the Ig superfamily. SIRPα is a ligand for CD47. Other names in the art for SIRPα include signal regulatory protein alpha, tyrosine-protein phosphatase non-receptor type substrate 1, BIT, CD172A, MFR, MYD-1, P84, PTPNS1, and SHPS1. An exemplary protein sequence for human SIRPα can be referenced in GENBANK® Accession no. AAH33092.1 (sequence including the signal peptide), and its nucleic acid sequence can be referenced in GENBANK® Accession no. BC033092.1.

[0046] As used herein, the term "SIRPγ" refers to another member of the SIRP / SHPS (CD172) family within the Ig superfamily, a cell surface type I transmembrane protein expressed on, for example, T cells and activated NK cells. SIRPγ can bind to CD47, but the signal transduction mechanism is unknown. Other names in the art for SIRPγ include signal regulatory protein gamma and SIRP beta 2. An exemplary protein sequence for human SIRPγ can be referenced in GENBANK® Accession no. NP_061026.2 (sequence including the signal peptide), and its nucleic acid sequence can be referenced in GENBANK® Accession no. NM_018556.3.

[0047] As used herein, the term "fragment" of a protein, peptide, or nucleic acid refers to a segment of a protein, peptide, or nucleic acid. A fragment of a protein, peptide, or nucleic acid according to some embodiments of the present invention may retain at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the functionality of the protein, peptide, or nucleic acid.

[0048] As used herein, the term "variant" of a protein, peptide, or nucleic acid refers to a protein, peptide, or nucleic acid that has at least one amino acid or nucleotide that differs from the protein, peptide, or nucleic acid. Variations of a protein, peptide, or nucleic acid include, but are not limited to, substitutions, deletions, frame shifts, or rearrangements of the protein, peptide, or nucleic acid. This term may be used interchangeably with the term "mutant." Variants of a protein, peptide, or nucleic acid according to some embodiments of the present invention may retain at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the function of the protein, peptide, or nucleic acid.

[0049] In the present invention, the term “homolog” means a protein having at least 95% sequence identity with a given protein.

[0050] The term "biologically active fragment" as used herein, when used in connection with a given molecule, refers to any fragment, derivative, homolog or analog that retains the activity of said molecule in vivo or in vitro.

[0051] In the present invention, the term "antibody" is used in the broadest sense. Specifically, it includes monoclonal antibodies (including monoclonal antibodies, full-length monoclonal antibodies), polyclonal antibodies (polyclonal antibodies), multispecific antibodies (e.g., bispecific antibodies), and antibody fragments (e.g., variable regions and other portions of antibodies exhibiting desired biological activity). The antibody fragment may be an antigen-binding fragment of the antibody, which means a fragment of the antibody that retains the binding affinity for the antigen to which the parent antibody binds, and preferably, the fragment may retain at least 20%, 50%, 70%, 80%, 90%, 95%, or 100% or more of the protein affinity of the parent antibody. The antibody fragment may specifically be in the form of, but is not limited to, diabody, Fab, Fab', F(ab)2, F(ab')2, Fv, scFv, etc.

[0052] In the present invention, the term "stem cell-derived extracellular particle" refers to an extracellular particle derived from a stem cell. The stem cell may be an embryonic stem cell or an adult stem cell. The adult stem cell may include a stem cell selected from the group consisting of mesenchymal stem cells, human tissue-derived mesenchymal stromal cells, human tissue-derived mesenchymal stem cells, embryonic stem cells, induced pluripotent stem cells, multipotent stem cells, and amniotic epithelial cells. The mesenchymal stem cells may be derived from one or more tissues selected from the group consisting of umbilical cord, umbilical cord blood, bone marrow, fat, muscle, nerve, skin, amniotic membrane, and placenta.

[0053] As used herein, the term "producer cell" or "host cell" refers to a cell used to produce an extracellular particle. In the present invention, the producer cell is preferably a stem cell, and more preferably a mesenchymal stem cell (MSC). The producer cell may be transformed or transfected with one or more vectors containing or containing exogenous sequence(s) or DNA construct(s). In some embodiments, the producer cell may be transformed or transfected with a single vector containing a DNA construct encoding a peptide comprising a CD47 ligand and an extracellular vesicle protein. In some embodiments, the producer cell may be transformed or transfected with a vector containing a DNA construct encoding a peptide comprising a CD47 ligand and an extracellular vesicle protein. In some embodiments, the producer cell may be transformed or transfected with at least one additional exogenous sequence or a DNA construct encoding another protein or peptide (e.g., a targeting moiety). Additional exogenous sequences may be introduced into a vector containing a DNA construct encoding a peptide comprising a CD47 ligand and an extracellular vesicle protein. In some embodiments, the exogenous sequence or DNA construct encoding the therapeutically active cargo, the additional exogenous or DNA construct sequence encoding another protein or peptide, or both, may be introduced into a producer cell to produce extracellular vesicles expressing the CD47 ligand. The therapeutically active cargo, the other protein or peptide, or both may be present on the surface of the extracellular vesicle or within the extracellular vesicle.

[0054] In the present invention, "target substance" is used interchangeably with "cargo" or "payload," and refers to a substance capable of being delivered to a target cell by an extracellular particle. In one embodiment, the target substance comprises a therapeutic agent and / or a diagnostic agent. In one embodiment, the target substance comprises a nucleic acid, a protein, a polypeptide, or a small molecule.

[0055] As used herein, the term "linker" refers to any molecular structure capable of conjugating a peptide or protein to another molecule (e.g., another peptide or protein, a small molecule, etc.). Suitable linkers are well known to those skilled in the art and include, but are not limited to, straight or branched chain carbon linkers, heterocyclic carbon linkers, or peptide linkers. Linkers may be linked to carboxyl and amino terminal amino acids via terminal carboxyl or amino groups or via reactive side groups. Additionally, in some embodiments, linkers may be classified as flexible or rigid and may be cleavable (e.g., comprising one or more protease-cleavable sites, which may be located within the linker sequence or flanked by the linker at either end).

[0056] The term “pharmaceutically acceptable” or “pharmaceutically acceptable” in the present invention means compounds and compositions suitable for administration to humans and / or animals without excessive side effects such as toxicity, irritation and / or allergic reactions commensurate with a reasonable benefit / risk ratio.

[0057] The term "biological activity" in the present invention means the ability of an active agent to modify a physiological system of an organism, regardless of how the active agent has a physiological effect.

[0058] The terms "subject," "subject," and "patient" are used interchangeably herein and are to be understood to encompass mammals and non-mammals. Examples of mammals include, but are not limited to, humans, chimpanzees, apes, cows, horses, sheep, goats, pigs, rabbits, dogs, cats, rats, mice, guinea pigs, and the like, while examples of non-mammals include, but are not limited to, birds, fish, and the like.

[0059] The term "treatment" in the present invention may mean alleviating or improving a disease, condition or symptom, preventing additional symptoms, improving or preventing the underlying metabolic cause of the symptom, inhibiting a disease, condition or symptom, arresting the development of a disease, condition or symptom, alleviating a disease, condition or symptom, causing regression of a disease, condition or symptom, or preventing or stopping a condition caused by a disease, condition or symptom, either prophylactically and / or therapeutically.

[0060] As used herein, the terms "administering" or "administering" a composition refers to providing the composition to a subject in need of treatment. According to embodiments of the present invention, the therapeutic composition may be administered alone or in combination with one or more additional therapeutic agents. Methods of administering such compositions may include, but are not limited to, intravenous administration, inhalation, oral administration, rectal administration, parenteral administration, intravitreal administration, subcutaneous administration, intramuscular administration, intranasal administration, dermal administration, topical administration, ophthalmic administration, buccal administration, tracheal administration, bronchial administration, sublingual administration, or optic nerve administration.

[0061] As used herein, the terms "therapeutic composition" and "pharmaceutical composition" refer to an active agent-containing composition that can be administered to a subject by any method known in the art or otherwise contemplated herein, wherein administration of the composition can produce a therapeutic effect as described elsewhere herein. Furthermore, the compositions of the present invention can be designed to provide delayed, controlled, extended, and / or sustained release using formulation techniques well known in the art. The compositions of the present invention can be administered via known pharmaceutical formulations including, but not limited to, tablets, pills, capsules, liquids, inhalants, nasal spray solutions, suppositories, solutions, gels, emulsions, ointments, and eye drops.

[0062] As used herein, the term "therapeutically effective amount" refers to a sufficient amount of the active ingredient(s) administered to alleviate to some extent one or more of the symptoms of the disease or condition being treated. The result may be a reduction and / or alleviation of the signs, symptoms, or causes of the disease, or other desired alterations in a biological system. For example, an "effective amount" for therapeutic purposes is the amount of the composition required to provide a clinically significant reduction in disease symptoms. The effective amount for a patient may vary depending on the patient type, the patient's physique and health, the nature and severity of the condition being treated, the method of administration, the duration of treatment, the nature of the formulation used, the nature of any concurrent therapies, and the like. Therefore, it is impossible to specify an exact effective amount in advance. However, the effective amount for a given situation can be determined by one of ordinary skill in the art using routine experimentation based on the information provided herein.

[0063] As used herein, the term "prophylactically effective amount" refers to an amount effective at the dosage and for the duration necessary to achieve the desired preventive result. Typically, prophylactic doses are administered to subjects at earlier or earlier stages of a disease, and therefore, in the context of a lower disease burden, the prophylactically effective amount may be lower than the therapeutically effective amount. In some aspects, the prophylactically effective amount may be higher than the therapeutically effective amount.

[0064] The present invention provides a method for delivering a target substance to a target cell, comprising a step of contacting extracellular particles loaded with a target substance, the extracellular particles comprising a ligand for CD47, a fragment thereof, a variant thereof, a variant of a fragment thereof, or a fragment of a variant thereof, with the target cell.

[0065] In some embodiments, the ligand for CD47 is a molecule or binding moiety that has a structure complementary to CD47 or can interact with CD47 to form a complex.

[0066] In some embodiments, the ligand for CD47 may mean a protein, peptide, or fragment thereof capable of forming a complex with CD47, comprising a binding motif of suitable length and amino acid sequence thereof.

[0067] In some embodiments, the ligand for CD47 may be characterized as a protein, peptide, antibody, antibody fragment, aptamer, small molecule compound, or a combination thereof that binds to CD47.

[0068] In some embodiments, the ligand for CD47 may be, but is not limited to, SIRP (signal regulatory protein), TSP-1 (Thrombospodin-1), ALX148, TTI-661, TTI-662, a variant thereof, or a homolog thereof.

[0069] In some embodiments, the SIRP is SIRPα, SIRPβ, SIRPγ, or all of them.

[0070] In a preferred embodiment, the SIRP is SIRPα or a functional fragment thereof. Preferably, the functional fragment of SIRPα is the ectodomain of SIRPα, or a biologically active fragment thereof. The biologically active fragment comprises any fragment of the ectodomain of SIRPα that specifically binds to CD47 in relation to the ectodomain of SIRPα and can inhibit CD47 binding to SIRPα, for example, in macrophages or cancer cells.

[0071] In some embodiments, the fragment of SIRPα can be a fragment of human SIRPα comprising at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, or 180 consecutive amino acids up to and including 508 consecutive amino acids of the amino acid sequence of human SIRPα protein (e.g., GENBANK® Accession no. AAH62197.1), wherein the fragment specifically binds to CD47 and has the functionality of inhibiting CD47 binding to SIRPα in, for example, a macrophage or a cancer cell.

[0072] In some embodiments, the variant of SIRPα may be a deletion, insertion, or substitution variant. The variant of SIRPα may comprise an amino acid sequence that is at least 50%, 60%, 70%, 80%, or 90% identical to a sequence of SIRPα disclosed, referenced, or disclosed in the art, including all values ​​and ranges therebetween.

[0073] In some embodiments, the variant of the SIRPα ectodomain may be a deletion, insertion, or substitution variant. The variant of the SIRPα ectodomain may comprise an amino acid sequence that is at least 50%, 60%, 70%, 80%, or 90% identical to a sequence of a SIRPα ectodomain disclosed, referenced, or published in the art, including all values ​​and ranges therebetween.

[0074] In the present invention, the "deletion" variant typically refers to one or more amino acids lacking in a native or wild-type protein or peptide. Individual amino acids may be deleted, or multiple consecutive amino acids may be deleted. The deleted amino acids may be deleted at a terminal or non-terminal position of the native or wild-type protein or peptide.

[0075] In the present invention, the “insertion” variant typically means the addition of one or more amino acid residues at a terminal or non-terminal point of the polypeptide.

[0076] The “substitution” variants herein generally involve exchanging one amino acid for another amino acid at one or more sites within a protein or polypeptide, and may be designed to modulate one or more properties of the polypeptide without loss or impairment of other functions or properties. Substitutions may be conservative, i.e., one amino acid may be replaced with another having similar chemical properties. A “conservative amino acid substitution” may involve exchanging a member of one amino acid family for another member of the same family. Conservative substitutions are well known in the art and include, for example, the following changes: alanine for serine; arginine for lysine; asparagine for glutamine or histidine; aspartate for glutamate; cysteine ​​for serine; glutamine for asparagine; glutamate for aspartate; glycine for proline; histidine for asparagine or glutamine; isoleucine for leucine or valine; Leucine to valine or isoleucine; lysine to arginine; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine, or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; and valine to isoleucine or leucine. Conservative amino acid substitutions may involve amino acid residues that do not occur naturally. Alternatively, the substitutions may be "non-conservative," meaning that the function or activity of the protein or polypeptide is affected. Non-conservative changes generally involve replacing amino acid residues with chemically dissimilar ones, such as substituting a polar or charged amino acid for a non-polar or uncharged amino acid, and vice versa. Non-conservative substitutions may involve exchanging a member of one amino acid class for a member of another class.

[0077] SIRPα is a type I membrane protein and has been sequenced in various species including but not limited to mouse: GENBANK® Accession no. AAH62197.1; human: GENBANK® Accession no. AAH33092.1; Pan troglodytes (chimpanzee): GENBANK® Accession no. JAA10535.1; Macaca mulatta (rhesus monkey): GENBANK® Accession no. AFE76783.1; gorilla: GENBANK® Accession no. XP_004061735.1; and Bos taurus: GENBANK® Accession no. NP_786982.1.

[0078] In a preferred embodiment, SIRPα is human SIRPα. Human SIRPα has 503 amino acids. There are at least 10 naturally occurring variants in wild-type human SIRPα.

[0079] In a preferred embodiment, the SIRPα ectodomain can be the D1 domain of SIRPα.

[0080] In a preferred embodiment, the SIRPα ectodomain can be the D1 domain of human SIRPα. The D1 domain of human SIRPα can be one of the D1 domains of a variant human SIRPα. Exemplary sequences for the human SIRPα ectodomain can comprise or consist of SEQ ID NOs: 1 to 6.

[0081] Sequence number 1:

[0082] EEELQVIQPDKSVLVAAGETATLRCTATSLIPVGPIQWFRGAGPGRELIYNQKEGHFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKP

[0083] Sequence number 2

[0084] EEELQVIQPDKSVSVAAGESAILHCTVTSLIPVGPIQWFRGAGPARELIYNQKEGHFPRVTTVSESTKRENMDFSISISNITPADAGTYYCVKFRKGSPDTEFKSGAGTELSVRAKP

[0085] Sequence number 3:

[0086] EEELQVIQPDKSVLVAAGETATLRCTATSLIPVGPIQWFRGAGPARELIYNQKEGHFPRVTTVSESTKRENMDFSISISNITPADAGTYYCVKFRKGSPDTEFKSGAGTELSVRAKP

[0087] Sequence number 4:

[0088] EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRVLIYNQRQGPFPRVTTVSDTTKRNNMDFSIRIGNITPADAGTYYCIKFRKGSPDDVEFKSGAGTELSVRAKP

[0089] Sequence number 5:

[0090] EEELQIIQPDKSVSVAAGESAILHCTITSLFPVGPIQWFRGAGPARVLIYNQRQGPFPRVTTVSETTKRENMDFSISISNITPADAGTYYCIKFRKGSPDTEFKSGAGTELSVRAKP

[0091] Sequence number 6:

[0092] EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPARVLIYNQRQGPFPRVTTVSETTKRENMDFSISISNITPADAGTYYCIKFRKGSPDTEFKSGAGTELSVRAKP

[0093] In a preferred embodiment, the SIRPα ectodomain comprises a deletion, insertion or substitution variant of SEQ ID NO: 1 to 6.

[0094] In some embodiments, the SIRP is SIRPγ, a functional fragment thereof, or a functional variant thereof. Preferably, the functional fragment or variant of SIRPγ is an ectodomain of SIRPγ, or a biologically active fragment or variant thereof. As used herein, with respect to the ectodomain of SIRPγ, the biologically active fragment or variant includes any fragment of the ectodomain that can specifically bind to CD47 and inhibits CD47 binding to SIRPγ, for example, in macrophages or cancer cells. An exemplary sequence for the ectodomain of human SIRPγ is residues 26 to 357 of the polypeptide sequence of GENBANK Accession No. NP_061026.2.

[0095] In a preferred embodiment, SIRPγ is human SIRPγ.

[0096] An exemplary sequence for the ectodomain of human SIRPγ comprises or consists of: (SEQ ID NOs: 7 to 9)

[0097] Sequence number 7:

[0098] EEELQMIQPEKLLLVTVGKTATLHCTVTSLLPVGPVLWFRGVGPGRELIYNQKEGHFPRVTTVSDLTKRNNMDFSIRISSITPADVGTYYCVKFRKGSPENVEFKSGPGTEMALGAKP

[0099] Sequence number 8:

[0100] EEELQIIQPEKLLLVTVGKTATLHCTVTSLFPVGPVLWFRGVGPGRVLIYNQRQGPFPRVTTVSDTTKRNNMDFSIRISSITPADVGTYYCIKFRKGSPENVEFKSGPGTEMALGAKP

[0101] Sequence number 9:

[0102] EEELQIIQPEKLLLVTVGKTATLHCTITSLFPVGPVLWFRGVGPGRVLIYNQRQGPFPRVTTVSDTTKRNNMDFSIRISSITPADVGTYYCIKFRKGSPENVEFKSGPGTEMALGAKP

[0103] In a preferred embodiment, the SIRPγ ectodomain comprises a deletion, insertion or substitution variant of SEQ ID NO: 7 to 9.

[0104] In some embodiments, the fragment of SIRPγ can be a fragment of human SIRPγ comprising at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, or 180 consecutive amino acids up to and including 386 consecutive amino acids of the amino acid sequence of human SIRPγ protein (e.g., GENBANK® Accession no. NP_061026.2), wherein the fragment specifically binds to CD47 and retains the functionality of inhibiting CD47 binding to SIRPγ, e.g., in macrophages or cancer cells.

[0105] In some embodiments, the ligand for CD47 may be an anti-CD47 antibody or antigen-binding fragment thereof. Non-limiting examples thereof may include anti-CD47 antibodies and antigen-binding fragments thereof, such as B6H12, 5F9, 8B6, C3, and Hu5F9-G4, as described in U.S. Patent No. 5,300,000.

[0106] In some embodiments, the extracellular particle may comprise a naturally occurring amphipathic lipid bilayer. The extracellular particle may comprise several different types of lipids, such as amphipathic lipids such as phospholipids. The extracellular particle may comprise a lipid bilayer as its outermost surface.

[0107] In some embodiments, the extracellular particle comprises a naturally occurring membrane, for example, a membrane vesicle manufactured from a cell or tissue.

[0108] In some embodiments, the extracellular particles can be selected from the group consisting of extracellular vesicles, non-vesicular extracellular particles, virus particles, virus-like particles, liposomes, artificial cell-derived vesicles, synthetic vesicles, extracellular vesicle mimetics, and platelet-like particles.

[0109] In some embodiments, the extracellular particles may be characterized as being stem cell derived.

[0110] In some embodiments, the stem cells are human stem cells. In some embodiments, the stem cells are surface-engineered stem cells. In some embodiments, the stem cells are surface-engineered human stem cells. In some embodiments, the stem cells are selected from the group consisting of adult stem cells, embryonic stem cells (ESCs), induced pluripotent stem cells, cord blood stem cells, and amniotic fluid stem cells. In some embodiments, the adult stem cells are selected from the group consisting of mesenchymal stem cells, human tissue-derived mesenchymal stromal cells, human tissue-derived mesenchymal stem cells, multipotent stem cells, and amniotic epithelial cells. In some embodiments, the mesenchymal stem cells are derived from one or more tissues selected from the group consisting of umbilical cord, cord blood, bone marrow, fat, muscle, nerve, skin, amniotic membrane, and placenta. In some embodiments, the adult stem cells are selected from the group consisting of neural stem cells, skin stem cells, epithelial stem cells, skeletal muscle satellite cells, mesenchymal stem cells, adipose-derived stem cells, endothelial stem cells, and dental pulp stem cells. Cells, hematopoietic stem cells (including bone marrow stem cells, bone marrow mesenchymal stem cells, etc.) and placental-derived stem cells (including placental-derived mesenchymal stem cells, etc.).

[0111] In some embodiments, the extracellular particles may be characterized as being derived from human embryonic kidney cells (HEK cells).

[0112] In some embodiments, the extracellular vesicles may be selected from the group consisting of exosomes, microvesicles, and apoptotic bodies.

[0113] In some embodiments, exemplary lipid bilayer particles are described, for example, in US2016137716, which is incorporated herein by reference in its entirety. In some embodiments, the fusosome comprises a vesicle obtainable from a cell, such as a microsome, an exosome, an apoptotic body (from an apoptotic cell), a microparticle (e.g., derived from platelets), an ectosome (e.g., derived from neutrophils and monocytes in serum), a prostatosome (e.g., derived from prostate cancer cells), a cardiosome (e.g., derived from cardiac cells), and the like.

[0114] In some embodiments, exemplary lipid bilayer particles are also described in WO / 2017 / 161010, WO / 2016 / 077639, US20160168572, US20150290343, and US20070298118, each of which is incorporated herein by reference in its entirety. In some embodiments, the lipid bilayer particle comprises an extracellular vesicle, a nanovesicle, or an exosome. In some embodiments, the lipid bilayer particle comprises an extracellular vesicle, e.g., a cell-derived vesicle comprising a membrane surrounding an interior space and having a diameter smaller than the cell from which it is derived. In some embodiments, the diameter of the extracellular vesicle is from 20 nm to 1000 nm. In some embodiments, the lipid bilayer particle comprises apoptotic bodies, cell fragments, vesicles derived from cells by direct or indirect manipulation, vesicled organelles, and vesicles generated from living cells (e.g., by direct plasma membrane budding or fusion of late endosomes with the plasma membrane). In some embodiments, the extracellular vesicles are derived from living or dead organisms, explanted tissues or organs, or cultured cells. In some embodiments, the lipid bilayer particle comprises a nanovesicle, e.g., a cell-derived small (e.g., 20 nm to 250 nm in diameter, or 30 nm to 150 nm in diameter) vesicle that encloses an interior space and comprises a membrane generated from said cell by direct or indirect manipulation. Generation of the nanovesicle, in some cases, results in the destruction of the source cell. The nanovesicle may comprise lipids or fatty acids and polypeptides. In some embodiments, the lipid bilayer particle comprises an exosome. In some embodiments, exosomes are cell-derived small (e.g., 20 nm to 300 nm in diameter, or 40 nm to 200 nm in diameter) membrane-containing vesicles that enclose an internal space and are generated in the cell by direct plasma membrane budding or fusion of late endosomes with the plasma membrane.

[0115] In some embodiments, exemplary lipid bilayer particles are also described in US 20160354313, which is incorporated herein by reference in its entirety. In some embodiments, the lipid bilayer particle comprises an exosome (e.g., having a diameter of about 30 nm to about 200 nm), a microvesicle (e.g., having a diameter of about 100 nm to about 2000 nm), an apoptotic body (e.g., having a diameter of about 300 nm to about 2000 nm), a membrane particle, a membrane vesicle, an exosome-like vesicle, an ectosome-like vesicle, an ectosome, or an exovesicle.

[0116] In some embodiments, the lipid bilayer particle may be composed of several different types of lipids, such as amphiphilic lipids such as phospholipids. The lipid bilayer particle may comprise a lipid bilayer as its outermost surface. This bilayer may be composed of one or more lipids of the same or different types. Examples include, but are not limited to, phospholipids such as phosphocholine and phosphoinositol. Examples include, but are not limited to, DMPC, DOPC, and DSPC.

[0117] In some embodiments, the extracellular particles may be surface-engineered to include a ligand for CD47 for intracellular delivery of a target agent (e.g., a therapeutic or diagnostic agent) loaded onto the extracellular particles in various diseases mediated by CD47. The term "surface engineering" refers to altering the surface composition of the extracellular particle. In some embodiments, the term "surface engineering" refers to altering the surface composition of an extracellular particle obtainable from a wild-type cell. For example, the surface-engineered extracellular particle may have a polypeptide structure on its surface at a higher (or lower) density than a naturally occurring extracellular particle. In the present invention, the surface-engineered extracellular particle may be produced from a genetically engineered producer cell or its progeny. For example, the surface-engineered extracellular particle may be produced from a stem cell transformed or transfected with a DNA construct encoding a ligand for CD47.

[0118] In some embodiments, the polypeptide construct comprising a ligand for CD47 may further comprise at least one extracellular particle protein to induce internalization of the polypeptide construct into an extracellular particle. Among these extracellular particle proteins, extracellular vesicle proteins include CD9, CD53, CD63, CD81, CD54, CD50, FLOT1, FLOT2, CD71, CD133, CD138, CD235a, syntenin-1, syntenin-2, Lamp2b, TSPAN8, TSPAN14, CD37, CD82, CD151, CD231, CD102, NOTCH1, NOTCH2, NOTCH3, NOTCH4, DLL1, DLL4, JAG1, JAG2, CD49d / ITGA4, ITGB5, ITGB6, ITGB7, CD11a, CD11b, CD11c, CD18 / ITGB2, CD41, CD49b, CD49c, CD49e, CD51, CD61, CD104, Tetraspanin, Fc receptor, interleukin receptor, immunoglobulin, MHC-I component, MHC-II component, CD2, CD3 epsilon, CD3 zeta, CD13, CD19, CD30, CD34, CD36, CD40, CD40L, CD44, CD45, CD45RA, CD47, CD86, CD110, CD111, CD115, CD117, CD125, CD135, CD184, CD200, CD279, CD273, CD274, CD362, COL6A1, AGRN, EGFR, GAPDH, GLUR2, GLUR3, HLA-DM, HSPG2, L1CAM, LAMB1, LAMC1, LFA-1, LGALS3BP, Mac-1 alpha, Mac-1 beta, MFGE8, SLIT2, STX3, TCRA, TCRB, TCRD, TCRG, VTI1A, VTI1B, PDGFR, GPI anchor protein, lactadherin, syndecan,Synaptotagmin, ALIX (apoptosis-linked gene 2-interacting protein X), PTGFRN (prostaglandin F2 receptor inhibitor), fragments thereof, mutants thereof, mutants of fragments thereof, fragments of mutants thereof, and any combination thereof. Non-vesicular extracellular particle proteins among these extracellular particle proteins include, but are not limited to, HSP90AA / B, TGFβ-induced (TGFBI), HSPA13, LDHA / B, ENO1, ENO2, ALDOA, GPI, ACTN4, AGO1, AGO2, HEXOKINASE I, fragments thereof, mutants thereof, mutants of fragments thereof, fragments of mutants thereof, and any combination thereof.

[0119] In some embodiments, the polypeptide construct may be a fusion protein comprising a ligand for CD47 and at least one extracellular particle protein. In some embodiments, the fusion protein may comprise a ligand for CD47 linked (fused) to at least one extracellular particle protein, either directly or via a linker.

[0120] In some embodiments, the extracellular particle protein in the polypeptide construct may be located in the lipid bilayer of the extracellular vesicle, and all or part of the ligand for CD47 may be exposed outside the lipid bilayer.

[0121] In some embodiments, the linker may be a peptide linker. In some embodiments, the peptide linker comprises at least about 2, at least about 3, at least about 4, at least about 5, at least about 10, at least about 15, at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least about 55, at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, at least about 90, at least about 95, or at least about 100 amino acids. In some embodiments, the peptide linker may be synthetic, i.e., non-naturally occurring. In some embodiments, the peptide linker can comprise a peptide (or polypeptide) (e.g., a naturally occurring or non-naturally occurring peptide) comprising an amino acid sequence that links or genetically fuses a first linear sequence of amino acids to a second linear sequence of amino acids. For example, in some embodiments, the peptide linker can comprise a non-naturally occurring polypeptide that is a modified form of a naturally occurring polypeptide (e.g., comprising a mutation such as an addition, substitution, or deletion).

[0122] In some embodiments, the SIRP can be fused directly or via a linker to the N-terminus, C-terminus, or both of the extracellular particle.

[0123] In a preferred embodiment, the SIRP can be fused directly to the N-terminus of the extracellular particle protein or via a linker.

[0124] In some embodiments, the polypeptide construct comprising a SIRP and at least one extracellular particle protein is or can be located within / on the membrane of the extracellular particle. In some embodiments, of the polypeptide construct comprising a SIRP and at least one extracellular particle, the extracellular particle is or can be located at least partially on the surface of the extracellular particle. In some embodiments, of the polypeptide construct comprising a SIRP and at least one extracellular particle protein, the SIRP is or can be located on the surface of the extracellular particle. In some embodiments, of the polypeptide construct comprising a SIRP and at least one extracellular particle, the SIRP is or can be expressed, displayed, or presented on the surface of the extracellular particle.

[0125] In some embodiments, the extracellular particles described herein are loaded with a target substance.

[0126] In some embodiments, the target substance may be exogenous or endogenous to the producing cell of the extracellular particle.

[0127] In some embodiments, the extracellular particle comprises a target substance associated with its membrane. In some embodiments, the extracellular particle comprises a target substance disposed within the extracellular particle. In some embodiments, the extracellular particle comprises a target substance associated with the extracellular particle and a target substance disposed within the extracellular particle.

[0128] In some embodiments, the target substance is not naturally expressed in the cell in which the extracellular particle is produced / induced. In some embodiments, the target substance is naturally expressed in the cell in which the extracellular particle is produced / induced.

[0129] In some embodiments, the target substance is loaded into the extracellular particle through expression in the cell in which the extracellular particle is produced / induced (e.g., expression from DNA introduced via transfection, transduction, or electroporation). In some embodiments, the target substance is expressed from DNA integrated into the genome of the cell in which the extracellular particle is produced / induced, or is maintained episomally in the cell in which the extracellular particle is produced / induced. In some embodiments, expression of the target substance is constitutive in the cell in which the extracellular particle is produced / induced. In some embodiments, expression of the target substance is produced / induced in the cell in which the extracellular particle is produced / induced. In some embodiments, expression of the target substance is produced / induced in the cell in which the extracellular particle is produced / induced immediately prior to production of the extracellular particle.

[0130] In some embodiments, the target substance is loaded into the extracellular particle, into the extracellular particle itself, or into the cell in which the extracellular particle is produced / induced via electroporation. In some embodiments, the target substance is loaded into the extracellular particle, into the extracellular particle itself, or into the cell in which the extracellular particle is produced / induced via transfection.

[0131] In some embodiments, the target agent may be a therapeutic agent, a diagnostic agent, or a combination thereof.

[0132] In some embodiments, the target substance may be selected from the group consisting of nucleic acids, proteins, polypeptides, small molecule compounds, and carbohydrates.

[0133] In some embodiments, the target substance may comprise one or more nucleic acid sequences, one or more polypeptides, a combination of nucleic acid sequences and / or polypeptides, one or more cellular organelles, and any combination thereof. In some embodiments, the target substance may comprise one or more cellular components. In some embodiments, the target substance may comprise one or more cytoplasmic and / or nuclear components.

[0134] In some embodiments, the target substance is a nucleic acid, for example, a transcription factor, DNA, nDNA (nuclear DNA), mtDNA (mitochondrial DNA), protein-coding DNA, a gene, an operon, a chromosome, a genome, a transposon, a retrotransposon, a viral genome, an intron, an exon, a modified DNA, ssDNA (single-stranded DNA), dsDNA (double-stranded DNA), mRNA (messenger RNA), sgRNA (single guide RNA), gRNA (guide RNA), pegRNA (prime editing guide RNA), tRNA (transfer RNA), a modified RNA, microRNA (miRNA), siRNA (small interfering RNA), tmRNA (transfer messenger RNA), rRNA (ribosomal RNA), mtRNA (mitochondrial RNA), snRNA (small nuclear RNA), small nucleolar RNA (snoRNA), SmY RNA (mRNA trans-splicing RNA), TERC (telomerase RNA component), aRNA (antisense RNA), cis-NAT (cis-native antisense transcript), CRISPR RNA (crRNA), tracrRNA (trans-activating CRISPR RNA), lncRNA (long noncoding RNA), piRNA (piwi interacting RNA), tasiRNA (trans-acting siRNA), eRNA (enhancer RNA), satellite RNA, pcRNA (protein coding RNA), RNAi (interfering RNA), circRNA (circular RNA), reprogramming RNA, aptamers, antisense oligonucleotides, shRNA (short hairpin RNA), dsRNA (double-stranded RNA), antisense RNA, ribozymes, and any combination thereof. In some embodiments, the nucleic acid is a wild-type nucleic acid. In some embodiments, the nucleic acid is a mutant nucleic acid. In some embodiments, the nucleic acid is a fusion or chimera of a plurality of nucleic acid sequences.

[0135] In some embodiments, the nucleic acid may contain a nuclear localization signal (NLS) that can enhance editing efficiency within the nucleus. An NLS is an amino acid sequence that tags a protein for nuclear transport into the cell nucleus.

[0136] In some embodiments, the target agent may comprise a nucleic acid. For example, the target agent may comprise RNA that enhances expression of an endogenous protein (e.g., endogenous to the cell producing the lipid bilayer particle, and endogenous to the target cell, in some embodiments), or siRNA or miRNA that inhibits protein expression of an endogenous protein.

[0137] In some embodiments, the target substance is a polypeptide, for example, an enzyme, a structural polypeptide, a signaling polypeptide, a regulatory polypeptide, a transport polypeptide, a sensory polypeptide, a motor polypeptide, a defense polypeptide, a storage polypeptide, a transcription factor, an antibody, a cytokine, a hormone, a catabolic polypeptide, an anabolic polypeptide, a proteolytic polypeptide, a metabolic polypeptide, a kinase, a transferase, a hydrolase, a lyase, an isomerase, a ligase, an enzyme modulator polypeptide, a protein binding polypeptide, a lipid binding polypeptide, a membrane fusion polypeptide, a cell differentiation polypeptide, an epigenetic polypeptide, an apoptotic polypeptide, a nuclear transport polypeptide, a nucleic acid binding polypeptide, a reprogramming polypeptide, a DNA editing polypeptide, a DNA repair polypeptide, a DNA recombination polypeptide, a transposase polypeptide, a DNA integrating polypeptide Polypeptides, targeting endonucleases (e.g., zinc finger nucleases, transcription activator-like nucleases (TALENs), cas9 and their homologs), recombinases, and any combination thereof. In some embodiments, the protein is a wild-type protein. In some embodiments, the protein is a mutant protein. In some embodiments, the protein is a fusion or chimeric protein.

[0138] In some embodiments, the target substance is a small molecule, for example, an ion (e.g., Ca 2+ , Cl - , Fe 2+ ), carbohydrates, lipids, reactive oxygen species, reactive nitrogen species, isoprenoids, signaling molecules, hemes, polypeptide cofactors, electron accepting compounds, electron donating compounds, metabolites, ligands, and any combination thereof.

[0139] In some embodiments, the target substance comprises a mixture of proteins, nucleic acids, or metabolites, e.g., multiple polypeptides, multiple nucleic acids, multiple small molecules; combinations of nucleic acids, polypeptides, and small molecules; ribonucleoprotein complexes (e.g., Cas9-gRNA complexes); multiple transcription factors, multiple epigenetic factors, reprogramming factors (e.g., Oct4, Sox2, cMyc, and Klf4); multiple regulatory RNAs; and any combination thereof.

[0140] In some embodiments, the target substance comprises one or more organelles, such as chondrosomes, mitochondria, lysosomes, nuclei, cell membranes, cytoplasm, endoplasmic reticulum, ribosomes, vacuoles, endosomes, spliceosomes, polymerases, capsids, acrosomes, autophagosomes, centrosomes, glycosomes, glyoxysomes, hydrogenosomes, melanosomes, mitosomes, myofibrils, nematodes, peroxisomes, proteasomes, vesicles, stress granules, networks of organelles, and any combination thereof.

[0141] In some embodiments, the target substance may be a DNA digesting agent capable of digesting DNA. This refers to an agent capable of cleaving bonds (e.g., phosphodiester bonds) between nucleotide subunits of a nucleic acid. In one embodiment, the DNA digesting agent is a nuclease. A nuclease is an enzyme that hydrolyzes nucleic acids. Nucleases can be classified as endonucleases or exonucleases. Endonucleases are a group of enzymes that catalyze the hydrolysis of bonds between nucleic acids within a DNA or RNA molecule. Exonucleases are a group of enzymes that catalyze the hydrolysis of single nucleotides at the end of a DNA or RNA chain. Nucleases can also be classified based on whether they specifically digest DNA or RNA. Nucleases that specifically catalyze the hydrolysis of DNA may be referred to as deoxyribonucleases, or DNases, while those that specifically catalyze the hydrolysis of RNA may be referred to as ribonucleases, or RNases. Some nucleases are specific for single-stranded or double-stranded nucleic acid sequences. Some enzymes possess both exonuclease and endonuclease properties. Furthermore, some enzymes can digest both DNA and RNA sequences.

[0142] In some embodiments, the target agent may be an endonuclease. Non-limiting examples of endonucleases include, but are not limited to, zinc finger nucleases (ZFNs), ZFN dimers, ZFNickases, transcription activator-like effector nucleases (TALENs), meganucleases, or RNA-guided DNA endonucleases (e.g., the CRISPR / Cas system). The endonuclease may be engineered, chimerized, or isolated from an organism. The endonuclease may be engineered to recognize a specific DNA sequence, for example, by mutagenesis.

[0143] In some embodiments, the target agent may be an RNA-guided DNA endonuclease (e.g., a CRISPR-Cas system). In this case, the lipid bilayer particle may additionally include a gRNA, a crRNA, a tracrRNA, etc. The terms "gRNA," "guide RNA," and "CRISPR guide sequence" may be used interchangeably and refer to a nucleic acid comprising a sequence that determines the specificity of the Cas DNA binding protein of the CRISPR / Cas system. The gRNA hybridizes (partially or fully complementarily) to a target nucleic acid sequence in the host cell genome. The gRNA or portion thereof that hybridizes to the target nucleic acid may be 15-25 nucleotides in length, 18-22 nucleotides in length, or 19-21 nucleotides in length. In some embodiments, the length of the gRNA sequence that hybridizes to the target nucleic acid is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides. A "scaffold sequence," also called tracrRNA (trans-activating CRISPR RNA), refers to a nucleic acid sequence that recruits a Cas endonuclease to a target nucleic acid that is bound to (hybridized with) a complementary gRNA sequence. In some embodiments, the gRNA sequence does not comprise a scaffold sequence, and the scaffold sequence is expressed as a separate transcript. In such embodiments, the gRNA sequence further comprises an additional sequence that is complementary to a portion of the scaffold sequence and functions to bind (hybridize) the scaffold sequence and recruit the endonuclease to the target nucleic acid.

[0144] In some embodiments, the RNA-guided DNA endonuclease is a Cas enzyme, i.e., a CRISPR-associated endonuclease. The Cas enzyme may be a naturally occurring Cas enzyme or a functional derivative thereof. In certain embodiments, the Cas enzyme may comprise one or more mutations. The Cas enzyme may be a type II, type I, type III, type IV, or type V CRISPR system enzyme. In some embodiments, the Cas enzyme is a Cas9 enzyme (also known as Cas5, Csn1, or Csx12). Cas9 may be wild-type or a mutant. In some embodiments, the endonuclease is a Cas9 homolog or ortholog. Cas9 may be any variant disclosed in U.S. Patent Publication No. US 2014 / 0068797 A1, which is incorporated herein by reference. Cas9 may be type II-A, type II-B, or type II-C. Cas9 may be derived from various species. Non-limiting examples of Cas9 enzymes may include Cas9 from Streptococcus pyogenes (S. pyogenes), Streptococcus pneumoniae (S. pneumoniae), Staphylococcus aureus, Neisseria meningitidis, Streptococcus thermophilus (S. thermophilus), or Treponema denticola.The Cas9 enzyme may also be derived from a microorganism of the genus Corynebacter, Sutterella, Legionella, Treponema, Filifactor, Eubacterium, Streptococcus, Lactobacillus, Mycoplasma, Bacteroides, Flaviivola, Flavobacterium, Sphaerochaeta, Azospirillum, Gluconacetobacter, Neisseria, Roseburia, Parvibaculum, Staphylococcus, Nitratifractor, Mycoplasma or campylobacter.

[0145] In some embodiments, the Cas enzyme is Cas9, Cpf1, C2c1, C2c2, C2c3, Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Cs f1, Csf2, Csf3, Csf4, It may be a homologue, an ortholog thereof, or a variant thereof.

[0146] In some embodiments, the target substance may comprise a diagnostic agent, including but not limited to a radiotracer or radionuclide used in positron emission tomography (PET) (e.g., carbon-11, nitrogen-13, oxygen-15, and fluorine-18). These agents can be more precisely delivered to the tissue of interest by loading them into engineered extracellular particles that target the tissue of interest, i.e., the tissue being scanned, either artificially or naturally, thereby reducing off-target delivery of the diagnostic agent.

[0147] In some embodiments, the extracellular particle may further comprise at least one targeting moiety. In some embodiments, the targeting moiety may be used to target the extracellular particle to a specific organ, tissue, or cell for delivery of a desired agent using the extracellular particle. In certain embodiments, the targeting moiety may bind to a marker (or target molecule) expressed on a cell or cell population. In certain embodiments, the marker may be expressed on various cell types, for example, all antigen-presenting cells (e.g., dendritic cells, macrophages, and B lymphocytes). In some embodiments, the marker may be expressed only on a specific cell population (e.g., dendritic cells). Non-limiting examples of markers expressed on a specific cell population (e.g., dendritic cells) include C-type lectin domain family 9 member A (CLEC9A) protein, dendritic cell-specific intercellular adhesion molecule-3-capture non-binding protein. Integrin (DC-SIGN), CD207, CD40, Clec6, dendritic cell immunoreceptor (DCIR), DEC-205, lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), MARCO, Clec12a, DC-asialoglycoprotein receptor (DC-ASGPR), DC immunoreceptor 2 (DCIR2), Dectin-1, macrophage mannose receptor (MMR), BDCA-1 (CD303, Clec4c), Dectin-2, Bst-2 (CD317), and any combination thereof. In some embodiments, the targeting moiety can be an antibody or an antigen-binding fragment thereof. Antibodies and antigen-binding fragments thereof include whole antibodies, polyclonal, monoclonal, and recombinant antibodies, and fragments thereof, and can further include single chain antibodies, humanized antibodies, murine antibodies, chimeric, mouse-human, mouse-primate, and primate-human. Monoclonal antibodies, anti-idiotypic antibodies, antibody fragments (e.g., scFv, (scFv)2, Fab, Fab' and F(ab')2, F(ab1)2, Fv, dAb and Fd fragments), diabodies, and antibody-related polypeptides.Antibodies and antigen-binding fragments thereof may include bispecific antibodies and multispecific antibodies, as long as they exhibit the desired biological activity or function.

[0148] In some embodiments, the target cell may be a CD47-positive cell, a CD47-overexpressing cell, or a combination thereof. "CD47-positive" is used in reference to the phenotype of a cell targeted by a lipid bilayer particle comprising a ligand for CD47. CD47-positive cells can be identified by flow cytometry using a CD47 antibody as an affinity ligand. Appropriately labeled CD47 antibodies are commercially available for this purpose. In some embodiments, the CD47-positive diseased cell of particular interest as a target for therapy using a lipid bilayer particle comprising a ligand for CD47 is a cell that overexpresses CD47. Such CD47-positive or CD47-overexpressing cells are generally diseased cells and display CD47 at a surface density that exceeds the normal CD47 density for a given cell type. CD47 overexpression can vary across different cell types, but herein refers to any CD47 level determined to be higher than a level measurable in a healthy counterpart, for example, by flow cytometry, immunostaining, or gene expression analysis.

[0149] In some embodiments, CD47 positive or overexpressing cells include, but are not limited to, cancer cells, virus-infected cells, and fibrotic cells.

[0150] In some embodiments, the CD47-positive or overexpressing cells include CD47-positive cancers or solid tumors, including CD47-overexpressing cancers. In some embodiments, the solid tumors also include CD47-positive or CD47-overexpressing tumors from the bladder, brain, breast, lung, colon, ovary, prostate, liver, and other tissues.

[0151] In some embodiments, the cancer includes a CD47-overexpressing cancer, particularly a CD47-positive cancer or liquid tumor. The term "liquid tumor" is used interchangeably herein with "hematological cancer." As used herein, the term "hematological cancer" refers to a blood cancer, particularly leukemia, lymphoma, and myeloma. "Leukemia" refers to a blood cancer in which white blood cells, which are ineffective in fighting infection, are produced in excess, crowding out other blood components such as platelets and red blood cells. Leukemia is known to be classified as acute or chronic. Specific forms of leukemia include, for example, acute lymphoblastic leukemia (ALL); acute myeloid leukemia (AML); chronic lymphocytic leukemia (CLL); chronic myeloid leukemia (CML); myeloproliferative disorders / neoplasms (MPDS); and myelodysplastic syndromes. "Lymphoma" may refer in particular to Hodgkin's lymphoma, indolent and aggressive non-Hodgkin's lymphoma, cutaneous T-cell lymphoma (CTCL), Burkitt's lymphoma, mantle cell lymphoma (MCL), and follicular lymphoma (small cell and large cell). Myelomas include multiple myeloma (MM), large cell myeloma, heavy chain myeloma, light chain myeloma, and Bence-Jones myeloma. In some embodiments, the hematological cancer is a CD47-positive or CD47-overexpressing leukemia, preferably selected from acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, and myelodysplastic syndrome, preferably human acute myeloid leukemia. In other embodiments, the blood cancer includes Hodgkin lymphoma, indolent and aggressive non-Hodgkin lymphoma, diffuse large-cell lymphoma (DLBCL), mantle cell lymphoma, T-cell lymphoma including mycosis fungoides, Sezary syndrome, Burkitt lymphoma, follicular lymphoma (small cell and large cell), multiple myeloma (MM), large cell myeloma, heavy chain myeloma, light chain or Bence-Jones myeloma, and leiomyosarcoma.

[0152] In some embodiments, the cancer is selected from the group consisting of melanoma, kidney cancer, prostate cancer, breast cancer, colon cancer, rectal adenocarcinoma, lung cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, skin cancer, and skin cancer. Or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal area, stomach cancer, testicular cancer, fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vaginal carcinoma of the vulva, Hodgkin's disease, non-Hodgkin's lymphoma, esophageal cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, chronic or acute leukemia including acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, pediatric solid tumors, lymphocytic lymphoma, bladder cancer, kidney cancer or ureter cancer, renal pelvic carcinoma, central nervous system neoplasms (CNS), non-small cell lung cancer (NSCLC), primary CNS lymphoma, tumor angiogenesis, spinal cord tumor, brainstem glioma, glioblastoma multiforme, low-grade glioma, pituitary adenoma, Kaposi's Sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, B-cell lymphoma, cholangiocarcinoma, thymoma, adrenocortical carcinoma, cervical cancer, uterine cancer, myxofibrosarcoma, undifferentiated pleomorphic sarcoma, dedifferentiated liposarcoma, dysembryonic neuroepithelial tumor, ependymoma, nasopharyngeal carcinoma, choroid plexus carcinoma, myoepithelial carcinoma, alveolar rhabdomyosarcoma, rhabdomyosarcoma, atypical teratoid / dermatous tumor, desmoplastic small round cell tumor, fibromatosis, synovial sarcoma, Wilm's tumor, myofibromatosis, Ewing's sarcoma, infantile fibrosarcoma, INI-deficient soft tissue sarcoma, medulloblastoma. And asbestos-induced cancers (e.g., mesothelioma).

[0153] In some embodiments, the extracellular particle enters the target cell by CD47-mediated endocytosis.

[0154] In some embodiments, the extracellular particle enters the target cell by clathrin-independent endocytosis.

[0155] In some embodiments, the extracellular particle may have an enhanced rate of clathrin-independent endocytosis in the target cell compared to a lipid bilayer particle that does not comprise a ligand for CD47, a fragment thereof, a variant thereof, a variant of a fragment thereof, or a fragment of a variant thereof.

[0156] In some embodiments, extracellular particles comprising a ligand for CD47 or the like may exhibit enhanced target substance delivery into target cells compared to native (or wild-type) extracellular particles that do not comprise a ligand for CD47 or the like. The target substance delivery into target cells may be evaluated using native (or wild-type) extracellular particles that are not loaded with the target substance as a control.

[0157] In some embodiments, the extracellular particle comprising a ligand for CD47 or the like can exhibit an improved ability to deliver a target substance into a target cell by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more compared to a native (or wild-type) extracellular particle that does not comprise a ligand for CD47 or the like.

[0158] In some embodiments, the extracellular particle comprising a ligand for CD47 or the like may have an improved rate of entering a target cell by clathrin-independent endocytosis by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more compared to a native (or wild-type) extracellular particle not comprising a ligand for CD47 or the like.

[0159] The present invention also provides a composition for delivering a target substance to a target cell, comprising a ligand for CD47, a fragment thereof, a variant thereof, a variant of a fragment thereof, or a fragment of a variant thereof, and comprising extracellular particles loaded with a target substance.

[0160] In some embodiments, the composition comprises a lipid bilayer particle comprising a ligand for CD47, a fragment thereof, a variant thereof, a variant of a fragment thereof, or a fragment of a variant thereof. The composition may further comprise a pharmaceutically acceptable carrier and / or excipient. The pharmaceutically acceptable excipient or carrier may be determined in part by the particular composition being administered as well as the particular method used to administer the composition. Accordingly, suitable formulations of the pharmaceutical composition vary widely. The pharmaceutical composition may generally be sterilized and formulated in full compliance with all Good Manufacturing Practice (GMP) regulations of the United States Food and Drug Administration (FDA).

[0161] In some embodiments, the pharmaceutically acceptable carrier may be a variety of oral or parenteral formulations. Diluents or excipients, such as fillers, bulking agents, binders, moisturizers, disintegrants, and surfactants, may be used in the manufacture of the formulations. Solid dosage forms for oral administration may include tablets, pills, powders, granules, and capsules, and these solid dosage forms may be manufactured by adding one or more excipients, such as starch, calcium carbonate, sucrose or lactose, and gelatin. In addition to simple excipients, lubricants, such as magnesium stearate and talc, may also be used. Liquid dosage forms for oral administration may include suspensions, solutions for oral administration, emulsions, and syrups, and in addition to commonly used simple diluents, various excipients, such as moisturizers, sweeteners, flavorings, and preservatives, may be used. Water and liquid paraffin. Formulations for parenteral administration may include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilizates, and suppositories. Non-aqueous solvents and suspending agents may include vegetable oils such as propylene glycol, polyethylene glycol, and olive oil; injectable esters such as ethyl oleate; and suppository bases such as withepsol, macrogol, Tween 61, cocoa butter, laurin, and glycerogelatin.

[0162] In some embodiments, the composition may have a formulation selected from the group consisting of tablets, pills, powders, granules, capsules, suspensions, solutions, emulsions, syrups, sterile aqueous solutions, non-aqueous solutions, lyophilized formulations, and suppositories.

[0163] In some embodiments, the composition may be administered orally or parenterally. When administered parenterally, the composition may be administered via various routes, including intravenous administration, intraarterial administration, epidural administration, intracerebral administration, intracerebroventricular administration, nasal administration, intramuscular administration, intraperitoneal administration, subcutaneous administration, intradermal administration, transdermal absorption, and transdermal administration.

[0164] In some embodiments, the composition may be administered in a therapeutically effective amount.

[0165] In some embodiments, the composition may be administered as a standalone treatment, in combination with another treatment, or sequentially or simultaneously with existing treatment(s), and may be administered once or multiple times. It is important to administer the amount that achieves maximum effect with the minimum amount possible without adverse effects, taking all factors into consideration, and these factors can be readily determined by those skilled in the art.

[0166]

[0167] The present invention also provides a method for improving the delivery efficiency of a target substance into a target cell, comprising the following steps:

[0168] (a) a step of loading a target substance into an extracellular particle comprising a ligand for CD47, a fragment thereof, a variant thereof, a variant of a fragment thereof, or a fragment of a variant thereof; and

[0169] (b) A step of bringing the extracellular particles into contact with a target cell.

[0170] In some embodiments, the method may be characterized by an improved delivery efficiency of the target substance into target cells by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more compared to a method in which the target substance is loaded into an extracellular particle that does not include a ligand for CD47 or the like.

[0171] In order to achieve another object of the present invention, the present invention provides the use of a ligand for CD47, a fragment thereof, a variant thereof, a variant of a fragment thereof, or a fragment of a variant thereof for preparing a composition for delivery of a target substance to a target cell.

[0172] In order to achieve another object of the present invention, the present invention provides the use of extracellular particles loaded with a target substance for preparing a composition for delivering a target substance to a target cell.

[0173]

[0174] Using the method and composition of the present invention, the target substance can be delivered into target cells very stably and effectively.

[0175]

[0176] Figure 1 shows the results of observing the cell uptake of SIRP-EV labeled with Cy5.5, and confirming the endocytosis pathway of SIRP-EV by treating it with Pitstop 2 (Pitstop-2, catalog: SML1169), a multi-pathway endocytosis inhibitor, and methyl-β-cyclodextrin (MβCD, catalog: C4555), a clathrin-independent endocytosis inhibitor, respectively.

[0177] Figures 2a to 2d show the results of observing the extent to which SIRP-EVs sink after binding to CD47 of cells after co-treatment of HEK297T cells with a clathrin-mediated endocytosis inhibitor, a clathrin-independent endocytosis inhibitor, or a macropinocytosis inhibitor.

[0178] Figures 3a and 3b show the results of observing the extent to which SIRP-EVs sink after binding to CD47 of the cells after co-treatment of HeLa cells with a clathrin-mediated endocytosis inhibitor, a clathrin-independent endocytosis inhibitor, or a macropinocytosis inhibitor.

[0179] Figure 4 shows the results of confirming the intracellular delivery ability of KRAS siRNA loaded into SIRP-EV through inhibition of KRAS expression, using lipofectamine as a control.

[0180] Figure 5 shows the results of confirming the intracellular delivery ability of SIRP-EVs by suppressing KRAS expression after loading KRAS siRNA into the EVs, and the results are obtained using naive EVs that do not express SIRPα as a control.

[0181] Figures 6a and 6b show the results comparing the cancer therapeutic effects of SIRP-EV and Naive-EV loaded with KRAS siRNA, respectively, in a CT26 tumor mouse model.

[0182] Figures 7a to 7c show the results comparing the cancer therapeutic effects of SIRP-EV loaded with KRAS siRNA and free KRAS siRNA in a CT26 tumor mouse model.

[0183] Figures 8a and 8b show the results comparing the anti-inflammatory effects of SIRP-EV loaded with NLRP3 antisense oligonucleotide (ASO) and free NLRP3 ASO in an LPS-induced acute inflammation mouse model.

[0184]

[0185] Hereinafter, the present invention will be described in detail with reference to the following examples. However, the following examples are intended only to illustrate the present invention and the present invention is not limited thereto.

[0186]

[0187] Example 1: Plasmid DNA construction

[0188] According to one embodiment of the present invention, a DNA construct capable of effectively localizing SIRPα protein to EV membranes was constructed. Commercial plasmid DNA was purchased from Origene, and additional vector constructs for the desired plasmid DNA sequences were obtained through a gene synthesis service (Cosmo Genetech Inc.). Vectors were based on pcDNA3.1, retroviral vectors, or lentiviral vectors. Empty vectors (pcDNA3.1 or retroviral vectors) were used as controls.

[0189] To maximize the efficiency of the existing structure, the SIRPα-ESM construct was prepared. The expression efficiency of the protein to be displayed in EVs was maximized through a sequence called the Extracellular Vesicle Sorting Motif (ESM). The ESM sequence and its preparation method are described in U.S. Application No. 18 / 322,723, which is incorporated herein by reference.

[0190]

[0191] The sequence for SIRPα-ESM comprises or consists of: (SEQ ID NO: 10 to 15)

[0192] Sequence number 10:

[0193] METDTLLLWVLLLWVPGSTGDEEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRVLIYNQRQGPFPRVTTVSDTTKRNNMDFSIRIGNITPADAGTYYCIKFRKGSPDDVEFKSGAGTELSVRAKPDVLNAFKYPLLIGIGLSAVIGLLSCLIGYCSSHWC

[0194] Sequence number 11:

[0195] MEPAGPAPGRLGPLLCLLLAASCAWSGVAGEEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRVLIYNQRQGPFPRVTTVSDTTKRNNMDFSIRIGNITPADAGTYYCIKFRKGSPDDVEFKSGAGTELSVRAKPDVLNAFKYPLLIGIGLSAVIGLLSCLIGYCSSHWC

[0196] Sequence number 12:

[0197] MMLQHLVIFCLGLVVQNFCSPEEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRVLIYNQRQGPFPRVTTVSDTTKRNNMDFSIRIGNITPADAGTYYCIKFRKGSPDDVEFKSGAGTELSVRAKPDVLNAFKYPLLIGIGLSAVIGLLSCLIGYCSSHWC

[0198] Sequence number 13:

[0199] METDTLLLWVLLLWVPGSTGDEEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRVLIYNQRQGPFPRVTTVSDTTKRNNMDFSIRIGNITPADAGTYYCIKFRKGSPDDVEFKSGAGTELSVRAKPKYPLLIGIGLSAVIGLLSCLIGYCSS

[0200] Sequence number 14:

[0201] MEPAGPAPGRLGPLLCLLLAASCAWSGVAGEEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRVLIYNQRQGPFPRVTTVSDTTKRNNMDFSIRIGNITPADAGTYYCIKFRKGSPDDVEFKSGAGTELSVRAKPKYPLLIGIGLSAVIGLLSCLIGYCSS

[0202] Sequence number 15:

[0203] MMLQHLVIFCLGLVVQNFCSPEEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRVLIYNQRQGPFPRVTTVSDTTKRNNMDFSIRIGNITPADAGTYYCIKFRKGSPDDVEFKSGAGTELSVRAKPKYPLLIGIGLSAVIGLLSCLIGYCSS

[0204] The above-mentioned plasmids were amplified and isolated according to the protocol of the Qiagen® Plasmid Maxi Kit.

[0205]

[0206] Example 2: Isolation of EVs

[0207] To generate SIRP-EVs from engineered HEK293 cells, various HEK293 cell lines (original or derivatives) were cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS) at 37°C with 5% CO2. When cells reached approximately 80% to 90% confluency, they were infected with specific plasmid DNA using appropriate infection reagents. Alternatively, cells were infected with retroviruses or lentiviruses to establish stable cell lines.

[0208] For transient transfection, cells were transfected using transfection agents such as Lipofectamine 2000, Lipofectamine 3000, or polyethyleneimine (PEI). The cell medium was replaced with DMEM, and the mixture of DNA and transfection agent was added to the cells. The cells were then cultured at 37°C with 5% CO2 for 24 hours. Twenty-four hours after transfection, the medium containing the transfection agent and plasmid was replaced with DMEM supplemented with 10% FBS and 1% antibiotic-antimycotic. The transiently transfected cells were cultured at 37°C with 5% CO2 for 24 hours.

[0209] For stable cell generation using retroviruses, Plat-E cells were used to produce retroviruses packaging a retroviral vector containing the DNA sequence of interest and the DNA sequence of the puromycin resistance gene. Specifically, Plat-E cells were cultured at 37°C with 5% CO2 in DMEM supplemented with 10% FBS. When the cells reached 80–90% confluence, the cells were transfected with the retroviral vector encoding the DNA sequence of interest using Lipofectamine 2000. After 24 hours, the culture medium was replaced with DMEM supplemented with 10% FBS and cultured for an additional 24 hours. Forty-eight hours after transfection, the culture medium containing viral particles was collected, centrifuged at 3,000 rpm, filtered through a 0.45 μm filter, and used for transfection of HEK293 cells.

[0210] To produce SIRP-EVs in engineered stem cells, lentiviral particles containing the desired DNA sequence were obtained from Genscript and Flash Therapeutics. The optimal multiplicity of infection (MOI) for the lentivirus was determined through titration studies. Human bone marrow-derived mesenchymal stem cells (hBM-MSCs, supplied by RoosterBio) were then transduced with the lentiviral particles for 24 hours using the RoosterGEM system (RoosterBio, catalog number: M40200). This process enabled the generation of stable cell lines producing SIRP-EVs.

[0211] To isolate EVs, cultures of transiently infected or stably expressing cells were placed in DMEM medium supplemented with insulin-transferrin-selenium (Gibco) or EV collection medium (RoosterCollect TM-EV, supplied by RoosterBio). The cells were then cultured at 37°C in 5% CO2 for 48 h. After culture, the cell supernatant was collected and sequentially centrifuged first at 300 g for 10 min, then at 2,000 g for 10 min, and finally at 10,000 g for 30 min. The supernatant was then filtered and concentrated using a tangential flow filtration (TFF) system or a 10 kDa Amicon Ultra-15 centrifugal filter device. After concentration, the supernatant was ultracentrifuged at 150,000 g for 1.5 h to pellet the EV. The resulting EV pellet was resuspended in phosphate-buffered saline (PBS) and stored at 4°C or -80°C until further use.

[0212]

[0213] Example 3: SIRP-EV cell uptake experiment (Fig. 1)

[0214] 1. Experimental method

[0215] - Cells: 293T wild type (CD47 wild type), 1.5 x 10 per well 5 Cells (24-well plate)

[0216] - Sample size: 3 (n = 3)

[0217] - EV group: naive-EV (MSC-derived EV), SIRP-EV (surface-engineered MSC-derived EV)

[0218] - Inhibitors: Pitstop 2 (25 μM), MβCD (3 mM)

[0219] - EV Labeling: 1 x 10 11 100ng Cy5.5 per EV

[0220] - EV: Cell Ratio: 1: 10,000

[0221] - Procedure: Cell seeding and overnight culture → Remove medium → Treat with inhibitor in serum-free medium for 15 minutes at 37℃ → Remove medium → Treat with EV for 3 hours at 37℃ → Dissociate cells with trypsin-EDTA → Wash with PBS x 2 times → Flow cytometry analysis

[0222]

[0223] 2. Experimental Results

[0224] - EVs were treated to CD47 wild-type 293T cells, and their uptake behavior was examined after 3 h. Naïve-EVs were not affected by inhibition of clathrin-independent endocytosis. In contrast, SIRP-EVs were confirmed to be internalized by both clathrin-independent endocytosis. Considering that clathrin-mediated endocytosis and macropinocytosis mostly cannot avoid the endolysosomal pathway, resulting in low intracellular delivery efficiency, these results suggest that SIRP-EVs can lead to cell uptake with higher delivery efficiency through clathrin-independent endocytosis than regular EVs.

[0225]

[0226] Example 4 (Figs. 2a to 3b)

[0227] 1. Experimental method

[0228] - Cells: HEK293T (1.5 x 10 per well) 5 cells (48-well plate)), HeLa (1 x 10 per well) 5 Cells (24 well plate)

[0229] - Sample size: 4 (n = 4)

[0230] - EV group: naive-EV (HEK293T-derived EV), SIRP-EV (surface-engineered HEK293T-derived EV)

[0231] - EV: Cell Ratio: 1: 10,000

[0232] - Procedure: Cell seeding and overnight culture → Remove medium → Treat with inhibitors in serum-free medium for 30 minutes at 37℃ → Remove medium → Treat with EV for 1 hour at 37℃, detach cells with trypsin-EDTA → Wash with acid (0.2 M acetic acid, 0.5 M NaCl) once → Wash with PBS x 2 times → Stain with APC anti-human CD47 (Catalog: 323124) antibody, wash with PBS x 2 times at 4℃ for 30 minutes → Flow cytometry analysis

[0233] - Inhibitors

[0234] Clathrin-mediated endocytosis inhibitor

[0235] : CPZ (chlorpromazine): 10μM

[0236] Macropinocytosis inhibitor

[0237] : EIPA (5-(N-ethyl-N-isopropyl)-amiloride): 50μM

[0238] Clathrin-independent endocytosis inhibitors

[0239] : MβCD (Methyl-β-cyclodextrin): 10mM

[0240] : Filip (Filipin lll): 1μg / mL

[0241] : Nys (Nystatin): 50μM

[0242] : 7-KC (7-ketocholesterol): 30μM

[0243]

[0244] 2. Experimental Results

[0245] Referring to Figures 2a to 2d, it was confirmed that SIRP-EVs in HEK293T cells were internalized into cells together with CD47 expressed on the cell surface through CD47-mediated endocytosis. This internalization was confirmed to be most significantly inhibited by MβCD among clathrin-independent endocytosis inhibitors.

[0246]

[0247] Referring to Figures 3a and 3b, we confirmed that SIRP-EVs were internalized into HeLa cells together with CD47 expressed on the cell surface through CD47-mediated endocytosis. This internalization was significantly inhibited by clathrin-independent endocytosis inhibitors Filip and MβCD.

[0248] These results imply that SIRP-EVs can bypass clathrin-mediated endocytosis and macropinocytosis and be delivered into cells, considering that endocytosis leads to lysosomal degradation.

[0249]

[0250] Example 5: KRAS siRNA delivery (Figures 4 and 5)

[0251] 1. Experimental method

[0252] - Cells: CT26 (CD47 overexpressing cell line) 1 x 10 per well 6 Cells (6-well plate)

[0253] - Sample size: 4 or 8 (n = 4 or 8)

[0254] - EV group: naive-EV (MSC-derived EV), SIRP-EV (surface-engineered MSC-derived EV)

[0255] - Conditions for chemically modified siRNA conjugation to EV

[0256] EV: chemocally modified siRNA = 1:100, incubated at 37°C for 1 hour

[0257] Culture volume: 500ul PBS

[0258] Remove unbound siRNA by centrifugation at 100,000 g for 30 minutes.

[0259] - KRAS siRNA information: (sense) GACCCUACGAUAGAGGACU (SEQ ID NO: 16) (antisense) AGUCCUCUAUCGUAGGGUC (SEQ ID NO: 17)

[0260] - Naive-EV and SIRP-EV treatment conditions: 10 per cell in CT26 4 Particles / cells treated with EV-Cholesterol-TEG-KRAS siRNA for 48 h

[0261] - Lipofectamine treatment conditions: CT26 cells were treated with the same amount of KRAS siRNA present in the relevant EV sample for 48 h.

[0262] - Procedure: Treat the siRNA group for 48 hours at 37°C under the above conditions for CT26 → Harvest cells and prepare RNA → Evaluate the KRAS gene using quantitative RT-PCR

[0263]

[0264] 2. Experimental Results

[0265] - SIRP-EV was confirmed to suppress KRAS expression in target cells more effectively than naive EV and lipofectamine via KRAS-siRNA. These results suggest that SIRP-EV may exhibit superior efficacy in terms of intracellular delivery efficiency.

[0266]

[0267] Example 6 (Fig. 6a and Fig. 6b)

[0268] To compare the anticancer activity of SIRP-EV and naive-EV loaded with KRAS siRNA in vivo, the following experiments were conducted.

[0269]

[0270] 1. Experimental method

[0271] - Mouse: Balb / c

[0272] - Sample size: 3-7 (n = 3-7)

[0273] - Sample groups: Naive-EV (MSC-derived EV), Naive-EV-siRNA (Naive-EV loaded with KRAS siRNA), SIRP-EV-siRNA (surface-engineered MSC-derived EV loaded with KRAS siRNA)

[0274] - EV treatment amount: 4 μg based on siRNA

[0275] - Procedure: Subcutaneous inoculation of CT26 cancer cell line into Balb / c mice → Direct injection of EV into tumor at 2-day intervals starting from day 8 after cancer cell inoculation (total 3 times) → 48 hours after the last EV administration, sacrifice the mouse and remove the tumor → Tumor weight measurement

[0276]

[0277] 2. Experimental Results

[0278] As shown in Figures 6a and 6b, when SIRP-EV-siRNA was directly administered intratumorally to a CT26 tumor mouse model, the antitumor effect of SIRP-EV-siRNA was confirmed to be superior to that of Naive-EV-siRNA. This indicates that SIRP-EV delivers the loaded drug to the tumor more efficiently than Naive-EV.

[0279]

[0280] Example 7 (Figs. 7a to 7c)

[0281] After loading KRAS siRNA into SIRP-EV, the following experiment was conducted to compare its therapeutic efficacy with that of free siRNA.

[0282]

[0283] 1. Experimental method

[0284] - Mouse: Balb / c

[0285] - Sample size: 4 (n = 4)

[0286] - Sample groups: siRNA (KRAS siRNA), SIRP-EV-siRNA (surface-engineered MSC-derived EV loaded with KRAS siRNA)

[0287] - Sample processing amount: 4 μg based on siRNA

[0288] - Procedure: Subcutaneous inoculation of CT26 cancer cell line into Balb / c mice → Tumor size measurement and intratumoral EV administration at 2-day intervals starting from day 6 after cancer cell inoculation (total 5 times) → 48 hours after the last EV administration, sacrifice the mouse and excise the tumor → RNA extraction using RNeasy Mini Kit (250) → Analysis of KRAS mRNA expression by RT-qPCR

[0289]

[0290] 2. Experimental Results

[0291] As shown in Figures 7a to 7c, when SIRP-EV-siRNA was directly administered intratumorally to a CT26 tumor mouse model, free siRNA showed no therapeutic effect at all, whereas SIRP-EV-siRNA was confirmed to effectively inhibit tumor growth. This indicates that SIRP-EV, unlike free siRNA, can effectively deliver siRNA into tumor cells.

[0292]

[0293] Example 8 (Figs. 8a and 8b)

[0294] To compare the anti-inflammatory response efficacy of SIRP-EV loaded with NLRP3 antisense oligonucleotide (ASO) with that of free NLRP3 ASO, the following experiment was conducted.

[0295]

[0296] 1. Experimental method

[0297] - Mouse: C57BL / 6

[0298] - Sample size: 3 (n = 3)

[0299] - Sample groups: Free ASO (NLRP3 ASO), SIRP-EV-ASO (surface-engineered MSC-derived EV loaded with NLRP3 ASO)

[0300] - Conditions for chemically modified ASO conjugation to EV

[0301] EV: chemically modified ASO = 1:1, room temperature, 30 min incubation

[0302] Culture volume: 400ul PBS

[0303] Removal of unbound ASO by ultracentrifugation for 20 minutes

[0304] NLRP3 ASO sequence information: GCTTGCAACGGACACTCGTC (SEQ ID NO: 18)

[0305] - Sample processing amount: 6 μg based on ASO

[0306] - Procedure: Administer samples into the mouse peritoneum every two days (2 times in total) → 24 hours after the last sample administration, induce acute inflammation by administering 200 μg of LPS into the mouse peritoneum → Collect mouse peritoneal lavage fluid 2 hours after LPS administration → Isolate cells from the collected peritoneal lavage fluid → Extract RNA using RNeasy Mini Kit (250) → Analyze NLRP3 mRNA expression using RT-qPCR

[0307]

[0308] 2. Experimental Results

[0309] As shown in Figures 8a and 8b, when SIRP-EV-ASO was intravenously administered to an LPS-induced acute inflammation mouse model, the level of NLRP3 mRNA expression in cells isolated from peritoneal lavage was evaluated, and unlike free ASO, SIRP-EV-ASO effectively inhibited NLRP3 expression.

[0310]

[0311] The method and composition of the present invention can deliver a target substance into a target cell very stably and effectively, and thus has a very high possibility of industrial application.

Claims

1. A method for delivering a target substance to a target cell, comprising a step of contacting extracellular particles loaded with a target substance, the extracellular particles comprising a ligand for CD47, a fragment thereof, a variant thereof, a variant of a fragment thereof, or a fragment of a variant thereof, with the target cell.

2. A method according to claim 1, characterized in that the ligand for CD47 is a protein, peptide, antibody, antibody fragment, aptamer, low-molecular-weight compound or a combination thereof that binds to CD47.

3. A method according to claim 1, characterized in that the ligand for CD47 is SIRP (signal regulatory protein), TSP-1 (Thrombospodin-1) or a combination thereof.

4. A method according to claim 3, characterized in that the SIRP is SIRPα, SIRPβ, SIRPγ or a combination thereof.

5. A method according to claim 1, wherein the extracellular particles are selected from the group consisting of extracellular vesicles, non-vesicular extracellular particles, virus particles, virus-like particles, liposomes, artificial cell-derived vesicles, synthetic vesicles, extracellular vesicle mimetics, and platelet-like particles.

6. A method according to claim 5, characterized in that the extracellular particles are derived from stem cells.

7. A method according to claim 6, characterized in that the stem cells are selected from the group consisting of mesenchymal stem cells, human tissue-derived mesenchymal stromal cells, human tissue-derived mesenchymal stem cells, multipotent stem cells, embryonic stem cells, induced pluripotent stem cells, multipotent stem cells, and amniotic epithelial cells.

8. A method according to claim 5, characterized in that the extracellular particles are derived from human embryonic kidney cells (HEK cells).

9. A method according to claim 5, characterized in that the extracellular vesicles are selected from the group consisting of exosomes, microvesicles, and apoptotic bodies.

10. A method according to claim 1, characterized in that the ligand for CD47, a fragment thereof, a variant thereof, a variant of a fragment thereof, or a fragment of a variant thereof is linked to at least one extracellular particle protein.

11. In the 10th paragraph, the extracellular particle protein is CD9, CD53, CD63, CD81, CD54, CD50, FLOT1, FLOT2, CD49d, CD71, CD133, CD138, CD235a, ALIX, syntenin-1, syntenin-2, Lamp2b, TSPAN8, TSPAN14, CD37, CD82, CD151, CD231, CD102, NOTCH1, NOTCH2, NOTCH3, NOTCH4, DLL1, DLL4, JAG1, JAG2, CD49d / ITGA4, ITGB5, ITGB6, ITGB7, CD11a, CD11b, CD11c, CD18 / ITGB2, CD41, CD49b, CD49c, CD49e, CD51, CD61, CD104, Tetraspanin, Fc receptor, interleukin receptor, immunoglobulin, MHC-I component, MHC-II component, CD2, CD3 epsilon, CD3 zeta, CD13, CD18, CD19, CD30, CD34, CD36, CD40, CD40L, CD44, CD45, CD45RA, CD47, CD86, CD110, CD111, CD115, CD117, CD125, CD135, CD184, CD200, CD279, CD273, CD274, CD362, COL6A1, AGRN, EGFR, GAPDH, GLUR2, GLUR3, HLA-DM, HSPG2, L1CAM, LAMB1, LAMC1, LFA-1, LGALS3BP, Mac-1 alpha, Mac-1 beta, MFGE8, SLIT2, STX3, TCRA, TCRB, TCRD, TCRG, VTI1A, VTI1B, PDGFR, GPI anchor protein, lactadherin, syndecan, synaptotagmin, ALIX (apoptosis-linked gene 2-interacting protein X),A method characterized in that it is selected from the group consisting of PTGFRN (prostaglandin F2 receptor inhibitor), HSP90AA / B, TGFβ-induced (TGFBI), HSPA13, LDHA / B, ENO1, ENO2, ALDOA, GPI, ACTN4, AGO1, AGO2, HEXOKINASE I, fragments thereof, variants thereof, variants of fragments thereof, and fragments of variants thereof.

12. A method according to claim 1, characterized in that the target substance is a therapeutic agent, a diagnostic agent, or a combination thereof.

13. A method according to claim 1, characterized in that the target substance is selected from the group consisting of nucleic acids, proteins, polypeptides, and small molecules.

14. A method according to claim 1, wherein the target substance is selected from the group consisting of a transcription factor, a messenger RNA (mRNA), a single-stranded DNA (ssDNA), a double-stranded DNA (dsDNA), a single guide RNA (sgRNA), a guide RNA (gRNA), a prime editing guide RNA (pegRNA), a microRNA (miRNA), a small interfering RNA (siRNA), an antisense oligonucleotide, a short hairpin RNA (shRNA), a double-stranded RNA (dsRNA), an antisense RNA, a ribozyme, a nuclease, and a CRISPR-Cas family protein and a CRISPR-associated endonuclease.

15. A method according to claim 1, characterized in that the target cell is a CD47 positive cell, a CD47 overexpressing cell, or a combination thereof.

16. A method according to claim 1, characterized in that the ligand for CD47, a fragment thereof, a variant thereof, a variant of a fragment thereof, or a fragment of a variant thereof is exposed to the outside of the extracellular particle.

17. A method according to claim 1, characterized in that the extracellular particle enters the target cell through CD47-mediated endocytosis.

18. A method according to claim 1, characterized in that the CD47-mediated endocytosis is clathrin-independent.

19. A method according to claim 1, wherein the extracellular particle has an improved clathrin-independent endocytosis rate in the target cell compared to an extracellular particle that does not include a ligand for CD47, a fragment thereof, a variant thereof, a variant of a fragment thereof, or a fragment of a variant thereof.

20. A composition for delivering a target substance to a target cell, comprising a ligand for CD47, a fragment thereof, a variant thereof, a variant of a fragment thereof, or a fragment of a variant thereof, and comprising extracellular particles loaded with a target substance.

21. A method for improving the delivery efficiency of a target substance into a target cell, comprising the following steps: (a) a step of loading a target substance into an extracellular particle comprising a ligand for CD47, a fragment thereof, a variant thereof, a variant of a fragment thereof, or a fragment of a variant thereof; and (b) A step of bringing the extracellular particles into contact with a target cell.

22. Use of a ligand for CD47, a fragment thereof, a variant thereof, a variant of a fragment thereof, or a fragment of a variant thereof for preparing a composition for delivering a target substance to a target cell.

23. Use of extracellular particles loaded with a target substance for preparing a composition for delivering a target substance to a target cell.

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