Adapter polypeptide and method of use thereof

Abstract

Providing an adapter polypeptide and a method for using it. [Solution] Compositions of extracellular vesicles, as well as methods and systems for producing extracellular vesicles, are described herein. Methods for using extracellular vesicles are also described herein. This disclosure provides extracellular vesicles designed to target a wide variety of cell types, including different cells and organs in the body, as well as cells associated with disease or disorder. In some cases, the extracellular vesicles provided herein can be readily modified to bind specifically to a target. For example, they may include an extracellular domain that binds to a cell surface marker (e.g., the extracellular domain of a transmembrane protein within the membrane of the extracellular vesicle). Generally, the extracellular vesicles provided herein include an adapter polypeptide having an extracellular domain and, optionally, a transmembrane domain that binds to a cell surface marker.

Description

[Technical Field]

[0001] [Cross-reference of related applications] This application claims the benefit of U.S. Provisional Application No. 63 / 061,749, filed on 5 August 2020. Priority is claimed pursuant to Section 119 of the U.S. Patent Act (35 U.S.C. § 119). The aforementioned patent application is incorporated by reference as if it were fully described herein.

[0002] Embedding by reference All publications, patents, and patent applications referenced herein are incorporated herein by reference in whole to the same extent that each individual publication, patent, or patent application is specifically and individually indicated as being incorporated by reference. [Background technology]

[0003] background Extracellular vesicles are secreted by a wide variety of cell types. Generally, extracellular vesicles such as exosomes, microvesicles, and apoptotic bodies are membrane-bound and can be loaded as therapeutic cargo. For example, exosomes are a type of membrane-bound extracellular vesicle secreted by most eukaryotic cells. Exosome biosynthesis can begin with the invagination of an endosome being constricted into a polytope to form an intraluminal vesicle. Once the polytope fuses with the cell's plasma membrane, the intraluminal vesicle can be released as an exosome. Microvesicles budding from the cell membrane surface. Apoptotic bodies, on the other hand, are released from dead cells. Exosomes, microvesicles, and apoptotic bodies can be released in vivo or in vitro, for example, in cell culture.

[0004] Extracellular vesicles have been studied as vehicles for encapsulating and delivering therapeutic agents. Targeting extracellular vesicles is generally difficult because most extracellular vesicles are degraded in the liver, spleen, and / or kidneys. Also, the design and manufacture of extracellular vesicles for encapsulating therapeutic agents for targeted delivery are time-consuming and costly. For example, extracellular vesicles designed to target one cell type may not effectively target another cell type. Thus, there is still a need for extracellular vesicles that can be easily modified to target multiple cell types. There is also still a need for extracellular vesicles that can encapsulate a sufficient amount and quality of therapeutic agents to be delivered to the targeted cells. Summary of the Invention Means for Solving the Problems

[0005] Overview The present disclosure provides extracellular vesicles designed to target a wide variety of cell types, including different cells and organs in the body, as well as cells associated with a disease or disorder. In some cases, the extracellular vesicles provided herein can be easily modified to specifically bind to a target. For example, they can include an extracellular domain that binds to a cell surface marker (e.g., the extracellular domain of a transmembrane protein within the membrane of the extracellular vesicle). Generally, the extracellular vesicles provided herein include an adapter polypeptide having an extracellular domain and, optionally, a transmembrane domain that binds to a cell surface marker.

[0006] In some aspects, described herein is a composition comprising at least one extracellular vesicle, wherein the extracellular vesicle is 10 -9A composition comprising at least one adapter polypeptide comprising a peptide sequence that binds to the Fc region of an antibody with a dissociation constant (Kd) less than or equal to M, wherein the adapter polypeptide comprises an extracellular domain; an antibody complexed with the adapter polypeptide, wherein the antibody binds to a first cell surface marker associated with an affected cell; and at least one therapeutic agent. In some embodiments, a composition comprising at least one extracellular vesicle is described herein, wherein the aforementioned extracellular vesicle comprises at least one adapter polypeptide comprising a peptide sequence that is at least 70% identical to an Fc receptor that specifically recognizes the Fc region of an antibody (wherein the aforementioned adapter polypeptide comprises an extracellular domain); the aforementioned antibody complexed with the aforementioned adapter polypeptide (wherein the aforementioned antibody binds to a first cell surface marker associated with an affected cell); and at least one therapeutic agent. In some embodiments, the aforementioned receptor comprises FcγRI(CD64), FcγRII(CD32), or FcγRIII(CD16). In some embodiments, the aforementioned Fc receptor is CD64. In some embodiments, the aforementioned adapter polypeptide further includes a targeting domain that binds to a second cell surface marker associated with the affected cell, the targeting domain being bound to the extracellular domain of the adapter polypeptide. In some embodiments, the aforementioned targeting domain is selected from the group consisting of tumor-homing peptides, tumor-targeting domains, tissue-targeting domains, cell-permeable peptides, viral membrane proteins, and any combination or fragment thereof. In some embodiments, the aforementioned affected cell is a cancer cell or a non-cancerous lesion cell. In some embodiments, the aforementioned first cell surface marker includes EGFR, PD-L1, or ROR1. In some embodiments, the aforementioned first cell surface marker and the second cell surface marker are different. In some embodiments, the aforementioned first cell surface marker and the second cell surface marker are identical. In some embodiments, the aforementioned antibody is a humanized monoclonal antibody.In some embodiments, the aforementioned antibody is selected from the group consisting of humanized anti-EGFR antibody clone C225, humanized anti-ROR1 antibody clone 2A2, and humanized anti-PD-L1 antibody clone SP142. In some embodiments, the aforementioned humanized monoclonal antibody contains IgG. In some embodiments, the aforementioned humanized monoclonal antibody contains IgG1 or IgG3. In some embodiments, the aforementioned antibody is non-covalently complexed with an adapter polypeptide. In some embodiments, the Fc region of the aforementioned antibody is configured to complex with the adapter polypeptide in an acidic environment. In some embodiments, the Fc region of the aforementioned antibody is configured to be released from the complex with the adapter polypeptide in an acidic environment. In some embodiments, the aforementioned at least one therapeutic agent is contained within an extracellular vesicle. In some embodiments, the aforementioned at least one therapeutic agent is expressed on the extracellular surface of the extracellular vesicle. In some embodiments, the aforementioned at least one therapeutic agent is bound to an extracellular domain. In some embodiments, the aforementioned at least one therapeutic agent includes a therapeutic polynucleotide, a therapeutic polypeptide, a therapeutic compound, an oncological drug, or a combination thereof. In some embodiments, the therapeutic polynucleotides described above include messenger RNA, microRNA, shRNA, or a combination thereof. In some embodiments, the extracellular vesicles described above are exosomes, microvesicles, or apoptotic bodies. In some embodiments, the extracellular vesicles described above are exosomes.

[0007] Described herein are methods for treating a subject, comprising administering a therapeutically effective amount of a pharmaceutical composition to the subject, wherein the pharmaceutical composition comprises the composition described herein. In some embodiments, the aforementioned pharmaceutical composition comprises at least one pharmaceutically acceptable excipient. In some embodiments, the aforementioned subject has a cancerous or non-cancerous lesion. In some embodiments, the aforementioned subject has a glioma. In some embodiments, the aforementioned subject has a muscular dystrophy. In some embodiments, the aforementioned muscular dystrophy is selected from the group consisting of Duchenne muscular dystrophy, Becker muscular dystrophy, facioscapulohumeral muscular dystrophy, congenital muscular dystrophy, and myotonic dystrophy. In some embodiments, the aforementioned subject has a retinal disease. In some embodiments, the aforementioned retinal disease is retinitis pigmentosa or Leber congenital amaurosis. In some embodiments, the aforementioned therapeutically effective amount of the aforementioned pharmaceutical composition comprises a therapeutically effective dose. In some embodiments, the aforementioned subject is administered the aforementioned therapeutically effective amount of the aforementioned pharmaceutical composition at a therapeutically effective frequency. In some embodiments, the subject is administered the aforementioned therapeutically effective dose of the aforementioned pharmaceutical composition at a frequency of at least once a year. In some embodiments, the subject is administered the aforementioned therapeutically effective dose of the aforementioned pharmaceutical composition at a frequency of at least once every six months. In some embodiments, the subject is administered the aforementioned therapeutically effective dose of the aforementioned pharmaceutical composition at a frequency of at least once a month. In some embodiments, the subject is administered the aforementioned therapeutically effective dose of the aforementioned pharmaceutical composition at a frequency of at least once a week. In some embodiments, the aforementioned pharmaceutical composition is an aqueous formulation. In some embodiments, the aforementioned pharmaceutical composition is formulated for injection. In some embodiments, the aforementioned pharmaceutical composition is administered to the subject intranasally, intrathecally, intraocularly, intravitrebellarly, intravenously, intramuscularly, intraventricularly, intracerebrally, intracerebellarly, intraventricularly, intraparenchymally, subcutaneously, or in combination thereof.

[0008] Described herein are, in some examples, methods for producing compositions, the methods comprising: transfecting extracellular vesicle donor cells with at least one heterologous polynucleotide encoding an adapter polypeptide, wherein the adapter polypeptide comprises a peptide sequence at least 70% identical to that of an Fc receptor, and the Fc receptor recognizes the Fc region of the antibody; recovering extracellular vesicles released from the extracellular vesicle donor cells, wherein the extracellular vesicles released from the extracellular vesicle donor cells express the adapter polypeptide, wherein the adapter polypeptide comprises an extracellular domain, and the extracellular vesicles contain at least one therapeutic agent; and conjugating an antibody with the extracellular domain, wherein the antibody binds to a first cell surface marker associated with the affected cell. In some embodiments, the aforementioned Fc receptor is an Fc-γ receptor, an Fc-α receptor, or an Fc-ε receptor. In some embodiments, the aforementioned Fc receptor is an FcγRI(CD64), an FcγRII(CD32), or an FcγRIII(CD16). In some embodiments, the aforementioned Fc receptor is CD64. In some embodiments, the aforementioned adapter polypeptide further includes a targeting domain that binds to a second cell surface marker associated with affected cells, the targeting domain being bound to an extracellular domain. In some embodiments, the aforementioned first or second cell surface marker is associated with cancer cells or non-cancerous lesion cells. In some embodiments, the aforementioned first cell surface marker includes EGFR, PD-L1, or ROR1. In some embodiments, the aforementioned first and second cell surface markers are different. In some embodiments, the aforementioned first and second cell surface markers are identical. In some embodiments, the aforementioned targeting domain is selected from the group consisting of tumor-homing peptides, tumor-targeting domains, tissue-targeting domains, cell-permeable peptides, viral membrane proteins, and any combination or fragment thereof. In some embodiments, the aforementioned at least one therapeutic agent is contained within an extracellular vesicle. In some embodiments, the aforementioned at least one therapeutic agent is expressed on the extracellular surface of the extracellular vesicle.In some embodiments, at least one of the aforementioned therapeutic agents is bound to the extracellular domain. In some embodiments, at least one of the aforementioned therapeutic agents includes a therapeutic polynucleotide, a therapeutic polypeptide, a therapeutic compound, a cancer drug, or a combination thereof. In some embodiments, the aforementioned therapeutic polynucleotide includes messenger RNA, microRNA, shRNA, or a combination thereof. In some embodiments, the extracellular vesicles released from the aforementioned extracellular vesicle donor cells are exosomes, microvesicles or apoptotic bodies. In some embodiments, the extracellular vesicles released from the aforementioned extracellular vesicle donor cells are exosomes. In some embodiments, the aforementioned extracellular vesicle donor cells include electroporation, microfluidic electroporation, microchannel electroporation, or nanochannel electroporation. In some embodiments, the aforementioned microchannel electroporation or nanochannel electroporation includes the use of a micro-pore patterned silicon wafer, a nano-pore patterned silicon wafer, a track-etched membrane, a ceramic micro-pore membrane, a ceramic nano-pore membrane, other porous materials, or a combination thereof. In some embodiments, the step of transfecting the aforementioned extracellular vesicle donor cells includes nanochannel electroporation, and at least one heterologous polynucleotide is nanoelectroporated into the extracellular vesicle donor cells through nanochannels located on a biochip. In some embodiments, the step of transfecting the aforementioned extracellular vesicle donor cells includes the use of a gene gun, a micro-needle array, a nano-needle array, sonication, or chemical permeation. In some embodiments, at least one of the aforementioned heterologous polynucleotides is a plasmid.

[0009] In some cases, 10 -9This specification describes a composition comprising at least one extracellular vesicle comprising at least one adapter polypeptide (wherein the adapter polypeptide comprises an extracellular domain) having a peptide sequence that binds to the Fc region of an antibody with a dissociation constant (Kd) less than or equal to M, the aforementioned antibody (wherein the aforementioned antibody binds to a first cell surface marker associated with immune cells) complexed with the aforementioned adapter polypeptide, and at least one viral-mimetic peptide. In some embodiments, the composition comprises at least one extracellular vesicle comprising at least one adapter polypeptide having a peptide sequence that is at least 70% identical to an Fc receptor that binds to the Fc region of an antibody, wherein the adapter polypeptide comprises an extracellular domain, an antibody complexed with the adapter polypeptide, wherein the antibody specifically binds to a first cell surface marker associated with immune cells, and at least one viral-mimetic peptide. In some embodiments, the aforementioned Fc receptor is an Fc-γ receptor, an Fc-α receptor, or an Fc-ε receptor. In some embodiments, the aforementioned Fc receptor includes FcγRI(CD64), FcγRII(CD32), or FcγRIII(CD16). In some embodiments, the aforementioned Fc receptor is CD64. In some embodiments, the aforementioned adapter polypeptide further includes a targeting domain that binds to a second cell surface marker associated with immune cells, the targeting domain being bound to the extracellular domain of the adapter polypeptide. In some embodiments, the aforementioned immune cells are T cells, B cells, dendritic cells, macrophages, or natural killer (NK) cells. In some embodiments, the aforementioned first cell surface marker includes LILRA 4, CD3, CD19, CD20, or CD28. In some embodiments, the aforementioned first cell surface marker and the aforementioned second cell surface marker are different. In some embodiments, the aforementioned first cell surface marker and the aforementioned second cell surface marker are identical. In some embodiments, the aforementioned antibody is a humanized monoclonal antibody. In some embodiments, the aforementioned antibody is IgG. In some embodiments, the aforementioned antibody includes IgG1 or IgG3.In some embodiments, the aforementioned antibody is non-covalently complexed with the adapter polypeptide. In some embodiments, the Fc region of the aforementioned antibody is configured to complex with the adapter polypeptide in an acidic environment. In some embodiments, the Fc region of the aforementioned antibody is configured to be released from complexation with the adapter polypeptide in an acidic environment. In some embodiments, the aforementioned at least one viral-mimetic peptide is expressed on the extracellular surface of an extracellular vesicle. In some embodiments, the aforementioned at least one viral-mimetic peptide is bound to an extracellular domain. In some embodiments, the aforementioned at least one viral-mimetic peptide contains a peptide sequence that is at least 70% identical to the SARS-CoV-2 viral protein. In some embodiments, the aforementioned SARS-CoV-2 viral protein includes an envelope (E) protein, a nucleocapsid (N) protein, a membrane (M) protein, or a spike (S) protein. In some embodiments, the aforementioned SARS-CoV-2 viral protein is the aforementioned S protein. In some embodiments, the aforementioned extracellular vesicle includes an exosome, a microvesicle, or an apoptotic body. In some embodiments, the aforementioned extracellular vesicles are exosomes.

[0010] Described herein are methods for vaccinating a subject, in some cases comprising administering a therapeutically effective amount of a pharmaceutical composition to the subject, wherein the pharmaceutical composition comprises the composition described herein. In some embodiments, the aforementioned pharmaceutical composition comprises at least one pharmaceutically acceptable excipient. In some embodiments, the aforementioned therapeutically effective amount of the aforementioned pharmaceutical composition comprises a therapeutically effective dose. In some embodiments, the aforementioned subject is administered the aforementioned therapeutically effective amount of the aforementioned pharmaceutical composition at a therapeutically effective frequency. In some embodiments, the aforementioned subject is administered the aforementioned therapeutically effective amount of the aforementioned pharmaceutical composition at a frequency of at least once a year. In some embodiments, the aforementioned subject is administered the aforementioned therapeutically effective amount of the aforementioned pharmaceutical composition at a frequency of at least once every six months. In some embodiments, the aforementioned subject is administered the aforementioned therapeutically effective amount of the aforementioned pharmaceutical composition at a frequency of at least once a month. In some embodiments, the aforementioned subject is administered the aforementioned therapeutically effective amount of the aforementioned pharmaceutical composition at a frequency of at least once a week. In some embodiments, the aforementioned pharmaceutical composition is an aqueous formulation. In some embodiments, the aforementioned pharmaceutical composition is formulated for injection. In some embodiments, the aforementioned pharmaceutical compositions are administered to the subject intranasally, intrathecally, intraocularly, intravitreously, intraretinally, intravenously, intramuscularly, intraventricularly, intracerebrally, intracerebellarly, intraventricularly, intraparenchymally, subcutaneously, or in combination thereof.

[0011] Described herein are methods for producing, in some cases, compositions described herein, the methods comprising: transfecting extracellular vesicle donor cells with at least one heterologous polynucleotide encoding an adapter polypeptide, wherein the adapter polypeptide comprises a peptide sequence at least 70% identical to that of an Fc receptor, and the Fc receptor recognizes the Fc region of an antibody; recovering extracellular vesicles released from the extracellular vesicle donor cells, wherein the extracellular vesicles released from the extracellular vesicle donor cells express an adapter polypeptide, wherein the adapter polypeptide comprises an extracellular domain, and the extracellular vesicles comprise at least one virus-mimicking peptide; and complexing an antibody with the extracellular domain, wherein the antibody binds to a first cell surface marker associated with an immune cell. In some embodiments, the aforementioned Fc receptor is an Fc-γ receptor, an Fc-α receptor, or an Fc-ε receptor. In some embodiments, the aforementioned Fc receptor includes FcγRI(CD64), FcγRII(CD32), or FcγRIII(CD16). In some embodiments, the aforementioned Fc receptor is CD64. In some embodiments, the aforementioned adapter polypeptide further includes a targeting domain that binds to a second cell surface marker associated with immune cells, the targeting domain being bound to an extracellular domain. In some embodiments, the aforementioned immune cells are T cells, B cells, dendritic cells, macrophages, or natural killer (NK) cells. In some embodiments, the aforementioned first cell surface marker includes LILRA 4, CD3, CD19, CD20, or CD28. In some embodiments, the aforementioned first cell surface marker and the second cell surface marker are different. In some embodiments, the aforementioned first cell surface marker and the second cell surface marker are identical. In some embodiments, the aforementioned antibody is a humanized monoclonal antibody. In some embodiments, the aforementioned antibody contains IgG. In some embodiments, the aforementioned antibody contains IgG1 or IgG3. In some embodiments, the aforementioned antibody is non-covalently complexed with the adapter polypeptide.In some embodiments, the Fc region of the aforementioned antibody is configured to complex with an adapter polypeptide in an acidic environment. In some embodiments, the Fc region of the aforementioned antibody is configured to be released from complexation with the adapter polypeptide in an acidic environment. In some embodiments, the aforementioned at least one viral-mimetic peptide is expressed on the extracellular surface of an extracellular vesicle. In some embodiments, the aforementioned at least one viral-mimetic peptide is bound to an extracellular domain. In some embodiments, the aforementioned at least one viral-mimetic peptide contains a peptide sequence that is at least 70% identical to that of the SARS-CoV-2 viral protein. In some embodiments, the aforementioned SARS-CoV-2 viral protein includes an envelope (E) protein, a nucleocapsid (N) protein, a membrane (M) protein, or a spike (S) protein. In some embodiments, the aforementioned SARS-CoV-2 viral protein is the aforementioned S protein. In some embodiments, the aforementioned extracellular vesicle includes an exosome, a microvesicle, or an apoptotic body. In some embodiments, the aforementioned extracellular vesicle is an exosome. In some embodiments, the step of transfecting the extracellular vesicle donor cells described above includes electroporation, microfluidic electroporation, microchannel electroporation, or nanochannel electroporation. In some embodiments, the aforementioned microchannel electroporation or nanochannel electroporation includes the use of a microporous patterned silicon wafer, a nanoporous patterned silicon wafer, a track etching film, a ceramic microporous film, a ceramic nanoporous film, other porous materials, or a combination thereof. In some embodiments, the step of transfecting the extracellular vesicle donor cells described above includes nanochannel electroporation, in which at least one heterologous polynucleotide is nanoelectroporated to the extracellular vesicle donor cells via nanochannels located on the biochip.In some embodiments, the step of transfecting the extracellular vesicle donor cells described above includes the use of a gene gun, microneedle array, nanoneedle array, sonication, or chemiosmosis. In some embodiments, the aforementioned at least one heterologous polynucleotide is a plasmid.

[0012] In some embodiments, a composition comprising at least one extracellular vesicle, wherein the said extracellular vesicle is 10 -9This specification describes a composition comprising at least one adapter polypeptide (wherein the adapter polypeptide comprises an extracellular domain) having a peptide sequence that binds to the Fc region of a binding molecule with a dissociation constant (Kd) less than or equal to M, the aforementioned binding molecule (wherein the aforementioned binding molecule binds to a first cell surface marker associated with the affected cell) complexed with the aforementioned adapter polypeptide, and at least one therapeutic agent. In some embodiments, this specification describes a composition comprising at least one extracellular vesicle, wherein the aforementioned extracellular vesicle comprises at least one adapter polypeptide having a peptide sequence that is at least 70% identical to an Fc receptor that specifically recognizes the Fc region of the binding molecule, the adapter polypeptide comprising an extracellular domain, a binding molecule complexed with the adapter polypeptide, the binding molecule specifically binds to a first cell surface marker associated with the affected cell, and at least one therapeutic agent. In some embodiments, the aforementioned Fc receptor is an Fc-γ receptor, an Fc-α receptor, or an Fc-ε receptor. In some embodiments, the aforementioned Fc receptor includes FcγRI(CD64), FcγRII(CD32), or FcγRIII(CD16). In some embodiments, the aforementioned Fc receptor is CD64. In some embodiments, the aforementioned adapter polypeptide further includes a targeting domain that binds to a second cell surface marker associated with the affected cell, the targeting domain being bound to the extracellular domain of the adapter polypeptide. In some embodiments, the aforementioned targeting domain is selected from the group consisting of tumor-homing peptides, tumor-targeting domains, tissue-targeting domains, cell-permeable peptides, viral membrane proteins, and any combination or fragment thereof. In some embodiments, the aforementioned first cell surface marker includes EGFR, PD-L1, or ROR1. In some embodiments, the aforementioned first cell surface marker and the second cell surface marker are different. In some embodiments, the aforementioned first cell surface marker and the second cell surface marker are identical. In some embodiments, the aforementioned binding molecule is a humanized monoclonal antibody.In some embodiments, the aforementioned binding molecule is selected from the group consisting of humanized anti-EGFR antibody clone C225, humanized anti-ROR1 antibody clone 2A2, and humanized anti-PD-L1 antibody clone SP142. In some embodiments, the aforementioned humanized monoclonal antibody contains IgG. In some embodiments, the aforementioned humanized monoclonal antibody contains IgG1 or IgG3. In some embodiments, the aforementioned binding molecule is non-covalently complexed with an adapter polypeptide. In some embodiments, the Fc region of the aforementioned binding molecule is configured to complex with the adapter polypeptide in an acidic environment. In some embodiments, the Fc region of the aforementioned binding molecule is configured to be released from the complex with the adapter polypeptide in an acidic environment. In some embodiments, the aforementioned at least one therapeutic agent is contained within an extracellular vesicle. In some embodiments, the aforementioned at least one therapeutic agent is expressed on the extracellular surface of the extracellular vesicle. In some embodiments, the aforementioned at least one therapeutic agent is bound to an extracellular domain. In some embodiments, the aforementioned at least one therapeutic agent includes a therapeutic polynucleotide, a therapeutic polypeptide, a therapeutic compound, an oncological drug, or a combination thereof. In some embodiments, the therapeutic polynucleotides described above include messenger RNA, microRNA, shRNA, or a combination thereof. In some embodiments, the extracellular vesicles described above are exosomes, microvesicles, or apoptotic bodies. In some embodiments, the extracellular vesicles described above are exosomes.

[0013] Described herein are methods for treating a subject, in some examples, comprising administering a therapeutically effective amount of a pharmaceutical composition to the subject, wherein the pharmaceutical composition comprises the composition described herein. In some embodiments, the aforementioned pharmaceutical composition comprises at least one pharmaceutically acceptable excipient. In some embodiments, the aforementioned subject has a cancerous or non-cancerous lesion. In some embodiments, the aforementioned subject has a glioma. In some embodiments, the aforementioned subject has a muscular dystrophy. In some embodiments, the aforementioned muscular dystrophy is selected from the group consisting of Duchenne muscular dystrophy, Becker muscular dystrophy, facioscapulohumeral muscular dystrophy, congenital muscular dystrophy, and myotonic dystrophy. In some embodiments, the aforementioned subject has a retinal disease. In some embodiments, the aforementioned retinal disease is retinitis pigmentosa or Leber congenital amaurosis. In some embodiments, the aforementioned therapeutically effective amount of the aforementioned pharmaceutical composition comprises a therapeutically effective dose. In some embodiments, the aforementioned subject is administered the aforementioned therapeutically effective amount of the aforementioned pharmaceutical composition at a therapeutically effective frequency. In some embodiments, the subject is administered the aforementioned therapeutically effective dose of the aforementioned pharmaceutical composition at a frequency of at least once a year. In some embodiments, the subject is administered the aforementioned therapeutically effective dose of the aforementioned pharmaceutical composition at a frequency of at least once every six months. In some embodiments, the subject is administered the aforementioned therapeutically effective dose of the aforementioned pharmaceutical composition at a frequency of at least once a month. In some embodiments, the subject is administered the aforementioned therapeutically effective dose of the aforementioned pharmaceutical composition at a frequency of at least once a week. In some embodiments, the aforementioned pharmaceutical composition is an aqueous formulation. In some embodiments, the aforementioned pharmaceutical composition is formulated for injection. In some embodiments, the aforementioned pharmaceutical composition is administered to the subject intranasally, intrathecally, intraocularly, intravitrebellarly, intravenously, intramuscularly, intraventricularly, intracerebrally, intracerebellarly, intraventricularly, intraparenchymally, subcutaneously, or in combination thereof.

[0014] Described herein are methods for producing compositions, in some cases, the method comprising: transfecting extracellular vesicle donor cells with at least one heterologous polynucleotide encoding an adapter polypeptide, wherein the adapter polypeptide comprises a peptide sequence at least 70% identical to that of an Fc receptor, and the Fc receptor recognizes the Fc region of the binding molecule; recovering extracellular vesicles released from the extracellular vesicle donor cells, wherein the extracellular vesicles released from the extracellular vesicle donor cells express the adapter polypeptide, wherein the adapter polypeptide comprises an extracellular domain, and the extracellular vesicles contain at least one therapeutic agent; and complexing the binding molecule with the extracellular domain, wherein the binding molecule binds to a first cell surface marker associated with the affected cell. In some embodiments, the aforementioned Fc receptor is an Fc-γ receptor, an Fc-α receptor, or an Fc-ε receptor. In some embodiments, the aforementioned Fc receptor is an FcγRI(CD64), an FcγRII(CD32), or an FcγRIII(CD16). In some embodiments, the aforementioned Fc receptor is CD64. In some embodiments, the aforementioned adapter polypeptide further includes a targeting domain that binds to a second cell surface marker associated with affected cells, the targeting domain being bound to an extracellular domain. In some embodiments, the aforementioned first or second cell surface marker is associated with cancer cells or non-cancerous lesion cells. In some embodiments, the aforementioned first cell surface marker includes EGFR, PD-L1, or ROR1. In some embodiments, the aforementioned first and second cell surface markers are different. In some embodiments, the aforementioned first and second cell surface markers are identical. In some embodiments, the aforementioned targeting domain is selected from the group consisting of tumor-homing peptides, tumor-targeting domains, tissue-targeting domains, cell-permeable peptides, viral membrane proteins, and any combination or fragment thereof. In some embodiments, the aforementioned at least one therapeutic agent is contained within an extracellular vesicle. In some embodiments, the aforementioned at least one therapeutic agent is expressed on the extracellular surface of the extracellular vesicle.In some embodiments, the aforementioned at least one therapeutic agent is bound to an extracellular domain. In some embodiments, the aforementioned at least one therapeutic agent comprises a therapeutic polynucleotide, a therapeutic polypeptide, a therapeutic compound, an oncological drug, or a combination thereof. In some embodiments, the aforementioned therapeutic polynucleotide comprises messenger RNA, microRNA, shRNA, or a combination thereof. In some embodiments, the extracellular vesicles released from the aforementioned extracellular vesicle donor cells are exosomes, microvesicles, or apoptotic bodies. In some embodiments, the extracellular vesicles released from the aforementioned extracellular vesicle donor cells are exosomes. In some embodiments, the aforementioned extracellular vesicle donor cells comprise electroporation, microfluidic electroporation, microchannel electroporation, or nanochannel electroporation. In some embodiments, the aforementioned microchannel electroporation or nanochannel electroporation comprises the use of microporous patterned silicon wafers, nanoporous patterned silicon wafers, track etching films, ceramic microporous films, ceramic nanoporous films, other porous materials, or a combination thereof. In some embodiments, the step of transfecting the extracellular vesicle donor cells comprises nanochannel electroporation, in which at least one heterologous polynucleotide is nanoelectroporated into the extracellular vesicle donor cells via nanochannels located on a biochip. In some embodiments, the step of transfecting the extracellular vesicle donor cells comprises the use of a gene gun, microneedle array, nanoneedle array, sonication, or chemiosmosis. In some embodiments, the at least one heterologous polynucleotide is a plasmid.

[0015] In some cases, 10 -9This specification describes a composition comprising at least one extracellular vesicle comprising at least one adapter polypeptide (wherein the adapter polypeptide comprises an extracellular domain) having a peptide sequence that binds to the Fc region of a binding molecule with a dissociation constant (Kd) less than or equal to M, the aforementioned binding molecule (wherein the aforementioned binding molecule binds to a first cell surface marker associated with immune cells) complexed with the aforementioned adapter polypeptide, and at least one viral-mimetic peptide. In some embodiments, the composition described herein comprises at least one extracellular vesicle comprising an adapter polypeptide having at least one adapter polypeptide having a peptide sequence that is at least 70% identical to an Fc receptor that binds to the Fc region of a binding molecule, the adapter polypeptide comprising an extracellular domain, an antibody complexed with the adapter polypeptide, the binding molecule specifically binding to a first cell surface marker associated with immune cells, and at least one viral-mimetic peptide. In some embodiments, the aforementioned Fc receptor is an Fc-γ receptor, an Fc-α receptor, or an Fc-ε receptor. In some embodiments, the aforementioned Fc receptor includes FcγRI(CD64), FcγRII(CD32), or FcγRIII(CD16). In some embodiments, the aforementioned Fc receptor is CD64. In some embodiments, the aforementioned adapter polypeptide further includes a targeting domain that binds to a second cell surface marker associated with immune cells, the targeting domain being bound to the extracellular domain of the adapter polypeptide. In some embodiments, the aforementioned immune cells are T cells, B cells, dendritic cells, macrophages, or natural killer (NK) cells. In some embodiments, the aforementioned first cell surface marker includes LILRA 4, CD3, CD19, CD20, or CD28. In some embodiments, the aforementioned first and second cell surface markers are different. In some embodiments, the aforementioned first and second cell surface markers are identical. In some embodiments, the aforementioned binding molecule is a humanized monoclonal antibody. In some embodiments, the aforementioned binding molecule is IgG.In some embodiments, the aforementioned binding molecule comprises IgG1 or IgG3. In some embodiments, the aforementioned binding molecule is non-covalently complexed with the adapter polypeptide. In some embodiments, the Fc region of the aforementioned binding molecule is configured to complex with the adapter polypeptide in an acidic environment. In some embodiments, the Fc region of the aforementioned binding molecule is configured to be released from complexation with the adapter polypeptide in an acidic environment. In some embodiments, the aforementioned at least one viral-mimetic peptide is expressed on the extracellular surface of an extracellular vesicle. In some embodiments, the aforementioned at least one viral-mimetic peptide is bound to an extracellular domain. In some embodiments, the aforementioned at least one viral-mimetic peptide contains a peptide sequence that is at least 70% identical to that of the SARS-CoV-2 viral protein. In some embodiments, the aforementioned SARS-CoV-2 viral protein comprises an envelope (E) protein, a nucleocapsid (N) protein, a membrane (M) protein, or a spike (S) protein. In some embodiments, the aforementioned SARS-CoV-2 viral protein is the aforementioned S protein. In some embodiments, the aforementioned extracellular vesicles include exosomes, microvesicles, or apoptotic bodies. In some embodiments, the aforementioned extracellular vesicles are exosomes.

[0016] Described herein are methods for vaccinating a subject, in some examples, comprising administering a therapeutically effective amount of a pharmaceutical composition to the subject, wherein the pharmaceutical composition comprises the composition described herein. In some embodiments, the aforementioned pharmaceutical composition comprises at least one pharmaceutically acceptable excipient. In some embodiments, the aforementioned therapeutically effective amount of the aforementioned pharmaceutical composition comprises a therapeutically effective dose. In some embodiments, the aforementioned subject is administered the aforementioned therapeutically effective amount of the aforementioned pharmaceutical composition at a therapeutically effective frequency. In some embodiments, the aforementioned subject is administered the aforementioned therapeutically effective amount of the aforementioned pharmaceutical composition at a frequency of at least once a year. In some embodiments, the aforementioned subject is administered the aforementioned therapeutically effective amount of the aforementioned pharmaceutical composition at a frequency of at least once every six months. In some embodiments, the aforementioned subject is administered the aforementioned therapeutically effective amount of the aforementioned pharmaceutical composition at a frequency of at least once a month. In some embodiments, the aforementioned subject is administered the aforementioned therapeutically effective amount of the aforementioned pharmaceutical composition at a frequency of at least once a week. In some embodiments, the aforementioned pharmaceutical composition is an aqueous formulation. In some embodiments, the aforementioned pharmaceutical composition is formulated for injection. In some embodiments, the aforementioned pharmaceutical compositions are administered to the subject intranasally, intrathecally, intraocularly, intravitreously, intraretinally, intravenously, intramuscularly, intraventricularly, intracerebrally, intracerebellarly, intraventricularly, intraparenchymally, subcutaneously, or in combination thereof.

[0017] Described herein are methods for producing, in some cases, compositions described herein, the methods comprising: transfecting extracellular vesicle donor cells with at least one heterologous polynucleotide encoding an adapter polypeptide, wherein the adapter polypeptide comprises a peptide sequence at least 70% identical to that of an Fc receptor, and the Fc receptor recognizes the Fc region of the binding molecule; recovering extracellular vesicles released from the extracellular vesicle donor cells, wherein the extracellular vesicles released from the extracellular vesicle donor cells express the adapter polypeptide, wherein the adapter polypeptide comprises an extracellular domain, and the extracellular vesicles comprise at least one virus-mimicking peptide; and complexing the binding molecule with the extracellular domain, wherein the binding molecule binds to a first cell surface marker associated with immune cells. In some embodiments, the aforementioned Fc receptor is an Fc-γ receptor, an Fc-α receptor, or an Fc-ε receptor. In some embodiments, the aforementioned Fc receptor includes FcγRI(CD64), FcγRII(CD32), or FcγRIII(CD16). In some embodiments, the aforementioned Fc receptor is CD64. In some embodiments, the aforementioned adapter polypeptide further includes a targeting domain that binds to a second cell surface marker associated with immune cells, the targeting domain being bound to an extracellular domain. In some embodiments, the aforementioned immune cells are T cells, B cells, dendritic cells, macrophages, or natural killer (NK) cells. In some embodiments, the aforementioned first cell surface marker includes LILRA 4, CD3, CD19, CD20, or CD28. In some embodiments, the aforementioned first cell surface marker and the second cell surface marker are different. In some embodiments, the aforementioned first cell surface marker and the second cell surface marker are identical. In some embodiments, the aforementioned binding molecule includes a humanized monoclonal antibody. In some embodiments, the aforementioned binding molecule is IgG. In some embodiments, the aforementioned binding molecule comprises IgG1 or IgG3. In some embodiments, the aforementioned binding molecule is non-covalently complexed with the adapter polypeptide.In some embodiments, the Fc region of the aforementioned binding molecule is configured to complex with the adapter polypeptide in an acidic environment. In some embodiments, the Fc region of the aforementioned binding molecule is configured to be released from complexation with the adapter polypeptide in an acidic environment. In some embodiments, the aforementioned at least one viral-mimetic peptide is expressed on the extracellular surface of the extracellular vesicle. In some embodiments, the aforementioned at least one viral-mimetic peptide is bound to the extracellular domain. In some embodiments, the aforementioned at least one viral-mimetic peptide contains a peptide sequence that is at least 70% identical to that of the SARS-CoV-2 viral protein. In some embodiments, the aforementioned SARS-CoV-2 viral protein includes an envelope (E) protein, a nucleocapsid (N) protein, a membrane (M) protein, or a spike (S) protein. In some embodiments, the aforementioned SARS-CoV-2 viral protein is the aforementioned S protein. In some embodiments, the aforementioned extracellular vesicle includes an exosome, a microvesicle, or an apoptotic body. In some embodiments, the aforementioned extracellular vesicle is an exosome. In some embodiments, the step of transfecting the extracellular vesicle donor cells comprises nanochannel electroporation, in which at least one heterologous polynucleotide is nanoelectroporated into the extracellular vesicle donor cells via nanochannels located on a biochip. In some embodiments, the step of transfecting the extracellular vesicle donor cells comprises the use of a gene gun, microneedle array, nanoneedle array, sonication, or chemiosmosis. In some embodiments, the at least one heterologous polynucleotide is a plasmid.

[0018] This patent application includes at least one drawing made in color. Copies of this patent or patent application accompanied by the color drawing will be provided by the Office upon request and payment of the necessary fees.

[0019] Novel features of this disclosure are described in detail in the attached claims. A better understanding of the features and advantages of this disclosure will be obtained by referring to the following detailed description which describes exemplary embodiments. In embodiments of the present invention, for example, the following items are provided. (Item 1) A composition comprising at least one extracellular vesicle, wherein the extracellular vesicle is a.10 -9 At least one adapter polypeptide comprising a peptide sequence that binds to the Fc region of an antibody with a dissociation constant (Kd) less than or equal to M, wherein the adapter polypeptide comprises an extracellular domain, b. The antibody complexed with the adapter polypeptide, wherein the antibody binds to a first cell surface marker associated with the affected cell, c. At least one therapeutic agent and A composition containing the following: (Item 2) The composition according to item 1, wherein the peptide sequence that binds to the Fc region of the antibody is at least 70% identical to that of the Fc receptor. (Item 3) The composition according to item 2, wherein the Fc receptor is an Fc-γ receptor, an Fc-α receptor, or an Fc-ε receptor. (Item 4) The composition according to item 2, wherein the Fc receptor comprises FcγRI(CD64), FcγRII(CD32), or FcγRIII(CD16). (Item 5) The composition according to item 4, wherein the Fc receptor is CD64. (Item 6) The composition according to item 1, wherein the adapter polypeptide further comprises a targeting domain that binds to a second cell surface marker associated with the affected cell, and the targeting domain is bound to the extracellular domain of the adapter polypeptide. (Item 7) The composition according to item 6, wherein the targeting domain is selected from the group consisting of tumor homing peptides, tumor targeting domains, tissue targeting domains, cell permeable peptides, viral membrane proteins, and any combination or fragment thereof. (Item 8) The composition according to item 1, wherein the affected cells are cancer cells or non-cancerous lesion cells. (Item 9) The composition according to item 1, wherein the first cell surface marker comprises EGFR, PD-L1, or ROR1. (Item 10) The composition according to item 6, wherein the first cell surface marker and the second cell surface marker are different. (Item 11) The composition according to item 6, wherein the first cell surface marker and the second cell surface marker are the same. (Item 12) The composition according to item 1, wherein the antibody is a humanized monoclonal antibody. (Item 13) The composition according to item 1, wherein the antibody is selected from the group consisting of humanized anti-EGFR antibody clone C225, humanized anti-ROR1 antibody clone 2A2, and humanized anti-PD-L1 antibody clone SP142. (Item 14) The composition according to item 12, wherein the humanized monoclonal antibody comprises IgG. (Item 15) The set according to item 14, wherein the humanized monoclonal antibody comprises IgG1 or IgG3. A finished product. (Item 16) The composition according to item 1, wherein the antibody is non-covalently complexed with the adapter polypeptide. (Item 17) The composition according to item 16, wherein the Fc region of the antibody is configured to complex with the adapter polypeptide in an acidic environment. (Item 18) The composition according to item 16, wherein the Fc region of the antibody is configured to be freed from complexation with the adapter polypeptide in an acidic environment. (Item 19) The composition according to item 1, wherein at least one therapeutic agent is located within the extracellular vesicle. (Item 20) The composition according to item 1, wherein at least one therapeutic agent is expressed on the extracellular surface of the extracellular vesicle. (Item 21) The composition according to item 1, wherein at least one therapeutic agent is bound to the extracellular domain. (Item 22) The composition according to item 1, wherein the at least one therapeutic agent comprises a therapeutic polynucleotide, a therapeutic polypeptide, a therapeutic compound, an anticancer drug, or a combination thereof. (Item 23) The composition according to item 22, wherein the therapeutic polynucleotide comprises messenger RNA, microRNA, shRNA, or a combination thereof. (Item 24) The composition according to item 1, wherein the extracellular vesicle is an exosome, a microvesicle, or an apoptotic body. (Item 25) The composition according to item 24, wherein the extracellular vesicle is an exosome. (Item 26) A method for treating a subject, comprising administering a therapeutically effective amount of a pharmaceutical composition to the subject, wherein the pharmaceutical composition comprises a composition described in any one of items 1 to 25. (Item 27) The method according to item 26, wherein the pharmaceutical composition comprises at least one pharmaceutically acceptable excipient. (Item 28) The method according to item 26, wherein the subject has cancer or a non-cancerous lesion. (Item 29) The method described in item 28, wherein the subject has a glioma. (Item 30) The method described in item 26, wherein the subject has muscular dystrophy. (Item 31) The method according to item 30, wherein the muscular dystrophy is selected from the group consisting of Duchenne muscular dystrophy, Becker muscular dystrophy, facioscapulohumeral muscular dystrophy, congenital muscular dystrophy, and myotonic dystrophy. (Item 32) The method described in item 26, wherein the subject has a retinal disease. (Item 33) The method according to item 32, wherein the retinal disease is retinitis pigmentosa or Leber congenital amaurosis. (Item 34) The method according to item 26, wherein the subject is administered the therapeutically effective amount of the pharmaceutical composition at a frequency of at least once a day, once a week, once a month, or once a year. (Item 35) The method according to item 26, wherein the pharmaceutical composition is an aqueous formulation. (Item 36) The method according to item 26, wherein the pharmaceutical composition is formulated for injection. (Item 37) The method according to item 26, wherein the pharmaceutical composition is administered to the subject intrathecally, intraocularly, intravitreously, intraretinally, intravenously, intramuscularly, intraventricularly, intracerebrally, intracerebellarly, intraventricularly, intraparenchymally, subcutaneously, intranasally, or in combination thereof. (Item 38) A method for producing a composition, wherein the method is a. A step of transfecting extracellular vesicle donor cells with at least one heterologous polynucleotide encoding an adapter polypeptide, wherein the adapter polypeptide comprises a peptide sequence that is at least 70% identical to that of the Fc receptor, and the Fc receptor recognizes the Fc region of the antibody. b. A step of recovering extracellular vesicles released from extracellular vesicle donor cells, wherein the extracellular vesicles released from the extracellular vesicle donor cells express the adapter polypeptide, the adapter polypeptide comprises an extracellular domain, and the extracellular vesicles contain at least one therapeutic agent. c. A step of complexing the antibody with the extracellular domain, wherein the antibody binds to a first cell surface marker associated with the affected cell. Methods that include... (Item 39) The method according to item 38, wherein the Fc receptor is an Fc-γ receptor, an Fc-α receptor, or an Fc-ε receptor. (Item 40) The method according to item 39, wherein the Fc receptor is FcγRI(CD64), FcγRII(CD32), or FcγRIII(CD16). (Item 41) The method according to item 40, wherein the Fc receptor is CD64. (Item 42) The method according to item 38, wherein the adapter polypeptide further comprises a targeting domain that binds to a second cell surface marker associated with the affected cell, and the targeting domain is bound to the extracellular domain. (Item 43) The method according to item 42, wherein the first cell surface marker or the second cell surface marker is associated with cancer cells or non-cancerous lesion cells. (Item 44) The method according to item 43, wherein the first cell surface marker comprises EGFR, PD-L1, or ROR1. (Item 45) The method according to item 43, wherein the first cell surface marker and the second cell surface marker are different. (Item 46) The method according to item 43, wherein the first cell surface marker and the second cell surface marker are the same. (Item 47) The aforementioned targeting domain is a tumor-homing peptide, a tumor-targeting domain, or a tissue-targeting domain. The method described in item 42, selected from the group consisting of ions, cell-permeable peptides, viral membrane proteins, and any combination or fragment thereof. (Item 48) The method according to item 38, wherein the at least one therapeutic agent is located within the extracellular vesicle. (Item 49) The method according to item 38, wherein at least one therapeutic agent is expressed on the extracellular surface of the extracellular vesicle. (Item 50) The method according to item 38, wherein at least one therapeutic agent is bound to the extracellular domain. (Item 51) The method according to item 38, wherein the at least one therapeutic agent comprises a therapeutic polynucleotide, a therapeutic polypeptide, a therapeutic compound, an oncological drug, or a combination thereof. (Item 52) The method according to item 51, wherein the therapeutic polynucleotide comprises messenger RNA, microRNA, shRNA, or a combination thereof. (Item 53) The method according to item 38, wherein the extracellular vesicles released from the extracellular vesicle donor cells are exosomes, microvesicles, or apoptotic bodies. (Item 54) The method according to item 53, wherein the extracellular vesicle released from the extracellular vesicle donor cell is an exosome. (Item 55) The method according to item 38, wherein the step of transfecting the extracellular vesicle donor cells comprises electroporation, microfluidic electroporation, microchannel electroporation, or nanochannel electroporation. (Item 56) The method according to item 55, wherein the microchannel electroporation or nanochannel electroporation includes the use of a microporous patterned silicon wafer, a nanoporous patterned silicon wafer, a track etching film, a ceramic microporous film, a ceramic nanoporous film, other porous materials, or a combination thereof. (Item 57) The method according to item 56, wherein the step of transfecting the extracellular vesicle donor cells comprises nanochannel electroporation, wherein the at least one heterologous polynucleotide is nanoelectroporated into the extracellular vesicle donor cells via nanochannels located on a biochip. (Item 58) The method according to item 38, wherein the step of transfecting the extracellular vesicle donor cells includes the use of a gene gun, a microneedle array, a nanoneedle array, sonication, or chemical permeation. (Item 59) The method according to item 38, wherein the at least one heterologous polynucleotide is a plasmid. (Item 60) A composition comprising at least one extracellular vesicle, a. At least one adapter polypeptide comprising a peptide sequence at least 70% identical to that of an Fc receptor that binds to the Fc region of an antibody, wherein the adapter polypeptide comprises an extracellular domain, b. The antibody complexed with the adapter polypeptide, wherein the antibody specifically binds to a first cell surface marker associated with immune cells, c. At least one viral mimetic peptide and A composition containing the following: (Item 61) The composition according to item 60, wherein the Fc receptor is an Fc-γ receptor, an Fc-α receptor, or an Fc-ε receptor. (Item 62) The composition according to item 61, wherein the Fc receptor comprises FcγRI(CD64), FcγRII(CD32), or FcγRIII(CD16). (Item 63) The composition according to item 62, wherein the Fc receptor is CD64. (Item 64) The composition according to item 60, wherein the adapter polypeptide further comprises a targeting domain that binds to a second cell surface marker associated with the immune cell, and the targeting domain is bound to the extracellular domain of the adapter polypeptide. (Item 65) The composition according to item 60, wherein the immune cells are T cells, B cells, dendritic cells, macrophages, or natural killer (NK) cells. (Item 66) The composition according to item 60, wherein the first cell surface marker comprises LILRA 4, CD3, CD19, CD20, or CD28. (Item 67) The composition according to item 64, wherein the first cell surface marker and the second cell surface marker are different. (Item 68) The composition according to item 64, wherein the first cell surface marker and the second cell surface marker are the same. (Item 69) The composition according to item 60, wherein the antibody is a humanized monoclonal antibody. (Item 70) The composition according to item 69, wherein the humanized monoclonal antibody is IgG. (Item 71) The composition according to item 70, wherein the IgG comprises IgG1 or IgG3. (Item 72) The composition according to item 60, wherein the antibody is non-covalently complexed with the adapter polypeptide. (Item 73) The composition according to item 70, wherein the Fc region of the antibody is configured to complex with the adapter polypeptide in an acidic environment. (Item 74) The composition according to item 70, wherein the Fc region of the antibody is configured to be freed from complexation with the adapter polypeptide in an acidic environment. (Item 75) The composition according to item 60, wherein at least one virus-mimicking peptide is expressed on the extracellular surface of the extracellular vesicle. (Item 76) The composition according to item 60, wherein at least one virus-mimicking peptide is bound to the extracellular domain. (Item 77) The composition according to item 60, wherein the at least one virus-mimicking peptide comprises a peptide sequence that is at least 70% identical to that of the SARS-CoV-2 virus protein. (Item 78) The composition according to item 77, wherein the SARS-CoV-2 virus protein comprises an envelope (E) protein, a nucleocapsid (N) protein, a membrane (M) protein, or a spike (S) protein. (Item 79) The composition according to item 78, wherein the SARS-CoV-2 virus protein is the S protein. (Item 80) The composition according to item 60, wherein the extracellular vesicles include exosomes, microvesicles, or apoptotic bodies. (Item 81) The composition according to item 80, wherein the extracellular vesicle is an exosome. (Item 82) A method for vaccinating a target, comprising administering a therapeutically effective amount of a pharmaceutical composition to the target, wherein the pharmaceutical composition comprises a composition described in any one of items 60 to 81. (Item 83) The method according to item 82, wherein the pharmaceutical composition comprises at least one pharmaceutically acceptable excipient. (Item 84) The method according to item 82, wherein the subject is administered the therapeutically effective amount of the pharmaceutical composition at a frequency of at least once a day, once a week, once a month, or once a year. (Item 85) The method according to item 82, wherein the pharmaceutical composition is an aqueous formulation. (Item 86) The method according to item 82, wherein the pharmaceutical composition is formulated for injection. (Item 87) The method according to item 82, wherein the pharmaceutical composition is administered to the subject intrathecally, intraocularly, intravitreously, intraretinally, intravenously, intramuscularly, intraventricularly, intracerebrally, intracerebellarly, intraventricularly, intraparenchymally, subcutaneously, intranasally, or in combination thereof. (Item 88) A method for producing a composition, wherein the method is a. A step of transfecting extracellular vesicle donor cells with at least one heterologous polynucleotide encoding an adapter polypeptide, wherein the adapter polypeptide comprises a peptide sequence that is at least 70% identical to that of the Fc receptor, and the Fc receptor recognizes the Fc region of the antibody. b. A step of recovering the extracellular vesicles released from the extracellular vesicle donor cells, wherein the extracellular vesicles released from the extracellular vesicle donor cells express the adapter polypeptide, the adapter polypeptide comprises an extracellular domain, and the extracellular vesicle comprises at least one virus-mimicking peptide, c. A step of complexing the antibody with the extracellular domain, wherein the antibody binds to a first cell surface marker associated with immune cells. Methods that include... (Item 89) The method according to item 88, wherein the Fc receptor is an Fc-γ receptor, an Fc-α receptor, or an Fc-ε receptor. (Item 90) The method according to item 89, wherein the Fc receptor comprises FcγRI(CD64), FcγRII(CD32), or FcγRIII(CD16). (Item 91) The method according to item 90, wherein the Fc receptor is CD64. (Item 92) The method according to item 88, wherein the adapter polypeptide further comprises a targeting domain that binds to a second cell surface marker associated with the immune cell, and the targeting domain is bound to the extracellular domain. (Item 93) The method according to item 88, wherein the immune cells are T cells, B cells, dendritic cells, macrophages, or natural killer (NK) cells. (Item 94) The method according to item 88, wherein the first cell surface marker comprises LILRA4, CD3, CD19, CD20, or CD28. (Item 95) The method according to item 92, wherein the first cell surface marker and the second cell surface marker are different. (Item 96) The method according to item 92, wherein the first cell surface marker and the second cell surface marker are the same. (Item 97) The method according to item 92, wherein the antibody comprises a humanized monoclonal antibody. (Item 98) The method according to item 97, wherein the antibody is IgG. (Item 99) The method according to item 98, wherein the IgG is IgG1 or IgG3. (Item 100) The method according to item 88, wherein the antibody is non-covalently complexed with the adapter polypeptide. (Item 101) The method according to item 100, wherein the Fc region of the antibody is configured to complex with the adapter polypeptide in an acidic environment. (Item 102) The method according to item 100, wherein the Fc region of the antibody is configured to be freed from complexation with the adapter polypeptide in an acidic environment. (Item 103) The method according to item 88, wherein at least one virus-mimicking peptide is expressed on the extracellular surface of the extracellular vesicle. (Item 104) The method according to item 88, wherein the at least one virus-mimicking peptide is bound to the extracellular domain. (Item 105) The method according to item 88, wherein the at least one virus-mimicking peptide contains a peptide sequence that is at least 70% identical to that of the SARS-CoV-2 virus protein. (Item 106) The method according to item 105, wherein the SARS-CoV-2 virus protein comprises an envelope (E) protein, a nucleocapsid (N) protein, a membrane (M) protein, or a spike (S) protein. (Item 107) The method according to item 106, wherein the SARS-CoV-2 virus protein is the S protein. (Item 108) The method according to item 88, wherein the extracellular vesicle comprises an exosome, a microvesicle, or an apoptotic body. (Item 109) The method according to item 108, wherein the extracellular vesicle is an exosome. (Item 110) The method according to item 88, wherein the step of transfecting the extracellular vesicle donor cells comprises electroporation, microfluidic electroporation, microchannel electroporation, or nanochannel electroporation. (Item 111) The method according to item 110, wherein the microchannel electroporation or nanochannel electroporation includes the use of a microporous patterned silicon wafer, a nanoporous patterned silicon wafer, a track etching film, a ceramic microporous film, a ceramic nanoporous film, other porous materials, or a combination thereof. (Item 112) The method according to item 111, wherein the step of transfecting the extracellular vesicle donor cells comprises nanochannel electroporation, wherein the at least one heterologous polynucleotide is nanoelectroporated into the extracellular vesicle donor cells via nanochannels located on a biochip. (Item 113) The method according to item 88, wherein the step of transfecting the extracellular vesicle donor cells includes the use of a gene gun, a microneedle array, a nanoneedle array, sonication, or chemical permeation. (Item 114) The method according to item 88, wherein the at least one heterologous polynucleotide is a plasmid. (Item 115) The method according to item 88, wherein the extracellular vesicle donor cells are selected from the group consisting of mouse embryonic fibroblasts (MEFs), human embryonic fibroblasts (HEFs), dendritic cells, mesenchymal stem cells, bone marrow-derived dendritic cells, bone marrow-derived stromal cells, adipocytes, endothelial cells, enucleated cells, neural stem cells, immature dendritic cells, and immune cells. (Item 116) The method according to item 88, wherein the extracellular vesicle donor cells are selected from the group consisting of primary animal cells, primary human cells, animal cell lines, and human cell lines. (Item 117) The method according to item 88, wherein the extracellular vesicle donor cells are selected from the group consisting of genetically modified primary animal cells, genetically modified primary human cells, genetically modified animal cell lines, and genetically modified human cell lines. [Brief explanation of the drawing]

[0020] [Figure 1]Figure 1 shows a schematic diagram of a targeted extracellular vesicle ("EXO") containing a monoclonal antibody (mAb) linked to CD64 on the surface of an extracellular vesicle and a tumor-homing peptide (THP). These extracellular vesicles (EVs) can target tumors and lesions, and exemplary targets are listed in Figure 1. EVs with CD64 or THP-CD64 can be generated by transfecting donor cells with human CD64 plasmid DNA or human THP-CD64 plasmid DNA, thereby expressing either human CD64 or human THP-CD64 on the surface of EVs (including exosomes) secreted from the transfected donor cells. CD64 provides a biological anchor for binding to humanized monoclonal antibodies (hmABs). The extracellular D1-D2 hinge of human CD64 binds to the lower hinge region of Fc in human IgG1 with high affinity (dissociation constant (Kd)) of approximately 10⁻⁹ M (nanomole concentration). In addition to the ability of bound hmAbs to specifically recognize cellular targets, targeting by small tumor-homing peptides (THPs) can also be manipulated at the N-terminus of CD64. Dual targeting of both hmAbs and THPs on the EV (or exosome) surface enhances targeting and delivery to tumors and other lesions in vivo. Examples of hmAbs for cancer / tumor targeting include, but are not limited to, anti-hEGFR, e.g., cetuximab, anti-hPD-L1, e.g., atezolizumab, and anti-humanized ROR1. Examples of THPs for cancer / tumor targeting include, but are not limited to, CKAAKN (CK), CREKA (CR), and ARRPKLD (AR).

[0021] [Figure 2]Figures 2A–2D show exemplary construct designs of plasmids encoding CD64 with additional tumor-homing peptides. Figure 2A. Plasmids were constructed using vectors containing genes for ampicillin resistance (AmpR) and EGFR markers for transformation and transfection, respectively. Functional CD64 was encoded by the CD64 coding sequence (CD64_CDS) driven by the EF-1α promoter. Figure 2B. CD64_CDS (355 amino acids) consisted of (i) a signal peptide (SP), (ii) extracellular domains (D1, D2, and D3), (iii) a transmembrane (TM) domain, and (iv) an intracellular (IC) domain, with THP inserted into the gap between the signal peptide and the extracellular D1 domain, enabling THP expression at the N-terminus of CD64. Figure 2C. THP was linked to the N-terminus of extracellular D1 by the Flag(DYKDDDK) linker, limiting conformational blocking of the Fc-binding region at the D1-D2 hinge of CD64. Figure 2D. List of exemplary peptides and nucleotide sequences of selected THP [Flag_control, CKAAKN(CK), CREKA(CR), ARRPKLD(AR)].

[0022] [Figure 3] Figures 3A-3C show how the addition of THP to CD64 does not affect its binding affinity to human IgG (hIgG). Figure 3A. Schematic diagram of purified CD64 proteins with different THPs bound to immobilized hIgG on a solid support and reacted with an ELISA substrate. Kd values ​​were determined by monovalent modeling between CD64 and hIgG. Figure 3B. Affinity index Kd of hIgG and recombinant wild-type CD64 (wt_CD64) was measured. Figure 3C. The affinity index Kd of different manipulated THP-CD64 proteins and hIgG suggested that manipulated CD64 with different THPs (Flag, CK, CR, AR) did not affect the high binding affinity to mAbs at the nM level compared to wt_CD64.

[0023] [Figure 4]Figures 4A-4B show the number and content of endogenous mRNA extracellular viable cells (EVs) from mouse embryonic fibroblasts (MEFs) transfected with THP-CD64 and therapeutic RNA plasmids via nanochannel electroporation (NEP). Figure 4A: Number of EVs per cell produced by untreated MEFs (control group) in PBS, MEFs transfected with both THP-CD64 and human TP53 plasmids via NEP, and MEFs transfected with both THP-CD64 and shKRAS G12D mutant plasmids via 24-hour NEP. Figure 4B: Fold change of TP53 mRNA in EVs produced by untreated and NEP-transfected MEFs, as determined by qRT-PCR.

[0024] [Figure 5] Figures 5A–5D show that THP-CD64-expressing exosomes maintained high binding affinity to hIgG. Figure 5A. Schematic diagram of purified exosomes containing engineered THP-CD64 captured by latex beads and incubated with anti-CD64-APC, anti-CD63-BV510, and FITC-conjugated hIgG for flow cytometry assay. Figure 5B. Surface expression profiling followed a standard protocol of gated singlet beads and CD63+ exosome populations to determine mean fluorescence intensity (MFI) of CD64 expression and hIgG binding. Figure 5C. Surface co-expression of CD64 within the CD63+ exosome population was determined by MFI of FITC to confirm exosome expression of engineered CD64 having either Flag, CK, CR, or AR THP. Figure 5D. Surface co-expression of hIgG and CD64 within the CD63+ exosome population was determined by FITC MFI, and high binding affinity of hIgG to exosomes expressing CD64 with either Flag, CK, CR, or AR THP was confirmed.

[0025] [Figure 6]Figure 6 shows the uptake of liposomes and extracellular viable cells (EVs) in cancer spheroids derived from the human pancreatic cancer cell line PANC-1. Purified EVs released from mouse embryonic fibroblast (MEF) cells after transfection with either Flag-CD64 or CK-CD64 plasmid DNA (CK-CD64) were formulated with either a humanized anti-EGFR mAb (cetuximab) or hIgG. Cancer spheroids were treated with PKH67 (green) labeled liposomes (lipofectamine 3000) and various EVs for 24 hours, followed by fixation, permeabilization, and staining with anti-hIgG-TRITC (red) and DAPI (blue). Cross-sections of cancer spheroids were imaged under a confocal microscope. All treatments of cancer spheroids with various EVs showed better spheroid uptake than commercially available lipofectamine 3000 based on fluorescence intensity and distribution. Among the various EVs, the dual-targeted EV (CK-CD64-Cet_Exo) demonstrated the highest spheroid uptake.

[0026] [Figure 7]Figures 7A–7C demonstrate that dual targeting with CK-CD64 and a humanized anti-EGFR mAb (cetuximab) enhances EV uptake in PANC-1 cancer spheroid cells, particularly in the CD24+CD44+ subpopulation. Figure 7A. PANC-1 cancer spheroids were formed and cultured for one week until they reached a diameter of approximately 500 μm, then treated with approximately 10⁹ PKH67-labeled exosomes in culture medium for 24 hours. Figure 7B. The treated spheroids were degraded into single-cell suspensions, and subpopulations were identified by CD24 and CD44 expression using flow cytometry. Figure 7C. The mean fluorescence intensity of PKH67 measured in the CD24low / CD44low or CD24+ / CD44+ subpopulations represents their EV uptake. All engineered extracellular genes (EVs) containing Flag-CD64, CK-CD64, CR-CD64, or AR-CD64 with humanized antibody binding (cetuximab: anti-EGFR, atezolizumab: anti-PD-L1, or hIgG) showed good cell uptake, particularly in the CD44+CD24+ subpopulation. Dual-targeted EVs using anti-hEGFR (cetuximab) and CK-CD64 provided the best cell uptake for both PANC-1 cell subpopulations.

[0027] [Figure 8] Figure 8 shows that targeting ROR1, which is highly expressed in 85% of pancreatic cancers, within spheroids formed from PANC-1 enhances the uptake of extracellular vesicles. PANC-1 spheroids were formed, cultured stably for one week until they reached a diameter of 300–500 μm, and then treated with 10¹¹ PKH67-labeled exosomes in culture medium for 24 hours.

[0028] [Figure 9]Figure 9 shows enhanced in vivo uptake of ROR1-targeted extracellular vesicles in a PANC-1 orthotopic model. Mice (4 weeks after xenografting of PANC-1 extracellular vesicles) were treated with 1.0E12 / 50μl (intraperitoneal) injection of extracellular vesicle solution (250μl) and sacrificed 24 hours later. PKH26 (excitation / emission: 535 / 580nm); GFP (465 / 540nm); IVIS (incident illumination, Bin: (M)1, FOV: 22, f2, 5s. Distribution: brain / heart / lung / liver / spleen / pancreas / kidney).

[0029] [Figure 10] Figure 10 shows enhanced uptake of extracellular vesicles targeted to ROR1 by penetration of tumor tissue. Anti-ROR1 targeting enhances the uptake of extracellular vesicles in tumor lesions, but the renewal of CK peptide extracellular vesicles is not significant compared to the flag control.

[0030] [Figure 11]Figures 11A–11D show exemplary designs of vacosomes and five proposed vaccine peptides (i.e., spike, S protein, and fragment) from epitope and structural predictions for COVID-19 vaccine development. Figure 11A. ACE2 acts as a receptor for the SARS-CoV-2 virus, allowing it to infect cells. Figure 11B. Bountiful vaccination via the T cell receptor (TCR) complex can be achieved synergistically by vaccination of a peptide on the N-terminus of CD64 and co-stimulation with pre-loaded anti-αCD3 / CD28 mAb on the hinges D1–D2 of CD64. Exosomes overexpressing various viral protein fragments fused to CD64 on the exosome surface can function as vaccines (called "vacosomes"). Figure 11C. The formation of immunological synapses between manipulated CD64 and TCRs can be confirmed by fluorescent tagging and T cell surface marker staining using a fluorescence-activated cell sorter (FACS). Similarly, simultaneous loading of mAbs targeting antigen-presenting cells (APCs) such as B cells (anti-αCD19 / CD20) and dendritic cells (DCs) (anti-αLILRA 4) should enhance the APC-T cell response. Figure 11D. Five candidate fusion S protein fragments likely to function as COVID-19 vaccine peptides are selected based on epitope and structural prediction. They can be expressed on vacosomes generated via donor cells transfected with NEPs such as human mesenchymal stem cells (MSCs) and DCs.

[0031] [Figure 12]Figures 12A-12B show the binding affinity strengths of human immunoglobulins and classical Fc receptors. Plasma membrane-embedded Fc receptors contain intracellular domains or subunits that can cause downstream activation or inhibition. Figure 12A. IgG affinity variants are highlighted for each human Fcγ receptor member, ranging from very high (dark orange), high (orange), moderate (yellow), low (light blue) to no binding (dark blue). The FcRn receptor binds to the IgG subclass under acidic conditions (e.g., pH=6), but its binding ability decreases under physiological conditions at pH=7.4. Figure 12B. IgE has very high binding affinity to the FcεRI receptor but low affinity to the FcεRII receptor. IgA has low binding affinity to the FcαRI receptor. ***The binding affinity between human immunoglobulins and Fc repeaters is expressed as a constant Kd at levels ranging from very high+++: approximately 10⁻⁹ M; high++: 10⁻⁹ to 10⁸ M; moderate: approximately 10⁷ M; or low: >10⁷ M.

[0032] [Figure 13] Figures 13A-13D show the dynamics of EV release in NEP. Figure 13A shows the time course of EV secretion after NEP. Figure 13B shows the multiplicative change in TP53 mRNA expression within EVs over time, as measured by qPCR. Figure 13C shows CD64 expression on the EV surface, measured by ELISA for EVs collected every 8 hours. Figure 13D shows the expression level of KRASG12D shRNA within EVs every 8 hours after NEP.

[0033] [Figure 14] Figures 14A–C show sequential NEP (sNEP) designs for TP53 mRNA / CD64EV. Figure 14A provides EV counts and TP53 mRNA expression in sNEP examples at (Figure 14A) 8 hours, (Figure 14B) 16 hours, and (Figure 14C) 24 hours. Ctrl represents a single NEP with the CD64 plasmid.

[0034] [Figure 15]Figures 15A–F provide characterization of as-prepared targeted extracellular proteins (tEVs). Figure 15A shows the size distribution of blank EVs (control) and manipulated EVs obtained by NEP, (Figure 15B) exosome biomarkers of as-prepared EVs, (Figure 15C) SEM and (Figure 15D) CryoTEM images of representative EVs, (Figure 15E) single EV capture and co-localization using an ILN biochip with fluorescently labeled anti-CD64 and molecular beacon for KRASG12D shRNA and TP53 mRNA, (Figure 15F) ratios of EVs containing CD64 protein, KRASG12D shRNA, and TP53 mRNA, as well as co-localization of CD64 / KRASG12D shRNA and CD64 / TP53 mRNA.

[0035] [Figure 16] Figures 16A–G show that binding to tumor-specific antibodies (αhROR1 and αhEGFR) on the surface of CD64 / EV can enhance the intracellular uptake of EVs in PANC-1 cells. Figure 16A provides the uptake efficiency of humanized antibodies on the CD64 flag peptide. Figure 16B provides the uptake efficiency of humanized antibodies on CD64 by the CK peptide. Figure 16C quantifies the relative EV uptake by each formulation. Figure 16D compares the staining of PANC-1 cells with no treatment (Con) and 4 hours of treatment with IgG_EV, αEGFR_EV, and αROR1_EV. Figures 16E–16F provide EV uptake assays for αEGFR_EV (Figure 16E) and αROR1_EV (Figure 16F) in PANC-1 cells against 3D tumor spheroids. Figure 16G provides a substitution assay with human serum (50%) at 37°C for 6 hours.

[0036] [Figure 17]Figures 17A–D show transcytosis assays and results based on TRANSWELL®. Figure 17A provides a schematic diagram of the assay. Figure 17B provides various inhibitors selected to block endocytosis and EV secretion (including pitstop 2, an inhibitor of clathrin-mediated endocytosis; methyl-β-cyclodextrin, an inhibitor of caveolae-mediated endocytosis; cytochalasin D, an inhibitor of micropinocytosis; and neticonazole, an inhibitor of exosome secretion). Figure 17C provides data from PANC-1 transcytosis assays using various inhibitors ("ctl": 1E10 untargeted EVs without inhibitors in upper PANC-1 cells; "Pos ctl": without upper cell layer). Figure 17D compares PANC-1 transcytosis levels using targeted hmAbs on the EV surface.

[0037] [Figure 18] Figures 18A-B demonstrate that human serum IgG does not affect human mAbs on the EV surface. Figures 18A-B show that αhEGFR_EV (left panel) and αhROR1_EV (right panel), incubated with human serum (50%) at 37°C for 6 hours and then treated with monolayer PANC-1 cells, maintained the same targeting ability after human serum incubation.

[0038] [Figure 19] Figures 19A and 19B show the in vivo distribution of targeted extravasation genes (EVs) in PANC-1 orthotopic NS mice. Figure 19A shows in vivo imaging (IVIS), and Figure 19B shows expression in the brain, heart, lungs, liver, spleen, pancreas, and kidneys. [Modes for carrying out the invention]

[0039] Detailed explanation While preferred embodiments of the present disclosure have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided only as examples. Numerous variations, modifications, and substitutions will be conceivable to those skilled in the art without departing from the present invention. It should be understood that various alternative embodiments to the embodiments of the present disclosure described herein may be used in carrying out the present disclosure. The following claims define the scope of the present disclosure, and the methods and structures within these claims, as well as their equivalents, are intended to be covered thereby.

[0040] The use of absolute or sequential terms, such as “do,” “do not,” “should,” “should not,” “must,” “must not,” “first,” “initially,” “next,” “followed by,” “before,” “after,” “finally,” and “ultimately,” is not intended to limit the scope of the embodiments disclosed herein, but is illustrative.

[0041] As used herein, the singular forms “a,” “an,” and “the” are intended to include the plural forms unless otherwise explicitly indicated in the context. Furthermore, the terms “including,” “includes,” “having,” “has,” and “with,” or their variations thereof, are intended to be as comprehensive as the term “comprising,” to the extent that they are used in either the detailed description and / or the claims.

[0042] As used herein, "or" can refer to "and," "or," or "and / or," and can be used both exclusively and comprehensively. For example, the term "A or B" can refer to "A or B," "A but not B," "B but not A," and "A and B." In some cases, the context may determine the specific meaning.

[0043] As used herein, the phrases “at least one,” “one or more,” and “and / or” are open-ended expressions that are both conjunctive and disjunctive in their function. For example, the expressions “at least one of A, B, and C,” “at least one of A, B, or C,” “one or more of A, B, and C,” and “one or more of A, B, or C” mean A only, B only, C only, A and B together, A and C together, B and C together, or A, B, and C together.

[0044] Any systems, methods, compositions, and platforms described herein are modular and not limited to sequential steps. Therefore, terms such as “first” and “second” do not necessarily imply priority, order of importance, or order of actions.

[0045] The terms “approximately” or “about” mean that a particular value is within an acceptable margin of error as determined by those skilled in the art, which in part depends on how the value is measured or determined, for example, the limits of the measuring system. For example, “approximately” may mean within or above one standard deviation, according to convention for a given value. Where a particular value is described in this application and claims, unless otherwise specified, the term “approximately” should be assumed to mean an acceptable margin of error for that particular value.

[0046] The terms “increased,” “increased,” or “increased” are used herein in general to mean an increase of a statically significant amount. In some cases, the terms “increased” or “increased” mean an increase of at least 10% compared to a baseline level, e.g., at least about 10%, at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or up to 100% (including 100%) compared to a baseline level, standard, or control, or any increase between 10% and 100%. Other examples of “increased” include increases of at least 2 times, at least 5 times, at least 10 times, at least 20 times, at least 50 times, at least 100 times, at least 1000 times, or more compared to a baseline level.

[0047] The terms “decreased,” “decrease,” or “decrease” are used herein in general to mean a decrease of a statistically significant amount. In some cases, “decreased” or “decrease” means a decrease of at least 10% compared to a baseline level, e.g., at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or up to 100% (including 100%) compared to a baseline level (e.g., a level that does not exist or is undetectable compared to a baseline level), or any decrease between 10% and 100%. In the context of markers or symptoms, these terms mean a statistically significant decrease of such a level. Other examples of “decrease” include decreases of at most half, at most one-fifth, at most one-tenth, at most one-twentieth, at most one-fiftieth, at most one-hundredth, at most one-thousandth, or more compared to a baseline level. The reduction may be, for example, at least 10%, at least 20%, at least 30%, at least 40%, or more, and preferably decreases to a level that is considered within the normal range for an individual without a given disease.

[0048] As used herein, “cell” generally refers to a biological cell. A cell is the basic structure, function, and / or biological unit of a living organism. Cells can originate from any organism that has one or more cells. Some non-limiting examples include prokaryotic cells, eukaryotic cells, bacterial cells, archaeal cells, cells of unicellular eukaryotes, protozoan cells, plant-derived cells (e.g., plant crops, fruits, vegetables, grains, soybeans, corn, maize, wheat, seeds, tomatoes, rice, cassava, sugarcane, pumpkins, hay, potatoes, cotton, hemp, tobacco, flowering plants, conifers, gymnosperms, ferns, clubmosses, hornworts, liverworts, moss-derived cells), algal cells (e.g., Botryococcus braunii, Chlamydomonas reinhardtii, Nannochloropsis gaditana, Chlorella), and algal cells (e.g., Botryococcus braunii, Chlamydomonas reinhardtii, Nannochloropsis gaditana, Chlorella). Examples of cell origins include cells from natural organisms (such as pyrenoidosa and Sargassum patens C.Agardh), seaweed (e.g., kelp), fungal cells (e.g., yeast cells, mushroom-derived cells), animal cells, invertebrate-derived cells (e.g., fruit flies, cnidarians, echinoderms, nematodes, etc.), vertebrate-derived cells (e.g., fish, amphibians, reptiles, birds, mammals), and mammal-derived cells (e.g., pigs, cattle, goats, sheep, rodents, rats, mice, non-human primates, humans, etc.). Cells may not originate from natural organisms (for example, cells are sometimes called artificial cells, which are synthetically produced). Cells may also originate from cell lines.

[0049] The terms “transfection” or “transfected” generally refer to the introduction of nucleic acid molecules into cells by non-viral or virus-based methods. Nucleic acid molecules can be gene sequences that encode complete proteins or their functional parts. In some cases, nucleic acid molecules can be non-coding sequences. In some cases, transfection methods are used to introduce nucleic acid molecules into cells in order to create transgenic animals. Such techniques may include pronuclear microinjection, retrovirus-mediated gene transfer into germline cells, gene targeting into embryonic stem cells, electroporation of embryos, sperm-mediated gene transfer, and in vitro transformation of somatic cells, e.g., cumulus cells or mammary gland cells, or adult, fetal, or embryonic stem cells, followed by nuclear transfer.

[0050] "Nanoelectroporation" or "nanochannel electroporation" refers to transfecting cells with at least one heterologous polynucleotide, such as a vector, by loading at least one heterologous polynucleotide into a nanochannel and generating an electric field to facilitate the entry of at least one heterologous polynucleotide into the cell. The cell to be transfected is located at the opening of the nanochannel, where the electric field of nanoelectroporation creates a pore in the cell membrane, allowing the introduction of at least one heterologous polynucleotide into the cell.

[0051] As used herein, “plasmid” generally refers to a non-viral expression vector, such as a nucleic acid molecule encoding a gene and / or a regulatory element necessary for gene expression. As used herein, the term “vector” generally refers to a nucleic acid molecule capable of importing or transporting a payload nucleic acid molecule. The payload nucleic acid molecule can generally be ligated, for example, inserted, into the vector nucleic acid molecule. A vector may contain sequences that direct autonomous replication within a cell, or may contain sequences sufficient to enable integration into host cell genes (e.g., host cell DNA). Examples of vectors include, but are not limited to, plasmids (e.g., DNA plasmids or RNA plasmids), transposons, cosmids, bacterial artificial chromosomes, and viral vectors. As used herein, “viral vector” generally refers to a viral nucleic acid capable of transporting another nucleic acid into a cell. A viral vector, when in a suitable environment, can direct the expression of one or more proteins encoded by one or more genes carried by the vector. Examples of viral vectors include, but are not limited to, gamma-retroviruses, alpha-retroviruses, foam viruses, lentiviruses, adenoviruses, or adeno-associated virus vectors. A vector in any embodiment of the present disclosure may include exogenous, endogenous, or heterogeneous regulatory sequences, such as promoters and / or enhancers.

[0052] As used herein, the term “nucleotide” generally refers to a combination of base-sugar-phosphate. Nucleotides may include synthetic nucleotides. Nucleotides may include synthetic nucleotide analogs. Nucleotides are monomeric units of nucleic acid sequences (e.g., deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)). The term nucleotide may include ribonucleoside triphosphates such as adenosine triphosphate (ATP), uridine triphosphate (UTP), cytosine triphosphate (CTP), guanosine triphosphate (GTP), and deoxyribonucleoside triphosphates, e.g., dATP, dCTP, dITP, dUTP, dGTP, dTTP, or derivatives thereof. Such derivatives include, for example, [αS]dATP, 7-deaza-dGTP, and 7-deaza-dATP, as well as nucleotide derivatives that confer nuclease resistance to nucleic acid molecules containing them. As used herein, the term nucleotide may refer to dideoxyribonucleoside triphosphates (ddNTPs) and their derivatives. Examples of dideoxyribonucleoside triphosphates include, but are not limited to, ddATP, ddCTP, ddGTP, ddITP, and ddTTP.

[0053] The terms “polynucleotide,” “oligonucleotide,” and “nucleic acid” are used interchangeably to refer to polymeric forms of nucleotides of any length, whether single-stranded, double-stranded, or multi-stranded, deoxyribonucleotides or ribonucleotides, or their analogues. In some cases, polynucleotides are exogenous to cells (e.g., heterologous polynucleotides). In some cases, polynucleotides are endogenous to cells. In some cases, polynucleotides can exist in a cell-free environment. In some cases, polynucleotides are genes or fragments thereof. In some cases, polynucleotides are DNA. In some cases, polynucleotides are RNA. Polynucleotides can have any three-dimensional structure and can perform any known or unknown function. In some cases, polynucleotides contain one or more analogues (e.g., modified skeletons, sugars, or nucleic acid bases). Modifications to the nucleotide structure, where present, may be conferred before or after the assembly of the polymer. Some non-limiting examples of analogs include 5-bromouracil, peptide nucleic acids, heteronucleotides, morpholino, locked nucleic acids, glycol nucleic acids, threose nucleic acids, dideoxynucleotides, cordycepin, 7-deaza-GTP, fluorophores (e.g., sugar-linked rhodamine or fluorescein), thiol-containing nucleotides, biotin-linked nucleotides, fluorescent base analogs, CpG islands, methyl-7-guanosine, methylated nucleotides, inosine, thiouridine, pseudouridine, dihydrouridine, keuosin, and waiosin.Non-limiting examples of polynucleotides include coding or non-coding regions of genes or gene fragments, one or more loci defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), short interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), non-coding RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, cell-free polynucleotides including cell-free DNA (cfDNA) and cell-free RNA (cfRNA), nucleic acid probes, and primers. The sequence of a nucleotide is interrupted by non-nucleotide components. The nucleotides or nucleic acids described herein may be modified to include modified nucleic acids, nucleic acid analogs, modified sugars, sugar analogs, modified nucleic acid bonds, skeletal phosphate modifications, or combinations thereof.

[0054] As used herein, the terms “polypeptide,” “peptide,” and “protein” are used interchangeably to refer to polymers of amino acid residues. In some cases, polypeptide refers to a full-length polypeptide that is translated from an open reading frame encoding it or processed into its mature form. In some cases, polypeptide or peptide may be a degraded or processing fragment of a protein that is still specifically or identifiablely mapped to a particular protein. In some cases, polypeptide may be a single linear polymer chain of amino acids linked to one another by peptide bonds between the carboxyl and amino groups of adjacent amino acid residues. Polypeptides can be modified, for example, by the addition of carbohydrates, phosphorylation, etc. As used herein, the term “fragment” or equivalent may refer to a portion of a protein that has less than the full length of a protein and, if necessary, maintains the function of the protein.

[0055] "Identity percentage" and "identity %" refer to the degree to which two sequences (nucleotides or amino acids) have the same residues at the same positions in the alignment. For example, "the amino acid sequence is X% identical to sequence number Y" refers to the identity % of the amino acid sequence with respect to sequence number Y, and more precisely expresses that X% of the residues in the amino acid sequence are identical to the residues in the sequence disclosed in sequence number Y. Generally, computer programs are used for such calculations. Exemplary programs for comparing and aligning pairs of sequences include ALIGN, FASTA, GAPBLAST, BLASTP, BLASTN, or GCG.

[0056] The terms “antibody” and “immunoglobulin” are used interchangeably herein and encompass fully assembled antibodies, antibody fragments capable of binding to antigens, such as Fab, F(ab')2, Fv, single-chain antibodies (scFv), diabodies, antibody chimeras, hybrid antibodies, and bispecific antibodies. In some cases, the antibody-binding domain is any domain that specifically binds to an antigen, including antibody-binding domains or non-antibody-binding domains. In some cases, the antibody-binding domain binds to tumor cells, such as antibodies against tumor cell surface receptors or tumor antigens. Antibodies may be monoclonal antibodies, polyclonal antibodies, recombinant antibodies, or their antigen-binding fragments, such as heavy chain variable domains (VH) and light chain variable domains (VL). In some cases, the binding domain of a non-antibody scaffold may be lipocalin, anticalin, "T-body," affibody, peptibody, DARPin, affimer, avimer, Nottin, monobody, affinity clamp, ectodomain, receptor ectodomain, receptor, cytokine, ligand, immune cytokine, centryin, T cell receptor, or recombinant T cell receptor. In some cases, the binding domain of an antibody construct is an antigen-binding domain derived from a monoclonal antibody, including light and heavy chains. In some cases, the antibody is a derivatized antibody, such as an antibody modified by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protective / blocking groups, or proteolytic cleavage. Antibodies can also be modified by defucosylation or deglycosylation, etc.

[0057] The terms “monoclonal antibody” and “mAb” are used interchangeably herein and refer to antibodies obtained from a substantially homogeneous population of antibodies. In some cases, individual monoclonal antibodies obtained from a substantially homogeneous population of antibodies are identical except for naturally occurring variations that may be present in small amounts. In some cases, monoclonal antibodies that bind to tumor antigens include a light chain and a heavy chain of tumor antigen antibodies. In some cases, monoclonal antibodies bind to antigens on the surface of immune cells (immune cell antigens) and include a light chain and a heavy chain of anti-immune cell antigen antibodies. In some cases, monoclonal antibodies specifically bind to antigens present on the surface of antigen-presenting cells (APC antigens) and include a light chain and a heavy chain of anti-APC antigen antibodies that bind to APC antigens.

[0058] As used herein, the term “antibody fragment” refers to a molecule that includes a portion of an intact antibody, preferably the antigen-binding or variable region of an intact antibody. Examples of antibody fragments include Fab, Fab, F(ab')2, Fv fragments, and single-chain variable fragments (scFv); diabodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. Digestion of an antibody with papain yields two identical antigen-binding fragments called “Fab” fragments, each having a single antigen-binding site, and a residual “Fc” fragment, named to reflect its ability to readily crystallize. Pepsin treatment yields an F(ab')2 fragment having two antigen-binding sites and still capable of crosslinking antigens. Depending on the amino acid sequence of the constant domain of the heavy chain, immunoglobulins can be assigned to different classes. Human immunoglobulins have five main classes: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known. Different isotypes have different effector functions.

[0059] The term "human antibody" includes all antibodies that have one or more variable and constant regions derived from human immunoglobulin sequences. In some cases, all of the variable and constant domains of an antibody are derived from human immunoglobulin sequences (referred to as "fully human antibodies").

[0060] As used herein, the term “recombinant human antibody” is intended to include all human antibodies prepared, expressed, produced, or isolated by recombinant methods, such as antibodies isolated from host cells, such as NSO or CHO cells, or from animals transgenic for human immunoglobulin genes (e.g., mice), or antibodies expressed using recombinant expression vectors transfected into host cells. Such recombinant human antibodies may have rearranged forms of variable and constant regions. In some cases, recombinant human antibodies have been subjected to in vivo somatic hypermutation. Therefore, the amino acid sequences of the VH and VL regions of recombinant antibodies are derived from and related to human germline VH and VL sequences, but are sequences that cannot naturally exist in vivo within the human antibody germline repertoire.

[0061] A "humanized" antibody is an antibody whose sequence has been altered in part from its original form to more closely resemble a human immunoglobulin. In some versions, the heavy (H) chain and light (L) chain constant (C) region are replaced with human sequences. This may be a fusion polypeptide containing a variable (V) region and a heterologous immunoglobulin C region. In some versions, the complementarity-determining region (CDR) contains a non-human antibody sequence, but the V framework region is also converted to a human sequence. In some versions, the V region is humanized by designing a consensus sequence of human and mouse V regions and converting different extra-CDR residues between the consensus sequences.

[0062] As used herein, the term "in vivo" is used to describe events occurring within the body of the subject.

[0063] As used herein, the term “ex vivo” is used to describe events occurring outside the body of the subject. “Ex vivo” assays cannot be performed on the subject; rather, they are performed on a sample separate from the subject. Ex vivo is used to describe events occurring in intact cells outside the body of the subject.

[0064] As used herein, the term “in vitro” is used to describe events that occur when experimental reagents are contained in a container for holding them and are isolated from the biological source organism from which the material is obtained. In vitro assays may encompass cell-based assays in which living or dead cells are used. In vitro assays may also encompass cell-free assays in which intact cells are not used.

[0065] As used herein, “microenvironment” refers to the extracellular environment in which cells targeted by the extracellular vesicles described herein are located. In some cases, the microenvironment may have an extracellular space in which proteases and other soluble proteins and factors are located. The microenvironment may include, for example, blood vessels, immune cells, fibroblasts, bone marrow-derived inflammatory cells, lymphocytes, signaling molecules, and the extracellular matrix (ECM).

[0066] "To treat" or "treatment" can refer to both therapeutic treatments and preventive or prophylactic measures, the purpose of which is to prevent or slow (reduce) the targeted pathological condition or disorder. Those who require treatment include those who already have the disorder, as well as those who are prone to developing the disorder or who should be prevented from developing the disorder. The therapeutic benefit can refer to the eradication of the disorder being treated or the improvement of the symptoms of the disorder being treated. The therapeutic benefit may also be achieved by the eradication or improvement of one or more physiological symptoms associated with the underlying disorder, so that improvement is observed in the subject, even though the subject may still be susceptible to the underlying disorder. Prophylactic effects can include delaying, preventing, or eliminating the onset of the disease or condition, delaying or eliminating the onset of symptoms of the disease or condition, slowing, stopping, or reversing the progression of the disease or condition, or any combination thereof. For the prophylactic benefit, subjects at risk of developing a particular disease, or those reporting one or more physiological symptoms of the disease, may receive treatment even if a diagnosis of the disease has not been made.

[0067] The terms “effective dose” and “therapeutic dose” are used interchangeably herein and generally refer to an amount of a pharmaceutical composition, for example, a pharmaceutical composition containing the composition described herein, that is sufficient to produce the desired activity when administered to a subject requiring the pharmaceutical composition. In the context of this disclosure, the term “therapeutically effective” refers to an amount of a pharmaceutical composition sufficient to delay the onset, halt the progression, reduce or alleviate at least one symptom of a disorder treated by the method of this disclosure.

[0068] The terms “pharmaceutically acceptable carrier,” “pharmaceutically acceptable excipient,” “physiologically acceptable carrier,” or “physiologically acceptable excipient” refer to a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material. A component is “pharmaceutically acceptable” in the sense that it is compatible with other components of a pharmaceutical formulation. It is also suitable for use in contact with human and non-human mammalian tissues or organs without excessive toxicity, irritation, allergic reactions, immunogenicity, or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0069] The term "pharmaceutical composition" refers to a composition disclosed herein that includes other chemical components such as diluents or carriers. Pharmaceutical compositions can facilitate administration to a subject. Multiple techniques exist in the art for administering compounds, including but not limited to oral, injectable, aerosol, parenteral, and topical administration.

[0070] The terms “patient” or “subject” are used interchangeably herein and encompass mammals. Non-exclusive examples of mammals include any member of the mammalian class: humans, non-human primates such as chimpanzees, and other ape and monkey species; farm animals such as cattle, horses, sheep, goats, and pigs; domestic animals such as rabbits, dogs, and cats; and laboratory animals, including rodents such as rats, mice, and guinea pigs.

[0071] overview This disclosure relates to the design and production of extracellular vesicles expressing at least one adapter polypeptide (e.g., Fc receptor, CD64) that can be conjugated with an antibody. The antibody can induce and target the extracellular vesicles to cells expressing a cell surface marker (e.g., an antigen to the antibody) that can be recognized and bound by the antibody. Cells expressing a cell surface marker may be diseased cells. In some cases, cells expressing a cell surface marker may be cancer cells, tumor cells, non-cancerous lesion cells, cells as part of damaged tissue, cells as part of healthy tissue, or immune cells.

[0072] In some cases, the adapter polypeptide can be further engineered to include additional targeting domains to enhance the targeting and accumulation of extracellular vesicles in targeted cells. These targeting domains can bind to the same or different cell surface markers expressed by the same targeted cells as those targeted by the antibody complexed with the adapter polypeptide. Extracellular vesicles can be configured to target different cells (e.g., different cell types, affected cells) or different cell markers by complexing the adapter polypeptide with another antibody that targets different cell surface markers expressed by different cells. Extracellular vesicles can be designed to carry payloads, such as therapeutic agents, delivered to targeted cells. Therapeutic agents delivered by extracellular vesicles may include therapeutic polynucleotides, therapeutic polypeptides, therapeutic compounds, or cancer drugs, or combinations thereof.

[0073] This disclosure also provides a method for producing extracellular vesicles containing large quantities of high-quality therapeutic agents, such as therapeutic polynucleotides (e.g., therapeutic messenger RNA), comprising at least one adapter polypeptide. Several approaches described herein involve transfecting a cell with at least one heterologous polynucleotide by nanoelectroporation, the at least one heterologous polynucleotide being transcribed and / or translated into at least one adapter polypeptide and / or at least one therapeutic agent. In some cases, the adapter polypeptide comprises an Fc receptor or a fragment thereof that can be complexed with the Fc region of an antibody. In some embodiments, the transfected cell is stimulated by nanoelectroporation to produce and secrete a large number of extracellular vesicles containing large quantities of therapeutic agents (e.g., therapeutic mRNA). The secreted extracellular vesicles can be complexed with any antibody containing an Fc region, and the complexed antibody targets and induces the extracellular vesicles to a targeted cell expressing a first cell surface marker that can be recognized and bound by the complexed antibody. At least one adapter polypeptide may include a targeting domain that binds to a second cell surface marker expressed by the same targeted cell. Therefore, the accumulation of extracellular vesicles containing dual targeting domains (e.g., antibody and targeting domain) in targeted cells may be increased compared to the accumulation of extracellular vesicles without targeting by antibody, targeting domain, or a combination thereof.

[0074] Extracellular vesicles Compositions comprising extracellular vesicles are described herein. Methods for producing extracellular vesicles are also described herein. In some cases, the extracellular vesicles comprise at least one adapter polypeptide, an antibody complexed with the adapter polypeptide, and at least one therapeutic agent. In some cases, the adapter polypeptide comprises a targeting domain. In some cases, at least one adapter polypeptide comprises a peptide sequence that is at least 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% identical to the peptide sequence of a cell surface protein. In some cases, the adapter polypeptide comprises a peptide that is at least 70% identical to the CD63 surface protein. In some cases, the adapter polypeptide comprises a peptide that is at least 70% identical to another cell surface protein. In some cases, the adapter polypeptide comprises a peptide that is at least 70% identical to a cell surface protein selected from the group ROR1, PD-L1, EpCAM, EGFR, EGFRIII, EGFRVIII, GPC1, GPC3, DLL3, L1CAM, GLAST, and CD138.

[0075] In some cases, at least one adapter polypeptide contains a peptide sequence that is at least 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% identical to the peptide sequence of the Fc-binding domain, Fc receptor, or fragment thereof that recognizes and binds to the Fc region of the antibody. In certain cases, the extracellular vesicle surface protein is the Fc receptor. Fc receptors include Fc-γ receptors, Fc-α receptors, or Fc-ε receptors. Exemplary Fc receptors include FcγRI(CD64), FcγRIIa(CD32a), FcγRIIb1(CD32b), FcγRIIb2(CD32b), FcγRIIc1(CD32c), FcγRIIc2(CD32c), FcγRIIc3(CD32c), FcγRIIc4(CD32c), FcγRIIc5(CD32c), FcγRIIIA(CD16a), FcγRIIIB(CD16b), FcεRI, FcεRII(CD23), FcαRI(CD89), Fcα / μR, FcRn, DC-SIGN, or plgR. In some cases, at least one adapter polypeptide contains a peptide sequence that is at least 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% identical to the peptide sequence of any one of the Fc receptors described herein. In some cases, at least one adapter polypeptide contains a peptide sequence that is at least 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% identical to the peptide sequence of any one of the Fc receptors FcγRI(CD64), FcγRII(CD32), or FcγRIII(CD16). In some cases, at least one adapter polypeptide contains a peptide sequence that is at least 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% identical to the peptide sequence of the Fc receptor FcγRI(CD64).

[0076] In some cases, the extracellular vesicles described herein comprise at least two adapter polypeptides. In some cases, one of the at least two adapter polypeptides comprises an adapter polypeptide comprising a peptide sequence of an Fc-binding domain, an Fc receptor, or a fragment thereof. In some cases, one of the at least two adapter polypeptides comprises a peptide sequence of CD47 or a fragment thereof. In some cases, the extracellular vesicles described herein comprise a first and a second adapter polypeptide, the first adapter polypeptide comprising a peptide sequence of an Fc-binding domain, an Fc receptor, or a fragment thereof, and the second adapter polypeptide comprising a peptide sequence of CD47 or a fragment thereof.

[0077] In some cases, the extracellular vesicle contains an antibody complexed with an adapter polypeptide containing a peptide sequence comprising an Fc-binding domain, an Fc receptor, or a fragment thereof. In some cases, the antibody complexed with the adapter polypeptide binds to a first cell surface marker expressed by the targeted cell. In some cases, the adapter polypeptide contains an extracellular domain. In some cases, the targeting domain binds to the extracellular domain, and the targeting domain binds to a second cell surface marker expressed by the same targeted cell expressing the first cell surface marker. In some cases, the targeting domain bound to the extracellular domain binds to a second cell surface marker expressed by the same targeted cell. In some cases, the targeted cell is a cell or immune cell as part of healthy tissue. In some cases, the targeted cell is a diseased cell. In some cases, the diseased cell is a cancer cell, tumor cell, non-cancerous lesion cell, or a cell as part of damaged tissue. In some cases, the first and second cell surface markers expressed by the targeted cell are identical. In some cases, the first and second cell surface markers expressed by the targeted cell are different.

[0078] In some cases, both the antibody and the targeting domain bind to the first and second cell surface markers expressed by the targeted cells, respectively. In certain cases, the antibody and the targeting domain bind to the first and second cell surface markers simultaneously. In certain cases, the antibody and the targeting domain bind to the first and second cell surface markers sequentially. In some cases, the antibody is released from complexation with the adapter polypeptide (i.e., no longer bound to the first cell surface marker), while the targeting domain remains bound to the second cell surface marker.

[0079] In some cases, the extracellular vesicles described herein are exosomes. In some cases, the exosomes include an adapter polypeptide comprising a peptide sequence of an Fc-binding domain, an Fc receptor, or a fragment thereof. The adapter polypeptide comprising an Fc-binding domain, an Fc receptor, or a fragment thereof can be complexed with the Fc region of any one of the antibodies described herein. In some cases, the adapter peptide includes a targeting domain. In some cases, the targeting domain is a tumor-homing peptide (THP), a tissue-homing peptide, a tissue-targeting domain, a cell-permeable peptide, a viral membrane protein, or a combination thereof. In some cases, the targeting domain is a tissue-homing peptide or a tumor-homing peptide. In some cases, the targeting domain is a tumor-homing peptide (THP). In some cases, the targeting domain is a tissue-homing peptide.

[0080] In some cases, the first cell surface marker may be any of the cell surface markers described herein (e.g., antigens or fragments thereof) that can be recognized and bound by the antibodies described herein. In some cases, the second cell surface marker may be recognized and bound by a targeting domain and may be the same as or different from the first cell surface marker. In some cases, the second cell surface marker may be any macromolecule or protein expressed on the surface of a cell. In some cases, the second cell surface marker may be expressed by cells of a particular tissue. In some cases, the second cell surface marker may be expressed by cancer cells or non-cancerous lesion cells. Non-limiting examples of the second cell surface marker include vascular receptors, fibronectin receptors, CD44, CD24, ESA, SSEA1, CD133, CD34, CD19, CD38, CD26, CD166, or CD90.

[0081] In some cases, the accumulation of extracellular vesicles containing antibodies complexed with at least one adapter polypeptide in cells expressing the first and second cell surface markers is higher than the accumulation of extracellular vesicles without antibodies complexed with at least one adapter polypeptide in cells expressing the first and second cell surface markers. In some cases, the accumulation of extracellular vesicles containing antibodies complexed with at least one adapter polypeptide in cells expressing the first and second cell surface markers is at least 2 times, 5 times, 10 times, 50 times, 100 times, or more, compared to the accumulation of extracellular vesicles without antibodies complexed with at least one adapter polypeptide in cells expressing the first and second cell surface markers. In certain specific cases, the accumulation of extracellular vesicles containing antibodies complexed with at least one adapter polypeptide in cells expressing the first and second cell surface markers is higher than the accumulation of extracellular vesicles without at least one adapter polypeptide in cells expressing the first and second cell surface markers. In some cases, the accumulation of extracellular vesicles containing antibodies complexed with at least one adapter polypeptide in cells expressing the first and second cell surface markers is at least 2-fold, 5-fold, 10-fold, 50-fold, 100-fold, or more, compared to the accumulation of extracellular vesicles without at least one adapter polypeptide in cells expressing the first and second cell surface markers.

[0082] An antibody complexed with an adapter polypeptide comprises either an antibody fragment or a binding domain of the antibody described herein. In some cases, the antibody is fused to an Fc region, which is recognized by the adapter polypeptide. In some cases, the Fc region contains IgA, IgD, IgE, IgG, or IgM. In some cases, the Fc region contains IgA, IgD, or IgG. In some cases, the Fc region contains IgG. IgG may be IgG1, IgG2, IgG3, or IgG4. In some cases, the Fc region contains IgG1 or IgG3. In some cases, the antibody described herein contains an Fc region containing IgG1 or IgG3, which is complexed with the adapter polypeptide described herein. In some cases, the antibody is complexed with the adapter polypeptide via non-covalent complexation of the adapter polypeptide to the Fc region of the antibody. In some cases, the antibody is a monoclonal antibody. In some cases, the antibody is a humanized antibody. In some cases, the antibody is a humanized monoclonal antibody.

[0083] In some cases, the extracellular vesicles described herein include at least one adapter polypeptide. In some cases, the adapter polypeptide includes at least one targeting domain bound to the extracellular domain of the adapter polypeptide. In some cases, the at least one targeting domain is a tumor-homing peptide (THP), a tissue-targeting domain, a cell-permeable peptide, a viral membrane protein, or a combination thereof. In some cases, the tissue-targeting domain is a tissue-homing peptide.

[0084] In some cases, at least one targeting domain is a tumor-homing peptide, and this tumor-homing peptide targets cancerous cells. In some cases, at least one targeting domain is a tumor-homing peptide, and this tumor-homing peptide targets cells as part of a tumor (such as a spheroid tumor). In some cases, at least one targeting domain is a tumor-homing peptide, where the tumor-homing peptide targets non-cancerous lesion cells.

[0085] In some cases, the adapter polypeptide comprises at least 1, 2, 3, 4, 5 or more targeting domains. In some cases, at least two targeting domains can be identical. In some cases, at least two targeting domains can be different. The targeting domain can be conjugated to the N-terminus of the adapter polypeptide. Alternatively, the targeting domain can be conjugated to the C-terminus of the adapter polypeptide. In some cases, the targeting domain can be incorporated into the adapter polypeptide. In some cases, the targeting domain is conjugated to the adapter polypeptide via a peptide linker. In some cases, the linker peptide comprises 5 to 200 amino acids. In other cases, the linker peptide comprises 5 to 25 amino acids. In some cases, the linker peptide is rigid (e.g., (EAAAK) 1~3 , A(EAAAK)4ALEA(EAAAK)4A, PAPAP, AEAAAKEAAAKA, or (AP) 10~34 ), flexible (e.g., (GGGGS) 1~4 or (Gly) 6~8), or may be cleavable (e.g., VSQTSKLTR↓AETVFPDV, PLG↓LWA, RVL↓AEA, EDVVCC↓SMSY, GGIEGR↓GS, TRHRQPR↓GWE, AGNRVRR↓SVG, RRRRRRR↓R↓R, or GFLG↓). In some cases, the linker peptide is a FLAG linker containing the peptide sequence DYKDDDDK. In certain specific cases, the FLAG linker may be a 3×FLAG linker containing the peptide sequence YKDHD-G-DYKDHD-I-DYKDDDDK.

[0086] In some cases, the adapter polypeptide contains at least one tumor-homing peptide. In some cases, the adapter polypeptide contains at least two, three, four, five or more tumor-homing peptides. In some cases, at least two tumor-homing peptides are identical. In some cases, at least two tumor-homing peptides are different. In some cases, the tumor-homing peptide is fused to the N-terminus of the adapter polypeptide. In some cases, the tumor-homing peptide is fused to the C-terminus of the adapter polypeptide. In some cases, the tumor-homing peptide can be incorporated into any peptide position of the adapter polypeptide. In some cases, the tumor-homing peptide contains at least three, four, five, six, seven, eight, nine, ten, fifteen, twenty, twenty-five, thirty, fourty, fifty, or one hundred amino acids. In some cases, the tumor-homing peptide is the CDX(FKESWREARGTRIERG) peptide. In some cases, the tumor-homing peptide is the CREKA peptide. In some cases, the tumor-homing peptide is the CKAAKN peptide. In some cases, the tumor-homing peptide is the ARRPKLD peptide.Other exemplary tumor-homing peptides include lung cancer (including SVSVGMKPSPRP, PRPSPKMGVSVS, TDSILRSYDWTY, CSNIDARAC, and ARRPKLD), gastric cancer (including CGNSNPKSC, GRRTRSRRLRRS, CTKNSYLMC, and AADNAKTKSFPV), pancreatic cancer (including CRGRRST, CRSRKG, and CKAAKN), prostate cancer (including FRPNRAQDYNTN, IAGLATPGWSHWLAL, CREAGRKAC, and CAGRRSAYC), and squamous cell carcinoma. Examples of targets include carcinoma (including CSRPRRSEC, CGKRK, and CDTRL), melanoma (including TAASGVRSMH, LTLRWVGLMS, CVNHPAFAC, and CLSDGKRKC), hepatocellular carcinoma (including KSLSRHDHIHHH and SFSIIHTPILPL), colon cancer (including CPHSKPCLC, CPIEDRPMC, CTPSPFSHC, and VHLGYAT), bladder cancer (including CSNRDARRC and CQDGRMGFC), breast cancer (including CREKA), glioma (including LWATFPPRPPWL and LLADTTHHRPWT), ovarian cancer (including CDGLGDDC, CDGWGPNC, and RLDTNRPLLPY), and head and neck cancer (including TSPLNIHNGQKL and SPRGDLAVLGHKY).

[0087] In some cases, the adapter polypeptide contains at least one tissue-targeting domain, which targets and guides the extracellular vesicle containing the adapter polypeptide to a specific tissue. In some cases, the tissue-targeting domain is a tissue-homing domain. In some cases, the adapter polypeptide contains at least two, three, four, five or more tissue-targeting peptides. In some cases, at least two tissue-targeting peptides are identical. In some cases, at least two tissue-targeting peptides are different. In some cases, the tissue-targeting peptide is fused to the N-terminus of the adapter polypeptide. In some cases, the tissue-targeting peptide is fused to the C-terminus of the adapter polypeptide. In some cases, the tissue-targeting peptide can be incorporated at any peptide position of the adapter polypeptide. In some cases, the tissue-targeting peptide contains at least three, four, five, six, seven, eight, nine, ten, fifteen, twenty, twenty-five, thirty, fourty, fifty, or one hundred amino acids. Examples of tissue-targeting domains that target endothelial or cardiac tissue include SIGYPLP, ​​LSIPPKA, FQTPPQL, LTPATAI, CNIWGVVLSWIGVFPEC, NTTTH, VHPKQHR (tetramer), CRKRLDRNCCRTLTVRKC, CLWTVGGGC, QPWLEQAYYSTF, YPHIDSLGHWRR, LLADTTHHRPWT, SAHGTSTGVPWP, VPWMEPAYQRFL, TLPWLEESYWRP, HWRR, CSTSMLKAC, DDTRHWG, CARPAR, CKRAVR, CRSTRANPC, CPKTRRVPC, CSGMARTKC, or CRPPR. Examples of tissue-targeting domains that target pancreatic tissue include CRVASVLPC, SWCEPGWCR, LSGTPERSGQAVKVKLKAIP, CHVLWSTRCCVSNPRWKC, or LSALPRT. Examples of tissue-targeting domains that target kidney tissue include CLPVASC, ELRGD(R / M)AX(W / L), GV(K / R)GX3(T / S)RDXR, HITSLLSHTTHREP, or ANTPCGPYTHDCPVKR.Examples of tissue-targeting domains that target lung tissue include CGFELETCCGFECVRQCPERC, QPFMQCLCLIYDASCRNVPPIFNDVYWIAF, VNTANST, CTSGTHPRC, or SGEWVIKEARGWKHW-VFYSCCPTTPYLDITYH. Examples of tissue-targeting domains that target intestinal tissue include YSGKWGW, LETTCASLCYPSYQCSYTMPHPPVVPPHPMTYSCQY, YPRLLTP, CSQSHPRHC, CSKSSDYQC, CKSTHPLSC, CTGKSCLRVG, SFKPSGLPAQSL, or CTANSSAQC. Examples of tissue-targeting domains that target brain tissue include CLSSRLDAC, GHKAKGPRK, HAIYPRH, THRPPMWSPVWP, HLNILSTLWKYRC, CAGALCY, CLEVSRKNC, RPRTRLHTHRNR(D-aa), ACTTPHAWLCG, GLAHSFSDFARDFV, GYRPVHNIRGHWAPG, TGNYKALHPHNG, CRTIGPSVC, CTSTSAPYC, CSYTSSTMC, CMPRLRGC, TPSYDTYAAELR, RLSSVDSDLSGC, CAQK, or SGVYKVAYDWQH. Further exemplary tissue-targeting domains that target various tissues include LMLPRAD (targeting the adrenal gland), CSCFRDVCC (targeting the retina), CRDVVSVIC (targeting the retina), CVALCREACGEGC (targeting the cutaneous subcutaneous vascular structure), GLSGGRS (targeting the uterus), WYRGRL (targeting cartilage), CPGPEGAGC (targeting the mammary vascular structure), SMSIARLVSFLEYR (targeting the prostate), GPEDTSRAPENQQKTGC (targeting the cutaneous Langerhans gland), CKGGRAKDC (targeting the white adipose vascular structure), CARSKNKDC (targeting wound or injured tissue), CHAQGSAEC (targeting the thymus), LEPRWGFGWWLKLSTHTTESRSMV (targeting the ear or cochlear tissue), ACSTEALRHCGGGS (targeting retinal blood vessels), or ASSLNIA (targeting muscle tissue).

[0088] In some cases, the adapter polypeptide contains at least 2, 3, 4, 5 or more cell-permeable peptides. In some cases, the adapter polypeptide containing cell-permeable peptides increases the rate at which extracellular vesicles are fused or endocytized by the targeted cell. In some cases, at least two cell-permeable peptides are identical. In some cases, at least two cell-permeable peptides are different. In some cases, the cell-permeable peptides are fused to the N-terminus of the adapter polypeptide. In some cases, the cell-permeable peptides are fused to the C-terminus of the adapter polypeptide. In some cases, the cell-permeable peptides can be incorporated at any peptide position of the adapter polypeptide. In some cases, the cell-permeable peptides contain at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, or 100 amino acids. Non-limiting examples of cell-permeable peptides include DSLKSYWYLQKFSWR, DWLKAFYDKVAEKLKEAF, KSKTEYYNAWAVWERNAP, GNGEQREMAVSRLRDCLDRQA, HTPGNSNKWKHLQENKKGRPRR, DWLKAFYDKVAEKLKEAF, R9GPLGLAGE8, Ac-GAFSWGSLWSGIKNFGSTVKNYG, RLRWR, LGQQQPFPPQQPY, ILGKLLSTAAGLLSNL, TFFYGGSRGKRNNFKTEEY, Ac-LRKLRKRLLRX-Bpg-G, Ac-LRKLRKRLLR, or MVRRFLVTLRIRRACGPPRVRV.

[0089] In some cases, the adapter polypeptide contains at least two, three, four, five or more viral membrane proteins or fragments thereof. In some cases, the adapter polypeptide containing viral membrane proteins increases the rate at which extracellular vesicles are fused or endocytized by the targeted cell. In some cases, at least two viral membrane proteins are identical. In some cases, at least two viral membrane proteins are different. In some cases, the viral membrane protein is fused to the N-terminus of the adapter polypeptide. In some cases, the viral membrane protein is fused to the C-terminus of the adapter polypeptide. In some cases, the viral membrane protein can be incorporated at any peptide position of the adapter polypeptide. In some cases, the viral membrane protein contains at least three, four, five, six, seven, eight, nine, ten, fifteen, twenty, two-fifth, three-fifth, four-fifth, five-fifth, or one-hundredth amino acids. Non-limiting examples of viral membrane proteins include hemagglutinin, glycoprotein 41, envelope protein, VSV G, HSV O1 gB, Ebola virus glycoprotein, or fusion-associated small transmembrane (FAST) protein.

[0090] In some cases, the extracellular vesicles described herein contain at least one therapeutic agent. In some cases, the therapeutic agent is a therapeutic polynucleotide. In some cases, the therapeutic agent is a therapeutic polypeptide. In some cases, the therapeutic agent is a therapeutic compound. In some cases, the therapeutic agent is an oncological drug comprising a therapeutic polynucleotide, a therapeutic polypeptide, a therapeutic compound, or a combination thereof. In some cases, the extracellular vesicles contain multiple therapeutic agents, and the multiple therapeutic agents comprise therapeutic polynucleotides, therapeutic polypeptides, therapeutic compounds, or a combination thereof.

[0091] In some cases, the extracellular vesicle contains at least one therapeutic agent, and at least one therapeutic agent is expressed on the extracellular surface of the extracellular vesicle. In some cases, at least one therapeutic agent is expressed on the surface of the extracellular vesicle by ligating at least one therapeutic agent to an adapter polypeptide. In some cases, at least one therapeutic agent is expressed and inserted into the membrane of the extracellular vesicle. In some cases, at least one therapeutic agent is located inside the extracellular vesicle.

[0092] In some cases, extracellular vesicles can be any membrane-bound particles. In some cases, extracellular vesicles can be any membrane-bound particles secreted by cells. In some cases, extracellular vesicles can be any membrane-bound particles produced in vitro. In some cases, extracellular vesicles can be any membrane-bound particles produced without cells. In some cases, extracellular vesicles can be exosomes, microvesicles, retrovirus-like particles, apoptotic bodies, apoptosomes, oncosomes, exophers, enveloped viruses, exomers, or other very large extracellular vesicles. In some cases, extracellular vesicles are exosomes.

[0093] In some cases, extracellular vesicles can have a diameter of approximately 10 nm to approximately 10,000 nm. Extracellular vesicles can have diameters of approximately 500 nm, 100 nm to 1,000 nm, 100 nm to 5,000 nm, 100 nm to 10,000 nm, 500 nm to 1,000 nm, 500 nm to 5,000 nm, 500 nm to 10,000 nm, 1,000 nm to 5,000 nm, 1,000 nm to 10,000 nm, or 5,000 nm to 10,000 nm. In some cases, extracellular vesicles can have diameters of approximately 10 nm, 50 nm, 100 nm, 500 nm, 1,000 nm, 5,000 nm, or 10,000 nm. In some cases, extracellular vesicles can have diameters of at least approximately 10 nm, 50 nm, 100 nm, 500 nm, 1,000 nm, or 5,000 nm. In some cases, extracellular vesicles can have diameters of up to approximately 50 nm, 100 nm, 500 nm, 1,000 nm, 5,000 nm, or 10,000 nm.

[0094] antibody Compositions comprising an extracellular vesicle containing at least one adapter polypeptide complexed with an antibody are described herein. In some cases, the antibody is complexed with the adapter polypeptide via non-covalent complexation between the adapter polypeptide and the Fc region of the antibody. In some cases, the antibody is a monoclonal antibody. In some cases, the antibody is a humanized antibody. In some cases, the antibody is a humanized monoclonal antibody.

[0095] In some cases, when complexed with at least one adapter polypeptide, the antibody induces extracellular vesicles into the cell by binding to cell surface markers expressed by the cell. In some cases, the antibody induces extracellular vesicles into the affected cell by binding to cell surface markers expressed by the affected cell. In some cases, the affected cell is a cancer cell. In some cases, the affected cell is a non-cancerous lesion cell. In some cases, the affected cell is a tumor cell. In some cases, the cell surface marker is an antigen associated with cancer cells or non-cancerous lesion cells. Exemplary cell surface markers associated with cancer cells or non-cancerous lesion cells that can be recognized and bound by the antibodies described herein include: 1-40-β-amyloid, 4-1BB (CD137), 5AC, 5'-nucleotidase, 5T4, activated F9, F10, activin receptor-like kinase 1, ACVR2B, adenocarcinoma antigen, α-fetoprotein, amyloid, angiopoietin 2, angiopoietin 3, anthrax toxin, protective antigen, AOC3 (VAP-1), AXL, B7-H3, Bacillus anthracis anthrax, BAFF, BAFF-R, BCMA, β-amyloid, B lymphoma cells, C1s, C242 antigen, C5, CA-125, CA-125 (imitation), calcitonin, calcitonin gene-related peptide, calcitonin gene-related peptide α, and Canis lupus familiaris IL31, carbonic anhydrase 9 (CA-IX), cardiac myosin, CCL11 (eotaxin-1), CCR2, CCR4, CCR5, CD11, CD18, CD123, CD125, CD134, CD147 (basidine), CD15, CD152, CD154 (CD40L), CD19, CD19, CD3E, CD2, CD20, CD200, CD22, CD23 (IgE receptor), CD25 (α chain of IL-2 receptor), CD27, CD276, CD278 (also known as ICOS), CD28, CD3, CD3ε, CD30 (TNFRSF8), CD319, CD33, CD37, CD38, CD3E, CD4, CD40, CD41 (integrin α-IIb), CD44v6, CD45, CD5, CD51, CD52, CD56, CD6, CD70, CD74, CD79B, CD80, CD97B, CEA, CEACAM5, CEA-related antigen, CFD, CGRP, Claudin 18 isoform 2, CLDN18.2, Clostridium difficile, Clamping factor A, c-Met, Coagulation factor III, Complement C5a, MCSF, CSF1, CSF1R, CSF2, CTGF, CTLA-4, CXCR 4 (CD184), cytomegalovirus, cytomegalovirus glycoprotein B, dabigatran, dendritic cell-associated lectin 2, DLL3, DLL4, DPP4, DR5, Escherichia coli Shiga toxin type 1, Escherichia coli Shiga toxin type 2, Ebola virus glycoprotein, EGFL7, EGFR, EGFR extracellular domain III, EGFR, cMet, EGFR, HER1, EGRF, ERBB1 HER1, endoglin, endotoxin, EpCAM, EPHA3, ephrin receptor A3, episialin, ERBB3 (HER3), ERBB3, HER3, Escherichia coli Hepatitis B (H. coli), F protein of respiratory multinucleated virus, FAP, FCGRT, FGF23, FGFR2, fibrin II, β chain, fibronectin extradomain-B, folate hydrolase, folate receptor 1, folate receptor α, Frizzled receptor, GCGR, GD2 ganglioside, GDF-8, gelatinase B, glypican 3, GMCSF, GMCSF receptor α chain, GPNMB, GPRC5D, CD3, growth differentiation factor 8, GUCY2C, hemagglutinin, hepatitis B surface antigen, hepatitis B virus, HER1, HER2, HER2 / neu, HER2 / neu, CD3, HGF, HGFR, HHGFR, histone complex, HIV-1, HLA-DR?, HNGF, Hsp90, human scatter factor receptor kinase kinase), human TNF, human β-amyloid, ICAM-1, ICOSL, IFN-α, IFN-γ, IgE, IgE Fc region, IGF-1 receptor (CD221), IGF1, IGF2, IGF1R, CD221, IGHE, IL-17A, IL-17A and IL-17F, IL-20, IL-3 receptor, IL-1, IL-12, IL-13, IL-17, IL-17A and IL-17F, IL1A, IL1β, IL2, IL-22, IL23, IL23A, IL31RA, IL-4, IL-4 Rα, IL-5, IL-6, IL6 receptor, IL6 receptor, IL6R, IL-6R, IL9, ILGF2, influenza A virus hemagglutinin, integrin α4β7, integrin α4, integrin α4β7, integrin α5β1, integrin αIIbβ3, integrin αvβ3, integrin β7, interferon γ, interferon receptor, interferon α / β receptor, interferon γ-inducible protein, interleukin 1α, interleukin 13, interleukin 17α, interleukin 17α, TNF, interleukin 17A, ITGA2 (CD49b), ITGB2 (CD18), kallikrein, KIR2D, LAG3, Lewis Y antigen, LFA-1 (CD11a), LINGO-1, lipoteichoic acid, LIV-1, LOXL 2, LRRC15, L-selectin (CD62L), LTA, LYPD3, MASP-2, MCAM, MCP-1, mesothelin, MIF, MS4A1, MSLN, MST1R (also known as RON), mucin CanAg, mucosal adrenergic cell adhesion molecule, myelin-related glycoprotein, myostatin, NACP, NCA-90 (granulocyte antigen), nectin-4, neuronal apoptosis regulatory proteinase 1, NGF, NGNA ganglioside, NKG2A, NOGO-A, Notch 1, Notch receptor, NRP1, OX-40, oxLDL, PCDC1, PCSK9, PD-1, PDCD1, PDCD1, CD279, PDGF-Rα, PDGFRA, PD-L1, sodium phosphate cotransporter, phosphatidylserine, platelet-derived growth factor receptor β, prostate cancer cells, Pseudomonas aeruginosa aeruginosa), Pseudomonas aeruginosa type III secretion system, PTK 7. Rabies virus G glycoprotein, RANKL, Respiratory polynulate virus, RGMA, RHD, Rhesus monkey factor, Buttress plate specific spongein 3, ROR1, RSV fusion glycoprotein, RSVFR, RTN4, Sclerostin, SDC1, Selectin P, Serum amyloid A protein, Serum amyloid P component, SLAMF7, SLITRK6, SOST, Sphingosine-1-phosphate, Staphylococcus aureus Examples include Staphylococcus aureus (Aureus), Staphylococcus aureus α-toxin, Staphylococcus aureus 2-component leucocidine, STEAP1, TAG-72, tau protein, T cell receptor, TEM1, tenascin C, TFPI, TGFβ1, TGFβ2, TGFβ, TIGIT, TNFR superfamily member 4, TNF-α, TRAIL-R1, TRAIL-R2, TRAP, TROP-2, TSLP, tumor antigen CTAA16, 88, tumor-specific glycosylation of MUC1, TWEAK receptor, TYRP1 (glycoprotein 75), VEGF-A, VEGF-A and Ang-2, VEGFR-1, VEGFR2, vimentin, VSIR, VWF, or Zaire Ebola virus glycoprotein. In some cases, cell surface markers recognized and bound by the antibodies described herein include EGFR, PD-L1, or ROR1.

[0096] In some cases, an antibody complexed with at least one adapter polypeptide contains either an antibody fragment or a binding domain of an antibody described herein. In some cases, an antibody complexed with at least one adapter polypeptide contains an Fc region containing one or more Fc binding domains or peptide sequences that bind to an Fc receptor or fragment thereof, as described herein. The Fc region may be antibody-derived. The Fc region of an antibody may be selected from the classes of immunoglobulin, IgA, IgD, IgE, IgG, or IgM. Some different classes can be further divided into isotypes such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant regions of Fc corresponding to different classes of immunoglobulin may be α, δ, ε, γ, and μ, respectively. The light chain may be either kappa, i.e. κ, or lambda, i.e. λ. Depending on the type of antibody, the Fc region may contain several Fc domains, CH1, CH2, CH3, and CH4.

[0097] Any antibody containing an Fc region comprising immunoglobulin IgA, IgD, IgE, IgG, or IgM can be conjugated with an adapter polypeptide containing an Fc-binding domain, an Fc receptor, or a fragment thereof. In some cases, the Fc region has the IgG1 isotype. In some cases, the Fc region has the IgG2 isotype. In some cases, the Fc region has the IgG3 isotype. In some cases, the Fc region has the IgG4 isotype. In some cases, the Fc region has a hybrid isotype comprising constant regions derived from two or more isotypes. The antibody conjugated with the adapter polypeptide may contain any antibody or antigen-binding fragment comprising an Fc domain. Exemplary antibodies conjugated with the adapter polypeptide described herein include cetuximab (including clone C225), atezolizumab, anti-PD-L1 (including clone SP142), or anti-ROR1 mAb (including clone 2A2).

[0098] In some cases, the Fc region is a K2 receptor for at least one adapter polypeptide comprising one of the Fc-binding domains, Fc receptors or fragments described herein. d It contains a peptide sequence having a K for at least one adapter polypeptide, which includes an Fc receptor or a fragment thereof. d It contains a peptide sequence having . In some cases, the Fc receptor includes FcγRI(CD64), FcγRIIa(CD32a), FcγRIIb1(CD32b), FcγRIIb2(CD32b), FcγRIIc1(CD32c), FcγRIIc2(CD32c), FcγRIIc3(CD32c), FcγRIIc4(CD32c), FcγRIIc5(CD32c), FcγRIIIA(CD16a), FcγRIIIB(CD16b), FcεRI, FcεRII(CD23), FcαRI(CD89), Fcα / μR, FcRn, DC-SIGN, or plgR. In some cases, the Fc region is K for at least one adapter polypeptide containing FcγRI(CD64), FcγRIIa(CD32a), FcγRIIb1(CD32b), FcγRIIb2(CD32b), FcγRIIc1(CD32c), FcγRIIc2(CD32c), FcγRIIc3(CD32c), FcγRIIc4(CD32c), FcγRIIc5(CD32c), FcγRIIIA(CD16a) or FcγRIIIB(CD16b). d It contains a peptide sequence having a K for at least one adapter polypeptide containing FcγRI(CD64). In some cases, the Fc region is K for at least one adapter polypeptide containing FcγRI(CD64). d It contains a peptide sequence having the following characteristics.

[0099] In some cases, the Fc region has a modified peptide sequence that alters the biological effector function mediated to at least one constant region compared to the wild-type Fc region and compared to the corresponding antibody containing the wild-type Fc region. For example, the dissociation constant (K d The Fc region can be modified by amino acid substitution to decrease or increase at least one Fc-mediated binding to the Fc receptor, as determined in response to changes in the Fc region.

[0100] In some cases, one of the antibodies described herein may contain a modified Fc region to enhance the biological effector function mediated by at least one Fc region compared to an antibody containing an unmodified Fc domain. For example, an antibody described herein having a modified Fc region that binds to either the Fc-binding domain or the Fc receptor described herein with higher affinity than the corresponding antibody containing a wild-type Fc region. Such modified Fc regions can be generated according to methods known to those skilled in the art. In some cases, the Fc regions described herein include a peptide sequence having at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid modifications.

[0101] In some cases, an Fc region containing at least one amino acid modification may have a K-type binding between wild-type Fc and the same Fc-binding domain or Fc receptor. d In comparison, K has reduced binding to either the Fc-binding domain or the Fc receptor as described herein. d This indicates that, in some cases, an Fc region containing at least one amino acid modification exhibits a K-value of binding between wild-type Fc and the same Fc-binding domain or Fc receptor over a pH range of 6.5–8.4. d Compared to the reduced K for either the Fc-binding domain or the Fc receptor described herein. d This demonstrates that, in some cases, an Fc region containing at least one amino acid modification is configured to complex with an adapter polypeptide containing an Fc receptor in an acidic pH or acidic microenvironment.

[0102] In some cases, an Fc region containing at least one amino acid modification may have a K-type binding between wild-type Fc and the same Fc-binding domain or Fc receptor. d Compared to the above, K has increased binding to either the Fc-binding domain or the Fc receptor as described herein. dThis indicates that, in some cases, an Fc region containing at least one amino acid modification exhibits a K-value of binding between wild-type Fc and the same Fc-binding domain or Fc receptor over a pH range of 6.5–8.4. d Compared to the increased K for either the Fc-binding domain or the Fc receptor described herein, d This indicates that, in some cases, the Fc region, which includes at least one amino acid modification, is configured to be freed from complexation with an adapter polypeptide containing the Fc receptor in an acidic pH or acidic microenvironment.

[0103] Fc binding In some cases, an adapter polypeptide containing an Fc-binding domain or an Fc receptor can be complexed with the Fc region of any one of the antibodies described herein. In some cases, the Fc-binding domain is a fragment of an Fc receptor that binds to the Fc region of the antibody. Exemplary Fc receptors include FcγRI(CD64), FcγRIIa(CD32a), FcγRIIb1(CD32b), FcγRIIb2(CD32b), FcγRIIc1(CD32c), FcγRIIc2(CD32c), FcγRIIc3(CD32c), FcγRIIc4(CD32c), FcγRIIc5(CD32c), FcγRIIIA(CD16a), FcγRIIIB(CD16b), FcεRI, FcεRII(CD23), FcαRI(CD89), Fcα / μR, FcRn, DC-SIGN, or plgR. In some cases, at least one adapter polypeptide contains a peptide sequence that is at least 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% identical to the peptide sequence of one of the Fc receptors FcγRI(CD64), FcγRII(CD32), or FcγRIII(CD16). In some cases, at least one adapter polypeptide contains a peptide sequence that is at least 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% identical to the peptide sequence of the Fc receptor FcγRI(CD64).

[0104] In some cases, the adapter polypeptide contains an Fc-binding domain that includes a bacterial protein that binds to the Fc region of the antibody. In some cases, the Fc-binding domain contains protein A, protein G, protein L, protein Z, protein LG, protein LA, protein AG, or fragments thereof. In some cases, the Fc-binding domain contains a peptide or peptide mimetic. Exemplary Fc-binding peptide sequences or peptide mimetic models include TWKTSRISIF, FGRLVSSIRY, EPIHRSTLTALL HWRGWV, HYFKFD, HFRRHL, HWCitGWV, D2AAG, DAAG, cyclo[(Nα-Ac)-Dap(A)-RWHYFK-Lact-E], cyclo[(Nα-Ac)-Dap(A)-RWHYFK-Lact-E], cyclo[Link-M-WFRHYK], NKFRGKYK, NARKFYKG, FYWHCLDE(1), FYCHWALE(2), FYCHTIDE, RRGW, KHRFNKD, APAR, PAM, Fc-III, FcBP-1, FcBP-2, Fc-III-4C, and FcRM.

[0105] Medical cargo In some cases, extracellular vesicles containing at least one therapeutic agent are described herein. In some cases, the at least one therapeutic agent is a therapeutic polynucleotide encoded by at least one heterologous polynucleotide or vector (e.g., plasmid) transfected into a cell that produces and secretes an extracellular vesicle. In some cases, the at least one therapeutic polynucleotide comprises a nucleic acid sequence that can be translated into a therapeutic polypeptide by a cell targeted and bound by an adapter polypeptide described herein.

[0106] In some cases, extracellular vesicles contain at least one therapeutic polynucleotide. In some cases, extracellular vesicles contain at least 1, 2, 5, 10, 50, 100, 500, 1,000, or more copies of therapeutic polynucleotides. In some cases, extracellular vesicles contain at least two therapeutic polynucleotides. In some cases, extracellular vesicles contain at least two therapeutic polynucleotides, and at least two of the therapeutic polynucleotides are different. In some cases, the at least two different therapeutic polynucleotides encapsulated by the extracellular vesicle contain different ratios. For example, the ratio of the first to the second of two different therapeutic polynucleotides may be 1:1,000, 1:500, 1:100, 1:50, 1:10, 1:5, 1:4, 1:3, 1:2, or 1:1.

[0107] In some cases, therapeutic polynucleotides include mRNA, rRNA, SRP RNA, tRNA, tmRNA, snRNA, snoRNA, gRNA, aRNA, crRNA, lncRNA, miRNA, ncRNA, piRNA, siRNA, shRNA, or combinations thereof. In some cases, therapeutic polynucleotides include mRNA. In some cases, the mRNA is intact, i.e., codes for the full length of a protein. In some cases, the mRNA codes for a portion of a protein. In some cases, the mRNA contains at least 100, 200, 500, 1,000, 5,000, or more RNA nucleotides. In some cases, therapeutic polynucleotides include DNA. In some cases, therapeutic polynucleotides include DNA such as a vector (e.g., plasmid) that codes for a therapeutic polypeptide.

[0108] In some cases, the copy number of the therapeutic polynucleotide encapsulated in the extracellular vesicle is at least 1, 2, 3, 5, 10, 100, or more copies of the therapeutic polynucleotide. In some cases, the copy number of the therapeutic polynucleotide containing RNA encapsulated in the extracellular vesicle is at least 1, 2, 3, 5, 10, 100, or more copies of the therapeutic polynucleotide. In some cases, the copy number of the therapeutic polynucleotide containing therapeutic messenger RNA encapsulated in the extracellular vesicle is at least 1, 2, 3, 5, 10, 100, or more copies of the therapeutic messenger RNA.

[0109] In some cases, the copy number of therapeutic polynucleotides (e.g., RNA therapeutics) encapsulated in extracellular vesicles produced from cells transfected by microchannel electroporation or nanochannel electroporation increases by at least 0.1 times, 0.2 times, 0.5 times, 2 times, 5 times, 10 times, 50 times, 100 times, 500 times, 1,000 times, 5,000 times, 10,000 times, or more compared to the copy number of therapeutic polynucleotides encapsulated in extracellular vesicles produced from cells transfected by other transfection methods. In some cases, the copy number of therapeutic polynucleotides (e.g., RNA therapeutics) encapsulated in extracellular vesicles produced from cells transfected by microchannel electroporation or nanochannel electroporation increases by at least 0.1 times, 0.2 times, 0.5 times, 2 times, 5 times, 10 times, 50 times, 100 times, 500 times, 1,000 times, 5,000 times, 10,000 times, or more compared to the copy number of therapeutic polynucleotides encapsulated in extracellular vesicles by direct introduction of therapeutic polynucleotides into extracellular vesicles (i.e., direct transfection of therapeutic polynucleotides into extracellular vesicles).

[0110] In some cases, therapeutic polynucleotides (e.g., RNA therapeutics) encapsulated in extracellular vesicles produced from cells transfected by microchannel electroporation or nanochannel electroporation as described herein are more intact than therapeutic polynucleotides encapsulated in extracellular vesicles produced from cells transfected by other transfection methods. For example, more copies of therapeutic RNA encapsulated in extracellular vesicles produced from cells transfected by microchannel electroporation or nanochannel electroporation are intact or full-length messenger RNAs that can be translated into therapeutic polynucleotides by targeted cells. In some cases, the copy number of intact RNA therapeutics encapsulated in extracellular vesicles produced from cells transfected by microchannel electroporation or nanochannel electroporation increases by at least 0.1 times, 0.2 times, 0.5 times, 2 times, 5 times, 10 times, 50 times, 100 times, 500 times, 1,000 times, 5,000 times, 10,000 times, or more compared to the copy number of intact RNA therapeutics encapsulated in extracellular vesicles produced from cells transfected by other transfection methods. In some cases, the copy number of intact RNA therapeutics encapsulated in extracellular vesicles produced from cells transfected by microchannel electroporation or nanochannel electroporation increases by at least 0.1 times, 0.2 times, 0.5 times, 2 times, 5 times, 10 times, 50 times, 100 times, 500 times, 1,000 times, 5,000 times, 10,000 times, or more compared to the copy number of intact RNA therapeutics encapsulated in extracellular vesicles produced from direct introduction of therapeutic polynucleotides into extracellular vesicles (i.e., direct transfection of therapeutic polynucleotides into extracellular vesicles).

[0111] In some cases, the extracellular vesicles described herein comprise at least one therapeutic polypeptide. In some cases, the at least one therapeutic polypeptide is encoded by at least one heterologous polynucleotide or vector (e.g., plasmid) transfected into the extracellular vesicle donor cell.

[0112] In some cases, a therapeutic polynucleotide can be translated by an extracellular vesicle donor cell to obtain at least one therapeutic polypeptide. In some cases, the therapeutic polypeptide is bound to an adapter polypeptide as described herein. In some cases, the therapeutic polypeptide is inserted into the membrane of an extracellular vesicle. In some cases, the therapeutic polypeptide encoded by the therapeutic polynucleotide can be encapsulated by an extracellular vesicle produced and secreted by an extracellular vesicle donor cell. In some cases, the extracellular vesicle can encapsulate both the therapeutic polynucleotide and the therapeutic polypeptide encoded by a nanoelectroporated vector (e.g., a plasmid). In some cases, the extracellular vesicle can be an exosome.

[0113] In some cases, the extracellular vesicles described herein may contain at least one therapeutic compound. In some cases, at least one therapeutic compound is complexed or immobilized by one of the adapter polypeptides described herein. In some cases, at least one therapeutic compound is located within the extracellular vesicle. Exemplary therapeutic compounds include cancer drugs.

[0114] Treatment with extracellular vesicles

[0115] This specification describes a method for treating a target disease by administering a therapeutically effective amount of a composition or pharmaceutical composition comprising extracellular vesicles as described herein. In some cases, the extracellular vesicle comprises at least one adapter polypeptide and at least one therapeutic agent as described herein. In some cases, the adapter polypeptide comprises an Fc receptor which is complexed with the Fc region of any one of the antibodies described herein. In some cases, the adapter polypeptide further comprises a targeting domain which comprises a tumor-homing peptide, a tissue-homing peptide, a tissue-targeting domain, a cell-permeable peptide, a viral membrane protein, and any combination or fragment thereof. In some cases, the antibody and the targeting domain bind to first and second cell surface markers associated with the affected cell, and upon binding to the affected cell, the extracellular vesicle delivers at least one therapeutic agent to the affected cell. In some cases, the affected cell is a cancer cell. In some cases, the affected cell is a non-cancerous lesion cell. In some cases, the affected cell is a tumor cell. In some cases, at least one therapeutic agent comprises a therapeutic polynucleotide, a therapeutic polypeptide, a therapeutic compound, an oncological drug, or a combination thereof.

[0116] In some cases, the uptake of a therapeutic agent delivered by an extracellular vesicle containing an antibody and a targeting domain complexed with at least one adapter polypeptide is increased by at least 0.1, 0.2, 0.5, 2, 5, 10, 50, 100, 500, 1,000, 5,000, 10,000, or more compared to the uptake of a therapeutic agent delivered by an extracellular vesicle without an antibody complexed with an adapter polypeptide. In some cases, targeted cells that show increased uptake of therapeutic agents delivered by extracellular vesicles containing antibodies and targeting domains complexed with at least one adapter polypeptide are cancerous cells, non-cancerous lesion cells, cells as part of a tumor, or cells as part of a tissue.

[0117] In some cases, what is described herein is a method of treating a disease using extracellular vesicles containing adapter polypeptides and therapeutic polynucleotides as described herein. In some cases, a method of treating a tumor with extracellular vesicles containing adapter polypeptides and therapeutic polynucleotides is described herein. In some cases, a method of treating a tumor includes delivering therapeutic polynucleotides, therapeutic polypeptides, therapeutic compounds, cancer drugs, or combinations thereof to tumor cells via extracellular vesicles. Non-limiting examples of tumor cells that can be treated by the methods described herein include cells of lung cancer, breast cancer, colorectal cancer, prostate cancer, skin cancer, gastric cancer, liver cancer, or brain cancer. In some cases, the cancer cells targeted by the extracellular vesicles represent a subpopulation within a cancer cell population, such as cancer stem cells.

[0118] In some cases, methods for treating a disease by administering extracellular vesicles containing adapter polypeptides and therapeutic polynucleotides to subjects requiring treatment of the disease are described herein. In some cases, the extracellular vesicles containing adapter polypeptides and therapeutic polynucleotides are administered daily, every day, every other day, five days a week, once a week, every other week, two weeks a month, three weeks a month, once a month, twice a month, three times a month, or more frequently. The extracellular vesicles containing adapter polypeptides and therapeutic polynucleotides may be administered for a period of at least one month, two months, three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, twelve months, eighteen months, two years, three years, or longer.

[0119] If the patient's condition improves, the dose of extracellular vesicles containing the adapter polypeptide and therapeutic polynucleotide administered may be temporarily reduced or interrupted for a period of time (i.e., a “drug-free period”). In some cases, the length of the drug-free period may vary between 2 days and 1 year, and examples include 2, 3, 4, 5, 6, 7, 10, 12, 15, 20, 28, 35, 50, 70, 100, 120, 150, 180, 200, 250, 280, 300, 320, 350, or 365 days. Dose reduction during drug-free periods may range from 10% to 100%, including, for example, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

[0120] In some cases, for subjects requiring an effective amount of extracellular vesicles containing the adapter polypeptide and therapeutic polynucleotide, an effective amount of extracellular vesicles containing the adapter polypeptide and therapeutic polynucleotide may be administered once a week, once every two weeks, once every three weeks, once every four weeks, once every five weeks, once every six weeks, once every seven weeks, once every eight weeks, once every nine weeks, once every ten weeks, once every eleven weeks, once every twelve weeks, or as a single dose over a longer period.

[0121] Once improvement in the target disease or disease-related condition occurs, a maintenance dose of extracellular vesicles should be administered as needed. Subsequently, the dose, frequency, or both can be reduced as a function of symptoms to a level at which the improved disease, disorder, or condition is maintained.

[0122] In some cases, the amount of extracellular vesicles containing adapter polypeptides and therapeutic polynucleotides corresponding to such amounts varies depending on factors such as the severity of the disease, the identity of the subject or host requiring treatment (e.g., weight), but nevertheless, it is routinely determined in the manner known in the art according to the specific circumstances surrounding the case, including, for example, the particular extracellular vesicles to be administered, the route of administration, and the subject or host being treated. In some cases, the desired dose is presented, conveniently, as a single dose or as divided doses administered simultaneously (or over a short period) or at appropriate intervals, for example, as two, three, four or more partial doses per day. In some cases, the dose can be determined at least in part by the occurrence or severity of grade 3 or grade 4 adverse events in the subject.

[0123] The number of variables related to individual treatment regimens is large, and deviations from these recommendations are not uncommon; therefore, the ranges above are merely suggestive. Such dosages will vary depending on several variables, including the activity of the compound used, the disease or condition being treated, the mode of administration, the requirements of the individual patient, the severity of the disease or condition being treated, and the judgment of the practitioner.

[0124] In some cases, the toxicity and therapeutic efficacy of such treatment regimens are determined by standard pharmaceutical procedures in cell culture or experimental animals, including, but not limited to, determining the LD50 (lethal dose for 50% of the population) and ED50 (therapeutably effective dose for 50% of the population). The dose-to-therapeutic ratio is the therapeutic index and is expressed as the ratio between LD50 and ED50. Compounds exhibiting a high therapeutic index are preferred. Data obtained from cell culture assays and animal studies are used when formulating a range of doses for use in humans. Doses of such compounds are preferably within the range of circulating concentrations containing the ED50 with minimal toxicity. Doses may vary within this range depending on the dosage form used and the route of administration utilized.

[0125] Production of extracellular vesicles In some cases, methods and systems for producing extracellular vesicles comprising the adapter polypeptides described herein are also described herein. In some cases, methods and systems for producing extracellular vesicles comprising the adapter polypeptides described herein and at least one therapeutic agent are also described herein.

[0126] In some cases, the method involves introducing at least one heterologous polynucleotide into a cell. In some cases, the at least one heterologous polynucleotide is a vector. In some cases, the vector is a plasmid. In some cases, the at least one heterologous polynucleotide introduced into the cell encodes at least one adapter polypeptide as described herein. In some cases, the at least one heterologous polynucleotide encodes at least one targeting domain. In some cases, the at least one heterologous polynucleotide comprises at least one therapeutic polynucleotide as described herein. In some cases, the at least one heterologous polynucleotide encodes at least one therapeutic polynucleotide as described herein. In some cases, the at least one heterologous polynucleotide encodes at least one therapeutic polypeptide as described herein.

[0127] In some cases, at least two heterologous polynucleotides are introduced into the same cell, and the first heterologous polynucleotide comprises a first vector (e.g., a plasmid) encoding at least one adapter polypeptide. In some cases, the second heterologous polynucleotide introduced into the same cell comprises a second vector (e.g., a plasmid) encoding at least one therapeutic polynucleotide or at least one therapeutic polypeptide. The first and second heterologous polynucleotides can be introduced into the same cell simultaneously or sequentially. In some cases, the first and second heterologous polynucleotides can be introduced into the same cell by the same transfection method. In some cases, the first and second heterologous polynucleotides can be introduced into the same cell by different transfection methods.

[0128] In some cases, heterologous polynucleotides can be introduced into cells via the use of expression vectors. In the context of expression vectors, the vectors can be readily introduced into cells by any method described herein in the art. For example, expression vectors can be introduced into cells by biological, chemical, or physical transfection methods.

[0129] Biological transfection methods for introducing a target heterologous polynucleotide into cells may include the use of DNA and RNA vectors. Viral vectors, particularly retroviral vectors, have become the most widely used method for inserting genes into non-human mammalian cells. Other viral vectors are, in some cases, derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, and adeno-associated viruses. Exemplary viral vectors include retroviral vectors, adenovirus vectors, adeno-associated virus vectors (AAV), pox vectors, parvovirus vectors, baculovirus vectors, measles virus vectors, or herpes simplex virus vectors (HSV). In some cases, retroviral vectors include gamma-retroviral vectors, such as those derived from Moloney's mouse leukemia virus (MoMLV, MMLV, MuLV, or MLV) or mouse stem cell virus (MSCV) genomes. In some cases, retroviral vectors also include lentiviral vectors, such as those derived from the human immunodeficiency virus (HIV) genome. In some cases, AAV vectors contain AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, or AAV9 serotypes. In some cases, viral vectors are chimeric viral vectors containing viral portions from two or more viruses. In further examples, viral vectors are recombinant viral vectors.

[0130] Chemical transfection methods for introducing heterologous polynucleotides into cells include colloidal dispersions, such as polymer complexes, nanocapsules, microspheres, beads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes. Exemplary colloidal systems for use as delivery vehicles in vitro and in vivo are liposomes (e.g., artificial membrane vesicles). Other state-of-the-art methods of targeted delivery of nucleic acids are available, such as delivery of polynucleotides using targeted nanoparticles or other suitable submicron-sized delivery systems.

[0131] When nonviral delivery systems are used, the exemplary delivery vehicle is the liposome. Lipid formulations are intended for the introduction of nucleic acids into host cells (in vitro, ex vivo, or in vivo). In another embodiment, the nucleic acid is associated with lipids. Lipid-associated nucleic acids may, in some cases, be encapsulated within the aqueous interior of a liposome, dispersed within the lipid bilayer of the liposome, bound to the liposome via linking molecules associated with both the liposome and oligonucleotides, captured by the liposome, complexed with the liposome, dispersed in a lipid-containing solution, mixed with the lipid, combined with the lipid, encapsulated as a suspension in the lipid, encapsulated in or complexed with a micelle, or otherwise associated with the lipid. Lipid, lipid / DNA, or lipid / expression vector-related compositions are not limited to any specific structure in solution. For example, in some cases they exist in a bilayer structure, as micelles, or in a "disintegrated" structure. Alternatively, they may simply be dispersed in solution, forming aggregates of non-uniform size or shape. Lipids are fatty substances that, in some cases, are naturally occurring or synthetic lipids. Examples of lipids include naturally occurring lipid droplets in the cytoplasm, as well as a class of compounds containing long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

[0132] Suitable lipids for use are obtained from commercial suppliers. For example, in some cases dimyristylphosphatidylcholine ("DMPC") is obtained from Sigma (St. Louis, Missouri), in some cases dicetyl phosphate ("DCP") is obtained from K&K Laboratories (Plainview, New York), cholesterol ("Choi") is obtained in some cases from Calbiochem-Behring, and dimyristylphosphatidylglycerol ("DMPG") and other lipids are often obtained from Avanti Polar Lipids, Inc. (Birmingham, Alabama). Stock solutions of lipids in chloroform or chloroform / methanol are often stored at approximately -20°C. Chloroform is used as the sole solvent because it evaporates more readily than methanol. "Liposomes" is a general term encompassing various monolayer and multilayer lipid vehicles formed by the formation of encapsulated lipid bilayers or aggregates. Liposomes are often characterized by having a vesicular structure with a phospholipid bilayer membrane and an internal aqueous medium. Multilayer liposomes have multiple lipid layers separated by an aqueous medium. They form spontaneously when phospholipids are suspended in an excess aqueous solution. The lipid components undergo self-rearrangement before the formation of a closed structure, trapping water and dissolved solute between the lipid bilayers (Ghosh et al., 1991 Glycobiology 5:505-10). However, compositions that have structures different from the usual vesicular structure in solution are also included. For example, lipids may, in some cases, take on a micelle structure or simply exist as heterogeneous aggregates of lipid molecules. Lipofectamine-nucleic acid complexes are also considered.

[0133] Physical transfection methods for introducing heterologous polynucleotides into cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, gene gun, electroporation, microneedle arrays, nanoneedle arrays, sonication, or chemical permeation. Electroporation methods include microfluidic electroporation, microchannel electroporation, or nanochannel electroporation. In certain cases, extracellular vesicle cells are transfected with at least one heterologous polynucleotide by microchannel electroporation or nanochannel electroporation. In some cases, microchannel electroporation or nanochannel electroporation involves the use of microporous patterned silicon wafers, nanoporous patterned silicon wafers, track-etched films, ceramic microporous films, ceramic nanoporous films, other porous materials, or combinations thereof. In some cases, at least one heterologous polynucleotide or at least one vector (e.g., plasmid) is nanoelectroporated into extracellular vesicle donor cells via nanochannels located on a biochip.

[0134] In some cases, extracellular vesicle donor cells can be grown and bound to the surface of a substrate. In some cases, the substrate includes a biochip. In some cases, the surface of the substrate includes a metallic material. In some cases, the substrate includes a metallic material. Non-limiting examples of metallic materials include aluminum (Al), indium tin oxide (ITO, In2O3:SnO2), chromium (Cr), gallium arsenide (GaAs), gold (Au), molybdenum (Mo), organic residues and photoresists, platinum (Pt), silicon (Si), silicon dioxide (SiO2), silicon-on-insulator (SOI), silicon nitride (Si3N4), tantalum (Ta), titanium (Ti), titanium nitride (TiN), and tungsten (W). In some cases, the metallic material can be processed or etched to fabricate arrays or channels. In some cases, metal surfaces can be treated or etched with phosphoric acid (H3PO4), acetic acid, nitric acid (HNO3), water (H2O), hydrochloric acid (HCl), (HNO3), cerium ammonium nitrate ((NH4)2Ce(NO3)6), citric acid (C6H8O7), hydrogen peroxide (H2O2), aqua regia, iodine solution, sulfuric acid (H2SO4), hydrofluoric acid (HF), potassium hydroxide (KOH), ethylenediamine pyrocatechol (EDP), tetramethylammonium hydroxide (TMAH), buffer oxides, ammonium fluoride (NH4F), SC1, Cl2, CCl4, SiCl4, BCl3, SiCl4, BCl3, CCl2F2, CF4, O2, CF4, SF6, NF3, CHF3, or combinations thereof.

[0135] In some cases, a metal surface can be treated with a gas or plasma to increase its hydrophilicity. In other cases, a metal surface can be treated with a gas or plasma to increase its hydrophobicity. Examples of gases or plasmas for increasing the hydrophilicity or hydrophobicity of a metal surface include oxygen, nitrogen, ammonia, argon, chlorine, fluorine, bromine, iodine, astatine, hydrogen, or combinations thereof.

[0136] In some cases, extracellular vesicle donor cells can be grown and bound to the surface of polymer substrates such as polypropylene, polyethylene, polystyrene, ABS, polyamide, polyethylene copolymer, epoxy, polyester, polyvinyl chloride, phenol, polytetrafluoroethylene, polyethylene copolymer, fluorinated ethylene propylene, polyvinylidene, silicone, natural rubber, latex, polyurethane, styrene-butadiene rubber, fluorocarbon copolymer elastomer, polyethylene terephthalate, polycarbonate, polyamide, polyaramid, polyarylether ketone, polyacetal, polyphenylene oxide, PBT, polysulfone, polyethersulfone, polyarylsulfone, polyphenylene sulfide, polytetrafluoroethylene, and beryllium oxide. In some cases, the polymer surface may be semipermeable, having at least one pore. In some cases, the pore size of the semipermeable polymer surface may be about 0.01 μm to about 10 μm. In some embodiments, the pore sizes of the semipermeable polymer surface are approximately 0.01 μm to 0.05 μm, 0.01 μm to 0.1 μm, 0.01 μm to 0.5 μm, 0.01 μm to 1 μm, 0.01 μm to 5 μm, 0.01 μm to 10 μm, 0.05 μm to 0.1 μm, 0.05 μm to 0.5 μm, and 0.05 μm to 1 μm. The pore sizes may be approximately 0.05 μm to 5 μm, approximately 0.05 μm to 10 μm, approximately 0.1 μm to 0.5 μm, approximately 0.1 μm to 1 μm, approximately 0.1 μm to 5 μm, approximately 0.1 μm to 10 μm, approximately 0.5 μm to 1 μm, approximately 0.5 μm to 5 μm, approximately 0.5 μm to 10 μm, approximately 1 μm to 5 μm, approximately 1 μm to 10 μm, or approximately 5 μm to 10 μm. In some embodiments, the pore size of the semipermeable polymer surface may be approximately 0.01 μm, approximately 0.05 μm, approximately 0.1 μm, approximately 0.5 μm, approximately 1 μm, approximately 5 μm, or approximately 10 μm. In some embodiments, the pore size of the semipermeable polymer surface may be at least between about 0.01 μm, about 0.05 μm, about 0.1 μm, about 0.5 μm, about 1 μm, or about 5 μm. In some embodiments, the pore size of the semipermeable polymer surface may be at most between about 0.05 μm, about 0.1 μm, about 0.5 μm, about 1 μm, about 5 μm, or about 10 μm.

[0137] In some cases, the surface of a polymer can be treated with a gas or plasma to increase its hydrophilicity. In other cases, the surface of a polymer can be treated with a gas or plasma to increase its hydrophobicity. Exemplary gases or plasmas for increasing the hydrophilicity or hydrophobicity of a metal surface include oxygen, nitrogen, ammonia, argon, chlorine, fluorine, bromine, iodine, astatine, hydrogen, or combinations thereof.

[0138] Nanoelectroporation In some cases, any cell can be electroporated by microchannel electroporation or nanochannel electroporation as described herein to become an extracellular vesicle donor cell and produce the extracellular vesicles described herein. Extracellular vesicle donor cells can be any cell that can be genetically modified or engineered to produce and secrete extracellular vesicles at levels higher than the basal level of extracellular vesicle secretion. Therefore, cells with low or negligible basal levels of extracellular vesicle secretion can also be transfected by microchannel electroporation or nanochannel electroporation to produce and secrete the extracellular vesicles described herein. In some cases, the extracellular vesicle donor cells can be autologous cells. In such cases, the extracellular vesicle donor cells are obtained from, for example, a subject that is also receiving the extracellular vesicles described herein. In some cases, the donor cells are genetically modified (e.g., genetically modified with targeted polypeptides on the EV surface and / or therapeutic RNA in the EV).

[0139] In some cases, extracellular vesicles produced and secreted from extracellular donor cells (calls) can induce a pro-inflammatory alloimmune response by T cells, thus raising challenges to the use of extracellular vesicles as therapeutic agents. In some cases, extracellular vesicle donor cells may be allogeneic cells, which are cells of the same species but derived from a genetically different source from the target receiving the extracellular vesicles described herein. In some cases, extracellular vesicle donor cells may be cell styles that produce and secrete allogeneic extracellular vesicles. For example, mesenchymal stem cells (MSCs) exhibit low immunogenicity due to the lack of expression of histocompatibility complex class II (MHC-II) and costimulatory molecules, which allows MSCs to act as extracellular vesicle donor cells for producing and secreting allogeneic extracellular vesicles that share similar anti-inflammatory and nutrient properties with parental MSCs that produce and secrete allogeneic extracellular vesicles.

[0140] In some cases, the extracellular vesicle donor cells electroporated by the microchannel electroporation or nanochannel electroporation described herein may be any eukaryotic cell. In some cases, the extracellular vesicle donor cells may be cells derived from cell lines, stem cells, primary cells, or differentiated cells. In some cases, the extracellular vesicle donor cells may be selected from the group consisting of mouse embryonic fibroblasts (MEFs), human embryonic fibroblasts (HEFs), dendritic mesenchymal stem cells, bone marrow-derived dendritic cells, bone marrow-derived stromal cells, adipocytes, endothelial cells, enucleated cells, neural stem cells, immature dendritic cells, and immune cells.

[0141] In some cases, extracellular vesicle donor cells may be genetically modified cells of any of the cells described herein, into which at least one heterologous polynucleotide is introduced. In some cases, at least one heterologous polynucleotide is transfected into extracellular vesicles by electroporation. In some cases, electroporation includes microchannel electroporation or nanochannel electroporation. In some cases, at least one heterologous polynucleotide is transfected into extracellular vesicles by nanochannel electroporation. In some cases, the heterologous polynucleotide transfected into extracellular vesicle donor cells is incorporated into the chromosomes of nanoelectroporated cells. In some cases, the heterologous polynucleotide transfected into extracellular vesicle donor cells is not incorporated into the chromosomes of nanoelectroporated cells. In some cases, nanoelectroporated extracellular vesicle donor cells are stably transfected with the heterologous polynucleotide. In some cases, nanoelectroporated extracellular vesicle donor cells are transiently transfected with the heterologous polynucleotide. In some cases, the transfected extracellular vesicle donor cells may be cells derived from a cell line. In some cases, at least one heterologous polynucleotide is the vector. In other cases, the vector is a plasmid.

[0142] In some cases, extracellular vesicle donor cells continuously produce and secrete extracellular vesicles at a steady or basal rate. In some cases, extracellular vesicle donor cells produce and secrete extracellular vesicles at a basal rate and can produce and secrete further extracellular vesicles by stimulating the cells. For example, by performing a heat shock on extracellular vesicle donor cells or by Ca2C on extracellular vesicle donor cells. 2+By contacting them, extracellular vesicle donor cells can be stimulated to produce and secrete extracellular vesicles at a rate faster than the basal rate. In some cases, extracellular vesicle donor cells can be stimulated to produce and secrete extracellular vesicles at a rate faster than the basal rate by electroporating at least one heterologous polynucleotide into the cell. In some cases, extracellular vesicle donor cells can be stimulated to produce and secrete extracellular vesicles at a rate faster than the basal rate by microchannel electroporation or nanochannel electroporation of at least one heterologous polynucleotide into the cell. In some cases, extracellular vesicle donor cells can be stimulated to produce and secrete extracellular vesicles at a rate faster than the basal rate by nanochannel electroporation of at least one heterologous polynucleotide into the cell. In some cases, extracellular vesicle donor cells stimulated by nanochannel electroporation can produce and secrete extracellular vesicles at rates at least 0.1 times, 0.2 times, 0.3 times, 0.4 times, 0.5 times, 0.6 times, 0.7 times, 0.8 times, 0.9 times, 2 times, 5 times, 10 times, 50 times, 100 times, 500 times, 1,000 times, 5,000 times, 10,000 times, 50,000 times, 100,000 times, or more than the basal rate of extracellular vesicle donor cells producing and secreting extracellular vesicles. In some cases, extracellular vesicle donor cells stimulated by nanochannel electroporation can produce and secrete extracellular vesicles at rates at least 0.1 times, 0.2 times, 0.3 times, 0.4 times, 0.5 times, 0.6 times, 0.7 times, 0.8 times, 0.9 times, 2 times, 5 times, 10 times, 50 times, 100 times, 500 times, 1,000 times, 5,000 times, 10,000 times, 50,000 times, 100,000 times, or more than those of extracellular vesicle donor cells stimulated by transfection methods other than nanoelectroporation for the production and secretion of extracellular vesicles.

[0143] Extracellular vesicles produced and secreted by extracellular vesicle donor cells can be recovered and purified by centrifugation or ultracentrifugation, and the extracellular vesicles are purified from other cellular debris or molecules. In some cases, extracellular vesicles produced and secreted by extracellular vesicle donor cells can be recovered and purified by tangential flow filtration, which can continuously remove other cellular debris or molecules other than the extracellular vesicles described herein.

[0144] In some cases, the heterologous polynucleotide transfected into extracellular vesicle donor cells encodes at least one adapter polypeptide as described herein. In some cases, the heterologous polynucleotide transfected into extracellular vesicle donor cells encodes at least one therapeutic agent as described herein. In some cases, the therapeutic agent is a therapeutic polynucleotide. In some cases, the therapeutic agent is a therapeutic polypeptide. In some cases, extracellular vesicle donor cells transfected with at least one heterologous polynucleotide produce and secrete extracellular vesicles containing at least one adapter polypeptide. In some cases, extracellular vesicle donor cells transfected with at least one heterologous polynucleotide produce and secrete extracellular vesicles containing at least one therapeutic agent. In some cases, extracellular vesicle donor cells transfected with at least one heterologous polynucleotide produce and secrete extracellular vesicles containing at least one adapter polypeptide and at least one therapeutic agent.

[0145] In some cases, the heterologous polynucleotide transfected into an extracellular vesicle donor cell is a vector (e.g., a plasmid). In some cases, the heterologous polynucleotide encodes at least one adapter polypeptide as described herein. In some cases, at least one adapter polypeptide comprises a peptide sequence of an Fc-binding domain, an Fc receptor, or a fragment thereof as described herein. In some cases, at least one adapter polypeptide comprises a peptide sequence of an extracellular domain. In some cases, at least one adapter polypeptide comprises a peptide sequence of a targeting domain bound to the extracellular domain of the adapter polypeptide.

[0146] In some cases, nanoelectroporated extracellular vesicle donor cells produce and secrete extracellular vesicles containing at least one therapeutic agent expressed on the extracellular surface of the extracellular vesicle. In some cases, nanoelectroporated extracellular vesicle donor cells produce and secrete extracellular vesicles containing at least one therapeutic agent expressed on the surface of the extracellular vesicle by conjugating at least one therapeutic agent to an adapter polypeptide. In some cases, nanoelectroporated extracellular vesicle donor cells produce and secrete extracellular vesicles containing at least one therapeutic agent expressed on the surface of the extracellular vesicle by conjugating at least one therapeutic agent to the extracellular domain of an adapter polypeptide. In some cases, nanoelectroporated extracellular vesicle donor cells produce and secrete extracellular vesicles containing at least one therapeutic agent expressed and inserted into the membrane of the extracellular vesicle. In some cases, nanoelectroporated extracellular vesicle donor cells produce and secrete extracellular vesicles containing at least one therapeutic agent within the extracellular vesicle.

[0147] In some cases, the extracellular vesicles produced and secreted by nanoelectroporated extracellular vesicle donor cells are any membrane-bound particles. In some cases, the extracellular vesicles produced and secreted by nanoelectroporated extracellular vesicle donor cells are exosomes, microvesicles, retrovirus-like particles, apoptotic bodies, apotosomes, oncosomes, exotheras, enveloped viruses, exomers, or other very large extracellular vesicles. In some cases, the extracellular vesicles produced and secreted by nanoelectroporated extracellular vesicle donor cells are exosomes.

[0148] In some cases, cells grown or bound to a metal or polymer surface can be nanoelectroporated by the nanoelectroporation systems described herein. In some cases, the system comprises a fluid chamber having an upper boundary and a lower boundary. The upper and lower chambers are formed by placing a substrate containing cells within the fluid chamber. In some cases, the system further comprises at least one nanochannel. In some cases, the nanochannel can be embedded in the substrate. In some cases, the nanochannel comprises pores in a semipermeable polymer substrate. In some embodiments, the nanochannel comprises a height of about 0.01 μm to about 500 μm.In some embodiments, the nanochannels are approximately 0.01 μm to 0.05 μm, 0.01 μm to 0.1 μm, 0.01 μm to 0.5 μm, 0.01 μm to 0.1 μm, 0.01 μm to 0.2 μm, 0.01 μm to 0.5 μm, 0.01 μm to 0.5 μm, 0.01 μm to 0.5 μm, 0.01 μm to 0.5 μm, and 0.01 μm to 0.5 μm. , about 0.05 μm to about 2 μm, about 0.05 μm to about 5 μm, about 0.05 μm to about 10 μm, about 0.05 μm to about 20 μm, about 0.05 μm to about 50 μm, about 0.05 μm to about 100 μm, about 0.05 μm to about 500 μm, about 0.1 μm to about 0. 5μm, about 0.1μm to about 1μm, about 0.1μm to about 2μm, about 0.1μm to about 5μm, about 0.1μm to about 10μm, about 0.1μm to about 20μm, about 0.1μm to about 50μm, about 0.1μm to about 100μm, about 0.1μm to about 500μm, about 0. 5μm to about 1μm, about 0.5μm to about 2μm, about 0.5μm to about 5μm, about 0.5μm to about 10μm, about 0.5μm to about 20μm, about 0.5μm to about 50μm, about 0.5μm to about 100μm, about 0.5μm to about 500μm, about 1μm to about 2μm , about 1 μm to about 5 μm, about 1 μm to about 10 μm, about 1 μm to about 20 μm, about 1 μm to about 50 μm, about 1 μm to about 100 μm, about 1 μm to about 500 μm, about 2 μm to about 5 μm, about 2 μm to about 10 μm, about 2 μm to about 20 μm, about 2 μm to about 50 μm , including heights of approximately 2 μm to 100 μm, approximately 2 μm to 500 μm, approximately 5 μm to 10 μm, approximately 5 μm to 20 μm, approximately 5 μm to 50 μm, approximately 5 μm to 100 μm, approximately 5 μm to 500 μm, approximately 10 μm to 20 μm, approximately 10 μm to 50 μm, approximately 10 μm to 100 μm, approximately 10 μm to 500 μm, approximately 20 μm to 50 μm, approximately 20 μm to 100 μm, approximately 20 μm to 500 μm, approximately 50 μm to 100 μm, approximately 50 μm to 500 μm, or approximately 100 μm to 500 μm. In some embodiments, the nanochannels include heights of approximately 0.01 μm, 0.05 μm, 0.1 μm, 0.5 μm, 1 μm, 2 μm, 5 μm, 10 μm, 20 μm, 50 μm, 100 μm, or 500 μm.In some embodiments, the nanochannels include heights of at least about 0.01 μm, about 0.05 μm, about 0.1 μm, about 0.5 μm, about 1 μm, about 2 μm, about 5 μm, about 10 μm, about 20 μm, about 50 μm, or about 100 μm. In some embodiments, the nanochannels include heights of up to about 0.05 μm, about 0.1 μm, about 0.5 μm, about 1 μm, about 2 μm, about 5 μm, about 10 μm, about 20 μm, about 50 μm, about 100 μm, or about 500 μm. In some cases, the heights of the nanochannels may be the same. In some cases, the heights of the nanochannels may be different. In some cases, the height of the nanochannels must be large enough to accelerate molecules being nanoelectroporated in the high-electric-field zone (e.g., inside the nanochannel), but small enough to allow larger molecules being nanoelectroporated to pass through with short electrical pulses.

[0149] In some embodiments, the nanochannels include diameters ranging from about 0.01 nm to about 10,000 nm.In some embodiments, the nanochannels are approximately 0.01nm to 0.1nm, 0.01nm to 0.5nm, 0.01nm to 1nm, 0.01nm to 5nm, 0.01nm to 10nm, 0.01nm to 50nm, 0.01nm to 100nm, 0.01nm to 500nm, 0.01nm to 1,000nm, 0.01nm to 5,000nm, 0.01nm to 10,000nm, 0.1nm to 0.5nm, 0.1nm to 1nm, 0.1nm to 5nm, 0.1nm to 10nm, and 0.1nm. ~50nm, approx. 0.1nm ~ approx. 100nm, approx. 0.1nm ~ approx. 500nm, approx. 0.1nm ~ approx. 1,000nm, approx. 0.1nm ~ approx. 5, 000nm, about 0.1nm to about 10,000nm, about 0.5nm to about 1nm, about 0.5nm to about 5nm, about 0.5nm to about 10nm, about 0.5nm to about 50nm, about 0.5nm to about 100nm, about 0.5nm to about 500nm, about 0.5nm to about 1,000nm, about 0.5n m ~ approx. 5,000 nm, approx. 0.5 nm ~ approx. 10,000 nm, approx. 1 nm ~ approx. 5 nm, approx. 1 nm ~ approx. 10 nm, approx. 1 nm ~ approx. 50 nm, approx. 1nm to about 100nm, about 1nm to about 500nm, about 1nm to about 1,000nm, about 1nm to about 5,000nm, about 1nm to about 10,0 00nm, about 5nm to about 10nm, about 5nm to about 50nm, about 5nm to about 100nm, about 5nm to about 500nm, about 5nm to about 1,00 0nm, about 5nm to about 5,000nm, about 5nm to about 10,000nm, about 10nm to about 50nm, about 10nm to about 100nm, about 10 nm ~ approx. 500nm, approx. 10nm ~ approx. 1,000nm, approx. 10nm ~ approx. 5,000nm, approx. 10nm ~ approx. 10,000nm, approx. 50nm Includes diameters of approximately ~100nm, approximately 50nm~500nm, approximately 50nm~1,000nm, approximately 50nm~5,000nm, approximately 50nm~10,000nm, approximately 100nm~500nm, approximately 100nm~1,000nm, approximately 100nm~5,000nm, approximately 100nm~10,000nm, approximately 500nm~1,000nm, approximately 500nm~5,000nm, approximately 500nm~10,000nm, approximately 1,000nm~5,000nm, approximately 1,000nm~10,000nm, or approximately 5,000nm~10,000nm.In some embodiments, the nanochannels include diameters of approximately 0.01 nm, approximately 0.1 nm, approximately 0.5 nm, approximately 1 nm, approximately 5 nm, approximately 10 nm, approximately 50 nm, approximately 100 nm, approximately 500 nm, approximately 1,000 nm, approximately 5,000 nm, or approximately 10,000 nm. In some embodiments, the nanochannels include diameters of at least approximately 0.01 nm, approximately 0.1 nm, approximately 0.5 nm, approximately 1 nm, approximately 5 nm, approximately 10 nm, approximately 50 nm, approximately 100 nm, approximately 500 nm, approximately 1,000 nm, or approximately 5,000 nm. In some embodiments, the nanochannels include diameters of up to approximately 0.1 nm, approximately 0.5 nm, approximately 1 nm, approximately 5 nm, approximately 10 nm, approximately 50 nm, approximately 100 nm, approximately 500 nm, approximately 1,000 nm, approximately 5,000 nm, or approximately 10,000 nm. In some cases, the diameters of the nanochannels can be the same. In other cases, the diameters of the nanochannels can be different.

[0150] In some cases, nanochannels can be arranged in a nanochannel array. In some cases, nanochannels can be arranged in a nanochannel array with spacing between them. In some cases, the spacing between nanochannels can range from about 0.01 μm to about 5,000 μm.In some cases, the spacing between nanochannels is approximately 0.01 μm to 0.05 μm, 0.01 μm to 0.1 μm, 0.01 μm to 0.5 μm, 0.01 μm to 1 μm, 0.01 μm to 5 μm, 0.01 μm to 10 μm, 0.01 μm to 50 μm, 0.01 μm to 100 μm, 0.01 μm to 500 μm, 0.01 μm to 1,000 μm, 0.01 μm to 5,000 μm, 0.05 μm to 0.1 μm, 0.05 μm to 0.5 μm, 0.05 μm to 1 μm, and 0.05 μm to 5 μm. m, about 0.05μm to about 10μm, about 0.05μm to about 50μm, about 0.05μm to about 100μm, about 0.05μm to about 500μm , about 0.05μm to about 1,000μm, about 0.05μm to about 5,000μm, about 0.1μm to about 0.5μm, about 0.1μm to about 1μm , about 0.1 μm to about 5 μm, about 0.1 μm to about 10 μm, about 0.1 μm to about 50 μm, about 0.1 μm to about 100 μm, about 0.1 μm ~about 500μm, about 0.1μm to about 1,000μm, about 0.1μm to about 5,000μm, about 0.5μm to about 1μm, about 0.5μm to about 5μm, about 0.5μm to about 10μm, about 0.5μm to about 50μm, about 0.5μm to about 100μm, about 0.5μm to about 500μm, about 0.5μm to about 1,000μm, about 0.5μm to about 5,000μm, about 1μm to about 5μm, about 1μm to about 10μm, about 1μm to about 50 μm, approximately 1 μm to approximately 100 μm, approximately 1 μm to approximately 500 μm, approximately 1 μm to approximately 1,000 μm, approximately 1 μm to approximately 5,000 μm, approximately 5 μm ~Approx. 10μm, approx. 5μm ~ approx. 50μm, approx. 5μm ~ approx. 100μm, approx. 5μm ~ approx. 500μm, approx. 5μm ~ approx. 1,000μm, approx. 5μm It could be approximately 5,000 μm, approximately 10 μm to approximately 50 μm, approximately 10 μm to approximately 100 μm, approximately 10 μm to approximately 500 μm, approximately 10 μm to approximately 1,000 μm, approximately 10 μm to approximately 5,000 μm, approximately 50 μm to approximately 100 μm, approximately 50 μm to approximately 500 μm, approximately 50 μm to approximately 1,000 μm, approximately 50 μm to approximately 5,000 μm, approximately 100 μm to approximately 500 μm, approximately 100 μm to approximately 1,000 μm, approximately 100 μm to approximately 5,000 μm, approximately 500 μm to approximately 1,000 μm, approximately 500 μm to approximately 5,000 μm, or approximately 1,000 μm to approximately 5,000 μm.In some cases, the spacing between nanochannels may be about 0.01 μm, about 0.05 μm, about 0.1 μm, about 0.5 μm, about 1 μm, about 5 μm, about 10 μm, about 50 μm, about 100 μm, about 500 μm, about 1,000 μm, or about 5,000 μm. In some cases, the spacing between nanochannels may be at least about 0.01 μm, about 0.05 μm, about 0.1 μm, about 0.5 μm, about 1 μm, about 5 μm, about 10 μm, about 50 μm, about 100 μm, about 500 μm, or about 1,000 μm. In some cases, the spacing between nanochannels can be up to approximately 0.05 μm, 0.1 μm, 0.5 μm, 1 μm, 5 μm, 10 μm, 50 μm, 100 μm, 500 μm, 1,000 μm, or 5,000 μm.

[0151] In some cases, the nanoelectroporation system includes upper and lower electrode layers for generating an electric field within the fluid chamber. In some cases, the electric field generated by the electrodes for nanoelectroporation includes electric field strengths ranging from approximately 0.1 volts / mm to approximately 50,000 volts / mm. In some cases, the electric field generated by the electrodes for nanoelectroporation includes electric field strengths ranging from approximately 0.1 volts / mm to approximately 0.5 volts / mm, approximately 0.1 volts / mm to approximately 1 volt / mm, approximately 0.1 volts / mm to approximately 5 volts / mm, approximately 0.1 volts / mm to approximately 10 volts / mm, approximately 0.1 volts / mm to approximately 50 volts / mm, approximately 0.1 volts / mm to approximately 100 volts / mm, approximately 0.1 volts / mm to approximately 500 volts / mm, approximately 0.1 volts / mm to approximately 1,000 volts / mm, approximately 0.1 volts / mm to approximately 5,000 volts / mm, approximately 0.1 volts / mm to approximately 10,000 volts / mm, approximately 0.1 volts / mm to approximately 50,000 volts / mm, approximately 0.5 volts / mm to approximately 1 volt / mm, approximately 0.5 volts / mm to approximately 5 volts / mm, approximately 0.5 volts / mm to approximately 10 volts / mm, approximately 0.5 volts / mm to approximately 50 volts / mm, approximately 0.5 volts / mm to approximately 100 volts / mm, approximately 0.5 volts / mm to approximately 500 volts / mm, approximately 0.5 volts / mm to approximately 1.0 00 volts / mm, approximately 0.5 volts / mm to approximately 5,000 volts / mm, approximately 0.5 volts / mm to approximately 10,000 volts / mm, approximately 0.5 volts / mm to approximately 50,000 volts / mm, approximately 1 volt / mm to approximately 5 volts / mm, approximately 1 volt / mm to approximately 10 volts / mm, approximately 1 volt / mm to approximately 50 volts / mm, approximately 1 volt / mm to approximately 100 volts / mm, approximately 1 volt / mm to approximately 500 volts / mm, approximately 1 volt / mm to approximately 1,000 volts / mm, approximately 1 volt / mm to approximately 5, 000 volts / mm, approximately 1 volt / mm to approximately 10,000 volts / mm, approximately 1 volt / mm to approximately 50,000 volts / mm, approximately 5 volts / mm to approximately 10 volts / mm, approximately 5 volts / mm to approximately 50 volts / mm, approximately 5 volts / mm to approximately 100 volts / mm, approximately 5 volts / mm to approximately 500 volts / mm, approximately 5 volts / mm to approximately 1,000 volts / mm, approximately 5 volts / mm to approximately 5,000 volts / mm, approximately 5 volts / mm to approximately 10,000 volts / mm, approximately 5 volts / mm to approximately 50,000 volts / mm, approximately 10 volts / mm to approximately 50 volts / mm, approximately 10 volts / mm to approximately 100 volts / mm, approximately 10 volts / mm to approximately 500 volts / mm, approximately 10 volts / mm to approximately 1,000 volts / mm, approximately 10 volts / mm to approximately 5,000 volts / mm, approximately 10 volts / mm to approximately 10,000 volts / mm, approximately 10 volts / mm to approximately 50,000 volts / mm, approximately 50 volts / mm to approximately 100 volts / mm, approximately 50 volts / mm to approximately 500 volts / mm, approximately 50 volts / mm to approximately 1,000 volts / mm, approximately 50 volts / mm to approximately 5,000 volts / mm, approximately 50 volts / mm to approximately 10,000 volts / mm, approximately 50 volts / mm to approximately 50,000 volts / mm, approximately 100 volts / mm to approximately 500 volts / mm, approximately 100 volts / mm to approximately 1,000 volts / m m, approximately 100 volts / mm to approximately 5,000 volts / mm, approximately 100 volts / mm to approximately 10,000 volts / mm, approximately 100 volts / mm to approximately 50,000 volts / mm, approximately 500 volts / mm to approximately 1,000 volts / mm, approximately 500 volts / mm to approximately 5,000 volts / mm, approximately 500 volts / mm to approximately 10,000 volts / mm, approximately 500 volts / mm to approximately 50,000 volts / mm , including electric field strengths of approximately 1,000 volts / mm to approximately 5,000 volts / mm, approximately 1,000 volts / mm to approximately 10,000 volts / mm, approximately 1,000 volts / mm to approximately 50,000 volts / mm, approximately 5,000 volts / mm to approximately 10,000 volts / mm, approximately 5,000 volts / mm to approximately 50,000 volts / mm, or approximately 10,000 volts / mm to approximately 50,000 volts / mm. In some cases, the electric field generated by the electrodes for nanoelectroporation includes approximately 0.1 volts / mm, approximately 0.5 volts / mm, approximately 1 volt / mm, approximately 5 volts / mm, approximately 10 volts / mm, approximately 500 volts / mm, approximately 1,000 volts / mm, approximately 5,000 volts / mm, approximately 10,000 volts / mm, or approximately 50,This includes an electric field strength of 000 volts / mm. In some cases, the electric field generated by the electrodes for nanoelectroporation includes electric field strengths of at least about 0.1 volts / mm, about 0.5 volts / mm, about 1 volt / mm, about 5 volts / mm, about 10 volts / mm, about 50 volts / mm, about 100 volts / mm, about 500 volts / mm, about 1,000 volts / mm, about 5,000 volts / mm, or about 10,000 volts / mm. In some cases, the electric field generated by the electrodes for nanoelectroporation includes electric field strengths of up to about 0.5 volts / mm, about 1 volt / mm, about 5 volts / mm, about 10 volts / mm, about 50 volts / mm, about 100 volts / mm, about 500 volts / mm, about 1,000 volts / mm, about 5,000 volts / mm, about 10,000 volts / mm, or about 50,000 volts / mm.

[0152] In some cases, the electric field generated by the electrodes for nanoelectroporation consists of multiple pulses with pulse durations ranging from approximately 0.01 milliseconds / pulse to approximately 5,000 milliseconds / pulse. In some cases, the electric field generated by the electrodes for nanoelectroporation consists of pulses ranging from approximately 0.01 milliseconds / pulse to approximately 0.05 milliseconds / pulse, approximately 0.01 milliseconds / pulse to approximately 0.1 milliseconds / pulse, approximately 0.01 milliseconds / pulse to approximately 0.5 milliseconds / pulse, approximately 0.01 milliseconds / pulse to approximately 1 millisecond / pulse, approximately 0.01 milliseconds / pulse to approximately 5 milliseconds / pulse, approximately 0.01 milliseconds / pulse to approximately 10 milliseconds / pulse, approximately 0.01 milliseconds / pulse to approximately 50 milliseconds / pulse, and approximately 0.01 milliseconds / pulse to approximately 50 milliseconds / pulse. Approximately 100 milliseconds / pulse, approximately 0.01 milliseconds / pulse to approximately 500 milliseconds / pulse, approximately 0.01 milliseconds / pulse to approximately 1,000 milliseconds / pulse, approximately 0.01 milliseconds / pulse to approximately 5,000 milliseconds / pulse, approximately 0.05 milliseconds / pulse to approximately 0.1 milliseconds / pulse, approximately 0.05 milliseconds / pulse to approximately 0.5 milliseconds / pulse, approximately 0.05 milliseconds / pulse to approximately 1 millisecond / pulse, approximately 0.05 milliseconds / pulse to approximately 5 milliseconds / pulse, approximately 0.05 milliseconds / pulse to approximately 10 milliseconds / pulse, approximately 0.05 milliseconds / pulse ~Approximately 50 milliseconds / pulse, approximately 0.05 milliseconds / pulse ~Approximately 100 milliseconds / pulse, approximately 0.05 milliseconds / pulse ~Approximately 500 milliseconds / pulse, approximately 0.05 milliseconds / pulse ~Approximately 1,000 milliseconds / pulse, approximately 0.05 milliseconds / pulse ~Approximately 5,000 milliseconds / pulse, approximately 0.1 milliseconds / pulse ~Approximately 0.5 milliseconds / pulse, approximately 0.1 milliseconds / pulse ~Approximately 1 millisecond / pulse, approximately 0.1 milliseconds / pulse ~Approximately 50 milliseconds / pulse, approximately 0.1 milliseconds / pulse ~Approximately 10 milliseconds / pulse, approximately 0.1 milliseconds / pulse ~Approximately 50 milliseconds / pulse milliseconds / pulse, approximately 0.1 milliseconds / pulse to approximately 100 milliseconds / pulse, approximately 0.1 milliseconds / pulse to approximately 500 milliseconds / pulse, approximately 0.1 milliseconds / pulse to approximately 1,000 milliseconds / pulse, approximately 0.1 milliseconds / pulse to approximately 5,000 milliseconds / pulse, approximately 0.5 milliseconds / pulse to approximately 1 millisecond / pulse, approximately 0.5 milliseconds / pulse to approximately 5 milliseconds / pulse, approximately 0.5 milliseconds / pulse to approximately 10 milliseconds / pulse, approximately 0.5 milliseconds / pulse to approximately 50 milliseconds / pulse, approximately 0.5 milliseconds / pulse to approximately 100 milliseconds / pulse, approximately 0.5 milliseconds / pulse to approximately 500 milliseconds / pulse, approximately 0.5 milliseconds / pulse to approximately 1,000 milliseconds / pulse, approximately 0.5 milliseconds / pulse to approximately 5,000 milliseconds / pulse, approximately 1 millisecond / pulse to approximately 5 milliseconds / pulse, approximately 1 millisecond / pulse to approximately 10 milliseconds / pulse, approximately 1 millisecond / pulse to approximately 50 milliseconds / pulse, approximately 1 millisecond / pulse to approximately 100 milliseconds / pulse, approximately 1 millisecond / pulse to approximately 500 milliseconds / pulse, approximately 1 millisecond / pulse to approximately 1,000 milliseconds / pulse, approximately 1 millisecond / pulse to approximately 5,000 milliseconds / pulse, approximately 5 milliseconds / pulse to approximately 10 milliseconds / pulse, approximately 5 milliseconds / pulse to approximately 50 milliseconds / pulse, approximately 5 milliseconds / pulse to approximately 100 milliseconds / pulse, approximately 5 milliseconds / pulse to approximately 500 milliseconds / pulse, approximately 5 milliseconds / pulse to approximately 1,000 milliseconds / pulse, approximately 5 milliseconds / pulse to approximately 5,000 milliseconds / pulse, approximately 10 milliseconds / pulse to approximately 50 milliseconds / pulse seconds / pulse, approximately 10 milliseconds / pulse to approximately 100 milliseconds / pulse, approximately 10 milliseconds / pulse to approximately 500 milliseconds / pulse, approximately 10 milliseconds / pulse to approximately 1,000 milliseconds / pulse, approximately 10 milliseconds / pulse to approximately 5,000 milliseconds / pulse, approximately 50 milliseconds / pulse to approximately 100 milliseconds / pulse, approximately 50 milliseconds / pulse to approximately 500 milliseconds / pulse, approximately 50 milliseconds / pulse to approximately 1,000 milliseconds / pulse, approximately 50 milliseconds / pulse to approximately 5,000 This includes multiple pulses having pulse durations of 0 milliseconds / pulse, approximately 100 milliseconds / pulse to approximately 500 milliseconds / pulse, approximately 100 milliseconds / pulse to approximately 1,000 milliseconds / pulse, approximately 100 milliseconds / pulse to approximately 5,000 milliseconds / pulse, approximately 500 milliseconds / pulse to approximately 1,000 milliseconds / pulse, approximately 500 milliseconds / pulse to approximately 5,000 milliseconds / pulse, or approximately 1,000 milliseconds / pulse to approximately 5,000 milliseconds / pulse. In some cases, the electric field generated by the electrode for nanoelectroporation is approximately 0.01 milliseconds / pulse, approximately 0.05 milliseconds / pulse, approximately 0.1 milliseconds / pulse, approximately 0.The electric field generated by the electrodes for nanoelectroporation includes multiple pulses having pulse durations of at least approximately 0.01 milliseconds / pulse, approximately 0.05 milliseconds / pulse, approximately 0.1 milliseconds / pulse, approximately 0.5 milliseconds / pulse, approximately 1 milliseconds / pulse, approximately 10 milliseconds / pulse, approximately 500 milliseconds / pulse, approximately 1,000 milliseconds / pulse, or approximately 5,000 milliseconds / pulse. In some cases, the electric field generated by the electrodes for nanoelectroporation involves multiple pulses with pulse durations of up to approximately 0.05 milliseconds / pulse, 0.1 milliseconds / pulse, 0.5 milliseconds / pulse, 1 millisecond / pulse, 5 milliseconds / pulse, 10 milliseconds / pulse, 50 milliseconds / pulse, 100 milliseconds / pulse, 500 milliseconds / pulse, 1,000 milliseconds / pulse, or 5,000 milliseconds / pulse. In some cases, nanoelectroporation involves 1 pulse, 2 pulses, 3 pulses, 4 pulses, 5 pulses, 6 pulses, 7 pulses, 8 pulses, 9 pulses, 10 pulses, 11 pulses, 12 pulses, 13 pulses, 14 pulses, 15 pulses, 16 pulses, 17 pulses, 18 pulses, 19 pulses, 20 pulses, or more.

[0153] In some cases, methods and systems for producing extracellular vesicles containing adapter polypeptides and therapeutic polynucleotides involve loading multiple heterogeneous polynucleotides (such as vectors) into nanochannels to be nanoelectroporated into cells. In some cases, molecules other than polynucleotides (e.g., proteins, biomolecules, compounds, etc.) can be loaded into nanochannels and nanoelectroporated into cells. In some cases, electric fields generated by upper and lower electrodes accelerate the vector (e.g., plasmid) into the cell. In some cases, the electric field generated for nanoelectroporation creates pores in the cell membrane, enabling the nanoelectroporation of the vector (e.g., plasmid). In some cases, pores in the membrane of the extracellular vesicle donor cell may be formed at a focus, e.g., at the exit of the nanochannel where the electric field is in direct contact with the cell membrane.

[0154] In some cases, nanoelectroporated extracellular vesicle donor cells may produce and secrete at least 10%, 50%, 1x, 5x, 10x, 50x, 100x, 500x, 1000x, 5000x, or more extracellular vesicles than extracellular vesicle donor cells transfected by non-nanoelectroporation (e.g., conventional bulk electroporation, gene guns, lipofectamine transfection).

[0155] In some cases, extracellular vesicles produced and secreted by nanoelectroporated extracellular vesicle donor cells contain at least 50%, 1x, 2x, 5x, 100x, 500x, 1000x, or more therapeutic polynucleotides compared to extracellular vesicles produced and secreted by non-nanoelectroporated extracellular vesicle donor cells. In some cases, therapeutic polynucleotides encapsulated by extracellular vesicles produced and secreted by nanoelectroporated extracellular vesicle donor cells are at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or more likely to be intact for encoding therapeutic polypeptides than therapeutic polynucleotides encapsulated by extracellular vesicles produced and secreted by non-nanoelectroporated extracellular vesicle donor cells.

[0156] vaccine Compositions comprising extracellular vesicles as described herein are described herein. In some cases, the extracellular vesicles comprise at least one adapter polypeptide comprising a peptide sequence that is 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% identical to the peptide sequence of any one of the Fc receptors described herein. In some cases, at least one adapter polypeptide comprises a peptide sequence that is 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% identical to the peptide sequence of any one of the Fc receptors FcγRI(CD64), FcγRII(CD32), or FcγRIII(CD16). In some cases, at least one adapter polypeptide comprises a peptide sequence that is 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% identical to the peptide sequence of the Fc receptor FcγRI(CD64). In some cases, the extracellular vesicle contains an antibody complexed with an adapter polypeptide. In some cases, the antibody binds to a primary cell surface marker of an immune cell. In some cases, the extracellular vesicle further contains at least one viral-mimetic peptide. At least one viral-mimetic peptide can trigger an immune response that results in adaptive immunity against the virus from which the viral-mimetic peptide originates. In some cases, the viral-mimetic peptide is bound to the extracellular domain of the adapter polypeptide.

[0157] In some cases, the adapter polypeptide contains a targeting domain bound to the extracellular domain of the adapter polypeptide. In some cases, the targeting domain binds to a second cell surface marker associated with the same immune cell. In some examples, the immune cells are myeloid cells, T cells, e.g., αβ cytotoxic T cells, γδ T cells, regulatory T cells, natural killer T cells, B cells, helper T cells, macrophages, mast cells, phagocytes, lymphoid cells, granulocytes, macrophages, or dendritic cells. In some cases, the immune cells are T cells, B cells, dendritic cells, macrophages, or natural killer (NK) cells.

[0158] In some cases, the first cell surface marker is C5aR, CD10, CD107, CD11, CD117, CD123, CD125, CD135, CD138 / Syndecan-1, CD14, CD16, CD163, CD18, CD19, CD193, CD20, CD203, CD206, CD21, CD22, CD23, CD235, CD25, CD3, CD32, CD33, CD34, CD36, CD38, CD4, CD41, CD42, CD44, CD45, CD45R, CD45RA, CD4 9, CD55, CD56, CD61, CD65, CD68, CD7, CD71, CD8, CD9, CD90 / Thy1, CD94, clusterin, CXCR3B specific, F4 / 80, FcεRI, glycophorin A, GP9, GZMB, HBE1 specific, HLA-DR, IL3Rα, integrin α-4, integrin β-1, integrin β-3, LILRA4, NKp4, P-selectin, Siglec-8, or VEGFR-1 / FLT-1. In some cases, the first cell surface marker includes LILRA 4, CD3, CD19, CD20, or CD28.

[0159] In some cases, the antibody complexed with at least one adapter polypeptide is a monoclonal antibody. In some cases, the antibody is a humanized antibody. In some cases, the antibody is a humanized monoclonal antibody. In some cases, the antibody is IgG. In some cases, the antibody is IgG1 or IgG3. In some cases, the antibody contains an Fc region that complexes with an adapter polypeptide containing an Fc receptor. In some cases, the antibody complexes with the adapter polypeptide noncovalently.

[0160] In some cases, at least one virus-mimicking peptide is expressed on the extracellular surface of the extracellular vesicle. In some cases, at least one virus-mimicking peptide is partially inserted into the membrane of the extracellular vesicle. In some cases, at least one virus-mimicking peptide is bound to the extracellular domain of the adapter polypeptide. In some cases, at least one virus-mimicking peptide and both the targeting domain and the targeting domain are bound to the same extracellular domain of the adapter polypeptide. In some cases, at least one virus-mimicking peptide is bound to the extracellular domain of the first adapter polypeptide, while the targeting domain is bound to a separate extracellular domain of the second adapter polypeptide.

[0161] In some cases, viral mimetic peptides are derived from the viral proteins of the virus. Viruses can be DNA viruses or RNA viruses. DNA viruses can be single-stranded (ss)DNA viruses, double-stranded (ds)DNA viruses, or DNA viruses containing both ss and ds DNA regions. RNA viruses can be single-stranded (ss)RNA viruses or double-stranded (ds)RNA viruses. ssRNA viruses can be further classified into positive-sense RNA viruses or negative-sense RNA viruses.

[0162] In some cases, the viral mimetic peptide is derived from a coronavirus protein of the Coronaviridae family. The Coronaviridae family may include alpha-coronavirus, beta-coronavirus, delta-coronavirus, or gamma-coronavirus. In some cases, coronaviruses include MERS-CoV, SARS-CoV, or SARS-CoV-2. In some cases, the coronavirus protein is a protein of the SARS-CoV-2 virus. In some cases, the viral mimetic peptide is derived from a viral protein encoded by the nucleic acid sequence provided in Sequence ID No. 1. In some cases, the viral mimetic peptides are derived from SARS-CoV-2 viral proteins and are selected from the group consisting of orf1a, orf1ab, spike protein (S protein), 3a, 3b, envelope protein (E protein), membrane protein (M protein), p6, 7a, 7b, 8b, 9b, nucleocapsid protein (N protein), orf14, nsp1 (leader protein), nsp2, nsp3, nsp4, nsp5 (3C-like proteinase), nsp6, nsp7, nsp8, nsp9, nsp10 (growth factor-like protein), nsp12 (RNA-dependent RNA polymerase or RdRp), nsp13 (RNA 5'-tryphosphatase), nsp14 (3'→5' exonuclease), nsp15 (endoRNAse), and nsp16 (2'-O-ribose-methyltransferase).

[0163] In some cases, the viral mimetic peptide is derived from a viral protein that is at least approximately 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to one of sequence numbers 2-5. In some cases, the viral mimetic peptide contains the peptide sequences of sequence numbers 6-10. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7]

[0164] Signal peptides (usually 16 - 30 amino acids long) that exist in front of the N-terminus of many precursor proteins can direct those proteins towards their intracellular fate in the process of maturation and secretory transport. In the case of the human transmembrane protein CD64 expressed in exosomes, the signal peptide can be MWFLTTLLLWVPVDG, amino acids 1 - 15 of UniProtKB_FCGR1_HUMAN (SEQ ID NO: 11). Similar signal peptides that can guide the expression of human target proteins on the exosome surface also include the following: LAMP1_amino acids 1 - 28: MAAPGSARRPLLLLLLLLLLGLMHCASA (SEQ ID NO: 12); LAMP 2_amino acids 1 - 28: MVCFRLFPVPGSGLVLVCLVLGAVRSYA (SEQ ID NO: 13); HLA-G_amino acids 1 - 24: MVVMAPRTLFLLLSGALTLTETWA (SEQ ID NO: 14); HLA-DRA_amino acids 1 - 25: MAISGVPVLGFFIIAVLMSAQESWA (SEQ ID NO: 15). The introduction of such signal peptide motifs can prevent the synthesized protein from being consumed in the cytosol and increase protein expression on the exosome surface.

[0165] In some cases, the composition containing extracellular vesicles can further contain an immunomodulatory agent or an adjuvant for enhancing the immune response induced by a virus-mimicking peptide that contacts immune cells. Exemplary immunomodulatory agents include pathogen-associated molecular pattern (PAMP) molecules, damage-associated molecular pattern (DAMP) molecules, Toll-like receptor agonists, STING agonists, RIG-I agonists, tumor necrosis factor (TNF) ligands, or cytokines (e.g., IL-2, IL-12, IL-15 or IL-21). Exemplary adjuvants include inorganic compounds (e.g., alum, aluminum hydroxide, aluminum phosphate, or calcium phosphate hydroxide), mineral oil, paraffin oil, peanut oil, bacterial products, such as inactivated Bordetella pertussis, non-bacterial organic substances, such as squalene, plant saponins, Freund's complete adjuvant, or Freund's incomplete adjuvant.

[0166] In some cases, a method of vaccinating a subject that requires vaccination is described herein, and the method includes administering a therapeutically effective amount of a pharmaceutical composition containing extracellular vesicles containing a virus-mimicking peptide. In some cases, the pharmaceutical composition containing a virus-mimicking peptide is administered to the subject at least once a day, at least once a week, at least once a month, at least once a year, or at least once for a period longer than one year.

[0167] If neutralizing antibodies against the virus-mimicking peptide are observed in the subject, administration can be stopped. Alternatively, a maintenance dose or a booster dose of the pharmaceutical composition containing extracellular vesicles containing a virus-mimicking peptide can be administered as needed. Thereafter, the dose or the frequency of administration, or both, can be decreased according to the level of neutralizing antibodies detected in the subject.

[0168] In some cases, methods for producing extracellular vesicles containing virus-mimicking peptides are described herein. In some cases, the methods involve introducing at least one heterologous polynucleotide into an extracellular vesicle donor cell. In some cases, the at least one heterologous polynucleotide is a vector (e.g., a plasmid). In some cases, the at least one heterologous polynucleotide introduced into the extracellular vesicle cell encodes at least one adapter polypeptide described herein. In some cases, the at least one heterologous polynucleotide encodes at least one targeting domain. In some cases, the at least one heterologous polynucleotide encodes a virus-mimicking peptide.

[0169] In some cases, heterologous polynucleotides can be introduced into cells via the use of expression vectors. In the context of expression vectors, the vectors can be readily introduced into cells as described herein by any method of the art. For example, expression vectors can be transferred into cells by the biological, chemical, or physical transfection methods described herein. In some cases, heterologous polynucleotides are transfected into extracellular donor cells by nanoelectroporation as described herein.

[0170] Pharmaceutical composition In some cases, extracellular vesicles can be formulated into pharmaceutical compositions. In some cases, pharmaceutical compositions containing extracellular vesicles contain at least one pharmaceutically acceptable excipient. In some cases, pharmaceutical compositions containing extracellular vesicles can be administered to a subject by multiple routes of administration, including but not limited to parenteral, oral, buccal, rectal, sublingual, or transdermal administration. In some cases, parenteral administration includes intravenous, subcutaneous, intramuscular, intracerebral, intranasal, intra-arterial, intra-articular, intradermal, intravitreous, intraosseous, intraperitoneal, or intrathecal administration. In some cases, pharmaceutical compositions are formulated for topical administration. In other cases, pharmaceutical compositions are formulated for systemic administration. In some cases, the pharmaceutical compositions and formulations described herein are administered to a subject by intravenous, subcutaneous, and intramuscular administration. In some cases, the pharmaceutical compositions and formulations described herein are administered to a subject by intravenous administration. In some cases, the pharmaceutical compositions and formulations described herein are administered to a subject by administration. In some cases, the pharmaceutical compositions and formulations described herein are administered to a subject by intramuscular administration.

[0171] Kits / Manufacturing Products In certain cases, kits and manufactured articles for use with one or more methods and compositions described herein are disclosed herein. Systems for producing extracellular vesicles described herein are also disclosed herein. In some cases, the system includes components for stimulating the production and secretion of extracellular vesicles by nano-electroporating extracellular vesicle donor cells, including adapter polypeptides and therapeutic agents described herein.

[0172] In some cases, a kit may include a carrier, package, or container that is compartmentalized to accept one or more containers, such as vials or tubes, each of which includes one of the distinct elements used in the manner described herein. Suitable containers include, for example, bottles, vials, syringes, and test tubes. In some cases, the containers may be formed from a variety of materials, such as glass or plastic. A kit typically includes a label listing the contents and / or instructions for use, and an accompanying document containing instructions for use. A set of instructions may also be included.

[0173] In one embodiment, the label is on the container or attached to the container. In one embodiment, the label is on the container when the letters, numbers, or other characters forming the label are attached to the container itself, molded, or etched, and the label is attached to the container, for example as an accompanying document, when the label is located within a receptacle or carrier that also holds the container. In one embodiment, the label is used to indicate that the contents are to be used for a particular therapeutic application. The label may also indicate instructions for the use of the contents, such as in the methods described herein.

[0174] In certain cases, extracellular vesicles containing adapter polypeptides and therapeutic agents may be provided in packs or dispenser devices containing one or more unit dosage forms containing the compounds provided herein. In certain cases, extracellular vesicles containing adapter polypeptides and therapeutic agents conjugated with any of the antibodies described herein may be presented in packs or dispenser devices containing one or more unit dosage forms containing the compounds provided herein. The packs include, for example, metal or plastic foil such as blister packs. In one embodiment, the packs or dispenser devices are accompanied by instructions for administration. In one embodiment, the packs or dispensers are also accompanied by notices relating to the form of container prescribed by government agencies that regulate the manufacture, use or sale of pharmaceuticals, the notices reflecting agency approval of the form of the drug for human or veterinary administration. Such notices are, for example, labels approved for the drug by the U.S. Food and Drug Administration, or approved product inserts. In one embodiment, extracellular vesicles containing adapter polypeptides and therapeutic conjugates provided herein, formulated on a suitable pharmaceutical carrier, are also prepared, placed in appropriate containers, and labeled for the treatment of the indicated condition. In some cases, the kit includes manufacturing articles useful for developing vaccines, therapeutics, adoptive therapies, and treatment methods described herein. [Examples]

[0175] The following illustrative examples represent, and are not intended to limit, any aspects of the stimuli, systems, and methods described herein.

[0176] Example 1. Design and testing of THP-CD64 plasmid DNA A novel platform for antibody-peptide-linked extracellular vesicles ("EVs"), particularly exosomes, was established by transfecting donor cells with plasmid DNA expressing CD64, CD64 peptide, or other Fc receptors on the surface of EVs and exosomes. CD64, also known as Fc-γ receptor 1 (FcγR1), is a dissociation constant K at nanomolar (nM) levels that hinges the Fc region of IgG1 and IgG3 via its extracellular D1 and D2 domains. d They bind with high affinity. Furthermore, plasmid DNA was co-transfected into donor cells to allow endogenous RNA and proteins to be added to EVs and exosomes to function as therapeutic agents. These therapeutic EVs (tEVs) and exosomes (tExos) functioned as targeted drug delivery vehicles to cancer cells and tumors, non-cancerous lesions, and damaged tissues. They were also designed for vaccine development and other medical procedures.

[0177] Tumor-homing peptides ("THP") designed to encode a FLAG tag, CKAAKN (CK), CREKA (CR), or ARRPKLD (AR) were added to the N-terminus of CD64, and various humanized monoclonal antibodies (mAbs) were conjugated to the D1 and D2 domains of CD64 to achieve dual targeting capabilities as shown in Figure 1. Extracellular vesicles ("EVs") containing CD64 or THP-CD64 were generated by transfecting donor cells with human CD64 plasmid DNA or human THP-CD64 plasmid DNA expressing either human CD64 or human THP-CD64 on the surface of EVs (e.g., exosomes) secreted from transfected donor cells. CD64 functioned as a biological anchor for binding to humanized monoclonal antibodies ("hmAbs"). The extracellular D1 and D2 domains of human CD64 exhibited high affinity to the lower hinge region of the Fc of human IgG1, e.g., at nanomolar concentration levels, and the dissociation constant (K) dThe hmAb was bound to the CD64 N-terminus. In addition to the specific recognition ability of the bound hmAb, targeting by the small tumor-homing peptide (THP) was also engineered to target the N-terminus of CD64. Dual targeting of both hmAb and THP on the EV (or exosome) surface enhanced the targeting of EV (or exosome) delivery to tumors and other lesions in vivo.

[0178] Plasmids were constructed using vectors containing genes for ampicillin resistance (AmpR) and EGFR markers for transformation and transfection, respectively. Functional CD64 was encoded by the CD64 coding sequence (CD64_CDS) driven by the EF1 promoter (Figure 2A). CD64_CDS (355 amino acids) consists of (i) a signal peptide (SP), (ii) extracellular (D1, D2, and D3) domains, (iii) a transmembrane (TM) domain, and (iv) an intracellular (IC) domain. THP was inserted into the gap between the signal peptide and the extracellular D1 domain and expressed at the N-terminus of CD64 (Figure 2B). THP was ligated to the N-terminus of the extracellular D1 domain by a Flag(DYKDDDK) linker, restricting conformational blocking on the Fc-binding region of the D1-D2 hinge of CD64 (Figure 2C). The peptide and nucleotide sequences of THP:Flag_control, CKAAKN(CK), CREKA(CR), and ARRPKLD(AR) are listed in Figure 2D.

[0179] To investigate whether tumor-homing peptides ("THPs") linked to the N-terminus of CD64 alter the interaction between CD64 and human immunoglobulin G (hIgG), engineered CD64 proteins with different THPs were purified and reacted with immobilized hIgG (coated on a 96-well plate) for sandwich enzyme-coupled immunosorbent assay (ELISA) (Figure 3A). The bound CD64 proteins with different THPs were reacted with anti-CD64 / Flag and a second HRP antibody, followed by the ELISA substrate (tetramethylbenzidine). Absorbance at 450 nm revealed the titration concentration with the engineered CD64 protein. The binding titration curve was fitted to a monovalent model between CD64 and hIgG to determine the dissociation constant K d (OD=[B max ×Con] / [K d +Con](where OD is the optical concentration of absorbance, B max The K of hIgG and recombinant wild-type CD64 (wt_CD64) was determined. d (K d =0.0456nM, Figure 3B). Affinity index K between differently manipulated THP-CD64 proteins and hIgG. d Flag-CD64(K d =0.0536nM), CK-CD64(K d =0.0588nM), CR-CD64(K d =0.0658nM) and AR-CD64(K d The value was determined to be 0.0506 nM (Figure 3C). These results indicate that manipulated CD64 with different THPs (Flag, CK, CR, or AR) does not affect the high binding affinity to mAbs at the nM level compared to wt_CD64.

[0180] Example 2. Nanochannel electroporation (NEP) induced EV and exosome release from transfected cells. THP-CD64 containing EVs and exosomes was produced using a nanochannel electroporation ("NEP") system. Donor cells were cultured on the chip surface. After 1 day of culture, the plasmid, pre-loaded in the cargo chamber, was injected into individual cells via nanochannels using a 25-250V electric field (dependent on cell type and source) with 10 pulses at 0.1-second intervals and 10 ms / pulse. After cell transfection, the released EVs (including exosomes) containing functional RNA and surface receptors were purified from the culture medium recovered by centrifugation to remove cells and large cell debris, followed by tangential flow filtration (TFF).

[0181] Figure 4 shows the number of extracellular viable cells (EVs) and endogenous RNA content from NEP-transfected mouse embryonic fibroblasts (MEFs) with THP-CD64 and therapeutic RNA plasmids. After 24 hours of NEP treatment, cell culture medium was collected for EV purification and recovery by centrifugation and TFF. After purification, the manipulated EVs were further purified to high concentration in a volume of approximately 200 μL using a spin column. As shown in Figure 4A, the number of EVs in both the human THP-CD64 + human TP53 group and the human THP-CD64 + shKRAS G12D mutant group showed an approximately 10-fold increase after NEP treatment compared to the control group (i.e., no NEP treatment). RT-qPCR of TP53 mRNA expression in Figure 4B revealed that EVs produced by NEP contained a large amount of transcribed mRNA compared to the control group without NEP treatment, and were estimated to have increased approximately 6,000-fold based on a Ct value of 27.5 compared to undetermined at 40.

[0182] Purified exosomes containing manipulated THP-CD64 were captured by latex beads and incubated with anti-CD64-APC, anti-CD63-BV510, and FITC-conjugated hIgG for flow cytometry assays (Figure 5A). Surface expression profiling was performed using singlet beads and CD63 to determine the mean fluorescence intensity (MFI) of CD64 expression and hIgG affiliation, as shown in Figure 5B. + We followed a standard protocol for gated exosome populations. CD63 + Surface co-expression of CD64 within the exosome population was determined by MFI of FITC, and exosome expression of manipulated CD64 having either Flag, CK, CR, or AR THP was confirmed, as shown in Figure 5C. + Surface co-expression of hIgG and CD64 within the exosome population was determined by FITC MFI, and as shown in Figure 5D, high binding affinity of hIgG to exosomes expressing CD64 with either Flag, CK, CR, or AR THP was confirmed.

[0183] Example 3. Incorporation of THP-CD64-containing exosomes with or without hmAb into PANC-1 spheroids. Spheroids of the pancreatic cancer cell line PANC-1 were formed using a suspension method with cellulose and type 1 collagen and cultured for one week until they reached a diameter of approximately 500-600 μm, as shown in Figure 7A. Pancreatic cancer stem cells (CSCs) are generally defined by their surface expression of CD44 and CD24. Spheroids exceeding 400 μm in diameter develop a hypoxic core, and this hypoxic microenvironment activates survival signaling pathways and reprograms them to maintain cell viability. When PANC-1 cells are stably cultured in spheroids, CD44 + CD24 + The group gradually increased in size.

[0184] Purified EVs released from mouse embryonic fibroblast (MEF) cells after transfection with either Flag-CD64 or CK-CD64 plasmid DNA (CK-CD64) were formulated with either humanized anti-EGFR mAb (cetuximab) or hIgG. Cancer spheroids formed from the human pancreatic cancer cell line PANC-1 were treated with PKH67 (green)-labeled liposomes (lipofectamine 3000) or various EVs for 24 h, and then processed by fixation, permeabilization, and staining with anti-hIgG-TRITC (red) and DAPI (blue). Cross-sections of cancer spheroids were imaged under a confocal microscope. Treatment of cancer spheroids with various EVs all showed better spheroid uptake than commercially available lipofectamine 3000 based on fluorescence intensity and distribution. Among various EVs, dual-targeted exosomes (CK-CD64-Cet_Exo) revealed the highest spheroid uptake as shown in Fig. 6.

[0185] Various THP-CD64 with or without human monoclonal antibody (hmAb) To further evaluate the cellular uptake of EVs, the treated spheroids were dissociated into single cell suspensions and subpopulations were identified by CD24 and CD44 expression using flow cytometry as shown in Fig. 7B. CD24 low CD44 low or CD24 + CD44 + The mean fluorescence intensity of PKH67 measured in the subpopulations represents their EV uptake. Engineered EVs containing Flag-CD64, CK-CD64, CR-CD64, or AR-CD64 with humanized antibody affinity (cetuximab: anti-EGFR, atezolizumab: anti-PD-L1, or hIgG) all showed good cellular uptake for the subpopulations, particularly for CD44 + CD24 + as shown in Fig. 7C. Dual-targeted EVs using anti-hEGFR (cetuximab) and CK-CD64 provided the best cellular uptake for both PANC-1 cell subpopulations.

[0186] Example 4: Uptake of CK-CD64 / anti-ROR1-containing exosomes in PANC-1 spheroids and orthotopic mouse models Purified extracellular viable cells (EVs) released from mouse embryonic fibroblast (MEF) cells after transfection with either Flag-CD64 or CK-CD64 plasmid DNA (CK-CD64) were formulated with humanized anti-ROR1. Cancer spheroids formed from the human pancreatic cancer cell line PANC-1 were treated for 24 hours with PKH67 (green) labeled liposomes (lipofectamine 3000) or various EVs, and then treated with fixation, permeabilization, and staining using anti-hIgG-TRITC (red) and DAPI (blue). Cross-sections of the cancer spheroids were imaged under a confocal microscope. Among the various EVs, the dual-targeted exosome (CK-CD64-ROR1_Exo) demonstrated the highest spheroid uptake, as shown in Figure 8.

[0187] PANC-1 cells were transduced with GP and luciferase to track their localization in vivo. Four weeks after orthotopic xenotransplantation of PANC-1 cancer cells, various EVs were injected via tail vein into NOD scid gamma (NSG) mice. The in vivo distribution of EVs in the brain, heart, lungs, liver, spleen, kidneys, and pancreas 24 hours after EV delivery was examined by PKH26 staining of the EV lipid bilayer. EV concentrations were approximately 10E12 / 50 μL each, and the donor cells were MEFs. Figures 9 and 19A-B show that CK-CD64-ROR1_Exo revealed the highest EV accumulation in the pancreas. Co-localization of PKH26, GFP, and luciferase intensities reflected the precision of CK-CD64-ROR1_Exo delivered to pancreatic tumor lesions.

[0188] Figure 10 compares the mean EV uptake in the liver, spleen, and pancreas, as well as the EV distribution in tumor tissue, as determined by PKH staining. It is clear that CK-CD64-ROR1_Exo can provide superior pancreatic targeting and tumor tissue uptake of EVs using CK-CD64-ROR1 targeting.

[0189] Example 5. Design concept of a Vacosome for vaccine development Coronaviruses (CoVs) are associated with significant global health risks, as evidenced by the various epidemics observed in several subtypes, including SARS-CoV-2, the causative agent of the current COVID-19 pandemic. The spike (S) protein is an essential structural component of the viral envelope and a strategic target for vaccine development. Exosomes overexpressing various viral S protein fragments fused to CD64 on the exosome surface can function as vaccines (called "vacosomes") (Figure 11). Robust vaccination via the T cell receptor (TCR) complex can be achieved synergistically by vaccination with a peptide on the N-terminus of CD64 and co-stimulation with pre-loaded anti-αCD3 / CD28 mAb on the CD64 hinges D1-D2. The formation of immunological synapses between the manipulated CD64 and TCR can be confirmed by fluorescent tagging and T cell surface marker staining using a fluorescence-activated cell sorter (FACS). Similarly, simultaneous loading of mAbs targeting antigen-presenting cells (APCs), such as B cells (anti-αCD19 / CD20) and dendritic cells (DCs) (anti-αLILRA 4), should enhance the APC-T cell response. Five candidate fusion S protein fragments likely to function as COVID-19 vaccine peptides are selected based on epitope and structural prediction. They can be expressed on vacosomes generated via NEP-transfected donor cells such as human mesenchymal stem cells (MSCs) and DCs.

[0190] Example 6. Binding affinity strength of human immunoglobulin and classical Fc receptor. In addition to Fcγ-IgG binding, other human immunoglobulins and Fc receptors are present. Plasma membrane-embedded Fc receptors contain intracellular domains or subunits that can induce downstream activation or inhibition. IgG affinity variants are highlighted in Figure 12A for each human Fcγ receptor member, ranging from very high (dark orange), high (orange), moderate (yellow), low (light blue) to no binding (dark blue). FcRn receptors bind to IgG subclasses under acidic conditions (e.g., pH=6), but their binding ability decreases under physiological conditions at pH=7.4. Figure 12B shows that IgE has very high binding affinity to the FcεRI receptor but low affinity to the FcεRII receptor. IgA has low binding affinity to the FcαRI receptor.

[0191] Example 7. KRAS with optimized NEP G12D Construction of siRNA / CD64 and TP53 mRNA / CD64-targeted extracellular genes (tEVs). To design engineered EVs for efficient targeted delivery in PDAC, the dynamics of cellularly stimulated EV release and therapeutic agents in secreted EVs (Kras G12D Load profiles of specific shRNA and hTP53 mRNA, as well as CD64 protein expression on the EV surface, were investigated. As shown in Figure 13, EV secreted from MEFs increased significantly, peaking approximately 16 hours after NEP using a 1 μm pore Transwell at 150 V with 10 10 ms pulses, and then rapidly decreasing. However, TP53-mRNA expression levels rapidly peaked at approximately 4 and 8 hours, and then expression decreased to very low levels 12 hours after NEP. KRAS G12DThe expression trend of shRNA targeting TP53 (Figure 13D) was similar to that of the EV secretion profile, i.e., significantly increased and peaked at approximately 16 hours. Compared to the expression of EV secretion and nucleotides encapsulated in EVs over time, the CD64 protein on the EV surface peaked at approximately 24 hours post-NEP and was able to maintain high levels of expression over a long period post-NEP. The release profile is thought to produce EVs containing both the CD64 protein and the desired nucleotides within the EV. Due to the large peak time difference between TP53 expression and EV secretion, a sequential transfection approach of parental cells is employed. Three sequential transfection designs were compared in which the CD64 plasmid was transfected first, followed by delivery of the TP53 plasmid at time lags of 8, 16, and 24 hours. As shown in Figure 14, in the 8-hour case, there was a dramatic increase in EV secretion after the second cell stimulation, but TP53 mRNA expression within the EV was very low, which may be due to excessive cell stimulation within a short period leading to insufficient cell viability. In contrast, the 16-hour and 24-hour cases resulted in much higher TP53 mRNA expression. Between these, the 16-hour case exhibited both the highest TP53 mRNA expression and very high EV secretion. Therefore, TP53 mRNA / CD64 EV was produced by sequential NEP using a TP53 plasmid delivered 16 hours after the initial transfection of the CD64 plasmid. (KRAS) G12D Regarding shRNA / CD64 EV, CD64 and KRAS in NEP G12D Simultaneous delivery of both shRNA plasmids worked well.

[0192] Example 8. Characterization of as-prepared targeted EV (tEV) qNANO, SEM, cryo-TEM, Western blotting, and immunolipoplex nanoparticle (ILN) biochip assays were performed to observe and characterize the generated tEVs. The qNANO results (Figure 15A) show that the average diameter of the blank EVs was approximately 110 nm, while EVs without cargo (PBS only) and EVs with therapeutic agents (KRAS) were also observed.G12D Engineered vesicles (tEVs), represented by shRNA / CD64 EVs, exhibited larger diameters, suggesting the encapsulation of large amounts of nucleotides and other biomolecules within the EVs. Figure 15B shows that typical exosomal proteins such as CD63, CD9, and TSG101 are highly expressed in the as-prepared EVs. As shown in SEM and cryo-TEM images (Figures 15C–15D), these engineered vesicles remained spherical. To confirm the encapsulated nucleic acids, surface proteins, and their co-localization within the tEVs, an ILN biochip on a TIRFM microscope was used for the capture and detection of single EVs. Calculated TIRFM results (Figures 15E–15F) indicate the encapsulated nucleotides (TP53 mRNA and Kras) within the tEVs. G12D The co-localization rates of shRNA and CD64-CK surface protein were 51.19% and 58.31%, respectively.

[0193] Example 9. Sequential Transwell electroporation (sTEP) protocol Mouse embryonic fibroblast (MEF) cells, human bone marrow-derived stem cells (hBMSCs), and other cell types can be used for therapeutic extracellular matrix (tEV) production. Here, we will explain using MEF as an example.

[0194] Mouse embryonic fibroblast (MEF) cells (obtained from Millipore Sigma) are used to generate cell clones for therapeutic extracellular life (tEV) production. In conventional cell culture using tissue culture flasks (Fisherbrand, catalog no. FB 012937), MEF cells are maintained in Dulbecco's Minimum Essential Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 100 U / mL penicillin / streptomycin, and incubated at 37°C in 5% CO2. MEF cells are seeded into Transwell electroporation (TEP) inserts (e.g., 12 mm) at a rate of 200,000–300,000 cells per insert. To grow the cells to 80% confluence, wash the cells three times with 1× DPBS and replace with serum-free medium for TEP treatment. The cells are then treated with TEP using an electroporation system (Bio-Rad Gene Pulser Xcell) with constant electroporation parameters. For optimization, the amount of EV release induced by TEP, therapeutic agents (CD64 protein, KRAS) G12D Load profiles of siRNA and hTP53 mRNA can be screened over time using the hCD64 ELISA kit (Biocompare) and qRT-PCR. Cell culture conditioned medium (CCM) is collected over time after TEP treatment and centrifuged at 200×g for 5 minutes to remove cells and debris. The qNANO system (Izon Science) counts nanoparticles in the 50-330 nm range in the CCM solution using an adjustable resistance pulse sensing (TRPS) method. To measure CD64 protein, EVs are lysed with RIPA buffer (Thermo Scientific® 89900) and the hCD64 ELISA kit (Biocompare), and then used according to the manufacturing protocol. TP53 mRNA and KRAS in EVs are also measured. G12DTo measure the expression level of siRNA targeting TP53, EVs are purified from cell culture medium using a total exosome isolation (TEI) kit (Invitrogen). Then, total RNA is isolated using an RNA purification kit (Norgen). Human TP53 mRNA and KRAS G12D The relative expression level of siRNA was measured by RT-PCR, followed by 2 △△Ct KRAS can be calculated using the Livak method (see qRT-PCR for tEV RNA below). G12D shRNA / CD64 therapeutic EVs are recovered after a single TEP treatment, while TP53 mRNA / CD64 therapeutic EVs are recovered after consecutive TEP treatments. KRAS G12D For shRNA / CD64 therapeutic EVs, KRAS G12D Cells treated with a single TEP with a mixture of shRNA and CD64 plasmids are incubated in serum-free medium for 16 hours. For TP53 mRNA / CD64 therapeutic EVs, cells are first treated with TEP with the CD64 plasmid and incubated for 16 hours. Then, cells are treated with TEP with the TP53 mRNA plasmid and incubated for another 16 hours in serum-free medium. The cell culture medium (CCM) is collected accordingly and centrifuged at 200×g for 5 minutes to remove cells and debris. The cell-removed CCM is centrifuged at 2000×g for 30 minutes to remove cell debris. The cell-removed CCM is transferred to a new tube without disturbing the pellet. The CCM is stored at 4°C for isolation and characterization of downstream tEVs, or frozen and stored at -80°C.

[0195] qRT-PCR of tEV RNA. Centrifuge the cell medium at 2000×g for 30 minutes to remove cells and debris. Transfer the supernatant containing the cell-free culture medium to a new tube without disturbing the pellet. After NTA measurement of the electroporated cell culture medium, purify the sample using the whole exosome isolation reagent (from cell culture medium, Invitrogen, catalog no. 4478359). If the EV concentration from the NTA data is less than 2e9 / mL, purify the sample using the whole exosome isolation reagent (from serum, Invitrogen, catalog no.) (proceed to the step below where the supernatant is discarded using a pipette). Transfer the required volume (1 mL) of cell-free culture medium to a new tube and add 0.5 volume (0.5 mL) of the whole exosome isolation (TEI from cell culture medium) reagent. Thoroughly mix the culture medium / reagent mixture by vortexing until a homogeneous solution is present and then aspirating and discharging with a pipette. Incubate the sample overnight at 4°C. After incubation, centrifuge the sample at 10,000 × g for 1 hour at 4°C. Discard the supernatant using a pipette. EV is contained in the pellet at the bottom of the tube (usually invisible). Resuspend the pellet in a convenient volume (100 μL) of RNase-free 1 × DPBS by vortexing until the solution becomes clear again and aspirating with a pipette. If the starting volume is 2 mL of culture medium, return the resuspended solution (100 μL) to another tube using the pellet. For EV RNA extraction, use the Plasma / Serum RNA Purification Mini Kit (Norgen Biotek, catalog number 55000). Warm lysis buffer A at 60°C for 20 minutes, and if precipitate is present, mix thoroughly until the solution becomes clear again. Place 100 μL of TEI reagent-treated sample into a 1.5 mL tube and add 300 μL of lysis buffer A. Mix thoroughly by vortexing for 10 seconds. Add 400 μL of 100% ethanol (200 proof). Mix thoroughly by vortexing for 10 seconds. Transfer the 400 μL mixture from the step of placing 100 μL of TEI reagent-treated sample into the microspin column. Centrifuge the mixture at 3,300 × g and room temperature (RT) for 2 minutes.Discard the flow-through and reassemble the spin column with its recovery tube. Add 400 μL of 100% ethanol, mix, and repeat the step of transferring the sample mixture to the spin column again. Apply 400 μL of washing solution A to the column and centrifuge at 3,300 × g and RT for 1 minute. Discard the flow-through and reassemble the spin column with its recovery tube. Discard the flow-through, reassemble, and repeat the step of adding ethanol two more times for a total of three washes. Rotate the empty column at 13,000 × g and RT for 2 minutes and discard the recovery tube. Transfer the spin column to a new 1.7 mL elution tube. Apply 12.5 μL of elution solution A to the column and let stand at room temperature for 2 minutes. Centrifuge the spin column using the elution tube at 400 × g for 1 minute, followed by 5,800 × g for 2 minutes. For maximum recovery, return the eluted buffer to the spin column and let stand at room temperature for 2 minutes. Centrifuge again at 400×g for 1 minute, then centrifuge again at 5,800×g for 2 minutes. Use a NanoDrop 2000C spectrophotometer (Thermo Scientific) to measure the concentration of the extracted total RNA solution (approximately 10 μL). A measurement volume of 1 μL relative to the nucleic acid aqueous solution is recommended. Select RNA mode (RNA-40) to measure the total RNA concentration. Raise the sampling arm and pipette 1× DPBS onto the lower measurement pedestal. Lower the sampling arm and start the spectral measurement using the software on the PC. Click on the blank to measure and save the reference spectrum. Analyze a new copy of the blank in the same way as the sample by selecting the measurement. The result should be a spectrum that changes below 0.04 A (equivalent to absorbance at 10 mm). Raise the sampling arm and pipette the sample onto the lower measurement pedestal. Lower the sampling arm and start the spectral measurement using the software on the PC. Once the measurement is complete, raise the sampling arm and wipe the sample from both the upper and lower pedestals using a lint-free, dry laboratory wipe. Verify the absorbance ratio of 260 / 280 at 260 nm and 280 nm. Use the absorbance ratio at 260 nm and 280 nm to assess the purity of DNA and RNA.A ratio of approximately 1.8 is generally considered "pure" for DNA, and a ratio of approximately 2.0 is generally considered "pure" for RNA. In either case, a significantly low ratio may indicate the presence of proteins, phenols, or other contaminants that strongly absorb at or around 280 nm. Check the absorbance ratio at 260 nm and 230 nm (260 / 230). This is a secondary measure of nucleic acid purity. The 260 / 230 value for "pure" nucleic acids is often higher than the respective 260 / 280 values, generally in the range of 1.8 to 2.2. A significantly low ratio may indicate the presence of co-purified contaminants. Adjust the sample volume from the total RNA concentration for normalization of RNA samples before RT-PCR. Do not exceed 1000 ng of total RNA (100 ng / μL in 10 μL). If using positive and negative reverse transcriptase (RNA) samples for amplification, prepare double-tube sets. Add the following to a 0.5 mL RNase-free microcentrifuge tube on ice: DNase I, amplification grade (Invitrogen, catalog no. 18-068-015); 1 μL of 10× DNase I reaction buffer; 1 μL of DNase I, amplification grade, 1 U / μL; use 1–8 μL of extracted total RNA sample with a volume adjusted for total RNA normalization. Typically, 100 ng of total RNA is added to 5 μL of 20 ng / μL DEPC-treated RNase-free water up to 10 μL. Incubate the tube at room temperature for 15 minutes. Inactivate DNase I by adding 1 μL of 25 mM EDTA solution to the reaction mixture. Heat at 65°C for 10 minutes. The RNA sample is ready for use in reverse transcription before PCR amplification. For the reverse transcription (RT) process, use a high-volume cDNA reverse transcription kit containing an RNase inhibitor (Applied Biosystems, catalog no. 43-749-66).Prepare a 2× reverse transcription master mix by quantitatively converting up to 2 μg (for a 20 μL reaction) of total RNA to cDNA: 2.0 μL of 10× RT buffer; 0.8 μL of 25× dNTP mixture (100 mM); 2.0 μL of 10× RT oligo(dT) primer or random primer; 1.0 μL of RNase inhibitor; 1.0 μL of MultiScribe® reverse transcriptase; 3.2 μL of nuclease-free H2O; and 10 μL of DNase-treated total RNA to 10 μL of the 2× RT master mix to produce 20 μL of 1× mix. Vortex briefly to mix. Centrifuge briefly to transfer the reaction mixture to the bottom of the tube and remove air bubbles. Reverse transcription is performed in a thermal cycler for 2 hours and 15 minutes using the oligo(dT) or random hexamer method. Prepare the reaction mixture for the qRT-PCR experiment: TaqMan® FastAdvanced Master Mix is ​​supplied at double the concentration and contains AmpliTaq® Fast DNA Polymerase; uracil-N glycosylase (UNG); dNTP with dUTP; ROX® dye (passive reference); and optimized buffer components. Keep the TaqMan® FastAdvanced Master Mix on ice. Thaw the TaqMan® assay on ice, then vortex, centrifuge briefly, and resuspend. Transfer an appropriate volume of the PCR reaction mixture to each well of an optical reaction plate (96-well plate). 10 μL of 2× master mix (Applied Biosystems, TaqMan Fast Advanced Master Mix, catalog no. 4444557); 1 μL of 20× TaqMan assay mix mixed with target probes and primers for the TP53, VEGFA, and COL1A1 genes; 2 μL of cDNA template (20 ng in 2 μL of 10 ng / μL in the RT sample); 7 μL of nuclease-free H2O. Seal the reaction plate with optical adhesive film, then briefly centrifuge to transfer the PCR reaction mixture to the bottom of the wells and remove air bubbles. Open the plate document or experiment file corresponding to the reaction plate in the system software. Load the reaction plate into the real-time PCR system.Start the qPCR reaction. After all reactions are complete, view the amplification plot of the reaction. Determine the threshold cycle (Ct) of the amplification curve using automatic baseline and automatic threshold setting. Use the comparative Ct (ΔCr) method with GAPDH reference to analyze the data. Analyze the qPCR data using the ΔΔCt method.

[0196] Example 10. Integration of enriched extracellular matrix (EV) into PANC-1 pancreatic cancer cells In addition to targeting, disclosed CD64-enriched EVs containing humanized monoclonal antibodies (hmAbs) and tissue-homing peptides (THPs) on the EV surface can substantially enhance intracellular integration and tissue permeability (including transcytosis). Due to the high surface expression of EGFR (epidermal growth factor receptor) and ROR1 (receptor tyrosine kinase-like orphan receptor 1), PANC-1 cells were used as a pancreatic cancer model. PANC-1 cells were cultured and incubated with CD64 / EV and one of the following targeted formulations: (i) flag-control (no targeted portion), (ii) IgG-free CK (CKAAKNK) peptide, (iii) CK peptide containing normal IgG (IgG without specificity), (iv) CK peptide and αhEGFR_IgG, and (v) CK peptide and αhROR1_IgG. CD64 / EV was pre-mixed with individual hmAbs at room temperature for 1 hour, and then unbound hmAbs were washed away. PANC-1 cells in monolayer culture were treated with formulated CD64 / EV containing each hmAb for 24 hours, then washed and suspended for flow cytometry. Flow assays were performed to quantify the amount of internalized EVs fluorescently labeled with PKH67. The first comparison was the uptake efficiency of flag peptide (Figure 16A) or CK peptide (Figure 16B) expressing EVs loaded with different hmAbs. Fluorescence intensity revealed that EVs targeted with αhROR1 were better taken up than EVs targeted with αhEGFR or IgG controls due to their selectivity for surface ROR1 or EGFR on PANC-1 cells. Binding of clinically available hmAbs on CD64 on the EV surface, particularly αhROR1, was able to increase the amount of internalized EVs in PANC-1 cancer cells by approximately 60% for αhROR and approximately 30% for αhEGFR compared to untargeted IgG_EVs (Figure 16C). Furthermore, additional CK peptides on the EV surface can nearly double the uptake of αhROR1_EV in PANC-1 cells (as shown in mean fluorescence intensity [MFI], Flag_αROR1: 5585±755.9; CK_αROR1: 112209±1914). Figure 16C summarizes the quantitative results from flow cytometry assays.To further quantify hmAb-assisted enrichment of EV uptake, surface EGFR and ROR1 expression on PANC-1 cells were stained with or without hmAb-targeted formulations. Staining of PANC-1 cells after 4 hours of αROR1_EV treatment revealed a decrease in surface ROR1 expression, suggesting that αhROR1 can induce stronger EV internalization than αhEGFR for enhanced drug delivery (Figure 16D). Subsequently, EV uptake assays were performed on 3D tumor spheroids using PANC-1 cells. Consistently, αhROR1_EV showed stronger surface ROR1 internalization and resulted in enriched EV uptake, while αhEGFR_EV was able to target the PANC-1 surface well but showed less internalization after 6 hours of incubation (Figures 16E and 16F). Since human IgG is naturally present in human serum at concentrations of 6–16 g / L, a substitution assay was performed to evaluate the stability of αhROR1 and αhROR1 on EVs. EVs were first loaded with targeted antibodies (i.e., αhEGFR and αhROR1) and then incubated with human serum (50%) at 37°C for 6 hours. The purpose of this substitution assay was to understand whether pre-loaded hmAbs could be replaced with serum human IgG in blood circulation. After comparing the targeting ability of EVs before and after the substitution assay, no significant loss of targeting ability was observed (Figure 16G and Figures 18A–18B). This data supports the stability of humanized antibodies on CD64 / EVs in clinical use.

[0197] Example 11. Enhancement of tissue penetration of targeted EVs For each EV formulation with a different targeting design, a transwell-based assay was established to quantify the permeability of PANC-1 cells through multiple layers. Transcytosis activity was determined by EV exchange between the upper and lower layers of PANC-1 cells separated by a 5 μm pore Transwell® membrane (Figure 17A). The upper layer consisted of PANC-1 cells with over 90% confluence in monolayer culture to mimic tight junctions in human pancreatic ductal epithelial cells. EVs were fluorescently labeled with PKH67 and incubated with the upper layer cells as the first recipient. As transcytosis gradually occurred, the fluorescently labeled EVs were taken up by the first recipient cells, but many were secreted into the extracellular region via exocytosis. Some of the fluorescently labeled EVs in the intermediate region were later taken up by the second recipient cells in the lower layer, yielding a detectable fluorescent signal as an indicator. Commercially available PEGylated liposomes, Invivofectamine (Thermofisher), were synthesized according to the manufacturer's instructions as a control. EVs were found to enter and exit the upper cell monolayer and be taken up by the lower cell monolayer 2–3 times more effectively than liposomes (Figure 17C). Various inhibitors selected to block endocytosis and EV secretion are shown in Figure 17B. Inhibition of clathrin and caveolae-mediated endocytosis significantly reduced EV transcytosis, suggesting that EV entry into recipient cells (in this case, PANC-1) is primarily mediated by clathrin and caveolae-mediated endocytosis. Interestingly, inhibition of EV secretion from upper PANC-1 cells with neticonazole strongly reduced transcytotic activity (Figure 17C). The presence of targeted hmAbs on the EV surface could enrich transcytotic activity 3–4 times compared to that of untargeted EVs (Figure 17D). Here again, the CD64 / EV surface loaded with αhROR1 best enhanced transcytosis of PANC-1 cells, and the combination of hmAb and CK-peptide on CD64 / EV would further improve transcytosis (Figure 17D).

[0198] While the above disclosure is described in some detail for clarity and understanding, it will be apparent to those skilled in the art from an understanding of this disclosure that various modifications of form and detail can be made without departing from the true scope of this disclosure. For example, all the technologies and apparatus described above can be used in various combinations. All publications, patents, patent applications, and / or other documents cited in this application are incorporated by reference in whole for all purposes to the same extent that each individual publication, patent, patent application, and / or other document is shown individually and separately so as to be incorporated by reference for all purposes.

Claims

[Claim 1] The invention described in the specification.

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