Membrane protein scaffolds for exosome engineering

Abstract

To provide membrane protein scaffolds for exosome engineering.SOLUTION: The present disclosure relates to heterologous exosomal vesicle proteins (HEVPs), engineered exosomes comprising HEVPs, and methods of preparing and using these compositions, including therapeutic applications. Aspects of the disclosure relate to novel EV (e.g., exosome) compositions, methods for preparing these compositions, and therapeutic methods using these compositions. Specifically, the composition and method relate to extracellular vesicles comprising a heterologous extracellular vesicle protein (HEVP) or a fragment thereof, where the extracellular vesicles are produced from a producer cell not naturally expressing the HEVP, and the HEVP is produced naturally by a donor cell.SELECTED DRAWING: None

Description

[Technical field]

[0001] 1. CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 62 / 801,065, filed February 4, 2019, No. 62 / 801,636, filed February 5, 2019, and No. 62 / 851,581, filed May 22, 2019, each of which is incorporated by reference in its entirety herein.

[0002] 2. Reference to a sequence listing submitted electronically via EFS-WEB The contents of the Sequence Listing, submitted electronically in an ASCII text file (Name: 4000_031PC03_SeqListing_ST25.txt; Size: 205,964; and Creation Date: February 4, 2020) filed with this application, are hereby incorporated by reference in their entirety.

[0003] 3. Field of disclosure The present disclosure relates to engineered extracellular vesicles (EVs) (e.g., exosomes) that contain one or more exogenous proteins that are not naturally expressed in the cell in which the EVs are produced (i.e., heterologous exosomal vesicle proteins), as well as methods of producing and using such EVs. [Background technology]

[0004] 4. Background of the Disclosure Extracellular vesicles (EVs) (e.g., exosomes) are important mediators of intercellular communication. They are also important biomarkers in the diagnosis and prognosis of many diseases, including cancer. As drug delivery vehicles, EVs (e.g., exosomes) offer many advantages over traditional drug delivery methods as a new therapeutic modality in many therapeutic areas.

[0005] The use of EVs (e.g., exosomes) for therapeutic purposes requires that the EVs (e.g., exosomes) are free or nearly free of impurities, including but not limited to contaminating proteins, DNA, carbohydrates, and lipids. Current purification methods do not provide sufficient selectivity to remove significant amounts of these impurities, so additional processes to improve purity are desirable.

[0006] Furthermore, as EVs (e.g., exosomes) become more frequently used in the treatment of human diseases, they may struggle to meet clinical expectations due to heterogeneity in their physicochemical parameters that confer molecular targeting, immune evasion, and controlled drug release. This is primarily due to the heterogeneity and complexity of EV (e.g., exosome) properties (e.g., composition, size, shape, rigidity, surface charge, hydrophilicity, stability, and ligand type and density), payload properties (e.g., drug type, solubility, loading, efficacy, dosing, immune response, and release kinetics), and in vivo physiological barriers to EV (e.g., exosome) trafficking (e.g., immune surveillance, particle extravasation, tissue targeting, tissue penetration, and cellular uptake). Although a significant amount of effort has been made, effective methods to obtain distinct subpopulations of therapeutic EVs (e.g., exosomes) with desired properties, e.g., EVs (e.g., exosomes) containing therapeutic payloads and with appropriate targeting moieties, are not yet readily available.

[0007] Suitable methods for generating, isolating, and purifying distinct subpopulations of EVs (e.g., exosomes) are needed to make therapeutic uses and other applications of EV (e.g., exosome)-based technologies more feasible. Summary of the Invention [Means for solving the problem]

[0008] 5. Disclosure Overview Aspects of the present disclosure relate to novel EV (e.g., exosome) compositions, methods for preparing these compositions, and therapeutic methods using the compositions. Specifically, the compositions and methods relate to extracellular vesicles comprising a heterologous extracellular vesicle protein (HEVP) or a fragment thereof, where the extracellular vesicle is produced from a producer cell that does not naturally express the HEVP, and the HEVP is naturally produced by a donor cell. In addition, the present disclosure relates to the use of HEVPs enriched on the surface of donor EVs (e.g., exosomes). Examples of HEVPs described herein include CD13, MME, ENPP1, and NRP1, particularly those recombinantly expressed on the surface of EVs (e.g., exosomes) produced by non-mesenchymal cells (e.g., CHO cells in the case of CD13, MME, ENPP1, and / or NRP1, and / or HEK cells in the case of CD13, MME, and NRP1). These HEVP proteins can be used to engineer EVs (e.g., exosomes) as described in U.S. Patent No. 10,195,290, which is incorporated by reference herein in its entirety.

[0009] In one embodiment, the present disclosure then provides extracellular vesicles comprising at least one heterologous extracellular vesicle protein (HEVP) or a fragment thereof, the extracellular vesicles being produced from a producer cell that does not naturally express HEVP, and the HEVP being naturally produced by donor cells. In certain embodiments, the extracellular vesicles comprise 2, 3, 4, 5, 6, 7, 8, 9, or 10 heterologous extracellular vesicle proteins (HEVPs) or fragments thereof, the extracellular vesicles being produced from a producer cell that does not naturally express HEVP, and the HEVP being naturally produced by donor cells. In general, unless otherwise specified, references to "at least one HEVP" and "2, 3, 4, 5... HEVPs" refer to the presence (if more than one) of different types or varieties of HEVPs (e.g., HEVPs with different amino acid sequences), and not the amount of HEVP molecules (of any type) on the EV (e.g., exosomes), which will be much greater.

[0010] In certain embodiments, the level of at least one HEVP in the EVs (e.g., exosomes) produced by the donor cells is about 20, about 80, about 125, or about 200 peptide spectral matches (PSMs) or more as measured using liquid chromatography with tandem mass spectrometry (LC-MS / MS). In some embodiments, the at least one HEVP is naturally produced by the donor cells producing the exosomes, and the level of the HEVP in the exosomes produced by the donor cells is about 20 peptide spectral matches (PSMs) or more as measured using liquid chromatography with tandem mass spectrometry (LC-MS / MS). In some embodiments, the at least one HEVP is naturally produced by the donor cells producing the exosomes, and the level of the HEVP in the exosomes produced by the donor cells is about 80 peptide spectral matches (PSMs) or more as measured using LC-MS / MS. In some embodiments, at least one HEVP is naturally produced by donor cells producing exosomes, and the level of HEVP in exosomes produced by donor cells is about 125 peptide spectral matches (PSMs) or more as measured using LC-MS / MS. In some embodiments, at least one HEVP is naturally produced by donor cells producing exosomes, and the level of HEVP in exosomes produced by donor cells is about 170 peptide spectral matches (PSMs) or more as measured using LC-MS / MS. In some embodiments, at least one HEVP is naturally produced by donor cells producing exosomes, and the level of HEVP in exosomes produced by donor cells is about 200 peptide spectral matches (PSMs) or more as measured using LC-MS / MS. In some embodiments, at least one HEVP is naturally produced by donor cells producing exosomes, and the level of HEVP in exosomes produced by donor cells is about 700 peptide spectral matches (PSMs) or more as measured using LC-MS / MS.In some embodiments, at least one HEVP is naturally produced by the donor cells producing exosomes, and the level of the HEVP in the exosomes produced by the donor cells is about 177 peptide spectral matches (PSMs) measured using LC-MS / MS. In some embodiments, at least one HEVP is naturally produced by the donor cells producing exosomes, and the level of the HEVP in the exosomes produced by the donor cells is about 742 peptide spectral matches (PSMs) measured using LC-MS / MS.

[0011] In certain embodiments, the level of at least one HEVP in the EVs (e.g., exosomes) produced by the donor cells is about 20 to about 80 PSM, or about 80 to about 200 PSM, as measured using LC-MS / MS. In some embodiments, the level of at least one HEVP in the exosomes produced by the donor cells is 20 to 80 peptide spectral matches (PSM), as measured using LC-MS / MS. In some embodiments, the level of at least one HEVP in the exosomes produced by the donor cells is 80 to 200 peptide spectral matches (PSM), as measured using LC-MS / MS. In some embodiments, the level of at least one HEVP in the exosomes produced by the donor cells is about 150 to about 750 peptide spectral matches (PSM), as measured using LC-MS / MS. In certain embodiments, the at least one HEVP is naturally produced by the exosome-producing donor cells, and the level of the at least one HEVP in the exosomes produced by the donor cells is greater than or equal to about 5% of the total protein content of the exosomes produced by the donor cells.

[0012] Additional embodiments include extracellular vesicles such as those described in the summary above, where at least one HEVP or fragment thereof is a fusion protein. As used herein, the term "fusion protein" refers to two or more proteins that are linked together. As described herein, in some aspects, the fusion protein includes a HEVP and an exogenous biologically active molecule (e.g., an antigen, a targeting moiety, an adjuvant, and / or an immunomodulator). In some aspects, the two or more proteins (e.g., a HEVP and an exogenous biologically active molecule) can be fused to each other. In some aspects, the two or more proteins (e.g., a HEVP and an exogenous biologically active molecule) can be linked to each other via a linker. In related embodiments, at least one HEVP or fragment thereof is modified by the addition of a functional moiety. In related embodiments, the functional moiety has affinity for a binding agent. In related embodiments, the functional moiety is an affinity tag. In certain embodiments, the affinity tag is a peptide. In other related embodiments, the functional moiety is a therapeutic compound. In still other related embodiments, the therapeutic compound is selected from the group consisting of a natural peptide, a recombinant peptide, a synthetic peptide, and any combination thereof. In yet other related embodiments, the therapeutic compound is selected from the group consisting of nucleotides, amino acids, lipids, carbohydrates, small molecules, and any combination thereof. In yet other related embodiments, the therapeutic compound is an antibody or a fragment thereof. In additional related embodiments, the therapeutic compound is an enzyme, a ligand, a receptor, or a fragment thereof. In other related embodiments, the functional moiety is a targeting moiety, which in some embodiments can be specific to an organ, tissue, or cell (for example). In related embodiments, at least one HEVP is a fusion protein, and the fusion protein comprises a linker. In related embodiments, the EV (e.g., exosome) further comprises a linker between at least one HEVP or a fragment thereof and the functional moiety bound thereto. In related embodiments, the linker is a flexible linker or a rigid linker.In other related embodiments, the linker is a straight chain carbon linker, a branched chain carbon linker, a heterocyclic carbon linker, or a peptide linker. In other related embodiments, the linker is a cleavable linker.

[0013] The present disclosure also includes the extracellular vesicles described in the summary above, wherein the producing cells are selected from the group consisting of HEK, CHO, MB-231, Raji, PER.C6, CAP, MSC cells, and any combination thereof. In a related embodiment, the at least one HEVP is selected from the group consisting of lung fibroblast EVP, aortic endothelial cell EVP, acute myeloid leukemia cell EVP, monocyte EVP, B cell lymphoma EVP, macrophage EVP, brain endothelial cell EVP, mesenchymal cell EVP, and any combination thereof. In a related embodiment, the at least one HEVP is human EVP, mouse EVP, rat VEP, dog EVP, or monkey EVP. In yet another related embodiment, the at least one HEVP is selected from the group consisting of CD13, MME, ENPP1, and NRP1, or fragments thereof. In yet another related embodiment, the at least one HEVP is selected from the group consisting of CD13, MME, ENPP1, and NRP1, or fragments thereof, and the producing cells are CHO cells. In another related embodiment, the at least one HEVP is selected from the group consisting of CD13, MME, and NRP1, or fragments thereof, and the producing cell is a CHO cell or a HEK cell.

[0014] In another related embodiment, at least one HEVP in the extracellular vesicle is selected from the group consisting of PTGFRN, BSG, IGSF3, ITGB1, ITGA4, SLC3A2, ATP transporter, or fragments thereof, and any combination thereof. In some embodiments, the producing cell is a cell that does not naturally produce these proteins. In a related embodiment, the extracellular vesicle described in the summary above further comprises an EVP selected from the group consisting of PTGFRN, BSG, IGSF3, ITGB1, ITGA4, SLC3A2, ATP transporter, or fragments thereof, and any combination thereof.

[0015] In a related embodiment, the present disclosure provides an extracellular vesicle (e.g., an exosome) as disclosed herein, wherein the producing cell is a non-human cell and at least one HEVP is a human HEVP. Alternatively, the producing cell can be a human cell and at least one HEVP is a non-human HEVP.

[0016] Additional related embodiments include pharmaceutical formulations comprising any of the above extracellular vesicles. Another aspect of the present disclosure includes kits comprising the extracellular vesicles or pharmaceutical formulations described in the Summary above, wherein the extracellular vesicles or pharmaceutical formulations are contained in a container or package, and the kits further include instructions describing the recommended use of the extracellular vesicles or pharmaceutical formulations.

[0017] The present disclosure also includes a method for treating a patient in need of treatment, comprising administering to the patient an effective amount of any of the above-described extracellular vesicles or pharmaceutical formulations.

[0018] The present disclosure further provides a method for expressing a non-naturally occurring protein in an EV derived from a cell, comprising transfecting a nucleic acid encoding at least one heterologous extracellular vesicle protein (HEVP) or a fragment thereof in a cell, and isolating an EV comprising a HEVP or a fragment thereof from the cell, wherein the HEVP is not naturally occurring in the EV derived from the cell. In certain embodiments, the EV comprises any of the EVs disclosed in the present disclosure. In some embodiments, the method for expressing a non-naturally occurring protein in an EV can further comprise characterizing the HEVP of the EV.

[0019] The HEVPs described herein can be used in various embodiments of the present disclosure. One aspect of the present disclosure relates to generating fusion proteins by conjugating HEVPs with functional compounds (e.g., exogenous biologically active molecules disclosed herein) and producing engineered EVs (e.g., exosomes) containing modified proteins on their surface. For example, native full-length or biologically active fragments of therapeutic proteins can be transported to the surface of EVs (e.g., exosomes) by conjugating them to HEVP-enriched proteins or fragments thereof. The methods of using HEVPs described herein are considered to be improved in certain aspects in some cases compared to other related systems (e.g., Lamp2B, PDGFR, lactadherin CD9, CD63 and / or CD81, or fragments thereof).

[0020] Another aspect of the present disclosure relates to the purification of EVs (e.g., exosomes) by affinity purification from heterogeneous solutions such as cell culture medium or plasma using HEVP. Some embodiments relate to the isolation of a subpopulation of EVs (e.g., exosomes) from total EVs (e.g., exosomes) by using surface markers specific for the subpopulation of EVs (e.g., exosomes).

[0021] Another aspect of the present disclosure relates to methods for removing EVs (e.g., exosomes) from a sample when EVs (e.g., exosomes) are a contaminating product. For example, natural or engineered viruses can be purified from contaminating EVs (e.g., exosomes). Thus, the HEVPs described herein can be used to selectively remove EVs (e.g., exosomes) from biological processes where other particles of similar size, shape, and / or charge are the desired product.

[0022] Another aspect of the present disclosure relates to the generation or use of surface engineered EVs (e.g., exosomes) designed for more efficient affinity purification or for presentation of targeting moieties or therapeutically relevant proteins (e.g., exogenous biologically active molecules as disclosed herein) on their surface. For example, the EV (e.g., exosome) surface can be engineered to contain full-length HEVP and / or fragments of HEVP or engineered proteins at a higher density on the surface.

[0023] The present disclosure further relates to a producer cell, or a method of generating a producer cell for producing such surface-engineered EVs (e.g., exosomes). An exogenous polynucleotide (e.g., encoding a HEVP) can be transiently or stably introduced into the producer cell to enable the producer cell to generate surface-engineered EVs (e.g., exosomes).

[0024] Specifically, aspects of the present disclosure relate to methods for isolating EVs (e.g., exosomes), comprising: (1) providing a sample containing EVs (e.g., exosomes); (2) contacting the sample with a binding agent having affinity for a target protein, the target protein comprising a HEVP (e.g., CD13, MME, ENPP1, or NRP1, or a fragment or variant thereof); and (3) isolating the EVs (e.g., exosomes) based on binding between the target protein and the binding agent.

[0025] In some embodiments, the sample is obtained from cells grown in vitro, optionally the cells are HEK293 cells, Chinese hamster ovary (CHO) cells, or mesenchymal stem cells (MSCs). In some embodiments, the sample is obtained from a bodily fluid of a subject.

[0026] In some embodiments, the cells are genetically modified to express a target protein. In some embodiments, the cells comprise an expression plasmid encoding the target protein. In some embodiments, the cells are genetically modified to include an exogenous sequence that expresses a tag with affinity to a binding agent, and the exogenous sequence is inserted into the genome of the cells. In some embodiments, the exogenous sequence is inserted into a genomic site located at the 3' or 5' end of the endogenous sequence encoding a HEVP (e.g., CD13, MME, ENPP1, or NRP1). In some embodiments, the endogenous sequence does not encode IGSF8. In some embodiments, the exogenous sequence is inserted into a genomic site located within the endogenous sequence encoding a HEVP (e.g., CD13, MME, ENPP1, or NRP1).

[0027] In some embodiments, the target protein is a fusion protein comprising a tag and CD13, MME, ENPP1, or NRP1, or a fragment or variant thereof. In some embodiments, the EV (e.g., exosome) comprises the target protein. In some embodiments, the target protein is not IGSF8 or a fragment or variant thereof. In some embodiments, the cell is genetically modified to have reduced expression of ADAM10.

[0028] In some embodiments, the EVs (e.g., exosomes) comprise a target protein. In some embodiments, the target protein is selected from CD13, MME, ENPP1, and NRP1. In some embodiments, the target protein comprises a fragment or variant of CD13, MME, ENPP1, or NRP1. In some embodiments, the target protein comprises a polypeptide of SEQ ID NO: 33. In some embodiments, the target protein is a fusion protein comprising CD13, MME, ENPP1, or NRP1, or a fragment or variant thereof, and an affinity tag, wherein the affinity tag has affinity for the binding agent. In some embodiments, the target protein does not comprise IGSF8 or a fragment or variant thereof.

[0029] In some embodiments, the binding agent comprises an immunoglobulin, a protein, a peptide, or a small molecule. In some embodiments, the binding agent is bound to a solid support, optionally, the solid support comprises porous agarose beads, a microtiter plate, a magnetic bead, or a membrane.

[0030] In some embodiments, the solid support forms a chromatographic column. In some embodiments, contacting the sample with the binding agent is carried out by applying the sample to the chromatographic column.

[0031] In some embodiments, the method further comprises: (1) contacting a subset of the sample with different binding agents having affinity for different target proteins; and (2) isolating EVs (e.g., exosomes) based on binding between the different target proteins and the different binding agents. In some embodiments, the different target proteins comprise CD13, MME, ENPP1, or NRP1, or fragments or variants thereof. In some embodiments, the target protein comprises CD13 (or a fragment or variant thereof). In some embodiments, the target protein comprises MME (or a fragment or variant thereof). In some embodiments, the target protein comprises ENPP1 (or a fragment or variant thereof). In some embodiments, the target protein comprises NRP1 (or a fragment or variant thereof). In some embodiments, the different target proteins comprise the polypeptide of SEQ ID NO: 33.

[0032] Another aspect of the present disclosure relates to EVs (e.g., exosomes) produced by the methods provided herein.

[0033] In yet another aspect, the present disclosure relates to a pharmaceutical composition comprising the EVs (e.g., exosomes) of the present disclosure and an excipient. In some embodiments, the pharmaceutical composition comprises a macromolecule at a lower concentration than a sample comprising a source of EVs (e.g., exosomes), the macromolecule being a nucleic acid, a contaminating protein, a lipid, a carbohydrate, a metabolite, or a combination thereof. In some embodiments, the pharmaceutical composition is substantially free of macromolecules.

[0034] Another aspect of the disclosure relates to an EV (e.g., an exosome) comprising a target protein, at least a portion of which is expressed from an exogenous sequence, and the target protein comprises a HEVP (e.g., CD13, MME, ENPP1, or NRP1, or a fragment or variant thereof). In some embodiments, the target protein comprises CD13 (or a fragment or variant thereof). In some embodiments, the target protein comprises MME (or a fragment or variant thereof). In some embodiments, the target protein comprises ENPP1 (or a fragment or variant thereof). In some embodiments, the target protein comprises NRP1 (or a fragment or variant thereof). In some embodiments, the target protein does not comprise IGSF8 or a fragment or variant thereof. In some embodiments, the target protein comprises a polypeptide of SEQ ID NO: 33.

[0035] In some embodiments, EVs (e.g., exosomes) are isolated based on binding between a target protein and a binding agent.

[0036] In some embodiments, the EVs (e.g., exosomes) are produced from cells genetically modified to contain exogenous sequences, and optionally the cells are HEK293 cells, Chinese hamster ovary (CHO) cells, or mesenchymal stem cells (MSCs). In some embodiments, the cells are genetically modified to have reduced expression of ADAM10.

[0037] In some embodiments, the cell comprises a plasmid comprising the exogenous sequence.

[0038] In some embodiments, the cell comprises an exogenous sequence inserted into the genome of the cell. In some embodiments, the exogenous sequence is inserted into a genomic site located at the 3' or 5' end of the genomic sequence encoding CD13, MME, ENPP1, or NRP1. In some embodiments, the exogenous sequence is inserted into the genomic sequence encoding CD13, MME, ENPP1, or NRP1. In some embodiments, the exogenous sequence does not encode IGSF8.

[0039] In some embodiments, the target protein is a fusion protein comprising CD13, MME, ENPP1, or NRP1, or a fragment or variant thereof, and an affinity tag, wherein the affinity tag has affinity for the binding agent. In some embodiments, the target protein does not comprise IGSF8 or a fragment thereof.

[0040] In some embodiments, the target protein is a fusion protein comprising CD13, MME, ENPP1, or NRP1, or a fragment or variant thereof, and a therapeutic compound. In some embodiments, the target protein does not comprise IGSF8 or a fragment thereof.

[0041] The therapeutic compound may be selected from the group consisting of natural peptides, recombinant peptides, and synthetic peptides. In some embodiments, the therapeutic compound comprises a linker. The therapeutic compound may be selected from the group consisting of nucleotides, amino acids, lipids, carbohydrates, and small molecules.

[0042] Functional moieties and related compounds, e.g., therapeutic compounds, capable of binding to HEVP or fragments thereof include antibodies or fragments or variants thereof. Functional compounds that are peptides can be enzymes, ligands, receptors, or fragments or variants thereof. Therapeutic peptides can be antimicrobial peptides or fragments or variants thereof.

[0043] In some embodiments, the targeting protein is a fusion protein comprising CD13, MME, ENPP1, or NRP1, or a fragment or variant thereof, and a targeting moiety. The targeting moiety can be organ, tissue, or cell specific. In some embodiments, the targeting protein does not comprise IGSF8 or a fragment thereof.

[0044] In some embodiments, the EVs (e.g., exosomes) further comprise a second different target protein, and the different target protein comprises CD13, MME, ENPP1, or NRP1, or a fragment or variant thereof. In some embodiments, the EVs (e.g., exosomes) are isolated based on the binding between the different target proteins and the different binding agents. In some embodiments, the target protein does not comprise IGSF8 or a fragment thereof.

[0045] In one aspect, the present disclosure relates to a pharmaceutical composition comprising an EV (e.g., an exosome) of the present disclosure and an excipient.

[0046] In some embodiments, the pharmaceutical composition is substantially free of macromolecules, where the macromolecules are selected from nucleic acids, contaminating proteins, lipids, carbohydrates, metabolites, and combinations thereof.

[0047] In one aspect, the present disclosure relates to cells for producing the EVs (e.g., exosomes) presented herein.

[0048] Specifically, some embodiments relate to cells for producing EVs (e.g., exosomes) comprising an exogenous sequence inserted into a genomic sequence encoding CD13, MME, ENPP1, or NRP1, where the exogenous sequence and the genomic sequence encode a fusion protein. In some embodiments, the genomic sequence does not encode IGSF8.

[0049] The exogenous sequence may encode an affinity tag.

[0050] The exogenous sequence can encode a therapeutic peptide. The therapeutic peptide can be selected from the group consisting of a natural peptide, a recombinant peptide, a synthetic peptide, or a linker to a therapeutic compound. The therapeutic compound can be selected from the group consisting of a nucleotide, an amino acid, a lipid, a carbohydrate, and a small molecule. The therapeutic peptide can be an antibody or a fragment or variant thereof. The therapeutic peptide can be an enzyme, a ligand, a receptor, or a fragment or variant thereof. The therapeutic peptide can be an antimicrobial peptide, or a fragment or variant thereof.

[0051] The exogenous sequence can encode a targeting moiety, which can be organ-, tissue-, or cell-specific.

[0052] In some embodiments, the cell lines are genetically modified to have reduced expression of ADAM10.

[0053] In one aspect, the disclosure provides EVs (e.g., exosomes) produced from a cell line of the disclosure. In some embodiments, the EVs (e.g., exosomes) contain a fusion protein on their surface at a higher density than the different fusion proteins on the surface of the different EVs (e.g., exosomes), and the different EVs (e.g., exosomes) are produced from different cell lines that contain an exogenous sequence inserted into a different genomic sequence that encodes a conventional EV (e.g., exosome) protein, and the exogenous sequence and the different genomic sequence encode the different fusion protein. In some embodiments, the conventional EV (e.g., exosome) protein is selected from the group consisting of CD9, CD63, CD81, PDGFR, GPI-anchored proteins, LAMP2, LAMP2B, and fragments thereof.

[0054] In another aspect, the disclosure relates to a method of isolating non-exosome material, the method comprising: providing a sample comprising EVs (e.g., exosomes) and non-EV (e.g., exosomes) material; contacting the sample with a binding agent having affinity for a target protein, the target protein comprising CD13, MME, ENPP1, or NRP1, or a fragment or variant thereof, thereby inducing EVs (e.g., exosomes) to bind to the binding agent; and isolating the non-EV (e.g., exosomes) material. In some embodiments, the target protein comprises CD13 (or a fragment or variant thereof). In some embodiments, the target protein comprises MME (or a fragment or variant thereof). In some embodiments, the target protein comprises ENPP1 (or a fragment or variant thereof). In some embodiments, the target protein comprises NRP1 (or a fragment or variant thereof).

[0055] In some embodiments, the non-exosome material is a virus or protein. In some embodiments, the non-exosome material is a lentivirus, retrovirus, adeno-associated virus, or other enveloped or non-enveloped virus. In some embodiments, the non-exosome material is a recombinant protein. In some embodiments, the isolated non-exosome material is substantially free of EVs (e.g., exosomes).

[0056] In some embodiments, the target protein further comprises an affinity tag, and the affinity tag has affinity for the binder. In some embodiments, the target protein comprises a polypeptide of SEQ ID NO: 33. In some embodiments, the binder comprises an immunoglobulin, a protein, a peptide, or a small molecule. In some embodiments, the binder is bound to a solid support, optionally comprising a porous agarose bead, a microtiter plate, a magnetic bead, or a membrane. In some embodiments, the solid support forms a chromatographic column. In some embodiments, the step of contacting the sample with the binder is performed by applying the sample to a chromatographic column.

[0057] In some embodiments, the purification methods described herein are used for the purification of nanovesicles. In some embodiments, the compositions and methods described herein relate to nanovesicles. Additional embodiments of the present disclosure are described in more detail below. In an embodiment of the present invention, for example, the following items are provided: (Item 1) An extracellular vesicle (EV) comprising at least one heterologous extracellular vesicle protein (HEVP) or a fragment thereof, wherein the EV is produced from a producer cell that does not naturally express the HEVP, and the HEVP is naturally produced by a donor cell. (Item 2) The EV of item 1, wherein the EV comprises 2, 3, 4, 5, 6, 7, 8, 9, or 10 heterologous extracellular vesicle proteins (HEVPs) or fragments thereof, the EV being produced from a producer cell that does not naturally express the HEVPs, and the HEVPs are naturally produced by a donor cell. (Item 3) The EV of any one of the preceding paragraphs, wherein the at least one HEVP is naturally produced by a donor cell that produces exosomes, and the level of the HEVP in the exosomes produced by the donor cell is greater than or equal to about 20 peptide spectral matches (PSMs) as measured using liquid chromatography with tandem mass spectrometry (LC-MS / MS). (Item 4) The EV of any one of the preceding paragraphs, wherein the at least one HEVP is naturally produced by a donor cell that produces exosomes, and the level of the HEVP in the exosomes produced by the donor cell is equal to or greater than about 80 peptide spectral matches (PSMs) as measured using LC-MS / MS. (Item 5) The EV of any one of the preceding paragraphs, wherein the at least one HEVP is naturally produced by a donor cell that produces exosomes, and the level of the HEVP in the exosomes produced by the donor cell is equal to or greater than about 125 peptide spectral matches (PSMs) as measured using LC-MS / MS. (Item 6) The EV of any one of the preceding paragraphs, wherein the at least one HEVP is naturally produced by a donor cell that produces exosomes, and the level of the HEVP in the exosomes produced by the donor cell is equal to or greater than about 170 peptide spectral matches (PSMs) as measured using LC-MS / MS. (Item 7) The at least one HEVP is naturally produced by exosome-producing donor cells. The EV of any one of the preceding paragraphs, wherein the level of the HEVP in the exosomes produced by the donor cells is greater than or equal to about 200 peptide spectral matches (PSMs) as measured using LC-MS / MS. (Item 8) The EV of any one of the preceding paragraphs, wherein the at least one HEVP is naturally produced by a donor cell that produces exosomes, and the level of the HEVP in the exosomes produced by the donor cell is greater than or equal to about 700 peptide spectral matches (PSMs) as measured using LC-MS / MS. (Item 9) 7. The EV according to any one of items 1 to 6, wherein the at least one HEVP is naturally produced by a donor cell that produces exosomes, and the level of the HEVP in the exosomes produced by the donor cell is about 177 peptide spectral matches (PSMs) measured using LC-MS / MS. (Item 10) 9. The EV according to any one of items 1 to 8, wherein the at least one HEVP is naturally produced by a donor cell that produces exosomes, and the level of the HEVP in the exosomes produced by the donor cell is about 742 peptide spectral matches (PSMs) measured using LC-MS / MS. (Item 11) 4. The EV according to any one of items 1 to 3, wherein the level of the at least one HEVP in the exosomes produced by the donor cells is about 20 to about 80 peptide spectral matches (PSMs) measured using LC-MS / MS. (Item 12) 6. The EV according to any one of items 1 to 5, wherein the level of the at least one HEVP in the exosomes produced by the donor cells is about 80 to about 200 peptide spectral matches (PSMs) measured using LC-MS / MS. (Item 13) 9. The EV according to any one of items 1 to 8, wherein the level of the at least one HEVP in the exosomes produced by the donor cells is about 150 to about 750 peptide spectral matches (PSMs) measured using LC-MS / MS. (Item 14) The EV of any one of the preceding paragraphs, wherein the at least one HEVP is naturally produced by a donor cell that produces exosomes, and the level of the at least one HEVP in the exosomes produced by the donor cell is about 5% or more of the total protein content of the exosomes produced by the donor cell. (Item 15) The EV of any one of the preceding claims, wherein the at least one HEVP or fragment thereof is a fusion protein. (Item 16) EV according to any one of the preceding claims, wherein the at least one HEVP or fragment thereof is modified by the addition of a functional moiety. (Item 17) Item 17. The EV according to item 16, wherein the functional moiety has affinity for a binding agent. (Item 18) Item 18. The EV according to item 17, wherein the functional moiety is an affinity tag. (Item 19) 19. The EV of item 18, wherein the affinity tag is a peptide. (Item 20) 17. The EV according to item 16, wherein the functional moiety is a therapeutic compound. (Item 21) 21. The EV of item 20, wherein the therapeutic compound is selected from the group consisting of natural peptides, recombinant peptides, synthetic peptides, and any combination thereof. (Item 22) 21. The EV of item 20, wherein the therapeutic compound is selected from the group consisting of nucleotides, amino acids, lipids, carbohydrates, small molecules, and any combination thereof. (Item 23) 21. The EV of item 20, wherein the therapeutic compound is an antibody or a fragment thereof. (Item 24) 21. The EV of item 20, wherein the therapeutic compound is an enzyme, a ligand, a receptor, or a fragment thereof. (Item 25) Item 17. The EV of item 16, wherein the functional moiety is a targeting moiety. (Item 26) 26. The EV of item 25, wherein the targeting moiety is organ, tissue, or cell specific. (Item 27) 27. The EV according to any one of items 15 to 26, wherein the fusion protein comprises a linker. (Item 28) 28. The EV according to any one of items 16 to 27, further comprising a linker between the at least one HEVP or a fragment thereof and the functional moiety. (Item 29) 29. The EV according to item 27 or 28, wherein the linker is a flexible linker or a rigid linker. (Item 30) 30. The EV according to any one of items 27 to 29, wherein the linker is a straight-chain carbon linker, a branched-chain carbon linker, a heterocyclic carbon linker, or a peptide linker. (Item 31) 31. The EV according to any one of items 27 to 30, wherein the linker is a cleavable linker. (Item 32) EV according to any one of the preceding items, wherein the producing cells are selected from the group consisting of HEK, CHO, MB-231, Raji, PER.C6, CAP, MSC cells, and any combination thereof. (Item 33) EV according to any one of the preceding claims, wherein the at least one HEVP is selected from the group consisting of lung fibroblast EVP, aortic endothelial cell EVP, acute myeloid leukemia cell EVP, monocyte EVP, B cell lymphoma EVP, macrophage EVP, brain endothelial cell EVP, mesenchymal cell EVP, and any combination thereof. (Item 34) 34. The EV of item 33, wherein the at least one HEVP is a human EVP, a mouse EVP, a rat EVP, a dog EVP, or a monkey EVP. (Item 35) EV according to any one of the preceding items, wherein the at least one HEVP is selected from the group consisting of CD13, MME, ENPP1, and NRP1, or fragments thereof. (Item 36) EV according to any one of the preceding items, wherein the at least one HEVP is selected from the group consisting of CD13, MME, ENPP1, and NRP1, or fragments thereof, and the producing cell is a CHO cell. (Item 37) The at least one HEVP is a polypeptide that binds to CD13, MME, and NRP1, or EV according to any one of the preceding claims, wherein the producer cell is selected from the group consisting of CHO cells or HEK cells. (Item 38) 35. The EV according to any one of items 1 to 34, wherein the at least one HEVP is selected from the group consisting of PTGFRN, BSG, IGSF3, ITGB1, ITGA4, SLC3A2, an ATP transporter, or a fragment thereof, and any combination thereof. (Item 39) 38. The EV according to any one of items 1 to 37, wherein the extracellular vesicles further comprise an EVP selected from the group consisting of PTGFRN, BSG, IGSF3, ITGB1, ITGA4, SLC3A2, an ATP transporter, or a fragment thereof, and any combination thereof. (Item 40) The EV of any one of the preceding claims, wherein the producing cell is a non-human cell and the at least one HEVP is a human HEVP. (Item 41) 40. The EV according to any one of items 1 to 39, wherein the producing cell is a human cell and the at least one HEVP is a non-human HEVP. (Item 42) A pharmaceutical formulation comprising an EV according to any one of the preceding items. (Item 43) A kit comprising the EV or pharmaceutical preparation of any one of the preceding paragraphs, wherein the EV or pharmaceutical preparation is contained within a container or package, and the kit further comprises instructions describing a recommended use of the EV or pharmaceutical preparation. (Item 44) 43. A method for treating a patient in need of treatment, comprising administering to said patient an effective amount of an EV or pharmaceutical preparation according to any one of items 1 to 42. (Item 45) A method for expressing a non-naturally occurring protein in an EV derived from a cell, comprising transfecting the cell with a nucleic acid encoding at least one heterologous extracellular vesicle protein (HEVP) or a fragment thereof, and isolating an EV containing the HEVP or a fragment thereof from the cell, wherein the HEVP does not naturally occur in the EV derived from the cell. (Item 46) Item 46. The method according to Item 45, wherein the EV comprises the EV according to any one of Items 1 to 41. (Item 47) 47. The method of claim 45 or 46, further characterizing the HEVP of the EV. 6. Brief description of the drawings [Brief description of the drawings]

[0058] [Figure 1] FIG. 1 provides a dot graph showing peptide-spectral matches of individual proteins identified in either mesenchymal stem cell (MSC)-derived (y-axis) or HEK293-derived (x-axis) EVs (e.g., exosomes). Individual dots correspond to different proteins. CD13 and MME (indicated using arrows) are both enriched in MSC-derived EVs but are not detected in HEK293-derived EVs. The table below the dot graph shows the molecular weight (MW) of CD13 and MME proteins and their relative expression in HEK293 (HEK) and MSC cells. [Diagram 2]1 shows the expression of CD13 and PTGFRN proteins in EVs (e.g., exosomes) derived from HEK293 producing cells that have been modified to overexpress either PTGFRN or CD13. A provides SDS-PAGE analysis of wild-type (WT) (i.e., unmodified), PTGFRN-overexpressing, and CD13-overexpressing HEK293-derived EVs. B provides quantification of CD13 protein expression in engineered HEK293-derived EVs by ELISA using a recombinant CD13 (rCD13) standard. The graph shown on the left is the standard. The graph shown on the right is for EVs (e.g., exosomes). [Diagram 3] Figure 1 provides a bioactivity analysis of CD13 protein expressed in engineered HEK293-derived EVs (e.g., exosomes). A shows the enzymatic activity of recombinant CD13 protein (rCD13) using a commercially available CD13 activity assay kit (BioVision K523). B shows the bioactivity of CD13 protein expressed in engineered HEK293-derived EVs (e.g., exosomes) using the same CD13 activity assay kit. Recombinant CD13 protein was tested at six different concentrations: (i) 880ng / mL, (ii) 440ng / mL, (iii) 293ng / mL, (iv) 220ng / mL, (v) 110ng / mL, and (vi) 55ng / mL. In B, EVs were tested at three different concentrations: (i) 1.6x1010p / mL, (ii) 7.9x109p / mL, and (iii) 4.0x109p / mL. Non-manipulated EVs (i.e., wild type) were used as a control. [Figure 4]Figure 1 provides an expression analysis of GFP conjugated to CD13 and MME proteins expressed in engineered HEK293-derived EVs (e.g., exosomes). A shows GFP expression measured using SDS-PAGE analysis. EVs expressing GFP conjugated to PTGFRN (PTGFRN-GFP) were used as a control. B shows a comparison of the expression of GFP conjugated to one of the following scaffold proteins: (i) LAMP2B, (ii) pDisplay, (iii) PTGFRN, (iv) MME, and (v) CD13. GFP was measured spectrophotometrically. Raw fluorescence measurements are shown for each of the different scaffold proteins. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0059] 7.1.Definition Unless otherwise defined, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this disclosure belongs. As used herein, the following terms have the meanings ascribed to them below.

[0060] As used herein, the term "extracellular vesicles" or "EVs" refers to cell-derived vesicles that contain a membrane that encloses an internal space. Extracellular vesicles include all membrane-bound vesicles (e.g., exosomes, nanovesicles) that have a diameter smaller than the cell from which they are derived. Generally, extracellular vesicles range from 20 nm to 1000 nm in diameter and can contain a variety of macromolecular cargo, either within the internal space (e.g., lumen) that is displayed on the external surface of the extracellular vesicle, and / or across the membrane. The cargo can include nucleic acids, proteins, carbohydrates, lipids, small molecules, and / or combinations thereof. In some embodiments, the EVs (e.g., exosomes) contain one or more cargoes or other exogenous biologically active molecules. In some embodiments, the EVs (e.g., exosomes) can include one or more scaffolding moieties. In certain embodiments, the one or more scaffolding moieties are not naturally expressed in the cell from which the EVs (e.g., exosomes) are produced. By way of example, but not by way of limitation, extracellular vesicles include apoptotic bodies, cell fragments, vesicles derived from cells by direct or indirect manipulation (e.g., by continuous extrusion or treatment with alkaline solution), vesiculated organelles, and vesicles produced by living cells (e.g., by direct budding of the plasma membrane or fusion of late endosomes with the plasma membrane). Extracellular vesicles can be derived from living or dead organisms, explanted tissues or organs, prokaryotic or eukaryotic cells, and / or cultured cells. As described herein, in some embodiments, the EVs (e.g., exosomes) disclosed herein are produced by cells that have been modified to express one or more transgene products. Thus, the EVs of the present disclosure do not include naturally occurring EVs (e.g., exosomes).

[0061] As used herein, the term "exosome" refers to small (20-300 nm in diameter, more preferably 40-200 nm in diameter) cell-derived vesicles that contain a membrane enclosing an interior space and are generated from cells by direct plasma membrane budding or by fusion with the plasma membrane of a late endosome. In certain embodiments, the exosomes of the present disclosure are about 20-290 nm, 20-280 nm, 20-270 nm, 20-260 nm, 20-250 nm, 20-240 nm, 20-230 nm, 20-220 nm, 20-210 nm, 20-200 nm, 20-190 nm, 20-180 nm, 20-170 nm, 20-160 nm, 20-150 nm, 20-140 nm, 20-130 nm, 20-120 nm, 20-110 nm, 20-100 nm, 20-90 nm, 20-140 nm, 20-150 nm, 20-160 nm, 20-170 nm, 20-180 nm, 20-190 nm, 20-200 nm, 20-210 nm, 20-220 nm, 20-230 nm, 20-240 nm, 20-250 nm, 20-260 nm, 20-270 nm, 20-280 nm, 20-290 nm, 20-300 nm, 20-320 nm, 20-340 nm, 20-360 nm, 20-380 nm, 20-390 nm, 20-400 nm, 20-410 nm, 20-420 nm, 20-430 nm, 20-440 nm, 20-450 nm, 20-460 nm, 20-470 nm, 20-480 nm, 20-490 nm, 20-500 nm, 20-510 nm, 20 ~80nm, 20~70nm, 20~60nm, 20~50nm, 20~40nm, 20~30nm, 30~300nm, 30~290nm, 30~280nm, 30~270nm, 30~260nm, 30~250nm, 30~240nm , 30~230nm, 30~220nm, 30~210nm, 30~200nm, 30~190nm, 30~180nm, 30~170nm, 30~160nm, 30~150nm, 30~140nm, 30~130nm, 30~120nm , 30~110nm, 30~100nm, 30~90nm, 30~80nm, 30~70nm, 30~60nm, 30~50nm, 30~40nm, 40~300nm, 40~290nm, 40~280nm, 40~270nm, 40~2 60nm, 40~250nm, 40~240nm, 40~230nm, 40~220nm, 40~210nm, 40~200nm, 40~190nm, 40~180nm, 40~170nm, 40~160nm, 40~150nm, 40~1 40nm, 40~130nm, 40~120nm, 40~110nm, 40~100nm, 40~90nm, 40~80nm, 40~70nm, 40~60nm, 40~50nm, 50~300nm, 50~290nm, 50~280nm, 50~270nm, 50~260nm, 50~250nm, 50~240nm, 50~230nm, 50~220nm, 50~210nm, 50~200nm, 50~190nm, 50~180nm, 50~170nm, 50~160nm,50~150nm、50~140nm、50~130nm、50~120nm、50~110nm、50~100nm、50~90nm、50~80nm、50~70nm、50~60nm、60~300nm、60~290nm、60~280nm、60~270nm、60~260nm、60~250nm、60~240nm、60~230nm、60~220nm、60~210nm、60~200nm、60~190nm、60~180nm、60~170nm、60~160nm、60~150nm、60~140nm、60~130nm、60~120nm、60~110nm、60~100nm、60~90nm、60~80nm、60~70nm、70~300nm、70~290nm、70~280nm、70~270nm、70~260nm、70~250nm、70~240nm、70~230nm、70~220nm、70~210nm、70~200nm、70~190nm、70~180nm、70~170nm、70~160nm、70~150nm、70~140nm、70~130nm、70~120nm、70~110nm、70~100nm、70~90nm、70~80nm、80~300nm、80~290nm、80~280nm、80~270nm、80~260nm、80~250nm、80~240nm、80~230nm、80~220nm、80~210nm、80~200nm、80~190nm、80~180nm、80~170nm、80~160nm、80~150nm、80~140nm、80~130nm、80~120nm、80~110nm、80~100nm、80~90nm、90~300nm、90~290nm、90~280nm、90~270nm、90~260nm、90~250nm、90~240nm、90~230nm、90~220nm、90~210nm、90~200nm、90~190nm、90~180nm、90~170nm、90~160nm、90~150nm、90~140nm、90~130nm、90~120nm、90~110nm、90~100nm、100~300nm、110~290nm、120~280nm、130~270nm、140~260nm、150~250nm、160~240nm、170~230nm、180~220nm、Or has a diameter of 190 to 210 nm. The size of the EVs (e.g., exosomes) described herein can be measured according to the methods described below.

[0062] In some embodiments, the EVs (e.g., exosomes) of the present disclosure comprise a bilipid membrane and comprise an internal surface and an external surface. In certain embodiments, the internal surface faces the inner core (i.e., the lumen) of the EV (e.g., exosome). In certain embodiments, the external surface may contact the endosome, multivesicular body, or membrane / cytoplasm of the producing or target cell.

[0063] In some embodiments, the EV (e.g., exosome) membrane comprises lipids and fatty acids. In some embodiments, the EV (e.g., exosome) membrane comprises phospholipids, glycolipids, fatty acids, sphingolipids, phosphoglycerides, sterols, cholesterol, and phosphatidylserine.

[0064] In some embodiments, the EV (e.g., exosome) membrane comprises an inner leaflet and an outer leaflet. The composition of the inner and outer leaflets can be determined by transbilayer distribution assays known in the art, see, e.g., Kuypers et al. Biochem. See Biophys Acta 1985 819:170. In some embodiments, the composition of the outer leaflet is approximately 70-90% choline phospholipid, approximately 0-15% acidic phospholipid, and approximately 5-30% phosphatidylethanolamine. In some embodiments, the composition of the inner leaflet is approximately 15-40% choline phospholipid, approximately 10-50% acidic phospholipid, and approximately 30-60% phosphatidylethanolamine.

[0065] In some embodiments, exosomes comprise lipids or fatty acids and polypeptides, and optionally comprise a payload (e.g., a therapeutic agent), a receiver (e.g., a targeting moiety), a polynucleotide (e.g., a nucleic acid, RNA, or DNA), a sugar (e.g., a simple sugar, a polysaccharide, or a glycan), or other molecules. As described herein, in some embodiments, exosomes of the present disclosure comprise one or more scaffold moieties. In certain embodiments, the one or more scaffold moieties are not naturally expressed in the cell in which the exosome is produced. Exosomes can be derived from producer cells and can be isolated from producer cells based on their size, density, biochemical parameters, or a combination thereof. Exosomes are a type of extracellular vesicle. Generally, exosome production / biogenesis does not result in the destruction of the producing cell. In some embodiments, exosomes of the present disclosure are produced by cells expressing one or more transgene products. Exosomes of the present disclosure are modified (e.g., engineered to overexpress a protein not naturally expressed in the producing cell) and therefore do not include naturally occurring exosomes.

[0066] As used herein, the term "heterologous exosomal vesicle protein" or "HEVP" refers to a protein that is heterologously expressed in EVs (e.g., exosomes) produced by a particular cell type, where that cell type does not naturally express the protein, but the protein is naturally expressed in EVs (e.g., exosomes) of a different cell type. The latter cell type is referred to herein as a "donor cell" and the former cell type is referred to herein as a "producer cell."

[0067] As used herein, the term "nanovesicles" refers to small (20-250 nm diameter, more preferably 30-150 nm diameter) vesicles of cell origin that include a membrane enclosing an internal space, and are generated from cells (e.g., producer cells) by direct or indirect manipulation; nanovesicles are not produced by producer cells without manipulation. Suitable manipulations of producer cells include, but are not limited to, continuous extrusion, treatment with alkaline solutions, sonication, or combinations thereof. Production of nanovesicles may, in some instances, result in the destruction of producer cells. In some embodiments, the population of nanovesicles described herein is substantially free of vesicles derived from producer cells, either by direct budding from the plasma membrane or by fusion with the plasma membrane of a late endosome. In some embodiments, nanovesicles include lipids or fatty acids and polypeptides, and optionally include payloads (e.g., therapeutic agents), receivers (e.g., targeting moieties), polynucleotides (e.g., nucleic acids, RNA, or DNA), sugars (e.g., simple sugars, polysaccharides, or glycans), or other molecules. In some embodiments, the nanovesicles described herein comprise one or more scaffolding moieties. In certain embodiments, one or more scaffolding moieties are not naturally expressed in the cell in which the nanovesicles are produced. Nanovesicles can be isolated from the producing cell based on their size, density, biochemical parameters, or combinations thereof, once the nanovesicles are derived from the producing cell according to the above-mentioned operations. Nanovesicles are a type of extracellular vesicles. As used herein, nanovesicles are modified (e.g., engineered to overexpress proteins that are not naturally expressed in the producing cell), and therefore do not include naturally occurring nanovesicles.

[0068] As used herein, the term "surface-modified exosome" refers to an exosome that has a membrane or surface modified in its composition, whereby the membrane or surface of the engineered exosome is different from that of the exosome prior to modification or naturally occurring exosome. The engineering can be performed at the surface of the exosome or within the membrane of the exosome, whereby the surface of the exosome is altered. For example, the membrane is modified in its composition, such as proteins, lipids, small molecules, carbohydrates, etc. The composition can be altered by chemical, physical, or biological methods, or by being produced from cells that have previously or simultaneously been modified by chemical, physical, or biological methods. Specifically, the composition can be altered by genetic engineering, or by being produced from cells that have already been modified by genetic engineering. As disclosed herein, in some embodiments, the surface-engineered exosomes disclosed herein include one or more exogenous proteins (e.g., scaffolding moieties, e.g., heterologous exosome vesicle proteins disclosed herein), or fragments or variants thereof, that may be exposed on the surface of the exosome or may be anchor points (attachments) for moieties exposed on the surface of the exosome. In certain embodiments, one or more scaffold moieties are not naturally expressed in the cells in which the exosomes are produced. In some embodiments, surface-engineered exosomes include higher expression (e.g., higher number) of native exosomal proteins (e.g., PTGFRN), or fragments or variants thereof, which may be exposed on the surface of the exosome or may be anchor points (binding points) of moieties exposed on the surface of the exosome. Although the above definitions are provided in the context of exosomes, other types of extracellular vesicles may also be surface-engineered in a similar manner. Thus, unless otherwise indicated, the disclosure regarding surface-engineered exosomes may be similarly applicable to other extracellular vesicles.

[0069] As used herein, the term "luminal engineered exosomes" refers to exosomes that have an exosomal membrane or lumen modified in its composition, such that the lumen of the engineered exosome is different from that of the pre-modified or naturally occurring exosome. The manipulation can be performed directly in the lumen or membrane of the exosome, thereby altering the lumen of the exosome. For example, the membrane is modified in its composition, such as with proteins, lipids, small molecules, carbohydrates, etc., thereby altering the lumen of the exosome. The composition can be altered by chemical, physical, or biological methods, or by being produced from cells that have already been modified by chemical, physical, or biological methods. Specifically, the composition can be altered by genetic engineering, or by being produced from cells that have already been modified by genetic engineering. In some embodiments, the luminal engineered exosomes include one or more exogenous biologically active molecules (e.g., scaffolding moieties, e.g., heterologous exosomal vesicle proteins disclosed herein). In certain embodiments, the exogenous biologically active molecule can include an exogenous protein (i.e., a protein not naturally expressed by EVs, e.g., exosomes), or a fragment or variant thereof, that can be exposed in the lumen of the exosome or can be an anchor point (junction) for a portion exposed to the inner layer of the exosome. In certain embodiments, one or more scaffolding moieties are not naturally expressed in the cell in which the exosome is produced. In some embodiments, luminally engineered exosomes include higher expression of a native exosomal protein, or a fragment or variant thereof, that can be exposed in the lumen of the exosome or can be an anchor point (junction) for a portion exposed to the lumen of the exosome. Although the above definition is provided in the context of exosomes, other types of extracellular vesicles can be luminally engineered in a similar manner. Thus, unless otherwise indicated, the disclosure regarding luminally engineered exosomes can be applied to other extracellular vesicles as well.

[0070] As used herein, the term "modified" when used in the context of a protein refers to a protein having at least about 15% sequence identity to the non-mutated amino acid sequence of the protein. Modifications of a protein include fragments or mutants of the protein. Modifications of a protein can further include chemical or physical modifications to the fragments or mutants of the protein.

[0071] The term "modified", when used in the context of EVs, e.g., exosomes, described herein, refers to the alteration or manipulation of EVs, e.g., exosomes, and / or their producing cells, such that the modified EVs, e.g., exosomes, differ from naturally occurring EVs, e.g., exosomes. In some embodiments, the modified EVs, e.g., exosomes, described herein, comprise a membrane that differs in composition, such as proteins, lipids, small molecules, carbohydrates, etc., compared to the membrane of naturally occurring EVs, e.g., exosomes (e.g., the membrane comprises a higher density or number of naturally occurring exosomal proteins, and / or the membrane comprises a plurality (e.g., at least two) biologically active molecules not naturally found in exosomes (e.g., therapeutic molecules (e.g., antigens), targeting moieties, adjuvants, and / or immunomodulators). As used herein, biologically active molecules not naturally found in exosomes are also described as "exogenous biologically active molecules." Non-limiting examples of such exogenous biologically active molecules include heterologous exosome vesicle proteins (e.g., CD13, MME, ENPP1, or NRP1) disclosed herein, therapeutic molecules (e.g., antigens), targeting moieties, adjuvants, immunomodulators, or combinations thereof. In certain embodiments, such modifications to the membrane alter the exterior surface of the EV, e.g., exosomes (e.g., surface-engineered EVs, e.g., exosomes described herein).

[0072] As used herein, the terms "scaffold moiety" and "scaffold" may be used interchangeably and refer to a molecule that can be used to anchor a cargo or any other exogenous biologically active molecule of interest (e.g., a targeting moiety, an adjuvant, and / or an immunomodulator) onto the external surface of an EV, e.g., an EV, e.g., an exosome. In certain embodiments, the scaffold moiety comprises a synthetic molecule. In some embodiments, the scaffold moiety comprises a non-polypeptide moiety. In some embodiments, the scaffold moiety comprises a lipid, carbohydrate, or protein that is naturally present in an EV, e.g., an exosome. In some embodiments, the scaffold moiety comprises a lipid, carbohydrate, or protein that is not naturally present in an EV, e.g., an exosome. In some embodiments, the scaffold moiety comprises a heterologous exosome vesicle protein disclosed herein. In some embodiments, the scaffold moiety can be an entire protein or a fragment thereof (e.g., a functional fragment, e.g., the smallest fragment that can anchor another moiety onto the external or luminal surface of an EV, e.g., an exosome). Non-limiting examples of other scaffold moieties that can be used in accordance with the present disclosure include scaffold X, scaffold Y, CD9, CD63, CD81, PDGFR, GPI-anchored proteins, lactadherin, LAMP2, and LAMP2B.

[0073] As used herein, the term "Scaffold X" refers to exosomal proteins recently identified on the surface of exosomes. See, for example, U.S. Patent No. 10,195,290, which is incorporated herein by reference in its entirety. Non-limiting examples of Scaffold X proteins include prostaglandin F2 receptor negative regulator ("PTGFRN protein"); basigin ("BSG protein"); immunoglobulin superfamily member 2 ("IGSF2 protein"); immunoglobulin superfamily member 3 ("IGSF3 protein"); immunoglobulin superfamily member 8 ("IGSF8 protein"); integrin beta-1 ("ITGB1 protein"); integrin alpha-4 ("ITGA4 protein"); 4F2 cell surface antigen heavy chain ("SLC3A2 protein"); and the class of ATP transporter proteins ("ATP1A1 protein", "ATP1A2 protein", "ATP1A3 protein", "ATP1A4 protein", "ATP1B3 protein", "ATP2B1 protein", "ATP2B2 protein", "ATP2B3 protein", "ATP2B protein").

[0074] As used herein, the term "scaffold Y" refers to a newly identified exosomal protein within the lumen of an exosome. See, for example, International Application PCT / US2018 / 061679, incorporated herein by reference in its entirety. Non-limiting examples of scaffold Y proteins include myristoylated alanine-rich protein kinase C substrate ("MARCKS protein"), myristoylated alanine-rich protein kinase C substrate 1 ("MARCKSL1 protein"), and brain acid-soluble protein 1 ("BASP1 protein").

[0075] As used herein, the term "fragment" of a protein refers to an amino acid sequence of a protein that is shorter than the naturally occurring sequence, e.g., the amino acid sequence of a protein that is deleted at the N-terminus and / or C-terminus and / or any other portion of the protein compared to the naturally occurring protein. Preferably, a fragment of a heterologous exosomal vesicle protein disclosed herein (e.g., CD13, MME, ENPP1, or NRP1) retains the ability to specifically target an EV (e.g., an exosome). In some embodiments, a fragment of a heterologous exosomal vesicle protein disclosed herein retains the ability to anchor another portion to the external or luminal surface of an EV (e.g., an exosome). Such a fragment is also referred to as a "functional fragment." As used herein, the term "functional fragment" can refer to a protein fragment that retains protein function. In some embodiments, the term "functional fragment" refers to a protein fragment that can be expressed in a cell line that does not naturally express the full-length protein. Whether a fragment is a functional fragment in this sense can be assessed by any method known in the art for determining the protein content of EVs (e.g., exosomes), including Western blot, FACS analysis, and fusion of the fragment with an autofluorescent protein, such as GFP. In some embodiments, the heterologous exosomal vesicle proteins disclosed herein retain at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% of the ability of naturally occurring heterologous exosomal vesicle proteins, e.g., the ability to anchor a moiety. In specific embodiments, a fragment of CD13, MME, ENPP1, or NRP1 retains at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% of the ability of naturally occurring CD13, MME, ENPP1, or NRP1 to specifically target EVs (e.g., exosomes) and / or anchor another moiety to an EV (e.g., to the external surface).

[0076] As used herein, the term "variant" of a protein refers to a protein that shares a certain amino acid sequence identity with another protein upon alignment by methods known in the art. A variant of a protein can include a substitution, insertion, deletion, frameshift, or rearrangement in another protein. In certain embodiments, the variant is a variant of CD13 (also known in the art as, e.g., membrane alanyl aminopeptidase, alanyl aminopeptidase (AAP), aminopeptidase N (AP-N), aminopeptidase M, GP150, LAP1, P150, PEPN, ANPEP, or microsome aminopeptidase), MME (also known in the art as, e.g., membrane metallo-endopeptidase, neprilysin, neuronal endopeptidase (NEP), cluster of differentiation 10 (CD10), common acute lymphoblastic leukemia antigen (CALLA), dermal fibroblast elastase, atriopeptidase, or enkephalinase). In some embodiments, the variant of CD13 is a variant having at least about 70% identity to CD13 according to SEQ ID NO:47, or a functional fragment thereof. In some embodiments, the MME variant or fragment variant shares at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% sequence identity with an MME according to SEQ ID NO:48 or a functional fragment thereof.In some embodiments, the ENPP1 variant or fragment variant shares at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% sequence identity with ENPP1 or a functional fragment thereof according to SEQ ID NO: 49. In some embodiments, the NRP1 variant or fragment variant shares at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% sequence identity with NRP1 or a functional fragment thereof according to SEQ ID NO: 50. In each of the above cases, the variant or fragment variant preferably retains the function of the protein (e.g., the ability to specifically target EVs (e.g., exosomes)).

[0077] A "conservative amino acid substitution" is one in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art and include basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, if an amino acid in a polypeptide is replaced with another amino acid from the same side chain family, the substitution is considered conservative. In another embodiment, a chain of amino acids can be conservatively replaced with a structurally similar chain that differs in the order and / or composition of the side chain family members.

[0078] The term "percent sequence identity" or "percent identity" between two polynucleotide or polypeptide sequences refers to the number of identical matching positions shared by the sequences over the comparison span, taking into account additions or deletions (i.e., gaps) that must be introduced for optimal alignment of the two sequences. A matching position is any position where an identical nucleotide or amino acid is present in both the target sequence and the reference sequence. Gaps presented in the target sequence are not counted, since gaps are not nucleotides or amino acids. Similarly, gaps presented in the reference sequence are not counted, since the target sequence nucleotides or amino acids are counted, and not the nucleotides or amino acids from the reference sequence.

[0079] Methods for aligning sequences for comparison are well known in the art. Various programs and alignment algorithms are described in the following: Smith and Waterman, Adv. Appl. Math. 2:482 (1981); Needleman, J. Med. 1999, 14:1311-1321 (1999); and Wunsch, J.Mol.Bio.48:443 (1970), Pearson and Lipman, Methods in Mol.Biol.24:307-31 (1988), Higgins and Sharp, Gene 73:15 237-44 (1988), Higgins and Sharp, CABIOS 5:151-3 (1989), Corpet et al. al., Nuc. 16:10881-90 (1988), Huang et al., Comp. Appl. 8: 155-65 (1992), and Pearson et al., Meth. Mol. Biol. 24: 307-31 (1994). NCBI Basic Local Alignment Search Tool (BLAST)[Altschul 20 et al., J. Mol. Biol. 215:403-10 (1990)] is available from several sources and is distributed to the National Institute of Standards and Technology (NIS) for use in conjunction with the sequence analysis programs blastp, blasm, blastx, tblastn, and tblastx. BLAST and a description of how to use the program to determine sequence identity can be accessed at the official website of the National Center for Biotechnology Information (NCBI) of the National Institutes of Health (NIH).

[0080] Any recitation of a protein provided herein encompasses functional variants of the protein. The term "functional variant" of a protein refers to a variant of the protein that retains the ability to specifically target EVs (e.g., exosomes).

[0081] Polynucleotide variants can contain mutations in coding regions, non-coding regions, or both. In one embodiment, polynucleotide variants contain mutations that result in silent substitutions, additions, or deletions, but do not change the properties or activities of the encoded polypeptide. In another embodiment, nucleotide variants are produced by silent substitutions due to the degeneracy of the genetic code. In other embodiments, variants have 5-10, 1-5, or 1-2 amino acids substituted, deleted, or added in any combination. Polynucleotide variants can be produced for a variety of reasons, such as optimizing codon expression for a particular host (changing codons in human mRNA for others, e.g., bacterial hosts such as E. coli).

[0082] Naturally occurring variants are called "allelic variants" and refer to one of several alternative forms of a gene occupying a given locus on an organism's chromosome (Genes II, Lewin, B., ed., John Wiley & Sons, New York (1985)). These allelic variants can vary at either the polynucleotide and / or polypeptide level and are included in the present disclosure. Alternatively, non-naturally occurring variants can be produced by mutagenesis techniques or by direct synthesis.

[0083] Using known methods of protein engineering and recombinant DNA technology, variants can be generated to improve or alter the characteristics of a polypeptide. For example, one or more amino acids can be deleted from the N-terminus or C-terminus of a secreted protein without substantial loss of biological function. Ron et al., J. Biol. Chem. 268:2984-2988 (1993), incorporated herein by reference in its entirety, reported mutant KGF proteins that retained heparin-binding activity even after deletion of 3, 8, or 27 amino acid residues. Similarly, interferon gamma exhibited up to 10-fold greater activity after deletion of 8-10 amino acid residues from the carboxy terminus of the protein. (Dobeli et al., J. Biotechnology 7:199-216 (1988), incorporated herein by reference in its entirety.)

[0084] Moreover, ample evidence demonstrates that variants often retain similar biological activity as the naturally occurring protein. For example, Gayle and colleagues (J. Biol. Chem 268:22105-22111 (1993), incorporated herein by reference in its entirety) performed an extensive mutational analysis of the human cytokine IL-1a. They used random mutagenesis to generate over 3,500 individual IL-1a variants that averaged 2.5 amino acid changes per variant over the entire length of the molecule. Multiple mutations were examined at all possible amino acid positions. The researchers acknowledged that "most of the molecules could be made with little effect on [binding or biological activity]." (See Abstract) Indeed, of the more than 3,500 nucleotide sequences examined, only 23 unique amino acid sequences produced proteins with significantly different activity from the wild type.

[0085] As described above, polypeptide variants include, for example, modified polypeptides. Modifications include, for example, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, pegylation (Mei et al., Blood 116:270-79 (2010), which is incorporated herein by reference in its entirety), proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer RNA-mediated addition of amino acids to proteins, such as arginylation, and ubiquitination. In some embodiments, scaffold X and / or scaffold Y are modified at any convenient position.

[0086] As used herein, the terms "linked to," "conjugated to," and "anchored to" are used interchangeably and refer to a covalent or non-covalent bond formed between a first moiety (e.g., a scaffold moiety, e.g., a heterologous exosome vesicle protein) and a second moiety (e.g., a payload).

[0087] As used herein, the term "producing cells" refers to cells used to generate EVs (e.g., exosomes). The producing cells can be cells cultured in vitro or in vivo. The producing cells include, but are not limited to, cells known to be effective in generating EVs, e.g., EVs (e.g., exosomes), e.g., HEK293 cells, Chinese Hamster Ovary (CHO) cells, mesenchymal stem cells (MSCs), BJ human foreskin fibroblasts, fHDF fibroblasts, AGE.HN® neuronal progenitor cells, CAP® amniotic fluid cells, adipose mesenchymal stem cells, RPTEC / TERT1 cells. In certain embodiments, the producing cells are not antigen-presenting cells. In some embodiments, the producing cells are not dendritic cells, B cells, mast cells, macrophages, neutrophils, Kupffer-Browicz cells, cells derived from any of these cells, or any combination thereof. As disclosed herein, in some embodiments, the producing cells of the present disclosure are modified to express one or more transgene products. In some embodiments, the producing cells are modified to express a protein that the producing cells do not naturally express (e.g., a heterologous exosome vesicle protein). In some embodiments, the EVs, e.g., exosomes, useful in the present disclosure do not carry antigens on MHC class I or class II molecules exposed on the surface of the EVs, e.g., exosomes, but instead can carry antigens within the lumen of the EVs, e.g., exosomes, or on the surface of the EVs, e.g., exosomes (e.g., exosomes) by binding to a scaffold moiety.

[0088] As used herein, "MHC class I molecule" refers to the protein product of a wild-type or mutant HLA class I gene that encodes an MHC class I molecule. Thus, "HLA class I molecule" and "MHC class I molecule" are used interchangeably herein.

[0089] MHC class I molecules are one of the two major classes of major histocompatibility complex (MHC) molecules (the other being MHC class II) and are found on the cell surface of all nucleated cells in the body of jawed vertebrates. They also occur on platelets but not on red blood cells. Their function is to present peptide fragments of proteins from within the cell to cytotoxic T cells, which triggers an immediate response from the immune system to specific non-self antigens presented with the help of MHC class I proteins. Because MHC class I molecules present peptides derived from cytoplasmic proteins, the pathway of MHC class I presentation is often referred to as the cytoplasmic or intrinsic pathway.

[0090] In humans, the HLAs corresponding to MHC class I are HLA-A, HLA-B, and HLA-C. MHC class I molecules contain two protein chains: an α chain and a β2-microglobulin (β2m) chain. Human β2m is encoded by the B2M gene. Class I MHC molecules bind peptides that are generated primarily from the degradation of cytoplasmic proteins by the proteasome. The MHC I:peptide complex is then inserted into the outer plasma membrane of the cell via the endoplasmic reticulum. Epitope peptides are bound to the extracellular portion of the class I MHC molecule. Thus, the function of class I MHC is to present intracellular proteins to cytotoxic T cells (CTLs). However, class I MHC can also present peptides generated from exogenous proteins in a process known as cross-presentation.

[0091] Normal cells present peptides from normal cellular protein turnover on their class I MHC, and CTLs are not activated in response to them due to central and peripheral tolerance mechanisms. When cells express foreign proteins, such as after viral infection, a portion of class I MHC presents these peptides on the cell surface. As a result, CTLs specific for the MHC:peptide complex will recognize and kill the presenting cell. Alternatively, class I MHC itself can function as an inhibitory ligand for natural killer cells (NK). Reduction of normal levels of surface class I MHC, a mechanism used by some viruses and certain tumors to evade CTL responses, activates NK cell killing.

[0092] As used herein, "MHC class II molecule" refers to the protein product of a wild-type or mutant HLA class II gene that encodes an MHC class II molecule. Thus, "HLA class II molecule" and "MHC class II molecule" are used interchangeably herein.

[0093] MHC class II molecules are a class of major histocompatibility complex (MHC) molecules that are normally found only on professional antigen-presenting cells such as dendritic cells, mononuclear phagocytes, some endothelial cells, thymic epithelial cells, and B cells. These cells are important in initiating immune responses. Antigens presented by class II peptides are derived from extracellular proteins (not cytoplasmic as in MHC class I).

[0094] Like MHC class I molecules, class II molecules are heterodimers, but in this case they consist of two homologous peptides, an α chain and a β chain, both of which are encoded by the MHC. The subnominae α1, α2, etc. refer to separate domains within the HLA gene, each domain usually being encoded by a different exon within the gene, with some genes having further domains encoding leader sequences, transmembrane sequences, etc. These molecules both have extracellular regions as well as transmembrane sequences and cytoplasmic tails. The α1 and β1 regions of the chain come together to create the membrane-distal peptide-binding domain, while the remaining extracellular portion of the chain, the α2 and β2 regions, form the membrane-proximal immunoglobulin-like domain. The antigen-binding groove, where the antigen or peptide binds, consists of two α-helical walls and a β sheet. The antigen-binding groove of MHC class II molecules is open at both ends, while the corresponding groove in class I molecules is closed at both ends, so that antigens presented by MHC class II molecules are long, typically 15-24 amino acid residues long. Loading of MHC class II molecules occurs by phagocytosis, where extracellular proteins are endocytosed and digested in lysosomes, and the resulting epitope peptide fragments are loaded onto MHC class II molecules before their transfer to the cell surface. In humans, MHC class II protein complexes are encoded by the human leukocyte antigen gene complex (HLA). The HLAs corresponding to MHC class II are HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQ, and HLA-DR. Mutations in the HLA gene complex can result in bare lymphocyte syndrome (BLS), a type of MHC class II deficiency.

[0095] As used herein, the term "target protein" refers to a protein that can be targeted to the surface of an EV (e.g., exosome). A target protein can be a non-mutated protein that naturally targets the EV (e.g., exosome) membrane, or a fragment or mutant of a non-mutated protein. A target protein can be a fusion protein containing a flag tag, a therapeutic peptide, a targeting moiety, or other peptide linked to a non-mutated protein or a mutant or fragment of a non-mutated protein. A target protein can include a transmembrane protein, an integral membrane protein, a peripheral protein, or a soluble protein linked to the membrane by a linker.

[0096] As used herein, the term "contaminating protein" refers to a protein that is not associated with an EV (e.g., an exosome). For example, contaminating proteins include proteins that are not packaged in an EV (e.g., an exosome) and are not bound to or incorporated into the membrane of an EV (e.g., an exosome).

[0097] As used herein, the terms "isolate", "isolated", and "isolating", or "purify", "purified", and "purifying", as well as "extracted" and "extracting", are used interchangeably and refer to the state of a preparation of desired EVs (e.g., exosomes) (e.g., multiple known or unknown amounts and / or concentrations) that has undergone one or more processes of purification, e.g., selection or enrichment of the desired EV (e.g., exosome) preparation. In some embodiments, isolating or purifying as used herein is a process of removing or partially removing EVs (e.g., exosomes) (e.g., a fraction) from a sample containing producing cells. In some embodiments, an isolated EV (e.g., exosome) composition has no detectable undesirable activity, or alternatively, the level or amount of undesirable activity is below an acceptable level or amount. In other embodiments, an isolated EV (e.g., exosome) composition has an amount and / or concentration of desired EVs (e.g., exosomes) that is equal to or greater than an acceptable amount and / or concentration. In other embodiments, the isolated EV (e.g., exosome) composition is enriched compared to the starting material (e.g., producer cell preparation) from which the composition is derived. This enrichment can be at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.9%, at least about 99.9%, at least about 99.99%, at least about 99.999%, or greater than about 99.9999% compared to the starting material. In some embodiments, the isolated EV (e.g., exosome) preparation is substantially free of residual biological products.In some embodiments, the isolated EV (e.g., exosome) preparation is about 100% free, about 99% free, about 98% free, about 97% free, about 96% free, about 95% free, about 94% free, about 93% free, about 92% free, about 91% free, or about 90% free of any contaminating biological material. Residual biological products can include non-biological materials (including chemicals) or unwanted nucleic acids, proteins, lipids, or metabolites. Substantially free of residual biological products can also mean that the EV (e.g., exosome) does not contain detectable producing cells, and only the EV (e.g., exosome) is detectable.

[0098] The term "excipient" or "carrier" refers to an inert substance added to a pharmaceutical composition to further facilitate administration of a compound. The term "pharmaceutical acceptable carrier" or "pharmaceutical acceptable excipient" includes any agent approved by a regulatory agency of the U.S. Federal Government for use in animals, including humans, or listed in the U.S. Pharmacopeia, as well as any carrier or diluent that does not cause significant irritation to the subject and does not abrogate the biological activity and properties of the administered compound. Excipients and carriers that are useful in preparing pharmaceutical compositions and are generally safe, non-toxic, and desirable are included.

[0099] As used herein, the term "payload" refers to a therapeutic agent that acts on a target (e.g., a target cell) that has come into contact with an EV (e.g., an exosome). Non-limiting examples of payloads that may be contained in an EV, e.g., an exosome, are therapeutic molecules (e.g., antigens or immunosuppressants), adjuvants, and / or immunomodulators. Payloads that can be introduced into EVs (e.g., exosomes) and / or producing cells include therapeutic agents such as nucleotides (e.g., nucleotides that contain a detectable moiety or a toxin or that disrupt transcription), nucleic acids (e.g., DNA or mRNA molecules that encode a polypeptide such as an enzyme, or an RNA molecule with a regulatory function such as miRNA, dsDNA, lncRNA, siRNA, antisense oligonucleotides, phosphorodiamidate morpholino oligomers (PMOs), or polypeptide-conjugated phosphorodiamidate morpholino oligomers (PPMOs)), amino acids (e.g., amino acids that contain a detectable moiety or a toxin or that disrupt translation), polypeptides (e.g., enzymes), lipids, carbohydrates, and small molecules (e.g., small molecule drugs and toxins).

[0100] As used herein, the term "biologically active molecule" refers to an agent having activity in a biological system (e.g., a cell or a human subject) and includes, but is not limited to, a protein, polypeptide or peptide, including, but not limited to, a structural protein, an enzyme, a cytokine (such as interferons and / or interleukins), an antibiotic, a polyclonal or monoclonal antibody, or an effective portion thereof, an antibody or an Fv fragment, where the antibody or a portion thereof may be natural, synthetic or humanized, a peptide hormone, a receptor, a signaling molecule or other protein; an oligonucleotide or modified oligonucleotide, an antisense oligonucleotide or modified antisense oligonucleotide, a cDNA, a genomic DNA, an artificial or natural chromosome (e.g., yeast artificial chromosome) or a portion thereof, a nucleic acid as defined below, an RNA, including an mRNA, a tRNA, a rRNA or a ribozyme, or a peptide nucleic acid (PNA); a virus or virus-like particle; a nucleotide or ribonucleotide or a synthetic analog thereof, which may be modified or unmodified; an amino acid or an analog thereof, which may be modified or unmodified; a non-peptide (e.g., steroid) hormone; a proteoglycan: a lipid; or a carbohydrate. In certain embodiments, the biologically active molecule includes a therapeutic molecule (e.g., an antigen), a targeting moiety (e.g., an antibody or an antigen-binding fragment thereof), an adjuvant, an immunomodulator, or any combination thereof. In some embodiments, the biologically active molecule includes a macromolecule (e.g., a protein, an antibody, an enzyme, a peptide, DNA, RNA, or any combination thereof). In some embodiments, the biologically active molecule includes a small molecule (e.g., an antisense oligomer (ASO), a phosphorodiamidate morpholino oligomer (PMO), a peptide-conjugated phosphorodiamidate morpholino oligomer (PPMO), an siRNA, STING, a pharmaceutical agent, or any combination thereof). In some embodiments, the biologically active molecule is exogenous to the EV, i.e., is not naturally found in the EV.

[0101] As used herein, the term "therapeutic compound" refers to any molecule capable of treating and / or preventing a disease or disorder in a subject (e.g., a human subject).

[0102] In some embodiments, the therapeutic molecule comprises an antigen. As used herein, the term "antigen" refers to any agent that, when introduced into a subject, induces an immune response (cellular or humoral) against itself. In some embodiments, the antigen is not expressed in major histocompatibility complex I and / or II molecules. In other embodiments, the antigen in the EV, e.g., exosome, is not expressed as an MHC class I or II complex, but the EV, e.g., exosome, can still contain MHC class I / II molecules on the surface of the EV, e.g., exosome. Thus, in certain embodiments, the EV, e.g., exosome disclosed herein induces an immune response against an antigen without directly interacting with a T cell receptor (TCR). Similarly, in certain embodiments, the EV, e.g., exosome, of the present disclosure does not directly transfer the antigen to the surface of a target cell (e.g., dendritic cell) by cross-dressing. Cross-dressing is a mechanism commonly used by EV, e.g., exosome, derived from dendritic cells (DEX) to induce T cell activation. See Pitt, JM, et al., J Clin Invest 126(4):1224-32 (2016). In other embodiments, the EVs, e.g., exosomes, of the present disclosure can be phagocytosed by antigen-presenting cells and expressed on the surface of the antigen-presenting cells as MHC class I and / or MHC class II complexes.

[0103] In some embodiments, the therapeutic molecule comprises an immunosuppressant.As used herein, the term "immunosuppressant" refers to any agent (e.g., therapeutic molecule) that slows or stops the immune response in a subject.The immunosuppressant can be administered to a subject to prevent the subject's immune system from mounting an immune response after organ transplantation, or to treat diseases caused by an overactive immune system. Examples of immunosuppressants include, but are not limited to, calcineurin inhibitors such as, but not limited to, cyclosporine, ISA(TX)247, tacrolimus or calcineurin, target of rapamycin such as, but not limited to, sirolimus, everolimus, FK778 or TAFA-93, interleukin-2α chain blockers such as, but not limited to, basiliximab and daclizumab, inhibitors of inosine monophosphate dehydrogenase such as, but not limited to, mycophenolate mofetil, inhibitors of dihydrofolate reductase such as, but not limited to, methotrexate, corticosteroids such as, but not limited to, prednisolone and methylprednisolone, or immunosuppressant antimetabolites such as, but not limited to, azathioprine. In certain embodiments, the immunosuppressant comprises an antisense oligonucleotide. In some embodiments, the EVs (e.g., exosomes) disclosed herein can comprise both an antigen and an immunosuppressant. Without being bound by any one theory, EVs (e.g., exosomes) that contain both an antigen and an immunosuppressant can be used to induce tolerance to an antigen.

[0104] As used herein, the term "antibody" includes immunoglobulins, whether naturally occurring or partially or wholly synthetically produced, and fragments thereof. The term also encompasses any protein having a binding domain that is homologous to an immunoglobulin binding domain. "Antibody" further includes a polypeptide comprising a framework region from an immunoglobulin gene or a fragment thereof that specifically binds and recognizes an antigen. The use of the term antibody is meant to include whole antibodies, polyclonal, monoclonal and recombinant antibodies, fragments thereof, and further includes single chain antibodies, humanized antibodies, murine antibodies, chimeric, mouse-human, mouse-primate, primate-human monoclonal antibodies, anti-idiotypic antibodies, antibody fragments such as, for example, scFv, (scFv)2, Fab, Fab', and F(ab')2, F(ab1)2, Fv, dAb, and Fd fragments, diabodies, and antibody-related polypeptides. Antibodies include bispecific and multispecific antibodies, so long as they exhibit the desired biological activity or function. In some embodiments, the antibody or antigen-binding fragment thereof comprises an scFv, scFab, scFab-Fc, nanobody, or any combination thereof. In some embodiments, the antibody or antigen-binding fragment thereof comprises an agonist antibody, a blocking antibody, a targeting antibody, a fragment thereof, or any combination thereof. In some embodiments, the agonist antibody is a CD40L agonist. In some embodiments, the blocking antibody binds to a target protein selected from programmed death 1 (PD-1), programmed death-ligand 1 (PD-L1), cytotoxic T-lymphocyte-associated protein 4, and any combination thereof.

[0105] As used herein, the term "immunomodulator" refers to an agent that acts on a target (e.g., a target cell) that is in contact with an extracellular vesicle and modulates the immune system. Non-limiting examples of immunomodulators that can be introduced into EVs (e.g., exosomes) and / or producing cells include agents such as regulators of checkpoint inhibitors, ligands of checkpoint inhibitors, cytokines, derivatives thereof, or any combination thereof. Immunomodulators also include agonists, antagonists, antibodies, antigen-binding fragments, polynucleotides such as siRNA, antisense oligonucleotides, phosphorodiamidate morpholino oligomers (PMOs), peptide-conjugated phosphorodiamidate morpholino oligomers (PPMOs), miRNA, lncRNA, mRNA, DNA, etc., or small molecules.

[0106] As used herein, a "mammalian subject" includes all mammals, including, but not limited to, humans, pet animals (e.g., dogs, cats, etc.), farm animals (e.g., cows, sheep, pigs, horses, etc.), and laboratory animals (e.g., monkeys, rats, mice, rabbits, guinea pigs, etc.).

[0107] The terms "individual," "subject," "host," and "patient" are used interchangeably herein and refer to any mammalian subject, particularly humans, for whom diagnosis, treatment, or therapy is desired. The methods described herein are applicable to both human therapy and veterinary applications. In some embodiments, the subject is a mammal, and in other embodiments, the subject is a human.

[0108] As used herein, the term "substantially free" means that a sample containing EVs (e.g., exosomes) contains less than about 10% macromolecules at a mass / volume (m / v) percentage concentration. Some fractions may contain less than about 0.001%, less than about 0.01%, less than about 0.05%, less than about 0.1%, less than about 0.2%, less than about 0.3%, less than about 0.4%, less than about 0.5%, less than about 0.6%, less than about 0.7%, less than about 0.8%, less than about 0.9%, less than about 1%, less than about 2%, less than about 3%, less than about 4%, less than about 5%, less than about 6%, less than about 7%, less than about 8%, less than about 9%, or less than about 10% (m / v) macromolecules.

[0109] As used herein, the term "macromolecule" means a nucleic acid, a contaminating protein, a lipid, a carbohydrate, a metabolite, or a combination thereof.

[0110] As used herein, the term "conventional exosomal protein" refers to proteins already known to be enriched in EVs (e.g., exosomes), including, but not limited to, CD9, CD63, CD81, PDGFR, GPI-anchored proteins, lactadherin, LAMP2, and LAMP2B, fragments thereof, or peptides that bind thereto.

[0111] As used herein, the term "linker" refers to any molecular structure that can link a peptide or protein to another molecule (e.g., a different peptide or protein, a small molecule, etc.). Suitable linkers are well known to those skilled in the art and include, but are not limited to, straight or branched chain carbon linkers, heterocyclic carbon linkers, or peptide linkers (see, for example, Chen et al., Advanced Drug Delivery Reviews, 2013, Vol. 65:10, pp. 1357-1369). Linkers can be attached to carboxyl and amino terminal amino acids through their terminal carboxyl or amino groups or through their reactive side groups. In addition, in some embodiments, linkers can be classified as flexible or rigid, and they can be cleavable (e.g., contain one or more protease cleavable sites, which can be located within the sequence of the linker or adjacent to the linker at either end of the linker sequence).

[0112] "Administering" as used herein means providing a composition comprising EVs, e.g., exosomes, disclosed herein to a subject via a pharma- ceutically acceptable route. The administration route can be intravenous, e.g., intravenous injection and intravenous infusion. Additional administration routes include, e.g., subcutaneous, intramuscular, oral, nasal, and pulmonary administration. The EVs, e.g., exosomes, can be administered as part of a pharmaceutical composition comprising at least one excipient.

[0113] As used herein, "immune response" refers to a biological response in a vertebrate to a foreign substance or an abnormal, e.g., cancerous, cell, which protects the organism from these substances and the diseases caused by them. The immune response is mediated by the action of one or more cells of the immune system (e.g., T lymphocytes, B lymphocytes, natural killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells, or neutrophils) and soluble macromolecules (including antibodies, cytokines, and complement) produced by either these cells or the liver, resulting in selective targeting, binding, damaging, destroying, and / or elimination of invading pathogens from the body of a vertebrate organism, pathogen-infected cells or tissues, cancer cells or other abnormal cells, or, in the case of autoimmunity or pathological inflammation, normal human cells or tissues. The immune response can include, for example, the activation or inhibition of T cells, e.g., effector T cells, Th cells, CD4+ cells, CD8+ T cells, or Treg cells, or the activation or inhibition of any other cells of the immune system, e.g., NK cells. Thus, an immune response can include a humoral immune response (e.g., mediated by B cells), a cellular immune response (e.g., mediated by T cells), or both a humoral and a cellular immune response. In some embodiments, the immune response is an "inhibitory" immune response. An inhibitory immune response is an immune response that blocks or reduces the effect of a stimulus (e.g., an antigen). In certain embodiments, an inhibitory immune response includes the production of inhibitory antibodies against the stimulus. In some embodiments, the immune response is a "stimulatory" immune response. A stimulatory immune response is an immune response that results in the generation of effector cells (e.g., cytotoxic T lymphocytes) that can destroy and eliminate a target antigen (e.g., a tumor antigen or a virus).

[0114] As used herein, "treat", "treatment" or "treating" refers to, for example, reducing the severity of a disease or condition, reducing the duration of a disease process, improving or eliminating one or more symptoms associated with a disease or condition, providing a beneficial effect to a subject with a disease or condition without necessarily curing the disease or condition. The term also includes prevention or prophylaxis of a disease or condition or its symptoms. In one embodiment, the term "treat" or "treatment" refers to inducing an immune response in a subject against an antigen.

[0115] As used herein, "prevent" or "preventing" refers to reducing or lessening the occurrence or severity of a particular outcome. In some embodiments, preventing an outcome is achieved through prophylactic treatment.

[0116] 7.2. Other Interpretive Conventions Ranges recited herein are understood to be shorthand notations for all values ​​within the range, including the recited endpoints. For example, the range of 1 to 50 is understood to include any number, combination of numbers, or subranges from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50.

[0117] The term "about" refers to and encompasses the indicated value and ranges above and below that value. In certain embodiments, the term "about" refers to the specified value ±10%, ±5%, or ±1%. In certain embodiments, where applicable, the term "about" refers to the specified value(s) ±1 standard deviation of that value(s).

[0118] Throughout this disclosure, the term "a" or "an" entity refers to one or more of that entity; for example, "an antibody" is understood to refer to one or more antibodies. Thus, the terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein.

[0119] Furthermore, as used herein, "and / or" should be considered as a specific disclosure of each of two particular features or components, whether or not the other is present. Thus, the term "and / or" used in phrases such as "A and / or B" is intended to include "A and B," "A or B," "A" (single), and "B" (single). Similarly, the term "and / or" used in phrases such as "A, B, and / or C" is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (single); B (single); and C (single).

[0120] 7.3.EV (e.g., exosome) proteins Aspects of the present disclosure relate to the identification, use, and modification of heterologous exosomal vesicle proteins (HEVPs) that are highly enriched in the membrane of EVs (e.g., exosomes) produced from specific producing cells. As described herein, different cells (e.g., mesenchymal stem cells and HEK293) naturally express different proteins. EVs (e.g., exosomes) produced from cells express one or more proteins that are naturally expressed in the producing cells. Thus, EVs (e.g., exosomes) produced from different producing cells can have different protein compositions. HEVPs can be identified by analyzing highly purified EVs (e.g., exosomes) using mass spectrometry or other methods known in the art.

[0121] The HEVPs of the present disclosure include various membrane proteins, such as transmembrane proteins, integral membrane proteins, and peripheral proteins, that are enriched in the EV (e.g., exosome) membrane. They include various CD proteins, transporters, integrins, lectins, and cadherins. Specifically, the proteins include, but are not limited to, CD13, MME, ENPP1, and NRP1. In some embodiments, the HEVP is CD13 (or a fragment or variant thereof). In some embodiments, the HEVP is MME (or a fragment or variant thereof). In some embodiments, the HEVP is ENPP1 (or a fragment or variant thereof). In some embodiments, the HEVP is NRP1 (or a fragment or variant thereof). In some embodiments, the HEVP is a type II transmembrane protein. Non-limiting examples of type II transmembrane proteins that may be used with the present disclosure include CD252, CD154, CD178, CD70, CD153, CD137, CD253, CD254, CD256, CD257, CD258, TL1, GITRL, and combinations thereof.

[0122] The present disclosure shows that exosomal vesicle proteins (e.g., HEVPs) that are expressed only in EVs derived from a certain cell type (e.g., EVs from MSCs) can be engineered to be expressed in EVs derived from other cell types that do not naturally express the exosomal vesicle protein (e.g., EVs from HEK293). One or more HEVPs identified herein can be selectively used depending on the producing cell, production conditions, purification method, or intended application of the EVs (e.g., exosomes). For example, HEVPs enriched in a particular population of EVs (e.g., exosomes) can be used to purify a particular population of EVs (e.g., exosomes). HEVPs enriched on the surface of a particular EV (e.g., exosomes) with a particular size range, targeting moiety, charge density, payload, etc. can be identified and used in some embodiments of the present disclosure. In some embodiments, two or more HEVPs can be used simultaneously or sequentially for the generation, purification, and isolation of therapeutic EVs (e.g., exosomes).

[0123] 7.4. Surface- and / or Luminally Engineered EVs (e.g., Exosomes) Another aspect of the present disclosure relates to the production and use of surface-engineered EVs (e.g., exosomes). In some embodiments, the present disclosure relates to the production and use of luminally engineered EVs (e.g., exosomes). Surface-engineered and / or luminally engineered EVs (e.g., exosomes) have membranes that are modified in composition. For example, their membrane composition can be modified by changing the protein, lipid, or glycan content of the membrane.

[0124] In some embodiments, surface-engineered and / or luminally engineered EVs (e.g., exosomes) are generated by chemical and / or physical methods, such as PEG-induced fusion and / or ultrasonic fusion.

[0125] In other embodiments, surface-engineered EVs and / or luminal engineered EVs (e.g., exosomes) are produced by genetic engineering. EVs (e.g., exosomes) produced from genetically modified producer cells or descendants of genetically modified cells can contain modified membrane compositions. In some embodiments, genetically modified producer cells or descendants of genetically modified cells contain one or more exogenous proteins that are not naturally found in the cell. In certain embodiments, the one or more exogenous proteins are scaffold moieties, such as heterologous exosome vesicle proteins (HEVPs) disclosed herein. In some embodiments, surface-engineered and / or luminal engineered EVs (e.g., exosomes) have a higher or lower density of HEVPs (compared to the density of HEVP expression in cells that naturally express HEVPs), or contain mutants or fragments of HEVPs.

[0126] For example, surface-engineered and / or lumen-engineered EVs (e.g., exosomes) can be produced from cells transformed with an exogenous sequence encoding a HEVP or a variant or fragment of a HEVP. EVs (e.g., exosomes) containing proteins expressed from the exogenous sequence can include modified membrane protein compositions.

[0127] Various modifications or fragments of HEVPs can be used for the embodiments of the present disclosure. For example, proteins modified to have enhanced affinity for binding agents can be used to generate surface-engineered and / or luminal-engineered EVs (e.g., exosomes) that can be purified using the binding agents. Proteins modified to target EVs (e.g., exosomes) and / or membranes more effectively can be used. Proteins modified to contain the minimal fragments required for specific and effective targeting to EV (e.g., exosome) membranes can also be used. In some embodiments, HEVPs (including fragments and variants thereof) capable of immobilizing cargo or any other exogenous biologically active molecules (e.g., those disclosed herein) can be used in constructing surface-engineered and / or luminal-engineered EVs (e.g., exosomes). In certain embodiments, HEVPs (including fragments and variants thereof) capable of immobilizing certain classes of proteins can be used.

[0128] For example, in some embodiments, the HEVP is a type I transmembrane protein, and such HEVP can be used to anchor the extracellular domain of a type I protein to an EV (e.g., an exosome). In certain embodiments, the expression of the extracellular domain of a type I protein on an EV (e.g., an exosome) is increased when anchored to a type I transmembrane HEVP compared to the corresponding expression when the protein is anchored to a different type of scaffold moiety (e.g., a non-type I transmembrane HEVP) or compared to the corresponding expression when the protein is overexpressed in an EV (e.g., exosome)-producing cell that naturally expresses the protein.

[0129] In some embodiments, the HEVP is a type II transmembrane protein, and such HEVP can be used to anchor the extracellular domain of a type II protein to an EV (e.g., an exosome). In certain embodiments, the expression of the extracellular domain of a type II protein on an EV (e.g., an exosome) is increased when anchored to a type II transmembrane HEVP, compared to the corresponding expression when the protein is anchored to a different type of scaffold moiety (e.g., a non-type II transmembrane HEVP), or compared to the corresponding expression when the protein is overexpressed in an EV (e.g., exosome)-producing cell that naturally expresses the protein.

[0130] In some embodiments, the HEVP is a type III transmembrane protein, and such HEVP can be used to anchor the extracellular domain of a type III protein to an EV (e.g., an exosome). In certain embodiments, the expression of the extracellular domain of a type III protein on an EV (e.g., an exosome) is increased when the protein is anchored to a type III transmembrane HEVP, compared to the corresponding expression when the protein is anchored to a different type of scaffold moiety (e.g., a non-type III transmembrane HEVP), or compared to the corresponding expression when the protein is overexpressed in an EV (e.g., an exosome)-producing cell that naturally expresses the protein.

[0131] In some embodiments, the HEVP is a type IV transmembrane protein, and such HEVP can be used to anchor the extracellular domain of a type IV protein to an EV (e.g., an exosome). In certain embodiments, the expression of the extracellular domain of a type IV protein on an EV (e.g., an exosome) is increased when the protein is anchored to a type IV transmembrane HEVP, compared to the corresponding expression when the protein is anchored to a different type of scaffold moiety (e.g., a non-type IV transmembrane HEVP), or compared to the corresponding expression when the protein is overexpressed in an EV (e.g., an exosome)-producing cell that naturally expresses the protein.

[0132] Fusion proteins can also be used, for example HEVP or fragments thereof fused to affinity tags (e.g., His tag, GST tag, glutathione-S-transferase, S-peptide, HA, Myc, FLAG™ (Sigma-Aldrich Co.), MBP, SUMO, and protein A) can be used for purification or removal of surface engineered EVs (e.g., exosomes) using binding agents specific for the affinity tag.

[0133] Fusion proteins with therapeutic activity can also be used for the generation of surface engineered EVs (e.g., exosomes). Thus, in some embodiments, the EVs (e.g., exosomes) disclosed herein are engineered or modified to express a fusion protein and can be used to deliver one or more (e.g., two, three, four, five or more) therapeutic molecules to a target. For example, the fusion protein can include a HEVP (e.g., CD13, MME, ENPP1, or NRP1, or a fragment or variant thereof) and a therapeutic compound (e.g., a peptide). In some embodiments, the fusion protein includes CD13 (or a fragment or variant thereof) and a therapeutic compound. In some embodiments, the fusion protein includes Mme (or a fragment or variant thereof) and a therapeutic compound. In some embodiments, the fusion protein includes ENPP1 (or a fragment or variant thereof) and a therapeutic compound. In some embodiments, the fusion protein includes NRP1 (or a fragment or variant thereof) and a therapeutic compound. In some embodiments, the fusion protein includes a therapeutic compound that is fused directly to the HEVP. In some embodiments, the therapeutic compound is attached to the HEVP via a linker (e.g., those disclosed herein).

[0134] In some embodiments, the linker is a peptide linker. In some embodiments, the peptide linker can comprise at least about 2, at least about 3, at least about 4, at least about 5, at least about 10, at least about 15, at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least about 55, at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, at least about 90, at least about 95, or at least about 100 amino acids.

[0135] In some embodiments, the peptide linker is synthetic, i.e., non-naturally occurring. In some embodiments, the peptide linker comprises a peptide (or polypeptide) (e.g., a naturally occurring or non-naturally occurring peptide) that comprises an amino acid sequence that links or genetically fuses a first linear sequence of amino acids to a second linear sequence of amino acids that is not naturally linked or genetically fused in nature. For example, in some embodiments, the peptide linker can comprise a non-naturally occurring polypeptide that is a modified form of a naturally occurring polypeptide (e.g., includes mutations such as additions, substitutions, or deletions).

[0136] The linker may be susceptible to cleavage (a "cleavable linker"), thereby facilitating release of the exogenous biologically active molecule (e.g., a targeting moiety, a therapeutic molecule, an adjuvant, or an immunomodulator).

[0137] In some embodiments, the linker is a "reduction sensitive linker." In some embodiments, the reduction sensitive linker contains a disulfide bond. In some embodiments, the linker is an "acid labile linker." In some embodiments, the acid labile linker contains a hydrazone. Suitable acid labile linkers also include, for example, a cis aconite linker, a hydrazide linker, a thiocarbamoyl linker, or any combination thereof.

[0138] In some embodiments, the linker comprises a non-cleavable linker.

[0139] In some embodiments, the therapeutic peptide is selected from the group consisting of a natural peptide, a recombinant peptide, a synthetic peptide, or a linker to the therapeutic compound. The therapeutic compound can be a nucleotide, an amino acid, a lipid, a carbohydrate, or a small molecule. The therapeutic peptide can be an antibody, an enzyme, a ligand, a receptor, an antimicrobial peptide, or a fragment or variant thereof. In some embodiments, the therapeutic peptide is a nucleic acid binding protein. The nucleic acid binding protein can be Dicer, an Argonaute protein, TRBP, or MS2 bacteriophage coat protein. In some embodiments, the nucleic acid binding protein additionally comprises one or more RNA or DNA molecules. The one or more RNAs can be miRNA, siRNA, antisense oligonucleotide, phosphorodiamidate morpholino oligomer (PMO), peptide-conjugated phosphorodiamidate morpholino oligomer (PPMO), guide RNA, lincRNA, mRNA, antisense RNA, dsRNA, or a combination thereof.

[0140] In some embodiments, the therapeutic peptide is part of a protein-protein interaction system. In some embodiments, the protein-protein interaction system is the FRB-FKBP interaction system, e.g., as described in Banaszynski et al. al., J Am Chem Soc. 2005 Apr 6;127(13):4715-21.

[0141] In some embodiments, the therapeutic molecule that can be immobilized in the HEVP and expressed in the EV (e.g., exosome) comprises an antigen. In certain embodiments, the antigen comprises a tumor antigen. Non-limiting examples of tumor antigens include alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), epithelial tumor antigen (ETA), mucin 1 (MUC1), Tn-MUC1, mucin 16 (MUC16), tyrosinase, melanoma associated antigen (MAGE), tumor protein p53 (p53), CD4, CD8, CD45, CD80, CD86, programmed death-ligand 1 (PD-L1), programmed death-ligand 2 (PD-L2), NY-ESO-1, PSMA, TAG-72, HER2, GD2, cMET, EGFR, mesothelin, VEGFR, alpha folate receptor, CE7R, IL-3, cancer testis antigen (CTA), MART-1 gp100, TNF-related apoptosis-inducing ligand, brachyury (preferably melanoma expressed antigen (PRAME)), or combinations thereof. In further embodiments, the antigen can comprise a neo-antigen. As used herein, the term "neo-antigen" refers to an antigen encoded by a tumor-specific mutated gene. In some embodiments, the antigen is derived from a bacterium, a virus, a fungus, a protozoan, or any combination thereof. In some embodiments, the antigen is derived from an oncogenic virus.In further embodiments, the antigen is selected from the group consisting of human gamma herpesvirus 4 (Epstein-Barr virus), influenza A virus, influenza B virus, cytomegalovirus, staphylococcus aureus, mycobacterium tuberculosis, chlamydia trachomatis, HIV-1, HIV-2, coronaviruses (e.g., MERS-CoV and SARS CoV), filoviruses (e.g., Marburg and Ebola), Streptococcus pyogenes, Streptococcus pneumoniae, Plasmodia species (e.g., vivax and falciparum), chikungunya virus, human papilloma virus (HPV), hepatitis B, hepatitis C, human herpes virus 8, herpes simplex virus 2 (HSV2), Klebsiella species, Pseudomonas aeruginosa, Enterococcus spp., Proteus spp., Enterobacter spp., Actinobacter spp., coagulase-negative staphylococci (CoNS), Mycoplasma spp., or combinations thereof.

[0142] In some embodiments, the therapeutic molecule comprises an immunosuppressant. Thus, in certain embodiments, the EV disclosed herein comprises a HEVP and an immunosuppressant.

[0143] Other non-limiting examples of suitable therapeutic molecules include pharmacologically active drugs and genetically active molecules, including anti-tumor agents, anti-inflammatory agents, hormones or hormone antagonists, ion channel modulators, and neuroactive agents. Examples of suitable payloads of therapeutic agents include those described in "The Pharmacological Basis of Therapeutics," Goodman and Gilman, McGraw-Hill, New York, NY, (1996), Ninth edition, in the section: Drugs Acting at Synaptic and Neuroeffector Junctional Sites, Drugs Acting on the Central Nervous System, Autacoids, Drug Therapy of Inflammation, Water, Salts and Ions, Drugs Affecting Renal Function and Electrolyte Metabolism, Cardiovascular Drugs, Drugs Affecting Gastrointestinal Function, Drugs Affecting Uterine Motility, Chemotherapy of Parasitic Infections, Chemotherapy of Microbial Diseases, Chemotherapy of Neoplastic Diseases, Drugs Used for Immunosuppression, Drugs Acting on Blood-Forming organs, Hormones and Hormone Antagonists, Vitamins, Dermatology; and Toxicology, all of which are incorporated herein by reference. Suitable payloads further include toxins, and biological and chemical warfare agents, see, for example, Somani, SM (ed.), Chemical Warfare Agents, Academic Press, New York (1992).

[0144] In some embodiments, the therapeutic molecule comprises an autoantigen. As used herein, the term "autoantigen" refers to an antigen expressed by a host cell or tissue. Under normal health conditions, such antigens are recognized by the body as self and do not elicit an immune response. However, under certain disease conditions, the body's own immune system may recognize autoantigens as foreign and initiate an immune response against them, resulting in autoimmunity. In certain embodiments, the EVs, e.g., exosomes, of the present disclosure can comprise an autoantigen (i.e., a self (germline) protein against which a T cell response is induced, resulting in autoimmunity). Such EVs, e.g., exosomes, can be used to target autoreactive T cells and suppress their activity. Non-limiting examples of autoantigens (including associated diseases or disorders) include beta cell proteins (type I diabetes), myelin oligodendrocyte glycoprotein (MOG, multiple sclerosis), synovial proteins (rheumatoid arthritis), or combinations thereof.

[0145] In some embodiments, the therapeutic molecule comprises an antibody or an antigen-binding fragment thereof. In some embodiments, the therapeutic molecule comprises at least two, at least three, at least four, or at least five antibodies or antigen-binding fragments thereof. In some embodiments, the antibody or antigen-binding fragment thereof comprises an scFv, scFab, scFab-Fc, nanobody, or any combination thereof. In some embodiments, the antibody or antigen-binding fragment thereof comprises an agonist antibody, a blocking antibody, a targeting antibody, a fragment thereof, or a combination thereof. In some embodiments, the agonist antibody is a CD40L agonist. In some embodiments, the blocking antibody binds to a target protein selected from programmed death 1 (PD-1), programmed death-ligand 1 (PD-L1), cytotoxic T-lymphocyte-associated protein 4, and any combination thereof. In some embodiments, the EV, e.g., exosome, comprises an anti-IL12 antibody, or an antigen-binding fragment thereof, and an anti-CD40L antibody, or an antigen-binding fragment thereof.

[0146] The fusion protein can target the surface of an EV (e.g., an exosome) and provide a therapeutic activity to the EV (e.g., an exosome). In some embodiments, the fusion protein does not include IGSF8 or a fragment or modification thereof.

[0147] In some embodiments, a fusion protein with a targeting moiety is used. For example, the fusion protein can include a HEVP (e.g., CD13, MME, ENPP1, or NRP1, or a fragment or variant thereof) and a targeting moiety. In some embodiments, the fusion protein includes CD13 and a targeting moiety. In some embodiments, the fusion protein includes MME and a targeting moiety. In some embodiments, the fusion protein includes ENPP1 and a targeting moiety. In some embodiments, the fusion protein includes NRP1 and a targeting moiety. The targeting moiety can be used to target EVs (e.g., exosomes) to a specific organ, tissue, or cell for therapy using EVs (e.g., exosomes). In certain embodiments, the targeting moiety binds to a marker (or target molecule) expressed on a cell or cell population. In certain embodiments, the marker is expressed on multiple cell types, for example, all antigen-presenting cells (e.g., dendritic cells, macrophages, and B lymphocytes). In some embodiments, the marker is expressed only on a specific cell population (e.g., dendritic cells). Non-limiting examples of markers expressed on specific cell populations (e.g., dendritic cells) include C-type lectin domain family 9 member A (CLEC9A) protein, dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN), CD207, CD40, Clec6, dendritic cell immunoreceptor (DCIR), DEC-205, lectin-like oxidized low density lipoprotein receptor-1 (LOX-1), MARCO, Clec12a, DC-asialoglycoprotein receptor (DC-ASGPR), DC immunoreceptor 2 (DCIR2), Dectin-1, macrophage mannose receptor (MMR), BDCA-1 (CD303, Clec4c), Dectin-2, Bst-2 (CD317), or any combination thereof.

[0148] In some embodiments, the targeting moiety is an antibody or an antigen-binding fragment thereof. Antibodies and their antigen-binding fragments include whole antibodies, polyclonal, monoclonal and recombinant antibodies, fragments thereof, and further include single chain antibodies, humanized antibodies, murine antibodies, chimeric, mouse-human, mouse-primate, primate-human monoclonal antibodies, anti-idiotypic antibodies, antibody fragments such as, for example, scFv, (scFv)2, Fab, Fab', and F(ab')2, and F(ab1)2, Fv, dAb, and Fd fragments, diabodies, and antibody-related polypeptides. Antibodies and their antigen-binding fragments also include bispecific and multispecific antibodies, so long as they exhibit the desired biological activity or function.

[0149] In some embodiments, the EVs (e.g., exosomes) described herein can comprise a HEVP (e.g., CD13, MME, ENPP1, or NRP1, or a fragment or variant thereof) and one or more exogenous biologically active molecules. In certain embodiments, the exogenous biologically active molecule that can be expressed in an EV (e.g., exosome) is an adjuvant. Thus, in some embodiments, the EVs (e.g., exosomes) comprise CD13 (or a fragment or variant thereof) and an adjuvant. In some embodiments, the EVs (e.g., exosomes) comprise MME (or a fragment or variant thereof) and an adjuvant. In some embodiments, the EVs (e.g., exosomes) comprise ENPP1 (or a fragment or variant thereof) and an adjuvant. In some embodiments, the EVs (e.g., exosomes) comprise NRP1 (or a fragment or variant thereof) and an adjuvant. In some embodiments, the EVs (e.g., exosomes) disclosed herein comprise two, three, four, five, or more different adjuvants. As used herein, the term "adjuvant" refers to any substance that enhances the therapeutic effect of a cargo (eg, increases the immune response to an antigen).

[0150] In some embodiments, the adjuvant useful for the present disclosure induces the activation of cytoplasmic pattern recognition receptors. Non-limiting examples of cytoplasmic pattern recognition receptors include stimulator of interferon genes (STING), retinoic acid-inducible gene I (RIG-1), melanoma differentiation-associated protein 5 (MDA5), nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain-containing (NLRP), inflammasome, or combinations thereof. In certain embodiments, the adjuvant is a STING agonist. Stimulator of interferon genes (STING) is a cytoplasmic sensor of cyclic dinucleotides typically produced by bacteria. When activated, it leads to the production of type I interferons, initiating an immune response. In certain embodiments, the STING agonist includes a cyclic dinucleotide STING agonist or a non-cyclic dinucleotide STING agonist.

[0151] In some embodiments, the adjuvant comprises a toll-like receptor (TLR) agonist. Non-limiting examples of TLR agonists include TLR2 agonists (e.g., lipoteichoic acid, untyped LPS, MALP-2 and MALP-404, OspA, porin, LcrV, lipomannan, GPI anchor, lysophosphatidylserine, lipophosphoglycan (LPG), glycophosphatidylinositol (GPI), zymosan, hsp60, gH / gL glycoprotein, hemagglutinin), TLR3 agonists (e.g., bifunctional agonists, stranded RNA, e.g., poly(I:C)), TLR4 agonists (e.g., lipopolysaccharide (LPS), lipoteichoic acid, beta-defensin 2, fibronectin EDA, HMGB1, snapin, tenascin C), TLR5 agonists (e.g., flagellin), TLR6 agonists, TLR7 / 8 agonists (e.g., single stranded RNA, CpG-A, polyG10, polyG3, resiquimod), TLR9 agonists (e.g., unmethylated CpG DNA), and combinations thereof. Non-limiting examples of TLR agonists can be found in WO2008 / 115319A2, US2013 / 0202707A1, US2012 / 0219615A1, US2010 / 0029585A1, WO2009 / 030996A1, WO2009 / 088401A2, and WO2011 / 044246A1, each of which is incorporated by reference in its entirety.

[0152] In some embodiments, the fusion protein does not include IGSF8 or a fragment or modification thereof.

[0153] In some embodiments, the EVs (e.g., exosomes) described herein can comprise a HEVP (e.g., CD13, MME, ENPP1, or NRP1, or a fragment or variant thereof) and one or more exogenous biologically active molecules, where the one or more exogenous biologically active molecules comprise one or more (e.g., two, three, four, five or more) immunomodulators. In some embodiments, the EVs (e.g., exosomes) comprise CD13 (or a fragment or variant thereof) and an immunomodulator. In some embodiments, the EVs (e.g., exosomes) comprise MME (or a fragment or variant thereof) and an immunomodulator. In some embodiments, the EVs (e.g., exosomes) comprise ENPP1 (or a fragment or variant thereof) and an immunomodulator. In some embodiments, the EVs (e.g., exosomes) comprise NRP1 (or a fragment or variant thereof) and an immunomodulator. In certain embodiments, the one or more immunomodulators are expressed in combination with other exogenous biologically active molecules (e.g., targeting moieties, therapeutic molecules, and / or adjuvants) disclosed herein.

[0154] In some embodiments, the immunomodulator comprises an inhibitor of a negative checkpoint regulator or an inhibitor of a binding partner of a negative checkpoint regulator. In certain embodiments, the negative checkpoint regulator comprises cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), programmed cell death protein 1 (PD-1), lymphocyte activation gene 3 (LAG-3), T-cell immunoglobulin mucin-containing protein 3 (TIM-3), B and T lymphocyte attenuation factor (BTLA), T cell immunoreceptor with Ig and ITIM domains (TIGIT), V domain Ig inhibitor of T cell activation (VISTA), adenosine A2a receptor (A2aR), killer cell immunoglobulin-like receptor (KIR), indoleamine 2,3-dioxygenase (IDO), CD20, CD39, CD73, or a combination thereof.

[0155] In some embodiments, the immunomodulator is an inhibitor of cytotoxic T-lymphocyte-associated protein 4 (CTLA-4). In certain embodiments, the CTLA-4 inhibitor is a monoclonal antibody to CTLA-4 ("anti-CTLA-4 antibody"). In certain embodiments, the inhibitor is a fragment of a monoclonal antibody to CTLA-4. In certain embodiments, the antibody fragment is an scFv, (scFv)2, Fab, Fab', and F(ab')2, F(ab1)2, Fv, dAb, or Fd of a monoclonal antibody to CTLA-4. In certain embodiments, the inhibitor is a nanobody, bispecific antibody, or multispecific antibody against CTLA-4. In some embodiments, the anti-CTLA-4 antibody is ipilimumab. In other embodiments, the anti-CTLA-4 antibody is tremelimumab.

[0156] In some embodiments, the immunomodulator is an inhibitor of programmed cell death protein 1 (PD-1). In some embodiments, the immunomodulator is an inhibitor of programmed death-ligand 1 (PD-L1). In some embodiments, the immunomodulator is an inhibitor of programmed death-ligand 2 (PD-L2). In certain embodiments, the PD-1, PD-L1, or PD-L2 inhibitor is a monoclonal antibody to PD-1 (an "anti-PD-1 antibody"), a monoclonal antibody to PD-L1 (an "anti-PD-L1 antibody"), or a monoclonal antibody to PD-L2 (an "anti-PD-L2 antibody"). In some embodiments, the inhibitor is a fragment of an anti-PD-1 antibody, an anti-PD-L1 antibody, or an anti-PD-L2 antibody. In certain embodiments, the antibody fragment is an scFv, (scFv)2, Fab, Fab', and F(ab')2, F(ab1)2, Fv, dAb, or Fd of a monoclonal antibody to PD-1, PD-L1, or PD-L2. In certain embodiments, the inhibitor is a nanobody, bispecific antibody, or multispecific antibody to PD-1, PD-L1, or PD-L2. In some embodiments, the anti-PD-1 antibody is nivolumab. In some embodiments, the anti-PD-1 antibody is pembrolizumab. In some embodiments, the anti-PD-1 antibody is pidilizumab. In some embodiments, the anti-PD-L1 antibody is atezolizumab. In other embodiments, the anti-PD-L1 antibody is avelumab.

[0157] In some embodiments, the immunomodulator is an inhibitor of lymphocyte activation gene 3 (LAG3). In certain embodiments, the inhibitor of LAG3 is a monoclonal antibody to LAG3 (an "anti-LAG3 antibody"). In some embodiments, the inhibitor is a fragment of an anti-LAG3 antibody, such as an scFv, (scFv)2, Fab, Fab', and F(ab')2, F(ab1)2, Fv, dAb, or Fd. In certain embodiments, the inhibitor is a nanobody, bispecific antibody, or multispecific antibody against LAG3.

[0158] In some embodiments, the immunomodulator is an inhibitor of T cell immunoglobulin mucin-containing protein 3 (TIM-3). In some embodiments, the immunomodulator is an inhibitor of B and T lymphocyte attenuation factor (BTLA). In some embodiments, the immunomodulator is an inhibitor of T cell immunoreceptor with Ig and ITIM domains (TIGIT). In some embodiments, the immunomodulator is an inhibitor of V domain Ig suppressor of T cell activation (VISTA). In some embodiments, the immunomodulator is an inhibitor of adenosine A2a receptor (A2aR). In some embodiments, the immunomodulator is an inhibitor of killer cell immunoglobulin-like receptor (KIR). In some embodiments, the immunomodulator is an inhibitor of indoleamine 2,3-dioxygenase factor (IDO). In some embodiments, the immunomodulator is an inhibitor of CD20, CD39, or CD73.

[0159] In some embodiments, the immunomodulator comprises an activator for a positive costimulatory molecule or an activator for a binding partner of a positive costimulatory molecule. In certain embodiments, the positive costimulatory molecule comprises a TNF receptor superfamily member (e.g., CD120a, CD120b, CD18, OX40, CD40, Fas receptor, M68, CD27, CD30, 4-1BB, TRAILR1, TRAILR2, TRAILR3, TRAILR4, RANK, OCIF, TWEAK receptor, TACI, BAFF receptor, ATAR, CD271, CD269, AITR, TROY, CD358, TRAMP, and XEDAR). In some embodiments, an activator for a positive costimulatory molecule is a TNF superfamily member (e.g., TNFα, TNF-C, OX40L, CD40L, FasL, LIGHT, TL1A, CD27L, Siva, CD153, 4-1BB ligand, TRAIL, RANKL, TWEAK, APRIL, BAFF, CAMLG, NGF, BDNF, NT-3, NT-4, GITR ligand, and EDA-2).

[0160] In some embodiments, the immunomodulator is an activator of TNF receptor superfamily member 4 (OX40). In certain embodiments, the activator of OX40 is an agonist anti-OX40 antibody. In further embodiments, the activator of OX40 is OX40 ligand (OX40L).

[0161] In some embodiments, the immunomodulator is an activator of CD27. In certain embodiments, the activator of CD27 is an agonist anti-CD27 antibody. In other embodiments, the activator of CD27 is CD27 ligand (CD27L).

[0162] In some embodiments, the immunomodulator is an activator of CD40. In certain embodiments, the activator of CD40 is an agonist anti-CD40 antibody. In some embodiments, the activator of CD40 is CD40 ligand (CD40L). In certain embodiments, the CD40L is monomeric CD40L. In other embodiments, the CD40L is trimeric CD40L.

[0163] In some embodiments, the immunomodulator is an activator of glucocorticoid-induced TNFR-related protein (GITR). In certain embodiments, the activator of GITR is an agonist anti-GITR antibody. In other embodiments, the activator of GITR is a natural ligand of GITR.

[0164] In some embodiments, the immunomodulator is an activator of 4-1BB. In a specific embodiment, the activator of 4-1BB is an agonist anti-4-1BB antibody. In a particular embodiment, the activator of 4-1BB is the natural ligand of 4-1BB.

[0165] In some embodiments, the immunomodulator is a Fas receptor (Fas). In such embodiments, the Fas receptor is presented on the surface of an EV, e.g., an exosome. In some embodiments, the immunomodulator is a Fas ligand (FasL). In certain embodiments, the Fas ligand is presented on the surface of an EV, e.g., an exosome. In some embodiments, the immunomodulator is an anti-Fas antibody or an anti-FasL antibody.

[0166] In some embodiments, the immunomodulator is an activator of CD28-superfamily costimulatory molecule. In certain embodiments, the CD28 superfamily costimulatory molecule is ICOS or CD28. In certain embodiments, the immunomodulator is ICOSL, CD80 or CD86.

[0167] In some embodiments, the immunomodulator is an activator of inducible T cell costimulator (ICOS). In certain embodiments, the activator of ICOS is an agonist anti-ICOS antibody. In other embodiments, the activator of ICOS is ICOS ligand (ICOSL).

[0168] In some embodiments, the immunomodulator is an activator of CD28. In some embodiments, the activator of CD28 is an agonist anti-CD28 antibody. In other embodiments, the activator of CD28 is a natural ligand of CD28. In certain embodiments, the ligand of CD28 is CD80.

[0169] In some embodiments, the immunomodulator comprises a cytokine or a binding partner of a cytokine. In certain embodiments, the cytokine comprises IL-2, IL-4, IL-7, IL-10, IL-12, IL-15, IL-21, or IFN-γ. In some embodiments, the immunomodulator comprises FLT-3 (CD135).

[0170] In some embodiments, the immunomodulator comprises a protein that supports the intracellular interaction required for germinal center response.In certain embodiments, such protein comprises signaling lymphocyte activation molecule (SLAM) family member or SLAM-associated protein (SAP).In some embodiments, the SLAM family member comprises SLAM, CD48, CD229 (Ly9), Ly108, 2B4, CD84, NTB-A, CRACC, BLAME, CD2F-10, or combinations thereof.

[0171] In some embodiments, the immunomodulator is a T cell receptor (TCR) or a derivative thereof. In certain embodiments, the immunomodulator is a TCR alpha chain or a derivative thereof. In other embodiments, the immunomodulator is a TCR beta chain or a derivative thereof. In further embodiments, the immunomodulator is a T cell co-receptor or a derivative thereof.

[0172] In some embodiments, the immunomodulator is a chimeric antigen receptor (CAR) or a derivative thereof. In certain embodiments, the CAR binds to one or more of the therapeutic molecules disclosed herein (e.g., tumor antigens such as alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), epithelial tumor antigen (ETA), mucin 1 (MUC1), Tn-MUC1, mucin 16 (MUC16), tyrosinase, melanoma-associated antigen (MAGE), tumor protein p53 (p53), CD4, CD8, CD45, CD80, CD86, programmed death-ligand 1 (PD-L1), programmed death-ligand 2 (PD-L2), NY-ESO-1, PSMA, TAG-72, HER2, GD2, cMET, EGFR, mesothelin, VEGFR, alpha folate receptor, CE7R, IL-3, cancer-testis antigen, MART-1 gp100, and TNF-related apoptosis-inducing ligand).

[0173] In certain embodiments, the immunomodulator is an activator of CD28. In certain embodiments, the active agent is a fragment of a monoclonal antibody to CD28. In certain embodiments, the antibody fragment is an scFv, (scFv)2, Fab, Fab', and F(ab')2, F(ab1)2, Fv, dAb, or Fd of a monoclonal antibody to CD28. In certain embodiments, the active agent is a nanobody, bispecific antibody, or multispecific antibody to CD28.

[0174] In some embodiments, the immunomodulator comprises an NF-κB inhibitor. Non-limiting examples of NF-κB inhibitors that can be used according to the present disclosure include IKK complex inhibitors (e.g., TPCA-1, NF-κB activation inhibitor VI (BOT-64), BMS345541, amlexanox, SC-514 (GK01140), IMD0354, IKK-16), IκB degradation inhibitors (e.g., BAY11-7082, MG-115, MG-132, lactacystin, epoxomicin, parthenolide, carfilzomib, MLN- 4924 (pevonedistat), NF-κB nuclear translocation inhibitors (e.g., JSH-23, rolipram), p65 acetylation inhibitors (e.g., gallic acid, anacardic acid), NF-κB-DNA binding inhibitors (e.g., GYY4137, p-XSC, CV3988, prostaglandin E2 (PGE2)), NF-κB transactivation inhibitors (e.g., LY294002, wortmannin, mesalamine), or combinations thereof. See Gupta, SC, et al. Biochim Biophys Acta 1799:775-787 (2010), which is incorporated by reference herein in its entirety. In further embodiments, the immunomodulator comprises a COX-2 inhibitor, an mTOR inhibitor (e.g., rapamycin and derivatives), a prostaglandin, a nonsteroidal anti-inflammatory drug (NSAID), an anti-leukotriene, or a combination thereof.

[0175] In some embodiments, the immunomodulator is an agonist. In certain embodiments, the agonist is an endogenous agonist, such as a hormone or neurotransmitter. In other embodiments, the agonist is an exogenous agonist, such as a drug. In some embodiments, the agonist is a physical agonist, which can create an agonist response without binding to the receptor. In some embodiments, the agonist is a superagonist, which can generate a greater maximum response than an endogenous agonist. In certain embodiments, the agonist is a full agonist, which has full potency at the receptor. In other embodiments, the agonist is a partial agonist, which has only partial potency at the receptor, compared to a full agonist. In some embodiments, the agonist is an inverse agonist, which can inhibit the constitutive activity of the receptor. In some embodiments, the agonist is a co-agonist, which acts with another co-agonist to produce an effect on the receptor. In certain embodiments, the agonist is an irreversible agonist, which permanently binds to the receptor through the formation of a covalent bond. In certain embodiments, the agonist is a selective agonist for a particular type of receptor.

[0176] In some embodiments, the immunomodulator is an antagonist. In a specific embodiment, the antagonist is a competitive antagonist, which reversibly binds to the receptor at the same binding site as the endogenous ligand or agonist without activating the receptor. A competitive antagonist can affect the amount of agonist required to achieve a maximum response. In other embodiments, the antagonist is a non-competitive antagonist, which binds to the active site of the receptor or the allosteric site of the receptor. A non-competitive antagonist can reduce the magnitude of the maximum response that can be achieved by any amount of agonist. In a further embodiment, the antagonist is an uncompetitive antagonist, which requires receptor activation by agonist before its binding to a separate allosteric binding site.

[0177] In some embodiments, the immunomodulator comprises an antibody or antigen-binding fragment. The immunomodulator can be a full-length protein or a fragment thereof. The antibody or antigen-binding fragment can be derived from a natural source or partially or wholly synthetically produced. In some embodiments, the antibody is a monoclonal antibody. In some of these embodiments, the monoclonal antibody is an IgG antibody. In certain embodiments, the monoclonal antibody is an IgG1, IgG2, IgG3, or IgG4. In some other embodiments, the antibody is a polyclonal antibody. In certain embodiments, the antigen-binding fragment is selected from Fab, Fab', and F(ab')2, F(ab1)2, Fv, dAb, and Fd fragments. In certain embodiments, the antigen-binding fragment is an scFv or (scFv)2 fragment. In certain other embodiments, the antibody or antigen-binding fragment is a NANOBODY® (single domain antibody). In some embodiments, the antibody or antigen-binding fragment is a bispecific or multispecific antibody.

[0178] In various embodiments, the antibody or antigen-binding fragment is fully human. In some embodiments, the antibody or antigen-binding fragment is humanized. In some embodiments, the antibody or antigen-binding fragment is chimeric. In some of these embodiments, the chimeric antibody has non-human V region domains and human C region domains. In some embodiments, the antibody or antigen-binding fragment is non-human, such as murine or veterinary.

[0179] In certain embodiments, the immunomodulator is a polynucleotide. In some of these embodiments, the polynucleotide includes, but is not limited to, mRNA, miRNA, siRNA, antisense oligonucleotide, phosphorodiamidate morpholino oligomer (PMO), peptide-conjugated phosphorodiamidate morpholino oligomer (PPMO), antisense RNA, shRNA, lncRNA, and dsDNA. In some embodiments, the polynucleotide is RNA (e.g., mRNA, miRNA, siRNA, antisense RNA, shRNA, or lncRNA). In some of these embodiments, if the polynucleotide is an mRNA, it can be translated into a desired polypeptide. In some embodiments, the polynucleotide is a microRNA (miRNA) or a pre-miRNA molecule. In some of these embodiments, the miRNA is delivered to the cytoplasm of the target cell, and the miRNA molecule can silence native mRNA in the target cell. In some embodiments, the polynucleotide is a small interfering RNA (siRNA) or a short hairpin RNA (shRNA) that can interfere with the expression of oncogenes or other dysregulated polypeptides. In some of these embodiments, siRNA is delivered to the cytoplasm of target cells, and the siRNA molecule can silence native mRNA in target cells. In some embodiments, the polynucleotide is an antisense RNA that is complementary to the mRNA. In some embodiments, the polynucleotide is a long non-coding RNA (lncRNA) that can control gene expression and regulate disease. In some embodiments, the polynucleotide is a DNA that can be transcribed into RNA. In some of these embodiments, the transcribed RNA can be translated into a desired polypeptide.

[0180] In some embodiments, the immunomodulator is a protein, peptide, glycolipid, or glycoprotein.

[0181] In some embodiments, the EVs (e.g., surface-engineered exosomes) described herein demonstrate superior characteristics compared to EVs (e.g., surface-engineered exosomes) known in the art. For example, the EVs (e.g., surface-engineered exosomes) produced using HEVPs provided herein contain engineered proteins that are more highly enriched on their surface than EVs (e.g., exosomes) in the prior art, e.g., those produced using conventional exosome proteins. In some embodiments, the expression level of the engineered protein is increased (i.e., enriched) by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, or at least about 300% or more compared to the expression of the corresponding protein using conventional exosome proteins.

[0182] Furthermore, in some embodiments, the EVs (e.g., surface engineered exosomes) of the present disclosure can have greater, more specific, or more controlled biological activity compared to EVs (e.g., surface engineered exosomes) known in the art. For example, surface engineered EVs (e.g., exosomes) containing therapeutic or biologically relevant exogenous sequences fused to HEVP proteins or fragments thereof (e.g., CD13, MME, ENPP1, or NRP1, or fragments thereof) described herein can have more desirable engineered properties than fusion to scaffolds known in the art. Scaffold proteins known in the art include tetraspanin molecules (e.g., CD63, CD81, CD9, etc.), lysosome-associated membrane protein 2 (LAMP2 and LAMP2B), platelet-derived growth factor receptor (PDGFR), GPI-anchored proteins, lactadherin and fragments thereof, and peptides having affinity to any of these proteins or fragments thereof. Previously, overexpression of exogenous proteins relied on stochastic or random placement of exogenous proteins on EVs (e.g., exosomes) to produce surface-engineered EVs (e.g., exosomes). This resulted in low levels and unpredictable densities of exogenous proteins (e.g., exosomes) on EVs. Thus, the HEVP proteins and their fragments described herein provide an important advance in novel EV (e.g., exosome) compositions and methods for their production.

[0183] In some embodiments, surface engineered EVs (e.g., exosomes) comprising an exogenous sequence identified herein (e.g., encoding an exogenous biologically active molecule, e.g., an antigen, adjuvant, targeting moiety, and / or immunomodulator) and a fusion protein containing a HEVP have a higher density of fusion protein than similarly engineered EVs (e.g., exosomes) comprising an exogenous sequence conjugated to a conventional EV (e.g., exosome) protein known in the art (e.g., CD9, CD63, CD81, PDGFR, GPI-anchored protein, lactadherin, LAMP2, and LAMP2B, fragments thereof, or peptides that bind thereto). In some embodiments, fusion proteins containing a HEVP described herein are present on the surface of EVs (e.g., exosomes) at about 2-fold, about 4-fold, about 8-fold, about 16-fold, about 32-fold, about 64-fold, about 100-fold, about 200-fold, about 400-fold, about 800-fold, about 1,000-fold, or higher density than fusion proteins on the surface of other EVs (e.g., exosomes) similarly modified using conventional EV (e.g., exosome) proteins. In some embodiments, a fusion protein containing a HEVP described herein is present on the surface of an EV (e.g., an exosome) at about 2 to about 4 times, about 4 to about 8 times, about 8 to about 16 times, about 16 to about 32 times, about 32 to about 64 times, about 64 to about 100 times, about 100 to about 200 times, about 200 to about 400 times, about 400 to about 800 times, about 800 to about 1000 times, or at a higher density than a fusion protein on the surface of another EV (e.g., an exosome) that has been similarly modified using conventional EV (e.g., exosome) proteins.

[0184] In some embodiments, fusion proteins of CD13, MME, ENPP1, or NRP1, variants, fragments, variants of fragments, or modifications thereof are present on the surface of EVs (e.g., exosomes) at about 2-fold, about 4-fold, about 8-fold, about 16-fold, about 32-fold, about 64-fold, about 100-fold, about 200-fold, about 400-fold, about 800-fold, about 1,000-fold, or higher density than other fusion proteins on the surface of EVs (e.g., exosomes) similarly modified with CD9. In some embodiments, fusion proteins of CD13, MME, ENPP1, or NRP1, variants, fragments, variants of fragments, or modifications thereof are present on the surface of EVs (e.g., exosomes) at about 2-fold, about 4-fold, about 8-fold, about 16-fold, about 32-fold, about 64-fold, about 100-fold, about 200-fold, about 400-fold, about 800-fold, about 1,000-fold, or higher density than other fusion proteins on the surface of EVs (e.g., exosomes) similarly modified with CD63. In some embodiments, fusion proteins of CD13, MME, ENPP1, or NRP1, variants, fragments, variants of fragments, or modifications thereof are present on the surface of EVs (e.g., exosomes) at about 2-fold, about 4-fold, about 8-fold, about 16-fold, about 32-fold, about 64-fold, about 100-fold, about 200-fold, about 400-fold, about 800-fold, about 1,000-fold, or higher density than other fusion proteins on the surface of EVs (e.g., exosomes) similarly modified with CD81. In some embodiments, fusion proteins of CD13, MME, ENPP1, or NRP1, variants, fragments, variants of fragments, or modifications thereof are present on the surface of EVs (e.g., exosomes) at about 2-fold, about 4-fold, about 8-fold, about 16-fold, about 32-fold, about 64-fold, about 100-fold, about 200-fold, about 400-fold, about 800-fold, about 1,000-fold, or higher density than other fusion proteins on the surface of EVs (e.g., exosomes) similarly modified using PDGFR.In some embodiments, fusion proteins of CD13, MME, ENPP1, or NRP1, variants, fragments, variants of fragments, or modifications thereof are present on the surface of EVs (e.g., exosomes) at about 2-fold, about 4-fold, about 8-fold, about 16-fold, about 32-fold, about 64-fold, about 100-fold, about 200-fold, about 400-fold, about 800-fold, about 1,000-fold, or higher density than other fusion proteins on the surface of EVs (e.g., exosomes) similarly modified using GPI-anchored proteins. In some embodiments, fusion proteins of CD13, MME, ENPP1, or NRP1, variants, fragments, variants of fragments, or modifications thereof are present on the surface of EVs (e.g., exosomes) at about 2-fold, about 4-fold, about 8-fold, about 16-fold, about 32-fold, about 64-fold, about 100-fold, about 200-fold, about 400-fold, about 800-fold, about 1,000-fold, or higher density than other fusion proteins on the surface of EVs (e.g., exosomes) similarly modified with lactadherin. In some embodiments, fusion proteins of CD13, MME, ENPP1, or NRP1, variants, fragments, variants of fragments, or modifications thereof are present on the surface of EVs (e.g., exosomes) at about 2-fold, about 4-fold, about 8-fold, about 16-fold, about 32-fold, about 64-fold, about 100-fold, about 200-fold, about 400-fold, about 800-fold, about 1,000-fold, or higher density than fusion proteins on the surface of other EVs (e.g., exosomes) similarly modified using LAMP2. In some embodiments, fusion proteins of CD13, MME, ENPP1, or NRP1, variants, fragments, variants of fragments, or modifications thereof are present on the surface of EVs (e.g., exosomes) at about 2-fold, about 4-fold, about 8-fold, about 16-fold, about 32-fold, about 64-fold, about 100-fold, about 200-fold, about 400-fold, about 800-fold, about 1,000-fold, or higher density than fusion proteins on the surface of other EVs (e.g., exosomes) similarly modified using LAMP2B.In some embodiments, the fusion protein of CD13, MME, ENPP1, or NRP1, or a variant, fragment, variant of a fragment, or modification thereof is present on the surface of an EV (e.g., an exosome) at about 2-fold, about 4-fold, about 8-fold, about 16-fold, about 32-fold, about 64-fold, about 100-fold, about 200-fold, about 400-fold, about 800-fold, about 1,000-fold, or higher density than a fusion protein on the surface of another EV (e.g., an exosome) that has been similarly modified using a fragment of a conventional EV (e.g., exosome) protein. In some embodiments, the fusion protein of CD13, MME, ENPP1, or NRP1, variants, fragments, variants of fragments, or modifications thereof is present on the surface of EVs (e.g., exosomes) at about 2-fold, about 4-fold, about 8-fold, about 16-fold, about 32-fold, about 64-fold, about 100-fold, about 200-fold, about 400-fold, about 800-fold, about 1,000-fold, or higher density than fusion proteins on the surface of other EVs (e.g., exosomes) that have been similarly modified using variants of conventional EV (e.g., exosome) proteins.

[0185] In certain embodiments, the fusion protein of CD13, MME, ENPP1, or NRP1, variants, fragments, variants of fragments, or modifications thereof is present on the surface of EVs (e.g., exosomes) at about 2-fold, about 4-fold, about 8-fold, about 16-fold, about 32-fold, about 64-fold, about 100-fold, about 200-fold, about 400-fold, about 800-fold, about 1,000-fold, or higher density than fusion proteins on the surface of other EVs (e.g., exosomes) similarly modified using conventional EV (e.g., exosome) proteins (e.g., tetraspanin molecules such as CD63).

[0186] The fusion proteins provided herein can include a HEVP (e.g., CD13, MME, ENPP1, or NRP1, or a fragment or variant thereof) and an additional peptide (e.g., an exogenous biologically active molecule disclosed herein, such as an antigen, a targeting moiety, an adjuvant, and / or an immunomodulator). The additional peptide can be attached to either the N-terminus or C-terminus of the HEVP or a fragment or variant thereof. The additional peptide can be located inside (in the luminal side) or outside of the EV (e.g., exosome) bound to the HEVP.

[0187] In some embodiments, the fusion protein provided herein comprises a HEVP (e.g., CD13, MME, ENPP1, or NRP1, or a fragment or variant thereof) and two additional peptides (e.g., an exogenous biologically active molecule disclosed herein, such as an antigen, a targeting moiety, an adjuvant, and / or an immunomodulator). For example, in some embodiments, the fusion protein comprises CD13 (or a fragment or variant thereof) and two additional peptides. In some embodiments, the fusion protein comprises MME (or a fragment or variant thereof) and two additional peptides. In some embodiments, the fusion protein comprises ENPP1 (or a fragment or variant thereof) and two additional peptides. In some embodiments, the fusion protein comprises NRP1 (or a fragment or variant thereof) and two additional peptides. Both of the two additional peptides can be attached to either the N-terminus or the C-terminus of the HEVP, or a fragment or variant thereof. In some embodiments, one of the two additional peptides is attached to the N-terminus and the other of the two additional peptides is attached to the C-terminus of the HEVP, or a fragment or variant thereof. The additional peptide can be located inside (in the luminal side) or outside, or both, of the EV (e.g., exosome) bound to the HEVP.

[0188] 7.5. Producer cells for production of surface engineered EVs (e.g., exosomes) The EVs (e.g., exosomes) of the present disclosure can be produced from in vitro grown cells or from subject's body fluids. When the EVs (e.g., exosomes) are produced from in vitro cell culture, various producer cells can be used for the present disclosure, such as HEK293 cells, Chinese hamster ovary (CHO) cells, mesenchymal stem cells (MSCs), HT-1080 cells, MB-231 cells, Raji cells, PER.C6 cells, and CAP cells. In some embodiments, the producer cells are HEK293 cell lines. In some embodiments, the producers are MSCs.

[0189] The producer cells can be genetically modified to include one or more exogenous sequences to produce surface engineered EVs (e.g., exosomes). In some embodiments, the one or more exogenous sequences encode a HEVP disclosed herein. In some embodiments, the one or more exogenous sequences encode an exogenous biologically active molecule (e.g., an antigen, a targeting moiety, an adjuvant, and / or an immunomodulator) disclosed herein. In some embodiments, the one or more exogenous sequences encode both a HEVP and an exogenous biologically active molecule disclosed herein. The genetically modified producer cells can include exogenous sequences introduced by transient or stable transformation. The exogenous sequences can be introduced into the producer cells as a plasmid. The exogenous sequences can be stably integrated into the genomic sequence of the producer cells, at targeted sites, or at random sites. In some embodiments, stable cell lines are generated for the production of surface engineered EVs (e.g., exosomes).

[0190] In some embodiments, the genetically modified production cells disclosed herein express endogenous levels of HEVP. In such embodiments, the exogenous sequence is inserted into the genomic sequence of the production cell and can be located in the middle, upstream (5' end) or downstream (3' end) of the endogenous sequence encoding HEVP. Various methods known in the art can be used for the introduction of exogenous sequence into the production cell. For example, cells modified using various gene editing methods (e.g., homologous recombination, transposon-mediated system, loxP-Cre system, CRISPR / Cas9 or TALEN method) are within the scope of the present disclosure.

[0191] The exogenous sequence can include a sequence encoding a variant or fragment of a HEVP or EV (e.g., exosome) protein. An extra copy of a sequence encoding a HEVP can be introduced to produce a surface-engineered EV (e.g., exosome) with a higher density of HEVP. An exogenous sequence encoding a variant or fragment of a HEVP can be introduced to produce a surface-engineered EV (e.g., exosome) containing a variant or fragment of a HEVP. An exogenous sequence encoding an affinity tag can be introduced to produce a surface-engineered EV (e.g., exosome) containing a fusion protein comprising an affinity tag bound to a HEVP. As described herein, in some embodiments, an exogenous sequence encoding an exogenous biologically active molecule (e.g., an antigen, a targeting moiety, an adjuvant, and / or an immunomodulator) can be introduced to produce a surface-engineered EV (e.g., exosome) containing a fusion protein comprising an exogenous biologically active molecule bound to a HEVP (e.g., directly or via a linker).

[0192] In some embodiments, surface engineered EVs (e.g., exosomes) have a higher density of HEVPs than native EVs (e.g., exosomes) isolated from the same or similar producing cell type. In some embodiments, HEVPs are present at about 2-fold, about 4-fold, about 8-fold, about 16-fold, about 32-fold, about 64-fold, about 100-fold, about 200-fold, about 400-fold, about 800-fold, about 1000-fold, or higher density in surface engineered EVs (e.g., exosomes) than in native EVs (e.g., exosomes). In some embodiments, HEVPs are present in surface-modified EVs (e.g., exosomes) at about 2 to about 4 times, about 4 to about 8 times, about 8 to about 16 times, about 16 to about 32 times, about 32 to about 64 times, about 64 to about 100 times, about 100 to about 200 times, about 200 to about 400 times, about 400 to about 800 times, about 800 to about 1,000 times, or higher density than in native EVs (e.g., exosomes). In some embodiments, the fusion protein comprising HEVP is present in surface-modified EVs (e.g., exosomes) at about 2 to about 4 times, about 4 to about 8 times, about 8 to about 16 times, about 16 to about 32 times, about 32 to about 64 times, about 64 to about 100 times, about 100 to about 200 times, about 200 to about 400 times, about 400 to about 800 times, about 800 to about 1000 times, or at a higher density than the unmodified HEVP in native EVs (e.g., exosomes). In some embodiments, the HEVP fragment or variant is present in a surface-modified EV (e.g., exosome) at about 2 to about 4 times, about 4 to about 8 times, about 8 to about 16 times, about 16 to about 32 times, about 32 to about 64 times, about 64 to about 100 times, about 100 to about 200 times, about 200 to about 400 times, about 400 to about 800 times, about 800 to about 1000 times, or higher density than the unmodified HEVP in the native EV (e.g., exosome).

[0193] In certain embodiments, CD13, a fragment or variant of CD13, or a modification thereof is present in a density that is about 2 to about 4 times, about 4 to about 8 times, about 8 to about 16 times, about 16 to about 32 times, about 32 to about 64 times, about 64 to about 100 times, about 100 to about 200 times, about 200 to about 400 times, about 400 to about 800 times, about 800 to about 1000 times, or higher, in surface engineered EVs (e.g., exosomes) than unmodified CD13 in native EVs (e.g., exosomes). In certain embodiments, the MME, fragment or variant of MME, or modification thereof is present in a density that is about 2 to about 4 times, about 4 to about 8 times, about 8 to about 16 times, about 16 to about 32 times, about 32 to about 64 times, about 64 to about 100 times, about 100 to about 200 times, about 200 to about 400 times, about 400 to about 800 times, about 800 to about 1000 times, or higher in the surface engineered EVs (e.g., exosomes) than the unmodified MME in the native EVs (e.g., exosomes). In certain embodiments, ENPP1, a fragment or variant of ENPP1, or a modification thereof is present in surface-engineered EVs (e.g., exosomes) at about 2 to about 4 times, about 4 to about 8 times, about 8 to about 16 times, about 16 to about 32 times, about 32 to about 64 times, about 64 to about 100 times, about 100 to about 200 times, about 200 to about 400 times, about 400 to about 800 times, about 800 to about 1000 times, or higher density than unmodified ENPP1 in native EVs (e.g., exosomes). In certain embodiments, NRP1, a fragment or variant of NRP1, or a modification thereof is present in surface-engineered EVs (e.g., exosomes) at about 2 to about 4 times, about 4 to about 8 times, about 8 to about 16 times, about 16 to about 32 times, about 32 to about 64 times, about 64 to about 100 times, about 100 to about 200 times, about 200 to about 400 times, about 400 to about 800 times, about 800 to about 1000 times, or at a higher density than unmodified NRP1 in native EVs (e.g., exosomes).

[0194] In some embodiments, the producing cells are further modified to include additional exogenous sequences. For example, additional exogenous sequences can be introduced to regulate endogenous gene expression or to produce EVs (e.g., exosomes) that include a certain polypeptide as a payload. In some embodiments, the producing cells are modified to include two exogenous sequences, one encoding a HEVP or a variant or fragment of a HEVP, and the other encoding a payload. In some embodiments, the producing cells can be further modified to include additional exogenous sequences that confer additional functionality to the EVs (e.g., exosomes), such as specific targeting capability, delivery function, enzymatic function, increased or decreased half-life in vivo, etc. In some embodiments, the producing cells are modified to include two exogenous sequences, one encoding a variant or fragment of a HEVP or EV (e.g., exosome) protein, and the other encoding a protein that confer additional functionality to the EVs (e.g., exosomes).

[0195] In some embodiments, the producing cells are modified to include two exogenous sequences, each of which encodes a fusion protein on the surface of the EV (e.g., exosome). In some embodiments, the surface-engineered EV (e.g., exosome) from the producing cells has a higher density of HEVPs compared to native EV (e.g., exosome) isolated from unmodified cells of the same or similar cell type. In some embodiments, the surface-engineered EV (e.g., exosome) contains HEVPs at about 2-fold, about 4-fold, about 8-fold, about 16-fold, about 32-fold, about 64-fold, about 100-fold, about 200-fold, about 400-fold, about 800-fold, about 1,000-fold, or higher density than native EV (e.g., exosome) isolated from unmodified cells of the same or similar cell type. In some embodiments, the producing cells are further modified to include 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more additional exogenous sequences. As described herein, in some embodiments, the exogenous sequence encodes one or more HEVPs disclosed herein. In some embodiments, the exogenous sequence encodes one or more exogenous biologically active molecules disclosed herein (e.g., antigens, targeting moieties, adjuvants, and / or immunomodulators). In some embodiments, the exogenous sequence encodes both a HEVP and an exogenous biologically active molecule disclosed herein.

[0196] More specifically, surface engineered EVs (e.g., exosomes) can be produced from cells transformed with sequences encoding one or more HEVPs or variants thereof, including, but not limited to, CD13, MME, ENPP1, and NRP1. Any of one or more HEVPs described herein can be expressed in the producing cells from a plasmid, an exogenous sequence inserted into the genome, or other exogenous nucleic acid, such as synthetic messenger RNA (mRNA).

[0197] In some embodiments, one or more HEVPs are expressed in cells transformed with an exogenous sequence encoding its full-length, endogenous form. In some embodiments, such exogenous sequence encodes the CD13 protein of SEQ ID NO: 47. In some embodiments, such exogenous sequence encodes the MME protein of SEQ ID NO: 48. In some embodiments, such exogenous sequence encodes the ENPP1 protein of SEQ ID NO: 49. In some embodiments, such exogenous sequence encodes the NRP1 protein of SEQ ID NO: 50.

[0198] Surface engineered EVs (e.g., exosomes) can be produced from cells transformed with sequences encoding one or more fragments of HEVP, including, but not limited to, CD13, MME, ENPP1, and NRP1. In some embodiments, EVs (e.g., exosomes) are produced from cells transformed with sequences encoding a fragment of CD13. In some embodiments, EVs (e.g., exosomes) are produced from cells transformed with sequences encoding a fragment of MME. In some embodiments, EVs (e.g., exosomes) are produced from cells transformed with sequences encoding a fragment of ENPP1. In some embodiments, EVs (e.g., exosomes) are produced from cells transformed with sequences encoding a fragment of NRP1. In some embodiments, the sequences encode a fragment of HEVP lacking at least about 5, about 10, about 50, about 100, about 200, about 300, about 400, about 500, about 600, about 700, or about 800 amino acids from the N-terminus of the native protein. In some embodiments, the sequence encodes a fragment of a HEVP lacking at least about 5, about 10, about 50, about 100, about 200, about 300, about 400, about 500, about 600, about 700, or about 800 amino acids from the C-terminus of the native protein. In some embodiments, the sequence encodes a fragment of a HEVP lacking at least about 5, about 10, about 50, about 100, about 200, about 300, about 400, about 500, about 600, about 700, or about 800 amino acids from both the N-terminus and the C-terminus of the native protein. In some embodiments, the sequence encodes a fragment of a HEVP lacking one or more functional or structural domains of the native protein.

[0199] In some embodiments, a fragment of a HEVP is fused to one or more heterologous proteins (e.g., exogenous biologically active molecules, such as antigens, targeting moieties, adjuvants, and / or immunomodulators). In some embodiments, the one or more heterologous proteins are fused to the N-terminus of the fragment. In some embodiments, the one or more heterologous proteins are fused to the C-terminus of the fragment. In some embodiments, the one or more heterologous proteins are fused to the N-terminus and C-terminus of the fragment. In some embodiments, the one or more heterologous proteins are mammalian proteins. In some embodiments, the one or more heterologous proteins are human proteins.

[0200] Surface engineered EVs (e.g., exosomes) can be produced from cells transformed with sequences encoding fragments of CD13. In some embodiments, the fragments of CD13 lack one or more functional or structural domains. In some embodiments, the fragments of CD13 are fused to one or more heterologous proteins (e.g., exogenous biologically active molecules, such as antigens, targeting moieties, adjuvants, and / or immunomodulators). One or more heterologous proteins can be fused to the N-terminus of the CD13 fragment. One or more heterologous proteins can be fused to the C-terminus of the CD13 fragment. In some embodiments, one or more heterologous proteins can be fused to both the N-terminus and the C-terminus of the CD13 fragment. In some embodiments, the heterologous protein is a mammalian protein. In some embodiments, the heterologous protein is a human protein. In some embodiments, the heterologous protein fused to the CD13 fragment additionally contains a signal sequence peptide.

[0201] Surface engineered EVs (e.g., exosomes) can be produced from cells transformed with sequences encoding fragments of MME. In some embodiments, the fragments of MME lack one or more functional or structural domains. In some embodiments, the fragments of MME are fused to one or more heterologous proteins (e.g., exogenous biologically active molecules, such as antigens, targeting moieties, adjuvants, and / or immunomodulators). In some embodiments, the one or more heterologous proteins are fused to the N-terminus of the MME fragment. In some embodiments, the one or more heterologous proteins are fused to the C-terminus of the MME fragment. In some embodiments, the one or more heterologous proteins are fused to both the N-terminus and C-terminus of the MME fragment. In some embodiments, the heterologous protein is a mammalian protein. In some embodiments, the heterologous protein is a human protein. In some embodiments, the heterologous protein fused to the MME fragment additionally contains a signal sequence peptide.

[0202] Surface engineered EVs (e.g., exosomes) can be produced from cells transformed with sequences encoding fragments of ENPP1. In some embodiments, the fragments of ENPP1 lack one or more functional or structural domains. In some embodiments, the fragments of ENPP1 are fused to one or more heterologous proteins (e.g., exogenous biologically active molecules, such as antigens, targeting moieties, adjuvants, and / or immunomodulators). In some embodiments, the one or more heterologous proteins are fused to the N-terminus of the ENPP1 fragment. In some embodiments, the one or more heterologous proteins are fused to the C-terminus of the ENPP1 fragment. In some embodiments, the one or more heterologous proteins are fused to both the N-terminus and the C-terminus of the ENPP1 fragment. In some embodiments, the heterologous protein is a mammalian protein. In some embodiments, the heterologous protein is a human protein. In some embodiments, the heterologous protein fused to the ENPP1 fragment additionally contains a signal sequence peptide.

[0203] Surface engineered EVs (e.g., exosomes) can be produced from cells transformed with sequences encoding fragments of NRP1. In some embodiments, the fragments of NRP1 are fused to one or more heterologous proteins (e.g., exogenous biologically active molecules, such as antigens, targeting moieties, adjuvants, and / or immunomodulators). In some embodiments, one or more heterologous proteins are fused to the N-terminus of the NRP1 fragment. In some embodiments, one or more heterologous proteins are fused to the C-terminus of the NRP1 fragment. In some embodiments, one or more heterologous proteins are fused to both the N-terminus and the C-terminus of the NRP1 fragment. In some embodiments, the heterologous protein is a mammalian protein. In some embodiments, the heterologous protein is a human protein. In some embodiments, the heterologous protein fused to the NRP1 fragment additionally contains a signal sequence peptide.

[0204] In some embodiments, the surface engineered EVs (e.g., exosomes) comprise a polypeptide of identical or similar sequence to a full length or fragment of a HEVP protein, including, but not limited to, CD13, MME, ENPP1, and NRP1. In some embodiments, the peptide is at least about 50% identical to a full length or fragment of a HEVP, e.g., at least about 50% identical to SEQ ID NO: 47-50 (i.e., SEQ ID NO: 47, 48, 49, or 50). In some embodiments, the polypeptide is at least about 60% identical to a full length or fragment of a HEVP, e.g., at least about 60% identical to SEQ ID NO: 47-50. In some embodiments, the polypeptide is at least about 70% identical to a full length or fragment of a HEVP, e.g., at least about 70% identical to SEQ ID NO: 47-50. In some embodiments, the polypeptide is at least about 80% identical to a full length or fragment of a HEVP, e.g., at least about 80% identical to SEQ ID NO: 47-50. In some embodiments, the polypeptide is at least about 90% identical to a full length or fragment of HEVP, e.g., at least about 90% identical to SEQ ID NOs: 47-50. In some embodiments, the polypeptide is at least about 95% identical to a full length or fragment of HEVP, e.g., at least about 95% identical to SEQ ID NOs: 47-50. In some embodiments, the polypeptide is at least about 96% identical to a full length or fragment of HEVP, e.g., at least about 96% identical to SEQ ID NOs: 47-50. In some embodiments, the polypeptide is at least about 97% identical to a full length or fragment of HEVP, e.g., at least about 97% identical to SEQ ID NOs: 47-50. In some embodiments, the polypeptide is at least about 98% identical to a full length or fragment of HEVP, e.g., at least about 98% identical to SEQ ID NOs: 47-50. In some embodiments, the polypeptide is at least about 99% identical to a full length or fragment of HEVP, e.g., at least about 99% identical to SEQ ID NOs: 47-50.In some embodiments, the polypeptide is at least about 99.9% identical to a full length or fragment of a HEVP, for example, at least about 99.9% identical to SEQ ID NOs:47-50.

[0205] 7.6.Affinity purification Some embodiments of the present disclosure relate to the isolation, purification, and subfractionation of EVs (e.g., exosomes) using specific binding interactions between proteins enriched in the EV (e.g., exosome) membrane and immobilized binding agents. These methods generally include the steps of (1) applying or loading a sample containing EVs (e.g., exosomes), (2) optionally washing away unbound sample components using an appropriate buffer that maintains the binding interaction between the EV target proteins (e.g., exosomes) and the binding agent, and (3) eluting (dissociating and recovering) the EVs (e.g., exosomes) from the immobilized binding agent by changing the buffer conditions such that the binding interaction no longer occurs.

[0206] Some embodiments relate to a method for removing EVs (e.g., exosomes) from a sample using specific binding interactions between proteins enriched in EV (e.g., exosome) membranes and immobilized binding agents. In this case, EVs (e.g., exosomes) bound to the binding agent are not eluted from the binding agent, and the fraction that does not bind to the binding agent can be collected. This method can be used to purify a sample containing EVs (e.g., exosomes) and non-EV (e.g., exosome) material, such as viruses (e.g., lentiviruses, retroviruses, adeno-associated viruses, or any other enveloped or non-enveloped viruses) or recombinant proteins (e.g., antibodies, enzymes, or other polypeptides), in which the EVs (e.g., exosomes) are contaminating particles. The bound EVs (e.g., exosomes) can remain bound to the binding agent, and the non-EV (e.g., exosome) material is collected, substantially free of EVs (e.g., exosomes).

[0207] The target protein used in this isolation, purification, subfractionation or removal process can be an endogenous protein produced from the genome of the producing cell, a protein introduced into the producing cell by genetic modification, or a protein modified by chemical, physical or other biological methods.In some cases, the protein is a non-mutant protein or a mutant protein, such as a mutant or fragment of an endogenous protein.In some cases, the protein is a fusion protein (such as those described herein).

[0208] Various binding agents with affinity to target proteins can be used for the embodiments of the present disclosure. For example, proteins, peptides, and small molecules with specific affinity to target proteins can be used as binding agents. In some embodiments, the binding agent is obtained from organic or inorganic sources. Examples of binding agents from organic sources include serum proteins, lectins, or antibodies. Examples of binding agents from inorganic sources include boronic acids, metal chelates, and triazine dyes.

[0209] The binding agent can be chemically fixed or bound to a solid support, so that EVs (e.g., exosomes) that have a specific affinity for the binding agent can be bound. Various forms of solid support can be used, such as porous agarose beads, microtiter plates, magnetic beads, or membranes. In some embodiments, the solid support can form a chromatography column and be used for affinity chromatography of EVs (e.g., exosomes).

[0210] In some cases, the isolation, purification, subfractionation and removal of EVs (e.g., exosomes) is performed by column chromatography using a column packed with a binder and a solid support. In some embodiments, a sample containing EVs (e.g., exosomes) is set to flow through the column, a wash buffer is run through the column, and then an elution buffer is applied to the column and collected. These steps can be performed at ambient pressure or with additional pressure.

[0211] In some cases, the isolation, purification, sub-fractionation, and removal of EVs (e.g., exosomes) is performed using batch processing, e.g., the sample is added to a binder bound to a solid support in a vessel, mixed, the solid support is separated, the liquid phase is removed, washed, centrifuged, elution buffer is added, recentrifuged, and the eluent is removed.

[0212] In some cases, hybrid methods can be used, for example, the sample is added to a binder bound to a solid support in a vessel, the solid support bound to the EVs (e.g., exosomes) is subsequently loaded onto a column, and washing and elution are performed on the column.

[0213] In some cases, isolation, purification, subfractionation, and removal of EVs (e.g., exosomes) is performed using binding agents bound to microtiter plates, magnetic beads, or membranes, where the sample is added to a binding agent bound to a solid support, followed by mixing, separating the solid support, removing the liquid phase, washing, removing the wash buffer, adding an elution buffer, and removing the eluent.

[0214] The binding between the binder and the target protein on the EV (e.g., exosome) is performed under various physiological conditions that are optimal for the specific interaction between the binder and the target protein on the EV (e.g., exosome). Elution of the bound entity (e.g., exosome) can be achieved by changing the salt concentration, pH, pI, charge, and ionic strength directly or through a gradient.

[0215] In some embodiments, a sample that has been isolated, purified, or sub-fractionated with one binding agent is subsequently treated with a different binding agent.

[0216] In some embodiments, two or more columns are used in series, with each of the multiple columns containing a different binding agent that is specific for a different target protein.

[0217] In some embodiments, a single column contains multiple binding agents, each specific for a different target protein.

[0218] In some cases, binders and solid supports are reused by the incorporation of periodic sterilization steps, for example, they can be sterilized with a combination of propylene glycol, isopropanol, high ionic strength, and / or sodium hydroxide.

[0219] 7.6.1. Sample preparation The methods described herein can be used to purify, isolate, sub-fractionate, or remove EVs (e.g., exosomes) from various samples that contain EVs (e.g., exosomes). In some embodiments, the sample is a clarified harvest material that contains EVs (e.g., exosomes). In some cases, the sample contains EVs (e.g., exosomes) that have been partially purified by purification methods known in the art. For example, EVs (e.g., exosomes) can be partially purified using ultrafiltration / diafiltration, hydroxylapatite chromatography, hydrophobic interaction chromatography, depth filtration, or ion exchange binding / elution chromatography before applying to a binder for affinity purification.

[0220] In some cases, the partially purified material is further processed to have specific physiological conditions (e.g., pH, temperature, salt concentration, salt type, polarity) for the desired interaction with the binding agent. Samples can be prepared by dilution or concentration to obtain a specific EV (e.g., exosome) concentration, or by adding excipients to change the structure of EV (e.g., exosome). In some cases, the partially purified material is applied to the binding agent without any manipulation.

[0221] 7.6.2.Join The methods described herein require a specific interaction between the target protein of EVs (e.g., exosomes) and the binder. High-throughput screening can be performed by varying salt concentration, pH, and / or reduction of polarity with organic modifiers, ethylene glycol, propylene glycol, or urea, to identify the ideal buffer conditions for specific binding. The interaction between the target protein and the binder can also vary depending on the sample conditions (e.g., amount of sample loaded per volume of chromatography resin, concentration of EVs (e.g., exosomes), concentration of impurities), loading buffer (e.g., pH, salt concentration, type of salt, polarity), and other physical conditions (e.g., temperature). Furthermore, adding excipients that change the structure of EVs (e.g., exosomes) can also change their interaction. In addition, the residence time can be adjusted based on the differential adsorption kinetics between impurities and EVs (e.g., exosomes). Thus, various purification conditions described herein can be tested to identify the ideal conditions for the steps.

[0222] Similar approaches can be used to improve purity and yield and to help enrich, deplete, or isolate subpopulations of EVs (e.g., exosomes). These properties, along with maximizing loading and applying more stringent elution conditions, can be used to further improve the concentration of EVs (e.g., exosomes).

[0223] Elution Elution of EVs (e.g., exosomes) can be achieved by altering salt concentration, pH, and / or polarity with organic modifiers, ethylene glycol, propylene glycol, or urea.

[0224] Selective elution of EVs (e.g., exosomes) can be achieved by increasing the concentration of monovalent cationic halide salts (e.g., sodium chloride, potassium chloride, sodium bromide, lithium chloride, sodium iodide, potassium bromide, lithium bromide, sodium fluoride, potassium fluoride, lithium fluoride, lithium iodide, sodium acetate, potassium acetate, lithium acetate, and potassium iodide), divalent or trivalent salts (e.g., calcium chloride, magnesium chloride, calcium sulfate, sodium sulfate, magnesium sulfate, chromium trichloride, chromium sulfate, sodium citrate, iron(III) chloride, yttrium(III) chloride, potassium phosphate, potassium sulfate, sodium phosphate, ferrous chloride, calcium citrate, magnesium phosphate, and ferric chloride) or combinations thereof in the elution buffer. This is accomplished by increasing at a fixed pH through the use of an increasing gradient (stepwise or linear) of a reactive halogen salt (e.g., sodium chloride, potassium chloride, sodium bromide, lithium chloride, sodium iodide, potassium bromide, lithium bromide, sodium fluoride, potassium fluoride, lithium fluoride, lithium iodide, sodium acetate, potassium acetate, lithium acetate, and potassium iodide), a divalent or trivalent salt (e.g., calcium chloride, magnesium chloride, calcium sulfate, sodium sulfate, magnesium sulfate, chromium trichloride, chromium sulfate, sodium citrate, ferric chloride, yttrium chloride, potassium phosphate, potassium sulfate, sodium phosphate, ferrous chloride, calcium citrate, magnesium phosphate, and ferric chloride), or a combination thereof.

[0225] Substantial EV (e.g., exosome) purity can be achieved by flowing through impurities during the column load step, eluting the impurities during the selective excipient wash, and selectively eluting the product during elution while leaving additional impurities bound to the column. The absorbance measured from the column eluate can indicate the purity of the EV (e.g., exosome) obtained by the method.

[0226] Elution can also be achieved by adjusting the pH range, salts, organic solvents, small molecules, detergents, zwitterions, amino acids, polymers, temperature, and any combination of the above. Similar eluents can be used to improve purity, improve yield, and / or isolate subpopulations of EVs (e.g., exosomes).

[0227] Elution can also be performed using multiple elution buffers with different properties, such as pH, salt, organic solvent, small molecule, detergent, zwitterion, amino acid, polymer, temperature, and any combination of the above. Multiple elution fractions can be collected, and the EVs (e.g., exosomes) collected in each fraction have different properties. For example, the EVs (e.g., exosomes) collected in one fraction have higher purity, smaller or larger average size, more favorable composition, etc. than the EVs (e.g., exosomes) in other fractions.

[0228] Elution buffers with different properties can be applied as a continuous flow, and multiple elution fractions are collected. The elution fractions can be collected during isocratic or gradient elution. Once at least one elution fraction is collected, the composition of the elution fraction can be analyzed. For example, the concentration of EVs (e.g., exosomes), host cell proteins, contaminating proteins, DNA, carbohydrates, or lipids can be measured in each elution fraction. Other properties of the EVs (e.g., exosomes) in each elution fraction can also be measured. These properties include average size, average charge density, and other physiological properties related to biodistribution, cellular uptake, half-life, pharmacodynamics, potency, dosing, immune response, loading efficiency, stability, or reactivity to other compounds.

[0229] Cleaning Optionally, the purity of EVs (e.g., exosomes) can be further improved by washing the sample before elution. In some embodiments, the excipient can be a washing buffer. The excipient can be a solution with a specific pH range, a salt, an organic solvent, a small molecule, a detergent, a zwitterion, an amino acid, a polymer, and any combination of the above.

[0230] More specifically, the excipient can include arginine, lysine, glycine, histidine, calcium, sodium, lithium, potassium, iodide, magnesium, iron, zinc, manganese, urea, propylene glycol, aluminum, ammonium, guanidinium, polyethylene glycol, EDTA, EGTA, surfactants, chloride, sulfate, carboxylate, sialic acid, phosphate, acetate, glycine, borate, formate, perchlorate, bromate, nitrate, dithiothreitol, beta mercaptoethanol, or tri-n-butyl phosphate.

[0231] The excipients may also include surfactants, such as cetyltrimethylammonium chloride, octoxynol-9, TRITON® X-100 (i.e., polyethylene glycol p-(1,1,3,3-tetramethylbutyl)-phenyl ether), and TRITON® CG-110, available from Sigma-Aldrich; sodium dodecyl sulfate; sodium lauryl sulfate; deoxycholic acid; polysorbate 80 (i.e., polyoxyethylene (20) sorbitan monooleate); polysorbate 20 (i.e., polyoxyethylene (20) sorbitan monolaurate); alcohol ethoxylates; alkyl polyethylene glycol ethers; decyl glucoside; octoglucoside; SafeCare; DOW the preferred tertiary ingredient is selected from the group consisting of ECOSURF™ EH9, ECOSURF™ EH6, ECOSURF™ EH3, ECOSURF™ SA7, and ECOSURF™ SA9 available from Lubrizol Chemical; LUTENSOL™ M5, LUTENSOL™ XL, LUTENSOL™ XP, and APG™ 325N available from BASF; TOMADOL™ 900 available from AIR PRODUCTS; NATSURF™ 265 available from CRODA; SAFECARE™ 1000 available from Bestchem; TERGITOL™ L64 available from DOW; caprylic acid; CHEMBETAINE™ LEC available from Lubrizol; and Mackol DG.

[0232] 7.6.3. Other ways to improve results The amount of EVs (e.g., exosomes) that can be loaded per volume of chromatography resin can be improved by adjusting the feed material, for example, by increasing the concentration of EVs (e.g., exosomes), decreasing the concentration of impurities, changing the pH, decreasing the salt concentration, decreasing the ionic strength, or changing a specific subpopulation of EVs (e.g., exosomes). Due to mass transfer limitations and the slow adsorption and desorption of EVs (e.g., exosomes) on the resin, the amount of EVs (e.g., exosomes) that can be loaded per volume of chromatography resin can be improved by slowing the flow rate during column loading, by using longer columns, and by increasing the residence time.

[0233] 7.7. Application 7.7.1. Purification of EVs (e.g., exosomes) The use of EVs (e.g., exosomes) for medical purposes requires that the EVs (e.g., exosomes) are free or nearly free of impurities, including but not limited to macromolecules such as nucleic acids, contaminating proteins, lipids, carbohydrates, metabolites, small molecules, metals, or combinations thereof. The present disclosure provides methods for purifying EVs (e.g., exosomes) from contaminating macromolecules. In some embodiments, the purified EVs (e.g., exosomes) are substantially free of contaminating macromolecules.

[0234] 7.7.2. Subfractionation of EVs (e.g., exosomes) Embodiments of the present disclosure further provide methods for subfractionating populations of EVs (e.g., exosomes) based on their membrane proteins, size, charge density, ligand type (e.g., tetraspanins), and heparin or other sulfated carbohydrate binding sites. Selection of affinity tags, loading and elution buffer compositions and protocols can result in the elution of different subpopulations of EVs (e.g., exosomes).

[0235] For example, embodiments of the present disclosure provide a method for purifying a population of EVs (e.g., exosomes) having smaller or larger sizes. The size of EVs (e.g., exosomes) can be determined by methods available in the art. For example, size can be measured by nanoparticle tracking analysis, multi-angle light scattering, single-angle light scattering, size exclusion chromatography, analytical ultracentrifugation, field-flow fractionation, laser diffraction, tunable resistive pulse sensing, or dynamic light scattering.

[0236] Embodiments of the present disclosure further relate to methods of sub-fractionating EVs (e.g., exosomes) based on their charge density, which can be determined by potentiometric titration, anion exchange, cation exchange, isoelectric focusing, zeta potential, capillary electrophoresis, capillary zone electrophoresis, gel electrophoresis, or any combination thereof.

[0237] Embodiments of the present disclosure also relate to sub-fractionating EVs (e.g., exosomes) based on other physiological properties such as biodistribution, cellular uptake, half-life, pharmacodynamics, efficacy, dosing, immune response, loading efficiency, stability, or responsiveness to other compounds. The methods allow for the isolation of a population of EVs (e.g., exosomes) that are suitable for a particular application.

[0238] 7.7.3.Use In some embodiments, the present disclosure provides a method of preventing and / or treating a disease or disorder in a subject in need thereof, comprising administering the EVs (e.g., exosomes) disclosed herein.

[0239] In some embodiments, the disease or disorder that can be treated by the method includes cancer, hemophilia, diabetes, growth factor deficiency, eye disease, graft-versus-host disease (GvHD), autoimmune disease, gastrointestinal disease, cardiovascular disease, respiratory disease, allergic disease, degenerative disease, infectious disease, fibrotic disease, or any combination thereof. In certain embodiments, the disease or disorder that can be treated is associated with chronic inflammation. In some embodiments, the treatment is preventative. In some embodiments, the EVs (e.g., exosomes) of the present disclosure are used to induce immune responses. In some embodiments, the EVs of the present disclosure are used to vaccinate a subject.

[0240] In some embodiments, the disease or disorder is cancer. In some embodiments, the disease or disorder is graft-versus-host disease (GvHD). In some embodiments, the disease or disorder is an infectious disease.

[0241] As described herein, a cell (e.g., a production cell) can be modified to express a protein (e.g., a HEVP) that is not naturally expressed in the cell (i.e., a heterologous protein). In some embodiments, such modification results in EVs (e.g., exosomes) being produced from the modified cell to express the heterologous protein. Thus, in some embodiments, the present disclosure provides a method of expressing a non-naturally occurring protein (i.e., a heterologous protein) in an EV (e.g., an exosome) derived from a cell. In some embodiments, the method includes transfecting a cell with a nucleic acid encoding at least one heterologous protein (e.g., a HEVP disclosed herein) (or a fragment or variant thereof) and isolating an EV (e.g., an exosome) produced from the cell, wherein the EV (e.g., an exosome) comprises at least one heterologous protein, and the heterologous protein is not naturally expressed in the EV derived from the cell.

[0242] In some embodiments, the EVs (e.g., exosomes) produced from the cells (i.e., transfected with a nucleic acid encoding at least one heterologous protein) contain at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten heterologous proteins. In certain embodiments, the heterologous proteins are different (e.g., exosomes are engineered to express both CD13 and MME proteins, and the exosomes are produced from cells that do not naturally express CD13 and MME proteins). In some embodiments, the heterologous proteins are the same, and the total amount of heterologous proteins expressed in the EVs (e.g., exosomes) is greater than EVs produced from cells that naturally express the heterologous proteins.

[0243] Any suitable method known in the art can be used to quantify the amount or level of a heterologous protein (e.g., HEVP) expressed in EVs (e.g., exosomes). In some embodiments, the amount of a protein (e.g., HEVP) expressed in EVs (e.g., exosomes) can be assessed by measuring the number of peptide spectral matches in a given sample containing EVs (e.g., exosomes) using liquid chromatography with tandem mass spectrometry (LC-MS / MS). See Examples 1 and 3. As used herein, "peptide spectral match" (PSM) refers to a system of assigning a numerical value to a peptide spectral pair (P,S) that represents the likelihood that fragmentation of a peptide with sequence P will be recorded in an experimental mass spectrum (S. Frank, A.M., J Proteome Res, 8(5):2241-2252 (May 2009). PSM correlates with protein abundance in a given sample.

[0244] In some embodiments, the level (or amount) of a heterologous protein (e.g., HEVP) expressed in EVs (e.g., exosomes) produced from a cell disclosed herein (e.g., transfected with a nucleic acid encoding at least one heterologous protein) is at least about 10 peptide spectral matches (PSMs), at least about 20 PSMs, at least about 30 PSMs, at least about 40 PSMs, at least about 50 PSMs, at least about 60 PSMs, at least about 70 PSMs, at least about 80 PSMs, at least about 90 PSMs, at least about 100 PSMs, at least about 110 PSMs, at least about 120 PSMs, at least about 130 PSMs, at least about 140 PSMs, at least about 150 PSMs, at least about 160 PSMs, at least about 170 PSM, at least about 180 PSM, at least about 190 PSM, at least about 200 PSM, at least about 210 PSM, at least about 220 PSM, at least about 230 PSM, at least about 240 PSM, at least about 250 PSM, at least about 260 PSM, at least about 270 PSM, at least about 280 PSM, at least about 290 PSM, at least about 300 PSM, at least about 350 PSM, at least about 400 PSM, at least about 450 PSM, at least about 500 PSM, at least about 550 PSM, at least about 600 PSM, at least about 650 PSM, at least about 700 PSM, at least about 750 PSM, at least about 800 PSM, at least about 850 PSM, at least about 900 PSM, at least about 950 PSM, or at least about 1,000 PSM or more.

[0245] In certain embodiments, the level of a heterologous protein (e.g., HEVP) expressed in an EV (e.g., exosome) is about 125 PSM or more when measured using LC-MS / MS. In certain embodiments, the level of a heterologous protein (e.g., HEVP) expressed in an EV (e.g., exosome) is about 150 PSM or more when measured using LC-MS / MS. In some embodiments, the level of a heterologous protein (e.g., HEVP) expressed in an EV (e.g., exosome) is about 200 PSM or more when measured using LC-MS / MS. In certain embodiments, the level of a heterologous protein (e.g., HEVP) expressed in an EV (e.g., exosome) is about 700 PSM or more when measured using LC-MS / MS. In some embodiments, the level of a heterologous protein (e.g., HEVP, e.g., MME) expressed in an EV (e.g., exosome) is about 177 PSM when measured using LC-MS / MS. In some embodiments, the level of a heterologous protein (e.g., a HEVP, e.g., CD13) expressed in an EV (e.g., an exosome) is about 742 PSM as measured using LC-MS / MS.

[0246] In some embodiments, the level of a heterologous protein (e.g., HEVP) expressed in an EV (e.g., exosome) is about 20 to about 80 PSM when measured using LC-MS / MS. In some embodiments, the level of a heterologous protein (e.g., HEVP) expressed in an EV (e.g., exosome) is about 80 to about 200 PSM when measured using LC-MS / MS. In some embodiments, the level of a heterologous protein (e.g., HEVP) expressed in an EV (e.g., exosome) is about 150 PSM to about 750 PSM when measured using LC-MS / MS.

[0247] 7.8. Characterization of EVs (e.g., exosomes) In some embodiments, the methods described herein further comprise characterizing the EVs (e.g., exosomes) contained in each collected fraction. In some embodiments, the contents of the EVs (e.g., exosomes) can be extracted for testing and characterization. In some embodiments, the EVs (e.g., exosomes) are isolated and characterized by metrics including, but not limited to, size, shape, morphology, or molecular composition, such as nucleic acids, proteins, metabolites, and lipids.

[0248] 7.8.1. Measurement of EV (e.g., exosome) content EVs (e.g., exosomes) can contain proteins, peptides, RNA, DNA, and lipids. Total RNA can be extracted using acid-phenol:chloroform extraction. RNA can then be purified using glass fiber filters under conditions that recover small RNA-containing total RNA or under conditions that separate small RNA species less than 200 nucleotides in length from longer RNA species such as mRNA. Because RNA is eluted in small amounts, in some embodiments, an alcohol precipitation step is not required for isolation of RNA.

[0249] EV (e.g., exome) composition can be assessed by methods known in the art, including but not limited to transcriptomics, sequencing, proteomics, mass spectrometry, or HP-LC.

[0250] The composition of nucleotides (including RNA and DNA) associated with isolated EVs (e.g., exosomes) can be measured using a variety of techniques well known to those skilled in the art (e.g., quantitative or semi-quantitative RT-PCR, Northern blot analysis, solution hybridization detection). In certain embodiments, the level of at least one RNA is measured by reverse transcribing RNA from an EV (e.g., exosome) composition to provide a set of target oligodeoxynucleotides, hybridizing the target oligodeoxynucleotides to one or more RNA-specific probe oligonucleotides (e.g., a microarray containing the RNA-specific probe oligonucleotides) to provide a hybridization profile of the EV (e.g., exosome) composition, and comparing the hybridization profile of the EV (e.g., exosome) composition to a hybridization profile generated from a control sample. A change in the signal of at least one RNA in the test sample relative to the control sample is indicative of the RNA composition.

[0251] Microarrays can also be prepared from gene-specific oligonucleotide probes generated from known RNA sequences. The array can contain two different oligonucleotide probes for each RNA, one containing the active, mature sequence and the other specific for the precursors of the RNA (e.g., miRNA and pre-miRNA). The array can also contain controls, such as one or more mouse sequences that differ from the human orthologues by only a few bases, which can serve as controls for stringency conditions of hybridization. tRNAs and other RNAs (e.g., rRNA, mRNA) from both species can also be printed on the microchip, providing an internal, relatively stable positive control for specific hybridization. One or more suitable controls for non-specific hybridization can also be included on the microchip. For this purpose, sequences are selected on the basis of the absence of any homology with any known RNA.

[0252] Microarrays can be assembled using techniques known in the art. For example, a probe oligonucleotide of suitable length, e.g., 40 nucleotides, is 5'-amine modified at position C6 and printed onto an activated slide using a commercially available microarray system, e.g., GeneMachine OmniGrid™ 100 Microarray and Amersham CodeLink™. A labeled cDNA oligomer corresponding to the target RNA is prepared by reverse transcribing the target RNA with a labeled primer. After first strand synthesis, the RNA / DNA hybrid is denatured to degrade the RNA template. The target cDNA thus prepared is then hybridized to the microarray chip under hybridization conditions, e.g., 6x SSPE / 30% formamide at 25°C for 18 hours, followed by 0.75x TNT at 37°C for 40 minutes. Hybridization occurs at positions on the array where the immobilized probe DNA recognizes a complementary target cDNA in the sample. The labeled target cDNA indicates the exact position on the array where binding occurs, allowing for automated detection and quantification. The output consists of a list of hybridization events, indicating the relative abundance of specific cDNA sequences, and therefore the relative abundance of the corresponding complementary RNA, in the EV (e.g., exosome) preparation. According to one embodiment, the labeled cDNA oligomer is a biotin-labeled cDNA, prepared from a biotin-labeled primer. The microarray is then processed by direct detection of biotin-containing transcripts, for example, using a streptavidin-Alexa647 conjugate, and scanned using conventional scanning methods. The image intensity of each spot on the array is proportional to the abundance of the corresponding RNA in the EV (e.g., exosome).

[0253] Data mining work is completed by bioinformatics including scanning chip, signal acquisition, image processing, normalization, statistical processing and data comparison, and pathway analysis. Therefore, microarrays can simultaneously profile hundreds and thousands of polynucleotides with high throughput performance. Microarray profiling analysis of mRNA expression has been successful in providing valuable data for gene expression testing in basic research. And this technology is further put into practical use in pharmaceutical industry and clinical diagnosis. As an increasing amount of miRNA data becomes available and evidence of the importance of miRNA in gene regulation accumulates, microarrays become a useful technology for high throughput miRNA testing. Analysis of miRNA levels utilizing polynucleotide probes can also be performed in various physical formats. For example, the use of microtiter plates or automation can be used to facilitate the processing of a large number of test samples.

[0254] 7.8.2. Measuring the size of EVs (e.g., exosomes) In some embodiments, the methods described herein include measuring the size of the EVs (e.g., exosomes) and / or populations of EVs (e.g., exosomes) contained in the purified fraction. In some embodiments, EV (e.g., exosome) size is measured as the longest measurable dimension. In general, the longest general dimension of an EV (e.g., exosome) is also referred to as its diameter.

[0255] EV (e.g., exosome) size can be measured using a variety of methods known in the art, such as nanoparticle tracking analysis, multi-angle light scattering, single-angle light scattering, size-exclusion chromatography, analytical ultracentrifugation, field-flow fractionation, laser diffraction, tunable resistive pulse sensing, or dynamic light scattering.

[0256] EV (e.g., exosome) size can be measured using dynamic light scattering (DLS) and / or multi-angle light scattering (MALS). Methods of using DLS and / or MALS to measure EV (e.g., exosome) size are known to those of skill in the art and include nanoparticle tracking assays (NTA, e.g., using a Malvern NanoSight NS300 nanoparticle tracking device). In a specific embodiment, EV (e.g., exosome) size is determined using a Malvern NanoSight NS300. In some embodiments, the EV (e.g., exosome) described herein has a longest dimension of about 20-1000 nm when measured by NTA (e.g., using a Malvern Nanosight NS300). In other embodiments, the EV (e.g., exosome) described herein has a longest dimension of about 40-1000 nm when measured by NTA (e.g., using a Malvern Nanosight NS300). In other embodiments, the EVs (e.g., exosomes) described herein comprise a population where 90% of the EVs (e.g., exosomes) have a longest dimension of about 20-1000 nm when measured by NTA (e.g., using a Malvern Nanosight NS300). In other embodiments, the EVs (e.g., exosomes) described herein comprise a population where 95% of the EVs (e.g., exosomes) have a longest dimension of about 20-1000 nm when measured by NTA (e.g., using a Malvern Nanosight NS300). In other embodiments, the EVs (e.g., exosomes) described herein comprise a population where 99% of the EVs (e.g., exosomes) have a longest dimension of about 20-1000 nm when measured by NTA (e.g., using a Malvern Nanosight NS300). In other embodiments, the EVs (e.g., exosomes) described herein comprise a population, where 90% of the EVs (e.g., exosomes) have a longest dimension of about 40-1000 nm as measured by NTA (e.g., using a Malvern Nanosight NS300).In other embodiments, the EVs (e.g., exosomes) described herein comprise a population where 95% of the EVs (e.g., exosomes) have a longest dimension of about 40-1000 nm as measured by NTA (e.g., using a Malvern Nanosight NS300). In other embodiments, the EVs (e.g., exosomes) described herein comprise a population where 99% of the EVs (e.g., exosomes) have a longest dimension of about 40-1000 nm as measured by NTA (e.g., using a Malvern Nanosight NS300).

[0257] EV (e.g., exosome) size can be measured using tunable resistive pulse sensing (TRPS). In a specific embodiment, EV (e.g., exosome) size measured by TRPS is determined using iZON qNANO Gold. In some embodiments, the EV (e.g., exosome) described herein has a longest dimension of about 20-1000 nm as measured by TRPS (e.g., using iZON qNano Gold). In other embodiments, the EV (e.g., exosome) described herein has a longest dimension of about 20-1000 nm as measured by TRPS (e.g., using iZON qNano Gold). In another embodiment, the EVs (e.g., exosomes) described herein comprise a population, where 90% of the EVs (e.g., exosomes) have a longest dimension of about 20-1000 nm when measured by TRPS (e.g., using iZON qNano Gold). In another embodiment, the EVs (e.g., exosomes) described herein comprise a population, where 95% of the EVs (e.g., exosomes) have a longest dimension of about 20-1000 nm when measured by TRPS (e.g., using iZON qNano Gold). In another embodiment, the EVs (e.g., exosomes) described herein comprise a population, where 99% of the EVs (e.g., exosomes) have a longest dimension of about 20-1000 nm when measured by TRPS (e.g., using iZON qNano Gold). In other embodiments, the EVs (e.g., exosomes) described herein comprise a population, where 90% of the EVs (e.g., exosomes) have a longest dimension of about 40-1000 nm when measured by TRPS (e.g., using iZON qNano Gold). In other embodiments, the EVs (e.g., exosomes) described herein comprise a population, where 95% of the EVs (e.g., exosomes) have a longest dimension of about 40-1000 nm when measured by TRPS (e.g., using iZON qNano Gold). In other embodiments, the EVs (e.g., exosomes) described herein comprise a population, where 99% of the EVs (e.g., exosomes) have a longest dimension of about 40-1000 nm when measured by TRPS (e.g., using iZON qNano Gold).

[0258] EV (e.g., exosome) size can be measured using electron microscopy. In some embodiments, the electron microscopy used to measure EV (e.g., exosome) size is transmission electron microscopy. In a specific embodiment, the transmission electron microscope used to measure EV (e.g., exosome) size is a Tecnai™ G2 Spirit BioTWIN. Methods for measuring EV (e.g., exosome) size using electron microscopy are well known to those of skill in the art, and any such method may be suitable for measuring EV (e.g., exosome) size. In some embodiments, the EV (e.g., exosome) described herein has a longest dimension of about 20-1000 nm as measured by scanning electron microscope (e.g., Tecnai™ G2 Spirit BioTWIN scanning electron microscope). In other embodiments, the EV (e.g., exosome) described herein has a longest dimension of about 40-1000 nm as measured by scanning electron microscope (e.g., Tecnai™ G2 Spirit BioTWIN scanning electron microscope). In other embodiments, the EV (e.g., exosome) populations described herein include populations where 90% of the EVs (e.g., exosomes) have a longest dimension of about 20-1000 nm as measured by scanning electron microscopy (e.g., a Tecnai™ G2 Spirit BioTWIN Scanning Electron Microscope). In other embodiments, the EV (e.g., exosome) populations described herein include populations where 95% of the EVs (e.g., exosomes) have a longest dimension of about 20-1000 nm as measured by scanning electron microscopy (e.g., a Tecnai™ G2 Spirit BioTWIN Scanning Electron Microscope). In other embodiments, the EV (e.g., exosome) populations described herein include populations where 99% of the EVs (e.g., exosomes) have a longest dimension of about 20-1000 nm as measured by scanning electron microscopy (e.g., a Tecnai™ G2 Spirit BioTWIN Scanning Electron Microscope).In other embodiments, the EV (e.g., exosome) populations described herein include populations where 90% of the EVs (e.g., exosomes) have a longest dimension of about 40-1000 nm as measured by scanning electron microscopy (e.g., a Tecnai™ G2 Spirit BioTWIN Scanning Electron Microscope). In other embodiments, the EV (e.g., exosome) populations described herein include populations where 95% of the EVs (e.g., exosomes) have a longest dimension of about 40-1000 nm as measured by scanning electron microscopy (e.g., a Tecnai™ G2 Spirit BioTWIN Scanning Electron Microscope). In other embodiments, the EV (e.g., exosome) populations described herein include populations where 99% of the EVs (e.g., exosomes) have a longest dimension of about 40-1000 nm as measured by scanning electron microscopy (e.g., a Tecnai™ G2 Spirit BioTWIN Scanning Electron Microscope).

[0259] 7.8.3. Measuring charge density of EVs (e.g., exosomes) In some embodiments, the methods described herein include measuring the charge density of the EVs (e.g., exosomes) and / or populations of EVs (e.g., exosomes) contained in the purified fraction. In some embodiments, the charge density is measured by potentiometric titration, anion exchange, cation exchange, isoelectric focusing, zeta potential, capillary electrophoresis, capillary zone electrophoresis, or gel electrophoresis.

[0260] 7.8.4. Measurement of EV (e.g., exosome) protein or HEVP density In some embodiments, the methods described herein include measuring the density of EV (e.g., exosome) proteins or HEVPs on the surface of EVs (e.g., exosomes). Surface density can be calculated or expressed as mass per unit area, number of proteins per area, number of molecules or intensity of molecular signal per EV (e.g., exosome), molar amount of protein, etc. Surface density can be measured experimentally by methods known in the art, for example, by biolayer interferometry (BLI), FACS, Western blotting, fluorescence (e.g., GFP fusion protein) detection, nanoflow cytometry, ELISA, αLISA, and / or densitometry by measuring bands on a protein gel.

[0261] Pharmaceutical Compositions Provided herein is a pharmaceutical composition comprising the EVs of the present disclosure, e.g., exosomes, having a desired purity, in a form suitable for administration to a subject, and a pharma- ceutically acceptable carrier or excipient. The pharma-ceutically acceptable excipient or carrier is determined, in part, by the particular composition to be administered, as well as by the particular method used to administer the composition. Thus, there are a wide variety of suitable formulations of pharmaceutical compositions comprising a plurality of extracellular vesicles. (See, e.g., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa. 21st ed. (2005)) Pharmaceutical compositions are generally formulated sterile and in full compliance with all Good Manufacturing Practice (GMP) regulations of the U.S. Food and Drug Administration.

[0262] In some embodiments, the pharmaceutical composition comprises one or more therapeutic agents described herein and EVs, e.g., exosomes. In certain embodiments, the EVs, e.g., exosomes, are co-administered with one or more additional therapeutic agents in a pharma- ceutically acceptable carrier. In some embodiments, the pharmaceutical composition comprising EVs, e.g., exosomes, is administered prior to administration of the additional therapeutic agent. In other embodiments, the pharmaceutical composition comprising EVs, e.g., exosomes, is administered after administration of the additional therapeutic agent. In further embodiments, the pharmaceutical composition comprising EVs, e.g., exosomes, is administered simultaneously with the additional therapeutic agent.

[0263] 7.10.Kit Also provided herein are kits comprising one or more exosomes described herein. In some embodiments, provided herein are pharmaceutical packs or kits comprising one or more containers filled with one or more of the components of the pharmaceutical compositions described herein, such as one or more exosomes provided herein, and optionally instructions for use. In some embodiments, the kit comprises a pharmaceutical composition described herein and any prophylactic or therapeutic agent, such as those described herein. EXAMPLES

[0264] 7.11 Example The following examples are presented to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present disclosure, and are not intended to limit the scope of what the inventors regard as their present disclosure, nor are they intended to represent that the following experiments are all or the only experiments actually performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.), but some experimental error and deviation should be accounted for. Unless otherwise indicated, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric pressure. Standard abbreviations can be used, such as bp, base pair(s); kb, kilobase(s); pl, picoliters(s); s or sec, seconds(s); min, minutes(s); h or hr, hours(s); aa, amino acid(s); nt, nucleotide(s), etc.

[0265] The practice of the present invention employs, unless otherwise indicated, conventional methods of protein chemistry, biochemistry, recombinant DNA technology, and pharmacology, within the skill of the art. Such techniques are fully explained in the literature, see, for example, TECreighton, Proteins: Structures and Molecular Properties (WH Freeman and Company, 1993); A.L. Lehninger, Biochemistry (Worth Publishers, Inc., current addition); Sambrook, et al., Molecular Cloning: A Laboratory Manual (2nd Edition, 1989); Methods In Enzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.); Remington's Pharmaceutical Sciences, 21st Edition (Easton, Pennsylvania: Mack Publishing Company, 2005); Carey and Sundberg Advanced Organic Chemistry 3rd Ed. (Plenum Press) Vols. See A and B(l992).

[0266] 7.11.1. Example 1: Identification of EV (e.g., exosome) proteins 7.11.1.1. Collection of EVs (e.g., exosomes) EVs (e.g., exosomes) were harvested from the supernatant of high-density suspension cultures of bone marrow-derived MSC cells after 4 days. The supernatant was filtered and concentrated by tangential flow filtration using a 1,000 kDa MWCO membrane (PXC01MC50). The concentrated cell culture supernatant was pelleted by ultracentrifugation using 100 mL Quick-Seal Ultra-Clear tubes (345778). The crude EV (e.g., exosomes) pellet was further fractionated on an OPTIPREP™ (60% iodixanol w / v) density gradient by ultracentrifugation.

[0267] The pelleted material was resuspended in 1 mL of PBS and 3 mL of OPTIPREP™ to a final iodixanol concentration of 45%. For the OPTIPREP™ gradient, a 4-layer sterile gradient was prepared in a 12 mL Ultra-Clear (344059) tube for a SW 41 Ti rotor with 4 mL of 45% iodixanol, 3 mL of 30% iodixanol, 2 mL of 22.5% iodixanol, 2 mL of 17.5% iodixanol, and 1 mL of PBS containing the resuspended material. The OPTIPREP™ was ultracentrifuged at 150,000×g for 16 hours at 4° C. to separate the EV (e.g., exosome) fraction. The EV (e.g., exosome) layer was then gently collected from the top approx. 3 mL of the tube.

[0268] The EV (e.g., exosome) fraction was diluted into approximately 32 mL of PBS in a 38.5 mL Ultra-Clear (344058) tube and ultracentrifuged at 133,900 x g for 3 hours at 4°C to pellet the purified EVs (e.g., exosomes). The pelleted EVs (e.g., exosomes) were then resuspended in a minimal volume of PBS (approximately 200 μL) and stored at 4°C.

[0269] 7.11.1.2. Sample preparation for LC-MS / MS analysis To determine proteins specific to EVs (e.g., exosomes), the top and bottom fractions of the Optiprep™ gradient were analyzed by liquid chromatography-tandem mass spectrometry. All samples were supplemented with either phosphate-buffered saline (PBS) buffer or PBS and 5% sucrose. Prior to analysis, the total protein concentration of each sample was determined by bicinchoninic acid (BCA) assay, and then each sample was appropriately diluted to 125 μg / mL with PBS buffer. Next, 50.0 μL of each sample was added to a separate 1.5 mL microcentrifuge tube containing an equal volume of EV (e.g., exosome) lysis buffer (60 mM Tris, 400 mM GdmCl, 100 mM EDTA, 20 mM TCEP, 1.0% TritonX-100), followed by the transfer of 2.0 μL of 1.0% TritonX-100 solution. All samples were then incubated at 55° C. for 60 minutes.

[0270] Protein precipitation was performed by adding 1250 μL of ethanol at −20° C. To improve efficiency, the samples were vortexed vigorously for about 10 minutes and then incubated at −20° C. for 60 minutes. After incubation, the samples were sonicated in a water bath for 5 minutes. The precipitated material was pelleted by centrifugation at 15,000 g for 5 minutes at 4° C. The supernatant was decanted and the pelleted material was completely dried using nitrogen gas. The pellet was resuspended in 30.0 μL of digestion buffer (30 mM Tris, 1.0 M GdmCl, 100 mM EDTA, 50 mM TCEP, pH 8.5) and disulfide bonds were reduced. Free cysteine ​​residues were alkylated by adding 5.0 μL of alkylation solution (375 mM iodoacetamide, 50 mM Tris, pH 8.5) and incubating the resulting solution at room temperature in the dark for at least 30 minutes.

[0271] After incubation, each sample was diluted using 30.0 μL of 50 mM Tris pH 8.5 and proteolytic digestion was initiated by adding 2.0 μg of trypsin. All samples were mixed and then incubated overnight at 37° C. After incubation, trypsin activity was stopped by adding 5.0 μL of 10% formic acid. Prior to analysis by LC-MS / MS, each sample was desalted using a Pierce C18 spin column. At the end of this process, each sample was dried, reconstituted with 50.0 μL of water containing 0.1% formic acid, and transferred to an HPLC vial for analysis.

[0272] 7.11.1.3.LC-MS / MS analysis Samples were analyzed using an UltiMate3000 RSCLnano (Thermo Fisher Scientific) The peptides were injected into a Thermo Fisher Scientific (TMS) low-flow chromatography system and the tryptic peptides were loaded onto an Acclaim PepMap100 C18 trapping column (75 μm×2 cm, 3 μm particle size, 100 Å pore size, Thermo Fisher Scientific) using a loading mobile phase (MPL: water, 0.1% formic acid) at a flow rate of 1.000 μL / min. The peptides were eluted and separated on an EASY-Spray C18 analytical column (75 μm×25 cm, 2 μm particle size, 100 Å pore size, Thermo Fisher Scientific) with a gradient of mobile phase A (MPA: water, 0.1% formic acid) and mobile phase B (MPB: acetonitrile, 0.1% formic acid) at a flow rate of 300 nL / min. The step gradient used for elution started with 2% MPB and was held for 8 min during loading. The percentage of MPB was then increased from 2 to 17% over 35 min, then increased again to 17 to 25% over 45 min, and finally increased to 25 to 40% over 10 min. Most hydrophobic species were removed by increasing to 98% MPB for 5 min followed by a 10 min hold. The total run time of the method was 135 min, allowing ample time for column re-equilibration. Wash cycles were performed between injections of non-identical analyses to minimize carryover.

[0273] Mass spectrometry was performed using a Q Exactive Basic (Thermo Fisher Scientific) mass spectrometer. Precursor ion mass spectra were measured at a resolution of 70,000 over an m / z range of 400-1600 Da. The 10 most intense precursor ions were selected and fragmented in an HCD cell using a collision energy of 27, and MS / MS spectra were measured at a resolution of 35,000 over an m / z range of 200-2000 Da. Ions with charge states between 2 and 4 were selected for fragmentation, and the dynamic exclusion time was set to 30 s. An exclusion list containing 14 common polysiloxanes was utilized to minimize false identification of known contaminants.

[0274] Data Processing Proteins were initially identified and quantified (unlabeled) using the Sequest HT algorithm combined with the Proteome Discoverer software (version 2.1.1.21, Thermo Fisher Scientific) and the Target Decoy PSM Validator. Searches were performed using the full Swiss-Prot Homo sapiens (taxonomy 9606 version 2017-05-10:42,153 entries) reference database, as well as a custom Uniprot database containing E1a proteins (7 entries). The following search parameters were used: enzyme, trypsin, maximum of two missing cleavages, minimum peptide length of 6 residues, precursor mass tolerance of 10 ppm, and fragment mass tolerance of 0.02 Da. The search also included specific dynamic modifications (oxidation of M, deamidation of N or Q, phosphorylation of S, T, or Y, pyroglutamination of peptide terminal E, and acetylation of protein N-terminus) and static modifications (carbamidomethylation of C).

[0275] For the Target Decoy PSM Validator, data were searched again using Scaffold software (version 4.8.2, Proteome Software Inc.), so that the maximum delta Cn and both the strict and relaxed target false discovery rates (FDR) were set to 1. For Scaffold, data were also searched using the X!Tandem open source algorithm to identify proteins using a protein threshold of 99.0%, a minimum of two peptides, and a peptide threshold of 95%.

[0276] To determine the identity of EV (e.g., exosome) specific proteins, total peptide spectral matches (PSMs) were compared for proteins found in the top EV (e.g., exosome) fraction of the Optiprep™ gradient with proteins found in the bottom fraction. Results showed that there were numerous membrane-associated proteins highly enriched in the EV (e.g., exosome) fraction. EV (e.g., exosome) proteins included CD13, MME, ENPP1, and NRP1.

[0277] 7.11.2. Example 2: Validation of Surface Protein Expression To confirm that the EV (e.g., exosome)-specific proteins identified in the mass spectrometry studies are highly enriched on the surface of EVs (e.g., exosomes), protein blotting is performed on whole cell lysates and purified EV (e.g., exosome) populations from bone marrow-derived MSCs. The total protein patterns are substantially different between whole cell lysates and EV (e.g., exosome) lysates. Specifically, there is a strong band present in the EV (e.g., exosome) lysates that is not present in the whole cell lysates. Western blotting of CD13 reveals that the strong band corresponds to CD13, indicating that CD13 is highly enriched in EVs (e.g., exosomes) produced from bone marrow-derived MSCs and can be visually detectable in whole EV (e.g., exosome) lysates.

[0278] 7.11.3. Example 3: Specific expression of exosomal proteins To determine the specificity of exosomal proteins, mass spectrometry studies and Western blot analysis (as described in Example 1) were performed to identify potential scaffolding proteins expressed in purified exosomes (see, e.g., FIG. 1) and to analyze the expression of several novel exosome-associated membrane proteins in exosomes produced from various cell types. Specifically, exosomes derived from mesenchymal stem cells (MSCs) and HEK293 cells were analyzed. EV (e.g., exosome) fractions from these cell types were purified on an Optiprep™ gradient and analyzed by Western blotting.

[0279] To determine the identity of the different exosomal proteins, we compared the total peptide spectral matches (PSMs) of proteins found in exosomes derived from MSCs and exosomes from HEK293 cells. As shown in Figure 1, when exosome samples from bone marrow-derived MSCs were analyzed, CD13 was observed to be highly enriched in the exosome fraction. However, when samples from HEK293 cells were analyzed, CD13 was not detected in the exosome fraction or any other fraction. Similar results were observed for MME (see Figure 1).

[0280] Each of the novel surface marker proteins was analyzed similarly, and the data indicate that certain exosomal proteins (e.g., CD13 and MME) are enriched on exosomes from a particular cell type (e.g., MSC) but are absent from exosomes produced from a different cell type (e.g., HEK293).

[0281] 7.11.4. Example 4: Overexpression of CD13 from producer cells The results in Example 3 demonstrated that CD13 and other exosomal proteins (e.g., MME) are not universally present in EVs (e.g., exosomes) produced from various cell types. For example, CD13 was highly enriched in exosomes produced from bone marrow-derived MSCs but was absent in exosomes produced from HEK293 cells.

[0282] To determine whether CD13 could be used as an exosome scaffold protein in producer cells that do not naturally produce CD13-bearing exosomes, HEK293 cells were stably transfected with a plasmid expressing full-length CD13 fused to a FLAG tag ("CD13-FLAG plasmid"). Exosomes were purified from wild-type HEK293SF cells and HEK293SF cells transfected with the CD13-FLAG plasmid. As a control, exosomes were purified from HEK293SF cells engineered to overexpress the PTGFRN scaffold protein.

[0283] The SDS-Page analysis results provided in Figure 2A show that CD13-FLAG was successfully overexpressed in HEK293SF cells transfected with CD13-FLAG plasmid. Overexpression of CD13 resulted in similar enrichment in HEK293-derived exosomes compared to PTGFRN. And as shown in Figure 2B (right graph), exosomes produced from HEK293SF cells transfected with CD13-FLAG plasmid also expressed high levels of CD13 on their surface. CD13 expressed in EVs was biologically active with activity levels comparable to recombinant CD13 protein (see Figures 3A and 3B). These results demonstrate that producer cells (e.g., HEK293SF) that do not naturally express exosomal proteins (e.g., CD13) can be modified to produce exosomes that overexpress exosomal proteins (e.g., CD13). These results indicate that exosomal proteins such as CD13 can be used to generate engineered EVs (e.g., exosomes) containing heterologous exosomal proteins, i.e., CD13. Furthermore, EVs (e.g., exosomes) purified from modified cells disclosed herein (e.g., HEK293SF cells transfected with CD13-FLAG plasmid) exhibit the native catalytic activity of heterologous exosomal proteins (e.g., CD13).

[0284] Similar procedures can be performed for CD13 and other HEVPs described herein using various cell types, for example, CD13 can be introduced into CHO cells (if it is not naturally expressed), MME can be introduced into CHO and HEK cells (if it is not naturally expressed), ENPP1 can be introduced into CHO cells (if it is not naturally expressed), and NRP1 can be introduced into CHO and HEK cells (if it is not naturally expressed).

[0285] 7.11.5. Example 5: Generation of engineered exosomal proteins As described herein, the exosomal proteins disclosed herein can be modified (e.g., truncated or conjugated to another moiety) to provide EVs (e.g., exosomes) expressing modified exosomal proteins that exhibit specific properties (e.g., increased targeting to a specific cell type). Polynucleotides encoding modified exosomal proteins are generated using polynucleotides encoding whole exosomal proteins or truncated exosomal proteins. Specific truncated exosomal proteins are selected by screening various truncated exosomal proteins to select truncated proteins with optimal ability to incorporate into exosomal membranes and interact with binding agents. Targeting of the truncated proteins to the exosomal membrane is tested by nanoflow cytometry.

[0286] Polynucleotides encoding modified exosomal proteins are generated by adding a polynucleotide encoding an affinity tag (such as glutathione-S-transferase, S-peptide, FLAG tag, GFP, etc.) to a polynucleotide encoding a complete or truncated exosomal protein (e.g., CD13, MME, ENPP1, and NRP1). The modified polynucleotides express fusion proteins. The polynucleotides are further modified to improve their targeting to the exosomal membrane and / or their affinity to binding agents.

[0287] Different types of polynucleotides encoding modified exosomal proteins are generated by adding a polynucleotide encoding a therapeutic peptide (e.g., an antibody, an enzyme, a ligand, a receptor, an antimicrobial peptide, a variant, or a fragment thereof) to a polynucleotide encoding an intact or truncated exosomal protein (e.g., CD13, MME, ENPP1, and NRP1). The modified polynucleotide expresses a fusion protein that is displayed on the surface of the exosome. The fusion protein maintains the therapeutic activity of the therapeutic peptide.

[0288] Different types of polynucleotides encoding modified exosomal proteins are generated by adding a polynucleotide encoding a targeting moiety (e.g., a targeting moiety specific for a particular organ, tissue, or cell) to a polynucleotide encoding a complete or truncated exosomal protein (e.g., CD13, MME, ENPP1, and NRP1). The modified polynucleotide expresses a fusion protein that is displayed on the surface of the exosome. The fusion protein allows the exosome to be targeted to a particular organ, tissue, or cell.

[0289] The localization of engineered exosomal proteins on the exosome surface will also be examined by nanoflow cytometry.

[0290] 7.11.6. Example 6: Generation of Surface Engineered Exosomes Producer cells that generate surface engineered exosomes are generated by introducing an exogenous sequence encoding an exosomal protein or a variant or fragment of an exosomal protein. A plasmid encoding the exosomal protein is transiently transfected to induce high level expression of the exosomal protein on the exosomal surface. A plasmid encoding the modified exosomal protein is transiently transfected to produce exosomes with the modified exosomal protein on their surface.

[0291] Surface engineered exosomes are produced by stably transforming the producing cells with a polynucleotide encoding an exosomal protein, a variant or fragment of an exosomal protein, or an exogenous sequence encoding an affinity tag, a therapeutic peptide, or a targeting moiety. The exogenous sequence encoding an affinity tag, a therapeutic peptide, or a targeting moiety is inserted into the genomic site encoding the exosomal protein to generate a fusion protein comprising the affinity tag bound to the exosomal protein. The polynucleotide encoding the modified exosomal protein is knocked-in into the genomic site encoding the exosomal protein.

[0292] The producing cell line is generated by stably transfecting at least two polynucleotides, each encoding an exosomal protein, a variant or fragment of an exosomal protein, or an exogenous peptide (e.g., affinity tag, targeting moiety, therapeutic peptide). Different producing cell lines are also generated by inserting two or more exogenous sequences (e.g., exogenous sequences encoding affinity tags, markers, targeting peptides, therapeutic peptides, etc.) into multiple genomic sites within or adjacent to the genomic sequence encoding the exosomal protein, generating surface-engineered exosomes containing multiple modified exosomal proteins. Each of the multiple modified exosomal proteins is targeted to the surface of the exosome. The exosomes have affinity for two different binding agents and are purified by either or both of the binding agents.

[0293] 7.11.7. Example 7: Isolation, Purification, and Subfractionation of Exosomes by Affinity Purification Binders for affinity purification of exosomes are developed by biopanning / directed expansion that elute under mild conditions.

[0294] The binding agent is attached to a solid support (e.g., porous agarose beads) to form a conventional chromatography system (e.g., GE AKTA). The sample containing the exosomes is applied to the column for affinity purification.

[0295] 7.11.8. Example 8: Analysis of Different Scaffold Moieties for Targeting Exosomes In addition to Example 6 above, the ability of CD13 and MME proteins was tested for their ability to target proteins to the surface of exosomes derived from HEK293 cells. Briefly, HEK293 cells were stably transfected with plasmids expressing full-length CD13 fused to green fluorescent protein (GFP) or full-length MME fused to GFP. For comparison purposes, HEK293 cells engineered to overexpress PTGFRN fused to GFP were also generated.

[0296] As shown in Figure 4A, HEK293 cells transfected with CD13-GFP or MME-GFP plasmids expressed the specific exosomal proteins of interest. The overall expression was similar to that of PTGFRN-GFP observed in HEK293 cells engineered to overexpress PTGFRN. And as shown in Figure 4B, both CD13-GFP and MME-GFP fusion proteins were properly folded and expressed in EVs generated from the respective HEK293 producer cells.

[0297] Taken together, the above results demonstrate that certain exosomal proteins (e.g., heterologous exosomal vesicle proteins disclosed herein, e.g., CD13 and MME) can be exogenously introduced into producing cells that do not naturally express the heterologous protein. The results further demonstrate that EVs (e.g., exosomes) produced from such engineered producing cells can express heterologous exosomal proteins, which can be used to anchor various molecules (e.g., antigens, targeting moieties, adjuvants, and / or immunomodulators) to the EVs (e.g., exosomes).

[0298] 8. Incorporated by Reference All publications, patents, patent applications, and other documents cited in this application are herein incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, or other document was individually indicated to be incorporated by reference for all purposes.

[0299] 9. Equivalents The present disclosure provides, inter alia, compositions of cannabinoids and associated compositions. The present disclosure also provides methods for treating neurodegenerative diseases by administering cannabinoids and associated compositions. Although various specific embodiments have been illustrated and described, the above specification is not intended to be limiting. It will be understood that various modifications can be made without departing from the spirit and scope of the present disclosure(s). Many modifications will be apparent to those skilled in the art upon review of this specification. Unofficial sequence listing SEQ ID NO:47 CD13>sp|P15144|AMPN_HUMAN Aminopeptidase N OS=Homo sapiens OX=9606 GN=ANPEP PE=1 SV=4 [ka] SEQ ID NO:48 MME >sp|P08473|NEP_HUMAN Neprilysin OS=Homo sapiens OX=9606 GN=MME PE=1 SV=2 [ka] SEQ ID NO:49 ENPP1 >sp|P22413|ENPP1_HUMAN Ectonucleotide pyrophosphatase / phosphodiesterase family member 1 OS=Homo sapiens OX=9606 GN=ENPP1 PE=1 SV=2 [ka] [ka] SEQ ID NO:50 NRP1 >sp|O14786|NRP1_HUMAN Neuropilin-1 OS=Homo sapiens OX=9606 GN=NRP1 PE=1 SV=3 [ka]

Claims

[Claim 1] The invention described in the specification.

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