Extracellular vesicles comprising a sting agonist

Abstract

This article provides compositions comprising an EV, such as an exogenous form, of a STING agonist, and methods for producing said compositions. This article also provides a method for modulating an immune response by administering a therapeutic amount of an EV, such as an exogenous form, of a STING agonist. The immune response may be an IENβ response or activation of myeloid dendritic cells (mDCs). This article further provides a method for modulating an immune response by administering an exogenous form of a STING agonist without inducing systemic inflammation.

Description

[0001] Cross-references to related applications

[0002] This application claims the benefit and priority of U.S. Provisional Application Serial Nos. 62 / 647,491, filed March 23, 2018; 62 / 680,501, filed June 4, 2018; 62 / 688,600, filed June 22, 2018; 62 / 756,247, filed November 6, 2018; and 62 / 822,019, filed March 21, 2019; the contents of each of these U.S. Provisional Applications are hereby incorporated herein by reference in their entirety.

[0003] References to sequence listings submitted electronically via EFS-WEB

[0004] The contents of the sequence list, which was submitted electronically as an ASCII text file (name: 4000_0210000_Seqlisting_ST25.txt; size: 238,061 bytes; and creation date: March 20, 2019) and submitted with this application, are incorporated herein by reference in their entirety. Background of the Invention

[0006] STING (Synthetic interferon gene stimulator) is a cytoplasmic sensor of a cyclic dinucleotide, typically produced by bacteria. Upon activation, it leads to the production of type I interferon and triggers an immune response. STING antagonism has shown promise as a preclinical method for generating an immune response against tumors. Unfortunately, given the broad expression profile of STING, systemic delivery of STING agonists leads to systemic inflammation. This limits the administerable dose and consequently, the therapeutic efficacy. An alternative to systemic delivery is direct injection of the STING agonist into the tumor. Intratumoral injection is quite effective; however, it is limited to solid tumors that can be accessed by needles and causes tissue damage. Therefore, improved methods for delivering STING agonists are needed. Summary of the Invention

[0007] This article provides compositions comprising an encapsulated STING agonist or an exogenous body associated with a STING agonist, which, upon administration to a subject in need, modulate the human immune system. Such compositions can be used to treat a variety of diseases or disorders in which modulation of the STING signaling pathway has a beneficial effect. For example, treating tumors or cancerous lesions in human subjects. Encapsulating the STING agonist within an exogenous body allows for selective activation of immune cells and provides a narrower biodistribution profile, thereby allowing for systemic delivery without the toxicity associated with administration of the agonist alone.

[0008] In some embodiments, the composition comprises a cyclic dinucleotide STING agonist or a non-cyclic dinucleotide STING agonist.

[0009] In one embodiment, the composition comprises an extracellular vesicle and a STING agonist, wherein the extracellular vesicle is an exosome, a nanovesicle, an apoptotic body, a microvesicle, a lysosome, an endosome, a liposome, a lipid nanoparticle, a micelle, a multilamellar structure, a revesiculated vesicle, or an extruded cell.

[0010] In some embodiments, the exosome overexpresses the protein PTGFRN. In one embodiment, the exosome is produced by a cell that overexpresses PTGFRN.

[0011] In some embodiments, the exosome overexpresses an IgV domain-containing protein. In one embodiment, the exosome is produced by a cell that overexpresses an IgV domain-containing protein. In some embodiments, the IgV-containing protein is Basigin, IGSF2, IGSF3, or IGSF8. In another embodiment, the exosome or cell producing the exosome overexpresses an exosome surface protein described in detail in U.S. Patent Application 62 / 656,956, which is incorporated by reference herein in its entirety. In some embodiments, the exosome is glycan-modified. In one embodiment, the glycan modification comprises an enzymatic or chemical modification. In another embodiment, the exosome is derived from a glycan-modified producer cell. In one embodiment, the glycan modification of the producer cell comprises an enzymatic or chemical modification. In one embodiment, the glycan modification of the producer cell comprises treatment with a kifunensine. In another embodiment, the glycan modification of the producer cell comprises a knockout of a sialyltransferase or a cytidylyltransferase gene. In one embodiment, the glycan modification of the producer cell comprises a CRISPR knockout of a sialyltransferase or a cytidylyltransferase gene. In one embodiment, the gene is cytidine monophosphate N-acetylneuraminic acid synthetase (CMAS). In other embodiments, the exosome is desialylated or deglycosylated.

[0012] In some embodiments, the exosome overexpressing PTGFRN or an IgV domain-containing protein is a glycan-modified exosome. In one embodiment, the exosome overexpressing PTGFRN or an IgV domain-containing protein is desialylated. In one embodiment, the exosome overexpressing PTGFRN or an IgV domain-containing protein is deglycosylated. In some embodiments, the exosome or producer cell is deglycosylated or desialylated by about or more than 95%, 90-95%, 85-90%, 80-85%, 75-80%, 70-75%, 65-70%, 60-65%, 50-60%, 40-50%, 30-40%, 20-30%, 10-20%, or 0-10%.

[0013] In some embodiments, the exosome further comprises an exosome expressing a ligand, a cytokine, or an antibody. In one embodiment, the ligand comprises CD40L, OX40L, or CD27L. In another embodiment, the cytokine comprises IL-7, IL-12, or IL-15. In one embodiment, the antibody comprises an antagonistic antibody or an agonistic antibody.

[0014] In one embodiment, the STING agonist comprises a cyclic dinucleotide STING agonist or a non-cyclic dinucleotide STING agonist comprising a lipid-binding tag. In another embodiment, the STING agonist comprises a cyclic dinucleotide STING agonist or a non-cyclic dinucleotide STING agonist that is physically or chemically modified, the modification comprising altering the polarity or charge of the agonist. In another embodiment, the STING agonist comprises a cyclic dinucleotide STING agonist or a non-cyclic dinucleotide STING agonist that is physically and / or chemically modified. In other embodiments, the STING agonist has a different polarity and / or charge than the STING agonist prior to modification (i.e., the corresponding unmodified STING agonist).

[0015] The concentration of the STING agonist associated with the exosome can be about 0.01 μm to 100 μm. In one embodiment, the concentration of the STING agonist associated with the exosome is about 0.01 μM to 0.1 μM, 0.1 μM to 1 μM, 1 μM to 10 μM, 10 μM to 50 μM, or 50 μM to 100 μM. In another embodiment, the concentration of the STING agonist associated with the exosome is about 1 μM to 10 μM.

[0016] Also provided herein is a kit comprising the composition of any of the above claims and instructions for use.

[0017] Also provided herein is a method of producing an exosome comprising a STING agonist, the steps comprising obtaining an exosome, mixing the exosome and the STING agonist in a solution, incubating the mixture of the exosome and the STING agonist in a solution comprising a buffer, and purifying the exosome comprising the STING agonist.

[0018] In some embodiments, the incubation step comprises incubating the exosomes and the STING agonist for about 2-24 hours. In one embodiment, the incubation step comprises incubating the exosomes and the STING agonist for about 6-12 hours. In one embodiment, the incubation step comprises incubating the exosomes and the STING agonist for about 12-20 hours. In one embodiment, the incubation step comprises incubating the exosomes and the STING agonist for about 14-18 hours. In one embodiment, the incubation step comprises incubating the exosomes and the STING agonist for about 16 hours.

[0019] In some embodiments, the incubation step comprises incubating the exosomes and the STING agonist at about 15-90 °C. In one embodiment, the incubation step comprises incubating the exosomes and the STING agonist at about 37 °C. In one embodiment, the incubation step comprises incubating the exosomes and the STING agonist at about 15-30 °C. In one embodiment, the incubation step comprises incubating the exosomes and the STING agonist at about 30-50 °C. In one embodiment, the incubation step comprises incubating the exosomes and the STING agonist at about 50-90 °C.

[0020] In some embodiments, the incubation step comprises at least 0.01 mM to 100 mM of the STING agonist. In one embodiment, the incubation step comprises at least 1 mM to 10 mM of the STING agonist.

[0021] In some embodiments, the incubation step comprises at least about 10 8 to at least about 10 16 total purified exosome particles. In one embodiment, the incubation step comprises at least about 10 12 total purified exosome particles.

[0022] In some embodiments, the buffer comprises phosphate buffered saline (PBS).

[0023] In some embodiments, the purification step comprises size exclusion chromatography or ion exchange chromatography. In one embodiment, the purification step comprises anion exchange chromatography. In some embodiments, the purification step comprises desalting, dialysis, tangential flow filtration, ultrafiltration, or diafiltration. In one embodiment, the purification step comprises one or more centrifugation steps. In one embodiment, the purification step comprises one or more centrifugation steps performed at about 100,000 x g.

[0024] Also provided herein are methods of inducing or modulating an immune or inflammatory response in a subject, the method comprising administering to a subject in need thereof a pharmaceutically effective amount of a composition comprising exosomes comprising a STING agonist, thereby inducing or modulating an immune or inflammatory response in the subject.

[0025] In some embodiments, the method activates dendritic cells. In one embodiment, the method activates myeloid dendritic cells. In some embodiments, the method results in reduced monocyte activation as compared to administration of a similar or identical level of free STING agonist. In one embodiment, the method does not induce monocyte activation.

[0026] In some embodiments, the method induces production of interferon-β (IFN-β).

[0027] In one embodiment, the method results in reduced systemic inflammation as compared to administration of a similar or identical level of free STING agonist. In some embodiments, the method results in non-massive systemic inflammation.

[0028] In some embodiments, administration is parenteral, oral, intravenous, intramuscular, intratumoral, intraperitoneal administration or administration by any other suitable route of administration. In one embodiment, administration is intravenous administration. In some embodiments, the immune response is an anti-tumor response.

[0029] Also provided herein are methods of inducing or modulating an immune or inflammatory response in a subject, the method comprising administering to a subject in need thereof a composition comprising exosomes comprising a STING agonist, the composition in an amount sufficient to induce IFN-β or activate dendritic cells, thereby inducing or modulating an immune or inflammatory response in the subject. In one embodiment, the method activates myeloid dendritic cells. In some embodiments, the method results in reduced monocyte activation as compared to administration of a similar or identical level of free STING agonist. In one embodiment, wherein the method does not induce monocyte activation. In one embodiment, the method results in reduced systemic inflammation as compared to administration of a similar or identical level of free STING agonist. In some embodiments, the method results in non-massive systemic inflammation.

[0030] In another aspect, also provided herein are methods of treating cancer in a subject, the method comprising administering to a subject in need thereof a composition comprising a therapeutically effective amount of exosomes comprising a STING agonist, thereby inducing or modulating an anti-tumor immune response in the subject.

[0031] In one embodiment, the method induces production of interferon-β (IFN-β).

[0032] In some embodiments, administration is parenteral, oral, intravenous, intramuscular, intratumoral, intraperitoneal administration or administration by any other suitable route of administration.

[0033] In various embodiments, the method further comprises administering an additional therapeutic agent. In some embodiments, the additional therapeutic agent is an immunomodulatory agent. In one embodiment, the additional therapeutic agent is an antibody or antigen binding fragment thereof. In one embodiment, the therapeutic antibody or antigen binding fragment thereof is an inhibitor of CTLA-4, PD-1, PD-L1, PD-L2, TIM-3, or LAG3.

[0034] In another aspect, provided herein are methods of preventing metastasis of a cancer in a subject, the method comprising administering to a subject in need thereof a composition comprising a therapeutically effective amount of exosomes comprising a STING agonist.

[0035] In some embodiments, the therapeutically effective amount of exosomes comprising a STING agonist is capable of preventing one or more tumors in one site of a subject from growing one or more tumors in another site of the subject.

[0036] In one embodiment, the method induces production of interferon-beta (IFN-β).

[0037] In various embodiments, the administration is parenteral, oral, intravenous, intramuscular, intratumoral, intraperitoneal route of administration or by any other suitable route of administration.

[0038] In some embodiments, the composition is administered intratumorally in a first tumor in one site, and wherein the composition administered in the first tumor prevents metastasis of one or more tumors in a second site.

[0039] In various embodiments, the method further comprises administering an additional therapeutic agent. In some embodiments, the additional therapeutic agent is an immunomodulatory agent. In one embodiment, the additional therapeutic agent is an antibody or antigen binding fragment thereof. In one embodiment, the therapeutic antibody or antigen binding fragment thereof is an inhibitor of CTLA-4, PD-1, PD-L1, PD-L2, TIM-3, or LAG3.

[0040] Provided herein is a composition comprising an extracellular vesicle and a stimulator of interferon genes (STING) agonist. In some embodiments, the extracellular vesicle is an exosome, nanovesicle, apoptotic body, microvesicle, lysosome, endosome, liposome, lipid nanoparticle, micelle, multilayered structure, re- vesiculated vesicle, or extruded cell. In certain embodiments, the extracellular vesicle is an exosome.

[0041] In some embodiments, the STING agonist is associated with the extracellular vesicle. In some embodiments, the STING agonist is encapsulated within the extracellular vesicle. In certain embodiments, the STING agonist is optionally linked to the lipid bilayer of the extracellular vesicle via a linker.

[0042] In some embodiments, the extracellular vesicle of the present disclosure overexpresses PTGFRN protein. In certain embodiments, the STING agonist is optionally linked to the PTGFRN protein via a linker.

[0043] In some embodiments, the extracellular vesicle is produced by a cell that overexpresses PTGFRN protein. In some embodiments, the extracellular vesicle is glycan-modified. In certain embodiments, the extracellular vesicle is sialylated. In further embodiments, the extracellular vesicle is deglycosylated.

[0044] In some embodiments, the extracellular vesicle further comprises a protein that binds to or enzymatically reacts with the STING agonist. In certain embodiments, the extracellular vesicle further comprises a ligand, a cytokine, or an antibody. In some embodiments, the ligand comprises CD40L, OX40L, and / or CD27L. In some embodiments, the cytokine comprises IL-7, IL-12, and / or IL-15. In certain embodiments, the antibody comprises an antagonistic antibody and / or an agonistic antibody.

[0045] In some embodiments, the STING agonist is a cyclic dinucleotide. In other embodiments, the STING agonist is a non-cyclic dinucleotide. In certain embodiments, the STING agonist comprises a lipid-binding tag. In some embodiments, the STING agonist is physically and / or chemically modified. In certain embodiments, the modified STING agonist has a different polarity and / or charge than the corresponding unmodified STING agonist.

[0046] In some embodiments, the concentration of the STING agonist associated with the extracellular vesicle is about 0.01 pm to 100 pm. In certain embodiments, the concentration of the STING agonist associated with the extracellular vesicle is about 0.01 pM to 0.1 pM, 0.1 pM to 1 pM, 1 pM to 10 pM, 10 pM to 50 pM, or 50 pM to 100 pM. In further embodiments, the concentration of the STING agonist associated with the extracellular vesicle is about 1 pM to 10 pM.

[0047] In some embodiments, the STING agonist comprises:

[0048] wherein:

[0049] X1is H, OH, or F;

[0050] X2 is H, OH, or F;

[0051] Z is OH, OR1, SH, or SR1, wherein:

[0052] i) R1 is Na or NH4, or

[0053] ii) R1 is an enzyme-labile group that provides OH or SH in vivo, such as neopentanoyloxymethyl;

[0054] Bi and B2 are bases selected from the group consisting of:

[0055]

[0056] with the proviso that:

[0057] - in Formula (I): X1 and X2 are not OH,

[0058] - in Formula (II): when X1 and X2 are OH, Bi is not adenine and B2 is not guanine, and

[0059] - in Formula (III): when X1 and X2 are OH, Bi is not adenine, B2 is not guanine and Z is not OH, or a pharmaceutically acceptable salt thereof.

[0060] In some embodiments, the STING agonist is selected from the group consisting of:

[0061]

[0062] and pharmaceutically acceptable salts thereof.

[0063] In some embodiments, the extracellular vesicle associated with the STING agonist exhibits one or more of the following characteristics: (i) activates dendritic cells, e.g., myeloid dendritic cells; (ii) activates monocytes to a lesser extent than the STING agonist alone (“free STING agonist”); (iii) does not activate monocytes; (iv) has a wider therapeutic index than the free STING agonist; (v) has lower systemic toxicity than the free STING agonist; (vi) has less immune cell killing than the free STING agonist; (vii) has higher cell selectivity than the free STING agonist; (viii) provides tumor protective immunity at lower doses than the free STING agonist; (ix) induces specific cellular responses in vivo in antigen presenting cells, e.g., dendritic cells; (x) is capable of inducing an immune response in a distant region after local administration; and (xi) is capable of being dosed at lower levels than the free STING agonist.

[0064] In some embodiments, the extracellular vesicles associated with a STING agonist do not deplete T cells and / or macrophages in a mammal when administered to the mammal. In other embodiments, the extracellular vesicles associated with a STING agonist deplete T cells and / or macrophages in a mammal to a lesser extent than a free STING agonist when administered to the mammal.

[0065] Disclosed herein is a pharmaceutical composition comprising a composition (e.g., comprising an extracellular vesicle described herein) and a pharmaceutically acceptable carrier.

[0066] Disclosed herein is a kit comprising a composition (e.g., comprising an extracellular vesicle described herein) and instructions for use.

[0067] Also provided herein is a method of producing an extracellular vesicle (EV) (e.g., exosome) comprising a STING agonist, the method comprising: (a) obtaining an EV, e.g., exosome; (b) mixing the EV (e.g., exosome) with the STING agonist in a solution; (c) incubating the mixture of the EV (e.g., exosome) and the STING agonist in a solution comprising a buffer under suitable conditions; and (d) purifying the EV (e.g., exosome) comprising the STING agonist.

[0068] In some embodiments, the suitable conditions comprise incubating the EV (e.g., exosome) and the STING agonist for about 2-24 hours. In certain embodiments, the suitable conditions comprise incubating the EV (e.g., exosome) and the STING agonist at about 15-90 °C. In some embodiments, the suitable conditions comprise incubating the EV (e.g., exosome) and the STING agonist at about 37 °C.

[0069] In some embodiments, the amount of the STING agonist in the mixing step comprises at least 0.01 mM to 100 mM. In certain embodiments, the amount of the STING agonist in the mixing step comprises at least 1 mM to 10 mM. In further embodiments, the amount of the exosome in the mixing step comprises at least about 10 8 to at least about 10 16 total particles. In some embodiments, the amount of the EV (e.g., exosome) in the mixing step comprises at least about 10 12 total particles.

[0070] In some embodiments, the buffer for producing the EV (e.g., exosome) disclosed herein comprises phosphate buffered saline (PBS).

[0071] In some embodiments, purifying EVs (e.g., exosomes) includes one or more centrifugation steps. In certain embodiments, the one or more centrifugation steps are performed at 100,000 x g.

[0072] The present disclosure also provides a method of inducing or modulating an immune response and / or an inflammatory response in a subject in need thereof, the method comprising administering to the subject a pharmaceutically effective amount of a composition or a pharmaceutical composition disclosed herein.

[0073] Also provided is a method of treating a tumor in a subject in need thereof, the method comprising administering to the subject a composition or a pharmaceutical composition disclosed herein.

[0074] In some embodiments, the administration induces or modulates an immune response and / or an inflammatory response in the subject. In certain embodiments, the administration activates dendritic cells. In some embodiments, the administration results in reduced monocyte activation as compared to a free STING agonist. In further embodiments, the administration does not induce monocyte activation. In some embodiments, the administration induces production of interferon-β (IFN-β). In some embodiments, the administration results in reduced systemic inflammation as compared to a free STING agonist. In some embodiments, the administration results in non-massive systemic inflammation.

[0075] In some embodiments, the administration is parenteral, oral, intravenous, intramuscular, intratumoral, intraperitoneal administration or by any other suitable route of administration. In certain embodiments, the administration is intravenous administration.

[0076] In some embodiments, the immune response (e.g., that can be induced or modulated by administration of a composition or a pharmaceutical composition disclosed herein) is an anti-tumor immune response.

[0077] In some embodiments, the amount of the composition (e.g., disclosed herein) is sufficient to induce IFN-β and / or activate dendritic cells. In some embodiments, the composition is administered intratumorally in a first tumor in one site, and wherein the composition administered in the first tumor prevents metastasis of one or more tumors in a second site.

[0078] In some embodiments, the method of inducing or modulating an immune response and / or an inflammatory response in a subject or the method of treating a tumor in a subject further comprises administering an additional therapeutic agent. In certain embodiments, the additional therapeutic agent is an immunomodulatory agent. In some embodiments, the additional therapeutic agent is an antibody or antigen-binding fragment thereof. In certain embodiments, the antibody or antigen-binding fragment thereof is an inhibitor of CTLA-4, PD-1, PD-L1, PD-L2, TIM-3, or LAG3.

[0079] In some embodiments, administration of the compositions or pharmaceutical compositions disclosed herein can prevent tumor metastasis in a subject.

[0080] Embodiments

[0081] Embodiment 1. A composition comprising an extracellular vesicle and a stimulator of interferon genes protein (STING) agonist.

[0082] Embodiment 2. The composition of embodiment 1, wherein the extracellular vesicle is an exosome, a nanovesicle, an apoptosome, a microvesicle, a lysosome, an endosome, a liposome, a lipid nanoparticle, a micelle, a multilayered structure, a re- vesiculated vesicle, or an extruded cell.

[0083] Embodiment 3. The composition of embodiment 2, wherein the extracellular vesicle is an exosome.

[0084] Embodiment 4. The composition of any one of the preceding embodiments, wherein the STING agonist is associated with the exosome.

[0085] Embodiment 5. The composition of any one of the preceding embodiments, wherein the STING agonist is associated with or encapsulated within a lipid bilayer of an exosome.

[0086] Embodiment 6. The composition of any one of the preceding embodiments, wherein the exosome overexpresses the protein PTGFRN.

[0087] Embodiment 7. The composition of any one of the preceding embodiments, wherein the exosome is produced by a cell that overexpresses PTGFRN.

[0088] Embodiment 8. The composition of any one of the preceding embodiments, wherein the exosome is glycan-modified.

[0089] Embodiment 9. The composition of embodiment 8, wherein the glycan modification comprises an enzymatic or chemical modification.

[0090] Embodiment 10. The composition of any one of embodiments 1-8, wherein the exosome is derived from a glycan-modified producer cell.

[0091] The composition of embodiment 10, wherein the glycan modification of the producer cell comprises an enzymatic or chemical modification.

[0092] Embodiment 12. The composition of embodiment 10, wherein the glycan modification of the producer cell comprises treatment with a kifunensine.

[0093] Embodiment 13. The composition of embodiment 10, wherein the glycan modification of the producer cell comprises a knock-out of a sialyltransferase or cytidylyltransferase gene.

[0094] Embodiment 14. The composition of embodiment 13, wherein the glycan modification of the producer cell comprises CRISPR knockout of a sialyltransferase or cytidylyltransferase gene.

[0095] Embodiment 15. The composition of embodiment 13 or 14, wherein the gene is cytidine monophosphate N-acetylneuraminic acid synthetase (CMAS).

[0096] Embodiment 16. The composition of any of the above embodiments, wherein the exosome is desialidated.

[0097] Embodiment 17. The composition of any of the above embodiments, wherein the exosome is deglycosylated.

[0098] Embodiment 18. The composition of any of the above embodiments, wherein the PTGFRN-overexpressing exosome is a glycan-modified exosome.

[0099] Embodiment 19. The composition of any of the above embodiments, wherein the PTGFRN-overexpressing exosome is desialidated.

[0100] Embodiment 20. The composition of any of the above embodiments, wherein the PTGFRN-overexpressing exosome is deglycosylated.

[0101] Embodiment 21. The composition of any of the above embodiments, wherein the exosome or producer cell is desialidated or deglycosylated by about or more than 95%, 90-95%, 85-90%, 80-85%, 75-80%, 70-75%, 65-70%, 60-65%, 50-60%, 40-50%, 30-40%, 20-30%, 10-20%, or 0-10%.

[0102] Embodiment 22. The composition of any of the above embodiments, wherein the exosome further comprises a protein bound to or enzymatically reacted with a cyclic dinucleotide STING agonist or a non-cyclic dinucleotide STING agonist.

[0103] Embodiment 23. The composition of any of the above embodiments, wherein the exosome further comprises an exosome expressing a ligand, a cytokine, or an antibody.

[0104] Embodiment 24. The composition of embodiment 23, wherein the ligand comprises CD40L, OX40L, or CD27L.

[0105] Embodiment 25. The composition of embodiment 23, wherein the cytokine comprises IL-7, IL-12, or IL-15.

[0106] Embodiment 26. The composition of embodiment 23, wherein the antibody comprises an antagonistic antibody or an agonistic antibody.

[0107] Embodiment 27. The composition of any of the above embodiments, wherein the STING agonist comprises a cyclic dinucleotide STING agonist or a non-cyclic dinucleotide STING agonist.

[0108] Embodiment 28. The composition of any of the above embodiments, wherein the STING agonist comprises a cyclic dinucleotide STING agonist or a non-cyclic dinucleotide STING agonist comprising a lipid-binding tag.

[0109] Embodiment 29. The composition of any of the above embodiments, wherein the STING agonist comprises a cyclic dinucleotide STING agonist or a non-cyclic dinucleotide STING agonist that is physically or chemically modified, the modification comprising altering the agonist polarity or charge.

[0110] Embodiment 30. The composition of any of the above embodiments, wherein the concentration of the STING agonist associated with the exosome is about 0.01 mM to 100 mM.

[0111] Embodiment 31. The composition of any of the above embodiments, wherein the concentration of the STING agonist associated with the exosome is about 0.01 mM to 0.1 mM, 0.1 mM to 1 mM, 1 mM to 10 mM, 10 mM to 50 mM, or 50 mM to 100 mM.

[0112] Embodiment 32. The composition of any of the above embodiments, wherein the concentration of the STING agonist associated with the exosome is about 1 mM to 10 mM.

[0113] Embodiment 33. A kit comprising the composition of any of the above embodiments and instructions for use.

[0114] Embodiment 34. A method of producing an exosome comprising a STING agonist, the method comprising:

[0115] a. obtaining an exosome;

[0116] b. mixing the exosome with a STING agonist in solution;

[0117] c. incubating the mixture of exosome and STING agonist in a solution comprising a buffer; and

[0118] d. purifying the exosome comprising the STING agonist.

[0119] Embodiment 35. The method of embodiment 34, wherein the incubating step comprises incubating the exosome and the STING agonist for about 2-24 hours.

[0120] Embodiment 36. The method of embodiment 34, wherein the incubating step comprises incubating the exosome and the STING agonist for about 6-12 hours.

[0121] Embodiment 37. The method of embodiment 34, wherein the incubating step comprises incubating the exosome and the STING agonist for about 12-20 hours.

[0122] Embodiment 38. The method of embodiment 34, wherein the incubating step comprises incubating the exosome and the STING agonist for about 14-18 hours.

[0123] Embodiment 39. The method of embodiment 34, wherein the incubating step comprises incubating the exosome and the STING agonist for about 16 hours.

[0124] Embodiment 40. The method of any one of embodiments 34-39, wherein the incubating step comprises incubating the exosome and the STING agonist at about 15-90 °C.

[0125] Embodiment 41. The method of any one of embodiments 34-39, wherein the incubating step comprises incubating the exosome and the STING agonist at about 37 °C.

[0126] Embodiment 42. The method of any one of embodiments 34-39, wherein the incubating step comprises incubating the exosome and the STING agonist at about 15-30 °C.

[0127] Embodiment 43. The method of any one of embodiments 34-39, wherein the incubating step comprises incubating the exosome and the STING agonist at about 30-50 °C.

[0128] Embodiment 44. The method of any one of embodiments 34-39, wherein the incubating step comprises incubating the exosome and the STING agonist at about 50-90 °C.

[0129] Embodiment 45. The method of any one of embodiments 34-44, wherein the incubating step comprises at least 0.01 mM to 100 mM of the STING agonist.

[0130] Embodiment 46. The method of any one of embodiments 34-44, wherein the incubating step comprises at least 1 mM to 10 mM of the STING agonist.

[0131] Embodiment 47. The method of any one of embodiments 34-46, wherein the incubation step comprises at least about 108to at least about 1016total purified exosome particles.

[0132] Embodiment 48. The method of any one of embodiments 34-46, wherein the incubation step comprises at least about 1012total purified exosome particles.

[0133] Embodiment 49. The method of any one of embodiments 34-47, wherein the buffer comprises phosphate buffered saline (PBS).

[0134] Embodiment 50. The method of any one of embodiments 34-49, wherein the purification step comprises size exclusion chromatography or ion chromatography.

[0135] Embodiment 51. The method of any one of embodiments 34-50, wherein the purification step comprises anion exchange chromatography.

[0136] Embodiment 52. The method of any one of embodiments 34-51, wherein the purification step comprises desalting, dialysis, tangential flow filtration, ultrafiltration, or diafiltration.

[0137] Embodiment 53. The method of any one of embodiments 34-49, wherein the purification step comprises one or more centrifugation steps.

[0138] Embodiment 54. The method of embodiment 53, wherein the purification step comprises one or more centrifugation steps performed at about 100,000 x g.

[0139] Embodiment 55. A method of inducing or modulating an immune or inflammatory response in a subject, the method comprising administering to a subject in need thereof a pharmaceutically effective amount of a composition comprising exosomes comprising a STING agonist, thereby inducing or modulating an immune or inflammatory response in the subject.

[0140] Embodiment 56. The method of embodiment 55, wherein the method activates dendritic cells.

[0141] Embodiment 57. The method of any one of embodiments 55-56, wherein the method activates myeloid dendritic cells.

[0142] Embodiment 58. The method of any one of embodiments 55-57, wherein the method results in reduced monocyte activation as compared to administration of a similar or identical level of free STING agonist.

[0143] Embodiment 59. The method of any one of embodiments 55-58, wherein the method does not induce monocyte activation.

[0144] Embodiment 60. The method of any one of embodiments 55-59, wherein the method induces production of interferon-beta (IFN-β).

[0145] Embodiment 61. The method of any one of embodiments 55-60, wherein the method results in reduced systemic inflammation as compared to administration of a similar or identical level of a free STING agonist.

[0146] Embodiment 62. The method of any one of embodiments 55-60, wherein the method results in systemic inflammation that is not substantial.

[0147] Embodiment 63. The method of any one of embodiments 55-62, wherein the administration is parenteral, oral, intravenous, intramuscular, intratumoral, intraperitoneal administration or administration by any other suitable route of administration.

[0148] Embodiment 64. The method of any one of embodiments 55-63, wherein the administration is intravenous administration.

[0149] Embodiment 65. The method of any one of embodiments 55-64, wherein the immune response is an anti-tumor response.

[0150] Embodiment 66. A method of inducing or modulating an immune or inflammatory response in a subject, the method comprising administering to a subject in need thereof a composition comprising exosomes comprising a STING agonist, the composition in an amount sufficient to induce IFN-β or activate dendritic cells, thereby inducing or modulating an immune or inflammatory response in the subject.

[0151] Embodiment 67. The method of embodiment 66, wherein the method activates myeloid dendritic cells.

[0152] Embodiment 68. The method of any one of embodiments 66-67, wherein the method results in reduced monocyte activation as compared to administration of a similar or identical level of a free STING agonist.

[0153] Embodiment 69. The method of any one of embodiments 66-67, wherein the method does not induce monocyte activation.

[0154] Embodiment 70. The method of any one of embodiments 66-69, wherein the method results in reduced systemic inflammation as compared to administration of a similar or identical level of a free STING agonist.

[0155] Embodiment 71. The method of any one of embodiments 66-69, wherein the method does not induce significant systemic inflammation.

[0156] Embodiment 72. The method of any one of embodiments 66-71, wherein the administration is parenteral, oral, intravenous, intramuscular, intratumoral, intraperitoneal administration or administration by any other suitable route of administration.

[0157] Embodiment 73. The method of any one of embodiments 66-71, wherein the administration is intravenous administration.

[0158] Embodiment 74. The method of any one of embodiments 66-73, wherein the immune response is an anti-tumor response.

[0159] Embodiment 75. A method of treating cancer in a subject, the method comprising administering to a subject in need thereof a composition comprising a therapeutically effective amount of exosomes containing a STING agonist, thereby inducing or modulating an anti-tumor immune response in the subject.

[0160] Embodiment 76. The method of embodiment 75, wherein the method induces production of interferon-β (IFN-β).

[0161] Embodiment 77. The method of embodiment 75 or 76, wherein the administration is parenteral, oral, intravenous, intramuscular, intratumoral, intraperitoneal administration or administration by any other suitable route of administration.

[0162] Embodiment 78. The method of any one of embodiments 75-77, further comprising administering an additional therapeutic agent.

[0163] Embodiment 79. The method of any one of embodiments 75-78, wherein the additional therapeutic agent is an immunomodulatory agent.

[0164] Embodiment 80. The method of embodiment 79, wherein the additional therapeutic agent is an antibody or antigen-binding fragment thereof.

[0165] Embodiment 81. The method of any one of embodiments 80, wherein the therapeutic antibody or antigen-binding fragment thereof is an inhibitor of CTLA-4, PD-1, PD-L1, PD-L2, TIM-3, or LAG3.

[0166] Embodiment 82. A method of preventing metastasis of a cancer in a subject, the method comprising administering to a subject in need thereof a composition comprising a therapeutically effective amount of exosomes containing a STING agonist.

[0167] Embodiment 83. The method of embodiment 81, wherein the therapeutically effective amount of exosomes containing a STING agonist is capable of preventing one or more tumors in one part of the subject from promoting growth of one or more tumors in another part of the subject.

[0168] Embodiment 84. The method of Embodiments 82 or 83, wherein the method induces production of interferon-beta (IFN-β).

[0169] Embodiment 85. The method of any one of Embodiments 81-84, wherein the administration is parenteral, oral, intravenous, intramuscular, intratumoral, intraperitoneal administration or administration by any other suitable route of administration.

[0170] Embodiment 86. The method of any one of Embodiments 81-85, wherein the composition is administered intratumorally in a first tumor in one site, and wherein the composition administered intratumorally in the first tumor prevents metastasis of one or more tumors in a second site.

[0171] Embodiment 87. The method of any one of Embodiments 81-86, further comprising administering an additional therapeutic agent.

[0172] Embodiment 88. The method of Embodiment 87, wherein the additional therapeutic agent is an immunomodulatory agent.

[0173] Embodiment 89. The method of Embodiment 88, wherein the additional therapeutic agent is an antibody or antigen-binding fragment thereof.

[0174] Embodiment 90. The method of Embodiment 89, wherein the additional therapeutic agent is a therapeutic antibody or antigen-binding fragment thereof that is an inhibitor of CTLA-4, PD-1, PD-L1, PD-L2, TIM-3, or LAG3. BRIEF DESCRIPTION OF DRAWINGS

[0175] Figure 1 A schematic showing a method of loading exosomes with a STING agonist.

[0176] Figure 2 A comparison of IFNβ response in peripheral blood mononuclear cells (PBMCs) treated with exosome-encapsulated STING agonist and free STING agonist as determined by relative luminescence (RLU) is shown.

[0177] Figure 3 A comparison of monocyte activation in cells treated with exosome-encapsulated STING agonist and free STING agonist as determined by CD86 mean fluorescence intensity (MFI) is shown.

[0178] Figure 4 A comparison of mDC activation in cells treated with exosome-encapsulated STING agonist and free STING agonist as determined by CD86 mean fluorescence intensity (MFI) is shown.

[0179] Figure 5A shows activation of mDCs in samples treated with exosome-encapsulated STING agonist (Exo-STING) or free STING agonist as determined by CD86 staining, while Figure 5B shows activation of monocytes in samples treated with exosome-encapsulated STING agonist (Exo-STING) or free STING agonist as determined by CD86 staining.

[0180] Figure 6A and Figure 6B shows the percentage of activation marker positive cells for different cell type populations (mDCs, pDCs, monocytes, NK cells, CD8+ T cells, and B cells) after treatment with free STING agonist (STING agonist).

[0181] Figure 7A shows the percentage of activation marker positive cells for different cell type populations (mDCs, pDCs, monocytes, NK cells, CD8+ T cells, and B cells) after treatment with free STING agonist (STING agonist). Figure 7B shows the percentage of activation marker positive cells for different cell type populations (mDCs, pDCs, monocytes, NK cells, CD8+ T cells, and B cells) after treatment with exosome-encapsulated STING agonist (STING exosomes).

[0182] Figure 8A and Figure 8B shows the dose-dependent IFN-β response in PBMCs from two donors after treatment with free STING agonist, exosome-encapsulated STING agonist (STING Exo), or STING agonist-encapsulated glycan-modified or overexpressing protein exosomes (Deglycated [Degly], Desialylated [Desialy], overexpressing PTGFRN [PTGFRN], deglycated and overexpressing PTGFRN [PTGFRN Degly], or desialylated and overexpressing PTGFRN [PTGFRN Desialy]).

[0183] Figure 9 shows the comparison of IFN-β production EC 50 for free STING agonist and exosome-encapsulated STING agonist as tested in Figure 8.

[0184] Figure 10A and Figure 10B shows the percentage of activation marker positive cells for different cell type populations (mDCs, pDCs, monocytes, NK cells, CD8+ T cells, and B cells) after treatment with free STING agonist (STING agonist). Figure 8A and Figure 8BThe dose-dependent CD86 expression response in monocytes from two donors after treatment with the free STING agonist, the exogenous encapsulated STING agonist (STING Exo), or the exogenous encapsulated STING agonist-modified or overexpressed protein.

[0185] Figure 11 The mononuclear cell activation of EC with free STING agonists and exogenously encapsulated STING agonists tested in Figure 10 is shown. 50 A comparison.

[0186] Figure 12A and Figure 12B It shows that in use such as Figure 8A and Figure 8B The dose-dependent CD86 expression response in mDC after treatment with free STING agonist, exogenous encapsulated STING agonist (STING Exo), or exogenous encapsulated STING agonist-modified or overexpressed protein.

[0187] Figure 13 The mDC-activated EC of free STING agonists and exogenously encapsulated STING agonists tested in Figure 12 are shown. 50 A comparison.

[0188] Figure 14 The concentration of STING agonist in the exogenous body was quantified.

[0189] Figure 15A and Figure 15B The dose-dependent IFNβ response was shown in two different donor samples after treatment with exogenous bodies containing encapsulated STING agonists, either with or without chif base treatment (Exo+Kif) or without chif base treatment (Exo).

[0190] Figure 16A and Figure 16B This study demonstrates dose-dependent activation of monocytes, as measured by CD86 signaling, in two different donor samples after treatment with exogenous bodies containing or without encapsulated STING agonists, either chifrine-treated (Exo+Kif) or untreated (Exo).

[0191] Figure 17A and Figure 17B This study demonstrates dose-dependent activation of mDCs, as measured by CD86 signal, in two different donor samples after treatment with exogenous bodies containing STING agonists with or without encapsulation, either treated with chif base (Exo+Kif) or untreated chif base (Exo).

[0192] Figure 18Aand Figure 18B Dose-dependent IFNβ responses in two different donor samples are shown after treatment with exosomes that have been incubated with STING agonist for different amounts of time (2h, 6h, overnight (O / N)) or not incubated with STING agonist (exo).

[0193] Figure 19 Dose-dependent IFNβ responses in human PBMCs treated with two different exosome-encapsulated STING agonists and free STING agonist (ML RR-S2 CDA and 3-3cAIMPdFSH) as determined by relative luminescence (RLU) are shown.

[0194] Figures 20A-20D Cytokine expression profiles (IFNβ, CXCL9, CXCL10, and IFN-γ, respectively) in tumors of B16F10 tumor-bearing mice after a single intratumoral injection of PBS, 20 pg free ML RR-S2 CDA, 0.2 pg free ML RR-S2 CDA, or 0.2 pg exosome-encapsulated ML RR-S2 CDA are shown.

[0195] Figures 21A-21C Cytokine expression profiles (IFNβ, CXCL9, and CXCL10, respectively) in draining lymph nodes of B16F10 tumor-bearing mice after a single intratumoral injection of PBS, 20 pg free ML RR-S2 CDA, 0.2 pg free ML RR-S2 CDA, or 0.2 pg exosome-encapsulated ML RR-S2 CDA are shown.

[0196] Figures 22A-22C Cytokine expression profiles (IFNβ, CXCL9, and CXCL10, respectively) in spleens of B16F10 tumor-bearing mice after a single intratumoral injection of PBS, 20 pg free ML RR-S2 CDA, 0.2 pg free ML RR-S2 CDA, or 0.2 pg exosome-encapsulated ML RR-S2 CDA are shown.

[0197] Figures 23A-23E Cytokine expression profiles (IFNβ, TNF-a, IL-6, MCP-1, and IFN-γ, respectively) in serum of B16F10 tumor-bearing mice after a single intratumoral injection of PBS, 20 pg free ML RR-S2 CDA, 0.2 pg free ML RR-S2 CDA, or 0.2 pg exosome-encapsulated ML RR-S2 CDA are shown.

[0198] Figures 24A-24DCytokine expression profiles (IFNβ, CXCL9, CXCL10, and IFN-γ, respectively) in the tumor of B16F10 tumor-bearing mice following a single intratumoral injection of PBS, 20 μg free 3-3cAIMPdFSH, 0.2 μg free 3-3cAIMPdFSH, or 0.2 μg exosome-encapsulated 3-3cAIMPdFSH.

[0199] Figures 25A-25D Cytokine expression profiles (IFNβ, CXCL9, CXCL10, and IFN-γ, respectively) in the tumor of B16F10 tumor-bearing mice following a single intratumoral injection of PBS, 20 μg free 3-3cAIMPdFSH, 0.2 μg free 3-3cAIMPdFSH, or 0.2 μg exosome-encapsulated 3-3cAIMPdFSH.

[0200] Figures 26A-26D Cytokine expression profiles (IFNβ, CXCL9, CXCL10, and IFN-γ, respectively) in the tumor of B16F10 tumor-bearing mice following a single intratumoral injection of PBS, 20 μg free 3-3cAIMPdFSH, 0.2 μg free 3-3cAIMPdFSH, or 0.2 μg exosome-encapsulated 3-3cAIMPdFSH.

[0201] Figures 27A-27D Cytokine expression profiles (IFNβ, TNF-α, IL-6, and MCP-1, respectively) in the serum of B16F10 tumor-bearing mice following a single intratumoral injection of PBS, 20 μg free 3-3cAIMPdFSH, 0.2 μg free 3-3cAIMPdFSH, or 0.2 μg exosome-encapsulated 3-3cAIMPdFSH.

[0202] Figure 28A Cytokine expression profiles (IFNβ, CXCL9, and CXCL10, respectively) in the tumor of B16F10 tumor-bearing mice following a single intraperitoneal injection of PBS, 20 μg free ML RR-S2 CDA, 0.2 μg free ML RR-S2 CDA, or 0.2 μg exosome-encapsulated ML RR-S2 CDA, or a single intratumoral injection of 0.2 μg exosome-encapsulated ML RR-S2 CDA.

[0203] Figure 29A-C shows the cytokine expression profile (IFNβ, CXCL9 and CXCL10, respectively) in the pancreas of B16F10 tumor-bearing mice after a single intraperitoneal injection of PBS, 20 pg free ML RR-S2 CDA, 0.2 pg free ML RR-S2 CDA or 0.2 pg exosome-encapsulated ML RR-S2 CDA, or a single intratumoral injection of 0.2 pg exosome-encapsulated ML RR-S2 CDA.

[0204] Figures 30A-30C shows the cytokine expression profile (IFNβ, CXCL9 and CXCL10, respectively) in the spleen of B16F10 tumor-bearing mice after a single intraperitoneal injection of PBS, 20 pg free ML RR-S2 CDA, 0.2 pg free ML RR-S2 CDA, 0.2 pg exosome-encapsulated ML RR-S2 CDA or an equal amount of exosomes.

[0205] Figures 31A-31C shows the cytokine expression profile (IFNβ, CXCL9 and CXCL10, respectively) in the lung of untreated mice after a single intraperitoneal injection of PBS, 20 pg free ML RR-S2 CDA, 0.2 pg free ML RR-S2 CDA, 0.2 pg exosome-encapsulated ML RR-S2 CDA or an equal amount of exosomes.

[0206] Figures 32A-32C shows the cytokine expression profile (IFNβ, CXCL9 and CXCL10, respectively) in the spleen of untreated mice after a single intraperitoneal injection of PBS, 20 pg free ML RR-S2 CDA, 0.2 pg free ML RR-S2 CDA, 0.2 pg exosome-encapsulated ML RR-S2 CDA or an equal amount of exosomes.

[0207] Figures 33A-33C shows the cytokine expression profile (IFNβ, CXCL9 and CXCL10, respectively) in the pancreas of untreated mice after a single intraperitoneal injection of PBS, 20 pg free ML RR-S2 CDA, 0.2 pg free ML RR-S2 CDA, 0.2 pg exosome-encapsulated ML RR-S2 CDA or an equal amount of exosomes.

[0208] Figures 34A-34Gshows the cytokine expression profile (IFN-β, IFN-γ, TNF-a, IL-6, MCP-1, IL-1a and IL-27, respectively) in serum of naive mice after a single intraperitoneal injection of PBS, 20 pg free ML RR-S2 CDA, 0.2 pg free ML RR-S2 CDA, 0.2 pg exosome-encapsulated ML RR-S2 CDA or an equal amount of exosomes.

[0209] Figure 35 shows the immune cell activation profile in the peritoneum 24 hours after a single intraperitoneal injection of PBS, 20 pg free ML RR-S2 CDA, 0.2 pg free ML RR-S2 CDA, 0.2 pg exosome-encapsulated ML RR-S2 CDA.

[0210] Figure 36 shows the immune cell activation profile in the spleen 24 hours after a single intraperitoneal injection of PBS, 20 pg free ML RR-S2 CDA, 0.2 pg free ML RR-S2 CDA, 0.2 pg exosome-encapsulated ML RR-S2 CDA.

[0211] Figure 37A shows the tumor growth curve of B16F10 tumor-bearing mice during the study described in Example 9 (i.e., an intratumoral injection study comparing the efficacy of PBS, 20 pg free ML RR-S2 CDA, 0.2 pg free ML RR-S2 CDA, 0.2 pg exosome-encapsulated ML RR-S2 CDA). Figures 37B-37E shows the tumor growth curve of each animal in the different groups (i.e., PBS, STING agonist (20 pg), STING agonist (0.2 pg) and Exo STING agonist (0.2 pg), respectively).

[0212] Figure 38A shows the tumor growth curve of B16F10 tumor-bearing mice previously treated with a STING agonist as described in Example 9 after a second challenge with a tumor cell inoculation. Figure 37A Figure 38B shows the tumor growth curve of each animal in the different groups. Figure 38C shows the tumor growth curve of each animal in the different groups. Figure 37A shows the survival of the animals in the study described in Example 10.

[0213] Figure 39 shows the tumor growth curve during the study described in Example 10 (an intratumoral injection dose titration study comparing the efficacy of 8 ng, 40 ng and 200 ng exosome-encapsulated ML RR-S2 CDA in B16F10 tumor-bearing mice). ​

[0214] Figures 40A-40D The tumor growth curves for each animal in the different groups (i.e. PBS, Exo STING agonist (8 ng), Exo STING agonist (40 ng) and Exo STING agonist (200 ng), respectively) are shown. Figure 39 The tumor growth curves for each animal in the different groups (i.e. PBS, Exo STING agonist (8 ng), Exo STING agonist (40 ng) and Exo STING agonist (200 ng), respectively) are shown.

[0215] Figures 41A-41E The experimental scheme and results of an antigen-specific T cell induction experiment using ovalbumin as antigen in untreated mice injected with 200 pg ovalbumin mixed with PBS, 20 pg free ML RR-S2 CDA, 0.2 pg free ML RR-S2 CDA or 0.2 pg exosome-encapsulated ML RR-S2 CDA are shown. The percentage of ovalbumin-reactive T cells and the number of IFN-gamma-producing splenocytes were determined.

[0216] Figure 42 The tumor growth curves during the study described in Example 12 (i.e. an intratumoral injection study comparing the anti-tumor effect and the induction of immune memory response in E.G7-OVA tumor-bearing mice treated with PBS, 20 pg free ML RR-S2 CDA, 0.2 pg free ML RR-S2 CDA or 0.2 pg exosome-encapsulated ML RR-S2 CDA) are shown.

[0217] Figures 43A-43D The tumor growth curves for each animal in the different groups (i.e. PBS, STING agonist (20 pg), STING agonist (0.2 pg) and Exo STING agonist (0.2 pg), respectively) are shown. Figure 43E The percentage of ovalbumin-reactive memory T cells isolated from the spleen of the animals of each group is shown.

[0218] Figures 44A-44B The potency of STING agonist loaded into native exosomes or exosomes overexpressing PTGFRN in PBMCs freshly prepared ( Figure 44A ) or frozen at -80°C for 7 days ( Figure 44B ) is shown. Potency was measured by IFN production. Figure 44C The decrease in potency of STING loaded into native exosomes or exosomes overexpressing PTGFRN after 7 days of storage at -80°C compared to exosomes freshly prepared is shown.

[0219] Figures 45A-45DRetention of uptake kinetics of PTGFRN overexpressing exosomes after 7 days of storage at -80°C compared to freshly prepared exosomes is shown. Figure 45A and Figure 45B Results from freshly prepared exosomes from two independent donors (donors 1 and 2, respectively) are shown. Figure 45C and Figure 45D Results from exosomes after storage from two independent donors (donors 5 and 6, respectively) are shown.

[0220] Figure 46A Tumor growth curves during the study described in Example 14 (i.e., an intratumoral injection study followed by a lung metastasis challenge comparing the antitumorigenic effect of B16F10 tumor-bearing mice treated with PBS, high or low doses of free 3-3cAIMPdFSH or one of three doses of 3-3cAIMPdFSH loaded into PTGFRN overexpressing exosomes) are shown. Figure 46B Images of representative lungs of animals in each group at the end of the study are shown.

[0221] Figure 47 Microscopic quantification of lung metastasis of animals in the study shown in Figure 46A and Figure 46B

[0222] Figure 48 Histological quantification of lung metastasis of animals in the study shown in Figure 46A and 46B

[0223] Figure 49A Tumor growth curves during the checkpoint blockade study described in Example 15 (i.e., an intratumoral injection study combined with systemic immune checkpoint inhibition (treatment with an anti-PD-1 antibody) in B16F10 tumor-bearing mice treated with 3 doses of 30 ng ML RR-S2 CDA loaded into PTGFRN overexpressing exosomes) are shown. Figure 49B Tumor growth curves during the T cell depletion study described in Example 15 (i.e., an intratumoral injection study combined with T cell depletion (treatment with an anti-CD8 antibody) in B16F10 tumor-bearing mice treated with 3 doses of 100 ng 3-3cAIMPdFSH loaded into PTGFRN overexpressing exosomes) are shown. Figure 49C ​​ELISPOT results showing the study described in Example 15 (i.e., an intratumoral injection study in combination in B16F10 tumor-bearing mice treated with 3 injections of high or low doses of free ML RR-S2 CDA or low dose of ML RR-S2 CDA loaded into PTGFRN-overexpressing exosomes, followed by tumor cell-specific ELISPOT to measure T cell reactivity to tumor antigens).

[0224] Figure 50A A comparison of IFNp responses in PBMCs treated with 3-3cAIMPdFSH, exosome-encapsulated 3-3cAIMPdFSH from wild-type exosomes, PTGFRN-overexpressing exosomes, or PTGFRN knockout exosomes as determined by relative luminescence (RLU) is shown. Figure 50B A comparison of maximum IFNp signals from PBMCs treated with exosomes in Figure 50A A comparison of maximum IFNp signals from PBMCs treated with exosomes in Figure 50C Growth curves of subcutaneously implanted B16F10 melanoma in mice after three intratumoral injections (day 6, day 9, and day 12 post-implantation) of PBS or 20 ng of 3-3cAIMPdFSH loaded into wild-type exosomes, PTGFRN-overexpressing exosomes, or PTGFRN knockout exosomes are shown.

[0225] Figure 51A Percent positive populations of different kinds of tumor infiltrating lymphocytes isolated from subcutaneous tumors injected with Alexa Fluor TM 488-labeled exosomes are shown. Figure 51B Relative populations of CD8 TM T cells isolated from subcutaneous tumors injected with PBS, 200 ng of ML RR-S2 CDA loaded in PTGFRN-overexpressing exosomes (EXOSTING + ), 200 ng of ML RR-S2 CDA, or 20 pg of ML RR-S2 CDA are shown. Figure 51C Relative populations of macrophages isolated from subcutaneous tumors injected with PBS, 200 ng of ML RR-S2 CDA loaded in PTGFRN-overexpressing exosomes (EXOSTING TM ), 200 ng of ML RR-S2 CDA, or 20 pg of ML RR-S2 CDA are shown. Figure 51D Relative populations of macrophages isolated from subcutaneous tumors injected with PBS, 200 ng of ML RR-S2 CDA loaded in PTGFRN-overexpressing exosomes (EXOSTING TMRelative population of dendritic cells isolated from subcutaneous tumors of 200 ng ML RR-S2 CDA or 20 μg ML RR-S2 CDA.

[0226] Figures 52A-52D Quantitative imaging results of IFN beta transcript ( Figure 52A ) or cleaved caspase 3 protein ( Figure 52B ) in mouse sarcoma cells injected with a minidose of free ML RR-S2 CDA or the indicated exosomes (with or without ML RR-S2 CDA) are shown. Figure 52C -D shows radial response analysis of IFN beta ( Figure 52C ) or CXCL10 ( Figure 52D ) transcripts after injection of a minidose of free 3-3-cAIMPdFSH or 3-3 cAIMPdFSH-loaded exosomes.

[0227] Figures 53A-53G Comparison of IFN beta responses in peripheral blood mononuclear cells (PBMCs) treated with exosome-encapsulated STING agonists and free STING agonists as determined by relative luminescence (RLU) is shown. Figure 53A Results of exosomes loaded with STING agonists ML RR-S2 CDA (“Exo ML RR-S2”) or 2-3cGAMP (“Exo 2-3cGAMP”) are shown. The corresponding free STING agonists are denoted as “Free ML RR-S2” and “Free 2-3cGAMP”, respectively. Figure 53B Results of exosomes loaded with STING agonists 3-3cAIMPdFSH (“exo 3-3cAIMPdFSH”) or 3-3cAIM(PS)2 (“exo 3-3 cAIM(PS)2”) are shown. “Free 3-3cAIMPdFSH” and “Free 3-3cAIM(PS)2” represent the free form of the respective agonists. Figure 53C Results of exosomes loaded with 3-3cAIMP (“exo 3-3cAIMP”) and 3-3cAIMPdF (“exo 3-3cAIMPdF”) are shown. The corresponding free STING agonists are shown as “Free 3-3cAIMP” and “Free 3-3cAIMPdF”, respectively. Figure 53D Results of exosomes loaded with STING agonist 3-3cAIMPmFSH (“exo 3-3cAIMPmFSH” and free STING agonist 3-3cAIMPmFSH (“Free 3-3cAIMPmFSH”) are shown. Figure 53EResults are shown for exosomes loaded with the STING agonist CP214 ("Exo-CP214"; open diamonds) and free CP214 STING agonist ("CP214"; closed diamonds). Figure 53F Results are shown for exosomes loaded with the STING agonist CP201 ("Exo-CP201"; open squares) and free CP201 STING agonist ("CP201"; closed squares). Figure 53G Results are shown for exosomes loaded with the STING agonist CP204 ("Exo-CP204"; open triangles) and free CP204 STING agonist ("CP204"; closed triangles). 3-3cAIMPdFSH, 3-3cAIM(PS)2, cAIMPdF, cAIMP correspond to compounds 53, 13, 52 and 51, respectively, in the paper (J Med Chem. 2016 Nov 23; 59(22): 10253-10267). CP214 is 2-3cAMPmFSH. CP201 and CP204 are analogs of compounds of patents WO2017 / 175156 and WO2017 / 175147, respectively.

[0228] Figures 54A-54C IFNβ expression profiles in tissues (tumor, draining lymph node and spleen, respectively) from B16F10 tumor-bearing C57BL / 6 mice (solid bars) or C57BL / 6-Tmem173 gt mice (open bars) following a single intratumoral injection of PBS, 20 pg free 3-3cAIMPdFSH or 0.1 pg exosome-encapsulated 3-3cAIMPdFSH.

[0229] Figures 55A-55C CXCL9 expression profiles in tissues (tumor, draining lymph node and spleen, respectively) from B16F10 tumor-bearing C57BL / 6 mice (solid bars) or C57BL / 6-Tmem173 gt mice (open bars) following a single intratumoral injection of PBS, 20 pg free 3-3cAIMPdFSH or 0.1 pg exosome-encapsulated 3-3cAIMPdFSH.

[0230] Figures 56A-56C CXCL10 expression profiles in tissues (tumor, draining lymph node and spleen, respectively) from B16F10 tumor-bearing C57BL / 6 mice (solid bars) or C57BL / 6-Tmem173 gt mice (open bars) following a single intratumoral injection of PBS, 20 pg free 3-3cAIMPdFSH or 0.1 pg exosome-encapsulated 3-3cAIMPdFSH.

[0231] Figures 57A-57C shows IFN-γ expression profiles in tissues (tumor, draining lymph node, and spleen, respectively) from B16F10 tumor-bearing C57BL / 6 mice (solid bars) or C57BL / 6-Tmem173 gt mice (open bars) following a single intratumoral injection of PBS, 20 pg free 3-3cAIMPdFSH, or 0.1 pg exosome-encapsulated 3-3cAIMPdFSH.

[0232] Figures 58A-58D shows serum cytokine expression profiles (IFN-β, TNF-a, IL-6, and MCP-1, respectively) in B16F10 tumor-bearing C57BL / 6 mice (solid bars) or C57BL / 6-Tmem173 gt mice (open bars) following a single intratumoral injection of PBS, 20 pg free 3-3cAIMPdFSH, or 0.1 pg exosome-encapsulated 3-3cAIMPdFSH.

[0233] Figure 59 shows tumor growth curves for B16F10 tumor-bearing C57BL / 6 mice or C57BL / 6-Tmem173 gt mice during the study described in Example 20 (i.e., a comparison of the efficacy of PBS, 20 pg free 3-3cAIMPdFSH, 0.2 pg exosome-encapsulated 3-3cAIMPdFSH, by intratumoral injection in B16F10 tumor-bearing C57BL / 6 mice or C57BL / 6-Tmem173 gt mice.

[0234] Figure 60 shows tumor growth curves for B16F10 tumor-bearing mice during the study described in Example 21.

[0235] Figures 61A-61E shows tumor growth curves for each animal in the different groups shown in Example 21. Figure 60 Figure 61A Figure 61B Figure 61C Figure 61D Figure 61E

[0236] Figure 62 shows tumor growth curves for CT26.CT25 tumor-bearing BALB / c mice during the study described in Example 22.​​​​​​

[0237] Figure 63 Tumor growth curves for CT26.wt tumor-bearing BALB / c mice during the study described in Example 22 are shown.

[0238] Figure 64 Tumor growth curves for injected B16F10 tumors during the study described in Example 23 are shown.

[0239] Figure 65 Tumor growth curves for uninjected contralateral B16F10 tumors during the study described in Example 23 are shown.

[0240] Figure 66 Tumor pharmacokinetics of 3-3cAIMPdFSH following intratumoral injection of 30 pg free 3-3cAIMPdFSH, 0.2 pg free 3-3cAIMPdFSH, and 0.2 pg exosome-encapsulated 3-3cAIMPdFSH in B16F10 tumors are shown. Table shows half-life for each sample.

[0241] Figure 67 Plasma pharmacokinetics of 3-3cAIMPdFSH in untreated C57BL / 6 mice following intravenous injection of 20 pg free 3-3cAIMPdFSH are shown.

[0242] Figure 68 Plasma pharmacokinetics of 3-3cAIMPdFSH in untreated C57BL / 6 mice following intravenous injection of 0.1 pg, 0.3 pg, and 0.6 pg exosome-encapsulated 3-3cAIMPdFSH are shown. Table shows half-life for each sample.

[0243] Figures 69A-69D Cytokine expression profiles (IFN-b, CXCL9, CXCL10, and IFN-g, respectively) in untreated C57BL / 6 mice livers over time following a single intravenous injection of 20 pg free 3-3cAIMPdFSH or 0.2 pg exosome-encapsulated 3-3cAIMPdFSH are shown.

[0244] Figures 70A-70D Cytokine expression profiles (IFN-b, CXCL9, CXCL10, and IFN-g, respectively) in untreated C57BL / 6 mice spleens over time following a single intravenous injection of 20 pg free 3-3cAIMPdFSH or 0.2 pg exosome-encapsulated 3-3cAIMPdFSH are shown.

[0245] Figures 71A-71ESerum cytokine expression profiles (IFN-β, TNF-α, IL-6, IFN-γ and MCP-1, respectively) in untreated C57BL / 6 mice over time following a single intravenous injection of 20 μg free 3-3cAIMPdFSH or 0.2 μg exosome-encapsulated 3-3cAIMPdFSH are shown.

[0246] Figures 72A-72C IFNβ expression profiles in tissues (lymph node, spleen and liver, respectively) from untreated C57BL / 6 mice following a single subcutaneous injection of PBS, exosomes, 20 μg free 3-3cAIMPdFSH or 0.2 μg exosome-encapsulated 3-3cAIMPdFSH are shown.

[0247] Figures 73A-73C CXCL9 expression profiles in tissues (lymph node, spleen and liver, respectively) from untreated C57BL / 6 mice following a single subcutaneous injection of PBS, exosomes, 20 μg free 3-3cAIMPdFSH or 0.2 μg exosome-encapsulated 3-3cAIMPdFSH are shown.

[0248] Figures 74A-74C CXCL10 expression profiles in tissues (lymph node, spleen and liver, respectively) from untreated C57BL / 6 mice following a single subcutaneous injection of PBS, exosomes, 20 μg free 3-3cAIMPdFSH or 0.2 μg exosome-encapsulated 3-3cAIMPdFSH are shown.

[0249] Figures 75A-75C IFN-γ expression profiles in tissues (lymph node, spleen and liver, respectively) from untreated C57BL / 6 mice following a single subcutaneous injection of PBS, exosomes, 20 μg free 3-3cAIMPdFSH or 0.2 μg exosome-encapsulated 3-3cAIMPdFSH are shown.

[0250] Figures 76A-76E Serum cytokine expression profiles (IFNβ, TNFα, IL-6, IFN-γ and MCP-1, respectively) in untreated C57BL / 6 mice following a single subcutaneous injection of PBS, exosomes, 20 μg free 3-3cAIMPdFSH or 0.2 μg exosome-encapsulated 3-3cAIMPdFSH are shown.

[0251] Figure 77A and Figure 77BQuantitative IFNβ expression profile in the tumor (A) or stromal (B) region from B16F10 tumor sections following intracranial injection of exosomes, 20 pg free 3-3cAIMPdFSH, 0.1 pg free 3-3cAIMPdFSH or 0.1 pg exosome-encapsulated 3-3cAIMPdFSH as described in Example 28. Figure 77A Figure 77B

[0252] Figure 78A Figure 78B Number of CD8-positive cells (A) and F4 / 80-positive cells (B) in tumor sections following intracranial injection of exosomes, 20 pg free 3-3cAIMPdFSH or 0.1 pg exosome-encapsulated 3-3cAIMPdFSH as described in Example 28. Figure 78A Figure 78B

[0253] Figure 79A Primary tumor growth curve of B16F10 tumor-bearing mice during the study described in Example 29 is shown. Figures 79B-79E Tumor growth curve of each animal in the different groups (i.e. PBS, exosomes, ADUS100 and exoCL656, respectively) is shown.

[0254] Figure 80A Re-challenge tumor growth curve of B16F10 tumor-bearing mice during the study described in Example 29 is shown. Figures 80B-80D Tumor growth curve of each animal in the different groups (i.e. PBS, ADUS100 and exoCL656, respectively) is shown. DETAILED DESCRIPTION

[0255] Before the application is described in further detail, it is to be understood that the application is not limited to the particular embodiments described and as such can of course vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting since the scope of the present application will be limited only by the appended claims.

[0256] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present application, representative illustrative methods and materials are now described.

[0257] ​​​​​All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited.

[0258] Those skilled in the art will appreciate, upon reading this disclosure, that each individual embodiment described and illustrated herein has discrete components and features which can be easily separated from the features of any of the other several embodiments without departing from the scope or spirit of the present application. Any recited method can be performed in the order of events recited or in any other order that is logically possible.

[0259] I. DEFINITIONS

[0260] It should be noted that, as used herein and in the appended claims, the singular form "a", "an", and "the" include plural references unless the context clearly dictates otherwise. As such, the terms "a" (or "an"), "one or more" and "at least one" are used interchangeably herein. It is also to be noted the claims can be drafted to exclude any optional element. Thus, this statement is intended to serve as antecedent basis for the use of such exclusive terminology as "solely," "only," and the like in

[0261] Further, as used herein, "and / or" shall, where used, be taken to exist as an explicit disclosure of each of the features or components listed in the respective clause to exist individually or in any combination with one another. As such, the term "and / or" as used in, for example, the phrase "A and / or B" is intended to encompass the following aspects of the disclosure: "A and B"; "A or B"; "A" (alone); and "B" (alone). Likewise, the use of the term "and / or" as used in, for example, the phrase "A, B, and / or C" is intended to encompass each of the following aspects of the disclosure: 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 (alone); B (alone); and C (alone).

[0262] It should be understood that wherever the word "comprising" is used in this text to describe aspects, it is also intended to provide other similar aspects described using the phrases "consisting of and / or "consisting essentially of.

[0263] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and the Oxford Dictionary Of Biochemistry And Molecular Biology, Revised, 2000, Oxford University Press, provide one of skill with a general dictionary of many of the terms used in this disclosure.

[0264] Units, prefixes, and symbols are denoted in their Systeme International de Unites (SI) accepted form. Numeric ranges are inclusive of the numbers defining the range. In instances where a range of values is recited, understanding is that every intervening integer value, as well as each fraction thereof (rounding to the nearest thousandth), between the stated range limits is specifically contemplated. The upper and lower limits of any range can independently be included in or excluded from the range, and each range where either, neither, or both extremes are included is also contemplated. It is therefore understood that any enumerated range is a shorthand manner of disclosing each and every set of values and subranges within the range. For example, a range of 1 to 10 is understood to include any number, combination, or sub-range from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.

[0265] In instances where a value is explicitly recited, it is understood that values approximately or about the same in quantity or amount as the recited value are also within the scope of the disclosure. In instances where a combination is disclosed, each subcombination of the elements of the combination is also specifically disclosed and within the scope of the disclosure. Conversely, in instances where different elements or groups of elements are disclosed singly, combinations of the elements or groups of elements are also disclosed. In instances where any element of the disclosure is disclosed as having a plurality of alternatives, examples of the disclosure in which each alternative is singly excluded or excluded in any combination with other alternatives are also specifically disclosed; more than one element of the disclosure can have such exclusions, and all combinations of elements having such exclusions are specifically disclosed.

[0266] Nucleotides are referred to by their generally accepted single-letter codes. Unless otherwise indicated, nucleotide sequences are written left to right in 5' to 3' orientation. Nucleotides are referred to herein by their well-known single-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Thus, A stands for adenine, C for cytosine, G for guanine, T for thymine, and U for uracil.

[0267] Amino acid sequences are written left to right in amino to carboxyl direction. Amino acids are referred to herein by their well-known three-letter symbols or by their one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.

[0268] The term "about" or "approximately" is used herein to mean roughly about, left and right around, or within... of. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries of the range by the equivalent of one unit in the stated range. The term as used herein means within 5% of the reference amount, e.g., about 50% is understood to cover the range of values from 47.5% to 52.5%.

[0269] As used herein, the term "extracellular vesicle" or "EV" refers to a cell-derived vesicle comprising a membrane that encloses an internal space. Extracellular vesicles include all membrane-bound vesicles (e.g., exosomes, nanovesicles) that are smaller in diameter than the cell from which they are derived. Typically, extracellular vesicles are in the range of 20 nm to 1000 nm in diameter, and can contain various macromolecular payloads within the internal space (i.e., lumen), displayed on the outer surface of the extracellular vesicle, and / or across the membrane. The payloads can include nucleic acids, proteins, carbohydrates, lipids, small molecules, and / or combinations thereof. In some embodiments, the extracellular vesicles comprise a scaffold moiety. By way of example and not limitation, extracellular vesicles include apoptotic bodies, cell debris, cell-derived vesicles obtained by direct or indirect manipulation (e.g., by serial extrusion or treatment with alkaline solution), budding organelles, and vesicles produced by living cells (e.g., by direct plasma membrane budding 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. In some embodiments, the extracellular vesicles are produced by cells expressing one or more transgene products.

[0270] As used herein, the term "exosome" refers to a small (between 20-300 nm in diameter, more preferably between 40-200 nm in diameter) vesicle of cellular origin that comprises a membrane that encloses an internal space (i.e., lumen) and is produced by the cell either by direct plasma membrane budding or by fusion of a late endosome with the plasma membrane. Exosomes are a type of extracellular vesicle. Exosomes comprise lipids or fatty acids and polypeptides, and optionally comprise a payload (e.g., a therapeutic agent), a receptor (e.g., a targeting moiety), a polynucleotide (e.g., a nucleic acid, RNA, or DNA), a sugar (e.g., a monosaccharide, polysaccharide, or glycan), or other molecule. In some embodiments, exosomes comprise a scaffold moiety. Exosomes can be derived from a producer cell and isolated from the producer cell according to their size, density, biochemical parameters, or a combination thereof. In some embodiments, the exosomes of the present disclosure are produced by a cell that expresses one or more transgene products.

[0271] As used herein, the term "nanovesicle" refers to a small (between 20-250 nm in diameter, more preferably between 30-150 nm in diameter) vesicle of cellular origin that comprises a membrane that encloses an internal space and is produced by the cell through direct or indirect manipulation such that the nanovesicle would not be produced by the producer cell in the absence of the manipulation. Suitable manipulation of the producer cell includes, but is not limited to, continuous extrusion, treatment with a basic solution, sonication, or a combination thereof. In some cases, production of nanovesicles can result in destruction of the producer cell. Preferably, the population of nanovesicles is substantially free of vesicles derived from the producer cell by direct budding from the plasma membrane or fusion of a late endosome with the plasma membrane. Nanovesicles comprise lipids or fatty acids and polypeptides, and optionally comprise a payload (e.g., a therapeutic agent), a receptor (e.g., a targeting moiety), a polynucleotide (e.g., a nucleic acid, RNA, or DNA), a sugar (e.g., a monosaccharide, polysaccharide, or glycan), or other molecule. In some embodiments, nanovesicles comprise a scaffold moiety. Nanovesicles, once they are derived from the producer cell according to the manipulation, can be isolated from the producer cell according to their size, density, biochemical parameters, or a combination thereof.

[0272] The term "modified," when used in the context of an exosome described herein, refers to an alteration or engineering of the EV such that the modified EV is different from a naturally occurring EV. In some embodiments, a modified EV described herein comprises a membrane that differs in protein, lipid, small molecule, carbohydrate, etc. composition compared to the membrane of a naturally occurring EV (e.g., the membrane comprises a higher density or amount of a native EV protein and / or the membrane comprises a protein that is not naturally occurring in an EV). In certain embodiments, such modifications to the membrane alter the outer surface of the EV. In certain embodiments, such modifications to the membrane alter the lumen of the EV.

[0273] As used herein, the term“scaffold portion” refers to a molecule that can be used to anchor a STING agonist or any other compound of interest (e.g., a payload) disclosed herein on the luminal surface or outer surface of an EV to an EV. In certain embodiments, the scaffold portion comprises a synthetic molecule. In some embodiments, the scaffold portion comprises a non-polypeptide moiety. In other embodiments, the scaffold portion comprises a lipid, carbohydrate, or protein that is naturally found in an EV. In some embodiments, the scaffold portion comprises a lipid, carbohydrate, or protein that is not naturally found in an exosome. In certain embodiments, the scaffold portion is Scaffold X. In some embodiments, the scaffold portion is Scaffold Y. In further embodiments, the scaffold portion comprises Scaffold X and Scaffold Y

[0274] As used herein, the term“scaffold X” refers to an exosome protein that has recently been identified on the surface of an exosome. See, e.g., U.S. Patent No. 10,195,290, which is incorporated by reference herein 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 a class of ATP transporters (“ATP1A1 protein,” “ATP1A2 protein,” “ATP1A3 protein,” “ATP1A4 protein,” “ATP1B3 protein,” “ATP2B1 protein,” “ATP2B2 protein,” “ATP2B3 protein,” “ATP2B protein”). In some embodiments, a Scaffold X protein can be an intact protein or a fragment thereof (e.g., a functional fragment, e.g., a minimal fragment that is capable of anchoring another moiety on the outer surface or luminal surface of an EV (e.g., an exosome)). In some embodiments, Scaffold X can anchor a moiety (e.g., a STING agonist) to the outer surface or luminal surface of an EV (e.g., an exosome).

[0275] As used herein, the term "scaffold Y" refers to a newly identified exosome protein within the luminal surface of an exosome. See, e.g., International Application No. PCT / US2018 / 061679, which is incorporated by reference herein in its entirety. Non-limiting examples of scaffold proteins Y include: myristoylated alanine-rich protein kinase C substrate ("MARCKS protein"); myristoylated alanine-rich protein kinase C substrate 1 ("MARCKS LI protein"); and brain acid-soluble protein 1 ("BASPl protein"). In some embodiments, a scaffold Y protein can be an intact protein or a fragment thereof (e.g., a functional fragment, e.g., a minimal fragment capable of anchoring a moiety to the luminal surface of an EV (e.g., an exosome)). In some embodiments, a scaffold Y can anchor a moiety (e.g., a STING agonist) to the lumen of an EV (e.g., an exosome).

[0276] As used herein, the term "fragment" of a protein (e.g., a therapeutic protein, scaffold X, or scaffold Y) refers to an amino acid sequence of a protein that is shorter than the naturally occurring sequence, has a deletion of the N- and / or C-terminus of the protein, or any portion thereof, as compared to the naturally occurring protein. As used herein, the term "functional fragment" refers to a fragment of a protein that retains the function of the protein. Thus, in some embodiments, a functional fragment of a scaffold X protein retains the ability to anchor a moiety to the luminal surface and / or the outer surface of an EV. Similarly, in certain embodiments, a functional fragment of a scaffold Y protein retains the ability to anchor a moiety to the luminal surface of an EV. Whether a fragment is a functional fragment can be assessed by any art-known method of assessing the protein content of an EV, including Western blotting, FACS analysis, and fusion of the fragment to a self-fluorescent protein such as, e.g., GFP. In certain embodiments, a functional fragment of a scaffold X protein retains at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100% of the ability, e.g., the ability to anchor a moiety, of a naturally occurring scaffold X protein. In some embodiments, a functional fragment of a scaffold Y protein retains at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100% of the ability, e.g., the ability to anchor another molecule, of a naturally occurring scaffold Y protein.

[0277] As used herein, the term "variant" of a molecule (e.g., a functional molecule, an antigen, a scaffold X, and / or a scaffold Y) refers to a molecule that shares certain structural and functional attributes with another molecule upon comparison by methods known in the art. For example, a variant of a protein can include a substitution, insertion, deletion, frameshift, or rearrangement in another protein.

[0278] In some embodiments, the variant of Scaffold X comprises a variant having at least about 70% identity to a full-length, mature PTGFRN, BSG, IGSF2, IGSF3, IGSF8, ITGB1, ITGA4, SLC3A2, or ATP transporter, or a fragment (e.g., a functional fragment) of PTGFRN, BSG, IGSF2, IGSF3, IGSF8, ITGB1, ITGA4, SLC3A2, or ATP transporter. In some embodiments, the variant of a variant of a fragment of PTGFRN shares at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to PTGFRN according to SEQ ID NO: 1, or a functional fragment thereof. In some embodiments, the variant of a variant of a fragment of BSG shares at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to BSG according to SEQ ID NO: 9, or a functional fragment thereof. In some embodiments, the variant of a variant of a fragment of IGSF2 shares at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to IGSF2 according to SEQ ID NO: 34, or a functional fragment thereof. In some embodiments, the variant of a variant of a fragment of IGSF3 shares at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to IGSF3 according to SEQ ID NO: 20, or a functional fragment thereof. In some embodiments, the variant of a variant of a fragment of IGSF8 shares at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to IGSF8 according to SEQ ID NO: 14, or a functional fragment thereof. In some embodiments, the variant of a variant of a fragment of ITGB1 shares at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to ITGB1 according to SEQ ID NO: 21, or a functional fragment thereof.In some embodiments, the variant of a variant or fragment of ITGA4 shares at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to ITGA4 according to SEQ ID NO:22, or a functional fragment thereof. In some embodiments, the variant of a variant or fragment of SLC3A2 shares at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SLC3A2 according to SEQ ID NO:23, or a functional fragment thereof. In some embodiments, the variant of a variant or fragment of ATP1A1 shares at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to ATP1A1 according to SEQ ID NO:24, or a functional fragment thereof. In some embodiments, the variant of a variant or fragment of ATP1A2 shares at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to ATP1A2 according to SEQ ID NO:25, or a functional fragment thereof. In some embodiments, the variant of a variant or fragment of ATP1A3 shares at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to ATP1A3 according to SEQ ID NO:26, or a functional fragment thereof. In some embodiments, the variant of a variant or fragment of ATP1A4 shares at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to ATP1A4 according to SEQ ID NO:27, or a functional fragment thereof. In some embodiments, the variant of a variant or fragment of ATP1B3 shares at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to ATP1B3 according to SEQ ID NO:28, or a functional fragment thereof. In some embodiments, the variant of a variant or fragment of ATP2B1 shares at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to ATP2B1 according to SEQ ID NO:29, or a functional fragment thereof.In some embodiments, a variant of a variant of a fragment of ATP2B2 shares at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to ATP2B2 according to SEQ ID NO: 30, or a functional fragment thereof. In some embodiments, a variant of a variant of a fragment of ATP2B3 shares at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to ATP2B3 according to SEQ ID NO: 31, or a functional fragment thereof. In some embodiments, a variant of a variant of a fragment of ATP2B4 shares at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to ATP2B4 according to SEQ ID NO: 32, or a functional fragment thereof. In some embodiments, a variant of a variant of a scaffold X protein disclosed herein retains the ability to specifically target EVs. In some embodiments, a scaffold X comprises one or more mutations, e.g., conservative amino acid substitutions.

[0279] In some embodiments, a variant of a variant of a fragment of MARCKS, MARCKSL1, BASP1, or a fragment of MARCKS, MARCKSL1, or BASP1 shares at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to MARCKS according to SEQ ID NO: 47, or a functional fragment thereof. In some embodiments, a variant of a variant of a fragment of MARCKSL1 shares at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to MARCKSL1 according to SEQ ID NO: 48, or a functional fragment thereof. In some embodiments, a variant of a variant of a fragment of BASP1 shares at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to BASP1 according to SEQ ID NO: 49, or a functional fragment thereof. In some embodiments, a variant of a variant of a scaffold Y protein retains the ability to specifically target the lumen of EVs. In some embodiments, a scaffold Y comprises one or more mutations, e.g., conservative amino acid substitutions.

[0280] A "conservative amino acid substitution" is one in which the 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, including 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), nonpolar 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 one amino acid in a polypeptide is replaced with another amino acid from the same side chain family, the substitution is considered to be conservative. In another embodiment, a string of amino acids can be conservatively replaced with a string of structurally similar amino acids that differ in the order and / or composition of side chain family members.

[0281] The term "percent sequence identity" or "percent identity" between two polynucleotide or polypeptide sequences means the percentage of matching identical positions in a comparison window, taking into account any additions or deletions (i.e., gaps) that must be introduced in order to achieve the best alignment of the two sequences. A matching position is one in which the same nucleotide or amino acid is present in both the target sequence and the reference sequence. Since gaps are not nucleotides or amino acids, a gap present in the target sequence is not counted. Likewise, since only the nucleotides or amino acids from the target sequence are counted, not those from the reference sequence, a gap present in the reference sequence is not counted.

[0282] The percent sequence identity is calculated by determining the number of positions where the same amino acid residue or nucleic acid base occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to yield the percent sequence identity. The comparison of sequences and the determination of the percent sequence identity between two sequences can be accomplished using software programs commonly available to those skilled in the art. Suitable software programs are available from various sources for the alignment of protein and nucleotide sequences. One suitable program for determining percent sequence identity is bl2seq, which is part of the BLAST suite of programs available from the National Center for Biotechnology Information BLAST website (blast.ncbi.nlm.nih.gov). Bl2seq uses either the BLASTN or BLASTP algorithm for comparison between two sequences. BLASTN is used for comparing nucleic acid sequences, while BLASTP is used for comparing amino acid sequences. Other suitable programs are, for example, Needle, Stretcher, Water, or Matcher, which are part of the EMBOSS suite of bioinformatics programs, and are also available from the European Bioinformatics Institute (EBI) at www.ebi.ac.uk / Tools / psa.

[0283] Different regions within a single polynucleotide or polypeptide target sequence aligned to a polynucleotide or polypeptide reference sequence can each have their own percent sequence identity. Note that percent sequence identity values are rounded to the nearest tenth. For example, 80.11, 80.12, 80.13, and 80.14 are rounded down to 80.1, while 80.15, 80.16, 80.17, 80.18, and 80.19 are rounded up to 80.2. Note also that length values will always be integers.

[0284] Those skilled in the art will appreciate that the generation of sequence alignments for the calculation of percent sequence identity is not limited to binary sequence-sequence comparisons driven by primary sequence data alone. Sequence alignments can be derived from multiple sequence alignments. One suitable program for generating multiple sequence alignments is ClustalW2, available at www.clustal.org. Another suitable program is MUSCLE, available at www.drive5.com / muscle / . ClustalW2 and MUSCLE are available, for example, from the EBI.

[0285] It will also be appreciated that sequence alignments can be generated by integrating sequence data with data from heterogeneous sources, such as structural data (e.g., crystallographic protein structures), functional data (e.g., positions of mutations), or phylogenetic data. Suitable programs for integrating heterogeneous data to generate multiple sequence alignments are available at www.tcoffee.org, and alternatively T-Coffee, which can be obtained, e.g., from EBI. It will also be appreciated that the final alignment for calculating percent sequence identity can be curated automatically or manually.

[0286] Polynucleotide variants can comprise alterations in the coding region, non-coding region, or both. In one embodiment, the polynucleotide variant comprises alterations that result in a silent substitution, addition, or deletion, but do not alter the properties or activity of the encoded polypeptide. In another embodiment, the nucleotide variant is created by a silent substitution due to the degeneracy of the genetic code. In other embodiments, variants that substitute, delete, or add 5-10, 1-5, or 1-2 amino acids in any combination. Polynucleotide variants can be generated for a variety of reasons, for example, to optimize codon expression for a particular host (changing codons in a human mRNA to other codons, e.g., for a bacterial host such as E. coli).

[0287] Naturally occurring variants, referred to as "allelic variants," refer to one of several alternative forms of a gene occupying the same locus on a chromosome of an organism (Genes II, Lewin, B., ed., John Wiley & Sons, New York (1985)). These allelic variants can vary in polynucleotide and / or polypeptide changes and are included in the present disclosure. Alternatively, non-naturally occurring variants can be produced by mutagenesis techniques or direct synthesis.

[0288] Using known methods of protein engineering and recombinant DNA technology, variants can be generated to improve or alter the characteristics of the polypeptide. For example, one or more amino acids can be deleted from the N- or C-terminus of a secreted protein without substantially losing biological function. Ron et al., J. Biol. Chem. 268:2984-2988 (1993) (incorporated by reference in its entirety) report that variant KGF proteins have heparin binding activity even after deletion of 3, 8, or 27 amino-terminal amino acid residues. Similarly, interferon gamma shows up to 10-fold 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 by reference in its entirety).

[0289] Furthermore, substantial evidence suggests that variants often retain biological activities similar to those of naturally occurring proteins. For example, Gayle et al. (J. Biol. Chem 268:22105-22111 (1993), incorporated herein by reference in its entirety) conducted an extensive mutational analysis of the human cytokine IL-1a. They generated over 3,500 individual IL-1a mutants using random mutagenesis, with each variant exhibiting an average of 2.5 amino acid changes across the entire length of the molecule. Multiple mutations were examined at every possible amino acid position. The researchers found that "most molecules could be altered with little effect on [binding or biological activity]" (see abstract). In fact, of the more than 3,500 nucleotide sequences examined, only 23 unique amino acid sequences produced proteins with significantly different activities from the wild-type.

[0290] As described above, peptide variants include, for example, modified peptides. Modifications include, for example, acetylation, acylation, ADP-ribosylation, amidation, covalent linkage of flavin, covalent linkage of heme moieties, covalent linkage of nucleotides or nucleotide derivatives, covalent linkage of lipids or lipid derivatives, covalent linkage of phosphatidylinositol, crosslinking, cyclization, disulfide bond formation, demethylation, formation of covalent crosslinks, formation of cysteine ​​residues, formation of pyroglutamate, formylation, γ-carboxylation, glycosylation, GPI anchoring, hydroxylation, iodination, methylation, myristylation, oxidation, polyethylene glycolation (Mei et al., Blood 116:270-79 (2010), which is incorporated herein by reference in its entirety), proteolytic processing, phosphorylation, isopreneation, racemization, selenoylation, sulfation, transfer RNA-mediated addition of amino acids to proteins such as argininoylation, and ubiquitous proteination. In some implementations, the bracket X and / or bracket Y can be modified in any convenient location.

[0291] As used herein, the term "producer cell" refers to cells used to produce EVs. Producer cells can be cells cultured in vitro or cells in vivo. Producer cells include, but are not limited to, cells known to be effective at producing EVs (e.g., exogenous cells) (e.g., HEK293 cells), Chinese hamster ovary (CHO) cells, mesenchymal stem cells (MSCs), BJ human foreskin fibroblasts, s9f cells, fHDF fibroblasts, etc. Neuronal precursor cells, Amniotic fluid cells, adipose mesenchymal stem cells, and RPTEC / TERT1 cells. In certain embodiments, the producer cell is an antigen presenting cell. In some embodiments, the producer cell is a bacterial cell. In some embodiments, the producer cell is a dendritic cell, a B cell, a mast cell, a macrophage, a neutrophil, a Kupffer-Browicz cell, or a cell derived from any of these, or any combination thereof. In some embodiments, the producer cell is not a bacterial cell. In other embodiments, the producer cell is not an antigen presenting cell.

[0292] As used herein, the term“associated with” refers to the encapsulation of a first moiety (e.g., a STING agonist) into a second moiety (e.g., an extracellular vesicle), or to the formation of a covalent or non-covalent bond between a first moiety and a second moiety (e.g., a STING agonist and an extracellular vesicle, respectively, e.g., a scaffold moiety and a STING agonist expressed in or on an extracellular vesicle, e.g., scaffold X (e.g., PTGFRN protein) on the luminal surface or the outer surface of an extracellular vesicle, respectively). In one embodiment, the term“associated with” means a covalent, non-peptide, or non-covalent bond. For example, the amino acid cysteine comprises a thiol that can form a disulfide bond or bridge with a thiol on a second cysteine residue. Examples of covalent bonds include, but are not limited to, a peptide bond, a metal bond, a hydrogen bond, a disulfide bond, a sigma bond, a pi bond, a delta bond, a glycosidic bond, an agnostic bond, a bent bond, a dipole bond, a pi backbone, a double bond, a triple bond, a quadruple bond, a quintuple bond, a sextuple bond, a complexation, a supercomplexation, an aromaticity, a hapticity, or an antibond. Non-limiting examples of non-covalent bonds include an ionic bond (e.g., a cation-pi bond or a salt bond), a metal bond, a hydrogen bond (e.g., a dihydrogen bond, a dihydrogen complex, a low-barrier hydrogen bond, or a symmetrical hydrogen bond), a van der Waals force, a London dispersion force, a mechanical bond, a halogen bond, aurophilicity, intercalation, stacking, an entropic force, or a chemical polarity. In other embodiments, the term“associated with” means the state of encapsulation of a second moiety (e.g., a STING agonist) by a first moiety (e.g., an extracellular vesicle). In the encapsulated state, the first moiety and the second moiety can be connected to each other. In other embodiments, encapsulation means that the first moiety and the second moiety are not physically and / or chemically connected to each other.

[0293] As used herein, the terms "linked to" or "conjugated to" are used interchangeably to refer to a covalent or non-covalent linkage between a first moiety and a second moiety (e.g., a STING agonist and an extracellular vesicle, e.g., a scaffold moiety and a STING agonist expressed in or on an extracellular vesicle, e.g., a scaffold X (e.g., PTGFRN protein) expressed on the luminal surface or the outer surface of an extracellular vesicle, respectively).

[0294] The term "encapsulated," or grammatically different forms of this term (e.g., encapsulation or encapsulating), refers to a state or process in which a first moiety (e.g., a STING agonist) is within a second moiety (e.g., an EV, e.g., an exosome) without the two moieties being chemically or physically linked. In some embodiments, the term "encapsulated" can be used interchangeably with "in the lumen of." Non-limiting examples of encapsulating a first moiety (e.g., a STING agonist) into a second moiety (e.g., an EV, e.g., an exosome) are disclosed elsewhere herein.

[0295] As used herein, the terms “isolate,” “isolated,” and “isolating” or “purify,” “purified,” and “purifying” and “extracted,” and “extracting” are used interchangeably to refer to the state of a preparation of desired EVs that has been subjected to one or more purification processes, e.g., selection or enrichment of a preparation of desired EVs. In some embodiments, isolating or purifying as used herein is a process of removing, partially removing (e.g., a portion of) EVs from a sample containing producer cells. In some embodiments, an isolated EV composition is free of detectable undesirable activity, or alternatively, the level or amount of undesirable activity is at or below an acceptable level or amount. In other embodiments, the amount and / or concentration of desired EVs of an isolated EV composition is at or above an acceptable amount and / or concentration. In other embodiments, an isolated EV composition is enriched compared to the starting material (e.g., a producer cell preparation) from which the composition is obtained. Such enrichment can be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99%, 99.999%, 99.9999%, or greater than 99.9999% compared to the starting material. In some embodiments, an isolated EV preparation is substantially free of residual biological products. In some embodiments, an isolated EV preparation is 100% free, 99% free, 98% free, 97% free, 96% free, 95% free, 94% free, 93% free, 92% free, 91% free, or 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 composition is free of detectable producer cells and only EVs are detectable.

[0296] As used herein, the term “agonist” refers to a molecule that binds to and activates a receptor to produce a biological response. A receptor can be activated by an endogenous or exogenous agonist. Non-limiting examples of endogenous agonists include hormones, neurotransmitters, and cyclic dinucleotides. Non-limiting examples of exogenous agonists include drugs, small molecules, and cyclic dinucleotides. An agonist can be a full, partial, or inverse agonist.

[0297] As used herein, the term "antagonist" refers to a molecule that blocks or inhibits an agonist-mediated response, but is not a molecule that itself elicits a biological response upon binding to a receptor. Many antagonists achieve their efficacy by competing with an endogenous ligand or substrate on a structurally defined binding site on the receptor. Non-limiting examples of antagonists include alpha blockers, beta blockers, and calcium channel blockers. An antagonist can be a competitive, non-competitive, or uncompetitive antagonist.

[0298] As used herein, the term "free STING agonist" means a STING agonist that is not associated with an extracellular vesicle, but is otherwise identical to a STING agonist associated with an extracellular vesicle. In particular, a free STING agonist is the same STING agonist associated with an extracellular vesicle when compared to the extracellular vesicle with which the STING agonist is associated. In some embodiments, a free STING agonist is the same amount as a STING agonist associated with an extracellular vesicle when the free STING agonist is compared to the extracellular vesicle containing the STING agonist in terms of efficacy, toxicity, and / or any other characteristic.

[0299] As used herein, the term "ligand" refers to a molecule that binds to a receptor and modulates the receptor to produce a biological response. The modulation can be activation, deactivation, blockade, or inhibition of a receptor-mediated biological response. The receptor can be modulated by an endogenous or exogenous ligand. Non-limiting examples of endogenous ligands include antibodies and peptides. Non-limiting examples of exogenous agonists include drugs, small molecules, and cyclic dinucleotides. A ligand can be a full ligand, a partial ligand, or a reverse ligand.

[0300] As used herein, the term "antibody" encompasses immunoglobulins, whether naturally produced or produced in part or wholly synthetically, and fragments thereof. The term also encompasses any protein having a binding domain homologous to that of an immunoglobulin binding domain. "Antibody" also includes polypeptides comprising a framework region from an immunoglobulin gene or fragment thereof that specifically binds and recognizes an antigen. The use of the term antibody is meant to include intact antibodies, polyclonal antibodies, monoclonal antibodies, and recombinant antibodies, fragments thereof, and further includes single chain antibodies, humanized antibodies, murine antibodies, chimeric antibodies, murine-human antibodies, murine-primate antibodies, 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 antibodies and multispecific antibodies, so long as they exhibit the desired biological activity or function.

[0301] As used herein, the term "therapeutically effective amount" refers to the amount of an agent or a pharmaceutical compound sufficient to effect a desired therapeutic, pharmacological, and / or physiological effect in a subject in need thereof. A therapeutically effective amount can be a "prophylactically effective amount" as prophylaxis can be considered a treatment.

[0302] As used herein, the term "pharmaceutical composition" refers to one or more compounds described herein, e.g., EVs, in admixture or in suspension with one or more other chemical components such as pharmaceutically acceptable carriers and excipients. One object of a pharmaceutical composition is to facilitate administration of the EV formulation to a subject. The term "excipient" or "carrier" refers to an inert substance added to a pharmaceutical composition to further facilitate administration of a compound. The terms "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" and grammatical variations thereof include any agent approved by a regulatory agency of the US Federal Government or listed in the US Pharmacopeia for use in animals, including humans, and any carrier or diluent that does not cause an undesirable physiological effect in a subject to whom the composition is administered and does not abrogate the biological activity and properties of the administered compound. Included are excipients and carriers that are safe, non-toxic, and desirable.

[0303] As used herein, the term "payload" refers to a therapeutic agent that acts on a target (e.g., a target cell) that comes into contact with the EV. Payloads that can be introduced into an EV and / or producer cell include therapeutic agents such as nucleotides (e.g., nucleotides that comprise a detectable moiety or a toxin or disrupt transcription), nucleic acids (e.g., DNA or mRNA molecules that encode polypeptides such as enzymes, or RNA molecules with regulatory functions such as miRNAs, dsDNA, IncRNAs, and siRNAs), amino acids (e.g., amino acids that comprise a detectable moiety or a toxin or disrupt translation), polypeptides (e.g., enzymes), lipids, carbohydrates, and small molecules (e.g., small molecule drugs and toxins).

[0304] The terms "administration," "administering," and variations thereof, refer to introducing a composition, such as an EV or an agent, into a subject and include simultaneous and sequential introduction of the composition or agent. The composition or agent is introduced into the subject by any suitable route, including intratumorally, orally, intrapulmonarily, intranasally, parenterally (intravenously, intraarterially, intramuscularly, intraperitoneally, or subcutaneously), rectally, intralymphatically, intrathecally, periocularly, or topically. Administration includes self-administration and administration by another person. A suitable route of administration allows the composition or agent to perform its intended function. For example, if a suitable route is intravenously, the composition is administered by introducing the composition or agent into a vein of the subject.

[0305] As used herein, the terms "treat," "treatment," or "treating" refer to, e.g., a reduction in severity; a shortening of the duration; an improvement or elimination of one or more symptoms associated with a disease or condition; the provision of a beneficial effect to a subject with a disease or condition, but not necessarily a cure of the disease or condition. The term also includes the prophylaxis or prevention of a disease or condition or a symptom thereof. In one embodiment, the terms "treating" or "treatment" refer to inducing an immune response against an antigen in a subject.

[0306] As used herein, the terms "prevent" or "preventing" refer to reducing or decreasing the occurrence or severity of a particular outcome. In some embodiments, prevention of an outcome is achieved by prophylactic treatment.

[0307] As used herein, the terms "modulate," "modulating," "modify," and / or "modulator" generally refer to the ability to increase or decrease, e.g., directly or indirectly facilitate / stimulate / upregulate or interfere / inhibit / downregulate, a particular concentration, level, expression, function, or behavior, such as, e.g., acting as an antagonist or agonist. In some instances, a modulator can increase and / or decrease a certain concentration, level, activity, or function relative to a control, or relative to an average activity level that would normally be expected, or relative to a control activity level.

[0308] As used herein, "mammalian subject" includes all mammals, including, but not limited to, humans, domestic 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.).

[0309] The terms "individual," "subject," "host," and "patient" are used interchangeably herein and refer to any mammalian subject for whom diagnosis, treatment, or therapy is desired, particularly humans. The methods described herein are applicable to both human therapy and veterinary applications. In some embodiments, the subject is a mammal, while in other embodiments, the subject is a human.

[0310] As used herein, the term“substantially free” means that a sample comprising EVs contains less than 10% of macromolecules by mass / volume (m / v) percent concentration. Some fractions can contain less than 0.001%, less than 0.01%, less than 0.05%, less than 0.1%, less than 0.2%, less than 0.3%, less than 0.4%, less than 0.5%, less than 0.6%, less than 0.7%, less than 0.8%, less than 0.9%, less than 1%, less than 2%, less than 3%, less than 4%, less than 5%, less than 6%, less than 7%, less than 8%, less than 9%, or less than 10% (m / v) of macromolecules.

[0311] As used herein, the term“macromolecule” means a nucleic acid, an exogenous protein, a lipid, a carbohydrate, a metabolite, or a combination thereof.

[0312] As used herein, the terms“non-substantial,”“reduced,” or“negligible” refer to the presence, level, or amount of an inflammatory response in a subject after administration of a sample comprising EVs encapsulating a STING agonist relative to a baseline inflammatory response in the subject or compared to an inflammatory response in the subject to administration of a free STING agonist. For example, a negligible or non-substantial presence, level, or amount of systemic inflammation can be less than 0.001%, less than 0.01%, less than 0.1%, less than 0.2%, less than 0.3%, less than 0.4%, less than 0.5%, less than 0.6%, less than 0.7%, less than 0.8%, less than 0.9%, less than 1%, less than 2%, less than 3%, less than 4%, less than 5%, less than 6%, less than 7%, less than 8%, less than 9%, less than 10%, less than 12%, less than 15%, less than 17%, less than 20%, or less than 25% of systemic inflammation relative to a baseline inflammation of the subject or compared to an immune response of the subject to administration of a free STING agonist. The level or amount of systemic inflammation can be less than 0.1-fold, less than 0.5-fold, less than 0.5-fold, less than 1-fold, less than 1.5-fold, less than 2-fold relative to the baseline or compared to the inflammatory response to administration of a free STING agonist.

[0313] Ranges recited herein are to be understood as shorthand for all values within the range, including the recited endpoints. For example, a range of 1 to 50 is to be understood as including any value, combination, or sub-range 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.

[0314] Unless otherwise indicated, reference to a compound having one or more stereocenters is intended to refer to each stereoisomer and all combinations of stereoisomers thereof.

[0315] II. Compositions Containing STING Agonists (Vesicles)

[0316] The innate immune system recognizes pathogen-associated molecular patterns (PAMPs) through pattern recognition receptors (PRRs) that induce immune responses. PRRs recognize a variety of pathogen molecules, including single- and double-stranded RNA and DNA. PRRs such as retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs) and some toll-like receptors (TLRs) recognize RNA ligands. DNA ligands are recognized by cyclic GMP-AMP synthase (cGAS), AIM2, and other TLRs. TLRs, RLRs, and AIM2 directly interact with other signaling cascade adaptor proteins to activate transcription factors, while cGAS produces cGAMP, a cyclic dinucleotide molecule that activates a stimulator of interferon genes (STING) receptor. Both STING and RLRs activate the adaptor kinase TBK1, which induces activation of the transcription factors IRF3 and NF-KB and leads to production of type I IFNs and proinflammatory cytokines.

[0317] Cyclic dinucleotides (CDNs) were first identified as bacterial signaling molecules, characterized by two 3', 5' phosphodiester linkages, such as in the molecule c-di-GMP. While STING can be activated by bacterial CDNs, the innate immune response in mammalian cells is also mediated by the CDN signaling molecule cGAMP, which is produced by cGAS. cGAMP is characterized by mixed 2', 5' and 3', 5' phosphodiester linkages. Both bacterial and mammalian CDNs directly interact with STING to induce a proinflammatory signaling cascade, resulting in production of type I IFNs, such as IFNa and IFN-β.

[0318] II.A. STING agonists

[0319] The STING agonists used in the present disclosure can be cyclic dinucleotide (CDN) agonists or non-cyclic dinucleotide agonists. Cyclic purine dinucleotides, such as, but not limited to, cGMP, cyclic di-GMP (c-di-GMP), cAMP, cyclic di-AMP (c-di-AMP), cyclic-di-GMP-AMP (cGAMP), cyclic di-IMP (c-di-IMP), cyclic AMP-IMP (cAIMP), and any analogs thereof, are known to stimulate or enhance the immune or inflammatory response in a patient. The CDNs can have 2'2', 2'3', 2'5', 3'3', or 3'5' linkages connecting the cyclic dinucleotides, or any combination thereof.

[0320] Cyclic purine dinucleotides can be modified by standard organic chemistry techniques to produce analogs of purine dinucleotides. Suitable purine dinucleotides include, but are not limited to, adenine, guanine, inosine, hypoxanthine, xanthine, isoguanine, or any other suitable purine dinucleotide known in the art. The cyclic dinucleotide can be a modified analog. Any suitable modification known in the art can be used, including but not limited to phosphorothioate, biphosphorothioate, fluorinated, and difluorinated modifications.

[0321] Non-cyclic dinucleotide agonists can also be used, such as 5,6-dimethylxanthenone-4-acetic acid (DMXAA), or any other non-cyclic dinucleotide agonist known in the art.

[0322] Any STING agonist is contemplated for use. Among the STING agonists are DMXAA, STING agonist-1, ML RR-S2CDA, ML RR-S2c-di-GMP, ML-RR-S2 cGAMP, 2’3’-c-di-AM(PS)2, 2’3’-cGAMP, 2’3’-cGAMPdFHS, 3'3'-cGAMP, 3'3'-cGAMPdFSH, cAIMP, cAIM(PS)2, 3’3’-cAIMP, 3’3’-cAIMPdFSH, 2’2’-cGAMP, 2’3’-cGAM(PS)2, 3'3'-cGAMP, c-di-AMP, 2'3'-c-di-AMP, 2’3’-c-di-AM(PS)2, c-di-GMP, 2’3’-c-di-GMP, c-di-IMP, c-di-UMP, or any combination thereof. In a preferred embodiment, the STING agonist is 3'3'-cAIMPdFSH, alternatively named 3-3cAIMPdFSH. Other STING agonists known in the art can also be used.

[0323] In some embodiments, STING agonists useful in the present disclosure include compounds having the formula:

[0324]

[0325] wherein:

[0326] X1is H, OH, or F;

[0327] X2is H, OH, or F;

[0328] Z is OH, OR1, SH, or SR1, wherein:

[0329] i) R1is Na or NH4, or

[0330] ii) R1is an enzyme-labile group that provides OH or SH in vivo, such as pivaloyloxymethyl;

[0331] Bi and B2are bases selected from:

[0332]

[0333] with the proviso that:

[0334] - in formula (I): X1and X2are not OH,

[0335] - in formula (II): when X1and X2are OH, Bi is not adenine and B2is not guanine, and

[0336] - in formula (III): when X1and X2are OH, Bi is not adenine, B2is not guanine and Z is not OH. See WO 2016 / 096174, the contents of which are incorporated by reference in their entirety.

[0337] In some embodiments, STING agonists useful for the present disclosure include:

[0338]

[0339] pharmaceutically acceptable salts thereof. See WO 2016 / 096174 Al.

[0340] In other embodiments, STING agonists useful for the present disclosure include compounds having the formula:

[0341]

[0342] or any pharmaceutically acceptable salt thereof.

[0343] In some embodiments, STING agonists useful for the present disclosure include compounds having the formula:

[0344]

[0345] wherein each symbol is defined in WO 2014 / 093936, the contents of which are incorporated by reference in their entirety.

[0346] In some embodiments, STING agonists useful for the present disclosure include compounds having the formula:

[0347]

[0348] wherein each symbol is defined in WO 2014 / 189805, the contents of which are incorporated herein by reference in their entirety.

[0349] In some embodiments, STING agonists useful in the present disclosure include compounds having the formula:

[0350]

[0351] wherein each symbol is defined in WO 2015 / 077354, the contents of which are incorporated herein by reference in their entirety. See also Cell reports 11, 1018-1030 (2015).

[0352] In some embodiments, STING agonists useful in the present disclosure include c-di-AMP, c-di-GMP, c-di-IMP, c-AMP-GMP, c-AMP-IMP, and c-GMP-IMP, as described in WO 2013 / 185052 and Sci. Transl. Med. 283, 283ra52 (2015), the contents of which are incorporated herein by reference in their entirety.

[0353] In some embodiments, STING agonists useful in the present disclosure include compounds having the formula:

[0354]

[0355] wherein each symbol is defined in WO 2014 / 189806, the contents of which are incorporated herein by reference in their entirety.

[0356] In some embodiments, STING agonists useful in the present disclosure include compounds having the formula:

[0357]

[0358] wherein each symbol is defined in WO 2015 / 185565, the contents of which are incorporated herein by reference in their entirety.

[0359] In some embodiments, STING agonists useful in the present disclosure include compounds having the formula:

[0360] or

[0361] wherein each symbol is defined in WO 2014 / 179760, the contents of which are incorporated herein by reference in their entirety.

[0362] In some embodiments, STING agonists useful for the present disclosure include compounds having the following formula:

[0363]

[0364] wherein each symbol is defined in WO 2014 / 179335, the contents of which are incorporated by reference in their entirety.

[0365] In some embodiments, STING agonists useful for the present disclosure include compounds having the following formula:

[0366]

[0367] described in WO 2015 / 017652, the contents of which are incorporated by reference in their entirety.

[0368] In some embodiments, STING agonists useful for the present disclosure include compounds having the following formula:

[0369]

[0370] described in WO 2016 / 096577, the contents of which are incorporated by reference in their entirety.

[0371] In some embodiments, STING agonists useful for the present disclosure include compounds having the following formula:

[0372]

[0373] wherein each symbol is defined in WO 2016 / 120305, the contents of which are incorporated by reference in their entirety.

[0374] In some embodiments, STING agonists useful for the present disclosure include compounds having the following formula:

[0375]

[0376] wherein each symbol is defined in WO 2016 / 145102, the contents of which are incorporated by reference in their entirety.

[0377] In some embodiments, STING agonists useful for the present disclosure include compounds having the following formula:

[0378]

[0379] wherein each symbol is defined in WO 2017 / 027646, the contents of which are incorporated by reference in their entirety.

[0380] In some embodiments, STING agonists useful for the present disclosure include compounds having the following formula:

[0381]

[0382] wherein each symbol is defined in WO 2017 / 075477, the contents of which are incorporated by reference in their entirety.

[0383] In some embodiments, STING agonists useful for the present disclosure include compounds having the following formula:

[0384]

[0385] wherein each symbol is defined in WO 2017 / 027645, the contents of which are incorporated by reference in their entirety.

[0386] In some embodiments, STING agonists useful for the present disclosure include compounds having the following formula:

[0387]

[0388] wherein each symbol is defined in WO 2018 / 100558, the contents of which are incorporated by reference in their entirety.

[0389] In some embodiments, STING agonists useful for the present disclosure include compounds having the following formula:

[0390]

[0391] wherein each symbol is defined in WO 2017 / 175147, the contents of which are incorporated by reference in their entirety.

[0392] In some embodiments, STING agonists useful for the present disclosure include compounds having the following formula:

[0393]

[0394] wherein each symbol is defined in WO 2017 / 175156, the contents of which are incorporated by reference in their entirety.

[0395] In some aspects, a STING agonist useful for the present disclosure is CL606, CL611, CL602, CL655, CL604, CL609, CL614, CL656, CL647, CL626, CL629, CL603, CL632, CL633, CL659, or a pharmaceutically acceptable salt thereof. In some aspects, a STING agonist useful for the present disclosure is CL606 or a pharmaceutically acceptable salt thereof. In some aspects, a STING agonist useful for the present disclosure is CL611 or a pharmaceutically acceptable salt thereof. In some aspects, a STING agonist useful for the present disclosure is CL602 or a pharmaceutically acceptable salt thereof. In some aspects, a STING agonist useful for the present disclosure is CL655 or a pharmaceutically acceptable salt thereof. In some aspects, a STING agonist useful for the present disclosure is CL604 or a pharmaceutically acceptable salt thereof. In some aspects, a STING agonist useful for the present disclosure is CL609 or a pharmaceutically acceptable salt thereof. In some aspects, a STING agonist useful for the present disclosure is CL614 or a pharmaceutically acceptable salt thereof. In some aspects, a STING agonist useful for the present disclosure is CL656 or a pharmaceutically acceptable salt thereof. In some aspects, a STING agonist useful for the present disclosure is CL647 or a pharmaceutically acceptable salt thereof. In some aspects, a STING agonist useful for the present disclosure is CL626 or a pharmaceutically acceptable salt thereof. In some aspects, a STING agonist useful for the present disclosure is CL629 or a pharmaceutically acceptable salt thereof. In some aspects, a STING agonist useful for the present disclosure is CL603 or a pharmaceutically acceptable salt thereof. In some aspects, a STING agonist useful for the present disclosure is CL632 or a pharmaceutically acceptable salt thereof. In some aspects, a STING agonist useful for the present disclosure is CL633 or a pharmaceutically acceptable salt thereof. In some aspects, a STING agonist useful for the present disclosure is CL659 or a pharmaceutically acceptable salt thereof.

[0396] In some aspects, an EV, e.g., an exosome, comprises a cyclic dinucleotide STING agonist and / or a non-cyclic dinucleotide STING agonist. In some aspects, when several cyclic dinucleotide STING agonists are present on the EVs (e.g., exosomes) disclosed herein, such STING agonists can be the same, or they can be different. In some aspects, when several non-cyclic dinucleotide STING agonists are present, such STING agonists can be the same, or they can be different. In some aspects, an EV (e.g., exosome) composition of the present disclosure can comprise two or more EV (e.g., exosome) populations, wherein each EV (e.g., exosome) population comprises a different STING agonist or combination thereof.

[0397] A STING agonist can also be modified to increase the encapsulation of the agonist in an extracellular vesicle or EV (e.g., not bound in a lumen). In some embodiments, the STING agonist is linked to a scaffold moiety (e.g., Scaffold Y). In certain embodiments, the modification allows for better expression of the STING agonist on the outer surface of an EV (e.g., exosome) (e.g., linked to a scaffold moiety disclosed herein (e.g., Scaffold X)). Such modifications can include the addition of a lipid-binding tag by treating the agonist with a chemical or enzyme, or by physically or chemically altering the polarity or charge of the STING agonist. The STING agonist can be modified by a single treatment or by a combination of treatments (e.g., addition of a lipid-binding tag only, or addition of a lipid-binding tag and alteration of polarity). The previous examples are meant to be non-limiting illustrative examples. Any combination of modifications is contemplated to be practiced. The modification can increase the encapsulation of the agonist in an EV by 2-fold to 10,000-fold, 10-fold to 1,000-fold, or 100-fold to 500-fold compared to the encapsulation of the unmodified agonist. The modification can increase the encapsulation of the agonist in an EV by at least 2-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 600-fold, 700-fold, 800-fold, 900-fold, 1000-fold, 2000-fold, 3000-fold, 4000-fold, 5000-fold, 6000-fold, 7000-fold, 8000-fold, 9000-fold, or 10,000-fold compared to the encapsulation of the unmodified agonist.

[0398] In some embodiments, a STING agonist can be modified to allow for better expression of the agonist on the outer surface of an EV (e.g., exosome) (e.g., linked to a scaffold moiety disclosed herein (e.g., Scaffold X)). Any of the above modifications can be used. The modification can increase the expression of the agonist on the outer surface of an EV (e.g., exosome) by about 2-fold to 10,000-fold, about 10-fold to 1,000-fold, or about 100-fold to 500-fold compared to the expression of the unmodified agonist. The modification can increase the expression of the agonist on the outer surface of an EV (e.g., exosome) by at least about 2-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 600-fold, 700-fold, 800-fold, 900-fold, 1000-fold, 2000-fold, 3000-fold, 4000-fold, 5000-fold, 6000-fold, 7000-fold, 8000-fold, 9000-fold, or 10,000-fold compared to the expression of the unmodified agonist.

[0399] The concentration of the STING agonist associated with the EV can be about 0.01 pm to 1000 pm. The concentration of the associated STING agonist can be about 0.01-0.05 pM, 0.05-0.1 pM, 0.1-0.5 pM, 0.5-1 pM, 1-5 pM, 5-10 pM, 10-15 pM, 15-20 pM, 20-25 pM, 25-30 pM, 30-35 pM, 35-40 pM, 45-50 pM, 55-60 pM, 65-70 pM, 70-75 pM, 75-80 pM, 80-85 pM, 85-90 pM, 90-95 pM, 95-100 pM, 100-150 pM, 150-200 pM, 200-250 pM, 250-300 pM, 300-350 pM, 250-400 pM, 400-450 pM, 450-500 pM, 500-550 pM, 550-600 pM, 600-650 pM, 650-700 pM, 700-750 pM, 750-800 pM, 800-850 pM, 805-900 pM, 900-950 pM, or 950-1000 pM. The concentration of the relevant STING agonist can be equal to or greater than about 0.01 pM, 0.1 pM, 0.5 pM, 1 pM, 5 pM, 10 pM, 15 pM, 20 pM, 25 pM, 30 pM, 35 pM, 40 pM, 45 pM, 50 pM, 55 pM, 60 pM, 65 pM, 70 pM, 75 pM, 80 pM, 85 pM, 90 pM, 95 pM, 100 pM, 150 pM, 200 pM, 250 pM, 300 pM, 350 pM, 400 pM, 450 pM, 500 pM, 550 pM, 600 pM, 650 pM, 700 pM, 750 pM, 800 pM, 850 pM, 900 pM, 950 pM, or 1000 pM.

[0400] II.B. Scaffold-X-engineered EVs, e.g., exosomes

[0401] In some embodiments, the EVs of the present disclosure include membranes whose composition is altered. For example, their membrane composition can be altered by changing the protein, lipid, or glycan content of the membrane.

[0402] In some embodiments, surface engineered EVs are produced by chemical and / or physical methods, such as PEG-induced fusion and / or sonication fusion. In other embodiments, surface engineered EVs (e.g., exosomes) are produced by genetic engineering. EVs produced from genetically modified producer cells or progeny of genetically modified cells can comprise an altered membrane composition. In some embodiments, surface engineered EVs (e.g., exosomes) have a higher or lower density (e.g., a higher number) of scaffold moieties (e.g., exosome proteins, e.g., Scaffold X), or variants or fragments comprising scaffold moieties.

[0403] For example, surface engineered EVs (e.g., Scaffold X-engineered EVs) can be produced from cells (e.g., HEK293 cells) transformed with an exogenous sequence encoding a scaffold moiety (e.g., an exosome protein, e.g., Scaffold X), or variants or fragments thereof. EVs comprising scaffold moieties expressed from the exogenous sequence can comprise an altered membrane composition.

[0404] Various modifications or fragments of scaffold moieties can be used in embodiments of the present disclosure. For example, scaffold moieties modified to have enhanced affinity for a binding agent can be used to produce surface engineered EVs that can be purified using the binding agent. Scaffold moieties modified to more effectively target EVs (e.g., exosomes) and / or membranes can be used. Scaffold moieties modified to comprise minimal fragments required to specifically and effectively target EVs (e.g., exosomes), membranes can also be used.

[0405] In some embodiments, the STING agonists disclosed herein are expressed on the surface of EVs (e.g., exosomes) as a fusion protein, e.g., a fusion protein of a STING agonist and Scaffold X. For example, the fusion protein can comprise a STING agonist disclosed herein linked to a scaffold moiety (e.g., Scaffold X). In certain embodiments, Scaffold X comprises a PTGFRN protein, a BSG protein, a IGSF2 protein, a IGSF3 protein, a IGSF8 protein, a ITGB1 protein, a ITGA4 protein, a SLC3A2 protein, an ATP transporter protein, or a fragment or variant thereof.

[0406] In some embodiments, the surface engineered EVs (e.g., exosomes) described herein (e.g., Scaffold X-engineered EVs (e.g., exosomes)) exhibit superior characteristics compared to EVs (e.g., exosomes) known in the art. For example, the surface (e.g., Scaffold X)-engineered EVs (e.g., exosomes) comprise a modified protein on their surface at a higher degree of enrichment than on naturally occurring EVs (e.g., exosomes) or EVs (e.g., exosomes) produced using conventional exosome proteins. Furthermore, the surface engineered EVs (e.g., exosomes) of the present application (e.g., Scaffold X-engineered EVs (e.g., exosomes)) can have greater, more specific, or more controllable biological activity compared to naturally occurring EVs (e.g., exosomes) or EVs (e.g., exosomes) produced using conventional exosome proteins.

[0407] In other embodiments, the EVs (e.g., exosomes) of the present disclosure comprise a STING agonist and Scaffold X, wherein the STING agonist is linked to Scaffold X. In some embodiments, the EVs (e.g., exosomes) of the present disclosure comprise a STING agonist and Scaffold X, wherein the STING agonist is not linked to Scaffold X.

[0408] In some embodiments, a scaffold X useful for the present disclosure comprises a prostaglandin F2 receptor negative regulator (PTGFRN polypeptide). The PTGFRN protein can also be referred to as CD9 partner 1 (CD9P-1), Glu-Trp-Ile EWI motif-containing protein F (EWI-F), prostaglandin F2-alpha receptor modulator protein, prostaglandin F2-alpha receptor-associated protein, or CD315. The full-length amino acid sequence of the human PTGFRN protein (Uniprot accession number Q9P2B2) is shown in Table 1 as SEQ ID NO: 1. The PTGFRN polypeptide comprises a signal peptide (amino acids 1 to 25 of SEQ ID NO: 1), an extracellular domain (amino acids 26 to 832 of SEQ ID NO: 1), a transmembrane domain (amino acids 833 to 853 of SEQ ID NO: 1), and a cytoplasmic domain (amino acids 854 to 879 of SEQ ID NO: 1). The mature PTGFRN polypeptide consists of the signal peptide-free SEQ ID NO: 1, i.e., amino acids 26 to 879 of SEQ ID NO: 1. In some embodiments, a PTGFRN polypeptide fragment useful for the present disclosure comprises the transmembrane domain of the PTGFRN polypeptide. In other embodiments, a PTGFRN polypeptide fragment useful for the present disclosure comprises the transmembrane domain of the PTGFRN polypeptide and (i) comprises at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150 amino acids N-terminal to the transmembrane domain, (ii) comprises at least 5, at least 10, at least 15, at least 20, or at least 25 amino acids C-terminal to the transmembrane domain, or both (i) and (ii).

[0409] In some embodiments, a fragment of a PTGFRN polypeptide lacks one or more functional or structural domains, such as IgV.

[0410] In other embodiments, Scaffold X comprises an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to amino acids 26 to 879 of SEQ ID NO: 1. In other embodiments, Scaffold X comprises an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO: 33. In other embodiments, Scaffold X comprises the amino acid sequence of SEQ ID NO: 33 except for 1 amino acid mutation, 2 amino acid mutations, 3 amino acid mutations, 4 amino acid mutations, 5 amino acid mutations, 6 amino acid mutations, or 7 amino acid mutations. The mutations can be substitutions, insertions, deletions, or any combination thereof. In some embodiments, Scaffold X comprises the amino acid sequence of SEQ ID NO: 33 and comprises 1 amino acid, 2 amino acids, 3 amino acids, 4 amino acids, 5 amino acids, 6 amino acids, 7 amino acids, 8 amino acids, 9 amino acids, 10 amino acids, 11 amino acids, 12 amino acids, 13 amino acids, 14 amino acids, 15 amino acids, 16 amino acids, 17 amino acids, 18 amino acids, 19 amino acids, or 20 amino acids or more amino acids at the N-terminus and / or C-terminus of SEQ ID NO: 33.

[0411] In other embodiments, Scaffold X comprises an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO: 2, 3, 4, 5, 6, or 7. In other embodiments, Scaffold X comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6, or 7 except for 1 amino acid mutation, 2 amino acid mutations, 3 amino acid mutations, 4 amino acid mutations, 5 amino acid mutations, 6 amino acid mutations, or 7 amino acid mutations. The mutations can be substitutions, insertions, deletions, or any combination thereof. In some embodiments, Scaffold X comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6, or 7 and comprises 1 amino acid, 2 amino acids, 3 amino acids, 4 amino acids, 5 amino acids, 6 amino acids, 7 amino acids, 8 amino acids, 9 amino acids, 10 amino acids, 11 amino acids, 12 amino acids, 13 amino acids, 14 amino acids, 15 amino acids, 16 amino acids, 17 amino acids, 18 amino acids, 19 amino acids, or 20 amino acids or more amino acids at the N-terminus and / or C-terminus of SEQ ID NO: 2, 3, 4, 5, 6, or 7.

[0412] Table 1.

[0413]

[0414]

[0415]

[0416]

[0417] In some embodiments, Scaffold X useful for the present disclosure comprises Basigin (BSG protein) set forth in SEQ ID NO: 9. BSG protein is also known as 5F7, collagenase-stimulating factor, extracellular matrix metalloproteinase inducer (EMMPRIN), leukocyte activation antigen M6, OK blood group antigen, tumor cell-derived collagenase-stimulating factor (TCSF), or CD147. The Uniprot number for human BSG protein is P35613. The signal peptide for BSG protein is amino acids 1-21 of SEQ ID NO: 9. Amino acids 138-323 of SEQ ID NO: 9 are the extracellular domain, amino acids 324-344 are the transmembrane domain, and amino acids 345-385 of SEQ ID NO: 9 are the cytoplasmic domain.

[0418] In other embodiments, Scaffold X comprises an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to amino acids 22 to 385 of SEQ ID NO: 9. In some embodiments, the fragment of a Basigin polypeptide lacks one or more functional or structural domains, such as an antibody, e.g., amino acids 221 to 315 of SEQ ID NO: 9. In other embodiments, Scaffold X comprises an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO: 10, 11, or 12. In other embodiments, Scaffold X comprises the amino acid sequence of SEQ ID NO: 10, 11, or 12 except for 1 amino acid mutation, 2 amino acid mutations, 3 amino acid mutations, 4 amino acid mutations, 5 amino acid mutations, 6 amino acid mutations, or 7 seven amino acid mutations. The mutations can be substitutions, insertions, deletions, or any combination thereof. In some embodiments, Scaffold X comprises the amino acid sequence of SEQ ID NO: 10, 11, or 12 and comprises 1 amino acid, 2 amino acids, 3 amino acids, 4 amino acids, 5 amino acids, 6 amino acids, 7 amino acids, 8 amino acids, 9 amino acids, 10 amino acids, 11 amino acids, 12 amino acids, 13 amino acids, 14 amino acids, 15 amino acids, 16 amino acids, 17 amino acids, 18 amino acids, 19 amino acids, or 20 amino acids or more amino acids at the N-terminus and / or C-terminus of SEQ ID NO: 10, 11, or 12.

[0419] In some embodiments, Scaffold X useful for the present disclosure comprises immunoglobulin superfamily member 8 (IgSF8 or IgSF8 protein), which is also known as CD81 partner 3, Glu-Trp-Ile EWI motif-containing protein 2 (EWI-2), keratinocyte-associated transmembrane protein 4 (KCT-4), LIR-D1, prostaglandin-regulatory-like protein (PGRL), or CD316. The full-length human IGSF8 protein has accession number Q969P0 in Uniprot, shown herein as SEQ ID NO: 14. The human IGSF8 protein has a signal peptide (amino acids 1 to 27 of SEQ ID NO: 14), an extracellular domain (amino acids 28 to 579 of SEQ ID NO: 14), a transmembrane domain (amino acids 580 to 600 of SEQ ID NO: 14), and a cytoplasmic domain (amino acids 601 to 613 of SEQ ID NO: 14).

[0420] In other embodiments, Scaffold X comprises an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to amino acids 28 to 613 of SEQ ID NO: 14. In some embodiments, the IGSF8 protein lacks one or more functional or structural domains, such as IgV. In other embodiments, Scaffold X comprises an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO: 15, 16, 17, or 18. In other embodiments, Scaffold X comprises the amino acid sequence of SEQ ID NO: 15, 16, 17, or 18 except for 1 amino acid mutation, 2 amino acid mutations, 3 amino acid mutations, 4 amino acid mutations, 5 amino acid mutations, 6 amino acid mutations, or 7 amino acid mutations. The mutations can be substitutions, insertions, deletions, or any combination thereof. In some embodiments, Scaffold X comprises the amino acid sequence of SEQ ID NO: 15, 16, 17, or 18 and comprises 1 amino acid, 2 amino acids, 3 amino acids, 4 amino acids, 5 amino acids, 6 amino acids, 7 amino acids, 8 amino acids, 9 amino acids, 10 amino acids, 11 amino acids, 12 amino acids, 13 amino acids, 14 amino acids, 15 amino acids, 16 amino acids, 17 amino acids, 18 amino acids, 19 amino acids, or 20 amino acids or more amino acids at the N-terminus and / or C-terminus of SEQ ID NO: 15, 16, 17, or 18.

[0421] In some embodiments, Scaffold X that can be used with the STING agonists disclosed herein comprises immunoglobulin superfamily member 3 (IgSF3 or IGSF3 protein), which is also known as protein with Glu-Trp-Ile EWI motif 3 (EWI-3) and is shown as the amino acid sequence of SEQ ID NO: 20. The human IGSF3 protein has a signal peptide (amino acids 1 to 19 of SEQ ID NO: 20), an extracellular domain (amino acids 20 to 1124 of SEQ ID NO: 20), a transmembrane domain (amino acids 1125 to 1145 of SEQ ID NO: 20), and a cytoplasmic domain (amino acids 1146 to 1194 of SEQ ID NO: 20).

[0422] In other embodiments, Scaffold X comprises an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to amino acids 28 to 613 of SEQ ID NO: 20. In some embodiments, the IGSF3 protein lacks one or more functional or structural domains, such as IgV.

[0423] In some embodiments, Scaffold X useful for the present disclosure comprises Integrin beta-1 (ITGB1 protein), which is also known as fibronectin receptor subunit beta, Glycoprotein Ila (GPIIA), VLA-4 subunit beta, or CD29, and is shown as the amino acid sequence of SEQ ID NO: 21. The human ITGB1 protein has a signal peptide (amino acids 1 to 20 of SEQ ID NO: 21), an extracellular domain (amino acids 21 to 728 of SEQ ID NO: 21), a transmembrane domain (amino acids 729 to 751 of SEQ ID NO: 21), and a cytoplasmic domain (amino acids 752 to 798 of SEQ ID NO: 21).

[0424] In other embodiments, Scaffold X comprises an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to amino acids 21 to 798 of SEQ ID NO: 21. In some embodiments, the ITGB1 protein lacks one or more functional or structural domains, such as IgV.

[0425] In other embodiments, Scaffold X comprises an ITGA4 protein comprising an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO: 22, but does not contain the signal peptide (amino acids 1 to 33 of SEQ ID NO: 22). In some embodiments, the ITGA4 protein lacks one or more functional or structural domains, such as IgV.

[0426] In other embodiments, Scaffold X comprises a SLC3A2 protein comprising an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO: 23, but does not comprise a signal peptide. In some embodiments, the SLC3A2 protein lacks one or more functional or structural domains, such as IgV.

[0427] In other embodiments, Scaffold X comprises an ATP1A1 protein comprising an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO: 24, but does not comprise a signal peptide. In some embodiments, the ATP1A1 protein lacks one or more functional or structural domains, such as IgV.

[0428] In other embodiments, Scaffold X comprises an ATP1A2 protein comprising an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO: 25, but does not comprise a signal peptide. In some embodiments, the ATP1A2 protein lacks one or more functional or structural domains, such as IgV.

[0429] In other embodiments, Scaffold X comprises an ATP1A3 protein comprising an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO: 26, but does not comprise a signal peptide. In some embodiments, the ATP1A3 protein lacks one or more functional or structural domains, such as IgV.

[0430] In other embodiments, Scaffold X comprises an ATP1A4 protein comprising an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO: 27, but does not comprise a signal peptide. In some embodiments, the ATP1A4 protein lacks one or more functional or structural domains, such as IgV.

[0431] In other embodiments, Scaffold X comprises an ATP1A5 protein comprising an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO: 28, but does not comprise a signal peptide. In some embodiments, the ATP1A5 protein lacks one or more functional or structural domains, such as IgV.

[0432] In other embodiments, Scaffold X comprises an ATP2B1 protein comprising an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO: 29, but does not comprise a signal peptide. In some embodiments, the ATP2B1 protein lacks one or more functional or structural domains, such as IgV.

[0433] In other embodiments, Scaffold X comprises an ATP2B2 protein comprising an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO: 30, but does not comprise a signal peptide. In some embodiments, the ATP2B2 protein lacks one or more functional or structural domains, such as IgV.

[0434] In other embodiments, Scaffold X comprises an ATP2B3 protein comprising an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO: 31, but does not comprise a signal peptide. In some embodiments, the ATP2B3 protein lacks one or more functional or structural domains, such as IgV.

[0435] In other embodiments, Scaffold X comprises an ATP2B4 protein comprising an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO: 32, but does not comprise a signal peptide. In some embodiments, the ATP2B4 protein lacks one or more functional or structural domains, such as IgV.

[0436] In other embodiments, the scaffold X comprises an IGSF2 protein comprising an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO: 34, but does not contain the signal peptide. In some embodiments, the IGSF2 protein lacks one or more functional or structural domains, such as IgV.

[0437] Non-limiting examples of other scaffold X proteins that can be used to link a STING agonist to the surface of an EV (e.g., an exosome) can be found in U.S. Patent No. 10,195,290 Bl, issued February 5, 2019, which is incorporated by reference in its entirety.

[0438] In some embodiments, scaffold X proteins useful in the present application lack at least 5, 10, 50, 100, 200, 300, 400, 500, 600, 700, or 800 amino acids from the N-terminus of the native protein. In some embodiments, scaffold X lacks at least 5, 10, 50, 100, 200, 300, 400, 500, 600, 700, or 800 amino acids from the C-terminus of the native protein. In some embodiments, scaffold X lacks at least 5, 10, 50, 100, 200, 300, 400, 500, 600, 700, or 800 amino acids from the N-terminus and C-terminus of the native protein. In some embodiments, scaffold X lacks one or more functional or structural domains of the native protein.

[0439] In some embodiments, the scaffold X described herein can also be used to link a STING agonist on the luminal surface of an EV (e.g., an exosome) and / or on the outer surface simultaneously. For example, in addition to the surface of an EV (e.g., an exosome), a PTGFRN polypeptide can be used to link a STING agonist inside the lumen. In some embodiments, scaffold X can be used to link a STING agonist and an additional therapeutic agent to an EV (e.g., an exosome (e.g., a payload)). Thus, in certain embodiments, the scaffold X disclosed herein can be used for dual purposes.

[0440] Scaffold-Y-engineered EVs, e.g., exosomes

[0441] In some embodiments, the EV (e.g., exogenous body) of this disclosure includes an internal space (i.e., a cavity) that differs from the internal space of a naturally occurring EV (e.g., exogenous body). For example, the EV (e.g., exogenous body) may be modified such that the composition in the cavity side of the EV (e.g., exogenous body) has a protein, lipid, or glycan content that differs from that of a naturally occurring EV (e.g., exogenous body).

[0442] In some embodiments, engineered EVs (e.g., exosomes) can be generated from cells transformed with exogenous sequences of scaffold portions encoding scaffold portions (e.g., exosomes) or scaffold portions that alter the composition or contents of the luminal side of an EV (e.g., an exosome). Various modifications or fragments of exosome proteins that can be expressed in the luminal side of an EV (e.g., an exosome) can be used in embodiments of the present invention.

[0443] In some embodiments, the STING agonist disclosed herein is encapsulated within the lumen of an EV (e.g., an exogenous organism). In some embodiments, the STING agonist is attached to the luminal surface of the EV (e.g., an exogenous organism). As used herein, when a molecule (e.g., an antigen or adjuvant) is described as being “intraluminal” in an EV (e.g., an exogenous organism), this means that the molecule is located within the EV (e.g., an exogenous organism) (e.g., associated) but is not attached to any molecule on the luminal surface of the EV. In other embodiments, the STING agonist is expressed on the luminal surface of the EV (e.g., an exogenous organism) as a fusion molecule (e.g., a fusion molecule of the STING agonist and a scaffold portion (e.g., scaffold Y)). In some embodiments, scaffold Y comprises the MARCKS protein, the MARCKSL1 protein, the BASP1 protein, or any combination thereof.

[0444] In other embodiments, the EV (e.g., exogenous body) of this disclosure comprises a STING agonist and a stent Y, wherein the STING agonist is connected to the stent Y. In some embodiments, the EV (e.g., exogenous body) of this disclosure comprises a STING agonist and a stent Y, wherein the STING agonist is not connected to the stent Y.

[0445] In some embodiments, the scaffold portion (e.g., Scaffold Y) that can be altered on the luminal side of the EV (e.g., exosome) includes, but is not limited to, a MARCKS protein, a MARCKSL1 protein, a BASP1 protein, or any combination thereof. In some embodiments, Scaffold Y comprises a brain acid soluble protein 1 (BASP1 protein). BASP1 protein is also known as 22 kDa neuronally enriched acidic protein or neuronal axoplasmic membrane protein NAP-22. The full-length human BASP1 protein sequence (isoform 1) is shown in Table 2. Isoforms produced by alternative splicing lack amino acids 88 to 141 of SEQ ID NO:XX (isoform 1).

[0446] Table 2.

[0447]

[0448] The mature BASP1 protein sequence lacks the first Met of SEQ ID NO:49, and thus contains amino acids 2 to 227 of SEQ ID NO:49.

[0449] In other embodiments, Scaffold Y useful for the present disclosure comprises an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to amino acids 2 to 227 of SEQ ID NO:49. In other embodiments, Scaffold X comprises an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO:50-155. In other embodiments, Scaffold Y useful for the present disclosure comprises the amino acid sequence of SEQ ID NO:50-155 except for 1 amino acid mutation, 2 amino acid mutations, 3 amino acid mutations, 4 amino acid mutations, 5 amino acid mutations, 6 amino acid mutations, or 7 amino acid mutations. The mutations can be substitutions, insertions, deletions, or any combination thereof. In some embodiments, Scaffold Y useful for the present disclosure comprises the amino acid sequence of SEQ ID NO:50-155 and comprises 1 amino acid, 2 amino acids, 3 amino acids, 4 amino acids, 5 amino acids, 6 amino acids, 7 amino acids, 8 amino acids, 9 amino acids, 10 amino acids, 11 amino acids, 12 amino acids, 13 amino acids, 14 amino acids, 15 amino acids, 16 amino acids, 17 amino acids, 18 amino acids, 19 amino acids, or 20 amino acids or more amino acids at the N-terminus and / or C-terminus of SEQ ID NO:50-155.

[0450] In some embodiments, a scaffold Y useful for the present disclosure is a MARCKS protein comprising an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO: 47, but does not contain the signal peptide. In certain embodiments, the MARCKS protein lacks one or more functional or structural domains.

[0451] In some embodiments, a scaffold Y comprises a MARCKSL1 protein comprising an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO: 48, but does not contain the signal peptide. In certain embodiments, the MARCKS protein lacks one or more functional or structural domains.

[0452] In some embodiments, a scaffold Y useful for the present disclosure comprises a peptide having MGXKLSKKK, where X is alanine or any other amino acid (SEQ ID NO: 163). In some embodiments, an EV (e.g., an exosome) comprises a peptide having the sequence (M)(G)(π)(ξ)(Φ / π)(S / A / G / N)(+)(+), where each bracketed position represents an amino acid, and where π is any amino acid selected from the group consisting of (Pro, Gly, Ala, Ser), ξ is any amino acid selected from the group consisting of (Asn, Gin, Ser, Thr, Asp, Glu, Lys, His, Arg), Φ is any amino acid selected from the group consisting of (Val, lie, Leu, Phe, Trp, Tyr, Met), and (+) is any amino acid selected from the group consisting of (Lys, Arg, His); and where position 5 is not (+) and position 6 is neither (+) nor (Asp or Glu). In further embodiments, an EV (e.g., an exosome) described herein (e.g., an engineered EV, e.g., an exosome) comprises a peptide having the sequence (M)(G)(π)(X)(Φ / π)(π)(+)(+), where each bracketed position represents an amino acid, and where π is any amino acid selected from the group consisting of (Pro, Gly, Ala, Ser), X is any amino acid, Φ is any amino acid selected from the group consisting of (Val, lie, Leu, Phe, Trp, Tyr, Met), and (+) is any amino acid selected from the group consisting of (Lys, Arg, His); and where position 5 is not (+) and position 6 is neither (+) nor (Asp or Glu).

[0453] In some embodiments, a scaffold Y useful for expressing a STING agonist on the luminal surface of an EV (e.g., an exosome) comprises an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to any one of SEQ ID NOs: 7-155.

[0454] Scaffold Y-engineered EVs (e.g., exosomes) described herein can be produced from cells transformed with the sequences set forth in SEQ ID NOs: 47-155.

[0455] II.C. Linkers

[0456] EVs of the present disclosure can comprise one or more linkers that connect a STING agonist to an EV or a scaffold moiety (e.g., a scaffold X on the outer surface of an EV). In some embodiments, a STING agonist is connected directly to an EV or is connected in a scaffold moiety on an EV via a linker. Linkers can be any chemical moiety known in the art.

[0457] In some embodiments, the term “linker” refers to a peptide or polypeptide sequence (e.g., a synthetic peptide or polypeptide sequence) or a non-polypeptide. In some aspects, two or more linkers can be connected in series. Generally, linkers provide flexibility or prevent / improve steric hindrance. Linkers are typically not cleaved; however, in certain aspects, such cleavage can be desirable. Thus, in some aspects, a linker can comprise one or more protease-cleavable sites, which can be located within the linker sequence or flanking the linker on either end of the linker sequence.

[0458] In some embodiments, a linker is a peptide linker. In some embodiments, a 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.

[0459] In some embodiments, the peptide linker is synthetic, i.e., not naturally occurring. In one aspect, the peptide linker comprises a peptide (or polypeptide) (e.g., a naturally or non-naturally occurring peptide) that comprises linking or genetically fusing a first linear amino acid sequence to a second linear amino acid sequence that are not naturally linked or genetically fused in nature. For example, in one aspect, the peptide linker can comprise a non-naturally occurring polypeptide that is a modified form of a naturally occurring polypeptide (e.g., comprising a mutation such as an addition, substitution, or deletion).

[0460] The linker can be susceptible to cleavage (“cleavable linker”), facilitating release of the STING agonist or other payload. In some aspects, the linker is a “reduction-sensitive linker”. In some aspects, the reduction-sensitive linker contains a disulfide bond. In some aspects, the linker is an “acid-labile linker”. In some aspects, the acid-labile linker contains a hydrazone. Suitable acid-labile linkers also include, for example, a cis-aconitic acid linker, a hydrazide linker, a thiocarbamoyl linker, or any combination thereof. In some aspects, the linker comprises a non-cleavable linker.

[0461] II.D. Producer cells and modifications

[0462] EVs (e.g., exosomes) can be produced from cells grown in vitro or from a subject’s bodily fluids. When EVs (e.g., exosomes) are produced from in vitro cell cultures, various producer cells can be used, for example, HEK293 cells. Other cell types that can be used to produce the lumen-engineered EVs (e.g., exosomes) described herein include, but are not limited to, mesenchymal stem cells, T cells, B cells, dendritic cells, macrophages, and cancer cell lines. Other examples include: Chinese hamster ovary (CHO) cells, mesenchymal stem cells (MSCs), BJ human foreskin fibroblasts, fHDF fibroblasts, neuronal precursor cells, amniotic cells, adipose mesenchymal stem cells, and RPTEC / TERT1 cells. In certain embodiments, the producer cell is not a dendritic cell, a macrophage, a B cell, a mast cell, a neutrophil, a Kupffer-Browicz cell, a cell derived from any of these cells, or any combination thereof.

[0463] Some embodiments can also include genetically modifying EVs (e.g., exosomes) to comprise one or more exogenous sequences, thereby producing modified EVs that express exogenous proteins on the surface of the vesicle. The exogenous sequences can comprise sequences that encode a modification or fragment of an EV (e.g., exosome) protein or an EV protein. Additional copies of sequences that encode an EV (e.g., exosome) protein can be introduced to produce surface engineered EVs with higher density of EV proteins. Exogenous sequences that encode a modification or fragment of an EV (e.g., exosome) protein can be introduced to produce modified EVs that comprise the modification or fragment of the EV protein. Exogenous sequences that encode an affinity tag can be introduced to produce modified EVs (e.g., exosomes) that contain a fusion protein comprising an affinity tag linked to an EV protein.

[0464] In some embodiments, the exogenous sequence encodes a scaffold X (e.g., a PTGFRN protein, a BSG protein, an IGSF2 protein, an IGSF3 protein, an IGSF8 protein, an ITGB1 protein, an ITGA4 protein, an SLC3A2 protein, an ATP transporter protein, or a fragment or variant thereof). In some embodiments, the modified EV (e.g., exosome) overexpresses a scaffold X (e.g., a PTGFRN protein, a BSG protein, an IGSF2 protein, an IGSF3 protein, an IGSF8 protein, an ITGB1 protein, an ITGA4 protein, an SLC3A2 protein, an ATP transporter protein, or a fragment or variant thereof). In other embodiments, the EV (e.g., exosome) is produced from a cell that overexpresses a scaffold X (e.g., a PTGFRN protein, a BSG protein, an IGSF2 protein, an IGSF3 protein, an IGSF8 protein, an ITGB1 protein, an ITGA4 protein, an SLC3A2 protein, an ATP transporter protein, or a fragment or variant thereof).

[0465] In some embodiments, the exogenous sequence encodes a scaffold Y (e.g., a MARCKS protein, a MARCKSL1 protein, a BASP1 protein, or a fragment or variant thereof). In some embodiments, the modified EV (e.g., exosome) overexpresses a scaffold Y (e.g., a MARCKS protein, a MARCKSL1 protein, a BASP1 protein, or a fragment or variant thereof). In other embodiments, the EV (e.g., exosome) is produced from a cell that overexpresses a scaffold Y (e.g., a MARCKS protein, a MARCKSL1 protein, a BASP1 protein, or a fragment or variant thereof).

[0466] The exogenous sequence can be transiently or stably expressed in the producer cell or cell line by transfection, transformation, transduction, electroporation, or any other suitable gene delivery method known in the art or combinations thereof. The exogenous sequence can be integrated into the genome of the producer cell or remain extrachromosomal. The exogenous sequence can be transformed in plasmid form. The exogenous sequence can be stably integrated into the genomic sequence of the producer cell at a target site or at a random site. The exogenous sequence can be inserted into the genomic sequence of the producer cell within, upstream (5’-end) or downstream (3’-end) of an endogenous sequence encoding an EV (e.g., exosome) protein. Various methods known in the art can be used to introduce the exogenous sequence into the producer cell. For example, cells modified using various gene editing methods (e.g., methods using homologous recombination, transposon-mediated systems, loxP-Cre systems, CRISPR / Cas9 CRISPR / Cfpl, CRISPR / C2cl, C2c2 or C2c3, CRISPR / CasY or CasX, TAL-effector nucleases or TALENs, or zinc-finger nuclease (ZFN) systems) are within the scope of various embodiments.

[0467] In some embodiments, the producer cell is further modified to comprise an additional exogenous sequence. For example, an additional exogenous sequence can be included to modulate endogenous gene expression, modulate immune response or immune signaling, or to produce an EV (e.g., exosome) including a certain polypeptide as a payload or additional surface expressed ligand. In some embodiments, the producer cell can be further modified to comprise an additional exogenous sequence that confers additional functionality to the EV (e.g., exosome), such as specific targeting ability, delivery function, enzymatic function, extended or shortened half-life in vivo, etc. In some embodiments, the producer cell is modified to comprise two exogenous sequences, one encoding an exosome protein or modification or fragment of an exosome protein, and the other encoding a protein that confers additional functionality to the exosome.

[0468] More specifically, EVs (e.g., exosomes) of the application can be produced from cells transformed with sequences encoding one or more additional exogenous proteins, including but not limited to ligands, cytokines, or antibodies, or any combination thereof. These additional exogenous proteins can enable the activation or modulation of additional immune stimulatory signals in combination with the STING agonist. Exemplary additional exogenous proteins contemplated for use include the proteins, ligands, and other molecules described in detail in U.S. Patent Application 62 / 611,140, which is incorporated by reference herein in its entirety. In some embodiments, the EVs (e.g., exosomes) are further modified with ligands including CD40L, OX40L, or CD27L. In some embodiments, the EVs (e.g., exosomes) are further modified with cytokines including IL-7, IL-12, or IL-15. Any of the one or more exosome proteins described herein can be expressed from a plasmid, an exogenous sequence inserted into the genome, or other exogenous nucleic acid such as a synthetic messenger RNA (mRNA).

[0469] In some embodiments, the EVs (e.g., exosomes) are further modified to display an antagonistic antibody or an agonistic antibody or fragments thereof on the surface of the EVs (e.g., exosomes) to direct EV uptake, activation, or block cellular pathways to enhance the combinatorial effect of the STING agonist. In some specific embodiments, the antibody or fragments thereof is an antibody against DEC205, CLEC9A, CLEC6, DCIR, DC-SIGN, LOX-1, or Langerin. The producer cell can be modified to include additional exogenous sequences encoding the antagonistic antibody or agonistic antibody. Alternatively, the antagonistic antibody or agonistic antibody can be covalently linked or conjugated to the EVs (e.g., exosomes) through any suitable linking chemistry known in the art. Non-limiting examples of suitable linking chemistries include amine-reactive groups, carboxyl-reactive groups, thiol-reactive groups, aldehyde-reactive groups, photoreactive groups, ClickIT substances, biotin-streptavidin or other avidin conjugates, or any combination thereof.

[0470] II.D.1. Glycan modification of producer cells or EVs (e.g., exosomes)

[0471] In some embodiments, the EV (e.g., exosome) is glycan-modified by enzymatic or chemical treatment. In one embodiment, the EV (e.g., exosome) is derived from a glycan-modified producer cell. In another embodiment, the glycan modification of the producer cell comprises an enzymatic or chemical modification. In various embodiments, the glycan modification of the producer cell is treatment with a kifunensine or knockout of a sialyltransferase or cytidylyltransferase gene. In one embodiment, the glycan modification of the producer cell comprises a knockout of the cytidylyltransferase gene cytidine monophosphate N-acetylneuraminic acid synthetase (CMAS). In one embodiment, the glycan modification of the producer cell comprises a knockout of the mannose biosynthesis gene mannosylase alpha class 1A member 1 (MAN1A1). In one embodiment, the glycan modification of the producer cell comprises a knockout of the mannose biosynthesis gene mannosylase alpha class 2A member 1 (MAN2A1).

[0472] The glycan modification can be deglycosylation or desialylation of the producer cell or the isolated or purified EV (e.g., exosome). The glycan modification of the EV (e.g., exosome) can be performed prior to encapsulating the STING agonist or after encapsulating the STING agonist. The producer cell or EV (e.g., exosome) can be glycan-modified (e.g., deglycosylated or desialylated) by about or more than 99%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% relative to the unmodified producer cell or EV (e.g., exosome). The producer cell or EV (e.g., exosome) can be glycan-modified (e.g., deglycosylated or desialylated) by about or more than 95% to 100%, 90% to 95%, 85% to 95%, 80% to 85%, 75% to 80%, 70% to 75%, 65% to 70%, 60% to 65%, 55% to 60%, 50% to 55%, 45% to 50%, 40% to 45%, 35% to 40%, 30% to 35%, 25% to 30%, 20% to 25%, 15% to 20%, 10% to 15%, or 5% to 10% relative to the unmodified producer cell or EV (e.g., exosome).

[0473] Glycan modification can be performed on producer cells or EVs (e.g., exosomes) by chemical, enzymatic, or gene editing techniques. Glycan modification can include treatment of producer cells with chemicals, small molecules, or enzymes that alter or inhibit glycosyltransferases, galactosyltransferases, sialyltransferases, or cytidyltransferases in the producer cells, resulting in glycan-modified EVs (e.g., exosomes) derived from the producer cells. Glycan modification can also include treatment of EVs (e.g., exosomes) with chemicals or enzymes that alter glycans on the surface of the EVs, such as small molecule inhibitors or glycoside hydrolases, such as sialidases or neuraminidases, as well as any other suitable chemical or enzymatic glycan modification treatment.

[0474] In some embodiments, the producer cells or EVs (e.g., exosomes) are glycan-modified by treatment with a kifunensine. Kifunensine is a mannosidase I inhibitor that inhibits the removal of mannose residues from a precursor glycoprotein by mannosidase I. Treatment of cells with kifunensine results in glycoproteins with terminal mannose residues. Another mannosidase I inhibitor that can be used is 1-deoxymannojirimycin. Other small molecules that inhibit a-mannosidase I or II or b-mannosidase, such as swainsonine, can also be used.

[0475] Some embodiments can also include treatment of the producer cells or EVs (e.g., exosomes) with a glycoside hydrolase, such as a sialidase, neuraminidase, or mannosidase. Any glycoside hydrolase known in the art can be used, including but not limited to exo-a-sialidase, endo-a-sialidase, N-acetylneuraminidase, sialidase 1, sialidase 2, sialidase 3, or sialidase 4, any other suitable sialidase, a-mannosidase, b-mannosidase, or any combination thereof.

[0476] Additionally, glycan modification can include genetic alteration of the producer cells by appropriate genome editing techniques to alter the expression of glycanases, such as knocking out or knocking down glycosyltransferases, galactosyltransferases, sialyltransferases, or cytidyltransferases in the producer cells. Any genome editing technique known in the art can be used, including but not limited to CRISPR / Cas9, CRISPR / Cfp1, CRISPR / C2c1, C2c2, or C2c3, CRISPR / CasY or CasX, TAL-effector nuclease or TALEN, or zinc-finger nuclease (ZFN) systems, or any combination thereof.

[0477] Exemplary genes that can be altered include cytidine monophosphate N-acetylneuraminic acid synthetase (CMAS), as well as the mannose biosynthesis genes mannosidase alpha class 1A member 1 (MAN1A1) and mannosidase alpha class 2A member 1 (MAN2A1).

[0478] Glycan-modified EVs (e.g., exosomes) can also be derived from producer cell lines that have been glycan-modified. In such examples, the producer cell line can be transformed, transfected, transduced, or otherwise genetically modified to express the PTGFRN gene and gene product, and to alter the expression of glycosyltransferases. In one embodiment, the producer cell is altered to overexpress the PTGFRN gene and gene product, and to knock down or knock out the cytidyltransferase gene CMAS. Alternatively, the producer cell line can be genetically modified to express the PTGFRN gene and gene product, and treated with a known inhibitor of a guanosine or other mannosyltransferase, glycosyltransferase, galactosyltransferase, sialyltransferase, or cytidyltransferase, or any combination thereof, to produce a producer cell that overexpresses the PTGFRN gene and gene product and has altered glycan expression.

[0479] III. Methods of producing EVs with STING agonists

[0480] III. A. Methods of encapsulating STING agonists in EVs

[0481] STING agonists can be encapsulated in EVs (e.g., exosomes) by any suitable technique known in the art. All known ways of loading biomolecules into EVs (e.g., exosomes) are contemplated as suitable for use herein. Such techniques include passive diffusion, electroporation, chemical or polymeric transfection, viral transduction, mechanical membrane disruption or mechanical shearing, or any combination thereof. The STING agonist and EVs (e.g., exosomes) can be incubated in a suitable buffer during encapsulation.

[0482] In one embodiment, the STING agonist is encapsulated by EVs (e.g., exosomes) by passive diffusion. The STING agonist and EVs (e.g., exosomes) can be mixed together and incubated for a period of time sufficient for the STING agonist to diffuse into the vesicle lipid bilayer, and thereby be encapsulated in the EV (e.g., exosome). The STING agonist and EVs (e.g., exosomes) can be incubated together for about 1 to 30 hours, 2 to 24 hours, 4 to 18 hours, 6 to 16 hours, 8 to 14 hours, 10 to 12 hours, 6 to 12 hours, 12 to 20 hours, 14 to 18 hours, or 20 to 30 hours. The STING agonist and EVs (e.g., exosomes) can be incubated together for about 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, 26 hours, or 30 hours.

[0483] The buffer conditions of the solution of EVs (e.g., exosomes) can also be altered to optimize the encapsulation of the STING agonist. In one embodiment, the buffer can be phosphate buffered saline (PBS) containing sucrose. PBS is a well-known buffer to those skilled in the art. Additional buffer modifications can also be used, such as a shear protection agent, a viscosity modifier, and / or a solute that affects the structural properties of the vesicles. Excipients can also be added to improve the efficiency of STING agonist encapsulation, such as membrane softening materials and molecular crowding agents. Other modifications to the buffer can include a specific pH range and / or concentration of salts, organic solvents, small molecules, detergents, zwitterions, amino acids, polymers, and / or any combination of the above, including multiple concentrations.

[0484] The temperature of the solution of EVs (e.g., exosomes) and STING agonist can be altered during incubation to optimize the encapsulation of the STING agonist. The temperature can be room temperature. The temperature can be about 15°C to 90°C, 15°C to 30°C, 30-50°C, 50°C to 90°C. The temperature can be about 15°C, 20°C, 35°C, 30°C, 35°C, 37°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, 80°C, 85°C, or 90°C

[0485] The concentration of the STING agonist during incubation of the agonist with EVs (e.g., exosomes) can also be altered to optimize the encapsulation of the STING agonist. The concentration of the agonist can be between at least 0.01 mM and 100 mM of the STING agonist. The concentration of the agonist can be at least 0.01-1 mM, 1-10 mM, 10-50 mM, or 50-100 mM. The concentration of the agonist can be at least 0.01 mM, 0.02 mM, 0.03 mM, 0.04 mM, 0.05 mM, 0.06 mM, 0.07 mM, 0.08 mM, 0.09 mM, 0.1 mM, 0.2 mM, 0.3 mM, 0.4 mM, 0.5 mM, 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 15 mM, 20 mM 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75 mM, 80 mM, 85 mM, 90 mM, 95 mM, or 100 mM.

[0486] The number of extracellular particles incubated with the STING agonist can also be altered to optimize the encapsulation of the STING agonist. The number of purified EV (e.g., exosome) particles can be between at least about 10 6 particles and at least about 10 20 particles of total purified vesicle particles. The number of purified particles can be 108 to 10 18 individuals, 10 10 to 10 16 individuals, 10 8 to 10 14 individuals, or 10 10 individuals, to 10 12 individuals, to 10 6 individuals, 10 8 individuals, 10 10 individuals, 10 12 individuals, 10 14 individuals, 10 16 individuals, 10 18 individuals, or 10 20 total purified vesicle particles.

[0487] In some embodiments, a synthetic macromolecule, such as a cationic lipid and a polymer, can be used to introduce one or more moieties into a suitable producer cell (Papapetrou et al., Gene Therapy 12:S118-S130 (2005)). In some embodiments, a cationic lipid forms a complex with one or more moieties through charge interactions. In some of these embodiments, the positively charged complex binds to the negatively charged cell surface and is taken up by the cell through endocytosis. In some other embodiments, a cationic polymer can be used to transfect the producer cell. In some of these embodiments, the cationic polymer is polyethylenimine (PEI). In certain embodiments, chemicals such as calcium phosphate, cyclodextrin, or polyaromatic hydrocarbons can be used to introduce one or more moieties into the producer cell. Physical methods such as particle-mediated transfection, "gene gun," biolistics, or particle bombardment techniques can also be used to introduce one or more moieties into the producer cell (Papapetrou et al., Gene Therapy 12:S118-S130 (2005)). Reporter genes such as beta-galactosidase, chloramphenicol acetyltransferase, luciferase, or green fluorescent protein can be used to assess the transfection efficiency of the producer cell.

[0488] In some embodiments, one or more moieties are introduced into the producer cell through viral transduction. Many viruses can be used as gene transfer vehicles, including Moloney murine leukemia virus (MMLV), adenovirus, adeno-associated virus (AAV), herpes simplex virus (HSV), lentivirus, and spumavirus. Viral-mediated gene transfer vehicles include vectors based on DNA viruses such as adenovirus, adeno-associated virus, and herpes virus, as well as vectors based on retrovirus.

[0489] In some embodiments, one or more moieties are introduced into producer cells by electroporation. Electroporation creates transient pores in the cell membrane, allowing the introduction of various molecules into the cell. In some embodiments, DNA and RNA, as well as polypeptide and non-polypeptide therapeutics, can be introduced into producer cells by electroporation.

[0490] In some embodiments, one or more moieties are introduced into producer cells by microinjection. In some embodiments, a glass micropipette can be used to inject one or more moieties into producer cells at the microscopic level.

[0491] In some embodiments, one or more moieties are introduced into producer cells by extrusion.

[0492] In some embodiments, one or more moieties are introduced into producer cells by sonication. In some embodiments, producer cells are exposed to high-intensity sound waves, causing transient disruption of the cell membrane, allowing loading of one or more moieties.

[0493] In some embodiments, one or more moieties are introduced into producer cells by cell fusion. In some embodiments, one or more moieties are introduced by electrocytofusion. In other embodiments, polyethylene glycol (PEG) is used to fuse producer cells. In additional embodiments, Sendai virus is used to fuse producer cells.

[0494] In some embodiments, one or more moieties are introduced into producer cells by hypotonic lysis. In such embodiments, producer cells can be exposed to low ionic strength buffers that cause them to rupture, allowing loading of one or more moieties. In other embodiments, controlled dialysis against a hypotonic solution can be used to swell producer cells and create pores in the producer cell membrane. The producer cells are then exposed to conditions that allow the membrane to reseal.

[0495] In some embodiments, one or more moieties are introduced into producer cells by detergent treatment. In certain embodiments, producer cells are treated with a mild detergent that transiently damages the producer cell membrane by creating pores, allowing loading of one or more moieties. After loading of the producer cells, the detergent is washed away, resealing the membrane.

[0496] In some embodiments, one or more moieties are introduced into producer cells by receptor-mediated endocytosis. In certain embodiments, producer cells have surface receptors that, upon binding one or more moieties, induce internalization of the receptor and associated moiety.

[0497] In some embodiments, the one or more moieties are introduced into the producer cell by filtration. In certain embodiments, the producer cell and the one or more moieties can be forced through a filter having a pore size smaller than the producer cell, resulting in a temporary disruption of the producer cell membrane and allowing the one or more moieties to enter the producer cell.

[0498] In some embodiments, the producer cell is subjected to several freeze-thaw cycles, resulting in a disruption of the cell membrane, allowing the loading of the one or more moieties.

[0499] IV. EV purification

[0500] EVs (e.g., exosomes) produced in accordance with the present disclosure can be isolated from the producer cell. It is contemplated that all known means of isolating EVs (e.g., exosomes) are considered suitable for use herein. For example, the physical properties of EVs (e.g., exosomes) can be used to isolate them from media or other source material, including separation based on charge (e.g., electrophoretic separation), size (e.g., filtration, molecular sieving, etc.), density (e.g., regular or gradient centrifugation), Svedberg constant (e.g., sedimentation with or without external force, etc.). Alternatively or additionally, separation can be based on one or more biological properties, and include methods that can use surface markers (e.g., for precipitation, reversible binding to a solid phase, FACS separation, specific ligand binding, non-specific ligand binding, etc.). In further contemplated methods, chemical and / or physical methods can also be used to fuse EVs (e.g., exosomes), including PEG-induced fusion and / or sonication fusion.

[0501] EVs (e.g., exosomes) can also be purified after incubation with the STING agonist to remove free, unencapsulated STING agonist from the composition. All means of previously disclosed methods are also considered suitable for use herein, including separation based on the physical or biological properties of the EVs (e.g., exosomes).

[0502] Isolation, purification, and enrichment can be performed in a general and non-selective manner, often including serial centrifugation. Alternatively, isolation, purification, and enrichment can be performed in a more specific and selective manner (e.g., using producer cell-specific surface markers). For example, specific surface markers can be used for immunoprecipitation, FACS sorting, affinity purification, bead-bound ligands for magnetic separation, etc.

[0503] In some embodiments, size exclusion chromatography can be used to isolate or purify EVs (e.g., exosomes). Size exclusion chromatography techniques are known in the art. Exemplary, non-limiting techniques are provided herein. In some embodiments, a void volume fraction is isolated that comprises EVs (e.g., exosomes) of interest. In some embodiments, for example, density gradient centrifugation can be used to further isolate EVs (e.g., exosomes). Still further, in some embodiments, it can be desirable to further isolate producer cell-derived EVs (e.g., exosomes) from other sources of EVs. For example, producer cell-derived EVs (e.g., exosomes) can be isolated from non-producer cell-derived EVs (e.g., exosomes) by immunoabsorbent capture using antibodies specific for antigens of the producer cell.

[0504] In some embodiments, isolation of EVs (e.g., exosomes) can involve size exclusion chromatography or ion chromatography, such as anion exchange, cation exchange, or mixed mode chromatography. In some embodiments, isolation of EVs (e.g., exosomes) can involve desalting, dialysis, tangential flow filtration, ultrafiltration, or diafiltration, or any combination thereof. In some embodiments, isolation of EVs (e.g., exosomes) can involve a combination of methods including, but not limited to, differential centrifugation, size-based membrane filtration, concentration, and / or rate zonal centrifugation. In some embodiments, isolation of EVs (e.g., exosomes) can involve one or more centrifugation steps. Centrifugation can be performed at about 50,000 to 150,000 x g. Centrifugation can be performed at about 50,000 x g, 75,000 x g, 100,000 x g, 125,000 x g, or 150,000 x g.

[0505] V. Therapeutic Administration

[0506] V. A. Immune modulation and dosing

[0507] Provided herein are methods of inducing and / or modulating an immune or inflammatory response in a subject by administering a pharmaceutically effective amount of EVs (e.g., exosomes) comprising a STING agonist.

[0508] Dendritic cells (DCs) are a group of antigen-presenting cells that originate from the hematopoietic cell lineage that links the innate and adaptive immune systems. DCs share common myeloid precursors with monocytes and macrophages, and are generally divided into two major classes: plasmacytoid DCs (pDCs) and myeloid DCs (mDCs), also known as conventional DCs (cDCs). mDCs are further classified according to their development from myeloid or lymphoid precursors and expression levels of CD8a, CD4, and C11b. A third DC population is monocyte-derived DCs (moDCs), which originate from monocyte precursors rather than DC progenitors such as pDCs and cDCs. moDCs are produced upon receiving inflammatory signals. Immature DCs exist in peripheral tissues before maturation. Several signaling pathways lead to DC maturation, including signaling cascades induced by pattern recognition receptors (PRRs). Each subset of immature DCs differs in the protein expression pattern of PRRs, which makes the population of immature DCs have different responses when activated by the same PRR. This leads to the modulation of immune responses mediated by DCs. Receptors present in DCs include Toll-like receptors (TLRs), C-type lectin receptors, retinoic acid-inducible gene (RIG)-I-like receptors (RLRs), NOD-like receptors (NLRs), and STING.

[0509] In mDCs and pDCs, the STING pathway is a major DNA sensing pathway. Activation of the STING pathway in DCs leads to type I IFN and proinflammatory cytokine production through TBK1, IRF3, and NF-KB signaling. Binding of IFN to its receptor on cells leads to the activation of IFN-stimulated response elements and transcription of IFN-sensitive genes that lead to immune and inflammatory responses. IFN signaling also cross-primes DCs to promote antigen persistence, changes the pool of antigens available for MHC I presentation, enhances MHC I presentation of antigens, and increases overall surface expression of MHC I, MHC II, and costimulatory molecules CD40, CD80, and CD86. These effects lead to increased priming of tumor-specific CD8+ T cells and initiation of adaptive immune responses.

[0510] In some embodiments, the method of administering an EV (e.g., an exosome) encapsulating or expressing on the surface a STING agonist to a subject in need thereof activates or induces dendritic cells, thereby inducing or modulating an immune or inflammatory response in the subject. In some embodiments, the activated dendritic cells are myeloid dendritic cells. In some embodiments, the dendritic cells are plasmacytoid dendritic cells.

[0511] In some embodiments, the method induces production of interferon (IFN)-beta. Administration of EVs (e.g., exosomes) comprising a STING agonist (e.g., encapsulated or expressed on the luminal surface or external surface) can result in induction of IFN-beta that is 2-fold to 10,000-fold greater than administration of the STING agonist alone. Administration of EVs (e.g., exosomes) comprising a STING agonist (e.g., encapsulated or expressed on the luminal surface or external surface) can result in induction of IFN-beta that is about 2-5-fold, 5-10-fold, 10-20-fold, 20-30-fold, 30-40-fold, 40-50-fold, 50-60-fold, 60-70-fold, 70-80-fold, 80-90-fold, 90-100-fold, 100-200-fold, 200-300-fold, 300-400-fold, 400-500-fold, 500-600-fold, 600-700-fold, 700-800-fold, 800-900-fold, 900-1000-fold, 1000-2000-fold, 2000-3000-fold, 3000-4000-fold, 4000-5000-fold, 5000-6000-fold, 6000-7000-fold, 7000-8000-fold, 8000-9000-fold, or 9000-10,000-fold greater than administration of the STING agonist alone. Administration of EVs (e.g., exosomes) comprising a STING agonist (e.g., encapsulated or expressed on the luminal surface or external surface) can result in induction of IFN-beta that is about 2-fold, >5-fold, >10-fold, >20-fold, >30-fold, >40-fold, >50-fold, >60-fold, >70-fold, >80-fold, >90-fold, >100-fold, >200-fold, >300-fold, >400-fold, >500-fold, >600-fold, >700-fold, >800-fold, >900-fold, >1000-fold, >2000-fold, >3000-fold, >4000-fold, >5000-fold, >6000-fold, >7000-fold, >8000-fold, >9000-fold, or >10,000-fold greater than administration of the STING agonist alone. Administration of EVs (e.g., exosomes) comprising a STING agonist (e.g., encapsulated or expressed on the luminal surface or external surface) can result in induction of IFN-beta that is 2-fold to 10,000-fold greater than the subject’s baseline IFN-beta production.Administration of EVs (e.g., exosomes) comprising a STING agonist (e.g., encapsulated or expressed on the luminal surface or external surface) can elicit induction of IFN-β that is about 2-5 fold, 5-10 fold, 10-20 fold, 20-30 fold, 30-40 fold, 40-50 fold, 50-60 fold, 60-70 fold, 70-80 fold, 80-90 fold, 90-100 fold, 100-200 fold, 200-300 fold, 300-400 fold, 400-500 fold, 500-600 fold, 600-700 fold, 700-800 fold, 800-900 fold, 900-1000 fold, 1000-2000 fold, 2000-3000 fold, 3000-4000 fold, 4000-5000 fold, 5000-6000 fold, 6000-7000 fold, 7000-8000 fold, 8000-9000 fold, 9000-10,000 fold of the baseline IFN-β production of the subject. Administration of EVs (e.g., exosomes) comprising a STING agonist can elicit induction of IFN-β that is about 2 fold, >5 fold, >10 fold, >20 fold, >30 fold, >40 fold, >50 fold, >60 fold, >70 fold, >80 fold, >90 fold, >100 fold, >200 fold, >300 fold, >400 fold, >500 fold, >600 fold, >700 fold, >800 fold, >900 fold, >1000 fold, >2000 fold, >3000 fold, >4000 fold, >5000 fold, >6000 fold, >7000 fold, >8000 fold, >9000 fold, or >10,000 fold of the baseline IFN-β production of the subject.

[0512] In some embodiments, administration of the EVs (e.g., exosomes) disclosed herein to a subject can also modulate the levels of other immunomodulatory agents (e.g., cytokines or chemokines). In certain embodiments, the methods disclosed herein can increase the levels of IFN-g, CXCL9, and / or CXCL10. In some embodiments, administration of the EVs (e.g., exosomes) described herein can produce amounts of IFN-g, CXCL9, and / or CXCL10 that are about 2-5 fold, 5-10 fold, 10-20 fold, 20-30 fold, 30-40 fold, 40-50 fold, 50-60 fold, 60-70 fold, 70-80 fold, 80-90 fold, 90-100 fold, 100-200 fold, 200-300 fold, 300-400 fold, 400-500 fold, 500-600 fold, 600-700 fold, 700-800 fold, 800-900 fold, 900-1000 fold, 1000-2000 fold, 2000-3000 fold, 3000-4000 fold, 4000-5000 fold, 5000-6000 fold, 6000-7000 fold, 7000-8000 fold, 8000-9000 fold, 9000-10,000 fold, of the free STING agonist.

[0513] In some embodiments, the method induces myeloid dendritic cell (mDC) activation. Administration of EVs (e.g., exosomes) comprising a STING agonist (e.g., encapsulated or expressed on the luminal surface or external surface) can result in mDC activation that is 2-fold to 50,000-fold greater than administration of the STING agonist alone. Administration of EVs (e.g., exosomes) comprising a STING agonist (e.g., encapsulated or expressed on the luminal surface or external surface) can result in mDC activation that is about 2-5-fold, 5-10-fold, 10-20-fold, 20-30-fold, 30-40-fold, 40-50-fold, 50-60-fold, 60-70-fold, 70-80-fold, 80-90-fold, 90-100-fold, 100-200-fold, 200-300-fold, 300-400-fold, 400-500-fold, 500-600-fold, 600-700-fold, 700-800-fold, 800-900-fold, 900-1000-fold, 1000-2000-fold, 2000-3000-fold, 3000-4000-fold, 4000-5000-fold, 5000-6000-fold, 6000-7000-fold, 7000-8000-fold, 8000-9000-fold, 9000-10,000-fold, 10,000-15,000-fold, 15,000-20,000-fold, 20,000-25,000-fold, 25,000-30,000-fold, 30,000-35,000-fold, 35,000-40,000-fold, 40,000-45,000-fold, or 45,000-50,000-fold greater than administration of the STING agonist alone. Administration of EVs (e.g., exosomes) comprising a STING agonist (e.g., encapsulated or expressed on the luminal surface or external surface) can result in mDC activation that is about 2-fold, >5-fold, >10-fold, >20-fold, >30-fold, >40-fold, >50-fold, >60-fold, >70-fold, >80-fold, >90-fold, >100-fold, >200-fold, >300-fold, >400-fold, >500-fold, >600-fold, >700-fold, >800-fold, >900-fold, >1000-fold, >2000-fold, >3000-fold, >4000-fold, >5000-fold, >6000-fold, >7000-fold, >8000-fold, >9000-fold, >10,000-fold, >15,000-fold, >20,000-fold, >25,000-fold, >30,000-fold, >35,000-fold, >40,000-fold, >45,000-fold, or >50,000-fold greater than administration of the STING agonist alone.

[0514] Administration of EVs (e.g., exogenous bodies) containing a STING agonist (e.g., encapsulated or expressed on the luminal or external surface) can induce mDC activation that is 2 to 10,000 times greater than the baseline mDC activation of the subject. Administration of EVs (e.g., exogenous forms) containing a STING agonist (e.g., encapsulated or expressed on the luminal or external surface) can induce approximately 2-5, 5-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-200, 200-300, 300-400, 400-500, 500-600, 600-700, 700-800, 800-900, 900-1000, 1000- mDC activation at 2000x, 2000-3000x, 3000-4000x, 4000-5000x, 5000-6000x, 6000-7000x, 7000-8000x, 8000-9000x, 9000-10,000x, 10,000-15,000x, 15,000-20,000x, 20,000-25,000x, 25,000-30,000x, 30,000-35,000x, 35,000-40,000x, 40,000-45,000x, or 45,000-50,000x. Administration of EVs (e.g., exogenous forms) containing a STING agonist (e.g., encapsulated or expressed on the luminal or external surface) can induce approximately 2-fold, >5-fold, >10-fold, >20-fold, >30-fold, >40-fold, >50-fold, >60-fold, >70-fold, >80-fold, >90-fold, >100-fold, >200-fold, >300-fold, >400-fold, >500-fold, >600-fold, >700-fold, >800-fold, and >800-fold increases in baseline mDC activation in subjects. mDC activation at multiples of 100x, 900x, 1000x, 2000x, 3000x, 4000x, 5000x, 6000x, 7000x, 8000x, 9000x, 10,000x, 15,000x, 20,000x, 25,000x, 30,000x, 35,000x, 40,000x, 45,000x, or 50,000x.

[0515] In some embodiments, the method of administering EVs (e.g., exosomes) comprising a STING agonist (e.g., encapsulated on or expressed on the luminal surface or the external surface) does not induce monocyte activation as compared to the subject’s baseline monocyte activation. In some embodiments, the method of administering EVs (e.g., exosomes) comprising a STING agonist (e.g., encapsulated on or expressed on the luminal surface or the external surface) induces a monocyte activation that is about 2-fold, <5-fold, <10-fold, <20-fold, <30-fold, <40-fold, <50-fold, <60-fold, <70-fold, <80-fold, <90-fold, <100-fold, <200-fold, <300-fold, <400-fold, <500-fold, <600-fold, <700-fold, <800-fold, <900-fold, <1000-fold, <2000-fold, <3000-fold, <4000-fold, <5000-fold, <6000-fold, <7000-fold, <8000-fold, <9000-fold, <10,000-fold, <15,000-fold, <20,000-fold, <25,000-fold, <30,000-fold, <35,000-fold, <40,000-fold, <45,000-fold, <50,000-fold, <55,000-fold, <60,000-fold, <65,000-fold, <70,000-fold, <75,000-fold, <80,000-fold, <85,000-fold, <90,000-fold, <95,000-fold, <100,000-fold, <200,000-fold, <300,000-fold, <400,000-fold, <500,000-fold, <600,000-fold, <700,000-fold, <800,000-fold, <900,000-fold, or <1,000,000-fold lower relative to the subject’s baseline monocyte activation.In some embodiments, administration of EVs (e.g., exosomes) comprising a STING agonist (e.g., encapsulated or expressed on the luminal surface or external surface) to a subject results in an induction of monocyte activation that is about 2-5 fold, 5-10 fold, 10-20 fold, 20-30 fold, 30-40 fold, 40-50 fold, 50-60 fold, 60-70 fold, 70-80 fold, 80-90 fold, 90-100 fold, 100-200 fold, 200-300 fold, 300-400 fold, 400-500 fold, 500-600 fold, 600-700 fold, 700-800 fold, 800-900 fold, 900-1000 fold, 1000-2000 fold, 2000-3000 fold, 3000-4000 fold, 4000-5000 fold, 5000-6000 fold, 6000-7000 fold, 7000-8000 fold, 8000-9000 fold, 9000-10,000 fold, 10,000-15,000 fold, 15,000-20,000 fold, 20,000-25,000 fold, 25,000-30,000 fold, 30,000-35,000 fold, 35,000-40,000 fold, 40,000-45,000 fold, 45,000-50,000 fold, 55,000-60,000 fold, 60,000-65,000 fold, 65,000-70,000 fold, 70,000-75,000 fold, 75,000-80,000 fold, 80,000-85,000 fold, 85,000-90,000 fold, 90,000-95,000 fold, 95,000-100,000 fold, 100,000-200,000 fold, 200,000-300,000 fold, 300,000-400,000 fold, 400,000-500,000 fold, 500,000-600,000 fold, 600,000-700,000 fold, 700,000-800,000 fold, 800,000-900,000 fold, or 900,000-1,000,000 fold lower relative to the baseline monocyte activation of the subject.

[0516] In some embodiments, the method of administering to a subject an EV (e.g., an exosome) comprising a STING agonist (e.g., encapsulated on or expressed on the luminal surface or the external surface) does not induce monocyte activation as compared to administration of a STING agonist alone. In some embodiments, administration of an EV (e.g., an exosome) comprising a STING agonist (e.g., encapsulated on or expressed on the luminal surface or the external surface) results in an induction of monocyte activation that is about 2-fold, <5-fold, <10-fold, <20-fold, <30-fold, <40-fold, <50-fold, <60-fold, <70-fold, <80-fold, <90-fold, <100-fold, <200-fold, <300-fold, <400-fold, <500-fold, <600-fold, <700-fold, <800-fold, <900-fold, <1000-fold, <2000-fold, <3000-fold, <4000-fold, <5000-fold, <6000-fold, <7000-fold, <8000-fold, <9000-fold, <10,000-fold, <15,000-fold, <20,000-fold, <25,000-fold, <30,000-fold, <35,000-fold, <40,000-fold, <45,000-fold, or <50,000-fold lower than the amount of monocyte activation following administration of a free STING agonist. In some embodiments, administration of an EV (e.g., an exosome) comprising a STING agonist (e.g., encapsulated on or expressed on the luminal surface or the external surface) results in an induction of monocyte activation that is about 2-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 600-fold, 700-fold, 800-fold, 900-fold, 1000-fold, 2000-fold, 3000-fold, 4000-fold, 5000-fold, 6000-fold, 7000-fold, 8000-fold, 9000-fold, 10,000-fold, 15,000-fold, 20,000-fold, 25,000-fold, 30,000-fold, 35,000-fold, 40,000-fold, 45,000-fold, or 50,000-fold lower than the amount of monocyte activation following administration of a free STING agonist. Monocyte activation can be measured by surface expression of CD86 on monocytes, or by any other suitable marker of monocyte activation known in the art

[0517] Due to the improved therapeutic effects associated with the EVs (e.g., exosomes) described herein, in some embodiments, a lower dose of EVs (e.g., exosomes) comprising a STING agonist (e.g., encapsulated or expressed on the luminal or external surface) can be delivered as compared to a free STING agonist. Furthermore, non-selective delivery of high doses of STING agonists can attenuate the desired immune stimulatory response. Thus, because the EVs (e.g., exosomes) described herein can be administered at lower doses, in some embodiments, they can operate within a wider therapeutic window and reduce adverse effects (e.g., systemic toxicity, immune cell killing, lack of cell selectivity) observed with free STING agonists.

[0518] The compositions described herein can be administered in a dose sufficient to ameliorate a disease, disorder, condition, or symptom in a subject in need thereof. In some embodiments, the dose of EVs (e.g., exosomes) comprising a STING agonist administered to a subject in need thereof is between about 0.01 mM and 0.1 mM, 0.1 mM and 1 mM, 1 mM and 10 mM, 10 mM and 100 mM, or 100 mM and 1000 mM. In certain embodiments, the dose of EVs (e.g., exosomes) comprising a STING agonist administered to a subject in need thereof is about 0.01 mM, 0.05 mM, 0.1 mM, 0.2 mM, 0.3 mM, 0.4 mM, 0.5 mM, 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 40 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75 mM, 80 mM, 85 mM, 90 mM, 95 mM, 100 mM, 150 mM, 200 mM, 250 mM, 300 mM, 350 mM, 400 mM, 450 mM, 500 mM, 550 mM, 600 mM, 650 mM, 700 mM, 750 mM, 800 mM, 850 mM, 900 mM, 950 mM, or 1000 mM.

[0519] In some embodiments, the amount of EVs (e.g., exosomes) comprising a STING agonist (e.g., encapsulated or expressed on a luminal surface or an external surface) administered to a subject in need thereof is 2-fold, <5-fold, <10-fold, <20-fold, <30-fold, <40-fold, <50-fold, <60-fold, <70-fold, <80-fold, <90-fold, <100-fold, <200-fold, <300-fold, <400-fold, <500-fold, <600-fold, <700-fold, <800-fold, <900-fold, <1000-fold, <2000-fold, <3000-fold, <4000-fold, <5000-fold, <6000-fold, <7000-fold, <8000-fold, <9000-fold, <10,000-fold, <15,000-fold, <20,000-fold, <25,000-fold, <30,000-fold, <35,000-fold, <40,000-fold, <45,000-fold, or <50,000-fold lower than the amount of free STING agonist required to achieve the same improved outcome in a subject in need thereof. In some embodiments, the amount of EVs (e.g., exosomes) comprising a STING agonist (e.g., encapsulated or expressed on a luminal surface or an external surface) administered to a subject in need thereof is about 2-5-fold, 5-10-fold, 10-20-fold, 20-30-fold, 30-40-fold, 40-50-fold, 50-60-fold, 60-70-fold, 70-80-fold, 80-90-fold, 90-100-fold, 100-200-fold, 200-300-fold, 300-400-fold, 400-500-fold, 500-600-fold, 600-700-fold, 700-800-fold, 800-900-fold, 900-1000-fold, 1000-2000-fold, 2000-3000-fold, 3000-4000-fold, 4000-5000-fold, 5000-6000-fold, 6000-7000-fold, 7000-8000-fold, 8000-9000-fold, 9000-10,000-fold, 10,000-15,000-fold, 15,000-20,000-fold, 20,000-25,000-fold, 25,000-30,000-fold, 30,000-35,000-fold, 35,000-40,000-fold, 40,000-45,000-fold, or 45,000-50,000-fold lower than the amount of free STING agonist required to achieve the same improved outcome in a subject in need thereof.

[0520] In some embodiments, the method of administering EVs (e.g., exosomes) comprising a STING agonist does not induce systemic inflammation compared to the subject’s baseline systemic inflammation. In some embodiments, the administration of EVs (e.g., exosomes) comprising a STING agonist results in an induction of systemic inflammation that is about 2-fold, <5-fold, <10-fold, <20-fold, <30-fold, <40-fold, <50-fold, <60-fold, <70-fold, <80-fold, <90-fold, <100-fold, <200-fold, <300-fold, <400-fold, <500-fold, <600-fold, <700-fold, <800-fold, <900-fold, <1000-fold, <2000-fold, <3000-fold, <4000-fold, <5000-fold, <6000-fold, <7000-fold, <8000-fold, <9000-fold, <10,000-fold, <15,000-fold, <20,000-fold, <25,000-fold, <30,000-fold, <35,000-fold, <40,000-fold, <45,000-fold, or <50,000-fold lower relative to the subject’s baseline systemic inflammation. In some embodiments, the administration of EVs, e.g., exosomes, comprising a STING agonist to a subject results in an induction of systemic inflammation that is about 2-5-fold, 5-10-fold, 10-20-fold, 20-30-fold, 30-40-fold, 40-50-fold, 50-60-fold, 60-70-fold, 70-80-fold, 80-90-fold, 90-100-fold, 100-200-fold, 200-300-fold, 300-400-fold, 400-500-fold, 500-600-fold, 600-700-fold, 700-800-fold, 800-900-fold, 900-1000-fold, 1000-2000-fold, 2000-3000-fold, 3000-4000-fold, 4000-5000-fold, 5000-6000-fold, 6000-7000-fold, 7000-8000-fold, 8000-9000-fold, 9000-10,000-fold, 10,000-15,000-fold, 15,000-20,000-fold, 20,000-25,000-fold, 25,000-30,000-fold, 30,000-35,000-fold, 35,000-40,000-fold, 40,000-45,000-fold, or 45,000-50,000-fold lower relative to the subject’s baseline systemic inflammation.

[0521] In some embodiments, the method of administering to a subject an EV (e.g., an exosome) comprising a STING agonist (e.g., encapsulated on or expressed on the luminal surface or the external surface) does not induce systemic inflammation compared to administration of a STING agonist alone. In some embodiments, administration of an EV (e.g., an exosome) comprising a STING agonist (e.g., encapsulated on or expressed on the luminal surface or the external surface) results in an induction of systemic inflammation that is about 2-fold, <5-fold, <10-fold, <20-fold, <30-fold, <40-fold, <50-fold, <60-fold, <70-fold, <80-fold, <90-fold, <100-fold, <200-fold, <300-fold, <400-fold, <500-fold, <600-fold, <700-fold, <800-fold, <900-fold, <1000-fold, <2000-fold, <3000-fold, <4000-fold, <5000-fold, <6000-fold, <7000-fold, <8000-fold, <9000-fold, <10,000-fold, <15,000-fold, <20,000-fold, <25,000-fold, <30,000-fold, <35,000-fold, <40,000-fold, <45,000-fold, or <50,000-fold lower in the amount of systemic inflammation relative to the induction of systemic inflammation after administration of a free STING agonist. In some embodiments, administration of an EV (e.g., an exosome) comprising a STING agonist (e.g., encapsulated on or expressed on the luminal surface or the external surface) results in an induction of systemic inflammation that is about 2-5-fold, 5-10-fold, 10-20-fold, 20-30-fold, 30-40-fold, 40-50-fold, 50-60-fold, 60-70-fold, 70-80-fold, 80-90-fold, 90-100-fold, 100-200-fold, 200-300-fold, 300-400-fold, 400-500-fold, 500-600-fold, 600-700-fold, 700-800-fold, 800-900-fold, 900-1000-fold, 1000-2000-fold, 2000-3000-fold, 3000-4000-fold, 4000-5000-fold, 5000-6000-fold, 6000-7000-fold, 7000-8000-fold, 8000-9000-fold, 9000-10,000-fold, 10,000-15,000-fold, 15,000-20,000-fold, 20,000-25,000-fold, 25,000-30,000-fold, 30,000-35,000-fold, 35,000-40,000-fold, 40,000-45,000-fold, or 45,000-50,000-fold lower in the amount of systemic inflammation relative to the induction of systemic inflammation after administration of a free STING agonist. Systemic inflammation can be quantified or measured by any suitable method known in the art.

[0522] In some embodiments, the method of administering to a subject an EV (e.g., an exosome) comprising a STING agonist (e.g., encapsulated or expressed on the luminal surface or the external surface) additionally comprises administering an additional therapeutic agent. In some embodiments, the additional therapeutic agent is an immunomodulatory agent. In some embodiments, the immunomodulatory component is an inhibitor of a negative checkpoint regulator or an inhibitor of a binding partner of a negative checkpoint regulator. In some of these embodiments, the negative checkpoint regulator is selected from the group consisting of 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 protein 3 (TIM-3), B- and T-lymphocyte attenuator (BTLA), T-cell immunoreceptor with Ig and ITIM domains (TIGIT), V-domain Ig suppressor of T cell activation (VISTA), adenosine A2a receptor (A2aR), killer-cell immunoglobulin-like receptor (KIR), indoleamine 2,3-dioxygenase (IDO), CD20, CD39, and CD73. In various embodiments, the additional therapeutic agent is an antibody or antigen-binding fragment thereof. In some embodiments, the antibody or antigen-binding fragment thereof is one or more of whole antibodies, polyclonal antibodies, monoclonal antibodies, and recombinant antibodies, fragments thereof, and further includes single-chain antibodies, humanized antibodies, murine antibodies, chimeric antibodies, murine-human antibodies, murine-primate antibodies, primate-human monoclonal antibodies, anti-idiotypic antibodies, antibody fragments such as scFv, (scFv)2, Fab, Fab' and F(ab')2, F(ab1)2, Fv, dAb, and Fd fragments, diabodies, and antibody-related polypeptides. The term antibody includes bispecific antibodies and multispecific antibodies, so long as they exhibit the desired biological activity or function. In some embodiments, the additional therapeutic agent is a therapeutic antibody or antigen-binding fragment thereof that is an inhibitor of CTLA-4, PD-1, PD-L1, PD-L2, TIM-3, or LAG3.

[0523] In some embodiments, the additional therapeutic agent is an agent that prevents or treats T cell exhaustion. Such agents can increase, decrease, or modulate the expression of genes associated with T cell exhaustion, including Prdm1, Bhlhe40, Irf4, Ikzf2, Zeb2, Lass6, Egr2, Tox, Eomes, Nfatcl, Nfatc2, Zbtb32, Rbpj, Hifla, Lag3, Tnfrsf9, Ptger2, Havcr2, Alcam, Tigit, Ctla4, Ptger4, Tnfrsib, Ccl4, CD109, CD200, Tnfsf9, Nrp1, Sema4c, Ptprj, Il21, Tspan2, Rgs16, Sh2d2a, Nucbl, Plscr1, Ptpnll, Prkca, Plscr4, Casp3, Gpd2, Gas2, Sh3rfl, Nhedc2, Plek, Tnfaip2, and Ctsb, or any combination thereof. The therapeutic agent can also increase, decrease, or modulate proteins associated with T cell exhaustion, including NFAT-1 or NFAT-2.

[0524] V.B. Methods of treating cancer

[0525] Provided herein are methods of treating cancer in a subject. The methods include administering to the subject a therapeutically effective amount of a composition disclosed herein, wherein the composition is capable of upregulating a STING-mediated immune response in the subject, thereby enhancing tumor targeting by the immune system of the subject. In some embodiments, the composition is administered intratumorally to the subject. In some embodiments, the composition is administered parenterally, orally, intravenously, intramuscularly, intraperitoneally, or by any other suitable route of administration.

[0526] Also provided herein are methods of preventing metastasis of cancer in a subject. The methods include administering to the subject a therapeutically effective amount of a composition disclosed herein, wherein the composition is capable of preventing one or more tumors in one site of the subject from promoting the growth of one or more tumors in another site of the subject. In some embodiments, the composition is administered intratumorally in a first tumor in one site, and the composition administered in the first tumor prevents metastasis of one or more tumors in a second site.

[0527] In some embodiments, administration of the EVs (e.g., exosomes) disclosed herein inhibits and / or reduces tumor growth in the subject. In some embodiments, the tumor growth (e.g., tumor volume or weight) is reduced by at least about 5%, by at least about 10%, by at least about 20%, by at least about 30%, by at least about 40%, by at least about 50%, by at least about 60%, by at least about 70%, by at least about 80%, by at least about 90%, or by about 100% as compared to a reference (e.g., tumor volume in a corresponding subject following administration of a free STING agonist or EVs (e.g., exosomes) without a STING agonist).

[0528] In some embodiments, the cancer treated is characterized by infiltration of white blood cells (T cells, B cells, macrophages, dendritic cells, monocytes) into the tumor microenvironment, or so-called “hot tumors” or “inflammatory tumors”. In some embodiments, the cancer treated is characterized by low or undetectable levels of infiltration of white blood cells into the tumor microenvironment, or so-called “cold tumors” or “non-inflammatory tumors”. In some embodiments, the EVs (e.g., exosomes) are administered in an amount and for a duration sufficient to convert a “cold tumor” to a “hot tumor”, i.e., the administration results in infiltration of white blood cells (such as T cells) into the tumor microenvironment. In certain embodiments, the cancer comprises bladder cancer, cervical cancer, renal cell carcinoma, testicular cancer, colorectal cancer, lung cancer, head and neck cancer, and ovarian cancer, lymphoma, liver cancer, glioblastoma, melanoma, myeloma, leukemia, pancreatic cancer, or a combination thereof. As used herein, the term “distal tumor” or “distant tumor” refers to a tumor that has spread from an original (or primary) tumor to a distant organ or distant tissue, e.g., a lymph node. In some embodiments, the EVs (e.g., exosomes) of the present disclosure treat tumors after metastatic spread.

[0529] Non-limiting examples of cancers (or tumors) that can be treated using the methods disclosed herein include squamous cell carcinoma, small cell lung cancer (SCLC), non-small cell lung cancer, squamous non-small cell lung cancer (NSCLC), non-squamous NSCLC, gastrointestinal cancer, renal cancer (e.g., clear cell carcinoma), ovarian cancer, liver cancer (e.g., hepatocellular carcinoma), colorectal cancer, endometrial cancer, kidney cancer (e.g., renal cell carcinoma (RCC)), prostate cancer (e.g., hormone refractory prostate adenocarcinoma), thyroid cancer, pancreatic cancer, cervical cancer, stomach cancer, bladder cancer, hepatoma, breast cancer, colon cancer, and head and neck cancer (cancer or carcinoma), gastric cancer, germ cell tumors, pediatric sarcomas, sinonasal natural killer cell cancer, melanoma (e.g., metastatic malignant melanoma, such as cutaneous or intraocular malignant melanoma), bone cancer, skin cancer, uterine cancer, anal region cancer, testicular cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, esophageal cancer (e.g., gastroesophageal junction cancer), small bowel cancer, endocrine system cancer, parathyroid cancer, adrenal gland cancer, soft tissue sarcoma, urethral cancer, penile cancer, pediatric solid tumors, ureter cancer, renal pelvis cancer, tumor angiogenesis, pituitary adenoma, Kaposi's sarcoma, epidermoid cancer, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers, including cancers induced by asbestos, virus-related or virus-derived cancers (e.g., human papilloma virus (HPV-associated or HPV-derived tumors)), and hematological malignancies derived from either of the two major blood cell lineages, i.e., the myeloid cell line (which gives rise to granulocytes, red blood cells, thrombocytes, macrophages, and mast cells) or the lymphoid cell line (which gives rise to B-cells, T-cells, NK cells, and plasma cells), such as all types of leukemia, lymphoma, and myeloma, e.g., acute, chronic, lymphocytic, and / or myelogenous leukemia, such as acute leukemia (ALL), acute myelogenous leukemia (AML), chronic lymphocytic leukemia (CLL), and chronic myelogenous leukemia (CML), undifferentiated AML (MO), myeloblasts leukemia (Ml), myeloblasts leukemia (M2; with cellular maturation), promyelocytic leukemia (M3 or M3 variant [M3V]), myelomonocytic leukemia (M4 or M4 variant with eosinophilia [M4E]), monocytic leukemia (M5), erytholeukemia (M6), megakaryoblastic leukemia (M7), isolated granulocytic sarcoma, and chloroma; lymphomas, such as Hodgkin's lymphoma (HL), non-Hodgkin's lymphoma (NHL), B-cell hematological malignancies, e.g., B-cell lymphoma, T-cell lymphoma, lymphoplasmacytoid lymphoma, monocytoid B-cell lymphoma, mucosa-associated lymphoid tissue (MALT) lymphoma, anaplastic (e.g., Ki1 +) large cell lymphoma, adult T-cell lymphoma / leukemia, mantle cell lymphoma, angioimmunoblastic T-cell lymphoma, angiocentric lymphoma, intestinal T-cell lymphoma, primary mediastinal B-cell lymphoma, precursor T-lymphoblastic lymphoma, T-lymphoblastic; and T-cell lymphoma / leukemia (T-Lbly / T-ALL), peripheral T-cell lymphoma, lymphoblastic lymphoma, post-transplant lymphoproliferative disorder, true histiocytic lymphoma, primary effusion lymphoma, B-cell lymphoma, lymphoblastic lymphoma (LBL), hematopoietic tumors of lymphoid lineage, acute lymphoblastic leukemia, diffuse large B-cell lymphoma, Burkitt's lymphoma, follicular lymphoma, diffuse histiocytic lymphoma (DHL), immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma, cutaneous T-cell lymphoma (CTLC) (also known as mycosis fungoides or Sezary syndrome), and lymphoplasmacytoid lymphoma (LPL) with Waldenstrom's macroglobulinemia; myeloma, such as IgG myeloma, light chain myeloma, non-secretory myeloma, smoldering myeloma (also known as indolent myeloma), solitary plasmacytoma, and multiple myeloma, chronic lymphocytic leukemia (CLL), hairy cell lymphoma; myeloid hematopoietic tumors, mesenchymal-derived tumors including fibrosarcoma and rhabdomyosarcoma; seminoma, teratoma, mesenchymal-derived tumors including fibrosarcoma, rhabdomyosarcoma, and osteosarcoma; and other tumors including melanoma, xeroderma pigmentosum, keratoacanthoma, seminoma, thyroid follicular cancer, and teratoma, hematopoietic tumors of lymphoid lineage, e.g., T-cell and B-cell tumors, including but not limited to T-cell disorders such as T-prolymphocytic leukemia (T-PLL), including small cell and convoluted cell types; large granular lymphocytic leukemia (LGL) of T-cell type; a / d T-NHL hepatosplenic lymphoma; peripheral / thymic post-T-cell lymphoma (polymorphic and immunoblastic subtypes); angiocentric (nasal) T-cell lymphoma; head and neck cancer, kidney cancer, rectal cancer, thyroid cancer; acute myeloid lymphoma and any combination thereof.

[0530] In some embodiments, the cancer (or tumor) that can be treated includes breast cancer, head and neck cancer, uterine cancer, brain cancer, skin cancer, kidney cancer, lung cancer, colorectal cancer, prostate cancer, liver cancer, bladder cancer, kidney cancer, peritoneal cancer, pancreatic cancer, thyroid cancer, esophageal cancer, eye cancer, stomach / gastric cancer, gastrointestinal cancer, carcinoma, sarcoma, leukemia, lymphoma, myeloma, or a combination thereof. In certain embodiments, the cancer that can be treated with the present disclosure is pancreatic cancer and / or peritoneal cancer.

[0531] In some embodiments, the methods described herein can also be used to treat metastatic cancer, unresectable refractory cancer (e.g., cancer refractory to previous cancer therapy), and / or recurrent cancer.

[0532] In some embodiments, the EVs (e.g., exosomes) disclosed herein can be used in combination with one or more additional anti-cancer and / or immunomodulatory agents. Such agents can include, for example, chemotherapeutic drugs, small molecule drugs, or antibodies that stimulate an immune response to a given cancer. In some embodiments, the methods described herein can be used in combination with standard-of-care treatments (e.g., surgery, radiation, and chemotherapy).

[0533] In some embodiments, the methods disclosed herein for treating cancer can include administering EVs (e.g., exosomes) comprising a STING agonist (e.g., encapsulated on or expressing on a luminal surface or an external surface) with one or more immunooncology agents, such that multiple elements of the immune pathway can be targeted. Non-limiting examples of such combinations include: therapies that enhance tumor antigen presentation (e.g., dendritic cell vaccines, GM-CSF secreting cell vaccines, CpG oligos, imiquimod); therapies that inhibit negative immune regulation (e.g., by inhibiting the CTLA-4 and / or PD1 / PD-L1 / PD-L2 pathways and / or depleting or blocking Tregs or other immunosuppressive cells (e.g., myeloid-derived suppressor cells)); therapies that stimulate positive immune regulation (e.g., with agonists that stimulate the CD137, OX-40, and / or CD40 or GITR pathways and / or stimulate T cell effector functions); therapies that systemically increase the frequency of anti-tumor T cells; therapies that deplete or inhibit Tregs (such as Tregs in the tumor) (e.g., using antagonists of CD25 (e.g., daclizumab) or by depleting with ex vivo anti-CD25 beads); therapies that affect suppressive myeloid cell function in the tumor; therapies that enhance tumor cell immunogenicity (e.g., anthracyclines); adoptive T cell or NK cell transfer, including genetically modified cells, e.g., cells modified with chimeric antigen receptors (CAR-T therapy); therapies that inhibit metabolic enzymes such as indoleamine dioxygenase (IDO), dioxygenase, arginase, or nitric oxide synthase; therapies that reverse / prevent T cell anergy or exhaustion; therapies that trigger innate immune activation and / or inflammation at the tumor site; administration of immune-stimulatory cytokines; or blockade of immune-suppressive cytokines.

[0534] In some embodiments, the immuno-oncology agent that can be used in combination with the EVs (e.g., exosomes) disclosed herein comprises an immune checkpoint inhibitor (i.e., blocks signaling through a particular immune checkpoint pathway). Non-limiting examples of immune checkpoint inhibitors that can be used in the methods of the application include a CTLA-4 antagonist (e.g., an anti-CTLA-4 antibody), a PD-1 antagonist (e.g., an anti-PD-1 antibody, an anti-PD-L1 antibody), a TIM-3 antagonist (e.g., an anti-TIM-3 antibody), or a combination thereof.

[0535] In some embodiments, the immuno-oncology agent comprises an immune checkpoint activator (i.e., promotes signaling through a particular immune checkpoint pathway). In certain embodiments, the immune checkpoint activator comprises an OX40 agonist (e.g., an anti-OX40 antibody), a LAG-3 agonist (e.g., an anti-LAG-3 antibody), a 4-1BB (CD137) agonist (e.g., an anti-CD137 antibody), a GITR agonist (e.g., an anti-GITR antibody), or any combination thereof.

[0536] In some embodiments, the combination of the EVs (e.g., exosomes) disclosed herein and the second agent (e.g., immune checkpoint inhibitor) discussed herein can be administered simultaneously as a single composition in a pharmaceutically acceptable carrier. In other embodiments, the combination of the EVs (e.g., exosomes) and the second agent (e.g., immune checkpoint inhibitor) discussed herein can be administered simultaneously as separate compositions. In further embodiments, the combination of the EVs (e.g., exosomes) and the second agent (e.g., immune checkpoint inhibitor) discussed herein can be administered sequentially. In some embodiments, the EVs (e.g., exosomes) are administered prior to administration of the second agent (e.g., immune checkpoint inhibitor).

[0537] V.C. Pharmaceutical Compositions

[0538] Provided herein are pharmaceutical compositions comprising EVs (e.g., exosomes) suitable for administration to a subject. The pharmaceutical compositions generally comprise a plurality of EVs (e.g., exosomes) comprising a STING agonist (e.g., encapsulated on or expressing on a luminal surface or an external surface) and a pharmaceutically acceptable excipient or carrier in a form suitable for administration to a subject. The pharmaceutically acceptable excipient or carrier is determined in part by the particular composition being administered, as well as the particular method used to administer the composition. Thus, a wide variety of suitable pharmaceutical composition formulations exist, comprising a plurality of EVs, e.g., exosomes. (See, e.g., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa. 18th Ed. (1990)). The pharmaceutical compositions are generally sterile, fully complies with all Good Manufacturing Practice (GMP) regulations of the U.S. Food and Drug Administration.

[0539] In some embodiments, the pharmaceutical composition comprises one or more STING agonists and an EV (e.g., exosome) described herein.

[0540] Pharmaceutically acceptable excipients include generally recognized as safe (GRAS), non-toxic, and desirable excipients, including excipients that are acceptable for veterinary use as well as human pharmaceutical use.

[0541] Examples of carriers or diluents include, but are not limited to, water, saline, Ringer's solutions, dextrose solutions, and 5% human serum albumin. Such media and compounds are known to the art, and their use is within the scope of the disclosure. Unless otherwise specified, any conventional media or compounds can be used in the compositions described herein, so long as they are compatible with the EVs (e.g., exosomes) described herein. Supplementary therapeutic agents can also be incorporated into the compositions. Generally, the pharmaceutical compositions are formulated to be compatible with their intended route of administration. The EVs (e.g., exosomes) can be administered by intratumoral, parenteral, topical, intravenous, oral, subcutaneous, intra-arterial, intradermal, transdermal, rectal, intracranial, intraperitoneal, intranasal, intramuscular routes or as an inhalant. In one embodiment, the pharmaceutical composition comprising EVs (e.g., exosomes) is administered intravenously (e.g., by injection). The EVs (e.g., exosomes) can be administered optionally in combination with other therapeutic agents that are at least partially effective in treating the disease, disorder, or condition for which the EVs (e.g., exosomes) are intended to treat.

[0542] The solutions or suspensions can include the following components: a sterile diluent such as water, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial compounds such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating compounds such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The formulations can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[0543] Pharmaceutical compositions adapted for injectable use include sterile aqueous solutions (if water soluble) or dispersions and sterile powders. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). The composition is generally sterile and fluid to the extent that easy syringeability exists. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal compounds, e.g., parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. If desired, isotonicity can be effected using TM (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). The composition is generally sterile and has fluidity to the extent that easy syringability exists. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal compounds, e.g., parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. If desired, isotonicity can be effected using

[0544] Sterile injectable solutions can be prepared by incorporating an EV (e.g., exosome) in the required amount in the appropriate solvent with one or a combination of ingredients enumerated herein, as required. Generally, dispersions are prepared by incorporating an EV (e.g., exosome) into a sterile vehicle which contains a basic dispersion medium and any required other ingredients. In the case of sterile powders for the preparation of sterile injectable solutions, methods of preparation are vacuum drying and freeze drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. An EV (e.g., exosome) can be administered in a form of a depot injection or implant preparation which can be formulated in such a manner as to permit a sustained or pulsatile release of the EV (e.g., exosome).

[0545] The compositions comprising EVs (e.g., exosomes) can also be administered systemically, either transmucosally or transdermally. For transmucosal or transdermal administration, suitable penetrants are used in the formulation to facilitate passage through the barrier. Such penetrants are well known in the art and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be achieved by the use of nasal sprays or suppositories. For transdermal administration, the modified EVs (e.g., exosomes) are formulated in an ointment, salve, gel, or cream as are well known in the art.

[0546] This PCT application claims priority to U.S. Provisional Application Nos. 62 / 647,491 filed March 23, 2018, 62 / 680,501 filed June 4, 2018, 62 / 688,600 filed June 22, 2018, and 62 / 756,247 filed November 6, 2018, each of which is incorporated by reference herein in its entirety.

[0547] EMBODIMENTS

[0548] The following examples are for illustrative purposes only and should not be construed as limiting the scope or content of the present application in any way. The practice of the present application will employ, unless otherwise indicated, conventional methods of protein chemistry, biochemistry, recombinant DNA techniques and pharmacology, within the skill of the art. Such techniques are explained fully in the literature. See, e.g., T.E. Creighton, Proteins: Structures and Molecular Properties (W.H. Freeman and Company, 1993); Green and Sambrook et al., Molecular Cloning: A Laboratory Manual, 4thEdition (Cold Spring Harbor Laboratory Press, 2012); Colowick and Kaplan, Methods In Enzymology (Academic Press); Remington: The Science and Practice of Pharmacy, 22ndEdition (Pharmaceutical Press, 2012); Sundberg and Carey, Advanced Organic Chemistry: Parts A and B, 5thEdition (Springer, 2007).

[0549] Methods

[0550] Exosome purification

[0551] HEK293SF cells were grown to high density in chemically defined media for 7 days. Conditioned cell culture media was collected and centrifuged at 300-800 x g for 5 min at room temperature to remove cells and large debris. The supernatant was then supplemented with 1000 U / L benzonase and incubated for 1 hour at 37°C water bath. The supernatant was collected and centrifuged at 16,000 x g for 30 min at 4°C to remove residual cell debris and other large contaminants. The supernatant was then ultracentrifuged at 133,900 x g for 3 hours at 4°C to pellet out the exosomes. The supernatant was discarded and any residual media was pipetted from the bottom of the tube. The pellet was resuspended in 200-1000 μΐ, PBS (-Ca-Mg).

[0552] To further enrich the exosome population, the pellet was treated by density gradient purification (sucrose or OPTIPREP® TM ) as defined in Table 3 below. For sucrose gradient purification, the exosome pellet was layered on top of a sucrose gradient as defined in Table 3 below as defined in Table 3 below.

[0553] Table 3:

[0554]

[0555] The gradient was spun at 200,000 x g for 16 hours at 4°C in a 12 mL Ultra-Clear (344059) tube placed in a SW 41 Ti rotor to isolate the exosome fraction.

[0556] The exosome layer was gently removed from the top layer and diluted in ~32.5 mL PBS in a 38.5 mL Ultra-Clear (344058) tube and ultracentrifuged again at 133,900 x g for 3 hours at 4°C to pellet the purified exosomes. The resulting pellet was resuspended in a minimal volume of PBS (~200 μΐ) and stored at 4°C.

[0557] For OPTIPREP® TM gradients, an equal volume of 10%, 30% and 45% OPTIPREP® TM was prepared in a 12 mL Ultra-Clear (344059) tube for a SW 41 Ti rotor. The pellet was added to the OPTIPREP® TM gradient and ultracentrifuged at 200,000 x g for 16 hours at 4°C to isolate the exosome fraction. The exosome layer was then gently collected from the top of the tube ~3 mL.

[0558] ​The exosome fraction was diluted in 38.5 mL Ultra-Clear (344058) tubes in approximately 32 mL of PBS and ultracentrifuged at 133,900 x g for 3 hours at 4°C to pellet the purified exosomes. The pelleted exosomes were then resuspended in a minimal volume of PBS (approximately 200 μΐ) and stored at 4°C.

[0559] In vivo intratumoral microinjection studies were performed Figure 1

[0560] Tumor cell culture

[0561] A20 cells (ATCC lot number 70006082) were cultured in RPMI 1640 containing L-glutamine (ThermoFisher), 10% fetal bovine serum (Thermofisher), and 50 nanomolar BME at 37 degrees Celsius, 5% CO2. IMPACT III testing (IDEXX Bioresearch) was performed to confirm mycoplasma and pathogen-free status. Upon receipt from the supplier, cells were expanded and cryopreserved after 2-3 passages. After thawing, cells were maintained for a maximum of 8 weeks by passaging three times per week, then replenished with fresh cryogenic stock.

[0562] In vivo studies

[0563] All mouse experiments were approved by the IACUC Board of Presage Biosciences, Seattle, WA (protocol number PR-001) and conducted in accordance with the relevant guidelines and regulations. All relevant procedures were performed under anesthesia and every effort was made to minimize pain and suffering. Female BALB / cAnNHsd mice (Envigo) with an average body weight of 18 grams were used for experiments at 5-7 weeks of age. To generate A20 allografts, mice were inoculated with 1 million A20 cells in a 100 μΐ inoculum volume.

[0564] Intratumoral microinjection

[0565] Intratumoral microinjection of CIVO was performed as described in Klinghoffer et al. (2016) Science Translational Medicine. Briefly, mice (n=6 per time point (4 and 24 hours)) were enrolled in the microinjection study when the implanted tumors reached the following approximate dimensions: 14 mm (length), 10 mm (width), and 7 mm (depth). The CIVO device was configured with 6 30-gauge injection needles for a total delivery volume of 2.0 μΐ. Presage’s fluorescent tracking marker (FTM, 5% by volume) was added to the injection contents for spatial localization. The following doses were microinjected: control PTGFRN++GFP exosomes, PTGFRN++GFP exosomes loaded with ML RR-S2 CDA, PTGFRN++GFP exosomes loaded with ML RR-S2 CDA desialyzed exosomes, native exosomes loaded with ML RR-S2 CDA, all doses at 10 ng / μΐ ML RR-S2 CDA, such that the total delivered amount was 20 ng. Free ML RR-S2 CDA was microinjected at 20 ng and 2 μg. Mice were euthanized using CO2 inhalation at 4 hours and 24 hours post-CIVO microinjection for biomarker analysis.

[0566] Histology, immunohistochemistry, and in situ hybridization

[0567] Excised tumors were cut into 2 mm thick sections perpendicular to the injection column and fixed in 10% buffered formalin for 48 hours. CIVO microinjection was confirmed using UV imaging based on the signal from the FTM injected at each CIVO site. The 2 mm thick tissue sections were then processed for standard paraffin embedding. 4 μιη thick sections were used for all histological analyses described below. Hematoxylin-eosin (H&E) staining was performed using standard methods.

[0568] Immunohistochemistry

[0569] Formalin-fixed paraffin-embedded tumors were cut at a thickness of 4 μιη onto glass slides. Slides were baked at 60°C for 1 hour, deparaffinated in xylene, and rehydrated through graded ethanol.

[0570] Slides were incubated in target retrieval solution at 100°C for 20 minutes, then cooled to room temperature for 20 minutes. Serum blocking (5% normal goat serum in TBST) was performed at room temperature for 1 hour. Primary antibody staining with appropriate primary antibody in 5% NGS TBS diluent was performed at room temperature, overnight. Corresponding isotype controls were included in each batch. Secondary antibody staining with appropriate secondary antibody in 5% NGS TBS diluent was performed at room temperature, overnight. Slides were counterstained with DAPI for 10 minutes and coverslipped with Prolong Gold mounting medium (Invitrogen). Stained slides were imaged with a digital, automated, high-resolution scanner.

[0571] In situ hybridization was performed using the RNAscope Multiplex Fluorescent Kit v2 (Advanced Cell Diagnostics). Formalin-fixed, paraffin-embedded tumors were cut at a thickness of 4 pm onto slides. Slides were baked at 60°C for 1 hour, deparaffinized in xylene, and rehydrated through graded ethanol. Hydrogen peroxide was added and left on for 10 minutes to quench endogenous peroxidase activity. Slides were incubated in target retrieval solution at 100°C for 15 minutes, followed by proteinase digestion at 40°C for 15 minutes. RNAscope ISH assay was performed with mouse Ifnb1 probe (Advanced Cell Diagnostics) and TSA plus Fast Red 5 detection (Perkin Elmer). Slides were counterstained with DAPI for 10 minutes and coverslipped with Prolong Gold mounting medium (Invitrogen). Stained slides were imaged with a digital, automated, high-resolution scanner.

[0572] Whole slide scanning and image analysis

[0573] Images of each cell from each stained tissue section were acquired by digital, automated, high-resolution whole tissue scanning (3D Histech Panoramic 250 Flash). Tumor responses were quantified from image files of each tissue section using the custom CIVO analyzer image analysis platform of Presage. Whole tissue section images captured by the slide scanner were automatically processed by the CIVO analyzer. Each cell from each tissue section was segmented based on nuclear (DAPI) signal and classified as biomarker negative or biomarker positive using Cell Profiler (Broad Institute). After cell segmentation and classification, circular regions of interest (ROIs) were positioned around each microinjection site at each location in each image around the FTM with a maximum ROI radius of no more than 2000 pm. To mitigate the effects of pre-existing necrosis on biomarker measurements, injection sites falling within mostly acellular tumor regions were excluded prior to quantitative analysis.

[0574] Example 1: Exosome-encapsulated STING agonists

[0575] Encapsulation of STING agonists

[0576] ImM STING agonists, including ML RR-S2 CDA ammonium salt (MedChem Express, Cat# HY-12885B) and (3-3cAIMPdFSH; InvivoGen, Cat# tlrl-nacairs), were incubated with purified exosomes (1E12 total particles) in 300 ul of PBS at 37°C overnight. The mixture was then washed twice in PBS and purified by ultracentrifugation at 100,000 x g. Figure 2 ).

[0577] Quantification of cyclic dinucleotide STING agonists

[0578] Sample preparation for LC-MS analysis

[0579] All samples were received in phosphate buffered saline (PBS) buffer or PBS and 5% sucrose. Particle concentration (P / mL) was measured by nanoparticle tracking analysis (NTA) on a NanoSight NS300 prior to analysis. All standards and samples were prepared so that each injection contained virtually the same number of particles. This was achieved by a combination of sample dilution and spiking exosomes in the standards (to achieve a final concentration of 1.0-4.0E+11 P / mL, depending on the initial particle concentration of the sample).

[0580] A standard curve was prepared by spiking known concentrations of STING agonist into PBS buffer, followed by serial dilution to prepare additional standards. Typically, separate standards were prepared such that the final concentration (after all sample preparation steps) was 25 nM, 50 nM, 250 nM, 500 nM, 1250 nM, 2500 nM, and 5000 nM STING agonist. First, 75.0 μL of each appropriately diluted sample and each matrix-matched standard was prepared in separate 1.5 mL microcentrifuge tubes. Next, 25.0 μL of exosome lysis buffer (60 mM Tris, 400 mM GdmCl, 100 mM EDTA, 20 mM TCEP, 1.0% Triton X-100) was added to each tube, then all tubes were vortex mixed and briefly centrifuged for sedimentation. Finally, 1.0 μL of concentrated proteinase K enzyme solution (Dako, ref S3004) was added to each tube, all tubes were vortexed again, then briefly centrifuged, followed by incubation at 55 °C for 60 minutes. Samples were allowed to cool to room temperature before injection on LC-MS, and were transferred to HPLC vials.

[0581] LC-MS analysis

[0582] Twenty 20.0 μL of standards and samples were injected neat into an UltiMate 3000 RSC Lnano (Thermo Fisher Scientific) low flow chromatography system without clean-up. Separation of the analytes was performed using a Phenomenex Kinetex EVO C18 core-shell analytical column (50 x 2.1 mm, 2.6 μm particle size, at a flow rate of 500 μL / min delivering a gradient of mobile phase A (MPA: water, 0.1% formic acid) and mobile phase B (MPB: acetonitrile, 0.1% formic acid). The gradient started at 2% MPB, held for 2 minutes to load the STING agonist analytes and to desalt them. The MPB percentage was then increased from 2% to 30% over 3 minutes to elute the STING agonist analytes. Then, the MPB percentage was increased from 30% to 95% over 1 minute, held at 95% for 3 minutes, decreased from 95% to 2% over 1 minute, then held at 2% for another 3 minutes to re-equilibrate the column. The total run time of the method was 13 minutes, with LC flow only flowing into the MS between 2.5 and 4.5 minutes. Typical carry-over was less than 0.05% of the peak area of the previous injection, so no blank injections were performed between analysis injections.

[0583] Mass analysis was performed with a Q Extractive Basic (Thermo Fisher Science) mass spectrometer with an Ion Max source and HESI-II probe operated in negative ion mode and using a full MS-SIM mode scan from 500 Da to 800 Da to collect mass spectra with an AGC target of 1E+6 ions, a maximum injection time of 200 ms, and a resolution of 35,000. STING agonist quantitation was performed using the monoisotopic-1 STING agonist peak by selectively extracting all ions in the m / z range of 688.97 Da to 689.13 Da and then integrating the resulting peak over a retention time of 3.80 min to 3.90 min. The concentration of STING agonist in a given sample was determined by comparing the STING agonist peak area in that sample to the STING agonist peak area generated from a standard, which is a typical relative quantitation.

[0584] Example 2: Increased potency of STING agonists loaded in exosomes

[0585] Exosome-encapsulated and free STING agonists were tested for activity in human peripheral blood mononuclear cells (PBMCs). PBMCs were isolated from fresh human blood by centrifugation at 1000 x g over a layer of Lymphoprep for 15 min. The resulting buffy coat was washed in PBS and counted. PBMCs were plated in 96-well U-bottom plates. Separate U-bottom plates were prepared with titrations of exosome-encapsulated (Exo-STING agonist) or free STING agonist for dose-response studies. Exosome-encapsulated or free STING agonist was added to the PBMCs and incubated overnight at 37 °C. The overall activation of PBMCs by STING agonists was detected by measuring the amount of IFNβ in the supernatant. As shown in Figure 3 , both free STING agonist and Exo-STING agonist induced maximal IFNβ to a similar extent. Interestingly, the EC 50 for Exo-STING agonist was approximately 65-fold lower than for free STING agonist, indicating that exosomes can increase the potency of STING agonist activity. To understand which cell types in the PBMCs were being differentially affected by Exo-STING agonist, the activation of monocytes and dendritic cells was measured. As shown in Figure 4 , maximal monocyte activation was attenuated in Exo-STING agonist-treated cells, while the EC 50 for free STING agonist was improved. In contrast, maximal activation of myeloid dendritic cells (mDCs) was higher, while the EC 50 for Exo-STING agonist was also improvedFigure 7A ). Upon treatment with free STING agonist or Exo-STING agonist, mDC and monocyte activation was measured in PBMCs from 13 donors, with significantly higher maximum mDC activation and significantly attenuated maximum monocyte activation compared to free STING agonist (Figure 5). These results indicate that only a fraction of myeloid dendritic cells are activated in the context of PBMCs. This result is saturable, exemplifying the limitation of STING agonist alone, as additional compound does not increase the amount of activation. In contrast, exosome-encapsulated STING agonist activates a significantly greater proportion of myeloid dendritic cells. Monocytes are robustly activated by STING agonist alone, with over 90% of monocytes activated at micromolar concentrations of agonist. However, exosome-encapsulated STING agonist results in significantly smaller proportion of monocytes being activated. Given that monocytes are much more abundant in circulation than myeloid dendritic cells, the lower activation of monocytes by exosome-encapsulated STING agonist compared to equivalent amounts of free compound can result in reduced systemic inflammation. Furthermore, initiation of adaptive immune responses against tumors is largely dependent on activation of dendritic cells; therefore, exosome-encapsulated STING agonist will likely result in enhanced anti-tumor immune responses with reduced toxicity compared to the compound alone.

[0586] To understand the extent to which different immune cell types are activated by STING agonist, specific activation of T cells, B cells, and NK cells was assessed by measuring the amount of CD69 on the cell surface by flow cytometry. Activation of antigen presenting cells (APCs), including monocytes, myeloid dendritic cells, plasmacytoid dendritic cells, and B cells, was assessed by measuring the amount of CD80, CD86, HLA-DR, or CD83 on the cell surface by flow cytometry. As shown in Figure 6, free STING agonist readily activated monocytes, NK cells, B cells, and CD8 T cells from two different donors. In contrast, Exo-STING agonist reduced activation of B cells and T cells while preserving activation of antigen presenting cells Figure 7B and Figure 2 ). These results indicate that antigen presenting cells can be specifically activated by exosomes loaded with STING agonist while reducing activation of T cells and B cells, which can limit systemic toxicity.

[0587] Example 3: Enhancement of STING exosome activity by PTGFRN overexpression and exosome glycan modification

[0588] The results in Examples 1 and 2 indicate that the exosome surface molecules can mediate an increase in the potency of the Exo-STING agonist compared to the free STING agonist. Previous results indicate that prostaglandin F2 receptor negative regulator (PTGFRN) is a rich glycoprotein located on the luminal or outer surface of exosomes. PTGFRN is the major glycoprotein on the luminal or outer surface of exosomes when overexpressed in producer cells. To determine if PTGFRN plays a role in mediating the activation of immune cells by Exo-STING agonists, exosomes with modified glycan profiles or engineered to express higher levels of PTGFRN were compared to free STING agonists. Similar to the results in Figure 8A , the Exo-STING agonists were more potent than the free STING agonists in inducing IFN production without changing the maximum level of IFN production in PBMCs. Loading of STING into exosomes that were first deglycosylated by PNGase F further enhanced this shift in potency, while delivery of STING agonists in exosomes that were first sialidated with sialidase resulted in further enhancement of potency and higher maximum levels of IFN production, indicating that glycan modifications of exosomes can alter the delivery of STING agonist molecules to immune cells. Surprisingly, exosomes overexpressing PTGFRN and loaded with STING agonists further enhanced the potency and maximum production of IFN compared to unmodified or glycan-engineered exosomes containing endogenous levels of PTGFRN. Further enhancement of potency by sialidation or deglycosylation of PTGFRN Exo-STING samples exceeded the effect of exosomes overexpressing PTGFRN alone Figure 8B and Figure 9 in two donors). Quantification of the levels of IFN as a result of STING agonist delivery demonstrated that glycan-modified exosomes overexpressing PTGFRN can make the potency of STING agonists more than 1000-fold greater than the free STING agonist and about 50-fold greater than the STING agonist loaded in unmodified exosomes Figure 9 .

[0589] Figure 9 The results in Example 3 indicate that the combination of glycan engineering and PTGFRN overexpression on exosomes can enhance the delivery of STING agonist molecules to immune cells. To understand the effects of these modifications on activation of specific cell types in PBMCs, the activation of monocytes and dendritic cells by the Exo-STING agonist formulations shown in Figure 11 were tested. Figure 10 demonstrates that glycan modification and / or PTGFRN overexpression of exosomes loaded with STING agonists resulted in enhanced potency of monocyte activation (as measured by EC 50but the maximal level of activation was reduced or unchanged (Figure 10). In comparison to free STING agonist, the sialidase-treated PTGFRN-overexpressing exosomes significantly enhanced the EC 50 of monocyte activation by up to 54,000-fold Figure 13 . In contrast, in both donors, the activation of mDCs by free STING agonist was poor, while glycan-engineered and / or PTGFRN-overexpressing exosomes significantly enhanced the EC 50 and maximal activation of mDCs (Figure 12). The EC 50 of mDCs was greater than 16,000-fold Figure 14 , while the maximal activation was about 4-10-fold for sialidase-treated PTGFRN-overexpressing STING agonist exosomes. Importantly, the effects observed in these experiments were not due to improved loading efficiency of PTGFRN-overexpressing or glycan-engineered exosomes, as the STING agonist quantification by LC-MS described above allowed for normalization of STING agonist in the exosome preparations. In fact, the loading efficiency on a per particle basis was lower for PTGFRN-overexpressing and / or glycan-engineered exosomes than for unmodified exosomes Figure 18A . Taken together, these results indicate that specific glycan modifications and / or overexpression of a single exosome surface protein can significantly enhance the potency of STING agonist-loaded exosomes and enhance the selectivity of cargo delivery to dendritic cells.

[0590] Pretreatment with a guanidinium agent that modulates STING activation

[0591] To determine whether any perturbation of exosome surface glycans could alter the uptake by immune cells, the producer cell line was treated with the alkaloid agent, guanidinium, which prevents trimming of high mannose residues during protein glycosylation and prevents complete glycosylation. The resulting exosomes from guanidinium-treated cells altered the glycosylation state and were enriched in high mannose. Guanidinium-treated exosomes were loaded with STING agonist and administered to PBMCs from two donors. This resulted in a partial attenuation of STING agonist activity in comparison to wild-type exosomes. Specifically, there was essentially no change in the activation of monocytes and mDCs (see Figures 16 and 17, respectively), while IFN production was significantly reduced (Figure 15). These results indicate that specific glycosylation patterns mediate the uptake of exosomes by immune cells at least in part, and that not all modifications of surface glycoproteins can enhance the activation of immune cells during exosome-mediated delivery of STING agonist molecules.

[0592] Example 4: Optimization of exosome loading with STING agonists

[0593] In the previous examples, exosomes were loaded with STING agonists by incubation overnight at 37°C. To determine the kinetics of STING agonist loading, exosomes were incubated with 1 mM STING agonist for 2 hours, 6 hours, or overnight, and added to PBMCs to measure IFN production. As shown in FIG. 1A, un-loaded exosomes failed to induce IFN production, while exosomes incubated in STING agonist for 2 hours either failed to induce IFN production or resulted in relatively low levels. Loading samples for 6 hours resulted in intermediate production of IFN, while overnight loading resulted in the highest levels of IFN production in both donors. These results indicate that loading of STING agonists into exosomes can be increased by increasing the incubation time. Figure 18B and Figure 19 As shown in FIG. 1A, un-loaded exosomes failed to induce IFN production, while exosomes incubated in STING agonist for 2 hours either failed to induce IFN production or resulted in relatively low levels. Loading samples for 6 hours resulted in intermediate production of IFN, while overnight loading resulted in the highest levels of IFN production in both donors. These results indicate that loading of STING agonists into exosomes can be increased by increasing the incubation time.

[0594] Example 5: Comparison of potency of different exosome-encapsulated STING agonist cyclic dinucleotides

[0595] HEK293SF cells overexpressing PTGFRN were grown in shake flasks as described in the Methods, and the resulting exosomes were purified by Optiprep TM density gradient ultracentrifugation. Purified exosomes were loaded with either STING agonist ML RR-S2CDA (MedChem Express, Catalog # HY-12885B) or 3-3cAIMPdFSH (InvivoGen, Catalog # tlrl-nacairs) as described in Example 1. Loading was quantified as described in Example 1. Exosome-encapsulated or free STING agonists were added to human PBMCs and incubated overnight at 37°C. Activation of PBMCs by STING agonists was detected by measuring the amount of IFN in the supernatant. As shown in FIG. 2A, both free STING agonists induced IFN to a similar extent, while both exosome-encapsulated STING agonists resulted in a change in potency as shown in Example 2. However, exosome-encapsulated 3-3cAIMPdFSH was more potent than exosome-encapsulated ML RR-S2CDA, indicating that fluorinated STING agonists can provide a potency advantage when delivered in an exosome formulation. Figure 20A As shown in FIG. 2A, both free STING agonists induced IFN to a similar extent, while both exosome-encapsulated STING agonists resulted in a change in potency as shown in Example 2. However, exosome-encapsulated 3-3cAIMPdFSH was more potent than exosome-encapsulated ML RR-S2CDA, indicating that fluorinated STING agonists can provide a potency advantage when delivered in an exosome formulation.

[0596] Example 6: In vivo potency and systemic effects of free STING agonists compared to exosome-encapsulated STING agonists in tumor-bearing mice

[0597] Tumor-bearing mice were injected with 5 x 107 5Four groups of C57BL / 6 mice (3-4 mice per group) were subcutaneously inoculated with 1 x 105B16F10 tumor cells. Eight days post-inoculation, mice were injected with a single intratumoral dose of PBS, 20 pg free ML RR-S2CDA, 0.2 pg free ML RR-S2CDA, or 0.2 pg ML RR-S2CDA loaded in exosomes overexpressing PTGFN (exo ML RR-S2CDA). Four hours post-injection, tumors, draining lymph nodes, spleens, and serum were collected and cytokine levels were measured. IFNp gene expression levels in the tumors were comparable in the 20 pg free STING agonist and 0.2 pg exosome-STING agonist groups, both of which were higher than the 0.2 pg free STING agonist and PBS groups Figure 20B ). Additionally, levels of IFNy and the T cell chemoattractants CXCL9 and CXCL10 were higher in the exosome-STING agonist group Figure 20C 、 Figure 20D and Figure 21A . These data indicate that 100-fold less STING agonist can induce comparable IFN gene expression signatures in the tumor when the STING agonist is encapsulated in exosomes.

[0598] STING agonists are very potent pro-inflammatory molecules, and one potential clinical disadvantage of these compounds is their induction of systemic toxicity due to the escape of free compounds from the tumor injection site and diffusion into the circulation. Draining lymph nodes of tumor-bearing mice treated with exosome-STING agonists showed comparable or slightly elevated IFNp Figure 21B ), CXCL9 Figure 21C , and CXCL10 Figure 22A gene expression compared to the free STING agonist groups that were matched for concentration, but significantly lower expression levels compared to the 100-fold free STING agonist treated group. These results were more pronounced in the spleen Figure 22B 、 Figure 22C and Figures 23A-23E and serum Figure 23A . Serum showed significantly lower levels of the pro-inflammatory cytokines IFNp Figure 23B , TNF-a Figure 23C , and IL-6 Figures 24A-24D in the exosome-STING agonist group compared to either of the free STING agonist groups.

[0599] To confirm that the effects observed in FIGS. 20-23 apply to other STING agonists, B16F10 subcutaneously tumor-bearing mice were injected with 20 pg free 3-3cAIMPdFSH, 0.2 pg free 3-3cAIMPdFSH, or 0.2 pg 3-3cAIMPdFSH loaded in PTGFRN-overexpressing exosomes (exo 3-3cAIMPdFSH). Tumor Figures 25A-25D ), draining lymph nodes Figures 26A-26D ), spleen Figures 27A-27D ), and serum Figure 29A ) showed similar expression patterns to those shown in FIGS. 20-23 for ML RR-S2 CDA. These results collectively indicate that exosome-encapsulated STING agonists are able to induce a potent IFN gene expression signature comparable to 100-fold more free STING agonist after intratumoral injection in vivo, and that this comparable gene expression pattern is largely confined to the tumor microenvironment and does not produce systemic inflammatory signals as observed for free STING agonists. Additionally, these effects were observed for two different STING agonists, demonstrating the broad applicability of using exosomes to deliver STING agonists to tumors.

[0600] Example 7: Comparison of local and systemic activation of the STING pathway after intratumoral and intraperitoneal administration of free and exosome-encapsulated STING agonists in tumor-bearing mice

[0601] Five groups of C57BL / 6 mice (n = 4 mice / group) were subcutaneously inoculated with 5 x 10 5 B16F10 murine melanoma cells. Eight days post-inoculation, mice were injected intraperitoneally (IP) with a single dose of PBS, 20 pg ML RR-S2, 0.2 pg ML RR-S2, 0.2 pg ML RR-S2 loaded in PTGFRN-overexpressing exosomes (Exo STING IP), or intratumorally (IT) injected with a single dose of 0.2 pg ML RR-S2 loaded in PTGFRN-overexpressing exosomes (Exo STING IT). Exosome-encapsulated STING agonist formulations were loaded and quantified as described in Example 1. The high-dose free STING agonist injected IP induced higher IFN expression in the tumor, pancreas, and spleen than the PBS-treated group. At 100-fold lower dose than the high-dose free STING, Exo STING IP resulted in higher IFN expression in the pancreas Figure 30A ) and spleen Figure 28A ) and lower IFN expression in the tumor Figure 28B). CXCL9 and CXCL10 expression was similar between the two groups in the lung ( Figure 30B -C) and spleen ( Figure 29B -C), but expression in the pancreas ( Figures 31A-31C -C) was enhanced in the Exo STING IP group. Exo STING IT showed much stronger activation of the STING pathway in the tumor compared to the other groups, but did not result in strong expression changes in the spleen compared to Exo STING IP or high dose free STING agonist groups, and showed similar expression in the pancreas compared to high dose free STING agonist. In the pancreas and spleen, activation of the STING pathway was consistently enhanced by Exo STING IP compared to the low dose free STING agonist at the same concentration, confirming the enhanced potency of the exosome-encapsulated STING agonist. Importantly, Exo STING IP resulted in comparable or in some cases enhanced potency in the pancreas and spleen compared to 100-fold dose of free STING agonist. These results indicate that regional IP administration of exosomes loaded with STING agonist can induce a potent immune response in tissues including the pancreas, providing an opportunity for regional administration to treat pancreatic and other peritoneal cancers.

[0602] Example 8: Differential STING pathway signaling in naive mice with exosome-encapsulated STING agonist and free STING agonist

[0603] Naive C57BL / 6 mice were injected intraperitoneally (IP) with a single dose of PBS, 20 pg ML RR-S2, 0.2 pg ML RR-S2, or 0.2 pg ML RR-S2 loaded in exosomes overexpressing PTGFRN (Exo STING), which were formulated and quantified as described in Example 1 (n=5 mice / group). Four hours post-injection, lungs, spleens, pancreas, and serum were isolated and analyzed for gene expression and cytokine production. Expression of IFNb, CXCL9, and CXCL10 was significantly higher in the lungs ( Figures 32A-32C ) and spleens ( Figures 33A-33C ) of Exo STING-treated mice compared to mice receiving 100-fold dose of free STING agonist, while the pancreas gene expression profile was similar between the two groups ( Figure 34A ). Similarly, serum cytokine levels were higher or equal in Exo STING-treated mice compared to mice treated with 100-fold dose of free STING agonist ( Figure 35- G). Taken together, these results indicate that exosomes loaded with STING agonists are significantly more potent activators of the STING pathway in vivo compared to an equivalent amount of free STING agonist, and thus exosome-loaded STING agonists can provide differential therapeutic applications, particularly in reducing systemic toxicity of high doses of free STING agonists and enhancing the expression of T cell chemoattractants.

[0604] A second experiment similar to the previous study was performed with a single dose IP administration and extended to 24 hours. Peritoneal and splenic cells were isolated from treated mice and cell activation was measured by detection of CD86. High doses of free STING agonist resulted in activation of peritoneal B cells, macrophages, monocytes, and conventional dendritic cells (cDCs), while a 100-fold lower dose of Exo STING induced greater activation of macrophages, similar activation of cDCs, and attenuated activation of B cells and monocytes Figure 36 ). In the spleen, high doses of STING agonist induced moderate levels of immune cell activation, while a 100-fold lower dose of Exo STING induced greater activation of macrophages and T cells, and significantly greater activation of cDCs, indicating a cell type uptake / delivery preference for exosomes in vivo in CdCs and macrophages Figure 37A . These results indicate that exosomes loaded with STING agonists can induce specific cellular responses in antigen presenting cells that are the primary mediators of STING pathway-induced anti-tumor and anti-pathogenic responses in vivo.

[0605] Example 9: Comparison of in vivo efficacy of exosomes loaded with STING agonists and free STING agonists in a mouse melanoma model

[0606] The results of the foregoing examples indicate that Exo STING can be a more potent anti-tumor agent compared to an equivalent or greater amount of soluble STING agonist. To verify this hypothesis, C57BL / 6 mice were subcutaneously inoculated with 5 x 10 5 B16F10 murine melanoma cells (n = 5 mice / group). On days 5, 8, and 11 post-inoculation, mice were injected intratumorally with PBS, 20 pg ML RR-S2, 0.2 pg ML RR-S2, or 0.2 pg ML RR-S2 loaded in exosomes overexpressing PTGFRN. Tumor volume was measured daily until day 39, when tumor volume reached 2000 mm 3Animals were sacrificed at the time. Tumor growth was moderately enhanced after treatment with 0.2 pg of free STING agonist compared to PBS control group, and almost completely eliminated after treatment with 20 pg of free STING agonist on day 25. Surprisingly, treatment with 0.2 pg of Exo STING resulted in significantly improved tumor regression compared to the concentration-matched free STING agonist group, and reached a similar degree as the high dose free STING agonist group on day 25. It is noteworthy that treatment with Exo STING and high dose free STING agonist resulted in complete response (defined as no detectable tumor at the inoculation site) in 3 out of 5 animals in each group. Figure 37A -E). Figure 37B Average tumor growth in animal groups is shown, Figure 38A -D shows tumor growth in individual mice in each treatment group.

[0607] STING pathway activation leads to recruitment of memory T cells and ultimately results in a durable adaptive immune response. To determine whether the anti-tumor effect in this study resulted in an immune response, five animals in the high dose STING agonist group and Exo STING group were re-challenged on day 21 by implanting 5x10 5 PBS to ensure cell viability and growth kinetics. By day 39 (18 days post-challenge), all mice in the PBS group were sacrificed. Tumors in 4 out of 5 animals from the high dose free STING agonist group did not grow, while strikingly, no tumor growth was detected in all 5 mice in the Exo STING group Figure 38A -D). Figure 38B Average tumor growth in animal groups is shown, Figure 38C Tumor growth in individual mice is shown. Figure 37A Survival rate for each treatment group is shown. It is noteworthy that although two animals in the Exo STING group were refractory to treatment of the primary tumor, these animals did not exhibit tumor growth at the re-challenge site, demonstrating the robustness of the immune response mediated by STING agonist loaded in exosomes Figure 38A -E and Figure 39 -C).

[0608] Example 10: Dose-dependent anti-tumor response of STING agonist loaded exosomes in a murine melanoma model

[0609] Results in Example 9 indicate that Exo STING can induce anti-tumor effects in vivo to a degree similar to 100-fold doses of free STING agonists. To determine the relationship between injected dose of Exo STING and tumor growth, an in vivo dose titration experiment was performed. C57BL / 6 mice were subcutaneously inoculated with 5 x 105B16F10 murine melanoma cells (n = 5 mice / group). Six, nine, and twelve days after inoculation, mice were intratumorally injected with PBS and 200 ng, 40 ng, or 8 ng of ML RR-S2 loaded in PTGFRN-overexpressing exosomes. Tumor volume was measured daily until day 18, when animals were sacrificed when tumor volume reached 2000 mm3. By day 18, four of the five mice in the PBS control group were sacrificed, while none of the Exo STING-treated mice in any group were sacrificed during the course of the study. There were two complete responses in the 200 ng Exo STING group and one complete response in the 40 ng Exo STING group. Surprisingly, tumor growth was significantly reduced in the 8 ng Exo STING group compared to the PBS group, indicating that very low doses of Exo STING can have a measurable pharmacological impact in an aggressive tumor model. 5 3 Figure 40A and Figure 39 -D). ​ Average tumor growth in animal groups is shown, Figure 40A -D shows tumor growth of individual mice in each treatment group. Low nanogram doses of STING agonists are unlikely to induce the deleterious systemic toxicities observed for higher doses (10-100 micrograms), and thus can be an attractive opportunity for combination therapy with other oncology or immuno-oncology agents (e.g., therapeutic antibodies against PD-1, PD-L1, and / or CTLA-4). Notably, the tumor growth curves for the 200 ng and 40 ng Exo STING groups are comparable, indicating that intermediate doses can be sufficient to induce a durable immune response, and that Exo STING can provide a therapeutic opportunity for intratumoral injection that reduces the dose of STING agonist by 100-fold to 1,000-fold.

[0610] Example 11: Induction of antigen-specific T cell responses by free STING agonists and STING agonist-loaded exosomes

[0611] Agonism of the STING pathway in dendritic cells enhances antigen presentation, IFN production, and recruitment of CD8+ memory T cells to elicit a durable adaptive immune response. To determine whether Exo STING can induce a memory T cell response to a defined antigen, an antigen-specific T cell response study was performed using purified ovalbumin (OVA). Figure 41A ​​The diagram shows a schematic of the experimental review. C57BL / 6 mice were intraperitoneally injected with 200 μg OVA (n = 4-10 mice / group) mixed with PBS, 20 μg ML RR-S2, 0.2 μg ML RR-S2, or 0.2 μg ML RR-S2 loaded in an exogenous body overexpressing PTGFRN. Six days post-injection, spleens and mesenteric lymph nodes were collected, homogenized into single-cell suspensions, and viable lymphocytes were enriched by density centrifugation. Flow cytometry was used to measure the binding of OVA peptides SIINFEKL and phycoerythrin (PE) (iTAg tetramer / PE–H-2OVA). Lymphocytes isolated using a fixed tetramer MHC class I binding assay (code T03000). OVA-responsive memory T cells were quantified by gating against PE, CD44, and CD8 positivity. Compared to PBS, low-dose free STING, and native exogenous cells, the high-dose free STING agonist and ExoSTING groups showed significantly increased activity in the spleen (…). Figure 41B ) and mesenteric lymph nodes ( Figure 41C A larger proportion of OVA-responsive T cells were detected in the exosome. Low-dose STING agonists matched to ExoSTING concentrations showed no activity in the spleen and only a moderate response in the mesenteric lymph nodes, indicating a significant increase in the potency of STING agonists loaded in the exosome. Mice treated with the unmodified exosome did not show an immune response, indicating that the exosome alone was non-immunogenic during the experimental time course.

[0612] As an orthogonal method for measuring antigen-specific immunity, ELISpot (based on a standard protocol) is used. Cellular Technology Limited measured IFNγ expression. Spleen cells were homogenized into a single-cell suspension and plated (200,000 cells / well) onto plates coated with anti-IFNγ antibody. The OVA peptide SIINFEKL was added to the cells and incubated for 18 hours to induce IFNγ production. Cells were washed from the plates, and the amount of IFNγ bound to the plates was detected using an orthogonal antibody. Figure 41D ).use The software (Cellular Technology Limited) counted the total number of reaction spots on each plate and compared them between groups. PBS, exogenous vesicles (EVs) alone, and the low-dose STING agonist group showed extremely low levels of OVA responsiveness. Both the high-dose STING agonist and ExoSTING groups were highly reactive, although the STING agonist dose was 100-fold lower in these groups, the ExoSTING group showed even higher responsiveness. Figure 41EThese results suggest that ExoSTING could be a differential therapeutic opportunity to elicit an immune response in oncology and infectious disease applications.

[0613] Example 12: Antitumor efficacy and antigen-specific immune response in a mouse T-cell lymphoma model

[0614] The in vivo efficacy results shown in Examples 9 and 10 and the induction of an immune response shown in Example 11 demonstrate that ExoSTING is sufficient to induce an antigen-specific tumor-killing response and subsequent in vivo immune response. To verify this hypothesis, C57BL / 6 mice were subcutaneously inoculated with 1x10 6 E.G7-OVA cells ( CRL-2113 TM (A mouse T-cell lymphoma cell line engineered to stably express OVA and allowing modeling of antigen-specific T-cell responses in mice) (n = 5 mice / group). Mice were intratumorally injected with PBS, 20 μg ML RR-S2, 0.2 μg ML RR-S2, or 0.2 μg ML RR-S2 loaded in exogenous form overexpressing PTGFRN at days 10, 13, and 16 post-inoculation. Similar to the effects observed in the B16F10 model (Figures 37-38, Example 9), low-dose free STING agonists moderately attenuated tumor growth compared to the PBS group, while high-dose free STING agonists and Exo STING significantly inhibited tumor growth. Figure 42 and 43A -D). Figure 42 The average tumor growth in the animal group was shown. Figure 43A -D indicates tumor growth in individual mice within each treatment group. As described in Example 11, splenic T cells were isolated from all groups, and OVA-specific responsiveness was measured. Low doses of free STING agonists induced a moderate memory T cell response, while high doses of both free STING agonists and ExoSTING induced a highly efficient memory T cell response. Figure 43E These data indicate that STING agonists loaded in exogenous bodies can simultaneously induce antitumor and memory T cell responses in vivo that are comparable to those induced by 100-fold greater free compounds.

[0615] Example 13: Compared with the natural exogenous form, the exogenous form overexpressing PTGFRN enhanced the stability of the STING agonist.

[0616] As described in Example 1, exogenous bodies from HEK293SF cells (natural exo STING) and HEK293SF cells overexpressing PTGFRN-GFP (PTGFRN exo STING) were loaded into ML RR-S2, purified, and quantified. Fresh samples of natural exo STING and PTGFRN exo STING induced similar IFNβ levels in single-donor PBMCs, and both provided a potency enhancement higher than that of free STING agonists. Figure 44A Aliquots of the exogenous STING agonist formulation were frozen at -80°C for 7 days, thawed, and added to PBMCs. PTGFRN exo STING induced an IFNβ production profile similar to the fresh formulation, while natural exo STING induced an inactive IFNβ expression profile, in which C... max Significantly reduced ( Figure 44B The potency loss of PTGFRN exo STING is moderate compared to that of natural exo STING. Figure 44C ).

[0617] Fresh and frozen formulations of PTGFRN exoSTING were incubated with PBMCs, and cellular uptake profiles of DCs, NK cells, and monocytes were measured using cell-specific surface markers and GFP positivity. Fresh ( Figures 45A-45B ) and frozen ( Figures 45C-45D The absorption curves of PTGFRN exo-STING showed no difference, indicating that a freeze-thaw cycle would not disrupt the absorption of the exosome. These results suggest that PTGFRN overexpression may be more suitable for the long-term storage and formulation of therapeutic exosomes loaded with STING agonists.

[0618] Example 14: Induction of protective immunity and reduction of metastasis by intratumoral administration of exogenous bodies loaded with STING agonists

[0619] As demonstrated in Examples 11 and 12, activation of the STING pathway promotes antigen presentation and induces a durable T-cell response. Therefore, after local application to primary tumors, EXOSTING... TM The induced immune memory response may be sufficient to prevent tumor metastasis. To verify this hypothesis, as described in Example 1, exogenous cells purified from HEK293SF cells overexpressing PTGFRN were loaded with cyclic dinucleotide 3-3cAIMPdFSH. On day 0, 1x10 6 C57BL / 6 mice were subcutaneously inoculated with 1 x 10 B16F10 melanoma cells and treated with 1 x 10 5Additional tail vein injections of B16F10 melanoma cells were used to challenge lung metastases (n = 8 mice / group). At days 5, 8, and 11 post-inoculation, mice were intratumorally injected with PBS, 20 μg 3-3cAIMPdFSH, 120 ng 3-3cAIMPdFSH, or 120 ng, 12 ng, or 1.2 ng of 3-3cAIMPdFSH (Exo STING agonist) loaded in an exogenous form overexpressing PTGFRN at the subcutaneous tumor site. By day 17, primary tumors showed no growth in the 20 μg STING agonist and 120 ng Exo STING agonist groups. A dose-response relationship was observed in the 12 ng and 1.2 ng Exo STING agonist groups, but tumor regression was not observed in the PBS or 120 ng STING agonist groups. Figure 46A Lungs of all mice were collected, imaged, and metastases were counted. Lung metastases were significantly reduced in the 120 ng and 12 ng Exo STING agonist groups and the 20 μg STING agonist group compared to the PBS injection group. As little as 12 ng of STING agonist provided the same level of protection against lung metastases as 20 μg of STING agonist. Figure 46B and Figure 47 Interestingly, when assessed histologically, the 20 μg STING agonist treatment group showed a significant amount of pulmonary lesions within the lungs, while the 120 ng and 12 ng Exo STING agonist groups each had 4 complete responses. Figure 48 These data suggest that exogenously encapsulated STING agonists can induce tumor-protective immunity at much lower doses (approximately 1,000 times) compared to free STING agonists.

[0620] Example 15: Synergistic effect of exogenous body-mediated STING agonist delivery and immune checkpoint blockade immunotherapy, and T cell-mediated tumor killing.

[0621] Activation of the STING pathway induces upregulation of immune pathway checkpoints, which subsequently reduces T cell-mediated cell killing, thereby mitigating the effects of STING pathway activation as a therapeutic principle (Cell Rep. 2015 May 19; 11(7):1018-30). Therefore, combining inhibitors of immune checkpoint regulation to further enhance immune-mediated clearance of tumor cells could be beneficial. To verify this hypothesis, as described in Example 1, exogenous material purified from HEK293SF cells overexpressing PTGFRN was loaded with cyclic dinucleotide MLRR-S2 CDA. C57BL / 6 mice were subcutaneously inoculated with 1x10 6B16F10 melanoma cells (n = 6 mice / group). At 5, 8, and 11 days post-inoculation, 30 ng of MLRR-S2 CDA (exosting agent) loaded with PTGFRN-overexpressing exogenous variants was injected intratumorally with or without injection. TM In the case of [unspecified condition], mice were injected intraperitoneally with either a control antibody (α-IgG; 10 mg / kg; BioLegend, catalog number 400559, clone RTK3758) or a PD-1 antagonist antibody 1 (αPD-1; 10 mg / kg; BioLegend, catalog number 114111, clone RMPI-14). B16F10 tumor immune cell infiltration was poor, and checkpoint blockade was difficult to achieve. 30 ng EXOSTING TM The suboptimal dose resulted in partial tumor reduction, and treatment with αPD-1 instead of αIgG amplified this effect. Figure 49A ).

[0622] In a separate study, C57BL / 6 mice were subcutaneously inoculated with 1x10 6 B16F10 melanoma cells (n = 6 mice / group). Mice were intraperitoneally injected with IgG (10 mg / kg) or anti-CD8 antibody (10 mg / kg) at 5, 8, 11, and 14 days post-inoculation. Mice were then treated intratumorally with exogenous or ExoSTING (3-3cAIMPdFSH, 100 ng) at 6, 9, and 12 days post-IP antibody administration.

[0623] Mice were treated with αCD8 antibody (10 mg / kg; BioLegend, catalog number 100769, clone 53-6.7) that depleted T cells, and then... Figure 49B The diagram shown illustrates intratumoral application of EXOSTING. TM (3-3cAIMPdFSH). Systemic depletion of T cells completely eliminated exosting. TM The effect of CD8 proves + T cells mediate exosting TM The key role of STING agonist-induced antitumor activity ( Figure 49B ).

[0624] In separate studies, C57BL / 6 mice (n=5 at each time point) were treated intratumorally with PBS (day 8 only), 0.2 μg MLRR-S2 CDA (days 5 and 8), 20 μg MLRR-S2 CDA (days 5 and 8), and 0.2 μg exoSTING (days 5 and 8). Forty-eight hours after injection on day 8, tumors and spleens were isolated using Miltenyi mouse digestion kits (catalog numbers 130-096-730 and 130-095-926, respectively) on a gentleMACS instrument according to the manufacturer's recommended protocol, and dissociated into single-cell suspensions. Cells were filtered, washed twice, and then subjected to flow cytometry analysis or ELISPOT culture to detect specific reactivity to antigens against B16F10 tumor cells. ELISPOT was performed using Mabtech mouse IFNγELISpot PLUS (HRP) according to the manufacturer's protocol. In short, 5x10 5 Each splenocyte was incubated with 10 μg / ml of three B16F10 peptides (i.e., GP100 amino acids 25-33 (AnaSpec, catalog number AS-62589), tyrosinase amino acids 368-376 (AnaSpec, catalog number AS-61456), and TRP2 amino acids 180-188 (AnaSpec, catalog number AS-61058)). Figure 49C As shown, compared with high-dose or low-dose free STING agonists, 200 ng of EXOSTING... TM Induced significantly more IFNγ-positive spots targeting the B16F10 peptide. In summary, these data suggest that T cells are key mediators of STING agonist-induced antitumor immunity, and that EXOSTING... TM It provides better activity than free STING agonists, either as a single agent or in combination with checkpoint blockade agents.

[0625] Example 16: The level of exogenous PTGFRN is associated with the potency of exogenous substances loaded with STING agonists.

[0626] The results in Examples 3 and 13 showed that PTGFRN overexpression enhanced the activity of exosomes loaded with the STING agonist. To determine whether PTGFRN levels are related to exosting... TMActivity-associated, HEK293SF cells were genetically engineered using CRISPR / Cas9 to delete the endogenous PTGFRN locus (as described in International Patent Application No. PCT / US2018 / 048026). Exogenous bodies were purified from WT HEK293SF cells (WT Exo), PTGFRN-overexpressing HEK293SF cells (PTGFRN O / E Exo), and PTGFRN knockout cells (PTGFRN KO Exo), and loaded with 3-3cAIMPdFSH as described above. All exostings showed improved activity compared to soluble 3-3cAIMPdFSH. TM The formulations were all potent activators of IFNβ production in PBMC cultures (n = 2 replicates). Interestingly, PTGFRN O / E exosting TM It is the most potent activator of IFNβ and leads to exosting. TM The maximum C of the formulation max With PTGFRN O / E EXOSTING TM In comparison, WT EXOSTING TM It was weakened, and PTGFRN KO EXOSTING TM This results in the mildest IFNβ response. Figure 50A The maximum IFNβ signal is also correlated with PTGFRN levels. Figure 50B To determine whether this difference in potency is consistent in the in vivo tumor environment, PBS or 20 ng WTEXOSTING were used. TM PTGFRN O / E EXOSTING TM Or PTGFRN KO EXOSTING TM Inject B16F10 into subcutaneous tumors (on days 6, 9, and 12). EXOSTING TM The extent to which treatments reduced tumor growth was also correlated with PTGFRN expression levels, indicating that elevated PTGFRN levels can induce a more favorable anti-tumor immune response, thus exosting... TM Therapeutic formulations can be optimized by increasing the expression of PTGFRN on the surface of exogenous organisms. Figure 50C ).

[0627] Example 17: Exogenous bodies loaded with the STING agonist were phagocytosed by antigen-presenting cells and showed no toxicity to tumor-resident immune effector cells.

[0628] Constitutive activation of the STING pathway leads to robust pro-inflammatory signaling and can be toxic to cells and tissues (N Engl J Med. 2014 Aug 7; 371(6):507–518). Non-selective delivery of STING agonists to the tumor microenvironment can result in a robust IFNβ response, but if the response is too strong or originates from unwanted cell populations, effector cells such as CD8+ may become toxic. + T cells can be killed or weakened in other ways. (Using Alexa Fluor) TM Exogenous PTGFRN overexpressing at 488 nm was injected into B16F10 melanoma tumors and removed 1 hour post-injection. Tumor-infiltrating lymphocytes were purified, and their fluorescence was measured at 488 nm to track exogenous uptake. Only about 20% of T cells phagocytosed the exogenous phagocytosis, while approximately 90% and 70% of macrophages and dendritic cells, respectively, phagocytosed the exogenous phagocytosis. Figure 51A These data suggest that antigen-presenting cells in the tumor microenvironment are natural target cells for human exogenous antibodies. To determine whether cell-specific uptake of exogenous antibodies leads to exosting... TM To compare the differential STING pathway activation with that of free STING agonists, a second injection was performed from the above-mentioned B16F10 melanoma tumors using PBS, 20 μg of free MLRR-S2 CDA, 0.2 μg of free MLRR-S2 CDA, or 200 ng of MLRR-S2 CDA loaded in exogenous bodies overexpressing PTGFRN. Twenty-four hours post-injection, the tumors were isolated, homogenized, and subjected to live CD45 assays. + Cell populations were counted. In the 20 μg ML RR-S2 CDA group, CD8... + T cells, macrophages, and dendritic cells were significantly reduced compared to the other groups. Figure 51B -D). These data indicate that high doses of free STING agonists can be toxic to antigen-presenting cells and T cells in the tumor microenvironment, which are precisely the cells required for antigen presentation and tumor cell killing. Therefore, the non-selective release of high doses of STING agonists can attenuate the desired immunostimulatory response. Since lower doses are required for a comparable therapeutic response, EXOSTING... TM It can exert its effects within a wider therapeutic window and reduce the adverse effects observed with free STING agonists (e.g., systemic toxicity, immune cell killing, lack of cell selectivity).

[0629] Example 18: High-resolution imaging of exogenous bodies loaded with STING agonists administered intratumorally showed increased potency and reduced toxicity compared to free STING agonists.

[0630] EXOSTING shown in the previous embodimentsTM Activity measurements indicated that exogenous isolates, particularly those overexpressing PTGFRN, enhanced the activity of the STING agonist molecule. Numerous measurements from homogenized tissue or isolated serum provided information on exosting. TM Meaningful data on efficacy and selectivity in various applications are available, but direct comparisons between samples from the same tumor or between local effects at the injection site are not permitted. To answer this question, a multi-injector device is used. Presage Biosciences (Seattle, WA) conducted a micro-dose intratumoral injection study. As described above, A20 lymphoma cells were subcutaneously implanted into mice, and up to six different agents were simultaneously injected. Single-dose injections were performed using 2 μg of free MLRR-S2 CDA, 200 ng of MLRR-S2 CDA, exogenous cells overexpressing PTGFRN, wild-type exogenous cells containing 20 ng of MLRR-S2 CDA, or exogenous cells overexpressing PTGFRN containing 20 ng of MLRR-S2 CDA. Tumors were collected at 4 and 24 hours post-injection, processed, and stained for the presence of IFNβ mRNA (via in situ hybridization), and caspase 3 protein was cleaved (Jackson Immunoresearch, antibody number 111-605-144). At 4 hours post-injection, the high-dose STING agonist group was treated with PTGFRN O / E EXOSTING. TM The IFNβ levels were comparable across groups, but significantly higher than in the low-dose free STING agonist group or the empty exogenous body group. Figure 52A By 24 hours post-treatment, IFNβ signaling had returned to baseline. Compared to all other groups, high-dose free STING agonists resulted in a significant increase in caspase 3 (CC3), a marker of apoptosis, at 4 and 24 hours, while exosting... TM The low-dose free STING agonist group showed a slight increase, which suggests a difference compared to EXOSTING. TM In contrast, high doses of free STING agonists led to more apoptosis and did not provide any benefit in enhancing IFNβ production. Figure 52B These data, combined with the selective cell type uptake described in Example 17, demonstrate that EXOSTING TM Selective targeting of immune cells leads to enhanced IFNβ secretion, without the non-selective cell killing observed with free STING agonists.

[0631] In another study, a single-dose injection was administered into A20 tumors using 2 μg of free 3-3-cAIMPdFSH, 20 ng of free 3-3-cAIMPdFSH, 0.4 ng, 2.2 ng, 6.6 ng, or 20 ng of 3-3-cAIMPdFSH loaded in exogenous bodies overexpressing PTGFN. Tumors were collected 4 hours post-injection, processed, and stained for the presence of IFNβ or CXCL10 mRNA (by in situ hybridization) and analyzed for radioactivity. When injected into the sample, IFNβ ( Figure 52C ) or CXCL10 ( Figure 52D mRNA expression was highest and gradually decreased with increasing radial distance.

[0632] Example 19: Comparison of the potency of STING agonists encapsulated by different exogenous entities in cyclic or acyclic dinucleotides

[0633] HEK293SF cells overexpressing PTGFRN were grown in shake flasks as described in the method, and then processed using Optiprep. TM Density gradient ultracentrifugation was used to purify the obtained exogenous body. The purified exogenous body was loaded with a STING agonist, comprising ML RR-S2 CDA, 2-3cGAMP, 3-3cAIMPdFSH, 3-3cAIM(PS)2, cAIMPmFSH, cAIMPdF, cAIMP, CP214, CP201, and CP204, according to the method in Example 1. 3-3cAIMPdFSH, 3-3cAIM(PS)2, cAIMPdF, and cAIMP correspond to compounds 53, 13, 52, and 51, respectively, in the paper (J Med Chem. 23 Nov 2016; 59(22):10253-10267). CP214 is 2-3cAMPmFSH. CP201 ​​and CP204 are analogues of compounds in patents WO2017 / 175156 and WO2017 / 175147, respectively. The loading was quantified as described in Example 1. Exogenous or free STING agonists were added to human PBMCs and incubated overnight at 37°C. The activation of PBMCs by the STING agonists was detected by measuring the amount of IFNβ in the supernatant. Figure 53A As shown in Example 2, all exogenously encapsulated STING agonists caused changes in potency compared to free STING agonists.

[0634] Example 20: In tumor-bearing mice (C57BL / 6) and STING knockout mice (C57BL / 6-Tmem173) gtIn this study, the in vivo potency of free STING agonists compared to exogenously encapsulated STING agonists was compared.

[0635] Three groups of C57BL / 6 mice and C57BL / 6-Tmem173 were given gt Mice (4-5 mice per group) were subcutaneously injected with 1x10 6 B16F10 tumor cells were collected. Eight days after inoculation, mice were injected with a single intratumoral dose of PBS, 20 μg of free 3-3cAIMPdFSH, or 0.1 μg of 3-3cAIMPdFSH (exeSTING) loaded in an exogenous form overexpressing PTGFN. Four hours after injection, tumors, draining lymph nodes, spleen, and serum were collected, and cytokine levels were measured. In the 20 μg free STING agonist and 0.1 μg exogenous STING agonist groups, the tumors of C57BL / 6 mice (solid strips) ( Figure 54A ), draining lymph nodes ( Figure 54B ) and spleen ( Figure 54C The expression levels of the IFNβ gene in C57BL / 6-Tmem173 are comparable, while those in C57BL / 6-Tmem173 are significantly different. gt Tumors in mice (hollow strips) Figure 54A ), draining lymph nodes ( Figure 54B ) and spleen ( Figure 54C The IFNβ gene expression level in the control group was similar to that in the control group. Additionally, in the exogenous STING agonist group of C57BL / 6 mice (solid strips), the levels of IFNγ and T cell chemical inducers CXCL9 and CXCL10 were both higher, but in C57BL / 6-Tmem173... gt In mice (hollow strips), the exogenous STING agonist group was not induced (Figs. 55, 56, and 57). Serum cytokine profiles, in addition to gene expression, showed the same trend (Fig. 58).

[0636] To confirm that the effects observed in Figures 54-58 translate into antitumor activity, C57BL / 6 mice and C57BL / 6-Tmem173 were administered the drugs. gt Mice subcutaneously injected with 1x10 6 Five B16F10 mouse melanoma cells (n = 5 mice / group) were injected intratumorally into mice at days 7, 10, and 13 post-inoculation. The cells were administered PBS, an exogenous cell line, 20 μg of free 3-3cAIMPdFSH, or 0.1 μg of 3-3cAIMPdFSH loaded in an exogenous cell line overexpressing PTGFRN. Tumor volume was measured daily until day 19, when the tumor volume reached 2000 mmHg. 3 The animals were euthanized. As expected, 0.1 μg of exosting was used. TMTreatment with 20 μg of free 3-3cAIMPdFSH induced significantly improved tumor regression in C57BL / 6 mice. Figure 59 However, in any process, C57BL / 6-Tmem173 gt No tumor regression was observed in mice. Figure 59 In summary, these data indicate that the activity of exogenous STING agonists is mediated by the STING pathway.

[0637] Example 21: Comparison of in vivo efficacy of exogenous and free STING agonists in an advanced mouse melanoma model.

[0638] Previous data on B16F10 tumors (Figures 37, 40) Figure 47 Figures 49, 50 and Figure 60 This indicates that the exogenous bodies loaded with the STING ...

Claims

1. A composition comprising an extracellular vesicle, the extracellular vesicle comprising a stimulator of interferon genes (STING) agonist, wherein, The extracellular vesicle is an exosome, and the STING agonist is a cyclic dinucleotide selected from the group consisting of: wherein: X1is H, OH, or F; X2is H, OH, or F; Z is OH, OR1, SH, or SR1, wherein: i) R1is Na or NH4, or ii) R1is neopentyl oxymethyl; B1and B2are bases selected from: with the proviso that: - in Formula (I): X1and X2are not OH, - in Formula (II): when X1and X2are OH, B1is not adenine and B2is not guanine, or a pharmaceutically acceptable salt thereof, wherein the STING agonist is encapsulated within the extracellular vesicle, and wherein the extracellular vesicle is produced by a cell that overexpresses a prostaglandin F2 receptor negative regulator (PTGFRN) protein.

2. The composition of claim 1, wherein the extracellular vesicle is glycan-modified.

3. The composition of claim 1, wherein the extracellular vesicle is desialidated.

4. The composition of claim 1, wherein the extracellular vesicle is deglycosylated.

5. The composition of claim 1, wherein the extracellular vesicle further comprises a protein that binds or enzymatically reacts with the STING agonist.

6. The composition of claim 1, wherein the extracellular vesicle further comprises a ligand, a cytokine, or an antibody.

7. The composition of claim 6, wherein the ligand comprises CD40L, OX40L, and / or CD27L.

8. The composition of claim 6, wherein the cytokine comprises IL-7, IL-12, and / or IL-15.

9. The composition of claim 6, wherein the antibody comprises an antagonistic antibody and / or an agonistic antibody.

10. The composition of claim 1, wherein the concentration of the STING agonist associated with the extracellular vesicle is 0.01 pm to 100 pm.

11. The composition of claim 10, wherein the concentration of the STING agonist associated with the extracellular vesicle is 0.01 pM to 0.1 pM, 0.1 pM to 1 pM, 1 pM to 10 pM, 10 pM to 50 pM, or 50 pM to 100 pM.

12. The composition of claim 11, wherein the concentration of the STING agonist associated with the extracellular vesicle is 1 pM to 10 pM.

13. The composition of claim 1, wherein the STING agonist is selected from the group consisting of: and pharmaceutically acceptable salts thereof.

14. The composition of claim 1, wherein the STING agonist is or a pharmaceutically acceptable salt.

15. The composition of claim 13 or 14, wherein the STING agonist is located in the lumen of the extracellular vesicle and is not attached to a scaffold moiety.

16. A pharmaceutical composition comprising the composition of claim 1 and a pharmaceutically acceptable carrier.

17. A kit comprising the composition of any one of claims 1 to 15 and instructions for use.

18. A method of producing exosomes comprising a STING agonist, the method comprising: a. obtaining exosomes from cells overexpressing Prostaglandin F2 Receptor Negative Regulator (PTGFRN); b. mixing the exosomes with a STING agonist in a solution, wherein the STING agonist is a cyclic dinucleotide; c. incubating the mixture of exosomes and STING agonist in a solution comprising a buffer under suitable conditions; and d. purifying the exosomes comprising the STING agonist, wherein the cyclic dinucleotide is selected from the group consisting of: wherein; X1is H, OH, or F; X2is H, OH, or F; Z is OH, OR1, SH, or SR1, wherein: i) R1is Na or NH4, or ii) R1is pivaloyloxymethyl; B1and B2are bases selected from: with the proviso that: - in Formula (I): X1and X2are not OH, - in Formula (II): when X1and X2are OH, B1is not adenine and B2is not guanine, or a pharmaceutically acceptable salt thereof.

19. The method of claim 18, wherein the suitable conditions comprise incubating the exosomes and the STING agonist for 2 to 24 hours.

20. The method of claim 18 or 19, wherein the suitable conditions comprise incubating the exosomes and the STING agonist at 15-90 °C.

21. The method of claim 20, wherein the suitable conditions comprise incubating the exosomes and the STING agonist at 37 °C.

22. The method of claim 18 or 19, wherein the amount of the STING agonist in the mixing step is 0.01 mM to 100 mM.

23. The method of claim 18 or 19, wherein the amount of the STING agonist in the mixing step is 1 mM to 10 mM.

24. The method of claim 18 or 19, wherein the amount of the exosome in the mixing step is at least 10 16 total particles.

25. The method of claim 18 or 19, wherein the amount of the exosome in the mixing step is at least 10 12 total particles.

26. The method of claim 18 or 19, wherein the buffer comprises phosphate buffered saline PBS.

27. The method of claim 18 or 19, wherein the purifying step comprises one or more centrifugation steps.

28. The method of claim 27, wherein the one or more centrifugation steps are performed at 100,000 x g.

29. Use of a composition of any one of claims 1 to 15 or a pharmaceutical composition of claim 16 in the manufacture of a medicament for (i) inducing or modulating an immune response and / or an inflammatory response in a subject in need thereof; and / or (ii) treating a tumor in a subject in need thereof.

30. The use of claim 29, wherein the medicament is configured for parenteral or oral administration.

31. The use of claim 30, wherein the medicament is configured for intravenous administration.

32. The use of claim 30, wherein the medicament is configured for intratumoral administration in a first tumor in one site, and wherein the medicament administered in the first tumor prevents metastasis of one or more tumors in a second site.

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