WO2022133301A1

WO2022133301A1 - Platforms, compositions, and methods for therapeutic delivery

Info

Publication number: WO2022133301A1
Application number: PCT/US2021/064186
Authority: WO
Inventors: Charles Cameron TAYLOR; Milad Riazifar; Todd SCHURR
Original assignee: Entelexo Biotherapeutics, Inc.
Priority date: 2020-12-18
Filing date: 2021-12-17
Publication date: 2022-06-23

Abstract

Described herein are compositions comprising an extracellular vesicle comprising at least one engineered Lysosomal Associated Membrane Protein 2 (LAMP2) comprising at least one immune checkpoint moiety. Also described herein are methods for utilizing the compositions described herein for delivery of therapeutics.

Description

PLATFORMS, COMPOSITIONS, AND METHODS FOR THERAPEUTIC DELIVERY

CROSS-REFERENCE

[001] This application claims benefit of US Provisional Application Serial Number 63/127,827 filed on December 18, 2020; US Provisional Application Serial Number 63/197,220 filed on June 4, 2021; and US Provisional Application Serial Number 63/220,226 filed on July 9, 2021, the entireties of which are hereby incorporated by reference herein.

INCORPORATION BY REFERENCE

[002] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

BACKGROUND

[003] Effective means of delivering therapeutics to a target cell is one of the cornerstones of modern medicine. Extracellular vesicles have been explored and utilized as carriers for delivering therapeutics to a target cell for treating diseases or disorders.

SUMMARY

[004] Current methods of delivering therapeutics via extracellular vesicles have drawbacks. There are no reliable methods for using extracellular vesicles for delivering a combination of different therapeutics to a cell for treating a disease or a disorder. There are also no methods for screening and validating the therapeutic effects of the combination of the different therapeutics. Accordingly, there remains a need for compositions and methods for delivering the combination of different therapeutics for treating a disease or a disorder.

[005] Described herein, in some aspects, is a composition comprising at least one extracellular vesicle, said at least one extracellular vesicle comprising at least one engineered Lysosomal Associated Membrane Protein 2 (LAMP2), wherein the at least one engineered LAMP2 comprises at least one immune checkpoint moiety. Described herein, in some aspects, is a composition comprising at least one extracellular vesicle, said at least one extracellular vesicle comprising at least one engineered Lysosomal Associated Membrane Protein 2 (LAMP2), wherein the at least one engineered LAMP2 comprises at least one immune checkpoint moiety, wherein said immune checkpoint moiety is flanked by at least a fragment of the engineered LAMP2, wherein the fragment comprises the N-terminus homology arm of the engineered LAMP2. In some embodiments, the immune checkpoint moiety comprises a polypeptide sequence that is at least 70% identical to VISTA, PD-L1, CTLA-4, PD-L2, B7-1 (CD80), B7-2 (CD86), B7-H3 (CD276), B7-H2, B7-H4 (VTCN1), HVEM (CD270, TNFRSF14), Galectin 9, Galectin3, CEACAM1 (CD66a), OX-2 (CD200), PVR (CD155), PVRL2 (Nectin-2, CD112), FGL-1, PECAM-1, TSG-6, CD47, Stabilin-1 (Clever-1), Neuropilin 1, Neuropilin 2, CD158 (family), IGSF2 (CD101), CD155, GITRL, CD137L, OX40L, LIGHT, CD70, PD-1, RGMB, CTLA-4 (CD 152), BTLA, CD 160, Tim-3, CD200R, TIGIT, CD112R (PVRIG), LAG-3 (CD223), PECAM-1, CD44, SIRP alpha (CD172a), IGSF11 (VSIG-3), or a combination thereof. In some embodiments, the immune checkpoint moiety comprises a polypeptide sequence that is at least 70% identical to VISTA, PD-L1, IGSF11 (VSIG-3), CTLA-4, OX-2 (CD200), or BTLA. In some embodiments, the immune checkpoint moiety comprises PD-L1. In some embodiments, the immune checkpoint moiety comprises IGSF11 (VSIG-3). In some embodiments, the at least one extracellular vesicle comprises a first immune checkpoint moiety comprising PD-L1 a second immune checkpoint moiety comprising IGSF11 (VSIG-3). In some embodiments, the immune checkpoint moiety comprises a polypeptide sequence that is at least 70% identical to PD-L1. In some embodiments, the immune checkpoint moiety comprises a polypeptide sequence that is at least 99% identical to PD-L1. In some embodiments, the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NOs: 7-9 or 24. In some embodiments, the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 70% identical to SEQ ID NOs: 7-9 or 24. In some embodiments, the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 99% identical to SEQ ID NOs: 7-9 or 24. In some embodiments, the immune checkpoint moiety comprises a polypeptide sequence that is at least 70% identical to IGSF11 (VSIG-3). In some embodiments, the immune checkpoint moiety comprises a polypeptide sequence that is at least 99% identical to IGSF11 (VSIG-3). In some embodiments, the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NOs: 10-12 or 26. In some embodiments, the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 70% identical to SEQ ID NOs: 10-12 or 26. In some embodiments, the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 99% identical to SEQ ID NOs: 10-12 or 26. In some embodiments, the immune checkpoint moiety comprises a polypeptide sequence that is at least 70% identical to VISTA. In some embodiments, the immune checkpoint moiety comprises a polypeptide sequence that is at least 99% identical to VISTA. In some embodiments, the immune checkpoint moiety comprises a polypeptide sequence that is at least 70% identical to CTLA-4. In some embodiments, the immune checkpoint moiety comprises a polypeptide sequence that is at least 99% identical to CTLA-4. In some embodiments, the immune checkpoint moiety comprises a polypeptide sequence that is at least 70% identical to OX-2 (CD200). In some embodiments, the immune checkpoint moiety comprises a polypeptide sequence that is at least 99% identical to OX-2 (CD200). In some embodiments, the immune checkpoint moiety comprises a polypeptide sequence that is at least 70% identical to BTLA. In some embodiments, the immune checkpoint moiety comprises a polypeptide sequence that is at least 99% identical to BTLA. In some embodiments, the engineered LAMP2 comprises engineered LAMP2A, LAMP2B, LAMP2C, or a combination thereof. In some embodiments, the engineered LAMP2 is the engineered LAMP2B. In some embodiments, the at least one extracellular vesicle comprises a targeting moiety. In some embodiments, the targeting moiety is covalently connected to the N-terminus of the at least one engineered LAMP2B. In some embodiments, the targeting moiety comprises a polypeptide that is at least 70% identical to an integrin comprising a6 1, a6 4, aL 2, aMp2, aXp2, or aDp2. In some embodiments, the targeting moiety comprises a polypeptide that is at least 70% identical to a single chain fragment variable (scFv) antibody. In some embodiments, the at least one extracellular vesicle comprises a signaling peptide. In some embodiments, the immune checkpoint moiety is covalently connected to the at least one engineered LAMP2B via a linker, said linker comprises a flexible linker, a rigid linker, or a cleavable linker. In some embodiments, the flexible linker comprises a polypeptide sequence comprising: (GGGGS)n where n=l, 2, 3, or 4; KESGSVSSEQLAQFRSLD; EGKSSGSGSESKST; GGGGGG; GGGGGGGG; GSAGSAAGSGEF; RKRR; SS; or LE. In some embodiments, the rigid linker comprises a polypeptide sequence comprising: (EAAAK)_n where n=l, 2, 3, or 4; A(EAAAK)_nA where n=2, 3, 4, or 5; A(EAAAK)4ALEA(EAAAK)₄A; AEAAAKEAAAKA; or LEAGCKNFFPRSFTSCGSLE. In some embodiments, the cleavable linker comprises a polypeptide sequence comprising: LEAGCKNFFPRSFTSCGSLE; CRRRRRREAEAC; GGIEGRGS; or TRHRQPRGWEQL. In some embodiments, the at least one engineered LAMP2B comprises an amino acid sequence GNSTM at N-Terminus of the at least one engineered LAMP2. In some embodiments, the at least one extracellular vesicle comprises a plurality of engineered LAMP2Bs. In some embodiments, the plurality of the engineered LAMP2Bs comprises a single species of the immune checkpoint moiety. In some embodiments, the plurality of the engineered LAMP2Bs comprises two or more species of the immune checkpoint moiety. In some embodiments, the two or more species of the immune checkpoint moiety comprises a combination of any two or more of PD-L1, CTLA-4, IGSF11 (VSIG-3), VISTA, OX-2 (CD200), or BTLA. In some embodiments, the two or more species of the immune checkpoint moiety are present on the extracellular vesicle at a ratio. In some embodiments, the composition comprises a plurality of extracellular vesicles, where the plurality of the extracellular vesicles comprises: at least a first population of the extracellular vesicles; and at least one additional population of the extracellular vesicles, wherein said first population of the extracellular vesicles comprises at least a first species immune checkpoint moiety and the at least one additional population of the extracellular vesicles comprises at least a second immune checkpoint moiety. In some embodiments, the extracellular vesicle comprises an exosome, a microvesicle, a retrovirus-like particle, an apoptotic body, an apoptosome, an oncosome, an exopher, an enveloped virus, an exomere, or a very large extracellular vesicle. In some embodiments, the extracellular vesicle is the exosome. In some embodiments, the at least one engineered LAMP2B comprises a homology arm comprising a nucleic acid sequence that is at least 70% identical to SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 21, or SEQ ID NO: 22 In some embodiments, the at least one engineered LAMP2B comprises an N-terminus homology arm comprising a nucleic acid sequence that is at least 70% identical to SEQ ID NO: 2. In some embodiments, the at least one engineered LAMP2B comprises an N-terminus homology arm comprising a nucleic acid sequence that is at least 70% identical to SEQ ID NO: 21. In some embodiments, the at least one engineered LAMP2B comprises a C-terminus homology arm comprising a nucleic acid sequence that is at least 70% identical to SEQ ID NO: 3. In some embodiments, the at least one engineered LAMP2B comprises a C-terminus homology arm comprising a nucleic acid sequence that is at least 70% identical to SEQ ID NO: 22. In some embodiments, the composition further comprises a fusogenic moiety. In some embodiments, the fusogenic moiety comprises a viral fusogenic moiety. In some embodiments, the fusogenic moiety comprises a mammalian fusogenic moiety. In some embodiments, the at least one extracellular vesicle further comprises an immune evasion moiety. In some embodiments, the immune evasion moiety comprises CD47. In some embodiments, the composition described herein does not comprise an enucleated cell. In some embodiments, the composition is derived from a cell. In some embodiments, the composition is cryopreserved. In some embodiments, the composition is lyophilized. In some embodiments, the composition is stable at 37°C for at least one hour.

[006] Described herein, in some aspects, is a cell genetically modified to produce the extracellular vesicle of any one of the preceding claims. In some embodiments, the cell is genetically modified by homologous recombination. In some embodiments, the cell is a stem cell. In some embodiments, the cell is a human cell. In some embodiments, the cell is a nonhuman cell. In some embodiments, the cell is a mesenchymal stem cell.

[007] Described herein, in some aspects, is a pharmaceutical composition comprises the composition described herein or the cell described herein. In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition comprises at least one additional active agent. In some embodiments, the pharmaceutical composition is formulated for administering intrathecally, intraocularly, intravitreally, retinally, intravenously, intramuscularly, intraventricularly, intracerebrally, intracerebellarly, intracerebroventricularly, intraperenchymally, subcutaneously, or a combination thereof.

[008] Described herein, in some aspects, is a kit comprising the composition described herein, the cell described herein, or the pharmaceutical composition described herein.

[009] Described herein, in some aspects, is a platform comprising components for generating the composition described herein, the cell described herein, or the pharmaceutical composition described herein 1, or the kit described herein.

[0010] Described herein, in some aspects, is a method of suppressing CD8+ CD25+ cells in a subject in need thereof, the method comprises administering the composition described herein. [0011] Described herein, in some aspects, is a method for generating the extracellular vesicle of any one of the preceding claims, said method comprising: contacting a cell with a polynucleotide encoding the immune checkpoint moiety; inducing homologous recombination in the cell, wherein the polynucleotide encoding the immune checkpoint moiety is inserted at a locus of the engineered LAMP2B, wherein the locus is flanked by one or both homology arms of the engineered LAMP2B; and harvesting the extracellular vesicle produced by the cell, wherein said extracellular vesicle comprises the engineered LAMP2B comprising the immune checkpoint moiety of any one of the preceding claims.

[0012] Described herein, in some aspects, is a method of treating an autoimmune disease, the method comprises administering the composition described herein, the cell described herein, or the pharmaceutical composition described herein 1, or the kit described herein to a subject in need thereof. In some embodiments, said autoimmune disease is Rheumatoid arthritis, Systemic lupus erythematosus, Psoriasis, Type 1 diabetes mellitus, Multiple sclerosis, Inflammatory bowel disease, Celiac disease, Crohn’s disease, Graves’ disease, Juvenile arthritis, Lyme disease chronic, Optic neuritis, Psoriatic arthritis, Scleritis, Scleroderma, Ulcerative colitis (UC), Uveitis, Sjogren’s syndrome, Inflammatory eye conditions, Idiopathic inflammatory myopathies, Vitiligo, COPD, complication from Organ transplantation, or graft-versus-host disease.

[0013] Described herein, in some aspects, is a method of treating Acute Respiratory Distress Syndrome (ARDS), especially as it pertains to COVID-19 due to unchecked viral lysis of host cells and ensuing cytokine storm, the method comprises administering the composition described herein, the cell described herein, or the pharmaceutical composition described herein 1, or the kit described herein to a subject in need thereof.

[0014] Described herein, in some aspects, is a method of inducing regulatory T-cells (Tregs) in a patient in need thereof, comprises administering the method comprises administering the composition described herein, the cell described herein, or the pharmaceutical composition described herein 1, or the kit described herein to a subject in need thereof.

[0015] Described herein, in some aspects, is a composition comprising at least one extracellular vesicle, said at least one extracellular vesicle comprising at least one engineered Lysosomal Associated Membrane Protein 2 (LAMP2), wherein the at least one engineered LAMP2 comprises at least one immune checkpoint moiety, wherein said immune checkpoint moiety is flanked by at least a fragment of the engineered LAMP2, wherein the fragment comprises the N- terminus homology arm of the engineered LAMP2. In some embodiments, the immune checkpoint moiety comprises a polypeptide sequence that is at least 70% identical to VISTA, PD-L1, CTLA-4, PD-L2, B7-1 (CD80), B7-2 (CD86), B7-H3 (CD276), B7-H2, B7-H4 (VTCN1), HVEM (CD270, TNFRSF14), Galectin 9, Galectin3, CEACAM1 (CD66a), OX-2 (CD200), PVR (CD155), PVRL2 (Nectin-2, CD112), FGL-1, PECAM-1, TSG-6, CD47, Stabilin-1 (Clever- 1), Neuropilin 1, Neuropilin 2, CD158 (family), IGSF2 (CD 101), CD 155, GITRL, CD137L, OX40L, LIGHT, CD70, PD-1, RGMB, CTLA-4 (CD 152), BTLA, CD 160, Tim-3, CD200R, TIGIT, CD112R (PVRIG), LAG-3 (CD223), PECAM-1, CD44, SIRP alpha (CD 172a), IGSF11 (VSIG-3), or a combination thereof. In some embodiments, the immune checkpoint moiety comprises a polypeptide sequence that is at least 70% identical to VISTA, PD-L1, IGSF11 (VSIG-3), CTLA-4, OX-2 (CD200), or BTLA. In some embodiments, the immune checkpoint moiety comprises a polypeptide sequence that is at least 70% identical to VISTA. In some embodiments, the immune checkpoint moiety comprises a polypeptide sequence that is at least 99% identical to VISTA. In some embodiments, the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 4 In some embodiments, the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 70% identical to SEQ ID NO: 4. In some embodiments, the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 99% identical to SEQ ID NO: 4 In some embodiments, the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 5. In some embodiments, the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 70% identical to SEQ ID NO: 5 In some embodiments, the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 99% identical to SEQ ID NO: 5. In some embodiments, the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 6. In some embodiments, the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 70% identical to SEQ ID NO: 6. In some embodiments, the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 99% identical to SEQ ID NO: 6. In some embodiments, the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 25. In some embodiments, the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 70% identical to SEQ ID NO: 25. In some embodiments, the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 99% identical to SEQ ID NO: 25. In some embodiments, the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 33. In some embodiments, the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 70% identical to SEQ ID NO: 33. In some embodiments, the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 99% identical to SEQ ID NO: 33. In some embodiments, the immune checkpoint moiety comprises a polypeptide sequence that is at least 70% identical to PD-L1. In some embodiments, the immune checkpoint moiety comprises a polypeptide sequence that is at least 99% identical to PD-L1. In some embodiments, the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 7. In some embodiments, the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 70% identical to SEQ ID NO: 7. In some embodiments, the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 99% identical to SEQ ID NO: 7. In some embodiments, the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 8. In some embodiments, the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 70% identical to SEQ ID NO: 8. In some embodiments, the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 99% identical to SEQ ID NO: 8. In some embodiments, the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 9. In some embodiments, the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 70% identical to SEQ ID NO: 9. In some embodiments, the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 99% identical to SEQ ID NO: 9. In some embodiments, the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 24. In some embodiments, the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 70% identical to SEQ ID NO: 24. In some embodiments, the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 99% identical to SEQ ID NO: 24. In some embodiments, the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 32. In some embodiments, the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 70% identical to SEQ ID NO: 32. In some embodiments, the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 99% identical to SEQ ID NO: 32. In some embodiments, the immune checkpoint moiety comprises a polypeptide sequence that is at least 70% identical to IGSF11 (VSIG-3). In some embodiments, the immune checkpoint moiety comprises a polypeptide sequence that is at least 99% identical to IGSF11 (VSIG-3). In some embodiments, the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 10. In some embodiments, the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 70% identical to SEQ ID NO: 10 In some embodiments, the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 99% identical to SEQ ID NO: 10. In some embodiments, the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 11. In some embodiments, the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 70% identical to SEQ ID NO: 11 In some embodiments, the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 99% identical to SEQ ID NO: 11. In some embodiments, the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 12. In some embodiments, the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 70% identical to SEQ ID NO: 12. In some embodiments, the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 99% identical to SEQ ID NO: 12. In some embodiments, the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 26. In some embodiments, the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 70% identical to SEQ ID NO: 26. In some embodiments, the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 99% identical to SEQ ID NO: 26. In some embodiments, the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 34 In some embodiments, the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 70% identical to SEQ ID NO: 34. In some embodiments, the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 99% identical to SEQ ID NO: 34 In some embodiments, the immune checkpoint moiety comprises a polypeptide sequence that is at least 70% identical to CTLA-4. In some embodiments, the immune checkpoint moiety comprises a polypeptide sequence that is at least 99% identical to CTLA-4. In some embodiments, the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 13. In some embodiments, the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 70% identical to SEQ ID NO: 13 In some embodiments, the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 99% identical to SEQ ID NO: 13. In some embodiments, the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 14. In some embodiments, the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 70% identical to SEQ ID NO: 14 In some embodiments, the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 99% identical to SEQ ID NO: 14. In some embodiments, the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 15. In some embodiments, the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 70% identical to SEQ ID NO: 15. In some embodiments, the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 99% identical to SEQ ID NO: 15. In some embodiments, the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 27. In some embodiments, the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 70% identical to SEQ ID NO: 27. In some embodiments, the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 99% identical to SEQ ID NO: 27. In some embodiments, the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 35. In some embodiments, the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 70% identical to SEQ ID NO: 35. In some embodiments, the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 99% identical to SEQ ID NO: 35. In some embodiments, the immune checkpoint moiety comprises a polypeptide sequence that is at least 70% identical to OX-2 (CD200). In some embodiments, the immune checkpoint moiety comprises a polypeptide sequence that is at least 99% identical to OX-2 (CD200). In some embodiments, the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 23. In some embodiments, the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 70% identical to SEQ ID NO: 23. In some embodiments, the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 99% identical to SEQ ID NO: 23. In some embodiments, the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 31. In some embodiments, the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 70% identical to SEQ ID NO: 31 In some embodiments, the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 99% identical to SEQ ID NO: 31. In some embodiments, the immune checkpoint moiety comprises a polypeptide sequence that is at least 70% identical to BTLA. In some embodiments, the immune checkpoint moiety comprises a polypeptide sequence that is at least 99% identical to BTLA. In some embodiments, the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 28. In some embodiments, the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 70% identical to SEQ ID NO: 28. In some embodiments, the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 99% identical to SEQ ID NO: 28. In some embodiments, the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 36. In some embodiments, the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 70% identical to SEQ ID NO: 36. In some embodiments, the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 99% identical to SEQ ID NO: 36. In some embodiments, the engineered LAMP2 comprises engineered LAMP2A, LAMP2B, LAMP2C, or a combination thereof. In some embodiments, the engineered LAMP2 is the engineered LAMP2B. In some embodiments, the at least one extracellular vesicle comprises a targeting moiety. In some embodiments, the targeting moiety is covalently connected to the N- terminus of the at least one engineered LAMP2B. In some embodiments, the targeting moiety comprises a polypeptide that is at least 70% identical to an integrin comprising a6pi, a6p4, aLp2, aMp2, aXp2, or aDp2. In some embodiments, the targeting moiety comprises a polypeptide that is at least 70% identical to a single chain fragment variable (scFv) antibody. In some embodiments, the at least one extracellular vesicle comprises a signaling peptide. In some embodiments, the immune checkpoint moiety is covalently connected to the at least one engineered LAMP2B via a linker, said linker comprises a flexible linker, a rigid linker, or a cleavable linker. In some embodiments, the flexible linker comprises a polypeptide sequence comprising: (GGGGS)n where n=l, 2, 3, or 4; KESGSVSSEQLAQFRSLD;

EGKSSGSGSESKST; GGGGGG; GGGGGGGG; GSAGSAAGSGEF; RKRR; SS; or LE. In some embodiments, the rigid linker comprises a polypeptide sequence comprising: (EAAAK)_n where n=l, 2, 3, or 4; A(EAAAK)nA where n=2, 3, 4, or 5; A(EAAAK)4ALEA(EAAAK)4A; AEAAAKEAAAKA; or LEAGCKNFFPRSFTSCGSLE. In some embodiments, the cleavable linker comprises a polypeptide sequence comprising: LEAGCKNFFPRSFTSCGSLE; CRRRRRREAEAC; GGIEGRGS; or TRHRQPRGWEQL. In some embodiments, the at least one engineered LAMP2B comprises an amino acid sequence GNSTM at N-Terminus of the at least one engineered LAMP2. In some embodiments, the at least one extracellular vesicle comprises a plurality of engineered LAMP2Bs. In some embodiments, the plurality of the engineered LAMP2Bs comprises a single species of the immune checkpoint moiety. In some embodiments, the plurality of the engineered LAMP2Bs comprises two or more species of the immune checkpoint moiety. In some embodiments, the two or more species of the immune checkpoint moiety comprises a combination of any two or more of PD-L1, CTLA-4, IGSF11 (VSIG-3), VISTA, OX-2 (CD200), or BTLA. In some embodiments, the two or more species of the immune checkpoint moiety are present on the extracellular vesicle at a ratio. In some embodiments, the plurality of the extracellular vesicles comprises: at least a first population of the extracellular vesicles; and at least one additional population of the extracellular vesicles, wherein said first population of the extracellular vesicles comprises at least a first species immune checkpoint moiety and the at least one additional population of the extracellular vesicles comprises at least a second immune checkpoint moiety. In some embodiments, the extracellular vesicle comprises an exosome, a microvesicle, a retrovirus-like particle, an apoptotic body, an apoptosome, an oncosome, an exopher, an enveloped virus, an exomere, or a very large extracellular vesicle. In some embodiments, the extracellular vesicle is the exosome. In some embodiments, the at least one engineered LAMP2B comprises a homology arm comprising a nucleic acid sequence that is at least 70% identical to SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 21, or SEQ ID NO: 22. In some embodiments, the at least one engineered LAMP2B comprises an N-terminus homology arm comprising a nucleic acid sequence that is at least 70% identical to SEQ ID NO: 2. In some embodiments, the at least one engineered LAMP2B comprises an N-terminus homology arm comprising a nucleic acid sequence that is at least 70% identical to SEQ ID NO: 21. In some embodiments, the at least one engineered LAMP2B comprises a C-terminus homology arm comprising a nucleic acid sequence that is at least 70% identical to SEQ ID NO: 3. In some embodiments, the at least one engineered LAMP2B comprises a C-terminus homology arm comprising a nucleic acid sequence that is at least 70% identical to SEQ ID NO: 22. In some embodiments, the composition further comprises a fusogenic moiety. In some embodiments, the fusogenic moiety comprises a viral fusogenic moiety. In some embodiments, the fusogenic moiety comprises a mammalian fusogenic moiety. In some embodiments, the at least one extracellular vesicle further comprises an immune evasion moiety. In some embodiments, the immune evasion moiety comprises CD47. In some embodiments, the composition described herein does not comprise an enucleated cell. In some embodiments, the composition is derived from a cell. In some embodiments, the composition is cryopreserved. In some embodiments, the composition is lyophilized. In some embodiments, the composition is stable at 37°C for at least one hour.

[0016] Described herein, in some embodiments, is a cell genetically modified to produce an extracellular vesicle described herein. In some embodiments, the cell is genetically modified by homologous recombination. In some embodiments, the cell is a stem cell. In some embodiments, the cell is a human cell. In some embodiments, the cell is a non-human cell. In some embodiments, the cell is a mesenchymal stem cell. [0017] Described herein, in some aspects, is a cell line comprising the cell described herein. [0018] Described herein, in some embodiments, is a pharmaceutical composition comprising the composition described herein, the cell described herein, or the cell line described herein. In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition comprises at least one additional active agent. In some embodiments, the pharmaceutical composition is formulated for administering intrathecally, intraocularly, intravitreally, retinally, intravenously, intramuscularly, intraventricularly, intracerebrally, intracerebellarly, intracerebroventricularly, intraperenchymally, subcutaneously, or a combination thereof.

[0019] Described herein is a kit comprising the composition, the cell, the cell line, or the pharmaceutical composition described herein.

[0020] Described herein is a platform comprising components for generating the composition, the cell, the cell line, the pharmaceutical composition, or the kit described herein.

[0021] Described herein, in some embodiments, is a method for generating the extracellular vesicle of any one of the preceding claims, said method comprising: contacting a cell with a polynucleotide encoding the immune checkpoint moiety; inducing homologous recombination in the cell, wherein the polynucleotide encoding the immune checkpoint moiety is inserted at a locus of the engineered LAMP2B, wherein the locus is flanked by one or both homology arms of the engineered LAMP2B; and harvesting the extracellular vesicle produced by the cell, wherein said extracellular vesicle comprises the engineered LAMP2B comprising the immune checkpoint moiety of any one of the preceding claims.

[0022] Described herein, in some aspects, is a method of treating an autoimmune disease, the method comprises administering the composition, the cell, the cell line, the pharmaceutical composition, or the kit described herein. In some embodiments, the method compromises administering the pharmaceutical composition described herein. In some embodiments, said autoimmune disease is Rheumatoid arthritis, Systemic lupus erythematosus, Psoriasis, Type 1 diabetes mellitus, Multiple sclerosis, Inflammatory bowel disease, Celiac disease, Crohn’s disease, Graves’ disease, Juvenile arthritis, Lyme disease chronic, Optic neuritis, Psoriatic arthritis, Scleritis, Scleroderma, Ulcerative colitis (UC), Uveitis, Sjogren’s syndrome, Inflammatory eye conditions, Idiopathic inflammatory myopathies, Vitiligo, COPD, complication from Organ transplantation, or graft-versus-host disease.

[0023] Described herein, in some aspects, is a method of treating Acute Respiratory Distress Syndrome (ARDS), especially as it pertains to COVID-19 due to unchecked viral lysis of host cells and ensuing cytokine storm, the method comprises administering the composition, the cell, the cell line, the pharmaceutical composition, or the kit described herein. In some embodiments, the method comprises administering the pharmaceutical composition described herein [0024] Described herein is a method of suppressing CD8+ CD25+ cells in a subject in need thereof, the method comprises administering the composition, the cell, the cell line, the pharmaceutical composition, or the kit described herein. In some embodiments, the method comprises administering the pharmaceutical composition described herein.

[0025] Described herein is a method of inducing regulatory T-cells (Tregs) in a patient in need thereof, comprises administering the composition, the cell, the cell line, the pharmaceutical composition, or the kit described herein. In some embodiments, the method comprises administering the pharmaceutical composition described herein.

[0026] Described herein, in some aspects, is a composition comprising a fusion protein or polypeptide, wherein the fusion protein or polypeptide comprises an immune checkpoint moiety and a transmembrane moiety. In some embodiments, the transmembrane moiety is a fragment of the transmembrane moiety. In some embodiments, the immune checkpoint moiety comprises a polypeptide sequence that is at least 70% identical to VISTA, PD-L1, CTLA-4, PD-L2, B7-1 (CD80), B7-2 (CD86), B7-H3 (CD276), B7-H2, B7-H4 (VTCN1), HVEM (CD270, TNFRSF14), Galectin 9, Galectin3, CEACAM1 (CD66a), OX-2 (CD200), PVR (CD 155), PVRL2 (Nectin-2, CD112), FGL-1, PECAM-1, TSG-6, CD47, Stabilin-1 (Clever-1), Neuropilin 1, Neuropilin 2, CD 158 (family), IGSF2 (CD101), CD 155, GITRL, CD137L, OX40L, LIGHT, CD70, PD-1, RGMB, CTLA-4 (CD152), BTLA, CD160, Tim-3, CD200R, TIGIT, CD112R (PVRIG), LAG-3 (CD223), PECAM-1, CD44, SIRP alpha (CD172a), IGSF11 (VSIG-3), or a combination thereof. In some embodiments, the immune checkpoint moiety comprises a polypeptide sequence that is at least 70% identical to VISTA, PD-L1, IGSF11 (VSIG-3), CTLA- 4, OX-2 (CD200), or BTLA. In some embodiments, the immune checkpoint moiety comprises a polypeptide sequence that is at least 70% identical to VISTA. In some embodiments, the immune checkpoint moiety comprises a polypeptide sequence that is at least 99% identical to VISTA. In some embodiments, the fusion protein or polypeptide comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 4. In some embodiments, the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 70% identical to SEQ ID NO: 4. In some embodiments, the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 99% identical to SEQ ID NO: 4. In some embodiments, the immune fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 5. In some embodiments, the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 70% identical to SEQ ID NO: 5 In some embodiments, the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 99% identical to SEQ ID NO: 5. In some embodiments, the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 6. In some embodiments, the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 70% identical to SEQ ID NO: 6 In some embodiments, the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 99% identical to SEQ ID NO: 6. In some embodiments, the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 25. In some embodiments, the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 70% identical to SEQ ID NO: 25. In some embodiments, the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 99% identical to SEQ ID NO: 25. In some embodiments, the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 33. In some embodiments, the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 70% identical to SEQ ID NO: 33. In some embodiments, the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 99% identical to SEQ ID NO: . In some embodiments, the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 70% identical to PD-L1. In some embodiments, the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 99% identical to PD-L1. In some embodiments, the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 7. In some embodiments, the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 70% identical to SEQ ID NO: 7. In some embodiments, the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 99% identical to SEQ ID NO: 7. In some embodiments, the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 8. In some embodiments, the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 70% identical to SEQ ID NO: 8. In some embodiments, the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 99% identical to SEQ ID NO: 8. In some embodiments, the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 9. In some embodiments, the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 70% identical to SEQ ID NO: 9. In some embodiments, the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 99% identical to SEQ ID NO: 9. In some embodiments, the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 24. In some embodiments, the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 70% identical to SEQ ID NO: 24. In some embodiments, the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 99% identical to SEQ ID NO: 24. In some embodiments, the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 32. In some embodiments, the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 70% identical to SEQ ID NO: 32. In some embodiments, the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 99% identical to SEQ ID NO: 32. In some embodiments, the immune checkpoint or polypeptide moiety comprises a polypeptide sequence that is at least 70% identical to IGSF11 (VSIG-3). In some embodiments, the immune checkpoint moiety comprises a polypeptide sequence that is at least 99% identical to IGSF11 (VSIG-3). In some embodiments, the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 10. In some embodiments, the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 70% identical to SEQ ID NO: 10. In some embodiments, the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 99% identical to SEQ ID NO: 10. In some embodiments, the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 11. In some embodiments, the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 70% identical to SEQ ID NO: 11. In some embodiments, the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 99% identical to SEQ ID NO: 11. In some embodiments, the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 12. In some embodiments, the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 70% identical to SEQ ID NO: 12. In some embodiments, the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 99% identical to SEQ ID NO: 12. In some embodiments, the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 26. In some embodiments, the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 70% identical to SEQ ID NO: 26. In some embodiments, the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 99% identical to SEQ ID NO: 26. In some embodiments, the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 34. In some embodiments, the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 70% identical to SEQ ID NO: 34. In some embodiments, the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 99% identical to SEQ ID NO: 34. In some embodiments, the immune checkpoint moiety comprises a polypeptide sequence that is at least 70% identical to CTLA-4. In some embodiments, the immune checkpoint moiety comprises a polypeptide sequence that is at least 99% identical to CTLA-4. In some embodiments, the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 13. In some embodiments, the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 70% identical to SEQ ID NO: 13. In some embodiments, the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 99% identical to SEQ ID NO: 13. In some embodiments, the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 14. In some embodiments, the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 70% identical to SEQ ID NO: 14. In some embodiments, the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 99% identical to SEQ ID NO: 14. In some embodiments, the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 15. In some embodiments, the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 70% identical to SEQ ID NO: 15. In some embodiments, the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 99% identical to SEQ ID NO: 15. In some embodiments, the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 27. In some embodiments, the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 70% identical to SEQ ID NO: 27. In some embodiments, the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 99% identical to SEQ ID NO: 27. In some embodiments, the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 35. In some embodiments, the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 70% identical to SEQ ID NO: 35. In some embodiments, the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 99% identical to SEQ ID NO: 35. In some embodiments, the immune checkpoint moiety comprises a polypeptide sequence that is at least 70% identical to OX-2 (CD200). In some embodiments, the immune checkpoint moiety comprises a polypeptide sequence that is at least 99% identical to OX-2 (CD200). In some embodiments, the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 23. In some embodiments, the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 70% identical to SEQ ID NO: 23. In some embodiments, the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 99% identical to SEQ ID NO: 23. In some embodiments, the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 31. In some embodiments, the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 70% identical to SEQ ID NO: 31. In some embodiments, the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 99% identical to SEQ ID NO: 31 In some embodiments, the immune checkpoint moiety comprises a polypeptide sequence that is at least 70% identical to BTLA. In some embodiments, the immune checkpoint moiety comprises a polypeptide sequence that is at least 99% identical to BTLA. In some embodiments, the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 28. In some embodiments, the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 70% identical to SEQ ID NO: 28. In some embodiments, the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 99% identical to SEQ ID NO: 28. In some embodiments, the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 36. In some embodiments, the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 70% identical to SEQ ID NO: 36. In some embodiments, the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 99% identical to SEQ ID NO: 36. In some embodiments, the transmembrane moiety is an engineered Lysosomal Associated Membrane Protein 2 (LAMP2). In some embodiments, the engineered LAMP2 comprises engineered LAMP2A, LAMP2B, LAMP2C, or a combination thereof. In some embodiments, the engineered LAMP2 is the engineered LAMP2B. In some embodiments, the immune checkpoint moiety is covalently connected to an at least one engineered LAMP2B via a linker, said linker comprises a flexible linker, a rigid linker, or a cleavable linker. In some embodiments, the flexible linker comprises a polypeptide sequence comprising: (GGGGS)n where n=l, 2, 3, or 4; KESGSVSSEQLAQFRSLD; EGKSSGSGSESKST; GGGGGG; GGGGGGGG;

GSAGSAAGSGEF; RKRR; SS; or LE. In some embodiments, the rigid linker comprises a polypeptide sequence comprising: (EAAAK)_n where n=l, 2, 3, or 4; A(EAAAK)_nA, where n=2, 3, 4, or 5; A(EAAAK)4ALEA(EAAAK)₄A; AEAAAKEAAAKA; or LEAGCKNFFPRSFTSCGSLE. In some embodiments, the cleavable linker comprises a polypeptide sequence comprising: LEAGCKNFFPRSFTSCGSLE; CRRRRRREAEAC; GGIEGRGS; or TRHRQPRGWEQL. In some embodiments, the at least one engineered LAMP2B comprises an amino acid sequence GNSTM of N-terminus of the at least one engineered LAMP2. In some embodiments, the at least one engineered LAMP2B comprises an amino acid sequence GNSTM of N-terminus of the at least one immune checkpoint moiety. [0027] Described herein, in some aspects, is a composition comprising at least one extracellular vesicle, said at least one extracellular vesicle comprises at least one polypeptide, and wherein said polypeptide comprises an immune check point moiety that is at least 70% identical to VISTA. In some embodiments, the polypeptide comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 4. In some embodiments, the polypeptide comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 5. In some embodiments, the polypeptide comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 6. In some embodiments, the polypeptide comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 25. In some embodiments, the polypeptide comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 33. In some embodiments, the at least one extracellular vesicle comprises at least one engineered transmembrane moiety. In some embodiments, the engineered transmembrane moiety comprises an engineered LAMP2. In some embodiments, the engineered transmembrane moiety comprises an engineered LAMP2A, LAMP2B, LAMP2C, or a combination thereof. In some embodiments, the engineered LAMP2 is an engineered LAMP2B. In some embodiments, the polypeptide is fused to the engineered transmembrane moiety. In some embodiments, the polypeptide is covalently connected to the N-terminus of the transmembrane moiety. In some embodiments, the extracellular vesicle comprises an exosome, a microvesicle, a retrovirus-like particle, an apoptotic body, an apoptosome, an oncosome, an exopher, an enveloped virus, an exomere, or a very large extracellular vesicle. In some embodiments, the extracellular vesicle is an exosome. [0028] Described herein, in some aspects, is a composition comprising at least one extracellular vesicle, said at least one extracellular vesicle comprises at least one polypeptide, and wherein said polypeptide comprises an immune check point moiety that is at least 70% identical to PD- Ll. In some embodiments, the polypeptide comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 7. In some embodiments, the polypeptide comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 8. In some embodiments, the polypeptide comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 9. In some embodiments, the polypeptide comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 24. In some embodiments, the polypeptide comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 32. In some embodiments, the at least one extracellular vesicle comprises at least one engineered transmembrane moiety. In some embodiments, the engineered transmembrane moiety comprises an engineered LAMP2. In some embodiments, the engineered transmembrane moiety comprises an engineered LAMP2A, LAMP2B, LAMP2C, or a combination thereof. In some embodiments, the engineered LAMP2 is an engineered LAMP2B. In some embodiments, the polypeptide is fused to the engineered transmembrane moiety. In some embodiments, the polypeptide is covalently connected to the N-terminus of the transmembrane moiety. In some embodiments, the extracellular vesicle comprises an exosome, a microvesicle, a retrovirus-like particle, an apoptotic body, an apoptosome, an oncosome, an exopher, an enveloped virus, an exomere, or a very large extracellular vesicle. In some embodiments, the extracellular vesicle is the exosome. [0029] Described herein, in some embodiments, is a composition comprising at least one extracellular vesicle, said at least one extracellular vesicle comprises at least one polypeptide, and wherein said polypeptide comprises an immune check point moiety that is at least 70% identical to IGSF11 (VSIG-3). In some embodiments, the polypeptide comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 10. In some embodiments, the polypeptide comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 11. In some embodiments, the polypeptide comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 12. In some embodiments, the polypeptide comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 26. In some embodiments, the polypeptide comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 34. In some embodiments, the at least one extracellular vesicle comprises at least one engineered transmembrane moiety. In some embodiments, the engineered transmembrane moiety comprises an engineered LAMP2. In some embodiments, the engineered transmembrane moiety comprises an engineered LAMP2A, LAMP2B, LAMP2C, or a combination thereof. In some embodiments, the engineered LAMP2 is an engineered LAMP2B. In some embodiments, the polypeptide is fused to the engineered transmembrane moiety. In some embodiments, the polypeptide is covalently connected to the N-terminus of the transmembrane moiety. In some embodiments, the extracellular vesicle comprises an exosome, a microvesicle, a retrovirus-like particle, an apoptotic body, an apoptosome, an oncosome, an exopher, an enveloped virus, an exomere, or a very large extracellular vesicle. In some embodiments, the extracellular vesicle is the exosome.

[0030] Described herein, in some embodiments, is a composition comprising at least one extracellular vesicle, said at least one extracellular vesicle comprises at least one polypeptide, and wherein said polypeptide comprises an immune check point moiety that is at least 70% identical to CTLA-4. In some embodiments, the polypeptide comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 13. In some embodiments, the polypeptide comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 14. In some embodiments, the polypeptide comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 15. In some embodiments, the polypeptide comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 27. In some embodiments, the polypeptide comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 35. In some embodiments, the at least one extracellular vesicle comprises at least one engineered transmembrane moiety. In some embodiments, the engineered transmembrane moiety comprises an engineered LAMP2. In some embodiments, the engineered transmembrane moiety comprises an engineered LAMP2A, LAMP2B, LAMP2C, or a combination thereof. In some embodiments, the engineered LAMP2 is an engineered LAMP2B. In some embodiments, the polypeptide is fused to the engineered transmembrane moiety. In some embodiments, the polypeptide is covalently connected to the N-terminus of the transmembrane moiety. In some embodiments, the extracellular vesicle comprises an exosome, a microvesicle, a retrovirus-like particle, an apoptotic body, an apoptosome, an oncosome, an exopher, an enveloped virus, an exomere, or a very large extracellular vesicle. In some embodiments, the extracellular vesicle is the exosome.

[0031] Described herein, in some embodiments, is a composition comprising at least one extracellular vesicle, said at least one extracellular vesicle comprises at least one polypeptide, and wherein said polypeptide comprises an immune check point moiety that is at least 70% identical to OX-2 (CD200). In some embodiments, the polypeptide comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 23. In some embodiments, the polypeptide comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 31. In some embodiments, the at least one extracellular vesicle comprises at least one engineered transmembrane moiety. In some embodiments, the engineered transmembrane moiety comprises an engineered LAMP2. In some embodiments, the engineered transmembrane moiety comprises an engineered LAMP2A, LAMP2B, LAMP2C, or a combination thereof. In some embodiments, the engineered LAMP2 is an engineered LAMP2B. In some embodiments, the polypeptide is fused to the engineered transmembrane moiety. In some embodiments, the polypeptide is covalently connected to the N-terminus of the transmembrane moiety. In some embodiments, the extracellular vesicle comprises an exosome, a microvesicle, a retrovirus-like particle, an apoptotic body, an apoptosome, an oncosome, an exopher, an enveloped virus, an exomere, or a very large extracellular vesicle. In some embodiments, the extracellular vesicle is the exosome. [0032] Described herein, in some aspects, is a composition comprising at least one extracellular vesicle, said at least one extracellular vesicle comprises at least one polypeptide, and wherein said polypeptide comprises an immune check point moiety that is at least 70% identical to BTLA. In some embodiments, the polypeptide comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 28. In some embodiments, the polypeptide comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 36. In some embodiments, the at least one extracellular vesicle comprises at least one engineered transmembrane moiety. In some embodiments, the engineered transmembrane moiety comprises an engineered LAMP2. In some embodiments, the engineered transmembrane moiety comprises an engineered LAMP2A, LAMP2B, LAMP2C, or a combination thereof. In some embodiments, the engineered LAMP2 is an engineered LAMP2B. In some embodiments, the polypeptide is fused to the engineered transmembrane moiety. In some embodiments, the polypeptide is covalently connected to the N-terminus of the transmembrane moiety. In some embodiments, the extracellular vesicle comprises an exosome, a microvesicle, a retrovirus-like particle, an apoptotic body, an apoptosome, an oncosome, an exopher, an enveloped virus, an exomere, or a very large extracellular vesicle. In some embodiments, the extracellular vesicle is the exosome.

[0033] Described herein, in some embodiments, is a method for generating the extracellular vesicle described herein, said method comprising: contacting a cell with a polynucleotide encoding a polypeptide comprising an immune checkpoint moiety that is at least 70% identical to VISTA; inducing homologous recombination in the cell, wherein the polynucleotide encoding the polypeptide is inserted at a locus of a transmembrane moiety, wherein the locus is flanked by one or both homology arms of the transmembrane moiety; and harvesting an extracellular vesicle produced by the cell, wherein the extracellular vesicle comprises the extracellular vesicle described herein.

[0034] Described herein is a method of treating an inflammatory disease, the method comprises administering the composition comprising the extracellular vesicle comprising a polypeptide comprising an immune checkpoint moiety that is at least 70% identical to VISTA.

[0035] Described herein is a method of treating an autoimmune disease, the method comprises administering the composition comprising the extracellular vesicle comprising a polypeptide comprising an immune checkpoint moiety that is at least 70% identical to VISTA.

[0036] Described herein is a method for generating the extracellular vesicle described herein, said method comprising: contacting a cell with a polynucleotide encoding a polypeptide comprising an immune checkpoint moiety that is at least 70% identical to PD-L1; inducing homologous recombination in the cell, wherein the polynucleotide encoding the polypeptide is inserted at a locus of a transmembrane moiety, wherein the locus is flanked by one or both homology arms of the transmembrane moiety; and harvesting an extracellular vesicle produced by the cell, wherein the extracellular vesicle comprises the extracellular vesicle described herein. [0037] Described herein is a method of treating an inflammatory disease, the method comprises administering the composition comprising the extracellular vesicle comprising a polypeptide comprising an immune checkpoint moiety that is at least 70% identical to PD-L1.

[0038] Described herein is a method of treating an autoimmune disease, the method comprises administering the composition comprising the extracellular vesicle comprising a polypeptide comprising an immune checkpoint moiety that is at least 70% identical to PD-L1.

[0039] Described herein is a method for generating the extracellular vesicle described herein, said method comprising: contacting a cell with a polynucleotide encoding a polypeptide comprising an immune checkpoint moiety that is at least 70% identical to IGSF11 (VSIG-3); inducing homologous recombination in the cell, wherein the polynucleotide encoding the polypeptide is inserted at a locus of a transmembrane moiety, wherein the locus is flanked by one or both homology arms of the transmembrane moiety; and harvesting an extracellular vesicle produced by the cell, wherein the extracellular vesicle comprises the extracellular vesicle described herein.

[0040] Described herein is a method of treating an inflammatory disease, the method comprises administering the composition comprising the extracellular vesicle comprising a polypeptide comprising an immune checkpoint moiety that is at least 70% identical to IGSF11 (VSIG-3). [0041] Described herein is a method of treating an autoimmune disease, the method comprises administering the composition comprising the extracellular vesicle comprising a polypeptide comprising an immune checkpoint moiety that is at least 70% identical to IGSF11 (VSIG-3). [0042] Described herein is a method for generating the extracellular vesicle described herein, said method comprising: contacting a cell with a polynucleotide encoding a polypeptide comprising an immune checkpoint moiety that is at least 70% identical to CTLA-4; inducing homologous recombination in the cell, wherein the polynucleotide encoding the polypeptide is inserted at a locus of a transmembrane moiety, wherein the locus is flanked by one or both homology arms of the transmembrane moiety; and harvesting an extracellular vesicle produced by the cell, wherein the extracellular vesicle comprises the extracellular vesicle described herein. [0043] Described herein is a method of treating an inflammatory disease, the method comprises administering the composition comprising the extracellular vesicle comprising a polypeptide comprising an immune checkpoint moiety that is at least 70% identical to CTLA-4. [0044] Described herein is a method of treating an autoimmune disease, the method comprises administering the composition comprising the extracellular vesicle comprising a polypeptide comprising an immune checkpoint moiety that is at least 70% identical to CTLA-4.

[0045] Described herein is a method for generating the extracellular vesicle described herein, said method comprising: contacting a cell with a polynucleotide encoding a polypeptide comprising an immune checkpoint moiety that is at least 70% identical to OX-2 (CD200); inducing homologous recombination in the cell, wherein the polynucleotide encoding the polypeptide is inserted at a locus of a transmembrane moiety, wherein the locus is flanked by one or both homology arms of the transmembrane moiety; and harvesting an extracellular vesicle produced by the cell, wherein the extracellular vesicle comprises the extracellular vesicle described herein.

[0046] Described herein is a method of treating an inflammatory disease, the method comprises administering the composition comprising the extracellular vesicle comprising a polypeptide comprising an immune checkpoint moiety that is at least 70% identical to OX-2 (CD200).

[0047] Described herein is a method of treating an autoimmune disease, the method comprises administering the composition comprising the extracellular vesicle comprising a polypeptide comprising an immune checkpoint moiety that is at least 70% identical to OX-2 (CD200).

[0048] Described herein is a method for generating the extracellular vesicle described herein, said method comprising: contacting a cell with a polynucleotide encoding a polypeptide comprising an immune checkpoint moiety that is at least 70% identical to BTLA; inducing homologous recombination in the cell, wherein the polynucleotide encoding the polypeptide is inserted at a locus of a transmembrane moiety, wherein the locus is flanked by one or both homology arms of the transmembrane moiety; and harvesting an extracellular vesicle produced by the cell, wherein the extracellular vesicle comprises the extracellular vesicle described herein. [0049] Described herein is a method of treating an inflammatory disease, the method comprises administering the composition comprising the extracellular vesicle comprising a polypeptide comprising an immune checkpoint moiety that is at least 70% identical to BTLA.

[0050] Described herein is a method of treating an autoimmune disease, the method comprises administering the composition comprising the extracellular vesicle comprising a polypeptide comprising an immune checkpoint moiety that is at least 70% identical to BTLA.

[0051] Another aspect of the disclosure is a composition comprising at least one extracellular vesicle, the at least one extracellular vesicle engineered to express at least one immune checkpoint moiety on a surface of said extracellular vesicle, wherein the at least one immune checkpoint moiety comprises PD-L1, or a fragment thereof. Another aspect of this present disclosure is a method of suppressing CD8+ CD25+ cells in a subject in need thereof, by administering the composition.

[0052] Another aspect of the disclosure is a composition comprising at least one extracellular vesicle, the at least one extracellular vesicle engineered to express at least one immune checkpoint moiety on a surface of said extracellular vesicle, wherein the at least one immune checkpoint moiety comprises PD-L1, or a fragment thereof. Another aspect of this present disclosure is a method of suppressing CD8+ CD25+ cells in a subject in need thereof, by administering the composition.

BRIEF DESCRIPTION OF THE DRAWINGS

[0053] This patent application contains at least one drawing executed in color. Copies of this patent or patent application with color drawing(s) can be provided by the Office upon request and payment of the necessary fee.

[0054] FIG. 1 illustrates features of the engineered extracellular vesicles or the engineered exosomes (termed Exo-MICA) described herein and their immunological implications that can be harnessed to make a tolerogenic or anti-inflammatory therapeutic. Features of endogenous extracellular vesicles or exosomes indicate how to enhance and combine them in engineering Exo-MICA to counter acute respiratory distress syndrome (ARDS). In some cases, the ARDS can be caused by coronavirus such as SARS-CoV-2.

[0055] FIG. 2 illustrates molecular and cellular mechanisms by which Exo-MICA modulates immune responses. FIG. 2A. Exo-MICA can deactivate hyperactive CD4 helper and CD8 cytotoxic T cells, which are thought to be the main source of inflammatory cytokines that cause CRS, as well as B cells which initiate the activation and sustain T cell responses in ectopic lymphoid follicles in bronchial tissue. In addition, Exo-MICA can distribute to the lung-draining lymph nodes, and via blood transport to the spleen, to additionally down-modulate the hyperactive immune response initiated by APCs like DC and B cells. FIG. 2B. The multivalent and mechano-active nature of Exo-MICA can bind to and trigger signaling of receptors engaged by their corresponding ligands in a manner that can be more effective than that induced by freely diffusing (soluble, not particulate) ligands.

[0056] FIG. 3 illustrates an exemplary overview of an approach for using engineered exosomes described herein with multiple immune checkpoint moieties (Exo-MICA) to treat severe inflammation arising in fulminant COVID-19. Exo-MICA with multiple different immunomodulatory surface proteins can be engineered to contain only one type of immune checkpoint moiety (singleplex, diamond), two or more Exo-MICA each with one type of immune checkpoint moiety (multiples, square ligands and circle ligands), and finally as mosaic containing multiple immune checkpoint moi eties on one type of Exo-MICA (multiplex). In this application, two or more immune checkpoint moieties can be examined In some cases, the two or more immune checkpoint moieties (e.g. immune checkpoint ligands or immune checkpoint inhibitory ligands) can be any of the following VISTA, CTLA-4, PD-L1, PD-1, and IGSF11 (VSIG-3). [0057] FIG. 4 illustrates an exemplary scheme of producing MSC lines engineered with multiple immune checkpoint moieties fused to LAMP2B through CRISPR/Cas9. FIG. 4A. Four MSC lines can be generated and engineered via CRISPR/Cas9 to fuse each of four checkpoint agonists to N-terminus of LAMP2B. Culture medium supernatants can be subjected to sequential steps for isolating exosomes. FIG. 4B illustrates map of the donor plasmid containing left arm of human LAMP2 (hLAMP2_LA) and right arm of human LAMP2 (hLAMP2_RA), which flank the PD- L1 gene insert. This plasmid can be co-transduced into the original MSC-Tert cells to create MSC cell lines engineered in their chromosome to express PD-L1 fused to the N-terminus of the LAMP2B gene (annotated MSC-Tert-PDL-l-LAMP2). Three other donor plasmids with the same map structure can be used to create the MSC-Tert-CTLA-4-LAMP2, MSC-Tert- VISTA- LAMP2, and MSC-Tert-IGSFl l (VSIG-3 )-LAMP2.

[0058] FIG. 5 illustrates workflow for producing MSC lines engineered with multiple immune checkpoint moieties and purifying their exosomes (Exo-MICA). FIG. 5A. Four MSC lines can be generated and engineered via CRISPR/Cas9 to fuse each of four checkpoint agonists to N- terminus of LAMP2B. Culture medium supernatants can be harvested and stored before isolating exosomes. FIG. 5B. Exo-MICA can be purified from supernatants using tangential flow filtration and polished with high molecular weight gel filtration chromatography, and finally stored via cry opreservation.

[0059] FIG. 6 illustrates combinations of Exo-MICA and their controls to be tested in in vitro assays. Exo-MICA can be tested in PBMC assays as described herein as single, double, triple, or all four checkpoint moieties combinations, together with control PBS and unmodified exosomes (vehicle).

[0060] FIG. 7 illustrates overview of in vivo studies testing Exo-MICA candidates in the murine ARDS and COVID-19 models. FIG. 7A. The murine model for human ARDS using LPS plus ventilator injury can be established and then used to test the potential immunomodulatory activity of the most promising EXO-MICA candidates screened from in vitro cell culture assays. In addition, the most promising EXO-MICA from the LPS plus ventilator injury murine model can be tested in the murine COVID-19 model involving transgenic mice expressing human ACE-2 receptor. In brief, a mouse adapted recombinant SARS-CoV-2 virus strain can infect mice and select Exo-MICA can be then administered. FIG. 7B. For both the LPS plus ventilator injury and murine COVID-19 model, the basic parameters measuring respiratory function and lung histology can be analyzed.

[0061]

[0062] FIG. 8 illustrates immune checkpoint moiety engineered to be expressed on extracellular vesicle surface.

[0063] FIG. 9 illustrates an exemplary experimental protocol for examining the therapeutic effect of the extracellular vesicle (e g., an exosome engineered to express immune checkpoint moiety on the described herein. This experimental protocol utilized an animal model, where inflammation was induced by administering Carrageenan (CG) in rat hind paw. The rat was then treated with the extracellular vesicle described herein at one hour after the CG treatment. Two measurements of the amount of edema of the hind paw were obtained at eight hour (8H) or 24 hour (24H) after the CG treatment.

[0064] FIG. 10 illustrates the measurements of the Carrageenan-induced paw edema model in rats described here FIG. 9. *** denotes p<0.0001 per Dunnett’s test. Engineered exosomes expressing IGSF11 (also known as VSIG-3) exhibited a significantly increase in antiinflammatory activity compared to controls (vehicle group or group treated with unmodified exosome).

[0065] FIG. 11 illustrates the presence of PD-L1 on the surface of engineered exosomes.

[0066] FIG. 12 illustrates engineered exosomes expressing PD-L1 significantly suppressed activated CD8 positive and CD25 positive T cell population (CD8+/CD25+) compared to T cell activation contacted with unmodified exosomes. Unpaired t tests were used to determine p values (***p < 0.001).

[0067] The novel features of the disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure can be obtained by reference to the following detailed description that sets forth illustrative embodiments.

DETAILED DESCRIPTION

Overview

[0068] Described herein are compositions and methods for treating diseases or disorders with engineered extracellular vesicles. In some cases, the engineered extracellular vesicle can express at least one immune checkpoint moiety on the surface of the engineered extracellular vesicle. In some instances, the engineered extracellular vesicle can express at least one species of immune checkpoint moieties on the surface of the engineered extracellular vesicles. In some instances, the engineered extracellular vesicle can express a plurality of the immune checkpoint moieties, where the immune checkpoint moieties are from a single species of the immune checkpoint moiety. For example, the engineered extracellular vesicle can express multiple copies of PD-L1 on its surface. In some embodiments, the engineered extracellular vesicle can express a plurality of the immune checkpoint moieties, where the immune checkpoint moieties are from different species of the immune checkpoint moieties. For example, a single engineered extracellular vesicle can express a mixture of copies of immune checkpoint moieties comprising PD-L1 and VISTA. In some embodiments, the engineered extracellular vesicle expressing the mixture of copies of different immune checkpoint moieties can exert synergistic therapeutic effects compared to treatment regimen utilizing engineered extracellular vesicles expressing only a single species of the immune checkpoint moieties.

[0069] The engineered extracellular vesicles described herein have been designed with optimal properties, including maximum packing density of one or more immune checkpoint moieties on the surface of the engineered extracellular vesicles. The genetic modification techniques described herein result in these optimal properties, including the maximum packing density. The desired effects of the engineered extracellular vesicles, including immune suppression, are augmented designs of the engineered extracellular vesicles described herein.

[0070] In some cases, the immune checkpoint moiety is inserted into a transmembrane moiety, where the transmembrane moiety is expressed on the surface (e.g. exterior) of the engineered extracellular vesicle. In some embodiments, the immune checkpoint moiety is encapsulated in the engineered extracellular vesicle. In some embodiments, the immune checkpoint moiety is released by the engineered extracellular vesicle. In some embodiments, the engineered extracellular vesicle can be used to treat diseases or disorders. In some instances, the diseases or disorders can be inflammatory or autoimmune diseases or disorders. In some embodiments, the engineered extracellular vesicles can be formulated into a composition or a pharmaceutical composition to be administered into a subject in need thereof. In some embodiments, a cell genetically modified to generate the engineered extracellular vesicles described herein can be formulated into a composition or a pharmaceutical composition to be administered into a subject in need thereof. FIG. 1 provides exemplary descriptions for the uses of the engineered extracellular vesicles described herein.

Compositions

[0071] Described herein, in some embodiments, are compositions comprising an engineered extracellular vesicle generated from the platforms and methods described herein. In some embodiments, the engineered extracellular vesicle is a membrane-bound particle secreted by a cell described herein. In some embodiments, the engineered extracellular vesicle is a membranebound particle secreted by a genetically modified cell described herein. In some embodiments, the engineered extracellular vesicle is a membrane-bound particle generated in vitro. In some embodiments, the engineered extracellular vesicle is a membrane-bound particle generated ex vivo. In some embodiments, the engineered extracellular vesicle is a membrane-bound particle generated without a cell. In some embodiments, the engineered extracellular vesicle is an engineered exosome, microvesicle, retrovirus-like particle, apoptotic body, apoptosome, oncosome, exopher, enveloped viruses, exomere, or other very large extracellular vesicle. In some embodiments, the engineered extracellular vesicle is an engineered exosome.

[0072] In some instances, the engineered extracellular vesicle comprises a diameter about 1 nm to about 10,000 nm. In some instances, the engineered extracellular vesicle comprises a diameter about 1 nm to about 5 nm, about 1 nm to about 10 nm, about 1 nm to about 20 nm, about 1 nm to about 50 nm, about 1 nm to about 100 nm, about 1 nm to about 200 nm, about 1 nm to about 500 nm, about 1 nm to about 1,000 nm, about 1 nm to about 2,000 nm, about 1 nm to about 5,000 nm, about 1 nm to about 10,000 nm, about 5 nm to about 10 nm, about 5 nm to about 20 nm, about 5 nm to about 50 nm, about 5 nm to about 100 nm, about 5 nm to about 200 nm, about 5 nm to about 500 nm, about 5 nm to about 1,000 nm, about 5 nm to about 2,000 nm, about 5 nm to about 5,000 nm, about 5 nm to about 10,000 nm, about 10 nm to about 20 nm, about 10 nm to about 50 nm, about 10 nm to about 100 nm, about 10 nm to about 200 nm, about 10 nm to about 500 nm, about 10 nm to about 1,000 nm, about 10 nm to about 2,000 nm, about 10 nm to about 5,000 nm, about 10 nm to about 10,000 nm, about 20 nm to about 50 nm, about 20 nm to about 100 nm, about 20 nm to about 200 nm, about 20 nm to about 500 nm, about 20 nm to about 1,000 nm, about 20 nm to about 2,000 nm, about 20 nm to about 5,000 nm, about 20 nm to about 10,000 nm, about 50 nm to about 100 nm, about 50 nm to about 200 nm, about 50 nm to about 500 nm, about 50 nm to about 1,000 nm, about 50 nm to about 2,000 nm, about 50 nm to about 5,000 nm, about 50 nm to about 10,000 nm, about 100 nm to about 200 nm, about 100 nm to about 500 nm, about 100 nm to about 1,000 nm, about 100 nm to about 2,000 nm, about 100 nm to about 5,000 nm, about 100 nm to about 10,000 nm, about 200 nm to about 500 nm, about 200 nm to about 1,000 nm, about 200 nm to about 2,000 nm, about 200 nm to about 5,000 nm, about 200 nm to about 10,000 nm, about 500 nm to about 1,000 nm, about 500 nm to about 2,000 nm, about 500 nm to about 5,000 nm, about 500 nm to about 10,000 nm, about 1,000 nm to about 2,000 nm, about 1,000 nm to about 5,000 nm, about 1,000 nm to about 10,000 nm, about 2,000 nm to about 5,000 nm, about 2,000 nm to about 10,000 nm, or about 5,000 nm to about 10,000 nm. In some instances, the engineered extracellular vesicle comprises a diameter about 1 nm, about 5 nm, about 10 nm, about 20 nm, about 50 nm, about 100 nm, about 200 nm, about 500 nm, about 1,000 nm, about 2,000 nm, about 5,000 nm, or about 10,000 nm. In some instances, the engineered extracellular vesicle comprises a diameter at least about 1 nm, about 5 nm, about 10 nm, about 20 nm, about 50 run, about 100 nm, about 200 nm, about 500 nm, about 1,000 nm, about 2,000 nm, or about 5,000 nm. In some instances, the engineered extracellular vesicle comprises a diameter at most about 5 nm, about 10 nm, about 20 nm, about 50 nm, about 100 nm, about 200 nm, about 500 nm, about 1,000 nm, about 2,000 nm, about 5,000 nm, or about 10,000 nm.

[0073] In some embodiments, the compositions described herein comprise engineered extracellular vesicles comprising at least one transmembrane moiety. In some embodiments, the transmembrane moiety can be genetically modified to express the at least one immune checkpoint moiety described herein. In some embodiments, the engineered extracellular vesicle can comprise an immune evasion moiety. In some embodiments, the immune evasion moiety comprises CD47.

[0074] In some embodiments, the compositions described herein comprise engineered extracellular vesicles comprising at least one immune checkpoint moiety. In some embodiments, the engineered extracellular vesicle comprises a plurality of immune checkpoint moieties, where the immune checkpoint moieties can be the same or different. In some embodiments, the immune checkpoint moiety is encapsulated by the engineered extracellular vesicle. In some embodiments, the immune checkpoint moiety is expressed on the surface of the engineered extracellular vesicle. In some embodiments, the immune checkpoint moiety is released by the engineered extracellular vesicle. In some embodiments, the immune checkpoint moiety is expressed on the surface of the engineered extracellular vesicle; released by the engineered extracellular vesicle; encapsulated by the engineered extracellular vesicle; delivered by the engineered extracellular to a target cell or a target microenvironment; or a combination thereof. In some embodiments, the immune checkpoint moiety comprises therapeutic properties for treating diseases or disorders. In some embodiments, the diseases or disorders can be inflammatory or autoimmune diseases or disorders.

[0075] In some embodiments, the composition can be cryopreserved. In some embodiments, the composition can be lyophilized. In some embodiments, the composition is stable at 4°C for at least one hour, two hours, six hours, 12 hours, one day, two days, three days, four days, five days, six days, one week, two weeks, three weeks, four weeks, one month, two months, three months, or longer period of time. In some embodiments, the composition is stable at room temperature for at least one hour, two hours, six hours, 12 hours, one day, two days, three days, four days, five days, six days, one week, two weeks, three weeks, four weeks, one month, two months, three months, or longer period of time. In some embodiments, the composition is stable at 37°C for at least one hour, two hours, six hours, 12 hours, one day, two days, three days, four days, five days, six days, one week, two weeks, three weeks, four weeks, one month, two months, three months, or longer period of time.

Transmembrane moiety

[0076] Discredited herein, in some embodiments, are compositions comprising engineered extracellular vesicles comprising at least one transmembrane moiety. In some embodiments, the transmembrane moiety comprises a full-length protein or a variation thereof or a fragment thereof. In some embodiments, the transmembrane moiety is endogenous to the cell that is generating the engineered extracellular vesicles. In some embodiments, the immune checkpoint moiety is heterologous to the cell that is generating the engineered extracellular vesicles. In some embodiments, the transmembrane moiety is selected from a group consisting of: 14-3-3 protein zeta/delta, 14-3-3 protein epsilon, 78 kDa glucose-regulated protein, acetylcholinesterase/ AChE- S, AChE-E, actin, cytoplasmic 1 (ACTA), ADAM 10, alkaline phosphatase, alpha-enolase, alpha- synuclein, aminopeptidase N, amyloid beta A4/APP, annexin 5 A, annexin A2, AP-1, ATF3, ATP citrate lyase, ATPase, beta actin (ACTB), beta-amyloid 42, caveolin-1, CD10, CD1 la, CD1 lb, CD l ie, CD 14, CD 142, CD 146, CD 163, CD24, CD26/DPP4, CD29/ITGB1, CD3, CD37, CD41, CD42a, CD44, CD45, CD47, CD49, CD49d, CD53, CD63, CD64, CD69, CD73 CD81, CD82, CD9, CD90, claudin, claudin-1 cofilin-1, complement-binding proteins CD55 and CD59, cytosolic heat shock protein 90 alpha, cytosolic heat shock protein 90 beta, EBV LMP1, EBV LMP2A, EF-lalpha-1, EF2, EFGR EGFR VIII, emmprin/CD147, enolase 1 alpha (ENO1), EPCAM, ERBB2, tetraspanins (CD9, CD63 and CD81), fatty acid synthase, fetuin-A, flotillin-1, flotillin-2, fructose-bisphosphate aldolase A, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), glycophorin A, GPC1, GPI-anchored 5'nucleotidase, GTPase, heat shock protein 8 (HSPA8), heat shock proteins (HSP70 and HSP90), heparan sulfate proteoglycans, heparinase, heterotrimeric G proteins, HIV Gag, HIV Nef, HLA-DRA, HLA-G, HSV gB, HTLV-1 Tax, huntingtin, ICAM1, integrins, lactadherin, LAMP 1/2, LAMP2A, LAMP2B, LAMP2C, leucine- rich receptor kinase 2, L-lactate dehydrogenase A chain, lysosome-associated membrane glycoprotein 1, lysosome-associated membrane glycoprotein 2, MHC class I, MHC class II, MUC1, multidrug resistance-associated protein, muscle pyruvate kinase (PKM2), N-cadherin, NKCC2, PDCD6IP/Alix, PEC AMI, phosphoglycerate kinase, placental prion proteins, prostatespecific antigen (PSA), pyruvate kinase (PKM), Rab-14, Rab-5a, Rab-5b, Rab-5c, Rab-7, Rap IB, resistin, sonic hedgehog (SHH), surviving, syndecan-1, syndecan-4, syntenin-1, transferrin receptor (TFR2), TSG101, TSPAN8, tumor-associated glycoprotein tetraspanin- 8, tyrosine 3 monooxygenase/tryptophan 5-monooxygenase activation protein, TYRP-2, or vacuolar-sorting- associated protein 35 (VPS35) [0077] In some embodiments, the transmembrane moiety comprises LAMP2. In some embodiments, the transmembrane moiety comprises LAMP-like domain 1 of LAMP2 In some embodiments, the transmembrane moiety comprises LAMP2B. In some embodiments, the transmembrane moiety comprises a polypeptide sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%. 85%. 90%, 95%, or 99% identical to SEQ ID NO: 1 (Table 1). In some embodiments, the transmembrane moiety can be encoded from a polynucleotide sequence comprising homology arms. In some instances, the immune checkpoint moiety described herein can be inserted into the transmembrane moiety by homologous recombination as induced by the homology arms of the transmembrane moiety. In some embodiments, the transmembrane moiety comprises an N-terminus homology arm or a C-terminus homology arm. In some embodiments, the N-terminus homology of the transmembrane moiety is encoded from a polynucleotide sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%. 85%. 90%, 95%, or 99% identical to SEQ ID NO: 2 (Table 1). In some embodiments, the C-terminus homology of the transmembrane moiety is encoded from a polynucleotide sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%. 85%. 90%, 95%, or 99% identical to SEQ ID NO: 3 (Table 1).

Table 1. LAMP2B transmembrane moiety sequences

[0078] In some embodiments, the transmembrane moiety can be complexed with the immune checkpoint moiety described herein. In some embodiments, the transmembrane moiety can be non-covalently complex with the immune checkpoint moiety described herein. In some embodiments, the transmembrane moiety can be covalently complexed with the immune checkpoint moiety described herein. In some embodiments, the transmembrane moiety can be fused to the immune checkpoint moiety described herein at the N-terminus of the transmembrane moiety. In some embodiments, the transmembrane moiety can be fused to the immune checkpoint moiety described herein at the C-terminus of the transmembrane moiety. In some embodiments, the immune checkpoint moiety can be inserted (e.g. via homology recombination) at any locus of the transmembrane moiety.

[0079] In some embodiments, the transmembrane moiety can be covalently connected to the immune checkpoint moiety by a peptidyl linker. In some cases, the linker can be a flexible linker, a rigid linker, or a cleavable linker. In some embodiments, the flexible linker can be (GGGGS)n where n=l, 2, 3, or 4; KESGSVSSEQLAQFRSLD; EGKSSGSGSESKST; GGGGGG; GGGGGGGG; GSAGSAAGSGEF; RKRR; SS; or LE. In some instances, the rigid linker can be (EAAAK)n where n=l, 2, 3, or 4; A(EAAAK)nA where n=2, 3, 4, or 5; A(EAAAK)₄ALEA(EAAAK)₄A; AEAAAKEAAAKA; or LEAGCKNFFPRSFTSCGSLE. In some aspects, the cleavable linker can be LEAGCKNFFPRSFTSCGSLE; CRRRRRREAEAC; GGIEGRGS; or TRHRQPRGWEQL.

Immune checkpoint moiety

[0080] Described herein, in some cases, are engineered extracellular vesicles comprising at least one immune checkpoint moiety. In some embodiments, the immune checkpoint moiety is endogenous to the cell that is generating the engineered extracellular vesicles. In some embodiments, the immune checkpoint moiety is heterologous to the cell that is generating the engineered extracellular vesicles. In some embodiments, the immune checkpoint moiety comprises therapeutic properties for treating diseases or disorders. In some embodiments, the immune checkpoint moiety comprises therapeutic properties for treating an inflammatory or autoimmune disease or disorder described herein. In some embodiments, the immune checkpoint moiety targets and modulates activities of immune cells. In some embodiments, the immune cells can be T cell, including cytotoxic T cell, Natural Killer T cell, Regulatory T cell, and T helper cells. In some embodiments, the immune cells can be CD8+ cells. In some embodiments, the immune cells can be CD25+ cells. In some embodiments, the immune cells can be CD4+ cells. In some embodiments, the immune cells can be CD8+ CD25+ cells. In some cases, the immune cell can be cell that expresses CD4. In some cases, the immune cell can be cell that expresses CD4 and CD 25 (CD4+CD25+). In some cases, the immune cell can be cell that expresses FOXP3. In some cases, the immune cell can be cell that expresses CD4, CD25, and FOXP3 (CD4+CD25+FOXP3+).

[0081] In some embodiments, the immune checkpoint moiety can comprise any one of VISTA, PD-L1, CTLA-4, PD-L2, B7-1 (CD80), B7-2 (CD86), B7-H3 (CD276), B7-H2, B7-H3, B7-H4 (VTCN1), IDO, KIR, LAG3, A2AR, HVEM (CD270, TNFRSF14), Galectin 9, Galectin3, CEACAM1 (CD66a), OX-2 (CD200), PVR (CD155), PVRL2 (Nectin-2, CD112), FGL-1, PECAM-1, TSG-6, CD47, Stabilin- 1 (Clever-1), Neuropilin 1, Neuropilin 2, CD158 (family), IGSF2 (CD101), CD155, GITRL, CD137L, OX40L, LIGHT, CD70, PD-1, RGMB, CTLA-4 (CD152), BTLA, CD160, Tim-3, CD200R, TIGIT, CD112R (PVRIG), LAG-3 (CD223), PECAM-1, CD44, IGSF11 (VSIG-3), or SIRP alpha (CD172a). In some embodiments, the immune checkpoint moiety comprises a polynucleotide sequence encoding a polypeptide or a variation thereof or a fragment thereof. In some embodiments, the immune checkpoint moiety comprises a polypeptide or a variation thereof or a fragment thereof. In some embodiments, the immune checkpoint moiety can be covalently connected to the transmembrane moiety. In some embodiments, the immune checkpoint moiety can be encoded by a polynucleotide sequence encoding VISTA, PD-L1, IGSF11 (VSIG-3), or CLLA-4. In some embodiments, the immune checkpoint moiety can be encoded by a polynucleotide sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%. 85%. 90%, 95%, or 99% identical to any one of SEQ ID NOs: 4-15 (Table 2)

Table 2. Exemplary polynucleotide sequence of immune checkpoint moiety

atggtggtgccgactacaagcgaattactgtgaaagtcaatgccccatacaacaaaatcaaccaaagaattttggttgtggatcc agtcacctctgaacatgaactgacatgtcaggctgagggctaccccaaggccgaagtcatctggacaagcagtgaccatcaa gtcctgagtggtaagaccaccaccaccaattccaagagagaggagaagcttttcaatgtgaccagcacactgagaatcaacac aacaactaatgagattttctactgcacttttaggagattagatcctgaggaaaaccatacagctgaattggtcatcccagaactac ctctggcacatcctccaaatgaaaggggcggcggcggcagcgaacttaatttgacagattcagaaaatgccacttgcctttatg caaaatggcagatgaatttcacagtacgctatgaaactacaaataaaacttatgtaagtatatattttaatttttctttgagttttatagt aggaatttaattgactttatcctttagatacacgtatacaaatgaaattccacgttgactgacttaatgctgaaactctgggatttttttt aagcatacacactcagaagttattctgtttagtgttagaatgaatatttcttctgattcattggttcttgggagttttgtaggacttgatt aagggcaaatgatctgaagacgactatatggtgttcaggtctaagtatttctgtaaagtgtttaaaatgtactagcaccaaagcttg cccttttaaaaagcagcttcccatctgggcatggtggctcatgcctgtgatcccagcattttggaaggccaaagcaggaggatt gcttgaacccagcccgggcaacatggtgagacctcatctctacaaaaaaactttaaaaattagccaggcatggtggcacacac atgtacctccagccactcgctgggccgaggctggaggattgcttgagctcaggaggttgagtctgcagtgagccatgattgtg ctactgcactccagcctgggcaacagagcgagactctatctcaaaaaaaaaaaaaatcgcttacttaggttggtacaaaagtaa ttgccattaaaggaacaaaccacaattacttttgcaccaacctaagtaagcgatttttttttttggcacaaactgcaattacttttgca ccaacctaacactaaaatctccccctttttgacatctcctcttaattcctttgccttcttattttctttttgttaaggtcctcactacttacaa cctaattttttaaaaaatcagtttaagaacagcagctcaagccagttttccttttctttttagacatacacgaa

atacacgtatacaaatgaaattccacgttgactgacttaatgctgaaactctgggattttttttaagcatacacactcagaagttattc tgtttagtgtagaatgaatatttcttctgattcattggttcttgggagtttttgtaggacttgattaagggcaaatgatctgaagacga ctatatggtgttcaggtctaagtatttctgtaaagtgtttaaaatgtactagcaccaaagcttgcccttttaaaaagcagcttcccatc tgggcatggtggctcatgcctgtgatcccagcattttggaaggccaaagcaggaggattgcttgaacccagcccgggcaacat ggtgagacctcatctctacaaaaaaactttaaaaattagccaggcatggtggcacacacatgtacctccagccactcgctgggc cgaggctggaggattgcttgagctcaggaggttgagtctgcagtgagccatgattgtgctactgcactccagcctgggcaaca gagcgagactctatctcaaaaaaaaaaaaaatcgcttacttaggttggtacaaaagtaattgccattaaaggaacaaaccacaat tactttgcaccaacctaagtaagcgatttttttttttggcacaaactgcaattacttttgcaccaacctaacactaaaatctcccccttt ttgacatctcctcttaattcctttgccttcttattttctttttgttaaggtcctcactacttacaacctaattttttaaaaaatcagtttaagaa cagcagctcaagccagttttccttttctttttagacatacacgaa

[0082] In some embodiments, the immune checkpoint moiety can be encoded by a polynucleotide sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%. 85%. 90%, 95%, or 99% identical to SEQ ID NO: 4 In some embodiments, the immune checkpoint moiety can be encoded by a polynucleotide sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%. 85%. 90%, 95%, or 99% identical to SEQ ID NO: 5. In some embodiments, the immune checkpoint moiety can be encoded by a polynucleotide sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%. 85%. 90%, 95%, or 99% identical to SEQ ID NO: 6. In some embodiments, the immune checkpoint moiety can be encoded by a polynucleotide sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%. 85%. 90%, 95%, or 99% identical to SEQ ID NO: 7. In some embodiments, the immune checkpoint moiety can be encoded by a polynucleotide sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%. 85%. 90%, 95%, or 99% identical to SEQ ID NO: 8. In some embodiments, the immune checkpoint moiety can be encoded by a polynucleotide sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%. 85%. 90%, 95%, or 99% identical to SEQ ID NO: 9. In some embodiments, the immune checkpoint moiety can be encoded by a polynucleotide sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%. 85%. 90%, 95%, or 99% identical to SEQ ID NO: 10. In some embodiments, the immune checkpoint moiety can be encoded by a polynucleotide sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%. 85%. 90%, 95%, or 99% identical to SEQ ID NO: 11. In some embodiments, the immune checkpoint moiety can be encoded by a polynucleotide sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%. 85%. 90%, 95%, or 99% identical to SEQ ID NO: 12. In some embodiments, the immune checkpoint moiety can be encoded by a polynucleotide sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%. 85%. 90%, 95%, or 99% identical to SEQ ID NO: 13. In some embodiments, the immune checkpoint moiety can be encoded by a polynucleotide sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%. 85%. 90%, 95%, or 99% identical to SEQ ID NO: 14. In some embodiments, the immune checkpoint moiety can be encoded by a polynucleotide sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%. 85%. 90%, 95%, or 99% identical to SEQ ID NO: 15. In some embodiments, the immune checkpoint moiety can be encoded by a polynucleotide sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%. 85%. 90%, 95%, or 99% identical to SEQ ID NO: 23. In some embodiments, the immune checkpoint moiety can be encoded by a polynucleotide sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%. 85%. 90%, 95%, or 99% identical to SEQ ID NO: 24. In some embodiments, the immune checkpoint moiety can be encoded by a polynucleotide sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%. 85%. 90%, 95%, or 99% identical to SEQ ID NO: 25. In some embodiments, the immune checkpoint moiety can be encoded by a polynucleotide sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%. 85%. 90%, 95%, or 99% identical to SEQ ID NO: 26. In some embodiments, the immune checkpoint moiety can be encoded by a polynucleotide sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%. 85%. 90%, 95%, or 99% identical to SEQ ID NO: 27. In some embodiments, the immune checkpoint moiety can be encoded by a polynucleotide sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%. 85%. 90%, 95%, or 99% identical to SEQ ID NO: 28.

[0083] In some embodiments, the immune checkpoint moiety can be VISTA and encoded from polynucleotide or a variation thereof or a fragment thereof that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%. 85%. 90%, 95%, or 99% identical to SEQ ID NOS: 4-6 or 25. In some embodiments, the immune checkpoint moiety can be PD-L1 and encoded from polynucleotide or a variation thereof or a fragment thereof that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%. 85%. 90%, 95%, or 99% identical to SEQ ID NOS: 7-9 or 24. In some embodiments, the immune checkpoint moiety can be IGSF11 (VSIG-3) and encoded from polynucleotide or a variation thereof or a fragment thereof that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%. 85%. 90%, 95%, or 99% identical to SEQ ID NOS: 10-12 or 26. In some embodiments, the immune checkpoint moiety can be CTLA-4 and encoded from polynucleotide or a variation thereof or a fragment thereof that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%. 85%. 90%, 95%, or 99% identical to SEQ ID NOS: 13-15 or 27. In some embodiments, the immune checkpoint moiety can be OX-2 (CD200) and encoded from polynucleotide or a variation thereof or a fragment thereof that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%. 85%. 90%, 95%, or 99% identical to SEQ ID NO: 23. In some embodiments, the immune checkpoint moiety can be BTLA and encoded from polynucleotide or a variation thereof or a fragment thereof that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%. 85%. 90%, 95%, or 99% identical to SEQ ID NO: 28

[0084] In some embodiments, the immune checkpoint moiety can be encoded by a heterologous polynucleotide. In some embodiments, the heterologous polynucleotide can be introduced into in any one of the cells described herein. In some embodiments, the heterologous polynucleotide comprises mRNA, rRNA, SRP RNA, tRNA, tmRNA, snRNA, snoRNA, gRNA, aRNA, crRNA, IncRNA, miRNA, ncRNA, piRNA, siRNA, and shRNA. In some cases, the heterologous polynucleotide comprises mRNA. In some embodiments, the heterologous polynucleotide comprises DNA. In some cases, the heterologous polynucleotide can be inserted into the transmembrane moiety described herein. In some embodiments, the heterologous polynucleotide can be inserted into the transmembrane moiety described herein via homologous recombination. In some embodiments, the heterologous polynucleotide comprises a nucleic sequence encoding VISTA, PD-L1, CTLA-4, PD-L2, B7-1 (CD80), B7-2 (CD86), B7-H3 (CD276), B7-H2, B7-H3, B7-H4 (VTCN1), IDO, KIR, LAG3, A2AR, HVEM (CD270, TNFRSF14), Galectin 9, Galectin3, CEACAM1 (CD66a), OX-2 (CD200), PVR (CD155), PVRL2 (Nectin-2, CD112), FGL-1, PECAM-1, TSG-6, CD47, Stabilin-1 (Clever-1), Neuropilin 1, Neuropilin 2, CD158 (family), IGSF2 (CD101), CD155, GITRL, CD137L, OX40L, LIGHT, CD70, PD-1, RGMB, CTLA-4 (CD 152), BTLA, CD 160, Tim-3, CD200R, TIGIT, CD112R (PVRIG), LAG-3

(CD223), PECAM-1, CD44, IGSF11 (VSIG-3), or SIRP alpha (CD 172a). In some embodiments, the immune checkpoint moiety can be encoded from a heterologous polynucleotide encoding VISTA. In some embodiments, the immune checkpoint moiety can be encoded from a heterologous polynucleotide encoding PD-L1. In some embodiments, the immune checkpoint moiety can be encoded from a heterologous polynucleotide encoding IGSF11 (VSIG-3). In some embodiments, the immune checkpoint moiety can be encoded from a heterologous polynucleotide encoding CTLA-4.

[0085] In some embodiments, the immune checkpoint moiety comprises a heterologous polynucleotide encoding a cytokine. In some embodiments, the immune checkpoint moiety comprises a polypeptide comprising a peptide sequence of the cytokine. Exemplary cytokines that can be utilized as the immune check point moiety includes 4-1BBL, acylation stimulating protein, adipokine, albinterferon, APRIL, Arh, BAFF, Bcl-6, CCL1, CCL1/TCA3, CCL11, CCL12/MCP-5, CCL13/MCP-4, CCL14, CCL15, CCL16, CCL17/TARC, CCL18, CCL19, CCL2, CCL2/MCP-1, CCL20, CCL21, CCL22/MDC, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CCL3, CCL3L3, CCL4, CCL4L1/LAG-1, CCL5, CCL6, CCL7, CCL8, CCL9, CCR10, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CD153, CD154, CD178, CD40LG, CD70, CD95L/CD178, Cerberus (protein), chemokines, CLCF1, CNTF, colony-stimulating factor, common b chain (CD131), common g chain (CD132), CX3CL1, CX3CR1, CXCL1, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCL17, CXCL2, CXCL2/MIP- 2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL9, CXCR3, CXCR4, CXCR5, EDA-A1, Epo, erythropoietin, FAM19A1, FAM19A2, FAM19A3, FAM19A4, FAM19A5, Flt-3L, FMS- like tyrosine kinase 3 ligand, Foxp3, GATA-3, GcMAF, G-CSF, GITRL, GM-CSF, granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, hepatocyte growth factor, IFNA1, IFNA10, IFNA13, IFNA14, IFNA2, IFNA4, IFNA5/IFNaG, IFNA7, IFNA8, IFNB1, IFNE, IFNG, IFNZ, IFN-a, IFN-p, IFN-y, IFNro/IFNWl, IL-1, IL- 10, IL- 10 family, IL- 10-like, IL-11, IL- 12, IL-13, IL-14, IL- 15, IL- 16, IL- 17, IL- 17 family, IL-17A-F, IL- 18, IL- 18BP, IL-19, IL-1A, IL-1B, IL-1F10, IL-1F3/IL-1RA, IL-1F5, IL-1F6, IL-1F7, IL-1F8, IL-1F9, IL-l-like, IL-IRA, IL-1RL2, IL-la, IL-lp, IL-2, IL-20, IL-21, IL-22, IL-23, IL-24, IL-28A, IL- 286, IL-29, IL-3, IL-31, IL-33, IL-35, IL-4, IL-5, IL-6, IL-6-like, IL-7, IL-8/CXCL8, IL-9, inflammasome, interferome, interferon, interferon beta-la, interferon beta-lb, interferon gamma, interferon type I, interferon type II, interferon type III, interferons, interleukin, interleukin 1 receptor antagonist, Interleukin 8, IRF4, Leptin, leukemia inhibitory factor (LIF), leukocytepromoting factor, LIGHT, LTA/TNFB, LT- , lymphokine, lymphotoxin, lymphotoxin alpha, lymphotoxin beta, macrophage colony-stimulating factor, macrophage inflammatory protein, macrophage-activating factor, M-CSF, MHC class III, miscellaneous hematopoietins, monokine, MSP, myokine, myonectin, nicotinamide phosphoribosyltransferase, oncostatin M (OSM), oprelvekin, OX40L, platelet factor 4, promegapoietin, RANKL, SCF, STAT3, STAT4, STAT6, stromal cell-derived factor 1, TALL-1, TBX21, TGF-a, TGF-p, TGF-pl, TGF-p2, TGF-p3, TNF, TNFSF10, TNFSF11, TNFSF12, TNFSF13, TNFSF14, TNFSF15, TNFSF4, TNFSF8, TNF-a, TNF-P, Tpo, TRAIL, TRANCE, TWEAK, vascular endothelial growth inhibitor, XCL1, or XCL2.

[0086] In some embodiments, the immune checkpoint moiety can be complexed with the transmembrane moiety described herein. In some embodiments, the immune checkpoint moiety can be non-covalently complexed with the transmembrane moiety described herein. In some embodiments, the immune checkpoint moiety can be covalently complexed with the transmembrane moiety described herein. In some embodiments, the immune checkpoint moiety can be expressed as part of a fusion protein comprising both the immune checkpoint moiety and the transmembrane moiety. In some embodiments, the immune checkpoint moiety can be expressed as part of a fusion protein comprising both the immune checkpoint moiety and a fragment of the transmembrane moiety, In some embodiments, the N-terminus of the immune checkpoint moiety can be fused to the transmembrane moiety. In some embodiments, the C- terminus of the immune checkpoint moiety can be fused to the transmembrane moiety described herein. In some embodiments, the immune checkpoint moiety can be fused and flanked by the transmembrane moiety on both N and C-terminus of the immune checkpoint moiety. For example, the immune checkpoint moiety can be inserted into a transmembrane moiety as part of a fusion peptide, where the N-terminus of the fusion peptide comprises a fragment of the transmembrane moiety, followed by the immune checkpoint moiety (or a variation there or a fragment thereof), and followed by the C-terminus of the fusion peptide comprising another fragment of the transmembrane moiety. In some embodiments, the immune checkpoint moiety comprises the immune checkpoint moiety complexed with the transmembrane moiety. In some embodiments, the immune checkpoint moiety comprises the immune checkpoint moiety non- covalently complexed with the transmembrane moiety. In some embodiments, the immune checkpoint moiety comprises the immune checkpoint moiety covalently complexed with the transmembrane moiety.

[0087] In some embodiments, the engineered extracellular vesicle comprises a plurality of the immune checkpoint moiety described herein. In some embodiments, the plurality of the immune checkpoint moieties is expressed on the surface of the engineered extracellular vesicle. In some embodiments, the plurality of the immune checkpoint moieties is expressed on the surface of the engineered extracellular vesicle as part of the fusion with the transmembrane moiety. In some embodiments, the engineered extracellular vesicle expresses at least one, ten, 100, 500, 1,000, 5,000, 10,000, 50,000, 100,000, 500,000, 1,000,000, 5,000,000, or more units of the immune checkpoint moiety. In some embodiments, the engineered extracellular vesicle delivers the expressed immune checkpoint moiety to a target cell or a target microenvironment.

[0088] In some embodiments, the engineered extracellular vesicle comprises a plurality of the immune checkpoint moiety described herein. In some embodiments, the plurality of the immune checkpoint moieties is encapsulated in the engineered extracellular vesicle. In some embodiments, the engineered extracellular vesicle encapsulates at least one, ten, 100, 500, 1,000, 5,000, 10,000, 50,000, 100,000, 500,000, 1,000,000, 5,000,000, or more units of the immune checkpoint moiety. In some embodiments, the engineered extracellular vesicle delivers the encapsulated immune checkpoint moiety to a target cell or a target microenvironment.

[0089] In some embodiments, the engineered extracellular vesicle secretes or releases a plurality of the immune checkpoint moiety described herein. In some embodiments, the engineered extracellular vesicle secretes at least one, ten, 100, 500, 1,000, 5,000, 10,000, 50,000, 100,000, 500,000, 1,000,000, 5,000,000, or more units of the immune checkpoint moiety. In some embodiments, the engineered extracellular vesicle secretes the immune checkpoint moiety to a target cell or a target environment.

[0090] In some embodiments, the plurality of the immune checkpoint moiety can be expressed on the surface of the engineered extracellular vesicle. In some embodiments, the plurality of the immune checkpoint moieties is expressed as part of the fusion peptide comprising immune checkpoint moiety and transmembrane moiety. In some instances, the engineered extracellular vesicle comprising the immune checkpoint moiety expressed on the surface of the engineered extracellular vesicle contacts with a target cell or a target environment.

[0091] In some embodiments, the number of units of the immune checkpoint moiety that can be expressed on the surface of the engineered extracellular vesicle is limited by a theoretical maximum as determined by the ratio between: the dimensions of the engineered extracellular vesicle; and the dimensions of the expressed immune checkpoint moiety or the expressed fusion peptide comprising the immune checkpoint moiety. In some embodiments, the platforms and methods described herein can generate and select for an extracellular vesicle expressing a number of units of immune checkpoint moiety that is at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of the theoretical maximum of number of units of immune checkpoint moiety that can expressed on the surface of the engineered extracellular vesicle. [0092] In some embodiments, the platforms and methods described herein can generate and select for a homogenous population of engineered extracellular vesicles. Homogenous population of engineered extracellular vesicles can express plurality of the immune checkpoint moiety, where the plurality of the immune checkpoint moiety comprises one species of the immune checkpoint moiety described herein (e g., singleplex as described in FIG. 3). For example, the homogenous population of engineered extracellular vesicles can express a plurality of PD-L1. In some embodiments, the homogenous population of engineered extracellular vesicles can express a plurality of immune checkpoint moieties, where the plurality of the immune checkpoint moiety comprises two or more of the species of the immune checkpoint moiety, where every engineered extracellular vesicle expresses the same plurality of the two or more species of the immune checkpoint moiety (e.g. mosaic as described in FIG. 3). In some cases, the two or more of the species of the immune checkpoint moiety can be expressed at a ratio. For example, the engineered extracellular vesicles in the homogenous population can express a first and a second species of the immune checkpoint moiety at a ratio of 1 to 1, 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 10, or 1 to 100. In some cases, the engineered extracellular vesicles in the homogenous population can express a first, a second, and third species of the immune checkpoint moiety at a ratio. In some instances, the engineered extracellular vesicles in the homogenous population can express a first, a second, a third, and a fourth species of the immune checkpoint moiety at a ratio. In some cases, the engineered extracellular vesicles in the homogenous population can express a first, a second, a third, a fourth, or more species of the immune checkpoint moiety at a ratio.

[0093] In some embodiments, the platforms and methods described herein can generate and select for a homogenous population of engineered extracellular vesicles expressing a number of units of immune checkpoint moiety that is at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of the theoretical maximum of number of units of immune checkpoint moiety that can expressed on the surface of the engineered extracellular vesicle. In some embodiments, the platforms and methods described herein can generate and select for a homogenous population of engineered extracellular vesicles expressing a number of units of immune checkpoint moiety on the surface of the engineered extracellular vesicle that is at least 30% of the theoretical maximum number of units of immune checkpoint moiety that can be expressed on the surface of the engineered extracellular vesicle. In some embodiments, the platforms and methods described herein can generate and select for a homogenous population of engineered extracellular vesicles expressing a number of units of immune checkpoint moiety on the surface of the engineered extracellular vesicle that is at least 70% of the theoretical maximum number of units of immune checkpoint moiety that can be expressed on the surface of the engineered extracellular vesicle. In some embodiments, the platforms and methods described herein can generate and select for a homogenous population of engineered extracellular vesicles expressing a number of units of immune checkpoint moiety on the surface of the engineered extracellular vesicle that is at least 75% of the theoretical maximum number of units of immune checkpoint moiety that can be expressed on the surface of the engineered extracellular vesicle. In some embodiments, the platforms and methods described herein can generate and select for a homogenous population of engineered extracellular vesicles expressing a number of units of immune checkpoint moiety on the surface of the engineered extracellular vesicle that is at least 80% of the theoretical maximum number of units of immune checkpoint moiety that can be expressed on the surface of the engineered extracellular vesicle. In some embodiments, the platforms and methods described herein can generate and select for a homogenous population of engineered extracellular vesicles expressing a number of units of immune checkpoint moiety on the surface of the engineered extracellular vesicle that is at least 85% of the theoretical maximum number of units of immune checkpoint moiety that can be expressed on the surface of the engineered extracellular vesicle. In some embodiments, the platforms and methods described herein can generate and select for a homogenous population of engineered extracellular vesicles expressing a number of units of immune checkpoint moiety on the surface of the engineered extracellular vesicle that is at least 90% of the theoretical maximum number of units of immune checkpoint moiety that can be expressed on the surface of the engineered extracellular vesicle. In some embodiments, the platforms and methods described herein can generate and select for a homogenous population of engineered extracellular vesicles expressing a number of units of immune checkpoint moiety on the surface of the engineered extracellular vesicle that is at least 95% of the theoretical maximum number of units of immune checkpoint moiety that can be expressed on the surface of the engineered extracellular vesicle. In some embodiments, the platforms and methods described herein can generate and select for a homogenous population of engineered extracellular vesicles expressing a number of units of immune checkpoint moiety on the surface of the engineered extracellular vesicle that is at least 95% of the theoretical maximum number of units of immune checkpoint moiety that can be expressed on the surface of the engineered extracellular vesicle.

[0094] In some embodiments, the platforms and methods described herein can generate and select for a heterogenous population of engineered extracellular vesicles. Heterogenous population of engineered extracellular vesicles can express plurality of the immune checkpoint moiety, where the plurality of the immune checkpoint moiety comprises two or more species of the immune checkpoint moiety described herein (e.g. multiplex as described in FIG. 3). For example, a subpopulation of the heterogenous population of engineered extracellular vesicles can express a plurality of PD-L1, while another subpopulation of the heterogenous population of engineered extracellular vesicles can express a plurality of VISTA. In some embodiments, the heterogenous population of engineered extracellular vesicles can express a plurality of immune checkpoint moieties, where the plurality of the immune checkpoint moiety comprises two or more of the species of the immune checkpoint moiety (e.g. mosaic as described in FIG. 3). For example, a subpopulation of the heterogenous population of engineered extracellular vesicles can express PD-L1 and VISTA, while another subpopulation of the heterogenous population of engineered extracellular vesicles can express VISTA and IGSF11 (VSIG-3). In some cases, the two or more of the species of the immune checkpoint moiety can be expressed at a ratio. For example, each extracellular vesicle in the heterogenous population can express two or more species of the immune checkpoint moiety at a ratio of 1 to 1, 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 10, or 1 to 100. In some cases, each engineered extracellular vesicle in the heterogenous population can express a first, a second, and third species of the immune checkpoint moiety at a ratio. In some instances, each engineered extracellular vesicle in the heterogenous population can express a first, a second, a third, and a fourth species of the immune checkpoint moiety at a ratio. In some cases, each engineered extracellular vesicle in the heterogenous population can express a first, a second, a third, a fourth, or more species of the immune checkpoint moiety at a ratio.

[0095] In some embodiments, the platforms and methods described herein can generate and select for a heterogeneous population of engineered extracellular vesicles. In some embodiments, the platforms and methods described herein can generate and select for a heterogeneous population of engineered extracellular vesicles expressing a number of units of immune checkpoint moiety that is at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of the theoretical maximum of number of units of immune checkpoint moiety that can expressed on the surface of the engineered extracellular vesicle. In some embodiments, the platforms and methods described herein can generate and select for a heterogeneous population of engineered extracellular vesicles expressing a number of units of immune checkpoint moiety on the surface of the engineered extracellular vesicle that is at least 30% of the theoretical maximum number of units of immune checkpoint moiety that can be expressed on the surface of the engineered extracellular vesicle. In some embodiments, the platforms and methods described herein can generate and select for a heterogeneous population of engineered extracellular vesicles expressing a number of units of immune checkpoint moiety on the surface of the engineered extracellular vesicle that is at least 70% of the theoretical maximum number of units of immune checkpoint moiety that can be expressed on the surface of the engineered extracellular vesicle. In some embodiments, the platforms and methods described herein can generate and select for a heterogeneous population of engineered extracellular vesicles expressing a number of units of immune checkpoint moiety on the surface of the engineered extracellular vesicle that is at least 75% of the theoretical maximum number of units of immune checkpoint moiety that can be expressed on the surface of the engineered extracellular vesicle. In some embodiments, the platforms and methods described herein can generate and select for a heterogeneous population of engineered extracellular vesicles expressing a number of units of immune checkpoint moiety on the surface of the engineered extracellular vesicle that is at least 80% of the theoretical maximum number of units of immune checkpoint moiety that can be expressed on the surface of the engineered extracellular vesicle. In some embodiments, the platforms and methods described herein can generate and select for a heterogeneous population of engineered extracellular vesicles expressing a number of units of immune checkpoint moiety on the surface of the engineered extracellular vesicle that is at least 85% of the theoretical maximum number of units of immune checkpoint moiety that can be expressed on the surface of the engineered extracellular vesicle. In some embodiments, the platforms and methods described herein can generate and select for a heterogeneous population of engineered extracellular vesicles expressing a number of units of immune checkpoint moiety on the surface of the engineered extracellular vesicle that is at least 90% of the theoretical maximum number of units of immune checkpoint moiety that can be expressed on the surface of the engineered extracellular vesicle. In some embodiments, the platforms and methods described herein can generate and select for a heterogeneous population of engineered extracellular vesicles expressing a number of units of immune checkpoint moiety on the surface of the engineered extracellular vesicle that is at least 95% of the theoretical maximum number of units of immune checkpoint moiety that can be expressed on the surface of the engineered extracellular vesicle. In some embodiments, the platforms and methods described herein can generate and select for a heterogeneous population of engineered extracellular vesicles expressing a number of units of immune checkpoint moiety on the surface of the engineered extracellular vesicle that is at least 95% of the theoretical maximum number of units of immune checkpoint moiety that can be expressed on the surface of the engineered extracellular vesicle.

[0096] In some embodiments, each extracellular vesicle expresses a number of units of immune checkpoint moiety on the surface of the engineered extracellular vesicle, where the number can be about 5 units to about 1,000,000 units. In some embodiments, each extracellular vesicle expresses a number of units of immune checkpoint moiety on the surface of the engineered extracellular vesicle, where the number can be about 5 units to about 10 units, about 5 units to about 50 units, about 5 units to about 100 units, about 5 units to about 500 units, about 5 units to about 1,000 units, about 5 units to about 5,000 units, about 5 units to about 10,000 units, about 5 units to about 50,000 units, about 5 units to about 100,000 units, about 5 units to about 500,000 units, about 5 units to about 1,000,000 units, about 10 units to about 50 units, about 10 units to about 100 units, about 10 units to about 500 units, about 10 units to about 1,000 units, about 10 units to about 5,000 units, about 10 units to about 10,000 units, about 10 units to about 50,000 units, about 10 units to about 100,000 units, about 10 units to about 500,000 units, about 10 units to about 1,000,000 units, about 50 units to about 100 units, about 50 units to about 500 units, about 50 units to about 1,000 units, about 50 units to about 5,000 units, about 50 units to about 10,000 units, about 50 units to about 50,000 units, about 50 units to about 100,000 units, about 50 units to about 500,000 units, about 50 units to about 1,000,000 units, about 100 units to about 500 units, about 100 units to about 1,000 units, about 100 units to about 5,000 units, about 100 units to about 10,000 units, about 100 units to about 50,000 units, about 100 units to about 100,000 units, about 100 units to about 500,000 units, about 100 units to about 1,000,000 units, about 500 units to about 1,000 units, about 500 units to about 5,000 units, about 500 units to about 10,000 units, about 500 units to about 50,000 units, about 500 units to about 100,000 units, about 500 units to about 500,000 units, about 500 units to about 1,000,000 units, about 1,000 units to about 5,000 units, about 1,000 units to about 10,000 units, about 1,000 units to about 50,000 units, about 1,000 units to about 100,000 units, about 1,000 units to about 500,000 units, about 1,000 units to about 1,000,000 units, about 5,000 units to about 10,000 units, about 5,000 units to about 50,000 units, about 5,000 units to about 100,000 units, about 5,000 units to about 500,000 units, about 5,000 units to about 1,000,000 units, about 10,000 units to about 50,000 units, about 10,000 units to about 100,000 units, about 10,000 units to about 500,000 units, about 10,000 units to about 1,000,000 units, about 50,000 units to about 100,000 units, about 50,000 units to about 500,000 units, about 50,000 units to about 1,000,000 units, about 100,000 units to about 500,000 units, about 100,000 units to about 1,000,000 units, or about 500,000 units to about 1,000,000 units. In some embodiments, each extracellular vesicle expresses a number of units of immune checkpoint moiety on the surface of the engineered extracellular vesicle, where the number can be about 5 units, about 10 units, about 50 units, about 100 units, about 500 units, about 1,000 units, about 5,000 units, about 10,000 units, about 50,000 units, about 100,000 units, about 500,000 units, or about 1,000,000 units. In some embodiments, each extracellular vesicle expresses a number of units of immune checkpoint moiety on the surface of the engineered extracellular vesicle, where the number can be at least about 5 units, about 10 units, about 50 units, about 100 units, about 500 units, about 1,000 units, about 5,000 units, about 10,000 units, about 50,000 units, about 100,000 units, or about 500,000 units. In some embodiments, each extracellular vesicle expresses a number of units of immune checkpoint moiety on the surface of the engineered extracellular vesicle, where the number can be at most about 10 units, about 50 units, about 100 units, about 500 units, about 1,000 units, about 5,000 units, about 10,000 units, about 50,000 units, about 100,000 units, about 500,000 units, or about 1,000,000 units. Targeting moiety

[0097] Described herein, in some embodiments, are engineered extracellular vesicles comprising at least one targeting moiety. In some embodiments, the targeting moiety can be expressed on the surface of the engineered extracellular vesicle. In some embodiments, the targeting moiety can be secreted by the engineered extracellular vesicle. The engineered extracellular vehicles comprising the targeting moiety localizes at the target cell or target environment is at least 2 fold, 5 fold, 10 fold, 50 fold, 100 fold, 200 fold, 500 fold, 1,000 fold, 5,000 fold, or 10,000 fold higher compared to localization of an extracellular vesicle lacking the targeting moiety. In some embodiments, the targeting moiety comprises EBV glycoprotein 350, which targets CD19+ B cells. In some embodiments, the targeting moiety comprises LAMP2B, which targets acetylcholine receptors on neurons. In some embodiments, the targeting moiety comprises C1C2 domain of lactadherin, which target immune cells or blood cells. In some embodiments, the targeting moiety comprises PDGFR, which targets EGFR or cells expressing EGFR. In some embodiments, the targeting moiety comprises GPI-anchored membrane proteins. In some embodiments, the targeting moiety can target a cell surface protein or a protein secreted by the target cell. Non-limiting examples of the cell surface or secreted proteins include any one of the chemokines described herein. In some embodiments, the targeting moiety can comprise an integrin or a fragment thereof. In some embodiments, the targeting moiety comprises a peptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to an integrin. In some embodiments, the targeting moiety comprises a peptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to an a or p subunit of an integrin. In some embodiments, the targeting moiety comprises a peptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to a combination of the a and subunit of the integrin. Integrin can comprise any combination of the a and P subunits. Integrin a subunit includes CD49, CD49b, CD49c, CD49d, CD49e, CD49f, ITGA7, ITGA8, ITGA9, ITGA10, ITGA11, CD11D, CD103, CDl la, CDl lb, CD51, CD41, or CDl lc. Integrin p subunit includes CD29, CD18, CD61, CD104, ITGB5, ITGB6, ITGB7, or ITGB8. Exemplary integrin can include aipi, a2pi, a3pi, a4pi, a5pi, a6pi, a7pi, aLp2, aMp2, allbp3, aVpl, aVp3, aVp5, aVp6, aVp8, aXp2, aDp2, or a6p4.

[0098] In some embodiments, the targeting moiety can be antibody. In some cases, the antibody can be a humanized antibody or binding fragment thereof, a chimeric antibody or binding fragment thereof, a monoclonal antibody or binding fragment thereof, or a bispecific antibody or binding fragment thereof. In some cases, the antibody comprises a monovalent Fab, a divalent Fab’2, a single-chain variable fragment (scFv), a diabody, a minibody, a nanobody, a singledomain antibody (sdAb), or a camelid antibody or binding fragment thereof. [0099] In some embodiments, the engineered extracellular vesicle comprising the targeting moiety exhibits decreased accumulation in liver, spleen, or kidney. In some embodiments, the engineered extracellular vesicle comprising the targeting moiety exhibits decreased off-target effects. For example, the engineered extracellular vesicle comprising the targeting moiety exhibits decreased accumulation at cells, sites, or microenvironments that are not in need of treatment.

Fusion Proteins

[00100] Described herein, in some embodiments, is a composition comprising a fusion protein or polypeptide, with the fusion protein or polypeptide comprising an immune checkpoint moiety and a transmembrane moiety.

[00101] In some embodiments, the fusion peptide or polypeptide comprises a plurality of immune checkpoint moieties. In some embodiments, the fusion protein or polypeptide comprises both an immune checkpoint and a transmembrane moiety comprising a full-length protein, a variation thereof, or a fragment thereof. In some embodiments, the fusion peptide of polypeptide comprises a plurality of transmembrane moieties. In some embodiments, there is a plurality of immune checkpoint moieties. In some embodiments, there is a single immune checkpoint moiety. [00102] In some embodiments, the immune checkpoint moiety is endogenous to the cell that is generating the engineered extracellular vesicles. In some embodiments, the immune checkpoint moiety is heterologous to the cell that is generating the engineered extracellular vesicles. In some embodiments, the immune checkpoint moiety comprises therapeutic properties for treating diseases or disorders. In some embodiments, the immune checkpoint moiety comprises therapeutic properties for treating an inflammatory or autoimmune disease or disorder described herein. In some embodiments, the immune checkpoint moiety targets and modulates activities of immune cells. In some embodiments, the immune cells can be T cell, including cytotoxic T cell, Natural Killer T cell, Regulatory T cell, and T helper cells. In some embodiments, the immune cells can be CD8+ cells. In some embodiments, the immune cells can be CD25+ cells. In some embodiments, the immune cells can be CD4+ cells. In some embodiments, the immune cells can be CD8+ CD25+ cells. In some cases, the immune cell can be cell that expresses CD4. In some cases, the immune cell can be cell that expresses CD4 and CD 25 (CD4+CD25+). In some cases, the immune cell can be cell that expresses FOXP3. In some cases, the immune cell can be cell that expresses CD4, CD25, and FOXP3 (CD4+CD25+FOXP3+).

[00103] In some embodiments, the one or more checkpoint moieties can be any of the following VISTA, CTLA-4, PD-L1, PD-1, and IGSF11 (VSIG-3). In some embodiments, the immune checkpoint moiety comprises a polypeptide sequence that is at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or from 60-100%, 60-90%, 60-80%, 60-70%, 70-100%, 70-90%, 70-80%, 80- 100%, 80-90%, 90-100% identical to VISTA, PD-L1, CTLA-4, PD-L2, B7-1 (CD80), B7-2 (CD86), B7-H3 (CD276), B7-H2, B7-H4 (VTCN1), HVEM (CD270, TNFRSF14), Galectin 9, Galectin3, CEACAM1 (CD66a), OX-2 (CD200), PVR (CD155), PVRL2 (Nectin-2, GDI 12), FGL-1, PECAM-1, TSG-6, CD47, Stabilin-1 (Clever-1), Neuropilin 1, Neuropilin 2, CD158 (family), IGSF2 (CD101), CD155, GITRL, CD137L, OX40L, LIGHT, CD70, PD-1, RGMB, CTLA-4 (CD 152), BTLA, CD 160, Tim-3, CD200R, TIGIT, CD112R (PVRIG), LAG-3 (CD223), PECAM-1, CD44, SIRP alpha (CD172a), IGSF11 (VSIG-3), or a combination thereof. In some embodiments, the immune checkpoint moiety can be encoded by a polynucleotide sequence encoding VISTA, PD-L1, IGSF11 (VSIG-3), or CLLA-4. In some embodiments, the immune checkpoint moiety comprises a polynucleotide sequence encoding a polypeptide or a variation thereof or a fragment thereof. In some embodiments, the immune checkpoint moiety comprises a polypeptide or a variation thereof or a fragment thereof. In some embodiments, the immune checkpoint moiety can be encoded by a polynucleotide sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%. 85%. 90%, 95%, or 99% identical to any one of SEQ ID NOS: 4-15 or 23-28 (Table 2 and Table 4).

[00104] In some embodiments, the immune checkpoint moiety can be encoded by a polynucleotide sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%. 85%. 90%, 95%, or 99% identical to SEQ ID NO: 4. In some embodiments, the immune checkpoint moiety can be encoded by a polynucleotide sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%. 85%. 90%, 95%, or 99% identical to SEQ ID NO: 5. In some embodiments, the immune checkpoint moiety can be encoded by a polynucleotide sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%. 85%. 90%, 95%, or 99% identical to SEQ ID NO: 6. In some embodiments, the immune checkpoint moiety can be encoded by a polynucleotide sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%. 85%. 90%, 95%, or 99% identical to SEQ ID NO: 7. In some embodiments, the immune checkpoint moiety can be encoded by a polynucleotide sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%. 85%. 90%, 95%, or 99% identical to SEQ ID NO: 8. In some embodiments, the immune checkpoint moiety can be encoded by a polynucleotide sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%. 85%. 90%, 95%, or 99% identical to SEQ ID NO: 9. In some embodiments, the immune checkpoint moiety can be encoded by a polynucleotide sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%. 85%. 90%, 95%, or 99% identical to SEQ ID NO: 10. In some embodiments, the immune checkpoint moiety can be encoded by a polynucleotide sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%. 85%. 90%, 95%, or 99% identical to SEQ ID NO: 11. In some embodiments, the immune checkpoint moiety can be encoded by a polynucleotide sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%. 85%. 90%, 95%, or 99% identical to SEQ ID NO: 12. In some embodiments, the immune checkpoint moiety can be encoded by a polynucleotide sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%. 85%. 90%, 95%, or 99% identical to SEQ ID NO: 13. In some embodiments, the immune checkpoint moiety can be encoded by a polynucleotide sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%. 85%. 90%, 95%, or 99% identical to SEQ ID NO: 14. In some embodiments, the immune checkpoint moiety can be encoded by a polynucleotide sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%. 85%. 90%, 95%, or 99% identical to SEQ ID NO: 15. In some embodiments, the immune checkpoint moiety can be encoded by a polynucleotide sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%. 85%. 90%, 95%, or 99% identical to SEQ ID NO: 23. In some embodiments, the immune checkpoint moiety can be encoded by a polynucleotide sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%. 85%. 90%, 95%, or 99% identical to SEQ ID NO: 24. In some embodiments, the immune checkpoint moiety can be encoded by a polynucleotide sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%. 85%. 90%, 95%, or 99% identical to SEQ ID NO: 25. In some embodiments, the immune checkpoint moiety can be encoded by a polynucleotide sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%. 85%. 90%, 95%, or 99% identical to SEQ ID NO: 26. In some embodiments, the immune checkpoint moiety can be encoded by a polynucleotide sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%. 85%. 90%, 95%, or 99% identical to SEQ ID NO: 27. In some embodiments, the immune checkpoint moiety can be encoded by a polynucleotide sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%. 85%. 90%, 95%, or 99% identical to SEQ ID NO: 28.

[00105] In some embodiments, the immune checkpoint moiety can be VISTA and encoded from polynucleotide or a variation thereof or a fragment thereof that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%. 85%. 90%, 95%, or 99% identical to SEQ ID NOs: 4-6 or 25. In some embodiments, the immune checkpoint moiety can be PD-L1 and encoded from polynucleotide or a variation thereof or a fragment thereof that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%. 85%. 90%, 95%, or 99% identical to SEQ ID NOs: 7-9 or 24. In some embodiments, the immune checkpoint moiety can be IGSF11 (VSIG-3) and encoded from polynucleotide or a variation thereof or a fragment thereof that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%. 85%. 90%, 95%, or 99% identical to SEQ ID NOs: 10-12 or 26. In some embodiments, the immune checkpoint moiety can be CTLA-4 and encoded from polynucleotide or a variation thereof or a fragment thereof that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%. 85%. 90%, 95%, or 99% identical to SEQ ID NOs: 13-15 or 27. In some embodiments, the immune checkpoint moiety can be OX-2 (CD200) and encoded from polynucleotide or a variation thereof or a fragment thereof that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%. 85%. 90%, 95%, or 99% identical to SEQ ID NO:23. In some embodiments, the immune checkpoint moiety can be BTLA and encoded from polynucleotide or a variation thereof or a fragment thereof that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%. 85%. 90%, 95%, or 99% identical to SEQ ID NO: 28

[00106] In some embodiments, the immune checkpoint moiety can be encoded by a heterologous polynucleotide. In some embodiments, the heterologous polynucleotide can be introduced into in any one of the cells described herein. In some embodiments, the heterologous polynucleotide comprises mRNA, rRNA, SRP RNA, tRNA, tmRNA, snRNA, snoRNA, gRNA, aRNA, crRNA, IncRNA, miRNA, ncRNA, piRNA, siRNA, and shRNA. In some cases, the heterologous polynucleotide comprises mRNA. In some embodiments, the heterologous polynucleotide comprises DNA. In some cases, the heterologous polynucleotide can be inserted into the transmembrane moiety described herein. In some embodiments, the heterologous polynucleotide can be inserted into the transmembrane moiety described herein via homologous recombination. In some embodiments, the heterologous polynucleotide comprises a nucleic sequence encoding VISTA, PD-L1, CTLA-4, PD-L2, B7-1 (CD80), B7-2 (CD86), B7-H3 (CD276), B7-H2, B7-H3, B7-H4 (VTCN1), IDO, KIR, LAG3, A2AR, HVEM (CD270, TNFRSF14), Galectin 9, Galectin3, CEACAM1 (CD66a), OX-2 (CD200), PVR (CD155), PVRL2 (Nectin-2, CD112), FGL-1, PECAM-1, TSG-6, CD47, Stabilin-1 (Clever-1), Neuropilin 1, Neuropilin 2, CD158 (family), IGSF2 (CD101), CD155, GITRL, CD137L, OX40L, LIGHT, CD70, PD-1, RGMB, CTLA-4 (CD 152), BTLA, CD 160, Tim-3, CD200R, TIGIT, CD112R (PVRIG), LAG-3 (CD223), PECAM-1, CD44, IGSF11 (VSIG-3), or SIRP alpha (CD 172a). In some embodiments, the immune checkpoint moiety can be encoded from a heterologous polynucleotide encoding VISTA. In some embodiments, the immune checkpoint moiety can be encoded from a heterologous polynucleotide encoding PD-L1. In some embodiments, the immune checkpoint moiety can be encoded from a heterologous polynucleotide encoding IGSF11 (VSIG-3). In some embodiments, the immune checkpoint moiety can be encoded from a heterologous polynucleotide encoding CTLA-4.

[00107] In some embodiments, the immune checkpoint moiety comprises a heterologous polynucleotide encoding a cytokine. In some embodiments, the immune checkpoint moiety comprises a polypeptide comprising a peptide sequence of the cytokine. Exemplary cytokines that can be utilized as the immune check point moiety includes 4-1BBL, acylation stimulating protein, adipokine, albinterferon, APRIL, Arh, BAFF, Bcl-6, CCL1, CCL1/TCA3, CCL11, CCL12/MCP-5, CCL13/MCP-4, CCL14, CCL15, CCL16, CCL17/TARC, CCL18, CCL19, CCL2, CCL2/MCP-1, CCL20, CCL21, CCL22/MDC, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CCL3, CCL3L3, CCL4, CCL4L1/LAG-1, CCL5, CCL6, CCL7, CCL8, CCL9, CCR10, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CD153, CD154, CD178, CD40LG, CD70, CD95L/CD178, Cerberus (protein), chemokines, CLCF1, CNTF, colony-stimulating factor, common b chain (CD131), common g chain (CD132), CX3CL1, CX3CR1, CXCL1, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCL17, CXCL2, CXCL2/MIP- 2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL9, CXCR3, CXCR4, CXCR5, EDA-A1, Epo, erythropoietin, FAM19A1, FAM19A2, FAM19A3, FAM19A4, FAM19A5, Flt-3L, FMS- like tyrosine kinase 3 ligand, Foxp3, GATA-3, GcMAF, G-CSF, GITRL, GM-CSF, granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, hepatocyte growth factor, IFNA1, IFNA10, IFNA13, IFNA14, IFNA2, IFNA4, IFNA5/IFNaG, IFNA7, IFNA8, IFNB1, IFNE, IFNG, IFNZ, IFN-a, IFN-p, IFN-y, IFNro/IFNWl, IL-1, IL- 10, IL- 10 family, IL- 10-like, IL-11, IL- 12, IL-13, IL-14, IL- 15, IL- 16, IL- 17, IL- 17 family, IL-17A-F, IL- 18, IL- 18BP, IL-19, IL-1A, IL-1B, IL-1F10, IL-1F3/IL-1RA, IL-1F5, IL-1F6, IL-1F7, IL-1F8, IL-1F9, IL-l-like, IL-IRA, IL-1RL2, IL-la, IL-lp, IL-2, IL-20, IL-21, IL-22, IL-23, IL-24, IL-28A, IL- 286, IL-29, IL-3, IL-31, IL-33, IL-35, IL-4, IL-5, IL-6, IL-6-like, IL-7, IL-8/CXCL8, IL-9, inflammasome, interferome, interferon, interferon beta-la, interferon beta-lb, interferon gamma, interferon type I, interferon type II, interferon type III, interferons, interleukin, interleukin 1 receptor antagonist, Interleukin 8, IRF4, Leptin, leukemia inhibitory factor (LIF), leukocytepromoting factor, LIGHT, LTA/TNFB, LT-p, lymphokine, lymphotoxin, lymphotoxin alpha, lymphotoxin beta, macrophage colony-stimulating factor, macrophage inflammatory protein, macrophage-activating factor, M-CSF, MHC class III, miscellaneous hematopoietins, monokine, MSP, myokine, myonectin, nicotinamide phosphoribosyltransferase, oncostatin M (OSM), oprelvekin, OX40L, platelet factor 4, promegapoietin, RANKE, SCF, STAT3, STAT4, STAT6, stromal cell-derived factor 1, TALL-1, TBX21, TGF-a, TGF-p, TGF-pl, TGF-p2, TGF-p3, TNF, TNFSF10, TNFSF11, TNFSF12, TNFSF13, TNFSF14, TNFSF15, TNFSF4, TNFSF8, TNF-a, TNF-P, Tpo, TRAIL, TRANCE, TWEAK, vascular endothelial growth inhibitor, XCL1, or XCL2.

[00108] In some embodiments, the immune checkpoint moiety can be complexed with the transmembrane moiety described herein to form the fusion proteins or polypeptides described herein. In some embodiments, the immune checkpoint moiety can be non-covalently complexed with the transmembrane moiety described herein. In some embodiments, the immune checkpoint moiety can be covalently complexed with the transmembrane moiety described herein. In some embodiments, the immune checkpoint moiety can be expressed as part of a fusion protein comprising both the immune checkpoint moiety and the transmembrane moiety. In some embodiments, the immune checkpoint moiety can be expressed as part of a fusion protein comprising both the immune checkpoint moiety and a fragment of the transmembrane moiety. In some embodiments, the N-terminus of the immune checkpoint moiety can be fused to the transmembrane moiety. In some embodiments, the C-terminus of the immune checkpoint moiety can be fused to the transmembrane moiety described herein. In some embodiments, the immune checkpoint moiety can be fused and flanked by the transmembrane moiety on both N and C- terminus of the immune checkpoint moiety. For example, the immune checkpoint moiety can be inserted into a transmembrane moiety as part of a fusion peptide, where the N-terminus of the fusion peptide comprises a fragment of the transmembrane moiety, followed by the immune checkpoint moiety (or a variation there or a fragment thereof), and followed by the C-terminus of the fusion peptide comprising another fragment of the transmembrane moiety. In some embodiments, the immune checkpoint moiety comprises the fusion peptide, where the immune checkpoint moiety is fused to the transmembrane moiety. In some embodiments, the immune checkpoint moiety comprises the immune checkpoint moiety complexed with the transmembrane moiety. In some embodiments, the immune checkpoint moiety comprises the immune checkpoint moiety non-covalently complexed with the transmembrane moiety. In some embodiments, the immune checkpoint moiety comprises the immune checkpoint moiety covalently complexed with the transmembrane moiety.

[00109] In some embodiments, a plurality of immune checkpoint moieties can be expressed on the surface of an extracellular vesicle. In some embodiments, the extracellular vesicle is an engineered extracellular vesicle. In some embodiments, the number of units of the immune checkpoint moiety that can be expressed on the surface of the engineered extracellular vesicle is limited by a theoretical maximum as determined by the ratio between: the dimensions of the engineered extracellular vesicle; and the dimensions of the expressed immune checkpoint moiety or the expressed fusion peptide comprising the immune checkpoint moiety

[00110] In some embodiments, the composition fusion protein or polypeptide is expressed on the surface of an extracellular vesicle.

[00111] In some embodiments, the fusion protein or polypeptide is encoded for by a heterologous polynucleotide.

[00112] In some embodiments, the fusion protein or polypeptide comprise at least one transmembrane moiety. In some embodiments, the transmembrane moiety comprises a full- length protein or a variation thereof or a fragment thereof. In some embodiments, the transmembrane moiety is endogenous to the cell that is generating the engineered extracellular vesicles. In some embodiments, the immune checkpoint moiety is heterologous to the cell that is generating the engineered extracellular vesicles. In some embodiments, the transmembrane moiety is selected from a group consisting of: 14-3-3 protein zeta/delta, 14-3-3 protein epsilon, 78 kDa glucose-regulated protein, acetylcholinesterase/AChE-S, AChE-E, actin, cytoplasmic 1 (ACTA), ADAMIO, alkaline phosphatase, alpha-enolase, alpha-synuclein, aminopeptidase N, amyloid beta A4/APP, annexin 5A, annexin A2, AP-1, ATF3, ATP citrate lyase, ATPase, beta actin (ACTB), beta-amyloid 42, caveolin-1, CD10, CDl la, GDI lb, CDl lc, CD14, CD142, CD146, CD163, CD24, CD26/DPP4, CD29/ITGB1, CD3, CD37, CD41, CD42a, CD44, CD45, CD47, CD49, CD49d, CD53, CD63, CD64, CD69, CD73 CD81, CD82, CD9, CD90, claudin, claudin-1 cofilin-1, complement-binding proteins CD55 and CD59, cytosolic heat shock protein 90 alpha, cytosolic heat shock protein 90 beta, EBV LMP1, EBV LMP2A, EF-lalpha-1, EF2, EFGR EGFR VIII, emmprin/CD147, enolase 1 alpha (ENO1), EPCAM, ERBB2, tetraspanins (CD9, CD63 and CD81), fatty acid synthase, fetuin-A, flotillin-1, flotillin-2, fructose- bisphosphate aldolase A, glyceraldehyde-3 -phosphate dehydrogenase (GAPDH), glycophorin A, GPC1, GPI-anchored 5 'nucleotidase, GTPase, heat shock protein 8 (HSPA8), heat shock proteins (HSP70 and HSP90), heparan sulfate proteoglycans, heparinase, heterotrimeric G proteins, HIV Gag, HIV Nef, HLA-DRA, HLA-G, HSV gB, HTLV-1 Tax, huntingtin, ICAM1, integrins, lactadherin, LAMP 1/2, LAMP2A, LAMP2B, LAMP2C, leucine-rich receptor kinase 2, L-lactate dehydrogenase A chain, lysosome-associated membrane glycoprotein 1, lysosome-associated membrane glycoprotein 2, MHC class I, MHC class II, MUC1, multi drug resistance-associated protein, muscle pyruvate kinase (PKM2), N-cadherin, NKCC2, PDCD6IP/Alix, PEC AMI, phosphoglycerate kinase, placental prion proteins, prostate-specific antigen (PSA), pyruvate kinase (PKM), Rab-14, Rab-5a, Rab-5b, Rab-5c, Rab-7, Rap IB, resistin, sonic hedgehog (SHH), surviving, syndecan-1, syndecan-4, syntenin-1, transferrin receptor (TFR2), TSG101, TSPAN8, tumor-associated glycoprotein tetraspanin- 8, tyrosine 3 monooxygenase/tryptophan 5- monooxygenase activation protein, TYRP-2, or vacuolar-sorting-associated protein 35 (VPS35) [00113] In some embodiments, the transmembrane moiety comprises LAMP2. In some embodiments, the transmembrane moiety comprises LAMP-like domain 1 of LAMP2. In some embodiments, the transmembrane moiety comprises LAMP2B. In some embodiments, the transmembrane moiety comprises a polypeptide sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%. 85%. 90%, 95%, or 99% identical to SEQ ID NO: 1 (Table 1). In some embodiments, the transmembrane moiety can be encoded from a polynucleotide sequence comprising homology arms. In some instances, the immune checkpoint moiety described herein can be inserted into the transmembrane moiety by homologous recombination as induced by the homology arms of the transmembrane moiety. In some embodiments, the transmembrane moiety comprises an N-terminus homology arm or a C-terminus homology arm. In some embodiments, the N-terminus homology of the transmembrane moiety is encoded from a polynucleotide sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%. 85%. 90%, 95%, or 99% identical to SEQ ID NO: 2 (Table 1). In some embodiments, the C-terminus homology of the transmembrane moiety is encoded from a polynucleotide sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%. 85%. 90%, 95%, or 99% identical to SEQ ID NO: 3 (Table 1). [00114] In some embodiments, the transmembrane moiety can be complexed with the immune checkpoint moiety described herein to generate the fusion proteins or polypeptides described herein. In some embodiments, the transmembrane moiety can be non-covalently complex with the immune checkpoint moiety described herein. In some embodiments, the transmembrane moiety can be covalently complexed with the immune checkpoint moiety described herein. In some embodiments, the transmembrane moiety can be fused to the immune checkpoint moiety described herein at the N-terminus of the transmembrane moiety. In some embodiments, the transmembrane moiety can be fused to the immune checkpoint moiety described herein at the C- terminus of the transmembrane moiety. In some embodiments, the immune checkpoint moiety can be inserted (e.g. via homology recombination) at any locus of the transmembrane moiety. [00115] In some embodiments, the transmembrane moiety can be covalently connected to the immune checkpoint moiety by a peptidyl linker. In some cases, the linker can be a flexible linker, a rigid linker, or a cleavable linker. In some embodiments, the flexible linker can be (GGGGS)n where n=l, 2, 3, or 4; KESGSVSSEQLAQFRSLD; EGKSSGSGSESKST; GGGGGG;

GGGGGGGG; GSAGSAAGSGEF; RKRR; SS; or LE. In some instances, the rigid linker can be (EAAAK)n where n=l, 2, 3, or 4; A(EAAAK)nA where n=2, 3, 4, or 5;

A(EAAAK)₄ALEA(EAAAK)₄A; AEAAAKEAAAKA; or LEAGCKNFFPRSFTSCGSLE. In some aspects, the cleavable linker can be LEAGCKNFFPRSFTSCGSLE; CRRRRRREAEAC; GGIEGRGS; or TRHRQPRGWEQL.

Methods

Generating engineered extracellular vesicles

[00116] Described herein, in some embodiments, are methods for generating the compositions descried herein utilizing the platforms described herein. In some embodiments, described herein are methods for generating the engineered extracellular vesicles described herein. In some instances, the engineered extracellular vesicles can be isolated from or released by a cell described herein. For example, engineered extracellular vesicles can be generated from lysing the cells to release the engineered extracellular vesicles. In some cases, the cells secrete or release the engineered extracellular vesicles, where the engineered extracellular vesicles can then be isolated. An exemplary workflow for generating and isolating the engineered extracellular vesicles described herein is illustrated in FIG. 5.

[00117] In some embodiments, the cells for generating the engineered extracellular vesicle can be cells from cell lines, stem cells, primary cells, or differentiated cells. In some embodiments, the cells can be selected from the group consisting of human embryonic fibroblasts (HEF), dendritic cells, mesenchymal stem cells, bone marrow-derived dendritic cells, bone marrow derived stromal cells, adipose stromal cells, endothelial cells, enucleated cells, neural stem cells, immature dendritic cells, and immune cells, bone marrow stromal cells, marrow derived adult progenitor cells (MAPCs), endothelial progenitor cells (EPC), blast cells, intermediate progenitor cells formed in the subventricular zone, neural stem cells, muscle stem cells, satellite cells, liver stem cells, hematopoietic stem cells, bone marrow stromal cells, epidermal stem cells, embryonic stem cells, umbilical cord stem cells, precursor cells, muscle precursor cells, myoblast, cardiomyoblast, neural precursor cells, glial precursor cells, neuronal precursor cells, or hepatoblasts.

[00118] In some embodiments, the cell for generating the engineered extracellular vesicles can be a genetically modified cell, where a genetic modification moiety is introduced into the modified cell. In some embodiments, at least one heterologous polynucleotide encoding any one of the immune checkpoint moieties described herein is introduced into the modified cell. In some embodiments, the heterologous polynucleotide encodes any one of the targeting moieties described herein. In some embodiments, the heterologous polynucleotide encodes any one of the transmembrane moieties described herein. In some embodiments, the heterologous polynucleotide encodes any one of the fusion peptides described herein. In some embodiments, the heterologous polynucleotide encodes any one of the immune evasion moieties described herein. In some embodiments, the heterologous polynucleotide can be integrated into the chromosome of the modified cell. In some embodiments, the heterologous polynucleotide is not integrated into the chromosome of the modified cell. In some embodiments, the heterologous polynucleotide can be a vector or plasmid comprising nucleic acid sequence encoding the transmembrane moiety. In some embodiments, the heterologous polynucleotide can be a vector or plasmid comprising nucleic acid sequences encoding both the transmembrane moiety and the immune checkpoint moiety. In some embodiments, the heterologous polynucleotide can be a vector or plasmid comprising nucleic acid sequences encoding the immune checkpoint moiety being flanked by the transmembrane moiety (FIG. 4). In such case, the immune checkpoint moiety can be inserted via homologous recombination.

[00119] In some embodiments, the heterologous polynucleotide can be a linear vector comprising single-stranded nucleic acid sequence encoding the transmembrane moiety or the immune checkpoint moiety. In some embodiments, the heterologous polynucleotide can be a linear vector comprising single- stranded nucleic acid sequence encoding the transmembrane moiety and the immune checkpoint moiety. In some embodiments, the heterologous polynucleotide can be a linear vector comprising double-stranded nucleic acid sequence encoding the transmembrane moiety or the immune checkpoint moiety. In some embodiments, the heterologous polynucleotide can be a linear vector comprising double-stranded nucleic acid sequence encoding the transmembrane moiety and the immune checkpoint moiety. In some embodiments, the heterologous polynucleotide comprising the linear vector comprises DNA nucleotides, RNA nucleotides, or a combination thereof. In some embodiments, the heterologous polynucleotide comprising the linear vector comprises single-stranded DNA. In some embodiments, the heterologous polynucleotide comprising the linear vector comprises singlestranded RNA. In some embodiments, the heterologous polynucleotide comprising the linear vector comprises double-stranded DNA. In some embodiments, the heterologous polynucleotide comprising the linear vector comprises double- stranded RNA. In some embodiments, the heterologous polynucleotide comprising the linear vector comprises a combination of singlestranded DNA and single-stranded RNA. In some embodiments, the heterologous polynucleotide comprising the linear vector comprises a combination of double-stranded DNA and doublestranded RNA. In some embodiments, the transmembrane moiety encoded by the linear vector comprises nucleic acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%. 85%. 90%, 95%, or 99% identical to any one of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 21, or SEQ ID NO: 22 (Table 3)

Table 3. Non-limiting example of nucleic acid sequence of homology arm

[00120] In some embodiments, the heterologous polynucleotide comprising the linear vector comprises nucleic acid sequence of at least one homology arm. In some embodiments, the heterologous polynucleotide comprising the linear vector comprises nucleic acid sequence of two homology arms. In some embodiments, the heterologous polynucleotide comprising the linear vector comprises nucleic acid sequence of at least one homology arm comprising nucleic acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%. 85%. 90%, 95%, or 99% identical to any one of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 21, or SEQ ID NO: 22 [00121] In some embodiments, the heterologous polynucleotide comprising the linear vector comprises nucleic acid sequence encoding an immune checkpoint moiety of any one of the immune checkpoint moiety described herein. In some embodiments, the immune checkpoint moiety can be encoded from nucleic acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%. 85%. 90%, 95%, or 99% identical to SEQ ID NOs: 4-15. In some embodiments, the immune checkpoint moiety can be encoded from nucleic acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%. 85%. 90%, 95%, or 99% identical to SEQ ID NOs: 23-28 (Table 4). In some embodiments, the immune checkpoint moiety can be encoded from nucleic acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%. 85%. 90%, 95%, or 99% identical to SEQ ID NO: 23. In some embodiments, the immune checkpoint moiety can be encoded from nucleic acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%. 85%. 90%, 95%, or 99% identical to SEQ ID NOs: 24. In some embodiments, the immune checkpoint moiety can be encoded from nucleic acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%. 85%. 90%, 95%, or 99% identical to SEQ ID NOs: 25. In some embodiments, the immune checkpoint moiety can be encoded from nucleic acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%. 85%. 90%, 95%, or 99% identical to SEQ ID NOs: 26. In some embodiments, the immune checkpoint moiety can be encoded from nucleic acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%. 85%. 90%, 95%, or 99% identical to SEQ ID NOs: 27. In some embodiments, the immune checkpoint moiety can be encoded from nucleic acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%. 85%. 90%, 95%, or 99% identical to SEQ ID NOs: 28.

Table 4. Non-limiting example of nucleic acid sequence of an immune checkpoint moiety

[00122] In some embodiments, the heterologous polynucleotide comprising the linear vector comprises nucleic acid sequence encoding at least one homology arm (e.g., a homology arm for the transmembrane moiety) and the immune checkpoint, some embodiments, the heterologous polynucleotide comprising the linear vector comprises nucleic acid sequence encoding two homology arms (e.g., a N-terminus and a C-terminus homology arm for the transmembrane moiety) and the immune checkpoint. In some embodiments, the heterologous polynucleotide comprising the linear vector comprises nucleic acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%. 85%. 90%, 95%, or 99% identical to SEQ ID NOs: 31-36 (Table 5). In some embodiments, the heterologous polynucleotide comprising the linear vector comprises nucleic acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%. 85%. 90%, 95%, or 99% identical to SEQ ID NO: 31. In some embodiments, the heterologous polynucleotide comprising the linear vector comprises nucleic acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%. 85%. 90%, 95%, or 99% identical to SEQ ID NO: 32. In some embodiments, the heterologous polynucleotide comprising the linear vector comprises nucleic acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%. 85%. 90%, 95%, or 99% identical to SEQ ID NO: 33. In some embodiments, the heterologous polynucleotide comprising the linear vector comprises nucleic acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%. 85%. 90%, 95%, or 99% identical to SEQ ID NO: 34. In some embodiments, the heterologous polynucleotide comprising the linear vector comprises nucleic acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%. 85%. 90%, 95%, or 99% identical to SEQ ID NO: 35. In some embodiments, the heterologous polynucleotide comprising the linear vector comprises nucleic acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%. 85%. 90%, 95%, or 99% identical to SEQ ID NO: 36. Table 5. Non-limiting example of nucleic acid sequence of linear vector encoding an immune checkpoint moiety

[00123] In some embodiments, the genetic modification moiety regulates the expressions of the heterologous polynucleotide encoding the transmembrane moiety and the immune checkpoint moiety. In some embodiments, the genetic modification moiety increases the expressions of the heterologous polynucleotide. In some embodiments, the genetic modification moiety comprises a CRISPR-Cas polypeptide. In some embodiments, the genetic modification moiety can be, for example, Class 1 CRISPR-associated (Cas) polypeptides, Class 2 Cas polypeptides, type I Cas polypeptides, type II Cas polypeptides, type III Cas polypeptides, type IV Cas polypeptides, type V Cas polypeptides, and type VI, CRISPR-associated RNA binding proteins, or a functional fragment thereof. Cas polypeptides suitable for use with the present disclosure can include Cas9, Casl2, Casl3, Cpfl (or Casl2a), C2C1, C2C2 (or Casl3a), Casl3b, Casl3c, Casl3d, C2C3, Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas5e (CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8a, Cas8al, Cas8a2, Cas8b, Cas8c, Csnl, Csxl2, Cas 10, CaslOd, CaslO, CaslOd, CasF, CasG, CasH, Csyl, Csy2, Csy3, Csel (CasA), Cse2 (CasB), Cse3 (CasE), Cse4 (CasC), Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4, or Cul966; any derivative thereof; any variant thereof; or any fragment thereof. In some embodiments, Cas l 3 can include, but are not limited to, Casl3a, Casl3b, Casl3c, and Cas 13d (e g., CasRx). CRISPR/Cas can be DNA and/or RNA cleaving or can exhibit reduced cleavage activity. Genetic modification moiety can be configured to complex with at least one heterologous RNA polynucleotide. In some cases, the genetic modification moiety can be fused with a transcription activator or transcription repressor.

[00124] Any suitable nuclease (e.g., endonuclease) can be used in as the genetic modification moiety. Suitable nucleases include, but are not limited to, CRISPR-associated (Cas) proteins or Cas nucleases including type I CRISPR-associated (Cas) polypeptides, type II CRISPR- associated (Cas) polypeptides, type III CRISPR-associated (Cas) polypeptides, type IV CRISPR- associated (Cas) polypeptides, type V CRISPR-associated (Cas) polypeptides, and type VI CRISPR-associated (Cas) polypeptides; zinc finger nucleases (ZFN); transcription activator-like effector nucleases (TALEN); meganucleases; RNA-binding proteins (RBP); CRISPR-associated RNA binding proteins; recombinases; flippases; transposases; Argonaute (Ago) proteins (e.g., prokaryotic Argonaute (pAgo), archaeal Argonaute (aAgo), eukaryotic Argonaute (eAgo), and Natronobacterium gregoryi Argonaute (NgAgo)); Adenosine deaminases acting on RNA (ADAR); CIRT, PUF, homing endonuclease, or any functional fragment thereof, any derivative thereof; any variant thereof; and any fragment thereof.

[00125] In some embodiments, the genetic modification moiety can be CRISPR/Cas9. In some embodiments, the CRISPR/Cas9 cleaves the nucleic acid sequence encoding the transmembrane moiety. Such cleavage event allows the heterologous polynucleotide encoding the immune checkpoint moiety to be inserted into a locus of the transmembrane moiety via homologous recombination.

[00126] A genetic modification moiety as disclosed herein can be coupled (e.g., linked or fused) to additional peptide sequences which are not involved in regulating gene expression, for example linker sequences, targeting sequences, etc. The term “targeting sequence,” as used herein, refers to a nucleotide sequence and the corresponding amino acid sequence which encodes a targeting polypeptide which mediates the localization (or retention) of a protein to a sub-cellular location, e.g., plasma membrane or membrane of a given organelle, nucleus, cytosol, mitochondria, endoplasmic reticulum (ER), Golgi, chloroplast, apoplast, peroxisome or other organelle. For example, a targeting sequence can direct a protein (e.g., a receptor polypeptide or an adaptor polypeptide) to a nucleus utilizing a nuclear localization signal (NLS); outside of a nucleus of a cell, for example to the cytoplasm, utilizing a nuclear export signal (NES); mitochondria utilizing a mitochondrial targeting signal; the endoplasmic reticulum (ER) utilizing an ER-retention signal; a peroxisome utilizing a peroxisomal targeting signal; plasma membrane utilizing a membrane localization signal; or combinations thereof. [00127] A genetic modification moiety as disclosed herein can be a part of a fusion construct (e.g., a fusion protein). As used herein, “fusion” can refer to a protein and/or nucleic acid comprising one or more non-native sequences (e.g., moieties). A fusion can comprise one or more of the same non-native sequences. A fusion can comprise one or more of different nonnative sequences. A fusion can be a chimera. A fusion can comprise a nucleic acid affinity tag. A fusion can comprise a barcode. A fusion can comprise a peptide affinity tag. A fusion can provide for subcellular localization of the site-directed polypeptide (e.g., a nuclear localization signal (NLS) for targeting to the nucleus, a mitochondrial localization signal for targeting to the mitochondria, a chloroplast localization signal for targeting to a chloroplast, an endoplasmic reticulum (ER) retention signal, and the like). A fusion can provide a non-native sequence (e.g., affinity tag) that can be used to track or purify. A fusion can be a small molecule such as biotin or a dye such as Alexa fluor dyes, Cyanine3 dye, Cyanine5 dye.

[00128] A fusion can refer to any protein with a functional effect. For example, a fusion protein can comprise methyltransferase activity, demethylase activity, dismutase activity, alkylation activity, depurination activity, oxidation activity, pyrimidine dimer forming activity, integrase activity, transposase activity, recombinase activity, polymerase activity (e.g., a reverse transcriptase activity), ligase activity, helicase activity, photolyase activity or glycosylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitinating activity, adenylation activity, deadenylation activity, SUMOylating activity, deSUMOylating activity, ribosylation activity, derib osylati on activity, myristoylation activity, remodelling activity, protease activity, oxidoreductase activity, transferase activity, hydrolase activity, lyase activity, isomerase activity, synthase activity, synthetase activity, or demyristoylation activity. An effector protein can modify a genomic locus. A fusion protein can be a fusion in a Cas protein. A fusion protein can be a non-native sequence in a Cas protein.

[00129] In some embodiments, the genetic modification moiety can be fused to one or more transcription repressor domains, activator domains, epigenetic domains, recombinase domains, transposase domains, flippase domains, nickase domains, or any combination thereof. The activator domain can include one or more tandem activation domains located at the carboxyl terminus of the protein. In some cases, the genetic modification moiety includes one or more tandem repressor domains located at the carboxyl terminus of the protein. Non-limiting exemplary activation domains include GAL4, herpes simplex activation domain VP 16, VP64 (a tetramer of the herpes simplex activation domain VP16), NF-KB p65 subunit, Epstein-Barr virus R transactivator (Rta). Non-limiting exemplary repression domains include the KRAB (Kruppel- associated box) domain of Koxl, the Mad mSIN3 interaction domain (SID), or ERF repressor domain (ERD). In some embodiments, the genetic modification moiety includes one or more tandem repressor domains located at the amino terminus of the protein.

[00130] In some embodiments, the nuclease disclosed herein can be a protein that lacks nucleic acid cleavage activity. In some cases, a Cas protein is a dead Cas protein. A dead Cas protein can be a protein that lacks nucleic acid cleavage activity A Cas protein can comprise a modified form of a wild type Cas protein. The modified form of the wild type Cas protein can comprise an amino acid change (e.g., deletion, insertion, or substitution) that reduces the nucleic acidcleaving activity of the Cas protein. For example, the modified form of the Cas protein can have less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, or less than 1% of the nucleic acidcleaving activity of the wild-type Cas protein (e.g., Cas9 from S. pyogenes). The modified form of Cas protein can have no substantial nucleic acid-cleaving activity. When a Cas protein is a modified form that has no substantial nucleic acid-cleaving activity, it can be referred to as enzymatically inactive and/or “dead” (abbreviated by “d”). A dead Cas protein (e.g., dCas, dCas9) can bind to a target polynucleotide but may not cleave the target polynucleotide. In some aspects, a dead Cas protein is a dead Cas9 protein.

[00131] In some embodiments, a dCas (e g., dCas9) polypeptide can associate with a single guide RNA (sgRNA) to activate or repress transcription of target DNA. sgRNAs can be introduced into cells expressing the engineered chimeric receptor polypeptide. In some cases, such cells contain one or more different sgRNAs that target the same nucleic acid. In other cases, the sgRNAs target different nucleic acids in the cell.

[00132] In some embodiments, the genetic modification moiety can comprise a catalytically inactive Cas polypeptide, where the nuclease activity of the Cas polypeptide is eliminated or substantially eliminated.

[00133] In some instances, the genetic modification moiety can comprise a catalytically inactivated Cas9 (dCas9), any derivative thereof; any variant thereof; or any fragment thereof. [00134] In some instances, the genetic modification moiety can comprise a catalytically inactivated Cas 12 (dCasl2), any derivative thereof; any variant thereof; or any fragment thereof. [00135] In some instances, the genetic modification moiety can comprise a catalytically inactivated Cas 13 (dCasl3); any derivative thereof; any variant thereof; or any fragment thereof. [00136] In some embodiments, the genetic modification moiety can be complexed with the at least one heterologous polynucleotide as described herein. In some embodiments, the at least one heterologous polynucleotide can be either heterologous DNA polynucleotide or heterologous RNA polynucleotide. In some embodiments, the genetic modification moiety can be complexed with at least one heterologous RNA polynucleotide. In some embodiments, the complexing with the at least one heterologous RNA polynucleotide direct and target the genetic modification moiety to the portion of the heterologous polynucleotide.

[00137] In some cases, the compositions and methods described herein comprise at least one heterologous polynucleotide. In some cases, the compositions and methods described herein comprise a plurality of heterologous nucleic acids. In some embodiments, the polynucleotide can be deoxyribonucleic acid (DNA). In some cases, the DNA sequence can be single-stranded or doubled-stranded. In some embodiments, the at least one heterologous nucleic acid polynucleotide can be ribonucleic acid (RNA).

[00138] In some embodiments, the genetic modification moiety can be complexed with the at least one heterologous RNA polynucleotide. The at least one heterologous RNA polynucleotide can comprise a nucleic-acid targeting region that comprises a complementary sequence to a nucleic acid sequence of the heterologous polynucleotide that encodes any one of the moieties described herein for specificity of the genetic modification moiety-dependent targeting. In some embodiments, the at least one heterologous RNA polynucleotide can be guide nucleic acid (or guide RNA) comprising two separate nucleic acid molecules, which can be referred to as a double guide nucleic acid or a single nucleic acid molecule, which can be referred to as a single guide nucleic acid (e.g., sgRNA). In some embodiments, the guide nucleic acid is a single guide nucleic acid comprising a fused CRISPR RNA (crRNA) and a transactivating crRNA (tracrRNA). In some embodiments, the guide nucleic acid is a single guide nucleic acid comprising a crRNA. In some embodiments, the guide nucleic acid is a single guide nucleic acid comprising a crRNA but lacking a tracrRNA. In some embodiments, the guide nucleic acid is a double guide nucleic acid comprising non-fused crRNA and tracrRNA. An exemplary double guide nucleic acid can comprise a crRNA-like molecule and a tracrRNA- like molecule. An exemplary single guide nucleic acid can comprise a crRNA-like molecule. An exemplary single guide nucleic acid can comprise a fused crRNA-like molecule and a tracrRNA-like molecule. [00139] A crRNA can comprise the nucleic acid-targeting segment (e.g., spacer region) of the guide nucleic acid and a stretch of nucleotides that can form one half of a double-stranded duplex of the Cas protein-binding segment of the guide nucleic acid.

[00140] A tracrRNA can comprise a stretch of nucleotides that forms the other half of the double-stranded duplex of the Cas protein-binding segment of the gRNA. A stretch of nucleotides of a crRNA can be complementary to and hybridize with a stretch of nucleotides of a tracrRNA to form the double-stranded duplex of the Cas protein-binding domain of the guide nucleic acid.

[00141] The crRNA and tracrRNA can hybridize to form a guide nucleic acid. The crRNA can also provide a single-stranded nucleic acid targeting segment (e.g., a spacer region) that hybridizes to a target nucleic acid recognition sequence (e.g., protospacer). The sequence of a crRNA, including spacer region, or tracrRNA molecule can be designed to be specific to the species in which the guide nucleic acid is to be used. In some embodiments, the nucleic acidtargeting region of a guide nucleic acid can be between 18 to 72 nucleotides in length.

[00142] The nucleotide sequence of the guide nucleic acid that is complementary to a nucleotide sequence (target sequence) of the target nucleic acid can have a length of, for example, at least about 12 nt, at least about 15 nt, at least about 18 nt, at least about 19 nt, at least about 20 nt, at least about 25 nt, at least about 30 nt, at least about 35 nt or at least about 40 nt. The nucleotide sequence of the guide nucleic acid that is complementary to a nucleotide sequence (target sequence) of the target nucleic acid can have a length of from about 12 nucleotides (nt) to about 80 nt, from about 12 nt to about 50 nt, from about 12 nt to about 45 nt, from about 12 nt to about

40 nt, from about 12 nt to about 35 nt, from about 12 nt to about 30 nt, from about 12 nt to about

25 nt, from about 12 nt to about 20 nt, from about 12 nt to about 19 nt, from about 19 nt to about

20 nt, from about 19 nt to about 25 nt, from about 19 nt to about 30 nt, from about 19 nt to about

35 nt, from about 19 nt to about 40 nt, from about 19 nt to about 45 nt, from about 19 nt to about

50 nt, from about 19 nt to about 60 nt, from about 20 nt to about 25 nt, from about 20 nt to about

30 nt, from about 20 nt to about 35 nt, from about 20 nt to about 40 nt, from about 20 nt to about

45 nt, from about 20 nt to about 50 nt, or from about 20 nt to about 60 nt. In some cases, the guide nucleic acid (e.g. guide RNA or gRNA) can direct the CRISPR/Cas9 described herein to cleave the transmembrane moiety to induce homologous recombination in order to insert the immune checkpoint moiety in the flanking regions of the transmembrane moiety.

[00143] A protospacer sequence of a targeted polynucleotide can be identified by identifying a PAM within a region of interest and selecting a region of a desired size upstream or downstream of the PAM as the protospacer. A corresponding spacer sequence can be designed by determining the complementary sequence of the protospacer region.

[00144] A spacer sequence can be identified using a computer program (e.g., machine readable code). The computer program can use variables such as predicted melting temperature, secondary structure formation, and predicted annealing temperature, sequence identity, genomic context, chromatin accessibility, % GC, frequency of genomic occurrence, methylation status, presence of SNPs, and the like.

[00145] The percent complementarity between the nucleic acid-targeting sequence (e.g., a spacer sequence of the at least one heterologous polypeptide as disclosed herein) and the target nucleic acid (e.g., a protospacer sequence of the heterologous polynucleotide encoding any one of the moieties described herein) can be at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%. The percent complementarity between the nucleic acid-targeting sequence and the target nucleic acid can be at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% over about 20 contiguous nucleotides.

[00146] The Cas protein-binding segment of a guide nucleic acid can comprise two stretches of nucleotides (e.g., crRNA and tracrRNA) that are complementary to one another. The two stretches of nucleotides (e.g., crRNA and tracrRNA) that are complementary to one another can be covalently linked by intervening nucleotides (e.g., a linker in the case of a single guide nucleic acid). The two stretches of nucleotides (e.g., crRNA and tracrRNA) that are complementary to one another can hybridize to form a double stranded RNA duplex or hairpin of the Cas proteinbinding segment, thus resulting in a stem-loop structure. The crRNA and the tracrRNA can be covalently linked via the 3’ end of the crRNA and the 5’ end of the tracrRNA. Alternatively, tracrRNA and crRNA can be covalently linked via the 5’ end of the tracrRNA and the 3’ end of the crRNA.

[00147] In some embodiments, the genetic modification moiety and the heterologous polynucleotide can be delivered into the cell via the use of expression vectors. In the context of an expression vector, the vector can be readily introduced into a host cell, e.g., mammalian, bacterial, yeast, or insect cell by any method in the art. For example, the expression vector can be transferred into a host cell by physical, chemical, or biological means. In some embodiments, the genetic modification moiety and the heterologous polynucleotide can be delivered into the cell via physical methods such as calcium phosphate precipitation, lipofection, particle bombardment, microinjection, gene gun, electroporation, and the like. Methods for producing cells comprising vectors and/or exogenous nucleic acids are suitable for methods herein. One method for the introduction of a polynucleotide into a host cell is calcium phosphate transfection. In some embodiments, the genetic modification moiety and the heterologous polynucleotide can be delivered into the cell via biological methods such as the use of DNA and RNA vectors. Viral vectors, and especially retroviral vectors, have become the most widely used method for inserting genes into mammalian, e.g., human cells. Other viral vectors, in some embodiments, are derived from lentivirus, poxviruses, herpes simplex virus I, adenoviruses and adeno-associated viruses, and the like. Exemplary viral vectors include retroviral vectors, adenoviral vectors, adeno- associated viral vectors (AAVs), pox vectors, parvoviral vectors, baculovirus vectors, measles viral vectors, or herpes simplex virus vectors (HSVs). In some instances, the retroviral vectors include gamma-retroviral vectors such as vectors derived from the Moloney Murine Keukemia Virus (MoMLV, MMLV, MuLV, or MLV) or the Murine Steam cell Virus (MSCV) genome. In some instances, the retroviral vectors also include lentiviral vectors such as those derived from the human immunodeficiency virus (HIV) genome. In some instances, AAV vectors include AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, or AAV9 serotype. In some instances, viral vector is a chimeric viral vector, comprising viral portions from two or more viruses. In additional instances, the viral vector is a recombinant viral vector. In some embodiments, the genetic modification moiety and the heterologous polynucleotide can be delivered into the cell via chemical means such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle). Other methods of state-of-the-art targeted delivery of nucleic acids are available, such as delivery of polynucleotides with targeted nanoparticles or other suitable sub-micron sized delivery system. In some embodiments, the genetic modification moiety and the heterologous polynucleotide can be delivered into the cell via a non-viral delivery system. Non-viral delivery system can be liposome. The use of lipid formulations is contemplated for the introduction of the nucleic acids into a host cell (in vitro, ex vivo or in vivo). In another aspect, the nucleic acid is associated with a lipid. The nucleic acid associated with a lipid, in some embodiments, is encapsulated in the aqueous interior of a liposome, interspersed within the lipid bilayer of a liposome, attached to a liposome via a linking molecule that is associated with both the liposome and the oligonucleotide, entrapped in a liposome, complexed with a liposome, dispersed in a solution containing a lipid, mixed with a lipid, combined with a lipid, contained as a suspension in a lipid, contained or complexed with a micelle, or otherwise associated with a lipid. Lipid, lipid/DNA or lipid/expression vector associated compositions are not limited to any particular structure in solution. For example, in some embodiments, they are present in a bilayer structure, as micelles, or with a “collapsed” structure. Alternately, they are simply be interspersed in a solution, possibly forming aggregates that are not uniform in size or shape. Lipids are fatty substances which are, in some embodiments, naturally occurring or synthetic lipids. For example, lipids include the fatty droplets that naturally occur in the cytoplasm as well as the class of compounds which contain long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes. Lipids suitable for use are obtained from commercial sources. Stock solutions of lipids in chloroform or chloroform/methanol are often stored at about -20 °C. Chloroform is used as the only solvent since it is more readily evaporated than methanol. “Liposome” is a generic term encompassing a variety of single and multilamellar lipid vehicles formed by the generation of enclosed lipid bilayers or aggregates. Liposomes are often characterized as having vesicular structures with a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers. However, compositions that have different structures in solution than the normal vesicular structure are also encompassed. For example, the lipids, in some embodiments, assume a micellar structure or merely exist as nonuniform aggregates of lipid molecules. Also contemplated are lipofectamine-nucleic acid complexes. In some embodiments, the genetic modification moiety and the heterologous polynucleotide can be delivered into the cell can be packaged and delivered to the cell via extracellular vesicles. The engineered extracellular vesicles can be any membrane-bound particles. In some embodiments, the engineered extracellular vesicles can be any membrane-bound particles secreted by at least one cell. In some instances, the engineered extracellular vesicles can be any membrane-bound particles synthesized in vitro. In some instances, the engineered extracellular vesicles can be any membrane-bound particles synthesized without a cell. In some cases, the engineered extracellular vesicles can be exosomes, microvesicles, retrovirus-like particles, apoptotic bodies, apoptosomes, oncosomes, exophers, enveloped viruses, exomeres, or other very large extracellular vesicles. Identifying and isolating homogeneous or heterogenous populations of engineered extracellular vesicles

[00148] Described herein, in some embodiments, are methods for utilizing the platforms described herein to generate compositions comprising a homogeneous or a heterogenous population of engineered extracellular vesicles. In some embodiments, the method identifies and isolates the homogeneous or the heterogenous population of engineered extracellular vesicles based on the dimensions (e.g. diameters or sizes) of the engineered extracellular vesicles. In some embodiments, the method identifies and isolates the homogeneous or the heterogenous population of engineered extracellular vesicles based on the mass of the engineered extracellular vesicles. In some embodiments, the method identifies and isolates the homogeneous or the heterogenous population of engineered extracellular vesicles based on the number of units of immune checkpoint moiety encapsulated, secreted, released, or expressed on the surface of the engineered extracellular vesicle. In some embodiments, the method identifies and isolates the homogeneous or the heterogenous population of engineered extracellular vesicles based on a combination of the dimensions and the number of units if immune checkpoint moiety encapsulated, secreted, or expressed on the surface of the extrasellar vesicle. In some embodiments, the method identifies and isolates the homogeneous or the heterogenous population of engineered extracellular vesicles based on a combination of dimensions and the number of units of immune checkpoint moiety expressed on the surface of the engineered extracellular vesicle. [00149] In some embodiments, the method of identifying and isolating the homogeneous or the heterogenous population of engineered extracellular vesicles comprises performing differential ultracentrifugation to isolate a homogeneous or a heterogenous population of engineered extracellular vesicle based on density. In some embodiments the method comprises performing filtration or ultrafiltration to isolate homogeneous or heterogenous population of engineered extracellular vesicles based on weights or sizes. In some embodiments, the method comprises performing HPLC. In some embodiments, the method comprises performing extracellular vesicle precipitation, where water-excluding polymers such as polyethylene glycol (PEG) can tie up water molecules and force less soluble components out of solution. As such, the precipitate containing extracellular vesicle cam ne isolated by means of either low-speed centrifugation or filtration. In some embodiments, the method comprises performing affinity -based capture by capturing the engineered extracellular vesicles by immunoaffinity. Examples of proteins or epitope displayed on the surface of the engineered extracellular vesicles include CD9, CD63. CD81. Alix, caveolin-1, CD41, CD4, flotillin, Rab5, HSC70, and Lamp-3. In some embodiments, the method comprises performing microfluidics-based isolation method for extracellular vesicle for identifying and isolating a homogeneous or a heterogenous population of engineered extracellular vesicle based on size, density, and immunoaffinity, innovative sorting mechanisms such as acoustic, electrophoretic and electromagnetic manipulations can be implemented. With the use of such devices, significant reductions in sample volume, reagent consumption, and isolation time are expected.

[00150] In some embodiments, the method of identifying and isolating the homogeneous or the heterogenous population of engineered extracellular vesicles comprises basing on the number of immune checkpoint moiety expressed on the surface of the engineered extracellular vesicle. In some embodiments, the method comprises immunoassay, where antibody recognizing the immune checkpoint moiety is used. In some embodiments, the antibody is conjugated to a detectable moiety. In some embodiments, the signal detected from the antibody recognizing and binding to the immune checkpoint moiety correlates with the number of immune checkpoint moiety expressed on the surface of the engineered extracellular vesicle. Exemplary detectable moiety includes an enzymatic moiety (e.g., horseradish peroxidase (HRP), beta-galactosidase, alkaline phosphatase, etc), fluorescent dye, luminescent moiety, radioactive moiety, colorimetric label, colored latex particle or nanoparticle, and metal -conjugated moiety such as metallic nanolayer, metallic nanoparticle, or metallic nanoshell-conjugated moiety. In some embodiments, the detectable moiety is directly or indirectly tagged for a colorimetric assay (e.g., for detection of HRP or beta-galactosidase activity), visual inspection using light microscopy, immunofluorescence microscopy, confocal microscopy, by flow cytometry (FACS), autoradiography electron microscopy, immunostaining, or subcellular fractionation. [00151] In some embodiments, the method of identifying and isolating the homogeneous or a heterogenous population of engineered extracellular vehicles comprises identifying and isolating the homogeneous or a heterogenous population of engineered extracellular vesicles based on both diameter and number of units of immune checkpoint moiety expressed on the surface of the engineered extracellular vesicle. For example, the method identifies and isolates a homogeneous or a heterogenous population of engineered extracellular vesicles comprising a diameter of about 50 nm and about 2000 units of immune checkpoint moiety expressed on the surface of the engineered extracellular vesicles. In some embodiments, the method identifies and isolates a homogeneous or a heterogenous population of engineered extracellular vesicles comprising a diameter of about 10 nm, 20 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70, nm, 80 nm, 90 nm, 100 nm, 110 nm, 120 nm, 130 nm, 140 nm, 150 nm, 160 nm, 170 nm, 180 nm, 190 nm, 200 nm, or more and 500 units, 1000 units, 1500 units, 2000 units, 2500 units, 3000 units, 3500 units, 4000 units, 4500 units, 5000 units, 5500 units, 6000 units, 7500 units, 8000 units, 8500 units, 9000 units, 9500 units, 10000 units, 11000 nuts, 12000 units, 13000 units, 14000 units, 15000 units, or more of the immune checkpoint expressed on the surface of the engineered extracellular vesicle. Treatment

[00152] Disclosed herein, in some embodiments, are methods of treating a disease or a disorder in a subject, comprising administrating of therapeutic effective amount of the compositions or pharmaceutical compositions described herein to the subject. In some embodiments, the disease or disorder is an autoimmune disease, including Rheumatoid arthritis, Systemic lupus erythematosus, Psoriasis, Type 1 diabetes mellitus, Multiple sclerosis, Inflammatory bowel disease, Celiac disease, Crohn’s disease, Graves’ disease, Juvenile arthritis, Lyme disease chronic, Optic neuritis, Psoriatic arthritis, Scleritis, Scleroderma, Ulcerative colitis (UC), Uveitis, Inflammatory eye conditions, Vitiligo, COPD, complication from Organ transplantation, or graft- versus-host disease. In some cases, the disease or disorder is Acute Respiratory Distress Syndrome (ARDS). In some cases, the ARDS is caused by infection of coronavirus. In some cases, the coronavirus is SARS-CoV-2.

[00153] In some embodiments, the method comprises steps of: contacting the cell with the compositions or pharmaceutical compositions as described herein; upon said contacting, the immune checkpoint moiety is delivered to the target cell. In some embodiments, the immune checkpoint moiety modulates immune response or of the target cell (FIG. 2). In some cases, the target cell can be immune cells such as monocyte, T cell, Regulatory T cell (Treg), B cell, dendritic cell, macrophage, NK cell, or NKT cell. In some cases, the immune cell can be cell that expresses CD8, CD25, or both CD8 and CD25 (CD8+CD25+). In some cases, the immune cell can be cell that expresses CD4. In some cases, the immune cell can be cell that expresses CD4 and CD 25 (CD4+CD25+). In some cases, the immune cell can be cell that expresses FOXP3 In some cases, the immune cell can be cell that expresses CD4, CD25, and FOXP3 (CD4+CD25+FOXP3+). In some embodiments, the contacting occurs in vivo, ex vivo, or in vitro. In some embodiment, the composition or pharmaceutical composition can directly be administered to the subject.

[00154] In some embodiments, the methods described herein comprise administering the engineered extracellular vesicles described herein to a patient and suppressing CD8 positive and CD25 positive T cell activation (CD8+/CD25+) compared to T cell activation contacted with unmodified exosomes. In some embodiments, the methods and compositions described herein result in about 30 % activation compared to activation when contacted with unmodified exosomes to about 140 % activation compared to activation when contacted with unmodified exosomes. In some embodiments, the methods and compositions described herein result in about 30 % activation compared to activation when contacted with unmodified exosomes to about 40 % activation compared to activation when contacted with unmodified exosomes, about 30 % activation compared to activation when contacted with unmodified exosomes to about 50 % activation compared to activation when contacted with unmodified exosomes, about 30 % activation compared to activation when contacted with unmodified exosomes to about 60 % activation compared to activation when contacted with unmodified exosomes, about 30 % activation compared to activation when contacted with unmodified exosomes to about 70 % activation compared to activation when contacted with unmodified exosomes, about 30 % activation compared to activation when contacted with unmodified exosomes to about 80 % activation compared to activation when contacted with unmodified exosomes, about 30 % activation compared to activation when contacted with unmodified exosomes to about 90 % activation compared to activation when contacted with unmodified exosomes, about 30 % activation compared to activation when contacted with unmodified exosomes to about 100 % activation compared to activation when contacted with unmodified exosomes, about 30 % activation compared to activation when contacted with unmodified exosomes to about 110 % activation compared to activation when contacted with unmodified exosomes, about 30 % activation compared to activation when contacted with unmodified exosomes to about 120 % activation compared to activation when contacted with unmodified exosomes, about 30 % activation compared to activation when contacted with unmodified exosomes to about 130 % activation compared to activation when contacted with unmodified exosomes, about 30 % activation compared to activation when contacted with unmodified exosomes to about 140 % activation compared to activation when contacted with unmodified exosomes, about 40 % activation compared to activation when contacted with unmodified exosomes to about 50 % activation compared to activation when contacted with unmodified exosomes, about 40 % activation compared to activation when contacted with unmodified exosomes to about 60 % activation compared to activation when contacted with unmodified exosomes, about 40 % activation compared to activation when contacted with unmodified exosomes to about 70 % activation compared to activation when contacted with unmodified exosomes, about 40 % activation compared to activation when contacted with unmodified exosomes to about 80 % activation compared to activation when contacted with unmodified exosomes, about 40 % activation compared to activation when contacted with unmodified exosomes to about 90 % activation compared to activation when contacted with unmodified exosomes, about 40 % activation compared to activation when contacted with unmodified exosomes to about 100 % activation compared to activation when contacted with unmodified exosomes, about 40 % activation compared to activation when contacted with unmodified exosomes to about 110 % activation compared to activation when contacted with unmodified exosomes, about 40 % activation compared to activation when contacted with unmodified exosomes to about 120 % activation compared to activation when contacted with unmodified exosomes, about 40 % activation compared to activation when contacted with unmodified exosomes to about 130 % activation compared to activation when contacted with unmodified exosomes, about 40 % activation compared to activation when contacted with unmodified exosomes to about 140 % activation compared to activation when contacted with unmodified exosomes, about 50 % activation compared to activation when contacted with unmodified exosomes to about 60 % activation compared to activation when contacted with unmodified exosomes, about 50 % activation compared to activation when contacted with unmodified exosomes to about 70 % activation compared to activation when contacted with unmodified exosomes, about 50 % activation compared to activation when contacted with unmodified exosomes to about 80 % activation compared to activation when contacted with unmodified exosomes, about 50 % activation compared to activation when contacted with unmodified exosomes to about 90 % activation compared to activation when contacted with unmodified exosomes, about 50 % activation compared to activation when contacted with unmodified exosomes to about 100 % activation compared to activation when contacted with unmodified exosomes, about 50 % activation compared to activation when contacted with unmodified exosomes to about 110 % activation compared to activation when contacted with unmodified exosomes, about 50 % activation compared to activation when contacted with unmodified exosomes to about 120 % activation compared to activation when contacted with unmodified exosomes, about 50 % activation compared to activation when contacted with unmodified exosomes to about 130 % activation compared to activation when contacted with unmodified exosomes, about 50 % activation compared to activation when contacted with unmodified exosomes to about 140 % activation compared to activation when contacted with unmodified exosomes, about 60 % activation compared to activation when contacted with unmodified exosomes to about 70 % activation compared to activation when contacted with unmodified exosomes, about 60 % activation compared to activation when contacted with unmodified exosomes to about 80 % activation compared to activation when contacted with unmodified exosomes, about 60 % activation compared to activation when contacted with unmodified exosomes to about 90 % activation compared to activation when contacted with unmodified exosomes, about 60 % activation compared to activation when contacted with unmodified exosomes to about 100 % activation compared to activation when contacted with unmodified exosomes, about 60 % activation compared to activation when contacted with unmodified exosomes to about 110 % activation compared to activation when contacted with unmodified exosomes, about 60 % activation compared to activation when contacted with unmodified exosomes to about 120 % activation compared to activation when contacted with unmodified exosomes, about 60 % activation compared to activation when contacted with unmodified exosomes to about 130 % activation compared to activation when contacted with unmodified exosomes, about 60 % activation compared to activation when contacted with unmodified exosomes to about 140 % activation compared to activation when contacted with unmodified exosomes, about 70 % activation compared to activation when contacted with unmodified exosomes to about 80 % activation compared to activation when contacted with unmodified exosomes, about 70 % activation compared to activation when contacted with unmodified exosomes to about 90 % activation compared to activation when contacted with unmodified exosomes, about 70 % activation compared to activation when contacted with unmodified exosomes to about 100 % activation compared to activation when contacted with unmodified exosomes, about 70 % activation compared to activation when contacted with unmodified exosomes to about 110 % activation compared to activation when contacted with unmodified exosomes, about 70 % activation compared to activation when contacted with unmodified exosomes to about 120 % activation compared to activation when contacted with unmodified exosomes, about 70 % activation compared to activation when contacted with unmodified exosomes to about 130 % activation compared to activation when contacted with unmodified exosomes, about 70 % activation compared to activation when contacted with unmodified exosomes to about 140 % activation compared to activation when contacted with unmodified exosomes, about 80 % activation compared to activation when contacted with unmodified exosomes to about 90 % activation compared to activation when contacted with unmodified exosomes, about 80 % activation compared to activation when contacted with unmodified exosomes to about 100 % activation compared to activation when contacted with unmodified exosomes, about 80 % activation compared to activation when contacted with unmodified exosomes to about 110 % activation compared to activation when contacted with unmodified exosomes, about 80 % activation compared to activation when contacted with unmodified exosomes to about 120 % activation compared to activation when contacted with unmodified exosomes, about 80 % activation compared to activation when contacted with unmodified exosomes to about 130 % activation compared to activation when contacted with unmodified exosomes, about 80 % activation compared to activation when contacted with unmodified exosomes to about 140 % activation compared to activation when contacted with unmodified exosomes, about 90 % activation compared to activation when contacted with unmodified exosomes to about 100 % activation compared to activation when contacted with unmodified exosomes, about 90 % activation compared to activation when contacted with unmodified exosomes to about 110 % activation compared to activation when contacted with unmodified exosomes, about 90 % activation compared to activation when contacted with unmodified exosomes to about 120 % activation compared to activation when contacted with unmodified exosomes, about 90 % activation compared to activation when contacted with unmodified exosomes to about 130 % activation compared to activation when contacted with unmodified exosomes, about 90 % activation compared to activation when contacted with unmodified exosomes to about 140 % activation compared to activation when contacted with unmodified exosomes, about 100 % activation compared to activation when contacted with unmodified exosomes to about 110 % activation compared to activation when contacted with unmodified exosomes, about 100 % activation compared to activation when contacted with unmodified exosomes to about 120 % activation compared to activation when contacted with unmodified exosomes, about 100 % activation compared to activation when contacted with unmodified exosomes to about 130 % activation compared to activation when contacted with unmodified exosomes, about 100 % activation compared to activation when contacted with unmodified exosomes to about 140 % activation compared to activation when contacted with unmodified exosomes, about 110 % activation compared to activation when contacted with unmodified exosomes to about 120 % activation compared to activation when contacted with unmodified exosomes, about 110 % activation compared to activation when contacted with unmodified exosomes to about 130 % activation compared to activation when contacted with unmodified exosomes, about 110 % activation compared to activation when contacted with unmodified exosomes to about 140 % activation compared to activation when contacted with unmodified exosomes, about 120 % activation compared to activation when contacted with unmodified exosomes to about 130 % activation compared to activation when contacted with unmodified exosomes, about 1 0 % activation compared to activation when contacted with unmodified exosomes to about 140 % activation compared to activation when contacted with unmodified exosomes, or about 130 % activation compared to activation when contacted with unmodified exosomes to about 140 % activation compared to activation when contacted with unmodified exosomes. In some embodiments, the methods and compositions described herein result in about 30 % activation compared to activation when contacted with unmodified exosomes, about 40 % activation compared to activation when contacted with unmodified exosomes, about 50 % activation compared to activation when contacted with unmodified exosomes, about 60 % activation compared to activation when contacted with unmodified exosomes, about 70 % activation compared to activation when contacted with unmodified exosomes, about 80 % activation compared to activation when contacted with unmodified exosomes, about 90 % activation compared to activation when contacted with unmodified exosomes, about 100 % activation compared to activation when contacted with unmodified exosomes, about 110 % activation compared to activation when contacted with unmodified exosomes, about 120 % activation compared to activation when contacted with unmodified exosomes, about 130 % activation compared to activation when contacted with unmodified exosomes, or about 140 % activation compared to activation when contacted with unmodified exosomes. In some embodiments, the methods and compositions described herein result in at least about 30 % activation compared to activation when contacted with unmodified exosomes, about 40 % activation compared to activation when contacted with unmodified exosomes, about 50 % activation compared to activation when contacted with unmodified exosomes, about 60 % activation compared to activation when contacted with unmodified exosomes, about 70 % activation compared to activation when contacted with unmodified exosomes, about 80 % activation compared to activation when contacted with unmodified exosomes, about 90 % activation compared to activation when contacted with unmodified exosomes, about 100 % activation compared to activation when contacted with unmodified exosomes, about 110 % activation compared to activation when contacted with unmodified exosomes, about 120 % activation compared to activation when contacted with unmodified exosomes, or about 130 % activation compared to activation when contacted with unmodified exosomes. In some embodiments, the methods and compositions described herein result in at most about 40 % activation compared to activation when contacted with unmodified exosomes, about 50 % activation compared to activation when contacted with unmodified exosomes, about 60 % activation compared to activation when contacted with unmodified exosomes, about 70 % activation compared to activation when contacted with unmodified exosomes, about 80 % activation compared to activation when contacted with unmodified exosomes, about 90 % activation compared to activation when contacted with unmodified exosomes, about 100 % activation compared to activation when contacted with unmodified exosomes, about 110 % activation compared to activation when contacted with unmodified exosomes, about 120 % activation compared to activation when contacted with unmodified exosomes, about 130 % activation compared to activation when contacted with unmodified exosomes, or about 140 % activation compared to activation when contacted with unmodified exosomes. In some embodiments, the methods and compositions described herein comprise extracellular vesicles wherein the immune checkpoint moiety on the surface of the extracellular vesicles is PD-L1. In some embodiments, the methods and compositions described herein comprise at least one extracellular vesicle wherein the immune checkpoint moiety on the surface of the extracellular vesicle is PD-L1. In some embodiments, the methods and compositions described herein comprise extracellular vesicles wherein the immune checkpoint moiety on the surface of the extracellular vesicles is VSIG3. In some embodiments, the methods and compositions described herein comprise at least one extracellular vesicle wherein the immune checkpoint moiety on the surface of the extracellular vesicle is VSIG3. [00155] In some embodiments, the composition or pharmaceutical composition to be administered to the subject can comprise different combinations of the engineered extracellular vesicles expressing a plurality of immune checkpoint moiety. FIG. 6 illustrates exemplary combinations of the immune checkpoint moieties comprising PD-L1, CTLA-4, VISTA, and IGSF11 (VSIG-3). In some embodiments, the methods described herein comprise administering a plurality of engineered extracellular vesicles wherein the plurality comprises subsets of engineered exosomes wherein each subset is defined by expression of one or more immune checkpoint moieties described herein. The therapeutic effects of the different combinations of the engineered extracellular vesicles can be determined by the methods described in Riazifar et al.; “Stem Cell-Derived Exosomes as Nanotherapeutics for Autoimmune and Neurodegenerative Disorders,” which is herein incorporated by reference in its entirety.

[00156] In some embodiments, the composition or pharmaceutical composition can be administered to the subject alone (e.g., standalone treatment). In some embodiments, the composition is administered in combination with an additional agent. In some embodiments, the composition is a first-line treatment for the disease or condition. In some embodiments, the composition is a second-line, third-line, or fourth-line treatment, for the autoimmune disease. [00157] In general, methods disclosed herein comprise administering a composition by oral administration. However, in some instances, methods comprise administering a composition by intraperitoneal injection. In some instances, methods comprise administering a composition in the form of an anal suppository. In some instances, methods comprise administering a composition by intravenous (“i.v.”) administration. It is conceivable that one can also administer compositions disclosed herein by other routes, such as subcutaneous injection, intramuscular injection, intradermal injection, transdermal injection percutaneous administration, intranasal administration, intralymphatic injection, rectal administration intragastric administration, or any other suitable parenteral administration. In some embodiments, routes for local delivery closer to site of injury or inflammation are preferred over systemic routes. Routes, dosage, time points, and duration of administrating therapeutics can be adjusted. In some embodiments, administration of therapeutics is prior to, or after, onset of either, or both, acute and chronic symptoms of the disease or condition.

[00158] An effective dose and dosage of the compositions to prevent or treat the autoimmune diseases herein is defined by an observed beneficial response related to the autoimmune disease or condition, or symptom of the autoimmune disease. In some instances, the beneficial response comprises reduction of symptoms of autoimmune disease. Additional beneficial response comprises preventing, alleviating, arresting, or curing the autoimmune disease. In instances where the composition is not therapeutically effective or is not providing a sufficient alleviation of the disease or condition, or symptom of the disease or condition, then the dosage amount and/or route of administration can be changed, or an additional agent can be administered to the subject, along with the composition. In some embodiments, as a patient is started on a regimen of a composition, the patient is also weaned off (e.g., stepwise decrease in dose) a second treatment regimen.

[00159] Suitable dose and dosage administrated to a subject is determined by factors including, but no limited to, the particular composition, disease condition and its severity, the identity (e.g., weight, sex, or age) of the subject in need of treatment, and can be determined according to the particular circumstances surrounding the case, including, e.g., the specific agent being administered, the route of administration, the condition being treated, and the subject being treated.

[00160] In some embodiments, the administration of the composition is hourly, once every 2 hours, 3 hours, 4 hours, 5 hours, 6 hours,? hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours 22 hours, 23 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, 3 years, 4 years, or 5 years, or 10 years. The effective dosage ranges can be adjusted based on subject’s response to the treatment. Some routes of administration will require higher concentrations of effective amount of therapeutics than other routes

[00161] In certain embodiments, where the patient’s condition does not improve, upon the doctor’s discretion the administration of composition is administered chronically, that is, for an extended period of time, including throughout the duration of the patient’s life in order to ameliorate or otherwise control or limit the symptoms of the patient’s disease or condition. In certain embodiments wherein a patient’s status does improve, the dose of composition being administered can be temporarily reduced or temporarily suspended for a certain length of time (i.e., a “drug holiday”). In specific embodiments, the length of the drug holiday is between 2 days and 1 year, including by way of example only, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 15 days, 20 days, 28 days, or more than 28 days. The dose reduction during a drug holiday is, by way of example only, by 10%-100%, including by way of example only 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, and 100%. In certain embodiments, the dose of drug being administered can be temporarily reduced or temporarily suspended for a certain length of time (i.e., a “drug diversion”). In specific embodiments, the length of the drug diversion is between 2 days and 1 year, including by way of example only, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 15 days, 20 days, 28 days, or more than 28 days. The dose reduction during a drug diversion is, by way of example only, by 10%-100%, including by way of example only 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, and 100%. After a suitable length of time, the normal dosing schedule is optionally reinstated.

[00162] In some embodiments, once improvement of the patient's conditions has occurred, a maintenance dose is administered if necessary. Subsequently, in specific embodiments, the dosage or the frequency of administration, or both, is reduced, as a function of the symptoms, to a level at which the improved disease, disorder or condition is retained. In certain embodiments, however, the patient requires intermittent treatment on a long-term basis upon any recurrence of symptoms.

[00163] Toxicity and therapeutic efficacy of such therapeutic regimens are determined by standard pharmaceutical procedures in cell cultures or experimental animals, including, but not limited to, the determination of the LD50 and the ED50. The dose ratio between the toxic and therapeutic effects is the therapeutic index and it is expressed as the ratio between LD50 and ED50. In certain embodiments, the data obtained from cell culture assays and animal studies are used in formulating the therapeutically effective daily dosage range and/or the therapeutically effective unit dosage amount for use in mammals, including humans. In some embodiments, the daily dosage amount of the composition described herein lies within a range of circulating concentrations that include the ED50 with minimal toxicity. In certain embodiments, the daily dosage range and/or the unit dosage amount varies within this range depending upon the dosage form employed and the route of administration utilized.

[00164] A composition can be used alone or in combination with an additional agent. In some cases, an “additional agent” as used herein is administered alone. The composition and the additional agent can be administered together or sequentially. The combination therapies can be administered within the same day, or can be administered one or more days, weeks, months, or years apart. Examples of additional agent can include other immune modulators such as antibodies targeting cytokines or small molecules.

Platforms

[00165] Described herein, in some embodiments, are platforms for generating the engineered extracellular vesicles described herein. In some embodiments, the platforms conform to good manufacturing practices (GMP) standard. In some embodiments, the compositions comprising the engineered extracellular vesicle can be generated according to good manufacturing practices (GMP). In some embodiments, the compositions comprise a pathogen level that is substantially free of pathogens. In some embodiments, the compositions comprise a contaminant level that is substantially free of contaminants. In some embodiments, the compositions comprise low immunogenicity.

[00166] In some embodiments, the compositions described herein can be generated and isolated via hypotonic treatment and centrifugation. In some embodiments, the engineered extracellular vesicles are isolated from mesenchymal stem cells (MSCs) expressing the engineered extracellular vesicles primarily by using hypotonic treatment such that the MSCs rupture and extracellular vesicles are released. In some instances, the MSCs are resuspended in hypotonic solution to induce cell swelling. In some embodiments, the platform comprises phase-contrast microscopy to monitor cell swelling. In some embodiments, the platform comprises cell culturing modules. In some embodiments, the platform comprises modules for culturing the cells described herein. In some embodiments, the platform comprises modules for collecting the engineered extracellular vesicles released by the cells described herein. In some embodiments, the platform comprises a homogenizer to rupture the swollen cells to release extracellular vesicles. In some embodiments, the platform comprises means for separating the ruptured cells in a gradient (e g., a sucrose gradient) to separate out the engineered extracellular vesicles. In some embodiments, the platform comprises other components to generate extracellular vesicles other approaches of lysing the MSC such as mild sonication, freeze- thaw, French-press, or needle-passaging. In some embodiments, the platform comprises centrifuges to centrifuge and isolate the fraction comprising the engineered extracellular vesicles. In some embodiments, the platform comprises means for separating a fraction comprising the engineered extracellular vesicle by floatation in a discontinuous sucrose density gradient.

[00167] In some embodiments, the platform comprises modules for generating the engineered extracellular vesicles by extrusion. In some embodiments, the extrusion process separates and isolates the engineered extracellular vesicles based on the sizes or diameters of the engineered extracellular vesicles. Exemplary extrusion process comprises the use of membranes with various pore sizes. The membranes can separate the engineered extracellular vesicles based on the sizes or diameters of the engineered extracellular vesicles from a solution comprising the ruptured MSC. Extracellular vesicles can be further isolated and reduced in size by continued extrusion following extrusion with increasingly smaller membrane pore sizes, ranging from 150 nm to 10 nm. When the final extrusion is complete, extracellular vesicle can be are pelleted by centrifugation. In some embodiments, the platform comprises components for performing sonication, extrusion, high pressure/homogenization, microfluidization, or detergent dialysis. In some embodiments, the platform comprises components for determining unit numbers of immune checkpoint moiety per extracellular vesicle.

Pharmaceutical compositions

[00168] Described herein are pharmaceutical compositions comprising the compositions described herein. In some cases, the pharmaceutical composition comprises the engineered extracellular vesicle described herein. In some embodiments, the pharmaceutical composition comprises both the composition comprising the engineered extracellular vesicle and the cells that release the engineered extracellular vesicles. A pharmaceutical composition, as used herein, refers to a mixture of a therapeutic agent comprising the engineered extracellular vesicle, with other chemical components (i.e., pharmaceutically acceptable inactive ingredients), such as carriers, excipients, binders, filling agents, suspending agents, flavoring agents, sweetening agents, disintegrating agents, dispersing agents, surfactants, lubricants, colorants, diluents, solubilizers, moistening agents, plasticizers, stabilizers, penetration enhancers, wetting agents, anti-foaming agents, antioxidants, preservatives, or one or more combination thereof. Optionally, the compositions include two or more therapeutic agent (e g., one or more therapeutic agents and one or more additional agents) as discussed herein. In practicing the methods of treatment or use provided herein, therapeutically effective amounts of therapeutic agents described herein are administered in a pharmaceutical composition to a mammal having a disease, disorder, or condition to be treated, e.g., an autoimmune disease. In some embodiments, the mammal is a human. A therapeutically effective amount can vary widely depending on the severity of the disease, the age and relative health of the subject, the potency of the therapeutic agent used and other factors. The therapeutic agents can be used singly or in combination with one or more therapeutic agents as components of mixtures.

[00169] The pharmaceutical formulations described herein are administered to a subject by appropriate administration routes, including but not limited to, intravenous, intraarterial, oral, parenteral, buccal, topical, transdermal, rectal, intramuscular, subcutaneous, intraosseous, transmucosal, inhalation, or intraperitoneal administration routes. The pharmaceutical formulations described herein include, but are not limited to, aqueous liquid dispersions, selfemulsifying dispersions, solid solutions, liposomal dispersions, aerosols, solid dosage forms, powders, immediate release formulations, controlled release formulations, fast melt formulations, tablets, capsules, pills, delayed release formulations, extended release formulations, pulsatile release formulations, multiparticulate formulations, and mixed immediate and controlled release formulations.

[00170] Pharmaceutical compositions including a therapeutic agent are manufactured in a conventional manner, such as, by way of example only, by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or compression processes.

[00171] The pharmaceutical compositions may include at least a therapeutic agent as an active ingredient in free-acid or free-base form, or in a pharmaceutically acceptable salt form. In addition, the methods and pharmaceutical compositions described herein include the use of N- oxides (if appropriate), crystalline forms, amorphous phases, as well as active metabolites of these compounds having the same type of activity. In some embodiments, therapeutic agents exist in unsolvated form or in solvated forms with pharmaceutically acceptable solvents such as water, ethanol, and the like. The solvated forms of the therapeutic agents are also considered to be disclosed herein.

[00172] In certain embodiments, compositions provided herein include one or more preservatives to inhibit microbial activity. Suitable preservatives include mercury-containing substances such as merfen and thiomersal; stabilized chlorine dioxide; and quaternary ammonium compounds such as benzalkonium chloride, cetyltrimethylammonium bromide and cetylpyridinium chloride. [00173] In some embodiments, formulations described herein benefit from antioxidants, metal chelating agents, thiol containing compounds and other general stabilizing agents. Examples of such stabilizing agents, include, but are not limited to: (a) about 0.5% to about 2% w/v glycerol, (b) about 0.1% to about 1% w/v methionine, (c) about 0.1% to about 2% w/v monothioglycerol, (d) about 1 mM to about 10 mM EDTA, (e) about 0.01% to about 2% w/v ascorbic acid, (f) 0.003% to about 0.02% w/v polysorbate 80, (g) 0.001% to about 0.05% w/v. polysorbate 20, (h) arginine, (i) heparin, (j) dextran sulfate, (k) cyclodextrins, (1) pentosan polysulfate and other heparinoids, (m) divalent cations such as magnesium and zinc; or (n) combinations thereof. [00174] The pharmaceutical compositions described herein are formulated into any suitable dosage form, including but not limited to, aqueous oral dispersions, liquids, gels, syrups, elixirs, slurries, suspensions, solid oral dosage forms, aerosols, controlled release formulations, fast melt formulations, effervescent formulations, lyophilized formulations, tablets, powders, pills, dragees, capsules, delayed release formulations, extended release formulations, pulsatile release formulations, multiparticulate formulations, and mixed immediate release and controlled release formulations. In one aspect, a therapeutic agent as discussed herein, e.g., therapeutic agent is formulated into a pharmaceutical composition suitable for intramuscular, subcutaneous, or intravenous injection. In one aspect, formulations suitable for intramuscular, subcutaneous, or intravenous injection include physiologically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Examples of suitable aqueous and non-aqueous carriers, diluents, solvents, or vehicles include water, ethanol, polyols (propyleneglycol, polyethylene-glycol, glycerol, cremophor and the like), suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters such as ethyl oleate. 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 dispersions, and by the use of surfactants. In some embodiments, formulations suitable for subcutaneous injection also contain additives such as preserving, wetting, emulsifying, and dispensing agents. Prevention of the growth of microorganisms can be ensured by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and the like. In some cases, it is desirable to include isotonic agents, such as sugars, sodium chloride, and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, such as aluminum monostearate and gelatin.

[00175] For intravenous injections or drips or infusions, a therapeutic agent described herein is formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank’s solution, Ringer’s solution, or physiological saline buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art. For other parenteral injections, appropriate formulations include aqueous or nonaqueous solutions, preferably with physiologically compatible buffers or excipients. Such excipients are known.

[00176] Parenteral injections may involve bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi dose containers, with an added preservative. The pharmaceutical composition described herein may be in a form suitable for parenteral injection as a sterile suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. In one aspect, the active ingredient is in powder form for constitution with a suitable vehicle, e g , sterile pyrogen-free water, before use.

[00177] For administration by inhalation, a therapeutic agent is formulated for use as an aerosol, a mist or a powder. Pharmaceutical compositions described herein are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebuliser, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, such as, by way of example only, gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the therapeutic agent described herein and a suitable powder base such as lactose or starch. Formulations that include a therapeutic agent are prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, fluorocarbons, and/or other solubilizing or dispersing agents known in the art. Preferably these compositions and formulations are prepared with suitable nontoxic pharmaceutically acceptable ingredients. The choice of suitable carriers is dependent upon the exact nature of the nasal dosage form desired, e.g., solutions, suspensions, ointments, or gels. Nasal dosage forms generally contain large amounts of water in addition to the active ingredient. Minor amounts of other ingredients such as pH adjusters, emulsifiers or dispersing agents, preservatives, surfactants, gelling agents, or buffering and other stabilizing and solubilizing agents are optionally present. Preferably, the nasal dosage form should be isotonic with nasal secretions.

[00178] Pharmaceutical preparations for oral use are obtained by mixing one or more solid excipient with one or more of the therapeutic agents described herein, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients include, for example, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methylcellulose, microcrystalline cellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose; or others such as: polyvinylpyrrolidone (PVP or povidone) or calcium phosphate. If desired, disintegrating agents are added, such as the cross linked croscarmellose sodium, polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate. In some embodiments, dyestuffs or pigments are added to the tablets or dragee coatings for identification or to characterize different combinations of active therapeutic agent doses. [00179] In some embodiments, pharmaceutical formulations of a therapeutic agent are in the form of a capsules, including push fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push fit capsules contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active therapeutic agent is dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In some embodiments, stabilizers are added. A capsule may be prepared, for example, by placing the bulk blend of the formulation of the therapeutic agent inside of a capsule. In some embodiments, the formulations (non-aqueous suspensions and solutions) are placed in a soft gelatin capsule. In other embodiments, the formulations are placed in standard gelatin capsules or non-gelatin capsules such as capsules comprising HPMC. In other embodiments, the formulation is placed in a sprinkle capsule, wherein the capsule is swallowed whole or the capsule is opened, and the contents sprinkled on food prior to eating.

[00180] All formulations for oral administration are in dosages suitable for such administration. In one aspect, solid oral dosage forms are prepared by mixing a therapeutic agent with one or more of the following: antioxidants, flavoring agents, and carrier materials such as binders, suspending agents, disintegration agents, filling agents, surfactants, solubilizers, stabilizers, lubricants, wetting agents, and diluents. In some embodiments, the solid dosage forms disclosed herein are in the form of a tablet, (including a suspension tablet, a fast-melt tablet, a bitedisintegration tablet, a rapid-disintegration tablet, an effervescent tablet, or a caplet), a pill, a powder, a capsule, solid dispersion, solid solution, bioerodible dosage form, controlled release formulations, pulsatile release dosage forms, multiparticulate dosage forms, beads, pellets, granules. In other embodiments, the pharmaceutical formulation is in the form of a powder. Compressed tablets are solid dosage forms prepared by compacting the bulk blend of the formulations described above. In various embodiments, tablets will include one or more flavoring agents. In other embodiments, the tablets will include a film surrounding the final compressed tablet. In some embodiments, the film coating can provide a delayed release of a therapeutic agent from the formulation. In other embodiments, the film coating aids in patient compliance (e.g., Opadry® coatings or sugar coating). Film coatings including Opadiy® typically range from about 1% to about 3% of the tablet weight. In some embodiments, solid dosage forms, e.g., tablets, effervescent tablets, and capsules, are prepared by mixing particles of a therapeutic agent with one or more pharmaceutical excipients to form a bulk blend composition. The bulk blend is readily subdivided into equally effective unit dosage forms, such as tablets, pills, and capsules. In some embodiments, the individual unit dosages include film coatings. These formulations are manufactured by conventional formulation techniques.

[00181] In another aspect, dosage forms include microencapsulated formulations. In some embodiments, one or more other compatible materials are present in the microencapsulation material. Exemplary materials include, but are not limited to, pH modifiers, erosion facilitators, anti-foaming agents, antioxidants, flavoring agents, and carrier materials such as binders, suspending agents, disintegration agents, filling agents, surfactants, solubilizers, stabilizers, lubricants, wetting agents, and diluents. Exemplary useful microencapsulation materials include, but are not limited to, hydroxypropyl cellulose ethers (HPC) such as Klucel® or Nisso HPC, low- substituted hydroxypropyl cellulose ethers (L-HPC), hydroxypropyl methyl cellulose ethers (HPMC) such as Seppifilm-LC, Pharmacoat®, Metolose SR, Methocel®-E, Opadry YS, PrimaFlo, Benecel MP824, and Benecel MP843, methylcellulose polymers such as Methocel®- A, hydroxypropylmethylcellulose acetate stearate Aqoat (HF-LS, HF -LG, HF -MS) and Metolose®, Ethylcelluloses (EC) and mixtures thereof such as E461, Ethocel®, Aqualon®-EC, Surelease®, Polyvinyl alcohol (PVA) such as Opadry AMB, hydroxyethylcelluloses such as Natrosol®, carboxymethylcelluloses and salts of carboxymethylcelluloses (CMC) such as Aqualon®-CMC, polyvinyl alcohol and polyethylene glycol co-polymers such as Kollicoat IR®, monoglycerides (Myverol), triglycerides (KLX), polyethylene glycols, modified food starch, acrylic polymers and mixtures of acrylic polymers with cellulose ethers such as Eudragit® EPO, Eudragit® L30D-55, Eudragit® FS 30D Eudragit® L100-55, Eudragit® L100, Eudragit® S100, Eudragit® RD100, Eudragit® E100, Eudragit® L12.5, Eudragit® S12.5, Eudragit® NE30D, and Eudragit® NE 40D, cellulose acetate phthalate, sepifilms such as mixtures of HPMC and stearic acid, cyclodextrins, and mixtures of these materials.

[00182] Liquid formulation dosage forms for oral administration are optionally aqueous suspensions selected from the group including, but not limited to, pharmaceutically acceptable aqueous oral dispersions, emulsions, solutions, elixirs, gels, and syrups. See, e.g., Singh et al., Encyclopedia of Pharmaceutical Technology, 2nd Ed., pp. 754-757 (2002). In addition to therapeutic agent the liquid dosage forms optionally include additives, such as: (a) disintegrating agents; (b) dispersing agents; (c) wetting agents; (d) at least one preservative, (e) viscosity enhancing agents, (f) at least one sweetening agent, and (g) at least one flavoring agent. In some embodiments, the aqueous dispersions further include a crystal-forming inhibitor.

[00183] In some embodiments, the pharmaceutical formulations described herein are selfemulsifying drug delivery systems (SEDDS). Emulsions are dispersions of one immiscible phase in another, usually in the form of droplets. Generally, emulsions are created by vigorous mechanical dispersion. SEDDS, as opposed to emulsions or microemulsions, spontaneously form emulsions when added to an excess of water without any external mechanical dispersion or agitation. An advantage of SEDDS is that only gentle mixing is required to distribute the droplets throughout the solution. Additionally, water or the aqueous phase is optionally added just prior to administration, which ensures stability of an unstable or hydrophobic active ingredient. Thus, the SEDDS provides an effective delivery system for oral and parenteral delivery of hydrophobic active ingredients. In some embodiments, SEDDS provides improvements in the bioavailability of hydrophobic active ingredients.

[00184] Buccal formulations that include a therapeutic agent are administered using a variety of formulations known in the art. In addition, the buccal dosage forms described herein can further include a bioerodible (hydrolysable) polymeric carrier that also serves to adhere the dosage form to the buccal mucosa. For buccal or sublingual administration, the compositions may take the form of tablets, lozenges, or gels formulated in a conventional manner.

[00185] For intravenous injections, a therapeutic agent is optionally formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank’s solution, Ringer’s solution, or physiological saline buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. For other parenteral injections, appropriate formulations include aqueous or nonaqueous solutions, preferably with physiologically compatible buffers or excipients.

[00186] Parenteral injections optionally involve bolus injection or continuous infusion. Formulations for injection are optionally presented in unit dosage form, e.g., in ampoules or in multi dose containers, with an added preservative. In some embodiments, a pharmaceutical composition described herein is in a form suitable for parenteral injection as a sterile suspensions, solutions or emulsions in oily or aqueous vehicles, and contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Pharmaceutical formulations for parenteral administration include aqueous solutions of an agent that modulates the activity of a carotid body in water soluble form. Additionally, suspensions of an agent that modulates the activity of a carotid body are optionally prepared as appropriate, e.g., oily injection suspensions. [00187] Conventional formulation techniques include, e g., one or a combination of methods: (1) dry mixing, (2) direct compression, (3) milling, (4) dry or non-aqueous granulation, (5) wet granulation, or (6) fusion. Other methods include, e.g., spray drying, pan coating, melt granulation, granulation, fluidized bed spray drying or coating (e.g., wurster coating), tangential coating, top spraying, tableting, extruding and the like.

[00188] In some embodiments, the pharmaceutical dosage forms are formulated to provide a controlled release of a therapeutic agent. Controlled release refers to the release of the therapeutic agent from a dosage form in which it is incorporated according to a desired profile over an extended period of time. Controlled release profiles include, for example, sustained release, prolonged release, pulsatile release, and delayed release profiles. In contrast to immediate release compositions, controlled release compositions allow delivery of an agent to a subject over an extended period of time according to a predetermined profile. Such release rates can provide therapeutically effective levels of agent for an extended period of time and thereby provide a longer period of pharmacologic response while minimizing side effects as compared to conventional rapid release dosage forms. Such longer periods of response provide for many inherent benefits that are not achieved with the corresponding short acting, immediate release preparations.

[00189] In other embodiments, the formulations described herein are delivered using a pulsatile dosage form. A pulsatile dosage form is capable of providing one or more immediate release pulses at predetermined time points after a controlled lag time or at specific sites. In one embodiment, the pulsatile dosage form includes at least two groups of particles, (i.e., multiparticulate) each containing the formulation described herein. The first group of particles provides a substantially immediate dose of a therapeutic agent upon ingestion by a mammal. The first group of particles can be either uncoated or include a coating and/or sealant. In one aspect, the second group of particles comprises coated particles. The coating on the second group of particles provides a delay of from about 2 hours to about 7 hours following ingestion before release of the second dose. Suitable coatings for pharmaceutical compositions are described herein or known in the art.

[00190] In some embodiments, pharmaceutical formulations are provided that include particles of a therapeutic agent and at least one dispersing agent or suspending agent for oral administration to a subject. The formulations may be powder and/or granule for suspension, and upon admixture with water, a substantially uniform suspension is obtained.

[00191] Furthermore, pharmaceutical compositions optionally include one or more pH adjusting agents or buffering agents, including acids such as acetic, boric, citric, lactic, phosphoric and hydrochloric acids; bases such as sodium hydroxide, sodium phosphate, sodium borate, sodium citrate, sodium acetate, sodium lactate and tri s-hydroxymethylaminom ethane; and buffers such as citrate/dextrose, sodium bicarbonate and ammonium chloride. Such acids, bases and buffers are included in an amount required to maintain pH of the composition in an acceptable range.

[00192] Additionally, pharmaceutical compositions optionally include one or more salts in an amount required to bring osmolality of the composition into an acceptable range. Such salts include those having sodium, potassium or ammonium cations and chloride, citrate, ascorbate, borate, phosphate, bicarbonate, sulfate, thiosulfate or bisulfite anions; suitable salts include sodium chloride, potassium chloride, sodium thiosulfate, sodium bisulfite and ammonium sulfate. [00193] Other pharmaceutical compositions optionally include one or more preservatives to inhibit microbial activity. Suitable preservatives include mercury-containing substances such as merfen and thiomersal; stabilized chlorine dioxide; and quaternary ammonium compounds such as benzalkonium chloride, cetyltrimethylammonium bromide and cetylpyridinium chloride.

[00194] In one embodiment, the aqueous suspensions and dispersions described herein remain in a homogenous state for at least 4 hours. In one embodiment, an aqueous suspension is resuspended into a homogenous suspension by physical agitation lasting less than 1 minute. In still another embodiment, no agitation is necessary to maintain a homogeneous aqueous dispersion. [00195] An aerosol formulation for nasal administration is generally an aqueous solution designed to be administered to the nasal passages in drops or sprays. Nasal solutions can be similar to nasal secretions in that they are generally isotonic and slightly buffered to maintain a pH of about 5.5 to about 6.5, although pH values outside of this range can additionally be used. Antimicrobial agents or preservatives can also be included in the formulation.

[00196] An aerosol formulation for inhalations and inhalants can be designed so that the agent or combination of agents is carried into the respiratory tree of the subject when administered by the nasal or oral respiratory route. Inhalation solutions can be administered, for example, by a nebulizer. Inhalations or insufflations, comprising finely powdered or liquid drugs, can be delivered to the respiratory system as a pharmaceutical aerosol of a solution or suspension of the agent or combination of agents in a propellant, e.g., to aid in disbursement. Propellants can be liquefied gases, including halocarbons, for example, fluorocarbons such as fluorinated chlorinated hydrocarbons, hydrochlorofluorocarbons, and hydrochlorocarbons, as well as hydrocarbons and hydrocarbon ethers.

[00197] Halocarbon propellants can include fluorocarbon propellants in which all hydrogens are replaced with fluorine, chlorofluorocarbon propellants in which all hydrogens are replaced with chlorine and at least one fluorine, hydrogen-containing fluorocarbon propellants, and hydrogencontaining chlorofluorocarbon propellants. Hydrocarbon propellants useful include, for example, propane, isobutane, n-butane, pentane, isopentane and neopentane. A blend of hydrocarbons can also be used as a propellant. Ether propellants include, for example, dimethyl ether as well as the ethers. An aerosol formulation can also comprise more than one propellant. For example, the aerosol formulation can comprise more than one propellant from the same class, such as two or more fluorocarbons; or more than one, more than two, more than three propellants from different classes, such as a fluorohydrocarbon and a hydrocarbon. Pharmaceutical compositions of the present disclosure can also be dispensed with a compressed gas, e.g., an inert gas such as carbon dioxide, nitrous oxide or nitrogen. [00198] Aerosol formulations can also include other components, for example, ethanol, isopropanol, propylene glycol, as well as surfactants or other components such as oils and detergents. These components can serve to stabilize the formulation and/or lubricate valve components.

[00199] The aerosol formulation can be packaged under pressure and can be formulated as an aerosol using solutions, suspensions, emulsions, powders and semisolid preparations. For example, a solution aerosol formulation can comprise a solution of an agent such as a transporter, carrier, or ion channel inhibitor in (substantially) pure propellant or as a mixture of propellant and solvent. The solvent can be used to dissolve the agent and/or retard the evaporation of the propellant. Solvents can include, for example, water, ethanol and glycols. Any combination of suitable solvents can be use, optionally combined with preservatives, antioxidants, and/or other aerosol components.

[00200] An aerosol formulation can be a dispersion or suspension. A suspension aerosol formulation can comprise a suspension of an agent or combination of agents, e.g., a transporter, carrier, or ion channel inhibitor, and a dispersing agent. Dispersing agents can include, for example, sorbitan trioleate, oleyl alcohol, oleic acid, lecithin and corn oil. A suspension aerosol formulation can also include lubricants, preservatives, antioxidant, and/or other aerosol components.

[00201] An aerosol formulation can similarly be formulated as an emulsion. An emulsion aerosol formulation can include, for example, an alcohol such as ethanol, a surfactant, water and a propellant, as well as an agent or combination of agents, e.g., a transporter, carrier, or ion channel. The surfactant used can be nonionic, anionic or cationic. One example of an emulsion aerosol formulation comprises, for example, ethanol, surfactant, water and propellant. Another example of an emulsion aerosol formulation comprises, for example, vegetable oil, glyceryl monostearate and propane.

Kits

[00202] Disclosed herein, in some embodiments, are kits for using the compositions, the pharmaceutical compositions, or the cells described herein. In some embodiments, the kits disclosed herein may be used to treat a disease or disorder in a subject; or select a subject for treatment and/or monitor a treatment disclosed herein. In some embodiments, the kit comprises the pharmaceutical compositions, or the cells described herein, which can be used to perform the methods described herein. Kits comprise an assemblage of materials or components, including at least one of the compositions. Thus, in some embodiments the kit contains a composition including of the pharmaceutical composition, for the treatment of the disease or disorder described herein. [00203] In some instances, the kits described herein comprise components for selecting for a homogenous population of the engineered extracellular vesicles. In some instances, the kits described herein comprise components for selecting for a heterogenous population of the engineered extracellular vesicles In some embodiments, the kit comprises the components for assaying the number of units of the immune checkpoint moiety expressed on the surface of the engineered extracellular vesicle. In some embodiments, the kit comprises components for performing assays such as enzyme-linked immunosorbent assay (ELISA), single-molecular array (Simoa), PCR, and qPCR. The exact nature of the components configured in the kit depends on its intended purpose. For example, some embodiments are configured for the purpose of treating a disease or condition disclosed herein (e.g., autoimmune disease) in a subject. In some embodiments, the kit is configured particularly for the purpose of treating mammalian subjects. In some embodiments, the kit is configured particularly for the purpose of treating human subjects.

[00204] Instructions for use may be included in the kit. Optionally, the kit also contains other useful components, such as, diluents, buffers, pharmaceutically acceptable carriers, syringes, catheters, applicators, pipetting or measuring tools, bandaging materials or other useful paraphernalia. The materials or components assembled in the kit can be provided to the practitioner stored in any convenient and suitable ways that preserve their operability and utility. For example, the components can be in dissolved, dehydrated, or lyophilized form; they can be provided at room, refrigerated or frozen temperatures. The components are typically contained in suitable packaging material(s). As employed herein, the phrase “packaging material” refers to one or more physical structures used to house the contents of the kit, such as compositions and the like. The packaging material is constructed by well-known methods, preferably to provide a sterile, contaminant-free environment. The packaging materials employed in the kit are those customarily utilized in gene expression assays and in the administration of treatments. As used herein, the term “package” refers to a suitable solid matrix or material such as glass, plastic, paper, foil, and the like, capable of holding the individual kit components. Thus, for example, a package can be a glass vial or prefilled syringes used to contain suitable quantities of the pharmaceutical composition. The packaging material has an external label which indicates the contents and/or purpose of the kit and its components.

[00205] While preferred embodiments of the present invention have been shown and described herein, it can be obvious to those skilled in the art that such embodiments are provided by way of example only. It is not intended that the invention be limited by the specific examples provided within the specification. While the invention has been described with reference to the aforementioned specification, the descriptions and illustrations of the embodiments herein are not meant to be construed in a limiting sense. Numerous variations, changes, and substitutions can now occur to those skilled in the art without departing from the invention. Furthermore, it shall be understood that all aspects of the invention are not limited to the specific depictions, configurations or relative proportions set forth herein which depend upon a variety of conditions and variables. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is therefore contemplated that the invention shall also cover any such alternatives, modifications, variations or equivalents. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby. [00206] Use of absolute or sequential terms, for example, “will,” “will not,” “shall,” “shall not,” “must,” “must not,” “first,” “initially,” “next,” “subsequently,” “before,” “after,” “lastly,” and “finally,” are not meant to limit scope of the present embodiments disclosed herein but as exemplary. While preferred embodiments of the present invention have been shown and described herein, it can be obvious to those skilled in the art that such embodiments are provided by way of example only. It is not intended that the invention be limited by the specific examples provided within the specification. While the invention has been described with reference to the aforementioned specification, the descriptions and illustrations of the embodiments herein are not meant to be construed in a limiting sense. Numerous variations, changes, and substitutions can now occur to those skilled in the art without departing from the invention. Furthermore, it shall be understood that all aspects of the invention are not limited to the specific depictions, configurations or relative proportions set forth herein which depend upon a variety of conditions and variables. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is therefore contemplated that the invention shall also cover any such alternatives, modifications, variations or equivalents. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby. [00207] Use of absolute or sequential terms, for example, “will,” “will not,” “shall,” “shall not,” “must,” “must not,” “first,” “initially,” “next,” “subsequently,” “before,” “after,” “lastly,” and “finally,” are not meant to limit scope of the present embodiments disclosed herein but as exemplary.

[00208] As used herein, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, to the extent that the terms “including”, “includes”, “having”, “has”, “with”, or variants thereof are used in either the detailed description and/or the claims, such terms are intended to be inclusive in a manner similar to the term “comprising.” [00209] As used herein, the phrases “at least one”, “one or more”, and “and/or” are open-ended expressions that are both conjunctive and disjunctive in operation. For example, each of the expressions “at least one of A, B and C”, “at least one of A, B, or C”, “one or more of A, B, and C”, “one or more of A, B, or C” and “A, B, and/or C” means A alone, B alone, C alone, A and B together, A and C together, B and C together, or A, B and C together.

[00210] As used herein, “or” may refer to “and”, “or,” or “and/or” and may be used both exclusively and inclusively. For example, the term “A or B” may refer to “A or B”, “A but not B”, “B but not A”, and “A and B”. In some cases, context may dictate a particular meaning.

[00211] Any systems, methods, software, and platforms described herein are modular. Accordingly, terms such as “first” and “second” do not necessarily imply priority, order of importance, or order of acts.

[00212] The term “about” when referring to a number or a numerical range means that the number or numerical range referred to is an approximation within experimental variability (or within statistical experimental error), and the number or numerical range may vary from, for example, from 1% to 15% of the stated number or numerical range. In examples, the term “about” refers to ±10% of a stated number or value.

[00213] The terms “increased”, “increasing”, or “increase” are used herein to generally mean an increase by a statically significant amount. In some aspects, the terms “increased,” or “increase,” mean an increase of at least 10% as compared to a reference level, for example an increase of at least about 10%, at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10-100% as compared to a reference level, standard, or control. Other examples of “increase” include an increase of at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, at least 1000-fold or more as compared to a reference level.

[00214] The terms “decreased”, “decreasing”, or “decrease” are used herein generally to mean a decrease by a statistically significant amount. In some aspects, “decreased” or “decrease” means a reduction by at least 10% as compared to a reference level, for example a decrease by at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% decrease (e.g., absent level or non-detectable level as compared to a reference level), or any decrease between 10-100% as compared to a reference level. In the context of a marker or symptom, by these terms is meant a statistically significant decrease in such level. The decrease can be, for example, at least 10%, at least 20%, at least 30%, at least 40% or more, and is preferably down to a level accepted as within the range of normal for an individual without a given disease.

[00215] The terms “individual,” “patient,” or “subject” are used interchangeably. None of the terms require or are limited to situation characterized by the supervision (e.g., constant or intermittent) of a health care worker (e g., a doctor, a registered nurse, a nurse practitioner, a physician’s assistant, an orderly, or a hospice worker).

[00216] The terms “expression” or “expressing” refers to one or more processes by which a polynucleotide is transcribed from a DNA template (such as into an mRNA or other RNA transcript) and/or the process by which a transcribed mRNA is subsequently translated into peptides, polypeptides, or proteins. Transcripts and encoded polypeptides can be collectively referred to as “gene product.” If the polynucleotide is derived from genomic DNA, expression can include splicing of the mRNA in a eukaryotic cell. “Up-regulated,” with reference to expression, generally refers to an increased expression level of a polynucleotide (e.g., RNA such as mRNA) and/or polypeptide sequence relative to its expression level in a wild-type state while “down-regulated” generally refers to a decreased expression level of a polynucleotide (e.g., RNA such as mRNA) and/or polypeptide sequence relative to its expression in a wild-type state. [00217] The term “gene,” as used herein, refers to a segment of nucleic acid that encodes an individual protein or RNA (also referred to as a “coding sequence” or “coding region”), optionally together with associated regulatory region such as promoter, operator, terminator and the like, which can be located upstream or downstream of the coding sequence. The term “gene” is to be interpreted broadly, and can encompass mRNA, cDNA, cRNA and genomic DNA forms of a gene. In some uses, the term “gene” encompasses the transcribed sequences, including 5' and 3' untranslated regions (5'-UTR and 3'-UTR), exons and introns. In some genes, the transcribed region can contain “open reading frames” that encode polypeptides. In some uses of the term, a “gene” comprises only the coding sequences (e g., an “open reading frame” or “coding region”) necessary for encoding a polypeptide. In some aspects, genes do not encode a polypeptide, for example, ribosomal RNA genes (rRNA) and transfer RNA (tRNA) genes. In some aspects, the term “gene” includes not only the transcribed sequences, but in addition, also includes nontranscribed regions including upstream and downstream regulatory regions, enhancers and promoters. The term “gene” can encompass mRNA, cDNA and genomic forms of a gene.

[00218] The terms “polynucleotide,” “oligonucleotide,” and “nucleic acid” are used interchangeably to refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof, either in single-, double-, or multistranded form. A polynucleotide can be exogenous or endogenous to a cell. A polynucleotide can exist in a cell-free environment. A polynucleotide can be a gene or fragment thereof. A polynucleotide can be DNA. A polynucleotide can be RNA. A polynucleotide can have any three-dimensional structure, and can perform any function, known or unknown A polynucleotide can comprise one or more analogs (e.g., altered backbone, sugar, or nucleobase). Non-limiting examples of polynucleotides include coding or non-coding regions of a gene or gene fragment, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), short interfering RNA (siRNA), short-hairpin RNA (shRNA), micro-RNA (miRNA), ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, cell-free polynucleotides including cell-free DNA (cfDNA) and cell-free RNA (cfRNA), nucleic acid probes, and primers. The sequence of nucleotides can be interrupted by non-nucleotide components.

[00219] As used herein, the terms “polypeptide,” “peptide” and “protein” can be used interchangeably herein in reference to a polymer of amino acid residues. A protein can refer to a full-length polypeptide as translated from a coding open reading frame, or as processed to its mature form, while a polypeptide or peptide can refer to a degradation fragment or a processing fragment of a protein that nonetheless uniquely or identifiably maps to a particular protein. A polypeptide can be a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of adjacent amino acid residues. Polypeptides can be modified, for example, by the addition of carbohydrate, phosphorylation, etc. Proteins can comprise one or more polypeptides.

[00220] As used herein, the terms “fragment,” or equivalent terms can refer to a portion of a protein that has less than the full length of the protein and optionally maintains the function of the protein. Further, when the portion of the protein is blasted against the protein, the portion of the protein sequence can align, for example, at least with 80% identity to a part of the protein sequence.

[00221] The terms “complement,” “complements,” “complementary,” and “complementarity,” as used herein, generally refer to a sequence that is fully complementary to and hybridizable to the given sequence. In some cases, a sequence hybridized with a given nucleic acid is referred to as the “complement” or “reverse-complement” of the given molecule if its sequence of bases over a given region is capable of complementarily binding those of its binding partner, such that, for example, A-T, A-U, G-C, and G-U base pairs are formed. In general, a first sequence that is hybridizable to a second sequence is specifically or selectively hybridizable to the second sequence, such that hybridization to the second sequence or set of second sequences is preferred (e.g., thermodynamically more stable under a given set of conditions, such as stringent conditions commonly used in the art) to hybridization with non-target sequences during a hybridization reaction. Typically, hybridizable sequences share a degree of sequence complementarity over all or a portion of their respective lengths, such as between 25%-100% complementarity, including at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100% sequence complementarity. Sequence identity, such as for the purpose of assessing percent complementarity, can be measured by any suitable alignment algorithm, including but not limited to the Needleman-Wunsch algorithm (see e.g., the EMBOSS Needle aligner available at www.ebi.ac.uk/Tools/psa/emboss_needle/nucleotide.html, optionally with default settings), the BLAST algorithm. Optimal alignment can be assessed using any suitable parameters of a chosen algorithm, including default parameters.

[00222] The term “percent (%) identity,” as used herein, generally refers to the percentage of amino acid (or nucleic acid) residues of a candidate sequence that are identical to the amino acid (or nucleic acid) residues of a reference sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent identity (i.e., gaps can be introduced in one or both of the candidate and reference sequences for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). Alignment, for purposes of determining percent identity, can be achieved in various ways that are commonly known. Percent identity of two sequences can be calculated by aligning a test sequence with a comparison sequence using BLAST, determining the number of amino acids or nucleotides in the aligned test sequence that are identical to amino acids or nucleotides in the same position of the comparison sequence, and dividing the number of identical amino acids or nucleotides by the number of amino acids or nucleotides in the comparison sequence.

[00223] As used herein, the term “/// vivo" can be used to describe an event that takes place in a subject’s body.

[00224] As used herein, the term “ex vivo" can be used to describe an event that takes place outside of a subject’s body. An “ex vivo" assay cannot be performed on a subject. Rather, it can be performed upon a sample separate from a subject. Ex vivo can be used to describe an event occurring in an intact cell outside a subject’s body.

[00225] As used herein, the term “in vitro” can be used to describe an event that takes places contained in a container for holding laboratory reagent such that it is separated from the living biological source organism from which the material is obtained. In vitro assays can encompass cell-based assays in which cells alive or dead are employed. In vitro assays can also encompass a cell-free assay in which no intact cells are employed.

[00226] “Treating” or “treatment” can refer to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) a targeted pathologic condition or disorder. Those in need of treatment include those already with the disorder, as well as those prone to have the disorder, or those in whom the disorder is to be prevented. A therapeutic benefit can refer to eradication or amelioration of symptoms or of an underlying disorder being treated. Also, a therapeutic benefit can be achieved with the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder such that an improvement is observed in the subject, notwithstanding that the subject can still be afflicted with the underlying disorder. A prophylactic effect can include delaying, preventing, or eliminating the appearance of a disease or condition, delaying or eliminating the onset of symptoms of a disease or condition, slowing, halting, or reversing the progression of a disease or condition, or any combination thereof. For prophylactic benefit, a subject at risk of developing a particular disease, or to a subject reporting one or more of the physiological symptoms of a disease can undergo treatment, even though a diagnosis of this disease cannot have been made.

[00227] The term “effective amount” and “therapeutically effective amount,” as used interchangeably herein, generally refer to the quantity of a composition, for example a composition comprising immune cells such as lymphocytes (e g., T lymphocytes and/or NK cells) comprising a system of the present disclosure, that is sufficient to result in a desired activity upon administration to a subject in need thereof. Within the context of the present disclosure, the term “therapeutically effective” refers to that quantity of a composition that is sufficient to delay the manifestation, arrest the progression, relieve or alleviate at least one symptom of a disorder treated by the methods of the present disclosure.

[00228] The term “pharmaceutically acceptable carrier,” “pharmaceutically acceptable excipient,” “physiologically acceptable carrier,” or “physiologically acceptable excipient” refers to a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material. A component can be “pharmaceutically acceptable” in the sense of being compatible with the other ingredients of a pharmaceutical formulation. It can also be suitable for use in contact with the tissue or organ of humans and animals without excessive toxicity, irritation, allergic response, immunogenicity, or other problems or complications, commensurate with a reasonable benefit/risk ratio.

[00229] The term “pharmaceutical composition” refers to a mixture of a compound disclosed herein with other chemical components, such as diluents or carriers. The pharmaceutical composition can facilitate administration of the compound to an organism. Multiple techniques of administering a compound exist in the art including, but not limited to, oral, injection, aerosol, parenteral, and topical administration. EMBODIMENTS

[00230] Embodiment 1 A composition comprising at least one extracellular vesicle, said at least one extracellular vesicle comprising at least one engineered Lysosomal Associated Membrane Protein 2 (LAMP2), wherein the at least one engineered LAMP2 comprises at least one immune checkpoint moiety, wherein said immune checkpoint moiety is flanked by at least a fragment of the engineered LAMP2, wherein the fragment comprises the N-terminus homology arm of the engineered LAMP2.

[00231] Embodiment 2. The composition of Embodiment 1, wherein the immune checkpoint moiety comprises a polypeptide sequence that is at least 70% identical to VISTA, PD-L1, CTLA- 4, PD-L2, B7-1 (CD80), B7-2 (CD86), B7-H3 (CD276), B7-H2, B7-H4 (VTCN1), HVEM (CD270, TNFRSF14), Galectin 9, Galectin3, CEACAM1 (CD66a), OX-2 (CD200), PVR (CD155), PVRL2 (Nectin-2, CD112), FGL-1, PECAM-1, TSG-6, CD47, Stabilin-1 (Clever-1), Neuropilin 1, Neuropilin 2, CD158 (family), IGSF2 (CD 101), CD 155, GITRL, CD137L, OX40L, LIGHT, CD70, PD-1, RGMB, CTLA-4 (CD 152), BTLA, CD 160, Tim-3, CD200R, TIGIT, CD112R (PVRIG), LAG-3 (CD223), PECAM-1, CD44, SIRP alpha (CD172a), IGSF11 (VSIG-3), or a combination thereof.

[00232] Embodiment 3. The composition of Embodiment 2, wherein the immune checkpoint moiety comprises a polypeptide sequence that is at least 70% identical to VISTA, PD-L1, IGSF11 (VSIG-3), CTLA-4, OX-2 (CD200), or BTLA.

[00233] Embodiment 4. The composition of Embodiment 3, wherein the immune checkpoint moiety comprises a polypeptide sequence that is at least 70% identical to VISTA.

[00234] Embodiment 5. The composition of Embodiment 3, wherein the immune checkpoint moiety comprises a polypeptide sequence that is at least 99% identical to VISTA.

[00235] Embodiment 6. The composition of Embodiment 3, wherein the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 4.

[00236] Embodiment 7. The composition of Embodiment 3, wherein the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 70% identical to SEQ ID NO: 4.

[00237] Embodiment 8. The composition of Embodiment 3, wherein the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 99% identical to SEQ ID NO: 4.

[00238] Embodiment 9. The composition of Embodiment 3, wherein the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 5.

[00239] Embodiment 10. The composition of Embodiment 3, wherein the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 70% identical to SEQ ID NO: 5. [00240] Embodiment 11. The composition of Embodiment 3, wherein the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 99% identical to SEQ ID NO: 5.

[00241] Embodiment 12. The composition of Embodiment 3, wherein the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 6

[00242] Embodiment 13. The composition of Embodiment 3, wherein the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 70% identical to SEQ ID NO: 6.

[00243] Embodiment 14. The composition of Embodiment 3, wherein the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 99% identical to SEQ ID NO: 6.

[00244] Embodiment 15. The composition of Embodiment 3, wherein the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 25.

[00245] Embodiment 16. The composition of Embodiment 3, wherein the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 70% identical to SEQ ID NO: 25.

[00246] Embodiment 17. The composition of Embodiment 3, wherein the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 99% identical to SEQ ID NO: 25.

[00247] Embodiment 18. The composition of Embodiment 3, wherein the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 33.

[00248] Embodiment 19. The composition of Embodiment 3, wherein the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 70% identical to SEQ ID NO: 33.

[00249] Embodiment 20. The composition of Embodiment 3, wherein the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 99% identical to SEQ ID NO: 33.

[00250] Embodiment 21. The composition of Embodiment 3, wherein the immune checkpoint moiety comprises a polypeptide sequence that is at least 70% identical to PD-L1.

[00251] Embodiment 22. The composition of Embodiment 3, wherein the immune checkpoint moiety comprises a polypeptide sequence that is at least 99% identical to PD-L1.

[00252] Embodiment 23. The composition of Embodiment 3, wherein the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 7.

[00253] Embodiment 24. The composition of Embodiment 3, wherein the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 70% identical to SEQ ID NO: 7.

[00254] Embodiment 25. The composition of Embodiment 3, wherein the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 99% identical to SEQ ID NO: 7. [00255] Embodiment 26. The composition of Embodiment 3, wherein the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 8.

[00256] Embodiment 27. The composition of Embodiment 3, wherein the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 70% identical to SEQ ID NO: 8.

[00257] Embodiment 28. The composition of Embodiment 3, wherein the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 99% identical to SEQ ID NO: 8.

[00258] Embodiment 29. The composition of Embodiment 3, wherein the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 9.

[00259] Embodiment 30. The composition of Embodiment 3, wherein the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 70% identical to SEQ ID NO: 9.

[00260] Embodiment 31. The composition of Embodiment 3, wherein the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 99% identical to SEQ ID NO: 9.

[00261] Embodiment 32. The composition of Embodiment 3, wherein the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 24.

[00262] Embodiment 33. The composition of Embodiment 3, wherein the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 70% identical to SEQ ID NO: 24.

[00263] Embodiment 34. The composition of Embodiment 3, wherein the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 99% identical to SEQ ID NO: 24.

[00264] Embodiment 35. The composition of Embodiment 3, wherein the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 32.

[00265] Embodiment 36. The composition of Embodiment 3, wherein the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 70% identical to SEQ ID NO: 32.

[00266] Embodiment 37. The composition of Embodiment 3, wherein the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 99% identical to SEQ ID NO: 32.

[00267] Embodiment 38. The composition of Embodiment 3, wherein the immune checkpoint moiety comprises a polypeptide sequence that is at least 70% identical to IGSF11 (VSIG-3).

[00268] Embodiment 39. The composition of Embodiment 3, wherein the immune checkpoint moiety comprises a polypeptide sequence that is at least 99% identical to IGSF11 (VSIG-3).

[00269] Embodiment 40. The composition of Embodiment 3, wherein the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 10. [00270] Embodiment 41. The composition of Embodiment 3, wherein the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 70% identical to SEQ ID NO: 10 [00271] Embodiment 42. The composition of Embodiment 3, wherein the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 99% identical to SEQ ID NO: 10.

[00272] Embodiment 43. The composition of Embodiment 3, wherein the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 11.

[00273] Embodiment 44. The composition of Embodiment 3, wherein the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 70% identical to SEQ ID NO: 11.

[00274] Embodiment 45. The composition of Embodiment 3, wherein the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 99% identical to SEQ ID NO: 11.

[00275] Embodiment 46. The composition of Embodiment 3, wherein the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 12.

[00276] Embodiment 47. The composition of Embodiment 3, wherein the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 70% identical to SEQ ID NO: 12.

[00277] Embodiment 48. The composition of Embodiment 3, wherein the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 99% identical to SEQ ID NO: 12.

[00278] Embodiment 49. The composition of Embodiment 3, wherein the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 26.

[00279] Embodiment 50. The composition of Embodiment 3, wherein the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 70% identical to SEQ ID NO: 26.

[00280] Embodiment 51. The composition of Embodiment 3, wherein the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 99% identical to SEQ ID NO: 26.

[00281] Embodiment 52. The composition of Embodiment 3, wherein the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 34.

[00282] Embodiment 53. The composition of Embodiment 3, wherein the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 70% identical to SEQ ID NO: 34.

[00283] Embodiment 54. The composition of Embodiment 3, wherein the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 99% identical to SEQ ID NO: 34.

[00284] Embodiment 55. The composition of Embodiment 3, wherein the immune checkpoint moiety comprises a polypeptide sequence that is at least 70% identical to CTLA-4. [00285] Embodiment 56. The composition of Embodiment 3, wherein the immune checkpoint moiety comprises a polypeptide sequence that is at least 99% identical to CTLA-4.

[00286] Embodiment 57. The composition of Embodiment 3, wherein the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 13

[00287] Embodiment 58. The composition of Embodiment 3, wherein the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 70% identical to SEQ ID NO: 13.

[00288] Embodiment 59. The composition of Embodiment 3, wherein the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 99% identical to SEQ ID NO: 13.

[00289] Embodiment 60. The composition of Embodiment 3, wherein the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 14.

[00290] Embodiment 61. The composition of Embodiment 3, wherein the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 70% identical to SEQ ID NO: 14.

[00291] Embodiment 62. The composition of Embodiment 3, wherein the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 99% identical to SEQ ID NO: 14.

[00292] Embodiment 63. The composition of Embodiment 3, wherein the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 15.

[00293] Embodiment 64. The composition of Embodiment 3, wherein the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 70% identical to SEQ ID NO: 15.

[00294] Embodiment 65. The composition of Embodiment 3, wherein the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 99% identical to SEQ ID NO: 15.

[00295] Embodiment 66. The composition of Embodiment 3, wherein the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 27.

[00296] Embodiment 67. The composition of Embodiment 3, wherein the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 70% identical to SEQ ID NO: 27.

[00297] Embodiment 68. The composition of Embodiment 3, wherein the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 99% identical to SEQ ID NO: 27.

[00298] Embodiment 69. The composition of Embodiment 3, wherein the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 35.

[00299] Embodiment 70. The composition of Embodiment 3, wherein the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 70% identical to SEQ ID NO: 35. [00300] Embodiment 71. The composition of Embodiment 3, wherein the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 99% identical to SEQ ID NO: 35 [00301] Embodiment 72. The composition of Embodiment 3, wherein the immune checkpoint moiety comprises a polypeptide sequence that is at least 70% identical to OX-2 (CD200).

[00302] Embodiment 73. The composition of Embodiment 3, wherein the immune checkpoint moiety comprises a polypeptide sequence that is at least 99% identical to OX-2 (CD200).

[00303] Embodiment 74. The composition of Embodiment 3, wherein the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 23.

[00304] Embodiment 75. The composition of Embodiment 3, wherein the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 70% identical to SEQ ID NO: 23.

[00305] Embodiment 76. The composition of Embodiment 3, wherein the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 99% identical to SEQ ID NO: 23.

[00306] Embodiment 77. The composition of Embodiment 3, wherein the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 31.

[00307] Embodiment 78. The composition of Embodiment 3, wherein the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 70% identical to SEQ ID NO: 31.

[00308] Embodiment 79. The composition of Embodiment 3, wherein the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 99% identical to SEQ ID NO: 31.

[00309] Embodiment 80. The composition of Embodiment 3, wherein the immune checkpoint moiety comprises a polypeptide sequence that is at least 70% identical to BTLA.

[00310] Embodiment 81. The composition of Embodiment 3, wherein the immune checkpoint moiety comprises a polypeptide sequence that is at least 99% identical to BTLA.

[00311] Embodiment 82. The composition of Embodiment 3, wherein the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 28.

[00312] Embodiment 83. The composition of Embodiment 3, wherein the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 70% identical to SEQ ID NO: 28.

[00313] Embodiment 84. The composition of Embodiment 3, wherein the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 99% identical to SEQ ID NO: 28.

[00314] Embodiment 85. The composition of Embodiment 3, wherein the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 36. [00315] Embodiment 86. The composition of Embodiment 3, wherein the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 70% identical to SEQ ID NO: 36 [00316] Embodiment 87. The composition of Embodiment 3, wherein the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 99% identical to SEQ ID NO: 36.

[00317] Embodiment 88. The composition of any one of the preceding Embodiments, wherein the engineered LAMP2 comprises engineered LAMP2A, LAMP2B, LAMP2C, or a combination thereof.

[00318] Embodiment 89. The composition of Embodiment 88, wherein the engineered LAMP2 is the engineered LAMP2B.

[00319] Embodiment 90. The composition of any one of the preceding Embodiments, wherein the at least one extracellular vesicle comprises a targeting moiety.

[00320] Embodiment 91. The composition of Embodiment 90, wherein the targeting moiety is covalently connected to the N-terminus of the at least one engineered LAMP2B.

[00321] Embodiment 92. The composition of Embodiment 90, wherein the targeting moiety comprises a polypeptide that is at least 70% identical to an integrin comprising a6pi, a6p4, aLp2, aMp2, aXp2, or aDp2.

[00322] Embodiment 93. The composition of Embodiment 90, wherein the targeting moiety comprises a polypeptide that is at least 70% identical to a single chain fragment variable (scFv) antibody.

[00323] Embodiment 94. The composition of any one of the preceding Embodiments, wherein the at least one extracellular vesicle comprises a signaling peptide.

[00324] Embodiment 95. The composition of any one of the preceding Embodiments, wherein the immune checkpoint moiety is covalently connected to the at least one engineered LAMP2B via a linker, said linker comprises a flexible linker, a rigid linker, or a cleavable linker.

[00325] Embodiment 96. The composition of Embodiment 95, wherein the flexible linker comprises a polypeptide sequence comprising: (GGGGS)n where n=l, 2, 3, or 4;

KESGSVSSEQLAQFRSLD; EGKSSGSGSESKST; GGGGGG; GGGGGGGG; GSAGSAAGSGEF; RKRR; SS; or LE.

[00326] Embodiment 97. The composition of Embodiment 95, wherein the rigid linker comprises a polypeptide sequence comprising: (EAAAK)n where n=l, 2, 3, or 4; A(EAAAK)nA where n=2, 3, 4, or 5; A(EAAAK)4ALEA(EAAAK)4A; AEAAAKEAAAKA; or LEAGCKNFFPRSFTSCGSLE

[00327] Embodiment 98. The composition of Embodiment 95, wherein the cleavable linker comprises a polypeptide sequence comprising: LEAGCKNFFPRSFTSCGSLE;

CRRRRRREAEAC; GGIEGRGS; or TRHRQPRGWEQL. [00328] Embodiment 99. The composition of any one of the preceding Embodiments, wherein the at least one engineered LAMP2B comprises an amino acid sequence GNSTM atN-Terminus of the at least one engineered LAMP2.

[00329] Embodiment 100. The composition of any one of the preceding Embodiments, wherein the at least one extracellular vesicle comprises a plurality of engineered LAMP2Bs

[00330] Embodiment 101. The composition of Embodiment 100, wherein the plurality of the engineered LAMP2Bs comprises a single species of the immune checkpoint moiety.

[00331] Embodiment 102. The composition of Embodiment 100, wherein the plurality of the engineered LAMP2Bs comprises two or more species of the immune checkpoint moiety.

[00332] Embodiment 103. The composition of Embodiment 102, wherein the two or more species of the immune checkpoint moiety comprises a combination of any two or more of PD-L1, CTLA-4, IGSF11 (VSIG-3), VISTA, OX-2 (CD200), or BTLA.

[00333] Embodiment 104. The composition of Embodiment 102, wherein the two or more species of the immune checkpoint moiety are present on the extracellular vesicle at a ratio. [00334] Embodiment 105. The composition of Embodiment 1 comprises a plurality of extracellular vesicles, where the plurality of the extracellular vesicles comprises: at least a first population of the extracellular vesicles; and at least one additional population of the extracellular vesicles, wherein said first population of the extracellular vesicles comprises at least a first species immune checkpoint moiety and the at least one additional population of the extracellular vesicles comprises at least a second immune checkpoint moiety.

[00335] Embodiment 106. The composition of any one of the preceding Embodiments, wherein the extracellular vesicle comprises an exosome, a microvesicle, a retrovirus-like particle, an apoptotic body, an apoptosome, an oncosome, an exopher, an enveloped virus, an exomere, or a very large extracellular vesicle.

[00336] Embodiment 107. The composition of Embodiment 106, wherein the extracellular vesicle is the exosome.

[00337] Embodiment 108. The composition of Embodiment 1, wherein the at least one engineered LAMP2B comprises a homology arm comprising a nucleic acid sequence that is at least 70% identical to SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 21, or SEQ ID NO: 22.

[00338] Embodiment 109. The composition of Embodiment 1, wherein the at least one engineered LAMP2B comprises an N-terminus homology arm comprising a nucleic acid sequence that is at least 70% identical to SEQ ID NO: 2.

[00339] Embodiment 110. The composition of Embodiment 1, wherein the at least one engineered LAMP2B comprises an N-terminus homology arm comprising a nucleic acid sequence that is at least 70% identical to SEQ ID NO: 21. [00340] Embodiment 111. The composition of Embodiment 1, wherein the at least one engineered LAMP2B comprises a C-terminus homology arm comprising a nucleic acid sequence that is at least 70% identical to SEQ ID NO: 3.

[00341] Embodiment 112. The composition of Embodiment 1, wherein the at least one engineered LAMP2B comprises a C-terminus homology arm comprising a nucleic acid sequence that is at least 70% identical to SEQ ID NO: 22.

[00342] Embodiment 113. The composition of any one of the preceding Embodiments, wherein the composition further comprises a fusogenic moiety.

[00343] Embodiment 114. The composition of Embodiment 113, wherein the fusogenic moiety comprises a viral fusogenic moiety.

[00344] Embodiment 115. The composition of Embodiment 113, wherein the fusogenic moiety comprises a mammalian fusogenic moiety.

[00345] Embodiment 116. The composition of any one of the preceding Embodiments, wherein the at least one extracellular vesicle further comprises an immune evasion moiety.

[00346] Embodiment 117. The composition of Embodiment 116, wherein the immune evasion moiety comprises CD47.

[00347] Embodiment 118. The composition of any one of the preceding Embodiments does not comprise an enucleated cell.

[00348] Embodiment 119. The composition of any one of the preceding Embodiments, wherein the composition is derived from a cell.

[00349] Embodiment 120. The composition of any one of the preceding Embodiments, wherein the composition is cryopreserved.

[00350] Embodiment 121. The composition of any one of the preceding Embodiments, wherein the composition is lyophilized.

[00351] Embodiment 122. The composition of any one of the preceding Embodiments, wherein the composition is stable at 37°C for at least one hour.

[00352] Embodiment 123. A cell genetically modified to produce the extracellular vesicle of any one of the preceding Embodiments.

[00353] Embodiment 124. The cell of Embodiment 123, wherein the cell is genetically modified by homologous recombination.

[00354] Embodiment 125. The cell of Embodiment 123, wherein the cell is a stem cell.

[00355] Embodiment 126. The cell of Embodiment 123, wherein the cell is a human cell.

[00356] Embodiment 127. The cell of Embodiment 123, wherein the cell is a non-human cell.

[00357] Embodiment 128. The cell of any one of Embodiments 123 to 127, wherein the cell is a mesenchymal stem cell. [00358] Embodiment 129. A cell line comprising the cell of any one of Embodiments 123-128. [00359] Embodiment 130. A pharmaceutical composition comprises the composition of any one of Embodiments 1-122, the cell of any one of Embodiment 123-128, or the cell line of Embodiment 129.

[00360] Embodiment 131. The pharmaceutical composition of Embodiment 130 comprises a pharmaceutically acceptable carrier.

[00361] Embodiment 132. The pharmaceutical composition of Embodiment 130 comprises at least one additional active agent.

[00362] Embodiment 133. The pharmaceutical composition of Embodiment 131, wherein the pharmaceutical composition is formulated for administering intrathecally, intraocularly, intravitreally, retinally, intravenously, intramuscularly, intraventricularly, intracerebrally, intracerebellarly, intracerebroventricularly, intraperenchymally, subcutaneously, or a combination thereof.

[00363] Embodiment 134. A kit comprising the composition of any one of Embodiments 1-122, the cell of any one of Embodiment 123-128, the cell line of Embodiment 129, or the pharmaceutical composition of any one of Embodiments 130-133.

[00364] Embodiment 135. A platform comprising components for generating the composition of any one of Embodiments 1-122, the cell of any one of Embodiment 123-128, the cell line of Embodiment 129, the pharmaceutical composition of any one of Embodiments 130-133, or the kit of Embodiment 134.

[00365] Embodiment 136. A method for generating the extracellular vesicle of any one of the preceding Embodiments, said method comprising: contacting a cell with a polynucleotide encoding the immune checkpoint moiety; inducing homologous recombination in the cell, wherein the polynucleotide encoding the immune checkpoint moiety is inserted at a locus of the engineered LAMP2B, wherein the locus is flanked by one or both homology arms of the engineered LAMP2B; and harvesting the extracellular vesicle produced by the cell, wherein said extracellular vesicle comprises the engineered LAMP2B comprising the immune checkpoint moiety of any one of the preceding Embodiments.

[00366] Embodiment 137. A method of treating an autoimmune disease, the method comprises administering the composition of any one of Embodiments 1-122, the cell of any one of Embodiment 123-128, the cell line of Embodiment 129, the pharmaceutical composition of any one of Embodiments 130-133, or the kit of Embodiment 134.

[00367] Embodiment 138. The method of Embodiment 137, wherein the method compromises administering the pharmaceutical composition of any one of Embodiments 130-133. [00368] Embodiment 139. The method of Embodiment 137, wherein said autoimmune disease is Rheumatoid arthritis, Systemic lupus erythematosus, Psoriasis, Type 1 diabetes mellitus, Multiple sclerosis, Inflammatory bowel disease, Celiac disease, Crohn’s disease, Graves’ disease, Juvenile arthritis, Lyme disease chronic, Optic neuritis, Psoriatic arthritis, Scleritis, Scleroderma, Ulcerative colitis (UC), Uveitis, Sjogren’s syndrome, Inflammatory eye conditions, Idiopathic inflammatory myopathies, Vitiligo, COPD, complication from Organ transplantation, or graft- versus-host disease.

[00369] Embodiment 140. A method of treating Acute Respiratory Distress Syndrome (ARDS), especially as it pertains to COVID- 19 due to unchecked viral lysis of host cells and ensuing cytokine storm, the method comprises administering the composition of any one of Embodiments 1-122, the cell of any one of Embodiment 123-128, the cell line of Embodiment 129, the pharmaceutical composition of any one of Embodiments 130-133, or the kit of Embodiment 134. [00370] Embodiment 141. The method of Embodiment 140 comprises administering the pharmaceutical composition of any one of Embodiments 130-133.

[00371] Embodiment 142. A method of suppressing CD8+ CD25+ cells in a subject in need thereof, the method comprises administering the composition of any one of Embodiments 1-122, the cell of any one of Embodiment 123-128, the cell line of Embodiment 129, the pharmaceutical composition of any one of Embodiments 130-133, or the kit of Embodiment 134.

[00372] Embodiment 143. The method of Embodiment 142 comprises administering the pharmaceutical composition of any one of Embodiments 130-133.

[00373] Embodiment 144. A method of inducing regulatory T-cells (Tregs) in a patient in need thereof, comprises administering the composition of any one of Embodiments 1-122, the cell of any one of Embodiment 123-128, the cell line of Embodiment 129, the pharmaceutical composition of any one of Embodiments 130-133, or the kit of Embodiment 134.

[00374] Embodiment 145. The method of Embodiment 144 comprises administering the pharmaceutical composition of any one of Embodiments 130-133.

[00375] Embodiment 146. A composition comprising a fusion protein or polypeptide, wherein the fusion protein or polypeptide comprises an immune checkpoint moiety and a transmembrane moiety.

[00376] Embodiment 147. The composition of Embodiment 146, wherein the transmembrane moiety is a fragment of the transmembrane moiety.

[00377] Embodiment 148. The composition of Embodiment 146, wherein the immune checkpoint moiety comprises a polypeptide sequence that is at least 70% identical to VISTA, PD-L1, CTLA-4, PD-L2, B7-1 (CD80), B7-2 (CD86), B7-H3 (CD276), B7-H2, B7-H4 (VTCN1), HVEM (CD270, TNFRSF14), Galectin 9, Galectin3, CEACAM1 (CD66a), OX-2 (CD200), PVR (CD155), PVRL2 (Nectin-2, CD112), FGL-1, PECAM-1, TSG-6, CD47, Stabilin-1 (Clever-1), Neuropilin 1, Neuropilin 2, CD158 (family), IGSF2 (CD101), CD155, GITRL, CD137L, OX40L, LIGHT, CD70, PD-1, RGMB, CTLA-4 (CD 152), BTLA, CD 160, Tim-3, CD200R, TIGIT, CD112R (PVRIG), LAG-3 (CD223), PECAM-1, CD44, SIRP alpha (CD 172a), IGSF11 (VSIG-3), or a combination thereof.

[00378] Embodiment 149. The composition of Embodiment 148, wherein the immune checkpoint moiety comprises a polypeptide sequence that is at least 70% identical to VISTA, PD-L1, IGSF11 (VSIG-3), CTLA-4, OX-2 (CD200), or BTLA.

[00379] Embodiment 150. The composition of Embodiment 149, wherein the immune checkpoint moiety comprises a polypeptide sequence that is at least 70% identical to VISTA.

[00380] Embodiment 151. The composition of Embodiment 149, wherein the immune checkpoint moiety comprises a polypeptide sequence that is at least 99% identical to VISTA.

[00381] Embodiment 152. The composition of Embodiment 149, wherein the fusion protein or polypeptide comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 4.

[00382] Embodiment 153. The composition of Embodiment 149, wherein the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 70% identical to SEQ ID NO: 4.

[00383] Embodiment 154. The composition of Embodiment 149, wherein the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 99% identical to SEQ ID NO: 4.

[00384] Embodiment 155. The composition of Embodiment 149, wherein the immune fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 5.

[00385] Embodiment 156. The composition of Embodiment 149, wherein the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 70% identical to SEQ ID NO: 5.

[00386] Embodiment 157. The composition of Embodiment 149, wherein the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 99% identical to SEQ ID NO: 5.

[00387] Embodiment 158. The composition of Embodiment 149, wherein the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 6. [00388] Embodiment 159. The composition of Embodiment 149, wherein the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 70% identical to SEQ ID NO: 6.

[00389] Embodiment 160. The composition of Embodiment 149, wherein the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 99% identical to SEQ ID NO: 6.

[00390] Embodiment 161. The composition of Embodiment 149, wherein the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 25.

[00391] Embodiment 162. The composition of Embodiment 149, wherein the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 70% identical to SEQ ID NO: 25.

[00392] Embodiment 163. The composition of Embodiment 149, wherein the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 99% identical to SEQ ID NO: 25.

[00393] Embodiment 164. The composition of Embodiment 149, wherein the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 33.

[00394] Embodiment 165. The composition of Embodiment 149, wherein the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 70% identical to SEQ ID NO: 33.

[00395] Embodiment 166. The composition of Embodiment 149, wherein the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 99% identical to SEQ ID NO: 33.

[00396] Embodiment 167. The composition of Embodiment 149, wherein the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 70% identical to PD-L1.

[00397] Embodiment 168. The composition of Embodiment 149, wherein the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 99% identical to PD-L1.

[00398] Embodiment 169. The composition of Embodiment 149, wherein the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 7.

[00399] Embodiment 170. The composition of Embodiment 149, wherein the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 70% identical to SEQ ID NO: 7. [00400] Embodiment 171. The composition of Embodiment 149, wherein the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 99% identical to SEQ ID NO: 7.

[00401] Embodiment 172. The composition of Embodiment 149, wherein the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 8.

[00402] Embodiment 173. The composition of Embodiment 149, wherein the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 70% identical to SEQ ID NO: 8.

[00403] Embodiment 174. The composition of Embodiment 149, wherein the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 99% identical to SEQ ID NO: 8.

[00404] Embodiment 175. The composition of Embodiment 149, wherein the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 9.

[00405] Embodiment 176. The composition of Embodiment 149, wherein the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 70% identical to SEQ ID NO: 9.

[00406] Embodiment 177. The composition of Embodiment 149, wherein the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 99% identical to SEQ ID NO: 9.

[00407] Embodiment 178. The composition of Embodiment 149, wherein the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 24.

[00408] Embodiment 179. The composition of Embodiment 149, wherein the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 70% identical to SEQ ID NO: 24.

[00409] Embodiment 180. The composition of Embodiment 149, wherein the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 99% identical to SEQ ID NO: 24.

[00410] Embodiment 181. The composition of Embodiment 149, wherein the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 32. [00411] Embodiment 182. The composition of Embodiment 149, wherein the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 70% identical to SEQ ID NO: 32.

[00412] Embodiment 183. The composition of Embodiment 149, wherein the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 99% identical to SEQ ID NO: 32.

[00413] Embodiment 184. The composition of Embodiment 149, wherein the immune checkpoint or polypeptide moiety comprises a polypeptide sequence that is at least 70% identical to IGSFH (VSIG-3).

[00414] Embodiment 185. The composition of Embodiment 149, wherein the immune checkpoint moiety comprises a polypeptide sequence that is at least 99% identical to IGSF11 (VSIG-3).

[00415] Embodiment 186. The composition of Embodiment 149, wherein the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 10.

[00416] Embodiment 187. The composition of Embodiment 149, wherein the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 70% identical to SEQ ID NO: 10.

[00417] Embodiment 188. The composition of Embodiment 149, wherein the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 99% identical to SEQ ID NO: 10.

[00418] Embodiment 189. The composition of Embodiment 149, wherein the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 11.

[00419] Embodiment 190. The composition of Embodiment 149, wherein the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 70% identical to SEQ ID NO: 11.

[00420] Embodiment 191. The composition of Embodiment 149, wherein the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 99% identical to SEQ ID NO: 11.

[00421] Embodiment 192. The composition of Embodiment 149, wherein the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 12. [00422] Embodiment 193. The composition of Embodiment 149, wherein the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 70% identical to SEQ ID NO: 12.

[00423] Embodiment 194. The composition of Embodiment 149, wherein the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 99% identical to SEQ ID NO: 12.

[00424] Embodiment 195. The composition of Embodiment 149, wherein the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 26.

[00425] Embodiment 196. The composition of Embodiment 149, wherein the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 70% identical to SEQ ID NO: 26.

[00426] Embodiment 197. The composition of Embodiment 149, wherein the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 99% identical to SEQ ID NO: 26.

[00427] Embodiment 198. The composition of Embodiment 149, wherein the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 34.

[00428] Embodiment 199. The composition of Embodiment 149, wherein the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 70% identical to SEQ ID NO: 34.

[00429] Embodiment 200. The composition of Embodiment 149, wherein the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 99% identical to SEQ ID NO: 34.

[00430] Embodiment 201. The composition of Embodiment 149, wherein the immune checkpoint moiety comprises a polypeptide sequence that is at least 70% identical to CTLA-4.

[00431] Embodiment 202. The composition of Embodiment 149, wherein the immune checkpoint moiety comprises a polypeptide sequence that is at least 99% identical to CTLA-4.

[00432] Embodiment 203. The composition of Embodiment 149, wherein the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 13.

[00433] Embodiment 204. The composition of Embodiment 149, wherein the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 70% identical to SEQ ID NO: 13. [00434] Embodiment 205. The composition of Embodiment 149, wherein the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 99% identical to SEQ ID NO: 13.

[00435] Embodiment 206. The composition of Embodiment 149, wherein the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 14.

[00436] Embodiment 207. The composition of Embodiment 149, wherein the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 70% identical to SEQ ID NO: 14.

[00437] Embodiment 208. The composition of Embodiment 149, wherein the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 99% identical to SEQ ID NO: 14.

[00438] Embodiment 209. The composition of Embodiment 149, wherein the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 15.

[00439] Embodiment 210. The composition of Embodiment 149, wherein the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 70% identical to SEQ ID NO: 15.

[00440] Embodiment 211. The composition of Embodiment 149, wherein the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 99% identical to SEQ ID NO: 15.

[00441] Embodiment 212. The composition of Embodiment 149, wherein the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 27.

[00442] Embodiment 213. The composition of Embodiment 149, wherein the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 70% identical to SEQ ID NO: 27.

[00443] Embodiment 214. The composition of Embodiment 149, wherein the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 99% identical to SEQ ID NO: 27.

[00444] Embodiment 215. The composition of Embodiment 149, wherein the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 35. [00445] Embodiment 216. The composition of Embodiment 149, wherein the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 70% identical to SEQ ID NO: 35.

[00446] Embodiment 217. The composition of Embodiment 149, wherein the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 99% identical to SEQ ID NO: 35.

[00447] Embodiment 218. The composition of Embodiment 149, wherein the immune checkpoint moiety comprises a polypeptide sequence that is at least 70% identical to OX-2 (CD200).

[00448] Embodiment 219. The composition of Embodiment 149, wherein the immune checkpoint moiety comprises a polypeptide sequence that is at least 99% identical to OX-2 (CD200).

[00449] Embodiment 220. The composition of Embodiment 149, wherein the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 23.

[00450] Embodiment 221. The composition of Embodiment 149, wherein the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 70% identical to SEQ ID NO: 23.

[00451] Embodiment 222. The composition of Embodiment 149, wherein the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 99% identical to SEQ ID NO: 23.

[00452] Embodiment 223. The composition of Embodiment 149, wherein the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 31.

[00453] Embodiment 224. The composition of Embodiment 149, wherein the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 70% identical to SEQ ID NO: 31.

[00454] Embodiment 225. The composition of Embodiment 149, wherein the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 99% identical to SEQ ID NO: 31.

[00455] Embodiment 226. The composition of Embodiment 149, wherein the immune checkpoint moiety comprises a polypeptide sequence that is at least 70% identical to BTLA.

[00456] Embodiment 227. The composition of Embodiment 149, wherein the immune checkpoint moiety comprises a polypeptide sequence that is at least 99% identical to BTLA. [00457] Embodiment 228. The composition of Embodiment 149, wherein the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 28.

[00458] Embodiment 229. The composition of Embodiment 149, wherein the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 70% identical to SEQ ID NO: 28.

[00459] Embodiment 230. The composition of Embodiment 149, wherein the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 99% identical to SEQ ID NO: 28.

[00460] Embodiment 231. The composition of Embodiment 149, wherein the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 36.

[00461] Embodiment 232. The composition of Embodiment 149, wherein the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 70% identical to SEQ ID NO: 36.

[00462] Embodiment 233. The composition of Embodiment 149, wherein the fusion protein or polypeptide moiety comprises a polypeptide sequence that is at least 99% identical to SEQ ID NO: 36.

[00463] Embodiment 234. The composition of any one of the preceding Embodiments, wherein the transmembrane moiety is an engineered Lysosomal Associated Membrane Protein 2 (LAMP2).

[00464] Embodiment 235. The composition of Embodiment 234, wherein the engineered LAMP2 comprises engineered LAMP2A, LAMP2B, LAMP2C, or a combination thereof. [00465] Embodiment 236. The composition of Embodiment 235, wherein the engineered LAMP2 is the engineered LAMP2B.

[00466] Embodiment 237. The composition of Embodiment 235, wherein the immune checkpoint moiety is covalently connected to an at least one engineered LAMP2B via a linker, said linker comprises a flexible linker, a rigid linker, or a cleavable linker.

[00467] Embodiment 238. The composition of Embodiment 237, wherein the flexible linker comprises a polypeptide sequence comprising: (GGGGS)n where n=l, 2, 3, or 4;

[00468] Embodiment 239. The composition of Embodiment 237 wherein the rigid linker comprises a polypeptide sequence comprising: (EAAAK)n where n=l, 2, 3, or 4; A(EAAAK)nA where n=2, 3, 4, or 5; A(EAAAK)4ALEA(EAAAK)4A; AEAAAKEAAAKA; or LEAGCKNFFPRSFTSCGSLE

[00469] Embodiment 240. The composition of Embodiment 237, wherein the cleavable linker comprises a polypeptide sequence comprising: LEAGCKNFFPRSFTSCGSLE;

CRRRRRREAEAC; GGIEGRGS; or TRHRQPRGWEQL

[00470] Embodiment 241. The composition of any one of the preceding Embodiments, wherein the at least one engineered LAMP2B comprises an amino acid sequence GNSTM of N-terminus of the at least one engineered LAMP2.

[00471] Embodiment 242. The composition of any one of the preceding Embodiments, wherein the at least one engineered LAMP2B comprises an amino acid sequence GNSTM of N-terminus of the at least one immune checkpoint moiety.

[00472] Embodiment 243. A composition comprising at least one extracellular vesicle, said at least one extracellular vesicle comprises at least one polypeptide, and wherein said polypeptide comprises an immune check point moiety that is at least 70% identical to VISTA.

[00473] Embodiment 244. The composition of Embodiment 243, wherein the polypeptide comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 4.

[00474] Embodiment 245. The composition of Embodiment 243, wherein the polypeptide comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 5.

[00475] Embodiment 246. The composition of Embodiment 243, wherein the polypeptide comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 6.

[00476] Embodiment 247. The composition of Embodiment 243, wherein the polypeptide comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 25.

[00477] Embodiment 248. The composition of Embodiment 243, wherein the polypeptide comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 33.

[00478] Embodiment 249. A composition comprising at least one extracellular vesicle, said at least one extracellular vesicle comprises at least one polypeptide, and wherein said polypeptide comprises an immune check point moiety that is at least 70% identical to PD-L1.

[00479] Embodiment 250. The composition of Embodiment 249, wherein the polypeptide comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 7. [00480] Embodiment 251. The composition of Embodiment 249, wherein the polypeptide comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 8.

[00481] Embodiment 252. The composition of Embodiment 249, wherein the polypeptide comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 9.

[00482] Embodiment 253. The composition of Embodiment 249, wherein the polypeptide comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 24.

[00483] Embodiment 254. The composition of Embodiment 249, wherein the polypeptide comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 32.

[00484] Embodiment 255. A composition comprising at least one extracellular vesicle, said at least one extracellular vesicle comprises at least one polypeptide, and wherein said polypeptide comprises an immune check point moiety that is at least 70% identical to IGSF11 (VSIG-3).

[00485] Embodiment 256. The composition of Embodiment 255, wherein the polypeptide comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 10.

[00486] Embodiment 257. The composition of Embodiment 255, wherein the polypeptide comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 11.

[00487] Embodiment 258. The composition of Embodiment 255, wherein the polypeptide comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 12.

[00488] Embodiment 259. The composition of Embodiment 255, wherein the polypeptide comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 26.

[00489] Embodiment 260. The composition of Embodiment 255, wherein the polypeptide comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 34.

[00490] Embodiment 261. A composition comprising at least one extracellular vesicle, said at least one extracellular vesicle comprises at least one polypeptide, and wherein said polypeptide comprises an immune check point moiety that is at least 70% identical to CTLA-4. [00491] Embodiment 262. The composition of Embodiment 261, wherein the polypeptide comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 13.

[00492] Embodiment 263. The composition of Embodiment 261, wherein the polypeptide comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 14.

[00493] Embodiment 264. The composition of Embodiment 261, wherein the polypeptide comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 15.

[00494] Embodiment 265. The composition of Embodiment 261, wherein the polypeptide comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 27.

[00495] Embodiment 266. The composition of Embodiment 261, wherein the polypeptide comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 35.

[00496] Embodiment 267. A composition comprising at least one extracellular vesicle, said at least one extracellular vesicle comprises at least one polypeptide, and wherein said polypeptide comprises an immune check point moiety that is at least 70% identical to OX-2 (CD200).

[00497] Embodiment 268. The composition of Embodiment 267, wherein the polypeptide comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 23.

[00498] Embodiment 269. The composition of Embodiment 267, wherein the polypeptide comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 31.

[00499] Embodiment 270. A composition comprising at least one extracellular vesicle, said at least one extracellular vesicle comprises at least one polypeptide, and wherein said polypeptide comprises an immune check point moiety that is at least 70% identical to BTLA.

[00500] Embodiment 271. The composition of Embodiment 270, wherein the polypeptide comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 28.

[00501] Embodiment 272. The composition of Embodiment 270, wherein the polypeptide comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 36.

[00502] Embodiment 273. The composition of any one of Embodiments 243-272, wherein the at least one extracellular vesicle comprises at least one engineered transmembrane moiety. [00503] Embodiment 274. The composition of Embodiment 273, wherein the engineered transmembrane moiety comprises an engineered LAMP2

[00504] Embodiment 275. The composition of Embodiment 273, wherein the engineered transmembrane moiety comprises an engineered LAMP2A, LAMP2B, LAMP2C, or a combination thereof.

[00505] Embodiment 276. The composition of Embodiment 274, wherein the engineered LAMP2 is an engineered LAMP2B.

[00506] Embodiment 277. The composition of any one of Embodiments 273-276, wherein the polypeptide is fused to the engineered transmembrane moiety.

[00507] Embodiment 278. The composition of Embodiment 277, wherein the polypeptide is covalently connected to the N-terminus of the transmembrane moiety.

[00508] Embodiment 279. The composition of any one of Embodiments 234-278, wherein the extracellular vesicle comprises an exosome, a microvesicle, a retrovirus-like particle, an apoptotic body, an apoptosome, an oncosome, an exopher, an enveloped virus, an exomere, or a very large extracellular vesicle.

[00509] Embodiment 280. The composition of Embodiment 279, wherein the extracellular vesicle is the exosome.

[00510] Embodiment 281. A method for generating the extracellular vesicle of any one of Embodiments 243-248, said method comprising: contacting a cell with a polynucleotide encoding a polypeptide comprising an immune checkpoint moiety that is at least 70% identical to VISTA; inducing homologous recombination in the cell, wherein the polynucleotide encoding the polypeptide is inserted at a locus of a transmembrane moiety, wherein the locus is flanked by one or both homology arms of the transmembrane moiety; and harvesting an extracellular vesicle produced by the cell, wherein the extracellular vesicle comprises the extracellular vesicle of any one of Embodiments 243-248.

[00511] Embodiment 282. A method of treating an inflammatory disease, the method comprises administering the composition of any one of Embodiments 243-248.

[00512] Embodiment 283. A method of treating an autoimmune disease, the method comprises administering the composition of any one of Embodiments 243-248.

[00513] Embodiment 284. A method for generating the extracellular vesicle of any one of Embodiments 249-254, said method comprising: contacting a cell with a polynucleotide encoding a polypeptide comprising an immune checkpoint moiety that is at least 70% identical to PD-L1; inducing homologous recombination in the cell, wherein the polynucleotide encoding the polypeptide is inserted at a locus of a transmembrane moiety, wherein the locus is flanked by one or both homology arms of the transmembrane moiety; and harvesting an extracellular vesicle produced by the cell, wherein the extracellular vesicle comprises the extracellular vesicle of any one of Embodiments 249-254.

[00514] Embodiment 285. A method of treating an inflammatory disease, the method comprises administering the composition of any one of Embodiments 249-254.

[00515] Embodiment 286. A method of treating an autoimmune disease, the method comprises administering the composition of any one of Embodiments 243-254.

[00516] Embodiment 287. A method for generating the extracellular vesicle of any one of Embodiments 255-260, said method comprising: contacting a cell with a polynucleotide encoding a polypeptide comprising an immune checkpoint moiety that is at least 70% identical to IGSF11 (VSIG-3); inducing homologous recombination in the cell, wherein the polynucleotide encoding the polypeptide is inserted at a locus of a transmembrane moiety, wherein the locus is flanked by one or both homology arms of the transmembrane moiety; and harvesting an extracellular vesicle produced by the cell, wherein the extracellular vesicle comprises the extracellular vesicle of any one of Embodiments 255-260.

[00517] Embodiment 288. A method of treating an inflammatory disease, the method comprises administering the composition of any one of Embodiments 255-260.

[00518] Embodiment 289. A method of treating an autoimmune disease, the method comprises administering the composition of any one of Embodiments 255-260.

[00519] Embodiment 290. A method for generating the extracellular vesicle of any one of Embodiments 261-266, said method comprising: contacting a cell with a polynucleotide encoding a polypeptide comprising an immune checkpoint moiety that is at least 70% identical to CTLA-4; inducing homologous recombination in the cell, wherein the polynucleotide encoding the polypeptide is inserted at a locus of a transmembrane moiety, wherein the locus is flanked by one or both homology arms of the transmembrane moiety; and harvesting an extracellular vesicle produced by the cell, wherein the extracellular vesicle comprises the extracellular vesicle of any one of Embodiments 261-266.

[00520] Embodiment 291. A method of treating an inflammatory disease, the method comprises administering the composition of any one of Embodiments 261-266.

[00521] Embodiment 292. A method of treating an autoimmune disease, the method comprises administering the composition of any one of Embodiments 261-266.

[00522] Embodiment 293. A method for generating the extracellular vesicle of any one of Embodiments 267-269, said method comprising: contacting a cell with a polynucleotide encoding a polypeptide comprising an immune checkpoint moiety that is at least 70% identical to OX-2 (CD200); inducing homologous recombination in the cell, wherein the polynucleotide encoding the polypeptide is inserted at a locus of a transmembrane moiety, wherein the locus is flanked by one or both homology arms of the transmembrane moiety; and harvesting an extracellular vesicle produced by the cell, wherein the extracellular vesicle comprises the extracellular vesicle of any one of Embodiments 267-269.

[00523] Embodiment 294. A method of treating an inflammatory disease, the method comprises administering the composition of any one of Embodiments 267-269.

[00524] Embodiment 295. A method of treating an autoimmune disease, the method comprises administering the composition of any one of Embodiments 267-269.

[00525] Embodiment 296. A method for generating the extracellular vesicle of any one of Embodiments 270-272, said method comprising: contacting a cell with a polynucleotide encoding a polypeptide comprising an immune checkpoint moiety that is at least 70% identical to BTLA; inducing homologous recombination in the cell, wherein the polynucleotide encoding the polypeptide is inserted at a locus of a transmembrane moiety, wherein the locus is flanked by one or both homology arms of the transmembrane moiety; and harvesting an extracellular vesicle produced by the cell, wherein the extracellular vesicle comprises the extracellular vesicle of any one of Embodiments 270-272.

[00526] Embodiment 297. A method of treating an inflammatory disease, the method comprises administering the composition of any one of Embodiments 270-272.

[00527] Embodiment 298. A method of treating an autoimmune disease, the method comprises administering the composition of any one of Embodiments 270-272.

EXAMPLES

[00528] The following illustrative examples are representative of embodiments of the stimulation, systems, and methods described herein and are not meant to be limiting in any way.

Example 1. Exemplary uses for the engineered extracellular vesicles

[00529] Since the winter 2019 emergence of the human SARS-CoV-2, and henceforth, the virus has been causing coronavirus-induced disease 2019 (COVID- 19). There have been major ongoing efforts to cure this infectious disease that is the source of the ongoing pandemic. COVID-19 pathology is thought to advance through two main pathways: direct damage to lung cells causing insufficient or fatal lack of oxygenation, and via auto-inflammatory attack on lung and other tissues due to elevated levels of inflammatory cytokines released in response to the virus and to dying cells (cytokine release syndrome, CRS). There are four main treatments for COVID-19 approved under emergency authorization by the FDA that seek to either inactivate the virus itself or seek to mitigate the inflammatory pathology that viral cell damage induces. These treatment classes are: small molecule drugs that combat virus replication; small molecule antiinflammatory drugs (e g. dexamethasone); biologic therapies such as convalescent plasma transfer from recovered individuals, and anti-viral antibody cocktail; and anti-cytokine antibodies to attenuate hyper-inflammation due to CRS.

[00530] Described herein are engineered extracellular vesicles or engineered exosomes as a multi -biologic to attenuate the acute respiratory distress syndrome (ARDS) as the main cardiovascular manifestation of CRS brought about by large-scale cell death and damage caused by viral infection4. This anti-inflammatory therapy can be applicable in a large subset of subjects with low to moderate viral loads, but with high levels of inflammation and inflammatory cytokines. Note that subjects with high viral loads and relatively controlled inflammation cannot be treated with anti-inflammatory agents since the main risk in this case can be uncontrolled viral growth itself, rather than its bystander, inflammation. While COVID-19 is a new disease that is not fully understood, there remains a pressing need for novel, potent, and selective antiinflammatory approaches. Finally, in addition to treating COVID-19, the engineered extracellular vesicles or engineered exosomes (e.g. Exo-MICA) can be applied to a variety of inflammatory, auto-inflammatory, and autoimmune diseases.

[00531] A current paradigm in stem cell therapy is that the exogenous cells exert their therapeutic functions not only by differentiating to parenchymal cells to replace the host’s injured or dying cells, but also via multiple secreted paracrine factors that can help injured cells to recover. These paracrine factors are not only soluble proteins, but they are also distributed on extracellular vesicles (EVs). The natural fabric of EV membranes enables the cells to communicate with other nearby and distant cells. It also empowers stem cells to contribute to tissue remodeling and regeneration in distant sites.

[00532] As described in the present disclosure, therapeutic effects of MSC-derived exosomes in mouse models of MS were evaluated and their main molecular mechanisms were investigated so that they can further be developed with increased potency and decreased side effects for clinical uses.

[00533] In some cases, the engineered EVs or engineered exosomes displaying on their surface multi -immune checkpoint agonists (abbreviated Exo-MICA), including PD-L1, CTLA-4, and VISTA can effectively crosslink their cognate receptors to turn on inhibitory signaling in lymphocytes and antigen-presenting cells (APCs), thereby presenting a novel anti-inflammatory platform to treat acute respiratory distress syndrome (ARDS) in the lungs and central lymphoid organs of patients with COVID-19. In addition, a targeting ligand can also be engineered on the surface of the EVs or exosomes to enhance their homing to lymph nodes, spleen or lung and thereby enhance their activity and specificity. Examples 2-4 described herein are to examine the compositions and methods of the engineered EVs and engineered exosomes (Exo-MICA) described herein. There examples specific can test a platform for immunomodulation based on the nano-scale size of the engineered EVs or engineered exosomes and their mechanoactive surface which can more efficiently crosslink and activate immune checkpoint receptors compared to soluble ligands or antibodies. The -100 nm size of EVs or exosomes enables them to efficiently penetrate peripheral tissues and accumulate in draining lymph nodes, whereas their human-cell derived membrane presents a biocompatible platform. This approach can also be adapted to target EVs and exosomes towards other bodily sites and for other immune-mediated diseases. Furthermore, by targeting the activated inflammatory cells rather than blocking their cytokines with antibodies, this therapeutic approach can be curative and have fewer side effects.

Example 2. Producing and characterizing human cell lines transfected with fusions of Exosomal-LAMP2B and one or more of the checkpoint proteins PD-L1, CTLA-4, VISTA, and IGSF11 (VSIG-3)

[00534] This example (Objective 1) produces several parent cell lines from which engineered EVs or engineered exosomes are produced. Production of the checkpoint fusion protein is first analyzed by immunoblotting of whole cell lysates from starting adipose-derived mesenchymal stem cells (MSCs) that have been immortalized with human telomerase reverse transcriptase (MSC-hTERT) as well as MSC-hTERT engineered to express the checkpoint fusions.

Recombinant proteins (PD-L1, CTLA-4, VISTA, and LAMP2B) can be used as standards. Objective 1A: Production of stable cell lines genetically engineered checkpoint immune genes fused to LAMP2B gene

[00535] An adipose-derived MSC line that has been immortalized with human telomerase reverse transcriptase (MSC-hTERT) is used as the source of the engineered extracellular vesicles or the engineered exosomes due to the excellent cell growth and phenotype stability characteristics and exosome secretion level. The cDNA of each of the genes for PD-L1 (Cd274), CTLA-4 (Ctla4), VISTA (C10orf54), and IGSF11 (VSIG-3) is fused at the N terminus of the native Lamp2 gene. CRISPR-Cas9 is used to knock in the portion of the immune checkpoint genes encoding the engineered extracellular binding domain directly downstream of the Lamp2 signaling peptide, resulting in a fusion at the N terminus of native LAMP2B.

Objective IB, Confirming genetic engineering of checkpoint fusions in whole cells

[00536] The gene editing is confirmed first by PCR, followed by DNA sequencing of the checkpoint genes, as well as reverse transcription PCR for DNA sequencing of the mRNA of each of the fusion proteins. The cells confirmed to contain the correct insert of each of the four checkpoint genes at the N-terminus of LAMP2B are expanded and inventoried for subsequent exosome production.

Objective 1C. Screening engineered cell lines to select optimal expression of checkpoint fusions [00537] Production of the checkpoint fusion protein is first analyzed by immunoblotting of whole cell lysates from starting MSC-hTERT as well as MSC-hTERT engineered to express the checkpoint fusions (MSC-Exo-MICA). Recombinant proteins (PD-L1, CTLA-4, VISTA, IGSF11, or VSIG-3), and LAMP2B) are used as standards. In brief, 10 pl of total cell lysates (total protein concentration brought to 0.5 mg/ml) are ran on 1-D SDS-PAGE followed by Western immunoblotting using an Owl P10DS system (Thermo Fisher). The membranes are incubated with primary antibodies against (PD-L1, CTLA-4, VISTA, IGSF11, or VSIG-3), LAMP2B, as well as household normalizing proteins beta-actin and GAPDH. Images are quantified with ImageJ and the cell lines with the highest expression of target checkpoint genes relative to LAMP2B are selected for subsequent experiments on their exosomes.

Example 3. Producing and characterizing Exo-MICA and test their immunomodulating activity in vitro

[00538] This example (Objective 2) tests various Exo-MICA forms bearing one, two, or three combinations of checkpoint molecule fusions as a surrogate for their immunomodulating ability from which to select the best candidates for further testing in animal models. The engineered extracellular vesicles or the engineered exosomes are purified from engineered MSC-hTERT using first sequential centrifugation to remove whole cells, apoptotic bodies, large debris and microvesicles, further purified and concentrated using tangential flow filtration (TFF) and finally polished using gel filtration on Sepharose 4B resin. The engineered extracellular vesicles or the engineered exosomes are analyzed for presence of total PD-L1, CTLA-4, VISTA, and LAMP2B by immunoblotting and flow cytometry. Next, the immunomodulating activity of Exo-MICA are tested in immune cell culture assays using naive and resting human PBMC. In brief, key inflammatory and anti-inflammatory cytokines using a flow cytometric cytokine bead array kit, and PBMC growth and proliferation are measured by flow cytometry by CFSE dye dilution. Objective 2A, Production of the engineered extracellular vesicles or the engineered exosomes from the engineered cell line and test for surface expression and function of checkpoint fusions [00539] The engineered extracellular vesicles or the engineered exosomes are harvested from conditioned starvation (MEM-a basal media + 0.4% human AB Serum) cell culture media produced by confluent MSC-Exo-MICA cells over a 48h period. The engineered extracellular vesicles or the engineered exosomes are purified from harvested starvation media using first centrifugation at 1,000 x g to remove whole cells, apoptotic bodies, large debris, and microvesicles. Smaller molecular weight contaminants are removed via Tangential Flow Filtration (TFF) through a Hollow fiber filter with a 300 kDa MW cut-off. Finally, the resulting suspension is polished using gel filtration on Capto Core 700 multimodal chromatography resin. The engineered extracellular vesicles or the engineered exosomes are analyzed for presence of total PD-L1, CTLA-4, VISTA, IGSF11, or VSIG-3), and LAMP2B by immunoblotting as described herein Finally, the engineered extracellular vesicles or the engineered exosomes are captured on beads with anti-CD63 mAb and then stained with fluorescent mAbs against PD-L1, CTLA-4, VISTA, IGSF11 (VSIG-3), and LAMP2B, washed, and measured by flow cytometry. In brief, antibodies conjugated with different fluorophores are used to stain the exosomes immobilized on anti-CD63 beads. After washing and resuspending in PBS, the beads containing immobilized exosomes are analyzed by flow cytometry for expression of each checkpoint protein and LAMP2B. The data is compensated using unstained and single-color stained control beads and processed to include only singlet events using the doublet mode discrimination technique of plotting FSC pulse height vs FSC pulse area (only diagonal events are considered singlets). The data is processed with Flowjo, figures exported to PowerPoint, and tables to Excel for further data analysis and rendering. The end result of this analysis generates the ranking of the candidate MSC-Exo-MICA cell lines for maximal expression of each of the checkpoint genes fused to LAMP2B.

Objective 2B, Testing immunomodulating activity of Exo-MICA in immune cell culture assays [00540] Resting and activated (anti-CD3 + anti-CD28 + IL-2 + IL-6) PBMC from healthy human donors are co-cultured with the engineered extracellular vesicles or the exosomes from nonengineered (original) MSCs as well as with engineered extracellular vesicles or Exo-MICA. After 6 h and 24 h, culture supernatant is harvested and stored for subsequent measurement of key inflammatory and anti-inflammatory cytokines using a flow cytometric cytokine bead array kit. After 48 h, PBMC growth and proliferation are measured by flow cytometry and data is analyzed with Flowjo as in sub-aim above. In select experiments, PBMCs are first labelled with the dye CFSE and cell proliferation is assessed by dye dilution. In additional experiments, human PBMCs are obtained from COVID-19 patients, and Exo-MICA are co-cultured with these using the same protocol above.

Objective 2C. Testing immunomodulating activity of Exo-MICA in immunodeficient mice receiving activated human PBMCs

[00541] This objective tests whether Exo-MICA are able to exert their immunosuppressive functions on human PBMC injected to immunodeficient NOD Scid Gamma (NSG) murine hosts. The objective also seeks to observe any adverse side effects of Exo-MICA injected into NSG mice in anticipation of their testing in the murine model of ARDS in subsequent objective. Immunodeficient (NSG) mice receive PBS (control), 1 million, 10 million, and 100 million activated human PBMC. Mice are then administered the most promising Exo-MICA from Objective 2B above, and the proliferation of PBMC and total serum mouse and human cytokines (a panel of 13 cytokines) are measured by flow cytometry. Example 4. Testing immunomodulating activity of Exo-MICA in a murine model of ARDS [00542] This example (Objective 3) tests the most promising Exo-MICA candidates from Example 3 above on a murine model of human ARDS, which closely mimics fulminant ARDS in COVID-19 patients. First, the ‘two hit’ model of lipopolysaccharide (LPS) combined with ventilator-induced lung injury (VILI) Then engineered extracellular vesicles and Exo-MICA immunological activity are tested in this murine model of ARDs. Underivatized exosomes and Exo-MICA candidates are injected in control (no ARDS) mice and in mice induce with ARDS. Serum is analyzed for common cytokine profdes using the CBA assay as described earlier. Immune cells from lungs, spleen and dissociated lung tissue are analyzed by flow cytometry for the status of macrophages, dendritic cells, B cells, and T cell subsets. Finally, lung sections are scored for histology.

Objective 3 A. Establishing the ‘two hit’ model of lipopolysaccharide (LPS) combined with ventilator-induced lung injury (VILI)

[00543] The above model is used in WT C57/BL6 mice, with increasing doses of LPS (1 pg, 10 pg, and 100 pg) administered (IP) or intranasally (IN) combined with increasing damage from a murine ventilator (1 h, 12 h, or 48 h duration). Bronchoalveolar lavage fluid (BAL), whole-lung tissue, and histology are performed to assess for inflammatory biomarkers of immune hyperactivation and tissue injury.

Objective 3B, Testing engineered extracellular vesicles and Exo-MICA activity in the LPS and ventilator injury murine model of ARDS

[00544] Underivatized exosomes (control), buffer (additional control), the engineered extracellular vesicles or the engineered exosomes (Exo-MICA) candidates and dexamethasone (positive control) are tested for their effects on the established murine ARDS model. Increasing doses (0.1 mg, 1 mg or 10 mg total exosomal protein content) of Exo-MICA (and their matched controls) are injected intravenously (IV), IP, or IN in control (no ARDS) mice, or mice induced with ARDS as established in the above aim. Then after 6 hr, 24 hr, and 72 hr the immunological and respiratory status of the mice is evaluated. In brief, serum is analyzed for common cytokine profiles using the CBA assay described herein. Immune cells from lungs, spleen, and dissociated lung tissue are analyzed by flow cytometry for the status of macrophages, dendritic cells, B cells, and ThO, Thl, Th2, Thl7 and Treg cells. Finally, lung sections are scored for histology as in Objective 3 A.

Objective 3C. Testing Exo-MICA activity in the humanized ACE2 and SARS-CoV2 murine model of human COVID-19

[00545] Since transgenic mice carrying the human ACE2 receptor bind to the spike (S) antigen SARS-CoV-2 and are susceptible to limited virus infection and mild symptoms, a murine model equivalent to late-stage severe ARDS is not yet available. However, the humanized ACE2 mice are valuable in testing Exo-MICA for reducing inflammation induced by SARS-CoV-2 and testing whether immunosuppression by the engineered extracellular vesicles or the engineered exosomes (Exo-MICA) is actually counterproductive in enabling more viral growth.

Nevertheless, this objective tests Exo-MICA influence in human transgenic ACE2 mice that are uninfected or infected with SARS-CoV-2. After injection of PBS (control) and increasing concentrations of Exo-MICA (determined from Example 3 above), viral replication is measured by rtPCR in murine nasal fluids and bronchoalveoli. A panel of the most common 13 mouse cytokines is also measured by the CBA assay as in Example 3 above.

Potential caveats and alternative approaches

[00546] If the murine transgenic ACE2 model is unavailable or inappropriate, the engineered extracellular vesicles or the engineered exosomes (Exo-MICA) candidates are tested in a more severe mouse viral infection model such as the respiratory syncytial virus (RSV) model. In brief, a mouse adapted recombinant SARS-CoV-2 virus strain infects mice, and the engineered extracellular vesicles or the engineered exosomes (Exo-MICA) are then administered. For both the LPS plus ventilator injury and murine COVID- 19 model, the basic parameters measuring respiratory function and lung histology are analyzed.

Addressing Biological Variables in Research Designs and Analyses

[00547] The following steps are taken to address biological variables in order to ensure rigor and reproducibility of the compositions and methods described herein. First, only one independent variable is changed at a time to ensure unambiguous data interpretation. For experiments where multiple variables are changed, they are set up and interpreted in a crossed format (as opposed to a nested format) such that each row and each column provides information on the variation of only one variable at a time.

[00548] Second, notes of all the biological reagents purchased, especially vendor information, catalog numbers, batch or lot numbers, are recorded. Most of these reagents are titrated where appropriate (e.g. Exo-MICA, cytokines, TLR ligands, PCR primers, etc.) for deciding on the optimal concentration (or range of concentrations), unless future experimental developments call for further optimization.

[00549] Third, experiments including large and independent data sets are performed in order to test them for statistical significance. Experiments are performed with five mice per group. The student’s t test is used to test for significance, with p values < 0.05 being considered significant at the 95% confidence level, and p values < 0.01 at the 99% confidence level.

[00550] Fourth, if DNA plasmid vectors, cells, cell lines, mouse strains are purchased from reputable vendors (such as ATCC, The Jackson Laboratory, Addgene, etc.). In the cases where such biological reagents are not commercially available, these biological reagents can be obtained from appropriate laboratories. Lastly, an equal number of female and male mice, and B cells from both female and male mice are used and analyzed in the in vitro assays to optimize the nanoparticles.

Example 5. Embodiments utilizing compositions and methods described herein

Innovation

[00551] Overall, the approach described herein combining the natural biocompatibility and multivalent platform of the engineered extracellular vesicles or the engineered exosomes (Exo- MICA) with immune checkpoint inhibitory ligands is innovative. The methodology of fusing checkpoint protein sequences to endosomal/exosomal LAMP2B protein via CRISPR/Cas9 is also an innovative and robust approach compared to traditional transient or stable cell transfection, in which case a significant amount of unfused LAMP2B competes with LAMP2B fusion proteins, and therefore lower the density of the latter on the exosomal membranes. While in this application, at least one, two, three, or four checkpoint protein fusions is tested and the engineered extracellular vesicles or the engineered exosomes (Exo-MICA) platform is compatible with loading other therapeutic agents, notably nucleic acids ( as described in Riazifar et al.; “Stem Cell-Derived Exosomes as Nanotherapeutics for Autoimmune and Neurodegenerative Disorders,” which is herein incorporated by reference in its entirety), which can be developed in future projects and enables the potential embodiment expansion and diversification.

[00552] In more detail, the approach described herein has the following innovative advantages. First, the average number of expressed recombinant checkpoint proteins per the engineered extracellular vesicles or the engineered exosomes (Exo-MICA) are measured and classified as low ligand density, medium density, or high density. This is critical to the biological efficacy of the exosomes since several classes of immune signals require a threshold of about 15 ligands for the signaling to be transmitted (known as the immunome concept). Next, two or more checkpoint proteins are combined on the same the engineered extracellular vesicles or the engineered exosomes (Exo-MICA), which enables synergistic action of the individual components. For example, by creating parental MSCs expressing two different checkpoint ligands, such as the PD- 1 ligand PD-L1 and the VISTA ligand IGSF11 (VSIG-3), EXO-PD-L1+IGSF11 (VSIG-3) activates the signaling from the corresponding heterologous receptors, which results in superior deactivation of hyperactive T cells and ultimately resulting in superior clinical scores in treating autoimmunity. Likewise, by combining a targeting ligand, such as an integrin, or single chain fragment variable (scFv) antibody together with the relevant checkpoint protein, the engineered extracellular vesicles or the engineered exosomes (Exo-MICA) are targeted to particular tissues to gain in efficacy (due to increased local concentration) while reducing off-targeting and side effects.

[00553] Moreover, the engineered extracellular vesicles or the engineered exosomes (Exo- MICA) target different types of immune cells simultaneously. For example, EXO-PD-L1 silences exhausted PD-1+ T cells, whereas EXO-PD-1 activates PD-L1 in CD4+ T lymphocytes to enhance their induction towards the regulatory T cell (Treg phenotype). Finally, unlike most currently marketed antibodies and biologies, the compositions and methods described here do not permanently block certain cytokine receptors, but rather to control the source cells making these inflammatory cytokines. This is expected to be a curative intervention, rather than constantly treating the symptoms. As such, the subjects being treated by the engineered extracellular vesicles or the engineered exosomes (Exo-MICA) described herein cannot be immunosuppressed systemically, unlike the systemic immunosuppression inherent in the current generation of anticytokine antibodies.

Application

[00554] The selectivity of the engineered extracellular vesicles or the engineered exosomes (exomica) platform changes the way immune-mediated diseases are treated. Because there is so much freedom to create myriad combinations of checkpoint proteins and targeting proteins, there is translational potential of exo-mica products for not only treating covid-19, but also treating other autoinflammatory and autoimmune disorders. The further freedom of manufacturing the engineered extracellular vesicles or the engineered exosomes (exo-mica) products across multiple modalities adds to the variety of products that could produce and potential commercial applications.

Example 6. Therapeutic efficacy of exosome expressing IGSF11 (VSIG-3)

[00555] This study examined: (1) whether IGSF11 (VSIG-3) could be engineered to be expressed on surface of an extracellular vesicle as an immune checkpoint moiety described herein; and whether the expressed IGSF11 (VSIG-3) could confer therapeutic effect to treat a disease or disorder, such as inflammation. To determine whether the extracellular vesicle described herein could be engineered to express IGSF11 (VSIG-3) on its surface, the engineered extracellular vesicles were attached to capture chip (which contained fixed receptors for CD63 or CD81, two classical markers for extracellular vesicles derived from MSCs) for imaging analysis. When extracellular vesicle samples were introduced to these chips, they bound to these receptors and were subsequently fixed to the chip as the remainder of the unbound sample was washed away. Fluorescent antibodies could then be introduced to bind the exposed surface molecules of the exosomes fixed on the chip. Such approach, unlike other methods such as western blotting, allowed examination of whether the IGSF11 (VSIG-3) was indeed displayed on the surface, and not inside or luminal region the extracellular vesicles

[00556] FIG. 8 shows that extracellular vesicles were indeed captured (left panel) and that the captured exosomes expressed IGSF11 (VSIG-3) on surface of the captured extracellular vesicles. [00557] FIG. 9 shows an exemplary experimental protocol of a Carrageenan-induced paw edema model in rats. In this animal model, Carrageenan (an inflammatory agent) was injected into the hind paw of the rat, which induced inflammation and swelling. The engineered extracellular vesicles expressing the IGSF11 (VSIG-3) were then injected one hour (Ih) later, and the antiinflammatory effect was quantified by measuring the volume of the paw at later time points (8 hour and 24 hour).

[00558] FIG. 10 illustrates the results of this study. The Normal Paw group refers to the measurement of the other hind paw that was not injected with Carrageenan. The Vehicle group was injected with Carrageenan and a PBS placebo. The engineered exosome group were injected with extracellular vesicles that was engineered to express IGSF11 (VSIG-3) on the surface. Finally, the Unmodified Exosome group was injected with extracellular vesicles derived from MSCs that were not engineered and cultured under the same conditions as the MSCs that were engineered to secret the extracellular vesicles that expressed IGSF11 (VSIG-3). These results demonstrate that the engineered exosome group possessed increased therapeutic antiinflammatory activity compared to unmodified exosomes.

Example 7. Engineered exosomes decreased activation of CD8+/CD25+ T cell

[00559] Flow Cytometry was used to confirm the presence of PD-L1 on the surface of engineered exosomes. Exosomes were immobilized onto magnetic beads and then marked with fluorescent antibodies, then the fluorescence activity was measured by flow cytometry. CD63, a common exosomal surface marker, was first used to demonstrate the shift in fluorescent activity from an isotype control (light gray peak, FIG. 11 left graph) to anti-human CD63 antibody (dark gray peak, FIG. 11 left graph). This experiment confirmed the presence of exosomes on the beads. This experiment was then repeated using an anti-PD-Ll antibody and two exosome groups: unmodified exosomes (light gray peak, FIG. 11 right graph) and engineered exosomes (dark gray peak, FIG. 11 right graph). The shift in fluorescence activity demonstrated that PD-L1 was displayed on the exosomal surface of the engineered exosome group and was not displayed on the surface of the unmodified exosomes. Anti-human CD63 Antibody: APC (BioLegend - 353007).

[00560] Anti-human CD274 Antibody: PE (BioLegend - 374512). Exosome-Human CD63 Isolation/Detecti on Beads: Invitrogen - 10606D. [00561] Human Peripheral Blood Mononuclear Cells (PBMCs) were activated and then cocultured with two groups of exosomes: unmodified exosomes and engineered exosomes After incubation, the cells were harvested and analyzed using flow cytometry. PBMCs co-cultured with engineered exosomes demonstrated significantly decreased numbers of activated T cells (CD8+/CD25+) when compared to the unmodified exosome group. As shown in FIG. 12, CD8+/CD25+ T cells contacted with the engineered exosomes expressing PD-L1 exhibited a 77.6% activation suppression compared to CD8+/CD25+ T cells contacted with unmodified exosomes. Anti-human CD8 Antibody: Brilliant Violet 711 (BioLegend - 344733). Anti-human CD25 Antibody: FITC (BioLegend - 356106).

[00562] While the foregoing disclosure has been described in some detail for purposes of clarity and understanding, it can be clear to one skilled in the art from a reading of this disclosure that various changes in form and detail can be made without departing from the true scope of the disclosure. For example, all the techniques and apparatus described above can be used in various combinations. All publications, patents, patent applications, and/or other documents cited in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, and/or other document were individually and separately indicated to be incorporated by reference for all purposes.

Claims

1. A composition comprising at least one extracellular vesicle, said at least one extracellular vesicle comprising at least one engineered Lysosomal Associated Membrane Protein 2 (LAMP2), wherein the at least one engineered LAMP2 comprises at least one immune checkpoint moiety.

2. A composition comprising at least one extracellular vesicle, said at least one extracellular vesicle comprising at least one engineered Lysosomal Associated Membrane Protein 2 (LAMP2), wherein the at least one engineered LAMP2 comprises at least one immune checkpoint moiety, wherein said immune checkpoint moiety is flanked by at least a fragment of the engineered LAMP2, wherein the fragment comprises the N-terminus homology arm of the engineered LAMP2.

3. The composition of claim 1 or 2, wherein the immune checkpoint moiety comprises a polypeptide sequence that is at least 70% identical to VISTA, PD-L1, CTLA-4, PD-L2, B7-1 (CD80), B7-2 (CD86), B7-H3 (CD276), B7-H2, B7-H4 (VTCN1), HVEM (CD270, TNFRSF14), Galectin 9, Galectin3, CEACAM1 (CD66a), OX-2 (CD200), PVR (CD 155), PVRL2 (Nectin-2, CD112), FGL-1, PECAM-1, TSG-6, CD47, Stabilin-1 (Clever-1), Neuropilin 1, Neuropilin 2, CD 158 (family), IGSF2 (CD101), CD 155, GITRL, CD137L, OX40L, LIGHT, CD70, PD-1, RGMB, CTLA-4 (CD152), BTLA, CD160, Tim-3, CD200R, TIGIT, CD112R (PVRIG), LAG-3 (CD223), PECAM-1, CD44, SIRP alpha (CD172a), IGSF11 (VSIG-3), or a combination thereof.

4. The composition of claim 3, wherein the immune checkpoint moiety comprises a polypeptide sequence that is at least 70% identical to VISTA, PD-L1, IGSF11 (VSIG-3), CTLA-4, OX-2 (CD200), or BTLA.

5. The composition of any one of claim 1-4, wherein the immune checkpoint moiety comprises PD-L1.

6. The composition of any one of claim 1-4, wherein the immune checkpoint moiety comprises IGSF11 (VSIG-3).

7. The composition of any one of claim 1-4, wherein the at least one extracellular vesicle comprises a first immune checkpoint moiety comprising PD-L1 and a second immune checkpoint moiety comprising IGSF11 (VSIG-3).

8. A method of suppressing CD8+ CD25+ cells in a subject in need thereof, the method comprises administering the composition of any one of claims 5-7.

9. The composition of claim 3, wherein the immune checkpoint moiety comprises a polypeptide sequence that is at least 70% identical to PD-L1.

10. The composition of claim 3, wherein the immune checkpoint moiety comprises a polypeptide sequence that is at least 99% identical to PD-L1.

11. The composition of claim 3, wherein the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NOs: 7- 9 or 24.

12. The composition of claim 3, wherein the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 70% identical to SEQ ID NOs: 7-9 or 24.

13. The composition of claim 3, wherein the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 99% identical to SEQ ID NOs: 7-9 or 24.

14. The composition of claim 3, wherein the immune checkpoint moiety comprises a polypeptide sequence that is at least 70% identical to IGSF11 (VSIG-3).

15. The composition of claim 3, wherein the immune checkpoint moiety comprises a polypeptide sequence that is at least 99% identical to IGSF11 (VSIG-3).

16. The composition of claim 3, wherein the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NOs: 10-12 or 26.

17. The composition of claim 3, wherein the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 70% identical to SEQ ID NOs: 10-12 or 26.

18. The composition of claim 3, wherein the at least one engineered LAMP2 comprises a polypeptide sequence that is at least 99% identical to SEQ ID NOs: 10-12 or 26.

19. The composition of claim 3, wherein the immune checkpoint moiety comprises a polypeptide sequence that is at least 70% identical to VISTA.

20. The composition of claim 3, wherein the immune checkpoint moiety comprises a polypeptide sequence that is at least 99% identical to VISTA.

21. The composition of claim 3, wherein the immune checkpoint moiety comprises a polypeptide sequence that is at least 70% identical to CTLA-4.

22. The composition of claim 3, wherein the immune checkpoint moiety comprises a polypeptide sequence that is at least 99% identical to CTLA-4.

23. The composition of claim 3, wherein the immune checkpoint moiety comprises a polypeptide sequence that is at least 70% identical to OX-2 (CD200).

24. The composition of claim 3, wherein the immune checkpoint moiety comprises a polypeptide sequence that is at least 99% identical to OX-2 (CD200).

25. The composition of claim 3, wherein the immune checkpoint moiety comprises a polypeptide sequence that is at least 70% identical to BTLA.

26. The composition of claim 3, wherein the immune checkpoint moiety comprises a polypeptide sequence that is at least 99% identical to BTLA.

27. The composition of any one of the preceding claims, wherein the engineered LAMP2 comprises engineered LAMP2A, LAMP2B, LAMP2C, or a combination thereof.

28. The composition of claim 27, wherein the engineered LAMP2 is the engineered LAMP2B.

29. The composition of any one of the preceding claims, wherein the at least one extracellular vesicle comprises a targeting moiety.

30. The composition of claim 29, wherein the targeting moiety is covalently connected to the N- terminus of the at least one engineered LAMP2B.

31. The composition of claim 30, wherein the targeting moiety comprises a polypeptide that is at least 70% identical to an integrin comprising ot6pi, a6p4, aLp2, aMp2, aXp2, or aDp2.

32. The composition of claim 31, wherein the targeting moiety comprises a polypeptide that is at least 70% identical to a single chain fragment variable (scFv) antibody.

33. The composition of any one of the preceding claims, wherein the at least one extracellular vesicle comprises a signaling peptide.

34. The composition of any one of the preceding claims, wherein the immune checkpoint moiety is covalently connected to the at least one engineered LAMP2B via a linker, said linker comprises a flexible linker, a rigid linker, or a cleavable linker.

35. The composition of claim 34, wherein the flexible linker comprises a polypeptide sequence comprising: i. (GGGGS)n where n=l, 2, 3, or 4; ii. KESGSVSSEQLAQFRSLD; iii. EGKSSGSGSESKST; iv. GGGGGG; v. GGGGGGGG; vi. GSAGSAAGSGEF; vii. RKRR; viii. SS; or ix. LE.

36. The composition of claim 34, wherein the rigid linker comprises a polypeptide sequence comprising: i. (EAAAK)n where n=l, 2, 3, or 4; ii. A(EAAAK)_nA where n=2, 3, 4, or 5; iii. A(EAAAK)₄ALEA(EAAAK)₄A; iv. AEAAAKEAAAKA; or v. LEAGCKNFFPRSFTSCGSLE

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37. The composition of claim 34, wherein the cleavable linker comprises a polypeptide sequence comprising: i. LEAGCKNFFPRSFTSCGSLE; ii. CRRRRRREAEAC; iii GGIEGRGS; or iv. TRHRQPRGWEQL.

38. The composition of any one of the preceding claims, wherein the at least one engineered LAMP2B comprises an amino acid sequence GNSTM at N-Terminus of the at least one engineered LAMP2.

39. The composition of any one of the preceding claims, wherein the at least one extracellular vesicle comprises a plurality of engineered LAMP2Bs.

40. The composition of claim 39, wherein the plurality of the engineered LAMP2Bs comprises a single species of the immune checkpoint moiety.

41. The composition of claim 39, wherein the plurality of the engineered LAMP2Bs comprises two or more species of the immune checkpoint moiety.

42. The composition of claim 41, wherein the two or more species of the immune checkpoint moiety comprises a combination of any two or more of PD-L1, CTLA-4, IGSF11 (VSIG-3), VISTA, OX-2 (CD200), or BTLA.

43. The composition of claim 42, wherein the two or more species of the immune checkpoint moiety are present on the extracellular vesicle at a ratio.

44. The composition of claim 1 comprises a plurality of extracellular vesicles, where the plurality of the extracellular vesicles comprises: a) at least a first population of the extracellular vesicles; and b) at least one additional population of the extracellular vesicles, wherein said first population of the extracellular vesicles comprises at least a first species immune checkpoint moiety and the at least one additional population of the extracellular vesicles comprises at least a second immune checkpoint moiety.

45. The composition of any one of the preceding claims, wherein the extracellular vesicle comprises an exosome, a microvesicle, a retrovirus-like particle, an apoptotic body, an apoptosome, an oncosome, an exopher, an enveloped virus, an exomere, or a very large extracellular vesicle.

46. The composition of claim 45, wherein the extracellular vesicle is the exosome.

47. The composition of claim 1, wherein the at least one engineered LAMP2B comprises a homology arm comprising a nucleic acid sequence that is at least 70% identical to SEQ ID NO:

2, SEQ ID NO: 3, SEQ ID NO: 21, or SEQ ID NO: 22

48. The composition of claim 1, wherein the at least one engineered LAMP2B comprises an N- terminus homology arm comprising a nucleic acid sequence that is at least 70% identical to SEQ ID NO: 2

49. The composition of claim 1, wherein the at least one engineered LAMP2B comprises an N- terminus homology arm comprising a nucleic acid sequence that is at least 70% identical to SEQ ID NO: 21

50. The composition of claim 1, wherein the at least one engineered LAMP2B comprises a C- terminus homology arm comprising a nucleic acid sequence that is at least 70% identical to SEQ ID NO: 3

51. The composition of claim 1, wherein the at least one engineered LAMP2B comprises a C- terminus homology arm comprising a nucleic acid sequence that is at least 70% identical to SEQ ID NO: 22

52. The composition of any one of the preceding claims, wherein the composition further comprises a fusogenic moiety.

53. The composition of claim 52, wherein the fusogenic moiety comprises a viral fusogenic moiety.

54. The composition of claim 52, wherein the fusogenic moiety comprises a mammalian fusogenic moiety.

55. The composition of any one of the preceding claims, wherein the at least one extracellular vesicle further comprises an immune evasion moiety.

56. The composition of claim 55, wherein the immune evasion moiety comprises CD47.

57. The composition of any one of the preceding claims does not comprise an enucleated cell.

58. The composition of any one of the preceding claims, wherein the composition is derived from a cell.

59. The composition of any one of the preceding claims, wherein the composition is cryopreserved.

60. The composition of any one of the preceding claims, wherein the composition is lyophilized.

61. The composition of any one of the preceding claims, wherein the composition is stable at 37°C for at least one hour.

62. A cell genetically modified to produce the extracellular vesicle of any one of the preceding claims.

63. The cell of claim 62, wherein the cell is genetically modified by homologous recombination.

64. The cell of claim 63, wherein the cell is a stem cell.

65. The cell of claim 63, wherein the cell is a human cell.

66. The cell of claim 63, wherein the cell is a non-human cell.

67. The cell of claim 63, wherein the cell is a mesenchymal stem cell.

68. A pharmaceutical composition comprises the composition of any one of claims 1-61 or the cell of any one of claim 62-67.

69. The pharmaceutical composition of claim 68 comprises a pharmaceutically acceptable carrier

70. The pharmaceutical composition of claim 68 comprises at least one additional active agent.

71. The pharmaceutical composition of claim 68, wherein the pharmaceutical composition is formulated for administering intrathecally, intraocularly, intravitreally, retinally, intravenously, intramuscularly, intraventricularly, intracerebrally, intracerebellarly, intracerebroventricularly, intraperenchymally, subcutaneously, or a combination thereof.

72. A kit comprising the composition of any one of claims 1-61, the cell of any one of claim 62- 67, or the pharmaceutical composition of any one of claims 68-71.

73. A platform comprising components for generating the composition of any one of claims 1-61, the cell of any one of claim 62-67, or the pharmaceutical composition of any one of claims 68- 71, or the kit of claim 72.

74. A method for generating the extracellular vesicle of any one of the preceding claims, said method comprising: a) contacting a cell with a polynucleotide encoding the immune checkpoint moiety; b) inducing homologous recombination in the cell, wherein the polynucleotide encoding the immune checkpoint moiety is inserted at a locus of the engineered LAMP2B, wherein the locus is flanked by one or both homology arms of the engineered LAMP2B; and c) harvesting the extracellular vesicle produced by the cell, wherein said extracellular vesicle comprises the engineered LAMP2B comprising the immune checkpoint moiety of any one of the preceding claims.

75. A method of treating an autoimmune disease, the method comprises administering the composition of any one of claims 1-61, the cell of any one of claim 62-67, or the pharmaceutical composition of any one of claims 68-71, or the kit of claim 72 to a subject in need thereof.

76. The method of claim 75, wherein said autoimmune disease is Rheumatoid arthritis, Systemic lupus erythematosus, Psoriasis, Type 1 diabetes mellitus, Multiple sclerosis, Inflammatory bowel disease, Celiac disease, Crohn’s disease, Graves’ disease, Juvenile arthritis, Lyme disease chronic, Optic neuritis, Psoriatic arthritis, Scleritis, Scleroderma, Ulcerative colitis (UC), Uveitis, Sjogren’s syndrome, Inflammatory eye conditions, Idiopathic inflammatory myopathies, Vitiligo, COPD, complication from Organ transplantation, or graft-versus-host disease.

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77. A method of inducing regulatory T-cells (Tregs) in a patient in need thereof, comprises administering the composition of any one of claims 1 composition of any one of claims 1-61, the cell of any one of claim 62-67, or the pharmaceutical composition of any one of claims 68-71, or the kit of claim 72.

78. A composition comprising at least one extracellular vesicle, said at least one extracellular vesicle engineered to express at least one immune checkpoint moiety on a surface of said extracellular vesicle, wherein said at least one immune checkpoint moiety comprises PD-L1, or a fragment thereof.

79. A composition comprising at least one extracellular vesicle, said at least one extracellular vesicle engineered to express at least one immune checkpoint moiety on a surface of said extracellular vesicle, wherein said at least one immune checkpoint moiety comprises IGSF11 (VSIG-3), or a fragment thereof.

80. A method of suppressing CD8+ CD25+ cells in a subject in need thereof, the method comprising administering the composition of claim 78 or 79.

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