Platforms, compositions, and methods for therapeutic delivery

Abstract

Descried herein are platforms for generating extracellular vesicles. Described herein are compositions of extracellular vesicles. Also described herein are methods of using the extracellular vesicles for therapeutics delivery.

1 . A composition comprising an extracellular vesicle, said extracellular vesicle comprising:
 a. an immune checkpoint moiety, wherein said immune checkpoint moiety comprises VISTA, PD-L1, CTLA-4, or any combination thereof; and   b. a transmembrane moiety, wherein said transmembrane moiety comprises CD63, wherein said CD63 comprises 3 transmembrane domains;   wherein said immune checkpoint moiety is coupled to said transmembrane moiety.   
     
     
         2 . The composition of any one of the preceding claims, wherein said immune checkpoint moiety is coupled to an extracellular loop of said CD63 to generate a modified CD63. 
     
     
         3 . The composition of any one of the preceding claims, wherein said extracellular loop is a large extracellular loop or the second extracellular loop of said modified CD63. 
     
     
         4 . The composition of any one of the preceding claims, wherein said modified CD63 comprises an amino acid sequence at least 90% identical to any one of the amino acid sequences set forth in SEQ ID NOS: 6-17. 
     
     
         5 . The composition of any one of the preceding claims, wherein said modified CD63 comprises an amino acid sequence at least 95% identical to any one of the amino acid sequences set forth in SEQ ID NOS: 6-17. 
     
     
         6 . The composition of any one of the preceding claims, wherein said modified CD63 comprises any one of the amino acid sequences set forth in SEQ ID NOS: 6-17. 
     
     
         7 . A composition comprising an extracellular vesicle, said extracellular vesicle comprising:
 a. an immune checkpoint moiety, wherein said immune checkpoint moiety comprises PD-L1; and   b. a transmembrane moiety, wherein said transmembrane moiety comprises lactadherin;   wherein said immune checkpoint moiety is coupled to said transmembrane moiety.   
     
     
         8 . A composition comprising an extracellular vesicle, said extracellular vesicle comprising:
 a. an immune checkpoint moiety, wherein said immune checkpoint moiety comprises V-domain Ig suppressor of T cell activation (VISTA), PD-L1, CTLA-4, or any combination thereof; and   b. a transmembrane moiety, wherein said transmembrane moiety comprises glycosylphosphatidylinositol (GPI);   wherein said immune checkpoint moiety is coupled to said transmembrane moiety.   
     
     
         9 . A composition comprising an extracellular vesicle, said extracellular vesicle comprising at least one of:
 a. an immune checkpoint moiety; and   b. a transmembrane moiety.   
     
     
         10 . The composition of any one of the previous claims, wherein the immune checkpoint moiety is encapsulated by the extracellular vesicle. 
     
     
         11 . The composition of any one of the previous claims, wherein the immune checkpoint moiety is expressed on a surface of the extracellular vesicle. 
     
     
         12 . The composition of any one of the previous claims, wherein the immune checkpoint moiety is secreted by the extracellular vesicle. 
     
     
         13 . The composition of any one of the previous claims, wherein the immune checkpoint moiety is complexed with the transmembrane moiety. 
     
     
         14 . The composition of any one of the previous claims, wherein the immune checkpoint moiety is covalently connected with the transmembrane moiety. 
     
     
         15 . The composition of any one of the previous claims, wherein the immune checkpoint moiety comprises 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 (CD152), BTLA, CD160, Tim-3, CD200R, TIGIT, CD112R (PVRIG), LAG-3 (CD223), PECAM-1, CD44, SIRP alpha (CD172a), or a combination thereof. 
     
     
         16 . The composition of any one of the previous claims, wherein the immune checkpoint moiety comprises VISTA, PD-L1, CTLA-4, or a combination thereof. 
     
     
         17 . The composition of any one of the previous claims, wherein the immune checkpoint moiety comprises PD-L1. 
     
     
         18 . The composition of any one of the previous claims, wherein the transmembrane moiety is selected from a group consisting of 14-3-3 protein zeta/delta, 4-3-3 protein epsilon, 78 kDa glucose-regulated protein, acetylcholinesterase/AChE-S, AChE-E, actin, cytoplasmic 1 (ACTA), ADAM10, 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, CD11a, CD11b, CD11c, 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-1alpha-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, LAMP1/2, leucine-rich receptor kinase 2, L-lactate dehydrogenase A chain, lysosome-associated membrane glycoprotein 1, lysosome-associated membrane glycoprotein 2, MEW class I, MHC class II, MUC1, multidrug resistance-associated protein, muscle pyruvate kinase (PKM2), N-cadherin, NKCC2, PDCD6IP/Alix, PECAM1, phosphoglycerate kinase, placental prion proteins, prostate-specific antigen (PSA), pyruvate kinase (PKM), Rab-14, Rab-5a, Rab-5b, Rab-5c, Rab-7, Rap 1B, 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, vacuolar-sorting protein 35, or zeta polypeptide (YWHAZ). 
     
     
         19 . The composition of any one of the previous claims, wherein the transmembrane moiety comprises lactadherin. 
     
     
         20 . The composition of any one of the previous claims, wherein the transmembrane moiety comprises LAMP2 or a variation thereof or a fragment thereof, said LAMP2 is at least 70% identical to a peptide sequence of SEQ ID NO: 4 
     
     
         21 . The composition of any one of the previous claims, wherein the transmembrane moiety comprises CD63 or a variation thereof or a fragment thereof, said CD63 is at least 70% identical to a peptide sequence of SEQ ID NO:5. 
     
     
         22 . The composition of  claim 21 , wherein the CD63 is a modified CD63. 
     
     
         23 . The composition of  claim 22 , wherein the modified CD63 is a truncated CD63. 
     
     
         24 . The composition of  claim 22 , wherein the modified CD63 is modified to comprise at least one additional CD63 transmembrane domain. 
     
     
         25 . The composition of  claim 22 , wherein the modified CD63 comprises 1 transmembrane domain. 
     
     
         26 . The composition of  claim 22 , wherein the modified CD63 comprises 2 transmembrane domains. 
     
     
         27 . The composition of  claim 22 , wherein the modified CD63 comprises 3 transmembrane domains. 
     
     
         28 . The composition of  claim 22 , wherein the modified CD63 comprises 4 transmembrane domains. 
     
     
         29 . The composition of  claim 22 , wherein the modified CD63 comprises 5 transmembrane domains. 
     
     
         30 . The composition of any one of the previous claims, wherein the immune checkpoint moiety is complexed with the modified CD63 at an extracellular loop of the modified CD63. 
     
     
         31 . The composition of any one of the previous claims, wherein the immune checkpoint moiety is complexed with the modified CD63 at a large extracellular loop of the modified CD63. 
     
     
         32 . The composition of any one of the previous claims, wherein the composition further comprises a targeting moiety. 
     
     
         33 . The composition of  claim 32 , wherein the targeting moiety comprises a peptide that targets cytokine. 
     
     
         34 . The composition of  claim 32  or  33 , wherein the targeting moiety comprises a peptide that targets a cancer cell marker. 
     
     
         35 . The composition of any one of the previous claims, wherein the composition further comprises a fusogenic moiety. 
     
     
         36 . The composition of  claim 35 , wherein the fusogenic moiety comprises a viral fusogenic moiety. 
     
     
         37 . The composition of  claim 35  or  36 , wherein the fusogenic moiety comprises a mammalian fusogenic moiety. 
     
     
         38 . The composition of any one of the previous claims, wherein the composition further comprises an immune evasion moiety. 
     
     
         39 . The composition of  claim 38  wherein the immune evasion moiety comprises CD47. 
     
     
         40 . The composition of any one of the previous claims does not comprise enucleated cell. 
     
     
         41 . The composition of any one of the previous claims, wherein the extracellular vesicle comprises exosome, microvesicle, retrovirus-like particle, apoptotic body, apoptosome, oncosome, exopher, enveloped virus, exomere, or other very large extracellular vesicle. 
     
     
         42 . The composition of any one of the previous claims, wherein the extracellular vesicle comprises exosome. 
     
     
         43 . The composition of any one of the previous claims, wherein the extracellular vesicle comprises a plurality of immune checkpoint moieties. 
     
     
         44 . The composition of  claim 43 , wherein the extracellular vesicle comprises at least 10,000 units of the immune checkpoint moiety per each extracellular vesicle, said extracellular vesicle comprises a diameter of 100 nm. 
     
     
         45 . The composition of  claim 43 , wherein the extracellular vesicle comprises at least 9,000 units of the immune checkpoint moiety per each extracellular vesicle, said extracellular vesicle comprises a diameter of 100 nm. 
     
     
         46 . The composition of  claim 43 , wherein the extracellular vesicle comprises at least 8,000 units of the immune checkpoint moiety per each extracellular vesicle, said extracellular vesicle comprises a diameter of 100 nm. 
     
     
         47 . The composition of  claim 43 , wherein the extracellular vesicle comprises at least 7,000 units of the immune checkpoint moiety per each extracellular vesicle, said extracellular vesicle comprises a diameter of 100 nm. 
     
     
         48 . The composition of  claim 43 , wherein the extracellular vesicle comprises at least 6,000 units of the immune checkpoint moiety per each extracellular vesicle, said extracellular vesicle comprises a diameter of 100 nm. 
     
     
         49 . The composition of  claim 43 , wherein the extracellular vesicle comprises at least 5,000 units of the immune checkpoint moiety per each extracellular vesicle, said extracellular vesicle comprises a diameter of 100 nm. 
     
     
         50 . The composition of  claim 43 , wherein the extracellular vesicle comprises at least 3,000 units of the immune checkpoint moiety per each extracellular vesicle, said extracellular vesicle comprises a diameter of 50 nm. 
     
     
         51 . The composition of  claim 43 , wherein the extracellular vesicle comprises at least 2,500 units of the immune checkpoint moiety per each extracellular vesicle, said extracellular vesicle comprises a diameter of 50 nm. 
     
     
         52 . The composition of  claim 43 , wherein the extracellular vesicle comprises at least 2,000 units of the immune checkpoint moiety per each extracellular vesicle, said extracellular vesicle comprises a diameter of 50 nm. 
     
     
         53 . The composition of  claim 43 , wherein the extracellular vesicle comprises at least 1,500 units of the immune checkpoint moiety per each extracellular vesicle, said extracellular vesicle comprises a diameter of 50 nm. 
     
     
         54 . The composition of any one of the previous claims, wherein the composition comprises a plurality of exosome. 
     
     
         55 . The composition of any one of the previous claims, wherein the composition comprises a therapeutically effective amount of exosome. 
     
     
         56 . The composition of  claim 54  or  55 , comprising at least 10{circumflex over ( )}6 exosomes. 
     
     
         57 . The composition of  claim 54  or  55 , comprising at least 10{circumflex over ( )}7 exosomes. 
     
     
         58 . The composition of  claim 54  or  55 , comprising at least 10{circumflex over ( )}8 exosomes. 
     
     
         59 . The composition of  claim 54  or  55 , comprising at least 1 μg of exosomes. 
     
     
         60 . The composition of  claim 54  or  55 , comprising at least 10 μg of exosomes. 
     
     
         61 . The composition of  claim 54  or  55 , comprising at least 20 μg of exosomes. 
     
     
         62 . The composition of  claim 54  or  55 , comprising at least 50 μg of exosomes. 
     
     
         63 . The composition of  claim 54  or  55 , comprising at least 100 μg of exosomes. 
     
     
         64 . The composition of  claim 54  or  55 , comprising at least 150 μg of exosomes. 
     
     
         65 . The composition of  claim 54  or  55 , comprising at least 200 μg of exosomes. 
     
     
         66 . The composition of  claim 54  or  55 , comprising at least 250 μg of exosomes. 
     
     
         67 . The composition of  claim 54  or  55 , comprising at least 500 μg of exosomes. 
     
     
         68 . The composition of  claim 54  or  55 , comprising at least 750 μg of exosomes. 
     
     
         69 . The composition of  claim 54  or  55 , comprising at least 1 mg of exosomes. 
     
     
         70 . The composition of  claim 54  or  55 , comprising at least 2 mg of exosomes. 
     
     
         71 . The composition of  claim 54  or  55 , comprising at least 3 mg of exosomes. 
     
     
         72 . The composition of  claim 54  or  55 , comprising at least 4 mg of exosomes. 
     
     
         73 . The composition of  claim 54  or  55 , comprising at least 5 mg of exosomes. 
     
     
         74 . The composition of  claim 54  or  55 , comprising at least 6 mg of exosomes. 
     
     
         75 . The composition of  claim 54  or  55 , comprising at least 7 mg of exosomes. 
     
     
         76 . The composition of  claim 54  or  55 , comprising at least 100 mg of exosomes. 
     
     
         77 . The composition of  claim 54  or  55 , comprising at least 200 mg of exosomes. 
     
     
         78 . The composition of  claim 54  or  55 , comprising at least 300 mg of exosomes. 
     
     
         79 . The composition of  claim 54  or  55 , comprising at least 400 mg of exosomes. 
     
     
         80 . The composition of  claim 54  or  55 , comprising at least 500 mg of exosomes. 
     
     
         81 . The composition of  claim 54  or  55 , comprising at least 600 mg of exosomes. 
     
     
         82 . The composition of  claim 54  or  55 , comprising at least 700 mg of exosomes. 
     
     
         83 . The composition of any one of the previous claims, wherein the composition is derived from a cell. 
     
     
         84 . The composition of any one of the previous claims, wherein the composition is cryopreserved. 
     
     
         85 . The composition of any one of the previous claims, wherein the composition is lyophilized. 
     
     
         86 . The composition of any one of the previous claims, wherein the composition is stable at 37° C. for 24 hours. 
     
     
         87 . The composition of any one of the previous claims, wherein the composition is stable at 37° C. for 48 hours. 
     
     
         88 . The composition of any one of the previous claims, wherein the composition is stable at 37° C. for 72 hours. 
     
     
         89 . A cell configured to generate extracellular vesicle of any one of  claims 1 - 88 . 
     
     
         90 . The cell of  claim 89 , wherein said cell is a stem cell. 
     
     
         91 . The cell of  claim 89 , wherein the cell is a human cell. 
     
     
         92 . The cell of  claim 89 , wherein the cell is a non-human cell. 
     
     
         93 . The cell of any one of  claims 89  to  92 , wherein the cell is a mesenchymal stem cell. 
     
     
         94 . The cell of any one of  claims 89  to  93 , wherein the cell is a genetically modified cell. 
     
     
         95 . A method of purifying extracellular vesicle configured to express one or more immune checkpoint moiety, said method comprising:
 a. obtaining a heterogenous population of extracellular vesicle;   b. subjecting the heterogenous population of extracellular vesicle to a detection assay solution, said detection assay solution comprising a detecting moiety for complexing with the immune checkpoint moiety; and   c. detecting a signal generated from complex formed between the immune checkpoint moiety and the detecting moiety, wherein intensity of the signal is proportional to units of immune checkpoint moiety expressed; and   d. isolating a subpopulation of the extracellular vesicle based on the intensity of the signal.   
     
     
         96 . The method of  claim 95 , wherein the detecting moiety comprises an antibody. 
     
     
         97 . The method of  claim 96 , wherein the detecting moiety comprises anti-VISTA antibody, anti-PD-L1 antibody, anti-CTLA-4 or a combination thereof. 
     
     
         98 . The method of  claim 95 , wherein the detecting moiety comprises a ligand of the immune checkpoint moiety. 
     
     
         99 . The method of  claim 95 , wherein the detection assay solution further comprises a peptide configured to bind to said immune checkpoint moiety. 
     
     
         100 . The method of  claim 99 , wherein the peptide configured to bind to said immune checkpoint moiety comprises PD-1, CD80, CD86, or a combination thereof. 
     
     
         101 . A pharmaceutical composition comprising the composition of any one of  claims 1 - 88 . 
     
     
         102 . The pharmaceutical composition of  claim 101  comprising a pharmaceutically acceptable carrier. 
     
     
         103 . The pharmaceutical composition of  claim 101  comprising at least one additional active agent. 
     
     
         104 . The pharmaceutical composition of  claim 101 , wherein the pharmaceutical composition is formulated for administering intrathecally, intraocularly, intravitreally, retinally, intravenously, intramuscularly, intraventricularly, intracerebrally, intracerebellarly, intracerebroventricularly, intraperenchymally, subcutaneously, or a combination thereof. 
     
     
         105 . A method of treating an autoimmune disease, the method comprising administering the pharmaceutical composition of any one of  claims 101  to  104 . 
     
     
         106 . The method of  claim 105 , 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, Inflammatory eye conditions, Vitiligo, COPD, complication from Organ transplantation, or graft-versus-host disease. 
     
     
         107 . The method of  claim 106 , wherein said autoimmune disease is Rheumatoid arthritis. 
     
     
         108 . A method of suppressing CD8+CD25+ cells in a patient in need thereof, the method comprising administering the composition of any one of  claims 1 - 88 . 
     
     
         109 . A kit comprising the pharmaceutical composition of  claims 101 - 104 . 
     
     
         110 . A platform comprising components for generating the composition of any one of  claims 1 - 88 .