WO2022182929A9

WO2022182929A9 - Anti-inflammatory extracellular vesicle compositions and methods

Info

Publication number: WO2022182929A9
Application number: PCT/US2022/017801
Authority: WO
Inventors: Stanley Hersh APPEL; Aaron Drew THOME; Jason Robert THONHOFF
Original assignee: The Methodist Hospital
Priority date: 2021-02-26
Filing date: 2022-02-25
Publication date: 2023-09-07
Also published as: WO2022182929A1

Abstract

The present disclosure provides anti-inflammatory extracellular vesicles (EVs) derived from ex vivo-cultured human suppressive immune cells, e.g., M2 macrophages. Such EVs are useful in the treatment of diseases such as amyotrophic lateral sclerosis (AES), Alzheimer's disease, and other neurological diseases, as well as inflammatory and autoimmune diseases or dysfunctions.

Description

ANTI-INFLAMMATORY EXTRACELLULAR VESICLE COMPOSITIONS AND METHODS

[0001] This application claims the benefit of U.S. Provisional Application No. 63/208,402, filed June 8, 2021, and U.S. Provisional Application No. 63/154,456, filed February 26, 2021, each of which is incorporated by reference herein in its entirety.

1. FIELD

[0002] The present disclosure provides anti-inflammatory extracellular vesicles (EVs) that are derived from ex vzvo-cultured human suppressive immune cells, e.g., M2 macrophages, and that are useful in the treatment of diseases such as amyotrophic lateral sclerosis (ALS), Alzheimer’s disease, and other neurological diseases, as well as inflammatory and autoimmune diseases or dysfunctions.

2. BACKGROUND

[0003] Inflammatory and neuroinflammatory mechanisms contribute to a wide variety of devastating diseases, including such neurodegenerative diseases as amyotrophic lateral sclerosis (ALS), Parkinson’s disease and multiple sclerosis. Neurodegenerative diseases such as this direct a tremendous health and economic burden that will only exacerbate further over time.

[0004] Currently, no disease-modifying treatments for such diseases are available. Antiinflammatory treatments have been utilized for decades in attempting to ameliorate a multitude of neurodegenerative diseases. Little progress, however, has been made with single drug/target approaches.

[0005] Increasingly, studies point to immune system involvement in the etiology of diseases such as this, and point to dysfunction of immune cells as a chief mediator of disease pathogenesis. The complex signaling mechanisms and built-in redundancies of the immune system and its constituents may help explain the ineffectiveness of such single drug/single target anti-inflammatory approaches.

[0006] Recently great promise has been demonstrated with regulatory T cell (Treg) cell therapy, which may represent a more global approach to suppressing immune system dysfunction contributing to disease. For example, clinical trials involving administration of expanded autologous Tregs to ALS patients report that the Treg therapy slowed progression rates during early and later stages of the disease, and that Treg suppressive function correlated with the slowing of disease progression (Thonhoff, J.R. et al., 2018, Neurology-Neuroimmunology Neuroinflammation 5(4)).

[0007] Nonetheless, there still exists a need for development of additional treatments that can suppress inflammatory and/or promote anti-inflammatory immune system components, and can do so in the pro-inflammatory, toxic microenvironment of the disease state.

3. SUMMARY

[0008] Presented herein are extracellular vesicles (EVs) that exhibit impressive antiinflammatory activity, both in vitro and in vivo. The EVs presented herein are derived from ex vivo-cultured human suppressive immune cells, for example M2 macrophages.

[0009] Results presented herein demonstrate that the EVs of the present disclosure are able to potently suppress T responder cell proliferation and pro-inflammatory macrophage activity in vitro, and also exert potent anti-inflammatory effects in vivo. In particular, results presented herein demonstrate that the EVs are able to suppress brain and peripheral inflammation in an in vivo model of neuroinflammation.

[0010] Thus, the EVs of the present disclosure retain the immune suppressive activities of the cells from which they are derived. Moreover, as EVs are not themselves cells, they avoid potential cell-based issues such as immune rejection and the possibility of polarization to a pro- inflammatory cell type. As such, the anti-inflammatory EVs presented herein are particularly useful for treatment of a variety of diseases such as, for example, neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS).

[0011] Presented herein are isolated, cell-free populations of anti-inflammatory EVs, wherein the anti-inflammatory EVs are derived from ex vivo-cultured human M2 macrophages, e.g., from M2 macrophages derived from inducible pluripotent stem cells (iPSC). Also presented herein are pharmaceutical compositions and cryopreserved compositions comprising an isolated, cell-free population of anti-inflammatory EVs described herein, methods of producing the EV populations and methods of using the EVs for treatment of diseases, such as neurodegenerative diseases, e.g., ALS. [0012] In one aspect, provided herein is an isolated, cell-free population of antiinflammatory extracellular vesicles (EVs), wherein the anti-inflammatory EVs are derived from ex vivo-cultured human suppressive immune cells. In some embodiments, the human suppressive immune cells are M2 macrophages. In some embodiments, the M2 macrophages are produced from induced pluripotent stems cells (iPSCs). In some embodiments, the precursors for the M2 macrophages are derived from multiple unrelated human subjects.

[0013] In some embodiments, the precursors for the M2 macrophages are iPSC. In some embodiments, the precursors for the M2 macrophages are derived from PBMCs.

[0014] In some embodiments, the anti-inflammatory EVs exhibit an ability to increase the expression of one or more anti-inflammatory markers in inflammatory cells. In some embodiments, the inflammatory cells are myeloid cells.

[0015] In some embodiments, the anti-inflammatory EVs exhibit an ability to increase the expression of IL-10, Argl and/or CD206 in inflammatory cells. In some embodiments, the antiinflammatory EVs exhibits an ability to suppress inflammatory cells, as measured by pro- inflammatory cytokine production by the inflammatory cells. In some embodiments, the inflammatory cells are myeloid cells. In some embodiments, the myeloid cells are monocytes, macrophages, or microglia. In some embodiments, macrophages are Ml macrophages. In some embodiments, the Ml macrophages are induced pluripotent stem cell (iPSC)-derived Ml macrophages.

[0016] In some embodiments, the ability to suppress inflammatory cells is measured by IL-6, IL-8, TNFa, ILip and/or Interferon-y production by the inflammatory cells.

[0017] In some embodiments, the anti-inflammatory EVs exhibit a suppressive function, as determined by suppression of proliferation of responder T cells. In some embodiments, the proliferation of responder T cells is determined by flow cytometry or thymidine incorporation. [0018] In some embodiments, the population of anti-inflammatory EVs is a saline-containing population of anti-inflammatory EVs. In some embodiments, the population is a physiological saline-containing population of anti-inflammatory EVs. In some embodiments, the population is a phosphate-buffered saline-containing population of anti-inflammatory EVs.

[0019] In some embodiments, the population of anti-inflammatory EVs comprises exosomes and microvesicles. In some embodiments, the majority of the EVs are exosomes. In some embodiments, at least about 80%, about 90%, or about 95% of the EVs are exosomes. In some embodiments, the majority of the EVs are microvesicles. In some embodiments, at least about 80%, about 90%, or about 95% of the EVs are microvesicles.

[0020] In some embodiments, the majority of the EVs in a population of anti-inflammatory EVs have diameters from about 30 nm to about 1000 nm. In some embodiments, the majority of the EVs have diameters from about 30 nm to about 100 nm, about 30 nm to about 150 nm, about 30 to about 200 nm, about 40 to about 100 nm, about 80 to about 100 nm, about 80 to about 110 nm, about 80 to about 125 nm, or about 100 to about 120 nm. In some embodiments, the majority of the EVs have diameters from about 60 nm to 1000 nm, about 70 nm to about 1000 nm, about 80 nm to about 1000 nm, 100 to about 1000 nm, about 200 to about 1000 nm, about 300 to about 1000 nm.

[0021] In another aspect, provided herein is a pharmaceutical composition comprising an isolated, cell-free population of anti-inflammatory EVs provided herein. In some embodiments, the population of anti-inflammatory EVs comprises about IxlO⁶ to about IxlO¹⁴ EVs, about IxlO⁸ to about IxlO¹⁴ EVs, about IxlO⁸ to about IxlO¹² EVs, about IxlO⁸ to about IxlO¹⁰ EVs, about IxlO¹⁰ to about IxlO¹⁴ EVs, or about IxlO¹⁰ to about IxlO¹² EVs. In some embodiments, the population of anti-inflammatory EVs comprises about IxlO⁶ to about IxlO¹⁴ EVs/ml, about IxlO⁸ to about IxlO¹⁴ EVs/ml, about IxlO⁸ to about IxlO¹² EVs/ml, about IxlO⁸ to about IxlO¹⁰ EVs/ml, about IxlO¹⁰ to about IxlO¹⁴ EVs/ml, or about IxlO¹⁰ to about IxlO¹² EVs/ml.

[0022] In some embodiments, the population of anti-inflammatory EVs in a pharmaceutical composition provided herein comprises about 1 pg to about 200 mg EVs. In some embodiments, the population of anti-inflammatory EVs in a pharmaceutical composition provided herein comprises about 1 pg to about 15 mg EVs. In some embodiments, the population of antiinflammatory EVs in a pharmaceutical composition provided herein comprises about 1 pg to about 15 mg EV/ml.

[0023] In some embodiments, a pharmaceutical composition provided herein is a cryopreserved pharmaceutical composition. In some embodiments, the pharmaceutical composition had previously been cryopreserved.

[0024] In another aspect, provided herein is a cryopreserved composition comprising an isolated, cell-free population of anti-inflammatory EVs provided herein.

[0025] In another aspect, provided herein is a method of producing an isolated, cell-free population of anti-inflammatory extracellular vesicles (EVs), said method comprising the steps of: (a) ex -vivo culturing a human suppressive immune cell population in culture media to produce a culture comprising the cells, the culture media and anti-inflammatory EVs; and (b) isolating the anti-inflammatory EVs from the culture. In some embodiments, the human suppressive immune cell population is a population of M2 macrophages.

[0026] In some embodiments, step b) comprises removing cells from the culture, followed by polyethylene glycol precipitation of the culture. In some embodiments, step b) comprises: (i) removing the cells from the culture to produce a cell-free, anti-inflammatory EV-containing solution; and (ii) isolating the anti-inflammatory EVs from the cell-free, anti-inflammatory EV- containing solution of (i). In some embodiments, step (i) comprises passing the culture through a filter such that the cells are retained by the filter, and thereby removed from the culture. In some embodiments, step (i) comprises microfiltration.

[0027] In some embodiments, step (ii) comprises step (ii-a): passing the cell-free, antiinflammatory EV-containing solution through a filter such that the anti-inflammatory EVs are retained by the filter. In some embodiments, the filter has a molecular weight cut-off (MWCO) of about 200 kilodaltons (kDa) to about 600 kDa. In some embodiments, the filter has an MWCO of about 500 kDa.

[0028] In some embodiments, step (ii) comprises ultrafiltration. In some embodiments, step (ii) further comprises step (ii-b): performing buffer exchange such that the isolated, cell-free population of anti-inflammatory EVs produced is a buffer-containing isolated, cell-free population of anti-inflammatory EVs. In some embodiments, the buffer is a saline-containing buffer. In some embodiments, the saline-containing buffer is physiological saline. In some embodiments, the saline-containing buffer is PBS.

[0029] In some embodiments, step (ii-b) comprises diafiltration.

[0030] In some embodiments, steps (ii-a) and (ii-b) are performed simultaneously.

[0031] In some embodiments, step (b) comprises tangential flow filtration.

[0032] In some embodiments, the culture media in step (a) is serum-free. In some embodiments, the culture media in step (a) comprises serum. In some embodiments, the serum is human AB serum. In some embodiments, the serum is depleted for serum-derived EVs.

[0033] In some embodiments, the method further comprises, prior to step (a), the step of culturing iPSC cells in the presence of cytokines to induce polarization to M2 macrophages. In some embodiments, the human suppressive immune cell population is a genetically engineered human suppressive immune cell population.

[0034] In another aspect, provided herein is a pharmaceutical composition comprising an isolated, cell-free population of anti-inflammatory EVs, wherein the population is made by any one of the methods provided herein..

[0035] In some embodiments, the method further comprises: (c) cryopreserving the isolated, cell-free population of anti-inflammatory EVs, thereby producing a cryopreserved, isolated, cell- free population of anti-inflammatory EVs. Also presented herein are cryopreserved compositions comprising an isolated, cell-free population of anti-inflammatory EVs, wherein the cryopreserved compositions are made using such methods.

[0036] In some embodiments, the method further comprises thawing the cryopreserved, isolated cell-free population of anti-inflammatory EVs after cryopreservation for about 1 week, 1 month, about 3 months, about 6 months, about 9 months, about 12 months, about 18 months or about 24 months. Also provided herein are compositions, for example, pharmaceutical compositions, comprising an isolated, cell-free population of anti-inflammatory EVs, wherein the compositions, for example, pharmaceutical compositions, are made using such methods.

[0037] In another aspect, provided herein is a method of treating a disorder associated with Treg dysfunction, the method comprising administering to a subject in need of said treatment a pharmaceutical composition provided herein.

[0038] In another aspect, provided herein is a method of treating a disorder associated with Treg deficiency, the method comprising administering to a subject in need of said treatment a pharmaceutical composition provided herein.

[0039] In another aspect, provided herein is a method of treating a disorder associated with over activation of the immune system, the method comprising administering to a subject in need of said treatment a pharmaceutical composition provided herein.

[0040] In another aspect, provided herein is a method of treating an inflammatory condition driven by a T cell response, the method comprising administering to a subject in need of said treatment a pharmaceutical composition provided herein.

[0041] In another aspect, provided herein is a method of treating an inflammatory condition driven by a myeloid cell response, the method comprising administering to a subject in need of said treatment a pharmaceutical composition provided herein. In some embodiments, the myeloid cell is a monocyte, macrophage or microglia.

[0042] In another aspect, provided herein is a method of treating a neurodegenerative disorder in a subject in need thereof, the method comprising administering to a subject in need of said treatment a pharmaceutical composition provided herein. In some embodiments, the neurodegenerative disease is ALS, Alzheimer’s disease, Parkinson’s disease, frontotemporal dementia or Huntington’s disease.

[0043] In another aspect, provided herein is a method of treating an autoimmune disorder in a subject in need thereof, the method comprising administering to a subject in need of said treatment a pharmaceutical composition provided herein. In some embodiments, the autoimmune disorder is polymyositis, ulcerative colitis, inflammatory bowel disease, Crohn’s disease, celiac disease, systemic sclerosis (scleroderma), multiple sclerosis (MS), rheumatoid arthritis (RA), Type I diabetes, psoriasis, dermatomy osititis, systemic lupus erythematosus, cutaneous lupus, myasthenia gravis, autoimmune nephropathy, autoimmune hemolytic anemia, autoimmune cytopenia, autoimmune encephalitis, autoimmune hepatitis, autoimmune uveitis, alopecia, thyroiditis or pemphigus.

[0044] In another aspect, provided herein is a method of treating graft-versus-host disease in a subject in need thereof, the method comprising administering to a subject in need of said treatment a pharmaceutical composition provided herein. In some embodiments, the subject has received a bone marrow transplant, kidney transplant or liver transplant.

[0045] In another aspect, provided herein is a method of improving islet graft survival in a subject in need thereof, the method comprising administering to a subject in need of said treatment a pharmaceutical composition provided herein.

[0046] In another aspect, provided herein is a method of treating cardio-inflammation in a subject in need thereof, the method comprising administering to a subject in need of said treatment a pharmaceutical composition provided herein. In some embodiments, the cardio- inflammation is associated with atherosclerosis, myocardial infarction, ischemic cardiomyopathy or heart failure.

[0047] In another aspect, provided herein is a method of treating neuroinflammation in a subject in need thereof, the method comprising administering to a subject in need of said treatment a pharmaceutical composition provided herein. In some embodiments, the neuroinflammation is associated with stroke, acute disseminated encephalomyelitis, acute optic neuritis, acute inflammatory demyelinating polyradiculoneuropathy, chronic inflammatory demyelinating polyradiculoneuropathy, Guillain-Barre syndrome, transverse myelitis, neuromyelitis optica, epilepsy, traumatic brain injury, spinal cord injury, encephalitis, central nervous system vasculitis, neurosarcoidosis, autoimmune or post-infectious encephalitis or chronic meningitis.

[0048] In another aspect, provided herein is a method of treating a Tregopathy in a subject in need thereof, comprising administering to a subject in need of said treatment a pharmaceutical composition provided herein. In some embodiments, the Tregopathy is caused by a FOXP3, CD25, cytotoxic T lymphocyte-associated antigen 4 (CTLA4), LPS-responsive and beige-like anchor protein (LRBA), or BTB domain and CNC homolog 2 (BACH2) gene loss-of-function mutation, or a signal transducer and activator of transcription 3 (STAT3) gain-of-function mutation.

[0049] In some embodiments, the anti-inflammatory EVs are derived from M2 macrophages that are autologous to the subject. In some embodiments, the anti-inflammatory EVs are derived from M2 macrophages that are allogeneic to the subject.

[0050] In some embodiments, the pharmaceutical composition is administered via intranasal administration. In some embodiments, the intranasal administration is via aerosol inhalation or nasal drip. In some embodiments, the pharmaceutical composition is administered intravenously. In some embodiments, the pharmaceutical composition is administered by local injection.

[0051] In some embodiments, the method further comprises administering to the subject a pharmaceutical composition comprising a therapeutic population of Tregs, for example a population of Tregs had been ex vivo expanded. In particular embodiments, the method further comprises administering to the subject a pharmaceutical composition comprising a therapeutic population of Tregs wherein the Tregs had been ex vivo expanded and cryopreserved, and wherein the Tregs are not further expanded prior to the administering. In some embodiments, the therapeutic population of Tregs is autologous to the subject. In some embodiments, the therapeutic population of Tregs is allogeneic to the subject. In some embodiments, the pharmaceutical composition comprising the therapeutic population of Tregs is administered intravenously. In some embodiments, the pharmaceutical composition comprising the anti- inflammatory EVs and the pharmaceutical composition comprising the therapeutic population of Tregs are administered to the patient on the same day.

[0052] In certain embodiments, the methods of treatment presented herein comprise administering to a subject in need of treatment a pharmaceutical composition comprising an isolated, cell-free population of anti-inflammatory EVs, wherein the EVs had been cryopreserved and thawed prior to being administered to the subject. In certain embodiments, the methods of treatment presented herein comprise administering to a subject in need of treatment a pharmaceutical composition comprising an isolated, cell-free population of anti-inflammatory EVs, wherein the EVs are stored at 4 °C, for example, are stored overnight at 4 °C, prior to being administered to the subject. In particular embodiments, the methods of treatment presented herein comprise administering to a subject in need of treatment a pharmaceutical composition comprising an isolated, cell-free population of anti-inflammatory EVs wherein the EVs had been cryopreserved, thawed and stored at 4 °C, for example, stored overnight at 4 °C, prior to being administered to the subject.

[0053] In certain embodiments, the methods of treatment presented herein comprise administering to a subject in need of treatment a pharmaceutical composition comprising an isolated, cell-free population of anti-inflammatory EVs wherein the EVs had undergone at least two freeze/thaw cycles prior to being administered to the subject, e.g., had undergone about 2 to about 20 freeze/thaw cycles prior to being administered to the subject.

[0054] Further illustrative embodiments are as follows:

1. An isolated, cell-free population of anti-inflammatory extracellular vesicles (EVs), wherein the anti-inflammatory EVs are derived from ex vi vo-cultured human suppressive immune cells.

2. The population of anti-inflammatory EVs of embodiment 1, wherein the human suppressive immune cells are M2 macrophages.

3. The population of anti-inflammatory EVs of embodiment 2, wherein the M2 macrophages are produced from induced pluripotent stems cells (iPSCs).

4. The population of anti-inflammatory EVs of any one of embodiments 1-3, wherein the precursors for the M2 macrophages are derived from multiple unrelated human subjects. The method of embodiment 4, wherein the precursors for the M2 macrophages are iPSC. The method of embodiment 4 or 5, wherein the precursors for the M2 macrophages are derived from PBMCs. The population of anti-inflammatory EVs of any one of embodiments 1-6, wherein the anti-inflammatory EVs exhibit an ability to increase the expression of one or more antiinflammatory markers in inflammatory cells. The population of anti-inflammatory EVs of embodiment 7, wherein the inflammatory cells are myeloid cells. The population of anti-inflammatory EVs of embodiment 7 or 8, wherein the antiinflammatory EVs exhibit an ability to increase the expression of IL-10, Argl and/or CD206 in inflammatory cells. The population of anti-inflammatory EVs of any one of embodiments 1-9, wherein the anti-inflammatory EVs exhibits an ability to suppress inflammatory cells, as measured by pro-inflammatory cytokine production by the inflammatory cells. The method of embodiment 10, wherein the inflammatory cells are myeloid cells. The population of anti-inflammatory EVs of embodiment 11, wherein the myeloid cells are monocytes, macrophages, or microglia. The population of anti-inflammatory EVs of embodiment 12, wherein macrophages are Ml macrophages. The population of anti-inflammatory EVs of embodiment 13, wherein the Ml macrophages are induced pluripotent stem cell (iPSC)-derived Ml macrophages. The population of anti-inflammatory EVs of any one of embodiments 7-14, wherein the ability to suppress inflammatory cells is measured by IL-6, IL-8, TNFa, ILip and/or Interferon-y production by the inflammatory cells. The population of anti-inflammatory EVs of any one of embodiments 1-14 wherein the anti-inflammatory EVs exhibit a suppressive function, as determined by suppression of proliferation of responder T cells. The population of anti-inflammatory EVs of embodiment 16, wherein the proliferation of responder T cells is determined by flow cytometry or thymidine incorporation. The population of anti-inflammatory EVs of any one of embodiments 1-17, wherein the population is a saline-containing population of anti-inflammatory EVs. The population of anti-inflammatory EVs of any one of embodiments 1-17, wherein the population is a physiological saline-containing population of anti-inflammatory EVs. The population of anti-inflammatory EVs of any one of embodiments 1-17, wherein the population is a phosphate-buffered saline-containing population of anti-inflammatory EVs. The population of anti-inflammatory EVs of any one of any one of embodiments 1-20, wherein the population of anti-inflammatory EVs comprises exosomes and microvesicles. The population of anti-inflammatory EVs of embodiment 21, wherein the majority of the EVs are exosomes. The population of anti-inflammatory EVs of embodiment 22, wherein at least about 80%, about 90%, or about 95% of the EVs are exosomes. The population of anti-inflammatory EVs of embodiment 21 wherein the majority of the EVs are microvesicles. The population of anti-inflammatory EVs of embodiment 24, wherein at least about 80%, about 90%, or about 95% of the EVs are microvesicles. The population of anti-inflammatory EVs of embodiment 21, wherein the majority of the EVs have diameters from about 30 nm to about 1000 nm. The population of anti-inflammatory EVs of embodiment 21, wherein the majority of the EVs have diameters from about 30 nm to about 100 nm, about 30 nm to about 150 nm, about 30 to about 200 nm, about 40 to about 100 nm, about 80 to about 100 nm, about 80 to about 110 nm, about 80 to about 125 nm, or about 100 to about 120 nm. The population of anti-inflammatory EVs of embodiment 21 wherein the majority of the EVs have diameters from about 60 nm to 1000 nm, about 70 nm to about 1000 nm, about 80 nm to about 1000 nm, 100 to about 1000 nm, about 200 to about 1000 nm, about 300 to about 1000 nm. A pharmaceutical composition comprising an isolated, cell-free population of antiinflammatory EVs of any one of embodiments 1-28. The pharmaceutical composition of embodiment 29, wherein the population of antiinflammatory EVs comprises about IxlO⁶ to about IxlO¹⁴ EVs, about IxlO⁸ to about IxlO¹⁴ EVs, about IxlO⁸ to about IxlO¹² EVs, about IxlO⁸ to about IxlO¹⁰ EVs, about IxlO¹⁰ to about IxlO¹⁴ EVs, or about IxlO¹⁰ to about IxlO¹² EVs. The pharmaceutical composition of embodiment 29, wherein the population of antiinflammatory EVs comprises about IxlO⁶ to about IxlO¹⁴ EVs/ml, about IxlO⁸ to about IxlO¹⁴ EVs/ml, about IxlO⁸ to about IxlO¹² EVs/ml, about IxlO⁸ to about IxlO¹⁰ EVs/ml, about IxlO¹⁰ to about IxlO¹⁴ EVs/ml, or about IxlO¹⁰ to about IxlO¹² EVs/ml. The pharmaceutical composition of embodiment 29, wherein the population of antiinflammatory EVs comprises about 1 pg to about 200 mg EVs. The pharmaceutical composition of embodiment 29, wherein the population of antiinflammatory EVs comprises about 1 pg to about 15 mg EVs. The pharmaceutical composition of embodiment 29, wherein the population of antiinflammatory EVs comprises about 1 pg to about 15 mg EV/ml. The pharmaceutical composition of any one of embodiments 29-34, wherein the pharmaceutical composition is a cryopreserved pharmaceutical composition. The pharmaceutical composition of any one of embodiments 29-34, wherein the pharmaceutical composition had previously been cryopreserved. A cryopreserved composition comprising an isolated, cell-free population of antiinflammatory EVs of any one of embodiments 1-36. A method of producing an isolated, cell-free population of anti-inflammatory extracellular vesicles (EVs), said method comprising the steps of: a. ex-vivo culturing a human suppressive immune cell population in culture media to produce a culture comprising the cells, the culture media and anti-inflammatory EVs; and b. isolating the anti-inflammatory EVs from the culture. The method of embodiment 38, wherein the human suppressive immune cell population is a population of M2 macrophages. The method of embodiment 38 or 39 wherein step b) comprises removing cells from the culture, followed by polyethylene glycol precipitation of the culture. The method of embodiment 38 or 39, wherein step b) comprises: i) removing the cells from the culture to produce a cell-free, anti-inflammatory EV- containing solution; and ii) isolating the anti-inflammatory EVs from the cell-free, anti-inflammatory EV- containing solution of i). The method of embodiment 41, wherein step i) comprises passing the culture through a filter such that the cells are retained by the filter, and thereby removed from the culture. The method of embodiment 41 or 42, wherein step i) comprises microfiltration. The method of any one of embodiments 41-43, wherein step ii) comprises step ii-a): passing the cell-free, anti-inflammatory EV-containing solution through a filter such that the anti-inflammatory EVs are retained by the filter. The method of embodiment 44, wherein the filter has a molecular weight cut-off (MWCO) of about 200 kilodaltons (kDa) to about 600 kDa. The method of embodiment 45, wherein the filter has an MWCO of about 500 kDa. The method of any one of embodiments 41-46, wherein step ii) comprises ultrafiltration. The method of any one of embodiments 44-47, wherein step ii) further comprises step ii- b: performing buffer exchange such that the isolated, cell-free population of antiinflammatory EVs produced is a buffer-containing isolated, cell-free population of antiinflammatory EVs. The method of embodiment 48, wherein the buffer is a saline-containing buffer. The method of embodiment 49, wherein the saline-containing buffer is physiological saline. The method of embodiment 50, wherein the saline-containing buffer is PBS. The method of any one of embodiments 49-51, wherein step ii-b) comprises diafiltration. The method of any one of embodiments 49-52 wherein steps ii-a) and ii-b) are performed simultaneously. The method of any one of embodiments 38-53, wherein step b) comprises tangential flow filtration. The method of any one of embodiments 38-54, wherein the culture media in step a) is serum-free. The method of any one of embodiments 38-55, wherein the culture media in step a) comprises serum. The method of embodiment 56, wherein the serum is human AB serum. The method of embodiment 56 or 57, wherein the serum is depleted for serum-derived EVs. The method of any one of embodiments 41-58 further comprising, prior to step a), the step of culturing iPSC cells in the presence of cytokines to induce polarization to M2 macrophages. The method of embodiment 38, wherein the human suppressive immune cell population is a genetically engineered human suppressive immune cell population. A pharmaceutical composition comprising an isolated, cell-free population of antiinflammatory EVs, wherein the population is made by any one of the methods of embodiment 38-60. The method of any one of embodiments 38-60, further comprising: c) cry opreserving the isolated, cell-free population of anti-inflammatory EVs, thereby producing a cryopreserved, isolated, cell-free population of anti-inflammatory EVs. The method of embodiment 62, further comprises thawing the cryopreserved, isolated cell-free population of anti-inflammatory EVs after cryopreservation for about 1 week, 1 month, about 3 months, about 6 months, about 9 months, about 12 months, about 18 months or about 24 months. A pharmaceutical composition comprising the isolated, cell-free population of antiinflammatory EVs of embodiment 63. A method of treating a disorder associated with Treg dysfunction, the method comprising administering to a subject in need of said treatment the pharmaceutical composition of any one of embodiments 29-36, 61, or 64. A method of treating a disorder associated with Treg deficiency, the method comprising administering to a subject in need of said treatment the pharmaceutical composition of any one of embodiments 29-36, 61, or 64. A method of treating a disorder associated with over activation of the immune system, the method comprising administering to a subject in need of said treatment the pharmaceutical composition of any one of embodiments 29-36, 61, or 64. A method of treating an inflammatory condition driven by a T cell response, the method comprising administering to a subject in need of said treatment the pharmaceutical composition of any one of embodiments 29-36, 61, or 64. A method of treating an inflammatory condition driven by a myeloid cell response, the method comprising administering to a subject in need of said treatment the pharmaceutical composition of any one of embodiments 29-36, 61, or 64. The method of embodiment 69, wherein the myeloid cell is a monocyte, macrophage or microglia. A method of treating a neurodegenerative disorder in a subject in need thereof, the method comprising administering to a subject in need of said treatment the pharmaceutical composition of any one of embodiments 29-36, 61, or 64. The method of embodiment 71, wherein the neurodegenerative disease is ALS, Alzheimer’s disease, Parkinson’s disease, frontotemporal dementia or Huntington’s disease. A method of treating an autoimmune disorder in a subject in need thereof, the method comprising administering to a subject in need of said treatment the pharmaceutical composition of any one of embodiments 29-36, 61, or 64. The method of embodiment 73, wherein the autoimmune disorder is polymyositis, ulcerative colitis, inflammatory bowel disease, Crohn’s disease, celiac disease, systemic sclerosis (scleroderma), multiple sclerosis (MS), rheumatoid arthritis (RA), Type I diabetes, psoriasis, dermatomy osititis, systemic lupus erythematosus, cutaneous lupus, myasthenia gravis, autoimmune nephropathy, autoimmune hemolytic anemia, autoimmune cytopenia, autoimmune encephalitis, autoimmune hepatitis, autoimmune uveitis, alopecia, thyroiditis or pemphigus. A method of treating graft-versus-host disease in a subject in need thereof, the method comprising administering to a subject in need of said treatment the pharmaceutical composition of any one of embodiments 29-36, 61, or 64. The method of embodiment 75, wherein the subject has received a bone marrow transplant, kidney transplant or liver transplant. A method of improving islet graft survival in a subject in need thereof, the method comprising administering to a subject in need of said treatment the pharmaceutical composition of any one of embodiments 29-36, 61, or 64. A method of treating cardio-inflammation in a subject in need thereof, the method comprising administering to a subject in need of said treatment the pharmaceutical composition of any one of embodiments 29-36, 61, or 64. The method of embodiment 78, wherein the cardio-inflammation is associated with atherosclerosis, myocardial infarction, ischemic cardiomyopathy or heart failure. A method of treating neuroinflammation in a subject in need thereof, the method comprising administering to a subject in need of said treatment the pharmaceutical composition of any one of embodiments 29-36, 61, or 64. The method of embodiment 80, wherein the neuroinflammation is associated with stroke, acute disseminated encephalomyelitis, acute optic neuritis, acute inflammatory demyelinating polyradiculoneuropathy, chronic inflammatory demyelinating polyradiculoneuropathy, Guillain-Barre syndrome, transverse myelitis, neuromyelitis optica, epilepsy, traumatic brain injury, spinal cord injury, encephalitis, central nervous system vasculitis, neurosarcoidosis, autoimmune or post-infectious encephalitis or chronic meningitis. A method of treating a Tregopathy in a subject in need thereof, comprising administering to a subject in need of said treatment the pharmaceutical composition of any one of embodiments 29-36, 61, or 64. The method of embodiment 82, wherein the Tregopathy is caused by a FOXP3, CD25, cytotoxic T lymphocyte-associated antigen 4 (CTLA4), LPS -responsive and beige-like anchor protein (LRBA), or BTB domain and CNC homolog 2 (BACH2) gene loss-of- function mutation, or a signal transducer and activator of transcription 3 (STAT3) gain- of-function mutation. The method of any one of embodiments 65-83, wherein the anti-inflammatory EVs are derived from M2 macrophages that are autologous to the subject. The method of any one of embodiments 65-83 wherein the anti-inflammatory EVs are derived from M2 macrophages that are allogeneic to the subject. The method of any one of embodiment 65-85, wherein the pharmaceutical composition is administered via intranasal administration. The method of embodiment 86, wherein the intranasal administration is via aerosol inhalation or nasal drip. The method of any one of embodiment 65-85, wherein the pharmaceutical composition is administered intravenously. The method of any one of embodiment 65-85, wherein the pharmaceutical composition is administered by local injection. The method of any one of embodiments 65-85, wherein the method further comprises administering to the subject a pharmaceutical composition comprising a therapeutic population of Tregs, wherein the Tregs had been ex vivo expanded and cryopreserved, and wherein the Tregs are not further expanded prior to the administering. The method of embodiment 90, wherein the therapeutic population of Tregs is autologous to the subject. The method of embodiment 91, wherein the therapeutic population of Tregs is allogeneic to the subject. The method of any one of embodiments 90-92, wherein the pharmaceutical composition comprising the therapeutic population of Tregs is administered intravenously. 94. The method of any one of embodiments 90-93, wherein the pharmaceutical composition comprising the anti-inflammatory EVs and the pharmaceutical composition comprising the therapeutic population of Tregs are administered to the patient on the same day.

95. The method of any one of embodiments 65-94, wherein the isolated, cell-free population of anti-inflammatory EVs had been cryopreserved and thawed prior to being administered to the subject.

96. The method of any one of embodiments 65-94, wherein the isolated, cell-free population of anti-inflammatory EVs are stored overnight at 4 °C prior to being administered to the subject.

97. The method of embodiment 96, wherein the isolated, cell-free population of antiinflammatory EVs had been cryopreserved then thawed and stored at 4 °C overnight prior to being administered to the subject.

98. The method of any one of embodiments 65-94, wherein the isolated, cell-free population of anti-inflammatory EVs had undergone at least two freeze/thaw cycles prior to being administered to the subject.

99. The method of embodiment 98, wherein the isolated, cell-free population of antiinflammatory EVs had undergone about 2 to about 20 freeze/thaw cycles prior to being administered to the subject.

4. BRIEF DESCRIPTION OF THE FIGURES

[0055] FIG. 1. Process flow diagram for an exemplary protocol for the generation of M2 macrophages from iPSCs.

[0056] FIG. 2A-2H. 10 pg of M2 EVs were added to in vitro cultures of 50,000 pro- inflammatory Ml cells in culture overnight (18 hr) followed by analysis of Ml RNA and cultured media. Ml activation used LPS and INFy combination treatment. 30pg of M2 EVs were given intranasally to mice 2 hours after 2mg/kg LPS injection. This acute neuroinflammation model produces systemic inflammation and also neuroinflammation in multiple regions of the murine brain. M2 EVs reduce pro-inflammatory IL-6 transcript (FIG. 2A). M2 EVs reduce IL6 protein production by Ml macrophages (FIG. 2B). M2 EV treatment of Ml cells increases IL-10 transcript, signaling potential shift to M2, anti-inflammatory phenotype (FIG. 2C). Argl is increased in Ml cells after M2 EV treatment. This further suggests M2 activation but also is a main mechanism of macrophages in reducing T cell proliferation (FIG. 2D). A dose escalation of M2 EVs in culture with T responder cells in CD3/CD28 stimulated cell proliferation assay shows that M2 EVs have a modest suppression of responder T cells (Tresp) proliferation (FIG. 2E). Intranasal M2 EVs reduce pro-inflammatory IL-6 transcript at 14 hours in the hippocampus and cortex of the LPS-induced acute pre-clinical model if neuroinflammation (FIG. 2F). The intranasal anti-inflammatory effects of M2 EVs in the same model are seen at 24 hours with IL-ip transcripts in hippocampus and cortex (FIG. 2G). Peripheral spleen-derived CD1 lb+ macrophages reduce their inflammatory markers at 24 hours post intranasal injection of M2 EVs following LPS injection (FIG. 2H).

5. DETAILED DESCRIPTION

[0057] Described herein are anti-inflammatory EV populations derived from ex vivo- cultured human suppressive immune cells, for example M2 macrophages. The EVs presented herein exhibit impressive anti-inflammatory activity, both in vitro and in vivo. For example, results presented herein demonstrate that the EVs of the present disclosure are able to potently suppress T responder cell proliferation and pro-inflammatory macrophage activity in vitro, and also exert potent anti-inflammatory effects in vivo. In particular, results presented herein demonstrate that the EVs are able to suppress brain and peripheral inflammation in an in vivo model of neuroinflammation.

[0058] Without wishing to be bound by theory or mechanism, it appears that EVs of the present disclosure retain the immune suppressive activities of the cells from which they are derived. Moreover, as EVs are not themselves cells, they avoid potential cell-based issues such as immune rejection and the possibility of polarization to a pro-inflammatory cell type. As such, the anti-inflammatory EVs presented herein are particularly useful for treatment of a variety of diseases such as, for example, neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS). [0059] Presented herein are isolated, cell-free populations of anti-inflammatory EVs, wherein the anti-inflammatory EVs are derived from ex vivo-cultured human suppressive immune cells, for example, M2 macrophages. Also presented herein are pharmaceutical compositions and cryopreserved compositions comprising an isolated, cell-free population of anti-inflammatory EVs described herein, methods of producing the EV populations and methods of using the EVs for treatment of diseases, such as neurodegenerative diseases, e.g., ALS.

[0060] Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein.

[0061] Unless specifically stated or apparent from context, as used herein, the terms “a”, “an”, and “the” are understood to be singular or plural, and denote “one or more.”

[0062] The terms “include,” “such as,” and the like are intended to convey inclusion without limitation, unless otherwise specifically indicated.

[0063] The terms “or” and “and” can be used interchangeably and can be understood to mean “and/or.”

[0064] The description herein of any aspect or embodiment of the invention using terms such as “comprising”, “having”, “including” or “containing” with reference to an element or elements is intended to provide support for a similar aspect or embodiment of the invention that “consists of, “consists essentially of, or “substantially comprises” that particular element or elements, unless otherwise stated or clearly contradicted by context (e.g., a composition described herein as comprising a particular element should be understood as also describing a composition consisting of that element, unless otherwise stated or clearly contradicted by context).

[0065] The terms “about” and “approximately” as used herein, are interchangeable, and should generally be understood to refer to a range of numbers around a given number, as well as to all numbers in a recited range of numbers (e.g., “about 5 to 15” means “about 5 to about 15” unless otherwise stated). Moreover, all numerical ranges herein should be understood to include each whole integer within the range. In particular, unless otherwise noted the terms mean within plus or minus 10% of a given value or range. In instances where an integer is required, the terms mean within plus or minus 10% of a given value or range, rounded either up or down to the nearest integer.

5.1 Anti-Inflammatory Extracellular Vesicles (EVs)

[0066] Presented herein are isolated, cell-free population of anti-inflammatory extracellular vesicles (EVs), wherein the anti-inflammatory EVs are derived from ex vi vo-cultured human suppressive immune cells. In certain embodiments, the anti-inflammatory EVs are derived from human M2 macrophages.

[0067] In certain embodiments, an isolated, cell-free population of anti-inflammatory EVs is produced by a method described herein, for example, as described in Section 5.2, below.

[0068] For ease of reference, unless otherwise noted, the terms “extracellular vesicles,” “EVs”, “extracellular vesicle particles,” and “EV particles” are used interchangeable herein. [0069] EVs are membrane-bound particles released by cells. Generally, EVs comprise one or more constituents from the cells from which they are released, e.g., one or more DNA, RNA (e.g., coding and/or non-coding RNA, for example, mRNA microRNA, and/or long non-coding RNA), protein (e.g., signaling proteins, receptors, other surface proteins, glycoproteins and/or enzymes) or non-protein, e.g., lipid, constituents. EVs generally range in size from about 30 nm to about 1000 nm in diameter. Larger EVs are sometimes referred to as “microvesicles.” Roughly speaking, microvesicles have size diameters larger than about 200 nm. Smaller EVs are sometimes referred to as “exosomes.” Roughly speaking, exosomes have size diameters that range from about 30-40 nm to about 150-200 nm. Methods for determining EV particle size and concentration are well known to those of skill in the art.

[0070] Methods for determining EV particle size, concentration and purity are well known, including determinations that use dynamic light scattering or single particle tracking analysis, or utilize techniques such as flow cytometry, ELISA, or electron microscopy. See, e.g., Balaj et al. (2015) Sci Rep 5, 10266, Nakai et al. (2016) Sci Rep 6, 33935 and Carnino et al. (2019) Respiratory Research 20:240. In a particular embodiment, routine determination may be performed using nanoparticle analyzers, e.g., NanoSight (Malvern Panalytical) nanoparticle analyzers.

[0071] In some embodiments, EVs may be analyzed for the presence of exosome markers and/or Treg markers (e.g., CD25) by protein analysis using Western blot, ELISA, and other protein-associated assays, or commercially available arrays such as the Exo-Check™ Exosome Antibody Array (System Biosciences). In certain embodiments, a population of EVs may be analyzed for the presence of proteins associated with serum. In particular embodiments, an EV population described herein is substantially free of proteins associated with serum.

[0072] In certain embodiments, the anti-inflammatory EVs described herein exhibit an ability to increase the expression of one or more anti-inflammatory markers in inflammatory cells. For example, in particular embodiments, the anti-inflammatory EVs described herein exhibit an ability to increase the transcription of and/or level of mRNA expression of one or more genes encoding anti-inflammatory protein in inflammatory cells. In another example, in particular embodiments, the anti-inflammatory EVs described herein exhibit an ability to increase translation, processing, secretion and/or activation of one or more anti-inflammatory protein produced by inflammatory cells.

[0073] In specific embodiments, the anti-inflammatory marker is IL-10, Argl, and/or CD206. In specific examples, the inflammatory cells are myeloid cells, for example, monocytes, macrophages or microglia, e.g., human inflammatory cells, for example, human monocytes, macrophages or microglia.

[0074] In certain embodiments, the anti-inflammatory EVs described herein exhibit an ability to suppress inflammatory cells. For example, in certain embodiments, the antiinflammatory EVs described herein exhibit an ability to suppress inflammatory cells as measured by pro-inflammatory cytokine production by the inflammatory cells.

[0075] In some embodiments, the ability to suppress inflammatory cells is measured by IL-6, TNFa, ILip, IL8, and/or Interferon-y production by the inflammatory cells. In some embodiments, the ability to suppress inflammatory cells is measured by IL-6 production by the inflammatory cells.

[0076] In particular embodiments, the inflammatory cells are myeloid cells, for example, monocytes, macrophages or microglia e.g., human inflammatory cells, for example, human myeloid cells, such as human monocytes, macrophages or microglia. In specific examples, the myeloid cells, e.g., monocytes, macrophages or microglia, are from human donors or generated from induced pluripotent stem cells. In certain embodiments, the macrophages are Ml macrophages, such as induced pluripotent stem cell (iPSC)-derived Ml macrophages.

[0077] In certain embodiments, the anti-inflammatory EVs described herein exhibit an ability to suppress inflammatory cells as determined by suppression of proliferation of responder T cells. In particular embodiments, the proliferation of responder T cells is determined by flow cytometry or thymidine incorporation, e.g., tritiated thymidine incorporation.

[0078] In certain embodiments, the anti-inflammatory EVs described herein exhibit an ability to suppress inflammatory cells (e.g., as measured by pro-inflammatory cytokine production and/or responder T cell proliferation) and an ability to increase expression of one or more inflammatory markers in inflammatory cells.

[0079] In certain aspects, a population of anti-inflammatory EVs as described herein comprises exosomes. In other aspects, a population of anti-inflammatory EVs described herein comprises microvesicles. In yet other aspects, a population of anti-inflammatory EVs as described herein comprises exosomes and microvesicles.

[0080] In certain embodiments, the majority of EVs of a population of anti-inflammatory EVs as described herein are exosomes. For example, in certain embodiments at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% or more of the EVs of a population of anti-inflammatory exosomes described herein are exosomes.

[0081] In certain embodiments, the majority of EVs of a population of anti-inflammatory EVs as described herein are microvesicles. For example, in certain embodiments at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% or more of the EVs of a population of anti-inflammatory exosomes described herein are microvesicles.

[0082] In certain embodiments, the EVs of a population of anti-inflammatory EVs as described herein have size diameters of about 5 nm to about 1000 nm. In certain embodiments, the EVs of a population of anti-inflammatory EVs as described herein have size diameters of about 10 nm to about 1000 nm. In certain embodiments, the EVs of a population of antiinflammatory EVs as described herein have size diameters of about 15 nm to about 1000 nm. In certain embodiments, the EVs of a population of anti-inflammatory EVs as described herein have size diameters of about 20 nm to about 1000 nm. In certain embodiments, the EVs of a population of anti-inflammatory EVs as described herein have size diameters of about 30 nm to about 1000 nm. [0083] In certain embodiments, the majority (e.g., at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% or more) of the EVs of a population of anti-inflammatory EVs as described herein have size diameters of about 5 nm to about 1000 nm, about 10 nm to about 1000 nm, about 15 nm to about 1000 nm, about 20 nm to about 1000 nm, or about 30 nm to about 1000 nm.

[0084] In certain embodiments, the majority of EVs of a population of anti-inflammatory EVs as described herein have size diameters less than about 300 nm, less than about 200 nm, less than about 150 nm or less than about 100 nm. For example, in certain embodiments at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% or more of the EVs of a population of anti-inflammatory exosomes described herein have size diameters less than about 300 nm, less than about 200 nm, less than about 150 nm, or less than about 100 nm.

[0085] In certain embodiments, the EVs of a population of anti-inflammatory EVs as described herein have size diameters of about 30 nm to about 300 nm, about 30 nm to about 250 nm, about 30 nm to about 200 nm, about 30 nm to about 160 nm, about 30 nm to about 150 nm, about 30 nm to about 100 nm, about 40 nm to about 300 nm, about 40 nm to about 200 nm, about 40 nm to about 160 nm, about 40 nm to about 150 nm, about 40 nm to about 100 nm, about 60 nm to about 300 nm, about 60 nm to about 200 nm, about 60 nm to about 160 nm, about 60 nm to about 150 nm, about 60 nm to about 125 nm, about 60 nm to about 110 nm, about 60 nm to about 100 nm, about 60 nm to about 80 nm, about 70 nm to about 300 nm, about 70 nm to about 200 nm, about 70 nm to about 160 nm, about 70 nm to about 150 nm, about 70 nm to about 125 nm, about 70 nm to about 110 nm, about 70 nm to about 100 nm, about 80 nm to about 300 nm, about 80 nm to about 200 nm, about 80 nm to about 160 nm, about 80 nm to about 150 nm, about 80 nm to about 125 nm, about 80 nm to about 110 nm, about 80 nm to about 100 nm, or about 110 nm to about 120 nm.

[0086] In certain embodiments, the majority (e.g., at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% or more) of the EVs of a population of anti-inflammatory EVs as described herein have size diameters of about 30 nm to about 300 nm, about 30 nm to about 250 nm, about 30 nm to about 200 nm, about 30 nm to about 160 nm, about 30 nm to about 150 nm, about 30 nm to about 100 nm, about 40 nm to about 300 nm, about 40 nm to about 200 nm, about 40 nm to about 160 nm, about 40 nm to about 150 nm, about 40 nm to about 100 nm, about 60 nm to about 300 nm, about 60 nm to about 200 nm, about 60 nm to about 160 nm, about 60 nm to about 150 nm, about 60 nm to about 125 nm, about 60 nm to about 110 nm, about 60 nm to about 100 nm, about 60 nm to about 80 nm, about 70 nm to about 300 nm, about 70 nm to about 200 nm, about 70 nm to about 160 nm, about 70 nm to about 150 nm, about 70 nm to about 125 nm, about 70 nm to about 110 nm, about 70 nm to about 100 nm, about 80 nm to about 300 nm, about 80 nm to about 200 nm, about 80 nm to about 160 nm, about 80 nm to about 150 nm, about 80 nm to about 125 nm, about 80 nm to about 110 nm, about 80 nm to about 100 nm, or about 110 nm to about 120 nm.

[0087] In certain embodiments, the majority (e.g., at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% or more) of the EVs of a population of anti-inflammatory EVs as described herein have a size diameter of about 30 nm, about 40 nm, about 50 nm, about 60 nm, about 65 nm, about 70 nm, about 75 nm, 80 nm, about 85 nm, about 90 nm, about 95 nm, about 100 nm, about 110 nm to about 120 nm, about 150 nm, about 175 nm, about 200 nm, about 250 nm or about 300 nm. [0088] In certain embodiments, the majority (e.g., at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% or more) of the EVs of a population of anti-inflammatory EVs as described herein have size diameters greater than about 300 nm, greater than about 400 nm, greater than about 500 nm, greater than about 500 nm, greater than about 700 nm, or greater than about 800 nm.

[0089] In certain embodiments, the EVs of a population of anti-inflammatory EVs as described herein have size diameters of about 200 nm to about 1000 nm, about 300 nm to about 1000 nm, about 400 nm to about 1000 nm, about 500 nm to about 1000 nm, about 600 nm to about 1000 nm, about 700 nm to about 1000 nm, about 800 nm to about 1000 nm, about 200 nm to about 800 nm, about 300 nm to about 800 nm, about 400 nm to about 800 nm, about 500 nm to about 800 nm, about 600 nm to about 800 nm, about 200 nm to about 600 nm, about 300 nm to about 600 nm, about 400 nm to about 600 nm, about 200 nm to about 500 nm, or about 300 nm to about 500 nm.

[0090] In certain embodiments, the majority (e.g., at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% or more) of EVs of a population of anti-inflammatory EVs as described herein have size diameters of about 200 nm to about 1000 nm, about 300 nm to about 1000 nm, about 400 nm to about 1000 nm, about 500 nm to about 1000 nm, about 600 nm to about 1000 nm, about 700 nm to about 1000 nm, about 800 nm to about 1000 nm, about 200 nm to about 800 nm, about 300 nm to about 800 nm, about 400 nm to about 800 nm, about 500 nm to about 800 nm, about 600 nm to about 800 nm, about 200 nm to about 600 nm, about 300 nm to about 600 nm, about 400 nm to about 600 nm, about 200 nm to about 500 nm, or about 300 nm to about 500 nm.

[0091] In certain embodiments, the majority (e.g., at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% or more) of the EVs of a population of anti-inflammatory EVs as described herein have a size diameter of about 400 nm, about 450 nm, about 500 nm, about 600 nm, about 650 nm, about 700 nm, about 750 nm, 800 nm, about 850 nm, about 900 nm, about 950 nm, or about 1000 nm. [0092] In certain embodiments, the EVs of a population of anti-inflammatory EVs as described herein have a mean size diameter of about 30 nm to about 1000 nm. In certain embodiments, the EVs of a population of anti-inflammatory EVs as described herein have a mean size diameter of less than about 300 nm, less than about 200 nm, less than about 150 nm or less than about 100 nm. In certain embodiments, the EVs of a population of anti-inflammatory EVs as described herein have a mean size diameter of about 30 nm to about 300 nm, about 30 nm to about 250 nm, about 30 nm to about 200 nm, about 30 nm to about 160 nm, about 30 nm to about 150 nm, about 30 nm to about 100 nm, about 40 nm to about 300 nm, about 40 nm to about 200 nm, about 40 nm to about 160 nm, about 40 nm to about 150 nm, about 40 nm to about 100 nm, about 60 nm to about 300 nm, about 60 nm to about 200 nm, about 60 nm to about 160 nm, about 60 nm to about 150 nm, about 60 nm to about 125 nm, about 60 nm to about 110 nm, about 60 nm to about 100 nm, about 60 nm to about 80 nm, about 70 nm to about

300 nm, about 70 nm to about 200 nm, about 70 nm to about 160 nm, about 70 nm to about 150 nm, about 70 nm to about 125 nm, about 70 nm to about 110 nm, about 70 nm to about 100 nm, about 80 nm to about 300 nm, about 80 nm to about 200 nm, about 80 nm to about 160 nm, about 80 nm to about 150 nm, about 80 nm to about 125 nm, about 80 nm to about 110 nm, about 80 nm to about 100 nm, or about 110 nm to about 120 nm.

[0093] In certain embodiments, the EVs of a population of anti-inflammatory EVs as described herein have a mean size diameter of about 30 nm, about 40 nm, about 50 nm, about 60 nm, about 65 nm, about 70 nm, about 75 nm, 80 nm, about 85 nm, about 90 nm, about 95 nm, about 100 nm, about 110 nm to about 120 nm, about 150 nm, about 175 nm, about 200 nm, about 250 nm or about 300 nm.

[0094] In certain embodiments, the EVs of a population of anti-inflammatory EVs as described herein have a mean size diameter greater than about 300 nm, greater than about 400 nm, greater than about 500 nm, greater than about 500 nm, greater than about 700 nm, or greater than about 800 nm. In certain embodiments, the EVs of a population of anti-inflammatory EVs as described herein have a mean size diameter of about 200 nm to about 1000 nm, about 300 nm to about 1000 nm, about 400 nm to about 1000 nm, about 500 nm to about 1000 nm, about 600 nm to about 1000 nm, about 700 nm to about 1000 nm, about 800 nm to about 1000 nm, about 200 nm to about 800 nm, about 300 nm to about 800 nm, about 400 nm to about 800 nm, about

500 nm to about 800 nm, about 600 nm to about 800 nm, about 200 nm to about 600 nm, about

300 nm to about 600 nm, about 400 nm to about 600 nm, about 200 nm to about 500 nm, or about 300 nm to about 500 nm.

[0095] In certain embodiments, the EVs of a population of anti-inflammatory EVs as described herein have a mean size diameter of about 400 nm, about 450 nm, about 500 nm, about 600 nm, about 650 nm, about 700 nm, about 750 nm, 800 nm, about 850 nm, about 900 nm, about 950 nm, or about 1000 nm.

[0096] In certain embodiments, the EVs of a population of anti-inflammatory EVs as described herein have a median size diameter of about 30 nm to about 1000 nm. In certain embodiments, the EVs of a population of anti-inflammatory EVs as described herein have a median size diameter of less than about 300 nm, less than about 200 nm, less than about 150 nm or less than about 100 nm. In certain embodiments, the EVs of a population of antiinflammatory EVs as described herein have a median size diameter of about 30 nm to about 300 nm, about 30 nm to about 250 nm, about 30 nm to about 200 nm, about 30 nm to about 160 nm, about 30 nm to about 150 nm, about 30 nm to about 100 nm, about 40 nm to about 300 nm, about 40 nm to about 200 nm, about 40 nm to about 160 nm, about 40 nm to about 150 nm, about 40 nm to about 100 nm, about 60 nm to about 300 nm, about 60 nm to about 200 nm, about 60 nm to about 160 nm, about 60 nm to about 150 nm, about 60 nm to about 125 nm, about 60 nm to about 110 nm, about 60 nm to about 100 nm, about 60 nm to about 80 nm, about 70 nm to about 300 nm, about 70 nm to about 200 nm, about 70 nm to about 160 nm, about 70 nm to about 150 nm, about 70 nm to about 125 nm, about 70 nm to about 110 nm, about 70 nm to about 100 nm, about 80 nm to about 300 nm, about 80 nm to about 200 nm, about 80 nm to about 160 nm, about 80 nm to about 150 nm, about 80 nm to about 125 nm, about 80 nm to about 110 nm, about 80 nm to about 100 nm, or about 110 nm to about 120 nm.

[0097] In certain embodiments, the EVs of a population of anti-inflammatory EVs as described herein have a median size diameter of about 30 nm, about 40 nm, about 50 nm, about 60 nm, about 65 nm, about 70 nm, about 75 nm, 80 nm, about 85 nm, about 90 nm, about 95 nm, about 100 nm, about 110 nm to about 120 nm, about 150 nm, about 175 nm, about 200 nm, about 250 nm or about 300 nm.

[0098] In certain embodiments, the EVs of a population of anti-inflammatory EVs as described herein have a median size diameter greater than about 300 nm, greater than about 400 nm, greater than about 500 nm, greater than about 500 nm, greater than about 700 nm, or greater than about 800 nm. In certain embodiments, the EVs of a population of anti-inflammatory EVs as described herein have a median size diameter of about 200 nm to about 1000 nm, about 300 nm to about 1000 nm, about 400 nm to about 1000 nm, about 500 nm to about 1000 nm, about 600 nm to about 1000 nm, about 700 nm to about 1000 nm, about 800 nm to about 1000 nm, about 200 nm to about 800 nm, about 300 nm to about 800 nm, about 400 nm to about 800 nm, about 500 nm to about 800 nm, about 600 nm to about 800 nm, about 200 nm to about 600 nm, about 300 nm to about 600 nm, about 400 nm to about 600 nm, about 200 nm to about 500 nm, or about 300 nm to about 500 nm.

[0099] In certain embodiments, the EVs of a population of anti-inflammatory EVs as described herein have a median size diameter of about 400 nm, about 450 nm, about 500 nm, about 600 nm, about 650 nm, about 700 nm, about 750 nm, 800 nm, about 850 nm, about 900 nm, about 950 nm, or about 1000 nm. [00100] In certain embodiments, the EVs of a population of anti-inflammatory EVs as described herein have a mode size diameter of about 30 nm to about 1000 nm. In certain embodiments, the EVs of a population of anti-inflammatory EVs as described herein have a mode size diameter of less than about 300 nm, less than about 200 nm, less than about 150 nm or less than about 100 nm. In certain embodiments, the EVs of a population of anti-inflammatory EVs as described herein have a mode size diameter of about 30 nm to about 300 nm, about 30 nm to about 250 nm, about 30 nm to about 200 nm, about 30 nm to about 160 nm, about 30 nm to about 150 nm, about 30 nm to about 100 nm, about 40 nm to about 300 nm, about 40 nm to about 200 nm, about 40 nm to about 160 nm, about 40 nm to about 150 nm, about 40 nm to about 100 nm, about 60 nm to about 300 nm, about 60 nm to about 200 nm, about 60 nm to about 160 nm, about 60 nm to about 150 nm, about 60 nm to about 125 nm, about 60 nm to about 110 nm, about 60 nm to about 100 nm, about 60 nm to about 80 nm, about 70 nm to about

[00101] In certain embodiments, the EVs of a population of anti-inflammatory EVs as described herein have a mode size diameter of about 30 nm, about 40 nm, about 50 nm, about 60 nm, about 65 nm, about 70 nm, about 75 nm, 80 nm, about 85 nm, about 90 nm, about 95 nm, about 100 nm, about 110 nm to about 120 nm, about 150 nm, about 175 nm, about 200 nm, about 250 nm or about 300 nm.

[00102] In certain embodiments, the EVs of a population of anti-inflammatory EVs as described herein have a mode size diameter greater than about 300 nm, greater than about 400 nm, greater than about 500 nm, greater than about 500 nm, greater than about 700 nm, or greater than about 800 nm. In certain embodiments, the EVs of a population of anti-inflammatory EVs as described herein have a mode size diameter of about 200 nm to about 1000 nm, about 300 nm to about 1000 nm, about 400 nm to about 1000 nm, about 500 nm to about 1000 nm, about 600 nm to about 1000 nm, about 700 nm to about 1000 nm, about 800 nm to about 1000 nm, about 200 nm to about 800 nm, about 300 nm to about 800 nm, about 400 nm to about 800 nm, about 500 nm to about 800 nm, about 600 nm to about 800 nm, about 200 nm to about 600 nm, about 300 nm to about 600 nm, about 400 nm to about 600 nm, about 200 nm to about 500 nm, or about 300 nm to about 500 nm.

[00103] In certain embodiments, the EVs of a population of anti-inflammatory EVs as described herein have a mode size diameter of about 400 nm, about 450 nm, about 500 nm, about 600 nm, about 650 nm, about 700 nm, about 750 nm, 800 nm, about 850 nm, about 900 nm, about 950 nm, or about 1000 nm.

[00104] In certain aspects a population of anti-inflammatory EVs as described herein is a buffer-containing population of anti-inflammatory EVs. The anti-inflammatory EVs described herein are derived from ex vivo-cultured human suppressive immune cells, e.g., M2 macrophages. As explained in detailed below, a population of such anti-inflammatory EVs may be isolated from a culture comprising ex vivo human suppressive immune cells, e.g., M2 macrophages and culture media. In certain instances, as part of the process of isolating EVs from the culture, the culture media may be replaced with a buffer, for example a sterile buffer, e.g., a buffer suitable for administration to a human, such as suitable for administration to a human for therapeutic use. In such instances, the resulting isolated, cell-free population of antiinflammatory EVs may be referred to as a buffer-containing population of anti-inflammatory EVs.

[00105] Similarly, in certain embodiments, a population of anti-inflammatory EVs as described herein is a saline-containing population of anti-inflammatory EVs. In particular embodiments, a population of anti-inflammatory EVs as described herein is a normal salinecontaining population of anti-inflammatory EVs. In particular embodiments, a population of anti-inflammatory EVs as described herein is a 0.9% saline-containing population of antiinflammatory EVs. In particular embodiments, a population of anti-inflammatory EVs as described herein is a phosphate-buffer saline-containing population of anti-inflammatory EVs. [00106] The isolated, cell-free populations of anti-inflammatory EVs described herein are substantially free of cellular material, microparticles or other contaminants (e.g., organelles, lipids, cholesterol) from the cell or tissue source from which the EVs are derived, e.g., from the human suppressive immune cells, for example, M2 macrophages, from which the EVs are derived. For example, the isolated, cell-free populations of anti-inflammatory EVs described herein generally contain less than about 5 weight percent, less than about 1 weight percent, less than about 0.5 weight percent, less than about 0.1 weight percent, or less than about 0.01 weight percent of free of cellular material, microparticles or other contaminants (e.g., organelles, lipids, cholesterol) from the cell or tissue source from which the EVs are derived, e.g., from the human suppressive immune cells, for example, M2 macrophages, from which the EVs are derived.

[00107] In certain embodiments, the isolated, cell-free populations of anti-inflammatory EVs described herein are present in a composition that is substantially free of other EVs. For example, in certain embodiments, the isolated, cell-free populations of anti-inflammatory EVs described herein are present in a composition that contains less than about 20%, less than about 10%, less than about 5%, or less than about 1% other EVs.

[00108] In certain embodiments, an isolated, cell-free population of anti-inflammatory EVs described herein is present in a composition that comprises other EVs, wherein the isolated, cell- free population of anti-inflammatory EVs makes up about 10%, about 20%, about 25%, about 30%, about 35%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or greater than about 95% of the EVs in the composition. In specific embodiments, the other EVs are serum EVs, for example, serum EVs or human serum EVs.

5.2 Methods of Producing Anti-Inflammatory EVs

[00109] In certain aspects, presented herein are methods for producing an isolated, cell-free population of anti-inflammatory EVs.

[00110] In certain embodiments, presented herein are methods for producing an isolated, cell- free population of anti-inflammatory EVs, wherein the method comprises: a) ex-vivo culturing a human suppressive immune cell population in culture media to produce a culture comprising the human suppressive immune cell population, the culture media and anti-inflammatory EVs (without wishing to be bound by theory or mechanism, it is presumed that the EVs are released by the human suppressive immune cells into the culture during the ex vivo culturing), and b) isolating the anti-inflammatory EVs from the culture. Ex vivo culturing of cells may, for example, include proliferation of cells being cultured. Ex vivo culturing of a cell population may, for example, include expansion of the cell population.

[00111] In certain embodiments, presented herein are methods for producing an isolated, cell- free population of anti-inflammatory EVs, wherein the method comprises: a) ex-vivo culturing a human suppressive immune cell population, wherein the human suppressive immune cell population is a population of M2 macrophages, in culture media to produce a culture comprising the population of M2 macrophages, the culture media and anti-inflammatory EVs, and b) isolating the anti-inflammatory EVs from the culture.

[00112] Methods for obtaining, optionally polarizing, and ex vivo culturing human suppressive immune cells, e.g., M2 macrophages, are well known. Exemplary methods are presented below.

[00113] For ease of description, methods for producing an isolated, cell-free population of anti-inflammatory EVs, may be presented herein as comprising: a) ex-vivo culturing a human suppressive immune cell population in culture media to produce a culture comprising the human suppressive immune cell population, the culture media and anti-inflammatory EVs, and b) isolating the anti-inflammatory EVs from the culture. It is to be understood however, that isolating the anti-inflammatory EVs from the culture may be performed at any point of the culturing, or may be performed repeatedly over the period of culturing.

[00114] In certain embodiments, the cell culturing period lasts about 24 h, 48h, or 72h. In certain embodiments, the cell culturing period lasts 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 1 week, 10 days, 14 days, 2 weeks, 3 weeks or more.

[00115] In certain embodiments, culture media is changed or replenished one or more times during the culturing period. In particular embodiments, EVs may be collected during culture at points when the culture media of the culture is replenished or changed.

[00116] In some embodiments, EVs may be isolated after about 24h, 48h, or 72h of culture. In some embodiments, EVs may be isolated about 24h, about 48h, or about 72h after the culture medium is replenished or changed. In some embodiments, EVs are isolated every 2, 3, 4, or 5 days.

[00117] In certain embodiments, culture media comprising EVs may be collected at one or more points during the culture process and the isolating of the EVs begins when the culture media is collected, e.g., within 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 8 hours or overnight after the culture media is collected. In particular embodiments, culture media comprising EVs may be collected at one or more points during the culture process and stored at 4°C prior to the isolating of the EVs. In specific embodiments, for example, culture media comprising EVs may be collected at one or more points during the culture process and may be stored at 4°C for about 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 8 hours or overnight prior to isolating of the EVs from the culture media.

[00118] In certain embodiments, culture media comprising EVs may be collected at one or more points during the culture process and stored, for example, frozen prior to isolating of the EVs.

[00119] In particular embodiments, EVs may be isolated from cell culture by centrifugation, for example differential centrifugation. In certain embodiments, differential centrifugation may be used to isolate a desired subpopulation of EVs. For example, differential centrifugation may be employed to isolate a subpopulation of EVs enriched for a smaller particle diameter size (e.g., exosomes; EVs with a particle size less than about 300 nm, less than about 200 nm, less than about 160 nm, less than about 150 nm, less than about 130 nm, less than about 100 nm, or less than about 80 nm). In a particular, non-limiting example, centrifugation steps at 2,000g (3,000rpm) for 20 min may be employed to remove cell debris and dead cells and at 16,500g (9,800rpm) for 45 min, or at 100,000g (26,450rpm) for 2 h, to specifically isolate exosomes.

[00120] EVs may also be purified using gradient density centrifugation, which separates EVs from the culture based on their based on their buoyant density in solutions of either sucrose, iohexol, or iodixanol.

[00121] Additional examples of methods used to isolate EVs include precipitation with organic solvents (e.g., polyethylene glycol, sodium acetate or protamine), immunoprecipitation, separation using antibody-coated magnetic beads, microfluidic devices, and ultrafiltration, which are described, for example, in Camino et al. Respiratory Research (2019) 20:240 and Momen- Heravi et al. Biol. Chem. 2013; 394(10): 1253-1262. Further exemplary methods are isolation using heparin-conjugated agarose beads (see, e.g., Balaj et al. (2015) Sci Rep 5, 10266) and purification using Tim4-affinity purification (see, e.g., Nakai et al. (2016) Sci Rep 6, 33935).

[00122] Commercial kits for the isolation of EVs are also available. Non-limiting examples include the exoEasy Kit (Qiagen), ExoQuick® kits (Systems Bioscience), and the EasySep™ Human Pan-Extracellular Vesicle Positive Selection Kit (Stem Cell Technologies).

[00123] In some embodiments, a method of producing an isolated, cell-free population of antiinflammatory EVs provided herein comprises the steps of (a) ex-vivo culturing a human suppressive immune cell population (e.g., an M2 macrophage population) in culture media to produce a culture comprising the cells, the culture media and anti-inflammatory EVs; and (b) isolating the anti-inflammatory EVs from the culture.

[00124] In certain embodiments, a method of producing an isolated, cell-free population of anti-inflammatory EVs, isolating the anti-inflammatory EVs from the culture comprises polyethylene glycol (PEG) precipitation. In a specific embodiment, PEG is added to the culture such that the EVs are precipitated out of the culture. Following removal of the EV-containing precipitate from the culture, the EVs are washed to produce an isolated, cell-free population of anti-inflammatory EVs.

[00125] An exemplary, non-limiting protocol for the isolation of EVs from cells (e.g., M2 macrophages) using PEG precipitation may comprise the steps of (i) centrifuging media from cell culture (e.g., ex vivo-cultured human suppressive immune cell, for example M2 macrophages, cell culture) at 3000 x g for 15 minutes to remove cells and debris; (ii) adding PEG reagent to the supernatant, for example at a 1 :5 ratio of PEG: supernatant; (iii) mixing thoroughly; (iv) refrigerating overnight at 4 °C; (v) centrifuging at 1500 x g for 30 minutes (vi) aspirating the supernatant (vii) centrifuging again at 1500 x g for 10 minutes; (viii) removing the supernatant, e.g., removing the supernatant via aspiration; and (ix) resuspending the resulting EV pellet in sterile buffer, e.g., sterile PBS.

[00126] EVs may also be isolated from cell culture using filtration, for example, tangential flow filtration (TFF). TFF, for example, may be utilized to efficiently isolate and concentrate EV populations in a scalable and reproducible manner even when beginning with a large culture volume.

[00127] In particular embodiments, for example, the isolation step (b) comprises removing the cells from the culture to produce a cell-free, population of anti-inflammatory EVs.

[00128] In particular embodiments, for example, the isolation step (b) comprises the steps of (i) removing the cells from the culture to produce a cell-free, anti-inflammatory EV-containing solution; and (ii) isolating the anti-inflammatory EVs from the cell-free, anti-inflammatory EV- containing solution of (i). Steps (i) and (ii) may be performed separately, e.g., sequentially, as separate steps, or may be accomplished as a single step.

[00129] In some embodiments, step (b) comprises filtration, for example, one or more filtration steps. In particular embodiments the filtration comprises TFF. For example, some or all of the filtration may utilize TFF. Filtration, for example, may be utilized to remove cell and debris from the culture. Filtration may also be used to isolate and concentrate EVs, for example, to isolate and concentrate EVs of a particular size or size range. In certain embodiments, removal of cell and debris and isolation of EVs, for example, a particular size or size range of EVs, may be accomplished using a single filtration step. In other embodiments, for example, a series (two or more) of filtration steps may be utilized to remove cell and debris and isolate EV, isolate a particular size range of EVs. For example, one or more filtration steps may be utilized to first remove cell and debris to produce an EV-containing solution, followed by one or more filtration steps that isolate and concentrate the EV population from the solution, e.g., isolate and concentrate a particular size or size range of EVs from the solution.

[00130] In some embodiments, step (b), for example, step (i), comprises filtration, e.g., microfiltration (for example, microfiltration by TFF). For example, the culture may be passed through a filter, e.g., a 0.05 pm, 0.1 pm, 0.2 pm, 0.45pm, 0.65 pm or 0.8 pm filter, to remove the cells and any debris from the culture to produce a cell-free anti-inflammatory EV-containing solution comprising the anti-inflammatory population. In a specific embodiment, the culture may be passed through a 0.65 pm filter to remove the cells and any debris from the culture to produce a cell-free anti-inflammatory EV-containing solution comprising the anti-inflammatory population. In particular embodiments, the culture may be circulated through a filter, e.g., a 0.05 pm, 0.1 pm, 0.2 pm, 0.45pm, 0.65 pm or 0.8 pm filter, using TFF to remove the cell and any debris from the culture to produce a cell-free anti-inflammatory EV-containing solution comprising the anti-inflammatory EV population. In a particular embodiment, the culture may be circulated through a 0.65 pm filter using TFF to remove the cell and any debris from the culture to produce a cell-free anti-inflammatory EV-containing solution comprising the antiinflammatory EV population. In specific embodiments, the filter used in step (i) has a membrane area of 85 cm². In specific embodiments, the filter used in step (i) is a hollow fiber filter. In a specific embodiment, the filter used in step (i) is a hollow fiber filter with a fiber diameter of 0.75 mm. One or more rounds of filtration may be utilized. One or more sizes of filter may be utilized. In addition to removal of cells and debris, it is to be understood that such filtration may also serve to isolate a particular size or size range of EVs.

[00131] In specific embodiments, the microfiltration in step (i) (for example, microfiltration by TFF) is performed at a flow rate of 20-1000 mL/min. In specific embodiments, the microfiltration in step (i) (for example, microfiltration by TFF) is performed at a flow rate of 50- 500 mL/min. In specific embodiments, the microfiltration in step (i) (for example, microfiltration by TFF) is performed at a flow rate of 100-200 mL/min. In a specific embodiment, the microfiltration in step (i) (for example, microfiltration by TFF) is performed at a flow rate of about 100 mL/min. In a specific embodiment, the microfiltration in step (i) (for example, microfiltration by TFF) is performed at a flow rate of about 150 mL/min. In a specific embodiment, the microfiltration in step (i) (for example, microfiltration by TFF) is performed at a flow rate of about 200 mL/min.

[00132] In specific embodiments, the microfiltration in step (i) (for example, microfiltration by TFF) is performed using a hollow fiber filter with a shear rate of about 2,000-5,000 s'¹. In specific embodiments, the microfiltration in step (i) (for example, microfiltration by TFF) is performed using a hollow fiber filter with a shear rate of about 2,000-3,000 s'¹. In specific embodiments, the microfiltration in step (i) (for example, microfiltration by TFF) is performed using a hollow fiber filter with a shear rate of about 3,000-4,000 s'¹. In specific embodiments, the microfiltration in step (i) (for example, microfiltration by TFF) is performed using a hollow fiber filter with a shear rate of about 4,000-5,000 s'¹. In a specific embodiment, the microfiltration in step (i) (for example, microfiltration by TFF) is performed using a hollow fiber filter with a shear rate of about 2,000 s'¹. In a specific embodiment, the microfiltration in step (i) (for example, microfiltration by TFF) is performed using a hollow fiber filter with a shear rate of about 3,000 s'¹. In a specific embodiment, the microfiltration in step (i) (for example, microfiltration by TFF) is performed using a hollow fiber filter with a shear rate of about 4,000 s' ^x. In a specific embodiment, the microfiltration in step (i) (for example, microfiltration by TFF) is performed using a hollow fiber filter with a shear rate of about 5,000 s'¹. Shear rate is a term used for hollow fiber membranes and is affected by flow rate and radius of the fiber. While the typical range of shear rate is 2000-12000 s'¹ , preferably the shear rate maintained in step (i) is about 2,000-5,000 s'¹ (and not higher) so as to avoid shredding of EVs and to result in a high efficiency of EV recovery (e.g., recovery of more than 90% or more than 95% EVs). In specific embodiments, the shear rate maintained in step (i) is about 2,000-5,000 s'¹, with a flow rate of 100-200 mL/min and using a hollow fiber filter that has a fiber diameter of 0.75 mm.

[00133] In certain embodiments, the retentate pressure of step (i) is maintained at about 5 psi. In a specific embodiment, the shear rate maintained in step (i) is about 2,000-5,000 s'¹, with a flow rate of 100-200 mL/min and using a hollow fiber filter that has a fiber diameter of 0.75 mm, resulting in a retentate pressure of about 5 psi.

[00134] In some embodiments, step (b) comprises step (ii), and step (ii) may comprise filtration, for example, ultrafiltration (for example, ultrafiltration by TFF). In particular embodiments, step (ii) comprises a step of passing the cell-free, anti-inflammatory EV- containing solution through a filter such that the anti-inflammatory EVs, for example, a particular size or size range of anti-inflammatory EVs, are retained by the filter. In particular embodiments, step (ii) comprises a step of circulating the cell-free, anti-inflammatory EV- containing solution through a filter using TFF such that the anti-inflammatory EVs are retained by the filter. One or more rounds of filtration may be utilized. One or more sizes of filter may be utilized. Step (ii) may also serve to concentrate the EVs. In specific embodiments, the final volume of the EV-containing solution after concentration is about 5-200 mLs. In specific embodiments, the final volume of the EV-containing solution after concentration is about 10-100 mLs. In specific embodiments, the final volume of the EV-containing solution after concentration is about 10-50 mLs. In a specific embodiment, the final volume of the EV- containing solution after concentration is about 10 mL. In a specific embodiment, the final volume of the EV-containing solution after concentration is about 15 mL. In a specific embodiment, the final volume of the EV-containing solution after concentration is about 20 mL. In a specific embodiment, the final volume of the EV-containing solution after concentration is about 25 mL. In a specific embodiment, the final volume of the EV-containing solution after concentration is about 30 mL.

[00135] In some embodiments, at least one filter used in step (ii) has a molecular weight cutoff (MWCO) of about 50 kilodaltons (kDa) to about 750 kDa, about 100 kDa to about 750 kDa, about 300 kDa to about 750 kDa, or about 300 kDa to about 500 kDa. In some embodiments, the filter has an MWCO of about 50 kDa, about 60 kDA, about 70 kDA, about 80 kDa, about 90 kDa, about lOOkDa, about 110 kDa, about 120 kDa, about 150 kDa, about 200 kDa, about 300 kDa, about 400 kDa, about 500 kDa about 600 kDa, about 700 kDa or about 750 kDa. In one embodiment, the filter has an MWCO of about 500 kDa. In some embodiments, a filter used in step (ii) has a pore size of about 0.3 pm, about 0.22 pm, about 0.2 pm or about 0.1 pm. In specific embodiments, the filter used in step (ii) has a membrane area of 115 cm². In specific embodiments, the filter used in step (ii) is a hollow fiber filter. In a specific embodiment, the filter used in step (ii) is a hollow fiber filter with a fiber diameter of 0.5 mm.

[00136] In certain embodiments, step (ii) is designed to retain EVs of a particle size or size range, e.g., to retain EVs greater than about 50 nm to about 60 nm, about 60 nm to about 70 nm, about 70 nm to about 80 nm, about 80 nm, about 100 nm, about 150 nm, or about 200 nm. [00137] In certain embodiments, step (ii) is designed to retain EVs greater than about 50 nm to about 60 nm and comprises use of a filter with an MWCO of about 300 kDa. In certain embodiments, step (ii) is designed to retain EVs greater than about 50 nm and comprises use of a filter with an MWCO of about 300 kDa. In certain embodiments, step (ii) is designed to retain EVs greater than about 70 nm to about 80 nm and comprises use of a filter with an MWCO of about 500 kDa. In certain embodiments, step (ii) is designed to retain EVs greater than about 70 nm and comprises use of a filter with an MWCO of about 500 kDa. In certain embodiments, step (ii) is designed to retain EVs greater than about 80 nm and comprises use of a filter with an MWCO of about 500 kDa. In certain embodiments, step (ii) is designed to retain EVs greater than about 60 nm and comprises use of a filter with an MWCO of about 500 kDa.

[00138] In some embodiments, step (b), for example, step (b)(ii), comprises performing buffer exchange such that the isolated, cell-free population of anti-inflammatory EVs produced is a buffer-containing isolated, cell-free population of anti-inflammatory EVs. In particular embodiments, buffer exchange comprises diafiltration. In specific embodiments, buffer exchange comprises TFF and diafiltration. In specific embodiments, the diafiltration is performed at 2X-100X. In specific embodiments, the diafiltration is performed at 5X-50X. In specific embodiments, the diafiltration is performed at 5X-20X. In a specific embodiment, the diafiltration is performed at 5X. In a specific embodiment, the diafiltration is performed at 10X. In a specific embodiment, the diafiltration is performed at 15X. In a specific embodiment, the diafiltration is performed at 20X.

[00139] For example, in some embodiments, step (b) comprises step (ii), and step (ii) may comprise a step of circulating the cell-free, anti-inflammatory EV-containing solution through a filter using TFF such that the anti-inflammatory EVs are retained by the filter, wherein the circulating comprises incorporation of a suitable buffer into the solution, so that over the course of the process the buffer replaces the solution, thereby results in a buffer-containing isolated, cell-free population of anti-inflammatory EVs. [00140] In certain embodiments, the buffer is a sterile buffer. In certain embodiments, the buffer is a sterile buffer suitable for administration to a human, e.g., is suitable for administration to a human for therapeutic use. In a specific embodiment, the buffer is a saline-containing buffer. In one embodiment, the buffer is saline. In one embodiment, the buffer is physiological saline. In one embodiment, the buffer is normal saline. In one embodiment, the buffer is 0.9% saline. In one embodiment, the buffer is phosphate-buffered saline (PBS).

[00141] In specific embodiments, the ultrafiltration (and optionally diafiltration) in step (ii) (for example, ultrafiltration, and optionally diafiltration, by TFF) is performed at a flow rate of 20-1000 mL/min. In specific embodiments, the ultrafiltration (and optionally diafiltration) in step (ii) (for example, ultrafiltration, and optionally diafiltration, by TFF) is performed at a flow rate of 50-500 mL/min. In specific embodiments, the ultrafiltration (and optionally diafiltration) in step (ii) (for example, ultrafiltration, and optionally diafiltration, by TFF) is performed at a flow rate of 80-200 mL/min. In specific embodiments, the ultrafiltration (and optionally diafiltration) in step (ii) (for example, ultrafiltration, and optionally diafiltration, by TFF) is performed at a flow rate of 80-175 mL/min. In a specific embodiment, the ultrafiltration (and optionally diafiltration) in step (ii) (for example, ultrafiltration, and optionally diafiltration, by TFF) is performed at a flow rate of about 80 mL/min. In a specific embodiment, the ultrafiltration (and optionally diafiltration) in step (ii) (for example, ultrafiltration, and optionally diafiltration, by TFF) is performed at a flow rate of about 100 mL/min. In a specific embodiment, the ultrafiltration (and optionally diafiltration) in step (ii) (for example, ultrafiltration, and optionally diafiltration, by TFF) is performed at a flow rate of about 125 mL/min. In a specific embodiment, the ultrafiltration (and optionally diafiltration) in step (ii) (for example, ultrafiltration, and optionally diafiltration, by TFF) is performed at a flow rate of about 150 mL/min. In a specific embodiment, the ultrafiltration (and optionally diafiltration) in step (ii) (for example, ultrafiltration, and optionally diafiltration, by TFF) is performed at a flow rate of about 175 mL/min. In a specific embodiment, the ultrafiltration (and optionally diafiltration) in step (ii) (for example, ultrafiltration, and optionally diafiltration, by TFF) is performed at a flow rate of about 200 mL/min.

[00142] In specific embodiments, the ultrafiltration (and optionally diafiltration) in step (ii) (for example, ultrafiltration, and optionally diafiltration, by TFF) is performed using a hollow fiber filter with a shear rate of about 2,000-8,000 s'¹. In specific embodiments, the ultrafiltration (and optionally diafiltration) in step (ii) (for example, ultrafiltration, and optionally diafiltration, by TFF) is performed using a hollow fiber filter with a shear rate of about 2,000-7,500 s'¹. In specific embodiments, the ultrafiltration (and optionally diafiltration) in step (ii) (for example, ultrafiltration, and optionally diafiltration, by TFF) is performed using a hollow fiber filter with a shear rate of about 2,000-7,000 s'¹. In specific embodiments, the ultrafiltration (and optionally diafiltration) in step (ii) (for example, ultrafiltration, and optionally diafiltration, by TFF) is performed using a hollow fiber filter with a shear rate of about 2,000-3,000 s'¹. In specific embodiments, the ultrafiltration (and optionally diafiltration) in step (ii) (for example, ultrafiltration, and optionally diafiltration, by TFF) is performed using a hollow fiber filter with a shear rate of about 3,000-4,000 s'¹. In specific embodiments, the ultrafiltration (and optionally diafiltration) in step (ii) (for example, ultrafiltration, and optionally diafiltration, by TFF) is performed using a hollow fiber filter with a shear rate of about 4,000-5,000 s'¹. In specific embodiments, the ultrafiltration (and optionally diafiltration) in step (ii) (for example, ultrafiltration, and optionally diafiltration, by TFF) is performed using a hollow fiber filter with a shear rate of about 5,000-6,000 s'¹. In specific embodiments, the ultrafiltration (and optionally diafiltration) in step (ii) (for example, ultrafiltration, and optionally diafiltration, by TFF) is performed using a hollow fiber filter with a shear rate of about 6,000-7,000 s'¹. In specific embodiments, the ultrafiltration (and optionally diafiltration) in step (ii) (for example, ultrafiltration, and optionally diafiltration, by TFF) is performed using a hollow fiber filter with a shear rate of about 7,000-8,000 s'¹. In a specific embodiment, the ultrafiltration (and optionally diafiltration) in step (ii) (for example, ultrafiltration, and optionally diafiltration, by TFF) is performed using a hollow fiber filter with a shear rate of about 2,000 s'¹. In a specific embodiment, the ultrafiltration (and optionally diafiltration) in step (ii) (for example, ultrafiltration, and optionally diafiltration, by TFF) is performed using a hollow fiber filter with a shear rate of about 3,000 s'¹. In a specific embodiment, the ultrafiltration (and optionally diafiltration) in step (ii) (for example, ultrafiltration, and optionally diafiltration, by TFF) is performed using a hollow fiber filter with a shear rate of about 4,000 s'¹. In a specific embodiment, the ultrafiltration (and optionally diafiltration) in step (ii) (for example, ultrafiltration, and optionally diafiltration, by TFF) is performed using a hollow fiber filter with a shear rate of about 5,000 s'¹. In a specific embodiment, the ultrafiltration (and optionally diafiltration) in step (ii) (for example, ultrafiltration, and optionally diafiltration, by TFF) is performed using a hollow fiber filter with a shear rate of about 6,000 s'¹. In a specific embodiment, the ultrafiltration (and optionally diafiltration) in step (ii) (for example, ultrafiltration, and optionally diafiltration, by TFF) is performed using a hollow fiber filter with a shear rate of about 7,000 s'¹. In a specific embodiment, the ultrafiltration (and optionally diafiltration) in step (ii) (for example, ultrafiltration, and optionally diafiltration, by TFF) is performed using a hollow fiber filter with a shear rate of about 7,500 s'¹. In a specific embodiment, the ultrafiltration (and optionally diafiltration) in step (ii) (for example, ultrafiltration, and optionally diafiltration, by TFF) is performed using a hollow fiber filter with a shear rate of about 8,000 s'¹. Shear rate is a term used for hollow fiber membranes and is affected by flow rate and radius of the fiber. While the typical range of shear rate is 2000-12000 s'¹ , preferably the shear rate maintained in step (ii) is about 2,000-8,000 s'¹, about 2,000-7,500 s' or about 2,000-7,000 s'¹ (and not higher) so as to avoid shredding of EVs and to result in a high efficiency of EV recovery (e.g., recovery of more than 90% or more than 95% EVs). In specific embodiments, the shear rate maintained in step (ii) is about 2,000-7,500 s'¹, with a flow rate of 80-200 mL/min and using a hollow fiber filter that has a fiber diameter of 0.5 mm. [00143] In certain embodiments, the transmembrane pressure of step (ii) is maintained at about 10 psi. In a specific embodiment, the shear rate maintained in step (ii) is about 2,000- 7,500 s'¹, with a flow rate of 80-200 mL/min and using a hollow fiber filter that has a fiber diameter of 0.5 mm, resulting in a transmembrane pressure of about 10 psi.

[00144] In certain embodiments, the isolation step (b) comprises: a step (i) that comprises microfiltration as described above, and a step (ii) that comprises ultrafiltration as described above and optionally diafiltration as described above.

[00145] In certain embodiments, the step (i) described above is performed using one or more pumps (e.g., one or more automated pumps), such as a main pump and an auxiliary pump. In certain embodiments, the step (ii) described above is performed using one or more pumps (e.g., one or more automated pumps), such as a main pump and an auxiliary pump. In certain embodiments, step (i) and step (ii) described above are each performed using one or more pumps (e.g., one or more automated pumps), such as a main pump and an auxiliary pump.

[00146] In a particular, non-limiting example, a Repligen KR2i TFF system can be used to isolate, concentrate, and diafiltrate the EVs from cell culture into an appropriate buffer for therapeutic use. For example, EV isolation using TFF may comprise the steps of (i) circulating the culture media using TFF and a Midi 20 cm 0.65 gm Spectrum mPES Hollow Fiber filter (D02-E65U-07-N) with a membrane area of 85 cm² and fiber diameter of 0.75 mm to filter out cells and debris (e.g, utilizing a flow rate of 100-200 mL/min that results in a shear rate of about 2,000-5,000 s'¹ while maintaining a variable transmembrane pressure (TMP) driven by a retentate pressure of 5 psi) and (ii) using the permeate of the process to concentrate and diafiltrate the EV product. For example, the process may utilize a TFF system and a Midi 20 cm 500kD Spectrum mPES Hollow Fiber filter (D02-E500-05-N) with a membrane area of 115 cm² filter and fiber diameter of 0.5 mm to retain/concentrate particles greater than about 60-80nm into the retentate with continuous circulation (e.g, utilizing a flow rate of 80-200 mL/min that results in a shear rate of 2,000-7,500 s'¹ while maintaining and driving the filtration at 10 psi TMP). Incorporation of a suitable buffer into the circulation (for example, sterile saline or sterile PBS) may be performed to diafiltrate and replace the existing solution so that the EVs end up in a sterile solution that is acceptable for therapeutic use.

[00147] In certain embodiments, the isolated EVs may be stored at -20°C. In particular embodiments, the isolated EVs may be stored at -20 °C while limiting freeze/thaw cycles. [00148] In certain embodiments, the isolated EVs may be stored at about 2°C to about 8 °C (e.g., at about 4°C), e.g., may be stored for up to about one week at about 2°C to about 8 °C (e.g., at about 4°C) , for example, may be stored about overnight, for up to about 1 day, up to about 2 days, up to about 3 days, up to about 4 days, up to about 5 days, up to about 6 days, or up to about 7 days at about 2°C to about 8 °C (e.g., at about 4°C) . In certain embodiments, the isolated EVs are stored at 4°C for less than about 2 weeks, e.g., are stored for less than about 14 days, less than about 13 days, less than about 12 days, less than about 11 days, less than about 10 days, less than about 9 days, less than about 8 days, at about 2°C to about 8 °C (e.g., at about 4°C) .

[00149] In certain embodiments, the methods presented herein for producing an isolated, cell- free population of anti-inflammatory EVs results in a yield of about IxlO⁸ to about IxlO¹⁰ EVs/ml of culture media. In certain embodiments, the methods presented herein for producing an isolated, cell-free population of anti-inflammatory EVs results in a yield of about 5xl0⁸ to about IxlO¹⁰ EVs/ml of culture media. In certain embodiments, the methods presented herein for producing an isolated, cell-free population of anti-inflammatory EVs results in a yield of about IxlO⁹ to about IxlO¹⁰ EVs/ml of culture media. In certain embodiments, the methods presented herein for producing an isolated, cell-free population of anti-inflammatory EVs results in a yield of about 5xl0⁹ to about IxlO¹⁰ EVs/ml of culture media. In certain embodiments, the methods presented herein for producing an isolated, cell-free population of anti-inflammatory EVs results in a yield of about IxlO⁹ EVs/ml, about 2xl0⁹ EVs/ml, about 3xl0⁹ EVs/ml, about 4xl0⁹ EVs/ml, about 5xl0⁹ EVs/ml, about 6xl0⁹ EVs/ml, about 7xl0⁹ EVs/ml, about 8xl0⁹ EVs/ml, about 9xl0⁹ EVs/ml, or about IxlO¹⁰ EVs/ml of culture media.

[00150] The anti-inflammatory EVs presented herein may be derived from ex vivo-cultured human suppressive immune cells, e.g., M2 macrophages. Exemplary methods for producing and culturing M2 macrophages are presented herein.

5.2.1. Culture, Enrichment, and Expansion of Human Suppressive Immune Cells

[00151] The isolated, cell-free populations of anti-inflammatory EVs presented herein are derived from ex vivo-cultured human suppressive immune cells. In certain aspects, the isolated, cell-free populations of anti-inflammatory EVs presented herein are derived from ex vivo- cultured human M2 macrophages.

[00152] M2 macrophages are an anti-inflammatory subset of macrophages. In certain embodiments, M2 macrophages useful as part of methods for producing an isolated, cell-free population of anti-inflammatory EVs presented herein may be obtained directly from one or more subjects, for example, for example, one or more human subjects, e.g., one or more unrelated human subjects.

[00153] In certain embodiments, precursors of M2 macrophages useful as part of methods for producing an isolated, cell-free population of anti-inflammatory EVs presented herein may be obtained from one or more subjects, for example, for example, one or more human subjects, e.g., one or more unrelated human subjects. M2 macrophages useful as part of methods for producing an isolated, cell-free population of anti-inflammatory EVs presented herein may then be produced from the M2 macrophage precursors.

[00154] In certain embodiments, an M2 macrophage or M2 macrophage precursor may be obtained from a donor sample, for example, a blood or a blood-derived sample. Exemplary samples include whole blood, serum, plasma, peripheral blood mononuclear cells (PBMCs), leukocytes, bone marrow, lymph node and thymus. [00155] In certain embodiments, the M2 macrophage precursor is a monocyte, for example, a naive, non-activated monocyte. In certain embodiments, the M2 macrophage precursor is a CD14+ monocyte. In some embodiments, the M2 macrophage precursor is an MO monocyte. [00156] In certain embodiments, the M2 macrophage precursor is a stem cell, for example, a hematopoietic stem cell. In particular embodiments, the stem cell is an induced pluripotent stem cell (iPSC). In specific embodiments, the iPSC has been produced from an adult, differentiated cell. In specific embodiments, the iPSC has been produced from a population of peripheral mononuclear cells (PBMCs), for example PBMCs isolated from blood tissues, or blood samples. In other specific embodiments, the iPSC may be produced from a population of fibroblasts, for example, skin fibroblasts. Methods for producing iPSCs, for example, producing iPSCs from adult, differentiated cells, are well-known.

[00157] In some embodiments, the precursors (e.g., iPSCs) for human suppressive immune cells, e.g., M2 macrophages, are isolated or generated from a sample, for example a blood sample, e.g., PBMCs are isolated from a blood sample, from a healthy human subject. In some embodiments, the precursors (e.g., iPSCs) for human suppressive immune cells, e.g., M2 macrophages, are isolated or generated from a sample, for example a blood sample, e.g., PBMCs are isolated from blood samples, from greater than one healthy human subject. In particular embodiments, for example, the precursors (e.g., iPSCs) for human suppressive immune cells, e.g., M2 macrophages, are isolated or generated from a sample, for example a blood sample, e.g., PBMCs are isolated from blood samples, from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50 or more healthy human subjects. In other particular embodiments, for example, the precursors (e.g., iPSCs) for human suppressive immune cells, e.g., M2 macrophages, are isolated or generated from a sample, for example a blood sample, e.g., PBMCs are isolated from blood samples, from 2-50, 2-5, 2-10, 5-10, 5-50, 5-25, 10-15, 10-50, 10-25, 15- 25, 25-30, 30-35, 35-40, 40-45, or 45-50 subjects. In some embodiments, the subjects are related. In some embodiments, the subject are not unrelated.

[00158] In particular embodiments where the precursors (e.g., iPSCs) for human suppressive immune cells, e.g., M2 macrophages, are isolated or generated from more than one human subject, e.g., are isolated from blood samples, for example PBMCs isolated from blood samples, a method of producing an isolated, cell-free population of anti-inflammatory EVs may comprise pooling the cells from the more than one human subject together prior to ex vivo-culturing the cells. In other particular embodiments where the precursors (e.g., iPSCs) for human suppressive immune cells, e.g., M2 macrophages, are isolated or generated from more than one human subject, e.g., are isolated from blood samples, for example PBMCs isolated from blood samples, a method of producing an isolated, cell-free population of anti-inflammatory EVs may comprise ex vivo-culturing the cells from one or more of the human subjects separately and pooling the anti-inflammatory EVs resulting from each culture.

[00159] In some embodiments, the precursors (e.g., iPSCs) for human suppressive immune cells, e.g., M2 macrophages, are isolated or generated from a sample, for example a blood sample, e.g., PBMCs from a blood sample, from a donor subject diagnosed with or is suspected of having a disorder associated with Treg dysfunction. In some embodiments, the donor subject is diagnosed with or is suspected of having a disorder associated with Treg deficiency. In some embodiments, the donor subject is diagnosed with or is suspected of having a condition driven by a T cell response.

[00160] In some embodiments, the precursors (e.g., iPSCs) for human suppressive immune cells, e.g., M2 macrophages, are isolated or generated from a sample, for example a blood sample, e.g., PBMCs from a blood sample, from a donor subject diagnosed with or is suspected of having a neurodegenerative disease. In some embodiments, the donor subject is diagnosed with or is suspected of having Alzheimer’s disease, Amyotrophic Lateral Sclerosis, multiple sclerosis (MS), Parkinson’s Disease, Huntington’s disease or frontotemporal dementia.

[00161] In some embodiments, the precursors (e.g., iPSCs) for human suppressive immune cells, e.g., M2 macrophages, are isolated or generated from a sample, for example a blood sample, e.g., PBMCs from a blood sample, from a donor subject diagnosed with or is suspected of having a disorder that would benefit from downregulation of the immune system.

[00162] In some embodiments, the precursors (e.g., iPSCs) for human suppressive immune cells, e.g., M2 macrophages, are isolated or generated from a sample, for example a blood sample, e.g., PBMCs from a blood sample, from a donor subject diagnosed with or suspected of having an autoimmune disease. The autoimmune disease may be, for example, systemic sclerosis (scleroderma), polymyositis, ulcerative colitis, inflammatory bowel disease, Crohn’s disease, celiac disease, multiple sclerosis (MS), rheumatoid arthritis (RA), Type I diabetes, psoriasis, dermatomyositis, systemic lupus erythematosus, cutaneous lupus, myasthenia gravis, autoimmune nephropathy, autoimmune hemolytic anemia, autoimmune cytopenia autoimmune hepatitis, autoimmune uveitis, alopecia, thyroiditis or pemhigus.

[00163] In some embodiments, the precursors (e.g., iPSCs) for human suppressive immune cells, e.g., M2 macrophages, are isolated or generated from a sample, for example a blood sample, e.g., PBMCs from a blood sample, from a donor subject diagnosed with or suspected of having heart failure or ischemic cardiomyopathy. In some embodiments, the donor subject is diagnosed with or suspected of having graft-versus-host disease, e.g., after undergoing organ transplantation (such as a kidney transplantation or a liver transplantation), or after undergoing stem cell transplantation (such as hematopoietic stem cell transplantation).

[00164] In some embodiments, the precursors (e.g., iPSCs) for human suppressive immune cells, e.g., M2 macrophages, are isolated or generated from a sample, for example a blood sample, e.g., PBMCs from a blood sample, from a donor subject diagnosed with or suspected of having neuroinflammation. Neuroinflammation may be associated, for example, with stroke, acute disseminated encephalitis, acute optic neuritis, transverse myelitis, neuromyelitis optica, epilepsy, traumatic brain injury, spinal cord injury, encephalitis central nervous system (CNS) vasculitis, neurosarcoidosis, autoimmune or post-infectious encephalitis or chronic meningitis. [00165] In some embodiments, the precursors (e.g., iPSCs) for human suppressive immune cells, e.g., M2 macrophages, are isolated or generated from a sample, for example a blood sample, e.g., PBMCs from a blood sample, from a donor subject diagnosed with or suspected of having chronic inflammatory demyelinating polyradiculoneuropathy (CIDP). In some embodiments, the donor subject is diagnosed with or suspected of having acute inflammatory demyelinating polyneuropathy (AIDP). In some embodiments, the donor subject is diagnosed with or suspected of having Guillain-Barre syndrome (GBS).

[00166] In some embodiments, the precursors (e.g., iPSCs) for human suppressive immune cells, e.g., M2 macrophages, are isolated or generated from a sample, for example a blood sample, e.g., PBMCs from a blood sample, from a donor subject diagnosed with or suspected of having cardo-inflammation, e.g., cardio-inflammation associated with myocardial infarction, ischemic cardiomyopathy, with heart failure.

[00167] In some embodiments, the precursors (e.g., iPSCs) for human suppressive immune cells, e.g., M2 macrophages, are isolated or generated from a sample, for example a blood sample, e.g., PBMCs from a blood sample, from a donor subject who has had a stroke. [00168] In some embodiments, the precursors (e.g., iPSCs) for human suppressive immune cells, e.g., M2 macrophages, are isolated or generated from a sample, for example a blood sample, e.g., PBMCs from a blood sample, from a donor subject diagnosed with or suspected of having cancer, e.g., a blood cancer.

[00169] In some embodiments, the precursors (e.g., iPSCs) for human suppressive immune cells, e.g., M2 macrophages, are isolated or generated from a sample, for example a blood sample, e.g., PBMCs from a blood sample, from a donor subject diagnosed with or suspected of having asthma.

[00170] In some embodiments, the precursors (e.g., iPSCs) for human suppressive immune cells, e.g., M2 macrophages, are isolated or generated from a sample, for example a blood sample, e.g., PBMCs from a blood sample, from a donor subject diagnosed with or suspected of having eczema.

[00171] In some embodiments, the precursors (e.g., iPSCs) for human suppressive immune cells, e.g., M2 macrophages, are isolated or generated from a sample, for example a blood sample, e.g., PBMCs from a blood sample, from a donor subject diagnosed with or suspected of having a disorder associated with over activation of the immune system.

[00172] In some embodiments, the precursors (e.g., iPSCs) for human suppressive immune cells, e.g., M2 macrophages, are isolated or generated from a sample, for example a blood sample, e.g., PBMCs from a blood sample, from a donor subject diagnosed with or suspected of having Tregopathy. The Tregopathy may, for example, be caused by a FOXP3, CD25, cytotoxic T lymphocyte-associated antigen 4 (CTLA4), LPS-responsive and beige-like anchor protein (LRBA), or BTB domain and CNC homolog 2 (BACH2) gene loss-of-function mutation, or a signal transducer and activator of transcription 3 (STAT3) gain-of-function mutation.

[00173] In some embodiments, the precursors (e.g., iPSCs) for human suppressive immune cells, e.g., M2 macrophages, are isolated or generated from a sample for example a blood sample, e.g., PBMCs from a blood sample, from one or more adult subjects, for example, one or more healthy adult subjects. In certain embodiments, the one or more subjects are of at least 18, 20, 25, 30, 35, 40, 45, 50 or 55 years of age. In particular embodiments, for example, the human suppressive immune cells or human suppressive immune cell precursors, e.g., M2 macrophages or M2 macrophage precursors, respectively, are from one or more adult subjects, wherein the one or more healthy adult subjects are about 18-55, about 18-50, about 18-45, about 18-40, about 18- 35, about 18-30, about 18-25, about 20-55, about 25-55, about 30-55, about 35-55, about 40-55, about 25-50, about 30-50, about 35-45, about 25-45 about 40-50 years of age.

[00174] In some embodiments, the M2 macrophage precursors (e.g., iPSCs) for human suppressive immune cells, e.g., M2 macrophages, are isolated or generated from a sample, for example a blood sample, e.g., PBMCs from a blood sample, from a geriatric subject, for example, a healthy geriatric subject, e.g., a subject of at least 65, at least 70, at least 75, at least 80, at least 85 or at least 90 years of age.

[00175] In some embodiments, the anti-inflammatory EVs provided herein are derived from a genetically engineered population of human suppressive immune cells, e.g., M2 macrophages, or M2 macrophage precursors.

[00176] In some embodiments, the isolation or generation of the human suppressive immune cells described herein (e.g., human M2 macrophages) or precursors thereof is or comprises an automated process. In some embodiments, the isolation or generation of the human suppressive immune cells described herein (e.g., human M2 macrophages) or precursors thereof takes place in a closed system. In some embodiments, the isolation or generation of the human suppressive immune cells described herein (e.g., human M2 macrophages) or precursors thereof is or comprises an automated process and takes place in a closed system. In some embodiments, the isolation or generation of the human suppressive immune cells described herein (e.g., human M2 macrophages) or precursors thereof is or comprises a process that takes place in a bioreactor.

5.3 Methods of Producing EVs from M2 Macrophages

[00177] M2 macrophages from which EVs can be isolated may be generated from any precursor. Non-limiting examples of M2 macrophage precursors include, for example, hemangioblasts, stem cells, e.g., hematopoietic stem cells, or other myeloid progenitor cells, monocytes, and M0 macrophages.

[00178] In certain embodiments, M2 macrophages from which EVs can be isolated may be generated from stem cells, for example, induced pluripotent stem cells (iPSCs). In some embodiments, the stem cells, e.g., iPSCs, are autologous to the patient to whom the M2-derived EV will ater be administered. In some embodiments, the stem cells, e.g., iPSCs, are allogeneic to the patient to whom the M2-derived EVs will later be administered. FIG. 1 shows an exemplary protocol for the generation of M2 macrophages from iPSC cells. [00179] In certain embodiments, M2 macrophages from which EVs can be isolated may be generated from hematopoietic stem cells. In particular embodiments, hematopoietic stem cells are isolated from a patient sample (e.g., a blood sample, a sample of lymphoid tissue, or a bone marrow sample), cultured, and differentiated into M2 macrophages. Methods of isolating hematopoietic stem cells from a subject sample are well known in the art. For example, methods of isolating hematopoietic stem cells from a bone marrow sample and expanding them in vitro are known in the art. See, e.g., Huss, Stem Cells 2000;18: 1-9. In a non-limiting example, hematopoietic stem cells may be isolated by positive selection of CD34-positive cells using magnetic separation and then cultured in media supplemented with cytokines to induce differentiation and M2 macrophage polarization.

[00180] In certain embodiments, M2 macrophages from which EVs can be isolated may be generated from CD14+ cells. In some embodiments, a population of CD14+ cells is isolated from a subject sample (e.g., a blood sample or a bone marrow sample) and differentiated into M2 macrophages. Methods for isolating CD14+ cells from a blood sample are known in the art. See, e.g., Weiss et al., Cytometry Part A 95A: 234-241, 2019. In a non-limiting example, CD14+ cells may be isolated using magnetic separation.

[00181] In certain embodiments, M2 macrophages from which EVs can be isolated may be generated from monocytes. In some embodiments, a population of monocytes is isolated from a subject sample (e.g., a blood sample or a bone marrow sample) and differentiated into M2 macrophages. Methods for isolating a population of monocytes from a sample are known in the art. See, e.g., Hopewell and Cox, Molecular Therapy: Methods & Clinical Development Vol. 16 March 2020 and Dagur and McCoy, 2016, Curr Protoc Cytom. ; 73: 5.1.1-5.1.16. In nonlimiting examples, monocytes may be isolated using monocyte adherence enrichment, gradient density centrifugation, or counterflow centrifugal elutriation (CCE) cell separation.

[00182] In certain embodiments, M2 macrophages from which EVs can be isolated may be generated from stem cells, for example embryonic stem cells (ES) or iPSCs, and may be differentiated into M2 macrophages using any suitable method known in the art.

[00183] In the case of iPSCs, methods of producing iPSCs are well known in the art. See, for example, Liu et al., 2020, Stem Cell Reviews and Reports 16:3-32; and Zakrzewski et al., 2019, Stem Cell Research & Therapy 10: 1-22. [00184] In one exemplary, non-limiting approach, stem cells, for example ES or iPSCs, may first be induced from an undifferentiated state to a primitive state, for example, induced to form primitive streak-like cells, by contacting the stem cells, e.g., ES or iPSCs, with bone morphogenic protein 4 (BMP4). The resulting cells may be contacted with VEGF, basic FGF and SCF to generate KDR+CD34+ hemangioblast-like cells. Hematopoietic stem cells may be generated by contacting the hemangioblast-like cells with hematopoietic cytokines, for example with SCF, IL3, TPO, M-CSF and/or Flt3 ligand. The resulting hematopoietic stem cells may be induced toward monocytic lineage (including CD14+ MO cells)-directed differentiation by contacting with Fit ligand, GM-CSF and M-CSF. CD14+ MO macrophages and differentiated into M2 macrophages.

[00185] In a more detailed, exemplary non-limiting protocol undifferentiated, primitive streak-like patient-derived cells are grown to a diameter of 1 mm in 6-well plates. Once the colonies reach a diameter of 1 mm (Day 0), all but a small number (e.g., 10) colonies are removed with a glass scraper or pipette. To induce the cells, the medium may be replaced with 2.5 mL TeSRl complete media supplemented with 16 pL BMP4 (80 ng/mL).

[00186] To generate KDR+CD34+ hemangioblast-like cells, the mTeSRl medium may be replaced by 19.3 mL StemPro-34 serum -free medium (Gibco) containing 0.5 mL StemPro Nutrient Supplement and 0.2 mL ImM Glutamax (Invitrogen) on day 4, and then supplemented with a step-2 cytokine cocktail composed of: VEGF 80 ng/mL, Basic FGF 25 ng/mL, SCF 100 ng/mL.

[00187] To generate hematopoietic cells, the medium may be replaced with 38.6 mL StemPro- 34 serum-free medium (Gibco) containing 1 mL StemPro Nutrient Supplement and 0.4 mL of 1 mM Glutamax (Invitrogen) on Day 6. Cytokines in StemPro-34 medium may be switched to a cytokine cocktail composed of: SCF 50 ng/mL, IL-3 50 ng/mL, TPO 5 ng/mL, MCSF 50 ng/mL, Flt-3 ligand 50 ng/mL. The medium is changed, e.g., at day 10.

[00188] To produce monocytic lineage-directed cells, the medium may be replaced with 96.5 mL StemPro-34 serum -free medium (Gibco) containing 2.5 mL StemPro Nutrient Supplement and 1 mL of 1 mM Glutamax (Invitrogen) on Day 13-15. The following cytokines may then be added to the media: Fit ligand 50 ng/mL, GMCSF 25 ng/mL, M-CSF 50 ng/mL.

[00189] To isolate CD14+ monocytes, floating cells may be isolated from the medium of the culture by centrifugation at 300 x g for 10 min at 4 °C. The resulting pellet may then be resuspended in 10 mL total running buffer and CD14+ can be isolated cells using a CD14 microbead kit and MS column. One column with a suitable amount (e.g., 80 pL) of buffer and a suitable amount (e.g., 20 pL) of CD14 beads per IxlO⁷ cells may be used. The MS column is primed with 500 pL Degas and washed three times with 500 pL Degas before the CD14+ cells may be pushed out and counted.

[00190] For differentiation into M0 cells, cells may be cultured in human 1640 medium supplemented with 10% serum and either 50 ng/ml GMCSF for cells which will later be differentiated into Ml cells or with 100 ng/ml MCSF for cells which will later be differentiated into M2 cells. A suitable amount of cells (e.g., 1x10⁶ cells per well) can be cultured in a 6-well plate with media changes every 3-4 days.

[00191] M0 cells may be obtained from sources other than differentiation from iPSCs, e.g., from human cell lines or from a blood sample.

[00192] For differentiation of M0 cells into Ml cells, the M0 cells can be detached from the culture plate after at least 5-7 days in culture using non-enzymatic cell dissociation buffer. M0 cells may then be isolated by centrifugation at 300 x g for 10 min at 4 °C and resuspended in in 10% 1640 Human media supplemented with 50 ng/mL GMCSF. Cells can be plated at, e.g., about 50,000 cells per well in a 96-well plate, or at about 50,000 to 70,000 cells in 24-well plates (number and plate can change depending on the experiment) for use in experiments.

[00193] Cells should be allowed to settle to culture bottom for a suitable amount of time (e.g., at least 30 min) before addition of a suitable amount of LPS and IFNy (e.g., 0.1 ng/mL to 1 ng/mL LPS and 0.2 ng/mL to 2 ng/mL IFNy).

[00194] Cells may be primed in activation media for Ml polarization (e.g., media containing a suitable amount of LPS and fFNy (e.g., 0.1 ng/mL to 1 ng/mL LPS and 0.2 ng/mL to 2 ng/mL IFNy) for a suitable amount of time (e.g., 1 hour) prior to additional cell/treatment exposure. After Ml polarization, cells may be used for further experiments.

[00195] For M0 cells to M2 differentiation, M0 cells may be detached from culture plate after at least 5-7 days in culture using non-enzymatic cell dissociation buffer. M0 cells may then be isolated by centrifugation at 300 x g for 10 min at 4 °C and resuspended in 10% 1640 human media supplemented with lOOng/mL MCSF. Cells should be plated at a suitable density (e.g., 50,000 cells per well in a 96-well plate, number and plate can change depending on the experiment) and allowed to settle to culture bottom for a suitable amount of time (e.g., at least 30 minutes, depending on well size) before addition of 40 ng/mL each of IL- 10, IL-4, and TGFP (M2 polarizing cytokine cocktail). Cells may be primed in the M2 polarization cytokine cocktail for a suitable amount of time (e.g., 1-3 hours, about Ih, about 2h, about 3h, or more than 3h) prior to additional cell/treatment exposure.

[00196] For M2 EV generation from M2 culture, the macrophages may be polarized in medium containing exosome-free FBS. For example, M2 cells may be activated using a cytokine cocktail containing 40ng/ml IL 10, 40ng/mL IL-4, and 40ng/mL TGFP for 48 hours. M0 cells should be at a suitable density, e.g., about IxlO⁶ cells per well in 6-well plates. Media may be replaced with 10% Exo-Free FBS + 1640 Human media + lOOng/mL MCSF (stock 0.1 mg/mL; 1 : 1000 dilution) with M2 polarizing cytokine cocktail. After this, the cells are polarized to M2 cells and begin to generate M2 EVs. After a suitable amount of time (e.g., about 48 hours) in this polarizing culture, the EV-containing media may be collected and the EVs may be isolated using a suitable technique (e.g., tangential flow filtration, ultracentrifugation or PEG precipitation).

[00197] To avoid contamination with EVs from serum, the cultures from which the EVs are isolated may comprise cells that are cultured in medium containing EV-depleted, for example, EV-free serum. For example, the cells may be cultured in medium containing EV-depleted or EV-free fetal bovine serum (FBS). In another example, the cells may be cultured in medium containing EV-depleted or EV-free human serum, e.g., human AB serum. In particular examples, the cells may be cultured in medium containing exosome-depleted, for example, exosome-free serum, e.g., exosome-depleted or exosome-free FBS, or exosome-depleted or exosome-free human serum, for example, human AB serum.

[00198] In particular, non -limiting examples, the cultures from which the EVs are isolated may comprise cells that are cultured in medium containing EV-depleted, for example, EV-free serum, for a period of 16 hrs, 24 hrs or 48 hrs preceding the isolation. For example, the cells may be cultured in medium containing EV-depleted or EV-free fetal bovine serum (FBS) for a period of 16 hrs, 24 hrs or 48 hrs preceding the isolation. In another example, the cells may be cultured in medium containing EV-depleted or EV-free human serum, e.g., human AB serum, for a period of 16 hrs, 24 hrs or 48 hrs preceding the isolation. In particular examples, the cells may be cultured in medium containing exosome-depleted, for example, exosome-free serum, e.g., exosome-depleted or exosome-free FBS, or exosome-depleted or exosome-free human serum, for example, human AB serum, for a period of 16hrs, 24hrs, or 48hrs preceding the isolation.

5.3.1. Compositions

[00199] In certain aspects, compositions are provided comprising an isolated, cell-free population of anti-inflammatory EVs as described herein. For example, provided herein are compositions comprising an isolated, cell-free population of anti-inflammatory EVs suitable for administration to a subject, for example, a human subject.

[00200] In certain aspects, provided are pharmaceutical compositions comprising an isolated, cell-free population of anti-inflammatory EVs described herein. In certain embodiments, provided herein is a pharmaceutical composition comprising an isolated, cell-free population of anti-inflammatory EVs and a buffer, for example, a sterile buffer, e.g., a saline-containing buffer. In particular embodiments, the pharmaceutical composition comprises an isolated, cell-free population of anti-inflammatory EVs and physiological saline. In particular embodiments, the pharmaceutical composition comprises an isolated, cell-free population of anti-inflammatory EVs and normal saline. In particular embodiments, the pharmaceutical composition comprises an isolated, cell-free population of anti-inflammatory EVs and 0.9% saline. In particular embodiments, the pharmaceutical composition comprises an isolated, cell-free population of anti-inflammatory EVs and phosphate-buffered saline.

[00201] In some embodiments, a composition provided herein is a pharmaceutical composition comprising a population of anti-inflammatory EVs provided herein and a pharmaceutically acceptable carrier, excipient, or diluent. In some embodiments, a composition provided herein is a pharmaceutical composition comprising an effective amount of a population of anti-inflammatory EVs provided herein and a carrier, excipient, or diluent, that is, an amount of a population of anti-inflammatory EVs provided herein which is sufficient to result in a desired outcome.

[00202] The term “pharmaceutically acceptable” as used herein means being approved by a regulatory agency of the Federal or a state government, or listed in United States Pharmacopeia, European Pharmacopeia, or other generally recognized Pharmacopeia for use in animals, and more particularly in humans. [00203] The carrier, excipient, or diluent may be any pharmaceutically acceptable carrier, excipient or diluent, known in the art. Examples of pharmaceutically acceptable carriers include non-toxic solids, semisolids, or liquid fillers, diluents, encapsulating materials, formulation auxiliaries or carriers. A pharmaceutically acceptable carrier can include all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solutions, dextrose solution, and 5% human serum albumin. Liposomes and non-aqueous vehicles such as fixed oils may also be used.

[00204] Excipients may include, for example, encapsulating materials or additives such as absorption accelerators, antioxidants, binders, buffers, coating agents, coloring agents, disintegrating agents, emulsifiers, extenders, fillers, flavoring agents, humectants, lubricants, perfumes, preservatives, propellants, releasing agents, sterilizing agents, sweeteners, solubilizers, wetting agents, and mixtures thereof. The term “excipient” may itself refer to a carrier or diluent. [00205] In some embodiments, the pharmaceutical composition comprises a population of anti-inflammatory EVs provided herein suspended in a sterile buffer. In some embodiments, a pharmaceutical composition provided herein comprises a population of anti-inflammatory EVs in a buffer suitable for administration to a human subject. Examples of buffers suitable for administration to a human subject include saline-containing buffers such as phosphate buffered saline, physiological saline, normal saline or 0.9 % saline.

[00206] A pharmaceutical composition may be formulated to be compatible with an intended route of administration. For example, pharmaceutical compositions may routinely be formulated to be suitable for administration by routes including intranasal, parenteral (e.g., subcutaneous, intravenous, intramuscular, intraperitoneal, intraarterial, intraventricular, intrathecal, intraurethral, intrastemal, and intrasynovial), intradermal, oral (e.g., ingestion, sublingual), inhalation, nasal, e.g., nasal drip, intracavity, intracranial, ocular, e.g., intraocular, and transdermal (topical).

[00207] In certain embodiments, for example, a pharmaceutical composition presented herein that comprises an isolated, cell-free population of anti-inflammatory EVs as described herein has been formulated to be suitable for intranasal administration to a subject, for example, a human subject. [00208] In certain embodiments, a pharmaceutical composition presented herein that comprises an isolated, cell-free population of anti-inflammatory EVs as described herein has been formulated to be suitable for injection, infusion or implantation to a subject, for example, a human subject.

[00209] In particular embodiments, a pharmaceutical composition presented herein that comprises an isolated, cell-free population of anti-inflammatory EVs as described herein has been formulated to be suitable for intravenous administration to a subject, for example, a human subject.

[00210] In another example, in particular embodiments, a pharmaceutical composition presented herein that comprises an isolated, cell-free population of anti-inflammatory EVs as described herein has been formulated to be suitable for subcutaneous administration to a subject, for example, a human subject.

[00211] In yet another example, in particular embodiments, a pharmaceutical composition presented herein that comprises an isolated, cell-free population of anti-inflammatory EVs as described herein has been formulated to be suitable for intramuscular administration to a subject, for example, a human subject.

[00212] In certain embodiments, a composition, for example a pharmaceutical composition presented herein that comprises an isolated, cell-free population of anti-inflammatory EVs as described herein has been formulated in solution, suspension, emulsion, micelle, liposome, microsphere, or nanosystem form.

[00213] In certain embodiments, a composition, for example a pharmaceutical composition, presented herein that comprises an isolated, cell-free population of anti-inflammatory EVs as described herein may be stored frozen, e.g., may be stored at -20°C or -80°C. For example, in particular embodiments, such a composition, e.g., pharmaceutical composition, may be stored frozen, for example, frozen at -20°C or -80°C, for about 1 week, 1 month, about 3 months, about 6 months, about 9 months, about 12 months, about 18 months or about 24 months. In specific embodiments, such a composition, e.g., pharmaceutical composition, may then be thawed and administered to a patient.

[00214] In certain embodiments, a composition, for example a pharmaceutical composition, presented herein that comprises an isolated, cell-free population of anti-inflammatory EVs as described herein may be stored frozen, e.g., may be stored at -20°C or -80°C, thawed, then refrozen. In particular embodiments, such a composition, e.g., pharmaceutical composition, may be thawed then refrozen one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen or twenty times In specific embodiments, such a composition, e.g., pharmaceutical composition, may then be thawed and administered to a patient.

[00215] In certain embodiments, a composition, for example a pharmaceutical composition, presented herein that comprises an isolated, cell-free population of anti-inflammatory EVs as described herein may be stored at about 2°C to about 8 °C (e.g., at about 4°C). For example, in particular embodiments, such a composition, e.g., pharmaceutical composition, may be stored at about 2°C to about 8 °C (e.g., at about 4°C) for less than about 2 weeks, less than about 1 week, less than about 14 days, less than about 13 days, less than about 12 days, less than about 11 days, less than about 10 days, less than about 9 days, less than about 8 days, less than about 7 days, less than about 6 days, less than about 5 days, less than about 4 days, less than about 3 days, less than about 2 days, less than about 1 day, or about overnight. In specific embodiments, such a composition, e.g., pharmaceutical composition, may be stored at 4°C prior to administration to a subject, for example, a human subject, e.g., may be thawed after being frozen, then stored at 4°C prior to administration to a subject, for example to a human subject.

[00216] In certain embodiments, provided herein is a cryopreserved composition, for example, pharmaceutical composition, comprising an isolated, cell-free population of antiinflammatory EVs as described herein. In particular embodiments, the cryopreserved, isolated cell-free population of anti-inflammatory EVs may be cryopreserved for about 1 week, 1 month, about 3 months, about 6 months, about 9 months, about 12 months, about 18 months or about 24 months, then may be thawed and administered to a patient after cry opreservation.

[00217] In certain embodiments, provided herein is a composition comprising an isolated, cell-free population of anti-inflammatory EVs as described herein, wherein the population comprises about IxlO⁶ to about IxlO¹⁶ EVs, about IxlO⁷ to about IxlO¹⁶ EVs, lxl0⁸to about IxlO¹⁶ EVs, about IxlO⁹ to about IxlO¹⁶ EVs, IxlO¹⁰ to about IxlO¹⁶ EVs, about IxlO¹¹ to about IxlO¹⁶ EVs, IxlO¹² to about IxlO¹⁶ EVs, about IxlO¹³ to about IxlO¹⁶ EVs, IxlO⁶ to about IxlO¹⁵ EVs, about IxlO⁷ to about IxlO¹⁵ EVs, IxlO⁸ to about IxlO¹⁵ EVs, about IxlO⁹ to about IxlO¹⁵ EVs, IxlO¹⁰ to about IxlO¹⁵ EVs, about IxlO¹¹ to about IxlO¹⁵ EVs, IxlO¹² to about IxlO¹⁵ EVs, about IxlO¹³ to about IxlO¹⁵ EVs, IxlO⁶ to about IxlO¹⁴ EVs, about IxlO⁷ to about 1X10¹⁴ EVS, IxlO⁸ to about 1X10¹⁴ EVS, about IxlO⁹ to about 1X10¹⁴ EVS, IxlO¹⁰ to about IxlO¹⁴ EVs, about IxlO¹¹ to about IxlO¹⁴ EVs, IxlO¹² to about IxlO¹⁴ EVs, about IxlO¹³ to about 1X10¹⁴ EVS, IxlO⁶ to about IxlO¹³ EVs, about IxlO⁷ to about IxlO¹³ EVs, IxlO⁸ to about IxlO¹³ EVs, about IxlO⁹ to about IxlO¹³ EVs, IxlO¹⁰ to about IxlO¹³ EVs, about IxlO¹¹ to about IxlO¹³ EVs, IxlO¹² to about IxlO¹³ EVs, IxlO⁶ to about IxlO¹² EVs, about IxlO⁷ to about IxlO¹² EVs, IxlO⁸ to about IxlO¹² EVs, about IxlO⁹ to about IxlO¹² EVs, IxlO¹⁰ to about IxlO¹² EVs, about IxlO¹¹ to about IxlO¹² EVs, about IxlO⁶ to about IxlO¹¹ EVs, about IxlO⁷ to about IxlO¹¹ EVs, IxlO⁸ to about IxlO¹¹ EVs, about IxlO⁹ to about IxlO¹¹ EVs, IxlO¹⁰ to about IxlO¹¹ EVs, about IxlO⁶ to about IxlO¹⁰ EVs, about IxlO⁷ to about IxlO¹⁰ EVs, IxlO⁸ to about IxlO¹⁰ EVs, about IxlO⁶ EVs, about IxlO⁷ EVs, IxlO⁸ to about IxlO⁹ EVs, about IxlO¹⁰ to about IxlO¹¹ EVs, IxlO¹² to about IxlO¹³ EVs, about IxlO⁶ EVs, about IxlO⁷ EVs, about IxlO⁸ EVs, about 2xl0⁸ EVs, about 3xl0⁸ EVs, about 4xl0⁸ EVs, about 5xl0⁸ EVs, about 6xl0⁸ EVs, about 7xl0⁸ EVs, about 8xl0⁸ EVs, about 9xl0⁸ EVs, about IxlO⁹ EVs, about 5xl0⁹ EVs, about IxlO¹⁰ EVs, about IxlO¹¹ EVs, about IxlO¹² EVs, about IxlO¹³ EVs, about IxlO¹⁴ EVs, about IxlO¹⁵ EVs, or about 1X10¹⁶ EVS.

[00218] In certain embodiments, provided herein is a composition comprising an isolated, cell-free population of anti-inflammatory EVs as described herein, wherein the population comprises IxlO⁶ to about IxlO¹⁶ EVs/ml, about IxlO⁷ to about IxlO¹⁶ EVs/ml, IxlO⁸ to about IxlO¹⁶ EVs/ml, about IxlO⁹ to about IxlO¹⁶ EVs/ml, IxlO¹⁰ to about IxlO¹⁶ EVs/ml, about IxlO¹¹ to about IxlO¹⁶ EVs/ml, IxlO¹² to about IxlO¹⁶ EVs/ml, about IxlO¹³ to about IxlO¹⁶ EVs/ml, IxlO⁶ to about IxlO¹⁵ EVs/ml, about IxlO⁷ to about IxlO¹⁵ EVs/ml, IxlO⁸ to about IxlO¹⁵ EVs/ml, about IxlO⁹ to about IxlO¹⁵ EVs/ml, IxlO¹⁰ to about IxlO¹⁵ EVs/ml, about IxlO¹¹ to about IxlO¹⁵ EVs/ml, IxlO¹² to about IxlO¹⁵ EVs/ml, about IxlO¹³ to about IxlO¹⁵ EVs/ml, about IxlO⁶ to about IxlO¹⁴ EVs/ml, about IxlO⁷ to about IxlO¹⁴ EVs/ml, IxlO⁸ to about IxlO¹⁴ EVs/ml, about IxlO⁹ to about IxlO¹⁴ EVs/ml, IxlO¹⁰ to about IxlO¹⁴ EVs/ml, about IxlO¹¹ to about IxlO¹⁴ EVs/ml, IxlO¹² to about IxlO¹⁴ EVs/ml, about IxlO¹³ to about IxlO¹⁴ EVs/ml, IxlO⁶ to about IxlO¹³ EVs/ml, about IxlO⁷ to about IxlO¹³ EVs/ml, IxlO⁸ to about IxlO¹³ EVs/ml, about IxlO⁹ to about IxlO¹³ EVs/ml, IxlO¹⁰ to about IxlO¹³ EVs/ml, about IxlO¹¹ to about IxlO¹³ EVs/ml, IxlO¹² to about IxlO¹³ EVs, /ml IxlO⁶ to about IxlO¹² EVs/ml, about IxlO⁷ to about IxlO¹² EVs/ml, IxlO⁸ to about IxlO¹² EVs/ml, about IxlO⁹ to about IxlO¹² EVs/ml, IxlO¹⁰ to about IxlO¹² EVs/ml, about IxlO¹¹ to about IxlO¹² EVs/ml, about IxlO⁶ to about IxlO¹¹ EVs/ml, about IxlO⁷ to about IxlO¹¹ EVs/rnl, lxl0⁸to about IxlO¹¹ EVs/rnl, about IxlO⁹ to about IxlO¹¹ EVs/ml, IxlO¹⁰ to about IxlO¹¹ EVs/ml, about IxlO⁶ to about IxlO¹⁰ EVs/ml, about IxlO⁷ to about IxlO¹⁰ EVs/ml, IxlO⁸ to about IxlO¹⁰ EVs/ml, about IxlO⁶ EVs/ml, about IxlO⁷ EVs/ml, IxlO⁸ to about IxlO⁹ EVs/ml, about IxlO¹⁰ to about IxlO¹¹ EVs/ml, IxlO¹² to about IxlO¹³ EVs/ml, about IxlO⁶ EVs/ml, about IxlO⁷ EVs/ml, about IxlO⁸ EVs/ml, about 2xl0⁸ EVs/ml, about 3xl0⁸ EVs/ml, about 4xl0⁸ EVs,/ml about 5xl0⁸ EVs/ml, about 6xl0⁸ EVs/ml, about 7xl0⁸ EVs/ml, about 8xl0⁸ EVs/ml, about 9xl0⁸ EVs/ml, about IxlO⁹ EVs/ml, about 5xl0⁹ EVs/ml, about IxlO¹⁰ EVs/ml, about IxlO¹¹ EVs/ml, about IxlO¹² EVs,/ml about IxlO¹³ EVs/ml, about IxlO¹⁴ EVs/ml, about IxlO¹⁵ EVs/ml, or about IxlO¹⁶ EV/mls

[00219] In certain embodiments, provided herein is a composition comprising an isolated, cell-free population of anti-inflammatory EVs as described herein, wherein the population comprises about 1 pg to about 200 mg EVs, about 1 pg to about 150 mg EVs, about 1 pg to about 100 mg EVs, about 1 pg to about 75 mg EVs, about 1 pg to about 50 mg EVs, about 1 pg to about 25 mg EVs, about 1 pg to about 20 mg EVs, about 1 pg to about 15 mg EVs, about 1 pg to about 10 mg EVs, about 1 pg to about 5 mg EVs, about 1 pg to about 1 mg EVs, about 1 pg to about 500 pg EVs, about 1 pg to about 250 pg EVs, about 1 pg to about 125 pg EVs, about 1 pg to about 100 pg EVs, about 1 pg to about 50 pg EVs, about 1 pg to about 25 pg EVs, about 1 pg to about 20 pg EVs, about 1 pg to about 10 pg EVs, about 1 pg to about 5 pg EVs, about 10 pg to about 500 pg EVs, about 10 pg to about 250 pg EVs, about 10 pg to about 125 pg EVs, about 10 pg to about 100 pg EVs, about 10 pg to about 50 pg EVs, about 10 pg to about 25 pg EVs, about 10 pg to about 20 pg EVs, about 100 pg to about 500 pg EVs, about 100 pg to about 250 pg EVs, or about 100 pg to about 125 pg EVs.

[00220] In certain embodiments, provided herein is a composition comprising an isolated, cell-free population of anti-inflammatory EVs as described herein, wherein the population comprises about 1 pg to about 200 mg EVs/ml, about 1 pg to about 150 mg EVs/ml, about 1 pg to about 100 mg EVs/ml, about 1 pg to about 75 mg EVs/ml, about 1 pg to about 50 mg EVs/ml, about 1 pg to about 25 mg EVs/ml, about 1 pg to about 20 mg EVs/ml, about 1 pg to about 15 mg EVs/ml, about 1 pg to about 10 mg EVs/ml, about 1 pg to about 5 mg EVs/ml, about 1 pg to about 1 mg EVs/ml, about 1 pg to about 500 pg EVs/ml, about 1 pg to about 250 pg EVs/ml, about 1 pg to about 125 pg EVs/ml, about 1 pg to about 100 pg EVs, /ml about 1 pg to about 50 pg EVs/ml, about 1 pg to about 25 pg EVs/rnl, about 1 pg to about 20 pg EVs/rnl, about 1 pg to about 10 pg EVs,/ml about 1 pg to about 5 pg EVs/ml, about 10 pg to about 500 pg EVs/ml, about 10 pg to about 250 pg EVs/ml, about 10 pg to about 125 pg EVs,/ml about 10 pg to about 100 pg EVs/ml, about 10 pg to about 50 pg EVs/ml, about 10 pg to about 25 pg EVs/ml, about 10 pg to about 20 pg EVs/ml, about 100 pg to about 500 pg EVs/ml, about 100 pg to about 250 pg EVs/ml, or about 100 pg to about 125 pg EVs/ml.

[00221] In certain embodiments, the isolated, cell-free populations of anti-inflammatory EVs described herein are present in a composition that is substantially free of other EVs. For example, in certain embodiments, the isolated, cell-free populations of anti-inflammatory EVs described herein are present in a composition that contains less than about 20%, less than about 10%, less than about 5%, or less than about 1% other EVs.

[00222] In certain embodiments, an isolated, cell-free population of anti-inflammatory EVs described herein is present in a composition that comprises other EVs, wherein the isolated, cell- free population of anti-inflammatory EVs makes up about 10%, about 20%, about 25%, about 30%, about 35%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or greater than about 95% of the EVs in the composition. In specific embodiments, the other EVs are serum EVs, for example, bovine serum EVs or human serum EVs.

[00223] In some embodiments, a composition comprising a population of anti-inflammatory EVs provided herein comprises no contaminants. In some embodiments, a composition comprising a population of anti-inflammatory EVs provided herein comprises a sufficiently low level of contaminants as to be suitable for administration, e.g., therapeutic administration, to a subject, for example a human subject. Contaminants include, for example, bacteria, fungus, mycoplasma, endotoxins or residual beads from the M2 macrophage culture. In some embodiments, a composition comprising a population of anti-inflammatory EVs provided herein comprises less than about 5 EU/kg endotoxins. In some embodiments, a composition comprising a population of anti-inflammatory EVs provided herein comprises about or less than about 100 beads per 3 x 10⁶ cells.

[00224] In some embodiments, a composition comprising a population of anti-inflammatory EVs provided herein is sterile. In some embodiments, isolation or enrichment of the cells is carried out in a closed or sterile environment, for example, to minimize error, user handling and/or contamination. In some embodiments, sterility may be readily accomplished, e.g., by filtration through sterile filtration membranes.

5.4 Methods of Treatment

[00225] Provided herein are methods of treatment comprising administering an effective amount of a population of anti-inflammatory EVs as described herein to a subject in need thereof.

[00226] In some embodiments, the subject is diagnosed with or is suspected of having a disorder associated with Treg dysfunction. In some embodiments, the subject is diagnosed with or is suspected of having a disorder associated with Treg deficiency. In some embodiments, the subject is diagnosed with or is suspected of having a condition (e.g., an inflammatory condition) driven by a T cell response. In some embodiments, the subject is diagnosed with or is suspected of having a condition (e.g., an inflammatory condition) driven by a myeloid cell response. In specific embodiments, the myeloid cell is a monocyte, macrophage or microglia.

[00227] In some embodiments the subject is diagnosed with or is suspected of having a neurodegenerative disease. In some embodiments, the subject is diagnosed with or is suspected of having Alzheimer’s disease, Amyotrophic Lateral Sclerosis, Huntington’s disease, Parkinson’s disease, or frontotemporal dementia.

[00228] In some embodiments, the subject is diagnosed with or is suspected of having a disorder that would benefit from downregulation of the immune system.

[00229] In some embodiments, the subject is diagnosed with or suspected of having an autoimmune disease. The autoimmune disease may be, for example, systemic sclerosis (scleroderma), polymyositis, ulcerative colitis, inflammatory bowel disease, Crohn’s disease, celiac disease, multiple sclerosis (MS), rheumatoid arthritis (RA), Type I diabetes, psoriasis, dermatomyositis, systemic lupus erythematosus, cutaneous lupus, myasthenia gravis, autoimmune nephropathy, autoimmune hemolytic anemia, autoimmune cytopenia, autoimmune encephalitis, autoimmune hepatitis, autoimmune uveitis, alopecia, thyroiditis or pemphigus.

[00230] In some embodiments, the subject is diagnosed with or suspected of having heart failure or ischemic cardiomyopathy. In some embodiments, the subject is diagnosed with or suspected of having graft-versus-host disease, e.g., after undergoing organ transplantation (such as a kidney transplantation or a liver transplantation), or after undergoing stem cell transplantation (such as hematopoietic stem cell transplantation including a bone marrow transplant).

[00231] In some embodiments, the subject is diagnosed with or suspected of having neuroinflammation. Neuroinflammation may be associated, for example, with stroke, acute disseminated encephalomyelitis (ADEM), acute optic neuritis, acute inflammatory demyelinating polyradiculoneuropathy, chronic inflammatory demyelinating polyradiculoneuropathy, Guillain- Barre syndrome, transverse myelitis, neuromyelitis optica (NMO), epilepsy, traumatic brain injury, spinal cord injury, encephalitis central nervous system (CNS) vasculitis, neurosarcoidosis, autoimmune or post-infectious encephalitis, or chronic meningitis.

[00232] In some embodiments, the subject is in need of improving islet graft survival, and the method comprises administering to the subject an effective amount of a population of antiinflammatory EVs as described herein or a pharmaceutical composition described herein in combination with the islet transplantation.

[00233] In some embodiments, the subject is diagnosed with or suspected of having cardoinflammation, e.g., cardio-inflammation associated with atheroscleorosis, myocardial infarction, ischemic cardiomyopathy, with heart failure.

[00234] In some embodiments, the subject is diagnosed with or suspected of having chronic inflammatory demyelinating polyradiculoneuropathy (CIDP). In some embodiments, the subject is diagnosed with or suspected of having acute inflammatory demyelinating polyneuropathy (AIDP). In some embodiments, the subject is diagnosed with or suspected of having Guillain- Barre syndrome (GBS).

[00235] In some embodiments, the subject has had a stroke.

[00236] In some embodiments, the subject is diagnosed with or suspected of having cancer, e.g., a blood cancer.

[00237] In some embodiments, the subject is diagnosed with or suspected of having asthma.

[00238] In some embodiments, the subject is diagnosed with or suspected of having eczema.

[00239] In some embodiments, the subject is diagnosed with or suspected of having a disorder associated with over activation of the immune system.

[00240] In some embodiments, the subject is diagnosed with or suspected of having

Tregopathy. The Tregopathy may be caused by a FOXP3, CD25, cytotoxic T lymphocyte- associated antigen 4 (CTLA4), LPS-responsive and beige-like anchor protein (LRBA), or BTB domain and CNC homolog 2 (BACH2) gene loss-of-function mutation, or a signal transducer and activator of transcription 3 (STAT3) gain-of-function mutation.

[00241] In some embodiments, the donor subject is a geriatric subject, e.g., a subject of at least 65, at least 70, at least 75, at least 80, at least 85 or at least 90 years of age.

[00242] In some embodiments, about IxlO⁸ to about IxlO¹⁴ EVs, about IxlO⁸ to about IxlO¹² EVs, about IxlO⁸ to about IxlO¹⁰ EVs, about IxlO¹⁰ to about IxlO¹⁴ EVs, about IxlO¹⁰ to about IxlO¹² EVs, about IxlO⁶ to about IxlO⁷ EVs, about IxlO⁷ to about IxlO⁸ EVs, about IxlO⁸ to about IxlO⁹ EVs, about IxlO⁹ to about IxlO¹⁰ EVs, about IxlO¹⁰ to about IxlO¹¹ EVs, about IxlO¹² to about IxlO¹³ EVs, about IxlO¹³ to about IxlO¹⁴ EVs, or about IxlO¹⁴ to about IxlO¹⁵ EVs are administered.

[00243] In some embodiments, about IxlO⁸ EVs, about 2xl0⁸ EVs, about 3xl0⁸ EVs, about 4xl0⁸ EVs, about 5xl0⁸ EVs, about 6xl0⁸ EVs, about 7xl0⁸ EVs, about 8xl0⁸ EVs, about 9xl0⁸ EVs, about IxlO⁹ EVs, about 2xl0⁹ EVs, about 3xl0⁹ EVs, about 4xl0⁹ EVs, about 5xl0⁹ EVs, about 6xl0⁹ EVs, about 7xl0⁹ EVs, about 8xl0⁹ EVs, about 9xl0⁹ EVs or about IxlO¹⁰ EVs are administered, for example, are administered per dose.

[00244] In some embodiments, about IxlO⁸ to about IxlO¹⁴ EVs/mL, about IxlO⁸ to about IxlO¹² EVs/mL, about IxlO⁸ to about IxlO¹⁰ EVs/mL, about IxlO¹⁰ to about IxlO¹⁴ EVs/mL, about IxlO¹⁰ to about IxlO¹² EVs/mL, about IxlO⁶ to about IxlO⁷ EVs/mL, about IxlO⁷ to about IxlO⁸ EVs/mL, about IxlO⁸ to about IxlO⁹ EVs/mL, about IxlO⁹ to about IxlO¹⁰ EVs/mL, about IxlO¹⁰ to about IxlO¹¹ EVs/mL, about IxlO¹² to about IxlO¹³ EVs/mL, about IxlO¹³ to about IxlO¹⁴ EVs/mL, or about IxlO¹⁴ to about lxl0¹⁵ EV/mL are administered.

[00245] In some embodiments, about IxlO⁸ EVs/ml, about 2xl0⁸ EVs/ml, about 3xl0⁸ EVs/ml, about 4xl0⁸ EVs/ml, about 5xl0⁸ EVs/ml, about 6xl0⁸ EVs/ml, about 7xl0⁸ EVs/ml, about 8xl0⁸ EVs/ml, about 9xl0⁸ EVs/ml, about IxlO⁹ EVs/ml, about 2xl0⁹ EVs/ml, about 3xl0⁹ EVs/ml, about 4xl0⁹ EVs/ml, about 5xl0⁹ EVs/ml, about 6xl0⁹ EVs/ml, about 7xl0⁹ EVs/ml, about 8xl0⁹ EVs/ml, about 9xl0⁹ EVs/ml or about IxlO¹⁰ EVs/ml are administered, for example, are administered per dose.

[00246] In some embodiments, about 1 pg to about 100 pg EVs, about 100 pg to about 200 pg EVs, about 200 pg to about 300 pg EVs, about 300 pg to about 400 pg EVs, about 400 pg to about 500 pg EVs, about 500 pg to about 600 pg EVs, about 600 pg to about 700 pg EVs, about 700 pg to about 800 pg EVs, about 800 pg to about 900 pg EVs, about 900 pg to about 1 mg EVs, about 1 mg to about 10 mg EVs, about 10 mg to about 20 mg EVs, about 20 mg to about 30 mg EVs, about 30 mg to about 40 mg EVs, about 40 mg to about 50 mg EVs, about 50 mg to about 60 mg EVs, about 60 mg to about 70 mg EVs, about 70 mg to about 80 mg EVs, about 80 mg to about 90 mg EVs, about 90 mg to about 100 mg EVs, about 100 mg to about 110 mg EVs, about 110 mg to about 120 mg EVs, about 120 mg to about 130 mg EVs, about 130 mg to about 140 mg EVs, about 150 mg to about 160 mg EVs, about 160 mg to about 170 mg EVs, about 170 mg to about 180 mg EVs, about 180 mg to about 190 mg EVs, or about 190 mg to about 200 mg EVs are administered.

[00247] In some embodiments, about 1 pg to about 100 pg EVs/mL, about 100 pg to about 200 pg EVs/mL, about 200 pg to about 300 pg EVs/mL, about 300 pg to about 400 pg EVs/mL, about 400 pg to about 500 pg EVs/mL, about 500 pg to about 600 pg EVs/mL, about 600 pg to about 700 pg EVs/mL, about 700 pg to about 800 pg EVs/mL, about 800 pg to about 900 pg EVs/mL, about 900 pg to about 1 mg EVs/mL, about 1 mg to about 10 mg EVs/mL, about 10 mg to about 20 mg EVs/mL, about 20 mg to about 30 mg EVs/mL, about 30 mg to about 40 mg EVs/mL, about 40 mg to about 50 mg EVs/mL, about 50 mg to about 60 mg EVs/mL, about 60 mg to about 70 mg EVs/mL, about 70 mg to about 80 mg EVs/mL, about 80 mg to about 90 mg EVs/mL, about 90 mg to about 100 mg EVs/mL, about 100 mg to about 110 mg EVs/mL, about 110 mg to about 120 mg EVs/mL, about 120 mg to about 130 mg EVs/mL, about 130 mg to about 140 mg EVs/mL, about 150 mg to about 160 mg EVs/mL, about 160 mg to about 170 mg EVs/mL, about 170 mg to about 180 mg EVs/mL, about 180 mg to about 190 mg EVs/mL, or about 190 mg to about 200 mg EVs/mL are administered.

[00248] A population of anti-inflammatory EVs may be administered to the subject by any suitable route. For example, a population of anti-inflammatory EVs may be administered to the subject by routes including intranasal, parenteral (e.g., subcutaneous, intravenous, intramuscular, intraperitoneal, intraarterial, intraventricular, intrathecal, intraurethral, intrasternal, and intrasynovial), intradermal, oral (e.g., ingestion, sublingual), inhalation, nasal, e.g., nasal drip, intracavity, intracranial, ocular, e.g., intraocular, and transdermal (topical). In particular embodiments, for example, a population of anti-inflammatory EVs may be administered to the subject in a pharmaceutical composition formulated for administration by a route that includes including intranasal, parenteral (e.g., subcutaneous, intravenous, intramuscular, intraperitoneal, intraarterial, intraventricular, intrathecal, intraurethral, intrasternal, and intrasynovial), intradermal, oral (e.g., ingestion, sublingual), inhalation, nasal, e.g., nasal drip, intracavity, intracranial, ocular, e.g., intraocular, and transdermal (topical).

[00249] In certain embodiments, for example, a method of treatment presented herein comprises administering to a subject in need of treatment a pharmaceutical composition that comprises an effective amount of an isolated, cell-free population of anti-inflammatory EVs as described herein and has been formulated to be suitable for intranasal administration to a subject, for example, a human subject.

[00250] In certain embodiments, for example, a method of treatment presented herein comprises administering to a subject in need of treatment a pharmaceutical composition that comprises an effective amount of an isolated, cell-free population of anti-inflammatory EVs as described herein and has been formulated to be suitable for injection, infusion or implantation to a subject, for example, a human subject.

[00251] In certain embodiments, for example, a method of treatment presented herein comprises administering to a subject in need of treatment a pharmaceutical composition that comprises an effective amount of an isolated, cell-free population of anti-inflammatory EVs as described herein and has been formulated to be suitable for intravenous administration to a subject, for example, a human subject.

[00252] In certain embodiments, for example, a method of treatment presented herein comprises administering to a subject in need of treatment a pharmaceutical composition that comprises an effective amount of an isolated, cell-free population of anti-inflammatory EVs as described herein and has been formulated to be suitable for subcutaneous administration to a subject, for example, a human subject.

[00253] In certain embodiments, for example, a method of treatment presented herein comprises administering to a subject in need of treatment a pharmaceutical composition that comprises an effective amount of an isolated, cell-free population of anti-inflammatory EVs as described herein and has been formulated to be suitable for intramuscular administration to a subject, for example, a human subject.

[00254] A population of anti-inflammatory EVs may be administered to the subject more than once. For example, a population of anti-inflammatory EVs may be administered to the subject every week, every other week, every three weeks, once a month, every other month, every 3 months, every 6 months, ever 12 months, every 18 months, every year, every other year, every 3 years, or every 5 years.

[00255] The population of anti-inflammatory EVs administered to the subject may be autologous to the subject. The population of anti-inflammatory EVs administered to the subject may be allogeneic to the subject. The population of anti-inflammatory EVs administered to the subject may be derived from human suppressive immune cell precursors or human suppressive immune cells, e.g., M2 macrophage precursors or M2 macrophages, respectively, from more than one individual. For example, the population of anti-inflammatory EVs administered to the subject may be a pooled population of anti-inflammatory EVs, wherein some or all of the population of anti-inflammatory EVs is allogeneic to the subject.

[00256] In certain embodiments, a population of anti-inflammatory EVs may be administered to the subject more than once. For example, a population of anti-inflammatory EVs may be administered to the subject every week, every other week, every three weeks, once a month, every other month, every 3 months, every 6 months, ever 12 months, every 18 months, every year, every other year, every 3 years, or every 5 years.

5.4.1. Additional Therapies

[00257] In some embodiments, In some embodiments, a subject treated in accordance with the method of treatment described herein further received one or more additional therapy or additional therapies.

[00258] In some embodiments, the subject is additionally administered an effective amount of an ex vi vo-expanded population of human Tregs, for example, CD4+CD25+ Treg cells. In some embodiments, the population of Tregs has been cryopreserved. In some embodiments, the cryopreserved population of Tregs is thawed and administered to the subject without further expansion.

[00259] In some embodiments, the population of Tregs or the cryopreserved population of Tregs is a population of Tregs described in International Patent Application No. PCT/US2020/63378, or is produced by a method described in International Patent Application No. PCT/US2020/63378, which is incorporated by reference herein in its entirety.

[00260] In some embodiments, about 1 x 10⁶to about 2 x 10⁶, about 2 x 10⁶ to about 3 x 10⁶, about 3 x 10⁶to about 4 x 10⁶, about 4 x 10⁶ to about 5 x 10⁶, about 5 x 10⁶ to about 6 x 10⁶, about 6 x 10⁶ to about 7 x 10⁶, about 7 x 10⁶ to about 8 x 10⁶, about 8 x 10⁶ to about 9 x 10⁶, about 9 x 10⁶ to about 1 x 10⁷, about 1 x 10⁷ to about 2 x 10⁷, about 2 x 10⁷ to about 3 x 10⁷, about 3 x 10⁷ to about 4 x 10⁷, about 4 x 10⁷ to about 5 x 10⁷, about 5 x 10⁷ to about 6 x 10⁷, about 6 x 10⁷to about 7 x 10⁷, about 7 x 10⁷ to about 8 x 10⁷, about 8 x 10⁷ to about 9 x 10⁷, about 9 x 10⁷to about 1 x 10⁸, about 1 x 10⁸ to about 2 x 10⁸, about 2 x 10⁸ to about 3 x 10⁸, about 3 x 10⁸ to about 4 x 10⁸, about 4 x 10⁸ to about 5 x 10⁸, about 5 x 10⁸ to about 6 x 10⁸, about 6 x 10⁸to about 7 x 10⁸, about 7 x 10⁸to about 8 x 10⁸, about 8 x 10⁸ to about 9 x 10⁸, about 9 x 10⁸to about 1 x 10⁹ Tregs, e.g., CD4+CD25+cells, per kg of body weight of the subject are administered. In some embodiments, 1 xlO⁶ Tregs, e.g., CD4+CD25+cells (+/- 10%) per kg of body weight of the subject are administered.

[00261] In some embodiments, about 1 x 10⁶to about 2 x 10⁶, about 2 x 10⁶ to about 3 x 10⁶, about 3 x 10⁶to about 4 x 10⁶, about 4 x 10⁶ to about 5 x 10⁶, about 5 x 10⁶ to about 6 x 10⁶, about 6 x 10⁶ to about 7 x 10⁶, about 7 x 10⁶ to about 8 x 10⁶, about 8 x 10⁶ to about 9 x 10⁶, about 9 x 10⁶ to about 1 x 10⁷, about 1 x 10⁷ to about 2 x 10⁷, about 2 x 10⁷ to about 3 x 10⁷, about 3 x 10⁷ to about 4 x 10⁷, about 4 x 10⁷ to about 5 x 10⁷, about 5 x 10⁷ to about 6 x 10⁷, about 6 x 10⁷to about 7 x 10⁷, about 7 x 10⁷ to about 8 x 10⁷, about 8 x 10⁷ to about 9 x 10⁷, about 9 x 10⁷to about 1 x 10⁸, about 1 x 10⁸ to about 2 x 10⁸, about 2 x 10⁸ to about 3 x 10⁸, about 3 x 10⁸ to about 4 x 10⁸, about 4 x 10⁸ to about 5 x 10⁸, about 5 x 10⁸ to about 6 x 10⁸, about 6 x 10⁸to about 7 x 10⁸, about 7 x 10⁸to about 8 x 10⁸, about 8 x 10⁸ to about 9 x 10⁸, about 9 x 10⁸to about 1 x 10⁹ Tregs, e.g., CD4⁺CD25⁺cells are administered to a patient.

[00262] In some embodiments, about 1 x 10⁶to about 2 x 10⁶, about 2 x 10⁶ to about 3 x 10⁶, about 3 x 10⁶to about 4 x 10⁶, about 4 x 10⁶ to about 5 x 10⁶, about 5 x 10⁶ to about 6 x 10⁶, about 6 x 10⁶ to about 7 x 10⁶, about 7 x 10⁶ to about 8 x 10⁶, about 8 x 10⁶ to about 9 x 10⁶, about 9 x 10⁶ to about 1 x 10⁷, about 1 x 10⁷ to about 2 x 10⁷, about 2 x 10⁷ to about 3 x 10⁷, about 3 x 10⁷ to about 4 x 10⁷, about 4 x 10⁷ to about 5 x 10⁷, about 5 x 10⁷ to about 6 x 10⁷, about 6 x 10⁷to about 7 x 10⁷, about 7 x 10⁷ to about 8 x 10⁷, about 8 x 10⁷ to about 9 x 10⁷, about 9 x 10⁷to about 1 x 10⁸, about 1 x 10⁸ to about 2 x 10⁸, about 2 x 10⁸ to about 3 x 10⁸, about 3 x 10⁸ to about 4 x 10⁸, about 4 x 10⁸ to about 5 x 10⁸, about 5 x 10⁸ to about 6 x 10⁸, about 6 x 10⁸to about 7 x 10⁸, about 7 x 10⁸to about 8 x 10⁸, about 8 x 10⁸ to about 9 x 10⁸, about 9 x 10⁸to about 1 x 10⁹ Tregs, e.g., CD4⁺CD25⁺cells are administered to a patient in one infusion. [00263] In some embodiments, a cryopreserved composition comprising a population of Tregs is administered within about 30 minutes, about Ih, about 2-3h, about 3-4h, about 4-5h, about 5-6, about 6-7h, about 7-8h, about 8-9h, or about 9-1 Oh of thawing the cryopreserved composition comprising a population of Tregs. The cryopreserved composition comprising a population of Tregs may be stored at about 2°C to about 8 °C (e.g., at about 4°) between thawing and administration.

[00264] In some embodiments, one dose of a population of Tregs or a composition comprising a population of Tregs is administered to a subject. In some embodiments, a population of Tregs or a composition comprising a population of Tregs is administered more than once. In some embodiments, a population of Tregs or a composition comprising a population of Tregs is administered two or more times. In some embodiments, a population of Tregs or a composition comprising a population of Tregs is administered every 1-2 weeks, 2-3 weeks, 3-4 weeks, 4-5 weeks, 5-6 weeks, 6-7 weeks, 7-8 weeks, 8-9 weeks, 9-10 weeks, 10-11 weeks, 11-12 weeks, every 1-2 months, 2-3 months, 3-4 months, 4-5 months, 5-6 months, 6-7 months, 7-8 months, 8-9 months, 9-10 months, 10-11 months, 11-12 months, 13-14 months, 14-15 months, 15-16 months, 16-17 months, 17-18 months, 18-19 months, 19-20 months, 20-21 months, 21-22 months, 22-23 months, 23-24 months, every 1-2 years, 2-3 years, 3-4 years or 4-5 years.

[00265] In some embodiments, about IxlO⁶ Tregs per kg of body weight of the subject are administered in the first administration and the number of Tregs administered is increased in the second third and subsequent administration. In some embodiments, about IxlO⁶ Tregs per kg of body weight of the subject are administered in the first two administrations, and the number of Tregs administered is increased in every other administration thereafter (e.g., the 4^th, 6^th, 8^th and 10^th administration). Thus, for example, about IxlO⁶ Tregs per kg of body weight of the subject may be administered per month for the first and second month, and about 2xl0⁶ Tregs per kg of body weight of the subject may be administered per month for the third and fourth month, and/or about 3xl0⁶ cells per kg of body weight of the subject are administered per month for the fifth and sixth month.

[00266] In some embodiments, a method of treatment provide herein comprises administering a population of autologous Tregs or a composition comprising a population of autologous Tregs to the subject. In other embodiments, a method of treating a neurodegenerative disorder in a subject comprises administering a population of allogeneic Tregs or a composition comprising a population of allogeneic Tregs to the subject.

[00267] In some embodiments, the subject is additionally administered IL-2. The dose of IL- 2 may be about 0.5-lxl0⁵IU/m², about l-1.5xlO⁵IU/m², about 1.5-2xlO⁵IU/m², about 2-2.5xl0⁵ IU/m², about 2.5-3xlO⁵IU/m², about 3 -3.5x10⁵IU/m², about 3.5-4xlO⁵IU/m², about 4-4.5xl0⁵ IU/m², about 4.5-5xlO⁵ IU/m², about 5-6xlO⁵IU/m², about 6-7xlO⁵IU/m², about 7-8xlO⁵ IU/m², about 8-9xlO⁵IU/m², about 9-10xl0⁵IU/m², about 10-15xl0⁵IU/m², about 15-20xl0⁵ IU/m², about 20-25x10⁵ IU/m², about 25-30xl0⁵ IU/m², about 30-35xl0⁵ IU/m², about 35 -40x10⁵ IU/m², about 40-45x10⁵ IU/m², about 45-50xl0⁵ IU/m², about 50-60xl0⁵ IU/m², about 60-70xl0⁵ IU/m², about 70-80xl0⁵ IU/m², about 80-90xl0⁵IU/m², or about 90-100xl0⁵ IU/m². In specific embodiments, the subject is administered 2xlO⁵IU/m² of IL-2.

[00268] The IL-2 may be administered one, two or more times a month. In some embodiments, the IL-2 is administered three times a month. In some embodiments, the IL-2 is administered subcutaneously. The IL-2 may be administered at least 2 weeks, at least 3 weeks, or at least 4 weeks prior to the population of anti-inflammatory EVs.

[00269] In some embodiments, the subjected treated in accordance with the methods described herein receives one or more additional therapies are for the treatment of Alzheimer’s. Addition therapies for the treatment of Alzheimer’s may include acetylcholinesterase inhibitors (e.g., donepezil (Aricept®), galantamine (Razadyne®), or rivastigmine (Exelon®)) or NMDA receptor antagonists (e.g., Memantine (Akatinol®, Axura®, Ebixa®/Abixa®, Memox® and Namenda®). Additional therapies may also include anti-inflammatory agents (e.g., nonsteroidal anti-inflammatory drugs (NS AID) such as ibuprofen, indomethacin, and sulindac sulfide)), neuronal death associated protein kinase (DAPK) inhibitors such as derivatives of 3-amino pyridazine, Cyclooxygenases (COX-1 and -2) inhibitors, or antioxidants such as vitamins C and E.

[00270] In some embodiments, a subject treated in accordance with the methods described herein receives on or more additional therapies for the treatment of ALS. Additional therapies for the treatment of ALS may include Riluzole (Rilutek®) or Riluzole (Rilutek®). 5.4.2. Methods of Determining Treatment Effect

[00271] The effect of a method of treatment provided herein may be assessed by monitoring clinical signs and symptoms of the disease to be treated.

[00272] The efficacy of a method of treatment described herein may be assessed at about 20 weeks, about 24 weeks, about 28 weeks, about 32 weeks, about 36 weeks, about 40 weeks, about 44 weeks, about 48 weeks, about 52 weeks, about 56 weeks, about 60 weeks, about 64 weeks, about 68 weeks, about 72 weeks, about 76 weeks, about 80 weeks, about 84 weeks, about 88 weeks, about 92 weeks, about 96 weeks, about 100 weeks, at about 2-3 months, 3-4 months, 4-5 months, 5-6 months, 6-7 months, 7-8 months, 8-9 months, about 9-10 months, about 10-11 months, about 11-12 months, about 12-18 months, about 18-24 months, about 1-2 years, about 2- 3 years, about 3-4 years, about 4-5 years, about 5-6 years, about 6-7 years, about 7-8 years, about 8-9 years, or about 9-10 years after initiation of treatment in accordance with the method described herein.

[00273] In some embodiments, method of treatment provided herein results in a change in the Appel ALS score compared to baseline. In the context of an assessment of the effect of a method of treatment, the term “baseline” refers to a measurement pre-treatment. The Appel ALS score measures overall progression of disability or altered function. In some embodiments, the Appel ALS score decreases in a subject treated in accordance with a method provided herein compared to baseline, indicating an improvement of symptoms. In other embodiments, the Appel ALS score remains unchanged ins a subject treated in accordance with a method provided herein compared to baseline.

[00274] In some embodiments, a method of treatment provided herein results in a change in the Amyotrophic Lateral Sclerosis Functional Rating Scale-revised (ALSFRS-R) score compared to baseline. The ALSFRS-R score assesses the progression of disability or altered function. In some embodiments, the ALSFRS-R score increases in a subject treated in accordance with a method provided herein compared to baseline, indicating an improvement of symptoms. In other embodiments, the Appel ALSFRS-R score remains unchanged in a subject treated in accordance with a method provided herein compared to baseline.

[00275] In some embodiments, a method of treatment provided herein results in a change in forced vital capacity (FVC; strength of muscles used with expiration) compared to baseline, where the highest number is the strongest measurement. In some embodiments, FVC increases in a subject treated in accordance with a method provided herein compared to baseline. In other embodiments, FVC remains unchanged in a subject treated in accordance with a method provided herein compared to baseline.

[00276] In some embodiments, a method of treatment provided herein results in a change in Maximum Inspiratory Pressure (MIP; strength of muscles used with inspiration) compared where the highest number is the strongest measurement. In some embodiments, MIP increases in a subject treated in accordance with a method provided herein compared to baseline. In other embodiments, MIP remains unchanged in a subject treated in accordance with a method provided herein compared to baseline.

[00277] In some embodiments, a method of treatment provided herein results in a change in Neuropsychiatric Inventory Questionnaire (NPI-Q) compared to baseline. The NPI-Q provides symptom Severity and Distress ratings for each symptom reported, and total Severity and Distress scores reflecting the sum of individual domain scores. In some embodiments, the NPI- Q score decreases in a subject treated in accordance with a method provided herein compared to baseline. In other embodiments, NPI-Q score remains unchanged in a subject treated in accordance with a method provided herein compared to baseline.

[00278] In some embodiments, a method of treatment provided herein results in a decrease in the frequency of GI symptoms, anaphylaxis or seizures compared to baseline.

[00279] In some embodiments, a method of treatment provided herein results in a change in a change in CSF amyloid and/or CSF tau protein (CSF-tau) compared to baseline. In some embodiments, the levels of CSF amyloid and/or CSF tau protein decreases in a subject treated in accordance with a method provided herein compared to baseline. In other embodiments, the levels of CSF amyloid and/or CSF tau protein remains unchanged in a subject treated in accordance with a method provided herein compared to baseline.

[00280] In some embodiments, a method of treatment provided herein results in a change in Clinical Dementia Rating (CDR) compared to baseline. The CDR rates memory, orientation, judgment and problem-solving, community affairs, home and hobbies, and personal care, and a global rating is then generated, ranging from 0-no impairment to 3-severe impairment. In some embodiments, the CDR decreases in a subject treated in accordance with the methods provided herein compared to baseline. In other embodiments, the CDR remains unchanged in a subject treated in accordance with a method provided herein compared to baseline. [00281] In some embodiments, a method of treatment provided herein results in a change in Alzheimer's Disease Assessment Scale (ADAS)-cogl3 score compared to baseline. ADAS-cog tests cognitive performance and has an upper limit is 85 (poor performance) and lower limit is zero (best performance). In some embodiments, the ADAS-cogl3 score decreases in a subject treated in accordance with a method provided herein compared to baseline. In other embodiments, the ADAS-cog 13 score remains unchanged in a subject treated in accordance with a method provided herein.

[00282] In some embodiments, wherein the method of treatment comprises administration of a population of anti-inflammatory EVs as well as administration of a population of Tregs , for example, a population of Tregs that had previously been cryopreserved, e.g., a population of Tregs that had been ex -vivo expanded prior to cry opreservation and were not further expanded prior to administration, the method results in an increase in the Treg suppressive function in the blood from baseline. In some embodiments, a method of treatment provided herein results in an increase in the Treg suppressive function in the blood from baseline to week 4, week 8, week 16, week 24, week 30 or week 36. In some embodiments, a method of treatment provided herein results in an increase in the Treg suppressive function in the blood from baseline to week 24. In some embodiments, a method of treatment provided herein results in an increase in the Treg numbers in the blood from baseline. In some embodiments, a method of treatment provided herein results in an increase in the Treg numbers in the blood from baseline to week 4, week 8, week 16, week 24, week 30 or week 36. In some embodiments, a method of treatment provided herein results in an increase in the Treg numbers in the blood from baseline to week 24.

5.5 Kits

[00283] Provided herein are kits comprising a therapeutic composition of a population of antiinflammatory EVs provided herein or a composition comprising a population of antiinflammatory EVs provided herein.

[00284] In some embodiments, a kit provided herein comprises instructions for use, additional reagents (e.g., sterilized water or saline solutions for dilution of the compositions), or components, such as tubes, containers or syringes for collection of biological samples, processing of biological samples, and/or reagents for quantitating the amount of one or more surface markers in a sample (e.g., detection reagents, such as antibodies). [00285] In some embodiments, the kits contain one or more containers containing a population of anti-inflammatory EVs provided herein or a composition comprising a population of anti-inflammatory EVs provided herein. The one or more containers holding the composition may be a single-use vial or a multi-use vial. In some embodiments, the article of manufacture or kit may further comprise a second container comprising a suitable diluent. In some embodiments, the kit contains instruction for use (e.g., dilution and/or administration) of a population of anti-inflammatory EVs provided herein.

6. EXAMPLES .1 Example 1: Extracellular vesicles derived from suppressive immune cells modulate in vitro and in vivo inflammation

6.1.1. Methods

6.1.1.1 M2 macrophage production.

6.1.1.2 iPSC-derived Ml and M2 myeloid cultures

[00286] Myeloid cells were generated using protocols previously developed/described (Thome et al., 2018, Molecular Neurodegeneration 13.1 : 1-11; Zhao et al., 2020, Iscience 23.6: 101192). An schematic showing the protocol for the differentiation of human M2 macrophages from iPSCs is shown in FIG. 1

[00287] Briefly, myeloid cells were produced using a 4-step culturing process that allows for the generation of CD14+ cells from control iPSC lines. CD14+ myeloid cells are isolated using positive, magnetic selection with Miltenyi Biotec CD14 beads, isolation columns, and magnet setup.

[00288] For Ml cells: CD14+ cells are cultured in complete RPMI media (10% fetal bovine serum, 25mM HEPES, ImM sodium pyruvate, lx nonessential amino acids, 55pM 2- mercaptoethanol, lOO units/ml penicillin, and lOOpg/ml streptomycin) supplemented with 50 ng/ml GMCSF (R&D systems) for 7 days to create M0 cells for Ml use. M0 cells are then primed with O. lng/ml lipopolysaccharides (LPS) (Sigma) and 0.2 ng/ml IFNy (Invitrogen) to polarize myeloid cells to be pro-inflammatory, Ml cells. [00289] For M2 polarization, CD14+ cells are cultured in complete RPMI media but supplemented with lOOng/ml M-CSF (R&D systems) for 7 days and then stimulated with IL-4 (40ng/ml, R&D systems), IL- 10 (40ng/ml, R&D systems), and TGF-P (40ng/ml, R&D systems) for the final 48 hours before M2 cell EV isolation. RPMI media fetal bovine serum (FBS) in the final 48 hours was replaced with exosome-depleted FBS to enrich the culture media with M2- derived EVs.

6, 1,1.3 Extracellular vesicle isolations

[00290] Extracellular vesicles (EVs) were isolated according to manufacturer’s protocols using a polyethylene glycol precipitation (PEG) method and ExoQuick-TC reagent (System Biosciences, SBI). Briefly, media from iPSC-M2 cultures were centrifuged at 3000 x g for 15 minutes to remove cells and debris. PEG reagent was added to spun supernatant at 1 :5 ratio of PEG: TC Media, mixed thoroughly, and refrigerated overnight at 4°C. The mixture was then centrifuged at 1500 x g for 30 minutes, the supernatant aspirated, spun again at 1500 x g for 10 minutes, and the supernatant aspirated again. The resulting EV pellet was resuspended in sterile PBS and diluted for Nanosight EV size/concentration analysis and for future use. EVs were stored at -20°C while limiting freeze/thaw cycles.

6.1.1.4 Nanosight EV size/concentration readings

[00291] EV readings were obtained using Nanosight NS300 (Malvern Panalytical) particle analyzer. EV samples were diluted for readings and analyzed using continual measurement at 50 pl per minute speed with 3 analysis recordings of 60 seconds each with the following parameters: camera level (levels 12-15), temperature (22°C), and detection threshold (level 5). Concentration was recorded as particles/ml and size statistics were recorded as mean and mode.

6.1.1.5 EV suppression assays with myeloid cells and Tresp proliferation assays

[00292] M0 (GM-CSF) cells were lifted with enzyme-free dissociation buffer, pelleted, and replated at 50,000 cells/well in 24 well plates. [00293] Ml cells were primed with O. lng/ml LPS (Sigma) and 0.2 ng/ml IFNy (Invitrogen) for 1 hour to polarize to Ml cells. M2 EVs (10pg) were spiked into the cultures following Ml polarization for overnight time point followed by collection of Ml cells for RNA analysis and cultured media for protein analysis.

[00294] For responder T cell (Tresp) proliferation assays, control Tresp were isolated using Miltenyi Biotec reagents and protocols to isolated CD4+CD25- T cells from peripheral blood. Tresps were plated at 50,000 cells per well in 96 well, round-bottom plates and stimulated with CD3/28 beads (Miltenyi Biotec).

[00295] M2 EVs were added to the cultures in escalating doses and remained in Tresp culture for the entire experiment. After 4 days in culture, Tresps were pulsed with tritium and proliferation was determined by examining tritium incorporation 18 hours after tritium pulsing.

6, 1,1, 6 RNA purification, RT-PCR analysis, and protein ELISAs

[00296] RNA was isolated from cells and tissues using Trizol reagent followed by Direct-zol RNA MiniPrep Plus Kit (Zymo Research) according to manufacturer’s recommendations. Quantitative RT-PCR was performed using one-step RT-PCR kit with SYBR Green (Bio-Rad) and an iQ5 Multicolor Real-Time PCR detection system (Bio-Rad). Primers for RT-PCR (IL-6, IL-ip, TNFa, IL-10, and Arg-1) were acquired from Bio-Rad and RT-PCRs were run according to the manufacturer’s protocols. The relative expression level of each mRNA was assessed using the AACt method and normalized to P-actin/controls. Supernatants were collected from coculture paradigms and IL-6 protein amounts were assessed using ELISA-based immunoassays (Invitrogen).

6.1.2. Results

6, 1,2, 1 EVs isolated from iPSC-derived M2 myeloid cells are immunosuppressive in vitro and in vivo.

[00297] The ability of EVs from M2 cultures (M2 EVs) to suppress pro-inflammatory myeloid cells and Tresp proliferation in culture was investigated. Following co-culture of M2 EVs with primed, pro-inflammatory myeloid cells, a 62% inhibition in LPS-induced IL-6 transcript production and an 86% inhibition in LPS-induced IL-6 protein was observed following overnight (18hr) treatment (Figs. 2A and 2B). Additionally, levels of anti-inflammatory IL- 10 and Argl transcripts increased in Ml cells treated with M2 EVs after overnight treatment (Figs. 2C and 2D). Moreover, a modest, dose-dependent increase in M2 EV suppression of Tresp proliferation was observed when M2 EVs were co-cultured with stimulated Tresp (Fig. 2E). [00298] The anti-inflammatory effects of intranasal M2 EVs was evaluated in an acute, LPS- induced neuroinflammation mouse model. Within 14 hours after LPS injection and intranasal delivery of M2 EVs, the increase in the amount of IL-6 and IL-ip transcript observed in mouse hippocampus and cortex with LPS alone was ameliorated using M2 EVs (Fig. 2F).

[00299] M2 EV-mediated suppression of LPS-induced IL-6 RNA production was also found in the hippocampus and cortex of injected mice within 24 hours, along with an even more robust activation of IL-ip transcripts at this time point (with LPS alone) that was suppressed in cortex tissues and in the hippocampus using M2 EVs (Fig. 2G).

[00300] The peripheral effects of M2 EVs in suppressing LPS-induced activation of myeloid cells was assessed by isolating CD1 Ib-positive myeloid cells from LPS-injected mouse spleens following M2 EV intranasal delivery. Although there was no robust inhibition of LPS-induced production of IL-6 and IL-ip in CD1 lb+ myeloid cell following M2 EV treatment, a significant inhibition in LPS-induced TNFa RNA production from these same myeloid cells was observed (Fig. 2H).

[00301] These data indicate that M2 EVs have anti-inflammatory properties that can suppress Ml cells and Tresp proliferation, while also potentially repolarizing Ml cells to become M2.

6.2 Evaluation of Anti-Inflammatory EVs in a SOD1 mouse model of ALS

[00302] To evaluate the effects of anti-inflammatory EVs in vivo, an LPS-induced mouse model of ALS is used. Transgenic mice harboring the SOD1-G93A mutation (see Gurney et al., 1994, Science 264.5166: 1772-1775) are used as a motor neuron degeneration model for ALS. For the acute LPS-induced neuroinflammation mouse model, C57B16 WT mice are injected intraperitoneally (IP) with 2mg/kg LPS (Sigma; Ol l i :B4) followed by either intranasal administration of M2 EV (30pg) 2 hours after LPS injection. Mice are then sacrificed at 12 hours or 22 hours post-intranasal administration and organs harvested for RNA and protein analyses, specifically brain components (hippocampus and cortex) and spleen. Myeloid cells are isolated from spleen by extracting single cells through a 40pm cell strainer and using mouse CD1 lb beads and magnetic columns (Miltenyi Biotec).

[00303] Phenotype analysis of SOD1 mice begins at day 70 and intranasal injections of EVs (IxlO⁹ particles) begins at day 90 and continued every two weeks until the mice reach their ethical endpoint, requiring them to be sacrificed.

[00304] Mouse phenotype is assessed using a modified “BASH scoring system” whereby SOD1 mice gain a degenerative point from 0-6 as phenotype worsens with disease progression. The phenotypes are assessed and points added as follows (but not necessarily in this order): +1 Tremulousness, +1 Gait abnormalities, +1 Hind limb weakness/paresis, +1 Weight loss of more than 10% adult weight, +1 Spasticity to one or both hind limbs, +1 Paralysis, which is terminal stage resulting in sacrificing the mouse and harvesting organs for RNA and protein analysis.

6.3 Isolation of Extracellular Vesicles Using TFF

[00305] EV populations isolated using tangential flow filtration (TFF) techniques are isolated using the protocol summarized herein.

[00306] Briefly, the isolation utilizes a Repligen KR2i TFF system that allows for isolation, concentration, and diafiltration of EV populations using a buffer appropriate for therapeutic use.

[00307] First, media from the culture of human suppressive immune cells (e.g., human M2 macrophages) is circulated using TFF and a Midi 20 cm 0.65pm Spectrum mPES 0.75 mm Hollow Fiber filter (D02-E65U-07-N) with a membrane area of 85 cm² and fiber diameter of 0.75 mm to filter out cells and debris. This process utilizes a flow rate of 100-200 mL/min that results in a shear rate of about 2,000-5,000 s'¹ while maintaining a variable transmembrane pressure (TMP) driven by a retentate pressure of 5 psi.

[00308] The permeate of this step is then subjected to a process designed to concentrate and diafiltrate the EV population into the retentate with continuous circulation. This process utilizes the TFF system and a Midi 20 cm 500kD Spectrum mPES 0.5 mm Hollow Fiber filter (D02- E500-05-N) with a membrane area of 115 cm² and fiber diameter of 0.5 mm to retain/concentrate all particles greater than approximately 60-80 nm into the retentate. This process utilizes a flow rate of 80-200 mL/min that results in a shear rate of 2,000-7,500 s'¹ while maintaining and driving the filtration at 10 psi TMP. The final volume after concentration is targeted to be around 20 mLs.

[00309] Sterile PBS is incorporated into the circulation, resulting in 10X diafiltration and replacement of the existing solution. 10X diafiltration effectively results in a full buffer volume exchange of 10 times and is performed to eliminate 99%+ of the soluble material or impurities that remains.

[00310] All publications, patents and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.

[00311] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

[00312] The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to fall within the scope of the appended claims.

Claims

What is claimed is:

2. The population of anti-inflammatory EVs of claim 1, wherein the human suppressive immune cells are M2 macrophages.

3. The population of anti-inflammatory EVs of claim 2, wherein the M2 macrophages are produced from induced pluripotent stems cells (iPSCs).

4. The population of anti-inflammatory EVs of any one of claims 1-3, wherein the precursors for the M2 macrophages are derived from multiple unrelated human subjects.

5. The method of claim 4, wherein the precursors for the M2 macrophages are iPSC.

6. The method of claim 4 or 5, wherein the precursors for the M2 macrophages are derived from PBMCs.

7. The population of anti-inflammatory EVs of any one of claims 1-6, wherein the antiinflammatory EVs exhibit an ability to increase the expression of one or more antiinflammatory markers in inflammatory cells.

8. The population of anti-inflammatory EVs of claim 7, wherein the inflammatory cells are myeloid cells.

9. The population of anti-inflammatory EVs of claim 7 or 8, wherein the anti-inflammatory EVs exhibit an ability to increase the expression of IL-10, Argl and/or CD206 in inflammatory cells.

10. The population of anti-inflammatory EVs of any one of claims 1-9, wherein the antiinflammatory EVs exhibits an ability to suppress inflammatory cells, as measured by pro- inflammatory cytokine production by the inflammatory cells. The method of claim 10, wherein the inflammatory cells are myeloid cells. The population of anti-inflammatory EVs of claim 11, wherein the myeloid cells are monocytes, macrophages, or microglia. The population of anti-inflammatory EVs of claim 12, wherein macrophages are Ml macrophages. The population of anti-inflammatory EVs of claim 13, wherein the Ml macrophages are induced pluripotent stem cell (iPSC)-derived Ml macrophages. The population of anti-inflammatory EVs of any one of claims 7-14, wherein the ability to suppress inflammatory cells is measured by IL-6, IL-8, TNFa, ILip and/or Interferon-y production by the inflammatory cells. The population of anti-inflammatory EVs of any one of claims 1-14 wherein the antiinflammatory EVs exhibit a suppressive function, as determined by suppression of proliferation of responder T cells. The population of anti-inflammatory EVs of claim 16, wherein the proliferation of responder T cells is determined by flow cytometry or thymidine incorporation. The population of anti-inflammatory EVs of any one of claims 1-17, wherein the population is a saline-containing population of anti-inflammatory EVs. The population of anti-inflammatory EVs of any one of claims 1-17, wherein the population is a physiological saline-containing population of anti-inflammatory EVs. The population of anti-inflammatory EVs of any one of claims 1-17, wherein the population is a phosphate-buffered saline-containing population of anti-inflammatory EVs. The population of anti-inflammatory EVs of any one of any one of claims 1-20, wherein the population of anti-inflammatory EVs comprises exosomes and microvesicles. The population of anti-inflammatory EVs of claim 21, wherein the majority of the EVs are exosomes. The population of anti-inflammatory EVs of claim 22, wherein at least about 80%, about 90%, or about 95% of the EVs are exosomes. The population of anti-inflammatory EVs of claim 21 wherein the majority of the EVs are microvesicles. The population of anti-inflammatory EVs of claim 24, wherein at least about 80%, about 90%, or about 95% of the EVs are microvesicles. The population of anti-inflammatory EVs of claim 21, wherein the majority of the EVs have diameters from about 30 nm to about 1000 nm. The population of anti-inflammatory EVs of claim 21, wherein the majority of the EVs have diameters from about 30 nm to about 100 nm, about 30 nm to about 150 nm, about 30 to about 200 nm, about 40 to about 100 nm, about 80 to about 100 nm, about 80 to about 110 nm, about 80 to about 125 nm, or about 100 to about 120 nm. The population of anti-inflammatory EVs of claim 21 wherein the majority of the EVs have diameters from about 60 nm to 1000 nm, about 70 nm to about 1000 nm, about 80 nm to about 1000 nm, 100 to about 1000 nm, about 200 to about 1000 nm, about 300 to about 1000 nm. A pharmaceutical composition comprising an isolated, cell-free population of antiinflammatory EVs of any one of claims 1-28. The pharmaceutical composition of claim 29, wherein the population of antiinflammatory EVs comprises about IxlO⁶ to about IxlO¹⁴ EVs, about IxlO⁸ to about IxlO¹⁴ EVs, about IxlO⁸ to about IxlO¹² EVs, about IxlO⁸ to about IxlO¹⁰ EVs, about IxlO¹⁰ to about IxlO¹⁴ EVs, or about IxlO¹⁰ to about IxlO¹² EVs. The pharmaceutical composition of claim 29, wherein the population of antiinflammatory EVs comprises about IxlO⁶ to about IxlO¹⁴ EVs/ml, about IxlO⁸ to about IxlO¹⁴ EVs/ml, about IxlO⁸ to about IxlO¹² EVs/rnl, about IxlO⁸ to about IxlO¹⁰ EVs/rnl, about IxlO¹⁰ to about IxlO¹⁴ EVs/ml, or about IxlO¹⁰ to about IxlO¹² EVs/ml. The pharmaceutical composition of claim 29, wherein the population of antiinflammatory EVs comprises about 1 pg to about 200 mg EVs. The pharmaceutical composition of claim 29, wherein the population of antiinflammatory EVs comprises about 1 pg to about 15 mg EVs. The pharmaceutical composition of claim 29, wherein the population of antiinflammatory EVs comprises about 1 pg to about 15 mg EV/ml. The pharmaceutical composition of any one of claims 29-34, wherein the pharmaceutical composition is a cryopreserved pharmaceutical composition. The pharmaceutical composition of any one of claims 29-34, wherein the pharmaceutical composition had previously been cryopreserved. A cryopreserved composition comprising an isolated, cell-free population of antiinflammatory EVs of any one of claims 1-36. A method of producing an isolated, cell-free population of anti-inflammatory extracellular vesicles (EVs), said method comprising the steps of: a. ex-vivo culturing a human suppressive immune cell population in culture media to produce a culture comprising the cells, the culture media and anti-inflammatory EVs; and b. isolating the anti-inflammatory EVs from the culture. The method of claim 38, wherein the human suppressive immune cell population is a population of M2 macrophages. The method of claim 38 or 39 wherein step b) comprises removing cells from the culture, followed by polyethylene glycol precipitation of the culture. The method of claim 38 or 39, wherein step b) comprises: i) removing the cells from the culture to produce a cell-free, anti-inflammatory EV- containing solution; and ii) isolating the anti-inflammatory EVs from the cell-free, anti-inflammatory EV- containing solution of i). The method of claim 41, wherein step i) comprises passing the culture through a filter such that the cells are retained by the filter, and thereby removed from the culture. The method of claim 41 or 42, wherein step i) comprises microfiltration. The method of any one of claims 41-43, wherein step ii) comprises step ii-a): passing the cell-free, anti-inflammatory EV-containing solution through a filter such that the antiinflammatory EVs are retained by the filter. The method of claim 44, wherein the filter has a molecular weight cut-off (MWCO) of about 200 kilodaltons (kDa) to about 600 kDa. The method of claim 45, wherein the filter has an MWCO of about 500 kDa. The method of any one of claims 41-46, wherein step ii) comprises ultrafiltration. The method of any one of claims 44-47, wherein step ii) further comprises step ii-b: performing buffer exchange such that the isolated, cell-free population of antiinflammatory EVs produced is a buffer-containing isolated, cell-free population of antiinflammatory EVs. The method of claim 48, wherein the buffer is a saline-containing buffer. The method of claim 49, wherein the saline-containing buffer is physiological saline. The method of claim 50, wherein the saline-containing buffer is PBS. The method of any one of claims 49-51, wherein step ii-b) comprises diafiltration. The method of any one of claim 49-52 wherein steps ii-a) and ii-b) are performed simultaneously. The method of any one of claims 38-53, wherein step b) comprises tangential flow filtration. The method of any one of claims 38-54, wherein the culture media in step a) is serum- free. The method of any one of claims 38-55, wherein the culture media in step a) comprises serum. The method of claim 56, wherein the serum is human AB serum. The method of claim 56 or 57, wherein the serum is depleted for serum-derived EVs. The method of any one of claims 41-58 further comprising, prior to step a), the step of culturing iPSC cells in the presence of cytokines to induce polarization to M2 macrophages. The method of claim 38, wherein the human suppressive immune cell population is a genetically engineered human suppressive immune cell population. A pharmaceutical composition comprising an isolated, cell-free population of antiinflammatory EVs, wherein the population is made by any one of the methods of claim 38-60. The method of any one of claims 38-60, further comprising: c) cry opreserving the isolated, cell-free population of anti-inflammatory EVs, thereby producing a cryopreserved, isolated, cell-free population of anti-inflammatory EVs. The method of claim 62, further comprises thawing the cryopreserved, isolated cell-free population of anti-inflammatory EVs after cryopreservation for about 1 week, 1 month, about 3 months, about 6 months, about 9 months, about 12 months, about 18 months or about 24 months. A pharmaceutical composition comprising the isolated, cell-free population of antiinflammatory EVs of claim 63. A method of treating a disorder associated with Treg dysfunction, the method comprising administering to a subject in need of said treatment the pharmaceutical composition of any one of claims 29-36, 61, or 64. A method of treating a disorder associated with Treg deficiency, the method comprising administering to a subject in need of said treatment the pharmaceutical composition of any one of claims 29-36, 61, or 64. A method of treating a disorder associated with over activation of the immune system, the method comprising administering to a subject in need of said treatment the pharmaceutical composition of any one of claims 29-36, 61, or 64. A method of treating an inflammatory condition driven by a T cell response, the method comprising administering to a subject in need of said treatment the pharmaceutical composition of any one of claims 29-36, 61, or 64. A method of treating an inflammatory condition driven by a myeloid cell response, the method comprising administering to a subject in need of said treatment the pharmaceutical composition of any one of claims 29-36, 61, or 64. The method of claim 69, wherein the myeloid cell is a monocyte, macrophage or microglia. A method of treating a neurodegenerative disorder in a subject in need thereof, the method comprising administering to a subject in need of said treatment the pharmaceutical composition of any one of claims 29-36, 61, or 64. The method of claim 71, wherein the neurodegenerative disease is ALS, Alzheimer’s disease, Parkinson’s disease, frontotemporal dementia or Huntington’s disease. A method of treating an autoimmune disorder in a subject in need thereof, the method comprising administering to a subject in need of said treatment the pharmaceutical composition of any one of claims 29-36, 61, or 64. The method of claim 73, wherein the autoimmune disorder is polymyositis, ulcerative colitis, inflammatory bowel disease, Crohn’s disease, celiac disease, systemic sclerosis (scleroderma), multiple sclerosis (MS), rheumatoid arthritis (RA), Type I diabetes, psoriasis, dermatomy osititis, systemic lupus erythematosus, cutaneous lupus, myasthenia gravis, autoimmune nephropathy, autoimmune hemolytic anemia, autoimmune cytopenia, autoimmune encephalitis, autoimmune hepatitis, autoimmune uveitis, alopecia, thyroiditis or pemphigus. A method of treating graft-versus-host disease in a subject in need thereof, the method comprising administering to a subject in need of said treatment the pharmaceutical composition of any one of claims 29-36, 61, or 64. The method of claim 75, wherein the subject has received a bone marrow transplant, kidney transplant or liver transplant. A method of improving islet graft survival in a subject in need thereof, the method comprising administering to a subject in need of said treatment the pharmaceutical composition of any one of claims 29-36, 61, or 64. A method of treating cardio-inflammation in a subject in need thereof, the method comprising administering to a subject in need of said treatment the pharmaceutical composition of any one of claims 29-36, 61, or 64. The method of claim 78, wherein the cardio-inflammation is associated with atherosclerosis, myocardial infarction, ischemic cardiomyopathy or heart failure. A method of treating neuroinflammation in a subject in need thereof, the method comprising administering to a subject in need of said treatment the pharmaceutical composition of any one of claims 29-36, 61, or 64. The method of claim 80, wherein the neuroinflammation is associated with stroke, acute disseminated encephalomyelitis, acute optic neuritis, acute inflammatory demyelinating polyradiculoneuropathy, chronic inflammatory demyelinating polyradiculoneuropathy, Guillain-Barre syndrome, transverse myelitis, neuromyelitis optica, epilepsy, traumatic brain injury, spinal cord injury, encephalitis, central nervous system vasculitis, neurosarcoidosis, autoimmune or post-infectious encephalitis or chronic meningitis. A method of treating a Tregopathy in a subject in need thereof, comprising administering to a subject in need of said treatment the pharmaceutical composition of any one of claims 29-36, 61, or 64. The method of claim 82, wherein the Tregopathy is caused by a F0XP3, CD25, cytotoxic T lymphocyte-associated antigen 4 (CTLA4), LPS-responsive and beige-like anchor protein (LRBA), or BTB domain and CNC homolog 2 (BACH2) gene loss-of-function mutation, or a signal transducer and activator of transcription 3 (STAT3) gain-of-function mutation. The method of any one of claims 65-83, wherein the anti-inflammatory EVs are derived from M2 macrophages that are autologous to the subject. The method of any one of claims 65-83 wherein the anti-inflammatory EVs are derived from M2 macrophages that are allogeneic to the subject. The method of any one of claim 65-85, wherein the pharmaceutical composition is administered via intranasal administration. The method of claim 86, wherein the intranasal administration is via aerosol inhalation or nasal drip. The method of any one of claim 65-85, wherein the pharmaceutical composition is administered intravenously. The method of any one of claim 65-85, wherein the pharmaceutical composition is administered by local injection. The method of any one of claims 65-85, wherein the method further comprises administering to the subject a pharmaceutical composition comprising a therapeutic population of Tregs, wherein the Tregs had been ex vivo expanded and cryopreserved, and wherein the Tregs are not further expanded prior to the administering. The method of claim 90, wherein the therapeutic population of Tregs is autologous to the subject. The method of claim 91, wherein the therapeutic population of Tregs is allogeneic to the subject. The method of any one of claims 90-92, wherein the pharmaceutical composition comprising the therapeutic population of Tregs is administered intravenously. The method of any one of claims 90-93, wherein the pharmaceutical composition comprising the anti-inflammatory EVs and the pharmaceutical composition comprising the therapeutic population of Tregs are administered to the patient on the same day. The method of any one of claims 65-94, wherein the isolated, cell-free population of antiinflammatory EVs had been cryopreserved and thawed prior to being administered to the subject. The method of any one of claims 65-94, wherein the isolated, cell-free population of antiinflammatory EVs are stored overnight at 4 °C prior to being administered to the subject. The method of claim 96, wherein the isolated, cell-free population of anti-inflammatory EVs had been cryopreserved then thawed and stored at 4 °C overnight prior to being administered to the subject. The method of any one of claims 65-94, wherein the isolated, cell-free population of antiinflammatory EVs had undergone at least two freeze/thaw cycles prior to being administered to the subject. The method of claim 98, wherein the isolated, cell-free population of anti-inflammatory EVs had undergone about 2 to about 20 freeze/thaw cycles prior to being administered to the subject.