TARGETED EXTRACELLULAR VESICLES AND METHODS OF USE THEREOF

CROSS-REFERENCE TO RELATED APPLICATION

[001] This application claims priority to United States Provisional Patent Application 63/281,226, filed November 19, 2021, which is incorporated by reference herein in its entirety.

FIELD OF INTEREST

[002] This invention relates to targeted extracellular vesicles (e.g., exosomes, ectosomes, micro vesicles/microparticles, apoptotic bodies, macro vesicles) and targeted micelles, as well as methods of using a targeted extracellular vesicle or a targeted micelle in a subject, the method comprising: tiised protein comprising a targeting moiety and a protein designed to treat an illness such as cancer and tumor (i.e., a therapeutic payload). The fused sequences may be tagged (e.g., by a selective marker) to allow differentiation of the treating extracellular vesicles. It also relates to a targeted extracellular vesicle comprising a targeting moiety and a therapeutic payload, as well as methods of making and using the targeted extracellular vesicle for treatment, e.g., of a tumor or a cancer, including in vivo. In some aspects, provided herein are methods for drug preparations to target tumors using extracellular vesicles or targeted micelles. In some aspects, provided herein are designs for extracellular vesicles or targeted micelles comprising drug compositions and methods of use thereof. In some aspects, an extracellular vesicle or a micelle is designed to regulate an innate and/or adaptive immune response against a tumor and/or metastasis development. In some embodiments, an extracellular vesicle or a micelle targets a malignant melanoma, a breast adenocarcinoma, a lung adenocarcinoma, or a pancreatic tumor.

BACKGROUND

[003] Cancers such as malignant melanoma, breast adenocarcinoma, lung adenocarcinoma, colon cancer, and pancreatic cancer affect many people, often resulting in deaths. Cancer impacts the United States and countries around the world. Cancer is among the leading causes of death worldwide. In 2018, there were 18.1 million diagnosed new cases and 9.5 million cancer-related death worldwide. In 2021, an estimated 1,806,590 new cancer cases will be diagnosed in the United States, and 606,520 people will die from the disease. Certain cancer diseases are caused by defined genetic abnormalities and resultant protein deficiencies. [004] Definitive cancer treatment includes chemotherapy, radiation, and surgery and their combination. The basic concept behind these treatments is cancer cells distraction and removal. The main disadvantages of these treatments are not specific and are associated with collateral toxicity and the death of normal cells. Cancer immunotherapy enhanced the specificity by targeting tumor biologic characteristics rather than treating the tumor site. However, current biologic formulations and delivery have yet to target the tumor site.

[005] Further, the effective treatment using biologies is limited due to tumor type, and the immunogenicity of biologies is associated with significant toxicity side effects affecting the quality of life. Treatments require dose reductions and discontinuation to decrease these side effects and significantly improve the poorer outcome with updated therapeutic development. Moreover, current chemo- and biotherapeutic delivery methods are limited in targeting multiple tumor types and/or cell types. Therefore, there is a need to increase the utility of proven drugs by masking the toxicity of high dose delivery to the tumor sites.

SUMMARY

[006] In some aspects, disclosed herein is a method for designing a targeted therapy to treat a subject in need thereof, the method comprising: a fluorescent tagged drug and target fusion sequencing method to produce a fused sequence plasmid, said plasmid is expressed in specific cell line, said cell line release exosomes or extracellular vesicles. The exosomes expressing tagged fused sequence are separated as the drug treatment.

[007] In some aspects, disclosed herein is a method of targeting a therapy to a tumor cell, a cancer cell, or an immune cell or to a tumor microenvironment in a subject in need thereof, the method comprising: identifying a target on the tumor cell, the cancer cell, or the immune cell, or a target of a component of the tumor microenvironment; providing an extracellular vesicle (EV) or a micelle, the extracellular vesicle or micelle comprising an expression plasmid expressing a fusion protein comprising at least one targeting moiety, the at least one targeting moiety directed to the target, at least one therapeutic payload, and at least one selectable marker fusion maker identifier, the at least one targeting moiety fused, linked, or attached to the at least one therapeutic payload and to the selectable marker identifier; and administering the extracellular vesicle or the micelle to the subject. [008] In some aspects, disclosed herein is a targeted extracellular vesicle comprising an expression plasmid expressing a fusion protein comprising at least one targeting moiety, the at least one targeting moiety directed to a target in a tumor cell, a cancer cell, an immune cell, or a component of a tumor microenvironment; at least one therapeutic payload; and at least one selectable marker, the at least one targeting moiety fused, linked, or attached to the at least one therapeutic payload and to the selectable marker, wherein the extracellular vesicle comprises an exosome, a microvesicle, a macrovesicle or an apoptotic body.

[009] In other aspects, disclosed herein is a targeted micelle comprising an expression plasmid expressing a fusion protein comprising at least one targeting moiety, the at least one targeting moiety directed to a target in a tumor cell, a cancer cell, an immune cell, or a component of a tumor microenvironment; at least one therapeutic payload; and at least one selectable marker, the at least one targeting moiety fused, linked, or attached to the at least one therapeutic payload and to the selectable marker, wherein the micelle comprises an exterior surface and a hydrophobic interior, wherein the exterior surface comprises the at least one targeting moiety or wherein the at least one targeting moiety is at least partially within the hydrophobic interior, and wherein the therapeutic payload is at least partially within the hydrophobic interior.

[0010] In some aspects, also disclosed herein is a method of making a targeted extracellular vesicle, the method comprising: providing an expression plasmid, the expression plasmid expressing a fusion protein comprising (i) at least one targeting moiety, the at least one targeting moiety directed to a target on a tumor cell, a cancer cell, or an immune cell, or to a target of a component of a tumor microenvironment, (ii) at least one therapeutic payload, and (iii) at least one selectable marker, the at least one targeting moiety fused, linked, or attached to the at least one therapeutic payload and to the selectable marker; transfecting a cell with the expression plasmid; isolating an extracellular vesicle from the cell, the extracellular vesicle comprising an exosome, a microvesicle, or a macrovesicle.

BRIEF DESCRIPTION OF THE DRAWINGS

[0011] The subject matter regarded as the invention is particularly pointed out and distinctly claimed in the concluding portion of the specification. The invention, however, both as to organization and method of operation, together with objects, features, and advantages thereof, may best be understood by reference to the following detailed description when read with the accompanying drawings.

[0012] FIGURE 1 is a schematic depicting a map of an exemplary plasmid with the location of the modifications for the production of the plasmid shown.

[0013] It will be appreciated that for simplicity and clarity of illustration, elements shown in the figures have not necessarily been drawn to scale. For example, the dimensions of some of the elements may be exaggerated relative to other elements for clarity. Further, where considered appropriate, reference numerals may be repeated among the figures to indicate corresponding or analogous elements.

DETAILED DESCRIPTION

[0014] In the following detailed description, numerous specific details are set forth to provide a thorough understanding of the invention. However, it will be understood by those skilled in the art that the present invention may be practiced without these specific details. In other instances, well-known methods, procedures, and components have not been described in detail so as not to obscure the present invention.

[0015] It would be desirable to have compositions and methods for making and using an extracellular vesicle or a micelle, the method for making the extracellular vesicle or micelle comprising a cell and/or a therapeutic payload and/or a targeting moiety, e.g., fused as one sequence producing a combined drug, optionally including a selectable marker. It would also be desirable to have methods of treating, reducing, inhibiting, ameliorating, or alleviating a tumor or a cancer in a subject or a growth or a metastasis thereof, comprising a step of administering the extracellular vesicle or the micelle to the subject.

[0016] Disclosed herein are extracellular vesicles, as well as micelles, comprising a therapeutic payload and/or a targeting moiety, methods of making the extracellular vesicles and micelles, and methods of use thereof.

[0017] Disclosed herein is an extracellular vesicle (e.g., an exosome, a microparticle, a microvesicle, an apoptotic body, or a macrovesicle) comprising a fused therapeutic payload (e.g., a protein, a cytokine, a chemokine, an antibody or fragment thereof, a nucleic acid [e.g., DNA or RNA], or a pharmaceutical or biopharmaceutical payload) and/or a targeting moiety, e.g., fused as one sequence producing a combined drug, optionally including a selectable marker. In other embodiments, the extracellular vesicle is designed to treat, reduce, ameliorate, or alleviate a tumor or a cancer or a growth or a metastasis thereof. In another aspect, disclosed herein is a micelle comprising a therapeutic payload (e.g., a protein, a cytokine, a chemokine, an antibody or a fragment thereof, a nucleic acid [e.g., DNA or RNA], or a pharmaceutical or biopharmaceutical payload) and/or a targeting moiety. In other embodiments, the micelle is designed to treat, reduce, ameliorate, or alleviate a tumor or a cancer or a growth or a metastasis thereof.

[0018] Disclosed herein is an extracellular vesicle or a micelle for treating, reducing, inhibiting, ameliorating, or alleviating a cancer or a tumor in a subject or a growth or a metastasis thereof. Disclosed herein is an extracellular vesicle or a micelle for treating, reducing, inhibiting, ameliorating, or alleviating a cancer or a tumor in a subject in need thereof. Also disclosed herein is an extracellular vesicle or a micelle for reducing or inhibiting metastases infiltration in a subject in need thereof.

[0019] Certain cancer diseases are caused by defined genetic abnormalities and resultant protein deficiencies. In some aspects, an extracellular vesicle is used to target and/or deliver a deficient protein as a therapeutic payload (i.e., a therapeutic cargo) and/or to target a tumor cell or a tumor microenvironment (TME) to treat a tumor type or a cancer type driven by a protein defect. Furthermore, extracellular vesicles and methods disclosed herein allow the delivery of multiple proteins to address multiple phenotypic deficiencies and can be applied equally to treating non- genetic cancer diseases that feature one or more protein defects. In some aspects, a micelle is used to target and/or deliver a deficient protein as a therapeutic payload and/or to target a tumor cell or a TME to treat a tumor type or a cancer type driven by a protein defect.

[0020] In some aspects, provided herein are methods for drug compositions or other therapeutic payloads to target tumors using extracellular vesicles. In some aspects, provided herein are designs for extracellular vesicles comprising compositions and/or targeting moieties and/or other therapeutic payloads and methods of use thereof.

[0021] In some aspects, disclosed herein is a method of targeting a therapy to a tumor cell, a cancer cell, or an immune cell, or to a tumor microenvironment in a subject in need thereof, the method comprising: identifying a target on the tumor cell, the cancer cell, or the immune cell, or a target of a component of the tumor microenvironment; providing an extracellular vesicle (EV) or a micelle, the extracellular vesicle or micelle comprising an expression plasmid expressing a fusion protein comprising at least one targeting moiety, the at least one targeting moiety directed to the target, at least one therapeutic payload, and at least one selectable marker fusion maker identifier, the at least one targeting moiety fused, linked, or attached to the at least one therapeutic payload and to the selectable marker identifier; and administering the extracellular vesicle or the micelle to the subject.

[0022] In some embodiments, the extracellular vesicle comprising an exosome, a micro vesicle, a macrovesicle, or an apoptotic body.

[0023] In some embodiments, the target comprises a biomarker on a tumor cell, a cancer cell, or an immune cell, or a biomarker of a component of the tumor microenvironment. In some embodiments, the biomarker comprises programmed cell death protein 1 (PD-1), programmed death ligand 1 (PD-L1), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), transforming growth factor beta (TGF-beta), epidermal growth factor receptor (EGFR), vascular endothelial growth factor receptors (VEGFR), or a combination of any of these.

[0024] In some embodiments, the extracellular vesicle comprises a lipid bilayer having an exterior surface and an interior surface, the interior surface of the lipid bilayer defining an interior space, wherein the exterior surface comprises the at least one targeting moiety, or wherein the at least one targeting moiety is at least partially within the interior space of the extracellular vesicle; and wherein the exterior surface or interior surface comprises the at least one therapeutic payload, or wherein the at least one therapeutic payload is within the interior space of the extracellular vesicle.

[0025] In some embodiments, the micelle comprises an exterior surface and a hydrophobic interior, wherein the exterior surface comprises the targeting moiety or wherein the at least one targeting moiety at least partially within the hydrophobic interior, and wherein the therapeutic payload is at least partially within the hydrophobic interior.

[0026] In some embodiments, the tumor cell or the cancer cell comprises a mutant nucleic acid encoding a defective protein, the defective protein associated with abnormal growth, carcinogenesis, angiogenesis, or metastasis, and wherein the at least one targeting moiety is directed to the defective protein or the mutant nucleic acid.

[0027] In some embodiments, the at least one targeting moiety comprises a cytokine or cytokine receptor, a chemokine or chemokine receptor, an antibody, an antigen-binding domain, an antigen, a chimeric antigen receptor (CAR), or a clathrin moiety. In some embodiments, the antibody or the antigen-binding domain comprises a single chain variable fragment (scFv), a bispecific single chain variable fragment (diabody), a trispecific single chain variable fragment (triabody), a monoclonal antibody (mAb), a bispecific antibody, an antibody-drug conjugate (ADC), or a combination of any of these. In some embodiments, the clathrin moiety is at least 95% identical to SEQ ID NO: 1, SEQ ID NO: 12, or SEQ ID NO: 13.

[0028] In some embodiments, the extracellular vesicle or the micelle further comprises a plurality of targeting moieties.

[0029] In some embodiments, the at least one therapeutic payload reduces, inhibits, ameliorates, or alleviates a cancer or growth thereof or a tumor or growth thereof or reduces or inhibits a metastasis of a cancer. In some embodiments, the at least one therapeutic payload comprises an antitumor payload. In some embodiments, the at least one therapeutic payload comprises an immunostimulant payload. In some embodiments, the tumor cell or the cancer cell comprises a mutant nucleic acid encoding a defective protein, the defective protein associated with abnormal growth, carcinogenesis, angiogenesis, or metastasis, and the at least one therapeutic pay load comprises the corresponding wild-type protein.

[0030] In some embodiments, the at least one therapeutic payload comprises an apoptotic protein, a cytokine or cytokine receptor, a chemokine or chemokine receptor, an antibody, an antigenbinding domain, a chimeric antigen receptor (CAR), or an antigen. In some embodiments, the antibody or the antigen-binding domain comprises a single chain variable fragment (scFv), a bispecific single chain variable fragment (diabody), a trispecific single chain variable fragment (triabody), a monoclonal antibody (mAb), a bispecific antibody, an antibody-drug conjugate (ADC), or a combination of any of these. In some embodiments, the antibody or the antigenbinding domain comprising a single chain variable fragment (scFv), a bispecific single chain variable fragment (diabody), a trispecific single chain variable fragment (triabody), a monoclonal antibody (mAb), a bispecific antibody, an antibody-drug conjugate (ADC), or a combination of any of these comprises an antigen binding site for programmed cell death protein 1 (PD-1), programmed death ligand 1 (PD-L1), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), transforming growth factor beta (TGF-beta), epidermal growth factor receptor (EGFR), vascular endothelial growth factor receptors (VEGFR), or a combination of any of these.

[0031] In some embodiments, the single chain variable fragment (scFv) comprises an antigen binding site for programmed cell death-1 (PD-1), the antigen binding site at least 95% identical to SEQ ID NO: 7. In some embodiments, the at least one therapeutic payload comprises an antitumor protein selected from the group consisting of granzyme B, perforin- 1, and tumor necrosis factor-alpha (TNF-alpha). In some embodiments, the at least one therapeutic payload comprises an immunostimulant protein selected from the group consisting of granzyme B, interferon-gamma (IFN-gamma), perforin- 1, interleukin- 1 -beta (IL- 1 -beta), interleukin-2 (IL2), interleukin-8 (IL-8), interleukin- 17 (IL-17), monocyte chemoattractant protein 1/chemokine (C- C motif) ligand 2 (MCP-1/CCL2), interferon gamma-induced protein 10/C-X-C motif chemokine ligand 10 (IP-10/CXCL10), and high mobility group box 1 protein (HMGB1).

[0032] In some embodiments, the extracellular vesicle or the micelle further comprises a plurality of therapeutic pay loads.

[0033] In some embodiments, the extracellular vesicle or the micelle further comprises a stabilizing moiety.

[0034] In some embodiments, the at least one targeting moiety is attached to the at least one therapeutic payload by a linker.

[0035] In some embodiments, the fusion protein comprises: (a) a targeting moiety comprising a Protin-101, the targeting moiety having a sequence at least 95% identical to SEQ ID NO: 1; (b) a therapeutic payload comprising a single chain variable fragment (scFv) comprising an antigen binding site for programmed cell death- 1 (PD-1), the therapeutic pay load having a sequence at least 95% identical to SEQ ID NO: 7; or (c) a combination thereof.

[0036] In some embodiments, the at least one targeting moiety or the at least one therapeutic payload is fused or linked to the at least one selectable marker. In some embodiments, the selectable marker comprises a fluorescent marker. [0037] In some embodiments, the fusion protein comprises: (a) a targeting moiety comprising a Protin-101 fused to a FLAG tag, the Protin-101 fused to a FLAG tag having a sequence at least 95% identical to SEQ ID NO: 3; (b) a therapeutic payload comprising a single chain variable fragment (scFv) comprising an antigen binding site for programmed cell death-1 (PD-1) fused to a red fluorescent protein marker, the single chain variable fragment (scFv) comprising an antigen binding site for programmed cell death- 1 (PD-1) fused to a red fluorescent protein marker having a sequence at least 95% identical to SEQ ID NO: 10; or (c) a combination thereof.

[0038] In some embodiments, the extracellular vesicle of step (b) is obtained from a cell. In some embodiments, the cell is a human embryonic kidney (HEK) cell.

[0039] In some embodiments, the cancer comprises a melanoma, a breast adenocarcinoma, a lung adenocarcinoma, a colon cancer, or a pancreatic cancer or pancreatic tumor, or a metastasis of any of these. In some embodiments, the tumor comprises a fibroma.

[0040] In some embodiments, the extracellular vesicle or the micelle is administered intravenously, intramuscularly, subcutaneously, or orally to the subject. In some embodiments, the subject is a human or non-human mammal or a bird.

[0041] In some aspects, disclosed herein is a targeted extracellular vesicle comprising an expression plasmid expressing a fusion protein comprising at least one targeting moiety, the at least one targeting moiety directed to a target in a tumor cell, a cancer cell, an immune cell, or a component of a tumor microenvironment; at least one therapeutic payload; and at least one selectable marker, the at least one targeting moiety fused, linked, or attached to the at least one therapeutic payload and to the selectable marker, wherein the extracellular vesicle comprises an exosome, a microvesicle, a macrovesicle or an apoptotic body.

[0042] In some embodiments, the extracellular vesicle comprises a lipid a lipid bilayer having an exterior surface and an interior surface, the interior surface of the lipid bilayer defining an interior space, wherein the exterior surface comprises the at least one targeting moiety or wherein the at least one targeting moiety is at least partially within the interior space of the extracellular vesicle; and wherein the exterior surface or interior surface comprises the at least one therapeutic payload or wherein the at least one therapeutic payload is within the interior space of the extracellular vesicle. [0043] In some embodiments, the tumor cell or the cancer cell comprises a mutant nucleic acid encoding a defective protein, the defective protein associated with abnormal growth, carcinogenesis, angiogenesis, or metastasis, and the at least one targeting moiety is directed to the defective protein or the mutant nucleic acid.

[0044] In some embodiments, the at least one targeting moiety comprises a cytokine or cytokine receptor, a chemokine or chemokine receptor, an antibody, an antigen-binding domain, an antigen, a chimeric antigen receptor (CAR), or a clathrin moiety. In some embodiments, the antibody or the antigen-binding domain comprises a single chain variable fragment (scFv), a bispecific single chain variable fragment (diabody), a trispecific single chain variable fragment (triabody), a monoclonal antibody (mAb), a bispecific antibody, an antibody-drug conjugate (ADC), or a combination of any of these. In some embodiments, the clathrin moiety is at least 95% identical to SEQ ID NO: 1, SEQ ID NO: 12, or SEQ ID NO: 13.

[0045] In some embodiments, the extracellular vesicle farther comprises a plurality of targeting moieties.

[0046] In some embodiments, the at least one therapeutic payload reduces, inhibits, ameliorates, or alleviates a cancer or growth thereof or a tumor or growth thereof or reduces or inhibits a metastasis of a cancer. In some embodiments, the at least one therapeutic payload comprises an antitumor payload. In some embodiments, the at least one therapeutic payload comprises an immunostimulant payload. In some embodiments, the tumor cell or the cancer cell comprises a mutant nucleic acid encoding a defective protein, the defective protein associated with abnormal growth, carcinogenesis, angiogenesis, or metastasis, and the at least one therapeutic pay load comprises the corresponding wild-type protein.

[0047] In some embodiments, the at least one therapeutic payload comprises an apoptotic protein, a cytokine or cytokine receptor, a chemokine or chemokine receptor, an antibody, an antigenbinding domain, a chimeric antigen receptor (CAR), or an antigen. In some embodiments, the antibody or the antigen-binding domain comprises a single chain variable fragment (scFv), a bispecific single chain variable fragment (diabody), a trispecific single chain variable fragment (triabody), a monoclonal antibody (mAb), a bispecific antibody, an antibody-drug conjugate (ADC), or a combination of any of these. In some embodiments, the antibody or the antigenbinding domain comprising a single chain variable fragment (scFv), a bispecific single chain variable fragment (diabody), a trispecific single chain variable fragment (triabody), a monoclonal antibody (mAb), a bispecific antibody, an antibody-drug conjugate (ADC), or a combination of any of these comprises an antigen binding site for programmed cell death protein 1 (PD-1), programmed death ligand 1 (PD-L1), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), transforming growth factor beta (TGF-beta), epidermal growth factor receptor (EGFR), vascular endothelial growth factor receptors (VEGFR), or a combination of any of these. In some embodiments, the single chain variable fragment (scFv) comprises an antigen binding site for programmed cell death-1 (PD-1), the antigen binding site at least 95% identical to SEQ ID NO: 7.

[0048] In some embodiments, the at least one therapeutic payload comprises an antitumor protein selected from the group consisting of granzyme B, perforin- 1, and tumor necrosis factor-alpha (TNF-alpha). In some embodiments, the at least one therapeutic payload comprises an immunostimulant protein selected from the group consisting of granzyme B, interferon-gamma (IFN-gamma), perforin-1, interleukin- 1 -beta (IL-l-beta), interleukin-2 (IL2), interleukin-8 (IL- 8), interleukin- 17 (IL- 17), monocyte chemoattractant protein 1/chemokine (C-C motif) ligand 2 (MCP-1/CCL2), interferon gamma-induced protein 10/C-X-C motif chemokine ligand 10 (IP- 10/CXCL10), and high mobility group box 1 protein (HMGB1).

[0049] In some embodiments, the extracellular vesicle further comprises a plurality of therapeutic payloads.

[0050] In some embodiments, the extracellular vesicle further comprises a stabilizing moiety.

[0051] In some embodiments, the fusion protein comprises: (a) a targeting moiety comprising a Protin-101, the targeting moiety having a sequence 95% identical to SEQ ID NO: 1; (b) a therapeutic payload comprising a single chain variable fragment (scFv) comprising an antigen binding site for programmed cell death- 1 (PD-1), the therapeutic payload having a sequence 95% identical to SEQ ID NO: 7; or (c) a combination thereof.

[0052] In some embodiments, the at least one targeting moiety or the at least one therapeutic payload is fused or linked to at least one selectable marker. In some embodiments, the at least one selectable marker comprising a fluorescent marker. [0053] In some embodiments, wherein the fusion protein comprises: (a) a targeting moiety comprising a Protin-101 fused to a FLAG tag, the Protin-101 fused to a FLAG tag having a sequence at least 95% identical to SEQ ID NO: 3; (b) a therapeutic pay load comprising a single chain variable fragment (scFv) comprising an antigen binding site for programmed cell death- 1 (PD-1) fused to a red fluorescent protein marker, the single chain variable fragment (scFv) comprising an antigen binding site for programmed cell death- 1 (PD-1) fused to a red fluorescent protein marker having a sequence at least 95% identical to SEQ ID NO: 10; or (c) a combination thereof.

[0054] In other aspects, disclosed herein is a targeted micelle comprising an expression plasmid expressing a fusion protein comprising at least one targeting moiety, the at least one targeting moiety directed to a target in a tumor cell, a cancer cell, an immune cell, or a component of a tumor microenvironment; at least one therapeutic payload; and at least one selectable marker, the at least one targeting moiety fused, linked, or attached to the at least one therapeutic payload and to the selectable marker, wherein the micelle comprises an exterior surface and a hydrophobic interior, wherein the exterior surface comprises the at least one targeting moiety or wherein the at least one targeting moiety is at least partially within the hydrophobic interior, and wherein the therapeutic payload is at least partially within the hydrophobic interior.

[0055] In some aspects, disclosed herein is a method of making a targeted extracellular vesicle, the method comprising: (a) providing an expression plasmid, the expression plasmid expressing a fusion protein comprising (i) at least one targeting moiety, the at least one targeting moiety directed to a target on a tumor cell, a cancer cell, or an immune cell, or to a target of a component of a tumor microenvironment, (ii) at least one therapeutic payload, and (iii) at least one selectable marker, the at least one targeting moiety fused, linked, or attached to the at least one therapeutic payload and to the selectable marker; (b) transfecting a cell with the expression plasmid; (c) isolating an extracellular vesicle from the cell, the extracellular vesicle comprising an exosome, a microvesicle, or a macrovesicle.

[0056] In some embodiments, the target comprises a biomarker on a tumor cell, a cancer cell, or an immune cell, or a biomarker of a component of the tumor microenvironment. In some embodiments, the biomarker comprises programmed cell death protein 1 (PD-1), programmed death ligand 1 (PD-L1), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), transforming growth factor beta (TGF-beta), epidermal growth factor receptor (EGFR), vascular endothelial growth factor receptors (VEGFR), or a combination of any of these.

[0057] In some embodiments, the tumor cell or the cancer cell comprises a mutant nucleic acid encoding a defective protein, the defective protein associated with abnormal growth, carcinogenesis, angiogenesis, or metastasis, and the at least one targeting moiety is directed to the defective protein or the mutant nucleic acid.

[0058] In some embodiments, the at least one targeting moiety comprises a cytokine or cytokine receptor, a chemokine or chemokine receptor, an antibody, an antigen-binding domain, an antigen, a chimeric antigen receptor (CAR), or a clathrin moiety. In some embodiments, the clathrin moiety is at least 95% identical to SEQ ID NO: 1, SEQ ID NO: 12, or SEQ ID NO: 13.

[0059] In some embodiments, the at least one therapeutic payload reduces, inhibits, ameliorates, or alleviates a cancer or growth thereof or a tumor or growth thereof or reduces or inhibits a metastasis of a cancer.

[0060] In some embodiments, the tumor cell or the cancer cell comprises a mutant nucleic acid encoding a defective protein, the defective protein associated with abnormal growth, carcinogenesis, angiogenesis, or metastasis, and the at least one therapeutic pay load comprises the corresponding wild-type protein.

[0061] In some embodiments, the at least one therapeutic pay load comprises an apoptotic protein, a cytokine or cytokine receptor, a chemokine or chemokine receptor, an antibody, an antigenbinding domain, a chimeric antigen receptor (CAR), or an antigen. In some embodiments, the antibody or antigen-binding domain comprises a single chain variable fragment (scFv), wherein the single chain variable fragment (scFv) comprises an antigen binding site for programmed cell death-1 (PD-1), the antigen binding site at least 95% identical to SEQ ID NO: 7.

[0062] In some embodiments, the at least one therapeutic payload comprises an antitumor protein selected from the group consisting of granzyme B, perforin- 1, and tumor necrosis factor-alpha (TNF-alpha). [0063] In some embodiments, the at least one therapeutic payload comprises an immunostimulant protein selected from the group consisting of granzyme B, interferon-gamma (IFN-gamma), perforin-1, interleukin- 1 -beta (IL-l-beta), interleukin-2 (IL2), interleukin-8 (IL-8), interleukin- 17 (IL- 17), monocyte chemoattractant protein 1/chemokine (C-C motif) ligand 2 (MCP-1/CCL2), interferon gamma-induced protein 10/C-X-C motif chemokine ligand 10 (IP-10/CXCL10), and high mobility group box 1 protein (HMGB1).

[0064] In some embodiments, the expression plasmid expressing a fusion protein comprises: (a) a targeting moiety comprising a Protin- 101, the targeting moiety having a sequence at least 95% identical to SEQ ID NO: 1; (b) a therapeutic payload comprising a single chain variable fragment (scFv) comprising an antigen binding site for programmed cell death- 1 (PD-1), the therapeutic pay load having a sequence at least 95% identical to SEQ ID NO: 7; or (c) a combination thereof.

[0065] In some embodiments, the expression plasmid expressing a fusion protein comprises at least one selectable marker.

[0066] In some embodiments, the expression plasmid expressing a fusion protein comprises: (a) a targeting moiety comprising a Protin-101 fused to a FLAG tag, the Protin-101 fused to a FLAG tag having a sequence at least 95% identical to SEQ ID NO: 3; (b) a therapeutic payload comprising a single chain variable fragment (scFv) comprising an antigen binding site for programmed cell death- 1 (PD-1) fused to a red fluorescent protein marker, the single chain variable fragment (scFv) comprising an antigen binding site for programmed cell death- 1 (PD-1) fused to a red fluorescent protein marker having a sequence at least 95% identical to SEQ ID NO: 10; or (c) a combination thereof.

[0067] In some embodiments, the cell comprises a human embryonic kidney (HEK) cell.

[0068] In some aspects, an extracellular vesicle is designed to regulate an innate and/or adaptive immune response against a tumor and/or a development of a metastasis. In some embodiments, an extracellular vesicle targets a malignant melanoma, a breast adenocarcinoma, a lung adenocarcinoma, or a pancreatic tumor.

[0069] In some aspects, a micelle is designed to regulate an innate and/or adaptive immune response against a tumor and/or a development of a metastasis. In some embodiments, a micelle targets a malignant melanoma, a breast adenocarcinoma, a lung adenocarcinoma, or a pancreatic tumor.

[0070] In some aspects, provided herein are compositions and methods for using a targeted extracellular vesicle to deliver a therapeutic payload to a tumor cell and/or tumor microenvironment (TME) to treat different types of tumors or cancers. In some embodiments, a therapeutic-loaded (e.g., drug-loaded) extracellular vesicle modulates the immune response against the tumor, slowing disease progression, extending survival time, and preventing, reducing, or slowing cancer metastasis spread, and in some embodiments, coupling an extracellular vesicle to one or more targeting moieties, such as biopharmaceutical based on single-chain variable fragments (scFvs), that specifically recognizes and binds one or more tumor cell markers. In other embodiments, the extracellular vesicle comprises an exosome, an ectosome, an endosome, a microparticle, a microvesicle, a liposome, an apoptotic body, a macrovesicle, or another type of extracellular vesicle loaded with an anti-tumor or immunostimulant protein or nucleic acid drawn to tumor cells within their microenvironment, in order to activate immune response cells and kill resistant tumor cells. In some embodiments, a micelle is used in place of an extracellular vesicle.

[0071] In some embodiments, disclosed herein are intravenous, intramuscular, and subcutaneous delivery methods. In other embodiments, the extracellular vesicle is engineered to be delivered orally and, upon metabolization, to reach the TME before targeting a tumor cell to deliver therapeutic pay load contained therein. In other embodiments, an extracellular vesicle delivers the therapeutic payload directly to specific targets in the TME.

[0072] In some aspects, the extracellular vesicle treats a cancer, such as malignant melanoma, breast adenocarcinoma, lung adenocarcinoma, colon cancer, pancreatic cancer, or another type of cancer.

[0073] In some embodiments, the extracellular vesicle comprises a composed combination drug or other therapeutic payload and targeting moiety expressed from a plasmid. In some embodiments, the plasmid comprises a fluorescent dye attached to one of the protein’s payloads to differentiate the produced extracellular vesicle. In some embodiments, the fluorescent dye is linked to another component in the assembled plasmid. An exemplary plasmid expressing a fusion of a targeting moiety and therapeutic payload with a selectable marker (a fluorescent marker) is depicted in FIGURE 1. [0074] In some embodiments, the fluorescent plasmid is constructed and transfected by methods known in the art and expressed in human embryonic kidney (HEK) cells. In some embodiments, the exosome released from the transfected HEK cells are collected and separated to the fluorescent exosomes comprising the targeted treatment composition. Additionally, in some embodiments, all these steps are used to produce the treatment. In some embodiments, the HEK cells comprise HEK 293 cells (HEK 293, HEK-293, 293 cells) or HEK 293T cells (HEK 293T, HEK-293T, 293T cells). HEK 293 cells are a specific immortalized cell line. HEK 293T cells are a derivative human cell line that expresses a mutant version of the SV40 large T antigen.

[0075] In some embodiments, extracellular vesicles are used as linking or connecting agents to bring immune cells and other cells and molecules close to tumor cells. In some embodiments, engineered exosomes may have express targeting moieties (e.g., scFvs, diabodies) that target both tumor cells and other molecules (e.g., cytotoxic T lymphocytes (CTL)). In some embodiments, dual-targeting mechanisms can still localize larger molecules to target tumor cells. In some embodiments, such linking or connecting exosomes are empty and contain additional cytokines and growth factors described herein for other therapeutic benefits.

[0076] In one aspect, engineering extracellular vesicles to express targeting moieties fused to a payload is accomplished by transfection of a cell with a DNA plasmid encoding a tumor-cell- targeting exosome. After transfection, the targeting exosomes can be isolated from the cells and loaded with therapeutic payload using electroporation, co-incubation with the therapeutic payload, sonication, extrusion, freeze/thaw cycling, and saponin-assisted loading. Thus, the engineered extracellular vesicle can contain a therapeutic payload, and the therapeutic load can include anti-tumor and immunostimulant proteins and nucleic acids. Anti-tumor and immunostimulant molecules include, but are not limited to, granzyme B, interferon-y (interferongamma; IFNy, IFN-gamma), perforin-1, tumor necrosis factor-alpha (TNF-a), interleukin- 1 -beta (IL-ip, IL-lbeta), interleukin-2 (IL-2), interleukin-8 (IL-8), interleukin- 17 (IL-17), monocyte chemoattractant protein 1/chemokine (C-C motif) ligand 2 (MCP-1/CCL2; small inducible cytokine A2), interferon gamma-induced protein 10/C-X-C motif chemokine ligand 10 (IP- 10/CXCL10; small inducible cytokine B10), and high mobility group box 1 protein (HMGB1; high-mobility group protein 1 [HMG-1]; amphoterin). [0077] The tumor-cell-specific targeting moiety can include an aptamer, an antibody, or any functional group thereof. In certain embodiments, the tumor-cell-specific targeting moiety is a single-chain variable fragment (scFv). One of ordinary skill should understand that, when referenced throughout the application, the term scFv further contemplates the use of divalent scFvs (diabodies) and tri valent scFvs (triabodies).

Extracellular Vesicles & Micelles

[0078] “Extracellular vesicles” (EVs) are lipid bilayer-delimited particles that are naturally released from almost all types of cells and, unlike a cell, cannot replicate. EVs range in diameter from near the size of the smallest physically possible unilamellar liposome (around 20-30 nanometers) to as large as 10 microns or more, although the vast majority of EVs are smaller than 200 nm.

[0079] There are three main types of extracellular vesicles generally characterized by their size or their origin, including “exosomes” (50-100 nm), “microparticles” (MPs) or “micro vesicles” (MVs) (200-1000 nm), “apoptotic bodies,” and “macro vesicles.” Engineered extracellular vesicles are described herein with primary reference to exosomes, but, depending on the size of the payload and payload, smaller microparticles and microvesicles are contemplated as well. For larger payloads, larger microparticles and microvesicles and even macrovesicles are contemplated. Naturally occurring EVs carry a payload of proteins, nucleic acids, lipids, metabolites, and even organelles from the parent cell.

[0080] Most cells that have been studied to date are thought to release EVs, including some archaeal, bacterial, fungal, and plant cells that are surrounded by cell walls. A wide variety of EV subtypes have been proposed, defined variously by size, biogenesis pathway, payload, cellular source, and function, leading to a historically heterogenous nomenclature including terms like exosomes, ectosomes, micro vesicles, microparticles, oncosomes, apoptotic bodies, exophers, migrasomes, exomeres, macro vesicles, and more. These EV subtypes have been defined by various, often overlapping, definitions, based mostly on biogenesis (cell pathway, cell or tissue identity, condition of origin), but EV subtypes may also be defined by size, constituent molecules, function, or method of separation. [0081] In some embodiments, engineered exosomes are used for targeted delivery of therapeutic payload to tumor cells. Naturally occurring exosomes are extracellular vesicles produced in endosomes of eukaryotic cells called multivesicular bodies. Intraluminal vesicles form within these multivesicular body endosomes and, upon fusion with a cell membrane, the intraluminal vesicles are released from the cell as exosomes. Exosomes are generally considered smaller than other extracellular vesicles, in the range of about 30 to 150 nanometers (nm) in diameter. Exosomes are thought to play a role in intercellular communication by carrying RNA, proteins, and other molecules from their origin cells to recipient cells via membrane vesicle trafficking. In addition, exosomes play active roles in mediating adaptive immune responses to pathogens and tumors by facilitating communication with dendritic cells and B cells. In certain embodiments, the extracellular vesicle or the micelle and methods of using the extracellular vesicle or the micelle harness these functions through tumor cell targeting to regulate innate or adaptive immune response against tumor development and metastasis.

[0082] The lipid bilayer (or phospholipid bilayer) that forms the membrane of a cell or an extracellular vesicle, is a thin polar membrane made of two layers of lipid molecules. In cells, the cell membranes are sheets that form a continuous barrier around all cells. The cell membranes of almost all organisms and many viruses are made of a lipid bilayer, as are the nuclear membrane surrounding the cell nucleus, and membranes of the membrane-bound organelles in the cell. Lipid bilayers are impermeable to most water-soluble (hydrophilic) molecules, especially ions, which allows cells to regulate salt concentrations and pH by transporting ions across their membranes using proteins called ion pumps. Biological bilayers are usually composed of amphiphilic phospholipids that have a hydrophilic phosphate head and a hydrophobic tail consisting of one or two fatty acid chains. When phospholipids are exposed to an aqueous environment, they selfassemble into a two-layered sheet with the hydrophobic tails pointing toward the center of the sheet. This arrangement results in two “leaflets” that are each a single molecular layer. The center of this bilayer is generally hydrophobic. The assembly process is driven by interactions between hydrophobic molecules (the hydrophobic effect).

[0083] In some bilayers, the compositions of the inner and outer membrane leaflets are different. In human red blood cells, the inner (cytoplasmic) leaflet is composed mostly of phosphatidylethanolamine, phosphatidylserine and phosphatidylinositol and its phosphorylated derivatives. By contrast, the outer (extracellular) leaflet is based on phosphatidylcholine, sphingomyelin and a variety of glycolipids. Lipid asymmetry arises, at least in part, from the fact that most phospholipids are synthesized and initially inserted into the inner monolayer: those that constitute the outer monolayer are then transported from the inner monolayer by a class of enzymes called flippases.

[0084] Lipid tails can be saturated or may have at least one unsaturated bond. Although lipid tails are responsible for modulating bilayer phase behavior, the headgroup determines the bilayer surface chemistry. Most natural bilayers are composed primarily of phospholipids, but sphingolipids and sterols such as cholesterol are also important components. Of the phospholipids, the most common headgroup is phosphatidylcholine (PC), accounting for about half the phospholipids in most mammalian cells. PC is a zwitterionic headgroup, as it has a negative charge on the phosphate group and a positive charge on the amine but, because these local charges balance, no net charge. Other headgroups can include phosphatidylserine (PS), phosphatidy lethanolamine (PE), and phosphatidylglycerol (PG). The presence of PS on the extracellular membrane face of erythrocytes is a marker of cell apoptosis, while PS in growth plate vesicles is required for the nucleation of hydroxyapatite crystals and subsequent bone mineralization. Unlike PC, some of the other headgroups carry a net charge, which can alter the electrostatic interactions of small molecules with the bilayer.

[0085] In situations in which molecules are too large or too hydrophilic to pass through a lipid bilayer or when rapid transport of molecules is required, these types of payload can be moved across the cell membrane through fusion or budding of vesicles. “Exocytosis” occurs when a vesicle is produced inside the cell and fuses with the plasma membrane to release its contents into the extracellular space. During “endocytosis,” a region of the cell membrane will dimple inwards and eventually pinch off, enclosing a portion of the extracellular fluid to transport it into the cell.

[0086] EVs are lipid bilayer-delimited particles having an aqueous interior compartment. The interior compartment of a naturally occurring EV can carry pay loads of proteins, nucleic acids, lipids, metabolites, and even organelles.

[0087] In some embodiments, the EV disclosed herein further comprises a payload (e.g., a therapeutic pay load). In some embodiments, the interior compartment of the EV disclosed herein comprises a payload. In some embodiments, the pay load is a hydrophilic pay load. In some embodiments, the hydrophilic pay load is on or is linked to the outer surface of the EV. In some embodiments, the interior compartment of the EV disclosed herein comprises a pay load. In some embodiments, the pay load comprises a hydrophilic pay load in the interior compartment of the EV. The hydrophilic payload may be free-floating within the interior compartment of the EV or is on or is linked to the surface of a headgroup lining the interior compartment of the EV. In some embodiments, the hydrophilic payload comprises a nucleic acid, a protein (e.g., a chemokine, a cytokine, an enzyme), a pharmaceutical composition (e.g., a chemotherapeutic or biologic agent), an antibody, a metabolite, a radionuclide, or a selectable marker [e.g., a fluorescent marker]).

[0088] In some embodiments, the hydrophobic region of the lipid bilayer of the EV further comprises a pay load. In some embodiments, the pay load comprises a hydrophobic pay load in the hydrophobic tail region of the lipid bilayer.

[0089] A “micelle” (micella) is an aggregate (supramolecular assembly) of surfactant phospholipid molecules dispersed in a liquid, forming a colloidal suspension (associated colloidal system). It is a particle of colloidal dimensions that exists in equilibrium with the molecules or ions in solution from which it is formed. A typical micelle in an aqueous solution forms an aggregate with the hydrophilic "head" regions in contact with surrounding solvent, thereby sequestering the hydrophobic tail regions (e.g., single- or double-tail) in the micelle center. (“Inverse micelles” have the head groups at the center with the tails extending out, such as in hydrophobic solvents.) A “supermicelle” is a hierarchical micelle structure wherein individual components are also micelles.

[0090] In some embodiments, a micelle further comprises a payload. In some embodiments, the payload is hydrophobic. In some embodiments, the payload comprises a hydrophobic payload in the interior tail region interior of the micelle. In some embodiments, the hydrophobic pay load comprises a pharmaceutical composition (e.g., a chemotherapeutic or biologic agent). In some embodiments, the payload is hydrophilic. In some embodiments, the hydrophilic payload is on or is linked to the outer surface of the micelle.

[0091] In some embodiments, the extracellular vesicle or the micelle comprises a pay load for delivery of the payload across the blood-brain barrier. In some embodiments, the micelle comprises a hydrophobic payload for delivery of the hydrophobic payload across the blood-brain barrier. [0092] In some embodiments, the extracellular vesicle or the micelle comprises a therapeutic payload. In some embodiments, the extracellular vesicle or the micelle comprises a therapeutic payload comprising a pharmaceutical composition, a cytokine or a chemokine, a modulating agent.

Therapeutic Payloads

[0093] A “therapeutic payload” or “therapeutic cargo” comprises a substance for treating, reducing, inhibiting, ameliorating, or alleviating a disease or aberrant physiological condition. Examples of therapeutic pay loads include, but are not limited to, a protein, a cytokine, a chemokine, an antibody or fragment thereof, an antigen, a nucleic acid (e.g., DNA or RNA) or enzymatic nucleic acid molecule, a pharmaceutical composition, a biopharmaceutical composition or payload, a radionuclide, or a combination of any of these.

[0094] In some aspects, the extracellular vesicle comprises a therapeutic payload. In some aspects, the micelle comprises a therapeutic payload. In some embodiments, the therapeutic payload comprises a protein, a cytokine, a chemokine, an antibody or fragment thereof, an antigen, a nucleic acid (e.g., DNA or RNA) or enzymatic nucleic acid molecule, a pharmaceutical composition, a biopharmaceutical payload, a radionuclide, or a combination of any of these. In some embodiments, the therapeutic payload comprises a biotherapeutic pay load.

[0095] In other embodiments, the extracellular vesicle comprises an anti-tumor moiety. In some embodiments, the antitumor moiety comprises a programmed death antibody, such as a PD1 antibody or a PDL1 antibody, as a therapeutic payload. In some embodiments, the programmed death antibody is fused and chemically attached to a cancer-targeting moiety.

[0096] In some embodiments, the therapeutic pay load delivered by the targeted extracellular vesicles includes a variety of molecules for treating cancer diseases. In certain embodiments, the compositions and methods recognize the presence of an immunosuppressive TME in cancers. In certain embodiments, the compositions and methods recognize the presence of immunostimulant molecules, protein modulators, enzyme inhibitors, and drug pathway modulators as a therapeutic payload. In certain embodiments, the therapeutic payload and methods can regulate immune cells, such as cytotoxic T lymphocytes (CTL), Type 1 and 17 T helper cells (Thl & Thl7), dendritic cells (DC), regulatory T cells (Treg), natural killer cells (NKC), tumor-associated macrophages (TAM), myeloid-derived suppressor cells (MDSC), RNA, siRNA, miRNA, mRNA, and antibodies. The therapeutic payload can thereby modulate the highly immunosuppressive TME in which tumor cells proliferate and metastasis.

[0097] While the extracellular vesicles and methods of making and/or use thereof are described concerning exosomes, other extracellular vesicles, such as ectosomes, macrovesicles, or microparticles, are contemplated. The type of extracellular vesicle used may depend on the size of the therapeutic payload to be delivered. For example, larger therapeutics may require a macro vesicle as opposed to smaller exosomes or microparticles.

[0098] “Targeted radionuclide therapy” (also called molecular radiotherapy) involves a radioactive drug called a radiopharmaceutical that targets cancer cells. “Radiopharmaceuticals” typically comprise a “radionuclide” (a radioactive atom) combined with a targeting moiety that seeks and destroys cancer cells. Some radionuclides have the ability to target specific cells on their own. Non-limiting examples of radionuclides include, radioiodine, radiozirconium, strontium-89 chloride (METASTRON®), samarium-153 (QUADRAMET®), or radium-223 dichloride (XOFIGO®).

[0099] A “stabilizing moiety” (“stabilizer,” “stabilizing excipient”) is a substance used to help the active pharmaceutical ingredient (API) maintain the desirable properties of the product until it is administered to the subject.

[00100] In some embodiments, the extracellular vesicle or micelle farther comprises a stabilizing moiety.

[00101] In some embodiments, the therapeutic payload is attached to, fused to, linked to, or otherwise in combination with, a targeting moiety as described herein.

[00102] In other embodiments, the therapeutic payload is fused, chemically attached, or linked (e.g., via a linker) to a targeting moiety. In a non-limiting example, a programmed death antibody (e.g., PD-1, and PD-L1) is used as a therapeutic payload and is fused, chemically attached, or linked to a cancer-targeting moiety. In other embodiments, the targeting moiety is a light chain of clathrin and analogs. [00103] In some embodiments, the composed combination (therapeutic payload and targeting moiety) is expressed in a plasmid. In some embodiments, the described plasmid comprises a selectable marker (e.g., a fluorescent dye marker or other cell surface selectable marker) attached to the protein to differentiate the produced exosome. In other embodiments, the fluorescent marker is linked to another component in the assembled plasmid, as described herein. In other embodiments, the plasmid production is as described herein (see, e.g., FIGURE 1).

[00104] In other embodiments, the fluorescent plasmid is transfected by methods known in the art and expressed in HEK cells. In a further embodiment, the exosomes released from the transfected HEK cells are collected, and the fluorescent exosomes comprising the targeted treatment composition are separated. In other embodiments, the exosomes are used for treating, reducing, inhibiting, alleviating, or ameliorating a tumor or a cancer, or a growth or metastasis thereof in a subject in need thereof.

Targeting Moieties

[00105] In some embodiments, the extracellular vesicle or the micelle comprises a targeting moiety, e.g., for targeting tumor cells to deliver anti-tumor and/or immunostimulant molecules to treat a tumor or a cancer.

[00106] A “targeting moiety” or “targeting agent” is a moiety or agent designed to direct the extracellular vesicle, the micelle, or the therapeutic payload to a tumor cell, a cancer cell, or a tumor microenvironment without affecting normal cells, or to direct the extracellular vesicle, the micelle, or the therapeutic payload to an immune cell to stimulate an immune response to a tumor or a cancer. A “targeting moiety” or “targeting agent” is a substance that binds to a specific target molecule. For example, a cytokine or chemokine targeting moiety may bind to a receptor (e.g., on the surface of a cell), and conversely, a receptor targeting moiety may bind to a cytokine or chemokine. An antibody or antigen-binding domain targeting moiety binds to an antigen, and conversely, an antigen targeting moiety binds to an antibody or antigen-binding domain. A fragment of DNA or RNA can be selected to bind to a complementary fragment.

[00107] In some embodiments, the targeting moiety comprises a protein, a nucleic acid, or a combination thereof. [00108] In some embodiments, the protein comprises a cytokine, a chemokine, an antibody or a fragment thereof, an antigen-binding domain, an antigen, or a clathrin moiety. In some embodiments, the targeting moiety comprises a clathrin moiety (e.g., a light chain of clathrin or an analog thereof, or a heavy chain of clathrin or an analog thereof).

[00109] A “signal peptide” (signal sequence, targeting signal, localization signal, localization sequence, transit peptide, leader sequence or leader peptide) is a short peptide (usually 16-30 amino acids long) present at the N-terminus (or occasionally C-terminus) of most newly synthesized proteins that are destined toward the secretory pathway. These proteins include those that reside either inside certain organelles (the endoplasmic reticulum, Golgi or endosomes), secreted from the cell, or inserted into most cellular membranes. Although most type I membranebound proteins have signal peptides, the majority of type II and multi-spanning membrane-bound proteins are targeted to the secretory pathway by their first transmembrane domain, which biochemically resembles a signal sequence except that it is not cleaved. They are a kind of target peptide.

[00110] In some embodiments in which the targeting moiety comprises a protein that is manufactured within a cell. In some embodiments, the protein comprises a signal peptide.

[00111] In some embodiments, a targeting moiety is attached to, fused to, linked to, or otherwise combined with, a modulating agent in order to deliver the modulating agent to the appropriate location (e.g., a targeting moiety that targets a receptor on a cancer cell can be linked with a modulating agent that inhibits the growth, division, or metastasis of the cancer cell or causes the cancer cell to undergo programmed cell death).

[00112] In some embodiments, the compounds and methods use antibodies, aptamers, and functional groups (such as scFv) that specifically bind tumor cell markers. Extracellular vesicles target glial cells based on charge, payload, protein, lipid, and glycan composition features in a specific embodiment. See Murphy, DE., de Jong, OG., Brouwer, M. et al. Extracellular vesiclebased therapeutics: natural versus engineered targeting and trafficking. Exp Mol Med 51, 32 (2019). Any known targeted method and combination of techniques may be used to preferentially direct extracellular vesicles of the invention to tumor cells to deliver therapeutic payload and recruit immune cells response. [00113] Tumor-cell-specific targeting moieties, such as antibodies, aptamers, and scFv, target proteins expressed by various tumor cells, such as programmed death receptors programmed cell death protein 1 (PD-1; CD279), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4, CTLA4; cluster of differentiation 152 [CD152]), transforming growth factor beta (TGF-J3; TGF-beta), epidermal growth factor receptor (EGFR; ErbB-1; HER1 in humans), vascular endothelial growth factor receptors (VEGFR, e.g., VEGFR1, VEGFR2, VEGFR3; membrane-bound VEGF [mb VEGFR]), and more. In certain embodiments, the above target proteins or other tumor-cell-specific molecules are targeted using nucleic acid aptamers engineered using systematic evolution of ligands by exponential enrichment (SELEX) methods. Engineered or selected aptamers bind their target molecules through electrostatic interactions, hydrophobic interactions, and complementary shapes. Aptamers are engineered using in vitro selection to identify and select aptamers that bind specific target peptides and whole tumor cells.

[00114] An exemplary targeting mechanism of scFv-linked extracellular vesicles is described (e.g., Longatti et al., High-affinity single-chain variable fragments are specific and versatile targeting motifs for extracellular vesicles, Nanoscale. 2018). In various embodiments, tumor-cell-targeting receptors and moieties include, but are not limited to, antibodies and functional fragments thereof. Such fragments include, but are not limited to, scFvs, antigenbinding fragments (Fab), and single-domain antibodies (such as VHH [VHH] fragments). In some embodiments, the binding moiety has an scFv as fusion proteins including, but not limited to, variable regions of the heavy (VH) and light chains (VL) of immunoglobulins. ScFvs are created by cloning VH and VL genes of mice and other animals immunized with the desired target molecule (e.g., PD-1). The VH and VL genes are expressed in multiple orientations and with various linkers to form a variety of scFvs that can provide the desired stability, expression levels, and binding affinity for tumor cells and specific markers thereof.

[00115] In some embodiments, the targeting moiety comprises an antibody (IgG or IgM based or their truncated forms). In some embodiments the antibody is attached to a clathrin moiety (e.g., Protin-101 [clathrin light chain]) and an extracellular vesicle or a micelle with or without a payload. In some embodiments, it is attached to another element in the construct directly. In some embodiments, it is tiised, attached or linked to another element in the construct via a linker. In some embodiments, the antibody targets a tumor cell or a cancer. Essentially, the antibody specifically targets a tumor antigen of interest on a tumor cell of interest. The antibody binds to the antigen on the surface of the tumor cell, triggering a signal in the tumor cell, which then absorbs or internalizes the antibody. The specific targeting of the cancer cell reduces side effects. Some embodiments include targeting by ProtinlOl-antibody providing even more specific discrimination of the target cell. Antibody linkers include, but are not limited to disulfides, hydrazones, peptides, or thioethers. In some embodiments, they are cleavable (e.g., peptides), while in other embodiments, they are non-cleavable (e.g., thioethers). Cleavable linkers may be engineered to be enzyme-sensitive. Non-cleavable linkers typically offer increased stability and maintain the drug within the cell. Longer linkers provide greater physical flexibility in the linker region, potentially altering cleavage kinetics.

[00116] In some embodiments , the targeting moiety comprises a Protin- 101 - antibody-drug conjugate (ADC). An “antibody-drug conjugate” comprises a Protin-101-antibody and a drug payload, optionally joined by an “ADC linker.” In some embodiments, the ADC comprises a Protin- 101- antibody that targets a cancer cell, and the drug payload comprises a cytotoxic drug that destroys the cancer cell. This type of bioconjugate/immunoconjugate combines the targeting capability of a monoclonal antibody with the cancer cell-destroying ability of a cytotoxic drug. Essentially, the antibody specifically targets a tumor antigen of interest on a tumor cell of interest. The antibody-Protin-101-Payload binds to the antigen on the surface of the tumor cell, triggering a signal in the tumor cell, which then absorbs or internalizes the antibody-Protin- 101 -pay load along with the linked cytotoxin, which in turn, kills the cancer cell. Some embodiments include targeting by both the T-cells and/or B-cells and/or Treg attached to an antibody providing even more specific discrimination of the target cell. ADC linkers include, but are not limited to disulfides, hydrazones, peptides, or thioethers. In some embodiments, they are cleavable (e.g., peptides), while in other embodiments, they are non-cleavable (e.g., thioethers). Cleavable ADC linkers may be engineered to be enzyme-sensitive. Non-cleavable ADC linkers typically offer increased stability and maintain the drug within the cell. Longer ADC linkers provide greater physical flexibility in the ADC linker region, potentially altering cleavage kinetics.

[00117] In some embodiments, the Protin-101 attached payload comprises programmed cell death protein 1 (PD-1; cluster of differentiation 279 [CD279]), a cell surface protein, a member of the immunoglobulin superfamily, is expressed on T cells and pro-B cells and promotes self-tolerance by suppressing T-cell inflammatory activity, preventing autoimmune diseases, but also inhibiting the immune system from killing cancer cells. Programmed cell death protein 1 (PD- 1 ; cluster of differentiation 279 [CD279]), is a cell surface protein that has a role in regulating the immune system's response to the cells of the human body by down-regulating the immune system and promoting self-tolerance by suppressing T cell inflammatory activity. An immune checkpoint protein, PD-1 promotes apoptosis of antigen-specific T-cells in lymph nodes and reduces apoptosis in regulatory T-cells (Tregs). PD-1 is a cell surface receptor that belongs to the immunoglobulin superfamily and is expressed on T cells and pro-B cells. PD-1 binds two ligands, programmed death-ligand 1 (PD-L1) and programmed death-ligand 2 (PD-L2). PD-L1 is highly expressed on the surface of cells of some types of cancers, including, but not limited to, melanoma, bladder cancer, and gastric cancer. As a result, PD-1 inhibitors block PD-1 and lower immune system activation when attacking tumors.

[00118] In certain embodiments, the invention relates to any of the compositions described herein, wherein the payload comprises an anti-PD-1 antibody or an anti-PD-1- antigen-binding domain.

[00119] Cytotoxic T-lymphocyte-associated protein 4 (CTLA4 or CTLA-4; CD152 [cluster of differentiation 152]) is a protein receptor that functions as an immune checkpoint and downregulates immune responses. CTLA-4 is constitutively expressed in regulatory T cells, but only upregulated in conventional T cells after activation. This situation is particularly observed in some cancers. For example, it can serve as an "off" switch when bound to CD80 or CD86 on the surface of antigen-presenting cells.

[00120] In certain embodiments, the invention relates to any of the composition described herein, wherein the payload comprises an anti-PD-1 antibody or an anti-PD-1 antigen-binding domain in combination with either (a) an anti-PD-Ll antibody or an anti-PD-Ll antigen-binding domain or (b) an anti-CTLA-4 antibody or an anti-CTLA-4 antigen-binding domain.

[00121] It will be beneficial that numerous different approaches may be used for targeting. Examples include, but are not limited to, extracellular vesicles targeting the surface expression of at least one aptamer, an antibody, and a fimctional group, functioning similarly to the chimeric antigen receptor (CAR) in CAR-T cells, conferring cell-specific affinity to extracellular vessels such as exosome. [00122] In some embodiments, a single-chain variable fragment (scFv) molecule may be present on the surface of engineered extracellular vesicles of the invention having specificity for specific surface proteins expressed on tumor cells. The preferential binding of the tumor-cell- specific targeting moiety to tumor-cell-specific surface proteins brings the exosome or other extracellular vesicle closer to the target tumor cells. The therapeutic payload can then be released into and near the TME to activate the immune response against tumors and kill resistant tumor cells.

Clathrins and derivative proteins

[00123] In some embodiments, the clathrin protein moiety or functional derivative thereof is a clathrin light chain and/or a clathrin heavy chain. Alternatively, it is a clathrin triskelion or a clathrin cage structure (including, but not limited to, a clathrin cage, a clathrin barrel, or a clathrin basket).

[00124] In some embodiments, the clathrin triskelion or the clathrin cage structure is selfassembled, namely, the clathrin protein(s) is exposed to conditions in which the triskelion or the cage structure assembles. In some embodiments, clathrin molecules exposed to, e.g., a buffer or biological fluid at an appropriate pH can be induced to assemble as a triskelion attached to the non-clathrin moiety (i.e., chimeric antibody), or to assemble as a three-dimensional clathrin structure (e.g., a cage, etc.) surrounding or enclosing all or part of the non-clathrin moiety (i.e., chimeric antibody). Alternatively, exposure to a different pH (e.g., a biological fluid or cellular microenvironment) can induce the complex clathrin structure to disassemble into its clathrin components, releasing the non-clathrin moiety (i.e., chimeric antibody), where the non-clathrin moiety (i.e., chimeric antibody), is inside a cage, barrel, or basket.

[00125] In some embodiments, clathrin molecules exposed to, e.g., a buffer or biological fluid at an appropriate pH can be induced to assemble as a triskelion attached to the non-clathrin moiety (i.e., chimeric antibody), or to assemble as a three-dimensional clathrin structure (e.g., a cage, etc.) surrounding or enclosing all or part of a payload (i.e., chimeric antibody). Alternatively, exposure to a different pH (e.g., a biological fluid or cellular microenvironment) can induce the complex clathrin structure to disassemble into its clathrin components, releasing the payload, such as chimeric antibody, or an additional chemotherapy or other antibody treatment (e.g., where attached to a clathrin triskelion) or releasing or exposing the payload (e.g., where the non-clathrin moiety is inside a cage, barrel, or basket).

[00126] In some embodiments, an adaptor protein is used for self-assembly. Examples of adaptor proteins include, but are not limited to anti-PD-1, anti PD-L1, anti-AP180 and/or anti- epsin.

[00127] In some embodiments, clathrin is fused, attached, or linked to anti-PD-1 and/or anti-PD-Ll antibody fragments, chemotherapy, CAR constructs, or an antibody or construct comprising an antigen-binding domain; a pharmaceutical compound; an antibody-drug conjugate; a biomarker; or an imaging agent at pH 6-7 to induce assembly into a clathrin cage, including a clathrin cage enclosing, e.g., anti-PD-1, anti-PD-Ll, chemotherapy, a CAR construct, or an antibody or construct comprising an antigen-binding domain; a pharmaceutical compound; an antibody-drug conjugate; a biomarker; or an imaging agent.

[00128] In some embodiments, the clathrin cage comprises a mini-coat (e.g., having 12 pentagons and 2 hexagons. In some embodiments, the clathrin cage comprises a mini-coat having tetrahedral symmetry (e.g., having 12 pentagons and 4 hexagons). In some embodiments, the clathrin cage comprises a hexagonal barrel (e.g., having 8 hexagons, 12 pentagons, and D6 symmetry). In some embodiments, the clathrin cage comprises a soccer ball (having 12 pentagons, 20 hexagons, and icosahedral symmetry, such as in a truncated icosahedron). In some embodiments, the clathrin comprises a triskelion.

[00129] In some embodiments, the clathrin moiety is fused, attached, or linked to an antibody specifically recognizing a tumor or cancer cell surface in order to target a tumor or cancer cell specifically.

[00130] Examples of clathrin sequences are shown in Table 1.

[00131] Table 1. Sequences Relating to Clathrin Proteins.

[00132] In some embodiments, the clathrin moiety is a clathrin light chain having SEQ ID NO: 12. [00133] In some embodiments, the clathrin moiety is a clathrin heavy chain having SEQ ID NO: 13.

[00134] In certain embodiments, the clathrin moiety comprises a clathrin light chain, wherein the light chain has greater than 85% sequence homology to SEQ ID NO: 12. In certain embodiments, the clathrin moiety comprises any of the proteins described herein, wherein the light chain has greater than 90% sequence homology to SEQ ID NO: 12. In certain embodiments, the clathrin moiety comprises any of the proteins described herein, wherein the light chain has greater than 95% sequence homology to SEQ ID NO: 12. In certain embodiments, the clathrin moiety comprises any of the proteins described herein, wherein the light chain has greater than 98% sequence homology to SEQ ID NO: 12. In certain embodiments, the clathrin moiety comprises any of the proteins described herein, wherein the light chain has greater than 99% sequence homology to SEQ ID NO: 12. In certain embodiments, the clathrin moiety comprises any of the clathrin proteins described herein, wherein the clathrin light chain has SEQ ID NO: 12.

[00135] In certain embodiments, the clathrin moiety comprises a clathrin heavy chain, the clathrin heavy chain having greater than 85% sequence homology to SEQ ID NO: 13. In certain embodiments, the clathrin moiety comprises any of the proteins described herein, wherein the heavy chain has greater than 90% sequence homology to SEQ ID NO: 13. In certain embodiments, the clathrin moiety comprises any of the proteins described herein, wherein the heavy chain has greater than 95% sequence homology to SEQ ID NO: 13. In certain embodiments, the clathrin moiety comprises any of the proteins described herein, wherein the heavy chain has greater than 98% sequence homology to SEQ ID NO: 13. In certain embodiments, the clathrin moiety comprises any of the proteins described herein, wherein the heavy chain has greater than 99% sequence homology to SEQ ID NO: 13. In certain embodiments, the clathrin moiety comprises any of the clathrin proteins described herein, wherein the clathrin heavy chain has SEQ ID NO: 13.

[00136] In certain embodiments, the clathrin moiety comprises any of the compositions described herein, wherein the light chain has a molecular weight from about 15 kDa to about 45 kDa. In certain embodiments, the clathrin moiety comprises any of the compositions described herein, wherein the light chain has a molecular weight of about 15 kDa, about 16 kDa, about 17 kDa, about 18 kDa, about 19 kDa, about 20 kDa, about 21 kDa, about 22 kDa, about 23 kDa, about 24 kDa, about 25 kDa, about 26 kDa, about 27 kDa, about 28 kDa, about 29 kDa, about 30 kDa, about 31 kDa, about 32 kDa, about 33 kDa, about 34 kDa, about 35 kDa, about 36 kDa, about 37 kDa, about 38 kDa, about 39 kDa, about 40 kDa, about 41 kDa, about 42 kDa, about 43 kDa, about 44 kDa, or about 45 kDa. In certain embodiments, the clathrin moiety comprises any of the compositions described herein, wherein the light chain has a molecular weight of about 28 kDa.

[00137] In certain embodiments, the clathrin moiety comprises any of the compositions described herein, wherein the heavy chain has a molecular weight from about 100 kDa to about 300 kDa. In certain embodiments, the clathrin moiety comprises any of the compositions described herein, wherein the heavy chain has a molecular weight of about 100 kDa, about 110 kDa, about 120 kDa, about 130 kDa, about 140 kDa, about 150 kDa, about 160 kDa, about 170 kDa, about 180 kDa, about 190 kDa, about 200 kDa, about 210 kDa, about 220 kDa, about 230 kDa, about 240 kDa, about 250 kDa, about 260 kDa, about 270 kDa, about 280 kDa, about 290 kDa, or about 300 kDa. In certain embodiments, the clathrin moiety comprises any of the compositions described herein, wherein the heavy chain has a molecular weight of about 190 kDa.

[00138] In certain embodiments, the clathrin moiety comprises any of the clathrin proteins described herein, wherein the clathrin protein has a heavy chain, a light chain, or a combination thereof.

[00139] In some embodiments, clathrin proteins are native. In other embodiments, clathrin proteins are truncated, elongated, mutated, or otherwise modified.

[00140] In some embodiments, scaffolding of truncated clathrin and their repeated sequences of these truncated peptides are used as payload carriers (e.g., of anti-tumor or anticancer moieties), such as for internalizing peptides.

[00141] In some embodiments, clathrin cages sequester toxic chemo-and bio-therapeutic drug payloads while reducing toxic exposure to the whole body and increasing delivery exclusivity to imaging, marking, tumor/other disease, or other target sites. For example, in some embodiments, the clathrin cage complex is targeted to a tumor, cancer, or other neoplasm, where the complex is internalized by the tumor/cancer/neoplastic cells, where the environment triggers the capsule to release its toxic drug payload. [00142] In certain embodiments, the clathrin moiety comprises any of the clathrin compositions described herein, wherein the three-dimensional cage structure has a diameter from about 10 nm to about 100 nm. In certain embodiments, the clathrin moiety comprises any of the compositions described herein, wherein the three-dimensional cage structure has a diameter of about 10 nm, about 20 nm, about 30 nm, about 40 nm, about 50 nm, about 60 nm, about 70 nm, about 80 nm, about 90 nm, or about 100 nm. In certain embodiments, the clathrin moiety comprises any of the compositions described herein, wherein the three-dimensional cage structures have an average diameter from about 10 nm to about 100 nm. In certain embodiments, the clathrin moiety comprises any of the compositions described herein, wherein the three- dimensional cage structures have an average diameter of about 10 nm, about 20 nm, about 30 nm, about 40 nm, about 50 nm, about 60 nm, about 70 nm, about 80 nm, about 90 nm, or about 100 nm. In certain embodiments, the diameter of the three-dimensional cage structures may be estimated or measured by techniques known in the art, such as dynamic light scattering or high- resolution NMR spectroscopy.

[00143] In certain embodiments, the clathrin moiety comprises any of the compositions described herein, wherein the three-dimensional cage structure is substantially spherical.

[00144] In certain embodiments, the clathrin moiety comprises any of the compositions described herein, wherein the three-dimensional cage structure is non-covalently assembled, for example, self-assembled.

[00145] In certain embodiments, the clathrin moiety comprises any of the compositions described herein, wherein the three-dimensional cage structure is substantially stable at about 37°C at about pH greater than or equal to 7. In certain embodiments, the clathrin moiety comprises any of the compositions described herein, wherein the three-dimensional cage structure is substantially stable at about 37°C at about pH 7. In certain embodiments, the clathrin moiety comprises any of the compositions described herein, wherein the three-dimensional cage structure is substantially stable at about 37°C at about pH 6.5 to about pH 8. In certain embodiments, the clathrin moiety comprises any of the compositions described herein, wherein the three- dimensional cage structure is substantially unstable at about 37°C at about pH less than or equal to 5.5. In certain embodiments, the clathrin moiety comprises any of the compositions described herein, wherein the three-dimensional cage structure is substantially stable at about 37°C and is substantially unstable at about pH 5.5.

[00146] Cage-like proteins such as clathrin, ferritins, DNA-binding proteins (dps), and heat shock proteins have three distinct surfaces (inside, outside, interface) that can be exploited to generate nanomaterials with multiple functionality by design. Protein cages are biological in origin and each cage exhibits extremely homogeneous size distribution. This homogeneity can be used to attain a high degree of homogeneity of the templated material and its associated property. A series of protein cages exhibiting diversity in size, functionality, and chemical and thermal stabilities can be utilized for materials synthesis under a variety of conditions. Because synthetic approaches to materials science often use harsh temperature and pH, in certain embodiments, it can be an advantage to utilize protein cages from extreme environments, such as acidic thermal hot springs.

[00147] Protein cage architectures, 10-100 nm in diameter, are self-assembled hollow spheres derived from viruses and other biological cages, including heat shock proteins (Hsp), DNA-binding proteins from starved cells (Dps), and ferritins. These architectures play critical biological roles. For example, heat shock proteins are thought to act as chaperones that prevent protein denaturation, and ferritins are known to store iron (which is both essential and toxic) as a nanoparticle of iron oxide. While each of these structures has evolved to perform a unique natural function, they are similar in that they are all essentially proteinaceous containers with three distinct surfaces (interior, exterior, and subunit interface) to which one can impart function by design. Protein cage architectures have demonstrated utility in nanotechnology with applications including inorganic nanoparticle synthesis and the development of targeted therapeutic and imaging delivery agents.

[00148] Protein cage architectures are naturally diverse; each has unique attributes (including size, structure, solvent accessibility, chemical and temperature stability, structural plasticity, assembly and disassembly parameters, and electrostatics) useful to particular applications. Importantly, one can capitalize on these features or alter them via genetic or chemical modification. Atomic level structural information identifies the precise location of amino acids within protein cage architectures and in turn allows for the rational inclusion, exclusion, and substitution of amino acid(s) (at the genetic level) resulting in protein cages with novel functional properties.

[00149] Protein cages isolated from thermophilic environments are desirable as building blocks for nanotechnology due to their potential stability in harsh reaction conditions including high temperature and pH extremes. Interestingly, one of the most stable protein cage architectures, ferritin, is commonly found in mesophilic organisms, including animals, plants, and microbes. For example, horse spleen ferritin exhibits broad pH (pH 2-8) and temperature stability (<70°C). Ferritins are involved in iron sequestration, which they accomplish through the oxidation of soluble Fe(II) using 02. This oxidation results in the formation of a nanoparticle of Fe2O3 encapsulated (and rendered nontoxic) within the protein cage. High charge density on the inner surface of the protein cage promotes this reaction, which is assisted by an enzymatic (ferroxidase) activity in some ferritin subunits. Ferritins are made up of 24 subunits, which form a spherical cage 12 nm in diameter. The ferritin family also includes the 24 subunit bacterioferritins and the Dps class of proteins, which assemble from 12 monomers.

[00150] A cavity forming protein cage is described in U.S. Pat. No. 7,393,924 (incorporated by reference). The cage is formed in vitro from a plurality of 3-legged triskelia, each triskelion having 6 protein subunits; 3 clathrin heavy chain and 3 clathrin light chain subunits. In certain embodiments, the 3-legged triskelia are not required (see, e.g., U.S. Patent Application Publication No. 2015/0307570, incorporated by reference). For example, the protein may be an isolated, synthetic or recombinant, protein comprising in whole or in part one or more types of clathrin proteins of one or more isoforms, including cloned isoforms.

[00151] Protein cage architectures have three surfaces (interior, subunit interface, and exterior) amenable to both genetic and chemical modification. Each surface can play a distinct role in the development of new targeted therapeutic and imaging agent delivery systems. The cage interior can house therapeutics, the subunit interface incorporates gadolinium (an MRI contrast agent) and the exterior presents cell-specific targeting ligands (such as peptides and antibodies).

[00152] Protein cages have many beneficial attributes that are useful in their development as targeted therapeutic and imaging agent delivery systems. Their size falls into the nanometer range shown to localize in tumors due to the enhanced permeability and retention effect. Their multivalent nature enables the incorporation of multiple functionalities (including targeting peptides and imaging agents) on a single protein cage. They are malleable to both chemical and genetic manipulation and can be produced in heterologous expression systems (including bacterial, yeast, and baculoviral systems). In addition, detailed atomic resolution structural information enables the rational design of genetic mutants with specific functions, including cellspecific targeting.

[00153] Another key component for the development of protein cage architectures as imaging and therapeutic agents is cell-specific targeting. In vivo application of the phage display library technique enabled the identification of peptides that bind specifically to the vasculature of particular organs as well as tumors. One of the most characterized of these targeting peptides is RGD-4C (CDCRGDCFC) (SEQ ID NO: 16), which binds alphaVbeta3 and alphaVbeta5 integrins that are more prevalently expressed within tumor vasculature. For example, RGD-4C and other targeting peptides may be incorporated on the exteriors of the proteins. Fluorescein labeling of cell-specific targeted cages enables their visualization by epifluorescence microscopy. In addition to genetic incorporation, cell-specific targeting ligands, including antibodies and peptides, have also been chemically coupled to protein cage platforms. For example, an anti-CD4 monoclonal antibody conjugated to a protein could enable targeting of CD4+ lymphocytes within a population of splenocytes. The multivalent nature of protein cage architectures results in the presentation of multiple targeting ligands on their surfaces and may potentially aid in the interaction of these protein cages with many surfaces including receptors on a variety of cell types.

Chimeric antigen receptors (CAR)

[00154] In some embodiments, the composition further comprises a chimeric antigen receptor (CAR). Generally, the CAR comprises an ectodomain, a transmembrane domain, and an endodomain.

[00155] When the CAR serves as a receptor on a cell surface, the ectodomain is the region of a receptor exposed to the outside of the cell, where its antigen-binding domain interacts with potential target molecules (e.g., potential antigens). In some embodiments, the CAR ectodomain comprises an antigen-binding domain. Examples of antigen-binding domains include, but are not limited to, domains that recognize and bind to a target cell of interest. Examples of a target cell of interest include, but are not limited to, a cell belonging to the subject but compromised (e.g., by damage, such as DNA damage) or a cell belonging to the subject but which expresses a particular cell-surface protein (e.g., a mutant cell, a tumor cell, or a cancer cell). Other examples of a target cell of interest include, but are not limited to, a regulatory cell, a secretory cell (e.g., a hormone-secreting cell), a cell that promotes growth, mutagenesis, or metastasis of a tumor or other neoplasm (e.g., a growth hormone producing or secreting cell), a cell that inhibits or promotes cell death, or an immune effector cell or a cell that regulates an immune effector cell.

[00156] In some embodiments, the CAR is a first-generation CAR. In some embodiments, the CAR is a second-generation CAR. In some embodiments, the CAR is a third generation CAR. In some embodiments, the CAR is a fourth generation CAR (also known as an armored CAR or TRUCK). In some embodiments, the CAR is a UniCAR, a dual-antigen receptor CAR, or is part of an on-switch system.

[00157] A “first generation CAR” typically comprises, e.g., an antigen binding domain (e.g., a single chain variable fragment [scFv]), an extracellular hinge (optionally), a transmembrane domain (TMD), and an intracellular signaling domain (CD3-zeta [CD3Q). A “second generation CAR” typically includes, e.g., an additional costimulatory domain (e.g., CD28, CD137, CD3-zeta, or 4-1BB). A “third generation CAR” typically includes, e.g., multiple costimulatory domains (e.g., CD28, CD137, CD3-zeta, CD3-epsilon, 4-1BB, 0X40). A “fourth generation CAR” (an “armored CAR” or a “TRUCK”) includes, e.g., an expression component (e.g., for expression of a cytokine [e.g., an interleukin, such as IL-2, IL-5, or IL-12; a costimulatory ligand; or an apoptosis or suicide inducer, such as caspase 9/inducible caspase 9 or HSV thymidine kinase]). The expression component is optionally inducible (e.g., iCasp9).

[00158] A “UniCAR” T cell recruitment system includes two components, namely, a universal CAR having an extracellular Pl (peptide or protein) domain attached to the hinge region and capable of binding to another peptide or protein P2, which is fused to an scFv recognizing a surface molecule on a target cell. “Dual-antigen receptor” CARs are engineered to express two antigen receptors at the same time (e.g., two tumor-associated antigens) to increase specificity of the T cells and to reduce side effects, including non-specific binding. In an “On-Switch” system, the CAR has a first receptor protein containing the antigen-binding domain and a second protein containing downstream signaling elements and costimulatory domains. The presence of an exogenous molecule causes dimerization of the binding and costimulatory proteins, e.g., to enable the CAR T cell to attack the tumor. Similarly, bispecific CAR molecules (e.g., CD20/CD3, fluorescein isothiocyanate [FITC]) can be used as switches, targeting a tumor-associated antigen and a surface molecule (e.g., CD3) on the surface of a T cell. In addition, a UniCAR T cell constructed to bind to a benign molecule (e.g., FITC) and coadministered with a bispecific small molecule drug conjugate (SMDC) adaptor molecule combining, e.g., a tumor-homing molecule with a FITC molecule with, e.g., anti-tumor activity induced only in the presence of both molecules.

[00159] “Antigens” (Ag) are structures or substances (e.g., proteins, polypeptides, polysaccharides) specifically bound by antibodies (Ab) (produced by a T cell) or by a B cell antigen receptor (BCR) (a surface receptor on a B cell), or in the case of CARs, are specifically bound by antigen-binding domains. With respect to CAR antigen-binding domains, examples include, but are not limited to, chimeric antigen receptor-polyclonal regulatory T cells (CAR-Treg cells) as immunosuppression regulatory cells that balance or regulate inflammation, as an example the neuroinflammation associated with neurodegenerative disease. CAR-T and B cells are part of the adaptive immune system. An antigen often includes multiple epitopes (i.e., distinct surface features of an antigen or antigenic determinant). Examples of antigens include, but are not limited to, interleukinlO (IL- 10), transforming growth factor beta (TGF-beta, TGF-J3), programmed cell death protein 1 (like PD-1), programmed death-ligand 1 (PD-L1; cluster of differentiation 274 [CD274] or B7 homolog 1 [B7-H1]), cytotoxic T lymphocyte-associated protein 4 (CTLA-4), and in the case of CAR-Treg, T helper 17 (Thl7), T helper 1 (Thl), cytotoxic T lymphocyte (CTL), Ml macrophage, and others.

[00160] In a CAR, the “antigen-binding site” or “antigen-binding domain” comprises the part of an CAR molecule comprised of the variable regions of an antigen-binding single-chain Fv (scFv) (e.g., a light chain [VL] and a heavy chain [VH], optionally linked by a scFv linker), such as an antigen-binding domain in a CAR.

Preparation of Extracellular Vesicles and Micelles

[00161] In certain embodiments, methods of the invention include the production of therapeutic extracellular vesicles for the treatment of cancers. See Faruqu FN, Xu L, Al-Janual KT. Preparation of Exosomes for siRNA Delivery to Cancer Cells. J. Vis. Exp. 2018; Fu W, Lei C, Liu S et al., CAR exosomes derived from effector CAR-T cells have potent antitumor effects and low toxicity. Nat Commun 10, 4355 (2019); Kriz et al., A plasmid-based multigene expression system for mammalian cells, Nat Common , 2010; Franzen et al., Characterization of Uptake and Internalization of Exosomes by the Bladder Cancer Cells, BioMed Research International, 2014: 619829.

[00162] Genetically engineering cells to produce exosomes or other EV expressing desired surface targeting moieties are accomplished using any known techniques, including molecular cloning, gene delivery (transformation, transfection, transduction), and genome editing (transcription activator-like effector nucleases [TALEN], zinc-fingers, clustered regularly interspaced short palindromic repeats [CRISPR]). For example, a cell may be transfected with an expression construct (e.g., plasmid) to express exosomes comprising the desired signaling and targeting moieties on their surface. See Cheng, 2018. Once accomplished, the engineered exosomes are isolated from the cells described in Cheng or other known techniques.

[00163] Therapeutic pay load is loaded into exosomes or other EV using any known techniques, including electroporation, co-incubation with the therapeutic payload, sonication, extrusion, freeze/thaw cycling, and saponin-assisted loading. See Antimisiaris, et al., 2018, Exosomes and Exosome-Inspired Vesicles for Targeted Drug Delivery. Pharmaceutics, 10(4):218. Once loaded, the engineered extracellular vesicles are delivered to cancer patients for treatment.

[00164] Micelle preparation methods include, but are not limited to: (1) simple dissolution (2) dialysis, (3) oil in water emulsion (4) solvent evaporation and (5) lyophilization or freeze drying. See, e.g., Kulthe et al. (2012) Polymeric micelles: authoritative aspects for drug delivery, Designed Monomers and Polymers, 15:5, 465-521, DOI: 10.1080/1385772X.2012.688328.

[00165] In some embodiments, the extracellular vesicle comprises a therapeutic payload comprising a biologic vaccine (e.g., a delayed release or slow-release biologic vaccine). In some embodiments, the biologic vaccine enhances a subject’s immunity to cancer spread and its treatment. In some other aspects, the extracellular vesicle and/or its pay load recruits a T-cell, a B-cell, or an NK-cell that expands cancer cell treatment.

[00166] The present disclosure relates to the extracellular vesicle or the micelle and uses thereof, such as the extracellular vesicle or the micelle comprising a therapeutic payload and/or a targeting moiety. In some embodiments, the extracellular vesicle or the micelle comprises a therapeutic payload (e.g., a chemokine, a cytokine, an antibody or fragment thereof, a CAR, a drug or pro-drug, a protein, a nucleic acid). In some embodiments, the extracellular vesicle or the micelle comprises a targeting moiety (e.g., a clathrin, a chemokine, a cytokine, an antibody or fragment thereof, a CAR, a protein, a nucleic acid). In some embodiments, the extracellular vesicle or the micelle can be used, e.g., as useful for the treatment of diseases or abnormal physiological conditions. In some embodiments, the therapeutic payload further comprises a stabilizing agent.

[00167] In other embodiments, provided herein is an extracellular vesicle or a micelle that is a potential therapeutic or biotherapeutic release for many antitumor and anticancer therapies, including, but not limited to, chemotherapies, monoclonal antibody (mAb, moAb) therapies, chimeric antigen receptor T-cells (CAR-T), apoptotic therapies (e.g., with apoptotic cells, supernatants, proteins), small interfering ribonucleic acid (siRNA) therapies, micro ribonucleic acid (miRNA) therapies, antisense therapies, metabolic therapies, and/or inhibitor therapies.

[00168] In some embodiments the technology is an antitumor or anticancer therapy using a specific extracellular vesicle or micelle (e.g., comprising one or more specific therapeutic pay loads and/or one or more specific targeting moieties) for enhancing a patient’s immunity to a tumor or a cancer or to cancer spread and for treatment of any of these.

[00169] In other embodiments, the product is an extracellular vesicle or a micelle that comprises anti-tumor or immunostimulant molecules (e.g., Granzyme B, Interferon-y, Perforin- 1, TNF-a, IL-ip, IL-2, IL-8, IL-17, MCP-1/CCL2, IP-10/CXCL10, and HMGB1).

[00170] In other embodiments, the extracellular vesicle or the micelle comprises a programmed death antibody such as an antibody to programmed cell death- 1 (PD-1) or an antibody to programmed death ligand 1 (PD-L1), and/or other biotherapeutic agents.

[00171] Disclosed herein is an extracellular vesicle or a micelle comprising a pharmaceutical composition, a cytokine or chemokine, an apoptotic protein or inhibitor of an anti- apoptotic protein, an antibody or antigen-binding domain, or a nucleic acid.

[00172] Some advantages of this technology over current cell therapy with biologies include, but are not limited to: (1) targeting of a treatment to a tumor, a cancer, or a tumor microenvironment; (2) specificity of treatment for a tumor, a cancer, or a tumor microenvironment; (3) decreased toxicity for non-tumor/non-cancer cells or other regions of the body outside the tumor microenvironment; and (4) potentially fewer side effects for the patient.

[00173] In some embodiments, the subject has a cancer or a tumor, and the method further comprises treating, reducing, inhibiting, ameliorating, or alleviating the cancer or the tumor or reducing or inhibiting growth, spread, or metastasis of the cancer or the tumor in the subject. In some embodiments, the cancer is a melanoma, a fibrosarcoma, a lymphoma, a breast tumor, a pancreatic tumor, or a metastasis of any of these. In some embodiments, the tumor is a fibroma. In some embodiments, the administration of the extracellular vesicle or the micelle comprises subcutaneous (SC) injection, intraperitoneal (IP) injection, intravenous injection (IV), intra-tumor injection, intra-lymph node injection, or tumor-adjacent injection in the subject. In some embodiments, the subject is a human or non-human mammal or a bird.

[00174] In some embodiments, the therapeutic payload comprises a pharmaceutical or biopharmaceutical composition, wherein the pharmaceutical or biopharmaceutical composition inhibits, ameliorates, or alleviates a cancer or growth thereof or a tumor or growth thereof or reduces or inhibits a metastasis of a cancer.

[00175] In some embodiments, the therapeutic payload comprises a modulating agent. In some embodiments, the modulating agent comprises an immunomodulating agent. In some embodiments, the modulating agent comprises a cytokine or a chemokine, an antibody or an antigen-binding domain, a nucleic acid, or a combination of any of these. In some embodiments, the therapeutic payload comprises an anti-tumor molecule or an immunostimulant molecule. In some embodiments, the therapeutic pay load comprises Granzyme B, Interferon-y, Perforin- 1, TNF-a, IL-10, IL-2, IL-8, IL-17, MCP-1/CCL2, IP-10/CXCL10, or HMGB1. In some embodiments, the cytokine comprises lymphotoxin-alpha (LT-alpha), lymphotoxin-beta (LT- beta), lyphotoxin-alphal-beta2 heterotrimer (LT-alphal-beta2), lymphotoxin-alpha2-betal heterotrimer (LT-alpha2-betal), or a combination of any of these. In some embodiments, the antibody or antigen-binding domain comprising a monoclonal antibody (mAb), a single chain variable fragment (scFv), abispecific single chain variable fragment (diabody), a trispecific single chain variable fragment (triabody), a bispecific antibody, an antibody-drug conjugate (ADC), or a combination of any of these. In some embodiments, the antibody or antigen-binding domain comprises an anti-cytotoxic T-lymphocyte-associated protein 5 (anti-CTLA-4) antibody or antigen-binding domain, an anti-programmed cell death protein 1 (anti-PD-1) antibody or antigen-binding domain, or an anti-programmed death-ligand 1 (anti-PD-Ll) antibody or antigenbinding domain, an anti-receptor activator of nuclear factor kappa-B ligand (anti-RANKL) antibody, or a combination of any of these. In some embodiments, the nucleic acid comprises a deoxyribonucleic acid (DNA) or a ribonucleic acid (RNA). In some embodiments, the DNA comprises an antisense DNA or a portion thereof, a complementary DNA (cDNA) or a portion thereof, or a genomic DNA or a portion thereof. In some embodiments, the RNA comprises a small interfering RNA (siRNA), microRNA, or messenger RNA (mRNA) or a portion thereof.

[00176] In some embodiments, the extracellular vesicle or the micelle comprises a targeting moiety. In some embodiments, the targeting moiety comprises a cytokine or a chemokine, an antibody or an antigen-binding domain, a nucleic acid, or a combination of any of these. In some embodiments, the antibody or antigen-binding domain comprises a monoclonal antibody (mAb), a single chain variable fragment (scFv), a bispecific single chain variable fragment (diabody), a trispecific single chain variable fragment (triabody), a bispecific antibody, an antibody-drug conjugate (ADC), or a combination of any of these. In some embodiments, the antibody or antigen-binding domain comprises an anti-cytotoxic T-lymphocyte-associated protein 5 (anti-CTLA-4) antibody or antigen-binding domain, an anti-programmed cell death protein 1 (anti-PD-1) antibody or antigen-binding domain, or an anti-programmed death-ligand 1 (anti-PD-Ll) antibody or antigen-binding domain, or a combination of any of these. In some embodiments, the nucleic acid comprising a deoxyribonucleic acid (DNA) or a ribonucleic acid (RNA). In some embodiments, the DNA comprises an antisense DNA or a portion thereof, a complementary DNA (cDNA) or a portion thereof, or a genomic DNA or a portion thereof. In some embodiments, the RNA comprises a small interfering RNA (siRNA), microRNA, or messenger RNA (mRNA) or a portion thereof.

[00177] In some embodiments, the extracellular vesicle or the micelle further comprises a, a stabilizing agent.

[00178] In some embodiments, the extracellular vesicle or the micelle inhibits, ameliorates, or alleviates a cancer or growth thereof or a tumor or growth thereof, or reduces or inhibits a growth or a metastasis of a cancer or a tumor.

[00179] In some embodiments, the extracellular vesicle or the micelle is a cancer vaccine. [00180] In some embodiments, the therapeutic comprises a modulating agent. In some embodiments, the modulating agent comprises an immunomodulating agent. In some embodiments, the modulating agent comprises a cytokine or a chemokine, an antibody or an antigen-binding domain, a nucleic acid, a or a combination of any of these. In some embodiments, the cytokine comprises lymphotoxin-alpha (LT-alpha), lymphotoxin-beta (LT-beta), lyphotoxin- alphal-beta2 heterotrimer (LT-alphal-beta2), lymphotoxin-alpha2-betal heterotrimer (LT- alpha2-betal), or a combination of any of these. In some embodiments, the chemokine comprising chemokine (C-C motif) ligand 20 (CCL20) or chemokine (C-X-C motif) ligand 13 (CXCL13). In some embodiments, the antibody or antigen-binding domain comprises a monoclonal antibody (mAb), a single chain variable fragment (scFv), a bispecific single chain variable fragment (diabody), a trispecific single chain variable fragment (triabody), a bispecific antibody, an antibody-drug conjugate (ADC), or a combination of any of these. In some embodiments, the antibody or antigen-binding domain comprises an anti-cytotoxic T- lymphocyte-associated protein 5 (anti-CTLA-4) antibody or antigen-binding domain, an antiprogrammed cell death protein 1 (anti-PD-1) antibody or antigen-binding domain, or an antiprogrammed death-ligand 1 (anti-PD-Ll) antibody or antigen-binding domain, an anti-receptor activator of nuclear factor kappa-B ligand (anti-RANKL) antibody or a combination of any of these. In some embodiments, the nucleic acid comprises a deoxyribonucleic acid (DNA) or a ribonucleic acid (RNA). In some embodiments, the DNA comprises an antisense DNA or a portion thereof, a complementary DNA (cDNA) or a portion thereof, or a genomic DNA or a portion thereof. In some embodiments, the RNA comprises a small interfering RNA (siRNA), microRNA, or messenger RNA (mRNA) or a portion thereof.

[00181] In some embodiments, the therapeutic payload comprises an inhibitor. In some embodiments, the therapeutic pay load comprises an activator.

[00182] In some embodiments, the therapeutic pay load comprises an immunomodulating agent. In some embodiments, the immunomodulating agent activates an immune response. In some embodiments, the immunomodulating agent activates an immune response of T-cells, B- cells, cytotoxic T lymphocytes (CTL), Th 1 or Th 17 helper cells, dendritic cells, regulatory T cells (Treg), natural killer (NK) cells, tumor-associated macrophages (TAM), or myeloid-derived suppressor cells (MDSC). [00183] In some embodiments, the therapeutic payload comprises a pharmaceutical composition. In some embodiments, the pharmaceutical composition comprises a therapeutic or a biotherapeutic. In some embodiments, the therapeutic payload comprises a cytokine or a chemokine. In some embodiments, the therapeutic payload comprises an apoptotic protein or an antibody or antigen-binding domain inhibiting tumor growth. In some embodiments, the therapeutic payload comprises a nucleic acid. In some embodiments, the therapeutic pay load comprises a ligand, an antibody, or an antigen-binding domain.

[00184] In some embodiments, the extracellular vesicle or the micelle comprises a targeting moiety. In some embodiments, the targeting moiety comprises a cytokine or a chemokine, an antibody or an antigen-binding domain, a nucleic acid, or a combination of any of these. In some embodiments, the antibody or antigen-binding domain comprises a monoclonal antibody (mAb), a single chain variable fragment (scFv), a bispecific single chain variable fragment (diabody), a trispecific single chain variable fragment (triabody), a bispecific antibody, an antibody-drug conjugate (ADC), or a combination of any of these. In some embodiments, the antibody or antigen-binding domain comprises an anti-cytotoxic T-lymphocyte-associated protein 5 (anti-CTLA-4) antibody or antigen-binding domain, an anti-programmed cell death protein 1 (anti-PD-1) antibody or antigen-binding domain, or an anti-programmed death-ligand 1 (anti-PD-Ll) antibody an anti-RANKL antibody or antigen-binding domain, or a combination of any of these. In some embodiments, the nucleic acid comprises a deoxyribonucleic acid (DNA) or a ribonucleic acid (RNA). In some embodiments, the DNA comprises an antisense DNA or a portion thereof, a complementary DNA (cDNA) or a portion thereof, or a genomic DNA or a portion thereof. In some embodiments, the RNA comprises a small interfering RNA (siRNA), microRNA, or messenger RNA (mRNA) or a portion thereof.

[00185] In some embodiments, the targeting moiety comprises a cytokine or chemokine. In some embodiments, the targeting moiety comprises a nucleic acid. In some embodiments, the targeting moiety comprises an antibody, or an antigen-binding domain.

Immune Cells

[00186] In certain embodiments, the targeting moiety and/or therapeutic payload and methods recognize the presence of immunostimulant molecules, protein modulators, enzyme inhibitors, and drug pathway modulators as a therapeutic payload. In certain embodiments, the therapeutic payload and methods can regulate immune cells, such as cytotoxic T lymphocytes (CTL), Type 1 and 17 T helper cells (Thl & Thl7), dendritic cells (DC), regulatory T cells (Treg), natural killer cells (NKC), tumor-associated macrophages (TAM), myeloid-derived suppressor cells (MDSC), DNA, RNA, siRNA, miRNA, mRNA, and antibodies. The therapeutic payload can thereby modulate the highly immunosuppressive TME in which tumor cells proliferate and metastasis.

[00187] In some embodiments, methods of using the extracellular vesicle or micelle comprise activation and/or recruitment of immune cells, e.g., for targeting a tumor or cancer cell.

[00188] The “lymphatic system” (“lymphoid system”) is an organ system in vertebrates that is part of the circulatory system and immune system. It comprises a large network of lymph, lymphatic vessels, lymph nodes, lymphatic or lymphoid organs, and lymphoid tissues.

[00189] Another major lunction of the lymphatic system is immune defense. Like blood plasma, lymph contains waste products and cellular debris, together with bacteria and proteins. The cells of the lymph are predominantly lymphocytes. Associated “lymphoid organs” are comprised of lymphoid tissue and are the sites either of lymphocyte production or of lymphocyte activation. The lymphoid organs also contain other types of cells, such as stromal cells, for support.

[00190] “Primary lymphoid organs” include the thymus and the bone marrow. These primary (or central) lymphoid organs generate lymphocytes from immature progenitor cells and are involved in the production and early clonal selection of lymphocyte tissues. In birds, the bursa of Fabricius is also a primary lymphoid organ.

[00191] “Bone marrow” is a semi-solid tissue found within the spongy or cancellous portions of bones. In birds and mammals, bone marrow is the primary site of new blood cell production or hematopoiesis. It is composed of hematopoietic cells, marrow adipose tissue, and supportive stromal cells. In adult humans, bone marrow is primarily located in the ribs, vertebrae, sternum, and bones of the pelvis. Bone marrow is also responsible for both the creation of T cell precursors and the production and maturation of B cells, which are important cell types of the immune system. From the bone marrow, B cells immediately join the circulatory system and travel to secondary lymphoid organs in search of pathogens. However, T cells travel from the bone marrow to the thymus, where they develop further and mature. Mature T cells then join B cells in search of pathogens. The other 95% of T cells begin a process of apoptosis, a form of programmed cell death. The bone marrow stroma contains mesenchymal stem cells (MSCs), which are also known as marrow stromal cells. These are multipotent stem cells that can differentiate into a variety of cell types. MSCs have been shown to differentiate, in vitro or in vivo, into osteoblasts, chondrocytes, myocytes, marrow adipocytes and beta-pancreatic islets cells.

[00192] The “bursa of Fabricius” is a primary lymphoid organ and epithelial organ exclusively found in birds. In birds, the bursa of Fabricius is the site of hematopoiesis and is essential for B cell development. The luminal (interior) surface of the bursa is plicated with up to 15 primary and 7 secondary plicae (folds) having hundreds of bursal follicles containing follicle-associated epithelial cells, lymphocytes, macrophages, and plasma cells. Lymphoid stem cells migrate from the fetal liver to the bursa during ontogeny. In the bursa, these stem cells acquire the characteristics of mature, immunocompetent B cells.

[00193] The “thymus” is also a primary lymphoid organ of the immune system and is the site of maturation for thymus cell lymphocytes or T cells. T cells are critical to the adaptive immune system, where the body adapts specifically to foreign invaders. The thymus is located in the upper front part of the chest, in the anterior superior mediastinum, behind the sternum, and in front of the heart. It is made up of two lobes, each consisting of a central medulla and an outer cortex, surrounded by a capsule. In response to postnatal antigen stimulation after birth, the thymus increases in size. The neonatal and pre-adolescent periods are when it is at its most active. At puberty, the thymus begins to atrophy and regress, with adipose tissue mostly replacing the thymic stroma. However, the thymus develops a severe immunodeficiency and subsequent high susceptibility to infection. In most species, the thymus consists of lobules divided by septa which are made up of epithelium, and as a result, it is often considered an epithelial organ. T cells, which provide cell-mediated immunity, mature from thymocytes, precursors of which have migrated from the bone marrow to the thymus. Thymocytes proliferate and undergo a selection process in the thymic cortex before entering the medulla to interact with epithelial cells. Thymocytes undergo a process of maturation, which involves ensuring the cells react against antigens ("positive selection"), but that they do not react against antigens found on body tissue ("negative selection"), e.g., to avoid autoimmunity. Positive selection occurs in the cortex and negative selection occurs in the medulla of the thymus. Once mature, T cells emigrate from the thymus to provide vital functions in the immune system. Further maturation occurs in the peripheral circulation. [00194] “Secondary lymphoid organs” (SLO; “peripheral lymphoid organs”) include the spleen, the lymph nodes (which have the highest lymphocyte concentration), the tonsils, and the appendix. The secondary lymphoid organs maintain mature naive lymphocytes and initiate an adaptive immune response and are the sites of lymphocyte activation by antigens. Activation leads to clonal expansion and affinity maturation. Mature lymphocytes recirculate between the blood and the peripheral lymphoid organs until they encounter their specific antigen.

[00195] The “spleen,” which is found in all vertebrates, acts primarily as a blood filter. The spleen plays important roles in red blood cells (erythrocytes) and the immune system. With respect to its role in the immune system, the spleen synthesizes antibodies in its white pulp and removes antibody-coated bacteria and antibody-coated blood cells by way of blood and lymph node circulation. These monocytes, upon moving to injured tissue, turn into dendritic cells and macrophages while promoting tissue healing. The spleen is a center of activity of the mononuclear phagocyte system and is sometimes compared to a large lymph node, as its absence causes a predisposition to certain infections. Up to a quarter of the body’s lymphocytes are stored in the spleen at any given time.

[00196] The "lymph nodes" ("lymph glands") have the highest lymphocyte concentration, however. A "lymph node" is a kidney-shaped organ of the lymphatic system and the adaptive immune system. A large number of lymph nodes are linked throughout the body by the lymphatic vessels. They are major sites of lymphocytes that include B and T cells. Lymph nodes are essential for the proper functioning of the immune system, acting as filters for foreign particles, including cancer cells, but have no detoxification function. A lymph node is enclosed in a fibrous capsule and comprises an outer cortex and an inner medulla. Lymph nodes become inflamed or enlarged in various diseases, from minor infections to cancers. The condition of lymph nodes is critical in cancer staging, which decides the treatment to be used and determines the prognosis, as the presence of cancer cells in lymph nodes near the primary cancer tumor indicates metastasis. After entering the lymph node from afferent lymphatic vessels, lymph flows into a space underneath the subcapsular sinus capsule, then into cortical sinuses. After passing through the cortex, lymph then collects in medullary sinuses and drains into the efferent lymph vessels to exit the node at the hilum on the concave side. There are about 450 lymph nodes in the adult human. A lymph node is divided into nodules (or lobules), each consisting of a region of cortex with combined follicle B cells, a paracortex of T cells, and a part of the nodule in the medulla. The substance of a lymph node is divided into the outer cortex and the inner medulla. The cortex of a lymph node is the outer portion of the node, underneath the capsule and the subcapsular sinus. It has an outer part and a deeper part known as the paracortex. The outer cortex consists of groups of mainly inactivated B cells called follicles. When activated, these may develop into what is called a germinal center. The deeper paracortex primarily consists of the T cells, which interact with dendritic cells, and the dense reticular network. The medullary cords are cords of lymphatic tissue and include plasma cells, macrophages, and B cells. Lymph nodes contain lymphocytes, a type of white blood cell, and are primarily B cells and T cells. B cells are mainly found in the outer cortex, clustered together as follicular B cells in lymphoid follicles, and T cells and dendritic cells are found primarily in the paracortex. The reticular network provides structural support and a surface for the adhesion of dendritic cells, macrophages, and lymphocytes. It also allows the exchange of material with blood through the high endothelial venules and provides the growth and regulatory factors necessary for activation and maturation of immune cells.

[00197] “Dendritic cells” (DCs) are antigen-presenting cells (APC, also known as accessory cells) of the mammalian immune system. Their main function is to process antigen material and present it on the cell surface to the T cells of the immune system. They act as messengers between the innate and the adaptive immune systems. Dendritic cells are present in those tissues that are in contact with the external environment, such as the skin and the inner lining of the nose, lungs, stomach and intestines. They can also be found in an immature state in the blood. Once activated, they migrate to the lymph nodes where they interact with T cells and B cells to initiate and shape the adaptive immune response. Dendritic cells can be classified as "conventional dendritic cells" (eDC, mDC) (myeloid) vs. "plasmacytoid dendritic cell" (pDC) (lymphoid). eDC can be divided farther into cDCl and cDC2. Three types of DCs have been defined in human blood: the CD1C+ myeloid DCs, the CD141+ myeloid DCs, and the CD303+ plasmacytoid DCs. The markers BDCA-2 (CLEC4C) (on pDC), BDCA-3 (thrombomodulin) (on cDC2), and BDCA-4 (neuropilin) can be used to discriminate among the types. eDCs produce interleukin (IL)- 12 (IL- 12), IL-6, tumor necrosis factor (TNF), and various chemokines. pDCs produce interferon-alpha (IFN-a). Lymphoid and myeloid DCs evolve from lymphoid and myeloid precursors, respectively, and thus are of hematopoietic origin. By contrast, follicular dendritic cells (FDC) are likely of mesenchymal rather than hematopoietic origin and do not express MHC class II.

[00198] The primary function of lymph nodes is the filtering of lymph to identify and fight infection. To accomplish this, lymph nodes contain lymphocytes, including B cells and T cells, which circulate through the bloodstream and enter and reside in lymph nodes. B cells produce antibodies. Each antibody has a single predetermined target, an antigen, to which it can bind. These circulate throughout the bloodstream, and if they find this target, the antibodies bind to it and stimulate an immune response. Each B cell produces different antibodies. B cells enter the bloodstream as "naive" cells produced in the bone marrow. After entering a lymph node, they enter a lymphoid follicle, where they divide, each with a different antibody. If a cell is stimulated, it will produce more antibodies (a plasma cell) or act as a memory cell to help the body fight future infection. If a cell is not stimulated, it will undergo apoptosis and die.

[00199] B cells acquire antigen directly from the afferent lymph. If a B cell binds its cognate antigen, it will be activated. Some B cells will immediately develop into antibodysecreting plasma cells and secrete IgM. Other B cells will internalize the antigen and present it to follicular helper T cells (follicular B helper T cells; TFH; TFH) on the B and T cell zone interface. If a cognate TFH is found, it will upregulate CD40L and promote somatic hypermutation and isotype class switching of the B cell, increasing its antigen-binding affinity and changing its effector function. The proliferation of cells within a lymph node will make the node expand. "Lymphadenopathy" refers to glands that are enlarged or swollen.

[00200] Antigens are molecules found on bacterial cell walls or on viruses, chemical substances secreted from bacteria, or sometimes even molecules present in body tissue itself. These are taken up by cells throughout the body called antigen-presenting cells, such as dendritic cells. These antigen-presenting cells enter the lymph system and then lymph nodes. They present the antigen to T cells and, if there is a T cell with the appropriate T cell receptor, it will be activated.

[00201] The "tonsils" are a set of secondary lymphoid organs facing into the aerodigestive tract and play an essential role in the immune system. On their surface, the tonsils have specialized antigen-capture cells called "Microfold cells" (M cells) that allow for the uptake of antigens produced by pathogens. These M cells then alert the B cells and T cells in the tonsil that a pathogen is present, and an immune response is stimulated. As a result, b cells are activated and proliferate in the germinal centers, where B memory cells are created, and secretory antibody (IgA) is produced of the tonsils. The tonsils also produce T cells like the thymus.

[00202] "Tertiary lymphoid organs" (TLOs) ("tertiary lymphoid structures" [TLS]; "ectopic lymphoid structures" [ELS]) are abnormal lymph node-like structures that form in peripheral tissues at sites of chronic inflammation, such as chronic infection, transplanted organs undergoing graft rejection, some cancers, and autoimmune and autoimmune-related diseases. TLOs are regulated differently from the normal process whereby lymphoid tissues are formed during ontogeny, dependent on cytokines and hematopoietic cells, but still, drain interstitial fluid and transport lymphocytes in response to the same chemical messengers and gradients.

[00203] "Lymph" is the fluid that flows through the lymphatic system. "Interstitial fluid," comprising the fluid between the cells in all body tissues, enters the lymph capillaries. This lymphatic fluid is then transported via progressively larger lymphatic vessels through lymph nodes, where tissue lymphocytes remove substances and circulating lymphocytes are added to the fluid, before emptying ultimately into the right or the left subclavian vein mixes with central venous blood. As a result, the composition of lymph continually changes. Lymph returns proteins and excess interstitial fluid to the bloodstream. It also transports fats from the digestive system to the blood via chylomicrons. Bacteria may enter the lymph channels and be transported to lymph nodes, where the bacteria are destroyed. Metastatic cancer cells can also be transported via lymph.

[00204] "Stromal cells" ("mesenchymal stromal cells") are differentiating cells that can become connective tissue cells of any organ but are commonly found in the bone marrow and other parts of the lymphatic system. Stromal cells, which have diverse functions, are an important part of the body's immune response and modulate inflammation through multiple pathways. They also aid in the differentiation of hematopoietic cells and forming necessary blood elements. Additionally, stromal cells play a role in inflammation responses and control the number of cells accumulating at an inflamed tissue region. Stromal cells originate from multipotent mesenchymal stem cells.

[00205] "Lymph node stromal cells" are essential to the structure and function of the lymph node. Their functions include, but are not limited to, creating an internal tissue scaffold for the support of hematopoietic cells; the release of small molecule chemical messengers that facilitate interactions between hematopoietic cells; the facilitation of the migration of hematopoietic cells; the presentation of antigens to immune cells at the initiation of the adaptive immune system; and the homeostasis of lymphocyte numbers. Types of lymph node stromal cells include fibroblastic reticular cells (FRC), follicular dendritic cells (FDC), marginal reticular cells (MRC), lymphatic endothelial cells (LEC), high endothelial cells (HEC), and alpha-7 integrin pericytes (AIP). [00206] "Fibroblastic reticular cells" ("fibroblast reticular cells"; FRC) are a type of lymph node stromal cell located in the T-cell zone of the cortex. FRCs produce collagen alpha- 1 (III) rich reticular fibers that form a dense network within the lymphoid tissue. These are connected by collagen XIV, small leucine-rich proteoglycans, and lysyl oxidase. The network of fibers supports and guides the movement of dendritic cells (DCs), T-cells, and B -cells and creates a porous molecular sieve in the lymph node. FRCs express chemokines that assist the activity of some T- cells and dendritic cells.

[00207] “Adipose cells” (“adipocytes”; “lipocytes”; “fat cells”) are the cells that primarily compose adipose tissue, specialized in storing energy as fat. Like stromal cells, adipocytes are derived from mesenchymal stem cells which give rise to adipocytes through adipogenesis. There are two types of adipose tissue, white adipose tissue (WAT) and brown adipose tissue (BAT), which are also known as white and brown fat, respectively, and comprise two types of fat cells. A third type of fat cells is the marrow adipocyte.

[00208] “Stem cells” are undifferentiated or partially differentiated cells that can differentiate into various types of cells and proliferate indefinitely to produce more of the same stem cell. They are the earliest type of cell in a cell lineage. They are found in both embryonic and adult organisms, but have slightly different properties in each. They are usually distinguished from progenitor cells, which cannot divide indefinitely, and precursor or blast cells, which are usually committed to differentiating into one cell type. Stem cell characteristics include selfrenewal and multipotency (multidifferentiative potential). “Adult stem cells” are found in a few specific locations in the body, known as niches, such as those in the bone marrow or gonads. They replenish rapidly lost cell types and are multipotent (differentiate into a number of cell types, but only those of a closely related family of cells), oligopotent (only differentiate into a few cell types, such as lymphoid or myeloid stem cells), or unipotent (only differentiate into one cell type while still retaining self-renewal properties). In mammals, they include, but are not limited to, hematopoietic stem cells (which replenish blood and immune cells), basal cells (which maintain the skin epithelium), and mesenchymal stem cells (which maintain bone, cartilage, muscle, and fat cells). Mesenchymal stem cells (MSC) are multipotent and are found, e.g., in the muscle, liver, bone marrow. MSC can differentiate into numerous cell categories, including, but not limited to, adipocytes, osteocytes, and chondrocytes, derived by the mesodermal layer. [00209] “Lymphocytes” are a type of white blood cell in the immune system of jawed vertebrates. Lymphocytes include natural killer (NK) cells (which function in cell-mediated, cytotoxic innate immunity), T-cells (for cell-mediated, cytotoxic adaptive immunity), and B -cells (for humoral, antibody-driven adaptive immunity), which are derived from a common progenitor. They are the main type of cell found in lymph and have a large nucleus. Lymphocytes comprise approximately 18%-42% of circulating white blood cells (“leukocytes”).

[00210] “T-cells” (“T cells”; “T lymphocytes”) are lymphocytes that play a central role in the adaptive immune response. T-cells can be distinguished from other lymphocytes by the presence of a “T-cell receptor” (TCR) on their cell surface. Subtypes of T-cells include, but are not limited to, helper CD4+ T-cells, cytotoxic CD8+ T-cells, memory T-cells, regulatory CD4+ T-cells, natural killer T-cells (not the same as natural killer cells), mucosal associated invariant T- cells, and gamma delta T-cells. A key function of T-cells is immune-mediated cell death, and it is carried out by two major subtypes: CD8+ "killer" and CD4+ "helper" T-cells.

[00211] “CD8+ T-cells,” more commonly known as “cytotoxic T lymphocytes” (CTL)

(also as “T-killer cells”, “cytolytic T cells,” "killer T-cells," “CD8+ cells,” and “CD8-postive cells”), are cytotoxic, because they are able to directly kill infected cells (especially virus-infected cells), as well as cancer cells and cells that are damaged in other ways. CD8+ T-cells are also able to use “cytokines” (small signaling proteins) to recruit other types of cells when mounting an immune response.

[00212] “CD4+ T-cells,” function as "T helper cells" (“helper cells,” “helper T cells,”

“CD4+ cells,” and “CD4-postive cells”). Unlike CD8+ killer T cells (cytotoxic T lymphocytes [CTL]), CD4+ helper T cells function by indirectly killing cells identified as foreign: they determine if and how other parts of the immune system respond to a specific, perceived threat. Helper T cells also use cytokine signaling to influence regulatory B cells directly, and other cell populations indirectly.

[00213] Proliferating helper T cells that develop into effector T cells differentiate into two major subtypes of cells known as Thl and Th2 cells (also known as Type 1 and Type 2 helper T cells, respectively). Thl helper cells lead to an increased cell-mediated response (primarily by macrophages and cytotoxic T cells), typically against intracellular bacteria and protozoa. They are triggered by the polarizing cytokine interleukin (IL)- 12 (IL- 12) and their effector cytokines are interferon-gamma (IFN-y, IFN-gamma) and IL-2. The main effector cells of Thl immunity are macrophages as well as CD8 T cells, immunoglobulin (IgG) B cells, and IFN-y CD4 T cells. Th2 helper cells lead to a humoral immune response, typically against extracellular parasites such as helminths. They are triggered by the polarizing cytokines interleukin (IL)-4 (IL-4) and IL-2, and their effector cytokines are IL-4, IL-5, IL-9, IL-10, IL-13 and IL-25. The main effector cells are eosinophils, basophils, and mast cells as well as B cells, and IL-4/IL-5 CD4 T cells. Thl7 helper cells are a subset of T helper cells developmentally distinct from Thl and Th2 lineages and producing interleukin- 17 (IL- 17). Thl7 cells produce IL- 17 which is a pro interleukin. THa[3 (TH-alpha-beta) helper cells provide the host immunity against viruses. Their differentiation is triggered by interferon-alpha-beta (IFNot/p, IFN-alpha-beta) or IL- 10. Their key effector cytokine is IL- 10. Their main effector cells are NK cells as well as CD8 T cells, IgG B cells, and IL- 10 CD4 T cells.

[00214] “Memory T-cells” are a long-lived antigen-specific subset of T-cells.

[00215] The “regulatory T-cells” (“T regulatory cells”; “regulatory CD4+ T-cells”; Treg cells; Treg), formerly known as “suppressor T-cells,” are a subpopulation of T-cells that modulate the immune system, maintain tolerance to self-antigens, and prevent autoimmune disease. Tregs are immunosuppressive and generally suppress or downregulate induction and proliferation of effector T cells. Tregs express the biomarkers CD4, FOXP3, and CD25.

[00216] “B-cells” (“B cells”; “B lymphocytes”) are lymphocytes that function in the humoral immunity component of the adaptive immune system. B-cells produce antibody molecules, but instead of being secreted, the antibody molecules are inserted into the plasma membrane where they serve as a part of B-cell receptors. When a naive or memory B-cell is activated by an antigen, it proliferates and differentiates into an antibody-secreting effector cell, known as a plasmablast or plasma cell. Additionally, B-cells present antigens (also classified as professional “antigen-presenting cells” (APCs)) and secrete cytokines. B-cells express “B-cell receptors” (BCRs) on their cell membrane. BCRs allow the B-cell to bind to a specific antigen, against which it will initiate an antibody response. Types of B-cells include, but are not limited to, plasmablasts, plasma cells, lymphoplasmacytoid cells, memory B-cells, B-2 cells (subtypes: follicular B-cells and marginal zone B-cells), B-l cells, and regulatory B-cells. “Memory B-cells” (MBC) are long-lived B-cells that develop within germinal centers of the secondary lymphoid organs. Memory B -cells circulate in the blood stream in a quiescent state, sometimes for decades. If the memory B-cell later encounters the same antigen during a subsequent infection, it triggers an accelerated and robust secondary immune response. Memory B-cells have “B cell receptors” (BCRs) on their cell membrane, identical to the one on their parent cell, which allow them to recognize antigen and mount a specific antibody response.

[00217] “Regulatory B-cells” (“B regulatory cells”; Breg cells; Breg) represent a small population of B-cells which participates in immunomodulations and in suppression of immune responses by various mechanisms. The main mechanism is a production of anti-inflammatory cytokine interleukin 10 (IL- 10).

[00218] “Natural killer cells” (“large granular lymphocytes” [LGL]; NK cells; NK), are a type of cytotoxic lymphocyte critical to the innate immune system that belong to the family of innate lymphoid cells (ILC). NK cells provide rapid responses to virus-infected cells and other intracellular pathogens acting at around 3 days after infection and respond to tumor formation. Typically, immune cells detect the “major histocompatibility complex” (MHC) presented on infected cell surfaces, triggering cytokine release, causing the death of the infected cell by lysis or apoptosis. NK cells are unique, however, as they have the ability to recognize and kill stressed cells in the absence of antibodies and MHC, allowing for a much faster immune reaction. NK cells can be classified as CD56bright or CD56dim. CD56bright NK cells release cytokines and constitute the majority of NK cells, being found in bone marrow, secondary lymphoid tissue, liver, and skin. CD56dim NK cells are primarily found in the peripheral blood and are characterized by their cell killing ability. CD56dim NK cells are always CD16 positive (CD16 is the key mediator of antibody-dependent cellular cytotoxicity [ADCC]), and CD56bright can transition into CD56dim by acquiring CD16. NK cells can eliminate virus-infected cells via CD16-mediated ADCC.

[00219] “Macrophages” (abbreviated as Mtp, M<5 or MP) are a type of white blood cell of the immune system that engulfs and digests anything that does not have, on its surface, proteins that are specific to healthy body cells, including cancer cells, microbes, cellular debris, foreign substances, etc. The process is called phagocytosis, which acts to defend the host against infection and injury. Macrophages have historically been described as falling into two categories: Ml and M2. Ml refers to macrophages that undergo “classical” activation by interferon-gamma (IFNy) with either lipopolysaccharide (LPS) or tumor necrosis factor (TNF), whereas M2 refers to macrophages that undergo “alternative” activation by IL-4. Ml macrophages are seen to have a pro-inflammatory and cytotoxic (anti-tumoral) function; M2 macrophages are anti-inflammatory (pro-tumoral) and promote wound healing. Ml "killer" macrophages are activated by LPS and IFN-gamma, and secrete high levels of IL-12 and low levels of IL-10. Ml macrophages have pro- inflammatory, bactericidal, and phagocytic functions. [30] In contrast, the M2 "repair" designation (also referred to as alternatively activated macrophages) broadly refers to macrophages that function in constructive processes like wound healing and tissue repair, and those that turn off damaging immune system activation by producing anti-inflammatory cytokines like IL- 10. M2 is the phenotype of resident tissue macrophages and can be further elevated by IL-4. M2 macrophages produce high levels of IL- 10, TGF-beta and low levels of IL- 12. Tumor-associated macrophages are mainly of the M2 phenotype, and seem to actively promote tumor growth. Macrophages can be identified using flow cytometry or immunohistochemical staining by their specific expression of proteins such as CD14, CD40, CDllb, CD64, F4/80 (mice)/EMRl (human), lysozyme M, MAC-l/MAC-3 and CD68.

[00220] “Tumor-associated macrophages” (TAMs) are a class of immune cells present in high numbers in the microenvironment of solid tumors. They are heavily involved in cancer- related inflammation. Macrophages are known to originate from bone marrow-derived blood monocytes (monocyte-derived macrophages) or yolk sac progenitors (tissue-resident macrophages), but the exact origin of TAMs in human tumors is certain. The composition of monocyte-derived macrophages and tissue-resident macrophages in the tumor microenvironment (TME) depends on the tumor type, stage, size, and location. TAM identity and heterogeneity may be the outcome of interactions between tumor-derived, tissue-specific, and developmental signals. Like macrophages, TAMs have historically been described as falling into two categories: Ml and M2. TAMs, which are believed to be tumor-promoting, affect most aspects of tumor cell biology and drive pathological phenomena including tumor cell proliferation, tumor angiogenesis, tumor lymphangiogenesis, invasion and metastasis, immunosuppression, and drug resistance. One of the major functions of TAMs is suppressing the T-cell mediated anti-tumor immune response. In mice, TAMs have immunosuppressive transcriptional profiles and express factors including IL- 10 and transforming growth factor [3 (TGF[3). In humans, TAMs have been shown to directly suppress T cell function through surface presentation of programmed death-ligand 1 (PD-L1) in hepatocellular carcinoma and B7-homologs in ovarian carcinoma, which activate programmed cell death protein 1 (PD-1) and cytotoxic T-lymphocyte antigen 4 (CTLA-4), respectively, on T cells. Inhibitory signals to PD-1 and CTLA-4 are immune checkpoints, and binding of these inhibitory receptors by their ligands prevents T cell receptor signaling, inhibits T cells cytotoxic function, and promotes T cell apoptosis. HIF-la also induces TAMs to suppress T cell function through arginase- 1. Siglec-15 has also been identified as an immune suppressive molecule that is solely expressed on TAMs.

[00221] “Myeloid-derived suppressor cells” (MDSC) are a heterogeneous group of immune cells from the myeloid lineage (a family of cells that originate from bone marrow stem cells). MDSCs strongly expand in pathological situations such as chronic infections and cancer, as a result of an altered haematopoiesis. MDSCs are discriminated from other myeloid cell types in which they possess strong immunosuppressive activities rather than immunostimulatory properties. Similar to other myeloid cells, MDSCs interact with other immune cell types including T cells, dendritic cells, macrophages and natural killer cells to regulate their functions. Cancer tissues with high infiltration of MDSCs are associated with poor patient prognosis and resistance to therapies. MDSCs can also be detected in the blood. In breast cancer patients, MDSC levels in the blood are about 10-fold higher than normal. The size of the myeloid suppressor compartment is considered to be an important factor in the clinical success or failure of cancer immunotherapy, highlighting the importance of this cell type for human pathophysiology. In mouse models, MDSCs are found as myeloid cells expressing high levels of CD1 lb (a classical myeloid lineage marker) and GR1 (granulocytic marker). The GR1 marker is made up of two cell membrane molecules, Ly6C and Ly6G, and according to their relative expression levels murine MDSCs are further classified into two subtypes, monocytic and granulocytic. Monocytic MDSCs express high levels of the Ly6C surface marker with low or no expression of the Ly6G marker, while granulocytic MDSCs express Ly6C and high levels of Ly6G. In humans, MDSCs are less characterized, and they are generally defined as myeloid cells expressing CD33, CD14 and low levels of HLA DR. The absence of the human equivalent to the murine GR1 marker makes it difficult to compare murine and human MDSCs. A combination of CD33 and CD 15 has been found to identify two major subsets of the MSDC in the peripheral blood of bladder cancer patients into granulocyte-type CD15(high) CD33(low) cells and monocyte-type CD15(low) CD33(high) cells. Regardless of whether they are murine or human, MDSC suppressor function lies in their ability to inhibit T cell proliferation and activation. In healthy individuals, immature myeloid cells formed in the bone marrow differentiate to dendritic cells, macrophages and neutrophils. However, under chronic inflammatory conditions (viral and bacterial infections) or cancer, myeloid differentiation is skewed towards the expansion of MDSCs. These MDSCs infiltrate inflammation sites and tumors, where they stop immune responses by inhibiting T cells and NK cells, for example. MDSCs also accelerate angiogenesis, tumor progression and metastasis through the expression of cytokines and factors such as transforming growth factor P (TGF-beta).

Cytokines, Chemokines, and Other Proteins

[00222] In some embodiments, the extracellular vesicle or the micelle comprises a therapeutic payload. In some embodiments, the extracellular vesicle or the micelle comprises a therapeutic payload comprising a pharmaceutical composition, a cytokine or a chemokine, a modulating agent.

[00223] In some embodiments, the modulating agent comprises an inhibitor or an activator. In some embodiments, the modulating agent comprises an immunomodulating agent.

[00224] A “modulating agent” is a substance that stimulates or suppresses physiological responses and may help in the treatment of a disease or abnormal physiological condition. For example, a cytokine or chemokine modulating agent may bind to a receptor (e.g., on the surface of a cell) to stimulate or suppress a physiological response (e.g., by activation or inhibition). An antibody or antigen-binding domain modulating agent binds to an antigen, thereby stimulating or suppressing a physiological response (e.g., stimulating an immune response). A fragment of DNA or RNA can be selected to bind to a complementary fragment, which may inhibit expression of the encoded protein.

[00225] An “immunomodulating agent” (“immune system modulator”) is a substance that stimulates or suppresses the immune system and may help the body fight cancer, infection, or other diseases. Specific immunomodulating agents, such as monoclonal antibodies, cytokines, and vaccines, affect specific parts of the immune system. Nonspecific immunomodulating agents, such as BCG and levamisole, affect the immune system in a general way.

[00226] An “immune checkpoint inhibitor” (“checkpoint inhibitor”) is a type of pharmaceutical or biopharmaceutical agent that blocks checkpoint proteins that are made by some types of immune system cells, such as T cells, and by some cancer cells. These checkpoints inhibit immune responses from being too strong and sometimes can inhibit T cells from killing cancer cells. When these checkpoints are blocked, T cells can kill cancer cells more efficiently. Examples of checkpoint proteins found on T cells or cancer cells include, but are not limited to, PD- 1/PD- L1 and CTLA-4/B7-1/B7-2.

[00227] Cytotoxic T-lymphocyte-associated protein 4 (CTLA4 or CTLA-4; CD152 [cluster of differentiation 152]) is a protein receptor that functions as an immune checkpoint and downregulates immune responses. CTLA-4 is constitutively expressed in regulatory T cells, but only upregulated in conventional T cells after activation. This situation is particularly observed in some cancers. For example, it can serve as an “off’ switch when bound to CD80 or CD86 on the surface of antigen-presenting cells. Inhibitors of CTLA-4 include, but are not limited to, ipilimumab.

[00228] In some embodiments, the extracellular vesicle or the micelle comprises an anti- CTLA-4 antibody or an anti-CTLA-4-antigen-binding domain. In some embodiments, the extracellular vesicle or the micelle comprises a biopharmaceutical composition comprising a specific antibody.

[00229] Programmed cell death protein 1 (PD-1; cluster of differentiation 279 [CD279]), a cell surface protein, a member of the immunoglobulin superfamily, is expressed on T cells and pro-B cells and promotes self-tolerance by suppressing T-cell inflammatory activity, preventing autoimmune diseases, but also inhibiting the immune system from killing cancer cells. Programmed cell death protein 1 (PD-1; cluster of differentiation 279 [CD279]), is a cell surface protein that has a role in regulating the immune system's response to the cells of the human body by down-regulating the immune system and promoting self-tolerance by suppressing T cell inflammatory activity. An immune checkpoint protein, PD-1 promotes apoptosis of antigenspecific T-cells in lymph nodes and reduces apoptosis in regulatory T-cells (Tregs). PD-1 is a cell surface receptor that belongs to the immunoglobulin superfamily and is expressed on T cells and pro-B cells. PD-1 binds two ligands, programmed death-ligand 1 (PD-L1) and programmed death-ligand 2 (PD-L2). Inhibitors of PD-1 include, but are not limited to, nivolumab, pembrolizumab, and cemiplimab.

[00230] In some embodiments, the extracellular vesicle or the micelle comprises an anti- PD-1 antibody or an anti-PD-1 -antigen-binding domain. In some embodiments, the extracellular vesicle or the micelle comprises a biopharmaceutical composition comprising nivolumab, pembrolizumab, or cemiplimab.

[00231] Programmed death-ligand 1 (PD-L1) is highly expressed on the surface of cells of some types of cancers, including, but not limited to, melanoma, bladder cancer, and gastric cancer. As a result, PD-1 inhibitors block PD-1 and lower immune system activation when attacking tumors. Inhibitors of PD-L1 include, but are not limited to, atezolizumab, avelumab, and durvalumab.

[00232] In some embodiments, the extracellular vesicle or the micelle comprises a payload comprising an anti-PD-Ll antibody or an anti-PD-Ll antigen-binding domain. In some embodiments, the extracellular vesicle or the micelle comprises a biopharmaceutical composition comprising atezolizumab, avelumab, or durvalumab.

[00233] In certain embodiments, the extracellular vesicle or the micelle comprises a pay load comprising an anti-PD-1 antibody or an anti-PD-1 antigen-binding domain in combination with either (a) an anti-PD-Ll antibody or an anti-PD-Ll antigen-binding domain, (b) an anti-CTLA-4 antibody or an anti-CTLA-4 antigen-binding domain, or (c) an anti-RANKL antibody or an anti-RANKL antigen-binding domain.

[00234] “Receptor activator of nuclear factor kappa-B ligand” (“RANKL”) (“tumor necrosis factor ligand superfamily member 11” [“TNFSF11”]; “TNF-related activation-induced cytokine” [“TRANCE”]; “osteoprotegerin ligand” [“OPGL”]; “osteoclast differentiation factor” [“ODE’]) is a type II membrane protein and is a member of the tumor necrosis factor (TNF) cytokine superfamily. RANKL is found on the surface of stromal cells, osteoblasts, and T cells. It affects the immune system and control bone regeneration and remodeling. RANKL is an apoptosis regulator gene, a binding partner of osteoprotegerin (OPG), a ligand for the receptor RANK and controls cell proliferation by modifying protein levels of Id4, Id2 and cyclin DI. RANKL also has a function in the immune system, where it is expressed by T helper cells and is thought to be involved in dendritic cell maturation. It is a dendritic cell survival factor and helps regulate T cell-dependent immune responses. T cell activation induces RANKL expression and can lead to an increase of osteoclastogenesis and bone loss. RANKL can also activate the antiapoptotic kinase AKT/PKB through a signaling complex involving SRC kinase and tumor necrosis factor receptor-associated factor 6 (TRAF6), indicating that RANKL may have a role in the regulation of apoptosis. A further role for RANKL in immunity was found in sinusoidal macrophages in lymph nodes that alert the immune system to lymph-home antigens. In some cancer patients, in addition to directly signaling through RANK for macrophage differentiation, RANKL activates the adjacent lymphatic endothelial cells to create a niche environment for these specialized immune cells. RANKL surface expression and secreted RANKL expression was reported to be increased, 80% and 50% respectively. Therefore, RANKL is considered to be a key signal regulator for cancer-induced bone loss. Overproduction of RANKL is also implicated in a variety of degenerative bone diseases, such as rheumatoid arthritis and psoriatic arthritis. The osteoclast cell surface “receptor activator of nuclear factor kappa-B” (“RANK”) binds to RANKL, and the osteocyte is the major source of RANKL regulating bone remodeling.

[00235] “Osteoprotegerin” (“OPG”) (“osteoclastogenesis inhibitory factor” [“OCIF”]; “tumor necrosis factor receptor superfamily member 11B” [“TNFRSF11B”]), is a cytokine receptor of the tumor necrosis factor (TNF) receptor superfamily. OPG has been identified as having a role in tumor growth and metastasis, heart disease, immune system development and signaling, mental health, diabetes, and the prevention of pre-eclampsia and osteoporosis during pregnancy. It serves as a decoy receptor for RANKL. Upregulation of OPG has been implicated in cancer metastasis and angiogenesis, as well as in multiple myeloma.

[00236] According to the vicious cycle hypothesis, after secondary tumors cells have migrated to bone, the tumor cell will secrete cytokines and growth factors that can act on osteoblast lineage cells. Since osteoblasts control the regulation of RANKL, the stimulation via cytokines and growth factors will then stimulate osteoblasts to increase the expression of RANKL, often while simultaneously reducing bone formation. The additional RANKL-mediated osteoclast frequency and activity will in turn increase secretion of growth factors, or matrix derived factors, which can ultimately increase tumor growth and bone destruction activity.

[00237] In some embodiments, the extracellular vesicle or the micelle comprises a payload comprising an anti-RANKL agent. In some embodiments, the anti-RANKL agent comprises an anti-RANKL antibody or antigen-binding domain. In some embodiments, the anti-RANKL agent comprises denosumab or a bisphosphonate.

[00238] “Granzyme B” (GrB) is a serine protease most commonly found in the granules of natural killer cells (NK cells) and cytotoxic T cells. It is secreted by these cells along with the pore forming protein perforin to mediate apoptosis in target cells. Granzyme B is also produced by a wide range of non-cytotoxic cells (e.g., basophils, mast cells, and smooth muscle cells) and has numerous secondary fiinctions (e.g., inducing inflammation via stimulating cytokine release and extracellular matrix remodeling).

[00239] When released with perforin, granzyme B is inserted into a target cell's plasma membrane via a pore formed by perforin. Alternatively, granzyme B can bind to negatively charged heparan sulphate containing receptors on a target cell and become endocytosed. Other mechanisms of entry have been proposed. Once inside the target cell, granzyme B can cleave and activate initiator caspases 8 and 10, and executioner caspases 3 and 7 which trigger apoptosis. Granzyme B can also cleave BID leading to B AX/B AK oligomerization and cytochrome c release from the mitochondria. Granzyme B can cleave ICAD leading to DNA fragmentation and the laddering pattern associated with apoptosis. Granzyme B has a potential of over 300 substrates and can cleave Mcl-1 in the outer mitochondrial membrane relieving its inhibition of Bim. Bim stimulates BAX/BAK oligomerization, mitochondrial membrane permeability and apoptosis. Granzyme B can also cleave HAX1 (Hs-1 associated protein X-l) to facilitate mitochondria polarization. It can also generate a cytotoxic level of mitochondrial reactive oxygen species (ROS) to mediate cell death. Other granzyme B -mediated pathways to cell death have also been proposed.

[00240] In some embodiments, the extracellular vesicle or the micelle comprises a payload comprising granzyme B (GrB). In some embodiments, the granzyme B is on or is linked to the outer surface of the EV. In some embodiments, the pay load comprises granzyme B in the interior compartment of the EV. The granzyme B may be free-floating within the interior compartment of the EV or is on or is linked to the surface of a headgroup lining the interior compartment of the EV. In some embodiments, the granzyme B is on or is linked to the outer surface of the micelle.

[00241] “Perforin-1” is a protein that in humans is encoded by the PRF1 gene and in mice by the Prfl gene. Perforin is a pore forming cytolytic protein found in the granules of cytotoxic T lymphocytes (CTLs) and natural killer cells (NK cells). Upon degranulation, perforin binds to the target cell's plasma membrane, and oligomerizes in a Ca2+ dependent manner to form pores on the target cell. The pore formed allows for the passive diffusion of a family of pro-apoptotic proteases, known as the granzymes, into the target cell. Perforin- 1 creates transmembrane tubules and is capable of non-specifically lysing a variety of target cells. It is one of the main cytolytic proteins of cytolytic granules and is a key effector molecule for T-cell- and natural killer-cell- mediated cytolysis. Perforin is thought to act by creating holes in the plasma membrane which triggers an influx of calcium and initiates membrane repair mechanisms. These repair mechanisms bring perforin and granzymes, such as granzyme B, into early endosomes.

[00242] In some embodiments, the extracellular vesicle or the micelle comprises a therapeutic pay load comprising perforin- 1. In some embodiments, the perforin- 1 is on or is linked to the outer surface of the EV. In some embodiments, the therapeutic payload comprises perforin- 1 in the interior compartment of the EV. The perforin- 1 may be free-floating within the interior compartment of the EV or is on or is linked to the surface of a headgroup lining the interior compartment of the EV. In some embodiments, the perforin- 1 is on or is linked to the outer surface of the micelle.

[00243] In some embodiments, the extracellular vesicle or the micelle comprises a therapeutic payload comprising perforin- 1 and granzyme B.

[00244] Additionally, a “targeting moiety” or “targeting agent” is a substance that binds to a specific target molecule. For example, a cytokine or chemokine targeting moiety may bind to a receptor (e.g., on the surface of a cell), and conversely, a receptor targeting moiety may bind to a cytokine or chemokine. An antibody or antigen-binding domain targeting moiety binds to an antigen, and conversely, an antigen targeting moiety binds to an antibody or antigen-binding domain. A fragment of DNA or RNA can be selected to bind to a complementary fragment.

[00245] In some embodiments, the extracellular vesicle or the micelle comprises a cytokine. In some embodiments, the extracellular vesicle or the micelle comprises a chemokine.

[00246] In some embodiments, immune cells, for example T-cells, are generated and expanded by the presence of cytokines in or on the surface of the extracellular vesicle or the micelle. In some embodiments, cytokines that affect generation and maintenance to T-helper cells in vivo comprise IL-2, IL-12, and IL-15. In some embodiments, T regulatory (Treg) cells are generated from naive T cells by cytokine induction in vivo. In some embodiments, transforming growth factor-beta (TGF-beta, TGF-J3) and/or IL-2 play a role in differentiating naive T cell to become Treg cells. [00247] “Cytokines” are a category of small proteins (-5-20 kDa) critical to cell signaling. Cytokines are peptides and usually are unable to cross the lipid bilayer of cells to enter the cytoplasm. Among other functions, cytokines may be involved in autocrine, paracrine and endocrine signaling as immunomodulating agents. Cytokines may be pro-inflammatory or antiinflammatory. Cytokines include, but are not limited to, chemokines (cytokines with chemotactic activities), interferons, interleukins (ILs; cytokines made by one leukocyte and acting on one or more other leukocytes), lymphokines (produced by lymphocytes), monokines (produced by monocytes), and tumor necrosis factors. Cells producing cytokines include, but are not limited to, immune cells (e.g., macrophages, B lymphocytes, T lymphocytes and mast cells), as well as endothelial cells, fibroblasts, and various stromal cells. A particular cytokine may be produced by more than one cell type. Cytokines have been shown to be involved in autocrine, paracrine and endocrine signaling as immunomodulating agents. They act through cell surface receptors and are especially important in the immune system, modulating the balance between humoral and cell-based immune responses. Adverse effects of cytokines have been implicated in Alzheimer’s disease.

[00248] A skilled artisan would appreciate that the term “cytokine” may encompass cytokines beneficial to enhancing an immune response targeted against a cancer or a pre- cancerous or non-cancerous tumor or lesion. A skilled artisan would also appreciate that the term “cytokine” may encompass cytokines beneficial to enhancing an immune response against a disease or inflammation (e.g., resulting from surgery, an injury, or damage from an autoimmune response) or that the term “cytokine” may encompass cytokines beneficial to reducing an abnormal autoimmune response.

[00249] In some embodiments, a cytokine encoded by the nucleic acid expands and maintains T-helper cells (helper T cells). In some embodiments, a cytokine encoded by the nucleic acid expands T-helper cells. In some embodiments, a cytokine encoded by the nucleic acid maintains T-helper cells. In some embodiments, a cytokine encoded by the nucleic acid expands cytotoxic T cells (CTLs). In some embodiments, a cytokine encoded by the nucleic acid activates cytotoxic T cells. In some embodiments, a cytokine encoded by the nucleic acid expands and activates cytotoxic T cells. In some embodiments, a cytokine encoded by the nucleic acid increases proliferation of a T-helper cell population. In some embodiments, a cytokine encoded by the nucleic acid increases proliferation of a cytotoxic T cell population. [00250] “Inflammatory cytokines” (or proinflammatory cytokines), as used herein, are a type of signaling molecule (a cytokine) that is secreted from immune cells like helper T cells (Th) and macrophages, and certain other cell types that promote inflammation. They include interleukin- 1 (IL-1), IL- 12, and IL- 18, tumor necrosis factor alpha (TNF-a), interferon-y (interferon-gamma; IFNy, IFN-gamma), and granulocyte-macrophage colony stimulating factor (GM-CSF) and play an important role in mediating the innate immune response. Inflammatory cytokines are predominantly produced by and involved in the upregulation of inflammatory reactions.

[00251] “Anti-inflammatory cytokines,” as used herein, are cytokines that counterbalance the effects of inflammatory cytokines. Major anti-inflammatory cytokines include interleukin (IL)-l receptor antagonist, IL-4, IL-6, IL-10, IL-11, and IL-13. Specific cytokine receptors for IL-1, tumor necrosis factor-alpha, and IL- 18 can also function as proinflammatory cytokine inhibitors.

[00252] “Cytokine release syndrome” (CRS), as used herein, is a form of systemic inflammatory response syndrome (SIRS) that can be triggered by a variety of factors such as infections and certain drugs. It refers to cytokine storm syndromes (CSS) and occurs when large numbers of white blood cells are activated and release inflammatory cytokines, which in turn activate yet more white blood cells. CRS is also an adverse effect of some monoclonal antibody medications, as well as adoptive T-cell therapies. When occurring as a result of a medication, it is also known as an infusion reaction. The term “cytokine storm” is often used interchangeably with CRS but, although they have similar clinical phenotypes, their characteristics are different. When occurring as a result of a therapy, CRS symptoms may be delayed until days or weeks after treatment. Immediate-onset CRS is a cytokine storm.

[00253] “Chemokines” are a family of small cytokines, or signaling proteins secreted by cells. They have the ability to induce directed chemotaxis in nearby responsive cells; they are chemotactic cytokines. Chemokines regulate cell migration, such as attracting immune cells to a site of infection or injury. Some chemokines are considered pro-inflammatory and can be induced during an immune response to recruit cells of the immune system to a site of infection, while others are considered homeostatic and are involved in controlling the migration of cells during normal processes of tissue maintenance or development. Chemokines are found in all vertebrates. Chemokines have been classified into four main subfamilies: CXC, CC, CX3C and C. All of these proteins exert their biological effects by interacting with G protein-linked transmembrane receptors called chemokine receptors, which are selectively found on the surfaces of their target cells. Their release is often stimulated by pro-inflammatory cytokines such as interleukin 1. Inflammatory chemokines function mainly as chemoattractants for leukocytes, recruiting monocytes, neutrophils and other effector cells from the blood to sites of infection or tissue damage. Certain inflammatory chemokines activate cells to initiate an immune response or promote wound healing. They are released by many different cell types and serve to guide cells of both innate immune system and adaptive immune system. “Homeostatic chemokines” are constitutively produced in certain tissues and are responsible for basal leukocyte migration, while “inflammatory chemokines” are formed under pathological conditions (on pro-inflammatory stimuli, such as IL-1, TNF-alpha, LPS, or viruses) and actively participate in the inflammatory response attracting immune cells to the site of inflammation, but some chemokines fall into both categories. Homeostatic chemokines are basal produced in the thymus and lymphoid tissues. Chemokines of various types attract monocytes/macrophages, T-cells, mast cells, eosinophils, and neutrophils.

[00254] In some embodiments, the cytokine comprises an interleukin (IL). A skilled artisan would appreciate that interleukins comprise a large family of molecules, including, but not limited to, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17A, IL-17F, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL- 27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-34, IL-35, and IL-36.

[00255] In some embodiments, the extracellular vesicle or the micelle comprises a payload comprising an immunostimulant molecule. In some embodiments, the immunostimulant molecule is on or is linked to the outer surface of the EV. In some embodiments, the immunostimulant molecule may be free-floating within the interior compartment of the EV or is on or is linked to the surface of a headgroup lining the interior compartment of the EV. In some embodiments, the immunostimulant molecule is on or is linked to the outer surface of the micelle.

[00256] Interleukin- 1-[3 (interleukin- 1 -beta; IL-ip, IL-1B; leukocytic pyrogen, leukocytic endogenous mediator, mononuclear cell factor, lymphocyte activating factor) is a member of the interleukin- 1 family of cytokines. It is produced by activated macrophages as a proprotein, which is proteolytically processed to its active form by caspase 1 (CASP1/ICE). Interleukin- 1 -beta is an important mediator of the inflammatory response, and is involved in a variety of cellular activities, including cell proliferation, differentiation, and apoptosis. The induction of cyclooxygenase-2 (PTGS2/COX2) by this cytokine in the central nervous system (CNS) is found to contribute to inflammatory pain hypersensitivity.

[00257] Interleukin-2 (IL-2) is a cytokine signaling molecule in the immune system. It regulates the activities of white blood cells (leukocytes, often lymphocytes) that are responsible for immunity. IL-2 is part of the body's natural response to microbial infection, and in discriminating between foreign ("non-self") and "self". IL-2 mediates its effects by binding to IL- 2 receptors, which are expressed by lymphocytes. The major sources of IL-2 are activated CD4+ T cells and activated CD8+ T cells. IL-2 has essential roles in key functions of the immune system, tolerance and immunity, primarily via its direct effects on T cells. IL-2 increases the cell killing activity of both natural killer cells and cytotoxic T cells. Through its role in the development of T cell immunologic memory, which depends upon the expansion of the number and function of antigen-selected T cell clones, IL-2 plays a key role in enduring cell-mediated immunity.

[00258] Interleukin 8 (IL-8; chemokine (C-X-C motif) ligand 8 [CXCL8]; neutrophil chemotactic factor) is a member of the CXC chemokine family produced by macrophages and other cell types such as epithelial cells, airway smooth muscle cells, and endothelial cells. IL-8 has several key functions. It induces chemotaxis in target cells, primarily neutrophils but also other granulocytes, causing them to migrate toward the site of infection. It also stimulates phagocytosis once they have arrived. IL- 8 is also known to be a potent promoter of angiogenesis. In target cells, IL-8 induces a series of physiological responses required for migration and phagocytosis, such as increases in intracellular Ca2+, exocytosis (e.g., histamine release), and the respiratory burst. Other cells that respond to IL- 8 include endothelial cells, macrophages, mast cells, and keratinocytes.

[00259] The interleukin- 17 (IL- 17) family is a family of pro-inflammatory cystine knot cytokines. Family members include IL- 17 A (IL- 17 in humans, CTLA8 in rodents), IL-17B, IL- 170, IL-17D, IL-17E (IL-25), and IL-17F. The biologically active IL-17 interacts with type I cell surface receptor IL-17R. In turn, there are at least three variants of IL-17R referred to as IL17RA, IL17RB, and IL17RC. After binding to the receptor, IL-17 activates several signaling cascades that, in turn, lead to the induction of chemokines. Acting as chemoattractants, these chemokines recruit the immune cells, such as monocytes and neutrophils to the site of inflammation. Typically, the signaling events mentioned above follow an invasion of the body by pathogens. Promoting the inflammation, IL- 17 acts in concert with tumor necrosis factor and interleukin- 1.

[00260] In some embodiments, the immunostimulant molecule comprises an interleukin (IL). In some embodiments, the interleukin is on or is linked to the outer surface of the EV. In some embodiments, the payload comprises interleukin in the interior compartment of the EV. The interleukin may be free-floating within the interior compartment of the EV or is on or is linked to the surface of a headgroup lining the interior compartment of the EV. In some embodiments, the interleukin is on or is linked to the outer surface of the micelle.

[00261] In some embodiments, the interleukin comprises an IL- 1 P (IL-lbeta), an IL-2, an IL- 8, or an IL- 17, or any combination thereof. In some embodiments, the interleukin comprises an IL-ip (IL-lbeta). In some embodiments, the interleukin comprises an IL-2. In some embodiments, the interleukin comprises an IL- 8. In some embodiments, the interleukin comprises an IL-17. In some embodiments, the IL-17 comprises IL-17A, IL-17B, IL-17C, IL- 17D, IL-17E, or IL-17F. In some embodiments the IL- 17 comprises IL-17A.

[00262] “Interferon-y” (Interferon-gamma; IFNy, IFN-gamma) is a dimerized soluble cytokine that is the only member of the type II class of interferons. IFNy, or type II interferon, is a cytokine that is critical for innate and adaptive immunity against viral, some bacterial and protozoan infections. IFNy is an important activator of macrophages and inducer of major histocompatibility complex class II molecule expression. Aberrant IFNy expression is associated with a number of autoinflammatory and autoimmune diseases. The importance of IFNy in the immune system arises in part from its ability to inhibit viral replication directly, especially from its immunostimulatory and immunomodulatory effects. IFNy is produced by natural killer cells (NK) and natural killer T cells (NKT) as part of the innate immune response, and by CD4 Thl and CD8 cytotoxic T lymphocyte (CTL) effector T cells once antigen-specific immunity develops as part of the adaptive immune response. IFNy is also produced by non-cytotoxic innate lymphoid cells (ILC). [00263] In some embodiments, the immunostimulant molecule comprises interferon-y (interferon-gamma). In some embodiments, the interferon-gamma is on or is linked to the outer surface of the EV. In some embodiments, the payload comprises interferon-gamma in the interior compartment of the EV. The interferon-gamma may be free-floating within the interior compartment of the EV or is on or is linked to the surface of a headgroup lining the interior compartment of the EV. In some embodiments, the interferon-gamma is on or is linked to the outer surface of the micelle.

[00264] “Tumor necrosis factor,” often called “tumor necrosis factor-a” (tumor necrosis factor-alpha; TNF-a, TNF-alpha; cachexin, cachectin) is an adipokine and a cytokine. TNF is a member of the TNF superfamily, which consists of various transmembrane proteins with a homologous TNF domain. As a cytokine, TNF-alpha is used by the immune system for cell signaling. For example, if macrophages detect an infection, they release TNF to alert other immune system cells as part of an inflammatory response. TNF signaling occurs through two receptors: TNFR1 and TNFR2. TNFR1 is constitutively expressed on most cell types, while TNFR2 is restricted primarily to endothelial, epithelial, and subsets of immune cells. TNF-a exists as a transmembrane form (mTNF-alpha) and as a soluble form (sTNF-alpha). sTNF-a results from enzymatic cleavage of mTNF-alpha. mTNF-alpha is mainly found on monocytes/macrophages where it interacts with tissue receptors by cell-to-cell contact. sTNF- alpha selectively binds to TNFR1, whereas mTNF-alpha binds to both TNFR1 and TNFR2. TNF- a binding to TNFR1 is irreversible, whereas binding to TNFR2 is reversible. The primary role of TNF-alpha is in the regulation of immune cells. TNF-alpha, as an endogenous pyrogen, is able to induce fever, apoptotic cell death, cachexia, and inflammation, inhibit tumorigenesis and viral replication, and respond to sepsis via IE-1 and IL-6-producing cells.

[00265] In some embodiments, the immunostimulant molecule comprises TNF-a (TNF- alpha). In some embodiments, the TNF-alpha is on or is linked to the outer surface of the EV. In some embodiments, the payload comprises TNF-alpha in the interior compartment of the EV. The TNF-alpha may be free-floating within the interior compartment of the EV or is on or is linked to the surface of a headgroup lining the interior compartment of the EV. In some embodiments, the TNF-alpha is on or is linked to the outer surface of the micelle. In some embodiments, the TNF-alpha comprises mTNF-alpha. In some embodiments, the mTNF-the lipid bilayer of the EV comprises mTNF-alpha. In some embodiments, the micelle comprises mTNF-alpha. In some embodiments, the TNF-alpha comprises sTNF-alpha. In some embodiments, the sTNF-alpha is on or is linked to the outer surface of the EV. In some embodiments, the sTNF-alpha is in the interior compartment of the EV. In some embodiments, the sTNF-alpha is on or is linked to the outer surface of the micelle.

[00266] “Monocyte chemoattractant protein 1/chemokine (C-C motif) ligand 2” (MCP-1, CCL2; small inducible cytokine A2) is a small cytokine that belongs to the CC chemokine family. CCL2 recruits monocytes, memory T cells, and dendritic cells to the sites of inflammation produced by either tissue injury or infection. CCL2 is anchored in the plasma membrane of endothelial cells by glycosaminoglycan side chains of proteoglycans. CCL2 is primarily secreted by monocytes, macrophages and dendritic cells. Platelet derived growth factor is a major inducer of CCL2 gene. CCL2 exhibits a chemotactic activity primarily for monocytes and basophils. After deletion of the N-terminal residue, CCL2 loses its attractivity for basophils and becomes a chemoattractant of eosinophils. Basophils and mast cells that are treated with CCL2 release their granules to the intercellular space. This effect can also be potentiated by a pre-treatment with IL- 3 or even by other cytokines. CCL2 augments monocyte anti- tumor activity and it is essential for formation of granulomas. CCL2 protein becomes a CCR2 (CCL2 receptor) antagonist when it is cleaved by metalloproteinase MMP-12.

[00267] In some embodiments, the immunostimulant molecule comprises monocyte chemoattractant protein 1/chemokine (C-C motif) ligand 2 (MCP-1/CCL2, MCP-1, CCL2; small inducible cytokine A2). In some embodiments, the MCP-1/CCL2 is on or is linked to the outer surface of the EV. In some embodiments, the pay load comprises MCP-1/CCL2 in the interior compartment of the EV. The MCP- 1/CCL2 may be free-floating within the interior compartment of the EV or is on or is linked to the surface of a headgroup lining the interior compartment of the EV. In some embodiments, the MCP-1/CCL2 is on or is linked to the outer surface of the micelle.

[00268] “Interferon gamma-induced protein 10/C-X-C motif chemokine ligand 10” (IP- 10/CXCL10, IP- 10, CXCL10; small inducible cytokine B10) is a small cytokine belonging to the CXC chemokine family. CXCL10 is secreted by several cell types in response to IFN-y (IFN- gamma). These cell types include monocytes, endothelial cells and fibroblasts. CXCL10 has been attributed to several roles, such as chemoattraction for monocytes/macrophages, T cells, NK cells, and dendritic cells, promotion of T cell adhesion to endothelial cells, antitumor activity, and inhibition of bone marrow colony formation and angiogenesis. It elicits its effects by binding to the cell surface chemokine receptor CXCR3.

[00269] In some embodiments, the immunostimulant molecule comprises interferon gamma-induced protein 10/C-X-C motif chemokine ligand 10 (IP-10/CXCL10, IP- 10, CXCL10; small inducible cytokine B10). In some embodiments, the IP-10/CXCL10 is on or is linked to the outer surface of the EV. In some embodiments, the payload comprises IP-10/CXCL10 in the interior compartment of the EV. The IP-10/CXCL10 may be free-floating within the interior compartment of the EV or is on or is linked to the surface of a headgroup lining the interior compartment of the EV. In some embodiments, the IP-10/CXCL10 is on or is linked to the outer surface of the micelle.

[00270] “High mobility group box 1 protein” (HMGB1; high-mobility group protein 1 [HMG-1]; amphoterin) is a type of chromatin protein and belongs to the high mobility group and contains a HMG-box domain. In the nucleus HMGB1 interacts with nucleosomes, transcription factors, and histones. This nuclear protein organizes the DNA and regulates transcription. After binding, HMGB1 bends DNA, which facilitates the binding of other proteins. HMGB1 supports transcription of many genes in interactions with many transcription factors. HMGB 1 is secreted by immune cells (like macrophages, monocytes and dendritic cells) through a leaderless secretory pathway. Activated macrophages and monocytes secrete HMGB1 as a cytokine mediator of inflammation. It interacts with p53. In vivo, HMGB 1 is a nuclear protein that binds to DNA and acts as an architectural chromatin-binding factor. It can also be released from cells, in which extracellular form it can bind the inflammatory receptor RAGE (Receptor for Advanced Glycation End-products) and Toll-like receptors (TLRs). Release from cells involves two distinct processes: necrosis, in which case cell membranes are permeabilized and intracellular constituents may diffuse out of the cell; and some form of active or facilitated secretion induced by signaling through the NF-KB (NF-kappaB). HMGB1 also translocates to the cytosol under stressful conditions such as increased ROS inside the cells. Under such conditions, HMGB1 promotes cell survival by sustaining autophagy through interactions with beclin-1. It is largely considered as an antiapoptotic protein.

[00271] In some embodiments, the immunostimulant compound comprises high mobility group box 1 protein (HMGB1; high-mobility group protein 1 [HMG-1]; amphoterin). In some embodiments, the HMGB 1 is on or is linked to the outer surface of the EV. In some embodiments, the payload comprises HMGB 1 in the interior compartment of the EV. The HMGB 1 may be free-floating within the interior compartment of the EV or is on or is linked to the surface of a headgroup lining the interior compartment of the EV. In some embodiments, the HMGB 1 is on or is linked to the outer surface of the micelle.

[00272] “Lymphotoxin-alpha” (LT-alpha, LT-a) (tumor necrosis factor-beta (TNF-P)), a member of the tumor necrosis factor superfamily, is a cytokine produced by lymphocytes. Belonging to the hematopoietic cell line, LT-alpha exhibits anti-proliferative activity and causes the cellular destruction of tumor cell lines. As a cytotoxic protein, LT-alpha performs a variety of important roles in immune regulation depending on the form that it is secreted as. Unlike other members of the TNF superfamily, LT-alpha is only found as a soluble homotrimer, when found at the cell surface it is found only as a heterotrimer with lymphotoxin-beta (LT-beta; LTP). LT- alpha has a significant impact on the maintenance of the immune system including the development of secondary lymphoid organs. LT-alpha mediates a large variety of inflammatory, immunostimulatory, and antiviral responses. As a signaling molecule, LT-alpha is involved in the regulation of cell survival, proliferation, differentiation, and apoptosis. LT-alpha plays an important role in innate immune regulation and its presence has been shown to prevent tumor growth and destroy cancerous cell lines. In contrast, unregulated expression of LT-alpha can result in a constantly active signaling pathway, thus leading to uncontrolled cellular growth and creation of tumors. Furthermore, LT-alpha effects depend on the type of organ it acts upon, type of cancer cells, cellular environment, gender, and time of effect during an immune response. “Lymphotoxin-beta” (LT-beta, LT-P) is a type II membrane protein of the tumor necrosis factor family. It anchors LT-alpha to the cell surface through heterotrimer formation. The predominant form on the lymphocyte surface is the lymphotoxin-alpha 1/beta 2 complex (LT-alphal-beta2, LT-al[32, LT-011P2) (e.g., 1 molecule alpha/2 molecules beta), and this complex is the primary ligand for the LT-beta receptor. The minor complex is LT-alpha 2/beta 1 (LT-alpha2-betal, LT- ot2pi, LT-a ₂ Pi ). LT-beta is an inducer of the inflammatory response system and is involved in normal development of lymphoid tissue. LT-alphal-beta2 can interact with receptors such as LT- beta receptors.

[00273] In some embodiments, the extracellular vesicle or the micelle comprises LT- alpha, LT-beta, LT-alphal-beta2, or LT-alpha2-betal. [00274] “Chemokine (C-C motif) ligand 20” (“CCL20”) (“liver activation regulated chemokine” [“LARC”]; “macrophage inflammatory protein-3” [“MIP3A”]) is a small cytokine belonging to the CC chemokine family. It is strongly chemotactic for lymphocytes and weakly attracts neutrophils. CCL20 is expressed in several tissues with highest expression observed in peripheral blood lymphocytes, lymph nodes, liver, appendix, and fetal lung and lower levels in thymus, testis, prostate and gut. CCL20 is implicated in the formation and function of mucosal lymphoid tissues via chemoattraction of lymphocytes and dendritic cells towards the epithelial cells surrounding these tissues. CCL20 elicits its effects on its target cells by binding and activating the chemokine receptor CCR6.

[00275] “Chemokine (C-X-C motif) ligand 13” (“CXCL13”) (“B lymphocyte chemoattractant” [“BLC”]; “B cell-attracting chemokine 1” [“BCA-1”]), is a protein ligand and is a small chemokine belonging to the CXC chemokine family. It is selectively chemotactic for B cells belonging to both the B-l and B-2 subsets and elicits its effects by interacting with chemokine receptor CXCR5. CXCL13 and its receptor CXCR5 control the organization of B cells within follicles of lymphoid tissues and is expressed highly in the liver, spleen, lymph nodes, and gut of humans. In T-cells, CXCL13 expression is thought to reflect a germinal center origin of the T cell, particularly a subset of T cells called follicular B helper T cells (or TFH cells).

[00276] In some embodiments, the extracellular vesicle or the micelle comprises a chemokine. In some embodiments, the chemokine comprises CCL20 or CXCL13.

[00277] In some embodiments, the extracellular vesicle or the micelle comprises a protein, a peptide, or an oligopeptide.

[00278] “Proteins” are large biomolecules and macromolecules that are comprised of one or more long chains of amino acid residues. Proteins differ from one another primarily in their sequence of amino acids, which is dictated by the nucleotide sequence of their genes, and which usually results in protein folding into a specific 3D structure that determines its activity. A linear chain of amino acid residues is called a “polypeptide.” A protein contains at least one long polypeptide. The individual amino acid residues are bonded together by peptide bonds and adjacent amino acid residues. The sequence of amino acid residues in a protein is defined by the sequence of a gene, which is encoded in the genetic code. In general, the genetic code specifies 20 standard amino acids; but in certain organisms the genetic code can include selenocysteine and — in certain archaea — pyrrolysine. Shortly after or even during synthesis, the residues in a protein are often chemically modified by post-translational modification, which alters the physical and chemical properties, folding, stability, activity, and ultimately, the function of the proteins. Some proteins have non-peptide groups attached, which can be called prosthetic groups or cofactors. Proteins can also work together to achieve a particular function, and they often associate to form stable protein complexes. A polypeptide that contains more than approximately fifty amino acids is known as a protein. “Peptides” are short polypeptides, typically containing more than 10-15 residues and fewer than 20-30 residues. “Oligopeptides” typically contain up to 10- 15 residues.

Antibodies, antigen-binding sites, and other immunogens

[00279] In certain embodiments, the targeting moiety and/or therapeutic pay load and methods recognize the presence of immunostimulant molecules, protein modulators, enzyme inhibitors, and drug pathway modulators as a therapeutic payload. In certain embodiments, the therapeutic payload and methods can regulate immune cells, such as cytotoxic T lymphocytes (CTL), Type 1 and 17 T helper cells (Thl & Thl7), dendritic cells (DC), regulatory T cells (Treg), natural killer cells (NKC), tumor-associated macrophages (TAM), myeloid-derived suppressor cells (MDSC), DNA, RNA, siRNA, miRNA, mRNA, and antibodies. The therapeutic payload can thereby modulate the highly immunosuppressive TME in which tumor cells proliferate and metastasis.

[00280] In some embodiments, the extracellular vesicle or the micelle further comprises a pharmaceutical composition, a cytokine or a chemokine, a nucleic acid, a targeting moiety, or a stabilizing agent.

[00281 ] In some embodiments, the modulating agent comprises an inhibitor or an activator

(e.g., a modified CAR-T activating domain). In some embodiments, the modulating agent comprises an immunomodulating agent. In some embodiment, the immunomodulating agent comprises an antibody, an antigen-binding domain, or an antigen.

[00282] In some embodiments, the targeting moiety comprises an antibody, an antigenbinding domain, or an antigen. [00283] Cytotoxic T-lymphocyte-associated protein 4 (CTLA4 or CTLA-4; CD152 [cluster of differentiation 152]) is a protein receptor that functions as an immune checkpoint and downregulates immune responses. CTLA-4 is constitutively expressed in regulatory T cells, but only upregulated in conventional T cells after activation. This situation is particularly observed in some cancers. For example, it can serve as an “off’ switch when bound to CD80 or CD86 on the surface of antigen-presenting cells.

[00284] In some embodiments, the extracellular vesicle or the micelle comprises an anti- CTLA-4 antibody or an anti-CTLA-4-antigen-binding domain. In some embodiments, the extracellular vesicle or the micelle comprises cytotoxic T-lymphocyte-associated protein 4 (CTLA-4).

[00285] Programmed cell death protein 1 (PD-1; cluster of differentiation 279 [CD279]), a cell surface protein, a member of the immunoglobulin superfamily, is expressed on T cells and pro-B cells and promotes self-tolerance by suppressing T-cell inflammatory activity, preventing autoimmune diseases, but also inhibiting the immune system from killing cancer cells. Programmed cell death protein 1 (PD-1; cluster of differentiation 279 [CD279]), is a cell surface protein that has a role in regulating the immune system's response to the cells of the human body by down-regulating the immune system and promoting self-tolerance by suppressing T cell inflammatory activity. An immune checkpoint protein, PD-1 promotes apoptosis of antigenspecific T-cells in lymph nodes and reduces apoptosis in regulatory T-cells (Tregs). PD-1 is a cell surface receptor that belongs to the immunoglobulin superfamily and is expressed on T cells and pro-B cells. PD-1 binds two ligands, programmed death-ligand 1 (PD-L1) and programmed death-ligand 2 (PD-L2).

[00286] In some embodiments, the extracellular composition or the micelle comprises an anti-PD-1 antibody or an anti-PD-1 -antigen-binding domain. In some embodiments, the extracellular composition or the micelle comprises programmed cell death protein 1 (PD-1).

[00287] Programmed death-ligand 1 (PD-L1) is highly expressed on the surface of cells of some types of cancers, including, but not limited to, melanoma, bladder cancer, and gastric cancer. As a result, PD-1 inhibitors block PD-1 and lower immune system activation when attacking tumors. [00288] In some embodiments, the extracellular composition or the micelle comprises an anti-PD-Ll antibody or an anti-PD-Ll-antigen-binding domain. In some embodiments, the extracellular composition or the micelle comprises programmed death-ligandl (PD-L1).

[00289] In some embodiments, the extracellular composition or the micelle comprises an anti-RANKL agent. In some embodiments, the anti-RANKL agent comprises an anti-RANKL antibody or antigen-binding domain.

[00290] In certain embodiments, the extracellular composition or the micelle comprises an anti-PD-1 antibody or an anti-PD-1 antigen-binding domain in combination with either (a) an anti-PD-Ll antibody or an anti-PD-Ll antigen-binding domain, (b) an anti-CTLA-4 antibody or an anti-CTLA-4 antigen-binding domain, or (c) an anti-RANKL antibody or an anti-RANKL antigen-binding domain.

[00291 ] In certain embodiments, the extracellular composition or the micelle comprises an immunogen, such as an antibody or other antigen-binding site moiety or an affinity reagent.

[00292] An “antibody,” an “antigen-binding site” or an “affinity reagent,” is a molecule that binds to an antigen or receptor or another molecule. In some embodiments, an antigenbinding site is a molecule that specifically binds to an antigen or receptor or other molecule. In certain embodiments, some or all of the immunogen is composed of amino acids (including natural, non-natural, and modified amino acids), nucleic acids, or saccharides. In certain embodiments, an antibody-binding site or affinity reagent or other immunogens or a small molecule. In certain embodiments, antibodies specifically bind to molecules or targets, such as a cell surface antigen, a cell surface receptor, or other cell surface molecule. Antibodies are discussed in more detail, infra.

[00293] In certain embodiments, the extracellular composition or the micelle comprises an immunogen, for example, an immunogenic antigen. An immunogen is an antigen or any substance that may be specifically bound by components of the immune system (e.g., antibody, lymphocytes). An immunogen is capable of inducing humoral or cell-mediated immune response rather than immunological tolerance. For example, the immunogen may be selected from the group consisting of keyhole limpet hemocyanin (KLH), concholepas concholepas hemacyanin (CCH), bovine serum albumin (BSA), and ovalbumin (OVA). Further information may be found in Chen et al. (2013) Immunity 39:1-10; and Chen et al. (2012) Clin Cancer Res. 18:6580-6587 (both incorporated by reference).

[00294] In some embodiments, the extracellular composition or the micelle comprises an antibody (including, e.g., IgG or IgM based or their truncated forms). In some embodiments, the antibody is attached to a pharmaceutical composition; in some embodiments, the antibody is attached to the chemokine or other cytokine; in some embodiment, the antibody is attached to a nucleic acid (e.g., an mRNA, an siRNA, an miRNA, an antisense DNA, or a cDNA); in some embodiments, the antibody is attached to a protein or peptide. In some embodiments, it is attached to another element in the composition, either directly or indirectly (e.g., via a linker). In some embodiments, the antibody targets a tumor cell or a cancer cell. Essentially, the antibody specifically targets a tumor or cancer antigen of interest on a tumor cell of interest or a cancer cell of interest, respectively. The antibody binds to the antigen on the surface of the tumor cell or cancer cell. The specific targeting of the tumor cell or the cancer cell reduces side effects. Some embodiments include targeting by an antibody to provide specific discrimination of the target cell for targeting of, e.g., a pharmaceutical composition, a cytokine, a chemokine, a nucleic acid, or a protein or peptide. Some embodiments include targeting by an antibody and a cytokine or chemokine to provide even more specific discrimination of the target cell. Antibody linkers include, but are not limited to disulfides, hydrazones, peptides, or thioethers. In some embodiments, they are cleavable (e.g., peptides), while in other embodiments, they are non- cleavable (e.g., thioethers). Cleavable linkers may be engineered to be enzyme-sensitive. Non- cleavable linkers typically offer increased stability and maintain the drug within the cell. Longer linkers provide greater physical flexibility in the linker region, potentially altering cleavage kinetics.

[00295] In some embodiments, the extracellular composition or the micelle comprises an antibody-drug conjugate (ADC). An “antibody-drug conjugate” comprises an antibody and a drug, optionally joined by an “ADC linker.” In some embodiments, the ADC comprises an antibody that targets a tumor cell or a cancer cell, and the drug comprises a cytotoxic drug that destroys the tumor cell or the cancer cell. This type of bioconjugate/immunoconjugate combines the targeting capability of a monoclonal antibody with the tumor cell-destroying or the cancer cell-destroying ability of a cytotoxic drug. Essentially, the antibody specifically targets a tumor antigen of interest or cancer antigen of interest on a tumor cell of interest or cancer cell of interest, respectively. The antibody-drug binds to the antigen on the surface of the tumor cell or cancer cell, and in turn, kills the tumor cell or the cancer cell. Some embodiments include targeting by both the ADC and a cytokine or chemokine, thereby providing even more specific discrimination of the target cell. ADC linkers include, but are not limited to disulfides, hydrazones, peptides, or thioethers. In some embodiments, they are cleavable (e.g., peptides), while in other embodiments, they are non-cleavable (e.g., thioethers). Cleavable ADC linkers may be engineered to be enzyme-sensitive. Non-cleavable ADC linkers typically offer increased stability and maintain the drug within the cell. Longer ADC linkers provide greater physical flexibility in the ADC linker region, potentially altering cleavage kinetics.

[00296] In some embodiments, an antigen-binding domain may be comprised of proteinaceous structures other than antibodies that are able to bind to protein targets specifically, including but not limited to avimers, ankyrin repeats and adnectins, and other such proteins with domains that can be evolved to generate specific affinity for antigens, collectively referred to as “antibody-like molecules.” Modifications of proteinaceous affinity reagents through the incorporation of unnatural amino acids during synthesis may be used to improve their properties. Such modifications may have several benefits, including the addition of chemical groups that facilitate subsequent conjugation reactions. In some embodiments, the antigen-binding domain may be a peptide. In some embodiments, the peptide chain is a bispecific peptide. Peptides can readily be made and screened to create affinity reagents that recognize and bind to macromolecules such as proteins.

[00297] Bispecific affinity reagents may be constructed by separate synthesis and expression of the first and second affinity reagents. A polypeptide bispecific reagent can be expressed as two separately encoded chains that are linked by disulfide bonds during production in the same host cell, such as, for example, a single chain variable fragment (scFv), a “bispecific single chain variable fragment” (“diabody”; “dibody”), or a trispecific single chain variable fragment (“triabody”; “tribody”). Similarly, standard and widely used solid-phase peptide synthesis technology can be used to synthesize peptides, and chimeric bispecific peptides are well known in the art. A bispecific peptide strategy may be used to combine the first and second affinity reagents in a single peptide chain. Alternatively, polypeptide chains or peptide chains can be expressed/synthesized separately, purified and then conjugated chemically to produce the bispecific affinity reagents useful in the compositions and methods described herein. Many different formats of antibodies may be used. Whole antibodies, F(ab')2, F(ab'), scFv, as well as smaller Fab and single-domain antibody fragments may all be used to create the first and second affinity reagents. Following their expression and purification, the targeting moieties can be chemically conjugated to the protein vehicle. Many conjugation chemistries may be used to effect this conjugation, including homofunctional or heterofunctional linkers that yield ester, amide, thioether, carbon-carbon, or disulfide linkages.

[00298] In some embodiments, a peptide aptamer is included. A peptide aptamer is a peptide molecule that specifically binds to a target protein and interferes with the functional ability of that target protein. Peptide aptamers consist of a variable peptide loop attached at both ends of a protein scaffold. Such peptide aptamers can often have a binding affinity comparable to that of an antibody (nanomolar range). Due to the highly selective nature of peptide aptamers, they can be used not only to target a specific protein, but also to target specific lunctions of a given protein (e.g., a signaling function). Peptide aptamers are usually prepared by selecting the aptamer for its binding affinity with the specific target from a random pool or library of peptides. Peptide aptamers can be isolated from random peptide libraries by yeast two-hybrid screens. They can also be isolated from phage libraries or chemically generated peptides/libraries.

[00299] In certain embodiments, the extracellular composition or the micelle further comprises an adjuvant. In certain embodiments, the extracellular composition or the micelle comprises an immunogen and an adjuvant: recruiting of professional antigen-presenting cells (APCs) to the site of antigen exposure; increasing the delivery of antigens by delayed/slow release (depot generation); immunomodulation by cytokine production (selection of Thl or Th2 response); inducing T-cell response (prolonged exposure of peptide-MHC complexes [signal 1] and stimulation of expression of T-cell-activating co-stimulators [signal 2] on the APCs' surface) and targeting (e. g. carbohydrate adjuvants which target lectin receptors on APCs). Examples of adjuvants include, but are not limited to Freund's Complete Adjuvant, lipopolysaccharides, muramyldipeptide from TB, synthetic polynucleotides, aluminum hydroxide, aluminum phosphate, cytokines, and squalene.

[00300] As used herein, an “antibody,” an “antigen-binding site” or an “affinity reagent,” is a molecule that binds to an antigen or receptor or another molecule. In some embodiments, an antibody, an antigen-binding site, an affinity reagent, or other immunogen is a molecule that specifically binds to an antigen or receptor or other molecule. In certain embodiments, some or all of an antibody, antigen-binding site, affinity reagent, or immunogen is composed of amino acids (including natural, non-natural, and modified amino acids), nucleic acids, or saccharides. In certain embodiments, an antigen-binding site, affinity reagent, or immunogen is a small molecule. In certain embodiments, antibodies specifically bind to molecules or targets, such as a cell surface antigen, a cell surface receptor, or other cell surface molecule.

[00301] As used herein, the term “antibody” encompasses the structure that constitutes the natural biological form of an antibody. In most mammals, including humans, and mice, this form is a tetramer and consists of two identical pairs of two immunoglobulin chains, each pair having one light and one heavy chain, each light chain comprising immunoglobulin domains VL and CL, and each heavy chain comprising immunoglobulin domains VH, C-gamma-1 (Cyl), C-gamma-2 (Cy2), and C-gamma-3 (Cy3). In each pair, the light and heavy chain variable regions (VL and VH) are together responsible for binding to an antigen, and the constant regions (CL, Cyl, Cy2, and Cy3, particularly Cy2, and Cy3) are responsible for antibody effector functions. In some mammals, for example in camels and llamas, full-length antibodies may consist of only two heavy chains, each heavy chain comprising immunoglobulin domains VH, Cy2, and Cy3. By “immunoglobulin (Ig)” herein is meant a protein consisting of one or more polypeptides substantially encoded by immunoglobulin genes. Immunoglobulins include but are not limited to antibodies. Immunoglobulins may have a number of structural forms, including but not limited to full-length antibodies, antibody fragments, and individual immunoglobulin domains including but not limited to VH, Cyl, Cy2, Cy3, VL, and CL.

[00302] Depending on the amino acid sequence of the constant domain of their heavy chains, intact antibodies can be assigned to different “classes”. There are five-major classes (isotypes) of intact antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into “subclasses”, e.g., IgGl, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy-chain constant domains that correspond to the different classes of antibodies are called alpha, delta, epsilon, gamma, and mu, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are known to one skilled in the art.

[00303] As used herein, the term “immunoglobulin G” or “IgG” refers to a polypeptide belonging to the class of antibodies that are substantially encoded by a recognized immunoglobulin gamma gene. In humans this class comprises IgGl, IgG2, IgG3, and IgG4. In mice this class comprises IgGl, IgG2a, IgG2b, IgG3. As used herein, the term “modified immunoglobulin G” refers to a molecule that is derived from an antibody of the “G” class. As used herein, the term “antibody” refers to a protein consisting of one or more polypeptides substantially encoded by all or part of the recognized immunoglobulin genes. The recognized immunoglobulin genes, for example in humans, include the kappa (K), lambda (X), and heavy chain genetic loci, which together comprise the myriad variable region genes, and the constant region genes mu (p), delta (5), gamma (y), sigma (o), and alpha (a) which encode the IgM, IgD, IgG, IgE, and IgA isotypes or classes, respectively.

[00304] The term “antibody” is meant to include full-length antibodies and may refer to a natural antibody from any organism, an engineered antibody, or an antibody generated recombinantly for experimental, therapeutic, or other purposes as further defined below. Furthermore, full-length antibodies comprise conjugates as described and exemplified herein. As used herein, the term “antibody” comprises monoclonal antibodies (mAb, moAb) and polyclonal antibodies. Antibodies can be antagonists, agonists, neutralizing, inhibitory, or stimulatory. Specifically included within the definition of “antibody” are full-length antibodies described and exemplified herein. By “full length antibody” herein is meant the structure that constitutes the natural biological form of an antibody, including variable and constant regions.

[00305] A “monoclonal antibody” (mAb, moAb) is an antibody made by cloning a unique white blood cell. All subsequent antibodies derived this way trace back to a unique parent cell. Monoclonal antibodies can have monovalent affinity, binding only to the same epitope (the part of an antigen that is recognized by the antibody). In contrast, “polyclonal antibodies” bind to multiple epitopes and are usually made by several different antibody secreting plasma cell lineages. “Bispecific monoclonal antibodies” can also be engineered, by increasing the therapeutic targets of one monoclonal antibody to two epitopes.

[00306] The “variable region” of an antibody contains the antigen binding determinants of the molecule, and thus determines the specificity of an antibody for its target antigen. The variable region is so named because it is the most distinct in sequence from other antibodies within the same isotype. The majority of sequence variability occurs in the complementarity determining regions (CDRs). There are 6 CDRs total, three each per heavy and light chain, designated VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3. The variable region outside of the CDRs is referred to as the framework (FR) region. Although not as diverse as the CDRs, sequence variability does occur in the FR region between different antibodies. Overall, this characteristic architecture of antibodies provides a stable scaffold (the FR region) upon which substantial antigen binding diversity (the CDRs) can be explored by the immune system to obtain specificity for a broad array of antigens.

[00307] Furthermore, antibodies may exist in a variety of other forms including, for example, Fv, Fab, and (Fab’)2, as well as bi-functional (i.e., bi-specific) hybrid antibodies (e.g., Lanzavecchia et al., (1987) Eur. J. Immunol. 17:105) and in single chains (e.g., Huston et al. (1988) Proc. Natl. Acad. Sci. U.S.A. 85: 5879-5883 and Bird et al. (1988) Science 242: 423-426 (and related Erratum (1989) Science 244: 409), which are incorporated herein by reference). (See, generally, Hood et al., “Immunology”, Benjamin, N.Y., 2nd ed. (1984), and Hunkapiller et al. 1(1986) Nature 323: 15-16). Bispecific antibodies are a technique for creating a single polypeptide that binds to two different determinants. Bispecific antibodies may be made in many different formats, including but not limited to quadroma, F(ab')2, tetravalent, heterodimeric scFv, bispecific scFv, tandem scFv, diabody and minibody formats, or scFvs appended to or recombinantly fused with whole antibodies.

[00308] The term “epitope” as used herein refers to a region of the antigen that binds to the antibody or antigen-binding fragment. It is the region of an antigen recognized by a first antibody wherein the binding of the first antibody to the region prevents binding of a second antibody or other bivalent molecule to the region. The region encompasses a particular core sequence or sequences selectively recognized by a class of antibodies. In general, epitopes are comprised by local surface structures that can be formed by contiguous or noncontiguous amino acid sequences.

[00309] As used herein, the terms “selectively recognizes”, “selectively bind” or “selectively recognized” mean that binding of the antibody, antigen-binding fragment or other bivalent molecule to an epitope is at least 2-fold greater, preferably 2-5 fold greater, and most preferably more than 5 -fold greater than the binding of the molecule to an unrelated epitope or than the binding of an antibody, antigen-binding fragment or other bivalent molecule to the epitope, as determined by techniques known in the art and described herein, such as, for example, ELISA or cold displacement assays.

[00310] As used herein, the term “Fc domain” encompasses the constant region of an immunoglobulin molecule. The Fc region of an antibody interacts with a number of Fc receptors and ligands, imparting an array of important functional capabilities referred to as effector functions, as described herein. For IgG the Fc region comprises Ig domains CH2 and CH3. An important family of Fc receptors for the IgG isotype are the Fc gamma receptors (FcyRs). These receptors mediate communication between antibodies and the cellular arm of the immune system.

[00311] As used herein, the term “Fab domain” encompasses the region of an antibody that binds to antigens. The Fab region is composed of one constant and one variable domain of each of the heavy and the light chains.

[00312] In one embodiment, the term “antibody” or “antigen-binding fragment” respectively refer to intact molecules as well as functional fragments thereof, such as Fab, a scFv- Fc bivalent molecule, F(ab’)2, and Fv that are capable of specifically interacting with a desired target. In some embodiments, the antigen-binding fragments comprise:

[00313] (1) Fab, the fragment which contains a monovalent antigen-binding fragment of an antibody molecule, which can be produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain;

[00314] (2) Fab’, the fragment of an antibody molecule that can be obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain; two Fab’ fragments are obtained per antibody molecule;

[00315] (3) (Fab’)2, the fragment of the antibody that can be obtained by treating whole antibody with the enzyme pepsin without subsequent reduction; F(ab’)2 is a dimer of two Fab’ fragments held together by two disulfide bonds;

[00316] (4) Fv, a genetically engineered fragment containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains; and [00317] (5) Single chain antibody (“SCA”), a genetically engineered molecule containing the variable region of the light chain and the variable region of the heavy chain, linked by a suitable polypeptide linker as a genetically fused single chain molecule.

[00318] (6) scFv-Fc, is produced in one embodiment, by lusing single-chain Fv (scFv) with a hinge region from an immunoglobulin (Ig) such as an IgG, and Fc regions.

[00319] In some embodiments, an antibody provided herein is a monoclonal antibody. In some embodiments, the antigen-binding fragment provided herein is a single chain Fv (scFv), a diabody, a tri(a)body, a di-or tri-tandem scFv, a scFv-Fc bivalent molecule, an Fab, Fab’, Fv, F(ab’)2 or an antigen binding scaffold (e.g., affibody, monobody, anticalin, DARPin, Knottin, etc.). “Affibodies” are small proteins engineered to bind to a large number of target proteins or peptides with high affinity, often imitating monoclonal antibodies, and are antibody mimetics.

[00320] As used herein, the terms “bivalent molecule” or “B V” refer to a molecule capable of binding to two separate targets at the same time. The bivalent molecule is not limited to having two and only two binding domains and can be a polyvalent molecule or a molecule comprised of linked monovalent molecules. The binding domains of the bivalent molecule can selectively recognize the same epitope or different epitopes located on the same target or located on a target that originates from different species. The binding domains can be linked in any of a number of ways including, but not limited to, disulfide bonds, peptide bridging, amide bonds, and other natural or synthetic linkages known in the art (Spatola et al., “Chemistry and Biochemistry of Amino Acids, Peptides and Proteins,” B. Weinstein, eds., Marcel Dekker, New York, p. 267 (1983); Morley (1980) Trends Pharrn Sci. 463-468; Hudson et al. (1979) Int. J. Pept. Prot. Res. 14: 177-185; Spatola et al. (1986) Life Sci. 38: 1243-1249; Hann (1982) J. Chem. Soc. Perkin Trans. I 307-314; Almquist et al. (1980) J. Med. Chem. 23: 1392-1398; Jennings-White et al. (1982) Tetrahedron Lett. 23: 2533-2534; Szelke et al., European Application EP 45665 Bl; Szelke et al. US Pat. 4,424,407; Chemical Abstracts 97, 39405 (1982); Holladay et al. (1983) Tetrahedron Lett. 24: 4401-4404; and Hruby (1982) Life Sci. 31: 189-199).

[00321] As used herein, the terms “binds” or “binding” or grammatical equivalents, refer to compositions having affinity for each other. “Specific binding” is where the binding is selective between two molecules. A particular example of specific binding is that which occurs between an antibody and an antigen. Typically, specific binding can be distinguished from non-specific when the dissociation constant (KD) is less than about 1x10-5 M or less than about 1x10-6 M or 1x10-7 M. Specific binding can be detected, for example, by ELISA, immunoprecipitation, coprecipitation, with or without chemical crosslinking, two-hybrid assays and the like. Appropriate controls can be used to distinguish between “specific” and “non-specific” binding.

[00322] In addition to antibody sequences, an antibody according to the present invention may comprise other amino acids, e.g., forming a peptide or polypeptide, such as a folded domain, or to impart to the molecule another functional characteristic in addition to ability to bind antigen. For example, antibodies of the invention may carry a detectable label, such as fluorescent or radioactive label, or may be conjugated to a toxin (such as a holotoxin or a hemitoxin) or an enzyme, such as beta-galactosidase or alkaline phosphatase (e.g., via a peptidyl bond or linker).

[00323] In one embodiment, an antibody of the invention comprises a stabilized hinge region. The term "stabilized hinge region" will be understood to mean a hinge region that has been modified to reduce Fab arm exchange or the propensity to undergo Fab arm exchange or formation of a half-antibody or a propensity to form a half-antibody. "Fab arm exchange" refers to a type of protein modification for human immunoglobulin, in which a human immunoglobulin heavy chain and attached light chain (half-molecule) is swapped for a heavy-light chain pair from another human immunoglobulin molecule. Thus, human immunoglobulin molecules may acquire two distinct Fab arms recognizing two distinct antigens (resulting in bispecific molecules). Fab arm exchange occurs naturally in vivo and can be induced in vitro by purified blood cells or reducing agents such as reduced glutathione. A "half-antibody" forms when a human immunoglobulin antibody dissociates to form two molecules, each containing a single heavy chain and a single light chain. In one embodiment, the stabilized hinge region of human immunoglobulin comprises a substitution in the hinge region.

[00324] In one embodiment, the term "hinge region" as used herein refers to a proline-rich portion of an immunoglobulin heavy chain between the Fc and Fab regions that confers mobility on the two Fab arms of the antibody molecule. It is located between the first and second constant domains of the heavy chain. The hinge region includes cysteine residues which are involved in inter-heavy chain disulfide bonds. In one embodiment, the hinge region includes cysteine residues which are involved in inter-heavy chain disulfide bonds. [00325] In some embodiments, the present invention comprises a first component protein comprising a first binding pair partner and a second component protein comprising a second binding pair partner, wherein the binding pair partners comprise two protein moieties that form a heterodimer.

[00326] A “dimer” is a macromolecular complex formed by two macromolecules, usually proteins (or portions thereof) or nucleic acids (or portions thereof). A “homodimer” is formed by two identical macromolecules (“homodimerization”), while a “heterodimer” is formed by two distinct macromolecules (“heterodimerization”). Many dimers are non-covalently linked, but some (e.g., NEMO homodimers) can link via, e.g., disulfide bonds. Some proteins comprise regions specialized for dimerization, known as “dimerization domains.” In some instances, a truncated protein containing or comprising a dimerization domain (or two truncated proteins containing or comprising corresponding dimerization domains) may be able to interact in the absence of one or both complete protein sequence(s). Similarly, a lusion protein comprising a dimerization domain (or two fusion proteins comprising corresponding dimerization domains) may be able to interact in the absence of one or both complete protein sequence(s). Mutations to these domains may increase, or alternatively reduce, the formation of a dimer. Examples of macromolecules that can form dimers include, but are not limited to, proteins, nucleic acids, antibodies, receptor tyrosine kinases, proteins with leucine zippers, peptide Velcro, nuclear receptors, 14-3-3 proteins, G proteins, G protein-coupled receptors, transcription factors, kinesin, triosephosphate isomerase (TIM), alcohol dehydrogenase, Toll-like receptors, fibrinogen, tubulin, some glycoproteins, and some clotting factors. Additional examples of particular pairs include, but are not limited to, c-Jun/c-Fos, RelA (or c-Rel or RelB)/p50 (or p51) (Rel/NF-kappaB), AP- 1, C/EBP, ATF/CREB, c-Myc, and NF-1.

[00327] The cell surface antigen may be any cell surface molecule that undergoes internalization, such as a protein, sugar, lipid head group or other antigen on the cell surface. Examples of cell surface antigens useful in the context of the invention include but are not limited to the transferrin receptor type 1 and 2, the EGF receptor (e.g., IMC-225), HER2/Neu (e.g., tastuzumab or pertuzumab), VEGF receptors, integrins, CD33, CD19, CD20, CD22, CD4 and the asialoglycoprotein receptor. [00328] In certain embodiments, the construct relates to any of the compositions described herein, wherein the antibody is an anti-PD-1 antibody or wherein the antigen-binding site is an anti-PD-1 antigen-binding site. In certain embodiments, the construct relates to any of the compositions described herein, wherein the antibody is an anti-PD-1 antibody or wherein the antigen-binding site is an anti-PD-1 antigen-binding site, each either alone or in combination with either (a) an anti-PD-Ll antibody or an anti-PD-Ll antigen-binding site or (b) an anti-CTLA-4 antibody or an anti-CTLA-4 antigen binding site.

[00329] Antibodies for use in the invention may be raised through any conventional method, such as through injection of immunogen into mice and subsequent fusions of lymphocytes to create hybridomas. Such hybridomas may then be used either (a) to produce antibody directly, which is purified and used for chemical conjugation to create a bispecific antibody, or (b) to clone cDNAs encoding antibody fragments for subsequent genetic manipulation. To illustrate one method employing the latter strategy, mRNA is isolated from the hybridoma cells, reverse-transcribed into cDNA using antisense oligo-dT or immunoglobulin gene-specific primers and cloned into a plasmid vector. Clones are sequenced and characterized. They may then be engineered according to standard protocols to combine the heavy and light chains of each antibody, separated by a short peptide linker, into a bacterial or mammalian expression vector as previously described to produce a recombinant bispecific antibody, which are then expressed and purified according to well-established protocols in bacteria or mammalian cells. Antibodies, or other proteinaceous affinity molecules or targeting moieties such as peptides, may also be created through display technologies that allow selection of interacting affinity reagents through the screening of very large libraries of, for example, immunoglobulin domains or peptides expressed by bacteriophage. Antibodies may also be humanized through grafting of human immunoglobulin domains or made from transgenic mice or bacteriophage libraries that have human immunoglobulin genes/cDNAs.

[00330] In some embodiments, a nucleic acid aptamer is included. Nucleic acid aptamers are nucleic acid oligomers that bind other macromolecules specifically; such aptamers that bind specifically to other macromolecules can be readily isolated from libraries of such oligomers by technologies such as SELEX. [00331] In some embodiments, an oligosaccharide is included. Certain oligosaccharides are known ligands for certain extracellular or cell surface receptors.

[00332] In one embodiment, the antibody or antigen-binding fragment binds its target with a KD of 0.1 nM - 10 mM. In one embodiment, the antibody or antigen-binding fragment binds its target with a KD of 0.1 nM - 1 mM. In one embodiment, the antibody or antigen-binding fragment binds its target with a KD within the 0.1 nM range. In one embodiment, the antibody or antigenbinding fragment binds its target with a KD of 0.1-2 nM. In another embodiment, the antibody or antigen-binding fragment binds its target with a KD of 0.1-1 nM. In another embodiment, the antibody or antigen-binding fragment binds its target with a KD of 0.05-1 nM. In another embodiment, the antibody or antigen-binding fragment binds its target with a KD of 0.1-0.5 nM. In another embodiment, the antibody or antigen-binding fragment binds its target with a KD of 0.1-0.2 nM.

[00333] In some embodiments, the antibody or antigen-binding fragment thereof provided herein comprises a modification. In another embodiment, the modification minimizes conformational changes during the shift from displayed to secreted forms of the antibody or antigen-binding fragment. It is to be understood by a skilled artisan that the modification can be a modification known in the art to impart a functional property that would not otherwise be present if it were not for the presence of the modification. Encompassed are antibodies which are differentially modified during or after translation, e.g., by glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, etc. Any of numerous chemical modifications may be carried out by known techniques, including but not limited, to specific chemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, NaBH4, acetylation, formylation, oxidation, reduction, metabolic synthesis in the presence of tunicamycin, etc.

[00334] In some embodiments, the modification is one as further defined herein below. In some embodiments, the modification is an N-terminus modification. In some embodiments, the modification is a C-terminal modification. In some embodiments, the modification is an N- terminus biotinylation. In some embodiments, the modification is a C-terminus biotinylation. In some embodiments, the secretable form of the antibody or antigen-binding fragment comprises an N-terminal modification that allows binding to an Immunoglobulin (Ig) hinge region. In some embodiments, the Ig hinge region is from but is not limited to, an IgA hinge region. In some embodiments, the secretable form of the antibody or antigen-binding fragment comprises an N- terminal modification that allows binding to an enzymatically biotinylatable site. In some embodiments, the secretable form of the antibody or antigen-binding fragment comprises a C- terminal modification that allows binding to an enzymatically biotinylatable site. In some embodiments, biotinylation of said site functionalizes the site to bind to any surface coated with streptavidin, avidin, avidin-derived moieties, or a secondary reagent.

[00335] It will be appreciated that the term “modification” can encompass an amino acid modification such as an amino acid substitution, insertion, and/or deletion in a polypeptide sequence.

[00336] In one embodiment, a variety of radioactive isotopes are available for the production of radioconjugate antibodies and other proteins and can be of use in the methods and compositions provided herein. Examples include, but are not limited to, At211, Cu64, 1131, 1125, Y90, Rel86, Rel88, Sml53, Bi212, P32, Zr89, F-18, 1-124 and radioactive isotopes of Lu. In a further embodiment, the amino acid sequences of the invention may be homologues, variants, isoforms, or fragments of the sequences presented. The term "homolog" as used herein refers to a polypeptide having a sequence homology of a certain amount, namely of at least 70%, e.g., at least 80%, 90%, 95%, 96%, 97%, 98%, 99% of the amino acid sequence it is referred to. Homology refers to the magnitude of identity between two sequences. Homolog sequences have the same or similar characteristics, in particular, have the same or similar property of the sequence as identified. The term 'variant' as used herein refers to a polypeptide wherein the amino acid sequence exhibits substantially 70, 80, 95, or 99% homology with the amino acid sequence as set forth in the sequence listing. It should be appreciated that the variant may result from a modification of the native amino acid sequences, or by modifications including insertion, substitution or deletion of one or more amino acids. The term “isoform” as used herein refers to variants of a polypeptide that are encoded by the same gene, but that differ in their isoelectric point (pl) or molecular weight (MW), or both. Such isoforms can differ in their amino acid composition (e.g., as a result of alternative splicing or limited proteolysis) and in addition, or in the alternative, may arise from differential post-translational modification (e.g., glycosylation, acylation, phosphorylation deamidation, or sulphation). As used herein, the term “isoform” also refers to a protein that exists in only a single form, i.e., it is not expressed as several variants. The term "fragment" as used herein refers to any portion of the full-length amino acid sequence of protein of a polypeptide of the invention which has less amino acids than the full-length amino acid sequence of a polypeptide of the invention. The fragment may or may not possess a functional activity of such polypeptides.

[00337] In an alternate embodiment, enzymatically active toxin or fragments thereof that can be used in the compositions and methods provided herein include, but are not limited, to diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, Sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes.

[00338] A chemotherapeutic or other cytotoxic agent may be conjugated to the protein, according to the methods provided herein, as an active drug or as a prodrug. The term “prodrug” refers to a precursor or derivative form of a pharmaceutically active substance that is less cytotoxic to tumor cells compared to the parent drug and is capable of being enzymatically activated or converted into the more active parent form. (See, for example Wilman, 1986, Biochemical Society Transactions, 615th Meeting Belfast, 14:375-382; and Stella et al., “Prodrugs: A Chemical Approach to Targeted Drug Delivery,” Directed Drug Delivery, Borchardt et al., (ed.): 247-267, Humana Press, 1985.) The prodrugs that may find use with the compositions and methods as provided herein include but are not limited to phosphate-containing prodrugs, thiophosphate-containing prodrugs, sulfate-containing prodrugs, peptide-containing prodrugs, D- amino acid-modified prodrugs, glycosylated prodrugs, beta-lactam-containing prodrugs, optionally substituted phenoxyacetamide-containing prodrugs or optionally substituted phenylacetamide-containing prodrugs, 5 -fluorocytosine and other 5 -fluorouridine prodrugs which can be converted into the more active cytotoxic free drug. Examples of cytotoxic drugs that can be derivatized into a prodrug form for use with the antibodies and Fc fusions of the compositions and methods as provided herein include but are not limited to any of the aforementioned chemotherapeutic.

Nucleic Acids [00339] In some embodiments, the extracellular vesicle or the micelle comprises a nucleic acid. In some embodiments, the therapeutic pay load comprises a nucleic acid. In some embodiments, the extracellular vesicle comprises an encoded therapeutic pay load. In some embodiments, the targeting moiety comprises a nucleic acid. In some embodiments, the extracellular vesicle comprises a plasmid encoding the targeting moiety. In some embodiments, the extracellular vesicle comprises a plasmid encoding the therapeutic payload fused, chemically attached, or linked to a targeting moiety. In some embodiments, the plasmid further comprises a selectable marker. In some embodiments, the plasmid further comprises a fluorescent marker or other marker as described herein.

[00340] In other embodiments, the therapeutic payload is fused, chemically attached, or linked (e.g., via a linker) to a targeting moiety. In a non-limiting example, a programmed death antibody (e.g., PD-1, and PD-L1) is used as a therapeutic payload and is fused, chemically attached, or linked to a cancer-targeting moiety. In other embodiments, the targeting moiety is a light chain of clathrin and analogs.

[00341] In some embodiments, the composed combination (therapeutic payload and targeting moiety) is expressed in an expression plasmid. In some embodiments, the described plasmid comprises a selectable marker (e.g., a fluorescent dye marker or other cell surface markers as described herein) attached to the protein to differentiate the produced exosome. In other embodiments, the fluorescent marker is linked to another component in the assembled plasmid, as described herein. In other embodiments, the plasmid production is as described herein (see, e g., FIGURE 1).

[00342] In some embodiments, the plasmid is transfected by methods known in the art and expressed in a cell selected for its ability to form exosomes or other extracellular vesicles.

[00343] In some embodiments, the exosomes or other extracellular vesicles are formed artificially as described herein.

[00344] In other embodiments, the fluorescent plasmid is transfected by methods known in the art and expressed in HEK cells. In a further embodiment, the exosomes released from the transfected HEK cells are collected, and the fluorescent exosomes comprising the targeted treatment composition are separated. In other embodiments, the exosomes are used for treating, reducing, inhibiting, alleviating, or ameliorating a tumor or a cancer, or a growth or metastasis thereof in a subject in need thereof.

[00345] Tumor-cell-specific targeting moieties such as antibodies, aptamers, and scFv target proteins expressed by various tumor cells, such as programmed death receptors PD-1, CTLA-4, TGF-J3, EGFR, VEGFR, and more. In certain embodiments, the above target proteins or other tumor-cell-specific molecules can be targeted using nucleic acid aptamers engineered using systematic evolution of ligands by exponential enrichment (SELEX) methods. SELEX (in vitro selection, in vitro evolution, SAAB [selected and amplified binding site], CASTing [cyclic amplification and selection of targets]), is a combinatorial chemistry technique in molecular biology for producing oligonucleotides of either single-stranded DNA or RNA that specifically bind to a target ligand or ligands. These single-stranded DNA or RNA are commonly referred to as aptamers. Engineered or selected aptamers bind their target molecules through electrostatic interactions, hydrophobic interactions, and complementary shapes. Aptamers are engineered using in vitro selection to identify and select aptamers that bind specific target peptides and whole tumor cells.

[00346] In certain embodiments, the targeting moiety and/or therapeutic payload and methods recognize the presence of immunostimulant molecules, protein modulators, enzyme inhibitors, and drug pathway modulators as a therapeutic payload. In certain embodiments, the therapeutic payload and methods can regulate immune cells, such as cytotoxic T lymphocytes (CTL), Type 1 and 17 T helper cells (Thl & Thl7), dendritic cells (DC), regulatory T cells (Treg), natural killer cells (NKC), tumor-associated macrophages (TAM), myeloid-derived suppressor cells (MDSC), DNA, RNA, siRNA, miRNA, mRNA, and antibodies. The therapeutic payload can thereby modulate the highly immunosuppressive TME in which tumor cells proliferate and metastasis.

[00347] In some embodiments, the extracellular vesicle or the micelle further comprises a therapeutic payload or a targeting moiety. In some embodiments, the extracellular vesicle or the micelle further comprises a stabilizing agent.

[00348] In some embodiments, the therapeutic payload comprises a nucleic acid. In some embodiments, the targeting moiety comprises a nucleic acid. [00349] In particular, the nucleic acid sequence, amino acid sequence, functional domain, structural domain, gene locus, and other identifying information for the signaling pathway targets described herein are well known in the art.

[00350] In some embodiments, the therapeutic payload comprises a nucleic acid (e.g., a deoxyribonucleic acid [DNA] or a ribonucleic acid [RNA]). In some embodiments, the targeting moiety comprises a nucleic acid (e.g., a deoxyribonucleic acid [DNA] or a ribonucleic acid [RNA]). In some embodiments, the nucleic acid is double-stranded (ds); in some embodiments, the nucleic acid is single-stranded (ss). In some embodiments, the nucleic acid comprises an antisense nucleic acid or a portion thereof. In some embodiments, the nucleic acid comprises an oligonucleotide.

[00351] In some embodiments, the nucleic acid comprises a DNA. In some embodiments, the DNA comprises a genomic DNA or a portion thereof or a complementary DNA (cDNA) or a portion thereof. In some embodiments, the DNA is double-stranded; in some embodiments, the DNA is single- stranded. In some embodiments, the nucleic acid comprises an antisense DNA. In some embodiment, the nucleic acid comprises a single-stranded antisense DNA.

[00352] In some embodiments, the nucleic acid comprises an RNA. In some embodiments, the RNA comprises a messenger RNA (mRNA), a small interfering RNA (siRNA), or a microRNA (miRNA). In some embodiments, the RNA is double-stranded; in some embodiments, the RNA is single-stranded. In certain embodiments, the therapeutic payload or the targeting moiety comprises an siRNA moiety comprised of a sense strand and an antisense strand; the sense strand comprising a 3' end and a 5' end; and the antisense strand comprising a 3' end and a 5' end.

[00353] “Antisense” nucleic acids refer to nucleic acids that specifically hybridize (e.g., bind) with a complementary sense nucleic acid, e.g., cellular mRNA and/or genomic DNA, under cellular conditions so as to inhibit expression (e.g., by inhibiting transcription and/or translation). The binding may be by conventional base pair complementarity or, for example, in the case of binding to DNA duplexes, through specific interactions in the major groove of the double helix.

[00354] Antisense technology is the process in which an antisense RNA or DNA molecule interacts with a target sense DNA or RNA strand. A sense strand is a 5' to 3' mRNA molecule or DNA molecule. The complementary strand, or mirror strand, to the sense is called an antisense. When an antisense strand interacts with a sense mRNA strand, the double helix is recognized as foreign to the cell and will be degraded, resulting in reduced or absent protein production. Although DNA is already a double-stranded molecule, antisense technology can be applied to it, building a triplex formation.

[00355] One skilled in the art would appreciate that the terms “complementary” or “complement thereof’ are used herein to encompass the sequences of polynucleotides which is capable of forming Watson & Crick base pairing with another specified polynucleotide throughout the entirety of the complementary region. This term is applied to pairs of polynucleotides based solely upon their sequences and not any particular set of conditions under which the two polynucleotides would actually bind.

[00356] RNA antisense strands can be either catalytic or non-catalytic. The catalytic antisense strands, also called ribozymes, cleave the RNA molecule at specific sequences. A non- catalytic RNA antisense strand blocks further RNA processing.

[00357] Antisense modulation of cells and/or tissue levels of the globulin genes of interest and/or desaturase genes of interest or any combination thereof may be effected by transforming the organism’s cells or tissues with at least one antisense compound, including antisense DNA, antisense RNA, a ribozyme, DNAzyme, a locked nucleic acid (LNA) and an aptamer. In some embodiments the molecules are chemically modified. In other embodiments the antisense molecule is antisense DNA or an antisense DNA analog.

[00358] Antisense modulation of cells and/or tissue levels of the globulin genes of interest and/or desaturase genes of interest or any combination thereof may be effected by transforming the organism’s cells or tissues with at least one antisense compound, including antisense DNA, antisense RNA, a ribozyme, DNAzyme, a locked nucleic acid (LNA), and an aptamer. In some embodiments, the molecules are chemically modified. In other embodiments, the antisense molecule is antisense DNA or an antisense DNA analog.

[00359] The term “RNA interference” or “RNAi” refers to the silencing or decreasing of gene expression mediated by small double stranded RNAs. It is the process of sequence-specific, post-transcriptional gene silencing in animals and plants, initiated by inhibitory RNA (iRNA) that is homologous in its duplex region to the sequence of the silenced gene. The gene may be endogenous or exogenous to the organism, present integrated into a chromosome or present in a transfection vector that is not integrated into the genome. The expression of the gene is either completely or partially inhibited. RNAi may also be considered to inhibit the function of a target RNA; the function of the target RNA may be complete or partial.

[00360] One of ordinary skill in the art would appreciate that the term RNAi molecule refers to single- or double-stranded RNA molecules comprising both a sense and antisense sequence. For example, the RNA interference molecule can be a double-stranded polynucleotide molecule comprising self-complementary sense and antisense regions, wherein the antisense region comprises complementarity to a target nucleic acid molecule. Alternatively the RNAi molecule can be a single-stranded hairpin polynucleotide having self-complementary sense and antisense regions, wherein the antisense region comprises complementarity to a target nucleic acid molecule or it can be a circular single-stranded polynucleotide having two or more loop structures and a stem comprising self-complementary sense and antisense regions, wherein the antisense region comprises complementarity to a target nucleic acid molecule, and wherein the circular polynucleotide can be processed either in vivo or in vitro to generate an active molecule capable of mediating RNAi.

[00361] RNAi refers to the introduction of homologous double-stranded RNA (dsRNA) to target a specific gene product, resulting in post transcriptional silencing of that gene. This phenomenon was first reported in Caenorhabditis elegans by Guo and Kemphues (1995, Cell, 81(4):611-620) and subsequently Fire et al. (1998, Nature 391:806-811) discovered that it is the presence of dsRNA, formed from the annealing of sense and antisense strands present in the in vitro RNA preps, that is responsible for producing the interfering activity.

[00362] In both plants and animals, RNAi is mediated by RNA-induced silencing complex (RISC), a sequence-specific, multicomponent nuclease that destroys messenger RNAs homologous to the silencing trigger. RISC is known to contain short RNAs (approximately 22 nucleotides) derived from the double-stranded RNA trigger. The short-nucleotide RNA sequences are homologous to the target gene that is being suppressed. Thus, the short-nucleotide sequences appear to serve as guide sequences to instruct a multicomponent nuclease, RISC, to destroy the specific mRNAs. [00363] The dsRNA used to initiate RNAi, may be isolated from native source or produced by known means, e.g., transcribed from DNA. Plasmids and vectors for generating RNAi molecules against target sequence are now readily available from commercial sources.

[00364] The dsRNA can be transcribed from the vectors as two separate strands. In other embodiments, the two strands of DNA used to form the dsRNA may belong to the same or two different duplexes in which they each form with a DNA strand of at least partially complementary sequence. When the dsRNA is thus produced, the DNA sequence to be transcribed is flanked by two promoters, one controlling the transcription of one of the strands, and the other that of the complementary strand. These two promoters may be identical or different. Alternatively, a single promoter can derive the transcription of single-stranded hairpin polynucleotide having self- complementary sense and antisense regions that anneal to produce the dsRNA.

[00365] One skilled in the art would appreciate that the terms “promoter element,” “promoter,” or “promoter sequence” may encompass a DNA sequence that is located at the 5' end (i.e., precedes) the coding region of a DNA polymer. The location of most promoters known in nature precedes the transcribed region. The promoter functions as a switch, activating the expression of a gene. If the gene is activated, it is said to be transcribed, or participating in transcription. Transcription involves the synthesis of mRNA from the gene. The promoter, therefore, serves as a transcriptional regulatory element and also provides a site for initiation of transcription of the gene into mRNA.

[00366] Inhibition is sequence-specific in that nucleotide sequences corresponding to the duplex region of the RNA are targeted for genetic inhibition. RNA molecules containing a nucleotide sequence identical to a portion of the target gene are preferred for inhibition. RNA sequences with insertions, deletions, and single point mutations relative to the target sequence have also been found to be effective for inhibition. Thus, sequence identity may be optimized by sequence comparison and alignment algorithms known in the art (see Gribskov and Devereux, Sequence Analysis Primer, Stockton Press, 1991, and references cited therein) and calculating the percent difference between the nucleotide sequences by, for example, the Smith-Waterman algorithm as implemented in the BESTHT software program using default parameters (e.g., University of Wisconsin Genetic Computing Group). Greater than 90% sequence identity, or even 100% sequence identity, between the inhibitory RNA and the portion of the target gene is preferred. Alternatively, the duplex region of the RNA may be defined functionally as a nucleotide sequence that is capable of hybridizing with a portion of the target gene transcript. The length of the identical nucleotide sequences may be at least 25, 50, 100, 200, 300 or 400 bases. There is no upper limit on the length of the dsRNA that can be used. For example, the dsRNA can range from about 21 base pairs (bp) of the gene to the full-length of the gene or more.

[00367] In some embodiments, the therapeutic payload or the targeting moiety comprises a small interfering RNA (siRNA). The siRNA moiety may further include a guanosine at the 5'- end.

[00368] The sense and/or antisense strands of the siRNA moiety may equal to or less than 30, 25, 24, 23, 22, 21, 20, 19, 18 or 17 nucleotides in length. An siRNA moiety may include one or more overhangs. For example, the siRNA moiety may include one or two 3' overhangs of 2-3 nucleotides. In certain embodiments, the invention relates to any of the compositions described herein, wherein the siRNA moiety is composed of 21 -nt sense and 21 -nt antisense strands, paired in a manner to have a 19-nucleotide duplex region and a 2-nt 3' overhang at each 3' terminus. In certain embodiments, the invention relates to any of the compositions described herein, wherein the 2-nt 3' overhang is either UU or dTdT. Symmetric 3'-overhangs ensure that the sequencespecific endonuclease complexes (siRNPs) are formed with approximately equal ratios of sense and antisense target RNA cleaving siRNPs. The 3'-overhang in the sense strand provides no contribution to recognition as it is believed the antisense siRNA strand guides target recognition. Therefore, the UU or dTdT 3'-overhang of the antisense sequences is complementary to the target mRNA but the symmetrical UU or dTdT 3'-overhang of the sense siRNA oligo does not need to correspond to the mRNA. The use of deoxythymidines in both 3'-overhangs may increase nuclease resistance, although siRNA duplexes with either UU or dTdT overhangs work equally well. 2'-Deoxynucleotides in the 3' overhangs are as efficient as ribonucleotides, but are often cheaper to synthesize.

[00369] The targeted region in the mRNA, and hence the sequence in the siRNA duplex, are chosen using the following guidelines. The open reading frame (ORF) region from the cDNA sequence is recommended for targeting, preferably at least 50 to 100 nucleotides downstream of the start codon, most preferably at least 75-100. Both the 5' and 3' untranslated regions (UTRs) and regions near the start codon are not recommended for targeting as these may be richer in regulatory protein binding sites. UTR-binding proteins and/or translation initiation complexes may interfere with binding of the siRNP endonuclease complex.

[00370] The sequence of the mRNA or cDNA is searched seeking the sequence AA(N19)TT. Sequences with approximately 50% G/C-content (30% to 70%) are used. If no suitable sequences are found, the search is extended to sequences AA(N21). The sequence of the sense siRNA corresponds to 5'-(N19)dTdT-3' or N21, respectively. In the latter case, the 3' end of the sense siRNA is converted to dTdT. The rationale for this sequence conversion is to generate a symmetric duplex with respect to the sequence composition of the sense and antisense 3' overhangs. It is believed that symmetric 3' overhangs help to ensure that the siRNPs are formed with approximately equal ratios of sense and antisense target RNA-cleaving siRNPs. The modification of the overhang of the sense sequence of the siRNA duplex is not expected to affect targeted mRNA recognition, as the antisense siRNA strand glides target recognition.

[00371] If the target mRNA does not contain a suitable AA(N21) sequence, it is recommended to search for NA(N21) The sequence of the sense and antisense strand may still be synthesized as 5' (N19)TT as the sequence of the 3' most nucleotide of the antisense siRNA does not appear to contribute to specificity.

[00372] It is further recommended to search the selected siRNA sequence against EST libraries in appropriate databases (e.g., NCBI BLAST database search) to ensure that only one gene is targeted.

[00373] The appropriately designed siRNAs are either obtained from commercial sources (such as DHARMACON RESEARCH™, Lafayette, Colo.; XERGON™, Huntsville, Ala.; AMBION™, Austin, Tex.) or chemically synthesized used appropriately protected ribonucleoside phosphoramidites and a conventional DNA/RNA synthesizer according to standard protocols. The RNA oligonucleotides are 2'-deprotected, desalted and the two strands annealed, according to manufacturer's specifications or conventional protocols, depending on how the siRNAs are obtained. All handling steps are conducted under strict sterile, RNase-free conditions.

[00374] In some embodiments, a nucleic acid aptamer is included. Nucleic acid aptamers are nucleic acid oligomers that bind other macromolecules specifically; such aptamers that bind specifically to other macromolecules can be readily isolated from libraries of such oligomers by technologies such as SELEX. In some embodiments, an oligosaccharide is included. Certain oligosaccharides are known ligands for certain extracellular or cell surface receptors.

Co-suppression Molecules

[00375] In some embodiments, the extracellular vesicle or the micelle comprises a cosuppression molecule. A co-suppression molecule is another agent capable of down-regulating the expression of a given gene, or a combination thereof. Co-suppression is a post-transcriptional mechanism where both the transgene and the endogenous gene are silenced.

Enzymatic Nucleic Acid Molecules

[00376] In some embodiments, the therapeutic payload or the targeting moiety comprises an enzymatic nucleic acid molecule. The terms “enzymatic nucleic acid molecule” or “enzymatic oligonucleotide” refers to a nucleic acid molecule which has complementarity in a substrate binding region to a specified gene target and also has an enzymatic activity which is active to specifically cleave target RNA of a given gene, thereby silencing each of the genes. The complementary regions allow sufficient hybridization of the enzymatic nucleic acid molecule to the target RNA and subsequent cleavage. The term enzymatic nucleic acid is used interchangeably with, for example, ribozymes, catalytic RNA, enzymatic RNA, catalytic DNA, aptazyme or aptamer-binding ribozyme, catalytic oligonucleotide, nucleozyme, DNAzyme, RNAenzyme. The specific enzymatic nucleic acid molecules described in the instant application are not limiting and an enzymatic nucleic acid molecule of this invention requires a specific substrate binding site which is complementary to one or more of the target nucleic acid regions, and that it have nucleotide sequences within or surrounding that substrate binding site which impart a nucleic acid cleaving and/or ligation activity to the molecule. US Patent No. 4,987,071 discloses examples of such molecules.

[00377] In some embodiments, the therapeutic payload or the targeting moiety is a DNAzyme molecule. A DNAzyme molecule is another agent capable of down-regulating the expression of a given gene, the DNAzyme molecule being capable of specifically cleaving an mRNA transcript or a DNA sequence of said gene. DNAzymes are single-stranded polynucleotides that are capable of cleaving both single- and double-stranded target sequences. A general model (the "10-23" model) for the DNAzyme has been proposed. "10-23” DNAzymes have a catalytic domain of 15 deoxyribonucleotides, flanked by two substrate-recognition domains of seven to nine deoxyribonucleotides each. This type of DNAzyme can effectively cleave its substrate RNA at purine:pyrimidine junctions (for review of DNAzymes, see: Khachigian, L. M. (2002) Curr Opin Mol Ther 4, 119-121).

[00378] Examples of construction and amplification of synthetic, engineered DNAzymes recognizing single- and double-stranded target cleavage sites are disclosed in U.S. Patent No. 6,326,174.

Pharmaceutical Compositions

[00379] The present invention further provides a pharmaceutical composition or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[00380] Disclosed herein is an extracellular vesicle or a micelle comprising a therapeutic payload, a targeting moiety, or a combination thereof, for use in the treatment of a disease or disorder in a subject in need thereof. In some embodiments, the subject is a human. In one embodiment, the disease or disorder is a cancer, cancer metastasis or a solid tumor.

[00381] Disclosed herein is a therapeutic pay load comprising a pharmaceutical composition or a pharmaceutically acceptable salt thereof, for use in the treatment of a cancer or a tumor.

[00382] In some embodiments, the therapeutic pay load comprises a pharmaceutical composition or a pharmaceutically acceptable salt thereof. In some embodiments, the pharmaceutical composition comprises a medicament (e.g., a drug).

[00383] In some embodiments, the extracellular vesicle or micelle further comprises a stabilizing agent.

[00384] Disclosed herein is a therapeutic pay load comprising a pharmaceutical composition or a pharmaceutically acceptable salt thereof, for use for treating, reducing, inhibiting, ameliorating, or alleviating a tumor or a cancer in a subject in need thereof. Also disclosed herein is a therapeutic payload comprising a pharmaceutical composition or a pharmaceutically acceptable salt thereof, for use for treating, reducing, inhibiting, ameliorating, or alleviating an immune disease or abnormal immune condition. Also disclosed herein is a therapeutic payload comprising a pharmaceutical composition or a pharmaceutically acceptable salt thereof, for use for treating, reducing, inhibiting, ameliorating, or alleviating a disease or abnormal physiological condition.

[00385] In some embodiments, the therapeutic payload comprises a pharmaceutical composition or a pharmaceutically acceptable salt thereof, for use in treating, reducing, inhibiting, ameliorating, or alleviating a tumor or cancer in a subject in need thereof. In some embodiments, the therapeutic pay load comprises a pharmaceutical composition or a pharmaceutically acceptable salt thereof, for use in treating, reducing, inhibiting, ameliorating, or alleviating metastasis of a cancer in a subject in need thereof. In some embodiments, the therapeutic pay load comprises a pharmaceutical composition or a pharmaceutically acceptable salt thereof, for use for treating, reducing, inhibiting, ameliorating, or alleviating an immune disease or abnormal immune condition. In some embodiments, the therapeutic pay load comprises a pharmaceutical composition or a pharmaceutically acceptable salt thereof, for use for treating, reducing, inhibiting, ameliorating, or alleviating a disease or abnormal physiological condition.

[00386] As used herein, "pharmaceutical composition" refers to a therapeutically effective amount of a compound of the present invention, or a pharmaceutically acceptable salt thereof, together with suitable diluents, preservatives, solubilizers, emulsifiers, adjuvant, carriers, or combinations of these. Such compositions are liquids or lyophilized or otherwise dried formulations and include diluents of various buffer content (e.g.; Tris-HCL, acetate, phosphate), pH and ionic strength, additives such as albumin or gelatin to prevent absorption to surfaces, detergents (e.g., Tween 20, Tween 80, Pluronic F68, bile acid salts), solubilizing agents (e.g., glycerol, polyethylene glycerol), anti-oxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives (e.g., Thimerosal, benzyl alcohol, parabens), bulking substances or tonicity modifiers (e.g., lactose, mannitol), covalent attachment of polymers such as polyethylene glycol to the protein, complexation with metal ions, or incorporation of the material into or onto particulate preparations of polymeric compounds such as polylactic acid, polglycolic acid, hydrogels, etc., or onto liposomes, microemulsions, micelles, unilamellar or multilamellar vesicles, erythrocyte ghosts, or spheroplasts. Such compositions will influence the physical state, solubility, stability, rate of in vivo release, and rate of in vivo clearance. Controlled or sustained release compositions include formulation in lipophilic depots (e.g., fatty acids, waxes, oils). [00387] In addition to the anti-tumor and immunostimulant molecules described herein (e.g., Granzyme B, Interferon-y, Perforin-1, TNF-a, IL-1J3, IL-2, IL-8, IL-17, MCP-1/CCL2, IP- 10/CXCL10, HMGB1, and others), pharmaceutical compositions also include, but are not limited to, paclitaxel (e.g., TAXOL™ [ABRAXANE™]) and DM4 (integrin-targeted immunoconjugate IMGN388).

[00388] A “stabilizing agent” (“stabilizer,” “stabilizing excipient”) is a substance used to help the active pharmaceutical ingredient (API) maintain the desirable properties of the product until it is administered to the subject.

[00389] A "therapeutically effective amount" as used herein refers to that amount which provides a therapeutic effect for a given indication and administration regimen.

[00390] Numerous standard references are available that describe procedures for preparing various compositions or formulations suitable for administration of the compounds of the invention. Examples of methods of making formulations and preparations can be found in the Handbook of Pharmaceutical Excipients, American Pharmaceutical Association (current edition); Pharmaceutical Dosage Forms: Tablets (Lieberman, Lachman and Schwartz, editors) current edition, published by Marcel Dekker, Inc., as well as Remington's Pharmaceutical Sciences (Arthur Osol, editor), 1553-1593 (current edition).

[00391] The mode of administration and dosage form are closely related to the therapeutic amounts of the compounds or compositions which are desirable and efficacious for the given treatment application.

[00392] The extracellular composition or the micelle disclosed herein can be administered to a subject by any method known to a person skilled in the art. These methods include, but are not limited to, orally, parenterally, intravascularly, paracancerally, transmucosally, transdermally, intramuscularly, intranasally, intravenously, intradermally, subcutaneously, sublingually, intraperitoneally, intraventricularly, intracranially, intravaginally, rectally, intratumorally, intralymph nodal, or adjacent to a tumor. These methods include any means in which the composition can be delivered to tissue (e.g. , needle or catheter). [00393] In some embodiments, disclosed herein are intravenous, intramuscular, and subcutaneous delivery methods. In other embodiments, the extracellular vesicle is engineered to be delivered orally and, upon metabolization, to reach the TME before targeting a tumor cell to deliver therapeutic pay load contained therein. In other embodiments, an extracellular vesicle delivers the therapeutic payload directly to specific targets in the TME.

[00394] In some embodiments, the extracellular vesicle or the micelle is administered intravenously (IV), intramuscularly, subcutaneously (SC), or orally. In some embodiments, the extracellular vesicle or the micelle is administered interperitoneally (IP), injected intra-lymph nodes, injected intra-tumorally, or injected adjacent to a tumor.

[00395] By “adjacent” to a tumor includes, but is not limited to, next to a tumor, adjoining a tumor, or in close proximity to a tumor. By “close proximity” to a tumor is meant that the closest part of the extracellular vesicle or the micelle is from about 1 mm to about 50 mm from a tumor, from about 1 mm to about 45 mm from a tumor, from about 1 mm to about 40 mm from a tumor, from about 1 mm to about 35 mm from a tumor, from about 1 mm to about 30 mm from a tumor, from about 1 mm to about 25 mm from a tumor, from about 1 mm to about 20 mm from a tumor, from about 1 mm to about 15 mm from a tumor, from about 1 mm to about 10 mm from a tumor, from about 1 mm to about 5 mm from a tumor.

[00396] Suitable dosage forms include, but are not limited to, subcutaneous (SC), intraperitoneal (IP), intra-tumor or intra-lymph node administration, tumor-adjacent administration and other dosage forms for systemic delivery of active ingredients.

[00397] As used herein "pharmaceutically acceptable carriers or diluents" are well known to those skilled in the art. The carrier or diluent may be a solid carrier or diluent for solid formulations, a liquid carrier or diluent for liquid formulations, or mixtures thereof.

[00398] Solid carriers/diluents include, but are not limited to, a protein or a salt.

[00399] For parenteral formulations, the carrier will usually comprise sterile water, though other ingredients may be included, such as ingredients that aid solubility or for preservation. Injectable suspensions may also be prepared in which case appropriate stabilizing agents may be employed. [00400] Formulations suitable for parenteral administration may comprise a sterile aqueous preparation of the active compound, which, in some embodiments, is isotonic with the blood of the recipient (e.g., physiological saline solution). Such formulations may include suspending agents and thickening agents and liposomes or other microparticulate systems which are designed to target the compound to blood components or one or more organs. The formulations may be presented in unit-dose or multi-dose form.

[00401] Parenteral administration may comprise any suitable form of systemic delivery. Administration may for example be intravenous, intra-arterial, intrathecal, intramuscular, subcutaneous, intramuscular, intra-abdominal (e.g., intraperitoneal), etc., and may be effected by infusion pumps (external or implantable) or any other suitable means appropriate to the desired administration modality.

[00402] In addition to the aforementioned ingredients, compositions of this invention may further include one or more ingredient selected from diluents, buffers, flavoring agents, binders, disintegrants, surface active agents, thickeners, lubricants, preservatives (including antioxidants), and the like.

[00403] The formulations may be of immediate release, sustained release, delayed-onset release or any other release profile known to one skilled in the art.

[00404] The methods of the invention comprise administration of a compound at a therapeutically effective amount. The therapeutically effective amount may include various dosages.

[00405] For administration to birds or to mammals, and particularly humans, it is expected that the physician will determine the actual dosage and duration of treatment, which will be most suitable for an individual and can vary with the age, weight, genetics and/or response of the particular individual. In some embodiments, the subject is a mammal (e.g., a human or nonhuman mammal). In some embodiments, the subject is a human.

[00406] The exact dose and regimen of administration of the composition will necessarily be dependent upon the therapeutic or nutritional effect to be achieved and may vary with the particular formula, the route of administration, and the age and condition of the individual subject to whom the composition is to be administered.

[00407] A dosage unit of the compounds as used herein may comprise a single compound or mixtures thereof with additional therapeutic agents. A “dose” or “dosage unit” or “unit dosage” of a compound of formula (I) of the invention as measured in milligrams refers to the milligrams of compound of formula (I) present in a preparation, regardless of the form of the preparation.

[00408] In some embodiments, a compound of the invention is administered at a dosage of 1-3000 mg per day. In some embodiments, a compound of the invention as described herein is administered at a dosage of 1-1000 mg per day. In some embodiments, a compound of the invention as described herein is administered at a dosage of 1-500 mg per day. In some embodiments, a compound of the invention as described herein is administered at a dosage of 10- 500 mg per day. In some embodiments, a compound of the invention as described herein is administered at a dosage of 25-500 mg per day. In some embodiments, a compound of the invention as described herein is administered at a dosage of 50-500 mg per day. In some embodiments, a compound of the invention as described herein is administered at a dosage of 5- 250 mg per day. In some embodiments, a compound of the invention as described herein is administered at a dosage of 10-250 mg per day. In some embodiments, a compound of the invention as described herein is administered at a dosage of 20-250 mg per day. In some embodiments, a compound of the invention as described herein is administered at a dosage of 25- 250 mg per day. In some embodiments, a compound of the invention as described herein is administered at a dosage of 25-200 mg per day. In some embodiments, a compound of the invention as described herein is administered at a dosage of 25-150 mg per day. In some embodiments, a compound of the invention as described herein is administered at a dosage of 25- 125 mg per day. In some embodiments, a compound of the invention as described herein is administered at a dosage of 25-100 mg per day.

[00409] In other embodiments, a compound of the invention is administered at a dose of 1-10 mg per day, 3-26 mg per day, 3-60 mg per day, 3-16 mg per day, 3-30 mg per day, 10-26 mg per day, 10-100 mg per day, 15-60 mg per day, 15-100 mg per day, 25-100 mg per day, 50- 100 mg per day, 50-200 mg per day, 100-200 mg per day, 100-250 mg per day, 125-300 mg per day, 20-50 mg per day, 5-50 mg per day, 200-500 mg per day, 125-500 mg per day, 500-1000 mg per day, 200-1000 mg per day, 1000-2000 mg per day, 1000-3000 mg per day, 125-3000 mg per day, 2000-3000 mg per day, 300-1500 mg per day or 100-1000 mg per day.

[00410] The methods may comprise administering a compound at various dosages. For example, the compound may be administered per day at a dosage of 3 mg, 10 mg, 30 mg, 40 mg, 50 mg, 80 mg, 100 mg, 120 mg, 125 mg, 200 mg, 250 mg, 300 mg, 450 mg, 500 mg, 600 mg, 900 mg, 1000 mg, 1500 mg, 2000 mg, 2500 mg or 3000 mg.

[00411] Alternatively, the compound may be administered at a dosage of 0.1 mg/kg/day. The compound may be administered at a dosage between 0.2 to 30 mg/kg/day, or 0.2 mg/kg/day, 0.3 mg/kg/day, 1 mg/kg/day, 3 mg/kg/day, 5 mg/kg/day, 10 mg/kg/day, 20 mg/kg/day, 30 mg/kg/day, 50 mg/kg/day or 100 mg/kg/day.

[00412] “Modified-release dosage” is a mechanism that (in contrast to “immediate-release dosage”) delivers a drug with a delay after its administration (delayed-release dosage) or for a prolonged period of time (extended-release [ER, XR, XL] dosage) or to a specific target in the body (targeted-release dosage).

[00413] “Sustained-release dosage” forms are dosage forms designed to release (liberate) a drug at a predetermined rate in order to maintain a constant drug concentration for a specific period of time with minimum side effects. This can be achieved through a variety of formulations, including liposomes and drug-polymer conjugates (an example being hydrogels). Sustained release's definition is more akin to a "controlled release" rather than "sustained".

[00414] “Extended-release dosage” (ER) consists of either “sustained-release” (SR) or “controlled-release” (CR) dosage. SR maintains drug release over a sustained period but not at a constant rate. CR maintains drug release over a sustained period at a nearly constant rate.

[00415] In some embodiments, the therapeutic payload has a sustained-release (SR) dosage or an extended-release (ER) dosage. In some embodiments, the SR or ER dosage extends at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 2 weeks, at least about 3 weeks, at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 9 months, or at least about 12 months. In some embodiments, the SR or ER dosage extends at least about 7 days.

[00416] As used herein, in some embodiments, the term “alkyl” refers to a saturated hydrocarbon group which is straight-chained or branched. Example alkyl groups include methyl (Me), ethyl (Et), propyl e.g., n-propyl and isopropyl), butyl e.g., n-butyl, isobutyl, t-butyl), pentyl (e.g., n-pentyl, isopentyl, neopentyl), and the like. An alkyl group can contain from 1 to about 20, from 2 to about 20, from 1 to about 10, from 1 to about 8, from 1 to about 6, from 1 to about 4, or from 1 to about 3 carbon atoms.

[00417] In some embodiments, “aryl” refers to monocyclic or polycyclic (e.g., having 2, 3 or 4 fused rings) aromatic hydrocarbons such as, for example, phenyl, naphthyl, anthracenyl, phenanthrenyl, and the like. In some embodiments, an aryl group has from 6 to about 20 carbon atoms. In some embodiments, “aryl” may be optionally substituted at any one or more positions.

[00418] In some embodiments, “heteroaryl” refers to an aromatic heterocycle having at least one heteroatom ring member such as sulfur, oxygen, or nitrogen. Heteroaryl groups include monocyclic and polycyclic (e.g., having 2, 3 or 4 fused rings) systems. Any ring-forming N atom in a heteroaryl group can also be oxidized to form an N-oxo moiety. Examples of heteroaryl groups include without limitation, pyridyl, N-oxopyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, triazinyl, furyl, quinolyl, isoquinolyl, thienyl, imidazolyl, thiazolyl, indolyl, pyrryl, oxazolyl, benzofuryl, benzothienyl, benzthiazolyl, isoxazolyl, pyrazolyl, triazolyl, tetrazolyl, indazolyl, 1,2,4-thiadiazolyl, isothiazolyl, benzothienyl, purinyl, carbazolyl, benzimidazolyl, indolinyl, and the like. In some embodiments, the heteroaryl group has from 1 to about 20 carbon atoms, and in further embodiments from about 3 to about 20 carbon atoms. In some embodiments, the heteroaryl group contains 3 to about 14, 3 to about 7, or 5 to 6 ring-forming atoms. In some embodiments, the heteroaryl group has 1 to about 4, 1 to about 3, or 1 to 2 heteroatoms. In some embodiments, “heteroaryl” may be optionally substituted at any one or more positions capable of bearing a hydrogen atom.

[00419] The term "heteroaryl" may be used interchangeably with the terms "heteroaryl ring," "heteroaryl group," or "heteroaromatic," any of which terms include rings that are optionally substituted. The term "heteroaralkyl" refers to an alkyl group substituted by a heteroaryl, wherein the alkyl and heteroaryl portions independently are optionally substituted. [00420] In some embodiments, “halo” or “halogen” includes fluoro, chloro, bromo, and iodo. A “halogen-substitution” or “halo” substitution designates replacement of one or more hydrogen atoms with F, CI, Br or I.

[00421] In some embodiments, “haloalkyl” refers to an alkyl group having one or more halogen substituents. Example haloalkyl groups include CF3, C2F5, CHF2, CCI3, CHCI2, C2CI5, and the like.

[00422] In some embodiments, the term “substituted” refers to the replacement of a hydrogen moiety with a non-hydrogen moiety in a molecule or group. It can refer to “monosubstituted” or “poly-substituted.” The term “mono-substituted” or “poly-substituted” means substituted with one or more than one substituent up to the valence of the substituted group. For example, a mono-substituted group can be substituted with 1 substituent, and a poly-substituted group can be substituted with 2, 3, 4, or 5 substituents. When a list of possible substituents is provided, the substituents can be independently selected from that group.

[00423] The term “optionally substituted,” in some embodiments, refers to that the groups in question are either unsubstituted or substituted with one or more of the substituents specified. When the groups in question are substituted with more than one substituent, the substituents may be the same or different. Such other functional groups illustratively include, but are not limited to, amino, hydroxyl, CN, halo, thiol, alkyl, haloalkyl, heteroalkyl, aryl, arylalkyl, arylheteroalkyl, heteroaryl, heteroarylalkyl, heteroarylheteroalkyl, nitro, sulfonic acids and derivatives thereof, carboxylic acids and derivatives thereof, and the like. Illustratively, any of amino, hydroxyl, CH, thiol, alkyl, haloalkyl, heteroalkyl, aryl, arylalkyl, arylheteroalkyl, heteroaryl, heteroarylalkyl, heteroarylheteroalkyl, and/or sulfonic acid is optionally substituted. In some embodiments, the functional groups are the substituents described herein for any one of variables. Furthermore, when using the terms “independently,” “independently are,” and “independently selected from” mean that the groups in question may be the same or different. Certain of the herein defined terms may occur more than once in the structure, and upon such occurrence each term shall be defined independently of the other.

[00424] In each of the foregoing and each of the following embodiments, it is to be understood that the formulas also include any and all hydrates and/or solvates of the compound formulas. It is appreciated that certain functional groups, such as the hydroxy, amino, and like groups form complexes and/or coordination compounds with water and/or various solvents, in the various physical forms of the compounds. Accordingly, the above formulas are to be understood to include and represent those various hydrates and/or solvates.

[00425] As used herein, the term “solvate” refers to compounds that further include a stoichiometric or non-stoichiometric amount of a solvent bound by non-covalent intermolecular forces. If the solvent is water, the solvate is referred to as "hydrate." Pharmaceutically acceptable solvates and hydrates are complexes that, for example, may include from 1 to about 100, or from 1 to about 10, or from one to about 2.3 or 4 molecules of water or a solvent. In some embodiments, the hydrate may be a channel hydrate. It should be understood that the term “compound” in this application covers the compound and solvates of the compound, as well as mixtures thereof.

[00426] In some embodiments, the term “hydrate” includes, but is not limited to, hemihydrate, monohydrate, dihydrate, trihydrate and the like.

[00427] Compounds of the invention can also include all isotopes of atoms occurring in the intermediates or final compounds. Isotopes include those atoms having the same atomic number but different mass numbers. For example, isotopes of hydrogen include tritium and deuterium.

[00428] Compounds described herein may contain one or more asymmetric centers and may thus give rise to diastereomers and optical isomers. The present invention includes all such possible optical isomers, diastereomers as well as their racemic mixtures, their substantially pure resolved enantiomers, all possible geometric isomers, and pharmaceutically acceptable salts thereof. The above Formula (I) is shown without a definitive stereochemistry at certain positions. The present invention includes all stereoisomers of Formula (I) and pharmaceutically acceptable salts thereof. Further, mixtures of stereoisomers as well as isolated specific stereoisomers are also included.

[00429] In some embodiments, this invention encompasses the use of various optical isomers of the compound of the invention. It will be appreciated by those skilled in the art that the compounds of the present invention contain at least one chiral center. Accordingly, the compounds used in the methods of the present invention may exist in, and be isolated in, optically- active or racemic forms. Some compounds may also exhibit polymorphism. It is to be understood that the present invention encompasses any racemic, optically-active, polymorphic, or stereoisomeric form, or any combination thereof, which form possesses properties useful in the treatment of androgen-related conditions described herein. In some embodiments, the compounds of the inventio are optically pure. In other embodiments, the compounds of the invention are a racemic mixture. It is well known in the art how to prepare optically-active forms (for example, by resolution of the racemic form by recrystallization techniques, by synthesis from optically- active starting materials, by chiral synthesis, or by chromatographic separation using a chiral stationary phase).

[00430] During the course of the synthetic procedures used to prepare such compounds, or in using racemization or epimerization procedures known to those skilled in the art, the products of such procedures can be a mixture of stereoisomers.

[00431] In some embodiments, the phrase “pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

[00432] The present invention also includes “pharmaceutically acceptable salts” of the compounds described herein. As used herein, “pharmaceutically acceptable salts” refers to derivatives of the disclosed compounds wherein the parent compound is modified by converting an existing acid or base moiety to its salt form. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like. The pharmaceutically acceptable salts of the compound of the invention include the conventional non-toxic salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. The pharmaceutically acceptable salts of the compound of the invention can be synthesized from the parent compound which contains a basic or acidic moiety by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two. In some embodiments, the solvent is a nonaqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile. [00433] The pharmaceutically acceptable salts of the compound of the invention can be formed by conventional means, such as by reacting the free base or free acid form of the product with one or more equivalents of the appropriate acid or base in a solvent or medium in which the salt is insoluble or in a solvent such as water, which is removed in vacuo or by freeze drying or by exchanging the ions of an existing salt for another ion or suitable ion-exchange resin.

[00434] Possible pharmaceutically acceptable salts are well known in the art. For example,

S. M. Berge, et al. describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 66: 1-19, 1977; incorporated herein by reference.

[00435] Typically, a pharmaceutically acceptable salt form of a compound can be prepared in situ during the final isolation and purification of the compound, or separately by reacting the free base functionality with a suitable organic or inorganic acid.

[00436] Suitable acids for preparation of the pharmaceutically acceptable salts include, but are not limited to, inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid, or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid or by using other methods used in the art such as ion exchange.

[00437] Other pharmaceutically acceptable salts can include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2- naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and the like.

[00438] Salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium and quaternary ammonium salts. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Further pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, loweralkyl sulfonate and aryl sulfonate.

[00439] Suitable bases for use in the preparation of pharmaceutically acceptable salts, including, but not limited to, inorganic bases, such as magnesium hydroxide, calcium hydroxide, potassium hydroxide, zinc hydroxide, or sodium hydroxide; and organic bases, such as primary, secondary, tertiary, and quaternary, aliphatic and aromatic amines, including L-arginine, benethamine, benzathine, choline, deanol, diethanolamine, diethylamine, dimethylamine, dipropylamine, diisopropylamine, 2-(diethylamino)-ethanol, ethanolamine, ethylamine, ethylenediamine, isopropylamine, N-methyl-glucamine, hydrabamine, IH-imidazole, L-lysine, morpholine, 4-(2-hydroxyethyl)-morpholine, methylamine, piperidine, piperazine, propylamine, pyrrolidine, l-(2-hydroxyethyl)-pyrrolidine, pyridine, quinuclidine, quinoline, isoquinoline, secondary amines, triethanolamine, trimethylamine, triethylamine, N-methyl-D-glucamine, 2- amino-2-(hydroxymethyl)-l,3-propanediol, and tromethamine.

[00440] The invention further includes derivatives of the compound of the invention. The term “derivatives” includes but is not limited to ether derivatives, acid derivatives, amide derivatives, ester derivatives and the like.

[00441] The invention further includes metabolites of the compound of the invention. The term “metabolite” means any substance produced from another substance by metabolism or a metabolic process.

[00442] The invention farther includes pharmaceutical products of the compound of the invention. The term “pharmaceutical product” means a composition suitable for pharmaceutical use (pharmaceutical composition), as defined herein.

[00443] The invention further includes prodrugs of the compound of the invention. The term “prodrug” means a substance which can be converted in vivo into a biologically active agent by such reactions as hydrolysis, esterification, de-esterification, activation, salt formation and the like.

[00444] In some embodiments, the extracellular vesicle further comprises a hydrophilic or hydrophobic pharmaceutical composition. In some embodiments, the extracellular vesicle further comprises a hydrophilic pharmaceutical composition, on the exterior surface of the extracellular vesicle or within the aqueous interior region of the extracellular vesicle.

[00445] In some embodiments, the micelle further comprises a hydrophilic or hydrophobic pharmaceutical composition. In some embodiments, micelle further comprises a hydrophobic pharmaceutical composition within the micelle.

Cell Therapies and Vaccines

[00446] In some embodiments, the extracellular vesicle or the micelle provides a method of therapy for a subject in need thereof. In some embodiments, the therapy is a vaccine.

[00447] A “vaccine” is a biological preparation that can be prophylactic (to prevent or ameliorate the effects of a future infection by a natural or "wild" pathogen), or therapeutic (to fight a disease that has already occurred, such as cancer). A prophylactic vaccine provides active acquired immunity to a particular infectious disease. It typically contains an agent that resembles a disease-causing microorganism and is often made from weakened or killed forms of the microbe, its toxins, or one of its surface proteins. The agent stimulates the body's immune system to recognize the agent as a threat, destroy it, and to further recognize and destroy any of the microorganisms associated with that agent that it may encounter in the future.

[00448] In some embodiments, the therapeutic payload comprises a vaccine. In some embodiments, the therapeutic pay load comprises a therapeutic vaccine.

Treatment Methods

[00449] Disclosed herein is a method for treating, reducing, inhibiting, ameliorating, or alleviating a tumor, a cancer, or a growth or metastasis thereof, in a subject in need thereof, comprising administering to the subject the extracellular vesicle or the micelle comprising a therapeutic payload, a targeting moiety, or a combination thereof.

[00450] In one embodiment, the method further comprises administering to the subject an extracellular vesicle or a micelle disclosed herein. In some embodiments, disclosed herein are intravenous, intramuscular, and subcutaneous delivery methods. In other embodiments, the extracellular vesicle is engineered to be delivered orally and, upon metabolization, to reach the TME before targeting a tumor cell to deliver therapeutic payload contained therein. In other embodiments, an extracellular vesicle delivers the therapeutic payload directly to specific targets in the TME.

[00451] In some embodiments, the extracellular vesicle or the micelle is administered intravenously (IV), intramuscularly, subcutaneously (SC), or orally. In some embodiments, the extracellular vesicle or the micelle is administered interperitoneally (IP), injected intra-lymph nodes, injected intra-tumorally, or injected adjacent to a tumor.

[00452] In some embodiments, the method further comprises administering to the subject at least once daily throughout the duration of therapy period: days, weeks, months, or years, with intervals that follow treatment outcome.

[00453] The exact dose and regimen of administration of the composition will necessarily be dependent upon the therapeutic or nutritional effect to be achieved and may vary with the particular formula, the route of administration, and the age and condition of the individual subject to whom the composition is to be administered.

[00454] In some embodiments, methods of using the extracellular vesicle or the micelle comprise treating, reducing, inhibiting, ameliorating, or alleviating a cancer or a tumor in a subject or a growth or a metastasis thereof, comprising a step of administering the extracellular vesicle or the micelle to the subject in a subject in need thereof. In some embodiments, methods of using the extracellular vesicle or the micelle comprise treating, reducing, inhibiting, ameliorating, or alleviating a cancer or a tumor in a subject in need thereof. In some embodiments, methods of using the extracellular vesicle or the micelle comprise treating, reducing, inhibiting, ameliorating, or alleviating metastasis of a cancer in a subject in need thereof. In some embodiments, methods of using the extracellular vesicle or the micelle further comprise increasing survival in the subject. In some embodiments, methods of using the extracellular composition or the micelle farther comprise reducing or inhibiting metastases infiltration.

[00455] In some embodiments, the method of treating, reducing, inhibiting, ameliorating, or alleviating a tumor or cancer comprises treatment of solid tumors or cancers. [00456] In some aspects, the method of treating, reducing, inhibiting, ameliorating, or alleviating a tumor or cancer comprises treating, as malignant melanoma, breast adenocarcinoma, lung adenocarcinoma, colon cancer, pancreatic cancer, or another type of cancer.

[00457] In some embodiments, the method comprises testing a tumor cell or a cancer cell to determine a biomarker on the cell surface of the tumor cell or the cancer cell or secreted by the tumor cell or the cancer cell (e.g., into the tumor microenvironment). In some embodiments, the method comprises selecting a targeting moiety corresponding to the biomarker on the cell surface of the tumor cell or the cancer cell. In some embodiments, the method comprises selecting a targeting moiety corresponding to the biomarker secreted by the tumor cell or the cancer cell. In some embodiments, the method comprises selecting a therapeutic payload for treating, reducing, inhibiting, ameliorating, or alleviating a tumor cell or a cancer cell comprising the biomarker.

[00458] In some embodiments, the method of treating, reducing, inhibiting, ameliorating, or alleviating a tumor or cancer comprises treatment of a fibroma or a fibrosarcoma.

[00459] Examples of biomarkers are numerous and may vary, not only from one patient to another, but also within a given patient from one tumor or cancer to another or from one tumor cell or cancer cell to another. Circulating tumor or cancer cells and metastatic tumors or cancers may also vary from the primary tumor or cancer site. Biomarkers may have lesser or greater degrees of specificity for the tumor or cancer cells as compared with other non-tumor or non- malignant cell types.

[00460] In some embodiments, the targeting moiety is directed to a biomarker of a tumor or cancer cell. In some embodiments, the targeting moiety is directed to a biomarker of a primary tumor or cancer cell. In some embodiments, the targeting moiety is directed to a biomarker on a circulating tumor or cancer cell or a metastatic tumor or cancer cell (e.g., secondary, tertiary, etc.).

[00461] Tumor-cell-specific targeting moieties such as antibodies, aptamers, and scFv target proteins expressed by various tumor cells, such as programmed death receptors PD-1, CTLA-4, TGF-P, EGFR, VEGFR, and more. In certain embodiments, the above target proteins or other tumor-cell-specific molecules can be targeted using nucleic acid aptamers engineered using systematic evolution of ligands by exponential enrichment (SEEEX) methods. SEEEX (in vitro selection, in vitro evolution, SAAB [selected and amplified binding site], CASTing [cyclic amplification and selection of targets]), is a combinatorial chemistry technique in molecular biology for producing oligonucleotides of either single-stranded DNA or RNA that specifically bind to a target ligand or ligands. These single-stranded DNA or RNA are commonly referred to as aptamers. Engineered or selected aptamers bind their target molecules through electrostatic interactions, hydrophobic interactions, and complementary shapes. Aptamers are engineered using in vitro selection to identify and select aptamers that bind specific target peptides and whole tumor cells.

[00462] A non-limiting example of a targeting mechanism of scFv-linked extracellular vesicles is described. For example, in Eongatti et al., High-affinity single-chain variable fragments are specific and versatile targeting motifs for extracellular vesicles, Nanoscale. 2018. In various embodiments, tumor-cell-targeting receptors and moieties may include antibodies and functional fragments thereof. Such fragments include scFvs, antigen-binding fragments (Fab), and singledomain antibodies such as VHH fragments. In preferred embodiments, the binding moiety has an scFvs are fusion proteins including variable regions of the heavy (VH) and light chains (VE) of immunoglobulins. ScFvs were created by cloning VH and VL genes of mice and other animals immunized with the desired target molecule (e.g., PD-1). The VH and VL genes are expressed in multiple orientations and with various linkers to form a variety of scFvs that provide the desired stability, expression levels, and binding affinity for tumor cells and specific markers thereof. In some embodiments, a divalent scFv (diabody) or divalent scFv (triabody) is used.

[00463] Some biomarkers are specific to certain cancer types. Others may be found on more than one cancer type. For example, nuclear protein Ki-67 (pKi67) is associated generally with tumor cell proliferation and growth.

[00464] Use of more than one targeting moiety can more specifically target a tumor cell, a cancer cell, or a tumor microenvironment, to maximize the dosage of the therapeutic payload to a given target and/or to reduce the incidence of damage to non- tumor or non-malignant cells.

[00465] In some embodiments, the extracellular vesicle or the micelle comprises more than one targeting moiety.

[00466] Non-limiting examples of melanoma biomarkers include, but are not limited to, human melanoma black-45 (HMB-45), melanotropin receptors, melan-A, tyronsinase, microphthalmia transcription factor, and S 100. Non-limiting examples of breast adenocarcinoma biomarkers include, but are not limited to, estrogen receptor, progesterone receptor, human epidermal growth factor 2 (HER2), ErbB2 protein, E-cadherin, Celsr2, Kail, CD9, NET6, Trop2, and serum biomarkers cancer antigen 15-3 (CA 15-3), cancer antigen 27.29 (CA 27.29), and carcinoembryonic antigen (CEA). Non-limiting examples of lung adenocarcinoma biomarkers include, but are not limited to, CA9, CA12, CXorf61, DSG3, FAT2, GPR87, KISS1R, LYPD3, SLC7A11, and TMPRSS4. Non-limiting example of colon cancer biomarkers include, but are not limited to, CD44+, CD133+, epithelial cell adhesion molecule ((EpCAM) ^hlgh ), CLDN1, LY6G6D/F, and TLR4. Non-limiting examples of pancreatic tumor or cancer biomarkers include, but are not limited to epithelial cell adhesion molecule ((EpCAM) ^hlgh ), CD109, CD133, C-Met, ALDH-1, and CD44+CD24+ESA+.

[00467] The subject may be administered more than one type of extracellular vesicle and/or micelle. For example, the subject may be administered one type of extracellular vesicle or micelle comprising a targeting moiety designed to target the tumor, the cancer, or the tumor microenvironment, e.g., while comprising an antitumor/anticancer and/or immunostimulant therapeutic payload and another type of extracellular vesicle or micelle comprising a targeting moiety designed to target an immune cell, e.g., while comprising an antitumor/anticancer and/or immunostimulant therapeutic payload. The two or more types of extracellular vesicles and/or micelles may be administered sequentially or simultaneously. The two or more types of extracellular vesicles and/or micelles may also be engineered to interact, e.g., via surface markers (e.g., a cytokine or chemokine on one with a receptor on another; an antibody on one with an antigen on another; and the like).

[00468] In certain embodiments, an extracellular vesicle or a micelle is used as a linking or connecting agent to bring immune cells and other cells and molecules close to tumor cells. For instance, engineered exosomes may have express targeting moieties (e.g., scFvs, diabodies, triabodies) that target both tumor cells and other molecules (e.g., cytotoxic T lymphocytes (CTL)).

[00469] “Inflammation,” as defined used, is part of the complex biological response of body tissues to harmful stimuli, such as pathogens, damaged cells, or irritants and is a protective response involving immune cells, blood vessels, and molecular mediators. The function of inflammation is to eliminate the initial cause of cell injury, clear out necrotic cells and tissues damaged from the original insult and the inflammatory process, and initiate tissue repair. The five classical signs of inflammation are heat, pain, redness, swelling, and loss of function. Inflammation is a generic response, and therefore, a mechanism of innate immunity. Causes of inflammation can be physical (e.g., injury/trauma, bum, frostbite, foreign body, ionizing radiation), biological (e.g., infection, immune reactions due to hypersensitivity, stress), chemical (e.g., chemical irritant, toxin, alcohol), or psychological (e.g., excitement). Inadequate inflammation can lead to tissue damage by the initial stimulus, while a prolonged response is associated with the development of chronic disease or disorder. Inflammation can be “acute” or “chronic.”

[00470] “Acute inflammation,” as used herein, is initiated by resident immune cells already present in the involved tissue, mainly resident macrophages, dendritic cells, histiocytes, Kupffer cells and mast cells. These cells possess surface receptors known as pattern recognition receptors (PRRs), which recognize and bind two subclasses of molecules: pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs). PAMPs are compounds that are associated with various pathogens, but which are distinguishable from host molecules. DAMPs are compounds that are associated with host-related injury and cell damage. In response to an infection, bum, or other injuries, these cells are activated (i.e., a PRR recognizes a PAMP or DAMP) and release inflammatory mediators responsible for the clinical signs of inflammation. Vasodilation and its resulting increased blood flow cause the redness and increased heat. Increased permeability of the blood vessels results in an exudation (leakage) of plasma proteins and fluid into the tissue (edema), resulting in swelling. Some of the released plasma- derived mediators (e.g., bradykinin) increase the sensitivity to pain. The mediator molecules also alter the blood vessels to permit the migration of leukocytes, mainly neutrophils and macrophages, outside of the blood vessels (extravasation) into the tissue. The neutrophils migrate along a chemotactic gradient created by the local cells to reach the site of injury. The loss of lunction may be a neurological reflex in response to pain. Plasma-derived mediators include, but are not limited to, C3, C5a, Factor XII, membrane attack complex, plasmin, thrombin. C3 and C5a, for example, stimulate histamine release by mast cells. This phase of inflammation is usually short-lived and is generally maintained by a cellular response. The cellular component involves leukocytes, which normally reside in blood and must move into the inflamed tissue via extravasation to aid in inflammation. Some act as phagocytes, ingesting bacteria, viruses, and cellular debris. Others release enzymatic granules that damage pathogenic invaders. The cellular response is mediated by leukocytes, which also release inflammatory mediators that develop and maintain the inflammatory response. In general, acute inflammation is mediated by granulocytes, whereas chronic inflammation is mediated by mononuclear cells such as monocytes and lymphocytes. Various leukocytes, particularly neutrophils, are critically involved in the initiation and maintenance of inflammation, resulting in a cascade of cytokines, chemokines, ligands, and receptors. Examples of cell-derived mediators include, but are not limited to, enzymes (e.g., lysosome granules, tryptase), monoamines (e.g., histamine), cytokines (e.g., interferon-gamma [IFN-y], interleukin-1 [IL-1], tumor necrosis factor-alpha [TNF-a]), chemokines (e.g., interleukin-8 [IL-8]), eicosanoids (e.g., leukotriene B4, LTC4, LTD4, 5-oxo-eicosatetraenoic acid, 5-HETE, prostaglandins), and soluble gases (e.g., nitric oxide). Typical outcomes of inflammation include resolution (complete restoration of the inflamed tissue to normal), fibrosis (scarring), abscess formation (pus-filled cavity), or chronic inflammation (e.g., due to persistence of the injurious agent or state, resulting in damage to the body’s own tissues by continued macrophage response).

[00471] “Chronic inflammation,” as used herein, may result from situations in which the injurious agent persists, leading to a continued inflammatory response. This process, marked by inflammation lasting many days, months or even years, may lead to the formation of a chronic wound, rash, or other chronic inflammatory condition. Chronic inflammation is characterized by the dominating presence of macrophages in the injured tissue. These cells are powerful defensive agents of the body, but the toxins they release (including reactive oxygen species) are injurious to the organism's own tissues as well as invading agents. In addition, other cells involved in the inflammatory response (e.g., mast cells) are often stabilized, continuously engaging in an inflammatory response, such as in the case of allergies, hypersensitivities, asthma, and chronic obstructive pulmonary disease. As a consequence, chronic inflammation is almost always accompanied by tissue destruction and may result in a chronic disease or other medical condition. Examples of diseases or other medical conditions in which chronic inflammation has been implicated, either as cause or effect, include, but are not limited to, allergic and/or hypersensitivity reactions, allergies, Alzheimer’s disease, arthritis and other joint diseases, asthma, atherosclerosis, acne vulgaris, autoimmune diseases, autoinflammatory diseases, bronchitis, cardiovascular disease (CVD; including ischemic heart disease), celiac disease, chronic obstructive pulmonary disease (COPD), chronic prostatitis, colitis, cystitis, dermatitis, diabetes, diverticulitis, familial Mediterranean fever, glomerulonephritis, chronic gout, gouty arthritis, hidradenitis suppurativa, inflammatory bowel diseases, interstitial cystitis, lichen planus, mast cell activation syndrome, mastocytosis, otitis, myopathies, pelvic inflammatory disease, chronic peptic ulcer, periodontitis, psoriasis, psoriatic arthritis, reflex sympathetic dystrophy/complex regional pain syndrome (RSD/CRPS), reperfusion injury, rheumatic fever, rheumatoid arthritis (as possibly chronic osteoarthritis), psoriasis, rhinitis, sarcoidosis, transplant rejection, vasculitis, and some cancers. In addition to injury, infections (e.g., bacterial, viral [COVID-19], fungal, parasitic), and other causes of chronic inflammation, obesity, smoking, periodontal disease, peptic ulcer, arthritis, bursitis, tendonitis, phlebitis, tonsilitis, reflex sympathetic dystrophy/complex regional pain syndrome (RSD/CRPS), asthma, tuberculosis, ulcerative colitis, Crohn’s disease, sinusitis, hepatitis, rheumatoid arthritis, and high cholesterol (e.g., high levels of low-density lipoprotein [LDL]), poor nutrition, stress, and insomnia have also been implicated as causal agents of chronic inflammation. Intestinal microbiota can fuel metabolic inflammation, e.g., by triggering an influx of bacteria-derived lipopolysaccharides (LPS) into systemic circulation. Alterations in gut microbiota composition are associated with a variety of disease states, including those associated with inflammation, such as obesity, diabetes, and inflammatory bowel disease (IBD). These diseases can have far-reaching effects, leading to increased risks of heart diseases, heart attack, and stroke (including ischemic stroke). It is noteworthy that some causes may also be effects, further contributing to the cycle of damage. Diagnosis of chronic inflammation can include a blood test measuring the amount of C-reactive protein (CRP), which rises in response to inflammation or an erythrocyte sedimentation rate. A CRP level between 1 and 3 milligrams per liter of blood often signals a low, yet chronic, level of inflammation.

[00472] Chronic “systemic inflammation” (SI), as used herein, is the result of release of pro-inflammatory cytokines from immune-related cells and the chronic activation of the innate immune system. It can contribute to the development or progression of certain conditions. Release of pro-inflammatory cytokines and activation of the innate immune system may be the result of either external (biological or chemical agents) or internal (genetic mutations/variations) factors, as well as aberrations in the microbiome (e.g., in the intestinal [gut] microbiota). Lack of control by tolerogenic dendritic cells (TDC) and T-regulatory cells (Treg) is a possible primary risk factor for the development of SI.

[00473] “Neuroinflammation,” as used herein, is inflammation of the nervous tissue. It may be initiated in response to a variety of cues, including infection, traumatic brain injury, toxic metabolites, or autoimmunity. In the central nervous system (CNS), including the brain and spinal cord, microglia are the resident innate immune cells that are activated in response to these cues. The CNS is typically an immunologically privileged site because peripheral immune cells are generally blocked by the blood-brain barrier (BBB), a specialized structure composed of astrocytes and endothelial cells. However, circulating peripheral immune cells may surpass a compromised or “leaky” BBB and encounter neurons and glial cells expressing major histocompatibility complex molecules, perpetuating the immune response. Although the response is initiated to protect the central nervous system from the infectious agent, the effect may be toxic and widespread inflammation as well as further migration of leukocytes through the blood-brain barrier. Neuroinflammation is usually chronic. While acute inflammation typically follows injury to the central nervous system immediately, and is characterized by inflammatory molecules, endothelial cell activation, platelet deposition, and tissue edema, chronic neuroinflammation is the sustained activation of glial cells and recruitment of other immune cells into the brain. It is chronic neuroinflammation that is typically associated with neurodegenerative diseases. Common causes of chronic neuroinflammation include, but are not limited to, toxic metabolites, autoimmunity, aging, microbes, viruses, traumatic brain injury, spinal cord injury, air pollution, smoking/passive smoke. The neuroimmune response relies primarily on glial cells and cytokines.

[00474] “Anti-inflammatory drugs,” as used herein, are pharmaceuticals that reduce inflammation. They include about half of analgesics, remedying pain by reducing inflammation in contrast to opioids, which affect the central nervous system to block pain signaling to the brain. Examples include, but are not limited to non-steroidal anti-inflammatory drugs (NSAID), COX- 2 inhibitors, antileukotrienes, ImSAIDs, etc.

[00475] “Non-steroidal anti-inflammatory drugs” (NSAID), as used herein, are drugs that alleviate pain by counteracting the cyclooxygenase (COX) enzyme. Inhibition of COX enzymes inhibits prostaglandin synthesis, thereby preventing PGs from inducing inflammation. Examples of NS AIDS include, but are not limited to, aspirin, ibuprofen, naproxen, and other NS AIDS.

[00476] “COX-2 inhibitors,” as used herein, are a subset of NSAIDs that specifically target cyclooxygenase-2 (COX-2), an enzyme responsible for inflammation and pain. Examples include, but are not limited to, celecoxib, rofecoxib, etoricoxib. [00477] “Antileukotrienes,” as used herein, are anti-inflammatory agents which function as leukotriene-related enzyme inhibitors (arachidonate 5 -lipoxygenase) or leukotriene receptor antagonists (cysteinyl leukotriene receptors) and consequently oppose the function of these inflammatory mediators. Although they are not used for analgesic benefits, they are widely utilized in the treatment of diseases related to inflammation of the lungs such as asthma and COPD, as well as sinus inflammation in allergic rhinitis. Examples include, but are not limited to, leukotriene receptor antagonists (e.g., montelukast, zafirlukast) and leukotriene synthesis inhibitors (e.g., zileuton), such as those blocking 5-lipooxygenase.

[00478] “Immune Selective Anti-Inflammatory Derivatives” (ImSAIDs)” as used herein, are a class of peptides which have diverse biological properties, including anti-inflammatory properties. ImSAIDs work by altering the activation and migration of inflammatory cells, which are immune cells responsible for amplifying the inflammatory response. Examples include, but are not limited to, phenylalanine-glutamine-glycine (FEG) and its D-isomeric form (feG).

[00479] A “neoplasm” is a type of abnormal and excessive growth of tissue. The process that occurs to form or produce a neoplasm is called neoplasia. The growth of a neoplasm is uncoordinated with that of the normal surrounding tissue, and persists in growing abnormally, even if the original trigger is removed. This abnormal growth usually forms a mass.

[00480] A “tumor” is a neoplasm that has formed a mass.

[00481 ] A “non-cancerous tumor” or “benign tumor” is one in which the cells demonstrate normal growth, but are produced, e.g., more rapidly, giving rise to an “aberrant lump” or “compact mass,” which is typically self-contained and does not invade tissues or metastasize to other parts of the body. Nevertheless, a non-cancerous tumor can have devastating effects based upon its location (e.g., a non-cancerous abdominal tumor that prevents pregnancy or causes a ureter, urethral, or bowel blockage, or a benign brain tumor that is inaccessible to normal surgery and yet damages the brain due to unrelieved pressure as it grows).

[00482] A “fibroma” is a benign tumor that is composed of fibrous or connective tissue. They can grow in all organs, arising from mesenchyme tissue. The term "fibroblastic" or "fibromatous" is used to describe tumors of the fibrous connective tissue. A “fibrosarcoma” is a malignant tumor (a malignant fibroma). [00483] A “pre-cancerous” condition, lesion, or tumor is a condition, lesion, or tumor comprising abnormal cells associated with a risk of developing cancer. Non-limiting examples of pre-cancerous lesions include colon polyps (which can progress into colon cancer), cervical dysplasia (which can progress into cervical cancer), and monoclonal monopathy (which can progress into multiple myeloma). Premalignant lesions comprise morphologically atypical tissue which appears abnormal when viewed under the microscope, and which are more likely to progress to cancer than normal tissue.

[00484] A “cancer” is one of a group of diseases characterized by uncontrollable growth and having the ability to invade normal tissues and to metastasize to other parts of the body. Cancers have many causes, including, but not limited to, diet, alcohol consumption, tobacco use, environmental toxins, heredity, and viral infections. In most instances, multiple genetic changes are required for the development of a cancer cell. Progression from normal to cancerous cells involves a number of steps to produce typical characteristics of cancer including, e.g., cell growth and division in the absence of normal signals and/or continuous growth and division due to failure to respond to inhibitors thereof; loss of programmed cell death (apoptosis); unlimited numbers of cell divisions (in contrast to a finite number of divisions in normal cells); aberrant promotion of angiogenesis; and invasion of tissue and metastasis.

[00485] In some embodiments, methods of using the extracellular composition or the micelle comprise treating, reducing, inhibiting, ameliorating, or alleviating a cancer or a tumor in a subject or a growth or a metastasis thereof, comprising a step of administering the extracellular composition or the micelle to the subject in a subject in need thereof. In some embodiments, methods of using the extracellular composition or the micelle comprise treating, reducing, inhibiting, ameliorating, or alleviating a cancer or a tumor in a subject in need thereof. In some embodiments, methods of using the extracellular composition or the micelle comprise treating, reducing, inhibiting, ameliorating, or alleviating metastasis of a cancer in a subject in need thereof. In some embodiments, methods of using the extracellular composition or the micelle farther comprise increasing survival in the subject. In some embodiments, methods of using the extracellular composition or the micelle further comprise reducing or inhibiting metastases infiltration. [00486] Non-limiting examples include esophageal cancer, pancreatic cancer, metastatic pancreatic cancer, metastatic adenocarcinoma of the pancreas, bladder cancer, stomach cancer, fibrotic cancer, glioma, malignant glioma, diffuse intrinsic pontine glioma, recurrent childhood brain neoplasm renal cell carcinoma, clear-cell metastatic renal cell carcinoma, kidney cancer, prostate cancer, metastatic castration resistant prostate cancer, stage IV prostate cancer, metastatic melanoma, melanoma, malignant melanoma, recurrent melanoma of the skin, melanoma brain metastases, stage IIIA skin melanoma; stage IIIB skin melanoma, stage IIIC skin melanoma; stage IV skin melanoma, malignant melanoma of head and neck, lung cancer, non-small cell lung cancer (NSCLC), squamous cell non-small cell lung cancer, breast cancer, recurrent metastatic breast cancer, hepatocellular carcinoma, Hodgkin’s lymphoma, follicular lymphoma, nonHodgkin’s lymphoma, advanced B-cell NHL, HL including diffuse large B-cell lymphoma (DLBCL), multiple myeloma, chronic myeloid leukemia, adult acute myeloid leukemia in remission; adult acute myeloid leukemia with Inv(16)(pl3.1q22); CBFB-MYH11; adult acute myeloid leukemia with t(16;16)(pl3.1;q22); CBFB-MYH11; adult acute myeloid leukemia with t(8;21)(q22;q22); RUNX1-RUNX1T1; adult acute myeloid leukemia with t(9;ll)(p22;q23); MLLT3-MLL; adult acute promyelocytic leukemia with t(15;17)(q22;ql2); PML-RARA; alkylating agent-related acute myeloid leukemia, chronic lymphocytic leukemia, Richter’s syndrome; Waldenstrom’s macroglobulinemia, adult glioblastoma; adult gliosarcoma, recurrent glioblastoma, recurrent childhood rhabdomyosarcoma, recurrent Ewing sarcoma/ peripheral primitive neuroectodermal tumor, recurrent neuroblastoma; recurrent osteosarcoma, colorectal cancer, MSI positive colorectal cancer; MSI negative colorectal cancer, nasopharyngeal nonkeratinizing carcinoma; recurrent nasopharyngeal undifferentiated carcinoma, cervical adenocarcinoma; cervical adenosquamous carcinoma; cervical squamous cell carcinoma; recurrent cervical carcinoma; stage IVA cervical cancer; stage IVB cervical cancer, anal canal squamous cell carcinoma; metastatic anal canal carcinoma; recurrent anal canal carcinoma, recurrent head and neck cancer; carcinoma, squamous cell of head and neck, head and neck squamous cell carcinoma (HNSCC), ovarian carcinoma, colon cancer, gastric cancer, advanced GI cancer, gastric adenocarcinoma; gastroesophageal junction adenocarcinoma, bone neoplasms, soft tissue sarcoma; bone sarcoma, thymic carcinoma, urothelial carcinoma, recurrent Merkel cell carcinoma; stage III Merkel cell carcinoma; stage IV Merkel cell carcinoma, myelodysplastic syndrome and recurrent mycosis fungoides and Sezary syndrome. In another related aspect, the tumor or cancer comprises a metastasis of a tumor or cancer. In some embodiments, a solid tumor treated using a method described herein, originated as a blood tumor or diffuse tumor.

[00487] A “tumor microenvironment” (TME) is the environment around a tumor, including the surrounding blood vessels, immune cells, fibroblasts, oxygen (hypoxia or hyperoxia) signaling molecules and the extracellular matrix (ECM). The tumor and the surrounding microenvironment are closely related and interact constantly. Tumors can influence the microenvironment by releasing extracellular signals, promoting tumor angiogenesis, and inducing peripheral immune tolerance, while the immune cells in the microenvironment can affect the growth and evolution of cancerous cells.

[00488] “Components of the tumor microenvironment” include, but are not limited to, cytokines, chemokines, angiogenesis factors, antigens, inflammatory proteins,

[00489] The “stroma” is defined as the non-malignant cells which are present in the tumor microenvironment. The stroma comprises a variable portion of the entire tumor; up to 90% of a tumor may be stroma, with the remaining 10% as cancer cells. Many types of cells are present in the stroma, but four abundant types are fibroblasts, T cells, macrophages, and endothelial cells. The stroma surrounding a tumor often reacts to intrusion via inflammation, similar to how it might respond to a wound. Inflammation can encourage angiogenesis, speed the cell cycle, and prevent cell death, all of which augments tumor growth.

[00490] Part of the stroma, “carcinoma associated fibroblasts” (CAFs) are a heterogenous group of fibroblasts whose function is pirated by cancer cells and redirected toward carcinogenesis. These cells are usually derived from the normal fibroblasts in the surrounding stroma but can also come from pericytes, smooth muscle cells, fibrocytes, mesenchymal stem cells (MSCs, often derived from bone marrow), or via epithelial-mesenchymal transition (EMT) or endothelial-mesenchymal transition (EndMT). Unlike their normal counterparts, CAFs do not retard cancer growth in vitro. CAFs perform several functions that support tumor growth, such as secreting vascular endothelial growth factor (VEGF), fibroblast growth factors (FGFs), platelet- derived growth factor (PDGF), and other pro-angiogenic signals to induce angiogenesis. CAFs can also secrete transforming growth factor beta (TGF-J3), which is associated with EMT, a process by which cancer cells can metastasize, and is associated with inhibiting cytotoxic T cells and natural killer T cells. As fibroblasts, CAFs are able to rework the ECM to include more paracrine survival signals such as IGF-1 and IGF-2, thus promoting survival of the surrounding cancer cells. CAFs are also associated with the Reverse Warburg Effect where the CAFs perform aerobic glycolysis and feed lactate to the cancer cells. Several markers identify CAFs, including expression of a smooth muscle actin (aSMA), vimentin, platelet-derived growth factor receptor a (PDGFR-a), platelet-derived growth factor receptor (PDGFR- ), fibroblast specific protein 1 (FSP-1) and fibroblast activation protein (FAP). Fibroblasts are in charge of laying down most of the collagens, elastin, glycosaminoglycans, proteoglycans (e.g., perlecan), and glycoproteins in the ECM. As many fibroblasts are transformed into CAFs during carcinogenesis, this reduces the amount of ECM produced and the ECM that is produced can be malformed, like collagen being loosely woven and non-planar, possibly even curved. In addition, CAFs produce matrix metalloproteinases (MMP) that cleave the proteins within the ECM. CAFs are also able to disrupt the ECM via force, generating a track that a carcinoma (cancer) cell can follow. In either case, destruction of the ECM allows cancer cells to escape from their in situ location and intravasate into the blood stream where they can metastasize systematically. It can also provide passage for endothelial cells to complete angiogenesis to the tumor site. Destruction of the ECM also modulates the signaling cascades controlled by the interaction of cell-surface receptors and the ECM, and it also reveals binding sites previously hidden, like the integrin alpha-v beta-3 (aVP3) on the surface of melanoma cells can be ligated to rescue the cells from apoptosis after degradation of collagen. In addition, the degradation products may have downstream effects as well that can increase cancer cell tumorigenicity and can serve as potential biomarkers. ECM destruction also releases the cytokines and growth factors stored therein (e.g., VEGF, basic fibroblast growth factor (bFGF), insulin-like growth factors (IGF1 and IGF2), TGF- , EGF, heparin-binding EGF- like growth factor (HB-EGF), and tumor necrosis factor (TNF)), which can increase the growth of the tumor. Cleavage of ECM components can also release cytokines that inhibit tumorigenesis, such as degradation of certain types of collagen can form endostatin, restin, canstatin and tumstatin, which have antiangiogenic functions.

[00491] In some embodiments, the biomarker for the tumor microenvironment is directed to a carcinoma associated fibroblast. In some embodiments, the biomarker for the tumor microenvironment is directed to a tumor microenvironment biomarker. Non-limiting examples of carcinoma associate fibroblast biomarkers include a smooth muscle actin (aSMA), vimentin, platelet-derived growth factor receptor a (PDGFR-a), platelet-derived growth factor receptor [3 (PDGFR-P), fibroblast specific protein 1 (FSP-1), fibroblast activation protein (FAP), vascular endothelial growth factor (VEGF), fibroblast growth factors (FGFs), platelet-derived growth factor (PDGF), and transforming growth factor-beta (TGF-J3). Other non-limiting examples of tumor microenvironments biomarkers include vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), insulin-like growth factors (IGF1 and IGF2), transforming growth factor-beta (TGF-P), epidermal growth factor (EGF), heparin-binding EGF-like growth factor (HB-EGF), and tumor necrosis factor (TNF).

[00492] In some embodiments, the method of treating, reducing, inhibiting, ameliorating, or alleviating a tumor or cancer comprises treatment of solid tumors or cancers. In some embodiments, the method of treating, reducing, inhibiting, ameliorating, or alleviating a tumor or cancer comprises treatment of a melanoma, a fibroma, or fibrosarcoma.

[00493] The method further provides a combination therapy. The term "combination therapy" means the administration of two or more therapeutic agents to treat a therapeutic disorder described herein. Such administration encompasses co-administration of these therapeutic agents in a substantially simultaneous manner, such as in a single capsule having a fixed ratio of active ingredients or in multiple, separate capsules for each active ingredient. In addition, such administration also encompasses use of each type of therapeutic agent in a sequential manner. In either case, the treatment regimen will provide beneficial effects of the drug combination in treating the disorders described herein.

[00494] The terms "subject" and "patient" are used interchangeably herein when referencing, for example, a mammalian or avian subject. The terms "subject" and "patient" are used interchangeably herein when referencing, for example, a mammalian subject, such as a human patient. In some embodiments, the subject in the method is a higher vertebrate (i.e., a mammal or a bird). In some embodiments, the subject in the method is a mammal. In some embodiments, the subject in the method is a human patient.

[00495] The term “treatment” or “treating” as used herein refers to the administering of a therapeutic effective amount of an extracellular vesicle or a micelle described herein which is effective to ameliorate undesired symptoms associated with a disease, to prevent the manifestation of such symptoms before they occur, to slow down the progression of the disease, slow down the deterioration of symptoms, to enhance the onset of remission period, slow down the irreversible damage caused in the progressive chronic stage of the disease, to delay the onset of said progressive stage, to lessen the severity or cure the disease, to improve survival rate or more rapid recovery, or to prevent the disease form occurring or a combination of two or more of the above.

[00496] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art.

[00497] Unless otherwise indicated, all numbers expressing quantities, ratios, and numerical properties of ingredients, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term "about". All parts, percentages, ratios, etc. herein are by weight unless indicated otherwise.

[00498] As used herein, the singular forms "a" or "an" or "the" are used interchangeably and intended to include the plural forms as well and fall within each meaning, unless expressly stated otherwise. Also as used herein, "at least one" is intended to mean "one or more" of the listed elements. For example, the term "a compound" or "at least one compound" may include a plurality of compounds, including mixtures thereof. Singular word forms are intended to include plural word forms and are likewise used herein interchangeably where appropriate and fall within each meaning, unless expressly stated otherwise. Except where noted otherwise, capitalized and noncapitalized forms of all terms fall within each meaning.

[00499] “Consisting of’ shall thus mean excluding more than traces of other elements. The skilled artisan would appreciate that while, in some embodiments the term “comprising” is used, such a term may be replaced by the term “consisting of’, wherein such a replacement would narrow the scope of inclusion of elements not specifically recited. The terms "comprises", "comprising", "includes", "including", “having” and their conjugates encompass "including but not limited to".

[00500] The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined. In some embodiments, the term “about” refers to a deviance of between 0.0001-5% from the indicated number or range of numbers. In some embodiments, the term “about” refers to a deviance of between 1-10% from the indicated number or range of numbers. In some embodiments, the term “about” refers to a deviance of up to 25% from the indicated number or range of numbers. In some embodiments, the term “about” refers to ± 10 %.

[00501] Throughout this application, various embodiments may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of certain embodiments. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[00502] Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases “ranging/ranges between” a first indicate number and a second indicate number and “ranging/ranges from” a first indicate number “to” a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween.

[00503] It is to be understood that the phraseology or terminology employed herein, and not otherwise defined, is for the purpose of description only and not of limitation. When a range of values is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent "about," it will be understood that the particular value forms another embodiment. All ranges are inclusive and combinable. Further, reference to values stated in ranges includes each and every value within that range. Certain features of the disclosed compositions and methods which are described herein in the context of separate aspects may also be provided in combination in a single aspect. Alternatively, various features of the disclosed compositions and methods that are, for brevity, described in the context of a single aspect, may also be provided separately or in any subcombination.

[00504] Any patent, patent application publication, or scientific publication, cited herein, is incorporated by reference herein in its entirety. [00505] The following examples are presented in order to more fully illustrate some embodiments of the invention. They should, in no way be construed, however, as limiting the broad scope of the invention. One skilled in the art can readily devise many variations and modifications of the principles disclosed herein without departing from the scope of the invention.

EXAMPLES

Example 1: Methods of Using a Targeted Extracellular Vesicle for Targeting a Therapy to a Tumor Cell or a Cancer Cell or to a Tumor Microenvironment

[00506] A subject is identified as having a tumor or a cancer, and a target (e.g., cell surface activation receptor, biomarker, or imaging) is identified on the tumor cell, the cancer cell, or the immune cell, or a target of a component of the tumor microenvironment (e.g., hypoxia).

[00507] An extracellular vesicle (EV) is constructed having at least one targeting moiety, which is directed to the target; and at least one therapeutic payload. The EV is then administered to the subject.

Example 2: Methods of Using a Targeted Micelle for Targeting a Therapy to a Tumor Cell or a Cancer Cell or to a Tumor Microenvironment

[00508] A subject is identified as having a tumor or a cancer, and a target (e.g., a biomarker) is identified on the tumor cell, the cancer cell, or the immune cell, or a target of a component of the tumor microenvironment.

[00509] A micelle is constructed having at least one targeting moiety, which is directed to the target; and at least one therapeutic payload. The micelle is then administered to the subject.

Example 3: Methods of Using a Targeted Extracellular Vesicle for Targeting a Therapy to a Tumor Cell or a Cancer Cell or to a Tumor Microenvironment Where the Tumor or Cancer is the Result of a Mutation

[00510] A subject is identified as having a tumor or a cancer, and a target (e.g., a biomarker) is identified on the tumor cell, the cancer cell, or the immune cell, or a target of a component of the tumor microenvironment. The tumor cell or the cancer cell comprises a mutant nucleic acid encoding a defective protein, the defective protein associated with abnormal growth, carcinogenesis, angiogenesis, or metastasis.

[00511] An extracellular vesicle (EV) is constructed having at least one targeting moiety, which is directed to the defective protein or to the mutant nucleic acid; and at least one therapeutic payload comprising the corresponding wild-type protein or an expression vector comprising a nucleic acid encoding the wild-type protein.

[00512] The EV is then administered to the subject.

Example 4: Methods of Using a Targeted Extracellular Vesicle or a Targeted Micelle for Targeting a Therapy to a Tumor Cell or a Cancer Cell or to a Tumor Microenvironment in a Subject with Melanoma

[00513] A subject is diagnosed with melanoma, and a biomarker (e.g., a human melanoma black-45 [HMB-45], melan-A, tyrosinase, microphthalmia transcription factor, S100, or melanotropin receptor) or other target is identified on the melanoma cell or in the tumor microenvironment.

[00514] An extracellular vesicle (EV) or a micelle is constructed having at least one targeting moiety, which is directed to the biomarker or other target; and at least one therapeutic payload. The EV or micelle is then administered to the subject.

Example 5: Methods of Using a Targeted Extracellular Vesicle or a Targeted Micelle for Targeting a Therapy to a Tumor Cell or a Cancer Cell or to a Tumor Microenvironment in a Subject with Breast Adenocarcinoma

[00515] A subject is diagnosed with breast adenocarcinoma, and a biomarker (e.g., an estrogen receptor, a progesterone receptor, human epidermal growth factor 2 [HER2], or another biomarker) or other target is identified on a breast adenocarcinoma cell or in the tumor microenvironment.

[00516] An extracellular vesicle (EV) is constructed having at least one targeting moiety, which is directed to the biomarker or other target; and at least one therapeutic payload. The EV is then administered to the subject.

Example 6: Methods of Using a Targeted Extracellular Vesicle or a Targeted Micelle for Targeting a Therapy to a Tumor Cell or a Cancer Cell or to a Tumor Microenvironment in a Subject with Lung Adenocarcinoma

[00517] A subject is diagnosed with lung adenocarcinoma, and a biomarker (e.g., CA9, CA12, CXorf61, DSG3, FAT2, GPR87, KISS1R, LYPD3, SLC7A11, TMPRSS4, or another biomarker) or other target is identified on a lung adenocarcinoma cell or in the tumor microenvironment.

[00518] An extracellular vesicle (EV) is constructed having at least one targeting moiety, which is directed to the biomarker or other target; and at least one therapeutic payload. The EV is then administered to the subject.

Example 7: Methods of Using a Targeted Extracellular Vesicle or a Targeted Micelle for Targeting a Therapy to a Tumor Cell or a Cancer Cell or to a Tumor Microenvironment in a Subject with Colon Cancer

[00519] A subject is diagnosed with colon cancer, and a biomarker (e.g., CD44+, CD133+, epithelial cell adhesion molecule [(EpCAM)high], CLDN1, LY6G6D/F, TLR4, or another biomarker) or other target is identified on a colon cancer cell or in the tumor microenvironment.

[00520] An extracellular vesicle (EV) is constructed having at least one targeting moiety, which is directed to the biomarker or other target; and at least one therapeutic payload. The EV is then administered to the subject.

Example 8: Methods of Using a Targeted Extracellular Vesicle or a Targeted Micelle for Targeting a Therapy to a Tumor Cell or a Cancer Cell or to a Tumor Microenvironment in a Subject with Pancreatic Cancer

[00521] A subject is diagnosed with pancreatic cancer, and a biomarker (e.g., epithelial cell adhesion molecule [(EpCAM)high], CD109, CD133, C-Met, ALDH-1,

CD44+CD24+ESA+, or another biomarker) or other target is identified on a pancreatic cancer cell or in the tumor microenvironment.

[00522] An extracellular vesicle (EV) is constructed having at least one targeting moiety, which is directed to the biomarker or other target; and at least one therapeutic payload. The EV is then administered to the subject.

Example 9: Preparation and Use of Exosomes for Treatment of Cancer [00523] A cancer cell or a sample of cancer cells is isolated from a patient and subjected to a biomarker study. Biomarkers for the cancer cells are identified. One or more biomarkers predominantly found on the cancer cells (as opposed to healthy, non-malignant cells in the patient) or biomarkers found in significantly larger numbers on cancer cells (a opposed to healthy, non- malignant cells in the patient) is selected, and a corresponding targeting moiety is selected (e.g., a cytokine or chemokine targeting moiety for a receptor biomarker (e.g., a surface receptor biomarker; a receptor targeting moiety for a cytokine biomarker or a chemokine biomarker; an antibody or antigen-binding domain targeting moiety for an antigen biomarker; an antigen targeting moiety for an antibody or antigen-binding domain biomarker; a CAR; or a clathrin moiety).

[00524] A programmed death antibody (e.g., anti-PD-1 and/or anti-PD-Ll) or antigenbinding domain is selected as a therapeutic payload.

[00525] An expression plasmid is constructed to express a protein in which the programmed death antibody or antigen binding domain is fused selected targeting moiety and one or more of these components is tagged with selectable marker(s). In some embodiments, more than one component is tagged, each with a different selectable marker.

[00526] Alternatively, the expression plasmid is constructed to express a protein in which the programmed death antibody or antigen binding domain is lused to both the selected targeting moiety and a selectable marker that allows the appropriate exosome separation following plasmid expression in cells and their release from the expression cells as follows below.

[00527] The plasmid is transfected and expressed in human embryonic kidney (HEK) cells. The exosomes released from the transfected HEK cells are collected, and the selectable fluorescent marker exosomes comprising the targeted treatment composition are separated from the bulk of the released exosomes.

[00528] The exosomes are administered to the patient (e.g., intravenously, intramuscularly, subcutaneously, or orally) to treat the targeted disease.

Example 10: Preparation of a Lentivector Plasmid for Use in Human Cells [00529] As shown in FIGURE 1, a CD512B-1 lentivector plasmid was used as a backbone. The fusion gene was synthesized and cloned into the MCS region of the plasmid. Using the above modified plasmid, the DNA sequence of anti-mouse PD-1 (CD279) ScFv - RFP was introduced as shown in FIGURE 1.

[00530] The DNA sequence of Protin-101 or the DNA sequence of the clathrin light chain as described in Table 1 with FLAG tag is synthesized and cloned to replace the RFP in the plasmid. The FLAG tag has the protein sequence DYKDDDDK (SEQ ID NO: 14). The sequences relating to Protin-101 with FLAG tag are described in Table 2.

[00531] Table 2. Sequences Relating to Protin-101 with FLAG tag

[00532] The sequences relating to anti-mouse PD-1 ScFv - RFP (red fluorescent protein) fusion are described in Table 3. See GenBank ID: AF506027 and Campbell et al. 2002. A monomeric red fluorescent protein. Proc. Natl. Acad. Sci. USA 99(12) 7877-7882.

[00533] Table 3. Sequences Relating to Anti-mouse PD-1 ScFv-RFP.

[00534] While certain features of the invention have been illustrated and described herein, many modifications, substitutions, changes, and equivalents will now occur to those of ordinary skill in the art. It is, therefore, to be understood that the appended claims are intended to cover all such modifications and changes as fall within the true spirit of the invention.