Extracellular vesicles for vaccine delivery

Abstract

This disclosure relates to extracellular vesicles (EVs), such as exosomes, comprising a payload (e.g., an antigen, adjuvant, and / or immunomodulator) and / or a targeting portion. Methods for generating EVs (e.g., exosomes) and methods for using EVs (e.g., exosomes) to treat and / or prevent diseases or conditions, such as cancer, graft-versus-host disease (GvHD), autoimmune diseases, infectious diseases, or fibrotic diseases, are also provided herein.

Description

[0001] Cross-reference to related applications

[0002] This PCT application claims priority to U.S. Provisional Application No. 62 / 822,008, filed March 21, 2019; No. 62 / 835,437, filed April 17, 2019; No. 62 / 840,348, filed April 29, 2019; No. 62 / 891,048, filed August 23, 2019; No. 62 / 901,166, filed September 16, 2019; No. 62 / 946,280, filed December 10, 2019; and No. 62 / 984,146, filed March 2, 2020, each of which is incorporated herein by reference in its entirety.

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

[0004] The contents of the electronically submitted sequence list (name: 4000_032PC07_Sequencelisting_ST25.txt, size: 283,505 bytes; and creation date: March 20, 2020) submitted in this application are incorporated herein by reference in their entirety. Technical Field

[0005] This disclosure relates to modified extracellular vesicles, such as exosomes (e.g., containing one or more payloads, such as antigens and adjuvants / immunomodulators), which can be used as vaccines for the treatment and / or prevention of a range of medical conditions, including but not limited to cancer, graft-versus-host disease (GvHD), autoimmune diseases, infectious diseases, and fibrotic diseases. This disclosure also relates to methods for generating such EVs (e.g., exosomes) and their uses. Technical Background

[0006] EVs (e.g., exosomes) are important mediators of intercellular communication. They are also important biomarkers for the diagnosis and prognosis of many diseases, such as cancer. As drug delivery mediators, EVs (e.g., exosomes) offer many advantages over conventional drug delivery methods (e.g., peptide immunization, DNA vaccines) as novel therapeutic modalities in many therapeutic areas. However, despite their advantages, the clinical efficacy of many EVs (e.g., exosomes) is limited. For example, in a phase II clinical trial, dendritic cell-derived exosomes (DEX) were investigated as maintenance immunotherapy following first-line chemotherapy in patients with inoperable non-small cell lung cancer (NSCLC). However, the trial was terminated because it did not meet the primary endpoint (at least 50% of patients had 4 months of progression-free survival (PFS) after chemotherapy cessation). (Besse, B. et al., Oncoimmunology 5(4):e1071008 (2015)).

[0007] Therefore, new and more effective engineered EVs (e.g., exogenous bodies) are needed to better realize the therapeutic uses and other applications of EV-based technologies. Summary of the Invention

[0008] This document provides isolated EVs (e.g., exogens) comprising (i) at least one antigen and (ii) at least one adjuvant. In some aspects, the EV comprises at least two, three, four, five, six, seven, eight, nine, ten, or more different antigens. In some aspects, the EV comprises at least two, three, four, five, six, seven, eight, nine, ten, or more different adjuvants. In some aspects, the antigen is not present on class I and / or class II MHC molecules.

[0009] In some respects, EVs (e.g., exogenous cells) do not originate from naturally occurring antigen-presenting cells. In other respects, EVs (e.g., exogenous cells) do not originate from naturally occurring dendritic cells, naturally occurring B cells, naturally occurring mast cells, naturally occurring macrophages, naturally occurring neutrophils, naturally occurring Kupffer-Browicz cells, cells derived from any of these cells, or any combination thereof.

[0010] In some respects, EVs (e.g., exogens) induce cellular immune responses, humoral immune responses, or cellular and humoral immune responses. In some respects, the induction of cellular immune responses, humoral immune responses, or cellular and humoral immune responses is increased by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100% or more compared to (i) a corresponding EV (e.g., an exogen) without an adjuvant or antigen, or (ii) an adjuvant or antigen without an EV (i.e., a non-EV delivery medium).

[0011] In some respects, the EVs (e.g., exogenous forms) described herein induce CD4+ T cell responses, CD8+ T cell responses, or CD4+ and CD8+ T cell responses. In other respects, the EVs (e.g., exogenous forms) do not directly interact with the T cell receptor (TCR) of T cells.

[0012] In some aspects, the EV (e.g., exogenous body) of this disclosure further includes a first stent portion. In some aspects, an antigen is connected to the first stent portion. In some aspects, an adjuvant is connected to the first stent portion. In some aspects, the EV (e.g., exogenous body) further includes a second stent portion. In some aspects, the antigen is connected to the first stent portion, and the adjuvant is connected to the second stent portion. In some aspects, the first stent portion and the second stent portion are identical. In other aspects, the first stent portion and the second stent portion are different.

[0013] In some respects, the first support component is support X. In other respects, the first support component is support Y. In some respects, the second support component is support X. In other respects, the second support component is support Y.

[0014] In some respects, the stent X is capable of: (i) anchoring an antigen to the luminal surface of an EV (e.g., an exogenous body); (ii) anchoring an antigen to the outer surface of an EV (e.g., an exogenous body); (iii) anchoring an adjuvant to the luminal surface of an EV (e.g., an exogenous body); (iv) anchoring an adjuvant to the outer surface of an EV (e.g., an exogenous body); or (v) combinations thereof. In some respects, scaffold X is selected from the group consisting of: prostaglandin F2 receptor negative regulator (PTGFRN protein); basigin (BSG protein); immunoglobulin superfamily member 2 (IGSF2 protein); immunoglobulin superfamily member 3 (IGSF3 protein); immunoglobulin superfamily member 8 (IGSF8 protein); integrin β-1 (ITGB1 protein); integrin α-4 (ITGA4 protein); 4F2 cell surface antigen heavy chain (SLC3A2 protein); a class of ATP transporters (ATP1A1, ATP1A2, ATP1A3, ATP1A4, ATP1B3, ATP2B1, ATP2B2, ATP2B3, ATP2B4 proteins) and any combination thereof.

[0015] In some respects, scaffold Y is capable of: (i) anchoring an antigen to the luminal surface of an EV (e.g., an exogenous body); (ii) anchoring an adjuvant to the luminal surface of an EV (e.g., an exogenous body); or (iii) both. In some respects, scaffold Y is selected from the group consisting of: myristyl alanine-rich protein kinase C substrate (MARCKS protein); myristyl alanine-rich protein kinase C substrate-like protein 1 (MARCKSL1 protein); brain acid-soluble protein 1 (BASP1 protein); and any combination thereof.

[0016] In some aspects, the antigen is attached to a first stent portion on the luminal surface of an EV (e.g., an exogenous body) (such as those described herein), and the adjuvant is attached to a second stent portion on the luminal surface of the EV (e.g., an exogenous body) (such as those described herein). In some such aspects, (a) each of the first and second stent portions is stent Y; (b) the first stent portion is stent Y and the second stent portion is stent X; (c) the first stent portion is stent X and the second stent portion is stent Y; or (d) each of the first and second stent portions is stent X.

[0017] In some aspects, the antigen is attached to a first scaffold portion on the luminal surface of the EV (e.g., an exogenous body), and the adjuvant is within the lumen of the EV. In some aspects, the antigen is within the lumen of the EV (e.g., an exogenous body), and the adjuvant is attached to a first scaffold portion on the luminal surface of the EV. In other aspects, the antigen is attached to a first scaffold portion on the luminal surface of the EV (e.g., an exogenous body), and the adjuvant is attached to a second scaffold portion on the outer surface of the exogenous body. In some of these aspects, (a) the first scaffold portion is scaffold Y, and the second scaffold portion is scaffold X; or (b) each of the first and second scaffold portions is scaffold X.

[0018] In some aspects, the antigen is attached to a first scaffold portion on the outer surface of the EV (e.g., an exogenous body), and the adjuvant is attached to a second scaffold portion within the luminal surface of the EV. In some aspects of these aspects, (a) the first scaffold portion is scaffold X and the second scaffold portion is scaffold Y; or (b) each of the first and second scaffold portions is scaffold X.

[0019] In some respects, the antigen is located within the lumen of the EV (e.g., an exogenous body) or attached to its lumen surface, and the adjuvant is located within the lumen of the EV (e.g., an exogenous body) or attached to its lumen surface.

[0020] In some aspects, the antigen is attached to a first scaffold portion on the outer surface of the EV (e.g., an exogenous organism), and the adjuvant is attached to a second scaffold portion on the outer surface of the EV (e.g., an exogenous organism). In some such aspects, the first and second scaffold portions are scaffold X.

[0021] In some aspects, the antigen is attached to a first scaffold portion on the outer surface of the EV (e.g., exogenous body), and the adjuvant is located within the lumen of the EV (e.g., exogenous body). In some of these aspects, the first scaffold is scaffold X.

[0022] In some aspects, the antigen is within the lumen of the EV (e.g., exogenous body), and the adjuvant is attached to a first scaffold portion on the outer surface of the EV (e.g., exogenous body). In some of these aspects, the first scaffold is X.

[0023] In some aspects, the antigen is attached to a first scaffold portion on the surface of the EV (e.g., an exogenous organism), and the adjuvant is attached to a first scaffold portion on the luminal surface of the EV (e.g., an exogenous organism). In some aspects, the antigen is attached to a first scaffold portion on the luminal surface of the EV (e.g., an exogenous organism), and the adjuvant is attached to a first scaffold portion on the outer surface of the EV (e.g., an exogenous organism). In some of these aspects, the first scaffold portion is scaffold X.

[0024] In some respects, (i) the antigen is linked to a first scaffold portion via a connector, (ii) the antigen is linked to a second scaffold portion via a connector, (iii) the adjuvant is linked to the first scaffold portion via a connector, (iv) the adjuvant is linked to the second portion via a connector, or (v) combinations thereof. In some respects, the connector is a polypeptide. In other respects, the connector is a non-polypeptide portion. In some respects, the connector contains a maleimide portion. In some respects, the connector contains a cholesterol portion.

[0025] In some aspects, the first or second scaffold portion is a PTGFRN protein. In some aspects, the first or second scaffold portion comprises an amino acid sequence as shown in SEQ ID NO:33. In other aspects, the first or second scaffold portion comprises an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or about 100% identity with SEQ ID NO:1.

[0026] In some aspects, the first scaffold portion or the second scaffold portion is the BASP1 protein. In some aspects, the first scaffold portion or the second scaffold portion comprises a peptide of (M)(G)(π)(X)(Φ / π)(π)(+)(+) or (G)(π)(X)(Φ / π)(π)(+)(+), wherein each bracket position represents an amino acid, and wherein π is any amino acid selected from the group consisting of Pro, Gly, Ala, and Ser, X is any amino acid, Φ is any amino acid selected from the group consisting of Val, Ile, Leu, Phe, Trp, Tyr, and Met, and (+) is any amino acid selected from the group consisting of Lys, Arg, and His; and wherein position five is not (+) and position six is ​​neither (+) nor (Asp or Glu). In some aspects, the first scaffold portion or the second scaffold portion comprises the amino acid sequence shown in any one of SEQ ID NO:50-155. In another aspect, the first or second stent portion comprises an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or about 100% identity with SEQ ID NO:3.

[0027] This document also provides an EV (e.g., an exogenous body) comprising (i) an antigen and (ii) an adjuvant, wherein: (a) the antigen is connected to a first scaffold Y on the luminal surface of the EV (e.g., an exogenous body), and the adjuvant is connected to a second scaffold Y on the luminal surface of the EV (e.g., an exogenous body); (b) the antigen is connected to the first scaffold Y on the luminal surface of the EV (e.g., an exogenous body), and the adjuvant is within the lumen of the EV (e.g., an exogenous body); (c) the antigen is within the lumen of the EV (e.g., an exogenous body), and the adjuvant is connected to a scaffold Y on the luminal surface of the EV (e.g., an exogenous body); (d) the antigen is connected to a scaffold Y on the luminal surface of the EV (e.g., an exogenous body), and the adjuvant is connected to a scaffold X on the outer surface of the EV (e.g., an exogenous body). (e) The antigen is within the lumen of the EV (e.g., exogenous body), and the adjuvant is attached to a scaffold X on the outer surface of the EV (e.g., exogenous body); (f) The antigen is attached to a scaffold Y on the lumen surface of the EV (e.g., exogenous body), and the adjuvant is attached to a scaffold X on the lumen surface of the EV (e.g., exogenous body); (g) The antigen is within the lumen of the EV (e.g., exogenous body), and the adjuvant is attached to a scaffold X on the lumen surface of the EV (e.g., exogenous body); (h) The antigen is attached to a scaffold X on the lumen surface of the EV (e.g., exogenous body), and the adjuvant is attached to a scaffold X on the outer surface of the EV (e.g., exogenous body); (i) The antigen is attached to a first scaffold X on the outer surface of the EV (e.g., exogenous body), and the adjuvant is attached to a scaffold X on the outer surface of the EV (e.g., exogenous body). (j) The antigen is connected to a second scaffold X on the outer surface of the EV (e.g., exogenous body); and the adjuvant is connected to a scaffold X on the outer surface of the EV (e.g., exogenous body); (k) The antigen is connected to a scaffold X on the outer surface of the EV (e.g., exogenous body), and the adjuvant is within the lumen of the EV (e.g., exogenous body); (l) The antigen is connected to a scaffold X on the outer surface of the EV (e.g., exogenous body), and the adjuvant is connected to a scaffold X on the lumen of the EV (e.g., exogenous body); (m) The antigen is connected to a first scaffold X on the lumen of the EV (e.g., exogenous body), and the adjuvant is connected to a second scaffold X on the lumen of the EV (e.g., exogenous body); (n) The antigen is connected to a second scaffold X on the outer surface of the EV (e.g., exogenous body); (o) An antigen is connected to a scaffold X on the luminal surface of an EV (e.g., an exogenous body), and an adjuvant is connected to a scaffold Y on the luminal surface of an EV (e.g., an exogenous body); (p) An antigen is connected to a first scaffold X on the outer surface of an EV (e.g., an exogenous body), and an adjuvant is connected to a second scaffold X on the luminal surface of an EV (e.g., an exogenous body); (q) An antigen is connected to a first scaffold X on the luminal surface of an EV (e.g., an exogenous body), and an adjuvant is connected to a second scaffold X on the outer surface of an EV (e.g., an exogenous body); (r) An antigen is in the lumen of an EV (e.g., an exogenous body), and an adjuvant is on the luminal surface of an EV (e.g., an exogenous body).(s) The antigen is directly attached to the luminal surface of the EV, and the adjuvant is also directly attached to the luminal surface of the EV; (t) The antigen is directly attached to the luminal surface of the EV, and the adjuvant is inside the lumen of the EV; (u) The antigen is directly attached to the luminal surface of the EV, and the adjuvant is attached to a scaffold Y on the luminal surface of the EV; (v) The antigen is directly attached to the luminal surface of the EV, and the adjuvant is attached to a scaffold X on the luminal surface of the EV; (w) The antigen is directly attached to the luminal surface of the EV, and the adjuvant is directly attached to the outer surface of the EV; (x) The antigen is directly attached to the luminal surface of the EV, and the adjuvant is attached to a scaffold X on the outer surface of the EV; (y) The antigen is attached to a scaffold Y on the luminal surface of the EV, and the adjuvant is directly attached to the luminal surface of the EV; (z) The antigen is attached to a scaffold Y on the luminal surface of the EV, and the adjuvant is directly attached to the outer surface of the EV; (aa) The antigen is attached to a scaffold X on the luminal surface of the EV, and the adjuvant is directly attached to the luminal surface of the EV; (bb) The antigen is attached to a scaffold X on the luminal surface of the EV, and the adjuvant is directly attached to the outer surface of the EV; (cc) The antigen is inside the lumen of the EV, and the adjuvant is directly attached to the luminal surface of the EV; or (dd) The antigen is inside the lumen of the EV, and the adjuvant is directly attached to the outer surface of the EV.

[0028] In some aspects, the EVs (e.g., exogenous bodies) disclosed herein also contain immunomodulators. In some aspects, the immunomodulators are directly attached to the luminal or outer surface of the EV. In some aspects, the immunomodulators are attached to a scaffold X on the outer surface or luminal surface of the EV (e.g., exogenous body). In some aspects, the immunomodulators are attached to a scaffold Y on the luminal surface of the EV (e.g., exogenous body). In other aspects, the immunomodulators are located on the luminal surface of the EV (e.g., exogenous body).

[0029] In some aspects, the EV comprises an antigen, an adjuvant, and an immunomodulator, wherein: (i) the antigen is directly attached to the luminal surface via a connector, (ii) the adjuvant is directly attached to the luminal surface via a connector, (iii) the immunomodulator is directly attached to the luminal surface via a connector, (iv) the antigen is directly attached to the outer surface via a connector, (v) the adjuvant is directly attached to the outer surface via a connector, (vi) the immunomodulator is directly attached to the outer surface via a connector, or (vii) a combination thereof. In some aspects, the EV comprises an antigen, an adjuvant, and an immunomodulator, wherein: (i) the antigen is attached to a stent X via a connector, (ii) the adjuvant is attached to a stent X via a connector, (iii) the immunomodulator is attached to a stent X via a connector, (iv) the antigen is attached to a stent Y via a connector, (v) the adjuvant is attached to a stent Y via a connector, (vi) the immunomodulator is attached to a stent Y via a connector, or (vii) a combination thereof. In some aspects, the immunomodulator is located within the lumen of the EV.

[0030] In some respects, the linker is a polypeptide. In some respects, the linker is a non-polypeptide moiety. In some respects, the linker contains a maleimide moiety. In some respects, the linker contains a cholesterol moiety.

[0031] In some respects, immunomodulators include inhibitors of negative checkpoint regulators or inhibitors of binding partners of negative checkpoint regulators. In some respects, negative checkpoint regulators include cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), programmed cell death protein 1 (PD-1), lymphocyte activation gene 3 (LAG-3), protein 3 containing T-cell immunoglobulin mucin (TIM-3), B-lymphocyte and T-lymphocyte attenuator (BTLA), T-cell immune receptor with Ig and ITIM domains (TIGIT), T-cell activation V-domain Ig repressor (VISTA), adenosine A2a receptor (A2aR), cytotoxic cell immunoglobulin-like receptor (KIR), indoleamine 2,3-dioxygenase (IDO), CD20, CD39, CD73, or any combination thereof.

[0032] In some respects, immunomodulators include activators of positive costimulatory molecules or activators of binding partners of positive costimulatory molecules. In some respects, positive costimulatory molecules are members of the TNF receptor superfamily (e.g., CD120a, CD120b, CD18, OX40, CD40, Fas receptor, M68, CD27, CD30, 4-1BB, TRAILR1, TRAILR2, TRAILR3, TRAILR4, RANK, OCIF, TWEAK receptor, TACI, BAFF receptor, ATAR, CD271, CD269, AITR, TROY, CD358, TRAMP, and XEDAR). In some respects, the activators of positive costimulatory molecules are members of the TNF superfamily (e.g., TNFα, TNF-C, OX40L, CD40L, FasL, LIGHT, TL1A, CD27L, Siva, CD153, 4-1BB ligand, TRAIL, RANKL, TWEAK, APRIL, BAFF, CAMLG, NGF, BDNF, NT-3, NT-4, GITR ligand, and EDA-2). In other respects, the positive costimulatory molecules are costimulators of the CD28 superfamily (e.g., ICOS or CD28). In some respects, the activators of positive costimulatory molecules are ICOSL, CD80, or CD86.

[0033] In some respects, immunomodulators include cytokines or cytokine conjugates. In some respects, cytokines include IL-2, IL-4, IL-7, IL-10, IL-12, IL-15, IL-21, IFN-γ, IL-1α, IL-1β, IL-1ra, IL-18, IL-33, IL-36α, IL-36β, IL-36γ, IL-36ra, IL-37, IL-38, IL-3, IL-5, IL-6, IL-11, IL-13, IL-23, granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte-macrophage colony-stimulating factor (G-CSF), leukemia suppressor factor (LIF), and stem cell factor (S). CF), thrombopoietin (TPO), macrophage-colony-stimulating factor (M-CSF), erythropoietin (EPO), Flt-3, IFN-α, IFN-β, IFN-γ, IL-19, IL-20, IL-22, IL-24, TNF-α, TNF-β, BAFF, APRIL, lymphotoxin β (TNF-γ), IL-17A, IL-17B, IL-17C, IL-17D, IL-17E, IL-17F, IL-25, TSLP, IL-35, IL-27, TGF-β, or combinations thereof.

[0034] In some respects, immunomodulators contain proteins that support intracellular interactions required for germinal center responses. These proteins include members of the signaling lymphocyte activation molecule (SLAM) family, SLAM-associated proteins (SAP), ICOS-ICOSL, CD40-40L, CD28 / B7, PD-1 / L1, IL-4 / IL4R, IL21 / IL21R, TLR4, TLR7, TLR8, TLR9, CD180, CD22, or combinations thereof. In some respects, SLAM family members include SLAM family member 1, CD48, CD229 (Ly9), Ly108, 2B4, CD84, NTB-A, CRACC, BLAME, CD2F-10, or combinations thereof.

[0035] This document also provides isolated EVs (e.g., exogenous organisms) comprising (i) an antigen and (ii) an immunomodulator, wherein: (a) the antigen is attached to a first scaffold Y on the luminal surface of the EV, and the immunomodulator is attached to a second scaffold Y on the luminal surface of the EV; (b) the antigen is attached to a scaffold Y on the luminal surface of the EV, and the immunomodulator is within the lumen of the EV; (c) the antigen is within the lumen of the EV, and the immunomodulator is attached to a scaffold Y on the luminal surface of the EV; (d) the antigen is attached to a scaffold Y on the luminal surface of the EV, and the immunomodulator is attached to a scaffold X on the outer surface of the EV; (e) the antigen is within the lumen of the EV, and the immunomodulator is attached to a scaffold X on the outer surface of the EV; (f) the antigen is attached to a scaffold Y on the luminal surface of the EV. (g) The antigen is inside the lumen of the EV, and the immunomodulator is connected to the scaffold X on the lumen surface of the EV; (h) The antigen is connected to the scaffold X on the lumen surface of the EV, and the immunomodulator is connected to the scaffold X on the outer surface of the EV; (i) The antigen is connected to the first scaffold X on the outer surface of the EV, and the immunomodulator is connected to the second scaffold X on the outer surface of the EV; (j) The antigen is connected to the scaffold X on the outer surface of the EV, and the immunomodulator is connected to the scaffold Y on the lumen surface of the EV; (k) The antigen is connected to the scaffold X on the lumen surface of the EV, and the immunomodulator is inside the lumen of the EV; (l) The antigen is connected to the scaffold Y on the outer surface of the EV. (m) The antigen is connected to a first stent X on the luminal surface of the EV, and the immunomodulator is connected to a second stent X on the luminal surface of the EV; (n) The antigen is connected to a stent X on the luminal surface of the EV, and the immunomodulator is connected to a stent Y on the luminal surface of the EV; (o) The antigen is connected to a stent X on the luminal surface of the EV, and the immunomodulator is inside the lumen of the EV; (p) The antigen is connected to a first stent X on the outer surface of the EV, and the immunomodulator is connected to a second stent X on the luminal surface of the EV; (q) The antigen is connected to a first stent X on the luminal surface of the EV, and the immunomodulator is connected to a second stent X on the outer surface of the EV; (r) The antigen is inside the lumen of the EV, and the immunomodulator is inside the lumen of the EV; (s) The antigen is directly attached to the lumen surface of the EV, and the immunomodulator is directly attached to the lumen surface of the EV; (t) The antigen is directly attached to the lumen surface of the EV, and the immunomodulator is inside the lumen of the EV; (u) The antigen is directly attached to the lumen surface of the EV, and the immunomodulator is attached to a scaffold Y on the lumen surface of the EV; (v) The antigen is directly attached to the lumen surface of the EV, and the immunomodulator is attached to a scaffold X on the lumen surface of the EV; (w) The antigen is directly attached to the lumen surface of the EV, and the immunomodulator is directly attached to the outer surface of the EV; (x) The antigen is directly attached to the lumen surface of the EV, and the immunomodulator is attached to a scaffold X on the outer surface of the EV.(y) The antigen is attached to scaffold Y on the luminal surface of the EV, and the immunomodulator is directly attached to the luminal surface of the EV; (z) The antigen is attached to scaffold Y on the luminal surface of the EV, and the immunomodulator is directly attached to the exterior of the EV; (aa) The antigen is attached to scaffold X on the luminal surface of the EV, and the immunomodulator is directly attached to the luminal surface of the EV; (bb) The antigen is attached to scaffold X on the luminal surface of the EV, and the immunomodulator is directly attached to the exterior of the EV; (cc) The antigen is inside the lumen of the EV, and the immunomodulator is directly attached to the luminal surface of the EV; or (dd) The antigen is inside the lumen of the EV, and the immunomodulator is directly attached to the exterior of the EV.

[0036] In some aspects, an EV (e.g., a foreign body) containing (i) an antigen and (ii) an immunomodulatory agent also contains an adjuvant (e.g., those described herein). In some of these aspects, the adjuvant is attached to a scaffold X on the outer surface of the EV (e.g., a foreign body) or on the luminal surface of the EV (e.g., a foreign body). In some of these aspects, the adjuvant is attached to a scaffold Y on the luminal surface of the EV (e.g., a foreign body). In other aspects, the adjuvant is within the lumen of the EV (e.g., a foreign body). In some aspects, the adjuvant is directly attached to the luminal or outer surface of the EV.

[0037] In some respects, antigens are tumor antigens. Tumor antigens include alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), epithelial tumor antigen (ETA), mucin 1 (MUC1), Tn-MUC1, mucin 16 (MUC16), tyrosinase, melanoma-associated antigen (MAGE), tumor protein p53 (p53), CD4, CD8, CD45, CD80, CD86, programmed death-ligand 1 (PD-L1), programmed death-ligand 2 (PD-L2), NY-ESO-1, PSMA, TAG-72, HER2, GD2, cMET, EGFR, mesothelin, VEGFR, α-folate receptor, CE7R, IL-3, testicular cancer antigen, MART-1gp100, TNF-associated apoptosis-inducing ligand, Brachyury (e.g., the antigen expressed in melanoma (PRAME)), Wilms tumor 1 (WT1), CD19, CD22, or any combination thereof.

[0038] In some respects, antigens originate from bacteria, viruses, fungi, protozoa, or any combination thereof. In other respects, antigens originate from carcinogenic viruses. In some respects, the antigens are derived from human gamma herpesvirus 4 (Epstein-Barr virus), influenza A virus, influenza B virus, cytomegalovirus, Staphylococcus aureus, Mycobacterium tuberculosis, Chlamydia trachomatis, HIV (e.g., HIV-1, HIV-2), coronaviruses (e.g., COVID-19, MERS-CoV, and SARS-CoV), filamentous viruses (e.g., Marburg virus and Ebola virus), Streptococcus pyogenes, Streptococcus pneumoniae, Plasmodium species (e.g., Plasmodium vivax and Plasmodium falciparum), chikungunya virus, human papillomavirus (HPV), hepatitis B, hepatitis C, human herpesvirus 8, and Merkel cell polyomavirus. Polyomavirus (MCV), Bunyavirus (e.g., Hantavirus), Arenavirus (e.g., LCMV and Lassa virus), Flavivirosis (e.g., Dengue virus, Zika virus, Japanese encephalitis virus, West Nile virus, and yellow fever virus), Enterovirus (e.g., poliovirus), Astrovirus (e.g., gastroenteritis virus), Rhabdoviridae (e.g., rabies virus), Borrelia burgdorferi and Mayo borborygmus (e.g., Lyme disease), Herpes simplex virus 2 (HSV2), Klebsiella sp., Pseudomonas aeruginosa, Enterococcus sp., Proteus sp., Enterobacter sp., Actinobacter sp. (sp.), coagulase-negative staphylococci (CoNS), a certain Mycoplasma sp., adenovirus, adeno-associated virus (AAV) or a combination thereof.

[0039] In some respects, adjuvants are interferon gene stimulator (STING) agonists, toll-like receptor (TLR) agonists, inflammatory mediators, RIG-I agonists, α-gal-cer (NKT agonists), heat shock proteins (e.g., HSP65 and HSP70), C-type lectin agonists (e.g., β-glucan (Dectin 1), chitosan and curdlan) or any combination thereof.

[0040] In some respects, the adjuvant is a STING agonist. In some respects, STING agonists include cyclic dinucleotide STING agonists or acyclic dinucleotide STING agonists.

[0041] In some respects, the adjuvant is a TLR agonist. In some respects, TLR agonists include TLR2 agonists (e.g., lipoteichoic acid, atypical LPS, MALP-2 and MALP-404, OspA, porin, LcrV, lipomannan, GPI anchor, lysophosphatidylserine, lipophosphatidylglycerol (LPG), glycophosphatidylinositol (GPI), zymosan, hsp60, gH / gL glycoprotein, hemagglutinin), TLR3 agonists (e.g., double-stranded RNA, such as poly(I:C)), TLR4 agonists (e.g., lipopolysaccharide (LPS), lipoteichoic acid, β-defensin 2, fibronectin EDA, HMGB1, snapin, tenascin C)), TLR5 agonists (e.g., flagellin), TLR6 agonists, and TLR7 / 8 agonists (e.g., single-stranded RNA, CpG-A, Poly G10, Poly G3, Resiquimod, TLR9 agonists (e.g., unmethylated CpG DNA), or any combination thereof.

[0042] In some respects, the EV disclosed in this article is an alien entity.

[0043] In some aspects, the EVs (e.g., exogenous forms) disclosed herein also include a targeting portion. In some aspects, the targeting portion specifically binds to a marker of dendritic cells. In some aspects, the marker is present only on dendritic cells. In some aspects, dendritic cells include plasmacytoid dendritic cells (pDCs), myeloid / conventional dendritic cells 1 (cDC1), myeloid / conventional dendritic cells 2 (cDC2), inflammatory monocyte-derived dendritic cells, Langerhans cells, dermal dendritic cells, lysozyme-expressing dendritic cells (LysoDCs), Kupffer cells, or any combination thereof. In some aspects, the dendritic cell is cDC1. In other aspects, the biomarkers include C-type lectin domain family 9 member A (Clec9a) protein, dendritic cell-specific intercellular adhesion molecule-3-capture integrin (DC-SIGN), CD207, CD40, Clec6, dendritic cell immune receptor (DCIR), DEC-205, lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), MARCO, Clec12a, Clec10a, DC-asialyl glycoprotein receptor (DC-ASGPR), DC immune receptor 2 (DCIR2), Dectin-1, macrophage mannose receptor (MMR), BDCA-1 (CD303, Clec4c), Dectin-2, Bst-2 (CD317), Langerin, CD206, CD11b, CD11c, CD123, CD304, XCR1, AXL, Siglec 6. CD209, SIRPA, CX3CR1, GPR182, CD14, CD16, CD32, CD34, CD38, CD10, or any combination thereof. In some respects, the biomarker is the Clec9a protein.

[0044] In some respects, it targets markers that specifically bind to T cells. These markers include the CD3 molecule.

[0045] In some aspects, the targeting portion is directly attached to the outer surface of the EV. In some aspects, the targeting portion is attached to a scaffold X on the outer surface of the EV. In some aspects, the targeting portion is directly attached to the outer surface of the EV via a connector. In some aspects, the targeting portion is attached to the scaffold X via a connector. In some aspects, the connector is a peptide. In some aspects, the connector is a non-peptide portion. In some aspects, the connector contains a maleimide portion. In some aspects, the connector contains a cholesterol portion.

[0046] In some aspects, the scaffold Y of the EV (e.g., exogenous organism) described herein comprises an N-terminal domain (ND) and an effector domain (ED), wherein the ND and / or ED associate with the luminal surface of the EV. In some aspects, the ND associates with the luminal surface of the exogenous organism via myristylation. In some aspects, the ED associates with the luminal surface of the exogenous organism via ion interactions. In some aspects, the ED contains (i) one basic amino acid or (ii) two or more basic amino acids in its sequence, wherein the basic amino acids are selected from the group consisting of Lys, Arg, His, and any combination thereof. In some aspects, the basic amino acid is (Lys)n, where n is an integer between 1 and 10. In some respects, ED includes Lys(K), KK, KKK, KKKK (SEQ ID NO:205), KKKKK (SEQ ID NO:206), Arg(R), RR, RRR, RRRR (SEQ ID NO:207); RRRRR (SEQ ID NO:208), KR, RK, KKR, KRK, RKK, KRR, RRK, (K / R)(K / R)(K / R)(K / R)(SEQ ID NO:209), (K / R)(K / R)(K / R)(K / R)(K / R)(SEQ ID NO:210) or any combination thereof.

[0047] In some respects, ND comprises an amino acid sequence as shown in G:X2:X3:X4:X5:X6, where G stands for Gly; where “:” represents a peptide bond; where each of X2 to X6 is an amino acid independently; and where X6 includes a basic amino acid. In some respects,

[0048] (i)X2 can be selected from the group consisting of Pro, Gly, Ala and Ser;

[0049] (ii) X4 selects the group consisting of Pro, Gly, Ala, Ser, Val, Ile, Leu, Phe, Trp, Tyr, Gln and Met;

[0050] (iii) X5 can be selected from the group consisting of Pro, Gly, Ala and Ser;

[0051] (iv) X6 selects the group consisting of Lys, Arg, and His; or

[0052] Any combination of (v)(i)-(iv).

[0053] In some respects, ND contains the amino acid sequence G:X2:X3:X4:X5:X6, where

[0054] (i) G stands for Gly;

[0055] (ii) “:” represents a peptide bond;

[0056] (iii) X2 is an amino acid selected from the group consisting of Pro, Gly, Ala and Ser;

[0057] (iv) X3 is an amino acid;

[0058] (v) X4 is an amino acid selected from the group consisting of Pro, Gly, Ala, Ser, Val, Ile, Leu, Phe, Trp, Tyr, Gln and Met;

[0059] (vi) X5 is an amino acid selected from the group consisting of Pro, Gly, Ala, and Ser; and

[0060] (vii) X6 is an amino acid selected from the group consisting of Lys, Arg, and His:

[0061] In some respects, X3 selects from the group consisting of Asn, Gln, Ser, Thr, Asp, Glu, Lys, His, and Arg.

[0062] In some respects, ND and ED are connected by a linker. In some respects, the linker contains one or more amino acids. In some respects, ND contains an amino acid sequence selected from the group consisting of: (i) GGKLSKK (SEQ ID NO:211), (ii) GAKLSKK (SEQ ID NO:212), (iii) GGKQSKK (SEQ ID NO:213), (iv) GGKLAKK (SEQ ID NO:214), or (v) GGKLSK (SEQ ID NO:215), or (vi) any combination thereof. In some aspects, ND comprises an amino acid sequence selected from the group consisting of: (i) GGKLSKKK (SEQ ID NO:238), (ii) GGKLSKKS (SEQ ID NO:239), (iii) GAKLSKKK (SEQ ID NO:240), (iv) GAKLSKKS (SEQ ID NO:241), (v) GGKQSKKK (SEQ ID NO:242), (vi) GGKQSKKS (SEQ ID NO:243), (vii) GGKLAKKK (SEQ ID NO:244), (viii) GGKLAKKS (SEQ ID NO:245), and (ix) any combination thereof. In some aspects, ND comprises the amino acid sequence GGKLSKK (SEQ ID NO:211).

[0063] In some respects, the length of stent Y is at least approximately 8, at least approximately 9, at least approximately 10, at least approximately 11, at least approximately 12, at least approximately 13, at least approximately 14, at least approximately 15, at least approximately 16, at least approximately 17, at least approximately 18, at least approximately 19, at least approximately 20, at least approximately 21, at least approximately 22, at least approximately 23, at least approximately 24, at least approximately 25, at least approximately 30, at least approximately 35, at least approximately 40, at least approximately 45, at least... Approximately 50, at least approximately 55, at least approximately 60, at least approximately 65, at least approximately 70, at least approximately 75, at least approximately 80, at least approximately 85, at least approximately 90, at least approximately 95, at least approximately 100, at least approximately 105, at least approximately 110, at least approximately 120, at least approximately 130, at least approximately 140, at least approximately 150, at least approximately 160, at least approximately 170, at least approximately 180, at least approximately 190, or at least approximately 200 amino acids. In some aspects, stent Y includes (i) GGKLSKKKKGYNVN (SEQ ID NO:246), (ii) GAKLSKKKKGYNVN (SEQ ID NO:247), (iii) GGKQSKKKKGYNVN (SEQ ID NO:248), (iv) GGKLAKKKKGYNVN (SEQ ID NO:249), (v) GGKLSKKKKGYSGG (SEQ ID NO:248) NO: 250), (vi) GGKLSKKKKGSGGS (SEQ ID NO: 251), (vii) GGKLSKKKKSGGSG (SEQ ID NO: 252), (viii) GGKLSKKKSGGSGG (SEQ ID NO: 253), (ix) GGKLSKKSGGSGGS (SEQ ID NO: 254), (x) GGKLSKSGGSGGSV (SEQ ID NO:255) or (xi)GAKKSKKRFSFKKS (SEQ ID NO:256).In certain aspects, Scaffold Y consists of the following sequences: (i) GGKLSKKKKGYNVN (SEQ ID NO:246), (ii) GAKLSKKKKGYNVN (SEQ ID NO:247), (iii) GGKQSKKKKGYNVN (SEQ ID NO:248), (iv) GGKLAKKKKGYNVN (SEQ ID NO:247) NO: 249), (v) GGKLSKKKKGYSGG (SEQ ID NO: 250), (vi) GGKLSKKKKGSGGS (SEQ ID NO: 251), (vii) GGKLSKKKKSGGSG (SEQ ID NO: 252), (viii) GGKLSKKKSGGSGG (SEQ ID NO: 253), (ix) GGKLSKKSGGSGGS (SEQ ID NO:254), (x)GGKLSKSSGGSGGSV(SEQ ID NO:255) or (xi)GAKKSKKRFSFKKS (SEQ ID NO:256).

[0064] In some respects, scaffold Y does not contain Met at the N-terminus. In some respects, scaffold Y contains myristylated amino acid residues at the N-terminus of the scaffold protein. In some respects, the amino acid residues at the N-terminus of scaffold Y are Gly. In some respects, the amino acid residues at the N-terminus of scaffold Y are synthetic. In some respects, the amino acid residues at the N-terminus of scaffold Y are glycine analogs.

[0065] This article provides pharmaceutical compositions comprising the EV (e.g., exogenous) described herein and a pharmaceutically acceptable carrier.

[0066] This document provides cells that generate EVs (e.g., exogens) of the present disclosure. The disclosure also provides cells comprising one or more vectors, wherein the vectors contain nucleic acid sequences encoding: (i) antigens (e.g., those described herein), (ii) adjuvants (e.g., those described herein), (iii) immunomodulators, (iv) targeting portions (e.g., those described herein), or (v) combinations thereof.

[0067] This document provides a kit containing the EV (e.g., exogenous form) described herein and instructions for use. This document also provides EV-drug conjugates containing any of the EVs (e.g., exogenous forms) described herein.

[0068] This article provides a method for preparing EVs (e.g., exogenous bodies), which includes culturing the cells disclosed herein under suitable conditions and obtaining EVs (e.g., exogenous bodies).

[0069] This article provides a method for inducing an immune response in a subject in need, which includes administering the EV (e.g., exogenous) of this disclosure to the subject.

[0070] This article provides methods for preventing or treating diseases in subjects in need, comprising administering an EV (e.g., an exogenous agent) as described herein, wherein the disease is associated with an antigen. In some aspects, the disease is cancer. In some aspects, the cancer includes bladder cancer, cervical cancer, renal cell carcinoma, testicular cancer, colorectal cancer, lung cancer, head and neck cancer, ovarian cancer, lymphoma, liver cancer, glioblastoma, melanoma, myeloma, leukemia, pancreatic cancer, or combinations thereof. In other aspects, the disease is an infection.

[0071] In some cases, EVs (e.g., exogenous EVs) can be administered parenterally, orally, intravenously, intramuscularly, intratumorally, intranasally, subcutaneously, or intraperitoneally.

[0072] In some respects, the methods disclosed herein (e.g., methods for inducing an immune response or for preventing or treating disease) involve the administration of additional therapeutic agents.

[0073] This article provides methods for inhibiting or reducing cancer metastasis in subjects in need, which include administering the EV (e.g., exogenous) of this disclosure to the subject. Attached Figure Description

[0074] Figure 1A An exemplary EV is shown that comprises one or more antigens, one or more adjuvants, one or more molecules for targeting a moiety, or any combination thereof.

[0075] Figure 1B A non-limiting example (ar) of an EV (e.g., exogenous body) comprising an antigen and an adjuvant is shown. “Ag” and “AD” represent the antigen and adjuvant, respectively. The arrow indicates the Y portion of the stent. “X” indicates the X portion of the stent. It is evident from this disclosure that… Figure 1B The EVs (e.g., exogens) shown may contain multiple antigens, multiple adjuvants, or multiple antigens and multiple adjuvants. EVs (e.g., exogens) may also contain one or more additional components (e.g., immunomodulators and / or targeting components). Further descriptions of such EVs (e.g., exogens) are provided throughout this disclosure.

[0076] Figure 2 Seven selected examples of exogenous bodies containing antigens and immunomodulators are shown. “Ag” and “IM” represent antigens and immunomodulators, respectively. Arrows indicate scaffold Y portion. “X” indicates scaffold X portion. As can be clearly seen from this disclosure, Figure 2The EVs (e.g., exogens) shown may comprise multiple antigens, multiple immunomodulators, or multiple antigens and multiple immunomodulators. EVs (e.g., exogens) may further comprise one or more additional portions (e.g., adjuvants and / or targeting portions). Further descriptions of such EVs (e.g., exogens) are provided throughout this disclosure.

[0077] Figure 3A and Figure 3B This demonstrates the ability of engineered EVs (e.g., exogenous bodies containing OVA-scaffold Y and loaded with a STING agonist (“Py-OVA-exoSTING”)) to induce an OVA-specific CD8 T cell immune response after intravenous administration to naïve C57 / BL6 mice. In the spleen (… Figure 3A ) and combined peripheral blood mononuclear cells (PBMCs) Figure 3B The induction of OVA-specific CD8 T cell immune responses was demonstrated in all studies. The following constructs were used as controls: (i) a combination of anti-CD40 antibody and soluble OVA protein (not part of the EV (e.g., exosome)) (“IP aCD40+OVA”); (ii) a combination of cAIM(PS)2Difluor(Rp / Sp) (“CL656”; STING agonist) and soluble OVA protein (not part of the EV (e.g., exosome)) (“CL656+OVA”); (iii) an EV overexpressing scaffold X, loaded with a combination of STING agonist and soluble OVA protein (OVA not part of the EV (e.g., exosome)), such as the exosome (“Px-exoSTING+OVA”); and (iv) an EV expressing only the OVA-scaffold Y fusion protein (“Py-OVA”) (e.g., the exosome). Data are presented individually, and the data are presented as mean ± SD. "***" indicates p < 0.0005 for a one-way ANOVA.

[0078] Figure 4A and Figure 4B This study demonstrated the ability of engineered EVs (e.g., exogenous organisms containing OVA-scaffold Y and loaded with a STING agonist (“Py-OVA-exoSTING”)) to induce OVA-specific CD8 T cell immune responses after intranasal administration to naïve C57 / BL6 mice. In the spleen (… Figure 4A ) and lungs ( Figure 4BAll of these studies demonstrated the induction of OVA-specific CD8 T cell immune responses. The following constructs were used as controls: (i) a combination of anti-CD40 antibody and soluble OVA protein (not part of the EV (e.g., exosome)); (ii) a combination of cAIM(PS)2Difluor(Rp / Sp) (“CL656”; STING agonist) and soluble OVA protein (“CL656+OVA”); (iii) an EV (e.g., exosome) overexpressing scaffold X and loaded with a combination of STING agonist and soluble OVA protein (OVA not part of the EV (e.g., exosome)); and (iv) an EV (e.g., exosome) expressing only OVA-scaffold Y fusion protein (“Py-OVA”). Data are presented individually, and data are presented as mean ± SD. “**” indicates p < 0.005 for one-way ANOVA. “***” indicates p < 0.0005 for one-way ANOVA.

[0079] Figure 5A and Figure 5B This study presents a comparison of OVA-specific T cell responses in the spleen of mice following intranasal administration of engineered EVs (e.g., exogenous organisms containing OVA-scaffold Y and loaded with a STING agonist ("Py-OVA-exoSTING")"). OVA-specific T cell responses were measured using IFN-γELISPOT assay one week post-administration. Figure 5A The CD8 T cell response was shown. Figure 5B CD4 T cell responses are shown. Control animals received one of the following: (i) soluble OVA protein alone (“OVA”); (ii) a combination of CL656 and soluble OVA protein (“OVA+CL656”); (iii) an EV expressing only the OVA-scaffold Y fusion protein (“Py-OVA”), e.g., an exogenous body; (iv) a combination of CL656 and an EV expressing only the OVA-scaffold Y fusion protein (e.g., an exogenous body) (“Py-OVA+CL656”). Data are shown individually, and data are presented as mean ± SD. “*” indicates p < 0.05 for one-way ANOVA.

[0080] Figure 6 A schematic diagram of the experimental design for evaluating the efficacy of Clec9a exogenous vaccine in a vaccination model is provided.

[0081] Figure 7A and Figure 7B The study demonstrated that engineered EVs (e.g., exogenous forms) induced superior CD8T cell responses compared to standard vaccine formulations. Figure 7AThe study demonstrated superior effect memory, particularly CD8 T cell response, following subcutaneous (SQ) administration of the standard vaccine (AddaVax), or subcutaneous (SQ), intranasal (IN), or intravenous (IV) administration of the engineered exogenous body. Figure 7B This study demonstrates the induction of tissue residency memory, particularly T-cell responses (defense lines) in the lungs, following intranasal inoculation with standard vaccines or engineered exogenous organisms.

[0082] Figure 8 This explains the use of EBV BZLF1 as a targeted antigen for post-transplant lymphoproliferative disorders in EBV transplant patients. Figure 8 This is a schematic diagram of an engineered EV (e.g., an exogenous body) containing an adjuvant (cyclic purine dinucleotide, such as CDN) and an EBV BZLF1 antigen attached to the luminal surface of the EV.

[0083] Figure 9A and Figure 9B CD4+ T cells were provided from wild-type mice immunized with soluble OVA or exogenous OVA in the presence or absence of STING adjuvant. Figure 9A ) and CD8+ T cells ( Figure 9B ) quantity comparison. As indicated ( Figure 9A and Figure 9B Wild-type mice were immunized with soluble OVA (ovalbumin), soluble OVA + CL656 (STING agonist), PyOVA (exogenous coelomic expression of OVA fused with BASP1), PyOVA + soluble CL656, PyOVA exoVacc (PyOVA exogenous body loaded with CL656), or soluble OVA + alum adjuvant. Antigen-specific cells were identified by IFN-g expression, and data are expressed as the number of IFN-g positive spot-forming units (SFUs) per 100,000 splenocytes after background (non-antigen-specific activation) (x-axis). Figure 9A and Figure 9B "Day 14" indicates the number of CD4+ and CD8+ T cells observed in the animal after a single immunization. "Day 28" indicates the number of CD4+ and CD8+ T cells observed in the animal after a second booster dose.

[0084] Figure 10 The number of OVA-specific CD8+ T cells in mouse lungs treated with the exogenous organisms disclosed herein (e.g., expressing OVA-scaffold Y and loaded with the STING agonist CL656) is shown. "Dose 1" indicates the effect memory (T cells) observed after a single administration of the exogenous organism. EMThe number of CD8+ T cells. "Dose 2" refers to the effector memory and / or resident memory (T cells) observed after a second booster dose. RM The number of CD8+ T cells.

[0085] Figure 11A and Figure 11B The effects of expressing the anti-Clec9a binding moiety in the EVs (e.g., exogens) disclosed herein are shown. Figure 11A This study illustrates the uptake of exogenous bodies expressing anti-Clec9a by different dendritic cell populations after administration to mice. The dendritic cell populations shown include: (i) conventional DC1 (“cDC1”), (ii) conventional DC2 (“cDC2”), and (iii) plasmacytoid DCs (“pDC”). Control animals received either PBS alone or exogenous bodies expressing scaffold X protein only (“PrXEV”). ****p<0.0001. Figure 11B A comparison of STING activity in mouse dendritic cells after stimulation with one of the following substances at three different doses (0.4 nM, 1 nM, or 4 nM) is provided: (i) a soluble STING agonist (“free STING”), (ii) an EV expressing only scaffold X protein (i.e., without an anti-Clec9a antibody fragment) and loaded with a STING agonist (e.g., exogenous bodies) (“PrX-STING”), (iii) an exogenous body expressing an anti-Clec9a antibody fragment linked to scaffold X protein (“aClec9a-STING”), and (iv) an EV expressing an unrelated antibody and loaded with a STING agonist (e.g., exogenous bodies) (“isotype-STING”). STING activity was indicated by the amount of IL-12 produced by the DCs.

[0086] Figure 12A and Figure 12B The administration pathway demonstrated for inducing OVA-specific CD8+ T2 in an engineered exogenous compound ("Py-OVA exoVACC") expressing OVA-scaffold Y and loaded with the STING agonist CL656. EM Effects on cells. The routes of administration shown include: (i) intravenous (“IV”), (ii) intranasal (“IN”), and (iii) subcutaneous (“SQ”). “SubQ AV” corresponds to the use of commercially available formulations (ADDAVAX). TM Animals treated with soluble OVA in InvioGen ("SubQ AV") Figure 12A A bar chart showing the average of the results is provided. ***, p = 0.0013; **, p = 0.0074; ns, by one-way ANOVA, compared with OVA+ADDAVAX TM The difference between the groups was not significant. Figure 12BFlow cytometry plots of representative samples from different treatment groups are provided. The percentages provided in the upper right quadrant of each flow cytometry plot represent the observed OVA-specific CD8. + T EM Percentage of cellular response. Different treatment groups are shown in the upper left quadrant of each flow cytometry plot.

[0087] Figure 13A , Figure 13B and Figure 13C This demonstrates OVA-specific resident memory (T) in mouse lungs. RM CD8+ T cells ( Figure 13A ) and CD4+ T cells ( Figure 13B In the induction of OVA-scaffold Y fusion protein, the mice were administered two doses of an exogenous protein expressing OVA-scaffold Y and loaded with the STING agonist CL656 ("Py-OVA exoVACC"). Control animals received one of the following: (i) soluble OVA ("OVA"), (ii) an exogenous protein expressing only OVA-scaffold Y fusion protein ("PyOVA"), (iii) soluble OVA + soluble poly I:C ("OVA+poly I:C"), and (iv) an exogenous protein expressing only OVA-scaffold Y fusion protein + soluble poly I:C ("PyOVA+poly I:C"). Figure 13C Provided Figure 13A and Figure 13B The data shown are flow cytometry plots of representative samples. The top row corresponds to CD8+ T cells. The bottom row corresponds to CD4+ T cells.

[0088] Figure 14A , Figure 14B , Figure 14C , Figure 14D , Figure 14E , Figure 14F , Figure 14G , Figure 14H , Figure 14I , Figure 14J , Figure 14K , Figure 14L , Figure 14M and Figure 14NAntitumor immune responses in mice received one of the following substances are illustrated: (i) an exogenous body expressing OVA-scaffold Y and loaded with the STING agonist CL656 via intranasal administration (“exoVACC(IN)”), (ii) an exogenous body expressing OVA-scaffold Y and loaded with the STING agonist CL656 via subcutaneous administration (“exoVACC(SQ)”), (iii) soluble OVA + soluble poly I:C via intranasal administration (“OVA+poly I:C(IN)”), and (iv) soluble OVA + soluble poly I:C via subcutaneous administration (“OVA+poly I:C(SC)”). Untreated animals served as controls. Figure 14A A schematic diagram of the experimental design is provided. Figure 14B and Figure 14N Survival data from two independent experiments are provided. Figure 14C and Figure 14I (Unprocessed) Figure 14D and Figure 14J (OVA+poly I:C(SC)) Figure 14E and Figure 14L (exoVACC(SQ)) Figure 14F and Figure 14K (OVA+poly I:C(IN)), Figure 14G and Figure 14M (exoVACC(IN)) provides tumor volume data from two independent experiments. Figure 14E and 14G The percentages shown represent the number of animals (total group) that are fully protected. Figure 14H The tumor growth rate is shown for each different treatment group. Figure 14H In the mean square, *, p = 0.028; ns, by one-way ANOVA, was not significant compared with the untreated control.

[0089] Figure 15A , Figure 15B , Figure 15C , Figure 15D , Figure 15E and Figure 15F The ability of the engineered EVs (e.g., exogens) disclosed herein to migrate to the mesenteric lymph nodes after intranasal administration is demonstrated. Figure 15A A schematic diagram of the experimental design is provided. Figure 15B , Figure 15C and Figure 15D The frequencies of OVA-specific CD4+ T cells (left block in each treatment group) and OVA-specific CD8+ T cells (right block in each treatment group) in the spleen, lung, and mesenteric lymph nodes, as measured by IFN-γELISPOT, are shown separately. Figure 15Eand Figure 15F The frequencies of OVA-specific effector memory CD8+ T cells in the lungs and spleen, as measured by flow cytometry, are shown separately.

[0090] Figure 16A , Figure 16B and Figure 16C The ability of surface-engineered EVs (e.g., exogens) containing scaffold X and loaded with STING agonists to induce antigen-specific immune responses has been demonstrated. Figure 16A A schematic diagram of the experimental design is provided. As shown, the CD4 peptide (Itgb1) and / or CD8 peptide (Lama4) are linked to the scaffold X of the EV (e.g., exogenous body). Figure 16B and Figure 16C The frequencies of Itgb1-specific CD4+ T cells and Lama4-specific CD8+ T cells in the spleens of animals in different treatment groups, as measured by IFN-γELISPOT, are shown respectively.

[0091] Figure 17A , Figure 17B and Figure 17C The ability of surface-engineered EVs (e.g., exogenous bodies) containing scaffold X and loaded with CpG adjuvant to induce antigen-specific immune responses has been demonstrated. Figure 17A A schematic diagram of the experimental design is provided. As shown, maleimide chemistry was used to link (i) a single CD8 peptide (Lama4) (Group 2) or (ii) a CD8 peptide and a CD4 peptide (Itgb1) (Group 3) to scaffold X. The control EV expressed only scaffold X (i.e., no peptide and no CpG adjuvant) (Group 1). Figure 17B and Figure 17C The frequencies of Itgb1-specific CD4+ T cells and Lama4-specific CD8+ T cells in the spleens of animals in different treatment groups, as measured by IFN-γELISPOT, are shown.

[0092] Figure 18A , Figure 18B , Figure 18C , Figure 18D , Figure 18E , Figure 18F , Figure 18G , Figure 18H , Figure 18I , Figure 18J , Figure 18K and Figure 18L The expression of E6 and E7 proteins of HPV16 and HPV18 in surface-engineered EVs (e.g., exogens) disclosed herein, as measured by protein blotting, is shown. Figure 18A , Figure 18B , Figure 18C , Figure 18D , Figure 18E and Figure 18F In this study, 293SF cells were transfected with plasmids encoding one of the following full-length proteins: (i) HPV16 E6, (ii) HPV16 E7, (iii) HPV16 E6 / E7, (iv) HPV 18 E6, (v) HPV18 E7, and (vi) HPV18 E6 / E7. Figure 18G , Figure 18H , Figure 18I , Figure 18J , Figure 18K and Figure 18L In this study, a splitting protein expression strategy was used. 293SF cells were transfected with one of the following plasmids: (i) pUC57-Kan-AAVS1HR-CAGGS-PTGFRN-FLAG-coHPV16nE6 ("pCB-2014"), (ii) pUC57-Kan-AAVS1HR-CAGGS-PTGFRN-FLAG-coHPV16cE6 ("pCB-2015"), and (iii) pUC57-Kan-AAVS1HR-CAGGS-coHPV16nE6-FLAG-PTGFRN ( The plasmids are pCB-2016 (iv), pUC57-Kan-AAVS1HR-CAGGS-coHPV16cE6-FLAG-PTGFRN (pCB-2017), pUC57-Kan-AAVS1HR-CAGGS-PrY-FLAG-coHPV16nE6 (pCB-2018), and pUC57-Kan-AAVS1HR-CAGGS-PrY-FLAG-coHPV16cE6 (pCB-2019). Detailed descriptions of the plasmids can be found in Example 23 (see also Table 11).

[0093] Figure 19A , Figure 19B and Figure 19C The ability of surface-engineered EVs (e.g., exogens) loaded with STING agonists and expressing (i) an anti-Clec9A targeting portion linked to scaffold X and (ii) an OVA linked to scaffold Y has been demonstrated. Figure 19A A schematic diagram of the experimental design is provided. Figure 19B and Figure 19C Showing from Figure 19A The number of OVA-specific CD8+ effector memory T cells observed in the spleens of animals in different treatment groups is shown. Figure 19B The results are shown one week after a single EV administration. Figure 19C The results are shown one week after the second dose of EV was administered.

[0094] Figure 20A , Figure 20B and Figure 20C This demonstrates the ability of engineered EVs (e.g., exogenous bodies) expressing OVA-scaffold Y and loaded with STING agonists to induce antigen-specific humoral immune responses upon in vivo administration. Figure 20A A schematic diagram of the experimental design is provided. As shown, the animals received one of the following substances: (i) soluble OVA alone (Group 1), (ii) soluble OVA in combination with a free STING agonist (Group 2), (iii) an EV (e.g., exogenous) expressing only OVA-scaffold Y (“PyOVA”) (Group 3), (iv) PyOVA in combination with a free STING agonist (Group 4), (v) an engineered exogenous expressing OVA-scaffold Y and loaded with the STING agonist CL656 ("Py-OVA exoVACC") (Group 5), and (vi) soluble OVA in combination with alum. Figure 20B A comparison of the amount of OVA-specific IgG1 antibody in serum was provided. Figure 20C A comparison of the amount of OVA-specific IgA antibody in serum was provided.

[0095] Figure 21 The chemical structures of AM152 (cyclopropanecarboxylic acid, 1-[4'-[3-methyl-4-[[[(1R)-1-phenylethoxy]carbonyl]amino]-5-isoxazolyl][[1,1'-biphenyl]-4-yl]) and AM095 (1,1′-biphenyl]-4-acetic acid, 4′-[3-methyl-4-[[[(1R)-1-phenylethoxy]carbonyl]amino]-5-isoxazolyl[]-) are shown. Arrows labeled 1 and 2 indicate positions suitable for derivatization to introduce maleimide reactive groups (carboxylic acids and carbamates). The corresponding positions shown in AM152 are also present in AM095.

[0096] Figure 22 A schematic diagram is provided, illustrating the conjugation of an LPA1 antagonist (AM152) with an exogenous entity to produce a group of exogenous entities containing multiple LPA1 antagonist molecules on their surface.

[0097] Figure 23 An example is shown of how a maleimide reactive group can be added to AM152 via its carboxylic acid group. This example shows that the maleimide group is part of a reactive complex containing an ala-val cleavable linker and a C5 spacer between the maleimide group and the carboxylic acid reactive chloromethylphenyl group.

[0098] Figure 24Two exemplary reagents that can be used to derive AM152 are shown. The top reagent comprises (i) a chloromethylphenyl group that reacts with the carboxylic acid group of AM152 and (ii) a maleimide group; and between them is a cleavable cit-val dipeptide and a C5 spacer. The bottom reagent comprises (i) a chloromethylphenyl group that reacts with the carboxylic acid group of AM152 and (ii) a maleimide group, and between them is a cleavable ala-val dipeptide and a C5 spacer.

[0099] Figure 25 The product shown is the cit-val or ala-val dipeptide (e.g., by cathepsin B) generated from the cleavage conjugate. This product, AM152 aniline ester, can be further processed by an endogenous esterase to produce the free acid AM152 product.

[0100] Figure 26 and Figure 27 Several AM152 derivatives containing free maleimide groups and different combinations of spacers are shown.

[0101] Figure 28 This demonstrates that, after protecting the carboxylic acid group, AM152 can be derived at its carbamate group using the same reagent used to derive the carboxylic acid group. The resulting product is then deprotected to release the carboxylic acid group.

[0102] Figure 29 An example is shown in which a complex having a maleimide group is attached to a urethane group of AM152 via a connector. Suitable connectors include any connectors disclosed in this specification.

[0103] Figure 30 It is shown that AM152 can be attached to a derived anchoring portion, rather than being derived and subsequently attached to the anchoring portion via a reactive maleimide group.

[0104] Figure 31 This is a schematic diagram illustrating how maleimide chemistry can be used, for example, to chemically link a bioactive molecule (BAM) to an EV (e.g., an exogenous body) via a scaffold portion described herein (e.g., scaffold X protein or a fragment or lipid thereof). The connectors described in the figures are optional and may include connectors (e.g., cleavable connectors) or combinations thereof, when present. Detailed Implementation

[0105] This disclosure relates to an engineered EV (e.g., exosome) that simultaneously delivers antigens and adjuvants to the same antigen-presenting cell. The EV platform allows for luminal expression of antigens and surface expression of immunostimulatory molecules, designed to generate a modular vaccination system. Various adjuvants can be incorporated into the EV (e.g., exosome) to enhance immune responses against multiple antigens. The engineered EV may contain one or more payloads and may improve at least one property of the EV (e.g., those disclosed herein) and its use. In some aspects, one or more payloads include antigens, adjuvants, and / or immunomodulators. In some aspects, the EV (e.g., exosome) includes one or more additional portions (e.g., targeting portions). In some aspects, one or more payloads (e.g., antigens, adjuvants, and / or immunomodulators) and / or one or more additional portions (e.g., targeting portions) may be attached (or connected) to one or more scaffold portions on the surface of the EV (e.g., exosome) or the luminal surface of the EV (e.g., exosome). Thus, the EV of this disclosure allows for platform delivery of vaccine media (i.e., exoVACC). TM The antigen on the EV and / or, in some respects, one or more payloads (e.g., antigens, adjuvants, and / or immunomodulators may be combined in a specific manner) and / or replaced by different antigens and / or one or more adjuvants or immunomodulators. Further additional portions (e.g., targeting portions) may be directly attached (or connected) to the outer surface and / or luminal surface of the EV (e.g., exosome). Non-limiting examples of various aspects are shown in this disclosure.

[0106] I. Definition

[0107] To make this instruction manual easier to understand, some terms are defined first. Additional definitions are presented throughout the detailed instruction manual.

[0108] It should be noted that the term "a / species(a)" or "a / species(an)" refers to one or more of that entity; for example, "a / species(a) nucleotide sequence" is understood to represent one or more nucleotide sequences. Therefore, the terms "a / species(a)" (or "a / species(an)"), "one / species or more / species", and "at least one / species" are used interchangeably herein.

[0109] Furthermore, the term “and / or” as used herein should be considered to specifically disclose that each of two specified features or components exists with or without the other. Therefore, the term “and / or” as used in phrases such as “A and / or B” herein is intended to include “A and B”, “A or B”, “A” (alone), and “B” (alone). Similarly, the term “and / or” as used in phrases such as “A, B, and / or C” is intended to include each of the following: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0110] It should be understood that wherever the term "comprising" is used to describe an aspect, other similar aspects described as "composed of" and / or "substantially composed of" are also provided.

[0111] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art relating to this disclosure. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd edition, 2002, CRC Press; the Dictionary of Cell and Molecular Biology, 3rd edition, 1999, Academic Press; and the Oxford Dictionary of Biochemistry and Molecular Biology, Revised, 2000, Oxford University Press provide a general dictionary for those skilled in the art of the use of many of the terms used in this disclosure.

[0112] Units, prefixes, and symbols are represented in their International System of Units (SI) recognized forms. Numerical ranges include values ​​within defined ranges. Unless otherwise specified, nucleotide sequences are written from left to right in a 5' to 3' direction. Amino acid sequences are written from left to right in an amino-to-carboxyl direction. The headings provided herein are not intended to limit the various aspects of this disclosure, and such headings are available by reference to the entire specification. Therefore, the terms defined immediately thereafter are defined more fully by reference to the entire specification.

[0113] The term "about" is used herein to mean approximately, roughly, around, or within a range. When the term "about" is used in conjunction with a numerical range, it modifies the range by extending the boundaries to be above and below the stated value. Generally, the term "about" can modify values ​​that are, for example, 10% higher or lower than the stated value.

[0114] As used herein, the term "extracellular vesicle" or "EV" refers to a cell-derived vesicle comprising a membrane encapsulating an internal space. Extracellular vesicles include all membrane-bound vesicles (e.g., exosomes, nanovesicles) with a diameter smaller than that of the cell from which they originate. In some aspects, extracellular vesicles have diameters ranging from 20 nm to 1000 nm and may contain various macromolecular payloads within an internal space (i.e., a lumen), displayed on the outer surface of the extracellular vesicle, and / or transmembrane. In some aspects, said payloads may include nucleic acids, proteins, carbohydrates, lipids, small molecules, and / or combinations thereof. In some aspects, the extracellular medium includes a scaffold portion. By way of example, and not limitation, extracellular vesicles include apoptotic bodies, cell debris, cell-derived vesicles obtained through direct or indirect manipulation (e.g., by continuous extrusion or treatment with an alkaline solution), vesicular organelles, and vesicles generated by living cells (e.g., by direct plasma membrane budding or late endosome fusion with the plasma membrane). Extracellular vesicles can originate from living or dead organisms, explant tissues or organs, prokaryotic or eukaryotic cells, and / or cultured cells. In some respects, extracellular vesicles are produced by cells expressing one or more transgenic products.

[0115] As used herein, the term "exogenous body" refers to an extracellular vesicle with a diameter between 20 and 300 nm (e.g., between 40 and 200 nm). Exogenous bodies comprise a membrane enclosing their internal space (i.e., lumen) and, in some respects, can be produced by cells (e.g., producer cells) via direct plasma membrane budding or via the fusion of late endosomes or multivesicles with the plasma membrane. In some respects, exogenous bodies comprise a scaffold portion. As described below, exogenous bodies can originate from producer cells and be isolated from them based on their size, density, biochemical parameters, or combinations thereof. In some respects, the EVs (e.g., exogenous bodies) of this disclosure are produced by cells expressing one or more transgenic products.

[0116] As used herein, the term "nanovesicle" refers to an extracellular vesicle with a diameter between 20 and 250 nm (e.g., between 30 and 150 nm) that is generated by said cells (e.g., producer cells) through direct or indirect manipulation, such that said nanovesicles would not be generated by said cells without said manipulation. Appropriate manipulation of cells to generate nanovesicles includes, but is not limited to, continuous extrusion, treatment with an alkaline solution, sonication, or combinations thereof. In some aspects, the generation of nanovesicles may lead to the destruction of said producer cells. In some aspects, the nanovesicle populations described herein are substantially free of vesicles obtained from said cells by direct budding from the plasma membrane or fusion of late endosomes with the plasma membrane. In some aspects, nanovesicles include a scaffold portion. Once nanovesicles are derived from producer cells, they can be isolated from the producer cells based on their size, density, biochemical parameters, or combinations thereof.

[0117] As used herein, the term "surface-engineered EV, e.g., exogenous" (e.g., scaffold-X-engineered EV, e.g., exogenous) refers to an EV (e.g., exogenous) in which the membrane or surface of the EV (e.g., exogenous) is modified in its composition such that the surface of the engineered EV (e.g., exogenous) differs from the surface of the unmodified EV (e.g., exogenous) or the surface of a naturally occurring EV (e.g., exogenous). The engineering can be present on the surface of the EV (e.g., exogenous) or within the membrane of the EV (e.g., exogenous), thereby altering the surface of the EV (e.g., exogenous). For example, the membrane may be modified in terms of its protein, lipid, small molecule, carbohydrate, etc. The composition can be altered by chemical, physical, or biological methods, or by production from cells previously or simultaneously modified by chemical, physical, or biological methods. Specifically, the composition can be altered by genetic engineering, or by production from cells previously modified by genetic engineering. In some aspects, surface-engineered EVs (e.g., exosomes) contain exogenous proteins (i.e., proteins not naturally expressed by the EV (e.g., exosomes)) or fragments or variants thereof, which may be exposed on the surface of the EV (e.g., exosome) or may be anchoring sites (attaches) of portions exposed on the surface of the EV (e.g., exosome). In other aspects, surface-engineered EVs (e.g., exosomes) contain higher expression (e.g., greater quantity) of native exosome proteins (e.g., scaffold X) or fragments or variants thereof, which may be exposed on the surface of the EV (e.g., exosome) or may be anchoring sites (attaches) of portions exposed on the surface of the EV (e.g., exosome).

[0118] As used herein, the term "cavity-engineered exogenous body" (e.g., scaffold Y-engineered exogenous body) refers to an EV (e.g., exogenous body) whose membrane or cavity is modified in its composition such that the cavity of the engineered EV (e.g., exogenous body) differs from that of the unmodified EV (e.g., exogenous body) or the cavity of a naturally occurring EV (e.g., exogenous body). The engineering can be directly present within the cavity or membrane of the EV (e.g., exogenous body), thereby altering the cavity of the EV (e.g., exogenous body). For example, the membrane is modified in its composition of proteins, lipids, small molecules, carbohydrates, etc., thereby modifying the cavity of the EV (e.g., exogenous body). The composition can be altered by chemical, physical, or biological methods, or by production from cells previously modified by chemical, physical, or biological methods. Specifically, the composition can be altered by genetic engineering, or by production from cells previously modified by genetic engineering. In some aspects, the cavity-engineered exosome contains a foreign protein (i.e., a protein not naturally expressed by the EV (e.g., the exosome)) or a fragment or variant thereof, which may be exposed within the lumen of the EV (e.g., the exosome) or may be an anchoring point (attachment) of a portion exposed on the inner layer of the EV (e.g., the exosome). In other aspects, the cavity-engineered EV (e.g., the exosome) contains higher expression of a native exosome protein (e.g., scaffold X or scaffold Y) or a fragment or variant thereof, which may be exposed within the lumen of the exosome or may be an anchoring point (attachment) of a portion exposed within the lumen of the exosome.

[0119] The term "modified," when used in the context of EVs (e.g., exosomes) as described herein, refers to alterations or engineering of an EV (e.g., exosome) and / or its producer cells such that the modified EV (e.g., exosome) differs from naturally occurring EVs (e.g., exosomes). In some aspects, the modified EVs (e.g., exosomes) described herein comprise membranes that differ in composition of proteins, lipids, small molecules, carbohydrates, etc., from those of naturally occurring EVs (e.g., exosomes) (e.g., membranes containing a higher density or number of natural exosome proteins and / or membranes containing proteins not naturally present in the exosome (e.g., antigens, adjuvants, and / or immunomodulators)). In some aspects, such modifications to the membrane alter the outer surface of the EV (e.g., exosome) (e.g., surface-engineered EVs, such as the exosomes described herein). In some aspects, such modifications to the membrane alter the cavity of the EV (e.g., exosome) (e.g., cavity-engineered EVs, such as the exosomes described herein).

[0120] As used herein, the term "scaffold portion" refers to a molecule that can be used to anchor a payload or any other compound of interest (e.g., antigen, adjuvant, and / or immunomodulator) to an EV (e.g., an exosome) (anchored on the luminal or outer surface of the EV). In some aspects, a scaffold portion comprises a synthetic molecule. In some aspects, a scaffold portion comprises a non-peptide portion. In other aspects, a scaffold portion comprises lipids, carbohydrates, or proteins naturally present in an EV (e.g., an exosome). In some aspects, a scaffold portion comprises lipids, carbohydrates, or proteins not naturally present in an EV (e.g., an exosome). In some aspects, a scaffold portion is scaffold X. In some aspects, a scaffold portion is scaffold Y. In still other aspects, a scaffold portion comprises both scaffold X and scaffold Y. Non-limiting examples of other scaffold components that may be used in this disclosure include: aminopeptidase N (CD13); neprilysin, also known as membrane metalloendopeptidase (MME); exonucleotide pyrophosphatase / phosphodiesterase family member 1 (ENPP1); neuropilin-1 (NRP1); CD9, CD63, CD81, PDGFR, GPI ankyrin, lactadherin, LAMP2, and LAMP2B.

[0121] As used herein, the term "scaffold X" refers to an exogenous protein recently identified on the surface of an exogenous body. See, for example, U.S. Patent No. 10,195,290, which is incorporated herein by reference in its entirety. Non-limiting examples of scaffold X proteins include: prostaglandin F2 receptor negative regulator (“PTGFRN protein”); basigin (“BSG protein”); immunoglobulin superfamily member 2 (“IGSF2 protein”); immunoglobulin superfamily member 3 (“IGSF3 protein”); immunoglobulin superfamily member 8 (“IGSF8 protein”); integrin β-1 (“ITGB1 protein”); integrin α-4 (“ITGA4 protein”); 4F2 cell surface antigen heavy chain (“SLC3A2 protein”); and a class of ATP transport proteins (“ATP1A1 protein”, “ATP1A2 protein”, “ATP1A3 protein”, “ATP1A4 protein”, “ATP1B3 protein”, “ATP2B1 protein”, “ATP2B2 protein”, “ATP2B3 protein”, “ATP2B protein”). In some aspects, scaffold X proteins can be complete proteins or fragments thereof (e.g., functional fragments, such as the smallest fragments capable of anchoring another portion to the outer or luminal surface of an EV (e.g., an exosome). In some respects, scaffold X can anchor a component (e.g., an antigen, adjuvant, and / or immunomodulator) to the outer or luminal surface of a foreign body.

[0122] As used herein, the term "scaffold Y" refers to a newly identified exosome protein within the lumen of an exosome. See, for example, International Application PCT / US2018 / 061679, which is incorporated herein by reference in its entirety. Non-limiting examples of scaffold Y proteins include: myristyl alanine-rich protein kinase C substrate ("MARCKS protein"); myristyl alanine-rich protein kinase C substrate-like protein 1 ("MARCKSL1 protein"); and encephalolysin 1 ("BASP1 protein"). In some aspects, scaffold Y protein may be a complete protein or a fragment thereof (e.g., a functional fragment, such as the smallest fragment capable of anchoring a portion to the luminal surface of an exosome). In some aspects, scaffold Y may anchor portions (e.g., antigens, adjuvants, and / or immunomodulators) to the luminal surface of an EV (e.g., an exosome).

[0123] As used herein, the term "fragment" of a protein (e.g., a therapeutic protein, scaffold X, or scaffold Y) refers to an amino acid sequence of such a protein that is shorter than the naturally occurring sequence and that, compared to the naturally occurring protein, lacks the N- and / or C-terminus or any portion thereof. As used herein, the term "functional fragment" refers to a protein fragment that retains the protein's function. Thus, in some aspects, a functional fragment of scaffold X protein retains the ability to anchor a portion to the luminal or outer surface of an EV (e.g., an exosome). Similarly, in some aspects, a functional fragment of scaffold Y protein retains the ability to anchor a portion to the luminal surface of an EV (e.g., an exosome). Whether a fragment is functional can be assessed by any method known in the art for determining the protein content of an EV, including Western blotting, FACS analysis, and fusion of the fragment with an autofluorescent protein (e.g., GFP). In some respects, the functional fragments of the scaffold X protein retain at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100% of the capabilities of the naturally occurring scaffold X protein, such as the ability to anchor to a part. In some respects, the functional fragments of the scaffold Y protein retain at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100% of the capabilities of the naturally occurring scaffold Y protein, such as the ability to anchor to another molecule.

[0124] As used herein, a “variant” of a molecule (e.g., a functional molecule, an antigen, scaffold X, and / or scaffold Y) is a molecule that shares certain structural and functional properties with another molecule when compared by methods known in the art. For example, a variant of a protein may include substitutions, insertions, deletions, frameshifts, or rearrangements in another protein.

[0125] In some aspects, variants of scaffold X include variants that share at least about 70% identity with full-length, mature PTGFRN, BSG, IGSF2, IGSF3, IGSF8, ITGB1, ITGA4, SLC3A2, or ATP transporter, or fragments (e.g., functional fragments) of PTGFRN, BSG, IGSF2, IGSF3, IGSF8, ITGB1, ITGA4, SLC3A2, or ATP transporter. In some aspects, variants of PTGFRN or variants of fragments share at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with PTGFRN or its functional fragment according to SEQ ID NO:1. In some respects, variants or fragments of BSG share at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with BSG or a functional fragment thereof according to SEQ ID NO:9. In some respects, variants or fragments of IGSF2 share at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with IGSF2 or a functional fragment thereof according to SEQ ID NO:34. In some aspects, variants or fragments of IGSF3 share at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with IGSF3 or a functional fragment thereof according to SEQ ID NO:20. In some aspects, variants or fragments of IGSF8 share at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with IGSF8 or a functional fragment thereof according to SEQ ID NO:14. In some aspects, variants or fragments of ITGB1 share at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with ITGB1 or a functional fragment thereof according to SEQ ID NO:21. In some aspects, variants or fragments of ITGA4 share at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with ITGA4 or a functional fragment thereof according to SEQ ID NO:22.In some respects, variants or fragments of SLC3A2 share at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with SLC3A2 or a functional fragment thereof according to SEQ ID NO:23. In some respects, variants or fragments of ATP1A1 share at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with ATP1A1 or a functional fragment thereof according to SEQ ID NO:24. In some respects, variants or fragments of ATP1A2 share at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with ATP1A2 or a functional fragment thereof according to SEQ ID NO:25. In some respects, variants or fragments of ATP1A3 share at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with ATP1A3 or a functional fragment thereof according to SEQ ID NO:26. In some respects, variants or fragments of ATP1A4 share at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with ATP1A4 or a functional fragment thereof according to SEQ ID NO:27. In some respects, variants or fragments of ATP1B3 share at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with ATP1B3 or a functional fragment thereof according to SEQ ID NO:28. In some respects, variants or fragments of ATP2B1 share at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with ATP2B1 or a functional fragment thereof according to SEQ ID NO:29. In some respects, variants or fragments of ATP2B2 share at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with ATP2B2 or a functional fragment thereof according to SEQ ID NO:30.In some aspects, variants or fragments of ATP2B3 share at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with ATP2B3 or a functional fragment thereof according to SEQ ID NO:31. In some aspects, variants or fragments of ATP2B4 share at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with ATP2B4 or a functional fragment thereof according to SEQ ID NO:32. In some aspects, variants or fragments of the scaffold X protein disclosed herein retain the ability to specifically target EVs (e.g., exogenous bodies). In some aspects, scaffold X includes one or more mutations, such as conserved amino acid substitutions.

[0126] In some aspects, variants of stent Y include variants that share at least about 70% identity with fragments of MARCKS, MARCKSL1, BASP1, or MARCKS, MARCKSL1, or BASP1. In some aspects, variants of MARCKS or its fragments share at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with MARCKS or its functional fragments according to SEQ ID NO:47. In some aspects, variants of MARCKSL1 or its fragments share at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with MARCKSL1 or its functional fragments according to SEQ ID NO:48. In some aspects, variants or fragments of BASP1 share at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with BASP1 or a functional fragment thereof according to SEQ ID NO:49. In some aspects, variants or fragments of the scaffold Y protein retain the ability to specifically target the luminal surface of EVs (e.g., exosomes). In some aspects, scaffold Y includes one or more mutations, such as conserved amino acid substitutions.

[0127] "Conservative amino acid substitution" refers to the substitution of an amino acid residue by an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, if an amino acid in a polypeptide is substituted by another amino acid from the same side chain family, such substitution is considered conserved. In another aspect, a string of amino acids can be conservatively substituted by a structurally similar string that differs in the order and / or composition of the side chain family members.

[0128] The term "sequence identity percentage" or "identity percentage" between two polynucleotide or polypeptide sequences refers to the number of identical matching positions shared by sequences within a comparison window, taking into account additions or deletions (i.e., vacancies) that must be introduced for optimal alignment of the two sequences. A matching position is any location where the same nucleotide or amino acid is present in both the target and reference sequences. Vacancies are not nucleotides or amino acids and are not counted in the target sequence. Similarly, vacancies in the reference sequence are not counted because nucleotides or amino acids from the target sequence are counted, but those from the reference sequence are not.

[0129] The percentage of sequence identity is calculated as follows: Determine the number of positions in both sequences where the same amino acid residue or nucleic acid base appears (matching positions), divide this number by the total number of positions in the comparison window, and multiply the result by 100 to obtain the percentage of sequence identity. Sequence comparison and determination of the percentage of sequence identity between two sequences can be accomplished using software that is readily available online and downloadable. Suitable software programs are available from various sources for protein and nucleotide sequence alignment. A suitable program for determining the percentage of sequence identity is bl2seq, which is part of the BLAST program suite available from the National Center for Biotechnology Information (NCBI) website (blast.ncbi.nlm.nih.gov). Bl2seq uses either the BLASTN or BLASTP algorithm for comparing two sequences. BLASTN is used for comparing nucleic acid sequences, while BLASTP is used for comparing amino acid sequences. Other suitable programs are, for example, Needle, Stretcher, Water, or Matcher, which are part of the EMBOSS suite of bioinformatics programs and are also available from the European Institute of Bioinformatics (EBI) at www.ebi.ac.uk / Tools / psa.

[0130] Different regions within a single polynucleotide or polypeptide target sequence aligned to a polynucleotide or polypeptide reference sequence can each have their own percentage of sequence identity. Note that the percentage of sequence identity values ​​are rounded to the nearest tenth. For example, 80.11, 80.12, 80.13, and 80.14 are rounded down to 80.1, while 80.15, 80.16, 80.17, 80.18, and 80.19 are rounded up to 80.2. It should also be noted that length values ​​will always be integers.

[0131] Those skilled in the art will understand that the generation of sequence alignments used to calculate the percentage of sequence identity is not limited to binary sequence-sequence comparisons driven solely by primary sequence data. Sequence alignments can originate from multiple sequence alignments. A suitable program for generating multiple sequence alignments is ClustalW2, available from www.clustal.org. Another suitable program is MUSCLE, available from www.drive5.com / muscle / . ClustalW2 and MUSCLE can be obtained, for example, from EBI.

[0132] It should also be understood that sequence alignment can be generated by integrating sequence data with data from heterogeneous sources, such as structural data (e.g., crystallographic protein structures), functional data (e.g., mutation locations), or phylogenetic data. Suitable procedures for integrating heterogeneous data to generate multiple sequence alignments are available at worldwideweb.tcoffee.org, and alternatively, T-Coffee, for example, from EBI. It should also be understood that the final alignment used to calculate the percentage of sequence identity can be programmed automatically or manually.

[0133] Polynucleotide variants can contain alterations in coding regions, non-coding regions, or both. On the one hand, polynucleotide variants contain changes that produce silent substitutions, additions, or deletions, but do not alter the properties or activity of the encoded polypeptide. On the other hand, nucleotide variants are generated through silent substitutions due to the degeneracy of the genetic code. In other respects, variants include those that substitute, delete, or add 5-10, 1-5, or 1-2 amino acids in any combination. Polynucleotide variants can be generated for a variety of reasons, such as to optimize codon expression for a specific host (changing codons in human mRNA to other codons, for example, in bacterial hosts such as E. coli).

[0134] Naturally occurring variants, referred to as “allele variants,” are one of several alternative forms of a gene occupying a given locus on an organism’s chromosome (Genes II, Lewin, B., editor, John Wiley & Sons, New York (1985)). These allele variants can vary at the polynucleotide and / or polypeptide level and are included in this disclosure. Alternatively, non-naturally occurring variants may be produced by mutagenesis or direct synthesis.

[0135] Known methods using protein engineering and recombinant DNA techniques can produce variants to improve or alter the characteristics of peptides. For example, one or more amino acids can be deleted from the N-terminus or C-terminus of a secretory protein without substantially losing its biological function. Ron et al., J. Biol. Chem. 268:2984-2988 (1993) (incorporated hereby by reference in its entirety) reported that variant KGF proteins retained heparin-binding activity even after deletion of 3, 8, or 27 amino acid residues from the N-terminus. Similarly, interferon-γ showed up to 10-fold activity after deletion of 8-10 amino acid residues from the C-terminus of the protein. (Dobeli et al., J. Biotechnology 7:199-216 (1988), incorporated hereby by reference in its entirety).

[0136] Furthermore, substantial evidence suggests that variants often retain biological activities similar to those of naturally occurring proteins. For example, Gayle et al. (J. Biol. Chem 268:22105-22111 (1993), incorporated herein by reference in its entirety) conducted an extensive mutational analysis of the human cytokine IL-1a. They generated over 3,500 individual IL-1a mutants using random mutagenesis, with each variant exhibiting an average of 2.5 amino acid changes across the entire length of the molecule. Multiple mutations were examined at every possible amino acid position. The researchers found that "most molecules could be altered with little effect on [binding or biological activity]" (see abstract). In fact, of the more than 3,500 nucleotide sequences examined, only 23 unique amino acid sequences produced proteins with significantly different activities from the wild-type.

[0137] As described above, peptide variants include, for example, modified peptides. Modifications include, for example, acetylation, acylation, ADP-ribosylation, amidation, covalent linkage of flavin, covalent linkage of heme moieties, covalent linkage of nucleotides or nucleotide derivatives, covalent linkage of lipids or lipid derivatives, covalent linkage of phosphatidylinositol, crosslinking, cyclization, disulfide bond formation, demethylation, formation of covalent crosslinks, formation of cysteine ​​residues, formation of pyroglutamate, formylation, γ-carboxylation, glycosylation, GPI anchoring, hydroxylation, iodination, methylation, myristylation, oxidation, polyethylene glycolation (Mei et al., Blood 116:270-79 (2010), which is incorporated herein by reference in its entirety), proteolytic processing, phosphorylation, isopreneation, racemization, selenylation, sulfation, transfer RNA-mediated addition of amino acids to proteins such as argininoylation, and ubiquitous proteination. In some respects, bracket X and / or bracket Y can be modified in any convenient location.

[0138] As used herein, the terms “connected to,” “fused to,” or “conjugated to” are used interchangeably and refer to a covalent or non-covalent bond formed between a first part and a second part (e.g., scaffold X and antigen (or adjuvant or immunomodulator), respectively, such as a scaffold portion expressed in or on an extracellular vesicle, such as scaffold X (e.g., PTGFRN protein) expressed in or on the luminal surface of an extracellular vesicle). In some aspects, the payload (e.g., antigen, adjuvant, and / or immunomodulator) and / or targeting portion disclosed herein may be directly connected to the outer and / or luminal surface of an EV (e.g., exogen). As used herein, the terms “direct connection,” “direct fusion,” or “direct conjugation to” refer to the process of connecting (fused to) a portion (e.g., payload and / or targeting portion) to the surface of an EV (e.g., exogen) without using the scaffold portion disclosed herein.

[0139] As used herein, the term "fusion protein" refers to two or more proteins that are interconnected or conjugated. For example, in some aspects, fusion proteins that can be expressed in EVs (e.g., exosomes) disclosed herein include (i) a payload (e.g., an antigen, adjuvant, and / or immunomodulator) and (ii) a scaffold portion (e.g., scaffold X and / or scaffold Y). In some aspects, fusion proteins that can be expressed in EVs (e.g., exosomes) available for use in this disclosure include (i) a targeting portion and (ii) a scaffold portion (e.g., scaffold X and / or scaffold Y). As described herein, in some aspects, EVs (e.g., exosomes) of this disclosure can express multiple fusion proteins, wherein a first fusion protein includes (i) a payload (e.g., an antigen, adjuvant, and / or immunomodulator) and (ii) a scaffold portion (e.g., scaffold X and / or scaffold Y), and wherein a second fusion protein includes (i) a targeting portion and (ii) a scaffold portion (e.g., scaffold X and / or scaffold Y).

[0140] The term “encapsulated,” or its grammatically different forms (e.g., encapsulation or encapsulating), refers to a state or process in which a first part (e.g., an antigen, adjuvant, or immunomodulator) is contained within a second part (e.g., an EV, for example, an exogenous body) and the two parts are not chemically or physically connected. In some aspects, the term “encapsulated” may be used interchangeably with the terms “in a cavity” and “loaded.” Non-limiting examples of encapsulating (or loading) a first part (e.g., a payload, such as an antigen, adjuvant, or immunomodulator) into a second part (e.g., an EV, for example, an exogenous body) are disclosed elsewhere herein.

[0141] As used herein, the term "producer cell" refers to cells used to produce EVs (e.g., exogenous bodies). Producer cells can be cells cultured in vitro or cells in vivo. Producer cells include, but are not limited to, cells known to be efficient at producing EVs (e.g., exogenous bodies), such as HEK293 cells, Chinese hamster ovary (CHO) cells, mesenchymal stem cells (MSCs), BJ human foreskin fibroblasts, fHDF fibroblasts, and AGEs. Neuronal precursor cells, Amniotic fluid cells, adipose-derived mesenchymal stem cells, and RPTEC / TERT1 cells. In some aspects, the producer cells are not antigen-presenting cells. In some aspects, the producer cells are not dendritic cells, B cells, mast cells, macrophages, neutrophils, Kupffer-Browicz cells, or cells derived from any of these cells, or any combination thereof. In some aspects, the producer cells are not naturally occurring antigen-presenting cells (i.e., modified). In some aspects, the producer cells are not naturally occurring dendritic cells, B cells, mast cells, macrophages, neutrophils, Kupffer-Browicz cells, or cells derived from any of these cells, or any combination thereof. Other disclosures relating to such producer cells are provided elsewhere in this disclosure. In some respects, the EVs (e.g., exogens) useful in this disclosure do not carry antigens on MHC class I or II molecules exposed on the surface of the EV (e.g., exogens) (i.e., the antigens are not present on MHC class I or II molecules), but can carry antigens in the cavity of the EV (e.g., exogens) or on the surface of the EV (e.g., exogens) by attaching to scaffold X and / or scaffold Y.

[0142] As used in this article, "MHC class I molecule" refers to the protein product of a wild-type or variant HLA class I gene encoding an MHC class I molecule. Therefore, "HLA class I molecule" and "MHC class I molecule" can be used interchangeably in this article.

[0143] MHC class I molecules are one of the two major classes of major histocompatibility complex (MHC) molecules (the other being MHC class II) and are present on the cell surface of all nucleated cells in jawed vertebrates. They are also present on platelets but not on red blood cells. Their function is to present peptide fragments of intracellular proteins to cytotoxic T cells; this triggers an immediate immune response to specific non-self antigens, which are presented with the help of MHC class I proteins. Because MHC class I molecules present peptides derived from cytosol proteins, the MHC class I presentation pathway is often referred to as the cytosol or endogenous pathway.

[0144] In humans, the HLA corresponding to MHC class I are HLA-A, HLA-B, and HLA-C. MHC class I molecules consist of two protein chains: an α-chain and a β2-microglobulin (β2m) chain. Human β2m is encoded by the B2M gene. Class I MHC molecules bind peptides primarily produced by the degradation of cytosol proteins by the proteasome. The MHC I:peptide complex then inserts into the cell's extracellular membrane via the endoplasmic reticulum. Epitope peptides bind to the extracellular portion of class I MHC molecules. Therefore, the function of class I MHC is to present intracellular proteins to cytotoxic T cells (CTLs). However, class I MHC can also present peptides derived from exogenous proteins in a process known as cross-presentation.

[0145] Normal cells display peptides from normal cellular protein turnover on their class I MHC, and CTLs are not activated in response to them due to central and peripheral tolerance mechanisms. When cells express foreign proteins, such as after viral infection, a portion of the class I MHC will display these peptides on the cell surface. Therefore, CTLs specific to the MHC:peptide complex will recognize and kill the presenting cell. Alternatively, class I MHC itself can act as an inhibitory ligand for natural killer (NK) cells. A decrease in the normal level of surface class I MHC (a mechanism used by some viruses and certain tumors to evade CTL responses) activates NK cell killing.

[0146] As used herein, “MHC class II molecule” refers to the protein product of a wild-type or variant HLA class II gene encoding an MHC class II molecule. Therefore, “HLA class II molecule” and “MHC class II molecule” are used interchangeably in this article.

[0147] MHC class II molecules are a class of major histocompatibility complex (MHC) molecules that are typically found only on professional antigen-presenting cells, such as dendritic cells, monocytes, some endothelial cells, thymic epithelial cells, and B cells. These cells are important in initiating the immune response. Antigens presented by class II peptides originate from extracellular proteins (unlike cytosol proteins in MHC class I).

[0148] Like MHC class I molecules, class II molecules are also heterodimers, but in this case, they consist of two homopeptides (an α chain and a β chain, both encoded in the MHC). Subdesignations such as α1, α2, etc., refer to independent domains within the HLA gene; each domain is typically encoded by a different exon within the gene, and some genes have additional domains encoding leader sequences, transmembrane sequences, etc. These molecules have extracellular regions, transmembrane sequences, and cytoplasmic tails. The α1 and β1 regions of the chain aggregate to form the distal membrane peptide-binding domain, while the α2 and β2 regions (the remaining extracellular portion of the chain) form the proximal membrane immunoglobulin-like domain. The antigen-binding groove for antigen or peptide binding consists of two α-helical walls and a β-sheet. Because the antigen-binding groove of MHC class II molecules is open at both ends, while the corresponding groove on class I molecules is closed at each end, the antigens presented by MHC class II molecules are longer, generally between 15 and 24 amino acid residues. Loading of MHC class II molecules occurs via phagocytosis; extracellular proteins are endocytosed, digested in lysosomes, and the resulting epitope peptide fragments are loaded onto MHC class II molecules before migrating to the cell surface. In humans, the MHC class II protein complex is encoded by the human leukocyte antigen gene complex (HLA). The HLAs corresponding to MHC class II are HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQ, and HLA-DR. Mutations in the HLA gene complex can lead to naked lymphocyte syndrome (BLS), a type of MHC class II deficiency.

[0149] As used herein, the terms “isolate,” “isolated,” and “isolating,” or “purify,” “purified,” and “purifying,” and “extracted” and “extracting” are used interchangeably to refer to the formulation state of a desired EV (e.g., multiple known or unknown amounts and / or concentrations) that has undergone one or more purification processes, such as selection or enrichment of the desired EV formulation. In some aspects, the isolation or purification referred to herein is the process of removing, partially removing (e.g., a portion), EV from a sample containing producer cells. In some aspects, the isolated EV composition has no detectable undesirable activity, or alternatively, the level or amount of undesirable activity is at or below an acceptable level or amount. In other aspects, the amount and / or concentration of the desired EV in the isolated EV composition is equal to or higher than an acceptable amount and / or concentration. In other aspects, the isolated EV composition is enriched compared to the raw material from which the composition was obtained (e.g., a producer cell formulation). Compared to the raw materials, this enrichment can be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99%, 99.999%, 99.9999%, or greater than 99.9999%. In some respects, the isolated EV formulation is substantially free of residual biological products. In some respects, the isolated EV formulation is 100% free, 99% free, 98% free, 97% free, 96% free, 95% free, 94% free, 93% free, 92% free, 91% free, or 90% free of any contaminating biological material. Residual biological products may include non-biological substances (including chemicals) or unwanted nucleic acids, proteins, lipids, or metabolites. Being substantially free of residual biological products can also mean that the EV composition does not contain detectable producer cells, and only EVs are detectable.

[0150] As used herein, the term "immunomodulator" refers to an agent (i.e., a payload) that acts on a target (e.g., a target cell) in contact with extracellular vesicles and modulates the immune system. Non-limiting examples of immunomodulators that can be introduced into EVs (e.g., exogenous organisms) and / or producer cells include agents such as modulators of checkpoint inhibitors, ligands of checkpoint inhibitors, cytokines, derivatives thereof, or any combination thereof. Immunomodulators may also include agonists, antagonists, antibodies, antigen-binding fragments, polynucleotides such as siRNA, antisense oligonucleotides, phosphodiesteromorpholino oligomers (PMOs), peptide-conjugated phosphodiesteromorpholino oligomers (PPMOs), miRNAs, lncRNAs, mRNAs, DNA, or small molecules.

[0151] As used herein, the term "biodistribution modifier" refers to an agent (i.e., payload) capable of altering the distribution of extracellular vesicles (e.g., exosomes, nanovesicles) in vivo or in vitro (e.g., in a mixed culture of different cell types). In some aspects, the term "targeting portion" may be used interchangeably with the term biodistribution modifier. In some aspects, the targeting portion alters the tropism of an EV (e.g., an exosome) ("tropy portion"). As used herein, the term "tropy portion" refers to a targeting portion that, when expressed on an EV (e.g., an exosome), alters and / or enhances the natural motility of the EV. For example, in some aspects, the tropy portion can promote the uptake of EVs by specific cells, tissues, or organs. Non-limiting examples of tropy portions that may be used with this disclosure include portions capable of binding to markers specifically expressed on dendritic cells (e.g., Clec9A or DEC205) or T cells (e.g., CD3). Unless otherwise stated, the term "targeting portion" as used herein encompasses tropy portions. Biodistributors can be biomolecules, such as proteins, peptides, lipids, or carbohydrates, or synthetic molecules. For example, biodistribution modifiers can be affinity ligands (e.g., antibodies, VHH domains, phage display peptides, fibronectin domains, camelids, VNAR), synthetic polymers (e.g., PEG), natural ligands / molecules (e.g., CD40L, albumin, CD47, CD24, CD55, CD59), recombinant proteins (e.g., XTEN), but are not limited to these.

[0152] In some aspects, biodistribution modifiers and / or targeting moieties are displayed on the surface of EVs (e.g., exogenous bodies). Biodistribution modifiers can be displayed on the EV surface by fusing with a scaffold protein (e.g., scaffold X) (e.g., as a genetically encoded fusion molecule). In some aspects, biodistribution modifiers can be displayed on the EV surface by a chemical reaction that attaches the biodistribution modifier to the EV surface molecule. A non-limiting example is PEGylation. In some aspects, in addition to antigens, adjuvants, or immunomodulators, the EVs (e.g., exogenous bodies) disclosed herein may also contain biodistribution modifiers. Non-limiting examples of biodistribution modifiers or targeting moieties that can be used in this disclosure include C-type lectin domain family 9 member A (Clec9a) protein, dendritic cell-specific intercellular adhesion molecule-3-capture integrin (DC-SIGN), CD207, CD40, Clec6, dendritic cell immune receptor (DCIR), DEC-205, lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), MARCO, Clec12a, DC-asialyl glycoprotein receptor (DC-ASGPR), DC immune receptor 2 (DCIR2), Dectin-1, macrophage mannose receptor (MMR), BDCA-1 (CD303, Clec4c), Dectin-2, Bst-2 (CD317), CD3, or any combination thereof. In some aspects, the targeting moieties are Clec9a protein. In some aspects, the targeting moieties are CD3 molecules.

[0153] As used herein, the term "C-type lectin domain family 9 member A" (Clec9a) protein refers to a group of C-type lectin-like receptors (CTLRs) that function as an activating receptor and are expressed on myeloid lineage cells (e.g., DCs). Huysamen et al., J Biol Chem 283(24):16693-701 (2008); U.S. Patent No. 9,988,431B2, each of which is incorporated herein by reference in its entirety. Synonyms for Clec9a are known and include CD370, DNGR-1, 5B5, HEEE9341, and C-type lectin domains containing 9a. In some respects, Clec9a protein is expressed on human cDC1 cells. In some respects, Clec9a protein is expressed on mouse cDC1 and pDC cells. Unless otherwise stated, as used herein, Clec9a may refer to Clec9a from one or more species (e.g., human, non-human primate, dog, cat, guinea pig, rabbit, rat, mouse, horse, cow, and bear).

[0154] As used herein, the term "CD3" or "differentiation cluster 3" refers to a protein complex that associates with the T cell receptor (TCR). The CD3 molecule consists of four distinct chains (CD3γ, CD3δ, and two CD3ε chains). These chains associate with the T cell receptor (TCR) and the ζ chain to generate activation signals in T lymphocytes. The TCR, the ζ chain, and the CD3 molecule together constitute the TCR complex. The CD3 molecule is expressed on all T cells, including CD4+ T cells and CD8+ T cells. Unless otherwise stated, as used herein, CD3 can refer to CD3 from one or more species (e.g., humans, non-human primates, dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, and bears).

[0155] As used herein, the term "payload" refers to an agent that acts on a target (e.g., target cell) in contact with an EV (e.g., exogenous organism). In some respects, unless otherwise stated, the term payload may be used interchangeably with the term "bioactive molecule." Non-limiting examples of payloads that may be contained on an EV (e.g., exogenous organism) are antigens, adjuvants, and / or immunomodulators. Payloads that may be introduced into EVs (e.g., exogenous organisms) and / or producer cells include agents such as nucleotides (e.g., nucleotides containing toxins that can detect partial or disrupt transcription), nucleic acids (e.g., DNA or mRNA molecules encoding polypeptides such as enzymes, or regulatory RNA molecules such as miRNA, dsDNA, lncRNA, siRNA, antisense oligonucleotides, phosphodiesteramide morpholino oligomers (PMOs), peptide-conjugated phosphodiesteramide morpholino oligomers (PPMOs), or combinations thereof), amino acids (e.g., amino acids containing toxins that can detect partial or disrupt translation), polypeptides (e.g., enzymes), lipids, carbohydrates, small molecules (e.g., small molecule drugs and toxins), and combinations thereof. In some respects, the payload includes antigens. As used herein, the term “antigen” refers to any agent that, when introduced into a subject, elicits an immune response (cellular or humoral) against itself.

[0156] As used herein, the term "affinity ligand" refers to a molecule capable of selectively and preferentially binding to a specific marker (e.g., expressed on target cells). Non-limiting examples of affinity ligands that can be used in this disclosure include antibodies, phage display peptides, fibronectin domains, camelids, VNAR, VHH domains, and combinations thereof. As used herein, the term "antibody" encompasses immunoglobulins (whether naturally occurring or partially or fully synthetically produced) and fragments thereof. The term also encompasses any protein having a binding domain homologous to an immunoglobulin binding domain. "Antibody" also includes polypeptides containing a framework region from an immunoglobulin gene or a fragment thereof that specifically binds to and recognizes an antigen. The term "antibody" is used to refer to complete antibodies, polyclonal antibodies, monoclonal antibodies, and recombinant antibodies, fragments thereof, and also includes single-chain antibodies, humanized antibodies, mouse antibodies, chimeric antibodies, mouse-human antibodies, mouse-primate antibodies, primate-human monoclonal antibodies, anti-idiotype antibodies, antibody fragments such as, for example, scFv, (scFv)2, Fab, Fab' and F(ab')2, F(ab1)2, Fv, dAb and Fd fragments, biantibodies, and antibody-associated peptides. Antibodies include both bispecific and multispecific antibodies, provided they exhibit the desired biological activity or function.

[0157] The terms “individual,” “subject,” “host,” and “patient” are used interchangeably herein to refer to any mammalian subject, particularly a human, in the presence of a diagnostic, therapeutic, or therapeutic agent. The compositions and methods described herein are suitable for therapeutic and veterinary applications in humans. In some respects, the subject is a mammal; in others, the subject is a human. As used herein, “mammal subject” includes all mammals, including but not limited to humans, domesticated animals (e.g., dogs, cats, etc.), farm animals (e.g., cattle, sheep, pigs, horses, etc.), and laboratory animals (e.g., monkeys, rats, mice, rabbits, guinea pigs, etc.).

[0158] As used herein, the term “substantially free” means that a sample containing EVs (e.g., exogens) contains less than 10% of macromolecules by mass / volume (m / v) percentage concentration. Some fractions may contain less than about 0.001%, less than about 0.01%, less than about 0.05%, less than about 0.1%, less than about 0.2%, less than about 0.3%, less than about 0.4%, less than about 0.5%, less than about 0.6%, less than about 0.7%, less than about 0.8%, less than about 0.9%, less than about 1%, less than about 2%, less than about 3%, less than about 4%, less than about 5%, less than about 6%, less than about 7%, less than about 8%, less than about 9%, or less than about 10% (m / v) of macromolecules.

[0159] As used in this article, the term "macromolecule" refers to nucleic acids, contaminating proteins, lipids, carbohydrates, metabolites, or combinations thereof.

[0160] As used herein, the term “common exosome protein” means a protein previously known to be enriched in exosomes, including but not limited to CD9, CD63, CD81, PDGFR, GPI-anchored proteins, lactoglucosins LAMP2 and LAMP2B, fragments thereof, or peptides bound to them.

[0161] As used herein, “administration” means providing a subject with a composition comprising an EV (e.g., exogenous form) disclosed herein via a pharmaceutically acceptable route. Routes of administration may be intravenous, such as intravenous injection and intravenous infusion. Other routes of administration include, for example, subcutaneous, intramuscular, oral, intranasal, and pulmonary administration. EVs (e.g., exogenous forms) may be administered as part of a pharmaceutical composition comprising at least one excipient.

[0162] As used herein, an “immune response” refers to a biological response in a vertebrate body against foreign factors or abnormal cells (e.g., cancer cells), which protects the organism from these factors and the diseases they cause. An immune response is mediated by the action of one or more cells of the immune system (e.g., T lymphocytes, B lymphocytes, natural killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells, or neutrophils) and soluble macromolecules (including antibodies, cytokines, and complement) produced by these cells or any of them in the liver. This immune response results in the selective targeting, binding, damage, destruction, and / or elimination from the vertebrate body of invading pathogens, pathogen-infected cells or tissues, cancerous or other abnormal cells, or, in cases of autoimmune or pathological inflammation. Immune responses include, for example, the activation or inhibition of T cells, such as effector T cells, Th cells, CD4+ cells, CD8+ T cells, or Treg cells, or the activation or inhibition of any other cells of the immune system, such as NK cells. Therefore, immune responses can include humoral immune responses (e.g., B cell-mediated), cellular immune responses (e.g., T cell-mediated), or a combination of humoral and cellular immune responses. In some aspects, immune responses are “suppressive” immune responses. A “suppressive” immune response is an immune response that blocks or attenuates the effects of a stimulus (e.g., an antigen). In some aspects, suppressive immune responses involve the production of inhibitory antibodies against the stimulus. In some aspects, immune responses are “stimulatory” immune responses. A “stimulatory” immune response is an immune response that results in the production of effector cells (e.g., cytotoxic T lymphocytes) capable of destroying and clearing target antigens (e.g., tumor antigens or viruses).

[0163] As used herein, the term “cellular immune response” is used interchangeably with the term “cell-mediated immune response” and refers to an immune response that does not primarily involve antibodies. Instead, a cellular immune response involves the activation of various immune cells (e.g., phagocytes and antigen-specific cytotoxic T lymphocytes) that, upon activation (e.g., upon antigen stimulation), produce a variety of effector molecules (e.g., cytokines, perforin, granzymes). As used herein, the term “humoral immune response” refers to an immune response primarily mediated by macromolecules found in extracellular fluids (such as secreted antibodies, complement proteins, and certain antimicrobial peptides). The term “antibody-mediated immune response” refers to one aspect of antibody-mediated humoral immune responses.

[0164] As used herein, the term "immune cell" refers to any cell of the immune system that participates in mediating an immune response. Non-limiting examples of immune cells include T lymphocytes, B lymphocytes, natural killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells, neutrophils, or combinations thereof. In some aspects, immune cells express CD3. In some aspects, immune cells expressing CD3 are T cells (e.g., CD4+ T cells or CD8+ T cells). In some aspects, immune cells that can be targeted by the targeting portions disclosed herein (e.g., anti-CD3) include primary CD4+ T cells. In some aspects, immune cells include memory CD4+ T cells. In some aspects, immune cells comprise effector CD4+ T cells. In some aspects, immune cells include primary CD8+ T cells. In some aspects, immune cells include memory CD8+ T cells. In some aspects, immune cells include effector CD8+ T cells. In some aspects, immune cells are dendritic cells. In some respects, dendritic cells include plasmacytoid dendritic cells (pDCs), conventional dendritic cell 1 (cDC1), conventional dendritic cell 2 (cDC2), inflammatory mononuclear cell-derived dendritic cells, Langerhans cells, dermal dendritic cells, lysozyme-expressing dendritic cells (LysoDCs), Kupffer cells, or any combination thereof. Therefore, in some respects, the EVs (e.g., exogens) disclosed herein can specifically target immune cells including conventional dendritic cell 1 (cDC1) and / or plasmacytoid dendritic cells (pDCs).

[0165] As used herein, the term "T cell" or "T-cell" refers to a type of lymphocyte that matures in the thymus. T cells play a crucial role in cell-mediated immunity and are distinguished from other lymphocytes, such as B cells, by the presence of T cell receptors on their cell surface. T cells encompass all types of CD3-expressing immune cells, including helper T cells (CD4+ cells), cytotoxic T cells (CD8+ cells), natural killer T cells, regulatory T cells (Tregs), and γ-δ T cells.

[0166] "Primitive" T cells refer to immune-undifferentiated (i.e., unactivated) mature T cells. Following positive and negative selection in the thymus, T cells emerge as CD4+ or CD8+ primitive T cells. In their primitive state, T cells express L-selectin (CD62L+), IL-7 receptor-α (IL-7R-α), and CD132, but they do not express CD25, CD44, CD69, or CD45RO. As used herein, "immature" can also refer to T cells exhibiting phenotypic characteristics of primitive or immature T cells (such as TSCM cells or TCM cells). For example, immature T cells may express one or more of L-selectin (CD62L+), IL-7Rα, CD132, CCR7, CD45RA, CD45RO, CD27, CD28, CD95, CXCR3, and LFA-1. Primitive or immature T cells can be associated with terminally differentiated effector T cells (such as T cells). EM Cells and T EFF (Cells) form a contrast.

[0167] As used in this article, the term "effective" T cell or "T cell" refers to... EFF "Effective T cells are T cells that can mediate the removal of pathogens or cells without further differentiation. Therefore, effector T cells are different from primary T cells and memory T cells, and these cells often need to differentiate and proliferate before becoming effector cells."

[0168] As used herein, the term "memory" T cell refers to a subset of T cells that have previously encountered and responded to their homologous antigens. In some respects, the term is synonymous with "antigen-stimulated" T cells. In some respects, memory T cells can be effector memory T cells or central memory T cells. In some respects, memory T cells are tissue-resident memory T cells. As used herein, the term "tissue-resident memory T cell" or "TRM cell" refers to a lineage of T cells that occupy tissues (e.g., skin, lungs, gastrointestinal tract) and are no longer circulating. TRM cells differ transcriptionally, phenotypedly, and functionally from central memory and effector memory T cells, which circulate in the blood, T cell zones of secondary lymphoid organs, and between lymphatic and non-lymphatic tissues. One of the roles of TRM cells is to provide immune protection against infection in extralymphatic tissues.

[0169] As used herein, the term "dendritic cell" or "DC" refers to a class of bone marrow-derived immune cells capable of processing extracellular and intracellular proteins and presenting antigens in the context of MHC molecules to initiate primordial T cells. In some respects, dendritic cells can be subdivided into further subtypes, such as conventional dendritic cell 1 (cDC1), conventional dendritic cell 2 (cDC2), plasmacytoid dendritic cells (pDC), inflammatory monocyte-derived dendritic cells, Langerhans cells, dermal dendritic cells, lysozyme-expressing dendritic cells (LysoDC), Kupffer cells, and combinations thereof. In some respects, different DC subgroups can be distinguished based on their phenotypic expression. For example, in some respects, human cDC1 cells are CD1c... - and CD141 + In some respects, human cDC2 cells are CD1c + and CD141 - In some respects, human pDC cells are CD123+. In some respects, mouse cDC1 cells are XCR1. + Clec9a + and Sirpa - In some respects, mouse cDC2 cells are CD8... + CD11b + Sirpa + XCR1 - and CD1c,b + In some respects, mouse pDC cells are CD137 + XCR1 - and Sirpa - Other phenotypic markers for distinguishing different DC subgroups are known in the art. See, for example, Collin et al., Immunology 154(1):3-20 (2018). In some respects, different DC subgroups can be distinguished based on their functional characteristics. For example, in some respects, pDCs produce large amounts of IFN-α, while cDC1 and cDC2 produce inflammatory cytokines such as IL-12, IL-6, and TNF-α. Other methods for distinguishing different DC subgroups are known in the art. See, for example, U.S. Patent Nos. 8,426,565B2 and 9,988,431, each of which is incorporated herein by reference in its entirety.

[0170] As used herein, the term "immunoconjugate" refers to a compound comprising a binding molecule (e.g., an antibody) and one or more portions (e.g., therapeutic or diagnostic portions) chemically conjugated to the binding molecule. Typically, immunoconjugates are defined by the general formula: A-(LM)n, where A is the binding molecule (e.g., an antibody), L is an optional linker, M is a heterologous portion, which can be, for example, a therapeutic agent, a detectable label, etc., and n is an integer. In some aspects, multiple heterologous portions may be chemically conjugated to different attachment sites in the same binding molecule (e.g., an antibody). In other aspects, multiple heterologous portions may be tandemly connected and attached to attachment sites in the binding molecule (e.g., an antibody). In some aspects, multiple heterologous portions (identical or different) may be conjugated to a binding molecule (e.g., an antibody).

[0171] Immunoconjugates can also be defined by the general formula in reverse order. In some aspects, an immunoconjugate is an "antibody-drug conjugate" ("ADC"). In this disclosure, the term "immunoconjugate" is not limited to chemical or enzymatic conjugate molecules. The term "immunoconjugate" as used in this disclosure also includes gene fusions. In some aspects of this disclosure, a bioactive molecule is an immunoconjugate. The terms "antibody-drug conjugate" and "ADC" are used interchangeably and refer to, for example, an antibody covalently linked to one or more therapeutic agents (sometimes referred to herein as an agent, drug, or active pharmaceutical ingredient). In some aspects of this disclosure, a bioactive molecule (i.e., the payload) is an antibody-drug conjugate.

[0172] As used herein, the terms “treat,” “treatment,” or “treating” refer to, for example, a reduction in the severity of a disease or ailment; a shortening of the duration of a disease or ailment; an improvement or elimination of one or more symptoms associated with a disease or ailment; or providing a beneficial effect to a subject suffering from a disease or ailment, but not necessarily curing the disease or ailment. The term also includes defense against or prevention of a disease or ailment or its symptoms. On the other hand, the term “treating” or “treatment” refers to inducing an immune response against an antigen in a subject.

[0173] As used in this article, the term "prevent" or "preventing" refers to reducing or mitigating the occurrence or severity of a particular outcome. In some respects, preventative measures are taken to achieve this.

[0174] II. Extracellular vesicles, for example, exogenous bodies

[0175] This document discloses EVs (e.g., exosomes) capable of modulating the immune system of a subject. EVs (e.g., exosomes) used in this disclosure have been engineered to produce multiple agents (i.e., payloads) together (e.g., antigen and adjuvant in a single EV (e.g., exosome); antigen and immunomodulator in a single EV (e.g., exosome); and antigen, adjuvant, and immunomodulator in a single EV (e.g., exosome); rather than a single agent, such as a single antigen, a single adjuvant, or a single immunomodulator). In some aspects, the EV (e.g., exosome) comprises (i) an antigen and (ii) an adjuvant. In other aspects, the EV (e.g., exosome) comprises (i) an antigen and (ii) an immunomodulator. In some aspects, the EV (e.g., exosome) comprises (i) an antigen, (ii) an adjuvant, and (iii) an immunomodulator. In some aspects, the EVs (e.g., exosomes) disclosed herein may also comprise additional portions, such as a targeting portion. In some aspects, the antigen is not expressed or presented on major histocompatibility complex I and / or II molecules. In other respects, although the antigen in the EV (e.g., exosome) is not expressed or presented as part of an MHC class I or II complex, the EV (e.g., exosome) may still contain MHC class I / II molecules on its surface. Therefore, in some respects, the EVs (e.g., exosomes) disclosed herein do not directly interact with the T cell receptor (TCR) of T cells to induce an immune response against the antigen. Similarly, in some respects, the EVs (e.g., exosomes) disclosed herein do not directly transfer the antigen to the surface of target cells (e.g., dendritic cells) through cross-dressing. “Cross-dressing” is a common mechanism by which EVs (e.g., dendritic cell-derived exosomes (DEX)) induce T cell activation. See Pitt, JM et al., J Clin Invest 126(4):1224-32 (2016). In other respects, the EVs (e.g., exogenous bodies) of this disclosure are phagocytosed by antigen-presenting cells and can be expressed as MHC class I and / or MHC class II complexes on the surface of antigen-presenting cells.

[0176] It will be apparent to those skilled in the art that the EVs (e.g., exogens) disclosed herein do not need to contain antigens, but may contain a variety of other payloads disclosed herein. For example, in some aspects, EVs (e.g., exogens) may contain a variety of different adjuvants. In some aspects, EVs (e.g., exogens) may contain a variety of different immunomodulators. In some aspects, EVs (e.g., exogens) may contain a combination of one or more adjuvants with one or more immunomodulators. Such antigen-free EVs (e.g., exogens) can be used to induce and / or enhance innate immune responses. Non-limiting examples of therapeutic settings in which such antigen-free EVs may be useful include: treating bacterial and / or viral infections, such as Pseudomonas aeruginosa for ventilator-associated pneumonia, influenza and RSV, SARS / MER, toxoplasmosis, sepsis, yellow fever, and Staphylococcus aureus for surgical site infections. In some aspects, such antigen-free EVs (e.g., exogens) may be used in combination with one or more additional therapeutic agents. In some respects, one or more additional therapeutic agents contain antigens, wherein the antigens are not expressed in EVs (e.g., exogenous bodies) (e.g., in a soluble form of the antigen)... Unless otherwise stated, the relevant disclosures provided herein also apply whether or not the EV (e.g., exogenous body) contains an antigen.

[0177] As described above, the EVs (e.g., exogenous vesicles) described herein are extracellular vesicles with a diameter between about 20 nm and 300 nm. In some aspects, the EVs (e.g., exogenous vesicles) of this disclosure have diameters between about 20 nm and 290 nm, about 20 nm and 280 nm, about 20 nm and 270 nm, about 20 nm and 260 nm, about 20 nm and 250 nm, about 20 nm and 240 nm, about 20 nm and 230 nm, about 20 nm and 220 nm, about 20 nm and 210 nm, about 20 nm and 200 nm, about 20 nm and 190 nm, about 20 nm and 180 nm, about 20 nm and 170 nm, about 20 nm and 160 nm, about 20 nm and 150 nm, and about 20 nm and 300 nm. Between 140nm and approximately 20nm and 130nm, between approximately 20nm and 120nm, between approximately 20nm and 110nm, between approximately 20nm and 100nm, between approximately 20nm and 90nm, between approximately 20nm and 80nm, between approximately 20nm and 70nm, between approximately 20nm and 60nm, between approximately 20nm and 50nm, between approximately 20nm and 40nm, between approximately 20nm and 30nm, between approximately 30nm and 300nm, between approximately 30nm and 290nm, between approximately 30nm and 280nm, between approximately 30nm and 270nm, between approximately 30nm and 260nm, between approximately 30nm and 250nm. Between nm, approximately 30nm and 240nm, approximately 30nm and 230nm, approximately 30nm and 220nm, approximately 30nm and 210nm, approximately 30nm and 200nm, approximately 30nm and 190nm, approximately 30nm and 180nm, approximately 30nm and 170nm, approximately 30nm and 160nm, approximately 30nm and 150nm, approximately 30nm and 140nm, approximately 30nm and 130nm, approximately 30nm and 120nm, approximately 30nm and 110nm, approximately 30nm and 100nm, approximately 30nm and 90nm, approximately 30nm and 80nm Between m, approximately 30nm and 70nm, approximately 30nm and 60nm, approximately 30nm and 50nm, approximately 30nm and 40nm, approximately 40nm and 300nm, approximately 40nm and 290nm, approximately 40nm and 280nm, approximately 40nm and 270nm, approximately 40nm and 260nm, approximately 40nm and 250nm, approximately 40nm and 240nm, approximately 40nm and 230nm, approximately 40nm and 220nm, approximately 40nm and 210nm, approximately 40nm and 200nm, approximately 40nm and 190nm, approximately 40nm and 180nmBetween approximately 40nm and 170nm, between approximately 40nm and 160nm, between approximately 40nm and 150nm, between approximately 40nm and 140nm, between approximately 40nm and 130nm, between approximately 40nm and 120nm, between approximately 40nm and 110nm, between approximately 40nm and 100nm, between approximately 40nm and 90nm, between approximately 40nm and 80nm, between approximately 40nm and 70nm, between approximately 40nm and 60nm, between approximately 40nm and 50nm, between approximately 50nm and 300nm, between approximately 50nm and 290nm, between approximately 50nm and 280nm, between approximately 50nm and 270nm, between approximately 50nm and 260nm, approximately 5 Between 0nm and 250nm, approximately 50nm and 240nm, approximately 50nm and 230nm, approximately 50nm and 220nm, approximately 50nm and 210nm, approximately 50nm and 200nm, approximately 50nm and 190nm, approximately 50nm and 180nm, approximately 50nm and 170nm, approximately 50nm and 160nm, approximately 50nm and 150nm, approximately 50nm and 140nm, approximately 50nm and 130nm, approximately 50nm and 120nm, approximately 50nm and 110nm, approximately 50nm and 100nm, approximately 50nm and 90nm, approximately 50nm and 80nm, approximately... Between 50nm and 70nm, approximately between 50nm and 60nm, approximately between 60nm and 300nm, approximately between 60nm and 290nm, approximately between 60nm and 280nm, approximately between 60nm and 270nm, approximately between 60nm and 260nm, approximately between 60nm and 250nm, approximately between 60nm and 240nm, approximately between 60nm and 230nm, approximately between 60nm and 220nm, approximately between 60nm and 210nm, approximately between 60nm and 200nm, approximately between 60nm and 190nm, approximately between 60nm and 180nm, approximately between 60nm and 170nm, approximately between 60nm and 160nm, approximately between 60nm and 150nm. Between approximately 60nm and 140nm, between approximately 60nm and 130nm, between approximately 60nm and 120nm, between approximately 60nm and 110nm, between approximately 60nm and 100nm, between approximately 60nm and 90nm, between approximately 60nm and 80nm, between approximately 60nm and 70nm, between approximately 70nm and 300nm, between approximately 70nm and 290nm, between approximately 70nm and 280nm, between approximately 70nm and 270nm, between approximately 70nm and 260nm, between approximately 70nm and 250nm, between approximately 70nm and 240nm, between approximately 70nm and 230nm, between approximately 70nm and 220nm, between approximately 70nm and 210nm.Between approximately 70nm and 200nm, between approximately 70nm and 190nm, between approximately 70nm and 180nm, between approximately 70nm and 170nm, between approximately 70nm and 160nm, between approximately 70nm and 150nm, between approximately 70nm and 140nm, between approximately 70nm and 130nm, between approximately 70nm and 120nm, between approximately 70nm and 110nm, between approximately 70nm and 100nm, between approximately 70nm and 90nm, between approximately 70nm and 80nm, between approximately 80nm and 300nm, between approximately 80nm and 290nm, between approximately 80nm and 280nm, between approximately 80nm and 270nm. Between m, approximately 80nm and 260nm, approximately 80nm and 250nm, approximately 80nm and 240nm, approximately 80nm and 230nm, approximately 80nm and 220nm, approximately 80nm and 210nm, approximately 80nm and 200nm, approximately 80nm and 190nm, approximately 80nm and 180nm, approximately 80nm and 170nm, approximately 80nm and 160nm, approximately 80nm and 150nm, approximately 80nm and 140nm, approximately 80nm and 130nm, approximately 80nm and 120nm, approximately 80nm and 110nm, approximately 80nm Between m and 100nm, approximately between 80nm and 90nm, approximately between 90nm and 300nm, approximately between 90nm and 290nm, approximately between 90nm and 280nm, approximately between 90nm and 270nm, approximately between 90nm and 260nm, approximately between 90nm and 250nm, approximately between 90nm and 240nm, approximately between 90nm and 230nm, approximately between 90nm and 220nm, approximately between 90nm and 210nm, approximately between 90nm and 200nm, approximately between 90nm and 190nm, approximately between 90nm and 180nm, approximately between 90nm and 170nm, approximately between 90nm and 160nm The sizes of the EVs (e.g., foreign bodies) described herein can be measured according to the methods described below. These range from approximately 90nm to 150nm, approximately 90nm to 140nm, approximately 90nm to 130nm, approximately 90nm to 120nm, approximately 90nm to 110nm, approximately 90nm to 100nm, approximately 100nm to 300nm, approximately 110nm to 290nm, approximately 120nm to 280nm, approximately 130nm to 270nm, approximately 140nm to 260nm, approximately 150nm to 250nm, approximately 160nm to 240nm, approximately 170nm to 230nm, approximately 180nm to 220nm, or approximately 190nm to 210nm.

[0178] In some aspects, the EV (e.g., exosome) of this disclosure includes a lipid bilayer membrane (“EV (e.g., exosome) membrane”) comprising an inner surface and an outer surface. In some aspects, the inner surface faces the core (i.e., lumen) of the EV (e.g., exosome). In some aspects, the outer surface may be in contact with the endosomes, multivesicles, or membrane / cytoplasm of the producer cell or target cell.

[0179] In some respects, EV (e.g., exogenous) membranes contain lipids and fatty acids. In other respects, EV (e.g., exogenous) membranes contain phospholipids, glycolipids, fatty acids, sphingolipids, glycerol phosphates, sterols, cholesterol, and phosphatidylserine.

[0180] In some aspects, the EV (e.g., exogenous) membrane comprises an inner lobule and an outer lobule. The composition of the inner and outer lobules can be determined by transbilayer distribution determinations known in the art, see, for example, Kuypers et al., Biohim Biophys Acta 1985 819:170. In some aspects, the outer lobule is composed of about 70-90% choline phospholipids, about 0-15% acidic phospholipids, and about 5-30% phosphatidylethanolamine. In some aspects, the inner lobule is composed of about 15-40% choline phospholipids, about 10-50% acidic phospholipids, and about 30-60% phosphatidylethanolamine.

[0181] In some respects, EV (e.g., exogenous) membranes contain one or more polysaccharides, such as polysaccharides.

[0182] In some aspects, the EV (e.g., exosome) membrane also includes one or more scaffold portions capable of anchoring, for example, antigens and / or adjuvants and / or immunomodulators to the EV (e.g., exosome) (e.g., on the luminal surface or on the outer surface). In some aspects, the scaffold portion is a polypeptide (“exosome protein”). In other aspects, the scaffold portion is a non-polypeptide portion. In some aspects, the exosome protein includes a variety of membrane proteins enriched on the exosome membrane, such as transmembrane proteins, integrase proteins, and peripheral proteins. These may include various CD proteins, transport proteins, integrins, lectins, and cadherins. In some aspects, the scaffold portion (e.g., exosome protein) includes scaffold X. In other aspects, the scaffold portion (e.g., exosome protein) includes scaffold Y. In other aspects, the scaffold portion (e.g., exosome protein) includes both scaffold X and scaffold Y.

[0183] In some respects, the EVs (e.g., exogens) disclosed herein are capable of delivering payloads (e.g., antigens, adjuvants, and / or immunomodulators) to a target. A payload is an agent that acts on a target (e.g., target cells) in contact with the EV. Contact can occur in vitro or in a subject. Non-limiting examples of payloads that can be introduced into an EV include agents such as nucleotides (e.g., nucleotides containing toxins that can detect partial or disrupt transcription), nucleic acids (e.g., DNA or mRNA molecules encoding polypeptides such as enzymes, or regulatory RNA molecules such as miRNA, dsDNA, lncRNA, siRNA, antisense oligonucleotides, phosphodiesteramide morpholino oligomers (PMOs), or peptide-conjugated phosphodiesteramide morpholino oligomers (PPMOs)), amino acids (e.g., amino acids containing toxins that can detect partial or disrupt translation), polypeptides (e.g., enzymes), lipids, carbohydrates, and small molecules (e.g., small molecule drugs and toxins).

[0184] As demonstrated herein (e.g., see Example 16), in some respects, the EVs (e.g., exogens) of this disclosure are capable of inducing effector cells and memory T cells. In some respects, memory T cells are tissue-resident memory T cells. Such EVs (e.g., exogens) can be used specifically as vaccines for certain infectious diseases. For example, most currently available influenza vaccines are inactivated and primarily focus on generating neutralizing antibodies against certain influenza surface antigens (e.g., hemagglutinin (HA) and neuraminidase (NA)). (See Wang et al., Science 367(6480):1-12 (February 21, 2020), which is incorporated herein by reference in its entirety). However, such antigens are constantly mutated, requiring annual vaccine updates. Even with annual updates, there are some years when influenza vaccines are ineffective because the HA and / or NA antigenicity of the vaccine virus strain does not match that of the circulating strain. Widespread immunization can be induced by natural viral infection or by live vector-engineered and attenuated vaccines, as these induce tissue (lung)-resident memory T cells in addition to humoral immunity. However, a delicate balance must be struck between the safety and immunogenicity of these “replicative” vaccines, and this balance is often only suitable for certain individuals. The EVs (e.g., exogenous vaccines) disclosed herein do not have such limitations. Therefore, in some aspects, the EVs (e.g., exogenous vaccines) disclosed herein (e.g., combinations of one or more influenza antigens with the payloads disclosed herein, such as STING agonists) can be used as “universal” vaccines against specific pathogens (e.g., different influenza subtypes).

[0185] In some respects, the EVs (e.g., exogens) disclosed herein are inherently capable of inducing activation of signaling pathways involved in immune responses. In some respects, signaling pathways involved in immune responses include toll-like receptors (TLRs), retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs), the interferon gene-stimulating factor (STING) pathway, or combinations thereof. In some respects, activation of such signaling pathways can lead to the production of type I interferon. For example, in some respects, the lipid bilayer membrane of the EVs (e.g., exogens) disclosed herein comprises one or more lipids sharing one of the following characteristics: (i) an unsaturated lipid tail, (ii) a dihydroimidazole linker, (iii) a cyclic amine head group, and (iv) combinations thereof. Lipids having such characteristics have been shown to activate the TLR / RLR-independent STING pathway. See Miao et al., Nature Biotechnology 37:1174-1185 (October 2019), which is incorporated herein by reference in its entirety.

[0186] II.A antigen

[0187] In some aspects, the payload is an antigen capable of inducing an immune response in a subject. In some aspects, the EVs (e.g., exosomes) disclosed herein contain a single antigen. In some aspects, the EVs (e.g., exosomes) disclosed herein contain multiple antigens. In some aspects, each of the multiple antigens is different. In some aspects, the EVs (e.g., exosomes) disclosed herein contain at least two, three, four, five, six, seven, eight, nine, ten, or more different antigens. As disclosed herein, scaffold portions (e.g., scaffold X and / or scaffold Y) can be used to attach antigens to the surface of the EV (e.g., exosome). In some aspects, antigens can be directly attached to the surface of the EV (e.g., exosome) (i.e., without using scaffold portions). In some aspects, antigens can be located within the lumen of the EV (e.g., exosome).

[0188] In some aspects, an EV (e.g., an exogenous body) comprises a combination of one or more antigens with one or more additional payloads (e.g., adjuvants and / or immunomodulators) as described herein. In some aspects, an EV (e.g., an exogenous body) may comprise one or more additional portions (e.g., targeting portions). For example, in some aspects, the EV (e.g., an exogenous body) disclosed herein may comprise (i) one or more additional antigens, (ii) one or more additional payloads (e.g., adjuvants and / or immunomodulators), and (iii) one or more targeting portions.

[0189] In some respects, antigens include tumor antigens. Non-limiting examples of tumor antigens include: alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), epithelial tumor antigen (ETA), mucin 1 (MUC1), Tn-MUC1, mucin 16 (MUC16), tyrosinase, melanoma-associated antigen (MAGE), tumor protein p53 (p53), CD4, CD8, CD45, CD80, CD86, programmed death-ligand 1 (PD-L1), programmed death-ligand 2 (PD-L2), NY-ESO-1, PSMA, TAG-72, HER2, GD2, cMET, EGFR, mesothelin, VEGFR, α-folate receptor, CE7R, IL-3, testicular cancer antigen (CTA), MART-1gp100, TNF-associated apoptosis-inducing ligand, Brachyury (an antigen preferentially expressed in melanoma (PRAME)), Wilms tumor 1 (WT1), CD19, CD22, or combinations thereof.

[0190] In some respects, the antigen is a universal tumor antigen. As used herein, the term "universal tumor antigen" refers to an immunogenic molecule (such as a protein) that is typically expressed in tumor cells at higher levels than in non-tumor cells and is also expressed in tumors of various origins. In some respects, universal tumor antigens are expressed in more than about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90% or more of cancers (e.g., human cancers). In some respects, universal tumor antigens may be expressed in non-tumor cells (e.g., normal cells), but at levels lower than those expressed in tumor cells. In some respects, the expression level of universal tumor antigens on tumor cells is about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10 or more times that on non-tumor cells. In some respects, universal tumor antigens are not expressed in normal cells and are expressed only in tumor cells. Non-limiting examples of general tumor antigens that may be used in this disclosure include endothelial lining antigens of the tumor vascular system, survivin, tumor protein D52 (TPD52), androgen receptor epitope, hepatic ligand A receptor 2 (EphA2), human telomerase reverse transcriptase (hTERT), survivin, mouse double minute 2-homolog (MDM2), cytochrome P450 1B1 (CYP1B), HER2 / neu, Wilms' tumor gene 1 (WT1), livin, alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), mucin 16 (MUC16), MUC1, prostate-specific membrane antigen (PSMA), p53, or cyclin (D1).

[0191] In another respect, antigens may include neoantigens. As used herein, the term "neoantigen" refers to an antigen encoded by a tumor-specific mutated gene.

[0192] In some respects, antigens originate from bacteria, viruses, fungi, protozoa, or any combination thereof. In some respects, antigens originate from oncogenic viruses (also referred to herein as cancer-associated viruses (CAVs)). In other respects, antigens originate from the following group: human gamma herpesvirus 4 (i.e., Epstein-Barr virus (EBV)), influenza A virus, influenza B virus, cytomegalovirus, Staphylococcus aureus, Mycobacterium tuberculosis, Chlamydia trachomatis, HIV (e.g., HIV-2), coronaviruses (e.g., COVID-19, MERS-CoV, and SARS). CoV), filamentous viruses (e.g., Marburg virus and Ebola virus), Streptococcus pyogenes, Streptococcus pneumoniae, Plasmodium species (e.g., Plasmodium vivax and Plasmodium falciparum), Chikungunya virus, human papillomavirus (HPV), hepatitis B virus (HBV), hepatitis C virus (HCV), human T-lymphotropic virus (HTLV1), human herpesvirus 8 (HHV8), Merkel cell polyomavirus (MCV), Bunyavirus (e.g., Hantavirus), arenavirus (e.g., LCMV virus and Lassa virus), flavivirus (e.g., dengue virus, Zika virus) Viruses, Japanese encephalitis virus, West Nile virus and yellow fever virus), enteroviruses (e.g. poliovirus), astroviruses (e.g. gastroenteritis virus), rhabdoviridae (e.g. rabies virus), Borrelia borborygius and Borrelia Mayo (e.g. Lyme disease), herpes simplex virus 2 (HSV-2), some Klebsiella spp., Pseudomonas aeruginosa, some Enterococcus spp., some Proteus spp., some Enterobacter spp., some Actinomyces spp., coagulase-negative Staphylococcus spp. (CoNS), some Mycoplasma spp., adenovirus, adeno-associated virus (AAV) or combinations thereof.

[0193] In some respects, the EBV-derived antigen is BZLF1. BZLF1 (also known as Zta or EB1) is an immediate early viral gene of EBV that can induce cancer and primarily infects B cells in 95% of the population. This gene (along with other genes) generates expression of other EBV genes at other stages of disease progression and is involved in converting the virus from its latent to its lytic form. ZEBRA (BamHI Z Epstein-Barr virus replication activator, also known as Zta and BZLF1) is an early lytic protein of EBV encoded by BZLF1. See Hartlage et al. (2015) Cancer Immunol. Res. 3(7):787-94 and Rist et al. (2015) J. Virology 70:703-12, both of which are incorporated herein by reference in their entirety. EVs (e.g., exogenous forms) containing EBV antigens such as BZLF1 disclosed herein may be used, for example, to treat post-transplant lymphoproliferative disease (PTLD). This EV can be administered to EBV-negative patients who have received EBV-positive grafts. BZLF1 is a dominant T-cell antigen associated with durable remission in patients with PTLD. The BZLF1-containing EVs (e.g., exogenous forms) disclosed herein can elicit potent CD8 T-cell-mediated immunity mediated by BZLF1. Therefore, mucosal immunity and tissue-resident memory cells (see figure) are also involved. Figure 7A and Figure 7B This can protect patients from developing PTLDF. Non-limiting exemplary antigens include, but are not limited to, those disclosed in U.S. Patent No. 8,617,564B2 (which is incorporated herein by reference in its entirety).

[0194] In some respects, the antigen is derived from Mycobacterium tuberculosis to induce cellular and / or humoral immune responses. In some respects, the antigen contains one or more epitopes of Mycobacterium tuberculosis (TB antigen). A variety of antigens are associated with Mycobacterium tuberculosis infection, including ESAT-6, TB10.4, CFP10, Rv2031 (hspX), Rv2654c (TB7.7), and Rv1038c (EsxJ). See, for example, Lindestam et al., J. Immunol. 188(10):5020-31 (2012), which is incorporated herein in its entirety. In some respects, the antigens used in this disclosure may contain one or more epitopes of ESAT6. In some respects, the antigens used in this disclosure may contain one or more epitopes of TB10.4. In some respects, the antigens used in this disclosure may contain one or more epitopes of CFP10. In some aspects, the antigens available for use in this disclosure may comprise one or more epitopes of Rv2031 (hspX). In some aspects, the antigens available for use in this disclosure may comprise one or more epitopes of Rv2654c (TB7.7). In some aspects, the antigens available for use in this disclosure may comprise one or more epitopes of Rv1038c (EsxJ). In some aspects, the antigens available for use in this disclosure may comprise epitopes selected from the group consisting of ESAT6, TB10.4 (ESAT-6-like protein EsxH; cfp7), CFP10, Rv2031 (hspX), Rv2654c (TB7.7), Rv1038c (EsxJ), and any combination thereof.

[0195] In some aspects, the TB antigen contains specific epitopes of the TB antigen, such as specific epitopes of ESAT6 or TB10.4. In some aspects, the ESAT6 antigen contains epitopes having at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, or at least fifteen amino acids of the amino acid sequence shown in MTEQQWNFAGIEAAASAIQGNVTSIHSLDEGKQSLTKLAAAWGGSGSEAYQGVQQKWDATATELNNALQNLARTISEAGQAMASTEGNVTGMFA (SEQ ID NO:370). In some aspects, the TB10.4 antigen contains epitopes having at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, or at least fifteen amino acids of the amino acid sequence shown in MSQIMYNYPAMLGHAGDMAGYAGTLQSLGAEIAVEQAALQSAWQGDTGITYQAWQAQWNQAMEDLVRAYHAMSSTHEANTMAMMARDTAEAAKWGG (SEQ ID NO: 371).

[0196] In some respects, antigens include self-antigens. As used herein, the term "self-antigen" refers to an antigen expressed by a host cell or tissue. Under normal health conditions, such antigens are recognized by the body as self and do not trigger an immune response. However, under certain disease conditions, the body's own immune system can recognize self-antigens as foreign substances and generate an immune response against them, resulting in autoimmunity. In some respects, the EVs (e.g., exogens) of this disclosure may contain self-antigens (i.e., self (germline) proteins that have induced a T-cell response and led to autoimmunity). Such EVs (e.g., exogens) can be used to target and inhibit the activity of self-reactive T cells. Non-limiting examples of autoantigens (including those associated with diseases or conditions) include: (i) β-cell proteins, insulin, islet antigen 2 (IA-2), glutamate decarboxylase (GAD65), and zinc transporter 8 (ZNT8) (type 1 diabetes), (ii) myelin oligodendrocyte glycoprotein (MOG), myelin basic protein (MBP), protein lipoprotein (PLP), and myelin-associated glycoprotein (MAG) (multiple sclerosis), (iii) citrullinated antigens and synovial proteins (rheumatoid arthritis), and (iv) aquaporin-4 (AQP4). (v) nicotinic acetylcholine receptor (nAChR) (myasthenia gravis), (vi) desmosome core protein-1 (DSG1) and desoglein-2 (DSG2) (pemphigus vulgaris), (v) thyroid-stimulating hormone receptor (Graves' disease), (vi) type IV collagen (Goodpasture syndrome), (vii) thyroglobulin, thyroid peroxidase and thyroid-stimulating hormone receptor (TSHR) (Hashimoto's thyroiditis), or (viii) a combination thereof.

[0197] II.B Adjuvants

[0198] As described above, the EV (e.g., exosome) of this disclosure may contain adjuvants (e.g., in combination with antigens and / or other payloads disclosed herein). In some aspects, the EV (e.g., exosome) disclosed herein contains multiple adjuvants. In some aspects, each of the multiple adjuvants is different. In some aspects, the EV (e.g., exosome) disclosed herein contains at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different adjuvants. As disclosed herein, adjuvants can be attached to the surface of the EV (e.g., exosome) using a scaffold portion (e.g., scaffold X and / or scaffold Y). In some aspects, adjuvants can be attached directly (i.e., without using a scaffold portion) to the surface of the EV (e.g., exosome). In some aspects, adjuvants can be intracavitary in the lumen of the EV (e.g., exosome).

[0199] In some aspects, an EV (e.g., an exogenous body) comprises a combination of one or more adjuvants and one or more additional payloads (e.g., antigens and / or immunomodulators). In some aspects, an EV (e.g., an exogenous body) may comprise one or more additional portions (e.g., targeting portions). For example, in some aspects, the EV (e.g., an exogenous body) disclosed herein may comprise (i) one or more additional adjuvants, (ii) one or more additional payloads (e.g., antigens and / or immunomodulators), and (iii) one or more targeting portions.

[0200] As used herein, the term "adjuvant" refers to any substance that enhances the therapeutic effect of a payload (e.g., enhances the immune response to an antigen). Therefore, the adjuvant-containing EVs (e.g., exogenous bodies) described herein, compared to references (e.g., corresponding EVs without adjuvants or non-EV delivery media containing antigens alone or in combination with adjuvants), are capable of enhancing, for example, the immune response against an antigen by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 250%, at least about 500%, at least about 750%, at least about 1,000%, or more. In some respects, incorporating the adjuvants disclosed herein into an EV (e.g., an exogenous body) compared to a reference (e.g., a corresponding EV containing a single antigen or a non-EV delivery medium containing a single antigen or in combination with an adjuvant) can enhance, for example, an immune response against the antigen by at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 20-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 60-fold, at least about 70-fold, At least approximately 80 times, at least approximately 90 times, at least approximately 100 times, at least approximately 200 times, at least approximately 300 times, at least approximately 400 times, at least approximately 500 times, at least approximately 600 times, at least approximately 700 times, at least approximately 800 times, at least approximately 900 times, at least approximately 1,000 times, at least approximately 2,000 times, at least approximately 3,000 times, at least approximately 4,000 times, at least approximately 5,000 times, at least approximately 6,000 times, at least approximately 7,000 times, at least approximately 8,000 times, at least approximately 9,000 times, at least approximately 10,000 times or more.

[0201] Non-limiting examples of adjuvants that may be used in this disclosure include: interferon gene stimulator (STING) agonists, toll-like receptor (TLR) agonists, inflammatory mediators, RIG-I agonists, α-gal-cer (NKT agonists), heat shock proteins (e.g., HSP65 and HSP70), C-type lectin agonists (e.g., β-glucan (Dectin 1), chitosan and gel polysaccharides) and combinations thereof.

[0202] In some respects, incorporating adjuvants (such as those disclosed herein) into EVs (e.g., exogenous bodies) can broaden EV-induced immune responses. As used herein, “broadening the immune response” means enhancing the diversity of the immune response. In some respects, the diversity of the immune response can be enhanced by epitope diffusion (i.e., inducing and / or enhancing immune responses (cellular and / or humoral immune responses) against a greater number / type of epitopes on the antigen). In some respects, the diversity of the immune response can be increased by generating different and / or multiple antibody isotypes (e.g., IgG, IgA, IgD, IgM, and / or IgE).

[0203] In some respects, adjuvants (e.g., those disclosed herein) can also help modulate the type of immune response induced by EVs (e.g., exogenous bodies). For example, in some respects, incorporating adjuvants into EVs (e.g., exogenous bodies) can help drive the immune response toward a more Th1 phenotype. As used herein, a “Th1” immune response is typically characterized by the production of IFN-γ, which can activate the bactericidal activity of innate cells (e.g., macrophages), help induce B cells to produce opsonizing (labeled for phagocytosis) and complement-binding antibodies, and / or lead to cell-mediated immunity (i.e., not antibody-mediated). Generally, Th1 responses are more effective against intracellular pathogens (viruses and bacteria within host cells) and / or cancer.

[0204] In some respects, incorporating adjuvants into EVs (e.g., exogenous bodies) can help drive the immune response toward a more Th2 phenotype. As used herein, a “Th2” immune response can be characterized by the release of certain cytokines, such as IL-5 (which induces eosinophil clearance of parasites) and IL-4 (which promotes B cell allotype conversion). Generally, Th2 responses are more effective against extracellular bacteria, parasites (including worms), and toxins.

[0205] In some respects, incorporating adjuvants into EVs (e.g., exogenous forms) can help drive the immune response toward a more Th17 phenotype. As used herein, the “Th17” immune response is mediated by Th17 cells. As used herein, “Th17 cells” refers to a subset of CD4+ T cells characterized by the production of pro-inflammatory cytokines such as IL-17A, IL-17F, IL-21, IL-22, and granulocyte-macrophage colony-stimulating factor (GM-CSF). Th17 cells are generally considered to play an important role in host defense against infection by recruiting neutrophils and macrophages to infected tissues.

[0206] In some respects, incorporating adjuvants into EVs (e.g., exogenous bodies) can help drive the immune response towards a more cellular immune response (e.g., T cell-mediated). In other respects, incorporating adjuvants into EVs (e.g., exogenous bodies) can help drive the immune response towards a more humoral immune response (e.g., antibody-mediated).

[0207] In some respects, adjuvants induce activation of cytosol pattern recognition receptors. Non-limiting examples of cytosol pattern recognition receptors include: interferon gene stimulators (STING), retinoic acid-inducible gene I (RIG-1), melanoma differentiation-associated protein 5 (MDA5), nucleotide-binding oligomerization domains, leucine-rich repeat-containing and Pyrin domains (NLRP), inflammasomes, or combinations thereof. In some respects, adjuvants are STING agonists. Interferon gene stimulators (STING) are cytosol sensors of cyclic dinucleotides typically produced by bacteria. Upon activation, they lead to the production of type I interferons (e.g., IFN-α (alpha), IFN-β (beta), IFN-κ (kappa), IFN-δ (delta), IFN-ε (epsilon), IFN-τ (tau), IFN-ω (omega), and IFN-ζ (zeta, also known as limitin)) and initiate an immune response. In some respects, STING agonists include cyclic dinucleotide STING agonists or acyclic dinucleotide STING agonists. As described herein, in some respects, STING agonists are loaded in the cavity of an EV (e.g., an exosome). In some respects, such an EV (e.g., an exosome) is referred to herein as “exoSTING”. Non-limiting examples of exoSTING are provided in International Publication No. WO2019183578A1 (which is incorporated herein by reference in its entirety). Further disclosures of useful STING agonists are also provided throughout this disclosure.

[0208] Cyclic purine dinucleotides, such as, but not limited to, cGMP, cyclic di-GMP (c-di-GMP), cAMP, cyclic di-AMP (c-di-AMP), cyclic di-GMP-AMP (cGAMP), cyclic di-IMP (c-di-IMP), cyclic AMP-IMP (cAIMP), and any analogues thereof, are known to stimulate or enhance an immune or inflammatory response in patients. CDNs may have 2'2', 2'3', 2'5', 3'3', or 3'5' bonds linking the cyclic dinucleotides, or any combination thereof.

[0209] Cyclic purine dinucleotides can be modified using standard organic chemistry techniques to produce purine dinucleotide analogs. Suitable purine dinucleotides include, but are not limited to, adenine, guanine, inosine, hypoxanthine, xanthine, isoguanine, or any other suitable purine dinucleotide known in the art. Cyclic dinucleotides can be modified analogs. Any suitable modifications known in the art can be used, including but not limited to thiophosphates, biphosphorothioates, fluorination, and difluorination.

[0210] Noncyclic dinucleotide agonists, such as 5,6-dimethylxanthonone-4-acetic acid (DMXAA), or any other noncyclic dinucleotide agonist known in the art, may also be used.

[0211] Non-limiting examples of STING agonists that can be used in this disclosure include: DMXAA, STING agonist-1, MLRR-S2 CDA, MLRR-S2c-bis-GMP, ML-RR-S2cGAMP, 2'3'-c-bis-AM(PS)2, 2'3'-cGAMP, 2'3'-cGAMPdFHS, 3'3'-cGAMP, 3'3'-cGAMPdFSH, cAIMP, cAIM(PS)2, 3'3'-cAIMP, 3'3'-cAIMPdFSH, 2'2'-cGAMP, 2'3'-cGAM(PS)2, 3'3'-cGAMP, and combinations thereof. Non-limiting examples of STING agonists can be found in U.S. Patent Nos. 9,695,212, 2014 / 189805 A1, 2014 / 179335 A1, 2018 / 100558 A1, 10,011,630B2, 2017 / 027646 A1, 2017 / 161349 A1 and 2016 / 096174 A1 (each of which is incorporated herein by reference in its entirety).

[0212] In some respects, the STING agonists available in this disclosure comprise compounds having the following formula:

[0213]

[0214] in:

[0215] X1 is H, OH, or F;

[0216] X2 is H, OH, or F;

[0217] Z can be OH, OR1, SH, or SR1, where:

[0218] i) R1 is Na or NH4, or

[0219] ii) R1 is an enzyme-unstable group that provides OH or SH in vivo, such as neopentyloxymethyl;

[0220] Bi and B2 are selected from the following bases:

[0221]

[0222] The conditions are:

[0223] - In equation (I): X1 and X2 are not OH,

[0224] - In formula (II): when X1 and X2 are OH, B1 is not adenine and B2 is not guanine, and

[0225] - In equation (III): when X1 and X2 are OH, B1 is not adenine, B2 is not guanine, and Z is not OH. See WO 2016 / 096174, the contents of which are incorporated herein by reference in their entirety.

[0226] In some respects, STING agonists that can be used in this disclosure include:

[0227]

[0228]

[0229] Its pharmaceutically acceptable salt. See WO 2016 / 096174 A1, which is incorporated herein by reference in its entirety.

[0230] In other respects, STING agonists that can be used in this disclosure include compounds having the following formula:

[0231]

[0232]

[0233] Or any pharmaceutically acceptable salt thereof.

[0234] In some respects, STING agonists that can be used in this disclosure include compounds having the following formula:

[0235]

[0236] Each of these symbols is defined in WO 2014 / 093936 (the contents of which are incorporated herein by reference in their entirety).

[0237] In some respects, STING agonists that can be used in this disclosure include compounds having the following formula:

[0238]

[0239] Each of these symbols is defined in WO 2014 / 189805 (the contents of which are incorporated herein by reference in their entirety).

[0240] In some respects, STING agonists that can be used in this disclosure include compounds having the following formula:

[0241]

[0242] Each of these symbols is defined in WO 2015 / 077354 (the contents of which are incorporated herein by reference in their entirety). See also Cell reports 11, 1018-1030 (2015), which is incorporated herein by reference in its entirety.

[0243] In some respects, STING agonists that can be used in this disclosure include c-di-AMP, c-di-GMP, c-di-IMP, c-AMP-GMP, c-AMP-IMP, and c-GMP-IMP, as described in WO 2013 / 185052 and Sci.Transl.Med.283,283ra52 (2015) (the aforementioned documents are incorporated herein by reference in their entirety).

[0244] In some respects, STING agonists that can be used in this disclosure include compounds having the following formula:

[0245]

[0246] Each of these symbols is defined in WO 2014 / 189806 (the contents of which are incorporated herein by reference in their entirety).

[0247] In some respects, STING agonists that can be used in this disclosure include compounds having the following formula:

[0248]

[0249] Each of these symbols is defined in WO 2015 / 185565 (the contents of which are incorporated herein by reference in their entirety).

[0250] In some respects, STING agonists that can be used in this disclosure include compounds having the following formula:

[0251]

[0252] Each of these symbols is defined in WO 2014 / 179760 (the contents of which are incorporated herein by reference in their entirety).

[0253] In some respects, STING agonists that can be used in this disclosure include compounds having the following formula:

[0254]

[0255]

[0256] Each of these symbols is defined in WO 2014 / 179335 (the contents of which are incorporated herein by reference in their entirety).

[0257] In some respects, STING agonists that can be used in this disclosure include compounds having the following formula:

[0258]

[0259] As described in WO 2015 / 017652, its contents are incorporated herein by reference in their entirety.

[0260] In some respects, STING agonists that can be used in this disclosure include compounds having the following formula:

[0261]

[0262] As stated in WO 2016 / 096577 (the contents of which are incorporated herein by reference in their entirety).

[0263] In some respects, STING agonists that can be used in this disclosure include compounds having the following formula:

[0264]

[0265] Each of these symbols is defined in WO 2016 / 120305 (the contents of which are incorporated herein by reference in their entirety).

[0266] In some respects, STING agonists that can be used in this disclosure include compounds having the following formula:

[0267]

[0268] Each of these symbols is defined in WO 2016 / 145102 (the contents of which are incorporated herein by reference in their entirety).

[0269] In some respects, STING agonists that can be used in this disclosure include compounds having the following formula:

[0270]

[0271] Each of these symbols is defined in WO 2017 / 027646 (the contents of which are incorporated herein by reference in their entirety).

[0272] In some respects, STING agonists that can be used in this disclosure include compounds having the following formula:

[0273]

[0274] Each of these symbols is defined in WO 2017 / 075477 (the contents of which are incorporated herein by reference in their entirety).

[0275] In some respects, STING agonists that can be used in this disclosure include compounds having the following formula:

[0276]

[0277] Each of these symbols is defined in WO 2017 / 027645 (the contents of which are incorporated herein by reference in their entirety).

[0278] In some respects, STING agonists that can be used in this disclosure include compounds having the following formula:

[0279]

[0280] Each of these symbols is defined in WO 2018 / 100558 (the contents of which are incorporated herein by reference in their entirety).

[0281] In some respects, STING agonists that can be used in this disclosure include compounds having the following formula:

[0282]

[0283] Each of these symbols is defined in WO 2017 / 175147 (the contents of which are incorporated herein by reference in their entirety).

[0284] In some respects, STING agonists that can be used in this disclosure include compounds having the following formula:

[0285]

[0286] Each of these symbols is defined in WO 2017 / 175156 (the contents of which are incorporated herein by reference in their entirety).

[0287] In some aspects, the STING agonists available for use in this disclosure are CL606, CL611, CL602, CL655, CL604, CL609, CL614, CL656, CL647, CL626, CL629, CL603, CL632, CL633, CL659, or pharmaceutically acceptable salts thereof. In some aspects, the STING agonists available for use in this disclosure are CL606 or pharmaceutically acceptable salts thereof. In some aspects, the STING agonists available for use in this disclosure are CL611 or pharmaceutically acceptable salts thereof. In some aspects, the STING agonists available for use in this disclosure are CL602 or pharmaceutically acceptable salts thereof. In some aspects, the STING agonists available for use in this disclosure are CL655 or pharmaceutically acceptable salts thereof. In some aspects, the STING agonists available for use in this disclosure are CL604 or pharmaceutically acceptable salts thereof. In some aspects, the STING agonists available for use in this disclosure are CL609 or pharmaceutically acceptable salts thereof. In some aspects, the STING agonist available for use in this disclosure is CL614 or a pharmaceutically acceptable salt thereof. In some aspects, the STING agonist available for use in this disclosure is CL656 or a pharmaceutically acceptable salt thereof. In some aspects, the STING agonist available for use in this disclosure is CL647 or a pharmaceutically acceptable salt thereof. In some aspects, the STING agonist available for use in this disclosure is CL626 or a pharmaceutically acceptable salt thereof. In some aspects, the STING agonist available for use in this disclosure is CL629 or a pharmaceutically acceptable salt thereof. In some aspects, the STING agonist available for use in this disclosure is CL603 or a pharmaceutically acceptable salt thereof. In some aspects, the STING agonist available for use in this disclosure is CL632 or a pharmaceutically acceptable salt thereof. In some aspects, the STING agonist available for use in this disclosure is CL633 or a pharmaceutically acceptable salt thereof. In some aspects, the STING agonist available for use in this disclosure is CL659 or a pharmaceutically acceptable salt thereof.

[0288] In some aspects, EVs (e.g., exogenous bodies) comprise cyclic dinucleotide STING agonists and / or acyclic dinucleotide STING agonists. In some aspects, when several cyclic dinucleotide STING agonists are present on the EVs (e.g., exogenous bodies) disclosed herein, such STING agonists may be the same or they may be different. In some aspects, when several acyclic dinucleotide STING agonists are present, such STING agonists may be the same or they may be different. In some aspects, the EV (e.g., exogenous body) compositions of this disclosure may comprise two or more EV (e.g., exogenous body) groups, wherein each EV (e.g., exogenous body) group comprises a different STING agonist or a combination thereof.

[0289] STING agonists can also be modified to increase the encapsulation (e.g., loading) of the agonist in extracellular vesicles or EVs (e.g., or not bound in the lumen). In some aspects, the STING agonist is attached to a scaffold portion (e.g., scaffold Y). In some aspects, the modification allows for better expression of the STING agonist on the outer surface of the EV (e.g., exosome) (e.g., attachment to a scaffold portion disclosed herein (e.g., scaffold X)). Such modification may include adding a lipid-binding tag by treating the agonist with a chemical or enzyme, or by physically or chemically altering the polarity or charge of the STING agonist. The STING agonist may be modified by a single treatment or a combination of treatments, such as adding only a lipid-binding tag, or adding a lipid-binding tag and altering the polarity. The foregoing examples are intended to be non-limiting and illustrative. Any combination of modifications is contemplated for practice. The modification may increase the encapsulation (i.e., loading) of the agonist in an EV (e.g., exogenous) by about 2 to about 10,000 times, about 10 to about 1,000 times, or about 100 to about 500 times compared to the encapsulation (i.e., loading) of an unmodified agonist. The modification may increase the encapsulation (i.e., loading) of the agonist in an EV by at least about 2, at least about 5, at least about 10, at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 100, at least about 200, at least about 30 ... 400 times, at least about 500 times, at least about 600 times, at least about 700 times, at least about 800 times, at least about 900 times, at least about 1,000 times, at least about 2,000 times, at least about 3,000 times, at least about 4,000 times, at least about 5,000 times, at least about 6,000 times, at least about 7,000 times, at least about 8,000 times, at least about 9,000 times, or at least about 10,000 times.

[0290] In some respects, the STING agonist can be modified to allow better expression of the agonist on the EV surface (e.g., the outer surface and / or cavity surface of the EV (e.g., connected to the stent portion disclosed herein (e.g., stent X and / or stent Y))). Any of the modifications described above can be used. Such modifications can increase the expression of the agonist in the EV (e.g., on the surface of the exogenous body and / or cavity surface) by about 2 to 10,000 times, about 10 to 1,000 times, or about 100 to 500 times compared to the corresponding expression of the unmodified agonist. The modification can increase the expression of the agonist on the outer surface of an EV (e.g., an exogenous organism) by at least about 2, at least about 5, at least about 10, at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 100, at least about 200, at least about 300, at least about 400 times, at least about 500 times, at least about 600 times, at least about 700 times, at least about 800 times, at least about 900 times, at least about 1,000 times, at least about 2,000 times, at least about 3,000 times, at least about 4,000 times, at least about 5,000 times, at least about 6,000 times, at least about 7,000 times, at least about 8,000 times, at least about 9,000 times, or at least about 10,000 times. The modification can increase the expression of the agonist on the luminal surface of an EV (e.g., an exogenous organism) by at least about 2, at least about 5, at least about 10, at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 100, at least about 200, at least about 300, at least about 400 times, at least about 500 times, at least about 600 times, at least about 700 times, at least about 800 times, at least about 900 times, at least about 1,000 times, at least about 2,000 times, at least about 3,000 times, at least about 4,000 times, at least about 5,000 times, at least about 6,000 times, at least about 7,000 times, at least about 8,000 times, at least about 9,000 times, or at least about 10,000 times.

[0291] The concentration of the STING agonist associated with EVs (e.g., exogenous entities) can be from about 0.01 μM to about 1000 μM. The concentrations of the associated STING agonist can be from about 0.01-0.05 μM, about 0.05-0.1 μM, about 0.1-0.5 μM, about 0.5-1 μM, about 1-5 μM, about 5-10 μM, about 10-15 μM, about 15-20 μM, about 20-25 μM, about 25-30 μM, about 30-35 μM, about 35-40 μM, about 45-50 μM, about 55-60 μM, about 65-70 μM, about 70-75 μM, about 75-80 μM, about 80-85 μM, about 85-90 μM, about 90-95 μM, about ... -100μM, approximately 100-150μM, approximately 150-200μM, approximately 200-250μM, approximately 250-300μM, approximately 300-350μM, approximately 250-400μM, approximately 400-450μM, approximately 450-500μM, approximately 500-550μM, approximately 550-600μM, approximately 600-650μM, approximately 650-700μM, approximately 700-750μM, approximately 750-800μM, approximately 800-850μM, approximately 805-900μM, approximately 900-950μM, or approximately 950-1000μM. The concentration of the associated STING agonist can be equal to or greater than approximately 0.01 μM, approximately 0.1 μM, approximately 0.5 μM, approximately 1 μM, approximately 5 μM, approximately 10 μM, approximately 15 μM, approximately 20 μM, approximately 25 μM, approximately 30 μM, approximately 35 μM, approximately 40 μM, approximately 45 μM, approximately 50 μM, approximately 55 μM, approximately 60 μM, approximately 65 μM, approximately 70 μM, approximately 75 μM, approximately 80 μM, and approximately 85 μM. Approximately 90 μM, approximately 95 μM, approximately 100 μM, approximately 150 μM, approximately 200 μM, approximately 250 μM, approximately 300 μM, approximately 350 μM, approximately 400 μM, approximately 450 μM, approximately 500 μM, approximately 550 μM, approximately 600 μM, approximately 650 μM, approximately 700 μM, approximately 750 μM, approximately 800 μM, approximately 850 μM, approximately 900 μM, approximately 950 μM, or approximately 1,000 μM.

[0292] In some respects, the adjuvant is a TLR agonist. Non-limiting examples of TLR agonists include: TLR2 agonists (e.g., lipoteichoic acid, atypical LPS, MALP-2 and MALP-404, OspA, porin, LcrV, lipomannan, GPI anchor, lysophosphatidylserine, lipophosphatidylglycerol (LPG), glycophosphatidylinositol (GPI), yeast polysaccharide, hsp60, gH / gL glycoprotein, hemagglutinin), TLR3 agonists (e.g., double-stranded RNA, such as poly(I:C)), TLR4 agonists (e.g., lipopolysaccharide (LPS)), lipoteichoic acid, β-defensin 2, fibronectin EDA, HMGB1, snapin, tendonin C, TLR5 agonists (e.g., flagellin), TLR6 agonists, TLR7 / 8 agonists (e.g., single-stranded RNA, CpG-A, Poly G10, Poly G3, requimomod), and TLR9 agonists (e.g., unmethylated CpG). DNA) and combinations thereof. Non-limiting examples of TLR agonists can be found in WO2008115319A2, US20130202707A1, US20120219615A1, US20100029585A1, WO2009030996A1, WO2009088401A2 and WO2011044246A1 (each of which is incorporated herein by reference in its entirety).

[0293] In some respects, adjuvants are inflammatory mediators.

[0294] In some respects, the antigen is expressed on the outer surface of the EV (e.g., exogenous body) or within the lumen (e.g., on the lumen surface). In some respects, the adjuvant is expressed on the outer surface of the EV (e.g., exogenous body) or in the lumen surface, directly linked to the lipid bilayer. In these respects, the antigen and / or adjuvant may be linked to a scaffold portion (e.g., scaffold X and / or scaffold Y).

[0295] In some aspects, the EV (e.g., exogenous body) described herein includes a first stent portion. In some aspects, an antigen is attached to the first stent portion. In other aspects, an adjuvant is attached to the first stent portion. In still other aspects, both the antigen and the adjuvant are attached to the first stent portion. In some aspects, the EV (e.g., exogenous body) also includes a second stent portion. In some aspects, the antigen is attached to the first stent portion, and the adjuvant is attached to the second stent portion. In some aspects, the first stent portion and the second stent portion are identical (e.g., both are stent X or both are stent Y). In other aspects, the first stent portion and the second stent portion are different (e.g., the first stent portion is stent X, and the second stent portion is stent Y; or the first stent portion is stent Y, and the second stent portion is stent X).

[0296] Non-limiting examples of scaffold X include: prostaglandin F2 receptor negative regulator (PTGFRN); basigin (BSG); immunoglobulin superfamily member 2 (IGSF2); immunoglobulin superfamily member 3 (IGSF3); immunoglobulin superfamily member 8 (IGSF8); integrin β-1 (ITGB1); integrin α-4 (ITGA4); 4F2 cell surface antigen heavy chain (SLC3A2); and a class of ATP transporters (ATP1A1, ATP1A2, ATP1A3, ATP1A4, ATP1B3, ATP2B1, ATP2B2, ATP2B3, ATP2B). In some respects, scaffold X is a complete protein. In other respects, scaffold X is a protein fragment (e.g., a functional fragment).

[0297] In other respects, the scaffold portions of this disclosure, the first scaffold portion, the second scaffold portion, and / or the third scaffold portion, may include conventional exogenous proteins, including but not limited to tetraspanin molecules (e.g., CD63, CD81, CD9, etc.), lysosomal associated membrane proteins 2 (LAMP2 and LAMP2B), platelet-derived growth factor receptor (PDGFR), GPI anchoring, lactoglucosins and fragments thereof, peptides with affinity for any of these proteins or fragments thereof, or any combination thereof.

[0298] Non-limiting examples of scaffold Y include: myristyl alanine-rich protein kinase C substrate (MARCKS) protein; myristyl alanine-rich protein kinase C substrate-like protein 1 (MARCKSL1) protein; and brain acid-soluble protein 1 (BASP1) protein. In some aspects, scaffold Y is a complete protein. In other aspects, scaffold Y is a protein fragment (e.g., a functional fragment).

[0299] In some aspects, the antigen is attached to a first scaffold portion on the luminal surface of the EV (e.g., exosome), and the adjuvant is within the lumen of the EV (e.g., exosome). As used herein, when a molecule (e.g., antigen or adjuvant) is described as being "within the lumen" of, for example, an EV (e.g., exosome), this means that the molecule is not attached to the scaffold portion described herein. In some aspects, the antigen is within the lumen of the EV (e.g., exosome), and the adjuvant is attached to a first scaffold portion on the luminal surface of the EV (e.g., exosome). In these aspects, the first scaffold portion may be scaffold X or scaffold Y.

[0300] In some aspects, the antigen is attached to a first stent portion on the luminal surface of the EV (e.g., an exogenous organism), and the adjuvant is attached to a second stent portion on the outer surface of the EV (e.g., an exogenous organism). In other aspects, the adjuvant is attached to a first stent portion on the luminal surface of the EV (e.g., an exogenous organism), and the antigen is attached to a second stent portion on the outer surface of the EV (e.g., an exogenous organism). In these aspects, the first stent portion may be stent Y, and the second stent portion may be stent X. In other aspects, each of the first and second stent portions may be stent X.

[0301] In some aspects, the antigen is attached to a first scaffold portion on the outer surface of the EV (e.g., an exogenous organism), and the adjuvant is attached to a second scaffold portion on the luminal surface of the EV (e.g., an exogenous organism). In other aspects, the adjuvant is attached to a first scaffold portion on the outer surface of the EV (e.g., an exogenous organism), and the antigen is attached to a second scaffold portion on the luminal surface of the EV (e.g., an exogenous organism). In these aspects, the first scaffold portion is scaffold X, and the second scaffold portion is scaffold Y; or each of the first and second scaffold portions is scaffold X.

[0302] In some respects, the antigen is inside the lumen of the EV (e.g., exogenous body), and the adjuvant is inside the lumen of the EV (e.g., exogenous body).

[0303] In some aspects, the antigen is attached to a first scaffold portion on the outer surface of the EV (e.g., exogenous organism), and the adjuvant is attached to a second scaffold portion on the outer surface of the EV (e.g., exogenous organism). In other aspects, the adjuvant is attached to a first scaffold portion on the outer surface of the EV (e.g., exogenous organism), and the antigen is attached to a second scaffold portion on the outer surface of the EV (e.g., exogenous organism). In some aspects, both the first and second scaffold portions are scaffold X.

[0304] In some aspects, the antigen is attached to a first scaffold portion on the outer surface of the EV (e.g., exogenous body), and the adjuvant is within the lumen of the EV (e.g., exogenous body). In some aspects, the antigen is within the lumen of the EV (e.g., exogenous body), and the adjuvant is attached to a first scaffold portion on the outer surface of the EV (e.g., exogenous body). In these aspects, the first scaffold portion may be scaffold X.

[0305] In some aspects, the antigen is attached to a first scaffold portion on the outer surface of the EV (e.g., an exogenous organism), and the adjuvant is attached to a first scaffold portion on the luminal surface of the EV (e.g., an exogenous organism). In other aspects, the antigen is attached to a first scaffold portion on the luminal surface of the EV (e.g., an exogenous organism), and the adjuvant is attached to a first scaffold portion on the outer surface of the EV (e.g., an exogenous organism). In these aspects, the first scaffold portion may be scaffold X.

[0306] Non-limiting examples of specific aspects include EVs containing (i) an antigen and (ii) an adjuvant, such as exogenous bodies, wherein:

[0307] (a) The antigen is attached to a first scaffold Y on the luminal surface of the EV (e.g., exogenous body), and the adjuvant is attached to a second scaffold Y on the luminal surface of the EV (e.g., exogenous body).

[0308] (b) The antigen is attached to a scaffold Y on the luminal surface of the EV (e.g., exogenous body), and the adjuvant is inside the lumen of the EV (e.g., exogenous body) and is not attached to any scaffold portion.

[0309] (c) The antigen is within the lumen of the EV (e.g., exogenous body) and is not attached to any scaffold portion, and the adjuvant is attached to the scaffold Y on the lumen surface of the EV (e.g., exogenous body).

[0310] (d) The antigen is attached to a scaffold Y on the luminal surface of the EV (e.g., exogenous body), and the adjuvant is attached to a scaffold X on the outer surface of the EV (e.g., exogenous body).

[0311] (e) The antigen is within the lumen of the EV (e.g., exogenous body) and is not attached to any scaffold portion, and the adjuvant is attached to the scaffold X on the outer surface of the EV (e.g., exogenous body);

[0312] (f) The antigen is attached to a scaffold Y on the luminal surface of the EV (e.g., exogenous body), and the adjuvant is attached to a scaffold X on the luminal surface of the EV (e.g., exogenous body).

[0313] (g) The antigen is located within the lumen of the EV (e.g., exogenous body) and is not attached to any scaffold portion, and the adjuvant is attached to scaffold X on the lumen surface of the EV (e.g., exogenous body).

[0314] (h) The antigen is attached to a scaffold X on the luminal surface of the EV (e.g., exogenous body), and the adjuvant is attached to a scaffold X on the outer surface of the EV (e.g., exogenous body).

[0315] (i) The antigen is attached to a first scaffold X on the outer surface of the EV (e.g., exogenous body), and the adjuvant is attached to a second scaffold X on the outer surface of the EV (e.g., exogenous body);

[0316] (j) The antigen is attached to a scaffold X on the outer surface of the EV (e.g., exogenous body), and the adjuvant is attached to a scaffold Y on the luminal surface of the EV (e.g., exogenous body).

[0317] (k) The antigen is attached to a scaffold X on the outer surface of the EV (e.g., exogenous body), and the adjuvant is located within the lumen of the EV (e.g., exogenous body) and is not attached to any part of the scaffold.

[0318] (l) The antigen is attached to a scaffold X on the outer surface of the EV (e.g., exogenous body), and the adjuvant is attached to a scaffold X on the luminal surface of the EV (e.g., exogenous body).

[0319] (m) The antigen is attached to a first scaffold X on the luminal surface of the EV (e.g., exogenous body), and the adjuvant is attached to a second scaffold X on the luminal surface of the EV (e.g., exogenous body).

[0320] (n) The antigen is attached to a scaffold X on the luminal surface of the EV (e.g., exogenous body), and the adjuvant is attached to a scaffold Y on the luminal surface of the EV (e.g., exogenous body).

[0321] (o) The antigen is attached to a scaffold X on the luminal surface of the EV (e.g., exogenous body), and the adjuvant is inside the lumen of the EV (e.g., exogenous body) and is not attached to any part of the scaffold.

[0322] (p) The antigen is attached to a first scaffold X on the outer surface of the EV (e.g., exogenous body), and the adjuvant is attached to a second scaffold X on the luminal surface of the EV (e.g., exogenous body).

[0323] (q) The antigen is attached to a first scaffold X on the luminal surface of the EV (e.g., exogenous body), and the adjuvant is attached to a second scaffold X on the outer surface of the EV (e.g., exogenous body);

[0324] (r) The antigen is within the lumen of the EV (e.g., exogenous body) and is not attached to any scaffold portion, and the adjuvant is within the lumen of the EV (e.g., exogenous body) and is not attached to any scaffold portion;

[0325] (s) The antigen is directly attached to the luminal surface of the EV (e.g., exogenous body), and the adjuvant is directly attached to the luminal surface of the EV (e.g., exogenous body);

[0326] (t) The antigen is directly attached to the luminal surface of the EV (e.g., exogenous body), and the adjuvant is inside the lumen of the EV (e.g., exogenous body);

[0327] (u) The antigen is directly attached to the luminal surface of the EV (e.g., exogenous body), and the adjuvant is attached to a scaffold Y on the luminal surface of the EV (e.g., exogenous body);

[0328] (v) The antigen is directly attached to the luminal surface of the EV (e.g., exogenous body), and the adjuvant is attached to a scaffold X on the luminal surface of the EV (e.g., exogenous body).

[0329] (w) The antigen is directly attached to the luminal surface of the EV (e.g., exogenous body), and the adjuvant is directly attached to the exterior of the EV (e.g., exogenous body);

[0330] (x) The antigen is directly attached to the luminal surface of the EV (e.g., exogenous body), and the adjuvant is attached to a scaffold X on the exterior of the EV (e.g., exogenous body);

[0331] (y) The antigen is attached to a scaffold Y on the luminal surface of the EV (e.g., exogenous body), and the adjuvant is attached directly to the luminal surface of the EV (e.g., exogenous body).

[0332] (z) The antigen is attached to a scaffold Y on the luminal surface of the EV (e.g., exogenous body), and the adjuvant is attached directly to the exterior of the EV (e.g., exogenous body);

[0333] (aa) The antigen is attached to a scaffold X on the luminal surface of the EV (e.g., exogenous body), and the adjuvant is attached directly to the luminal surface of the EV (e.g., exogenous body).

[0334] (bb) The antigen is attached to a scaffold X on the luminal surface of the EV (e.g., exogenous body), and the adjuvant is attached directly to the exterior of the EV (e.g., exogenous body).

[0335] (cc) The antigen is located within the lumen of the EV (e.g., exogenous organism), and the adjuvant is directly attached to the luminal surface of the EV (e.g., exogenous organism); or

[0336] (dd) The antigen is inside the lumen of the EV (e.g., exogenous body), and the adjuvant is directly attached to the outside of the EV (e.g., exogenous body).

[0337] In some aspects, the EV (e.g., exogenous body) of this disclosure comprises (i) an antigen and (ii) an adjuvant, wherein the antigen is directly connected to a first stent Y on the luminal surface of the EV (e.g., exogenous body), and the adjuvant is connected to a second stent Y on the luminal surface of the EV (e.g., exogenous body). In some aspects, the EV (e.g., exogenous body) of this disclosure comprises (i) an antigen and (ii) an adjuvant, wherein the antigen is connected to a stent Y on the luminal surface of the EV (e.g., exogenous body), and the adjuvant is within the lumen of the EV (e.g., exogenous body) and is not connected to any stent portion. In some aspects, the EV (e.g., exogenous body) of this disclosure comprises (i) an antigen and (ii) an adjuvant, wherein the antigen is within the lumen of the EV (e.g., exogenous body) and is not connected to any stent portion, and the adjuvant is connected to a stent Y on the luminal surface of the EV (e.g., exogenous body). In some aspects, the EV (e.g., exogenous body) of this disclosure comprises (i) an antigen and (ii) an adjuvant, wherein the antigen is attached to a scaffold Y on the luminal surface of the EV (e.g., exogenous body), and the adjuvant is attached to a scaffold X on the outer surface of the EV (e.g., exogenous body). In some aspects, the EV (e.g., exogenous body) of this disclosure comprises (i) an antigen and (ii) an adjuvant, wherein the antigen is within the lumen of the EV (e.g., exogenous body) and is not attached to any scaffold portion, and the adjuvant is attached to a scaffold X on the outer surface of the EV (e.g., exogenous body). In some aspects, the EV (e.g., exogenous body) of this disclosure comprises (i) an antigen and (ii) an adjuvant, wherein the antigen is attached to a scaffold Y on the luminal surface of the EV (e.g., exogenous body), and the adjuvant is attached to a scaffold X on the luminal surface of the EV (e.g., exogenous body). In some aspects, the EV (e.g., exosome) of this disclosure comprises (i) an antigen and (ii) an adjuvant, wherein the antigen is located within the lumen of the EV (e.g., exosome) and is not attached to any scaffold portion, and the adjuvant is attached to a scaffold X on the lumen surface of the EV (e.g., exosome). In some aspects, the EV (e.g., exosome) of this disclosure comprises (i) an antigen and (ii) an adjuvant, wherein the antigen is attached to a scaffold X on the lumen surface of the EV (e.g., exosome), and the adjuvant is attached to a scaffold X on the outer surface of the EV (e.g., exosome). In some aspects, the EV (e.g., exosome) of this disclosure comprises (i) an antigen and (ii) an adjuvant, wherein the antigen is attached to a first scaffold X on the outer surface of the EV (e.g., exosome), and the adjuvant is attached to a second scaffold X on the outer surface of the EV (e.g., exosome). In some aspects, the EV (e.g., exogenous body) of this disclosure comprises (i) an antigen and (ii) an adjuvant, wherein the antigen is connected to a scaffold X on the outer surface of the EV (e.g., exogenous body) and the adjuvant is connected to a scaffold Y on the luminal surface of the EV (e.g., exogenous body).In some aspects, the EV (e.g., exogenous body) of this disclosure comprises (i) an antigen and (ii) an adjuvant, wherein the antigen is attached to a scaffold X on the outer surface of the EV (e.g., exogenous body), and the adjuvant is located within the lumen of the EV (e.g., exogenous body) and is not attached to any scaffold portion. In some aspects, the EV (e.g., exogenous body) of this disclosure comprises (i) an antigen and (ii) an adjuvant, wherein the antigen is attached to a scaffold X on the outer surface of the EV (e.g., exogenous body), and the adjuvant is attached to a scaffold X on the luminal surface of the EV (e.g., exogenous body). In some aspects, the EV (e.g., exogenous body) of this disclosure comprises (i) an antigen and (ii) an adjuvant, wherein the antigen is attached to a first scaffold X on the luminal surface of the EV (e.g., exogenous body), and the adjuvant is attached to a second scaffold X on the luminal surface of the EV (e.g., exogenous body). In some aspects, the EV (e.g., exogenous body) of this disclosure comprises (i) an antigen and (ii) an adjuvant, wherein the antigen is connected to a stent X on the luminal surface of the EV (e.g., exogenous body), and the adjuvant is connected to a stent Y on the luminal surface of the EV (e.g., exogenous body). In some aspects, the EV (e.g., exogenous body) of this disclosure comprises (i) an antigen and (ii) an adjuvant, wherein the antigen is connected to a stent X on the luminal surface of the EV (e.g., exogenous body), and the adjuvant is located within the lumen of the EV (e.g., exogenous body) and is not connected to any stent portion. In some aspects, the EV (e.g., exogenous body) of this disclosure comprises (i) an antigen and (ii) an adjuvant, wherein the antigen is connected to a first stent X on the outer surface of the EV (e.g., exogenous body), and the adjuvant is connected to a second stent X on the luminal surface of the EV (e.g., exogenous body). In some aspects, the EV (e.g., exogenous body) of this disclosure comprises (i) an antigen and (ii) an adjuvant, wherein the antigen is attached to a first stent X on the luminal surface of the EV (e.g., exogenous body), and the adjuvant is attached to a second stent X on the outer surface of the EV (e.g., exogenous body). In some aspects, the EV (e.g., exogenous body) of this disclosure comprises (i) an antigen and (ii) an adjuvant, wherein the antigen is within the lumen of the EV (e.g., exogenous body) and is not attached to any stent portion, and the adjuvant is within the lumen of the EV (e.g., exogenous body) and is not attached to any stent portion. In some aspects, the EV (e.g., exogenous body) comprises (i) an antigen and (ii) an adjuvant, wherein the antigen is directly attached to the luminal surface of the EV, and the adjuvant is directly attached to the luminal surface of the EV. In some aspects, the EV (e.g., exogenous body) comprises (i) an antigen and (ii) an adjuvant, wherein the antigen is directly attached to the luminal surface of the EV, and the adjuvant is within the lumen of the EV. In some aspects, an EV (e.g., an exogenous body) comprises (i) an antigen and (ii) an adjuvant, wherein the antigen is directly attached to the luminal surface of the EV, and the adjuvant is attached to a scaffold Y on the luminal surface of the EV. In some aspects, an EV (e.g., an exogenous body) comprises (i) an antigen and (ii) an adjuvant, wherein the antigen is directly attached to the luminal surface of the EV, and the adjuvant is attached to a scaffold X on the luminal surface of the EV.In some aspects, an EV (e.g., an exogenous body) comprises (i) an antigen and (ii) an adjuvant, wherein the antigen is directly attached to the luminal surface of the EV, and the adjuvant is directly attached to the exterior of the EV. In some aspects, an EV (e.g., an exogenous body) comprises (i) an antigen and (ii) an adjuvant, wherein the antigen is directly attached to the luminal surface of the EV, and the adjuvant is attached to a scaffold X on the exterior of the EV. In some aspects, an EV (e.g., an exogenous body) comprises (i) an antigen and (ii) an adjuvant, wherein the antigen is attached to a scaffold Y on the luminal surface of the EV, and the adjuvant is directly attached to the luminal surface of the EV. In some aspects, an EV (e.g., an exogenous body) comprises (i) an antigen and (ii) an adjuvant, wherein the antigen is attached to a scaffold X on the luminal surface of the EV, and the adjuvant is directly attached to the luminal surface of the EV. In some aspects, an EV (e.g., an exogenous body) comprises (i) an antigen and (ii) an adjuvant, wherein the antigen is attached to a scaffold X on the luminal surface of the EV, and the adjuvant is directly attached to the exterior of the EV. In some aspects, an EV (e.g., an exogenous body) comprises (i) an antigen and (ii) an adjuvant, wherein the antigen is within the lumen of the EV, and the adjuvant is directly attached to the luminal surface of the EV. In some aspects, an EV (e.g., an exogenous body) comprises (i) an antigen and (ii) an adjuvant, wherein the antigen is within the lumen of the EV, and the adjuvant is directly attached to the exterior of the EV.

[0338] In some aspects, adjuvants and / or antigens may be modified to increase encapsulation (i.e., loading) in EVs (e.g., exosomes). Such modifications may include adding lipid-binding tags by treating the agonist (i.e., adjuvant and / or antigen) with chemicals or enzymes, or by physically or chemically altering the polarity or charge of the adjuvant and / or antigen. Adjuvants and / or antigens may be modified by a single treatment or by a combination of treatments, such as adding only a lipid-binding tag, or adding a lipid-binding tag and altering its polarity. The foregoing examples are intended to be non-limiting and illustrative. Any combination of modifications is contemplated to be practiced. Such modifications may increase the encapsulation (i.e., loading) of the adjuvant and / or antigen in the EV (e.g., exosome) by about 2 to about 10,000 times, about 10 to 1,000 times, or about 100 to about 500 times compared to the encapsulation (i.e., loading) of an unmodified agonist (i.e., adjuvant and / or antigen). The modification may increase the encapsulation (i.e., loading) of adjuvants and / or antigens in EVs (e.g., exogens) by at least about 2, about 5, about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 200, about 300, about 400, about 500, about 600, about 700, about 800, about 900, about 1,000, about 2,000, about 3,000, about 4,000, about 5,000, about 6,000, about 7,000, about 8,000, about 9,000, or about 10,000 times compared to the encapsulation (i.e., loading) of unmodified adjuvants and / or antigens.

[0339] In some respects, adjuvants and / or antigens may be modified to allow for better expression on the surface of the EV (e.g., the outer and / or luminal surfaces of the EV, (e.g., connected to the scaffold portions disclosed herein (e.g., scaffold X and / or scaffold Y))). Any of the modifications described above may be used. Such modifications may increase the expression of the agonist in the EV (e.g., on the surface and / or luminal surfaces of the exogenous body) by approximately 2 to 10,000 times, approximately 10 to 1,000 times, or approximately 100 to 500 times compared to the corresponding expression of the unmodified agonist. The modification can increase the expression of the agonist on the outer surface of an EV (e.g., an exogenous organism) by at least about 2, at least about 5, at least about 10, at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 100, at least about 200, at least about 300, at least about 400 times, at least about 500 times, at least about 600 times, at least about 700 times, at least about 800 times, at least about 900 times, at least about 1,000 times, at least about 2,000 times, at least about 3,000 times, at least about 4,000 times, at least about 5,000 times, at least about 6,000 times, at least about 7,000 times, at least about 8,000 times, at least about 9,000 times, or at least about 10,000 times. The modification can increase the expression of the agonist on the luminal surface of an EV (e.g., an exogenous organism) by at least about 2, at least about 5, at least about 10, at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 100, at least about 200, at least about 300, at least about 400 times, at least about 500 times, at least about 600 times, at least about 700 times, at least about 800 times, at least about 900 times, at least about 1,000 times, at least about 2,000 times, at least about 3,000 times, at least about 4,000 times, at least about 5,000 times, at least about 6,000 times, at least about 7,000 times, at least about 8,000 times, at least about 9,000 times, or at least about 10,000 times.

[0340] In some aspects, EVs (e.g., exosomes) are further modified to exhibit additional proteins (or fragments thereof) that can help guide EV uptake (e.g., targeting moieties), activate or block cellular pathways to enhance EV-related combined effects (e.g., the effect of a payload loaded into the exosome, such as a STING agonist). In some aspects, the EVs (e.g., exosomes) disclosed herein also include targeting moieties capable of altering the distribution of EVs in vivo or in vitro. In some aspects, the targeting moieties can be biomolecules, such as proteins, peptides, lipids, or synthetic molecules.

[0341] In some aspects, the targeting portion of this disclosure specifically binds to markers of dendritic cells. In some aspects, the markers are expressed only on dendritic cells. In some aspects, dendritic cells include pre-dendritic cells, inflammatory monodendritic cells, plasmacytoid dendritic cells (pDCs), myeloid / conventional dendritic cell 1 (cDC1), myeloid / conventional dendritic cell 2 (cDC2), inflammatory mononuclear cell-derived dendritic cells, Langerhans cells, dermal dendritic cells, lysozyme-expressing dendritic cells (LysoDCs), Kupffer cells, non-classical mononuclear cells, or any combination thereof. Markers expressed on these dendritic cells are known in the art. See, for example, Collin et al., Immunology 154(1):3-20 (2018). In some respects, the target component is a protein, wherein the protein is an antibody or fragment thereof capable of specifically binding to a marker selected from the following: DEC205, CLEC9A, CLEC6, DCIR, DC-SIGN, LOX-1, MARCO, Clec12a, Clec10a, DC-asialic acid glycoprotein receptor (DC-ASGPR), DC immune receptor 2 (DCIR2), Dectin-1, macrophage mannose receptor (MMR), BDCA-2 (CD303, Clec4c), Dectin-2, Bst-2 (CD317), Langerin, CD206, CD11b, CD11c, CD123, CD304, XCR1, AXL, Siglec 6, CD209, SIRPA, CX3CR1, GPR182, CD14, CD16, CD32, CD34, CD38, CD10, or any combination thereof. In some respects, biomarkers useful for use in this disclosure include C-type lectin-like domains. In some respects, the biomarker is Clec9a, and the dendritic cell is cDC1.

[0342] In some aspects, the targeting portion disclosed herein can bind to both human and mouse Clec9a (including any variants thereof). In some aspects, the targeting portion disclosed herein can bind to Clec9a from other species, including but not limited to chimpanzees, rhesus monkeys, dogs, cattle, horses, or rats. The sequence of such Clec9a protein is known in the art. See, for example, U.S. Patent No. 8,426,565B2, which is incorporated herein by reference in its entirety.

[0343] In some respects, the targeting portion of this disclosure specifically binds to T cell markers. In some respects, the T cells are CD4+ T cells. In some respects, the T cells are CD8+ T cells.

[0344] In some respects, the targeting portions disclosed herein bind to human CD3 protein or fragments thereof. The sequence of human CD3 protein is known in the art.

[0345] In some respects, the targeting portion disclosed herein can bind to both human and mouse CD3 (including any variants thereof). In some respects, the targeting portion disclosed herein can bind to CD3 from other species (including, but not limited to, chimpanzees, rhesus monkeys, dogs, cattle, horses, or rats). The sequence of such CD3 protein is also known in the art.

[0346] In some aspects, the targeting portion disclosed herein allows cells expressing markers specific to the targeting portion (e.g., CD3:CD4+ T cells and / or CD8+ T cells; Clec9a: dendritic cells) to take up more EVs (e.g., exogenous organisms). In some aspects, EV uptake is increased by at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 100 compared to a reference (e.g., a corresponding EV without the targeting portion or a non-EV delivery medium). At least 200 times, at least 300 times, at least 400 times, at least 500 times, at least 600 times, at least 700 times, at least 800 times, at least 900 times, at least 1,000 times, at least 2,000 times, at least 3,000 times, at least 4,000 times, at least 5,000 times, at least 6,000 times, at least 7,000 times, at least 8,000 times, at least 9,000 times, at least 10,000 times, or more. In some aspects, references include EVs (e.g., exogens) that do not express the target portion disclosed herein.

[0347] In some respects, increased uptake of the EVs (e.g., exosomes) disclosed herein can allow for a greater immune response. Therefore, in some respects, EVs (e.g., exosomes) expressing the target motifs disclosed herein can increase an immune response (e.g., against tumor antigens loaded on the exosome) by at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70 At least 80 times, at least 90 times, at least 100 times, at least 200 times, at least 300 times, at least 400 times, at least 500 times, at least 600 times, at least 700 times, at least 800 times, at least 900 times, at least 1,000 times, at least 2,000 times, at least 3,000 times, at least 4,000 times, at least 5,000 times, at least 6,000 times, at least 7,000 times, at least 8,000 times, at least 9,000 times, at least 10,000 times or more. In some aspects, reference includes EVs (e.g., exogenous bodies) that do not express the target portion disclosed herein. In some aspects, the immune response is mediated by T cells (e.g., CD8+ T cells or CD4+ T cells) and / or B cells.

[0348] As described above, the targeted portion disclosed herein may include peptides, antibodies or their antigen-binding fragments, compounds or any combination thereof.

[0349] In some aspects, the targeting portion is a peptide capable of specifically binding to Clec9a. See, for example, Yan et al., Oncotarget 7(26):40437-40450 (2016). For example, in some aspects, the peptide comprises a soluble fragment of Clec9a. Non-limiting examples of such peptides are described in U.S. Patent No. 9,988,431B2 (which is incorporated herein by reference in its entirety). In some aspects, the peptide comprises a ligand of Clec9a (natural or synthetic), such as those described in Ahrens et al., Immunity 36(4):635-45 (2012); and Zhang et al., Immunity 36(4):646-57 (2012). Non-limiting examples of peptides comprising Clec9a ligands are described in International Publication No. WO 2013 / 053008 A2 (which is incorporated herein by reference in its entirety).

[0350] In some respects, the targeting portion is a peptide capable of specifically binding to CD3. For example, in some respects, the peptide comprises a soluble fragment of CD3. In some respects, the peptide comprises a CD3 ligand (natural or synthetic).

[0351] In some respects, the targeting moiety is an antibody or its antigen-binding fragment. In some respects, the targeting moiety is a single-chain Fv antibody fragment. In some respects, the targeting moiety is a single-chain F(ab) antibody fragment. In some respects, the targeting moiety is a nanobody. In some respects, the targeting moiety is a monomer.

[0352] In some aspects, the EVs (e.g., exogenous bodies) disclosed herein comprise one or more (e.g., two, three, four, five, or more) targeting moieties. In some aspects, one or more targeting moieties are expressed in combination with other exogenous bioactive molecules (e.g., therapeutic molecules, adjuvants, or immunomodulators) disclosed herein. In some aspects, one or more targeting moieties may be expressed on the outer surface of the EV (e.g., exogenous body). Thus, in some aspects, one or more targeting moieties are attached to a scaffold portion (e.g., scaffold X) on the outer surface of the EV (e.g., exogenous body). When one or more targeting moieties are expressed in combination with other exogenous bioactive molecules (e.g., therapeutic molecules, adjuvants, or immunomodulators), said other exogenous bioactive molecules may be expressed on the surface (e.g., the outer surface or the luminal surface) or within the lumen of the EV (e.g., exogenous body).

[0353] Producer cells may be modified to include additional exogenous sequences encoding additional proteins or fragments thereof. Alternatively, the additional protein or fragment thereof may be covalently linked or conjugated to the EV (e.g., exogenous organism) using any suitable linker chemistry method known in the art. Non-limiting examples of suitable linker chemistry include amine reactive groups, carboxyl reactive groups, thiol reactive groups, aldehyde reactive groups, photoreactive groups, ClickIT chemistry, biotin-streptavitin or other avidin conjugates, or any combination thereof.

[0354] II.C Immunomodulators

[0355] In some aspects, the EVs (e.g., exogenous bodies) disclosed herein may contain immunomodulatory agents (e.g., together with the antigens and / or other payloads disclosed herein). In some aspects, the EVs (e.g., exogenous bodies) disclosed herein contain multiple immunomodulatory agents. In some aspects, each of the multiple immunomodulatory agents is different. In some aspects, the EVs (e.g., exogenous bodies) disclosed herein contain at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different immunomodulatory agents.

[0356] In some aspects, an EV (e.g., an exogenous body) comprises a combination of one or more immunomodulators with one or more additional payloads (e.g., antigens and / or adjuvants). In some aspects, an EV (e.g., an exogenous body) may comprise one or more additional portions (e.g., targeting portions). For example, in some aspects, the EV (e.g., an exogenous body) disclosed herein may comprise (i) one or more immunomodulators, (ii) one or more additional payloads (e.g., antigens and / or adjuvants), and (iii) one or more targeting portions.

[0357] In some aspects, immunomodulators may be expressed on the surface (e.g., the outer surface or the luminal surface) or within the lumen of an EV (e.g., an exosome). Thus, in some aspects, the immunomodulator is attached to a scaffold portion (e.g., scaffold X) on the outer surface or luminal surface of the EV (e.g., an exosome). In other aspects, the immunomodulator is attached to a scaffold portion (e.g., scaffold Y) on the luminal surface of the EV (e.g., an exosome). In still other aspects, the immunomodulator is within the lumen of the exosome (i.e., not attached to scaffold X or scaffold Y). In some aspects, the immunomodulator may be attached directly (i.e., without using a scaffold portion) to the outer surface and / or luminal surface of the EV (e.g., an exosome).

[0358] Non-limiting examples of these aspects include EVs (e.g., exogens) that contain (i) an antigen and (ii) an immunomodulator, wherein:

[0359] (a) The antigen is attached to a first scaffold Y on the luminal surface of the EV (e.g., exogenous body), and the immunomodulator is attached to a second scaffold Y on the luminal surface of the EV (e.g., exogenous body).

[0360] (b) The antigen is attached to a scaffold Y on the luminal surface of the EV (e.g., exogenous body), and the immunomodulator is inside the lumen of the EV (e.g., exogenous body) and is not attached to any scaffold portion.

[0361] (c) The antigen is inside the lumen of the EV (e.g., exogenous body) and is not attached to any scaffold portion, and the immunomodulator is attached to the scaffold Y on the lumen surface of the EV (e.g., exogenous body);

[0362] (d) The antigen is attached to a scaffold Y on the luminal surface of the EV (e.g., exogenous body), and the immunomodulator is attached to a scaffold X on the outer surface of the EV (e.g., exogenous body).

[0363] (e) The antigen is attached to a scaffold X on the luminal surface of the EV (e.g., exogenous body), and the immunomodulator is attached to a scaffold Y on the luminal surface of the EV (e.g., exogenous body).

[0364] (f) The antigen is attached to a scaffold X on the luminal surface of the EV (e.g., an exogenous organism), and the immunomodulator is attached to a scaffold Y on the luminal surface of the EV (e.g., an exogenous organism); or

[0365] (g) The antigen is attached to a scaffold X on the outer surface of the EV (e.g., exogenous body), and the immunomodulator is attached to a scaffold Y on the luminal surface of the EV (e.g., exogenous body).

[0366] Non-limiting examples of specific aspects include EVs (e.g., exogens) that comprise (i) an antigen and (ii) an immunomodulator, wherein:

[0367] (a) The antigen is attached to a first scaffold Y on the luminal surface of the EV (e.g., exogenous body), and the immunomodulator is attached to a second scaffold Y on the luminal surface of the EV (e.g., exogenous body).

[0368] (b) The antigen is attached to a scaffold Y on the luminal surface of the EV (e.g., exogenous body), and the immunomodulator is inside the lumen of the EV (e.g., exogenous body) and is not attached to any scaffold portion.

[0369] (c) The antigen is inside the lumen of the EV (e.g., exogenous body) and is not attached to any scaffold portion, and the immunomodulator is attached to the scaffold Y on the lumen surface of the EV (e.g., exogenous body);

[0370] (d) The antigen is attached to a scaffold Y on the luminal surface of the EV (e.g., exogenous body), and the immunomodulator is attached to a scaffold X on the outer surface of the EV (e.g., exogenous body).

[0371] (e) The antigen is within the lumen of the EV (e.g., exogenous body) and is not attached to any scaffold portion, and the immunomodulator is attached to scaffold X on the outer surface of the EV (e.g., exogenous body);

[0372] (f) The antigen is attached to a scaffold Y on the luminal surface of the EV (e.g., exogenous body), and the immunomodulator is attached to a scaffold X on the luminal surface of the EV (e.g., exogenous body).

[0373] (g) The antigen is located within the lumen of the EV (e.g., exogenous body) and is not attached to any scaffold portion, and the immunomodulator is attached to scaffold X on the lumen surface of the EV (e.g., exogenous body).

[0374] (h) The antigen is attached to a scaffold X on the luminal surface of the EV (e.g., exogenous body), and the immunomodulator is attached to a scaffold X on the outer surface of the EV (e.g., exogenous body).

[0375] (i) The antigen is attached to a first scaffold X on the outer surface of the EV (e.g., exogenous body), and the immunomodulator is attached to a second scaffold X on the outer surface of the EV (e.g., exogenous body);

[0376] (j) The antigen is attached to a scaffold X on the outer surface of the EV (e.g., exogenous body), and the immunomodulator is attached to a scaffold Y on the luminal surface of the EV (e.g., exogenous body).

[0377] (k) The antigen is attached to a scaffold X on the outer surface of the EV (e.g., exogenous body), and the immunomodulator is located within the lumen of the EV (e.g., exogenous body) and is not attached to any part of the scaffold.

[0378] (l) The antigen is attached to a scaffold X on the outer surface of the EV (e.g., exogenous body), and the immunomodulator is attached to a scaffold X on the luminal surface of the EV (e.g., exogenous body).

[0379] (m) The antigen is attached to a first scaffold X on the luminal surface of the EV (e.g., exogenous body), and the immunomodulator is attached to a second scaffold X on the luminal surface of the EV (e.g., exogenous body).

[0380] (n) The antigen is attached to a scaffold X on the luminal surface of the EV (e.g., exogenous body), and the immunomodulator is attached to a scaffold Y on the luminal surface of the EV (e.g., exogenous body).

[0381] (o) The antigen is attached to a scaffold X on the luminal surface of the EV (e.g., exogenous body), and the immunomodulator is located within the lumen of the EV (e.g., exogenous body) and is not attached to any part of the scaffold.

[0382] (p) The antigen is attached to a first scaffold X on the outer surface of the EV (e.g., exogenous body), and the immunomodulator is attached to a second scaffold X on the luminal surface of the EV (e.g., exogenous body).

[0383] (q) The antigen is attached to a first scaffold X on the luminal surface of the EV (e.g., exogenous body), and the immunomodulator is attached to a second scaffold X on the outer surface of the EV (e.g., exogenous body);

[0384] (r) The antigen is within the lumen of the EV (e.g., exogenous body) and is not attached to any scaffold portion, and the immunomodulator is within the lumen of the EV (e.g., exogenous body) and is not attached to any scaffold portion;

[0385] (s) The antigen is directly attached to the luminal surface of the EV (e.g., exogenous body), and the immunomodulator is directly attached to the luminal surface of the EV (e.g., exogenous body);

[0386] (t) The antigen is directly attached to the luminal surface of the EV (e.g., exogenous body), and the immunomodulator is inside the lumen of the EV (e.g., exogenous body);

[0387] (u) The antigen is directly attached to the luminal surface of the EV (e.g., exogenous body), and the immunomodulator is attached to a scaffold Y on the luminal surface of the EV (e.g., exogenous body);

[0388] (v) The antigen is directly attached to the luminal surface of the EV (e.g., exogenous body), and the immunomodulator is attached to a scaffold X on the luminal surface of the EV (e.g., exogenous body).

[0389] (w) The antigen is directly attached to the luminal surface of the EV (e.g., exogenous body), and the immunomodulator is directly attached to the exterior of the EV (e.g., exogenous body);

[0390] (x) The antigen is directly attached to the luminal surface of the EV (e.g., exogenous body), and the immunomodulator is attached to a scaffold X on the outer surface of the EV (e.g., exogenous body);

[0391] (y) The antigen is attached to a scaffold Y on the luminal surface of the EV (e.g., exogenous body), and the immunomodulator is attached directly to the luminal surface of the EV (e.g., exogenous body).

[0392] (z) The antigen is attached to a scaffold Y on the luminal surface of the EV (e.g., exogenous body), and the immunomodulator is attached directly to the exterior of the EV (e.g., exogenous body);

[0393] (aa) The antigen is attached to a scaffold X on the luminal surface of the EV (e.g., exogenous body), and the immunomodulator is attached directly to the luminal surface of the EV (e.g., exogenous body).

[0394] (bb) The antigen is attached to a scaffold X on the luminal surface of the EV (e.g., exogenous body), and the immunomodulator is attached directly to the exterior of the EV (e.g., exogenous body).

[0395] (cc) The antigen is located within the lumen of the EV (e.g., exogenous organism), and the immunomodulator is directly attached to the luminal surface of the EV (e.g., exogenous organism); or

[0396] (dd) The antigen is located within the lumen of the EV (e.g., exogenous body), and the immunomodulator is directly attached to the exterior of the EV (e.g., exogenous body).

[0397] In some aspects, the EV (e.g., exosome) of this disclosure comprises (i) an antigen and (ii) an immunomodulator, wherein the antigen is attached to a first scaffold Y on the luminal surface of the EV (e.g., exosome), and the immunomodulator is attached to a second scaffold Y on the luminal surface of the EV (e.g., exosome). In some aspects, the EV (e.g., exosome) of this disclosure comprises (i) an antigen and (ii) an immunomodulator, wherein the antigen is attached to a scaffold Y on the luminal surface of the EV (e.g., exosome), and the immunomodulator is located within the lumen of the EV (e.g., exosome) and is not attached to any scaffold portion. In some aspects, the EV (e.g., exosome) of this disclosure comprises (i) an antigen and (ii) an immunomodulator, wherein the antigen is located within the lumen of the EV (e.g., exosome) and is not attached to any scaffold portion, and the immunomodulator is attached to a scaffold Y on the luminal surface of the EV (e.g., exosome). In some aspects, the EV (e.g., exosome) of this disclosure comprises (i) an antigen and (ii) an immunomodulator, wherein the antigen is attached to a scaffold Y on the luminal surface of the EV (e.g., exosome), and the immunomodulator is attached to a scaffold X on the outer surface of the EV (e.g., exosome). In some aspects, the EV (e.g., exosome) of this disclosure comprises (i) an antigen and (ii) an immunomodulator, wherein the antigen is within the lumen of the EV (e.g., exosome) and is not attached to any scaffold portion, and the immunomodulator is attached to a scaffold X on the outer surface of the EV (e.g., exosome). In some aspects, the EV (e.g., exosome) of this disclosure comprises (i) an antigen and (ii) an immunomodulator, wherein the antigen is attached to a scaffold Y on the luminal surface of the EV (e.g., exosome), and the immunomodulator is attached to a scaffold X on the luminal surface of the EV (e.g., exosome). In some aspects, the EV (e.g., exosome) of this disclosure comprises (i) an antigen and (ii) an immunomodulator, wherein the antigen is located within the lumen of the EV (e.g., exosome) and is not attached to any scaffold portion, and the immunomodulator is attached to a scaffold X on the luminal surface of the EV (e.g., exosome). In some aspects, the EV (e.g., exosome) of this disclosure comprises (i) an antigen and (ii) an immunomodulator, wherein the antigen is attached to a scaffold X on the luminal surface of the EV (e.g., exosome), and the immunomodulator is attached to a scaffold X on the outer surface of the EV (e.g., exosome). In some aspects, the EV (e.g., exosome) of this disclosure comprises (i) an antigen and (ii) an immunomodulator, wherein the antigen is attached to a first scaffold X on the outer surface of the EV (e.g., exosome), and the immunomodulator is attached to a second scaffold X on the outer surface of the EV (e.g., exosome). In some aspects, the EV (e.g., exogenous body) of this disclosure comprises (i) an antigen and (ii) an immunomodulator, wherein the antigen is connected to a scaffold X on the outer surface of the EV (e.g., exogenous body) and the immunomodulator is connected to a scaffold Y on the luminal surface of the EV (e.g., exogenous body).In some aspects, the EV (e.g., exosome) of this disclosure comprises (i) an antigen and (ii) an immunomodulator, wherein the antigen is attached to a scaffold X on the outer surface of the EV (e.g., exosome), and the immunomodulator is located within the lumen of the EV (e.g., exosome) and is not attached to any scaffold portion. In some aspects, the EV (e.g., exosome) of this disclosure comprises (i) an antigen and (ii) an immunomodulator, wherein the antigen is attached to a scaffold X on the outer surface of the EV (e.g., exosome), and the immunomodulator is attached to a scaffold X on the luminal surface of the EV (e.g., exosome). In some aspects, the EV (e.g., exosome) of this disclosure comprises (i) an antigen and (ii) an immunomodulator, wherein the antigen is attached to a first scaffold X on the luminal surface of the EV (e.g., exosome), and the immunomodulator is attached to a second scaffold X on the luminal surface of the EV (e.g., exosome). In some aspects, the EV (e.g., exosome) of this disclosure comprises (i) an antigen and (ii) an immunomodulator, wherein the antigen is connected to a scaffold X on the luminal surface of the EV (e.g., exosome), and the immunomodulator is connected to a scaffold Y on the luminal surface of the EV (e.g., exosome). In some aspects, the EV (e.g., exosome) of this disclosure comprises (i) an antigen and (ii) an immunomodulator, wherein the antigen is connected to a scaffold X on the luminal surface of the EV (e.g., exosome), and the immunomodulator is located within the lumen of the EV (e.g., exosome) and is not connected to any scaffold portion. In some aspects, the EV (e.g., exosome) of this disclosure comprises (i) an antigen and (ii) an immunomodulator, wherein the antigen is connected to a first scaffold X on the outer surface of the EV (e.g., exosome), and the immunomodulator is connected to a second scaffold X on the luminal surface of the EV (e.g., exosome). In some aspects, the EV (e.g., exosome) of this disclosure comprises (i) an antigen and (ii) an immunomodulator, wherein the antigen is attached to a first scaffold X on the luminal surface of the EV (e.g., exosome), and the immunomodulator is attached to a second scaffold X on the outer surface of the EV (e.g., exosome). In some aspects, the EV (e.g., exosome) of this disclosure comprises (i) an antigen and (ii) an immunomodulator, wherein the antigen is within the lumen of the EV (e.g., exosome) and is not attached to any scaffold portion, and the immunomodulator is within the lumen of the EV (e.g., exosome) and is not attached to any scaffold portion. In some aspects, the EV (e.g., exosome) comprises (i) an antigen and (ii) an immunomodulator, wherein the antigen is directly attached to the luminal surface of the EV, and the immunomodulator is directly attached to the luminal surface of the EV. In some aspects, the EV (e.g., exosome) comprises (i) an antigen and (ii) an immunomodulator, wherein the antigen is directly attached to the luminal surface of the EV, and the immunomodulator is within the lumen of the EV. In some respects, an EV (e.g., an exogenous body) comprises (i) an antigen and (ii) an immunomodulator, wherein the antigen is directly attached to the luminal surface of the EV and the immunomodulator is attached to a scaffold Y on the luminal surface of the EV.In some aspects, an EV (e.g., an exogenous body) comprises (i) an antigen and (ii) an immunomodulator, wherein the antigen is directly attached to the luminal surface of the EV, and the immunomodulator is attached to a scaffold X on the luminal surface of the EV. In some aspects, an EV (e.g., an exogenous body) comprises (i) an antigen and (ii) an immunomodulator, wherein the antigen is directly attached to the luminal surface of the EV, and the immunomodulator is directly attached to the exterior of the EV. In some aspects, an EV (e.g., an exogenous body) comprises (i) an antigen and (ii) an immunomodulator, wherein the antigen is directly attached to the luminal surface of the EV, and the immunomodulator is attached to a scaffold X on the exterior of the EV. In some aspects, an EV (e.g., an exogenous body) comprises (i) an antigen and (ii) an immunomodulator, wherein the antigen is attached to a scaffold Y on the luminal surface of the EV, and the immunomodulator is directly attached to the luminal surface of the EV. In some aspects, an EV (e.g., an exogenous body) comprises (i) an antigen and (ii) an immunomodulator, wherein the antigen is attached to a scaffold Y on the luminal surface of the EV, and the immunomodulator is directly attached to the exterior of the EV. In some aspects, an EV (e.g., an exogenous body) comprises (i) an antigen and (ii) an immunomodulator, wherein the antigen is attached to a scaffold X on the luminal surface of the EV, and the immunomodulator is directly attached to the luminal surface of the EV. In some aspects, an EV (e.g., an exogenous body) comprises (i) an antigen and (ii) an immunomodulator, wherein the antigen is within the lumen of the EV, and the immunomodulator is directly attached to the luminal surface of the EV. In some aspects, an EV (e.g., an exogenous body) comprises (i) an antigen and (ii) an immunomodulator, wherein the antigen is within the lumen of the EV, and the immunomodulator is directly attached to the luminal surface of the EV. In some aspects, an EV (e.g., an exogenous body) comprises (i) an antigen and (ii) an immunomodulator, wherein the antigen is within the lumen of the EV, and the immunomodulator is directly attached to the exterior of the EV.

[0398] Non-limiting examples of specific aspects include EVs (e.g., exogens) that comprise (i) an antigen, (ii) an adjuvant, and (iii) an immunomodulator, wherein:

[0399] (a) The antigen is attached to a first scaffold Y on the luminal surface of the EV (e.g., exogenous body), the adjuvant is attached to a second scaffold Y on the luminal surface of the EV (e.g., exogenous body), and the immunomodulator (a1) is in the lumen of the EV (e.g., exogenous body) and is not attached to any scaffold portion, or (a2) is attached to a third scaffold portion (e.g., scaffold X on the outer surface of the exogenous body or in the lumen of the exogenous body or scaffold Y on the luminal surface of the EV (e.g., exogenous body)).

[0400] (b) The antigen is attached to a scaffold Y on the luminal surface of the EV (e.g., exogenous body), the adjuvant is inside the lumen of the EV (e.g., exogenous body) and is not attached to any scaffold portion, and the immunomodulator (b1) is inside the lumen of the EV (e.g., exogenous body) and is not attached to any scaffold portion, or (b2) is attached to a scaffold portion (e.g., scaffold X on the outer surface of the exogenous body or the luminal surface of the exogenous body or scaffold Y on the luminal surface of the EV (e.g., exogenous body)).

[0401] (c) The antigen is in the lumen of the EV (e.g., exogenous body) and is not attached to any scaffold portion, the adjuvant is attached to scaffold Y on the lumen surface of the EV (e.g., exogenous body), and the immunomodulator (c1) is in the lumen of the EV (e.g., exogenous body) and is not attached to any scaffold portion, or (c2) is attached to a scaffold portion (e.g., scaffold X on the outer surface of the exogenous body or on the lumen surface of the exogenous body or scaffold Y on the lumen surface of the EV (e.g., exogenous body)).

[0402] (d) The antigen is attached to a scaffold Y on the luminal surface of the EV (e.g., exogenous body), the adjuvant is attached to a scaffold X on the outer surface of the EV (e.g., exogenous body), and the immunomodulator (d1) is in the lumen of the EV (e.g., exogenous body) and is not attached to any scaffold portion, or (d2) is attached to a third scaffold portion (e.g., scaffold X on the outer surface of the exogenous body or on the luminal surface of the exogenous body or scaffold Y on the luminal surface of the EV (e.g., exogenous body)).

[0403] (e) The antigen is in the lumen of the EV (e.g., exogenous body) and is not attached to any scaffold portion, the adjuvant is attached to scaffold X on the outer surface of the EV (e.g., exogenous body), and the immunomodulator (e1) is in the lumen of the EV (e.g., exogenous body) and is not attached to any scaffold portion, or (e2) is attached to a scaffold portion (e.g., scaffold X on the surface of the exogenous body or the surface of the exogenous body lumen or scaffold Y on the surface of the EV (e.g., exogenous body) lumen).

[0404] (f) The antigen is attached to a scaffold Y on the luminal surface of the EV (e.g., exogenous body), the adjuvant is attached to a scaffold X on the luminal surface of the EV (e.g., exogenous body), and the immunomodulator (f1) is in the lumen of the EV (e.g., exogenous body) and is not attached to any scaffold portion, or (f2) is attached to a third scaffold portion (e.g., scaffold X on the outer surface of the exogenous body or on the luminal surface of the exogenous body or scaffold Y on the luminal surface of the EV (e.g., exogenous body)).

[0405] (g) The antigen is in the lumen of the EV (e.g., exogenous body) and is not attached to any scaffold portion, the adjuvant is attached to scaffold X on the lumen surface of the EV (e.g., exogenous body), and the immunomodulator (g1) is in the lumen of the EV (e.g., exogenous body) and is not attached to any scaffold portion, or (g2) is attached to a scaffold portion (e.g., scaffold X on the outer surface of the exogenous body or on the lumen surface of the exogenous body or scaffold Y on the lumen surface of the EV (e.g., exogenous body);

[0406] (h) The antigen is attached to a scaffold X on the luminal surface of the EV (e.g., exogenous body), the adjuvant is attached to a scaffold X on the outer surface of the EV (e.g., exogenous body), and the immunomodulator (h1) is in the lumen of the EV (e.g., exogenous body) and is not attached to any scaffold portion, or (h2) is attached to a scaffold portion (e.g., scaffold X on the outer surface of the exogenous body or on the luminal surface of the exogenous body or scaffold Y on the luminal surface of the EV (e.g., exogenous body)).

[0407] (i) The antigen is attached to a first scaffold X on the outer surface of the EV (e.g., exogenous body), the adjuvant is attached to a second scaffold X on the outer surface of the EV (e.g., exogenous body), and the immunomodulator (i1) is in the lumen of the EV (e.g., exogenous body) and is not attached to any scaffold portion, or (i2) is attached to a third scaffold portion (e.g., scaffold X on the outer surface of the exogenous body or on the lumen surface of the exogenous body or scaffold Y on the lumen surface of the EV (e.g., exogenous body)).

[0408] (j) The antigen is attached to a scaffold X on the outer surface of the EV (e.g., exogenous body), the adjuvant is attached to a scaffold Y on the luminal surface of the EV (e.g., exogenous body), and the immunomodulator (j1) is in the lumen of the EV (e.g., exogenous body) and is not attached to any scaffold portion, or (j2) is attached to a third scaffold portion (e.g., scaffold X on the outer surface of the exogenous body or scaffold Y on the luminal surface of the EV (e.g., exogenous body)).

[0409] (k) The antigen is attached to a scaffold X on the outer surface of the EV (e.g., exogenous body), the adjuvant is in the lumen of the EV (e.g., exogenous body) and is not attached to any scaffold portion, and the immunomodulator (k1) is in the lumen of the EV (e.g., exogenous body) and is not attached to any scaffold portion, or (k2) is attached to a scaffold portion (e.g., scaffold X on the outer surface of the exogenous body or the lumen surface of the exogenous body or scaffold Y on the lumen surface of the EV (e.g., exogenous body)).

[0410] (l) The antigen is attached to a scaffold X on the outer surface of the EV (e.g., exogenous body), the adjuvant is attached to a scaffold X on the luminal surface of the EV (e.g., exogenous body), and the immunomodulator (l1) is in the lumen of the EV (e.g., exogenous body) and is not attached to any scaffold portion, or (l2) is attached to a scaffold portion (e.g., scaffold X on the outer surface of the exogenous body or scaffold Y on the luminal surface of the EV (e.g., exogenous body)).

[0411] (m) The antigen is attached to a first scaffold X on the luminal surface of the EV (e.g., exogenous body), the adjuvant is attached to a second scaffold on the luminal surface of the EV (e.g., exogenous body), and the immunomodulator (m1) is in the lumen of the EV (e.g., exogenous body) and is not attached to any scaffold portion, or (m2) is attached to a third scaffold portion (e.g., scaffold X on the outer surface of the exogenous body or the luminal surface of the exogenous body or scaffold Y on the luminal surface of the EV (e.g., exogenous body)).

[0412] (n) The antigen is attached to a scaffold X on the luminal surface of the EV (e.g., exogenous body), the adjuvant is attached to a scaffold Y on the luminal surface of the EV (e.g., exogenous body), and the immunomodulator (n1) is in the lumen of the EV (e.g., exogenous body) and is not attached to any scaffold portion, or (n2) is attached to a third scaffold portion (e.g., scaffold X on the outer surface of the exogenous body or on the luminal surface of the exogenous body or scaffold Y on the luminal surface of the EV (e.g., exogenous body)).

[0413] (o) The antigen is attached to a scaffold X on the luminal surface of an EV (e.g., an exogenous body), the adjuvant is inside the lumen of the EV (e.g., an exogenous body) and is not attached to any scaffold portion, and the immunomodulator (o1) is inside the lumen of the EV (e.g., an exogenous body) and is not attached to any scaffold portion, or (o2) is attached to a scaffold portion (e.g., scaffold X on the outer surface of the exogenous body or the luminal surface of the exogenous body or scaffold Y on the luminal surface of the EV (e.g., an exogenous body);

[0414] (p) The antigen is attached to a first scaffold X on the outer surface of the EV (e.g., exogenous body), the adjuvant is attached to a second scaffold X on the luminal surface of the EV (e.g., exogenous body), and the immunomodulator (p1) is in the lumen of the EV (e.g., exogenous body) and is not attached to any scaffold portion, or (p2) is attached to a third scaffold portion (e.g., scaffold X on the surface of the exogenous body or in the lumen of the exogenous body or scaffold Y on the luminal surface of the EV (e.g., exogenous body)).

[0415] (q) The antigen is attached to a first scaffold X on the luminal surface of the EV (e.g., exogenous body), the adjuvant is attached to a second scaffold X on the outer surface of the EV (e.g., exogenous body), and the immunomodulator (q1) is in the lumen of the EV (e.g., exogenous body) and is not attached to any scaffold portion, or (q2) is attached to a third scaffold portion (e.g., scaffold X on the outer surface of the exogenous body or the luminal surface of the exogenous body or scaffold Y on the luminal surface of the EV (e.g., exogenous body)).

[0416] (r) The antigen is in the lumen of the EV (e.g., exogenous body) and is not attached to any scaffold portion; the adjuvant is in the lumen of the EV (e.g., exogenous body) and is not attached to any scaffold portion; and the immunomodulator (r1) is in the lumen of the exogenous body, or (r2) is attached to a scaffold portion (e.g., scaffold X on the outer surface of the exogenous body or on the lumen surface of the exogenous body, or scaffold Y on the lumen surface of the EV (e.g., exogenous body)).

[0417] In some aspects, the exogenous body of this disclosure comprises: (i) an antigen, (ii) an adjuvant, and (iii) an immunomodulator, wherein the antigen is attached to a first scaffold Y on the luminal surface of the EV (e.g., the exogenous body), the adjuvant is attached to a second scaffold Y on the luminal surface of the EV (e.g., the exogenous body), and the immunomodulator is located within the lumen of the EV (e.g., the exogenous body) and is not attached to any scaffold portion. In some aspects, the exogenous body of this disclosure comprises: (i) an antigen, (ii) an adjuvant, and (iii) an immunomodulator, wherein the antigen is attached to a first scaffold Y on the luminal surface of the EV (e.g., the exogenous body), the adjuvant is attached to a second scaffold Y on the luminal surface of the EV (e.g., the exogenous body), and the immunomodulator is attached to a scaffold X on the outer surface of the EV (e.g., the exogenous body). In some aspects, the exogenous body of this disclosure comprises: (i) an antigen, (ii) an adjuvant, and (iii) an immunomodulator, wherein the antigen is connected to a first scaffold Y on the luminal surface of the EV (e.g., the exogenous body), the adjuvant is connected to a second scaffold Y on the luminal surface of the EV (e.g., the exogenous body), and the immunomodulator is connected to a scaffold X on the luminal surface of the EV (e.g., the exogenous body). In some aspects, the exogenous body of this disclosure comprises: (i) an antigen, (ii) an adjuvant, and (iii) an immunomodulator, wherein the antigen is connected to a first scaffold Y on the luminal surface of the EV (e.g., the exogenous body), the adjuvant is connected to a second scaffold Y on the luminal surface of the EV (e.g., the exogenous body), and the immunomodulator is connected to a third scaffold Y on the luminal surface of the EV (e.g., the exogenous body).

[0418] In some aspects, the exogenous body of this disclosure comprises: (i) an antigen, (ii) an adjuvant, and (iii) an immunomodulator, wherein the antigen is attached to a scaffold Y on the luminal surface of the EV (e.g., the exogenous body), the adjuvant is located within the lumen of the EV (e.g., the exogenous body) and is not attached to any scaffold portion, and the immunomodulator is located within the lumen of the EV (e.g., the exogenous body) and is not attached to any scaffold portion. In some aspects, the exogenous body of this disclosure comprises: (i) an antigen, (ii) an adjuvant, and (iii) an immunomodulator, wherein the antigen is attached to a scaffold Y on the luminal surface of the EV (e.g., the exogenous body), the adjuvant is located within the lumen of the EV (e.g., the exogenous body) and is not attached to any scaffold portion, and the immunomodulator is attached to a scaffold X on the outer surface of the EV (e.g., the exogenous body). In some aspects, the exogenous body of this disclosure comprises: (i) an antigen, (ii) an adjuvant, and (iii) an immunomodulator, wherein the antigen is attached to a scaffold Y on the luminal surface of the EV (e.g., the exogenous body), the adjuvant is located within the lumen of the EV (e.g., the exogenous body) and is not attached to any scaffold portion, and the immunomodulator is attached to a scaffold X on the luminal surface of the EV (e.g., the exogenous body). In some aspects, the exogenous body of this disclosure comprises: (i) an antigen, (ii) an adjuvant, and (iii) an immunomodulator, wherein the antigen is attached to a first scaffold Y on the luminal surface of the EV (e.g., the exogenous body), the adjuvant is located within the lumen of the EV (e.g., the exogenous body) and is not attached to any scaffold portion, and the immunomodulator is attached to a second scaffold Y on the luminal surface of the EV (e.g., the exogenous body).

[0419] In some aspects, the exogenous body of this disclosure comprises: (i) an antigen, (ii) an adjuvant, and (iii) an immunomodulator, wherein the antigen is located within the lumen of the EV (e.g., the exogenous body) and is not attached to any scaffold portion, the adjuvant is attached to a scaffold Y on the luminal surface of the EV (e.g., the exogenous body), and the immunomodulator is located within the lumen of the EV (e.g., the exogenous body) and is not attached to any scaffold portion. In some aspects, the exogenous body of this disclosure comprises: (i) an antigen, (ii) an adjuvant, and (iii) an immunomodulator, wherein the antigen is located within the lumen of the EV (e.g., the exogenous body) and is not attached to any scaffold portion, the adjuvant is attached to a scaffold Y on the luminal surface of the EV (e.g., the exogenous body), and the immunomodulator is attached to a scaffold X on the outer surface of the EV (e.g., the exogenous body). In some aspects, the exogenous body of this disclosure comprises: (i) an antigen, (ii) an adjuvant, and (iii) an immunomodulator, wherein the antigen is located within the lumen of the EV (e.g., the exogenous body) and is not attached to any scaffold portion, the adjuvant is attached to a scaffold Y on the luminal surface of the EV (e.g., the exogenous body), and the immunomodulator is attached to a scaffold X on the luminal surface of the EV (e.g., the exogenous body). In some aspects, the exogenous body of this disclosure comprises: (i) an antigen, (ii) an adjuvant, and (iii) an immunomodulator, wherein the antigen is located within the lumen of the EV (e.g., the exogenous body) and is not attached to any scaffold portion, the adjuvant is attached to a first scaffold Y on the luminal surface of the EV (e.g., the exogenous body), and the immunomodulator is attached to a second scaffold Y on the luminal surface of the EV (e.g., the exogenous body).

[0420] In some aspects, the exogenous body of this disclosure comprises: (i) an antigen, (ii) an adjuvant, and (iii) an immunomodulator, wherein the antigen is attached to a scaffold Y on the luminal surface of the EV (e.g., the exogenous body), the adjuvant is attached to a scaffold X on the outer surface of the EV (e.g., the exogenous body), and the immunomodulator is located within the lumen of the EV (e.g., the exogenous body) and is not attached to any scaffold portion. In some aspects, the exogenous body of this disclosure comprises: (i) an antigen, (ii) an adjuvant, and (iii) an immunomodulator, wherein the antigen is attached to a scaffold Y on the luminal surface of the EV (e.g., the exogenous body), the adjuvant is attached to a first scaffold X on the outer surface of the EV (e.g., the exogenous body), and the immunomodulator is attached to a second scaffold X on the outer surface of the EV (e.g., the exogenous body). In some aspects, the exogenous body of this disclosure comprises: (i) an antigen, (ii) an adjuvant, and (iii) an immunomodulator, wherein the antigen is attached to a scaffold Y on the luminal surface of the EV (e.g., the exogenous body), the adjuvant is attached to a first scaffold X on the outer surface of the EV (e.g., the exogenous body), and the immunomodulator is attached to a second scaffold X on the luminal surface of the EV (e.g., the exogenous body). In some aspects, the exogenous body of this disclosure comprises: (i) an antigen, (ii) an adjuvant, and (iii) an immunomodulator, wherein the antigen is attached to a first scaffold Y on the luminal surface of the EV (e.g., the exogenous body), the adjuvant is attached to a scaffold X on the outer surface of the EV (e.g., the exogenous body), and the immunomodulator is attached to a second scaffold Y on the luminal surface of the EV (e.g., the exogenous body).

[0421] In some aspects, the exogenous body of this disclosure comprises: (i) an antigen, (ii) an adjuvant, and (iii) an immunomodulator, wherein the antigen is located within the lumen of the EV (e.g., the exogenous body) and is not attached to any scaffold portion, the adjuvant is attached to a scaffold X on the outer surface of the EV (e.g., the exogenous body), and the immunomodulator is located within the lumen of the EV (e.g., the exogenous body) and is not attached to any scaffold portion. In some aspects, the exogenous body of this disclosure comprises: (i) an antigen, (ii) an adjuvant, and (iii) an immunomodulator, wherein the antigen is located within the lumen of the EV (e.g., the exogenous body) and is not attached to any scaffold portion, the adjuvant is attached to a first scaffold X on the outer surface of the EV (e.g., the exogenous body), and the immunomodulator is attached to a second scaffold X on the outer surface of the EV (e.g., the exogenous body). In some aspects, the exogenous body of this disclosure comprises: (i) an antigen, (ii) an adjuvant, and (iii) an immunomodulator, wherein the antigen is located within the lumen of the EV (e.g., the exogenous body) and is not attached to any scaffold portion, the adjuvant is attached to a first scaffold X on the outer surface of the EV (e.g., the exogenous body), and the immunomodulator is attached to a second scaffold X on the luminal surface of the EV (e.g., the exogenous body). In some aspects, the exogenous body of this disclosure comprises: (i) an antigen, (ii) an adjuvant, and (iii) an immunomodulator, wherein the antigen is located within the lumen of the EV (e.g., the exogenous body) and is not attached to any scaffold portion, the adjuvant is attached to a first scaffold X on the outer surface of the EV (e.g., the exogenous body), and the immunomodulator is attached to a scaffold Y on the luminal surface of the EV (e.g., the exogenous body).

[0422] In some aspects, the exogenous body of this disclosure comprises: (i) an antigen, (ii) an adjuvant, and (iii) an immunomodulator, wherein the antigen is attached to a scaffold Y on the luminal surface of the EV (e.g., the exogenous body), the adjuvant is attached to a scaffold X on the luminal surface of the EV (e.g., the exogenous body), and the immunomodulator is located within the lumen of the EV (e.g., the exogenous body) and is not attached to any scaffold portion. In some aspects, the exogenous body of this disclosure comprises: (i) an antigen, (ii) an adjuvant, and (iii) an immunomodulator, wherein the antigen is attached to a scaffold Y on the luminal surface of the EV (e.g., the exogenous body), the adjuvant is attached to a first scaffold X on the luminal surface of the EV (e.g., the exogenous body), and the immunomodulator is attached to a second scaffold X on the outer surface of the EV (e.g., the exogenous body). In some aspects, the exogenous body of this disclosure comprises: (i) an antigen, (ii) an adjuvant, and (iii) an immunomodulator, wherein the antigen is attached to a scaffold Y on the luminal surface of the EV (e.g., the exogenous body), the adjuvant is attached to a first scaffold X on the luminal surface of the EV (e.g., the exogenous body), and the immunomodulator is attached to a second scaffold X on the luminal surface of the EV (e.g., the exogenous body). In some aspects, the exogenous body of this disclosure comprises: (i) an antigen, (ii) an adjuvant, and (iii) an immunomodulator, wherein the antigen is attached to a first scaffold Y on the luminal surface of the EV (e.g., the exogenous body), the adjuvant is attached to a scaffold X on the luminal surface of the EV (e.g., the exogenous body), and the immunomodulator is attached to a second scaffold Y on the luminal surface of the EV (e.g., the exogenous body).

[0423] In some aspects, the exogenous body of this disclosure comprises: (i) an antigen, (ii) an adjuvant, and (iii) an immunomodulator, wherein the antigen is located within the lumen of the EV (e.g., the exogenous body) and is not attached to any scaffold portion, the adjuvant is attached to a scaffold X on the luminal surface of the EV (e.g., the exogenous body), and the immunomodulator is located within the lumen of the EV (e.g., the exogenous body) and is not attached to any scaffold portion. In some aspects, the exogenous body of this disclosure comprises: (i) an antigen, (ii) an adjuvant, and (iii) an immunomodulator, wherein the antigen is located within the lumen of the EV (e.g., the exogenous body) and is not attached to any scaffold portion, the adjuvant is attached to a first scaffold X on the luminal surface of the EV (e.g., the exogenous body), and the immunomodulator is attached to a second scaffold X on the outer surface of the EV (e.g., the exogenous body). In some aspects, the exogenous body of this disclosure comprises: (i) an antigen, (ii) an adjuvant, and (iii) an immunomodulator, wherein the antigen is located within the lumen of the EV (e.g., the exogenous body) and is not attached to any scaffold portion, the adjuvant is attached to a first scaffold X on the luminal surface of the EV (e.g., the exogenous body), and the immunomodulator is attached to a second scaffold X on the luminal surface of the EV (e.g., the exogenous body). In some aspects, the exogenous body of this disclosure comprises: (i) an antigen, (ii) an adjuvant, and (iii) an immunomodulator, wherein the antigen is located within the lumen of the EV (e.g., the exogenous body) and is not attached to any scaffold portion, the adjuvant is attached to a scaffold X on the luminal surface of the EV (e.g., the exogenous body), and the immunomodulator is attached to a scaffold Y on the luminal surface of the EV (e.g., the exogenous body).

[0424] In some aspects, the exogenous body of this disclosure comprises: (i) an antigen, (ii) an adjuvant, and (iii) an immunomodulator, wherein the antigen is attached to a scaffold X on the luminal surface of the EV (e.g., the exogenous body), the adjuvant is attached to a scaffold X on the outer surface of the EV (e.g., the exogenous body), and the immunomodulator is located within the lumen of the EV (e.g., the exogenous body) and is not attached to any scaffold portion. In some aspects, the exogenous body of this disclosure comprises: (i) an antigen, (ii) an adjuvant, and (iii) an immunomodulator, wherein the antigen is attached to a scaffold X on the luminal surface of the EV (e.g., the exogenous body), the adjuvant is attached to a scaffold X on the outer surface of the EV (e.g., the exogenous body), and the immunomodulator is attached to a second scaffold X on the outer surface of the EV (e.g., the exogenous body). In some aspects, the exogenous body of this disclosure comprises: (i) an antigen, (ii) an adjuvant, and (iii) an immunomodulator, wherein the antigen is attached to a scaffold X on the luminal surface of the EV (e.g., the exogenous body), the adjuvant is attached to a scaffold X on the outer surface of the EV (e.g., the exogenous body), and the immunomodulator is attached to a second scaffold X on the luminal surface of the EV (e.g., the exogenous body). In some aspects, the exogenous body of this disclosure comprises: (i) an antigen, (ii) an adjuvant, and (iii) an immunomodulator, wherein the antigen is attached to a scaffold X on the luminal surface of the EV (e.g., the exogenous body), the adjuvant is attached to a scaffold X on the outer surface of the EV (e.g., the exogenous body), and the immunomodulator is attached to a scaffold Y on the luminal surface of the EV (e.g., the exogenous body).

[0425] In some aspects, the exogenous body of this disclosure comprises: (i) an antigen, (ii) an adjuvant, and (iii) an immunomodulator, wherein the antigen is attached to a first scaffold X on the outer surface of the EV (e.g., the exogenous body), the adjuvant is attached to a second scaffold X on the outer surface of the EV (e.g., the exogenous body), and the immunomodulator is located within the lumen of the EV (e.g., the exogenous body) and is not attached to any scaffold portion. In some aspects, the exogenous body of this disclosure comprises: (i) an antigen, (ii) an adjuvant, and (iii) an immunomodulator, wherein the antigen is attached to a first scaffold X on the outer surface of the EV (e.g., the exogenous body), the adjuvant is attached to a second scaffold X on the outer surface of the EV (e.g., the exogenous body), and the immunomodulator is attached to a third scaffold X on the outer surface of the EV (e.g., the exogenous body). In some aspects, the exogenous body of this disclosure comprises: (i) an antigen, (ii) an adjuvant, and (iii) an immunomodulator, wherein the antigen is attached to a first stent X on the outer surface of the EV (e.g., the exogenous body), the adjuvant is attached to a second stent X on the outer surface of the EV (e.g., the exogenous body), and the immunomodulator is attached to a third stent X on the luminal surface of the EV (e.g., the exogenous body). In some aspects, the exogenous body of this disclosure comprises: (i) an antigen, (ii) an adjuvant, and (iii) an immunomodulator, wherein the antigen is attached to a first stent X on the outer surface of the EV (e.g., the exogenous body), the adjuvant is attached to a second stent X on the outer surface of the EV (e.g., the exogenous body), and the immunomodulator is attached to a stent Y on the luminal surface of the EV (e.g., the exogenous body).

[0426] In some aspects, the exogenous body of this disclosure comprises: (i) an antigen, (ii) an adjuvant, and (iii) an immunomodulator, wherein the antigen is attached to a scaffold X on the outer surface of the EV (e.g., the exogenous body), the adjuvant is attached to a scaffold Y on the luminal surface of the EV (e.g., the exogenous body), and the immunomodulator is located within the lumen of the EV (e.g., the exogenous body) and is not attached to any scaffold portion. In some aspects, the exogenous body of this disclosure comprises: (i) an antigen, (ii) an adjuvant, and (iii) an immunomodulator, wherein the antigen is attached to a first scaffold X on the outer surface of the EV (e.g., the exogenous body), the adjuvant is attached to a scaffold Y on the luminal surface of the EV (e.g., the exogenous body), and the immunomodulator is attached to a second scaffold X on the outer surface of the EV (e.g., the exogenous body). In some aspects, the exogenous body of this disclosure comprises: (i) an antigen, (ii) an adjuvant, and (iii) an immunomodulator, wherein the antigen is attached to a first scaffold X on the outer surface of the EV (e.g., the exogenous body), the adjuvant is attached to a scaffold Y on the luminal surface of the EV (e.g., the exogenous body), and the immunomodulator is attached to a second scaffold X on the luminal surface of the EV (e.g., the exogenous body). In some aspects, the exogenous body of this disclosure comprises: (i) an antigen, (ii) an adjuvant, and (iii) an immunomodulator, wherein the antigen is attached to a scaffold X on the outer surface of the EV (e.g., the exogenous body), the adjuvant is attached to a first scaffold Y on the luminal surface of the EV (e.g., the exogenous body), and the immunomodulator is attached to a second scaffold Y on the luminal surface of the EV (e.g., the exogenous body).

[0427] In some aspects, the exogenous body of this disclosure comprises: (i) an antigen, (ii) an adjuvant, and (iii) an immunomodulator, wherein the antigen is attached to a scaffold X on the outer surface of the EV (e.g., the exogenous body), the adjuvant is located within the lumen of the EV (e.g., the exogenous body) and is not attached to any scaffold portion, and the immunomodulator is located within the lumen of the EV (e.g., the exogenous body) and is not attached to any scaffold portion. In some aspects, the exogenous body of this disclosure comprises: (i) an antigen, (ii) an adjuvant, and (iii) an immunomodulator, wherein the antigen is attached to a first scaffold X on the outer surface of the EV (e.g., the exogenous body), the adjuvant is located within the lumen of the EV (e.g., the exogenous body) and is not attached to any scaffold portion, and the immunomodulator is attached to a second scaffold X on the outer surface of the EV (e.g., the exogenous body). In some aspects, the exogenous body of this disclosure comprises: (i) an antigen, (ii) an adjuvant, and (iii) an immunomodulator, wherein the antigen is attached to a first scaffold X on the outer surface of the EV (e.g., the exogenous body), the adjuvant is located within the lumen of the EV (e.g., the exogenous body) and is not attached to any scaffold portion, and the immunomodulator is attached to a second scaffold X on the luminal surface of the EV (e.g., the exogenous body). In some aspects, the exogenous body of this disclosure comprises: (i) an antigen, (ii) an adjuvant, and (iii) an immunomodulator, wherein the antigen is attached to a scaffold X on the outer surface of the EV (e.g., the exogenous body), the adjuvant is located within the lumen of the EV (e.g., the exogenous body) and is not attached to any scaffold portion, and the immunomodulator is attached to a scaffold Y on the luminal surface of the EV (e.g., the exogenous body).

[0428] In some aspects, the exogenous body of this disclosure comprises: (i) an antigen, (ii) an adjuvant, and (iii) an immunomodulator, wherein the antigen is attached to a scaffold X on the outer surface of the EV (e.g., the exogenous body), the adjuvant is attached to a scaffold X on the luminal surface of the EV (e.g., the exogenous body), and the immunomodulator is located within the lumen of the EV (e.g., the exogenous body) and is not attached to any scaffold portion. In some aspects, the exogenous body of this disclosure comprises: (i) an antigen, (ii) an adjuvant, and (iii) an immunomodulator, wherein the antigen is attached to a first scaffold X on the outer surface of the EV (e.g., the exogenous body), the adjuvant is attached to a scaffold X on the luminal surface of the EV (e.g., the exogenous body), and the immunomodulator is attached to a second scaffold X on the outer surface of the EV (e.g., the exogenous body). In some aspects, the exogenous body of this disclosure comprises: (i) an antigen, (ii) an adjuvant, and (iii) an immunomodulator, wherein the antigen is attached to a first scaffold X on the outer surface of the EV (e.g., the exogenous body), the adjuvant is attached to a scaffold X on the luminal surface of the EV (e.g., the exogenous body), and the immunomodulator is attached to a second scaffold X on the luminal surface of the EV (e.g., the exogenous body). In some aspects, the exogenous body of this disclosure comprises: (i) an antigen, (ii) an adjuvant, and (iii) an immunomodulator, wherein the antigen is attached to a scaffold X on the outer surface of the EV (e.g., the exogenous body), the adjuvant is attached to a scaffold X on the luminal surface of the EV (e.g., the exogenous body), and the immunomodulator is attached to a scaffold Y on the luminal surface of the EV (e.g., the exogenous body).

[0429] In some aspects, the exogenous body of this disclosure comprises: (i) an antigen, (ii) an adjuvant, and (iii) an immunomodulator, wherein the antigen is attached to a first scaffold X on the luminal surface of the EV (e.g., the exogenous body), the adjuvant is attached to a second scaffold X on the luminal surface of the EV (e.g., the exogenous body), and the immunomodulator is located within the lumen of the EV (e.g., the exogenous body) and is not attached to any scaffold portion. In some aspects, the exogenous body of this disclosure comprises: (i) an antigen, (ii) an adjuvant, and (iii) an immunomodulator, wherein the antigen is attached to a first scaffold X on the luminal surface of the EV (e.g., the exogenous body), the adjuvant is attached to a second scaffold X on the luminal surface of the EV (e.g., the exogenous body), and the immunomodulator is attached to a third scaffold X on the outer surface of the EV (e.g., the exogenous body). In some aspects, the exogenous body of this disclosure comprises: (i) an antigen, (ii) an adjuvant, and (iii) an immunomodulator, wherein the antigen is attached to a first scaffold X on the luminal surface of the EV (e.g., the exogenous body), the adjuvant is attached to a second scaffold X on the luminal surface of the EV (e.g., the exogenous body), and the immunomodulator is attached to a third scaffold X on the luminal surface of the EV (e.g., the exogenous body). In some aspects, the exogenous body of this disclosure comprises: (i) an antigen, (ii) an adjuvant, and (iii) an immunomodulator, wherein the antigen is attached to a first scaffold X on the luminal surface of the EV (e.g., the exogenous body), the adjuvant is attached to a second scaffold X on the luminal surface of the EV (e.g., the exogenous body), and the immunomodulator is attached to a scaffold Y on the luminal surface of the EV (e.g., the exogenous body).

[0430] In some aspects, the exogenous body of this disclosure comprises: (i) an antigen, (ii) an adjuvant, and (iii) an immunomodulator, wherein the antigen is attached to a scaffold X on the luminal surface of the EV (e.g., the exogenous body), the adjuvant is attached to a scaffold Y on the luminal surface of the EV (e.g., the exogenous body), and the immunomodulator is located within the lumen of the EV (e.g., the exogenous body) and is not attached to any scaffold portion. In some aspects, the exogenous body of this disclosure comprises: (i) an antigen, (ii) an adjuvant, and (iii) an immunomodulator, wherein the antigen is attached to a first scaffold X on the luminal surface of the EV (e.g., the exogenous body), the adjuvant is attached to a scaffold Y on the luminal surface of the EV (e.g., the exogenous body), and the immunomodulator is attached to a second scaffold X on the outer surface of the EV (e.g., the exogenous body). In some aspects, the exogenous entity of this disclosure comprises: (i) an antigen, (ii) an adjuvant, and (iii) an immunomodulator, wherein the antigen is attached to a first scaffold X on the luminal surface of the EV (e.g., the exogenous entity), the adjuvant is attached to a scaffold Y on the luminal surface of the EV (e.g., the exogenous entity), and the immunomodulator is attached to a second scaffold X on the luminal surface of the EV (e.g., the exogenous entity). In some aspects, the exogenous entity of this disclosure comprises: (i) an antigen, (ii) an adjuvant, and (iii) an immunomodulator, wherein the antigen is attached to a scaffold X on the luminal surface of the EV (e.g., the exogenous entity), the adjuvant is attached to a first scaffold Y on the luminal surface of the EV (e.g., the exogenous entity), and the immunomodulator is attached to a second scaffold Y on the luminal surface of the EV (e.g., the exogenous entity).

[0431] In some aspects, the exogenous body of this disclosure comprises: (i) an antigen, (ii) an adjuvant, and (iii) an immunomodulator, wherein the antigen is attached to a scaffold X on the luminal surface of the EV (e.g., the exogenous body), the adjuvant is located within the lumen of the EV (e.g., the exogenous body) and is not attached to any scaffold portion, and the immunomodulator is located within the lumen of the EV (e.g., the exogenous body) and is not attached to any scaffold portion. In some aspects, the exogenous body of this disclosure comprises: (i) an antigen, (ii) an adjuvant, and (iii) an immunomodulator, wherein the antigen is attached to a first scaffold X on the luminal surface of the EV (e.g., the exogenous body), the adjuvant is located within the lumen of the EV (e.g., the exogenous body) and is not attached to any scaffold portion, and the immunomodulator is attached to a second scaffold X on the outer surface of the EV (e.g., the exogenous body). In some aspects, the exogenous body of this disclosure comprises: (i) an antigen, (ii) an adjuvant, and (iii) an immunomodulator, wherein the antigen is attached to a first scaffold X on the luminal surface of the EV (e.g., the exogenous body), the adjuvant is located within the lumen of the EV (e.g., the exogenous body) and is not attached to any scaffold portion, and the immunomodulator is attached to a second scaffold X on the luminal surface of the EV (e.g., the exogenous body). In some aspects, the exogenous body of this disclosure comprises: (i) an antigen, (ii) an adjuvant, and (iii) an immunomodulator, wherein the antigen is attached to a scaffold X on the luminal surface of the EV (e.g., the exogenous body), the adjuvant is located within the lumen of the EV (e.g., the exogenous body) and is not attached to any scaffold portion, and the immunomodulator is attached to a scaffold Y on the luminal surface of the EV (e.g., the exogenous body).

[0432] In some aspects, the exogenous body of this disclosure comprises: (i) an antigen, (ii) an adjuvant, and (iii) an immunomodulator, wherein the antigen is attached to a first stent X on the outer surface of the EV (e.g., the exogenous body), the adjuvant is attached to a second stent X on the luminal surface of the EV (e.g., the exogenous body), and the immunomodulator is located within the lumen of the EV (e.g., the exogenous body) and is not attached to any stent portion. In some aspects, the exogenous body of this disclosure comprises: (i) an antigen, (ii) an adjuvant, and (iii) an immunomodulator, wherein the antigen is attached to a first stent X on the outer surface of the EV (e.g., the exogenous body), the adjuvant is attached to a second stent X on the luminal surface of the EV (e.g., the exogenous body), and the immunomodulator is attached to a third stent X on the outer surface of the EV (e.g., the exogenous body). In some aspects, the exogenous body of this disclosure comprises: (i) an antigen, (ii) an adjuvant, and (iii) an immunomodulator, wherein the antigen is attached to a first stent X on the outer surface of the EV (e.g., the exogenous body), the adjuvant is attached to a second stent X on the luminal surface of the EV (e.g., the exogenous body), and the immunomodulator is attached to a third stent X on the luminal surface of the EV (e.g., the exogenous body). In some aspects, the exogenous body of this disclosure comprises: (i) an antigen, (ii) an adjuvant, and (iii) an immunomodulator, wherein the antigen is attached to a first stent X on the outer surface of the EV (e.g., the exogenous body), the adjuvant is attached to a second stent X on the luminal surface of the EV (e.g., the exogenous body), and the immunomodulator is attached to a stent Y on the luminal surface of the EV (e.g., the exogenous body).

[0433] In some aspects, the exogenous body of this disclosure comprises: (i) an antigen, (ii) an adjuvant, and (iii) an immunomodulator, wherein the antigen is attached to a first stent X on the luminal surface of the EV (e.g., the exogenous body), the adjuvant is attached to a second stent X on the outer surface of the EV (e.g., the exogenous body), and the immunomodulator is located within the lumen of the EV (e.g., the exogenous body) and is not attached to any stent portion. In some aspects, the exogenous body of this disclosure comprises: (i) an antigen, (ii) an adjuvant, and (iii) an immunomodulator, wherein the antigen is attached to a first stent X on the luminal surface of the EV (e.g., the exogenous body), the adjuvant is attached to a second stent X on the outer surface of the EV (e.g., the exogenous body), and the immunomodulator is attached to a third stent X on the outer surface of the EV (e.g., the exogenous body). In some aspects, the exogenous body of this disclosure comprises: (i) an antigen, (ii) an adjuvant, and (iii) an immunomodulator, wherein the antigen is attached to a first scaffold X on the luminal surface of the EV (e.g., the exogenous body), the adjuvant is attached to a second scaffold X on the outer surface of the EV (e.g., the exogenous body), and the immunomodulator is attached to a third scaffold X on the luminal surface of the EV (e.g., the exogenous body). In some aspects, the exogenous body of this disclosure comprises: (i) an antigen, (ii) an adjuvant, and (iii) an immunomodulator, wherein the antigen is attached to a first scaffold X on the luminal surface of the EV (e.g., the exogenous body), the adjuvant is attached to a second scaffold X on the outer surface of the EV (e.g., the exogenous body), and the immunomodulator is attached to a scaffold Y on the luminal surface of the EV (e.g., the exogenous body).

[0434] In some aspects, the exogenous body of this disclosure comprises: (i) an antigen, (ii) an adjuvant, and (iii) an immunomodulator, wherein the antigen is located within the lumen of the EV (e.g., the exogenous body) and is not attached to any scaffold portion, the adjuvant is located within the lumen of the EV (e.g., the exogenous body) and is not attached to any scaffold portion, and the immunomodulator is located within the lumen of the EV (e.g., the exogenous body) and is not attached to any scaffold portion. In some aspects, the exogenous body of this disclosure comprises: (i) an antigen, (ii) an adjuvant, and (iii) an immunomodulator, wherein the antigen is located within the lumen of the EV (e.g., the exogenous body) and is not attached to any scaffold portion, the adjuvant is located within the lumen of the EV (e.g., the exogenous body) and is not attached to any scaffold portion, and the immunomodulator is attached to a scaffold X on the outer surface of the EV (e.g., the exogenous body). In some aspects, the exogenous body of this disclosure comprises: (i) an antigen, (ii) an adjuvant, and (iii) an immunomodulator, wherein the antigen is located within the lumen of the EV (e.g., the exogenous body) and is not attached to any scaffold portion, the adjuvant is located within the lumen of the EV (e.g., the exogenous body) and is not attached to any scaffold portion, and the immunomodulator is attached to a scaffold X on the luminal surface of the EV (e.g., the exogenous body). In some aspects, the exogenous body of this disclosure comprises: (i) an antigen, (ii) an adjuvant, and (iii) an immunomodulator, wherein the antigen is located within the lumen of the EV (e.g., the exogenous body) and is not attached to any scaffold portion, the adjuvant is located within the lumen of the EV (e.g., the exogenous body) and is not attached to any scaffold portion, and the immunomodulator is attached to a scaffold Y on the luminal surface of the EV (e.g., the exogenous body).

[0435] In some aspects, an EV (e.g., an exogenous body) comprises: (i) an antigen, (ii) an adjuvant, and (iii) an immunomodulator, wherein (a1) the antigen is directly attached to the luminal surface of the EV, (a2) the adjuvant is directly attached to the luminal surface of the EV, and (a3) ​​the immunomodulator is directly attached to the luminal surface of the EV. In some aspects, an EV (e.g., an exogenous body) comprises: (i) an antigen, (ii) an adjuvant, and (iii) an immunomodulator, wherein (a1) the antigen is directly attached to the luminal surface of the EV, (a2) the adjuvant is directly attached to the luminal surface of the EV, and (a3) ​​the immunomodulator is directly attached to the outer surface of the EV. In some aspects, an EV (e.g., an exogenous body) comprises: (i) an antigen, (ii) an adjuvant, and (iii) an immunomodulator, wherein (a1) the antigen is directly attached to the luminal surface of the EV, (a2) the adjuvant is directly attached to the luminal surface of the EV, and (a3) ​​the immunomodulator is connected to a scaffold Y within the EV lumen. In some aspects, an EV (e.g., an exogenous body) comprises: (i) an antigen, (ii) an adjuvant, and (iii) an immunomodulator, wherein (a1) the antigen is directly attached to the luminal surface of the EV, (a2) the adjuvant is directly attached to the luminal surface of the EV, and (a3) ​​the immunomodulator is attached to a scaffold X within the EV lumen. In some aspects, an EV (e.g., an exogenous body) comprises: (i) an antigen, (ii) an adjuvant, and (iii) an immunomodulator, wherein (a1) the antigen is directly attached to the luminal surface of the EV, (a2) the adjuvant is directly attached to the luminal surface of the EV, and (a3) ​​the immunomodulator is attached to a scaffold X in the outer surface of the EV. In some aspects, an EV (e.g., an exogenous body) comprises: (i) an antigen, (ii) an adjuvant, and (iii) an immunomodulator, wherein (a1) the antigen is directly attached to the luminal surface of the EV, (a2) the adjuvant is directly attached to the luminal surface of the EV, and (a3) ​​the immunomodulator is within the EV lumen.

[0436] In some aspects, an EV (e.g., an exosome) comprises: (i) an antigen, (ii) an adjuvant, and (iii) an immunomodulator, wherein (a1) the antigen is directly attached to the luminal surface of the EV, (a2) the adjuvant is directly attached to the outer surface of the EV, and (a3) ​​the immunomodulator is directly attached to the luminal surface of the EV. In some aspects, an EV (e.g., an exosome) comprises: (i) an antigen, (ii) an adjuvant, and (iii) an immunomodulator, wherein (a1) the antigen is directly attached to the luminal surface of the EV, (a2) the adjuvant is attached to a scaffold Y on the luminal surface of the EV, and (a3) ​​the immunomodulator is directly attached to the luminal surface of the EV. In some aspects, an EV (e.g., an exosome) comprises: (i) an antigen, (ii) an adjuvant, and (iii) an immunomodulator, wherein (a1) the antigen is directly attached to the luminal surface of the EV, (a2) the adjuvant is attached to a scaffold X on the luminal surface of the EV, and (a3) ​​the immunomodulator is directly attached to the luminal surface of the EV. In some aspects, an EV (e.g., an exogenous body) comprises: (i) an antigen, (ii) an adjuvant, and (iii) an immunomodulator, wherein (a1) the antigen is directly attached to the luminal surface of the EV, (a2) the adjuvant is attached to a scaffold X on the outer surface of the EV, and (a3) ​​the immunomodulator is directly attached to the luminal surface of the EV. In some aspects, an EV (e.g., an exogenous body) comprises: (i) an antigen, (ii) an adjuvant, and (iii) an immunomodulator, wherein (a1) the antigen is directly attached to the luminal surface of the EV, (a2) the adjuvant is within the EV lumen, and (a3) ​​the immunomodulator is directly attached to the luminal surface of the EV.

[0437] In some aspects, an EV (e.g., an exosome) comprises: (i) an antigen, (ii) an adjuvant, and (iii) an immunomodulator, wherein (a1) the antigen is directly attached to the outer surface of the EV, (a2) the adjuvant is directly attached to the luminal surface of the EV, and (a3) ​​the immunomodulator is directly attached to the luminal surface of the EV. In some aspects, an EV (e.g., an exosome) comprises: (i) an antigen, (ii) an adjuvant, and (iii) an immunomodulator, wherein (a1) the antigen is attached to a scaffold Y on the luminal surface of the EV, (a2) the adjuvant is directly attached to the luminal surface of the EV, and (a3) ​​the immunomodulator is directly attached to the luminal surface of the EV. In some aspects, an EV (e.g., an exosome) comprises: (i) an antigen, (ii) an adjuvant, and (iii) an immunomodulator, wherein (a1) the antigen is attached to a scaffold X on the luminal surface of the EV, (a2) the adjuvant is directly attached to the luminal surface of the EV, and (a3) ​​the immunomodulator is directly attached to the luminal surface of the EV. In some aspects, an EV (e.g., an exosome) comprises: (i) an antigen, (ii) an adjuvant, and (iii) an immunomodulator, wherein (a1) the antigen is attached to a scaffold X on the outer surface of the EV, (a2) the adjuvant is directly attached to the luminal surface of the EV, and (a3) ​​the immunomodulator is directly attached to the luminal surface of the EV. In some aspects, an EV (e.g., an exosome) comprises: (i) an antigen, (ii) an adjuvant, and (iii) an immunomodulator, wherein (a1) the antigen is within the lumen of the EV, (a2) the adjuvant is directly attached to the luminal surface of the EV, and (a3) ​​the immunomodulator is directly attached to the luminal surface of the EV.

[0438] In some aspects, an EV (e.g., an exosome) comprises: (i) an antigen, (ii) an adjuvant, and (iii) an immunomodulator, wherein (a1) the antigen is directly attached to the outer surface of the EV, (a2) the adjuvant is directly attached to the outer surface of the EV, and (a3) ​​the immunomodulator is directly attached to the outer surface of the EV. In some aspects, an EV (e.g., an exosome) comprises: (i) an antigen, (ii) an adjuvant, and (iii) an immunomodulator, wherein (a1) the antigen is directly attached to the outer surface of the EV, (a2) the adjuvant is directly attached to the outer surface of the EV, and (a3) ​​the immunomodulator is directly attached to the luminal surface of the EV. In some aspects, an EV (e.g., an exosome) comprises: (i) an antigen, (ii) an adjuvant, and (iii) an immunomodulator, wherein (a1) the antigen is directly attached to the outer surface of the EV, (a2) the adjuvant is directly attached to the outer surface of the EV, and (a3) ​​the immunomodulator is attached to a scaffold Y on the luminal surface of the EV. In some aspects, an EV (e.g., an exosome) comprises: (i) an antigen, (ii) an adjuvant, and (iii) an immunomodulator, wherein (a1) the antigen is directly attached to the outer surface of the EV, (a2) the adjuvant is directly attached to the outer surface of the EV, and (a3) ​​the immunomodulator is attached to a scaffold X on the luminal surface of the EV. In some aspects, an EV (e.g., an exosome) comprises: (i) an antigen, (ii) an adjuvant, and (iii) an immunomodulator, wherein (a1) the antigen is directly attached to the outer surface of the EV, (a2) the adjuvant is directly attached to the outer surface of the EV, and (a3) ​​the immunomodulator is attached to a scaffold X on the outer surface of the EV. In some aspects, an EV (e.g., an exosome) comprises: (i) an antigen, (ii) an adjuvant, and (iii) an immunomodulator, wherein (a1) the antigen is directly attached to the outer surface of the EV, (a2) the adjuvant is directly attached to the outer surface of the EV, and (a3) ​​the immunomodulator is within the EV lumen.

[0439] In some aspects, an EV (e.g., an exosome) comprises: (i) an antigen, (ii) an adjuvant, and (iii) an immunomodulator, wherein (a1) the antigen is directly attached to the outer surface of the EV, (a2) the adjuvant is directly attached to the luminal surface of the EV, and (a3) ​​the immunomodulator is directly attached to the outer surface of the EV. In some aspects, an EV (e.g., an exosome) comprises: (i) an antigen, (ii) an adjuvant, and (iii) an immunomodulator, wherein (a1) the antigen is directly attached to the outer surface of the EV, (a2) the adjuvant is attached to a scaffold Y on the luminal surface of the EV, and (a3) ​​the immunomodulator is directly attached to the outer surface of the EV. In some aspects, an EV (e.g., an exosome) comprises: (i) an antigen, (ii) an adjuvant, and (iii) an immunomodulator, wherein (a1) the antigen is directly attached to the outer surface of the EV, (a2) the adjuvant is attached to a scaffold X on the luminal surface of the EV, and (a3) ​​the immunomodulator is directly attached to the outer surface of the EV. In some aspects, an EV (e.g., an exosome) comprises: (i) an antigen, (ii) an adjuvant, and (iii) an immunomodulator, wherein (a1) the antigen is directly attached to the outer surface of the EV, (a2) the adjuvant is attached to a scaffold X on the outer surface of the EV, and (a3) ​​the immunomodulator is directly attached to the outer surface of the EV. In some aspects, an EV (e.g., an exosome) comprises: (i) an antigen, (ii) an adjuvant, and (iii) an immunomodulator, wherein (a1) the antigen is directly attached to the outer surface of the EV, (a2) the adjuvant is within the EV cavity, and (a3) ​​the immunomodulator is directly attached to the outer surface of the EV.

[0440] In some aspects, an EV (e.g., an exosome) comprises: (i) an antigen, (ii) an adjuvant, and (iii) an immunomodulator, wherein (a1) the antigen is directly attached to the luminal surface of the EV, (a2) the adjuvant is directly attached to the outer surface of the EV, and (a3) ​​the immunomodulator is directly attached to the outer surface of the EV. In some aspects, an EV (e.g., an exosome) comprises: (i) an antigen, (ii) an adjuvant, and (iii) an immunomodulator, wherein (a1) the antigen is attached to a scaffold Y on the luminal surface of the EV, (a2) the adjuvant is directly attached to the outer surface of the EV, and (a3) ​​the immunomodulator is directly attached to the outer surface of the EV. In some aspects, an EV (e.g., an exosome) comprises: (i) an antigen, (ii) an adjuvant, and (iii) an immunomodulator, wherein (a1) the antigen is attached to a scaffold X on the luminal surface of the EV, (a2) the adjuvant is directly attached to the outer surface of the EV, and (a3) ​​the immunomodulator is directly attached to the outer surface of the EV. In some aspects, an EV (e.g., an exosome) comprises: (i) an antigen, (ii) an adjuvant, and (iii) an immunomodulator, wherein (a1) the antigen is attached to a scaffold X on the outer surface of the EV, (a2) the adjuvant is directly attached to the outer surface of the EV, and (a3) ​​the immunomodulator is directly attached to the outer surface of the EV. In some aspects, an EV (e.g., an exosome) comprises: (i) an antigen, (ii) an adjuvant, and (iii) an immunomodulator, wherein (a1) the antigen is within the lumen of the EV, (a2) the adjuvant is directly attached to the outer surface of the EV, and (a3) ​​the immunomodulator is directly attached to the outer surface of the EV.

[0441] In some aspects, immunomodulators that can be used with EVs (e.g., exogens) described herein have antitumor activity. In other aspects, immunomodulators that can be used with the present disclosure have tolerogenic activity. In some aspects, immunomodulators can modulate innate immune responses. In some aspects, immunomodulators modulate innate immune responses by targeting natural killer cells. In some aspects, immunomodulators can modulate adaptive immune responses. In some aspects, immunomodulators modulate adaptive immune responses by targeting cytotoxic T cells. In other aspects, immunomodulators modulate adaptive immune responses by targeting B cells. In some aspects, the immunomodulators disclosed herein can modulate the distribution of exogens to cytotoxic T cells or B cells (i.e., biodistribution modifiers).

[0442] In some respects, immunomodulators include inhibitors of negative checkpoint regulators or inhibitors of binding partners of negative checkpoint regulators. In some respects, negative checkpoint regulators include cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), programmed cell death protein 1 (PD-1), lymphocyte activation gene 3 (LAG-3), protein 3 containing T-cell immunoglobulin mucin (TIM-3), B-lymphocyte and T-lymphocyte attenuator (BTLA), T-cell immune receptor with Ig and ITIM domains (TIGIT), T-cell activation V-domain Ig repressor (VISTA), adenosine A2a receptor (A2aR), cytotoxic cell immunoglobulin-like receptor (KIR), indoleamine 2,3-dioxygenase (IDO), CD20, CD39, CD73, or any combination thereof.

[0443] In some respects, the immunomodulator is an inhibitor of cytotoxic T-lymphocyte-associated protein 4 (CTLA-4). In some respects, the CTLA-4 inhibitor is a monoclonal antibody against CTLA-4 (“anti-CTLA-4 antibody”). In some respects, the inhibitor is a fragment of a monoclonal antibody against CTLA-4. In some respects, the antibody fragment is scFv, (scFv)2, Fab, Fab', and F(ab')2, F(ab1)2, Fv, dAb, or Fd of a monoclonal antibody against CTLA-4. In some respects, the inhibitor is a nanobody, bispecific antibody, or multispecific antibody against CTLA-4. In some respects, the anti-CTLA-4 antibody is ipilimumab. In other respects, the anti-CTLA-4 antibody is trimemumab.

[0444] In some aspects, the immunomodulator is an inhibitor of programmed cell death protein 1 (PD-1). In some aspects, the immunomodulator is an inhibitor of programmed death ligand 1 (PD-L1). In some aspects, the immunomodulator is an inhibitor of programmed death ligand 2 (PD-L2). In some aspects, the inhibitor of PD-1, PD-L1, or PD-L2 is a monoclonal antibody of PD-1 (“anti-PD-1 antibody”), PD-L1 (“anti-PD-L1 antibody”), or PD-L2 (“anti-PD-L2 antibody”). In some aspects, the inhibitor is a fragment of an anti-PD-1 antibody, anti-PD-L1 antibody, or anti-PD-L2 antibody. In some aspects, the antibody fragment is scFv, (scFv)2, Fab, Fab', and F(ab')2, F(ab1)2, Fv, dAb, or Fd of a monoclonal antibody of PD-1, PD-L1, or PD-L2. In some respects, the inhibitor is a nanobody, bispecific antibody, or multispecific antibody targeting PD-1, PD-L1, or PD-L2. In some respects, the anti-PD-1 antibody is nivolumab. In some respects, the anti-PD-1 antibody is pembrolizumab. In some respects, the anti-PD-1 antibody is pidilimumab. In some respects, the anti-PD-L1 antibody is atezolizumab. In other respects, the anti-PD-L1 antibody is avelumab.

[0445] In some respects, immunomodulators are inhibitors of lymphocyte activation gene 3 (LAG3). In some respects, LAG3 inhibitors are monoclonal antibodies against LAG3 (“anti-LAG3 antibodies”). In some respects, inhibitors are fragments of anti-LAG3 antibodies, such as scFv, (scFv)2, Fab, Fab', and F(ab')2, F(ab1)2, Fv, dAb, or Fd. In some respects, inhibitors are nanobodies, bispecific antibodies, or multispecific antibodies targeting LAG3.

[0446] In some respects, immunomodulators are inhibitors of T-cell immunoglobulin mucin 3 (TIM-3). In some respects, immunomodulators are inhibitors of B-lymphocyte and T-lymphocyte attenuating factor (BTLA). In some respects, immunomodulators are inhibitors of T-cell immune receptors (TIGIT) with Ig and ITIM domains. In some respects, immunomodulators are inhibitors of T-cell activation V-domain Ig repressor factor (VISTA). In some respects, immunomodulators are inhibitors of adenosine A2a receptor (A2aR). In some respects, immunomodulators are inhibitors of cytotoxic cell immunoglobulin-like receptors (KIR). In some respects, immunomodulators are inhibitors of indoleamine 2,3-dioxygenase (IDO). In some respects, immunomodulators are inhibitors of CD20, CD39, or CD73.

[0447] In some respects, immunomodulators include activators of positive costimulatory molecules or activators of binding partners of positive costimulatory molecules. In some respects, positive costimulatory molecules include members of the TNF receptor superfamily (e.g., CD120a, CD120b, CD18, OX40, CD40, Fas receptor, M68, CD27, CD30, 4-1BB, TRAILR1, TRAILR2, TRAILR3, TR...

Claims

1. An isolated extracellular vesicle (EV) comprising (i) an antigen, (ii) an adjuvant, and (iii) a first scaffold portion, wherein the first scaffold portion is a prostaglandin F2 receptor negative regulator (PTGFRN) protein or a functional fragment thereof. The antigens mentioned therein are tumor antigens or human papillomavirus (HPV) antigens; The adjuvant is an interferon gene-stimulating factor (STING) agonist or a Toll-like receptor (TLR) agonist, and The antigen is connected to the first scaffold portion.

2. The EV of claim 1, wherein the antigen is not present on MHC class I and / or class II molecules.

3. The EV as described in claim 1, wherein it is not derived from naturally occurring antigen-presenting cells.

4. The EV as described in claim 1, wherein it is not derived from naturally occurring dendritic cells, naturally occurring B cells, naturally occurring mast cells, naturally occurring macrophages, naturally occurring neutrophils, naturally occurring Kupffer-Browicz cells, or any combination thereof.

5. The EV of claim 1, which is capable of inducing cellular immune response, humoral immune response, or cellular and humoral immune response in a subject.

6. The EV of claim 5, which, compared with (i) a corresponding EV that does not contain the adjuvant or the antigen or (ii) an adjuvant or antigen without the EV, is capable of increasing the cellular immune response, the humoral immune response, or the combination of cellular and humoral immune responses in a subject by at least 5%.

7. The EV of claim 5, wherein the cellular immune response comprises a CD4+ T cell response, a CD8+ T cell response, or a CD4+ T cell response and a CD8+ T cell response.

8. The EV as described in claim 1, which does not directly interact with the T cell receptor of T cells.

9. The EV of claim 1, further comprising a second support portion.

10. The EV of claim 9, wherein the adjuvant is connected to the second support portion.

11. The EV of claim 9, wherein the first bracket portion and the second bracket portion are identical.

12. The EV of claim 9, wherein the first support portion and the second support portion are different.

13. The EV of claim 9, wherein the second support portion is a support Y or a support X.

14. The EV of claim 13, wherein the scaffold X is selected from the group consisting of: prostaglandin F2 receptor negative regulator (PTGFRN protein); basigin (BSG protein); immunoglobulin superfamily member 2 (IGSF2 protein); immunoglobulin superfamily member 3 (IGSF3 protein); immunoglobulin superfamily member 8 (IGSF8 protein); integrin β-1 (ITGB1 protein); integrin α-4 (ITGA4 protein); 4F2 cell surface antigen heavy chain (SLC3A2 protein); ATP transporter or fragments thereof, or any combination thereof.

15. The EV of claim 13, wherein the scaffold Y is selected from the group consisting of: protein kinase C substrate rich in myristyl alanine (MARCKS protein); protein kinase C substrate-like protein 1 rich in myristyl alanine (MARCKSL1 protein); brain acid-soluble protein 1 (BASP1 protein) or fragments thereof and any combination thereof.

16. The EV of claim 9, wherein the antigen is connected to a first scaffold portion on the luminal surface of the EV, and the adjuvant is connected to a second scaffold portion on the luminal surface of the EV.

17. The EV of claim 1, wherein the antigen is connected to a first scaffold portion on the luminal surface of the EV, and the adjuvant is located within the cavity of the EV.

18. The EV of claim 9, wherein the antigen is connected to a first scaffold portion on the luminal surface of the EV, and the adjuvant is connected to a second scaffold portion on the outer surface of the EV.

19. The EV of claim 9, wherein the antigen is connected to a first scaffold portion on the outer surface of the EV, and the adjuvant is connected to a second scaffold portion on the luminal surface of the EV.

20. The EV of claim 9, wherein the antigen is connected to a first scaffold portion on the outer surface of the EV, and the adjuvant is connected to a second scaffold portion on the outer surface of the EV.

21. The EV of claim 1, wherein the antigen is connected to a first scaffold portion on the outer surface of the EV, and the adjuvant is located within the cavity of the EV.

22. The EV of claim 9, wherein: (i) the antigen is connected to the first scaffold portion via a connector, (ii) the adjuvant is connected to the second scaffold portion via a connector, or (iii) a combination thereof.

23. The EV of claim 22, wherein the connector is a polypeptide.

24. The EV of claim 22, wherein the connector is a non-peptide portion.

25. The EV of claim 22, wherein the connector comprises a maleimide portion.

26. The EV of claim 22, wherein the connector comprises a cholesterol portion.

27. The EV of claim 14, wherein the scaffold X is a PTGFRN protein or a functional fragment thereof.

28. The EV of claim 27, wherein the scaffold X is composed of the amino acid sequence shown in SEQ ID NO:

33.

29. The EV of claim 27, wherein the scaffold X is composed of an amino acid sequence having at least 70% sequence identity with amino acids 26-879 shown in SEQ ID NO:

1.

30. The EV of claim 27, wherein the stent X is composed of amino acids 26 to 879 of SEQ ID NO:

1.

31. The EV of claim 15, wherein the scaffold Y is a BASP1 protein or a functional fragment thereof.

32. The EV of claim 15, wherein the scaffold Y is composed of the amino acid sequence shown in any one of SEQ ID NO: 50-155 and 246-256.

33. The EV of claim 31, wherein the scaffold Y is composed of an amino acid sequence having at least 70% sequence identity with amino acids 2-227 shown in SEQ ID NO:

49.

34. The EV according to any one of claims 1 to 33, further comprising an immunomodulator.

35. The EV of claim 34, wherein the immunomodulator is directly connected to the cavity surface of the EV, the outer surface of the EV, or both.

36. The EV of claim 34, wherein the immunomodulator is... (i) The bracket X on the outer surface of the EV, (ii) the bracket X on the cavity surface of the EV, or (iii) both (i) and (ii) are connected by a connector.

37. The EV of claim 34, wherein the immunomodulator is connected to the scaffold Y on the luminal surface of the EV via a connector.

38. The EV of claim 36 or 37, wherein the connector is a polypeptide.

39. The EV of claim 36 or 37, wherein the connector is a non-peptide portion.

40. The EV of claim 36 or 37, wherein the connector comprises a maleimide portion.

41. The EV of claim 36 or 37, wherein the connector comprises a cholesterol portion.

42. The EV of claim 34, wherein the immunomodulator is located within the cavity of the EV.

43. The EV of claim 34, wherein the immunomodulator comprises an inhibitor of a negative checkpoint regulator or an inhibitor of a binding partner of a negative checkpoint regulator.

44. The EV of claim 43, wherein the negative checkpoint regulator comprises cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), programmed cell death protein 1 (PD-1), lymphocyte activation gene 3 (LAG-3), protein 3 containing T-cell immunoglobulin mucin (TIM-3), B-lymphocyte and T-lymphocyte attenuator (BTLA), T-cell immune receptor with Ig and ITIM domains (TIGIT), T-cell activation V-domain Ig repressor (VISTA), adenosine A2a receptor (A2aR), cytotoxic cell immunoglobulin-like receptor (KIR), indoleamine 2,3-dioxygenase (IDO), CD20, CD39, CD73, or any combination thereof.

45. The EV of claim 34, wherein the immunomodulator comprises an activator of a positive costimulatory molecule or an activator of a binding partner of a positive costimulatory molecule.

46. ​​The EV of claim 45, wherein the positive co-stimulatory molecule is a member of the TNF receptor superfamily.

47. The EV of claim 45, wherein the activator of the positive co-stimulatory molecule is a member of the TNF superfamily.

48. The EV of claim 45, wherein the positive co-stimulatory molecule is a CD28 superfamily co-stimulatory molecule.

49. The EV of claim 45, wherein the activator of the positive co-stimulatory molecule is ICOSL, CD80, or CD86.

50. The EV of claim 34, wherein the immunomodulator comprises a cytokine or a binding partner of a cytokine.

51. The EV of claim 50, wherein the cytokines comprise IL-2, IL-4, IL-7, IL-10, IL-12, IL-15, IL-21, IFN-γ, IL-1α, IL-1β, IL-1ra, IL-18, IL-33, IL-36α, IL-36β, IL-36γ, IL-36ra, IL-37, IL-38, IL-3, IL-5, IL-6, IL-11, IL-13, IL-23, granulocytes-... Macrophage colony-stimulating factor (GM-CSF), granulocyte-colony-stimulating factor (G-CSF), leukemia inhibitory factor (LIF), stem cell factor (SCF), thrombopoietin (TPO), macrophage colony-stimulating factor (M-CSF), erythropoietin (EPO), Flt-3, IFN-α, IFN-β, IFN-γ, IL-19, IL-20, IL-22, IL-24, TNF-α, TNF-β, BAFF, APRIL, lymphotoxin β (TNF-γ), IL-17A, IL-17B, IL-17C, IL-17D, IL-17E, IL-17F, IL-25, TSLP, IL-35, IL-27, TGF-β, or combinations thereof.

52. The EV of claim 34, wherein the immunomodulator comprises proteins that support intracellular interactions required for germinal center responses.

53. The EV of claim 52, wherein the proteins supporting intracellular interactions required for germinal center responses include members of the signaling lymphocyte activation molecule (SLAM) family, SLAM-associated protein (SAP), ICOS-ICOSL, CD40-40L, CD28 / B7, PD-1 / L1, IL-4 / IL4R, IL21 / IL21R, TLR4, TLR7, TLR8, TLR9, CD180, CD22, or combinations thereof.

54. The EV of claim 53, wherein the SLAM family member includes SLAM, CD48, CD229 (Ly9), Ly108, 2B4, CD84, NTB-A, CRACC, BLAME, CD2F-10, or combinations thereof.

55. The EV of claim 1, wherein the tumor antigen comprises CD8.

56. The EV of claim 1, wherein the adjuvant is a STING agonist.

57. The EV of claim 56, wherein the STING agonist comprises a cyclic dinucleotide STING agonist or a non-cyclic dinucleotide STING agonist.

58. The EV as claimed in any one of claims 1 to 33, wherein the EV is an external body.

59. The EV as claimed in any one of claims 1 to 33, wherein the EV further comprises a targeting portion.

60. The EV of claim 59, wherein the targeting portion specifically binds to a marker of dendritic cells.

61. The EV of claim 60, wherein the marker is present only on the dendritic cells.

62. The EV of claim 60 or 61, wherein the dendritic cells comprise plasmacytoid dendritic cells (pDC), myeloid / conventional dendritic cells 1 (cDC1), myeloid / conventional dendritic cells 2 (cDC2), inflammatory mononuclear cell-derived dendritic cells, Langerhans cells, dermal dendritic cells, lysozyme-expressing dendritic cells (LysoDC), Kupffer cells, or any combination thereof.

63. The EV of claim 62, wherein the dendritic cells are cDC1.

64. The EV of claim 60, wherein the biomarkers include C-type lectin domain family 9 member A (Clec9a) protein, dendritic cell-specific intercellular adhesion molecule-3-capture non-integrin (DC-SIGN), CD207, CD40, Clec6, dendritic cell immune receptor (DCIR), DEC-205, lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), MARCO, Clec12a, Clec10a, DC-asialyl glycoprotein receptor (DC-ASGPR), DC immune receptor 2 (DCIR2), Dectin-1, macrophage mannose receptor (MMR), BDCA-1 (CD303, Clec4c), Dectin-2, Bst-2 (CD317), Langerin, CD206, CD11b, CD11c, CD123, CD304, XCR1, AXL, Siglec 6. CD209, SIRPA, CX3CR1, GPR182, CD14, CD16, CD32, CD34, CD38, CD10 or any combination thereof.

65. The EV of claim 64, wherein the marker is the Clec9a protein.

66. The EV of claim 59, wherein the targeting portion specifically binds to a marker of a T cell.

67. The EV of claim 66, wherein the marker comprises a CD3 molecule.

68. The EV of claim 59, wherein the targeting portion is directly connected to the outer surface of the EV.

69. The EV of claim 59, wherein the targeting portion is connected to a bracket X on the outer surface of the EV.

70. The EV of claim 68, wherein the targeting portion is directly connected to the outer surface of the EV via a connector.

71. The EV of claim 69, wherein the targeting portion is connected to the bracket X via a connector.

72. The EV of claim 70 or 71, wherein the connector is a polypeptide.

73. The EV of claim 70 or 71, wherein the connector is a non-peptide portion.

74. The EV of claim 70 or 71, wherein the connector comprises a maleimide portion.

75. The EV of claim 70 or 71, wherein the connector comprises a cholesterol portion.

76. A pharmaceutical composition comprising the EV of any one of claims 1 to 75 and a pharmaceutically acceptable carrier.

77. A cell that produces EV according to any one of claims 1 to 75.

78. A pillbox comprising the EV according to any one of claims 1 to 75 and instructions for use.

79. A method for preparing EVs, the method comprising culturing the cells of claim 77 under suitable conditions and obtaining the EVs.

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