Exosomes for immuno-oncology and anti-inflammatory therapies

Abstract

Disclosed herein are extracellular vesicles comprising immunomodulatory components. Also provided are methods of producing extracellular vesicles and methods of using extracellular vesicles to treat cancer, GvHD, and autoimmune diseases.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application Nos. 62 / 723,267, filed August 27, 2018; and 62 / 611,140, ​​filed December 28, 2017, the entire contents of each of which are incorporated herein by reference. Field of the Invention

[0003] The present invention relates to compositions for interacting and modulating the human immune system, methods of making the compositions, and methods of using the compositions to treat cancer, GvHD, and autoimmune diseases. Background Art

[0004] Immunotherapy is the treatment of disease by inducing, enhancing or suppressing immune responses. Immunotherapy can stimulate the patient's own immune system to attack cancer cells. Cancer immunotherapy generally has fewer side effects than traditional cancer therapies such as chemotherapy and radiotherapy. Anti-inflammatory immunotherapy can downregulate the patient's immune system to treat autoimmune diseases and graft-versus-host disease (GvHD). Improved methods for delivering immunomodulatory molecules to cells and tissues of the body are needed. Summary of the Invention

[0005] As drug delivery vehicles, extracellular vesicles (EVs) offer numerous advantages over traditional drug delivery methods, particularly for gene therapy. Systemic delivery of EVs results in the distribution of these lipid nanoparticles to various tissues. Studies have shown that EVs can interact with various cells involved in regulating the human immune system. EVs selected, enriched, or engineered to deliver therapeutic molecules that activate, inhibit, or influence the human immune system may be effective therapeutic agents for cancer and other immune-related diseases.

[0006] Provided herein are compositions comprising extracellular vesicles selected, enriched, or engineered to have immunomodulatory components that can upregulate or downregulate the human immune system, either enhancing the patient's immune system to fight cancer or suppressing the patient's immune system to alleviate symptoms of GvHD and autoimmune diseases.

[0007] Also provided are methods of producing and using extracellular vesicles to modulate the human immune system.

[0008] Thus, in a first aspect, provided herein is a composition comprising: an extracellular vesicle comprising a cell membrane defining an enclosed volume, the cell membrane having an inner surface and an outer surface; and a first immunomodulatory component associated with the cell membrane or enclosed within the enclosed volume.

[0009] In various embodiments, the first immunomodulatory component is an inhibitor of a negative checkpoint regulator or an inhibitor of a binding partner of a negative checkpoint regulator. In some of these embodiments, the negative checkpoint regulator is selected from the group consisting of 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 and T lymphocyte attenuation protein (BTLA), T cell immunoreceptor with Ig and ITIM domains (TIGIT), V domain Ig inhibitor of T cell activation (VISTA), adenosine A2a receptor (A2aR), killer cell immunoglobulin-like receptor (KIR), indoleamine 2,3-dioxygenase (IDO), CD20, CD39 and CD73.

[0010] In various embodiments, the first immunomodulatory component is an activator of a positive costimulatory molecule or an activator of a binding partner of a positive costimulatory molecule. In some embodiments, the positive costimulatory molecule is a TNF receptor superfamily member. In some of these embodiments, the TNF receptor superfamily member is selected from the group consisting of: 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 embodiments, the activator of a positive costimulatory molecule is a TNF superfamily member. In some of these embodiments, the TNF superfamily member is selected from the group consisting of 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 certain embodiments, the TNF superfamily member is CD40L. In certain embodiments, the TNF superfamily member is CD27L. In certain embodiments, the TNF superfamily member is OX40L.

[0011] In some embodiments, the positive costimulatory molecule is a CD28 superfamily costimulatory molecule. In some of these embodiments, the CD28 superfamily costimulatory molecule is ICOS or CD28. In some embodiments, the activator of the positive costimulatory molecule is ICOSL, CD80 or CD86. In certain embodiments, the activator of the positive costimulatory molecule is CD80.

[0012] In some embodiments, the first immunomodulatory component is a cytokine or a binding partner of a cytokine. In some embodiments, the cytokine is selected from the group consisting of IL-2, IL-7, IL-10, IL-12, and IL-15. In certain embodiments, the cytokine is IL-7. In certain embodiments, the cytokine is IL-12. In certain embodiments, the cytokine is IL-15.

[0013] In some embodiments, the first immunomodulatory component is a T cell receptor (TCR), a T cell coreceptor, a major histocompatibility complex (MHC), a human leukocyte antigen (HLA), or a derivative thereof.

[0014] In some embodiments, the first immunomodulatory component is an activator of a T cell receptor or coreceptor. In certain embodiments, the activator of a T cell receptor or coreceptor is an activator of CD3, optionally an agonist antibody of CD3.

[0015] In some embodiments, the first immunomodulatory component is a tumor antigen. In some embodiments, the tumor antigen is selected from the group consisting of: 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, cancer-testis antigen, MART-1 gp100, and TNF-related apoptosis-inducing ligand. In certain embodiments, the tumor antigen is derived from a reference genome sequence. In certain embodiments, the tumor antigen is derived from a genomic sequence of a subject.

[0016] In some embodiments, the first immunomodulatory component is an agonist or antagonist of a selected target or activity.

[0017] In some embodiments, the first immunomodulatory component is an antibody or antigen-binding fragment.

[0018] In some embodiments, the first immunomodulatory component is a polynucleotide. In some of these embodiments, the polynucleotide is selected from the group consisting of: mRNA, miRNA, siRNA, antisense RNA, shRNA, lncRNA, and dsDNA.

[0019] In some embodiments, the first immunomodulatory component is a protein, peptide, glycolipid, or glycoprotein.

[0020] In some embodiments, the first immunomodulatory component is expressed as a fusion protein displayed on the outer surface of the extracellular vesicle. In some embodiments, the fusion protein comprises PTGFRN or a fragment or variant thereof. In some embodiments, the sequence of the fusion protein is SEQ ID NO: 3.

[0021] In some embodiments, the extracellular vesicles are exosomes. In some other embodiments, the extracellular vesicles are nanovesicles.

[0022] In certain embodiments, the composition further comprises a pharmaceutically acceptable carrier.

[0023] In some embodiments, the extracellular vesicle further comprises a second immunomodulatory component.

[0024] In various embodiments, the second immunomodulatory component is an inhibitor for a negative checkpoint regulator or an inhibitor for a binding partner of a negative checkpoint regulator. In some of these embodiments, the negative checkpoint regulator is selected from the group consisting of 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 and T lymphocyte attenuation protein (BTLA), T cell immunoreceptor with Ig and ITIM domains (TIGIT), V domain Ig inhibitor of T cell activation (VISTA), adenosine A2a receptor (A2aR), killer cell immunoglobulin-like receptor (KIR), indoleamine 2,3-dioxygenase (IDO), CD20, CD39 and CD73.

[0025] In various embodiments, the second immunomodulatory component is an activator of a positive costimulatory molecule or an activator of a binding partner of a positive costimulatory molecule. In some embodiments, the positive costimulatory molecule is a TNF receptor superfamily member. In some of these embodiments, the TNF receptor superfamily member is selected from the group consisting of: 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 embodiments, the activator of a positive costimulatory molecule is a TNF superfamily member. In some of these embodiments, the TNF superfamily member is selected from the group consisting of 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 certain embodiments, the TNF superfamily member is CD40L. In certain embodiments, the TNF superfamily member is CD27L. In certain embodiments, the TNF superfamily member is OX40L.

[0026] In some embodiments, the positive costimulatory molecule is a CD28 superfamily costimulatory molecule. In some of these embodiments, the CD28 superfamily costimulatory molecule is ICOS or CD28. In some embodiments, the activator of the positive costimulatory molecule is ICOSL, CD80 or CD86. In certain embodiments, the activator of the positive costimulatory molecule is CD80.

[0027] In some embodiments, the second immunomodulatory component is a cytokine or a binding partner for a cytokine. In some embodiments, the cytokine is selected from the group consisting of IL-2, IL-7, IL-10, IL-12, and IL-15. In certain embodiments, the cytokine is IL-7. In certain embodiments, the cytokine is IL-12. In certain embodiments, the cytokine is IL-15.

[0028] In some embodiments, the second immunomodulatory component is a T cell receptor (TCR), a T cell coreceptor, a major histocompatibility complex (MHC), a human leukocyte antigen (HLA), or a derivative thereof.

[0029] In some embodiments, the second immunomodulatory component is an activator of a T cell receptor or coreceptor. In certain embodiments, the activator of a T cell receptor or coreceptor is an activator of CD3, optionally an agonist antibody of CD3.

[0030] In some embodiments, the second immunomodulatory component is a tumor antigen. In some embodiments, the tumor antigen is selected from the group consisting of: 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, cancer-testis antigen, MART-1 gp100, and TNF-related apoptosis-inducing ligand. In certain embodiments, the tumor antigen is derived from a reference genome sequence. In certain embodiments, the tumor antigen is derived from a genomic sequence of a subject.

[0031] In some embodiments, the second immunomodulatory component is an agonist or antagonist of a selected target or activity.

[0032] In some embodiments, the second immunomodulatory component is an antibody or antigen-binding fragment.

[0033] In some embodiments, the second immunomodulatory component is a polynucleotide. In some of these embodiments, the polynucleotide is selected from the group consisting of: mRNA, miRNA, siRNA, antisense RNA, shRNA, lncRNA, and dsDNA.

[0034] In some embodiments, the second immunomodulatory component is a protein, peptide, glycolipid, or glycoprotein.

[0035] In some embodiments, the second immunomodulatory component is expressed as a fusion protein displayed on the outer surface of the extracellular vesicle. In some embodiments, the fusion protein comprises PTGFRN or a fragment or variant thereof. In some embodiments, the sequence of the fusion protein is SEQ ID NO: 3.

[0036] In some embodiments, the second immunomodulatory component is different from the first immunomodulatory component.

[0037] In some embodiments, the extracellular vesicle further comprises a third immunomodulatory component. In some embodiments, the third immunomodulatory component is different from the first and second immunomodulatory components.

[0038] In another aspect, provided herein is a method for producing a composition. In some embodiments, the method comprises modifying a producer cell with a first, second, and / or third immunomodulatory component; obtaining extracellular vesicles from the producer cell; and optionally isolating the obtained extracellular vesicles. In some other embodiments, the method comprises obtaining extracellular vesicles from the producer cell; isolating the obtained extracellular vesicles; and modifying the isolated extracellular vesicles with a first, second, and / or third immunomodulatory component. In certain embodiments, the method further comprises formulating the isolated extracellular vesicles into a pharmaceutical composition.

[0039] In another aspect, provided herein is a method for treating cancer in a subject, comprising administering to the subject a therapeutically effective amount of a composition, wherein the composition is capable of upregulating the subject's immune response, thereby enhancing tumor targeting by the subject's immune system.

[0040] In another aspect, the present invention provides a method for treating graft-versus-host disease (GvHD) in a subject. The method comprises administering to the subject a therapeutically effective amount of a composition, wherein the composition is capable of downregulating the subject's immune response, thereby alleviating the symptoms of GvHD.

[0041] In another aspect, provided herein is a method for treating an autoimmune disease in a subject, comprising administering to the subject a therapeutically effective amount of a composition, wherein the composition is capable of downregulating the subject's immune response, thereby suppressing the subject's immune activity.

[0042] In another aspect, provided herein is a method for treating or preventing cancer in a subject, comprising administering to the subject a therapeutically effective amount of a composition comprising a tumor antigen, wherein the composition is capable of enhancing an immune response to the tumor antigen, thereby enhancing the subject's immune response to cancer.

[0043] In some embodiments, the tumor antigen is selected from the group consisting of 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, alpha-folate receptor, CE7R, IL-3, cancer-testis antigen, MART-1 gplOO, and TNF-related apoptosis-inducing ligand.

[0044] In certain embodiments, the tumor antigen is derived from a reference genomic sequence. In certain embodiments, the tumor antigen is derived from a genomic sequence of a subject. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1A and 1B Shown is the time course of mice injected with radiolabeled exosomes. Figure 1A Intravenous administration route is indicated. Figure 1B The intraperitoneal route of administration is indicated.

[0046] Figure 2 Quantification of exosome distribution in different mouse tissues following intravenous and intraperitoneal administration of radiolabeled exosomes.

[0047] Figure 3A and 3B Shown are the effects of B cell activation in peripheral blood mononuclear cells (PBMCs) from two human donors following incubation with CD40L-expressing exosomes.

[0048] Figure 4A and 4B Shown are the effects of B cell activation on purified B cells from two human donors following incubation with CD40L-expressing exosomes.

[0049] Figure 5A is a schematic diagram of the CD40 reporter cell line. Figure 5B Shown is the concentration-dependent activation of the CD40 reporter cell line treated with anti-CD40 agonistic antibody or recombinant human CD40L. Figure 5C Shown are the effects of CD40L-expressing exosomes on a CD40 reporter cell line.

[0050] Figure 6A and 6BShown are the effects of T cell activation in peripheral blood mononuclear cells (PBMCs) with CD80-expressing exosomes. Figure 6A Shows that CD80-expressing exosomes are positive for CD8 + Effect of T cell numbers. Figure 6B Shows that CD80-expressing exosomes are positive for CD4 + Effect of T cell numbers.

[0051] Figure 7A and 7B Shown are the effects of CD80-expressing exosomes on IFNγ expression in human PBMCs.

[0052] Figure 8A and 8B Shown are the effects of CD27L-expressing exosomes on IFNγ expression in human PBMCs from two donors.

[0053] Figure 9A and 9B Shown are the effects of CD27L-expressing exosomes on IL-2 expression in human PBMCs from two donors.

[0054] Figure 10A and 10B Shown are the effects of OX40L-expressing exosomes on IFNγ expression in human PBMCs from two donors.

[0055] Figure 11A and 11B Shown are the effects of OX40L-expressing exosomes on IL-2 expression in human PBMCs from two donors.

[0056] Figure 12A is a schematic diagram of the OX40 reporter cell line. Figure 12B Shown is the concentration-dependent activation of OX40 reporter cell lines treated with anti-OX40 agonistic antibodies or recombinant human OX40L. Figure 12C Shown are the effects of OX40L-expressing exosomes on an OX40 reporter cell line.

[0057] Figure 13A and 13B Shown are the effects of IL-7 expressing exosomes in combination with anti-CD3 antibodies on IFNγ expression in human PBMCs.

[0058] Figure 14A is a schematic diagram of the IL-7 receptor reporter cell line. Figure 14B Shown is concentration-dependent activation of the IL-7 receptor reporter cell line treated with recombinant human IL-7. Figure 14C Shown are the effects of IL-7 expressing exosomes on an IL-7 receptor reporter cell line.

[0059] Figure 15A and 15B Shown are the effects of IL-7 expressing exosomes on T cell proliferation in mice in vivo, as measured by EdU incorporation. Figure 15A Shown are the effects of IL-7 expressing exosomes on CD8+ T cells. Figure 15B Shown are the effects of IL-7 expressing exosomes on memory CD8+ T cells.

[0060] Figure 16A and 16B Shown are the effects of IL-7 expressing exosomes on T cell proliferation in mice in vivo, as measured by CD71 positivity. Figure 16A Shown are the effects of IL-7 expressing exosomes on CD8+ T cells. Figure 16B Shown are the effects of IL-7 expressing exosomes on memory CD8+ T cells.

[0061] Figure 17A Schematic diagram of the PTGFRN / IL-7 fusion protein expressed at high density on the surface of exosomes, as well as variants of the fusion protein. Figure 17B It is the sequence of the optimized PTGFRN / IL-7 fusion protein.

[0062] Figure 18A is a Western blot showing the relative expression of different IL-7 fusion proteins on the surface of purified exosomes. Figure 18B Shown are the effects of IL-7 expressing exosomes on IL-7 receptor downregulation as a model of IL-7 mediated T cell activation.

[0063] Figure 19A Shown are the effects of anti-CD3 scFv exosomes on T cell activation in PBMCs. Figure 19B Shown are the effects of anti-CD3scFv exosomes on B cell activation in PBMCs.

[0064] Figure 20A Shown are the effects of anti-CD3 scFab exosomes on T cell activation in PBMCs. Figure 20B Shown are the effects of anti-CD3 scFab exosomes on B cell activation in PBMCs.

[0065] Figure 21A is a histogram showing the extent of T cell activation after treatment with anti-CD3 scFv exosomes. Figure 21B is a histogram showing the extent of B cell activation after treatment with anti-CD3 scFv exosomes.

[0066] Figure 22AShown are the effects of anti-CD3 scFab exosomes on T cell activation in a plate-coated activation assay compared to soluble anti-CD3 antibody or plate-coated anti-CD3 antibody. Figure 22B It is a bar graph that quantifies Figure 22A Results of separate experiments conducted in .

[0067] Figure 23A A schematic diagram of the full-length PTGFRN / IL-12 fusion protein is shown. Figure 23B A schematic diagram of the shortened PTGFRN / IL-12 fusion protein is shown.

[0068] Figure 24A Shown are the effects of recombinant human IL-12 or exosomes overexpressing short or full-length PTGFRN-IL-12 on induction of IFNγ in human PBMCs. Figure 24B is a table summarizing the efficacy of recombinant IL-12 and IL-12-containing exosomes.

[0069] Figure 25 Shown are the effects of recombinant IL-12 and IL-12-PTGFRN exosomes on reducing tumor growth in a murine model of melanoma.

[0070] Figure 26A Shown are the Figure 25 Tumor growth curves for each tumor-bearing mouse are shown in . Figure 26B Shown are the Figure 25 Tumor growth curves for each tumor-bearing mouse are shown in . Figure 26C Shown are exosomes treated with IL-12-PTGFRN Figure 25 Tumor growth curves for each tumor-bearing mouse are shown in .

[0071] Figure 27 Shown Figure 25 Images of all B16F10 tumor-bearing mice in the efficacy study are shown in .

[0072] Figure 28 Shown Figure 25 Survival curves of B16F10 tumor-bearing mice are shown in .

[0073] Figure 29A Shown are the levels of IFNγ gene expression in tumors of mice treated with PBS, rIL-12, or IL-12-PTGFRN exosomes. Figure 29B Shown are the levels of CXCL9 gene expression in tumors of mice treated with PBS, rIL-12, or IL-12-PTGFRN exosomes. Figure 29CShown are the levels of CXCL10 gene expression in tumors of mice treated with PBS, rIL-12, or IL-12-PTGFRN exosomes. Figure 29D Shown are the levels of TGFβ gene expression in tumors of mice treated with PBS, rIL-12, or IL-12-PTGFRN exosomes.

[0074] Figure 30 Shown are the percentages of IFNγ-positive CD8+ splenic T cells in tumor-bearing mice treated with PBS, rIL-12, or IL-12-PTGFRN exosomes.

[0075] Figure 31A Schematic representation of full-length PTGFRN fused to an IFNγ monomer is shown. Figure 31B Schematic diagram of full-length PTGFRN fused to IFNγ tandem dimer is shown.

[0076] Figure 32 Shown are the results of PAGE analysis of purified human and mouse monomeric (m) and tandem dimer (td) PTGFRN IFNγ exosomes.

[0077] Figure 33 Figure 2 shows monocyte PD-L1 expression after addition of native exosomes (WT), monomeric IFNγPTGFRN exosomes (m-IFNγ-PTGFRN), and tandem dimeric IFNγPTGFRN exosomes (td-IFNγ-PTGFRN). LPS-induced PD-L1 activation was used as a positive control.

[0078] Figure 34 Schematic diagram of the 15 / IL-15Rα fusion protein fused to the transmembrane domain of PDGFR is shown.

[0079] Figure 35 Shown is NK cell activation measured by the percentage of CD69-positive NK cells after addition of pDisplay IL-15 exosomes.

[0080] Figure 36A Schematic representation of IL-15 fused to full-length PTGFRN and IL-15N72D fused to full-length PTGFRN is shown. Figure 36B Shown are Western blots of IL-15 fused to full-length PTGFRN and IL-15N72D fused to full-length PTGFRN.

[0081] Figure 37 Shown are NK cell activation measured by the percentage of CD69-positive NK cells after addition of IL-15 fused to full-length PTGFRN and IL-15N72D fused to full-length PTGFRN.

[0082] Figure 38 Schematic representation of anti-CD3 antibody fragments fused to the PDGFR transmembrane region (exoCD3-PD), full-length PTGFRN (exoCD3-long), and PTGFRN fragment (exoCD3-short), respectively.

[0083] Figure 39 Shown are the results of biolayer interferometry (BLI) after addition of native exosomes (WT), exosomes with anti-CD3 antibody fragments fused to the PDGFR transmembrane region (pDisplay), exosomes with anti-CD3 antibody fragments fused to full-length PTGFRN (FLPTGFRN), and exosomes with anti-CD3 antibody fragments fused to PTGFRN fragments (short PTGFRN), respectively.

[0084] Figure 40A Shown is CD4+ T cell activation measured by the percentage of CD69-positive CD4+ T cells after addition of anti-CD3 antibody fragments. Figure 40B Shown are CD4+ T cell activation measured by the percentage of CD69-positive CD4+ T cells following addition of native exosomes (exoNative) and exosomes with an anti-CD3 antibody fragment fused to a PTGFRN fragment (exoCD3-short), respectively.

[0085] Figure 41 CD40L-GFP PTGFRN fusion protein and EC for each construct in a B cell activation assay measured by CD69 positivity on B cells are shown. 50 Schematic diagram of .

[0086] Figure 42A Shown are B cell activation measured by the percentage of CD69-positive B cells after addition of native exosomes, exosomes with the trimeric CD40L-PTGFRN construct pCB-527, and exosomes with the trimeric CD40L-PTGFRN construct pCB-766, respectively. Figure 42B Shown are B cell activation measured by the percentage of CD69-positive B cells following addition of exosomes harboring the trimeric CD40L-PTGFRN constructs pCB-527 and pCB-766, respectively, compared to concentration-matched CD40L.

[0087] Figure 43A Shown is B cell activation in donor 1 as measured by the percentage of CD69-positive B cells following addition of exosomes harboring the trimeric CD40L-PTGFRN construct pCB-527. Figure 43BShown is B cell activation in donor 2 measured by the percentage of CD69-positive B cells after addition of exosomes with the trimeric CD40L-PTGFRN construct pCB-527.

[0088] Figure 44A Shown is FACS analysis of native exosomes isolated with anti-CD40L decorated beads and labeled with fluorescent antibodies against IL-12 and CD40L. Figure 44B Shown are FACS analyses of native exosomes isolated with anti-CD40L decorated beads and labeled with fluorescent antibodies against CD81 and CD40L.

[0089] Figure 45A Shown is FACS analysis of PTGFRN-CD40L / IL-12 dual-engineered exosomes isolated with anti-CD40L decorated beads and labeled with fluorescent antibody against CD81. Figure 45B Shown is FACS analysis of PTGFRN-CD40L / IL-12 dual-engineered exosomes isolated with anti-CD40L decorated beads and labeled with fluorescent antibodies against IL-12 and CD40L.

[0090] Figure 46A Shown is FACS analysis of PTGFRN-CD40L / IL-12 dual-engineered exosomes isolated with anti-IL-12 decorated beads and labeled with fluorescent antibodies against IL-12 and CD40L. Figure 46B Shown is FACS analysis of PTGFRN-CD40L / IL-12 dual-engineered exosomes isolated with anti-IL-12 decorated beads and labeled with fluorescent antibody against CD81.

[0091] Figure 47A Shown are the IFNγ responses in human PBMCs from donor 1 after addition of recombinant IL-12, a mixture of recombinant IL-12 and recombinant CD40L, PTGFRN-IL-12 exosomes, double-positive PTGFRN-CD40L / IL-12 exosomes, and a mixture of PTGFRN-IL-12 exosomes and PTGFRN-CD40L exosomes, respectively. Figure 47B Shown are the IFNγ responses in human PBMCs from two donors after the addition of recombinant IL-12, a mixture of recombinant IL-12 and recombinant CD40L, PTGFRN-IL-12 exosomes, double-positive PTGFRN-CD40L / IL-12 exosomes, and a mixture of PTGFRN-IL-12 exosomes and PTGFRN-CD40L exosomes, respectively.

[0092] Figure 48The EC values ​​of IFNγ responses in human PBMCs from donors 1 and 2 after addition of recombinant IL-12, a mixture of recombinant IL-12 and recombinant CD40L, PTGFRN-IL-12 exosomes, double-positive PTGFRN-CD40L / IL-12 exosomes, and a mixture of PTGFRN-IL-12 exosomes and PTGFRN-CD40L exosomes, respectively, are shown. 50 .

[0093] Figure 49A Shown are B cell activation in human PBMCs from donor 1 after addition of recombinant CD40L, a mixture of recombinant IL-12 and recombinant CD40L, PTGFRN-CD40L exosomes, double-positive PTGFRN-CD40L / IL-12 exosomes, and a mixture of PTGFRN-IL-12 exosomes and PTGFRN-CD40L exosomes, respectively. Figure 49B Shown are B cell activation in human PBMCs from donor 2 after addition of recombinant CD40L, a mixture of recombinant IL-12 and recombinant CD40L, PTGFRN-CD40L exosomes, double-positive PTGFRN-CD40L / IL-12 exosomes, and a mixture of PTGFRN-IL-12 exosomes and PTGFRN-CD40L exosomes, respectively.

[0094] Figure 50 The EC values ​​of IFNγ responses in human PBMCs from donors 1 and 2 after addition of recombinant CD40L, a mixture of recombinant IL-12 and recombinant CD40L, PTGFRN-CD40L exosomes, double-positive PTGFRN-CD40L / IL-12 exosomes, and a mixture of PTGFRN-IL-12 exosomes and PTGFRN-CD40L exosomes are shown. 50 .

[0095] Figure 51A Shown is FACS analysis of PTGFRN-CD40L / IL-12 / FLT3L triple engineered exosomes isolated with anti-IL-12 decorated beads and labeled with fluorescent antibodies against IL-12 and CD40L. Figure 51B Shown is FACS analysis of PTGFRN-CD40L / IL-12 / FLT3L triple engineered exosomes isolated with anti-IL-12 decorated beads and labeled with fluorescent antibodies against IL-12 and FLT3L. Figure 51C Shown is FACS analysis of PTGFRN-CD40L / IL-12 / FLT3L triple engineered exosomes isolated with anti-IL-12 decorated beads and labeled with fluorescent antibodies against CD40L and FLT3L.

[0096] Figure 52AShown is FACS analysis of PTGFRN-CD40L / IL-12 / FLT3L triple engineered exosomes isolated with anti-CD40L decorated beads and labeled with fluorescent antibodies against IL-12 and CD40L. Figure 52B Shown is FACS analysis of PTGFRN-CD40L / IL-12 / FLT3L triple engineered exosomes isolated with anti-CD40L decorated beads and labeled with fluorescent antibodies against IL-12 and FLT3L. Figure 52C Shown is FACS analysis of PTGFRN-CD40L / IL-12 / FLT3L triple engineered exosomes isolated with anti-CD40L decorated beads and labeled with fluorescent antibodies against CD40L and FLT3L. DETAILED DESCRIPTION

[0097] Disclosed herein are extracellular vesicles capable of modulating the human immune system. Also provided are methods for producing extracellular vesicles, and methods for using these extracellular vesicles to treat cancer and other immune system-related diseases.

[0098] Before describing the present invention in more detail, it should be understood that the present invention is not limited to the particular embodiments described, as they may, of course, vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be restrictive, as the scope of the present invention will be limited only by the appended claims.

[0099] If a range of values ​​is provided, it is understood that each intervening value between the upper and lower limits of the range (up to one-tenth of the unit of the lower limit, unless the context clearly indicates otherwise) and any other stated or intervening value in the stated range are encompassed within the present invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the present invention, subject to any explicitly excluded limits within the stated ranges. If the stated range includes one or both of the stated limits, ranges excluding one or both of these included limits are also encompassed within the present invention.

[0100] 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 to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, representative illustrative methods and materials are now described.

[0101] All publications and patents cited in this specification are herein incorporated by reference to the same extent as if each individual publication or patent was specifically and individually indicated to be incorporated by reference and are incorporated herein to disclose and describe the methods and / or materials to which the publication is cited.

[0102] It should be noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. It should be further noted that the claims may be drafted to exclude any optional elements. Thus, this statement is intended to serve as antecedent basis for use of such exclusive terminology as "solely," "only," and the like in connection with the recitation of claim elements, or for use of a "negative" limitation.

[0103] It will be apparent to those skilled in the art after reading this disclosure that the individual embodiments described and illustrated herein each have discrete components and features that can be readily separated or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present teachings. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.

[0104] In further describing the subject invention, the subject systems for practicing the subject methods will be discussed in more detail, followed by a review of related methods.

[0105] As used herein, the term "extracellular vesicle" refers to a vesicle of cell origin that includes a membrane that encloses an internal space. Extracellular vesicles include all membrane-bound vesicles whose diameter is smaller than the cell from which they are derived. Typically, the diameter of an extracellular vesicle ranges from 20 nm to 1000 nm and may include various macromolecular cargoes that are displayed on the outer surface of the extracellular vesicle and / or span the membrane within the internal space. The cargo may include nucleic acids, proteins, carbohydrates, lipids, small molecules and / or combinations thereof. For example, but not limited to, extracellular vesicles include apoptotic bodies, cell debris, vesicles derived from cells by direct or indirect manipulation (e.g., by continuous extrusion or treatment with an alkaline solution), vesicles produced by vesicled organelles and living cells (e.g., by direct plasma membrane budding or late endosome fusion with the plasma membrane). Extracellular vesicles can be derived from living or dead organisms, explanted tissues or organs and / or cultured cells.

[0106] As used herein, the term "exosome" refers to a small vesicle of cell origin (with a diameter between 20-300 nm, more preferably between 40-200 nm), which contains a membrane that encloses the internal space and is produced from the cell by direct plasma membrane budding or fusion of late endosomes with the plasma membrane. Exosomes are an extracellular vesicle. Exosomes contain lipids or fatty acids and polypeptides, and may also contain a payload (e.g., a therapeutic agent), a receptor (e.g., a targeting moiety), a polynucleotide (e.g., a nucleic acid, RNA or DNA), a sugar (e.g., a simple sugar, a polysaccharide or a glycan) or other molecules. Exosomes can be derived from production cells and isolated from production cells based on their size, density, biochemical parameters, or a combination thereof.

[0107] As used herein, the term "nanovesicle" refers to a small vesicle (with a diameter of 20-250 nm, more preferably 30-150 nm) derived from a cell, which comprises a membrane that encloses an internal space and is produced from the cell by direct or indirect manipulation so that nanovesicles will not be produced by the production cell without manipulation. Suitable manipulations of the production cell include, but are not limited to, continuous extrusion, treatment with an alkaline solution, ultrasonic treatment, or a combination thereof. In some cases, the production of nanovesicles can lead to the destruction of the production cell. Preferably, the nanovesicle population is substantially free of vesicles derived from the production cell by directly budding from the plasma membrane or by fusion of late endosomes with the plasma membrane. The nanovesicle is an extracellular vesicle. The nanovesicle comprises lipids or fatty acids and polypeptides, and optionally comprises a payload (e.g., a therapeutic agent), a receptor (e.g., a targeting moiety), a polynucleotide (e.g., a nucleic acid, RNA, or DNA), a sugar (e.g., a monosaccharide, a polysaccharide, or a polysaccharide) or other molecules. Once the nanovesicle is derived from the production cell according to the manipulation, the nanovesicle can be isolated from the production cell based on its size, density, biochemical parameters, or a combination thereof.

[0108] The term "extracellular vesicle delivery" or "delivery of extracellular vesicles" refers to the administration and localization of extracellular vesicles to target tissues, cells, and / or organs of a subject. In some embodiments, the immunomodulatory components can be delivered to the cytoplasm of a target cell. In other embodiments, the immunomodulatory components can be delivered to the membrane of a target cell. In some embodiments, the membrane of the extracellular vesicle fuses with the membrane of the target cell.

[0109] As used herein, the term "producer cell" refers to any cell from which extracellular vesicles can be isolated. Producer cells are cells that serve as a source of extracellular vesicles. Producer cells can share protein, lipid, sugar, or nucleic acid components with extracellular vesicles. In some embodiments, producer cells are modified or synthetic cells. In some embodiments, producer cells are cultured or isolated cells. In certain embodiments, producer cells are cell lines. In certain other embodiments, producer cells are primary cells. In some specific embodiments, producer cells are immune cells.

[0110] As used herein, a "membrane" is a boundary layer that separates an interior space from an exterior space and comprises one or more biological compounds, typically lipids, and optionally polypeptides and / or carbohydrates. In some embodiments, the membrane comprises lipids and fatty acids. In some embodiments, the membrane comprises phospholipids, glycolipids, fatty acids, sphingolipids, phosphoglycerides, sterols, cholesterol, and phosphatidylserine. In some of these embodiments, the membrane further comprises one or more polypeptides and / or one or more polysaccharides, such as glycans. Extracellular vesicles comprise a membrane as defined herein.

[0111] As used herein, the term "immunomodulatory component" refers to a therapeutic agent that acts on a target (e.g., a target cell) in contact with an extracellular vesicle and regulates the immune system. The immunomodulatory components that can be introduced into extracellular vesicles and / or production cells include therapeutic agents, such as regulators of checkpoint inhibitors or ligands of checkpoint inhibitors, surface antigens and derivatives thereof, cytokines and derivatives thereof. The immunomodulatory components may also include agonists, antagonists, antibodies and antigen-binding fragments, or polynucleotides, such as siRNA, miRNA, lncRNA, and DNA.

[0112] The term "receptor" refers to a molecule that directs extracellular vesicles to a target and / or facilitates the interaction of extracellular vesicles with a target in a subject. In some embodiments, the receptor is a polypeptide. In some embodiments, the receptor is capable of increasing the concentration of an immunomodulatory component in a tissue of a subject. Examples of receptors include, but are not limited to, those listed in Table 3.

[0113] The term "target" refers to a cell, pathogen, metabolite, polypeptide complex, or any molecule or structure present in a tissue or circulating in the circulatory or lymphatic system of a subject, such as an immune cell or cancer cell. Examples of targets include, but are not limited to, those listed in Table 4.

[0114] A "therapeutic agent" or "therapeutic molecule" includes a compound or molecule that, when present in an effective amount, produces a desired therapeutic, pharmacological, and / or physiological effect on a subject in need thereof. This includes any compound, such as a small molecule drug or a biopharmaceutical (e.g., a polypeptide drug or a nucleic acid drug), which, when administered to a subject, has a measurable or communicable effect on the subject, e.g., it alleviates or alleviates the symptoms of a disease, disorder, or condition.

[0115] As used herein, the term "antibody" encompasses immunoglobulins and fragments thereof produced naturally or partially or entirely synthetically. The term also encompasses any protein having a binding domain homologous to an immunoglobulin binding domain. "Antibody" further includes polypeptides comprising a framework region from an immunoglobulin gene or its fragment that specifically binds and recognizes an antigen. The use of the term antibody is intended to include complete antibodies, polyclonal, monoclonal and recombinant antibodies, fragments thereof, and also includes single-chain antibodies, humanized antibodies, murine antibodies, chimeric, mouse-human, mouse-primate, primate-human monoclonal antibodies, anti-idiotypic antibodies, antibody fragments, such as, for example, scFv, (scFv) 2, Fab, Fab' and F(ab') 2, F(ab1) 2, Fv, dAb and Fd fragments, diabodies and antibody-related polypeptides. Antibodies include bispecific antibodies and multispecific antibodies, as long as they exhibit the desired biological activity or function.

[0116] As used herein, the term "antigen-binding fragment" refers to a fragment of an intact immunoglobulin, as well as any part of a polypeptide, including an antigen-binding region that has the ability to specifically bind to an antigen. For example, an antigen-binding fragment may be a F(ab')2 fragment, a Fab' fragment, a Fab fragment, an Fv fragment, or a scFv fragment, but is not limited thereto. A Fab fragment has one antigen-binding site and comprises the variable regions of the light and heavy chains, the constant region of the light chain, and the first constant region, CH1, of the heavy chain. The difference between a Fab' fragment and a Fab fragment is that the Fab' fragment additionally comprises the hinge region of the heavy chain and at least one cysteine ​​residue at the C-terminus of the CH1 region of the heavy chain. F(ab')2 fragments are produced whereby the cysteine ​​residues of the Fab' fragment are linked by disulfide bonds in the hinge region. Fv fragments are the smallest antibody fragments having only the heavy and light chain variable regions, and recombinant techniques for producing Fv fragments are well known in the art. A two-chain Fv fragment may have a structure in which the heavy chain variable region is linked to the light chain variable region by a non-covalent bond. Single-chain Fv (scFv) fragments can generally have a dimer structure like a two-chain Fv fragment, wherein the heavy chain variable region is covalently bound to the light chain variable region via a peptide linker, or the heavy chain and light chain variable regions are directly connected to each other at their C-termini. Antigen-binding fragments can be obtained using proteases (e.g., complete antibodies are digested with papain to obtain Fab fragments, and digested with pepsin to obtain F(ab')2 fragments), and can be prepared by genetic recombination techniques. dAb fragments are composed of VH domains. Single-chain antibody molecules can comprise polymers having multiple individual molecules, such as dimers, trimers, or other polymers.

[0117] The phrase "nucleic acid molecule" refers to a single- or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases. This includes chromosomal DNA and self-replicating plasmids, vectors, mRNA, tRNA, siRNA, miRNA, and the like. Nucleic acid molecules can be recombinant and can express exogenous polypeptides when the nucleic acid is introduced into a cell.

[0118] The term "agonist" refers to a molecule that binds to a receptor and activates it to produce a biological response. Receptors can be activated by endogenous or exogenous agonists. Non-limiting examples of endogenous agonists include hormones and neurotransmitters. Non-limiting examples of exogenous agonists include drugs. Agonists can be full, partial, or inverse agonists.

[0119] The term "antagonist" refers to a molecule that, when bound to a receptor, blocks or attenuates an agonist-mediated response rather than itself causing a biological response. Many antagonists achieve their efficacy by competing with endogenous ligands or substrates at a structurally defined binding site on the receptor. Non-limiting examples of antagonists include alpha blockers, beta blockers, and calcium channel blockers. Antagonists can be competitive, noncompetitive, or uncompetitive antagonists.

[0120] As used herein, the term "fragment" of a protein refers to a protein that is deleted at the N- and / or C-terminus compared to the naturally occurring protein. Preferably, the fragment of PTGFRN, BSG, IGSF2, IGSF3, IGSF8, ITGB1, ITGA4, SLC3A2, or an ATP transporter retains the ability to specifically target exosomes. Such fragments are also referred to as "functional fragments." In a sense, whether a fragment is functional can be assessed by determining the protein content of exosomes by any method known in the art, including Western blotting, FACS analysis, and fusion of the fragment with an autofluorescent protein such as GFP. In a specific embodiment, the fragment of PTGFRN, BSG, IGSF2, IGSF3, IGSF8, ITGB1, ITGA4, SLC3A2, or an ATP transporter retains at least 50%, 60%, 70%, 80%, 90%, or 100% of the ability of the naturally occurring PTGFRN, BSG, IGSF2, IGSF3, IGSF8, ITGB1, ITGA4, SLC3A2, or an ATP transporter to specifically target exosomes.

[0121] As used herein, the term "variant" of a protein refers to a protein that has certain amino acid sequence identity with another protein after alignment by methods known in the art. Variants of a protein can include substitutions, insertions, deletions, frameshifts, or rearrangements in another protein. In a specific embodiment, the variant is a variant that has at least 70% identity to PTGFRN, BSG, IGSF2, IGSF3, IGSF8, ITGB1, ITGA4, SLC3A2, an ATP transporter, or a fragment of PTGFRN, BSG, IGSF2, IGSF3, IGSF8, ITGB1, ITGA4, SLC3A2, or an ATP transporter. In some embodiments, the variant of PTGFRN or a variant of a fragment has at least 70%, 80%, 85%, 90%, 95%, or 99% sequence identity to PTGFRN according to SEQ ID NO: 1, or a functional fragment thereof. In some embodiments, the variant of BSG or a variant of a fragment thereof has at least 70%, 80%, 85%, 90%, 95% or 99% sequence identity with BSG according to SEQ ID NO: 9 or a functional fragment thereof. In some embodiments, the variant of IGSF2 or a variant of a fragment thereof has at least 70%, 80%, 85%, 90%, 95% or 99% sequence identity with IGSF2 according to SEQ ID NO: 34 or a functional fragment thereof. In some embodiments, the variant of IGSF3 or a variant of a fragment thereof has at least 70%, 80%, 85%, 90%, 95% or 99% sequence identity with IGSF3 according to SEQ ID NO: 20 or a functional fragment thereof. In some embodiments, the variant of IGSF8 or a variant of a fragment thereof has at least 70%, 80%, 85%, 90%, 95% or 99% sequence identity with IGSF8 according to SEQ ID NO: 14 or a functional fragment thereof. In some embodiments, the variant or variant of an ITGB1 fragment has at least 70%, 80%, 85%, 90%, 95% or 99% sequence identity with ITGB1 according to SEQ ID NO: 21, or a functional fragment thereof. In some embodiments, the variant or variant of an ITGA4 fragment has at least 70%, 80%, 85%, 90%, 95% or 99% sequence identity with ITGA4 according to SEQ ID NO: 22, or a functional fragment thereof. In some embodiments, the variant or variant of an SLC3A2 fragment has at least 70%, 80%, 85%, 90%, 95% or 99% sequence identity with SLC3A2 according to SEQ ID NO: 23, or a functional fragment thereof. In some embodiments, the variant or variant of an ATP1A1 fragment has at least 70%, 80%, 85%, 90%, 95% or 99% sequence identity with ATP1A1 according to SEQ ID NO: 24, or a functional fragment thereof.In some embodiments, the variants or variants of ATP1A2 have at least 70%, 80%, 85%, 90%, 95% or 99% sequence identity with ATP1A2 according to SEQ ID NO: 25, or a functional fragment thereof. In some embodiments, the variants or variants of ATP1A3 have at least 70%, 80%, 85%, 90%, 95% or 99% sequence identity with ATP1A3 according to SEQ ID NO: 26, or a functional fragment thereof. In some embodiments, the variants or variants of ATP1A4 have at least 70%, 80%, 85%, 90%, 95% or 99% sequence identity with ATP1A4 according to SEQ ID NO: 27, or a functional fragment thereof. In some embodiments, the variants or variants of ATP1B3 have at least 70%, 80%, 85%, 90%, 95% or 99% sequence identity with ATP1B3 according to SEQ ID NO: 28, or a functional fragment thereof. In some embodiments, the variants or variants of ATP2B1 have at least 70%, 80%, 85%, 90%, 95% or 99% sequence identity with ATP2B1 according to SEQ ID NO: 29, or a functional fragment thereof. In some embodiments, the variants or variants of ATP2B2 have at least 70%, 80%, 85%, 90%, 95% or 99% sequence identity with ATP2B2 according to SEQ ID NO: 30, or a functional fragment thereof. In some embodiments, the variants or variants of ATP2B3 have at least 70%, 80%, 85%, 90%, 95% or 99% sequence identity with ATP2B3 according to SEQ ID NO: 31, or a functional fragment thereof. In some embodiments, the variants or variants of ATP2B4 have at least 70%, 80%, 85%, 90%, 95% or 99% sequence identity with ATP2B4 according to SEQ ID NO: 32, or a functional fragment thereof. In each of the above cases, it is preferred that the variant or variant of the fragment retains the ability to specifically target exosomes.

[0122] Methods of alignment of sequences for comparison are well known in the art. Various programs and alignment algorithms are described in Smith and Waterman, Adv. Appl. Math. 2:482 (1981); Needleman and Wunsch, J. Mol. Bio. 48:443 (1970); Pearson and Lipman, Methods in Mol. Biol. 24:307-31 (1988); Higgins and Sharp, Gene 73:15 237-44 (1988); Higgins and Sharp, CABIOS 5:151-3 (1989); Corpet et al., Nuc. Acids Res. 16:10881-90 (1988); Huang et al., Comp. Appl. BioSci. 8:155-65 (1992); and Pearson et al., Meth. Mol. Biol. 24:307-31 (1994). The NCBI Basic Local Alignment Search Tool (BLAST) [Altschul et al., J. Mol. Biol. 215:403-10 (1990)] is available from a variety of sources, including the National Center for Biotechnology Information (NBC1, Bethesda, Maryland) and the internet, for use in conjunction with the sequence analysis programs blastp, blasm, blastx, tblastn, and tblastx. BLAST and instructions for determining sequence identity using the program can be accessed on the official website of the NCBI (National Center for Biotechnology Information) at the NIH (National Institutes of Health).

[0123] The description of any protein provided herein includes functional variants of that protein. The term "functional variant" of a protein refers to a variant of the protein that retains the ability to specifically target exosomes.

[0124] As used herein, the term "pharmaceutical composition" refers to one or more compounds described herein, such as extracellular vesicles, mixed or admixed with or suspended in one or more other chemical components, such as pharmaceutically acceptable carriers and excipients. One purpose of a pharmaceutical composition is to facilitate the administration of a formulation of extracellular vesicles to a subject. The term "pharmaceutically acceptable" and its grammatical variations refer to compositions, carriers, diluents, and agents that can be administered to or on a subject without producing undesirable physiological effects to the extent that administration of the composition is prohibited. The term "excipient" or "carrier" refers to an inert substance added to a pharmaceutical composition to further facilitate administration of a compound. The term "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" encompasses any pharmaceutical agent approved by a U.S. federal government regulatory agency or listed in the U.S. Pharmacopoeia for use in animals (including humans), as well as any carrier or diluent that does not cause significant irritation to the subject and does not abrogate the biological activity and properties of the administered compound. Excipients and carriers that are generally safe, non-toxic, and desirable for preparing pharmaceutical compositions are included.

[0125] As used herein, the terms "isolate," "isolated," and "isolating," or "purify," "purified," and "purifying," and "extracted" and "extracting" are used interchangeably and refer to a state (e.g., a plurality of known or unknown amounts and / or concentrations) of a desired extracellular vesicle preparation that has undergone one or more purification processes, such as selection or enrichment for a desired extracellular vesicle preparation. In some embodiments, isolation or purification as used herein is a process of removing, partially removing (e.g., a portion) extracellular vesicles from a sample containing producer cells. In some embodiments, the isolated extracellular vesicle composition has no detectable undesirable activity, or the level or amount of the undesirable activity is at or below an acceptable level or amount. In other embodiments, the isolated extracellular vesicle composition has an amount and / or concentration of the desired extracellular vesicles at or above an acceptable amount and / or concentration. In other embodiments, the isolated extracellular vesicle composition is enriched compared to the starting material (e.g., producer cell preparation) from which the composition is obtained. Compared to the starting material, such enrichment can be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.999%, 99.9999%, or greater than 99.9999%. In some embodiments, the isolated extracellular vesicle preparation is substantially free of residual biological products. In some embodiments, the isolated extracellular vesicle preparation is 100% free, 99% free, 98% free, 97% free, 96% free, or 95% free of any contaminating biological substances. Residual biological products may include non-biological materials (including chemicals) or unwanted nucleic acids, proteins, lipids, or metabolites. Substantially free of residual biological products can also mean that the extracellular vesicle composition does not contain detectable producer cells and only extracellular vesicles are detectable.

[0126] The terms "administration," "administering," and variations thereof refer to the introduction of a composition, such as an extracellular vesicle or an agent, into a subject, and include simultaneous or sequential introduction of the composition or agent. The composition or agent is introduced into the subject by any suitable route, including oral, intrapulmonary, intranasal, parenteral (intravenous, intraarterial, intramuscular, intraperitoneal, or subcutaneous), rectal, intralymphatic, intrathecal, intratumoral, intraocular, or topical. Administration includes self-administration and administration by another person. A suitable route of administration allows the composition or agent to perform its intended function. For example, if the suitable route is intravenous, the composition is administered by introducing the composition or agent into a vein of the subject.

[0127] As used herein, the terms "modulate," "modulating," "modify," and / or "modulator" generally refer to the ability to change by increasing or decreasing, e.g., directly or indirectly, to promote / stimulate / upregulate or interfere with / inhibit / downregulate a specific concentration, level, expression, function, or behavior, e.g., act as an antagonist or agonist. In some cases, a modulator can increase and / or decrease a certain concentration, level, activity, or function relative to a control, or relative to a generally expected average activity level or relative to a control activity level.

[0128] The term "sufficient amount" refers to an amount sufficient to produce a desired effect, eg, an amount sufficient to modulate a condition in a subject.

[0129] The term "therapeutically effective amount" is an amount effective to ameliorate symptoms of a disease. A therapeutically effective amount can be a "prophylactically effective amount" since prophylaxis can be considered treatment.

[0130] As used herein, the term "substantially" or "substantial" refers to, for example, the presence, level, or concentration of an entity in a particular space, the effect of one entity on another entity, or the effect of a treatment. For example, the activity, level, or concentration of an entity is substantially increased if it increases by 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 50-fold, 100-fold, or 1000-fold relative to a baseline. The activity, level, or concentration of an entity is also substantially increased if it increases by 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, or 500% relative to a baseline.

[0131] The term "in vivo" refers to processes that occur in a living organism.

[0132] As used herein, the term "mammal" includes human and non-human mammals.

[0133] Abbreviations used in this application include: “mRNA” refers to messenger RNA, “miRNA” refers to microRNA, “siRNA” refers to small interfering RNA, “antisense RNA” refers to single-stranded RNA complementary to mRNA, “shRNA” refers to small or short hairpin RNA, “lncRNA” refers to long noncoding RNA, and “dsDNA” refers to double-stranded DNA.

[0134] Composition

[0135] Various aspects of the present disclosure include compositions capable of modulating the immune system. The compositions comprise extracellular vesicles comprising a cell membrane and an immunomodulatory component associated with the cell membrane or enclosed within a membrane-bound enclosed volume.

[0136] Extracellular vesicles

[0137] In various embodiments, the composition comprises an extracellular vesicle.In certain embodiments, the extracellular vesicle is a cell-derived vesicle comprising a membrane enclosing an interior space.

[0138] In various embodiments, the extracellular vesicles can be membrane-bound vesicles that have a diameter smaller than that of the cell from which they originate. In some embodiments, the longest dimension of the extracellular vesicle is about 20-1000 nm, e.g., 20-100 nm, 20-200 nm, 20-300 nm, 20-400 nm, 20-500 nm, 20-600 nm, 20-700 nm, 20-800 nm, 20-900 nm, 30-100 nm, 30-200 nm, 30-300 nm, 30-400 nm, 30-500 nm, 30-600 nm. , 30-700nm, 30-800nm, 30-900nm, 40-100nm, 40-200nm, 40-300nm, 40-400nm, 40-500nm, 40-600nm, 40-700nm, 40-800nm, 40-900nm, 50-150nm, 50-500nm, 50-750nm, 100-200nm, 100-500nm or 500-1000nm.

[0139] In certain embodiments, the extracellular vesicle is an exosome. In certain embodiments, the extracellular vesicle is a nanovesicle. In certain embodiments, the extracellular vesicle is an apoptotic body. In certain embodiments, the extracellular vesicle is a cell fragment. In certain embodiments, the extracellular vesicle is a vesicle derived from a cell by direct or indirect manipulation. In certain embodiments, the extracellular vesicle is a vesicled organelle. In various embodiments, the extracellular vesicle is a vesicle produced by a living cell.

[0140] In some embodiments, the extracellular vesicles are derived from living organisms. In some embodiments, the extracellular vesicles are derived from dead organisms. In some embodiments, the extracellular vesicles are derived from explanted tissues. In some embodiments, the extracellular vesicles are derived from explanted organs. In some embodiments, the extracellular vesicles are derived from cultured cells. In some of these embodiments, when the extracellular vesicles are produced in a cell culture system, the extracellular vesicles are further separated (for example, by separating the extracellular vesicles from the cultured cells). Separation can be achieved by sedimentation. For example, the specific density of the extracellular vesicles can be 0.5-2.0, 0.6-1.0, 0.7-1.0, 0.8-1.0, 0.9-1.0, 1.0-1.1, 1.1-1.2, 1.2-1.3, 1.4-1.5, 1.0-1.5, 1.5-2.0 and 1.0-2.0 kg / m3 . Separation can also be achieved by affinity purification. For example, extracellular vesicles can be purified by binding a population comprising extracellular vesicles to a resin comprising multiple ligands with specific affinity for one or more target proteins on the surface of the extracellular vesicles. The target protein can be a tetraspanin (e.g., CD63, CD81, CD9), an EWI protein / immunoglobulin superfamily member (e.g., PTGFRN, IGSF8, IGSF3), an integrin (e.g., ITGB1, ITGA4), an ATP transporter (e.g., ATP1A1, ATP1A2, ATP1A3, ATP1A4, ATP1B3, ATP2B1, ATP2B2, ATP2B3, ATP2B4), SLC3A2, BSG or CD98hc. The target protein can additionally be an immunomodulatory component displayed on the surface of the exosome.

[0141] In various embodiments, the extracellular vesicle comprises a lipid or fatty acid and a polypeptide. In certain embodiments, the extracellular vesicle further comprises a sugar. In certain embodiments, the extracellular vesicle further comprises a polynucleotide.

[0142] In various embodiments, the extracellular vesicle membrane comprises an inner surface and an outer surface, and encloses an interior space. In some embodiments, the extracellular vesicle further comprises a payload. In certain embodiments, the payload is enclosed within the interior space. In certain embodiments, the payload is displayed on the outer surface of the extracellular vesicle. In certain embodiments, the payload spans the membrane of the extracellular vesicle. In various embodiments, the payload comprises nucleic acids, proteins, carbohydrates, lipids, small molecules and / or combinations thereof. In some embodiments, the extracellular vesicle further comprises a receptor.

[0143] exosomes

[0144] In various embodiments, the extracellular vesicles are exosomes. In certain embodiments, exosomes are small membrane-bound vesicles secreted by production cells.

[0145] In some embodiments, the longest dimension of the exosomes from the producer cells is about 20-300 nm, e.g., about 20-290 nm, 20-280 nm, 20-270 nm, 20-260 nm, 20-250 nm, 20-240 nm, 20-230 nm, 20-220 nm, 20-210 nm, 20-200 nm, 20-190 nm, 20-180 nm, 20-170 nm, 20-160 nm, 20-150 nm, 20-140 nm, 20-130 nm, 20-120 nm, 20-110 nm, 20-100 nm, 20-90 nm, 20-80 nm, 20-70 nm, 20-60 nm , 20-50nm, 20-40nm, 20-30nm, 30-300nm, 30-290nm, 30-280nm, 30-270nm, 3 0-260nm, 30-250nm, 30-240nm, 30-230nm, 30-220nm, 30-210nm, 30-200nm, 3 0-190nm, 30-180nm, 30-170nm, 30-160nm, 30-150nm, 30-140nm, 30-130nm, 30-120nm, 30-110nm, 30-100nm, 30-90nm, 30-80nm, 30-70nm, 30-60nm, 30-5 0nm, 30-40nm, 40-300nm, 40-290nm, 40-280nm, 40-270nm, 40-260nm, 40-25 0nm, 40-240nm, 40-230nm, 40-220nm, 40-210nm, 40-200nm, 40-190nm,, 40- 180nm, 40-170nm, 40-160nm, 40-150nm, 40-140nm, 40-130nm, 40-120nm, 40 -110nm, 40-100nm, 40-90nm, 40-80nm, 40-70nm, 40-60nm, 40-50nm, 50-300n m, 50-290nm, 50-280nm, 50-270nm, 50-260nm, 50-250nm, 50-240nm, 50-230 nm, 50-220nm, 50-210nm, 50-200nm, 50-190nm, 50-180nm, 50-170nm, 50-160 nm, 50-150nm, 50-140nm, 50-130nm, 50-120nm, 50-110nm, 50-100nm, 50-90 nm, 50-80nm, 50-70nm, 50-60nm, 60-300nm, 60-290nm, 60-280nm, 60-270nm,60-260nm, 60-250nm, 60-240nm, 60-230nm, 60-220nm, 60-210nm, 60-200nm, 60-190nm, 60-180nm, 60-170nm, 60-160nm, 60-150nm, 60-140nm, 60-130nm, 60-120nm, 60-110nm, 60-100nm, 60-90nm, 60-80nm, 60-70nm, 70-300nm, 70-290nm, 70-280nm, 70-270nm, 70-2 60nm, 70-250nm, 70-240nm, 70-230nm, 70-220nm, 70-210nm, 70-200nm, 70-190nm, 70-180nm, 70-170nm, 70-160nm, 70-150nm, 70-1 40nm, 70-130nm, 70-120nm, 70-110nm, 70-100nm, 70-90nm, 70-80nm, 80-300nm, 80-290nm, 80-280nm, 80-270nm, 80-260nm, 80-250n m, 80-240nm, 80-230nm, 80-220nm, 80-210nm, 80-200nm, 80-190nm, 80-180nm, 80-170nm, 80-160nm, 80-150nm, 80-140nm, 80-130n m, 80-120nm, 80-110nm, 80-100nm, 80-90nm, 90-300nm, 90-290nm, 90-280nm, 90-270nm, 90-260nm, 90-250nm, 90-240nm, 90-230nm, 90-220nm, 90-210nm, 90-200nm, 90-190nm, 90-180nm, 90-170nm, 90-160nm, 90-150nm, 90-140nm, 90-130nm, 90-120nm, 90-110nm, 90-100nm, 100-300nm, 110-290nm, 120-280nm, 130-270nm, 140-260nm, 150-250nm, 160-240nm, 170-230nm, 180-220nm or 190-210nm.

[0146] In a particularly preferred embodiment, the longest dimension of the exosomes from the production cells described herein is about 30-100 nm. In another preferred embodiment, the longest dimension of the exosomes from the production cells is about 20-300 nm. In another preferred embodiment, the longest dimension of the exosomes from the production cells is about 40-200 nm. In another embodiment, the exosome population described herein includes a population in which 90% of the exosomes have a longest dimension of 20-300 nm. In another embodiment, the exosome population described herein includes a population in which 95% of the exosomes have a longest dimension of 20-300 nm. In another embodiment, the exosome population described herein includes a population in which 99% of the exosomes have a longest dimension of 20-300 nm. In another embodiment, the exosome population described herein includes a population in which 90% of the exosomes have a longest dimension of 40-200 nm. In another embodiment, the exosome population described herein includes a population in which 95% of the exosomes have a longest dimension of 40-200 nm. In another embodiment, the population of exosomes described herein comprises a population wherein 99% of the exosomes are between 40-200 nm in their longest dimension. In other preferred embodiments, the size of an exosome or exosome population described herein is measured according to the methods described below.

[0147] In some embodiments, allochthon is produced by production cell.In some embodiments, allochthon's film comprises one or more molecules derived from production cell.In some embodiments, allochthon is produced and separated (for example, by separating allochthon from production cell) in cell culture system.Separation can be achieved by sedimentation.For example, the specific density of allochthon can be 0.5-2.0, 0.6-1.0, 0.7-1.0, 0.8-1.0, 0.9-1.0, 1.0-1.1, 1.1-1.2, 1.2-1.3, 1.4-1.5, 1.0-1.5, 1.5-2.0 and 1.0-2.0kg / m 3. Separation can also be achieved by affinity purification. For example, extracellular vesicles can be purified by binding a colony comprising extracellular vesicles to a resin comprising multiple ligands with specific affinity for one or more target proteins on the surface of the extracellular vesicles. One or more target proteins can be tetraspanins (e.g., CD63, CD81, and / or CD9), EWI protein / immunoglobulin superfamily members (e.g., PTGFRN, IGSF8, and / or IGSF3), integrins (e.g., ITGB1 and / or ITGA4), ATP transporters (e.g., ATP1A1, ATP1A2, ATP1A3, ATP1A4, ATP1B3, ATP2B1, ATP2B2, ATP2B3, and / or ATP2B4), SLC3A2, BSG, or CD98hc. The target protein can additionally be an immunomodulatory component displayed on the surface of the exosome.

[0148] In some embodiments, the exosome membrane comprises an inner surface and an outer surface. In certain embodiments, the inner surface faces the inner core of the exosome. In certain embodiments, the outer surface can contact the endosome, multivesicular body, or membrane / cytoplasm of the producer cell or target cell.

[0149] In some embodiments, the exosome membrane comprises lipids and fatty acids. In some embodiments, the exosome membrane comprises phospholipids, glycolipids, fatty acids, sphingolipids, phosphoglycerides, sterols, cholesterol, and phosphatidylserine. In some embodiments, the lipids and fatty acids can be one or more of those listed in Table 1.

[0150] In certain embodiments, the exosome comprises a lipid bilayer composed of an inner leaflet and an outer leaflet. The composition of the inner leaflet and the outer leaflet can be determined by transbilayer distribution assays known in the art, see, for example, Kuypers et al. Biohim Biophys Acta 1985 819:170. In some embodiments, the composition of the outer leaflet is about 70-90% choline phospholipids, about 0-15% acidic phospholipids, and about 5-30% phosphatidylethanolamine. In some embodiments, the composition of the inner leaflet is about 15-40% choline phospholipids, about 10-50% acidic phospholipids, and about 30-60% phosphatidylethanolamine.

[0151] In some embodiments, the exosome membrane further comprises one or more polypeptides. In certain embodiments, the exosome comprises one or more polypeptides selected from the following list, including but not limited to spectrin, myosin-like polypeptide, band 3, SLC4A1, actin, actin-like polypeptide, glyceraldehyde 3-P dehydrogenase (G3PD), tetraspanins (e.g., CD63, CD81, and / or CD9), Alix and TSG101, integrins (e.g., ITGB1 and / or ITGA4), selectins, CR1, TNFRI, proteolytic enzymes, glycosylphosphatidylinositol (GPI)-related proteins or histones, EWI proteins / immunoglobulin superfamily members (e.g., PTGFRN, IGSF8, and / or IGSF3), ATP transporters (e.g., ATP1A1, ATP1A2, ATP1A3, ATP1A4, ATP1B3, ATP2B1, ATP2B2, ATP2B3, and / or ATP2B4), SLC3A2, BSG, or CD98hc. In some embodiments, the exosome comprises at least one polypeptide selected from Table 2.

[0152] In some embodiments, allochthon comprises polypeptide on its surface. In some embodiments, allochthon is modified to comprise one or more polypeptides. In some embodiments, production cell is modified to comprise one or more polypeptides. In some embodiments, production cell naturally comprises one or more polypeptides, and the allochthon derived therefrom also comprises polypeptide. The level of any desired surface marker can be modified directly on allochthon (for example, by making complex and recombinant polypeptide contact to insert or conjugate the membrane of complex). Alternatively or in addition, the level of any desired surface marker can be modified directly on production cell (for example, by making complex and recombinant polypeptide contact to insert or conjugate the membrane of cell). Alternatively, production cell can be modified by transducing exogenous nucleic acid into production cell to express required surface marker. Surface marker can already be naturally present on production cell, in which case, exogenous construct can cause overexpression of marker and increase the concentration of marker in production cell or on production cell. Alternatively, naturally expressed surface marker can be removed from production cell (for example, by inducing gene silencing in production cell). Polypeptide can give allochthon different functions (for example, specific targeting ability, delivery function (for example, fusion molecule), enzyme function, half-life in vivo increase or decrease etc.). In some embodiments, polypeptides include, but are not limited to, CD47, CD55, CD49, CD40, CD133, CD59, syndecans-1, CD9, CD63, CD81, integrins, selectins, lectins, and cadherins.

[0153] In certain embodiments, the exosomes comprise one or more polypeptides on their surface, wherein the polypeptides are selected from a group of proteins recently identified as being enriched on the surface of exosomes (described in detail in U.S. patent application 62 / 550,543, which is incorporated herein by reference in its entirety). This group of polypeptides includes prostaglandin F2 receptor negative regulator (PTGFRN); basic immunoglobulin (basigin; BSG); immunoglobulin superfamily member 3 (IGSF3); immunoglobulin superfamily member 8 (IGSF8); integrin beta-1 (ITGB1); integrin alpha-4 (ITGA4); 4F2 cell surface antigen heavy chain (SLC3A2); and a class of ATP transporters (ATP1A1, ATP1A2, ATP1A3, ATP1A4, ATP1B3, ATP2B1, ATP2B2, ATP2B3, ATP2B4).

[0154] In some embodiments, the exosome membrane further comprises one or more polysaccharides, such as glycans.

[0155] In some embodiments, allochthon is delivered to target by payload (therapeutic agent).Payful load is the therapeutic agent that acts on the target (such as target cell) contacted with allochthon.Contact can occur in vitro or in subject.The payload that can introduce allochthon and / or production cell includes therapeutic agent, such as nucleotide (for example, comprising detectable part or toxin or destroying nucleotide of transcription), nucleic acid (for example, DNA or mRNA molecule of encoding polypeptide such as enzyme, or RNA molecule with regulatory function, such as miRNA, dsDNA, lncRNA or siRNA), amino acid (for example, comprising detectable part amino acid or destroying the toxin of translation), polypeptide (for example, enzyme), lipid, carbohydrate and small molecule (for example, small molecule drug and toxin).

[0156] Exosomes can interact with target cells through membrane fusion and deliver the payload (e.g., therapeutic agent) in the exosome composition to the surface or cytoplasm of the target cell. In some embodiments, membrane fusion occurs between the plasma membrane of the exosome and the target cell. In other embodiments, membrane fusion occurs between the endosomal membrane of the exosome and the target cell.

[0157] In some embodiments, allochthon comprises receptor polypeptide.Receptor polypeptide can be synthetic.In some embodiments, receptor polypeptide is introduced into production cell (for example, exogenous nucleic acid encoding receptor polypeptide is introduced into production cell) or in the recombinant receptor polypeptide (for example, synthesized by protein expression system) prepared outside production cell.In some embodiments, receptor polypeptide (for example, polypeptide produced by recombinant production) is directly introduced into allochthon (for example, after allochthon is separated from production cell).In some embodiments, receptor polypeptide can be on the surface of allochthon.In some embodiments, receptor polypeptide can be targeted to the specific target (for example, target such as pathogen, metabolite, polypeptide complex or cell such as non-functional cell or cancer cell) that circulates in the circulatory system of subject by allochthon, for example blood or target located in tissue (for example diseased tissue).

[0158] In some embodiments, allochthon is synthetic.For example, allochthon can comprise payload, such as for example therapeutic polypeptide, nucleic acid (such as DNA or RNA) or other polynucleotide, polysaccharide or polysaccharide, lipid or fatty acid, large organism, small molecule or toxin, so that allochthon is not naturally occurring.In some embodiments, allochthon is modified (for example, by introducing payload or otherwise modifying the content of complex, for example, by changing the protein, lipid or polysaccharide content of membrane).For example, allochthon is first isolated from production cell, then modified as needed, so as to produce synthetic allochthon.In some embodiments, production cell is modified.For example, exogenous nucleic acid, exogenous polypeptide or small molecule or toxin can be introduced into production cell.Alternatively or additionally, production cell can be modified in other ways (for example, by changing the content of cell or membrane, for example, by changing the lipid or polysaccharide content of cell membrane).The allochthon produced from modified production cell comprises one or more modifications of production cell.This process produces synthetic allochthon.In some embodiments, production cell and the allochthon separated from production cell are all modified as described herein.

[0159] Nanovesicles

[0160] In various embodiments, the extracellular vesicles are nanovesicles. In certain embodiments, the nanovesicles are small vesicles of cell origin that contain a membrane enclosing an interior space and are produced from cells by direct or indirect manipulation, such that nanovesicles are not produced by the production cells without manipulation. Suitable manipulations of the cells include, but are not limited to, continuous extrusion, treatment with alkaline solutions, sonication, or a combination thereof, and in some cases can result in the destruction of the production cells.

[0161] In various embodiments, the longest dimension of the nanovesicle is about 20-250 nm, e.g., about 20-100 nm, 20-150 nm, 20-200 nm, 30-100 nm, 30-150 nm, 30-200 nm, 30-250 nm, 40-100 nm, 40-150 nm, 40-200 nm, 40-250 nm, 50-100 nm, 50-150 nm, 50-200 nm, 50-250 nm, 100-200 nm, or 150-250 nm.

[0162] In various embodiments, the nanovesicles are derived from producer cells. In certain embodiments, the nanovesicles are generated from the producer cells by direct or indirect manipulation. Suitable manipulations include, but are not limited to, continuous extrusion, treatment with an alkaline solution, sonication, or a combination thereof. In some of these embodiments, the manipulations may result in the destruction of the producer cells. In some preferred embodiments, the population of nanovesicles is substantially free of vesicles derived from the producer cells by direct budding from the plasma membrane or fusion of late endosomes with the plasma membrane.

[0163] In some embodiments, the nanovesicles are separated from the production cells based on their size, density, biochemical parameters, or a combination thereof. In certain embodiments, separation can be achieved by sedimentation. For example, the specific density of the nanovesicles can be 0.5-2.0, 0.6-1.0, 0.7-1.0, 0.8-1.0, 0.9-1.0, 1.0-1.1, 1.1-1.2, 1.2-1.3, 1.4-1.5, 1.0-1.5, 1.5-2.0, and 1.0-2.0 kg / m 3 .

[0164] In various embodiments, the nanovesicle comprises a lipid or fatty acid and a polypeptide. In certain embodiments, the nanovesicle further comprises a sugar. In certain embodiments, the nanovesicle further comprises a polynucleotide. In some embodiments, the nanovesicle further comprises a receptor. In some embodiments, the nanovesicle further comprises a payload. In some of these embodiments, the payload comprises a nucleic acid, a protein, a carbohydrate, a lipid, a small molecule, and / or a combination thereof.

[0165] Immunomodulatory components

[0166] In various embodiments, the composition further comprises an immunomodulatory component.

[0167] In some embodiments, immunomodulatory compounds are proteins expressed as translational fusion proteins with exosome surface proteins so that the proteins are retained on the exosome surface. In certain embodiments, immunomodulatory compounds are membrane proteins. In certain embodiments, immunomodulatory compounds are soluble proteins. In some embodiments, exosome surface proteins are tetraspanins (e.g., CD63, CD81, CD9), EWI protein / immunoglobulin superfamily members (e.g., PTGFRN, IGSF8, IGSF3), integrins (e.g., ITGB1, ITGA4), ATP transporters (e.g., ATP1A1, ATP1A2, ATP1A3, ATP1A4, ATP1B3, ATP2B1, ATP2B2, ATP2B3, ATP2B4), SLC3A2, BSG or CD98hc or its fragment or variant.

[0168] In some embodiments, the immunomodulatory compound is a soluble protein expressed as a translational fusion protein with an exosome surface protein such that the soluble protein is retained on the exosome surface. In some embodiments, the exosome surface protein is a tetraspanin (e.g., CD63, CD81, CD9), an EWI protein / immunoglobulin superfamily member (e.g., PTGFRN, IGSF8, IGSF3), an integrin (e.g., ITGB1, ITGA4), an ATP transporter (e.g., ATP1A1, ATP1A2, ATP1A3, ATP1A4, ATP1B3, ATP2B1, ATP2B2, ATP2B3, ATP2B4), SLC3A2, BSG or CD98hc or a fragment or variant thereof.

[0169] In certain embodiments, the immunomodulatory component has anti-tumor activity. In some embodiments, the immunomodulatory component modulates the innate immune response. In some of these embodiments, the immunomodulatory component targets natural killer cells. In some other embodiments, the immunomodulatory component modulates the adaptive immune response. In some of these embodiments, the immunomodulatory component targets cytotoxic T cells.

[0170] In some embodiments, immunomodulatory components are expressed in production cells in their full-length form. In other embodiments, immunomodulatory components are expressed as translational fusion proteins with exosome surface proteins, which results in a higher level of expression of the biologically active portion of the immunomodulatory compound on the exosome surface. In some embodiments, immunomodulatory compounds are soluble proteins expressed as translational fusion proteins with exosome surface proteins, such that the soluble proteins are retained on the exosome surface. In some embodiments, exosome surface proteins are tetraspanins (e.g., CD63, CD81, CD9), EWI protein / immunoglobulin superfamily members (e.g., PTGFRN, IGSF8, IGSF3), integrins (e.g., ITGB1, ITGA4), ATP transporters (e.g., ATP1A1, ATP1A2, ATP1A3, ATP1A4, ATP1B3, ATP2B1, ATP2B2, ATP2B3, ATP2B4), SLC3A2, BSG or CD98hc or its fragment or variant.

[0171] In some embodiments, the immunomodulatory component is an inhibitor of a negative checkpoint modulator. In some embodiments, the immunomodulatory component is an inhibitor of a binding partner of a negative checkpoint modulator.

[0172] In certain embodiments, the immunomodulatory component is an inhibitor of cytotoxic T lymphocyte-associated protein 4 (CTLA-4). In some of these embodiments, the CTLA-4 inhibitor is a monoclonal antibody to CTLA-4. In certain embodiments, the inhibitor is a fragment of a monoclonal antibody to CTLA-4. In certain embodiments, the antibody fragment is scFv, (scFv)2, Fab, Fab', and F(ab')2, F(ab1)2, Fv, dAb or Fd of a monoclonal antibody to CTLA-4. In certain embodiments, the inhibitor is a nanobody, bispecific antibody or multispecific antibody against CTLA-4. In some specific embodiments, the monoclonal antibody is ipilimumab. In some specific embodiments, the monoclonal antibody is tesimumab.

[0173] In certain embodiments, the immunomodulatory component is an inhibitor of programmed cell death protein 1 (PD-1). In certain embodiments, the immunomodulatory component is an inhibitor of programmed death ligand 1 (PD-L1). In certain embodiments, the immunomodulatory component is an inhibitor of programmed death ligand 2 (PD-L2). In some embodiments, the inhibitor of PD-1, PD-L1 or PD-L2 is a monoclonal antibody to PD-1, PD-L1 or PD-L2. In certain embodiments, the inhibitor is a fragment of a monoclonal antibody to PD-1, PD-L1 or PD-L2. In certain embodiments, the antibody fragment is scFv, (scFv)2, Fab, Fab' and F(ab')2, F(ab1)2, Fv, dAb or Fd of a monoclonal antibody to PD-1, PD-L1 or PD-L2. In certain embodiments, the inhibitor is a nanobody, bispecific antibody or multispecific antibody against PD-1, PD-L1 or PD-L2. In some specific embodiments, the monoclonal antibody is nivolumab. In some specific embodiments, the monoclonal antibody is pembrolizumab. In some specific embodiments, the monoclonal antibody is pidilizumab. In some specific embodiments, the monoclonal antibody is atezolizumab. In some specific embodiments, the monoclonal antibody is avelumab.

[0174] In certain embodiments, the immunomodulatory component is an inhibitor of lymphocyte activation gene 3 (LAG3). In some of these embodiments, the inhibitor of LAG3 is a monoclonal antibody to LAG3.

[0175] In certain embodiments, the immunomodulatory component is an inhibitor of protein 3 (TIM-3) containing T cell immunoglobulin mucin. In certain embodiments, the immunomodulatory component is an inhibitor of B and T lymphocyte attenuation protein (BTLA). In certain embodiments, the immunomodulatory component is an inhibitor of T cell immune receptor (TIGIT) with Ig and ITIM domains. In certain embodiments, the immunomodulatory component is an inhibitor of V domain Ig inhibitor (VISTA) of T cell activation. In certain embodiments, the immunomodulatory component is an inhibitor of adenosine A2a receptor (A2aR). In certain embodiments, the immunomodulatory component is an inhibitor of killer cell immunoglobulin-like receptor (KIR). In certain embodiments, the immunomodulatory component is an inhibitor of indoleamine 2,3-dioxygenase (IDO). In certain embodiments, the immunomodulatory component is an inhibitor of CD20, CD39 or CD73.

[0176] In some embodiments, the immunomodulatory component is an activator of a positive co-stimulatory molecule. In some embodiments, the immunomodulatory component is an activator of a binding partner of a positive co-stimulatory molecule.

[0177] In some embodiments, the immunomodulatory component is a TNF receptor superfamily member of the activator. In certain embodiments, the TNF receptor superfamily member is selected from the group consisting of: 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, GITR, TROY, CD358, TRAMP and XEDAR. In some embodiments, the immunomodulatory component is a TNF superfamily member. In certain embodiments, the TNF superfamily member is selected from the group consisting of 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.

[0178] In some embodiments, the activator of the TNF receptor superfamily member is expressed as a monomeric protein. In some embodiments, the activator of the TNF receptor superfamily member is expressed as a trimeric protein. In some embodiments, the TNF receptor superfamily member is expressed as a monomeric protein. In some embodiments, the TNF receptor superfamily member is expressed as a trimeric protein.

[0179] In certain embodiments, the immunomodulatory component is an activator of TNF receptor superfamily member 4 (OX40). In some of these embodiments, the activator of OX40 is an agonist antibody of OX40. In some other of these embodiments, the activator of OX40 is OX40 ligand (OX40L).

[0180] In certain embodiments, the immunomodulatory component is an activator of CD27. In some of these embodiments, the activator of CD27 is an agonist antibody of CD27. In some other of these embodiments, the agonist of CD27 is a CD27 ligand (CD27L).

[0181] In certain embodiments, the immunomodulatory component is an activator of CD40. In some of these embodiments, the activator of CD40 is an agonist antibody of CD40. In some other of these embodiments, the agonist of CD40 is CD40 ligand (CD40L). In some embodiments, CD40L is monomeric CD40L. In some embodiments, CD40L is trimeric CD40L.

[0182] In some embodiments, trimeric CD40L is fused to PTGFRN or a fragment thereof. In some embodiments, trimeric CD40L is fused to the N-terminus of PTGFRN or a fragment thereof. In some embodiments, trimeric CD40L is expressed as a fusion protein with PTGFRN, wherein the polypeptide has the sequence of SEQ ID NO: 19 or SEQ ID NO: 20.

[0183] In certain embodiments, the immunomodulatory component is an activator of glucocorticoid-induced TNFR-related protein (GITR). In some of these embodiments, the activator of GITR is an agonist antibody of GITR. In some other of these embodiments, the activator of GITR is a natural ligand of GITR.

[0184] In certain embodiments, the immunomodulatory component is an activator of 4-1BB. In some of these embodiments, the activator of 4-1BB is an agonist antibody of 4-1BB. In some other of these embodiments, the activator of 4-1BB is a natural ligand of 4-1BB.

[0185] In some embodiments, the immunomodulatory component is a Fas receptor (Fas). In some of these embodiments, the Fas receptor is displayed on the surface of an extracellular vesicle. In some other embodiments, the immunomodulatory component is a Fas ligand (FasL). In some of these embodiments, the Fas ligand is displayed on the surface of an extracellular vesicle. In certain embodiments, the immunomodulatory component is an antibody to the Fas receptor. In certain embodiments, the immunomodulatory component is an antibody to the Fas ligand.

[0186] In some embodiments, the immunomodulatory component is an activator of a CD28 superfamily costimulatory molecule. In certain embodiments, the CD28-superfamily costimulatory molecule is ICOS or CD28. In certain embodiments, the immunomodulatory component is ICOSL, CD80, or CD86.

[0187] In certain embodiments, the immunomodulatory component is an activator of inducible T cell co-stimulator (ICOS). In some of these embodiments, the activator of ICOS is an agonist antibody of ICOS. In some other of these embodiments, the activator of ICOS is ICOS ligand (ICOSL).

[0188] In certain embodiments, the immunomodulatory component is an activator of CD28. In some of these embodiments, the activator of CD28 is an agonist antibody of CD28. In some other of these embodiments, the activator of CD28 is a natural ligand of CD28. In certain embodiments, the ligand of CD28 is CD80.

[0189] In certain embodiments, the composition comprises an inhibitor of a negative checkpoint modulator or an inhibitor of a binding partner of a negative checkpoint modulator and an activator of a positive co-stimulatory molecule or an activator of a binding partner of a positive co-stimulatory molecule.

[0190] In certain embodiments, the immunomodulatory component is a cytokine. In some embodiments, the cytokine is a soluble cytokine that has been fused to an exosome surface protein or a fragment thereof in a translational manner. In some embodiments, the cytokine is interleukin 2 (IL-2). In some embodiments, the cytokine is interleukin 7 (IL-7). In some embodiments, the cytokine is interleukin 12 (IL-12). In some embodiments, the cytokine is interleukin 15 (IL-15).

[0191] In certain embodiments, the cytokine is fused to PTGFRN or a fragment thereof. In some embodiments, IL-7 is fused to PTGFRN or a fragment thereof. In some embodiments, IL-7 is fused to the N-terminus of PTGFRN or a fragment thereof. In some embodiments, IL-7 is expressed as a fusion protein with PTGFRN, wherein the polypeptide has the sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

[0192] In certain embodiments, the cytokine is fused to PTGFRN or a fragment thereof. In some embodiments, IL-12 is fused to PTGFRN or a fragment thereof. In some embodiments, IL-12 is fused to the N-terminus of PTGFRN or a fragment thereof. In some embodiments, IL-12 is expressed as a fusion protein with PTGFRN, wherein the polypeptide has the sequence of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6.

[0193] In certain embodiments, the cytokine is fused to PTGFRN or a fragment thereof. In some embodiments, IL-15 is fused to PTGFRN or a fragment thereof. In some embodiments, IL-15 is fused to the N-terminus of PTGFRN or a fragment thereof. In some embodiments, IL-15 is expressed as a fusion protein with PTGFRN, wherein the polypeptide has the sequence of SEQ ID NO: 15 or SEQ ID NO: 16.

[0194] In some embodiments, the cytokine is an interferon (IFN). In certain embodiments, the interferon is fused to PTGFRN or a fragment thereof. In certain embodiments, the interferon is interferon gamma (IFNγ). In some embodiments, IFNγ is fused to PTGFRN or a fragment thereof. In some embodiments, IFNγ is fused to the N-terminus of PTGFRN or a fragment thereof. In some embodiments, IFNγ is expressed as a fusion protein with PTGFRN, wherein the polypeptide has the sequence of SEQ ID NO:7 or SEQ ID NO:8.

[0195] In some embodiments, the immunomodulatory component is a T cell receptor (TCR) or a derivative thereof. In certain embodiments, the immunomodulatory component is a TCR alpha chain or a derivative thereof. In certain embodiments, the immunomodulatory component is a TCR beta chain or a derivative thereof. In some embodiments, the immunomodulatory component is a T cell co-receptor or a derivative thereof.

[0196] In some embodiments, the immunomodulatory component is a tumor antigen. In certain embodiments, the tumor antigen is selected from the group consisting of 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, cancer-testis antigen, MART-1 gpl00, and TNF-related apoptosis-inducing ligand.

[0197] In certain embodiments, the tumor antigen is carcinoembryonic antigen (CEA).In certain embodiments, the tumor antigen is epithelial tumor antigen (ETA).

[0198] In certain embodiments, the tumor antigen is a mucin. In some of these embodiments, the mucin is a secreted mucin. In some other of these embodiments, the mucin is a transmembrane mucin. In a specific embodiment, the tumor antigen is mucin 1 (MUC1). In a specific embodiment, the tumor antigen is Tn-MUC1. In a specific embodiment, the tumor antigen is mucin 16 (MUC16).

[0199] In certain embodiments, the tumor antigen is a melanoma-associated antigen (MAGE). In some of these embodiments, the MAGE is a type I MAGE. In some other of these embodiments, the MAGE is a type II MAGE. In specific embodiments, the type I MAGE is MAGE-A2. In specific embodiments, the type I MAGE is MAGE-A4.

[0200] In certain embodiments, the tumor antigen is alpha-fetoprotein (AFP). In certain embodiments, the tumor antigen is tumor protein p53 (p53). In certain embodiments, the tumor antigen is tyrosinase. In certain embodiments, the tumor antigen is tyrosinase-related protein (TRP). In some embodiments, the tumor antigen is programmed death ligand 1 (PD-L1) or programmed death ligand 2 (PD-L2). In various embodiments, the tumor antigen is selected from the group consisting of CD4, CD8, CD45, CD80, and CD86.

[0201] In some embodiments, the immunomodulatory component is a chimeric antigen receptor (CAR) or a derivative thereof. In some embodiments, CAR binds to 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, cancer-testis antigen, MART-1gp100 and TNF-related apoptosis-inducing ligand One or more.

[0202] In some embodiments, the immunomodulatory component is an activator of a T cell receptor or coreceptor. In certain embodiments, the immunomodulatory component is an activator of CD3. In certain embodiments, the activator is a fragment of a monoclonal antibody against CD3. In certain embodiments, 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 CD3. In certain embodiments, the activator is a nanobody, bispecific antibody, or multispecific antibody against CD3. In some embodiments, the anti-CD3 antibody fragment is fused to PTGFRN or a fragment thereof. In some embodiments, the anti-CD3 antibody fragment is fused to the N-terminus of PTGFRN or a fragment thereof. In some embodiments, the anti-CD3 antibody fragment is expressed as a fusion protein with PTGFRN, wherein the polypeptide has the sequence of SEQ ID NO:18 or SEQ ID NO:21. In certain embodiments, the immunomodulatory component is an activator of CD28. In certain embodiments, the inhibitor is a fragment of a monoclonal antibody against CD28. In certain embodiments, the antibody fragment is scFv, (scFv)2, Fab, Fab', and F(ab')2, F(ab1)2, Fv, dAb or Fd of a monoclonal antibody to CD28. In certain embodiments, the inhibitor is a nanobody, bispecific antibody or multispecific antibody to CD28.

[0203] In some embodiments, the immunomodulatory component is a major histocompatibility complex (MHC) or a derivative thereof. In some of these embodiments, the immunomodulatory component is class I MHC or a derivative thereof. In some of these embodiments, the immunomodulatory component is class II MHC or a derivative thereof. In some of these embodiments, the immunomodulatory component is class III MHC or a derivative thereof.

[0204] In some embodiments, the immunomodulatory component is a human leukocyte antigen (HLA) or a derivative thereof. In some of these embodiments, the immunomodulatory component is HLA-A, HLA-B, HLA-C or a derivative thereof. In some of these embodiments, the immunomodulatory component is HLA-E, HLA-F, HLA-G or a derivative thereof. In some of these embodiments, the immunomodulatory component is HLA-DP, HLA-DQ, HLA-DR or a derivative thereof.

[0205] In various embodiments, the immunomodulatory component can be a polypeptide, a polynucleotide, a polysaccharide, a lipid, a small molecule, or a toxin.

[0206] In some embodiments, the immunomodulatory component can be a protein, peptide, glycolipid, or glycoprotein.

[0207] In certain embodiments, the immunomodulatory component is an agonist. In some of these embodiments, the agonist is an endogenous agonist, such as a hormone or neurotransmitter. In some other of these embodiments, the agonist is an exogenous agonist, such as a drug. In some embodiments, the agonist is a physical agonist that can produce an agonist response without binding to a receptor. In some embodiments, the agonist is a superagonist that can produce a maximum response greater than that of an endogenous agonist. In certain embodiments, the agonist is a full agonist that has complete efficacy on a receptor. In certain other embodiments, the agonist is a partial agonist that has only partial efficacy on a receptor relative to a full agonist. In some embodiments, the agonist is an inverse agonist that can inhibit the constitutive activity of a receptor. In some embodiments, the agonist is a co-agonist that acts together with other co-agonists to produce an effect on a receptor. In certain embodiments, the agonist is an irreversible agonist that permanently binds to a receptor by forming a covalent bond. In certain embodiments, the agonist is a selective agonist for a specific type of receptor.

[0208] In certain embodiments, immunomodulatory components are antagonists. In some of these embodiments, antagonist is a competitive antagonist, which reversibly binds to receptors and does not activate receptors at the binding site identical with endogenous ligands or agonists. Competitive antagonists can affect the amount of the agonist necessary to the maximum response. In some other of these embodiments, antagonist is a noncompetitive antagonist, which is combined with the active site of receptor or the allosteric site of receptor. Noncompetitive antagonists can reduce the amplitude of the maximum response that the agonist of any amount can reach. In some other embodiments, antagonist is a noncompetitive antagonist, which needs to be activated by an agonist before being combined with an independent allosteric binding site.

[0209] In various embodiments, the immunomodulatory component comprises an antibody or antigen binding fragment. The immunomodulatory component can be a full-length protein or a fragment thereof. The antibody or antigen binding fragment can be derived from a natural source, or partially or completely synthetically produced. In some embodiments, the antibody is a monoclonal antibody. In some of these embodiments, the monoclonal antibody is an IgG antibody. In certain embodiments, the monoclonal antibody is IgG1, IgG2, IgG3 or IgG4. In some other embodiments, the antibody is a polyclonal antibody. In certain embodiments, the antigen binding fragment is selected from Fab, Fab' and F(ab')2, F(ab1)2, Fv, dAb and Fd fragments. In certain embodiments, the antigen binding fragment is a scFv or (scFv)2 fragment. In certain other embodiments, the antibody or antigen binding fragment is (Single domain antibodies.) In some embodiments, the antibody or antigen-binding fragment is a bispecific or multispecific antibody.

[0210] In various embodiments, the antibody or antigen-binding fragment is fully human. In some embodiments, the antibody or antigen-binding fragment is humanized. In some embodiments, the antibody or antigen-binding fragment is chimeric. In some of these embodiments, the chimeric antibody has a non-human V region domain and a human C region domain. In some embodiments, the antibody or antigen-binding fragment is non-human, such as murine or mammalian.

[0211] In certain embodiments, the immunomodulatory component is a polynucleotide. In some of these embodiments, the polynucleotide includes but is not limited to mRNA, miRNA, siRNA, antisense RNA, shRNA, lncRNA and dsDNA. In some embodiments, the polynucleotide is RNA (e.g., mRNA, miRNA, siRNA, antisense RNA, shRNA or lncRNA). In some of these embodiments, when the polynucleotide is mRNA, it can be translated into the desired polypeptide. In some embodiments, the polynucleotide is microRNA (miRNA) or miRNA precursor molecule. In some of these embodiments, miRNA is delivered to the cytoplasm of the target cell so that the miRNA molecule can silence the natural mRNA in the target cell. In some embodiments, the polynucleotide is a small interfering RNA (siRNA) or short hairpin RNA (shRNA) that can interfere with the expression of oncogenes or other dysregulated polypeptides. In some of these embodiments, siRNA is delivered to the cytoplasm of the target cell so that the siRNA molecule can silence the natural mRNA in the target cell. In some embodiments, the polynucleotide is an antisense RNA complementary to mRNA. In some embodiments, the polynucleotide is a long non-coding RNA (lncRNA) that can regulate gene expression and regulate disease. In some embodiments, the polynucleotide is a DNA that can be transcribed into RNA. In some of these embodiments, the transcribed RNA can be translated into a desired polypeptide.

[0212] In some embodiments, the immunomodulatory component is a protein, peptide, glycolipid, or glycoprotein.

[0213] In various embodiments, the composition comprises two or more of the above-mentioned immunomodulatory components, including mixtures, fusions, combinations and conjugates of atoms, molecules, etc. In some embodiments, the composition comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 different immunomodulatory components associated with the membrane or enclosed in the closed volume of the extracellular vesicle. In certain embodiments, the composition comprises a nucleic acid bound to a polypeptide. In certain embodiments, the composition comprises two or more polypeptides conjugated to each other. In certain embodiments, the composition comprises a protein conjugated to a bioactive molecule. In some of these embodiments, the bioactive molecule is a prodrug.

[0214] In some embodiments, the composition comprises two different immunomodulatory components associated with a membrane or enclosed within the enclosed volume of the extracellular vesicle. In certain embodiments, the two different immunomodulatory components are IL-12 and CD40L. In some embodiments, CD40L and IL-12 are fused to PTGFRN or a fragment thereof, respectively. In some embodiments, CD40L and IL-12 are fused to the N-terminus of PTGFRN or a fragment thereof, respectively. In some embodiments, CD40L and IL-12 are expressed as a fusion protein with PTGFRN, wherein the polypeptides have the sequences of SEQ ID NO: 20 and SEQ ID NO: 3, respectively.

[0215] In some embodiments, compositions include three kinds of different immunomodulatory components associated with membrane or enclosed in the closed volume of the extracellular vesicle. In certain embodiments, two different immunomodulatory components are IL-12, CD40L and FMS sample tyrosine kinase 3 ligands (FLT3L). In some embodiments, CD40L, IL-12 and FLT3L are fused with PTGFRN or its fragment respectively. In some embodiments, CD40L, IL-12 and FLT3L are fused with the N-terminal of PTGFRN or its fragment respectively. In some embodiments, CD40L, IL-12 and FLT3L are expressed as fusion protein with PTGFRN, wherein polypeptide has the sequence of SEQ ID NO:20, SEQ ID NO:3 and SEQ ID NO:22 respectively.

[0216] Pharmaceutical composition

[0217] The pharmaceutical composition typically comprises a plurality of extracellular vesicles and a pharmaceutically acceptable excipient or carrier in a form suitable for administration to a subject. The pharmaceutically acceptable excipient or carrier depends in part on the specific composition being administered, as well as the specific method used to administer the composition. Thus, there are a variety of suitable formulations of pharmaceutical compositions comprising a plurality of extracellular vesicles. (See, for example, Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa. 21st ed. (2005)). Pharmaceutical compositions are typically formulated as sterile and in full compliance with all Good Manufacturing Practice (GMP) regulations of the U.S. Food and Drug Administration.

[0218] In some embodiments, the pharmaceutical composition comprises one or more therapeutic agents and an extracellular vesicle as described herein. In some embodiments, the extracellular vesicle is co-administered with one or more separate therapeutic agents, wherein co-administration includes administering the separate therapeutic agent before, after, or simultaneously with the administration of the extracellular vesicle.

[0219] Pharmaceutically acceptable excipients include excipients that are generally safe, non-toxic and desirable, including excipients that are acceptable for veterinary use as well as human pharmaceutical use.

[0220] Examples of carriers or diluents include, but are not limited to, water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. The use of such media and compounds for pharmaceutically active substances is well known in the art. Unless any conventional media or compounds are incompatible with the extracellular vesicles described herein, they may be considered for use in the composition. Supplementary therapeutic agents may also be incorporated into the composition. Typically, the pharmaceutical composition is formulated to be compatible with its intended route of administration. Extracellular vesicles can be administered parenterally, topically, intravenously, orally, subcutaneously, intraarterially, intradermally, transdermally, rectally, intracranially, intraperitoneally, intranasally, intratumorally, intramuscularly, or as an inhalant. In certain embodiments, the pharmaceutical composition comprising the extracellular vesicles is administered intravenously, for example, by injection. The extracellular vesicles may optionally be administered in combination with other therapeutic agents that are at least partially effective in treating the disease, disorder, or condition for which the extracellular vesicles are intended.

[0221] The solution or suspension may include the following components: a sterile diluent such as water, saline solution, fixed oil, polyethylene glycol, glycerol, propylene glycol or other synthetic solvents; an antimicrobial compound such as benzyl alcohol or methyl paraben; an antioxidant such as ascorbic acid or sodium bisulfite; a chelating compound such as ethylenediaminetetraacetic acid (EDTA); a buffer such as acetate, citrate or phosphate, and a compound for adjusting tonicity such as sodium chloride or dextrose. The pH may be adjusted with an acid or base such as hydrochloric acid or sodium hydroxide. The formulation may be packaged in an ampoule, disposable syringe or multiple-dose vial made of glass or plastic.

[0222] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (if water soluble) or dispersions and sterile powders. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL TM (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). Compositions are typically sterile and are mobile to a certain extent so as to be easily injected. Carriers can be solvents or dispersion media, which include, for example, water, ethanol, polyols (such as glycerol, propylene glycol and liquid polyethylene glycol, etc.) and suitable mixtures thereof. Can, for example, be by using a coating such as lecithin, in the case of a dispersion, by maintaining the required particle size and by using a surfactant to maintain suitable fluidity. The effects of microorganisms can be prevented by various antibacterial and antifungal compounds, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. If desired, isotonic compounds (such as sugars, polyols such as mannitol, sorbitol and sodium chloride) can be added to the composition. The extended absorption of injectable compositions can be achieved by including in the composition a compound that delays absorption, such as aluminum monostearate and gelatin.

[0223] As needed, sterile injectable solutions can be prepared by incorporating an effective amount of extracellular vesicles into a suitable solvent in combination with one or more of the ingredients listed herein. Typically, dispersions are prepared by incorporating the extracellular vesicles into a sterile vehicle containing a basic dispersion medium and any desired other ingredients. In the case of sterile powders for the preparation of sterile injectable solutions, the preparation methods are vacuum drying and freeze drying, which produce a powder of the active ingredient plus any additional desired ingredients from a previously sterile-filtered solution thereof. The extracellular vesicles can be administered in the form of a depot injection or implant formulation, which can be formulated in a manner that allows for sustained or pulsatile release of the extracellular vesicles.

[0224] Systemic administration of the composition comprising extracellular vesicles can also be by transmucosal means. For transmucosal administration, penetrants suitable for the barrier to be crossed can be used in the formulation. Such penetrants are generally known in the art and include, for example, detergents, bile salts, and fusidic acid derivatives for transmucosal administration. Transmucosal administration can be accomplished using, for example, nasal sprays.

[0225] In certain embodiments, the pharmaceutical composition comprising extracellular vesicles is administered intravenously to a subject who would benefit from the pharmaceutical composition. In certain other embodiments, the composition is administered to the lymphatic system, for example, by intralymphatic injection or by intranodal injection (see, e.g., Senti et al., PNAS 105(46):17908 (2008)), or by intramuscular injection, by subcutaneous administration, by intratumoral injection, by direct injection into the thymus or liver.

[0226] In certain embodiments, the pharmaceutical composition comprising extracellular vesicles is administered in the form of a liquid suspension. In certain embodiments, the pharmaceutical composition is administered in the form of a formulation capable of forming a depot after administration. In certain preferred embodiments, the depot slowly releases the extracellular vesicles into the circulation, or remains in depot form.

[0227] Typically, pharmaceutically acceptable compositions are highly purified to be free of contaminants, are biocompatible and non-toxic, and are suitable for administration to a subject. If water is a component of the carrier, the water is highly purified and processed to be free of contaminants, such as endotoxins.

[0228] Pharmaceutically acceptable carriers can be lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinyl pyrrolidone, cellulose, water, syrup, methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and / or mineral oil, but are not limited thereto. The pharmaceutical composition may further comprise a lubricant, a wetting agent, a sweetener, a flavor enhancer, an emulsifier, a suspending agent and / or a preservative.

[0229] The pharmaceutical compositions described herein comprise the extracellular vesicles described herein and optionally a pharmaceutically active or therapeutic agent. The therapeutic agent can be a biological agent, a small molecule agent, or a nucleic acid agent.

[0230] Provided are dosage forms comprising a pharmaceutical composition comprising the extracellular vesicles described herein. In some embodiments, the dosage form is formulated as a liquid suspension for intravenous injection. In some embodiments, the dosage form is formulated as a liquid suspension for intratumoral injection.

[0231] In certain embodiments, the preparation of extracellular vesicles is subjected to radiation, e.g., X-rays, gamma rays, beta particles, alpha particles, neutrons, protons, elemental nuclei, UV rays, to destroy residual replication-competent nucleic acids.

[0232] In certain embodiments, the preparation of extracellular vesicles is gamma irradiated using an irradiation dose of greater than 1, 5, 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, or greater than 100 kGy.

[0233] In some embodiments, the preparation of extracellular vesicles is subjected to X-ray irradiation using an irradiation dose of greater than 0.1, 0.5, 1, 5, 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, or greater than 10000 mSv.

[0234] method

[0235] Various aspects of the present disclosure also include methods for producing compositions comprising extracellular vesicles and immunomodulatory components. In some embodiments, the method comprises: obtaining extracellular vesicles from production cells, wherein the production cells naturally contain immunomodulatory components; and optionally isolating the obtained extracellular vesicles. In some embodiments, the method comprises: modifying the production cells with immunomodulatory components; obtaining extracellular vesicles from the modified production cells; and optionally isolating the obtained extracellular vesicles. In some other embodiments, the method comprises: obtaining extracellular vesicles from production cells; isolating the obtained extracellular vesicles; and modifying the isolated extracellular vesicles with immunomodulatory components. In certain embodiments, the method further comprises formulating the isolated extracellular vesicles into a pharmaceutical composition.

[0236] Methods for producing extracellular vesicles

[0237] Methods for modifying producer cells with immunomodulatory components

[0238] In various embodiments, the method comprises modifying the producer cell with an immunomodulatory component.

[0239] The production cell can be a mammalian cell line, a plant cell line, an insect cell line, a fungal cell line or a prokaryotic cell line. In certain embodiments, the production cell is a mammalian cell line. Mammalian cell lines include, but are not limited to, human embryonic kidney (HEK) cell lines, Chinese hamster ovary (CHO) cell lines, HT-1080 cell lines, HeLa cell lines, PERC-6 cell lines, CEVEC cell lines, fibroblast cell lines, amniotic cell lines, epithelial cell lines and mesenchymal stem cell (MSC) cell lines. In some preferred embodiments, the mammalian cell line can be HEK-293 cells, BJ human foreskin fibroblasts, fHDF fibroblasts, Neuronal precursor cells, Amniotic membrane cells, adipose-derived mesenchymal stem cells, or RPTEC / TERT1 cells. The production cells can also be primary cells. In various embodiments, the primary cells can be primary mammalian cells, primary plant cells, primary insect cells, primary fungal cells, or primary prokaryotic cells.

[0240] In certain preferred embodiments, the producer cell is an immune cell, such as a dendritic cell, a T cell, a B cell, a natural killer cell (NK cell), an antigen presenting cell, a macrophage, a T helper cell, or a regulatory T cell (Treg cell).

[0241] In various embodiments, the immunomodulatory components can be expressed in producer cells from a transgene or mRNA introduced into the producer cells by transfection, viral transduction, electroporation, extrusion, sonication, cell fusion, or other methods known to those skilled in the art.

[0242] In certain embodiments, immunomodulatory components are introduced into production cells by transfection. In some embodiments, synthetic macromolecules such as cationic lipids and polymers can be used to introduce immunomodulatory components into suitable production cells (Papapetrou et al., Gene Therapy 12: S118-S130 (2005)). In some embodiments, cationic lipids form complexes with immunomodulatory components by charge interaction. In some of these embodiments, positively charged complexes are bound to negatively charged cell surfaces and are absorbed by cells through endocytosis. In some other embodiments, cationic polymers can be used for transfection production cells. In some of these embodiments, cationic polymers are polyethyleneimine (PEI). In certain embodiments, chemicals such as calcium phosphate, cyclodextrin or polybrene can be used to introduce immunomodulatory components into production cells. Physical methods such as particle-mediated transfection, "gene guns", bioprojectiles or particle bombardment techniques can also be used to introduce immunomodulatory components into production cells (Papapetrou et al., Gene Therapy 12: S118-S130 (2005)). The transfection efficiency of producer cells can be assessed using reporter genes such as, for example, β-galactosidase, chloramphenicol acetyltransferase, luciferase, or green fluorescent protein.

[0243] In certain embodiments, the immunomodulatory components are introduced into the production cells by viral transduction. Many viruses can be used as gene transfer vectors, including Moloney murine leukemia virus (MMLV), adenovirus, adeno-associated virus (AAV), herpes simplex virus (HSV), lentivirus, and foamy virus. Viral-mediated gene transfer vectors include vectors based on DNA viruses (e.g., adenovirus, adeno-associated virus, and herpes virus), as well as vectors based on retroviruses.

[0244] In certain embodiments, immunomodulatory components are introduced into production cells by electroporation. Electroporation creates transient holes in the cell membrane, thereby allowing various molecules to be introduced into cells. In some embodiments, DNA and RNA, as well as polypeptide and non-polypeptide therapeutic agents, can be introduced into production cells by electroporation.

[0245] In certain embodiments, the immunomodulatory components are introduced into the production cells by microinjection.In some embodiments, the immunomodulatory components can be injected into the production cells at the microscopic level using a glass micropipette.

[0246] In certain embodiments, the immunomodulatory components are introduced into the producer cells by extrusion.

[0247] In certain embodiments, the immunomodulatory components are introduced into the producer cells by sonication.In some embodiments, the producer cells are exposed to high intensity sound waves, causing transient disruption of the cell membrane, thereby allowing loading of the immunomodulatory components.

[0248] In certain embodiments, immunomodulatory components are introduced into production cells by cell fusion. In some embodiments, immunomodulatory components are introduced by electrical cell fusion. In some other embodiments, polyethylene glycol (PEG) is used to fuse production cells. In some other embodiments, Sendai virus is used to fuse production cells.

[0249] In some embodiments, immunomodulatory components are introduced into production cells by hypotonic lysis. In some of these embodiments, production cells are exposed to a buffer of low ionic strength to break it, thereby allowing the loading of immunomodulatory components. In some alternative embodiments, controlled dialysis of a hypotonic solution is used to swell the production cells and produce holes in the production cell membrane. Subsequently, the production cells are exposed to conditions that allow the membrane to be sealed again.

[0250] In some embodiments, the immunomodulatory components are introduced into the production cells by detergent treatment. In certain embodiments, the production cells are treated with a mild detergent that temporarily disrupts the production cell membrane by creating pores, thereby allowing the loading of the immunomodulatory components. After the production cells have been loaded, the detergent is washed off, thereby resealing the membrane.

[0251] In some embodiments, the immunomodulatory component is introduced into the producer cell by receptor-mediated endocytosis.In certain embodiments, the producer cell has a surface receptor that, upon binding of the immunomodulatory component, induces internalization of the receptor and the associated immunomodulatory component.

[0252] In some embodiments, the immunomodulatory components are introduced into the production cells by filtration. In certain embodiments, the production cells and immunomodulatory components can be forced through a filter with a pore size smaller than that of the production cells, thereby causing transient disruption of the production cell membrane and allowing the immunomodulatory components to enter the production cells.

[0253] In some embodiments, the producer cells are subjected to several freeze-thaw cycles, resulting in rupture of the cell membrane, thereby allowing loading of immunomodulatory components.

[0254] Methods for modifying extracellular vesicles with immunomodulatory components

[0255] In various alternative embodiments, the immunomodulatory components are introduced directly into the extracellular vesicles after isolation of the extracellular vesicles.

[0256] In certain embodiments, immunomodulatory components are introduced into extracellular vesicles by transfection. In some embodiments, synthetic macromolecules such as cationic lipids and polymers can be used to introduce immunomodulatory components into extracellular vesicles (Papapetrou et al., Gene Therapy 12: S118-S130 (2005)). In certain embodiments, chemicals such as calcium phosphate, cyclodextrin or polybrene can be used to introduce immunomodulatory components into extracellular vesicles.

[0257] In certain embodiments, the immunomodulatory components are introduced into the extracellular vesicles by electroporation. In some embodiments, the extracellular vesicles are exposed to an electric field that causes transient pores in the extracellular vesicle membrane, thereby allowing the loading of the immunomodulatory components.

[0258] In certain embodiments, the immunomodulatory components are introduced into extracellular vesicles by microinjection. In some embodiments, the immunomodulatory components can be injected directly into extracellular vesicles at a microscopic level using a glass micropipette.

[0259] In certain embodiments, the immunomodulatory components are introduced into the extracellular vesicles by extrusion.

[0260] In certain embodiments, the immunomodulatory components are introduced into the extracellular vesicles by sonication. In some embodiments, the extracellular vesicles are exposed to high-intensity sound waves, causing transient disruption of the extracellular vesicle membrane, thereby allowing loading of the immunomodulatory components.

[0261] In some embodiments, the immunomodulatory component can be conjugated to the surface of the extracellular vesicle. Conjugation can be achieved chemically or enzymatically by methods known in the art.

[0262] In some embodiments, the extracellular vesicle comprises a chemically conjugated immunomodulatory component. Chemical conjugation can be achieved by covalent bonding of the immunomodulatory component to another molecule, with or without a linker. The formation of such conjugates is within the technical scope of the technician, and various techniques are known to be used for completing conjugation, wherein the selection of a particular technique is guided by the material to be conjugated. In certain embodiments, the polypeptide is conjugated to the extracellular vesicle. In certain other embodiments, non-polypeptides (e.g., lipids, carbohydrates, nucleic acids, and small molecules) are conjugated to the extracellular vesicle.

[0263] In some embodiments, immunomodulatory components are introduced into extracellular vesicles by hypotonic lysis. In some of these embodiments, the extracellular vesicles are exposed to a buffer of low ionic strength to rupture them, thereby allowing the loading of immunomodulatory components. In some alternative embodiments, controlled dialysis against a hypotonic solution is used to expand the extracellular vesicles and create holes in the extracellular vesicle membrane. The extracellular vesicles are then exposed to conditions that allow the membrane to be sealed again.

[0264] In some embodiments, the immunomodulatory component is introduced into the extracellular vesicle by detergent treatment. In certain embodiments, the extracellular vesicle is treated with a mild detergent that temporarily disrupts the extracellular vesicle membrane by creating pores, thereby allowing the loading of the immunomodulatory component. After the extracellular vesicle is loaded, the detergent is washed off, thereby resealing the membrane.

[0265] In some embodiments, the immunomodulatory component is introduced into the extracellular vesicle by receptor-mediated endocytosis. In certain embodiments, the extracellular vesicle has a surface receptor that, upon binding of the immunomodulatory component, induces internalization of the receptor and the associated immunomodulatory component.

[0266] In some embodiments, the immunomodulatory components are introduced into the extracellular vesicles by mechanical firing. In certain embodiments, the extracellular vesicles can be bombarded with immunomodulatory components attached to heavy or charged particles, such as gold microcarriers. In some of these embodiments, the particles can be accelerated mechanically or electrically so that they traverse the extracellular vesicle membrane.

[0267] In some embodiments, the immunomodulatory components are introduced into the extracellular vesicles by filtration. In certain embodiments, the extracellular vesicles and the immunomodulatory components can be forced through a filter having a pore size smaller than that of the extracellular vesicles, thereby causing transient disruption of the extracellular vesicle membrane and allowing the immunomodulatory components to enter the extracellular vesicles.

[0268] In some embodiments, the extracellular vesicles are subjected to several freeze-thaw cycles, resulting in rupture of the extracellular vesicle membrane, thereby allowing loading of immunomodulatory components.

[0269] Methods for isolating extracellular vesicles

[0270] Extracellular vesicles can be separated from production cells. In certain embodiments, extracellular vesicles are released into cell culture medium by production cells. It is expected that all known methods of separating extracellular vesicles are considered to be applicable to this article. For example, the physical properties of extracellular vesicles can be used to separate them from media or other source materials, including separation based on charge (e.g., electrophoretic separation), size (e.g., filtration, molecular sieves, etc.), density (e.g., conventional or gradient centrifugation), Svedberg constant (e.g., sedimentation with or without external force, etc.). Alternatively or in addition, separation can be based on one or more biological properties, and includes methods that can use surface labeling (e.g., for precipitation, reversible binding to a solid phase, FACS separation, specific ligand binding, non-specific ligand binding, affinity purification, etc.).

[0271] Isolation and enrichment can be performed in a general, non-selective manner, typically involving continuous centrifugation. Alternatively, isolation and enrichment can be performed in a more specific and selective manner, such as using extracellular vesicles or producer cell-specific surface markers. For example, specific surface markers can be used for immunoprecipitation, FACS sorting, affinity purification, and magnetic separation of bead-bound ligands.

[0272] In some embodiments, size exclusion chromatography can be used to separate extracellular vesicles. Size exclusion chromatography technology is known in the art. Exemplary non-limiting techniques are provided herein. In some embodiments, the void volume portion is separated and contains the extracellular vesicles of interest. In addition, in some embodiments, as is generally known in the art, after chromatographic separation, the extracellular vesicles can be further separated by centrifugation techniques (of one or more chromatographic fractions). In some embodiments, for example, density gradient centrifugation can be used to further separate the extracellular vesicles. In certain embodiments, it may be desirable to further separate the extracellular vesicles of the production cell source from the extracellular vesicles of other sources. For example, the extracellular vesicles of the production cell source can be separated from the extracellular vesicles of non-production cell sources by immunoabsorption capture using antigen-antibodies specific to the production cells.

[0273] In some embodiments, isolation of extracellular vesicles can involve a combination of methods including, but not limited to, differential centrifugation, size-based membrane filtration, immunoprecipitation, FACS sorting, and magnetic separation.

[0274] Methods for measuring extracellular vesicle size

[0275] In some embodiments, the methods described herein include measuring the size of an extracellular vesicle and / or a population of extracellular vesicles. Typically, the size of an extracellular vesicle is measured as the longest measurable dimension. Typically, the longest measurable dimension of an extracellular vesicle is also referred to as its diameter.

[0276] Extracellular vesicle size can be measured using dynamic light scattering (DLS) and / or multi-angle light scattering (MALS). Methods of measuring extracellular vesicle size using DLS and / or MALS are well known to those skilled in the art and include nanoparticle tracking assays (NTA, for example, using a Malvern NanoSight NS300 nanoparticle tracking device). In a specific embodiment, extracellular vesicle size is measured using Malvern NanoSight NS300. In some embodiments, the extracellular vesicles described herein have a longest dimension of about 20-300 nm as measured by NTA (for example, using a Malvern NanoSight NS300). In other embodiments, the extracellular vesicles described herein have a longest dimension of about 40-200 nm as measured by NTA (for example, using a Malvern NanoSight NS300). In other embodiments, the extracellular vesicle populations described herein include a population in which 90% of the extracellular vesicles have a longest dimension of about 20-300 nm as measured by NTA (for example, using a Malvern NanoSight NS300). In other embodiments, the extracellular vesicle populations described herein include a population in which 95% of the extracellular vesicles have a longest dimension of about 20-300 nm as measured by NTA (e.g., using a Malvern NanoSight NS300). In other embodiments, the extracellular vesicle populations described herein include a population in which 99% of the extracellular vesicles have a longest dimension of about 20-300 nm as measured by NTA (e.g., using a Malvern NanoSight NS300). In other embodiments, the extracellular vesicle populations described herein include a population in which 90% of the extracellular vesicles have a longest dimension of about 40-200 nm as measured by NTA (e.g., using a Malvern NanoSight NS300). In other embodiments, the extracellular vesicle populations described herein include a population in which 95% of the extracellular vesicles have a longest dimension of about 40-200 nm as measured by NTA (e.g., using a Malvern NanoSight NS300). In other embodiments, a population of extracellular vesicles described herein comprises a population in which 99% of the extracellular vesicles have a longest dimension of about 40-200 nm as measured by NTA (e.g., using a Malvern NanoSight NS300).

[0277] Extracellular vesicle size can be measured using tunable resistive pulse sensing (TRPS). In a specific embodiment, the extracellular vesicle size measured by TRPS is determined using iZON qNANO Gold. In some embodiments, the extracellular vesicles described herein have a longest dimension of about 20-300 nm measured by TRPS (e.g., using iZON qNano Gold). In other embodiments, the extracellular vesicles described herein have a longest dimension of about 40-200 nm measured by TRPS (e.g., using iZON qNanoGold). In other embodiments, the extracellular vesicle populations described herein include a population in which 90% of the extracellular vesicles have a longest dimension of about 20-300 nm measured by TRPS (e.g., using iZON qNano Gold). In other embodiments, the extracellular vesicle populations described herein include a population in which 95% of the extracellular vesicles have a longest dimension of about 20-300 nm measured by TRPS (e.g., using iZON qNano Gold). In other embodiments, the extracellular vesicle populations described herein include a population in which 99% of the extracellular vesicles have a longest dimension of about 20-300 nm as measured by TRPS (e.g., using iZON qNano Gold). In other embodiments, the extracellular vesicle populations described herein include a population in which 90% of the extracellular vesicles have a longest dimension of about 40-200 nm as measured by TRPS (e.g., using iZON qNano Gold). In other embodiments, the extracellular vesicle populations described herein include a population in which 95% of the extracellular vesicles have a longest dimension of about 40-200 nm as measured by TRPS (e.g., using iZON qNano Gold). In other embodiments, the extracellular vesicle populations described herein include a population in which 99% of the extracellular vesicles have a longest dimension of about 40-200 nm as measured by TRPS (e.g., using iZON qNano Gold).

[0278] The size of extracellular vesicles can be measured using electron microscopy. In some embodiments, the electron microscopy method used to measure the size of extracellular vesicles is a transmission electron microscopy. In a specific embodiment, the transmission electron microscopy used to measure the size of extracellular vesicles is a Tecnai TM G 2 Spirit BioTWIN. Methods for measuring the size of extracellular vesicles using electron microscopy are well known to those skilled in the art, and any such method may be suitable for measuring the size of extracellular vesicles. In some embodiments, the extracellular vesicles described herein have a size that can be measured by scanning electron microscopy (e.g., Tecnai TM G2 In other embodiments, the extracellular vesicles described herein have a longest dimension of about 20-300 nm as measured by a scanning electron microscope (e.g., a Tecnai BioTWIN scanning electron microscope). TM G 2 In other embodiments, the extracellular vesicle populations described herein include those wherein 90% of the extracellular vesicles have a longest dimension of about 40-200 nm as measured by a scanning electron microscope (e.g., a Tecnai TM G 2 In other embodiments, the extracellular vesicle populations described herein include those wherein 95% of the extracellular vesicles have a longest dimension of about 20-300 nm as measured by a scanning electron microscope (e.g., a Tecnai BioTWIN scanning electron microscope). TM G 2 In other embodiments, the extracellular vesicle populations described herein include those wherein 99% of the extracellular vesicles have a longest dimension of about 20-300 nm as measured by a scanning electron microscope (e.g., Tecnai BioTWIN scanning electron microscope). TM G 2 In other embodiments, the extracellular vesicle populations described herein include those wherein 90% of the extracellular vesicles have a longest dimension of about 20-300 nm as measured by a scanning electron microscope (e.g., a Tecnai BioTWIN scanning electron microscope). TM G 2 In other embodiments, the extracellular vesicle populations described herein include a population wherein 95% of the extracellular vesicles have a longest dimension of about 40-200 nm as measured by a scanning electron microscope (e.g., a Tecnai BioTWIN scanning electron microscope). TM G 2 In other embodiments, the extracellular vesicle populations described herein include a population wherein 99% of the extracellular vesicles have a longest dimension of about 40-200 nm as measured by a scanning electron microscope (e.g., a Tecnai BioTWIN scanning electron microscope). TM G 2 The populations were approximately 40-200 nm in their longest dimension as measured by a Spirit BioTWIN scanning electron microscope.

[0279] Methods for treating cancer, GvHD, and autoimmune diseases

[0280] Additionally, provided herein are methods of treating cancer, graft-versus-host disease (GvHD), and autoimmune diseases in a subject.

[0281] In various embodiments, the composition is applied to a subject with cancer. In some of these embodiments, the composition can raise the immune response and enhance the tumor targeting of the subject's immune system. In some embodiments, the cancer treated is characterized by leukocytes (T cells, B cells, macrophages, dendritic cells, monocytes) infiltrating into the tumor microenvironment, or so-called "hot tumors" or "inflammatory tumors". In some embodiments, the cancer treated is characterized by leukocytes infiltrating into low levels or undetectable levels in the tumor microenvironment, or so-called "cold tumors" or "non-inflammatory tumors". In some embodiments, the composition is applied in an amount and time sufficient to convert "cold tumors" into "hot tumors", that is, the application causes leukocytes (such as T cells) to infiltrate into the tumor microenvironment.

[0282] In some embodiments, the composition comprising an extracellular vesicle and an immunomodulatory component is administered to a subject as a cancer vaccine. In some of these embodiments, the composition is administered to a subject as a personalized cancer vaccine. In some embodiments, the immunomodulatory component is a tumor antigen or a peptide derived from a tumor antigen. Examples of suitable 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, cancer-testis antigen, MART-1 gp100, and TNF-related apoptosis-inducing ligand. In certain embodiments, the tumor antigen is derived from a reference genome sequence. In certain embodiments, the tumor antigen is derived from the genomic sequence of the subject receiving the composition.

[0283] Cancers that can be treated with the composition include, but are not limited to, those listed in Table 5.

[0284] In certain embodiments, the composition is administered to a subject suffering from graft-versus-host disease (GvHD). In some of these embodiments, the composition can downregulate the immune response and alleviate the symptoms of GvHD. In some specific embodiments, the composition alleviates the symptoms of GvHD by activating apoptosis signaling. In certain embodiments, the composition for treating GvHD comprises Fas ligand (FasL). In some of these embodiments, FasL is expressed on the surface of extracellular vesicles.

[0285] In various embodiments, the composition is administered to a subject having an autoimmune disease. In some of these embodiments, the composition can downregulate the immune response and suppress the immune activity of the subject.

[0286] Autoimmune diseases include, but are not limited to, multiple sclerosis, peripheral neuritis, Sjögren's syndrome, rheumatoid arthritis, alopecia, autoimmune pancreatitis, Behçet's disease, bullous pemphigus, celiac disease, Deweker's disease (neuromyelitis optica), glomerulonephritis, IgA nephropathy, mixed vasculitis, scleroderma, diabetes mellitus, arteritis, vitiligo, ulcerative colitis, irritable bowel syndrome, psoriasis, uveitis, and systemic lupus erythematosus.

[0287] In some embodiments, the composition is administered intravenously to the subject's circulatory system. In some embodiments, the composition is infused in a suitable fluid and administered into a vein of the subject.

[0288] In some embodiments, the composition is administered intra-arterially to the subject's circulatory system. In some embodiments, the composition is infused in a suitable fluid and administered into an artery of the subject.

[0289] In some embodiments, the composition is administered to the subject by intrathecal administration.In some embodiments, the composition is administered by injection into the spinal canal or subarachnoid space so that it reaches the cerebrospinal fluid (CSF).

[0290] In some embodiments, the composition is administered intratumorally into one or more tumors in a subject.

[0291] In some embodiments, the composition is administered to a subject via intranasal administration. In some embodiments, the composition may be insufflated into the nose in the form of a topical or systemic administration. In certain embodiments, the composition is administered in the form of a nasal spray.

[0292] In some embodiments, the composition is administered to the subject by intraperitoneal administration. In some embodiments, the composition is injected into a suitable liquid and injected into the peritoneum of the subject. In some embodiments, the intraperitoneal administration causes the composition (e.g., the extracellular vesicle in the composition) to be distributed to the lymphatic vessels. In some embodiments, the intraperitoneal administration causes the composition (e.g., the extracellular vesicle in the composition) to be distributed to the thymus, spleen and / or bone marrow. In some embodiments, the intraperitoneal administration causes the composition (e.g., the extracellular vesicle in the composition) to be distributed to one or more lymph nodes. In some embodiments, the intraperitoneal administration causes the composition (e.g., the extracellular vesicle in the composition) to be distributed to one or more lymph nodes, inguinal lymph nodes, mediastinal lymph nodes or sternal lymph nodes. In some embodiments, the intraperitoneal administration causes the composition (e.g., the extracellular vesicle in the composition) to be distributed to the pancreas.

[0293] In some embodiments, the composition is administered to a subject via periocular administration. In some embodiments, the composition is injected into periocular tissue. Periocular drug administration includes subconjunctival, anterior subtenon, posterior subtenon, and retrobulbar routes of administration.

[0294] In some embodiments, the composition is administered to the same subject via multiple routes of administration. In some embodiments, the multiple routes of administration include intravenous administration, intraarterial administration, intrathecal administration, intranasal administration, intratumoral administration, intraperitoneal administration, and / or periocular administration. In a preferred embodiment, the multiple routes of administration include intravenous administration and intraperitoneal administration.

[0295] In certain embodiments, the dose of extracellular vesicles is 1 ng to 10 ng, 10 ng to 100 ng, 100 ng to 1 μg, 1 μg to 5 μg, 5 μg to 10 μg, 10 μg to 50 μg, 50 μg to 75 μg, 75 μg to 100 μg, 100 μg to 150 μg, 150 μg to 200 μg, 200 μg to 300 μg, 300 μg to 500 μg, 500 μg to 1 mg, or 1 mg to 10 mg.

[0296] The composition can be administered to the subject once. Alternatively, multiple administrations can be performed over a period of time. For example, the subject can be administered two, three, four, five, or more times. In some embodiments, administrations can be given as needed, for example, as long as symptoms associated with the disease, disorder, or condition persist. In some embodiments, repeated administrations can be indicated for the remainder of the subject's life. The treatment period can vary and can, for example, be no more than one year, six months, three months, two months, one month, two weeks, one week, three days, two days, or no more than one day.

[0297] In certain embodiments, the dose of extracellular vesicles is administered at intervals, such as daily, every other day, weekly, twice weekly, monthly, or twice monthly.

[0298] In some embodiments, the pharmaceutical composition is administered at a frequency sufficient to effectively increase the concentration of the immunomodulatory component in the target cell or tissue above a level associated with symptoms of the disease, disorder, or condition.

[0299] In some embodiments, the composition is administered at least twice during the treatment period to treat the disease, disorder or condition, or to improve its symptoms. In some embodiments, the composition is administered at least twice during the treatment period to treat the disease, disorder or condition, or to prevent its symptoms. In some embodiments, the pharmaceutical composition is administered a sufficient number of times during the treatment period so that a sufficient amount of immunomodulatory components are delivered to the target cell or tissue during the treatment period. In some embodiments, the pharmaceutical composition is administered a sufficient number of times during the treatment period so that a sufficient amount of immunomodulatory components are delivered to the target cell or tissue during the treatment period to prevent, reduce, improve or delay one or more symptoms of the disease, disorder or condition. In some embodiments, increasing the concentration of immunomodulatory components in the target cell or tissue includes increasing the peak concentration, and in other embodiments, it includes increasing the average concentration. In some embodiments, a significant increase during the treatment period can be determined by comparing the early or late treatment period of the subject, or by comparing the measurements performed in the treated population with the matched untreated control population.

[0300] In some embodiments, the pharmaceutical composition is administered a sufficient number of times during each treatment period to increase the concentration of the immunomodulatory component in the target cell or tissue for at least about one week, two weeks, three weeks, four weeks, one month, two months, three months, four months, five months, six months, or more than six months. In some embodiments, the pharmaceutical composition is administered a sufficient number of times during each treatment period to increase the concentration of the immunomodulatory component in the target cell or tissue for a period of time at least as long as the treatment period.

[0301] In some embodiments, the time interval between repeated administrations during the treatment period is no greater than the time period during which the number of extracellular vesicles in the circulation decreases to less than about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of the number of extracellular vesicles present in the administered pharmaceutical composition.

[0302] In some embodiments, the method further comprises one or more doses of non-therapeutic extracellular vesicles prior to injecting a suitable therapeutic dose of extracellular vesicles carrying a therapeutic agent. In certain embodiments, the non-therapeutic extracellular vesicles are administered separately from the therapeutic extracellular vesicles and at different doses. In certain embodiments, the dose of non-therapeutic extracellular vesicles is greater than the dose of therapeutic extracellular vesicles. In certain other embodiments, the dose of non-therapeutic extracellular vesicles is less than the dose of therapeutic extracellular vesicles. In certain embodiments, the dose of non-therapeutic extracellular vesicles is the same as the dose of therapeutic extracellular vesicles. In various embodiments, the method of injecting non-therapeutic extracellular vesicles prior to injecting a suitable dose of therapeutic extracellular vesicles reduces the turnover of therapeutic extracellular vesicles in the liver, lungs and / or spleen.

[0303] An effective amount of the composition is provided based, at least in part, on the target tissue, target cell type, mode of administration, physical characteristics of the extracellular vesicles (e.g., size, and in some cases, the extent of the molecule to be delivered), and other determinants. Generally, an effective amount of the composition provides an effective cellular response in the target cells. Increased efficiency can be demonstrated by increased cell transfection (i.e., the percentage of cells transfected with the extracellular vesicle components), increased cellular response, or decreased innate immune response in the host subject.

[0304] The dosage and frequency of administration of the extracellular vesicles and their pharmaceutical compositions can be determined, for example, by the attending physician based on various factors, such as the severity of the disease, the patient's age, sex, and diet, the severity of any inflammation, the time of administration, and other clinical factors. In one example, intravenous administration is initiated at the minimum effective dose and the dose is increased over a preselected time period until a positive effect is observed. Subsequently, the dose is increased to a level that produces a corresponding increase in effect, taking into account any side effects that may occur.

[0305] Example

[0306] The following examples are set forth so as to provide one of ordinary skill in the art with a complete disclosure and description of how to make and use the invention, and are not intended to limit the scope of what the inventors regard as their invention, nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental errors and deviations should be taken into account. Unless otherwise indicated, parts are parts by weight, molecular weights are weight average molecular weights, temperatures are in degrees Celsius, and pressures are at or near atmospheric pressure. Standard abbreviations may be used, for example, bp, base pairs; kb, kilobases; p1, picoliters; s or sec, seconds; min, minutes; h or hr, hours; aa, amino acids; nt, nucleotides, etc.

[0307] Unless otherwise indicated, the practice of the present invention will employ conventional methods of protein chemistry, biochemistry, recombinant DNA technology, and pharmacology within the skill of the art. These techniques are fully explained in the literature. See, for example, T. C. Creighton, Proteins: Structures and Molecular Properties (W. H. Freeman and Company, 1993); A. L. Lehninger, Biochemistry (Worth Publishers, Inc., current addition); Sambrook, et al., Molecular Cloning: A Laboratory Manual (2nd Edition, 1989); Methods In Enzymology (S. Colowick and N. Kaplan, eds., Academic Press, Inc.); Remington's Pharmaceutical Sciences, 21st edition (Easton, Pennsylvania: Mack Publishing Company, 2005); Carey and Sundberg Advanced Organic Chemistry, 3rd edition (Plenum Press), Volumes A and B (1992).

[0308] method

[0309] Exosome purification

[0310] The conditioned medium was collected and centrifuged at 300-800 × g for 5 minutes at room temperature to remove cells and large debris. The culture supernatant was then supplemented with 1000 U / L of Incubate in a 37°C water bath for 1 hour. Collect the supernatant and centrifuge at 16,000 × g for 30 minutes at 4°C to remove residual cell debris and other large contaminants. Then, ultracentrifuge the supernatant at 133,900 × g for 3 hours at 4°C to pellet the exosomes. Discard the supernatant and aspirate any remaining culture medium from the bottom of the tube. Resuspend the pellet in 200–1000 μL of PBS (Ca-Mg).

[0311] To further enrich the exosome population, the cells were purified by density gradient purification (sucrose or Optiprep TM ) was treated with the precipitate. For sucrose gradient purification, the exosome precipitate was layered on top of the sucrose gradient, as shown in Table 6 below:

[0312] Table 6, sucrose density gradient:

[0313] Working percentage (%) 65% stock solution volume (mL) Milli-Q volume (mL) 50 3.85 1.15 40 3.08 1.92 25 1.92 3.08 10 0.46 2.54

[0314] The gradient was spun at 200,000 x g in 12 mL Ultra-Clear (344059) tubes placed in a SW 41 Ti rotor at 4°C for 16 hours to separate the exosome fraction.

[0315] The exosome layer was gently removed from the top layer and diluted in approximately 32.5 mL of PBS in a 38.5 mL Ultra-Clear (344058) tube and ultracentrifuged again at 133,900 × g for 3 hours at 4°C to pellet the purified exosomes. The resulting pellet was resuspended in a minimum volume of PBS (approximately 200 μL) and stored at 4°C.

[0316] For Optiprep TM Gradients: Prepare a 3-layer sterile gradient with equal volumes of 10%, 30%, and 45% Optiprep in a 12 mL Ultra-Clear (344059) tube for a SW 41Ti rotor. Add the precipitate to the Optiprep TM The exosome fraction was isolated by ultracentrifugation at 200,000 × g for 16 h at 4° C. The exosome layer was then gently collected from the top 3 mL of the tube.

[0317] The exosome fraction was diluted in approximately 32 mL of PBS in a 38.5 mL Ultra-Clear (344058) tube and ultracentrifuged at 133,900 × g for 3 hours at 4°C to pellet the purified exosomes. The pelleted exosomes were then resuspended in a minimum volume of PBS (approximately 200 μL) and stored at 4°C.

[0318] Example 1: Engineering exosomes to display immune checkpoint modulator antibodies

[0319] Human embryonic kidney (HEK) cell lines are grown to high density, and the resulting exosomes are isolated from the culture medium according to methods known to those skilled in the art (e.g., methods described herein). Exosomes engineered with cytotoxic T lymphocyte-associated protein 4 (CTLA-4) antibodies are prepared by chemical conjugation according to techniques known in the art. Exosomes modified with CTLA4 antibodies are selected by flow cytometry. Simultaneously, unmodified exosomes are isolated according to the same standard methods.

[0320] Two exosome populations were labeled with a radioactive tracer and 150 μg of each preparation was injected into live mice (e.g., a mouse model of melanoma). Mice that received exosomes displaying CTLA-4 antibodies or unmodified exosomes were continuously monitored for 30 minutes and then again at four-hour intervals using whole-animal PET / CT. Whole-animal imaging allowed real-time, high-resolution tracking of labeled exosomes in various tissues.

[0321] Without first labeling with a radioactive tracer, 150 μg of each exosome population was injected intravenously into two cohorts of mice. Five weeks after administration, the mice were euthanized. Tumor samples were collected and analyzed by immunohistochemistry and real-time PCR.

[0322] Example 2: Engineering exosomes to display Fas ligand

[0323] Human antigen-presenting cells are transfected with a plasmid encoding a puromycin resistance selectable marker and Fas ligand. The transfected cells are treated with puromycin, resistant colonies are selected, and surface expression of Fas ligand is determined by flow cytometry. Stable cells expressing Fas ligand are grown to a high concentration, and the resulting exosomes are isolated from the culture medium according to methods known to those skilled in the art (e.g., methods described herein). Concurrently, untransfected producer cells are cultured and the resulting exosomes are isolated according to the same standard methods.

[0324] Two exosome populations were labeled with a radioactive tracer, and 150 μg of each preparation was injected into live mice (e.g., a mouse model of GvHD). Mice receiving exosomes derived from unmodified cells or exosomes derived from Fas ligand-expressing cells were continuously monitored for 30 minutes and again at four-hour intervals using whole-animal PET / CT. Whole-animal imaging allowed real-time, high-resolution tracking of labeled exosomes in various tissues.

[0325] Purified exosome populations from unmodified producer cells and producer cells engineered to express Fas ligand were purified according to the methods described herein. Without first labeling with a radioactive tracer, 150 μg of each exosome population was injected into two cohorts of mice. Three to five weeks after administration, animals from both cohorts were euthanized for immunohistochemistry analysis and real-time PCR.

[0326] Example 3: Lymphatic Uptake of Exosomes Following Intraperitoneal Administration

[0327] To determine the biodistribution of purified exosomes in vivo, the following experiments were performed:

[0328] Conditioned medium from 293T cells was collected and centrifuged at 300-800 × g for 5 minutes at room temperature to remove cells and large debris. The culture supernatant was then supplemented with 1000 U / L of Incubate in a 37°C water bath for 1 hour. Collect the supernatant and centrifuge at 16,000 × g for 30 minutes at 4°C to remove residual cell debris and other large contaminants. Ultracentrifuge the supernatant at 133,900 × g for 3 hours at 4°C to pellet the exosomes. Discard the supernatant and aspirate the remaining culture medium from the bottom of the tube. Resuspend the pellet in 200–1000 μL of PBS (Ca-Mg).

[0329] To further enrich the exosome population, the pellet was processed by sucrose density gradient purification as defined in Table 6.

[0330] The gradient was spun at 200,000 x g in 12 mL Ultra-Clear (344059) tubes placed in a SW 41 Ti rotor at 4°C for 16 hours to separate the exosome fraction.

[0331] The exosome layer was gently removed from the top layer and diluted in approximately 32.5 mL of PBS in a 38.5 mL Ultra-Clear (344058) tube and ultracentrifuged again at 133,900 × g for 3 hours at 4°C to pellet the purified exosomes. The resulting pellet was resuspended in a minimum volume of PBS (approximately 200 μL) and stored at 4°C.

[0332] To radiolabel purified exosomes for in vivo imaging, 1x10 11 Each purified exosome was diluted with HEPES (200 μL, 0.1 M, pH 8.5) and conjugated with p-SCN-Bn-DFO (5 μg) at 37°C for 1 hour, and then incubated at 4°C overnight. DFO-exosomes were incubated with 89Zr (7.5 mCi) diluted in HEPES (100 μL, 1 M, pH 7.3) at 37°C for 1 hour and purified on a qEv column. 100 μCi / 1x10 10 The total yield of exosomes was 0.4 mCi of 89Zr-DFO-exosomes in up to 0.8 mL PBS. Quality control (HPLC) was performed before release to ensure >95% RCP.

[0333] In vitro stability

[0334] Exosomes (20 μCi / 2x10 10 ) were incubated at room temperature under the following conditions:

[0335] a. Prepare buffer

[0336] b. Mouse serum (if possible, 10% exosome solution in serum)

[0337] Two hours after the start of incubation, the solution was injected into HPLC to determine the stability of the tracer.

[0338] In vivo imaging

[0339] Mice (SKH-1, n=8, 5-8 weeks old) were randomly divided into two groups, weighed and injected (the first group was injected with the second group immediately after the dynamic scanning) with 1x10 10 The first group received an intravenous (IV) injection, and the second group received an intraperitoneal (IP) injection.

[0340] Mice were subjected to whole-body PET / CT scanning in four mouse houses using the following schedule: 1 hour of dynamic imaging (5x60, 5x180, 8x300 seconds) and static imaging at 4 hours (20 minutes), 24 hours (Thursday, 20 minutes), and 48 hours (Friday, 30 minutes). CT examinations were performed after each imaging time point to provide anatomical reference.

[0341] After the last imaging time point, mice were euthanized and the following organs were collected, weighed, and counted in a gamma counter: blood, lung (one), liver (lobes), spleen, pancreas, kidney (one), liver, colon, and other high uptake organs.

[0342] If the count is very high, allow the organs to decay for 2-3 days and then count again.

[0343] Table 7

[0344]

[0345] result

[0346] The two treated mouse cohorts were imaged at 4, 24, and 48 hours post-treatment. Whole-body PET / CT imaging showed stable delivery of ( Figure 1A ), the mice in group 2 treated with IP had a clear non-overlapping distribution ( Figure 1B Organs were dissected and analyzed by radiographic gamma counter, which showed significant liver and spleen uptake in IV treated mice ( Figure 2 ). In contrast, for mice treated with IP, uptake was primarily observed in the pancreas, spleen, thymus, and lymph nodes, with additional uptake in the liver and ovaries. These results suggest that different routes of administration result in substantially different biodistribution profiles. Importantly, IP administration resulted in significant uptake in lymphatic vessels, suggesting that IP administration may be a suitable route of administration for reaching immune cells.

[0347] Example 4: Engineering CD40L exosomes to activate B cells

[0348] CD40L is a member of the tumor necrosis factor (TNF) superfamily that is primarily expressed on T cells. The CD40L receptor, CD40, is expressed on antigen-presenting cells, including macrophages, dendritic cells, and B cells. Signaling through CD40 activates B cells and induces antigen-specific responses. Therefore, activation of the CD40 pathway has an impact on the development of anti-tumor immunity in various tumor types. To determine whether engineered exosomes can be produced to induce specific immune effects, exosomes were produced from HEK293SF cells transfected with a plasmid containing full-length human CD40L. The transfected cells were placed under puromycin selection, and the resistant cell population was grown to a high density. The resulting exosomes were collected from the conditioned medium as described above and purified by Optiprep. TM Gradient purification. Exosomes from unmodified HEK293SF cells were also isolated and used as a control. Human peripheral blood mononuclear cells (PBMCs) were seeded into 96-well plates at 150,000 cells per well and incubated with purified CD40L exosomes or natural exosomes at 37°C overnight. A PBMC sample was incubated with 1 μg / mL of soluble recombinant CD40L-Fc as a positive control. Figure 3A and 3B As shown, CD40L exosomes activated B cells in a dose-dependent manner, as measured by CD69 expression in samples from two different donors. Natural exosomes failed to induce B cell activation. Importantly, the level of B cell activation by CD40L exosomes was comparable to that caused by CD40L-Fc.

[0349] To determine whether the observed exosome-mediated B cell activation was due to direct activation of B cells or through trans-acting immune cells, similar experiments were performed using purified human B cells. 50,000 purified human B cells were seeded in 96-well plates and incubated with CD40L exosomes, native exosomes, or CD40L-Fc. A high-concentration CD40L exosome sample was freeze-thaw cycled (CD40L-EVs [F / T]) and tested for B cell activation. Figure 4A and 4B As shown, CD40L exosomes activated purified B cells from two donors to a similar extent as CD40L-Fc. Natural exosomes failed to activate B cells, while frozen-thawed samples of CD40L exosomes successfully activated B cells, suggesting that the effects of CD40L exosomes are mediated directly through B cells and that the presence of CD40L is sufficient for B cell activation. Furthermore, the engineered exosomes remained stable and active through at least one freeze-thaw cycle.

[0350] To further validate the CD40L exosomes, a reporter system was used to measure the activity of the engineered exosomes. Activation of the CD40 pathway leads to activation of NF-κB. Using a modified U2OS cell line (Promega Corporation) engineered to overexpress CD40 on its surface and containing a luciferase reporter gene downstream of the NF-κB promoter, CD40 activation was confirmed by incubating cells in the presence of an agonist anti-CD40 antibody (BioLegend, Inc.) cross-linked with an anti-Fc antibody (Jackson ImmunoResearch, Inc.) or recombinant human CD40L (ACROBiosystems) cross-linked with an anti-IgG antibody (Jackson ImmunoResearch, Inc.). Figure 5A and 5B Incubation of CD40L-engineered exosomes with engineered cells resulted in a robust increase in luciferase activity comparable to that achieved with anti-CD40+anti-Fc. Importantly, the engineered exosomes did not require cross-linking antibodies, suggesting that CD40L on the exosome surface can form functional CD40L trimers sufficient to activate CD40.

[0351] Example 5: Engineered CD80 exosomes activate T cells.

[0352] CD80 is expressed on antigen presenting cells and binds to CD28 and CTLA-4 on the surface of T cells. Stimulation of CD80 (and CD86) by CD28 and CTLA-4 activates T cells during the initiation of an immune response. To determine whether exosomes can be engineered to activate T cells, CD80-containing exosomes were generated by transfecting and selecting HEK293SF cells as described in Example 4. To verify the activity of CD80 exosomes, human PBMCs were seeded into 96-well plates at 150,000 cells per well and incubated with (i) purified CD80 exosomes and anti-CD3 antibodies, (ii) native exosomes and anti-CD3 antibodies, (iii) anti-CD3 antibodies alone, or (iv) a combination of anti-CD28 and anti-CD3 antibodies. The samples were incubated at 37°C for three days and analyzed for CD4 + T cells ( Figure 6A ) and CD8 + T cells ( Figure 6B ) T cell counts. CD80 exosomes activated T cells in a dose-dependent manner, to an extent comparable to positive controls of CD3 and CD28 antibodies. In contrast, native exosomes had no effect on T cell proliferation.

[0353] To confirm that CD80 exosomes induce functional activation of T cells, IFNγ levels were measured by AlphaLISA in PBMCs incubated with native exosomes and CD80 exosomes in the presence of additional anti-CD3 antibodies. Figure 7A As shown in Figure 3, there was a dose-dependent increase in IFNγ levels for CD80 exosomes, but not for native exosomes. Figure 7B As shown, the highest concentration of CD80 exosomes resulted in higher levels of IFNγ than any other condition, including the positive control (anti-CD28 / anti-CD3). These results suggest that exosomes can be engineered to have specific activities that lead to immune cell activation.

[0354] Example 6: Engineered CD27L and OX40L exosomes produce pro-inflammatory cytokines

[0355] CD27L (CD70) and OX40L are members of the TNF superfamily and bind to cognate receptors on T cells (CD27 and OX40, respectively). CD27L is expressed by certain T and B cell populations, while OX40L is expressed by certain antigen-presenting cell populations. Therefore, signaling through CD27 or OX40 has implications in immuno-oncology, particularly as a method for activating anergic T cells. To determine whether exosomes can be engineered to induce proinflammatory cytokine production in PBMCs, exosomes containing CD27L and OX40L were generated by transfection and selection of HEK293SF cells as described in Example 4. To validate the activity of CD27L exosomes, human PBMCs were seeded in 96-well plates and incubated with purified CD27L exosomes and anti-CD3 antibodies, native exosomes and anti-CD3 antibodies, anti-CD3 antibodies alone, or a combination of anti-CD28 and anti-CD3 antibodies. The samples were incubated at 37°C for two days and interferon gamma (IFNγ) production was determined in two different donors ( Figure 8A and 8B ) and IL-2 production ( Figure 9A and 9B CD27L exosomes induced IFNγ and IL-2 production in a dose-dependent manner, to an extent comparable to (donor 1) or significantly greater than (donor 2) the positive controls for CD3 and CD28 antibodies. In contrast, native exosomes had no effect on IFNγ or IL-2 production. Similarly, OX40L exosomes were sufficient to induce IFNγ and IL-2 production to a similar or greater extent in two different donors ( Figure 10A and 10B as well as Figure 11A and 11B ).

[0356] To further validate the OX40L exosomes, a reporter system was used to measure the activity of the engineered exosomes. Activation of the OX40 pathway leads to activation of NF-κB. Using a modified Jurkat T cell line (Promega Corporation) engineered to overexpress OX40 on its surface and containing a luciferase reporter gene downstream of the NF-κB promoter, the activation of OX40 was confirmed by incubating cells in the presence of an agonist anti-OX40 antibody (Biolegend) cross-linked with an anti-Fc antibody (Jackson ImmunoResearch, Inc.) or recombinant human OX40L (ACROBiosystems) cross-linked with an anti-IgG antibody (Jackson Immunoresearch). Figure 12A and 12B Anti-OX40L antibodies cross-linked with anti-IgG failed to activate reporter cells, whereas recombinant OX40L cross-linked with anti-Fc resulted in robust activation of the reporter gene ( Figure 12B Strikingly, engineered OX40L exosomes induced reporter gene expression to a greater extent than either anti-OX40 antibodies or recombinant OX40L ( Figure 12C Importantly, the engineered exosomes did not require cross-linking antibodies, suggesting that OX40L on the exosome surface can form functional OX40L trimers sufficient to activate OX40.

[0357] Example 7: Activation of T cells by IL-7 engineered exosomes

[0358] IL-7 is a cytokine involved in B cell and T cell proliferation and has implications for immunotherapy. Specifically, IL-7 may activate T cells and induce tumor antigen responses in tumors that are difficult for leukocytes to infiltrate or in tumor microenvironments that induce T cell anergy. IL-7 induces interferon gamma (IFNγ) signaling through heterodimeric IL-7 receptors, which can enhance tumor-specific antigen responses of T cells. To determine whether exosomes can be engineered to induce T cell activation, we used pDisplay™, which encodes a fusion of IL-7 and PDGF receptors, to express IFNγ in the tumor. TM HEK293SF cells were transfected with plasmids (ThermoFisher) and selected to generate IL-7-containing exosomes. The engineered exosomes were purified as described in the methods. To verify the activity of IL-7 exosomes, human PBMCs were seeded in 96-well plates and incubated with purified IL-7 exosomes and anti-CD3 antibodies, native exosomes and anti-CD3 antibodies, anti-CD3 antibodies alone, or a combination of anti-CD28 and anti-CD3 antibodies. The samples were incubated at 37°C for two days and IFNγ ( Figure 13A and 13BThe combination of IL-7 exosomes and anti-CD3 antibodies induced a greater degree of peak IFNγ production compared to anti-CD3 alone ( Figure 13A Furthermore, IL-7 exosomes induced IFNγ in a dose-dependent manner, to an extent comparable to that of positive controls of CD3 and CD28 antibodies. In contrast, native exosomes had no effect on IFNγ production ( Figure 13B ).

[0359] The IL-7 receptor is a heterodimeric complex composed of IL-7R and IL-2RG, which forms a ternary complex in the presence of IL-7 and induces downstream signaling through the JAK / STAT pathway, leading to cell proliferation. A synthetic cell-based assay was used to measure IL-7 signaling through the IL-7 receptor to evaluate the functional activity of engineered IL-7 exosomes (DiscoverX Corporation) ( Figure 14A Recombinant human IL-7 (rhIL-7) is sufficient to increase signaling through the IL-7 receptor ( Figure 14B ), while engineered IL-7 exosomes were able to induce signaling through the IL-7 receptor, whereas native exosomes were not ( Figure 14C These data demonstrate that exosomes expressing IL-7 are sufficient to induce signaling through the IL-7 receptor in vitro.

[0360] To determine whether the effects of IL-7 exosomes observed in vitro could be reproduced in an in vivo model, IL-7 exosomes were administered to C57BL / 6 mice. Cohorts of 20 mice were divided into the following groups: (1) PBS, (2) recombinant human IL-7 (rhIL-7), (3) IL-7 engineered exosomes, and (4) unmodified natural exosomes. Each group of five mice received an intraperitoneal (IP) injection of 1 mg EdU and PBS once daily, 1x10 11 The mice were sacrificed, spleens were isolated, and EdU levels in splenocytes were measured by flow cytometry. Figure 15A As shown, the percentage of positive CD8+ T cells was significantly increased in the IL-7 exosome mice and rhIL-7 mice compared to the control cohort. Although the T cell counts in the IL-7 exosome mice were lower than those in the rhIL-7 cohort, it was estimated that the IL-7 molecules administered in the IL-7 exosome cohort were reduced by five times (data not shown). A similar trend was observed for memory CD8+ T cells by measuring the levels of the memory marker CD45RO ( Figure 15B ).

[0361] As an orthogonal approach, the levels of CD71 (transferrin receptor) were measured in splenocytes isolated from exosome-treated mice. CD71 is required for proliferation, and the levels of CD71 correlate with T cell numbers. Figure 15A and 15B As shown, the number of CD8+ T cells and memory CD-8+ T cells followed the Figure 16A and 16B Together, these data suggest that engineered exosomes can induce specific immune cell effectors in vivo and that such activation may be more potent on a per-molecule basis compared with recombinant agonists.

[0362] Example 8: Fusion of IL-7 to a Proprietary Scaffold Enhances Specific Activity

[0363] To enhance the activity of IL-7 engineered exosomes, the IL-7 sequence was fused to a truncated portion of PTGFRN, a novel exosomal transmembrane protein that is highly expressed on the surface of HEK293SF exosomes. IL-7 was expressed as a translational fusion upstream of a short fragment of PTGFRN that included the C-terminal IgV domain of PTGFRN, the region preceding the transmembrane domain and the intracellular domain, and a FLAG tag. A series of expression constructs ( Figure 17A The resulting constructs were numbered pX-1 to pX-4 ( Figure 17B The complete sequence of pX-4 is shown in Figure 2). As shown by Western blot analysis using anti-IL-7 antibodies, constructs pX-3 and pX-4 showed the highest expression levels. The IL-7 expression level in the PTGFRN backbone was significantly higher than that of pDisplay-IL-7 ( Figure 18A ). Increased IL-7 expression suggests that these novel fusion proteins can induce higher levels of IL-7-mediated T cell activation. To determine the efficacy of the PTGFRN-IL-7 fusion, an in vitro model of T cell activation was performed. Following IL-7-mediated T cell activation, IL-7 receptor (IL-7R) levels decreased in a dose-dependent manner within 24 hours (Ghawazi et al., Immunol Cell Biol. 2013 Feb; 91(2): 149-58). Therefore, after incubation of PBMCs with various IL-7 engineered exosomes, IL-7R levels were monitored. Figure 18BAs shown in Figure 2, natural exosomes failed to reduce IL-7R levels, while pDisplay-IL-7 exosomes (IL-7-pD) only reduced IL-7R levels at high doses. On the contrary, PTGFRN-IL-7 exosomes (IL-7-pX3 to pX4) completely reduced IL-7R levels with much lower dosages, which shows that the effectiveness of these engineered exosomes is enhanced. As a measure of IC50, the effectiveness of PTGFRN-IL-7 exosomes is 20 to 76 times higher than that of IL-7-pD exosomes (Table 2), which shows that the increased ligand density is sufficient to increase biological effectiveness. In addition, these results indicate that specifically truncated PTGFRN may be an ideal scaffold for engineering therapeutic exosomes.

[0364] Table 8

[0365]

[0366] Example 9: Exosomes engineered with anti-CD3 antibody fragments

[0367] As shown in previous examples, exosomes can be engineered to overexpress functional endogenous sequences of immunomodulatory proteins. To determine whether synthetic agonists can be engineered on the surface of exosomes, anti-CD-3 antibodies were expressed as fusions with the transmembrane domains of pDisplay or CD80 as described in Example 4. Human PBMCs were seeded at 100,000 cells per well in 96-well plates and incubated with exosomes engineered to express anti-CD3 single-chain Fv (scFv) ( Figure 19A and 19B ) or single-chain Fab (scFab) ( Figure 20A and 20B As a positive control, PBMCs were incubated with ImmunoCult® according to the manufacturer's protocol. TM In the presence of anti-CD28 co-stimulation, all engineered exosome-induced T cells ( Figure 19A and 20A ) and B cells ( Figure 19B and 20B ) were comparable to the positive control, while the non-engineered exosome control had no effect. To measure the effect of anti-CD3 exosomes on immune cell populations, CD69 positivity of T and B cells was determined by flow cytometry. Figure 21A As shown, incubation of PBMCs with exosomes expressing anti-CD3 scFv fused to the CD80 transmembrane domain resulted in approximately 40% activation of T cells. Similar effects were observed for B cell activation ( Figure 21B ).

[0368] To determine whether anti-CD3 exosome-mediated T cell activation was due to direct T cell activation or through trans-acting immune cells, the activation of purified T cells was measured. 100,000 purified human T cells were plated in a 96-well format in wells pre-coated with non-targeting antibodies or anti-CD3 exosomes in the presence or absence of anti-CD28 antibodies, or in wells incubated with soluble anti-CD3 exosomes in the presence or absence of anti-CD28 antibodies. Figure 22A As shown, both soluble and plate-coated anti-CD3 scFv exosomes activated T cells in the presence of anti-CD28 antibodies as measured by CD69 expression. Figure 22B As shown, plate-coated anti-CD3 antibodies activated T cells to the same extent as plate-coated anti-CD3 scFv in the presence of anti-CD28 antibodies. Surprisingly, while soluble anti-CD3 antibodies were sufficient to activate approximately 30% of T cells in the presence of anti-CD28 antibodies, soluble anti-CD3 scFv exosomes activated a significantly higher proportion of T cells in the presence of anti-CD28 antibodies, suggesting that exosomes engineered to overexpress antibody fragments can induce higher levels of T cell activation compared to soluble antibodies. Taken together, these results demonstrate that exosomes can be engineered to overexpress antibody fragments that are functionally active against specific cell types.

[0369] Example 10: IL-12-PTGFRN exosomes have potent immunomodulatory activity both in vitro and in vivo.

[0370] IL-12 is an effective immunostimulatory cytokine produced by antigen-presenting cells in response to infection and other antigenic stimuli. Activated dendritic cells, macrophages, and neutrophils produce IL-12, which induces CD8+ and CD4+ T cells to produce IFNγ and induces the cytotoxic effect of natural killer (NK) cells. The combined effects of IL-12 secretion in the tumor microenvironment lead to the secretion of Th1 cytokines (including IFNγ), resulting in tumor cell killing, reprogramming of myeloid-derived suppressor cells (MDSCs), and anti-angiogenic effects. IL-12-mediated anti-tumor effects can produce lasting T cell responses and anti-tumor immunity in many animal models. IL-12 has previously been tested as an immunotherapeutic agent in humans, but despite the detectable induction of robust IFNγ responses, it still produced significant toxicity in patients with renal cell carcinoma (Leonard et al., Blood. October 1, 1997; 90(7): 2541-8). Therefore, exosomes may represent an ideal delivery method for IL-12 due to the high local concentration of the cytokine and the presumed pharmacological effects of tumor retention.

[0371] IL-12 is composed of two domains, p35 and p40. Human IL-12 dimer is encoded as a ligand with full-length PTGFRN ( Figure 23A , construct 871, SEQ ID NO: 3) or a shortened fragment of PTGFRN that enables high-density surface display ( Figure 23B , construct 873, SEQ ID NO: 5) and the construct was stably expressed in HEK293SF cells. The stable cell line was grown in chemically defined medium and cultured by Optiprep as described in the methods. TM Exosomes were gradient purified from culture supernatants. The amount of IL-12 protein on the exosome surface was measured by ELISA and concentration-matched with rIL-12 in all functional studies. Purified full-length and short hIL-12-PTGFRN exosomes or recombinant hIL-12 (rhIL-12; BioLegend, catalog number 573004) were titrated in human PBMCs in the presence of suboptimal concentrations of anti-CD3 antibodies to induce IFNγ expression. rhIL-12 resulted in robust IFNγ expression, EC 50 The efficacy was 0.029 ng / ml, which was comparable to that of full-length IL12-PTGFRN. Both were approximately 10-fold more potent than IL12-short-PTGFRN ( Figure 24A These results suggest that IL-12 displayed on the full-length PTGFRN scaffold may be a more potent immunomodulator than the shorter PTGFRN construct.

[0372] Mouse and human IL-12 proteins do not cross-react, and the in vitro data shown in Figure 24 indicate that mIL-12 fused to full-length PTGFRN is more effective than a shorter scaffold using PTGFRN. To determine the efficacy of mIL-12-PTGFRN exosomes in an in vivo cancer model, C57BL / 6 mice were subcutaneously implanted with 1x10 6 B16F10 mouse melanoma cells (n = 5 mice per group). On days 5, 6, and 7 after tumor inoculation, animals were injected intratumorally with PBS, 0.2 μg recombinant mouse IL-12 (mIL12; BioLegend, catalog number 577004), or 1×10 11 Once the tumor volume reached 2,000 mm 3 , kill the animals. Figures 25-27As shown, tumors in the PBS group grew rapidly, while tumors in the rmIL12 and mIL12-Exo groups decreased significantly (volume reduction of approximately 65-80%). Importantly, by day 16, tumors in the mIL12-Exo group were smaller than those in the rmIL12 group, demonstrating the greater efficacy of IL-12 when displayed on the surface of exosomes compared to soluble cytokines. The IL-12-treated group also had a survival advantage compared to the PBS-treated group ( Figure 28 ).

[0373] To understand the mechanistic advantage of IL-12-PTGFRN-exosomes over rmIL12, Th1 gene expression was analyzed in tumors from control and treated groups. IFNγ( Figure 29A ), T cell chemokine CXCL9 ( Figure 29B ) and CXCL10( Figure 29C ) and TGFβ( Figure 29D ) were increased. In most cases, cytokine signals were higher in animals treated with mIL12-Exo compared to rmIL-12. By measuring IFNγ levels in splenic CD8+ T cells by flow cytometry, mice treated with Exo-mIL-12 showed significantly higher signals than those in the PBS or rmIL-12 groups ( Figure 30 Together, these data demonstrate that IL-12 displayed on the surface of exosomes represents a novel and potent immunomodulatory strategy that promotes robust T cell activation in vitro and can be used to elicit potent antitumor effects in vivo in an aggressive model of mouse melanoma. Mechanistically, IL-12 exosomes demonstrate superiority over rIL-12 and thus represent a novel, differentiated therapeutic modality in cancer immunotherapy.

[0374] Example 11: Exosomes displaying interferon gamma are potent immune cell activators

[0375] Interferon gamma (IFNγ) is a cytokine involved in initiating both innate and adaptive immune responses. It is expressed by a variety of cell types in response to a variety of signals, including IL-12, and is sufficient to activate NK cells, drive antigen presentation in antigen-presenting cells, and promote leukocyte activation and invasion. IFNγ is naturally expressed as a homodimer and secreted as a soluble factor. Exosomes expressing IFNγ were generated by stably transfecting HEK293SF cells with full-length PTGFRN fused to monomeric or dimeric human and mouse IFNγ (respectively). Figure 31A and 31B). Exosomes from suspension cell cultures were purified as described above and analyzed by PAGE. Monomeric (m) and tandem dimer (td) PTGFRN IFNγ exosomes expressed at comparable levels at predicted molecular weights (arrows) ( Figure 32 Purified exosomes were analyzed by ELISA and compared to a standard curve using recombinant IFNγ (Biolegend, catalog number 570206) to calculate the number of IFNγ molecules per exosome. The results in Table 9 show the number of IFNγ molecules in each of the four types of purified exosomes. Notably, the tandem dimeric IFNγPTGFRN exosomes contained at least twice as many IFNγ molecules as the monomeric IFNγPTGFRN exosomes, indicating that the tandem dimeric exosomes appropriately expressed the dimeric IFNγ construct.

[0376] Table 9

[0377] Construct IFNγ molecules / exosomes h-mIFNγ-PTGFRN 53 h-tdIFNγ-PTGFRN 173 m-mIFNγ-PTGFRN 47 m-tdIFNγ-PTGFRN 113

[0378] Human monomeric and tandem dimer PTGFRN-IFNγ exosomes were incubated with human PBMCs at increasing concentrations for 24 hours. Monocyte activation was measured by PD-L1 expression, a downstream surface protein induced by IFNγ signaling. Figure 33 As shown, native HEK293SF exosomes (WT) failed to induce PD-L1 expression, whereas both monomeric and tandem dimeric IFNγPTGFRN exosomes induced PD-L1 in a dose-dependent manner, with tandem dimeric IFNγPTGFRN exosomes having a greater activation effect. Exosome-mediated PD-L1 activation was comparable to LPS-induced activation ( Figure 33 These data demonstrate that soluble cytokines in monomeric or dimeric form can be functionally expressed on the surface of exosomes and induce immune cell activation. The use of exosomes expressing IFNγ in immuno-oncology can be used to induce NK and T cell responses against tumor cells.

[0379] Example 12: Exosomes expressing IL-15 induce NK cell activation

[0380] Interleukin 15 (IL-15) is a cytokine produced by monocytes following pathogenic infection. IL-15 can be secreted as a soluble protein or presented as a dimeric membrane-anchored protein that binds to IL-15Rα. IL-15 activates NK cells and T cells and is considered a potential therapeutic molecule in immuno-oncology and other immunointervention therapies. Exosomes expressing IL-15 were generated by stably transfecting HEK293SF cells with an expression plasmid encoding the transmembrane domain of PDGFR (pDisplay) fused to IL-β. 15 / IL-15Rα fusion protein ( Figure 34As described above, exosomes were purified by Optiprep TM Purified by density gradient ultracentrifugation. Purified exosomes were incubated with human PBMC for 24 hours, and NK cell activation was measured by flow cytometry as the percentage of CD69 positivity. pDisplay IL-15 exosomes were not expressed at a maximum of 10 cells per cell in PBMC cultures. 5 The dose of exosomes induced NK cell activation ( Figure 35 ; Exosome construct quantity as Figure 34 To investigate whether a higher density of IL-15 display is required to induce NK cell activation, HEK293SF cells were stably transfected with an expression plasmid encoding IL-15 fused to the full-length PTGFRN. In addition, HEK293SF cells were stably transfected with an expression plasmid encoding a more potent IL-15 fused to the full-length PTGFRN (IL-15N72D, as described in J Immunol. 2009 Sep 15;183(6):3598-607; Figure 36A Expression was confirmed by anti-PTGFRN Western blotting ( Figure 36B ). IL-15 levels were quantified by ELISA (R&D Systems, catalog number D1500) and normalized to recombinant IL-15 (Biolegend, catalog number 570302). IL-15PTGFGN exosomes were added to two separate PBMC cultures overnight and compared with concentration-matched recombinant IL-15. All three IL-15 sources induced NK cell activation in PBMCs in a dose-dependent manner, as measured by the percentage of NK cells positive for CD69. In addition, all constructs were comparable between the two donors, demonstrating meaningful comparative efficacy ( Figure 37 ; the number of exosome constructs is shown in Figure 36). These data indicate that IL-15 can be efficiently and robustly displayed on the surface of exosomes, but this requires high expression levels, such as those conferred by PTGFRN.

[0381] Example 13: Activation of T cells by exosomes displaying anti-CD-3 antibody fragments on PTGFRN scaffolds

[0382] The results in Example 9 demonstrate that exosomes displaying anti-CD3 antibody fragments can activate T cells. To determine whether the PTGFRN scaffold supports this activity, anti-CD3 antibody fragments (OKT3 variants) were fused to the PDGFR transmembrane region (exoCD3-PD), full-length PTGFRN (exoCD3-long), or a PTGFRN fragment (exoCD3-short) and stably expressed in HEK293SF cells ( Figure 38 ). Exosome binding is the use of RED96 (Pall) was used to confirm the presence of CD3 fragments by biolayer interferometry (BLI). Figure 39 , ii), washing ( Figure 39 , iii), and adding exosome constructs ( Figure 39 iv). Exosomes from WT HEK293SF cells did not bind to the BLI probe, but all engineered constructs did. Both PTGFRN fragments bound to the probe with high affinity and maintained stable binding ( Figure 39 , v). Anti-CD3 displaying exosomes were tested In vitro T cell activation was measured by CD69 positivity on CD4+ T cells, as measured by flow cytometry. Exosomes harboring anti-CD3 fused to a PTGFRN fragment (exoCD3-short) efficiently stimulated CD3 expression in vitro compared to unmodified native exosomes (exoNative). Live CD4+ T cells (Figure 40).

[0383] Example 14: Exosomes displaying CD40L are potent activators of B cells

[0384] CD40 ligand (CD40L) is a ligand of the tumor necrosis superfamily (TNFSF) that binds to the co-stimulatory receptor CD40, which is highly expressed on B cells and other antigen-presenting cells. TNFSF ligand-mediated cell activation requires the formation of a trimeric ligand complex that forms on the cell surface and binds to the cognate receptor. To investigate whether exosomes displaying different conformations of CD40L on their surface are sufficient to activate B cells, more than 40 different CD40L expression constructs were designed and transfected into HEK293SF cells. CD40L was expressed as a fusion to the transmembrane domain of PDGFR, full-length PTGFRN, and a short single-domain fragment of PTGFRN ( Figure 41 , bottom). CD40L-GFP PTGFRN fusions were expressed as monomers (pCB-518 to pCB-526) or as forced trimers (pCB-607 and pCB-527) ( Figure 41 , bottom). To promote trimerization of monomeric CD40L, constructs expressing fusions with the multimerization domains from TRAF2 (pCB-521 to pCB-523) or collagen XV (pCB-524 to pCB-526) were designed. Of the monomeric CD40L constructs, pCB-518 / 521 / 524 contained the full-length N-terminal stem sequence from endogenous CD40L; pCB-519 / 522 / 525 contained a truncated N-terminal stem sequence from endogenous CD40L; and pCB-520 / 523 / 526 contained only the soluble portion of CD40L. Each engineered exosome population was then immunized with a 500 μg / mL exosome using RosetteSep. TMPurified B cells isolated from human peripheral blood were incubated with human B cell enrichment cocktail (Stemcell Technologies #15064), and B cell activation was measured by flow cytometry to detect CD4+ positivity of B cells. 50 Calculated as a function of the particle concentration of the cell culture and plotted in Figure 41 Interestingly, all monomeric CD40L constructs were moderately potent, while the trimeric constructs were at least ten times more potent than the monomer ( Figure 41 , upper panel). These results suggest that monomeric CD40L is a weak activator of B cells when present on the surface of exosomes, but forced trimeric CD40L can induce robust B cell activation. Furthermore, PTGFRN has been shown to form dimeric structures (PCT / US2018 / 048026), suggesting that higher-order multimeric structures may form on the surface of exosomes to further promote target engagement and immune cell activation.

[0385] Figure 41 The results shown in all used exosomes containing lumenal GFP fused to the C-terminus of PTGFRN. To generate untagged CD40L exosomes, a trimeric CD40L-PTGFRN construct identical to the lead construct pCB-527 but lacking the C-terminal GFP was stably expressed in HEK293SF cells (pCB-766). The absolute concentration of CD40L on the surface of the engineered exosomes was quantified using ELISA (R&D Systems, catalog number DCDL40) as shown in Table 10 below.

[0386] Table 10

[0387] EC50 pCB-0766 pCB-0527 rhCD40L Particles / mL 6.63E+08 4.53E+08 N / A ng / mL 1.68 1.89 28.51

[0388] Purified CD40L-PTGFRN exosomes were tested in a B cell activation assay as described above compared to concentration-matched recombinant human CD40L (Biolegend, catalog number 591702). GFP-containing and untagged CD40L exosomes were comparable B cell activators when measured as a function of particle number or CD40L concentration ( Figure 42A ), and both exosome preparations were more effective than concentration-matched CD40L ( Figure 42B ). Native non-engineered exosomes from HEK293SF cells failed to activate B cells, indicating that the engineered CD40L trimer construct on the exosome surface is sufficient to effectively activate B cells.

[0389] Another way to agonize CD40 and activate B cells is to use an agonistic antibody cross-linked with a secondary antibody. To compare the efficacy of exosomes expressing trimeric CD40L with an agonistic CD40L antibody, PBMC cultures were incubated with 2 μg / ml of an anti-CD40L antibody ( Clone 5C3) and incubate together. Figure 43A and 43B In the figure, the dotted line shows the maximum B cell activation. Figure 43A and 43B ), pCB-527 exosomes (PTGFRN-trimeric CD40L-GFP) induced greater maximal B cell activation than cross-linked agonistic antibodies ( Figure 43A and 43B ), which suggests that trimeric CD40L exosomes have superiority in activating immune cells.

[0390] Example 15: Simultaneous display of multiple immuno-oncology molecules on a single exosome

[0391] Previous examples have demonstrated that a single immunomodulatory protein can be displayed on the surface of exosomes and induce functional changes in one or more immune cell types. In certain applications, it may be desirable to use combinatorially engineered exosomes, i.e., exosomes containing more than one molecule on the exosome surface, each capable of conducting a different immune cell pathway. HEK293SF cells were stably transfected with plasmids expressing PTGFRN-IL-12 and PTGFRN-CD40L fusion proteins. Exosomes were isolated and purified as described above. Exosomes from unmodified HEK293SF cells were used as a negative control.

[0392] To demonstrate simultaneous loading of different ligands, a pull-down co-staining assay was developed:

[0393] Reagents:

[0394] Dynabeads (Thermofisher Exosome-Streptavidin Isolation / Detection Reagent, Cat. No. 10608D): 1x10 7 Beads / mL, 50% slurry

[0395] Separation buffer: 0.5% BSA / PBS (1:4 2% BSA)

[0396] Blocking buffer: 2% BSA / PBS (1 gr / 50 mL, filter)

[0397] Wash 0.5 ml of beads with 0.5 ml of separation buffer and resuspend in 0.5 mL of separation buffer

[0398] Add 1 μg of biotinylated capture antibody (2.2 μl of 0.5 μg / μl stock solution)

[0399] ● Rotate for 1 hour at room temperature

[0400] Wash with 500 μl separation buffer

[0401] Resuspend in 500 μl blocking buffer and rotate at room temperature for 10 minutes

[0402] ● Incubate in 500 μl separation buffer (1x10 7 beads / mL, 50% slurry)

[0403] ●Store at 4°C

[0404] A. Exosome capture and mobilization

[0405] 1x10 5 Beads / sample (10 μl beads, 20 μl slurry)

[0406] 50,000 exosomes / bead; 5x10 9 Exosomes / sample (1.2x10 9 exosomes / μL stock solution)

[0407] 5 μl of each fluorescently labeled detection antibody was flow cytometry tested.

[0408] Mixed 5x10 9 Exosomes + 20 μl Dynabeads slurry + 0.7 ml 0.1% BSA / PBS

[0409] program:

[0410] 1. 120 μl slurry beads, remove supernatant, add 0.7 ml blocking buffer, mix, rotate at room temperature for 10 minutes, remove supernatant

[0411] 2. Resuspend the beads in 0.7 ml isolation buffer + 25.2 μl exosomes and spin at 4°C

[0412] 3. Day 2: Quickly spin the exosomes and beads for 5 seconds

[0413] 4. Place the tube on the magnet and remove the supernatant

[0414] 5. Seal 700μl and rotate at room temperature for 10 minutes

[0415] 6. Place the tube on the magnet and remove the supernatant

[0416] 7. Resuspend in 600 μl separation buffer: 6 x 100 μl per tube

[0417] 8. Add 1 μl of labeled detection antibody, mix, and incubate at 4°C in the dark for 30 minutes

[0418] 9. Spin at 500g for 2 minutes and remove the supernatant

[0419] 10. Wash 2x with separation buffer

[0420] 11. Resuspend in 200 μl separation buffer and run the flow cytometry.

[0421] Beads decorated with anti-CD40L and antibodies against IL-12 and CD40L ( Figure 44A ) or CD81 (exosomal marker present on natural and engineered exosomes) and CD40L ( Figure 44B ) were used to isolate natural exosomes. Since no fluorescent signals for IL-12, CD40L, or CD81 were detected, CD40L beads did not pull down any natural exosomes. In contrast, PTGFRN-CD40L / IL-12 dual-engineered exosomes were incubated with anti-CD40L beads and isolated as described above. CD81 ( Figure 45A ), IL-12 or CD40L ( Figure 45B ), indicating that CD40L-mediated isolation can also isolate IL-12 exosomes. Similarly, anti-IL-12 decorated beads were incubated with IL-12 / CD40L engineered exosomes and stained for IL-12, CD40L, and CD81. Greater than 98% of all beads were positive for CD40L and either IL-12 or CD81 ( Figure 46A and 46B ), which indicates that exosomes contain both IL-12 and CD40L on their surface.

[0422] The concentration of IL-12 and CD40L was quantified by ELISA (Abcam catalog number ab119517) to test the in vitro efficacy of engineered exosomes. Equal concentrations of recombinant IL-12, a mixture of recombinant IL-12 and recombinant CD40L, PTGFRN-IL-12 exosomes, double positive PTGFRN-CD40L / IL-12 exosomes or a mixture of PTGFRN-IL-12 exosomes and PTGFRN-CD40L exosomes were added to human PBMCs (rhIL-12-BioLegend, catalog number 573004; rhCD40L-Biolegend, catalog number 591702) at increasing concentrations. Cells were co-stimulated with anti-CD3 antibodies and the production of IFNγ was measured by (PerkinElmer, catalog number AL217C). Figure 47A and47B As shown, all IL-12-containing exosome preparations elicited IFNγ responses comparable to those of the recombinant cytokine. Calculation of the EC50 under various conditions indicated that exosome-associated IL-12 was more potent than concentration-matched IL-12, whether expressed alone or in combination on the exosome surface ( Figure 48 In the context of B cell activation, similar results were obtained with recombinant CD40L and single- or double-engineered CD40L exosomes ( Figure 49A and B). Again, CD40L-engineered exosomes were more potent than soluble recombinant cytokines, in this case dually engineered exosomes were the most potent construct tested in the assay ( Figure 50 ).

[0423] To further explore the possibility of combinatorial surface display of exosomes, HEK293SF cells were stably transfected with three separate constructs expressing either PTGFRN-IL-12, PTGFRN-CD40L, or PTGFRN-FLT3L fusion proteins. Exosomes were purified and isolated by the affinity bead method as described above, but were also interrogated for the presence of surface FLT3L using an anti-FLT3L-PE conjugated antibody. Exosomes isolated with anti-IL-12 beads expressed IL-12 and CD40L ( Figure 51A ), IL-12 and FLT3L ( Figure 51B ), and CD40L and FLT3L ( Figure 51C ) were double positive. Exosomes isolated with anti-CD40L beads were positive for IL-12 and CD40L ( Figure 52A ), IL-12 and FLT3L ( Figure 52B ), and CD40L and FLT3L ( Figure 52C ), confirming that each exosome expressed three immunomodulatory ligands. These results suggest that multiply engineered immunomodulatory exosomes are a viable therapeutic approach and are comparable or more effective than soluble cytokines in activating immune cells.

[0424] ***

[0425] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be apparent to those skilled in the art in light of the teachings of this disclosure that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

[0426] Therefore, the above merely illustrates the principles of the present invention. It should be understood that those skilled in the art will be able to design various arrangements that, although not explicitly described or shown herein, embody the principles of the present invention and are included within its spirit and scope. In addition, all embodiments and conditional language described herein are primarily intended to help readers understand the principles of the present invention without limiting the embodiments and conditions of such specific descriptions. In addition, all statements describing the principles, aspects, and embodiments of the present invention and its specific embodiments herein are intended to encompass their structural equivalents and functional equivalents. In addition, it is intended that such equivalents include currently known equivalents and equivalents developed in the future, that is, any element that performs the same function, regardless of structure. Therefore, the scope of the present invention is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of the present invention are embodied by the appended claims.

[0427] Sequence Listing

[0428] >SEQ ID NO:1

[0429]

[0430] >SEQ ID NO:2

[0431]

[0432] >hIL-12-PTGFRN;871(SEQ ID NO:3)

[0433]

[0434] >mIL-12-PTGFRN;872(SEQ ID NO:4)

[0435]

[0436] >hIL-12-short PTGFRN; 873 (SEQ ID NO: 5)

[0437]

[0438] >mIL-12-short PTGFRN; 874 (SEQ ID NO: 6)

[0439]

[0440] SEQ ID NO:7PTGFRN_IFN_γ monomer

[0441]

[0442] SEQ ID NO:8PTGFRN_IFN_γ dimer

[0443]

[0444]

[0445] SEQ ID NO:10PTGFRN_IFN_γ mouse dimer

[0446]

[0447] SEQ ID NO:11 IL-15 441

[0448] MAPRRARGCRTLGLPALLLLLLLRPPATRGITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTTGGSGGGSGGGGSGGGGSGGGSGGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSSADYKDDDDKFEGGGGSGGGGSAVGQDTQEVIVVPHSLPFKVVVISAILALVVLTIISLIILIMLWQKKPRSGLLTGRT

[0449] SEQ ID NO:12 IL-15 442

[0450] MAPRRARGCRTLGLPALLLLLLLRPPATRGHHHHHHITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTTGGSGGGSGGGGSTLDPRSFLLRNPNDKYEPFWEDEEKNESGGGGSGGGSGGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSSADYKDDDDKFEGGGGSGGGGSAVGQDTQEVIVVPHSLPFKVVVISAILALVVLTIISLIILIMLWQKKPRSGLLTGRT

[0451] SEQ ID NO:13 IL-15 443

[0452] METDTLLLWVLLLWVPGSTGNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSGGSGGGSGGGGSGGGGSGGGSGGSITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTTSADYKDDDDKFEGGGGSGGGGSAVGQDTQEVIVVPHSLPFKVVVISAILALVVLTIISLIILIMLWQKKPRSGLLTGRT

[0453] SEQ ID NO:14 IL-15 444

[0454] METDTLLLWVLLLWVPGSTGNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSDYKDDDDKGGSGGGSGGGGSTLDPRSFLLRNPNDKYEPFWEDEEKNESGGGGSGGGSGGSITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTTSAFEGGGGSGGGGSAVGQDTQEVIVVPHSLPFKVVVISAILALVVLTIISLIILIMLWQKKPRSGLLTGRTHHHHHH

[0455] SEQ ID NO:15 IL-15 1009

[0456]

[0457] SEQ ID NO:16 IL-15 1010

[0458]

[0459] SEQ ID NO:17 pDisplay - anti - CD3

[0460] MKIICLALVALLLTAQPAMAEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGSSGSGSGSTGTSSSGTGTSAGTTGTSASTSGSGSGGGGGSGGGGSAGGTATAGASSGSQVQLVESGGGVVQPGRSLRLSCAASGFKFSGYGMHWVRQAPGKGLEWVAVIWYDGSKKYYVDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARQMGYWHFDLWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTGGSGGGSGGGGSGGGGSGGGSGGSAVGQDTQEVIVVPHSLPFKVVVISAILALVVLTIISLIILIMLWQKKPRDYKDDDDK

[0461] SEQ ID NO:18 PTGFRN - anti - CD3

[0462]

[0463] SEQ ID NO:19PTGFRN_CD40L trimer mouse

[0464]

[0465] SEQ ID NO:20PTGFRN_CD40L trimer human

[0466]

[0467] MKIICLALVALLLTAQPAMAEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGSSGSGSGSTG TSSSGTGTSAGTTGTSASTSGSGSGGGGGSGGGGSAGGTATAGASSGSQVQLVESGGGVVQPGRSLRLSCAASGFKFSGYGMHWVRQAPGKGLEWVAVIWYDGSKYYVDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARQMGYWHFDLWGRGTLVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI CNVNHKPSNTKVDKKVEPKSCDKTHTGGSGGGSGGGSGGGSGGGSGGSGGSGPIFNASVHSDTPSVIRGDLIKLFCIITVEGAALDPDDMAFDVSWFAVHSFGLDKAPVLLSSLDRKGIVTTSRRDWKSDLSLERVSVLEFLLQVHGSEDQDFGNYYCSVTPWVKSPTGSWQKEAEIHSKPVFITVKMDVLNAFKYPLLIGVGLSTVIGLLSCLIGYCSSHWCCKKEVQETRRERRRLMSMEMDTGGSGGSVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICCTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKDYKDDDDK

[0468] SEQ ID NO:22FLT3L-PTGFRN

[0469]

[0470] surface

[0471]

[0472]

[0473]

[0474]

[0475]

[0476]

[0477]

[0478]

[0479]

[0480]

[0481]

[0482]

[0483]

[0484]

[0485]

[0486]

[0487]

[0488]

[0489]

[0490]

[0491]

[0492]

[0493]

[0494]

[0495]

Claims

1. A composition comprising: An extracellular vesicle comprising (i) a cell membrane defining an enclosed volume, the cell membrane having an inner surface and an outer surface; and (ii) a protein fused to a prostaglandin F2 receptor negative regulator (PTGFRN), wherein the PTGFRN is: Full-length PTGFRN; or A functional fragment of PTGFRN selected from: (i) amino acid residues 1222 to 1418 of SEQ ID NO: 3; or (i) amino acid residues 1226 to 1418 of SEQ ID NO: 3, wherein the protein comprises an immunomodulatory component selected from the group consisting of CD80, CD27L, OX40L, IL-7, IL-12, IL-15, IFNγ, CD40L, FLT3L, and an anti-CD3 antibody fragment, and wherein the protein is displayed on the outer surface of the extracellular vesicle.

2. The composition of claim 1, wherein the immunomodulatory component is CD40L.

3. The composition according to claim 2, wherein the amino acid sequence of the protein comprising CD40L is shown in SEQ ID NO: 19 or SEQ ID NO:

20.

4. The composition of claim 1, wherein the immunomodulatory component comprises IL-12. 5 . The composition according to claim 4 , wherein the amino acid sequence of the protein comprising the IL-12 is shown in SEQ ID NO: 3, 4, 5 or 6. The composition of claim 1 , wherein the immunomodulatory component comprises FLT3L. 7 . The composition according to claim 6 , wherein the amino acid sequence of the protein comprising FLT3L is shown in SEQ ID NO:

22.

8. A composition according to any one of claims 1 to 7, wherein the immunomodulatory component is fused to the N-terminus of PTGFRN.

9. The composition of any one of claims 1 to 7, wherein the PTGFRN is full-length PTGFRN.

10. The composition according to any one of claims 1 to 7, wherein the PTGFRN is a functional fragment of PTGFRN.

11. The composition of any one of claims 1 to 7, wherein the extracellular vesicles are exosomes.

12. The composition according to any one of claims 1 to 7, wherein the extracellular vesicles are nanovesicles.

13. The composition according to any one of claims 1 to 7, further comprising a pharmaceutically acceptable carrier.

14. The composition of any one of claims 1 to 7, wherein the extracellular vesicles further comprise one or more additional immunomodulatory components.

15. A composition comprising (i) extracellular vesicles and (ii) interleukin 12 (IL-12) protein, wherein the IL-12 is expressed as a fusion protein fused to prostaglandin F2 receptor negative regulator (PTGFRN), wherein the PTGFRN is: Full-length PTGFRN; or A functional fragment of PTGFRN selected from: (i) amino acid residues 1222 to 1418 of SEQ ID NO: 3; or (i) amino acid residues 1226 to 1418 of SEQ ID NO: 3, The fusion protein is expressed on the outer surface of the extracellular vesicles. The composition according to claim 15 , wherein the amino acid sequence of the fusion protein is shown in SEQ ID NO: 3, 4, 5 or 6.

17. A composition comprising (i) extracellular vesicles and (ii) CD40L, wherein the CD40L is expressed as a fusion protein fused to prostaglandin F2 receptor negative regulator (PTGFRN) or a functional fragment or variant thereof, wherein the PTGFRN is: Full-length PTGFRN; or A functional fragment of PTGFRN selected from: (i) amino acid residues 1222 to 1418 of SEQ ID NO: 3; or (i) amino acid residues 1226 to 1418 of SEQ ID NO:

3. The composition according to claim 17 , wherein the amino acid sequence of the fusion protein is shown in SEQ ID NO: 19 or 20.

19. A composition comprising (i) extracellular vesicles and (ii) FLT3L, wherein the FLT3L is expressed as a fusion protein fused to prostaglandin F2 receptor negative regulator (PTGFRN), wherein the PTGFRN is: Full-length PTGFRN, or A functional fragment of PTGFRN selected from: (i) amino acid residues 1222 to 1418 of SEQ ID NO: 3; or (i) amino acid residues 1226 to 1418 of SEQ ID NO: 3, The fusion protein is expressed on the outer surface of the extracellular vesicles.

20. The composition according to claim 19, wherein the amino acid sequence of the fusion protein is shown in SEQ ID NO:

22.

21. A composition comprising (i) extracellular vesicles, (ii) IL-12, and (iii) CD40L, wherein the IL-12, CD40L, or both are expressed as a fusion protein fused to a prostaglandin F2 receptor negative regulator (PTGFRN), wherein the PTGFRN is: Full-length PTGFRN, or A functional fragment of PTGFRN selected from: (i) amino acid residues 1222 to 1418 of SEQ ID NO: 3; or (i) amino acid residues 1226 to 1418 of SEQ ID NO: 3, The fusion protein is expressed on the outer surface of the extracellular vesicles.

22. A composition comprising (i) extracellular vesicles, (ii) IL-12, (iii) CD40L, and (iv) FLT3L, wherein the IL-12, CD40L, FLT3L, or a combination thereof, is expressed as a fusion protein fused to prostaglandin F2 receptor negative regulator (PTGFRN), wherein the PTGFRN is: Full-length PTGFRN, or A functional fragment of PTGFRN selected from: (i) amino acid residues 1222 to 1418 of SEQ ID NO: 3; or (i) amino acid residues 1226 to 1418 of SEQ ID NO: 3, The fusion protein is expressed on the outer surface of the extracellular vesicles.

23. A method for producing extracellular vesicles comprising a fusion protein comprising an immunomodulatory component fused to a prostaglandin F2 receptor negative regulator (PTGFRN), wherein the PTGFRN is: Full-length PTGFRN, or A functional fragment of PTGFRN selected from: (i) amino acid residues 1222 to 1418 of SEQ ID NO: 3; or (i) amino acid residues 1226 to 1418 of SEQ ID NO: 3, wherein the protein is expressed on the outer surface of the extracellular vesicle, and wherein the method comprises: modifying a production cell with the fusion protein; as well as The extracellular vesicles are obtained from the producer cells.

24. Use of a composition according to any one of claims 1 to 22 in the preparation of a medicament for treating a disease or disorder in a subject in need thereof.

25. Use of a composition according to any one of claims 1 to 22 in the preparation of a medicament for treating cancer in a subject in need thereof.

26. Use of the composition of any one of claims 1 to 22 in the preparation of a medicament for treating graft-versus-host disease (GvHD) in a subject in need thereof.

27. Use of a composition according to any one of claims 1 to 22 in the preparation of a medicament for treating an autoimmune disease in a subject in need thereof.

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