Exosomes for immunooncology and anti-inflammatory therapy

Abstract

Disclosed herein are extracellular vesicles comprising an immunomodulatory component. Methods of producing the extracellular vesicles and methods of using the extracellular vesicles to treat cancer, GvHD, and autoimmune diseases are also provided.

Description

[0001] This application is a divisional application of patent application No. 201880083335.0, filed on December 28, 2018, filed by Lonsa Sales Co., Ltd., entitled "Exogenous Body for Immuno-oncology and Anti-inflammatory Therapy".

[0002] Cross-references to related applications

[0003] This application claims the benefit of U.S. Provisional Application No. 62 / 723,267, filed August 27, 2018; and U.S. Provisional Application No. 62 / 611,140, ​​filed December 28, 2017, the entire contents of which are incorporated herein by reference. Invention Field

[0004] This invention relates to compositions for interacting with and modulating the human immune system, methods for preparing said compositions, and methods for using said compositions to treat cancer, GvHD, and autoimmune diseases. Background Technology

[0005] Immunotherapy treats disease by inducing, enhancing, or suppressing the immune response. Immunotherapy can stimulate a patient's own immune system to attack cancer cells. Cancer immunotherapy generally has fewer side effects than traditional cancer therapies such as chemotherapy and radiation therapy. Anti-inflammatory immunotherapy can downregulate a patient's immune system to treat autoimmune diseases and graft-versus-host disease (GvHD). Improved methods are needed for delivering immune-modulating molecules to the body's cells and tissues. Summary of the Invention

[0006] Extracellular vesicles offer numerous advantages over traditional drug delivery methods, particularly for gene therapy. Systemic delivery of extracellular vesicles allows these lipid nanoparticles to distribute throughout various tissues. Studies have shown that extracellular vesicles can interact with a wide range of cells involved in the regulation of the human immune system. Selecting, enriching, or engineering extracellular vesicles to deliver therapeutic molecules to activate, inhibit, or influence the human immune system may be effective therapeutic agents for cancer and other immune system-related diseases.

[0007] This article provides compositions comprising extracellular vesicles that are selected, enriched, or engineered to have immunomodulatory components that can upregulate or downregulate the human immune system, thereby enhancing a patient’s immune system to fight cancer or suppressing a patient’s immune system to alleviate symptoms of GvHD and autoimmune diseases.

[0008] It also provides methods for generating and utilizing extracellular vesicles to regulate the human immune system.

[0009] Therefore, in a first aspect, this document provides a composition comprising: an extracellular vesicle comprising a cell membrane defining a closed volume, the cell membrane having an inner surface and an outer surface; and a first immunomodulatory component associated with or enclosed within the closed volume.

[0010] In various embodiments, the first immunomodulatory component is an inhibitor of a negative checkpoint modulator or an inhibitor of a binding partner of a negative checkpoint modulator. In some of these embodiments, the negative checkpoint modulator 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 immune receptor with Ig and ITIM domains (TIGIT), inhibitor of V-domain Ig of T-cell activation (VISTA), adenosine A2a receptor (A2aR), cytotoxic cell immunoglobulin-like receptor (KIR), indoleamine 2,3-dioxygenase (IDO), CD20, CD39, and CD73.

[0011] In various embodiments, the first immunomodulatory component is an activator of the positive co-stimulatory molecule or an activator of the binding chaperone of the positive co-stimulatory molecule. In some embodiments, the positive co-stimulatory molecule is a member of the TNF receptor superfamily. In some of these embodiments, the TNF receptor superfamily members are 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 the positive co-stimulatory molecule is a member of the TNF superfamily. 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 some embodiments, the TNF superfamily member is CD40L. In some embodiments, the TNF superfamily member is CD27L. In some embodiments, the TNF superfamily member is OX40L.

[0012] In some embodiments, the positive co-stimulatory molecule is a CD28 superfamily co-stimulatory molecule. In some of these embodiments, the CD28 superfamily co-stimulatory molecule is ICOS or CD28. In some embodiments, the activator of the positive co-stimulatory molecule is ICOS, CD80, or CD86. In some embodiments, the activator of the positive co-stimulatory molecule is CD80.

[0013] In some embodiments, the first immunomodulatory component is a cytokine or a cytokine binding partner. In some embodiments, the cytokine is selected from the group consisting of IL-2, IL-7, IL-10, IL-12, and IL-15. In some embodiments, the cytokine is IL-7. In some embodiments, the cytokine is IL-12. In some embodiments, the cytokine is IL-15.

[0014] In some implementations, the first immunomodulatory component is a T-cell receptor (TCR), a T-cell helper receptor, a major histocompatibility complex (MHC), a human leukocyte antigen (HLA), or a derivative thereof.

[0015] In some embodiments, the first immunomodulatory component is an activator of a T-cell receptor or co-receptor. In some embodiments, the activator of the T-cell receptor or co-receptor is a CD3 activator, optionally a CD3 agonist antibody.

[0016] 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-1gp100, and TNF-associated apoptosis-inducing ligand. In some embodiments, the tumor antigen is derived from a reference genome sequence. In some embodiments, the tumor antigen is derived from the subject's genome sequence.

[0017] In some implementations, the first immunomodulatory component is a selected target or active agonist or antagonist.

[0018] In some implementations, the first immunomodulatory component is an antibody or antigen-binding fragment.

[0019] In some implementations, the first immunomodulatory component is a polynucleotide. In some of these implementations, the polynucleotide is selected from the group consisting of: mRNA, miRNA, siRNA, antisense RNA, shRNA, lncRNA, and dsDNA.

[0020] In some implementations, the first immunomodulatory component is a protein, peptide, glycolipid, or glycoprotein.

[0021] In some embodiments, the first immunomodulatory component is expressed as a fusion protein displayed on the outer surface of the extracellular vesicles. 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.

[0022] In some embodiments, extracellular vesicles are foreign bodies. In other embodiments, extracellular vesicles are nanovesicles.

[0023] In some embodiments, the composition also includes a pharmaceutically acceptable carrier.

[0024] In some implementations, the extracellular vesicles also contain a second immunomodulatory component.

[0025] In various embodiments, the second immunomodulatory component is an inhibitor of the negative checkpoint modulator or an inhibitor of the binding partner of the negative checkpoint modulator. In some of these embodiments, the negative checkpoint modulator 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 immune receptor with Ig and ITIM domains (TIGIT), inhibitor of V-domain Ig of T-cell activation (VISTA), adenosine A2a receptor (A2aR), cytotoxic cell immunoglobulin-like receptor (KIR), indoleamine 2,3-dioxygenase (IDO), CD20, CD39, and CD73.

[0026] In various embodiments, the second immunomodulatory component is an activator of the positive co-stimulatory molecule or an activator of the binding chaperone of the positive co-stimulatory molecule. In some embodiments, the positive co-stimulatory molecule is a member of the TNF receptor superfamily. In some of these embodiments, the TNF receptor superfamily members are 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 the positive co-stimulatory molecule is a member of the TNF superfamily. 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 some embodiments, the TNF superfamily member is CD40L. In some embodiments, the TNF superfamily member is CD27L. In some embodiments, the TNF superfamily member is OX40L.

[0027] In some embodiments, the positive co-stimulatory molecule is a CD28 superfamily co-stimulatory molecule. In some of these embodiments, the CD28 superfamily co-stimulatory molecule is ICOS or CD28. In some embodiments, the activator of the positive co-stimulatory molecule is ICOS, CD80, or CD86. In some embodiments, the activator of the positive co-stimulatory molecule is CD80.

[0028] In some embodiments, the second immunomodulatory component is a cytokine or a cytokine binding partner. In some embodiments, the cytokine is selected from the group consisting of IL-2, IL-7, IL-10, IL-12, and IL-15. In some embodiments, the cytokine is IL-7. In some embodiments, the cytokine is IL-12. In some embodiments, the cytokine is IL-15.

[0029] In some implementations, the second immunomodulatory component is a T-cell receptor (TCR), a T-cell helper receptor, a major histocompatibility complex (MHC), a human leukocyte antigen (HLA), or a derivative thereof.

[0030] In some embodiments, the second immunomodulatory component is an activator of a T-cell receptor or co-receptor. In some embodiments, the activator of a T-cell receptor or co-receptor is a CD3 activator, optionally a CD3 agonist antibody.

[0031] 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-1gp100, and TNF-associated apoptosis-inducing ligand. In some embodiments, the tumor antigen is derived from a reference genome sequence. In some embodiments, the tumor antigen is derived from the subject's genome sequence.

[0032] In some implementations, the second immunomodulatory component is a selected target or active agonist or antagonist.

[0033] In some implementations, the second immunomodulatory component is an antibody or antigen-binding fragment.

[0034] 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.

[0035] In some implementations, the second immunomodulatory component is a protein, peptide, glycolipid, or glycoprotein.

[0036] In some embodiments, the second immunomodulatory component is expressed as a fusion protein displayed on the outer surface of the extracellular vesicles. 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.

[0037] In some implementations, the second immunomodulatory component is different from the first immunomodulatory component.

[0038] In some embodiments, the extracellular vesicles further comprise a third immunomodulatory component. In some embodiments, the third immunomodulatory component differs from the first and second immunomodulatory components.

[0039] In another embodiment, this document provides a method for producing a composition. In some embodiments, the method includes modifying production cells with a first, second, and / or third immunomodulatory component; obtaining extracellular vesicles from the production cells; and optionally isolating the obtained extracellular vesicles. In some other embodiments, the method includes obtaining extracellular vesicles from production cells; isolating the obtained extracellular vesicles; and modifying the isolated extracellular vesicles with a first, second, and / or third immunomodulatory component. In some embodiments, the method further includes formulating the isolated extracellular vesicles into a pharmaceutical composition.

[0040] On the other hand, this article provides a method for treating cancer in a subject. The method includes administering a therapeutically effective amount of a composition to the subject, wherein the composition is capable of upregulating the subject's immune response, thereby enhancing the tumor-targeting ability of the subject's immune system.

[0041] On the other hand, this article provides a method for treating graft-versus-host disease (GvHD) in a subject. The method includes administering a therapeutically effective amount of a composition to the subject, wherein the composition is capable of downregulating the subject's immune response, thereby alleviating the symptoms of GvHD.

[0042] On the other hand, this article provides a method for treating an autoimmune disease in a subject. The method includes administering a therapeutically effective amount of a composition to the subject, wherein the composition is capable of downregulating the subject's immune response, thereby inhibiting the subject's immune activity.

[0043] On the other hand, this article provides a method for treating or preventing cancer in a subject, the method comprising administering to the subject a therapeutically effective amount of a composition containing 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.

[0044] In some implementations, 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-1gp100, and TNF-associated apoptosis-inducing ligand.

[0045] In some implementations, the tumor antigen is derived from a reference genome sequence. In other implementations, the tumor antigen is derived from the subject's genome sequence. Attached Figure Description

[0046] FIG. 1A and 1B The time course of mice injected with radiolabeled exogenous bodies is shown. FIG. 1A The intravenous administration route is shown. FIG. 1B The intraperitoneal administration route is shown.

[0047] FIG. 2 It is a quantitative measure of the distribution of exogenous bodies in different mouse tissues after intravenous and intraperitoneal administration of radiolabeled exogenous bodies.

[0048] FIG. 3A and 3B The effect of incubation with exogenous CD40L-expressing cells on B cell activation in peripheral blood mononuclear cells (PBMCs) from two donors was demonstrated.

[0049] FIG. 4A and 4B The effect of incubation with exogenous cells expressing CD40L on B cell activation from purified B cells from two donors was demonstrated.

[0050] FIG. 5A This is a schematic diagram of the CD40 reporting cell line. FIG. 5B Concentration-dependent activation of CD40 reporter cell lines treated with anti-CD40 agonist antibody or recombinant human CD40L is shown. FIG. 5C The effects of exogenous CD40L expression on CD40 reporter cell lines are shown.

[0051] FIG. 6A and 6BThe effect of T cell activation in peripheral blood mononuclear cells (PBMCs) expressing CD80 was demonstrated. FIG. 6A The exogenous expression of CD80 is shown to be paired with CD8 + The effect of T cell number. FIG. 6B The exogenous expression of CD80 is shown to be paired with CD4. + The effect of T cell number.

[0052] FIG. 7A and 7B The effect of the CD80-expressing exogenous agent on IFNγ expression in human PBMCs was demonstrated.

[0053] FIG. 8A and 8B The effect of exogenous CD27L expression on IFNγ expression in human PBMCs from two donors is shown.

[0054] FIG. 9A and 9B The effect of exogenous CD27L expression on IL-2 expression in human PBMCs from two donors is shown.

[0055] FIG. 10A and 10B The effect of exogenous OX40L expression on IFNγ expression in human PBMCs from two donors is shown.

[0056] FIG. 11A and 11B The effect of exogenous OX40L expression on IL-2 expression in human PBMCs from two donors is shown.

[0057] FIG. 12A This is a schematic diagram of the OX40 reporting cell line. FIG. 12B Concentration-dependent activation of OX40 reporter cell lines treated with anti-OX40 agonist antibody or recombinant human OX40L is shown. FIG. 12C The effects of exogenous OX40L expression on OX40 reporter cell lines are shown.

[0058] FIG. 13A and 13B The effect of the combination of an IL-7-expressing exogenous body and an anti-CD3 antibody on IFNγ expression in human PBMCs was demonstrated.

[0059] FIG. 14A This is a schematic diagram of an IL-7 receptor reporter cell line. FIG. 14B Concentration-dependent activation of the IL-7 receptor reporter cell line treated with recombinant human IL-7 is shown. FIG. 14C The effects of exogenous IL-7 expression on IL-7 receptor reporter cell lines are shown.

[0060] FIG. 15A and 15B The effect of exogenous IL-7 expressing cells on T cell proliferation in mice in vivo is shown, as measured by EdU incorporation. FIG. 15A The effect of exogenous IL-7 expression on CD8+ T cells was demonstrated. FIG. 15B The effect of an IL-7-expressing exogenous variant on memory CD8+ T cells was demonstrated.

[0061] FIG. 16A and 16B The effect of exogenous IL-7 expressing cells on T cell proliferation in mice in vivo was shown, as measured by CD71 positivity. FIG. 16A The effect of exogenous IL-7 expression on CD8+ T cells was demonstrated. FIG. 16B The effect of an IL-7-expressing exogenous variant on memory CD8+ T cells was demonstrated.

[0062] FIG. 17A A schematic diagram of the PTGFRN / IL-7 fusion protein expressed at high density on the surface of an exogenous organism, as well as a variant of the fusion protein, is shown. FIG. 17B It is the sequence of the optimized PTGFRN / IL-7 fusion protein.

[0063] FIG. 18A It is a protein blot that shows the relative expression of different IL-7 fusion proteins on the surface of purified exogenous organisms. FIG. 18B The effect of IL-7-expressing exogenous organisms on IL-7 receptor downregulation was demonstrated as a model of IL-7-mediated T cell activation.

[0064] FIG. 19A The effect of anti-CD3 scFv exogenous material on T cell activation in PBMCs was demonstrated. FIG. 19B The effect of anti-CD3scFv exogenous material on B cell activation in PBMCs was demonstrated.

[0065] FIG. 20A The effect of anti-CD3 scFab exogenous material on T cell activation in PBMCs was demonstrated. FIG. 20B The effect of anti-CD3scFab exogenous material on B cell activation in PBMCs was demonstrated.

[0066] FIG. 21A This is a histogram showing the degree of T cell activation after treatment with anti-CD3 scFv exogenous body. FIG. 21B This is a histogram showing the degree of B cell activation after treatment with anti-CD3 scFv exogenous body.

[0067] FIG. 22AThe effect of anti-CD3scFab exogenous body on T cell activation in plate-coated activation assays was shown compared to soluble anti-CD3 antibody or plate-coated anti-CD3 antibody. FIG. 22B It is a bar chart, which quantifies as follows: FIG. 22A The results of individual experiments conducted in [the context].

[0068] FIG. 23A A schematic diagram of the full-length PTGFRN / IL-12 fusion protein is shown. FIG. 23B A schematic diagram of the shortened PTGFRN / IL-12 fusion protein is shown.

[0069] FIG. 24A The effects of recombinant human IL-12 or exogenous organisms overexpressing short or full-length PTGFRN-IL-12 on IFNγ in human PBMCs were demonstrated. FIG. 24B This is a table summarizing the efficacy of recombinant IL-12 and IL-12-containing exogenous forms.

[0070] FIG. 25 The effects of recombinant IL-12 and IL-12-PTGFRN exogenous forms on reducing tumor growth were demonstrated in a mouse model of melanoma.

[0071] FIG. 26A The treatment with PBS is shown. FIG. 25 The tumor growth curves for each tumor-bearing mouse are shown in the image. FIG. 26B The treatment with recombinant IL-12 is shown. FIG. 25 The tumor growth curves for each tumor-bearing mouse are shown in the image. FIG. 26C The treatment with IL-12-PTGFRN exogenous body is shown. FIG. 25 The tumor growth curves for each tumor-bearing mouse are shown in the image.

[0072] FIG. 27 It shows FIG. 25 Images of all B16F10 tumor-bearing mice shown in the efficacy study.

[0073] FIG. 28 It shows FIG. 25 The survival curve of B16F10 tumor-bearing mice is shown in the figure.

[0074] FIG. 29A The levels of IFNγ gene expression in tumors of mice treated with PBS, rIL-12, or IL-12-PTGFRN exogenous bodies are shown. FIG. 29B The levels of CXCL9 gene expression in tumors of mice treated with PBS, rIL-12, or IL-12-PTGFRN exogenous bodies are shown. FIG. 29CThe levels of CXCL10 gene expression in tumors of mice treated with PBS, rIL-12, or IL-12-PTGFRN exogenous bodies are shown. FIG. 29D The levels of TGFβ gene expression in tumors of mice treated with PBS, rIL-12, or IL-12-PTGFRN exogenous bodies are shown.

[0075] FIG. 30 The percentage of IFNγ-positive CD8+ spleen T cells in tumor-bearing mice treated with PBS, rIL-12, or IL-12-PTGFRN exogenous bodies is shown.

[0076] FIG. 31A A schematic diagram of the full-length PTGFRN fused to the IFNγ monomer is shown. FIG. 31B A schematic diagram of the full-length PTGFRN fused into the IFNγ tandem dimer is shown.

[0077] FIG. 32 PAGE analysis results of purified human and mouse monomers (m) and tandem dimers (td) of PTGFRN IFNγ exogenous organisms are shown.

[0078] FIG. 33 The images show PD-L1 expression in monocytes after the addition of natural exogenous IFNγPTGFRN (WT), monomeric IFNγ-PTGFRN (m-IFNγ-PTGFRN), and tandem dimeric IFNγPTGFRN (td-IFNγ-PTGFRN), respectively. LPS-induced PD-L1 activation was used as a positive control.

[0079] FIG. 34 A schematic diagram of the 15 / IL-15Rα fusion protein fused to the transmembrane domain of PDGFR is shown.

[0080] FIG. 35 The figure shows NK cell activation as a percentage of CD69-positive NK cells after the addition of pDisplay IL-15 exogenous cells.

[0081] FIG. 36A A schematic diagram of IL-15 fused to full-length PTGFRN and IL-15N72D fused to full-length PTGFRN is shown. FIG. 36B Western blots of IL-15 fused to full-length PTGFRN and IL-15N72D fused to full-length PTGFRN are shown.

[0082] FIG. 37 The results show NK cell activation as measured by the percentage of CD69-positive NK cells after the addition of IL-15 fused to full-length PTGFRN and IL-15N72D fused to full-length PTGFRN.

[0083] FIG. 38 A schematic diagram of anti-CD3 antibody fragments fused to the PDGFR transmembrane region (exoCD3-PD), the full-length PTGFRN (exoCD3-long), and the PTGFRN fragment (exoCD3-short) is shown.

[0084] FIG. 39 The results of the added biological layer interferometry (BLI) are shown: native exosomes (WT) with anti-CD3 antibody fragments fused to the PDGFR transmembrane region (pDisplay), exosomes with anti-CD3 antibody fragments fused to the full-length PTGFRN (FLPTGFRN), and exosomes with anti-CD3 antibody fragments fused to PTGFRN fragments (short PTGFRN).

[0085] FIG. 40A The figure shows CD4+ T cell activation as measured by the percentage of CD69-positive CD4+ T cells after the addition of an anti-CD3 antibody fragment. FIG. 40B The diagram shows CD4+ T cell activation as measured by the percentage of CD69-positive CD4+ T cells after the addition of a natural exosome (exoNative) and an exosome with an anti-CD3 antibody fragment fused to the PTGFRN fragment (exoCD3-short).

[0086] FIG. 41 The CD40L-GFP PTGFRN fusion protein and EC of each construct in the B cell activation assay, measured by CD69 positivity on B cells, are shown. 50 A schematic diagram.

[0087] FIG. 42A The results show B cell activation as measured by the percentage of CD69-positive B cells after the addition of a natural exogenous body, an exogenous body with the trimer CD40L-PTGFRN construct pCB-527, and an exogenous body with the trimer CD40L-PTGFRN construct pCB-766, respectively. FIG. 42B The results show B cell activation as measured by the percentage of CD69-positive B cells after the addition of exogenous constructs pCB-527 and pCB-766, respectively, with CD40L at concentration-matched levels.

[0088] FIG. 43A The activation of B cells in donor 1, as measured by the percentage of CD69-positive B cells, is shown after the addition of a foreign body containing the trimer CD40L-PTGFRN construct pCB-527. FIG. 43BThe activation of B cells in donor 2, measured by the percentage of CD69-positive B cells, is shown after the addition of a foreign body containing the trimeric CD40L-PTGFRN construct pCB-527.

[0089] FIG. 44A The FACS analysis of natural exogens separated from beads decorated with anti-CD40L and labeled with fluorescent antibodies against IL-12 and CD40L is shown. FIG. 44B The results show the separation of natural exogenous bodies using beads decorated with anti-CD40L and FACS analysis using fluorescent antibodies against anti-CD81 and CD40L.

[0090] FIG. 45A FACS analysis of PTGFRN-CD40L / IL-12 double-engineered exogens separated from anti-CD40L-decorated beads and labeled with a fluorescent antibody against CD81 is shown. FIG. 45B FACS analysis of PTGFRN-CD40L / IL-12 dual-engineered exogens separated from beads decorated with anti-CD40L and labeled with fluorescent antibodies against IL-12 and CD40L is shown.

[0091] FIG. 46A FACS analysis of PTGFRN-CD40L / IL-12 dual-engineered exogens separated from beads decorated with anti-IL-12 and labeled with fluorescent antibodies against IL-12 and CD40L is shown. FIG. 46B FACS analysis of PTGFRN-CD40L / IL-12 double-engineered exogens separated from beads decorated with anti-IL-12 and labeled with a fluorescent antibody against CD81 is shown.

[0092] FIG. 47A The IFNγ response in donor 1 human PBMCs was demonstrated after the addition of recombinant IL-12, a mixture of recombinant IL-12 and recombinant CD40L, PTGFRN-IL-12 exogene, double-positive PTGFRN-CD40L / IL-12 exogene, and a mixture of PTGFRN-IL-12 exogene and PTGFRN-CD40L exogene, respectively. FIG. 47B The IFNγ response in donor 2 human PBMCs was demonstrated after the addition of recombinant IL-12, a mixture of recombinant IL-12 and recombinant CD40L, PTGFRN-IL-12 exogene, double-positive PTGFRN-CD40L / IL-12 exogene, and a mixture of PTGFRN-IL-12 exogene and PTGFRN-CD40L exogene, respectively.

[0093] FIG. 48The following figures illustrate the IFNγ response in PBMCs of donor 1 and donor 2 after the addition of recombinant IL-12, a mixture of recombinant IL-12 and recombinant CD40L, PTGFRN-IL-12 exogene, double-positive PTGFRN-CD40L / IL-12 exogene, and a mixture of PTGFRN-IL-12 exogene and PTGFRN-CD40L exogene, respectively. 50 .

[0094] FIG. 49A The activation of B cells in donor 1 human PBMCs was demonstrated after the addition of recombinant CD40L, a mixture of recombinant IL-12 and recombinant CD40L, PTGFRN-CD40L exogene, double-positive PTGFRN-CD40L / IL-12 exogene, and a mixture of PTGFRN-IL-12 exogene and PTGFRN-CD40L exogene, respectively. FIG. 49B The activation of B cells in donor 2 human PBMCs was demonstrated after the addition of recombinant CD40L, a mixture of recombinant IL-12 and recombinant CD40L, PTGFRN-CD40L exogene, double-positive PTGFRN-CD40L / IL-12 exogene, and a mixture of PTGFRN-IL-12 exogene and PTGFRN-CD40L exogene, respectively.

[0095] FIG. 50 The following figures illustrate the IFNγ response in PBMCs of donor 1 and donor 2 after the addition of recombinant CD40L, a mixture of recombinant IL-12 and recombinant CD40L, PTGFRN-CD40L exogene, double-positive PTGFRN-CD40L / IL-12 exogene, and a mixture of PTGFRN-IL-12 exogene and PTGFRN-CD40L exogene, respectively. 50 .

[0096] FIG. 51A FACS analysis of the PTGFRN-CD40L / IL-12 / FLT3L triple-engineered exogenous organism separated from anti-IL-12-decorated beads and labeled with fluorescent antibodies against IL-12 and CD40L is shown. FIG. 51B FACS analysis of PTGFRN-CD40L / IL-12 / FLT3L triple-engineered exogens separated from beads decorated with anti-IL-12 and labeled with fluorescent antibodies against IL-12 and FLT3L is shown. FIG. 51C FACS analysis of PTGFRN-CD40L / IL-12 / FLT3L triple-engineered exogens separated from beads decorated with anti-IL-12 and labeled with fluorescent antibodies against CD40L and FLT3L is shown.

[0097] FIG. 52AFACS analysis of PTGFRN-CD40L / IL-12 / FLT3L triple-engineered exogens separated from anti-CD40L-decorated beads and labeled with fluorescent antibodies against IL-12 and CD40L is shown. FIG. 52B FACS analysis of PTGFRN-CD40L / IL-12 / FLT3L triple-engineered exogens separated from anti-CD40L-decorated beads and labeled with fluorescent antibodies against IL-12 and FLT3L is shown. FIG. 52C FACS analysis of PTGFRN-CD40L / IL-12 / FLT3L triple-engineered exogens separated from anti-CD40L-decorated beads and labeled with fluorescent antibodies against CD40L and FLT3L is shown. Detailed Implementation

[0098] This article discloses extracellular vesicles capable of modulating the human immune system. It also provides methods for generating these extracellular vesicles and methods for using them to treat cancer and other immune system-related diseases.

[0099] Before describing the invention in more detail, it should be understood that the invention is not limited to the specific embodiments described, as they can certainly be varied. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to be limiting, as the scope of the invention will be limited only by the appended claims.

[0100] If a range of values ​​is provided, it should be understood that every intermediate value between the upper and lower limits of the range (up to one-tenth of the lower limit unit, unless the context clearly indicates otherwise) and any other values ​​or intermediate values ​​within the range are included within this invention. The upper and lower limits of these smaller ranges may be independently included within the smaller ranges and also within this invention, subject to any explicitly excluded limits within the range. If the range contains one or both of the included limits, the range excluding one or both of these included limits is also included within this invention.

[0101] 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 this invention pertains. While any methods and materials similar to or equivalent to those described herein may also be used in the practice or testing of this invention, representative exemplary methods and materials are described here.

[0102] All publications and patents cited in this specification are incorporated herein by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference, and are incorporated herein by reference to disclose and describe the methods and / or materials involved when the publication is cited.

[0103] It should be noted that, as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural objects unless the context clearly indicates otherwise. It should be further noted that the claims may be drafted to exclude any optional elements. Therefore, this statement is intended to serve as a priori basis for the use of exclusive terms such as “alone,” “only,” etc., or for the use of “negative” limitations in conjunction with elements of the stated claims.

[0104] It will be apparent to those skilled in the art upon reading this disclosure that the individual embodiments described and illustrated herein each have discrete components and features that can be readily separated from or combined with features of any of the other several embodiments without departing from the scope or spirit of the teachings of the invention. Any stated methods may be performed in the order of the stated events or in any other logically possible order.

[0105] In a further description of the invention, the subject system used to practice the subject method will be discussed in more detail, and then the relevant methods will be reviewed.

[0106] As used herein, the term "extracellular vesicle" refers to a cell-derived vesicle containing a membrane that encloses its internal space. Extracellular vesicles include all membrane-bound vesicles with a diameter smaller than the cell from which they originate. Typically, extracellular vesicles range in diameter from 20 nm to 1000 nm and may include various macromolecular cargoes displayed on and / or spanning the membrane within their internal space. Cargoes 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 through direct or indirect manipulation (e.g., by continuous extrusion or treatment with an alkaline solution), vesicularized organelles, and vesicles generated by living cells (e.g., by direct plasma membrane budding or late endosome fusion with the plasma membrane). Extracellular vesicles can originate from living or dead organisms, explanted tissues or organs, and / or cultured cells.

[0107] As used herein, the term "exosome" refers to a small, cell-derived vesicle (with a diameter between 20 and 300 nm, more preferably between 40 and 200 nm) that comprises a membrane enclosing its internal space and is generated from the cell via direct plasma membrane budding or fusion of late endosomes with the plasma membrane. An exosome is an extracellular vesicle. Exosomes contain lipids or fatty acids and polypeptides, and may also contain a payload (e.g., a therapeutic agent), a recipient (e.g., a target moiety), polynucleotides (e.g., nucleic acids, RNA, or DNA), sugars (e.g., simple sugars, polysaccharides, or glycans), or other molecules. Exosomes may be derived from production cells and isolated from production cells based on their size, density, biochemical parameters, or combinations thereof.

[0108] As used herein, the term "nanovesicle" refers to small vesicles (with a diameter between 20 and 250 nm, more preferably between 30 and 150 nm) derived from cells, containing a membrane that encloses an internal space, and generated from cells by direct or indirect manipulation such that no nanovesicles will be generated from the producing cells without such manipulation. Suitable manipulation of the producing cells includes, but is not limited to, continuous extrusion, treatment with an alkaline solution, sonication, or combinations thereof. In some cases, the generation of nanovesicles can lead to the destruction of the producing cells. Preferably, the nanovesicle population is substantially free of vesicles derived from the producing cells by direct budding from the plasma membrane or fusion of late endosomes with the plasma membrane. A nanovesicle is an extracellular vesicle. Nanovesicles contain lipids or fatty acids and polypeptides, and optionally contain a payload (e.g., a therapeutic agent), a receptor (e.g., a targeting moiety), a polynucleotide (e.g., nucleic acid, RNA, or DNA), a sugar (e.g., a monosaccharide, polysaccharide, or glycan), or other molecules. Once nanovesicles are derived from the producing cells according to the manipulation, they can be isolated from the producing cells based on their size, density, biochemical parameters, or combinations thereof.

[0109] 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, immunomodulatory components may be delivered to the cytoplasm of target cells. In other embodiments, immunomodulatory components may be delivered to the membrane of target cells. In some embodiments, the membrane of the extracellular vesicle fuses with the membrane of the target cell.

[0110] As used herein, the term "productive cell" refers to any cell from which extracellular vesicles can be isolated. A productive cell is a cell that serves as a source of extracellular vesicles. Productive cells may share protein, lipid, glycoprotein, or nucleic acid components with extracellular vesicles. In some embodiments, the productive cell is a modified or synthetic cell. In some embodiments, the productive cell is a cultured or isolated cell. In some embodiments, the productive cell is a cell line. In some other embodiments, the productive cell is a primary cell. In some specific embodiments, the productive cell is an immune cell.

[0111] As used herein, a “membrane” is a boundary layer separating an internal space from an external space, comprising 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, glycerol phosphates, sterols, cholesterol, and phosphatidylserine. In some of these embodiments, the membrane also comprises one or more polypeptides and / or one or more polysaccharides, such as glycans. Extracellular vesicles comprise membranes as defined herein.

[0112] 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 extracellular vesicles and modulates the immune system. Immunomodulatory components that can be introduced into extracellular vesicles and / or producing cells include therapeutic agents, such as modulators of checkpoint inhibitors or ligands of checkpoint inhibitors, surface antigens and their derivatives, cytokines and their derivatives. Immunomodulatory components may also include agonists, antagonists, antibodies and antigen-binding fragments, or polynucleotides, such as siRNA, miRNA, lncRNA, and DNA.

[0113] The term "receptor" refers to a molecule in a subject that directs extracellular vesicles to a target and / or promotes the interaction between extracellular vesicles and the target. In some embodiments, the receptor is a peptide. In some embodiments, the receptor is capable of increasing the concentration of immunomodulatory components in the subject's tissues. Examples of receptors include, but are not limited to, those listed in Table 3.

[0114] The term "target" refers to cells, pathogens, metabolites, polypeptide complexes, or any molecule or structure present in tissues or circulating in the circulatory or lymphatic system of a subject, such as immune cells or cancer cells. Examples of targets include, but are not limited to, those listed in Table 4.

[0115] "Therapeutic agent" or "therapeutic molecule" includes compounds or molecules that, when present in an effective amount, produce a desired therapeutic, pharmacological, and / or physiological effect on a subject in need of it. It includes any compound, such as a small molecule drug or a biological drug (e.g., a peptide drug or a nucleic acid drug), that, when administered to a subject, has a measurable or communicable effect on the subject, such as relieving or reducing symptoms of a disease, disorder, or symptom.

[0116] As used herein, the term "antibody" encompasses naturally occurring or partially or wholly synthetic immunoglobulins and fragments thereof. The term also encompasses any protein having a binding domain homologous to an immunoglobulin binding domain. "Antibody" further includes polypeptides comprising a framework region derived from an immunoglobulin gene or a fragment thereof that specifically binds to 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, mouse antibodies, chimeric, mouse-human, mouse-primate, primate-human monoclonal antibodies, anti-idiotype antibodies, antibody fragments such as, for example, scFv, (scFv)2, Fab, Fab' and F(ab')2, F(ab1)2, Fv, dAb and Fd fragments, biantibodies, and antibody-associated polypeptides. Antibodies include both bispecific and multispecific antibodies, provided they exhibit the desired biological activity or function.

[0117] As used herein, the term "antigen-binding fragment" refers to a fragment of a complete immunoglobulin, as well as any portion of a polypeptide, including an antigen-binding region capable of specifically binding to an antigen. For example, an antigen-binding fragment can be an 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 an antigen-binding site and contains variable regions of both the light and heavy chains, a constant region of the light chain, and a first constant region CH1 of the heavy chain. A Fab' fragment differs from a Fab fragment in that it additionally includes a hinge region of the heavy chain, with at least one cysteine ​​residue at the C-terminus of the CH1 region of the heavy chain. An F(ab')2 fragment is generated whereby the cysteine ​​residues of the Fab' fragment are linked by disulfide bonds in the hinge region. An Fv fragment is the smallest antibody fragment containing only the variable regions of the heavy and light chains, and recombinant techniques for generating Fv fragments are well known in the art. Two-chain Fv fragments can have a structure in which the variable region of the heavy chain is linked to the variable region of the light chain by non-covalent bonds. Single-chain Fv (scFv) fragments can typically have a dimer structure, similar to two-chain Fv fragments, where the heavy chain variable region is covalently linked to the light chain variable region via a peptide linker, or the heavy and light chain variable regions are directly linked to each other at their C-termini. Antigen-binding fragments can be obtained using proteases (e.g., digestion of intact antibodies with papain to obtain the Fab fragment, and digestion with pepsin to obtain the F(ab')2 fragment), and can be prepared using genetic recombination techniques. dAb fragments consist of VH domains. Single-chain antibody molecules can contain polymers comprising multiple individual molecules, such as dimers, trimers, or other polymers.

[0118] The phrase "nucleic acid molecule" refers to a single- or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases. These include chromosomal DNA, self-replicating plasmids, vectors, mRNA, tRNA, siRNA, miRNA, etc. Nucleic acid molecules can be recombinant; when introduced into cells, they can express exogenous polypeptides.

[0119] The term "agonist" refers to a molecule that binds to a receptor and activates the receptor 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.

[0120] The term "antagonist" refers to a molecule that blocks or attenuates an agonist-mediated response upon binding to a receptor, rather than generating a biological response itself. Many antagonists exert their efficacy by competing with endogenous ligands or substrates for binding sites on the receptor. Non-limiting examples of antagonists include alpha blockers, beta blockers, and calcium channel blockers. Antagonists can be competitive, non-competitive, or non-competitive.

[0121] As used herein, the term "fragment" for protein refers to a protein lacking the N- and / or C-terminus compared to a naturally occurring protein. Preferably, fragments of PTGFRN, BSG, IGSF2, IGSF3, IGSF8, ITGB1, ITGA4, SLC3A2, or ATP transporters retain the ability to specifically target exogenous bodies. Such fragments are also referred to as "functional fragments." In a sense, whether a fragment is functional can be evaluated by any method known in the art to determine the protein content of the exogenous body, including Western blotting, FACS analysis, and fusion of the fragment with an autofluorescent protein such as GFP. In a particular embodiment, fragments of PTGFRN, BSG, IGSF2, IGSF3, IGSF8, ITGB1, ITGA4, SLC3A2, or ATP transporters retain at least 50%, 60%, 70%, 80%, 90%, or 100% of the ability of naturally occurring PTGFRN, BSG, IGSF2, IGSF3, IGSF8, ITGB1, ITGA4, SLC3A2, or ATP transporters to specifically target exogenous bodies.

[0122] As used herein, the term "variant" refers to a protein that, upon alignment by methods known in the art, shares certain amino acid sequence identity with another protein. Protein variants may include substitutions, insertions, deletions, frameshifts, or rearrangements in another protein. In one particular embodiment, a variant is a variant having at least 70% identity with PTGFRN, BSG, IGSF2, IGSF3, IGSF8, ITGB1, ITGA4, SLC3A2, the ATP transporter, or a fragment of PTGFRN, BSG, IGSF2, IGSF3, IGSF8, ITGB1, ITGA4, SLC3A2, or the ATP transporter. In some embodiments, a variant of PTGFRN or a variant of a fragment has at least 70%, 80%, 85%, 90%, 95%, or 99% sequence identity with PTGFRN or a functional fragment thereof according to SEQ ID NO: 1. In some embodiments, variants or fragments of BSG have at least 70%, 80%, 85%, 90%, 95%, or 99% sequence identity with the BSG or functional fragment thereof according to SEQ ID NO: 9. In some embodiments, variants or fragments of IGSF2 have at least 70%, 80%, 85%, 90%, 95%, or 99% sequence identity with the IGSF2 or functional fragment thereof according to SEQ ID NO: 34. In some embodiments, variants or fragments of IGSF3 have at least 70%, 80%, 85%, 90%, 95%, or 99% sequence identity with the IGSF3 or functional fragment thereof according to SEQ ID NO: 20. In some embodiments, variants or fragments of IGSF8 have at least 70%, 80%, 85%, 90%, 95%, or 99% sequence identity with the IGSF8 or functional fragment thereof according to SEQ ID NO: 14. In some embodiments, variants or fragments of ITGB1 have at least 70%, 80%, 85%, 90%, 95%, or 99% sequence identity with ITGB1 or a functional fragment thereof according to SEQ ID NO: 21. In some embodiments, variants or fragments of ITGA4 have at least 70%, 80%, 85%, 90%, 95%, or 99% sequence identity with ITGA4 or a functional fragment thereof according to SEQ ID NO: 22. In some embodiments, variants or fragments of SLC3A2 have at least 70%, 80%, 85%, 90%, 95%, or 99% sequence identity with SLC3A2 or a functional fragment thereof according to SEQ ID NO: 23. In some embodiments, variants or fragments of ATP1A1 have at least 70%, 80%, 85%, 90%, 95%, or 99% sequence identity with ATP1A1 or a functional fragment thereof according to SEQ ID NO: 24.In some embodiments, variants or fragments of ATP1A2 have at least 70%, 80%, 85%, 90%, 95%, or 99% sequence identity with ATP1A2 or a functional fragment thereof according to SEQ ID NO: 25. In some embodiments, variants or fragments of ATP1A3 have at least 70%, 80%, 85%, 90%, 95%, or 99% sequence identity with ATP1A3 or a functional fragment thereof according to SEQ ID NO: 26. In some embodiments, variants or fragments of ATP1A4 have at least 70%, 80%, 85%, 90%, 95%, or 99% sequence identity with ATP1A4 or a functional fragment thereof according to SEQ ID NO: 27. In some embodiments, variants or fragments of ATP1B3 have at least 70%, 80%, 85%, 90%, 95%, or 99% sequence identity with ATP1B3 or a functional fragment thereof according to SEQ ID NO: 28. In some embodiments, variants or fragments of ATP2B1 have at least 70%, 80%, 85%, 90%, 95%, or 99% sequence identity with ATP2B1 or a functional fragment thereof according to SEQ ID NO: 29. In some embodiments, variants or fragments of ATP2B2 have at least 70%, 80%, 85%, 90%, 95%, or 99% sequence identity with ATP2B2 or a functional fragment thereof according to SEQ ID NO: 30. In some embodiments, variants or fragments of ATP2B3 have at least 70%, 80%, 85%, 90%, 95%, or 99% sequence identity with ATP2B3 or a functional fragment thereof according to SEQ ID NO: 31. In some embodiments, variants or fragments of ATP2B4 have at least 70%, 80%, 85%, 90%, 95%, or 99% sequence identity with ATP2B4 or a functional fragment thereof according to SEQ ID NO: 32. In each of the above cases, it is preferable that the variant or fragment retains the ability to specifically target the foreign body.

[0123] The sequence alignment methods used for comparison are well known in the art. Various procedures and alignment algorithms are described in the following: 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 20 et al., J.Mol.Biol.215:403-10(1990)] is available from multiple sources, including the National Center for Biotechnology Information (NBCI, Bethesda, Maryland) and the Internet, for use in conjunction with the sequence analysis programs blastp, blasm, blastx, tblastn, and tblastx. Instructions on how to use the program to determine sequence identification can be accessed on the official website of the NCBI (National Center for Biotechnology Information) at the NIH (National Institutes of Health).

[0124] The description of any protein provided herein includes functional variants of that protein. The term "functional variant" refers to a protein variant that retains the ability to specifically target foreign bodies.

[0125] As used herein, the term "pharmaceutical composition" refers to one or more compounds described herein, such as extracellular vesicles mixed, blended, or suspended therein with 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 extracellular vesicles to a subject. The term "pharmaceuticalally acceptable" and its grammatical variations refer to compositions, carriers, diluents, and reagents that can be administered to or on a subject without producing undesirable physiological effects to the extent that the composition is prohibited from being administered. The term "excipient" or "carrier" refers to an inert substance added to a pharmaceutical composition to further facilitate the administration of the compound. The terms "pharmaceuticalally acceptable carrier" or "pharmaceuticalally acceptable excipient" encompass any agent approved by a U.S. federal regulatory agency or listed in the United States Pharmacopeia for use in animals (including humans), and any carrier or diluent that does not cause significant irritation to the subject and does not eliminate the biological activity and properties of the administered compound. This includes excipients and carriers that can be used to prepare pharmaceutical compositions and are generally safe, non-toxic, and desirable.

[0126] As used herein, the terms “isolate,” “isolated,” and “isolating,” or “purify,” “purified,” and “extracted” are used interchangeably and refer to a state of desired extracellular vesicle formulation (e.g., multiple known or unknown amounts and / or concentrations) undergoing one or more purification processes, such as selecting or enriching a desired extracellular vesicle formulation. In some embodiments, the isolation or purification referred to herein is a process of removing, or partially removing (e.g., a portion), extracellular vesicles from a sample containing production cells. In some embodiments, the isolated extracellular vesicle composition does not have detectable undesirable activity, or the level or amount of undesirable activity is at or below an acceptable level or amount. In other embodiments, the isolated extracellular vesicle composition has 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 from which the composition is obtained (e.g., the production cell formulation). Compared to the starting material, this enrichment can be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99%, 99.999%, 99.9999%, or greater than 99.9999%. In some embodiments, the isolated extracellular vesicle formulation is substantially free of residual biological products. In some embodiments, the isolated extracellular vesicle formulation is 100% free, 99% free, 98% free, 97% free, 96% free, or 95% free of any contaminating biological material. 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 producing cells, and only extracellular vesicles are detectable.

[0127] The terms "administration," "administering," and variations thereof refer to the introduction of a composition, such as extracellular vesicles or a drug, into a subject, including simultaneous or sequential introduction of the composition or drug. The composition or drug is introduced into the subject via any suitable route, including oral, intrapulmonary, intranasal, parenteral (intravenous, intraarterial, intramuscular, intraperitoneal, or subcutaneous), rectal, intralymphatic, intrathecal, intratumoral, intraocular, intraocular, or topical administration. Administration includes self-administration and administration by another person. A suitable route of administration allows the composition or drug to perform its intended function. For example, if the suitable route is intravenous, the composition is administered by introducing the composition or drug into the subject's vein.

[0128] As used herein, the terms “modulate,” “modifying,” “modify,” and / or “modifier” generally refer to the ability to alter, for example, by increasing or decreasing, a particular concentration, level, expression, function, or behavior, either directly or indirectly, by promoting / stimulating / upregulating or interfering with / inhibiting / downregulating it, such as acting as an antagonist or agonist. In some cases, modifiers may increase and / or decrease a concentration, level, activity, or function relative to a control, or relative to the generally expected mean activity level or relative to the control activity level.

[0129] The term "sufficient amount" refers to an amount that is sufficient to produce the desired effect, such as an amount sufficient to regulate the symptoms of a subject.

[0130] The term "therapeutic effective dose" is the amount that effectively improves the symptoms of a disease. A therapeutic effective dose can also be a "preventive effective dose," since prevention can be considered treatment.

[0131] As used herein, the term "substantially" or "substantial" means, for example, the presence, level, or concentration of an entity in a particular space, the effect of one entity on another, or the therapeutic effect. For example, if an increase of 2, 3, 4, 5, 10, 50, 100, or 1000 times relative to baseline, the activity, level, or concentration of the entity is substantially increased. If an increase of 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, or 500% relative to baseline, the activity, level, or concentration of the entity is also substantially increased.

[0132] The term "in vivo" refers to processes that occur within a living organism.

[0133] As used in this article, the term “mammal” includes both human and non-human mammals.

[0134] The 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 a single-stranded RNA complementary to mRNA, "shRNA" refers to small or short hairpin RNA, "lncRNA" refers to long non-coding RNA, and "dsDNA" refers to double-stranded DNA.

[0135] Composition

[0136] Several aspects of this disclosure include compositions capable of modulating the immune system. The compositions comprise extracellular vesicles containing a cell membrane and immunomodulatory components associated with or enclosed within a membrane-bound closed volume.

[0137] extracellular vesicles

[0138] In various embodiments, the composition comprises extracellular vesicles. In some embodiments, the extracellular vesicles are cell-derived vesicles that contain a membrane that encloses their internal space.

[0139] In various embodiments, extracellular vesicles can be membrane-bound vesicles with a diameter smaller than that of the cell from which they originate. In some embodiments, the longest dimension of the extracellular vesicles is approximately 20-1000 nm, such as 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, and 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.

[0140] In some embodiments, extracellular vesicles are foreign bodies. In some embodiments, extracellular vesicles are nanovesicles. In some embodiments, extracellular vesicles are apoptotic bodies. In some embodiments, extracellular vesicles are cell fragments. In some embodiments, extracellular vesicles are vesicles derived from cells through direct or indirect manipulation. In some embodiments, extracellular vesicles are vesicled organelles. In various embodiments, extracellular vesicles are vesicles produced by living cells.

[0141] In some embodiments, extracellular vesicles are derived from living organisms. In some embodiments, extracellular vesicles are derived from dead organisms. In some embodiments, extracellular vesicles are derived from explanted tissues. In some embodiments, extracellular vesicles are derived from explanted organs. In some embodiments, extracellular vesicles are derived from cultured cells. In some of these embodiments, when extracellular vesicles are generated in a cell culture system, the extracellular vesicles are further isolated (e.g., by isolating extracellular vesicles from cultured cells). Isolation can be achieved by sedimentation. For example, the specific density of 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 / m³.3 Separation can also be achieved through affinity purification. For example, extracellular vesicles can be purified by binding a population containing extracellular vesicles to a resin containing multiple ligands with specific affinity for one or more target proteins on the surface of the extracellular vesicles. Target proteins can be tetraspan proteins (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. Target proteins can also be immunomodulatory components displayed on the surface of exogenous organisms.

[0142] In various embodiments, extracellular vesicles comprise lipids or fatty acids and peptides. In some embodiments, extracellular vesicles also comprise sugars. In some embodiments, extracellular vesicles also comprise polynucleotides.

[0143] In various embodiments, the extracellular vesicle membrane includes an inner surface and an outer surface, and encloses an internal space. In some embodiments, the extracellular vesicle also contains a payload. In some embodiments, the payload is enclosed within the internal space. In some embodiments, the payload is displayed on the outer surface of the extracellular vesicle. In some embodiments, the payload spans the membrane of the extracellular vesicle. In various embodiments, the payload includes nucleic acids, proteins, carbohydrates, lipids, small molecules, and / or combinations thereof. In some embodiments, the extracellular vesicle also contains a receptor.

[0144] Exosome

[0145] In various implementations, extracellular vesicles are foreign bodies. In some implementations, the foreign body is a membrane-bound small vesicle secreted by the producing cell.

[0146] In some implementations, the longest size of the exogenous body from the production cell is approximately 20-300 nm, such as approximately 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, and 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.

[0147] In a particularly preferred embodiment, the longest size of the exogenous organisms from the production cells described herein is about 30-100 nm. In another preferred embodiment, the longest size of the exogenous organisms from the production cells is about 20-300 nm. In another preferred embodiment, the longest size of the exogenous organisms from the production cells is about 40-200 nm. In another embodiment, the exogenous organism population described herein comprises a population in which 90% of the exogenous organisms have a longest size of 20-300 nm. In another embodiment, the exogenous organism population described herein comprises a population in which 95% of the exogenous organisms have a longest size of 20-300 nm. In another embodiment, the exogenous organism population described herein comprises a population in which 99% of the exogenous organisms have a longest size of 20-300 nm. In another embodiment, the exogenous organism population described herein comprises a population in which 90% of the exogenous organisms have a longest size of 40-200 nm. In another embodiment, the exogenous organism population described herein comprises a population in which 95% of the exogenous organisms have a longest size of 40-200 nm. In another embodiment, the exogenous population described herein comprises a population in which 99% of the exogenous organisms have a maximum size of 40-200 nm. In other preferred embodiments, the size of the exogenous organisms or exogenous populations described herein is measured according to the methods described below.

[0148] In some embodiments, the exogenous body is generated by the production cells. In some embodiments, the membrane of the exogenous body comprises one or more molecules derived from the production cells. In some embodiments, the exogenous body is generated and separated in a cell culture system (e.g., by separating the exogenous body from the production cells). Separation can be achieved by sedimentation. For example, the specific density of the exogenous body 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. 3Separation can also be achieved through affinity purification. For example, extracellular vesicles can be purified by binding a population containing extracellular vesicles to a resin containing 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 tetraspan proteins (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. Target proteins can also be immunomodulatory components displayed on the surface of exogenous organisms.

[0149] In some embodiments, the exogenous body membrane includes an inner surface and an outer surface. In some embodiments, the inner surface faces the core of the exogenous body. In some embodiments, the outer surface may be in contact with the endosomes, multivesicles, or membrane / cytoplasm of the producing or target cells.

[0150] In some embodiments, the exosome membrane comprises lipids and fatty acids. In some embodiments, the exosome membrane comprises phospholipids, glycolipids, fatty acids, sphingolipids, glycerol phosphates, sterols, cholesterol, and phosphatidylserine. In some embodiments, the lipids and fatty acids may be one or more of those listed in Table 1.

[0151] In some embodiments, the exogenous body comprises a lipid bilayer consisting of an inner lobule and an outer lobule. The composition of the inner and outer lobules 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 outer lobule consists of about 70-90% choline phospholipids, about 0-15% acidic phospholipids, and about 5-30% phosphatidylethanolamine. In some embodiments, the inner lobule consists of about 15-40% choline phospholipids, about 10-50% acidic phospholipids, and about 30-60% phosphatidylethanolamine.

[0152] In some embodiments, the exosome membrane further comprises one or more polypeptides. In some 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), tetraspan proteins (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 protein / 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 exogenous body comprises at least one polypeptide selected from Table 2.

[0153] In some embodiments, the exogenous body contains a polypeptide on its surface. In some embodiments, the exogenous body is modified to contain one or more polypeptides. In some embodiments, the production cell is modified to contain one or more polypeptides. In some embodiments, the production cell naturally contains one or more polypeptides, and the exogenous body derived therefrom also contains a polypeptide. The level of any desired surface marker can be directly modified on the exogenous body (e.g., by contacting the complex with a recombinant-derived polypeptide to insert into or conjugate the membrane of the complex). Alternatively or additionally, the level of any desired surface marker can be directly modified on the production cell (e.g., by contacting the complex with a recombinant-derived polypeptide to insert into or conjugate the cell membrane). Alternatively, the production cell can be modified by transducing a foreign nucleic acid into the production cell to express the desired surface marker. The surface marker may already be naturally present on the production cell, in which case the foreign construct can lead to overexpression of the marker and increase the concentration of the marker within or on the production cell. Alternatively, the naturally expressed surface marker can be removed from the production cell (e.g., by inducing gene silencing in the production cell). The polypeptide can confer different functions on the exogenous body (e.g., specific targeting ability, delivery function (e.g., fusion molecule), enzymatic function, increased or decreased in vivo half-life, etc.). In some implementations, the peptides include, but are not limited to, CD47, CD55, CD49, CD40, CD133, CD59, glycan-1, CD9, CD63, CD81, integrin, selectin, lectin, and cadherin.

[0154] In a particular embodiment, the exogenous body comprises one or more polypeptides on its surface, said polypeptides being selected from a group of proteins recently identified as enriched on the surface of the exogenous body (detailed 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); basigin (BSG); immunoglobulin superfamily member 3 (IGSF3); immunoglobulin superfamily member 8 (IGSF8); integrin β-1 (ITGB1); integrin α-4 (ITGA4); 4F2 cell surface antigen heavy chain (SLC3A2); and a class of ATP transporters (ATP1A1, ATP1A2, ATP1A3, ATP1A4, ATP1B3, ATP2B1, ATP2B2, ATP2B3, ATP2B4)).

[0155] In some implementations, the exogenous membrane also contains one or more polysaccharides, such as polysaccharides.

[0156] In some implementations, the exogenous body delivers a payload (therapeutic agent) to a target. The payload is a therapeutic agent that acts on a target (e.g., target cells) that comes into contact with the exogenous body. Contact can occur in vitro or in a subject. Payloads that can be introduced into the exogenous body and / or produced cells include therapeutic agents such as nucleotides (e.g., nucleotides containing detectable portions or toxins or disrupting transcription), nucleic acids (e.g., DNA or mRNA molecules encoding polypeptides such as enzymes, or RNA molecules with regulatory functions such as miRNA, dsDNA, lncRNA, or siRNA), amino acids (e.g., amino acids containing detectable portions or toxins that disrupt translation), polypeptides (e.g., enzymes), lipids, carbohydrates, and small molecules (e.g., small molecule drugs and toxins).

[0157] Exosomes can interact with target cells via membrane fusion, delivering the payload (e.g., a therapeutic agent) from the exosome composition to the surface or cytoplasm of the target cells. In some embodiments, membrane fusion occurs between the exosome and the plasma membrane of the target cell. In other embodiments, membrane fusion occurs between the exosome and the endosome membrane of the target cell.

[0158] In some embodiments, the exogenous body comprises a receptor polypeptide. The receptor polypeptide may be synthetic. In some embodiments, the receptor polypeptide is introduced into production cells (e.g., by introducing a foreign nucleic acid encoding the receptor polypeptide into production cells) or a recombinant receptor polypeptide prepared outside production cells (e.g., synthesized via a protein expression system). In some embodiments, the receptor polypeptide (e.g., a recombinantly generated polypeptide) is introduced directly into the exogenous body (e.g., after isolating the exogenous body from production cells). In some embodiments, the receptor polypeptide may be on the surface of the exogenous body. In some embodiments, the receptor polypeptide is capable of targeting the exogenous body to specific targets circulating in the subject's circulatory system (e.g., targets such as pathogens, metabolites, polypeptide complexes, or cells such as nonfunctional cells or cancer cells), such as blood or targets located in tissues (e.g., diseased tissue).

[0159] In some embodiments, the exogenous body is synthetic. For example, the exogenous body may contain a payload, such as, for example, a therapeutic peptide, nucleic acid (e.g., DNA or RNA) or other polynucleotide, polysaccharide or glycan, lipid or fatty acid, macroorganism, small molecule, or toxin, such that the exogenous body is not naturally occurring. In some embodiments, the exogenous body is modified (e.g., by introducing a payload or otherwise modifying the content of the complex, such as by altering the protein, lipid, or glycan content of the membrane). For example, the exogenous body is first isolated from the production cell and then modified as needed to produce a synthetic exogenous body. In some embodiments, the production cell is modified. For example, exogenous nucleic acids, exogenous peptides, or small molecules or toxins may be introduced into the production cell. Alternatively or additionally, the production cell may be modified in other ways (e.g., by altering the cell or membrane content, such as by altering the lipid or glycan content of the cell membrane). The exogenous body generated from the modified production cell contains one or more modifications of the production cell. This process produces a synthetic exogenous body. In some embodiments, both the production cell and the exogenous body isolated from the production cell are modified as described herein.

[0160] Nanovesicle

[0161] In various embodiments, extracellular vesicles are nanovesicles. In some embodiments, nanovesicles are small, cell-derived vesicles containing a membrane that encloses their internal space, and are generated from cells through direct or indirect manipulation, such that nanovesicles will not be generated from the producing cells without manipulation. Appropriate manipulation of the cells includes, but is not limited to, continuous squeezing, treatment with an alkaline solution, sonication, or combinations thereof, and in some cases may result in the destruction of the producing cells.

[0162] In various implementations, the longest size of the nanovesicles is about 20-250 nm, such as 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.

[0163] In various embodiments, nanovesicles are derived from production cells. In some embodiments, nanovesicles are generated from production cells through direct or indirect manipulation. Suitable manipulations include, but are not limited to, continuous extrusion, treatment with an alkaline solution, sonication, or combinations thereof. In some of these embodiments, the manipulation may result in the destruction of the production cells. In some preferred embodiments, the population of nanovesicles is substantially free of vesicles derived from production cells via direct budding from the plasma membrane or fusion of late endosomes with the plasma membrane.

[0164] In some embodiments, nanovesicles are separated from the production cells based on their size, density, biochemical parameters, or combinations thereof. In some embodiments, separation can be achieved by sedimentation. For example, the specific density of 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 / m³. 3 .

[0165] In various embodiments, nanovesicles comprise lipids or fatty acids and peptides. In some embodiments, nanovesicles also comprise sugars. In some embodiments, nanovesicles also comprise polynucleotides. In some embodiments, nanovesicles also comprise receptors. In some embodiments, nanovesicles also comprise payloads. In some of these embodiments, the payload includes nucleic acids, proteins, carbohydrates, lipids, small molecules, and / or combinations thereof.

[0166] Immunomodulatory components

[0167] In various embodiments, the composition also includes an immunomodulatory component.

[0168] In some embodiments, the immunomodulatory compound is a protein expressed as a translational fusion protein with a foreign body surface protein, such that the protein remains on the foreign body surface. In some embodiments, the immunomodulatory compound is a membrane protein. In some embodiments, the immunomodulatory compound is a soluble protein. In some embodiments, the foreign body surface protein is a tetraspan membrane protein (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 fragments or variants thereof.

[0169] In some embodiments, the immunomodulatory compound is a soluble protein expressed as a translational fusion protein with a foreign body surface protein, such that the soluble protein is retained on the foreign body surface. In some embodiments, the foreign body surface protein is a tetraspan membrane protein (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 fragments or variants thereof.

[0170] In some embodiments, the immunomodulatory component has antitumor 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.

[0171] In some embodiments, the immunomodulatory component is expressed in its full-length form in the production cells. In other embodiments, the immunomodulatory component is expressed as a translational fusion protein with a foreign body surface protein, resulting in a higher level of expression of the biologically active portion of the immunomodulatory compound on the foreign body surface. In some embodiments, the immunomodulatory compound is a soluble protein expressed as a translational fusion protein with a foreign body surface protein, such that the soluble protein is retained on the foreign body surface. In some embodiments, the foreign body surface protein is a tetraspan membrane protein (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 fragments or variants thereof.

[0172] In some embodiments, the immunomodulatory component is an inhibitor of the negative checkpoint modulator. In some embodiments, the immunomodulatory component is an inhibitor of the binding partner of the negative checkpoint modulator.

[0173] In some 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 against CTLA-4. In some embodiments, the inhibitor is a fragment of a monoclonal antibody against CTLA-4. In some 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 CTLA-4. In some 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.

[0174] In some embodiments, the immunomodulatory component is an inhibitor of programmed cell death protein 1 (PD-1). In some embodiments, the immunomodulatory component is an inhibitor of programmed death ligand 1 (PD-L1). In some 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 of PD-1, PD-L1, or PD-L2. In some embodiments, the inhibitor is a fragment of a monoclonal antibody of PD-1, PD-L1, or PD-L2. In some 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 of PD-1, PD-L1, or PD-L2. In some embodiments, the inhibitor is a nanobody, bispecific antibody, or multispecific antibody targeting 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 pidizumab. In some specific embodiments, the monoclonal antibody is atezolizumab. In some specific embodiments, the monoclonal antibody is averumab.

[0175] In some embodiments, the immunomodulatory component is an inhibitor of lymphocyte activation gene 3 (LAG3). In some of these embodiments, the LAG3 inhibitor is a monoclonal antibody against LAG3.

[0176] In some embodiments, the immunomodulatory component is an inhibitor of protein 3 (TIM-3), which contains T-cell immunoglobulin mucin. In some embodiments, the immunomodulatory component is an inhibitor of B and T lymphocyte attenuation protein (BTLA). In some embodiments, the immunomodulatory component is an inhibitor of the T-cell immune receptor (TIGIT) having both Ig and ITIM domains. In some embodiments, the immunomodulatory component is an inhibitor of the T-cell activation V-domain Ig inhibitor (VISTA). In some embodiments, the immunomodulatory component is an inhibitor of adenosine A2a receptor (A2aR). In some embodiments, the immunomodulatory component is an inhibitor of cytotoxic cell immunoglobulin-like receptor (KIR). In some embodiments, the immunomodulatory component is an inhibitor of indoleamine 2,3-dioxygenase (IDO). In some embodiments, the immunomodulatory component is an inhibitor of CD20, CD39, or CD73.

[0177] In some embodiments, the immunomodulatory component is an activator of the positive co-stimulatory molecule. In some embodiments, the immunomodulatory component is an activator of the binding chaperone of the positive co-stimulatory molecule.

[0178] In some embodiments, the immunomodulatory component is a member of the TNF receptor superfamily of activators. In some embodiments, the TNF receptor superfamily members are selected from the following groups: 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 member of the TNF superfamily. In some implementations, the TNF superfamily members are 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.

[0179] In some embodiments, activators of the TNF receptor superfamily members are expressed as monomeric proteins. In some embodiments, activators of the TNF receptor superfamily members are expressed as trimeric proteins. In some embodiments, TNF receptor superfamily members are expressed as monomeric proteins. In some embodiments, TNF receptor superfamily members are expressed as trimeric proteins.

[0180] In some 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 against OX40. In some other of these embodiments, the activator of OX40 is an OX40 ligand (OX40L).

[0181] In some embodiments, the immunomodulatory component is an activator of CD27. In some of these embodiments, the activator of CD27 is a CD27 agonist antibody. In some other of these embodiments, the CD27 agonist is a CD27 ligand (CD27L).

[0182] In some embodiments, the immunomodulatory component is a CD40 activator. In some of these embodiments, the CD40 activator is a CD40 agonist antibody. In some other of these embodiments, the CD40 agonist is a CD40 ligand (CD40L). In some embodiments, CD40L is monomeric CD40L. In some embodiments, CD40L is trimeric CD40L.

[0183] In some embodiments, the trimer CD40L is fused to PTGFRN or a fragment thereof. In some embodiments, the trimer CD40L is fused to the N-terminus of PTGFRN or a fragment thereof. In some embodiments, the trimer 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.

[0184] In some embodiments, the immunomodulatory component is an activator of glucocorticoid-induced TNFR-associated protein (GITR). In some of these embodiments, the activator of GITR is a GITR agonist antibody. In some other of these embodiments, the activator of GITR is a natural ligand of GITR.

[0185] In some embodiments, the immunomodulatory component is an activator of 4-1BB. In some of these embodiments, the activator of 4-1BB is a 4-1BB agonist antibody. In some other of these embodiments, the activator of 4-1BB is a natural ligand of 4-1BB.

[0186] In some embodiments, the immunomodulatory component is the Fas receptor (Fas). In some of these embodiments, the Fas receptor is displayed on the surface of extracellular vesicles. In some other embodiments, the immunomodulatory component is the Fas ligand (FasL). In some of these embodiments, the Fas ligand is displayed on the surface of extracellular vesicles. In some embodiments, the immunomodulatory component is an antibody against the Fas receptor. In some embodiments, the immunomodulatory component is an antibody against the Fas ligand.

[0187] In some embodiments, the immunomodulatory component is an activator of a CD28 superfamily co-stimulatory molecule. In some embodiments, the CD28-superfamily co-stimulatory molecule is ICOS or CD28. In some embodiments, the immunomodulatory component is ICOSL, CD80, or CD86.

[0188] In some embodiments, the immunomodulatory component is an activator of inducible T cell co-stimulatory agents (ICOS). In some of these embodiments, the ICOS activator is an ICOS agonist antibody. In some other of these embodiments, the ICOS activator is an ICOS ligand (ICOSL).

[0189] In some embodiments, the immunomodulatory component is an activator of CD28. In some of these embodiments, the activator of CD28 is a CD28 agonist antibody. In some other of these embodiments, the activator of CD28 is a natural ligand of CD28. In some embodiments, the ligand of CD28 is CD80.

[0190] In some 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 costimulatory molecule or an activator of a binding partner of a positive costimulatory molecule.

[0191] In some embodiments, the immunomodulatory component is a cytokine. In some embodiments, the cytokine is a soluble cytokine that has been fused to a foreign body surface protein or a fragment thereof via translation. 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).

[0192] In some 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.

[0193] In some 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.

[0194] In some 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 SEQ ID NO:15 or SEQ ID NO:16.

[0195] In some embodiments, the cytokine is interferon (IFN). In some embodiments, interferon is fused to PTGFRN or a fragment thereof. In some embodiments, interferon is interferon-γ (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.

[0196] In some embodiments, the immunomodulatory component is a T-cell receptor (TCR) or a derivative thereof. In some embodiments, the immunomodulatory component is a TCRα chain or a derivative thereof. In some embodiments, the immunomodulatory component is a TCRβ chain or a derivative thereof. In some embodiments, the immunomodulatory component is a T-cell co-receptor or a derivative thereof.

[0197] In some embodiments, the 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-associated apoptosis-inducing ligand.

[0198] In some embodiments, the tumor antigen is carcinoembryonic antigen (CEA). In some embodiments, the tumor antigen is epithelial tumor antigen (ETA).

[0199] In some 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 particular embodiment, the tumor antigen is mucin 1 (MUC1). In a particular embodiment, the tumor antigen is Tn-MUC1. In a particular embodiment, the tumor antigen is mucin 16 (MUC16).

[0200] In some embodiments, the tumor antigen is melanoma-associated antigen (MAGE). In some of these embodiments, the MAGE is type I MAGE. In some other of these embodiments, the MAGE is type II MAGE. In a particular embodiment, type I MAGE is MAGE-A2. In a particular embodiment, type I MAGE is MAGE-A4.

[0201] In some embodiments, the tumor antigen is alpha-fetoprotein (AFP). In some embodiments, the tumor antigen is tumor protein p53 (p53). In some embodiments, the tumor antigen is a tyrosinase. In some embodiments, the tumor antigen is tyrosinase-associated 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.

[0202] In some embodiments, the immunomodulatory component is a chimeric antigen receptor (CAR) or a derivative thereof. In some embodiments, the CAR binds to one or more of the following: 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-associated apoptosis-inducing ligands.

[0203] In some embodiments, the immunomodulatory component is an activator of a T-cell receptor or co-receptor. In some embodiments, the immunomodulatory component is an activator of CD3. In some embodiments, the activator is a fragment of a monoclonal antibody against CD3. In some 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 some 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 some embodiments, the immunomodulatory component is an activator of CD28. In some embodiments, the inhibitor is a fragment of a monoclonal antibody against CD28. In some 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 CD28. In some embodiments, the inhibitor is a nanobody, bispecific antibody, or multispecific antibody targeting CD28.

[0204] 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 a class I MHC or a derivative thereof. In some of these embodiments, the immunomodulatory component is a class II MHC or a derivative thereof. In some of these embodiments, the immunomodulatory component is a class III MHC or a derivative thereof.

[0205] In some embodiments, the immunomodulatory component is 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.

[0206] In various implementation schemes, the immunomodulatory component may be a polypeptide, polynucleotide, polysaccharide, lipid, small molecule, or toxin.

[0207] In some implementations, the immunomodulatory component may be a protein, peptide, glycolipid, or glycoprotein.

[0208] In some 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 the receptor. In some embodiments, the agonist is a hyperagonist that can produce a maximal response greater than that of an endogenous agonist. In some embodiments, the agonist is a full agonist with complete efficacy against the receptor. In some other embodiments, the agonist is a partial agonist with only partial efficacy against the receptor relative to a full agonist. In some embodiments, the agonist is a reverse agonist that can inhibit the constitutive activity of the receptor. In some embodiments, the agonist is a co-agonist that acts together with other co-agonists to produce an effect on the receptor. In some embodiments, the agonist is an irreversible agonist that permanently binds to the receptor by forming a covalent bond. In some embodiments, the agonist is a selective agonist targeting a specific type of receptor.

[0209] In some embodiments, the immunomodulatory component is an antagonist. In some of these embodiments, the antagonist is a competitive antagonist that reversibly binds to the receptor at the same binding site as an endogenous ligand or agonist without activating the receptor. A competitive antagonist can affect the amount of agonist necessary to achieve a maximal response. In some other of these embodiments, the antagonist is a non-competitive antagonist that binds to the active site or allosteric site of the receptor. A non-competitive antagonist can reduce the magnitude of the maximal response achievable with any amount of agonist. In some other embodiments, the antagonist is a non-competitive antagonist that requires receptor activation by an agonist before binding to a separate allosteric binding site.

[0210] In various embodiments, the immunomodulatory component comprises an antibody or an 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 be partially or wholly synthesized. In some embodiments, the antibody is a monoclonal antibody. In some of these embodiments, the monoclonal antibody is an IgG antibody. In some embodiments, the monoclonal antibody is IgG1, IgG2, IgG3, or IgG4. In some other embodiments, the antibody is a polyclonal antibody. In some embodiments, the antigen-binding fragment is selected from Fab, Fab' and F(ab')2, F(ab1)2, Fv, dAb, and Fd fragments. In some embodiments, the antigen-binding fragment is scFv or (scFv)2 fragment. In some other embodiments, the antibody or antigen-binding fragment is... (Single-domain antibody). In some implementations, the antibody or antigen-binding fragment is a bispecific or multispecific antibody.

[0211] 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 mouse or animal.

[0212] In some 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 a desired polypeptide. In some embodiments, the polynucleotide is a microRNA (miRNA) or a miRNA precursor molecule. In some of these embodiments, the miRNA is delivered to the cytoplasm of the target cell such that the miRNA molecule can silence the native mRNA in the target cell. In some embodiments, the polynucleotide is a small interfering RNA (siRNA) or short hairpin RNA (shRNA) capable of interfering with the expression of oncogenes or other dysregulated polypeptides. In some of these embodiments, the siRNA is delivered to the cytoplasm of the target cell such that the siRNA molecule can silence the native mRNA in the target cell. In some embodiments, the polynucleotide is an antisense RNA complementary to the mRNA. In some embodiments, the polynucleotide is a long non-coding RNA (lncRNA) capable of regulating gene expression and modulating disease. In some embodiments, the polynucleotide is DNA that can be transcribed into RNA. In some of these embodiments, the transcribed RNA can be translated into the desired polypeptide.

[0213] In some implementations, the immunomodulatory component is a protein, peptide, glycolipid, or glycoprotein.

[0214] In various embodiments, the composition comprises two or more of the above-described 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 that are associated with or enclosed within a closed volume of the extracellular vesicle. In some embodiments, the composition comprises a nucleic acid bound to a polypeptide. In some embodiments, the composition comprises two or more polypeptides conjugated to each other. In some embodiments, the composition comprises a protein conjugated to a bioactive molecule. In some of these embodiments, the bioactive molecule is a prodrug.

[0215] In some embodiments, the composition comprises two distinct immunomodulatory components that are membrane-associated or enclosed within a sealed volume of the extracellular vesicle. In some embodiments, the two distinct 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 fusion proteins with PTGFRN, wherein the peptides have the sequences SEQ ID NO:20 and SEQ ID NO:3, respectively.

[0216] In some embodiments, the composition comprises three distinct immunomodulatory components that are membrane-associated or enclosed within a sealed volume of the extracellular vesicle. In some embodiments, two distinct immunomodulatory components are IL-12, CD40L, and FMS-like tyrosine kinase 3 ligand (FLT3L). In some embodiments, CD40L, IL-12, and FLT3L are fused to PTGFRN or a fragment thereof, respectively. In some embodiments, CD40L, IL-12, and FLT3L are fused to the N-terminus of PTGFRN or a fragment thereof, respectively. In some embodiments, CD40L, IL-12, and FLT3L are expressed as a fusion protein with PTGFRN, wherein the polypeptide has the sequences SEQ ID NO:20, SEQ ID NO:3, and SEQ ID NO:22, respectively.

[0217] Pharmaceutical Composition

[0218] Pharmaceutical compositions typically comprise multiple 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 and the specific method of administration. Therefore, there are various suitable formulations of pharmaceutical compositions comprising multiple extracellular vesicles. (See, for example, Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa. 21st ed. (2005)). Pharmaceutical compositions are typically formulated aseptically and fully compliant with all Good Manufacturing Practices (GMP) requirements of the U.S. Food and Drug Administration.

[0219] In some embodiments, the pharmaceutical composition comprises one or more therapeutic agents and the extracellular vesicles described herein. In some embodiments, the extracellular vesicles are co-administered with one or more individual therapeutic agents, wherein co-administration includes administering the individual therapeutic agent before, after, or simultaneously with the administration of the extracellular vesicles.

[0220] Pharmaceutically acceptable excipients include those that are generally safe, non-toxic, and suitable for use, including those acceptable for veterinary and human pharmaceutical applications.

[0221] Examples of carriers or diluents include, but are not limited to, water, saline, Ringer's solution, dextran 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 compound is incompatible with the extracellular vesicles described herein, its use in the composition may be considered. Supplemental therapeutic agents may also be incorporated into the composition. Typically, pharmaceutical compositions are formulated to be compatible with their intended route of administration. Extracellular vesicles can be administered via parenteral, topical, intravenous, oral, subcutaneous, intra-arterial, intradermal, transdermal, rectal, intracranial, intraperitoneal, intranasal, intratumoral, intramuscular, or as an inhaler. In some embodiments, the pharmaceutical composition comprising extracellular vesicles is administered intravenously, for example by injection. 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 to which the extracellular vesicles are intended.

[0222] The solution or suspension may include the following components: a sterile diluent, such as water, a salt solution, a non-volatile oil, polyethylene glycol, glycerol, propylene glycol, or other synthetic solvents; an antimicrobial compound, such as benzyl alcohol or methylparaben; 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 tension-regulating compound, such as sodium chloride or dextrose. The pH may be adjusted using an acid or base, such as hydrochloric acid or sodium hydroxide. The formulation may be packaged in glass or plastic ampoules, disposable syringes, or multi-dose vials.

[0223] Pharmaceutical compositions suitable for injection include sterile aqueous solutions (if water-soluble) or dispersions and sterile powders. For intravenous administration, suitable carriers include physiological saline, antibacterial water, Cremophor EL... TM (BASF, Parsippany, NJ) or phosphate-buffered saline (PBS). The composition is typically sterile and to some extent fluid to facilitate injection. The carrier can be a solvent or dispersion medium comprising, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. Appropriate flowability can be maintained, for example, by using a coating such as lecithin, in the case of a dispersion, by maintaining the desired particle size, and by using surfactants. Microbial activity can be prevented by various antimicrobial and antifungal compounds, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. Isotonic compounds (e.g., sugars, polyols such as mannitol, sorbitol, and sodium chloride) can be added to the composition if desired. Extended absorption of the injectable composition can be achieved by including delayed-absorption compounds such as aluminum monostearate and gelatin in the composition.

[0224] As needed, sterile injectable solutions can be prepared by incorporating an effective amount of extracellular vesicles with one or more of the components listed herein into a suitable solvent. Typically, dispersions are prepared by incorporating extracellular vesicles into a sterile medium containing a basic dispersion medium and any other desired components. In the case of sterile powders used to prepare sterile injectable solutions, preparation methods include vacuum drying and freeze-drying, methods that produce a powder of the active ingredient plus any additional desired components from a previously sterile filtered solution. Extracellular vesicles can be administered in the form of reservoir injections or implantable formulations, which can be formulated to allow for continuous or pulsatile release of extracellular vesicles.

[0225] Systemic administration of compositions containing extracellular vesicles can also be performed transmucosally. For transmucosal administration, a penetrant 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 by using, for example, a nasal spray.

[0226] In some embodiments, the pharmaceutical composition comprising extracellular vesicles is administered intravenously to a subject who will benefit from the pharmaceutical composition. In some other embodiments, the composition is administered to the lymphatic system, for example, by intralymphatic injection or intranodal injection (see, for example, Senti et al., PNAS 105(46):17908(2008)), or by intramuscular injection, by subcutaneous administration, by intratumoral injection, or by direct injection into the thymus or liver.

[0227] In some embodiments, the pharmaceutical composition comprising extracellular vesicles is administered in the form of a liquid suspension. In some embodiments, the pharmaceutical composition is administered in the form of a formulation capable of forming a reservoir after administration. In some preferred embodiments, the reservoir slowly releases the extracellular vesicles into circulation or remains in reservoir form.

[0228] Typically, pharmaceutically acceptable compositions are highly purified to be free of contaminants, biocompatible, non-toxic, and suitable for administration to subjects. If water is a component of the carrier, it is highly purified and processed to be free of contaminants such as endotoxins.

[0229] Pharmaceutically acceptable carriers may include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylparaben, propylparaben, talc, magnesium stearate, and / or mineral oil. The pharmaceutical composition may further comprise lubricants, humectants, sweeteners, flavor enhancers, emulsifiers, suspending agents, and / or preservatives.

[0230] The pharmaceutical compositions described herein comprise the extracellular vesicles described herein and optional pharmaceutical actives or therapeutic agents. The therapeutic agents may be biological agents, small molecule agents, or nucleic acid agents.

[0231] Dosage forms comprising pharmaceutical compositions comprising the extracellular vesicles described herein are provided. 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.

[0232] In some implementations, the preparation of extracellular vesicles is irradiated with, for example, X-rays, gamma rays, beta particles, alpha particles, neutrons, protons, elemental nuclei, or UV rays, in order to destroy any remaining replicable nucleic acids.

[0233] In some embodiments, the formulation of extracellular vesicles is subjected to gamma irradiation with an irradiation dose greater than 1, 5, 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100 or greater than 100 kGy.

[0234] In some embodiments, the formulation of extracellular vesicles is irradiated with X-rays at doses 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.

[0235] Method

[0236] Several aspects of this disclosure also include methods for generating compositions comprising extracellular vesicles and immunomodulatory components. In some embodiments, the method includes: obtaining extracellular vesicles from production cells, wherein the production cells naturally contain the immunomodulatory component; and optionally isolating the obtained extracellular vesicles. In some embodiments, the method includes: modifying production cells with the immunomodulatory component; obtaining extracellular vesicles from the modified production cells; and optionally isolating the obtained extracellular vesicles. In some other embodiments, the method includes: obtaining extracellular vesicles from production cells; isolating the obtained extracellular vesicles; and modifying the isolated extracellular vesicles with the immunomodulatory component. In some embodiments, the method further includes formulating the isolated extracellular vesicles into a pharmaceutical composition.

[0237] extracellular vesicle production methods

[0238] Methods of modifying producer cells with immunomodulatory components

[0239] In various implementations, the method includes modifying production cells with immunomodulatory components.

[0240] The production cells can be mammalian cell lines, plant cell lines, insect cell lines, fungal cell lines, or prokaryotic cell lines. In some embodiments, the production cells are mammalian cell lines. 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, etc. Neuronal precursor cells, Amniotic cells, adipose-derived mesenchymal stem cells, or RPTEC / TERT1 cells. The producing 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.

[0241] In some preferred embodiments, the producing cells are immune cells, such as dendritic cells, T cells, B cells, natural killer cells (NK cells), antigen-presenting cells, macrophages, T helper cells, or regulatory T cells (Treg cells).

[0242] In various implementations, the immunomodulatory component can be expressed in the production cells from transgenic or mRNA introduced into the production cells by transfection, viral transduction, electroporation, extrusion, sonication, cell fusion, or other methods known to those skilled in the art.

[0243] In some embodiments, immunomodulatory components are introduced into production cells via 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 the immunomodulatory components through charge interactions. In some of these embodiments, the positively charged complex binds to the negatively charged cell surface and is absorbed by the cell via endocytosis. In some other embodiments, cationic polymers can be used to transfect production cells. In some of these embodiments, the cationic polymer is polyethyleneimine (PEI). In some 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," bio-ejection, or particle bombardment techniques can also be used to introduce immunomodulatory components into production cells (Papapetrou et al., Gene Therapy 12:S118-S130 (2005)). Reporter genes, such as β-galactosidase, chloramphenicol acetyltransferase, luciferase, or green fluorescent protein, can be used to assess the transfection efficiency of production cells.

[0244] In some implementations, immunomodulatory components are introduced into production cells via 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. Virus-mediated gene transfer vectors include vectors based on DNA viruses (such as adenovirus, adeno-associated virus, and herpesvirus) as well as vectors based on retroviruses.

[0245] In some embodiments, immunomodulatory components are introduced into production cells via electroporation. Electroporation creates transient pores in the cell membrane, allowing the introduction of various molecules into the cells. In some embodiments, DNA and RNA, as well as peptide and non-peptide therapeutic agents, can be introduced into production cells via electroporation.

[0246] In some embodiments, immunomodulatory components are introduced into production cells via microinjection. In some embodiments, glass micropipettes can be used to inject immunomodulatory components into production cells at a microscopic level.

[0247] In some implementations, immunomodulatory components are introduced into production cells by extrusion.

[0248] In some embodiments, immunomodulatory components are introduced into production cells via ultrasound treatment. In other embodiments, production cells are exposed to high-intensity sound waves, causing transient disruption of the cell membrane, thereby allowing the loading of immunomodulatory components.

[0249] In some embodiments, immunomodulatory components are introduced into production cells via cell fusion. In some embodiments, immunomodulatory components are introduced via electrocellular 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.

[0250] In some embodiments, immunomodulatory components are introduced into production cells via hypotonic lysis. In some of these embodiments, production cells are exposed to a low-ionic-strength buffer solution, causing them to rupture and allowing loading of immunomodulatory components. In some alternative embodiments, controlled dialysis against a hypotonic solution is used to swell the production cells and create pores in the production cell membrane. The production cells are then exposed to conditions that allow for resealing of the membrane.

[0251] In some embodiments, immunomodulatory components are introduced into the production cells via detergent treatment. In some embodiments, the production cells are treated with a mild detergent that temporarily disrupts the cell membrane through the production pores, thereby allowing the loading of immunomodulatory components. After loading, the production cells are washed off the detergent, thereby resealing the membrane.

[0252] In some embodiments, immunomodulatory components are introduced into production cells via receptor-mediated endocytosis. In some embodiments, the production cells have surface receptors that induce the internalization of the receptors and associated immunomodulatory components upon binding to the immunomodulatory components.

[0253] In some embodiments, immunomodulatory components are introduced into the production cells via filtration. In other embodiments, the production cells and immunomodulatory components may 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.

[0254] In some implementations, the production cells are subjected to several freeze-thaw cycles, causing the cell membrane to rupture, thereby allowing the loading of immunomodulatory components.

[0255] Methods of modifying extracellular vesicles with immunomodulatory components

[0256] In various alternative implementations, immunomodulatory components are introduced directly into the extracellular vesicles after they are isolated.

[0257] In some embodiments, immunomodulatory components are introduced into extracellular vesicles via 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 some embodiments, chemicals such as calcium phosphate, cyclodextrin, or polybrenes can be used to introduce immunomodulatory components into extracellular vesicles.

[0258] In some embodiments, immunomodulatory components are introduced into extracellular vesicles via electroporation. In some embodiments, the extracellular vesicles are exposed to an electric field that induces transient pores in the extracellular vesicle membrane, thereby allowing loading of immunomodulatory components.

[0259] In some embodiments, immunomodulatory components are introduced into extracellular vesicles via microinjection. In other embodiments, glass micropipettes can be used to inject immunomodulatory components directly into extracellular vesicles at a microscopic level.

[0260] In some implementations, immunomodulatory components are introduced into extracellular vesicles by squeezing.

[0261] In some embodiments, immunomodulatory components are introduced into extracellular vesicles via ultrasound treatment. In some embodiments, extracellular vesicles are exposed to high-intensity sound waves, causing transient disruption of the extracellular vesicle membrane, thereby allowing the loading of immunomodulatory components.

[0262] In some embodiments, immunomodulatory components can be conjugated to the surface of extracellular vesicles. Conjugation can be achieved chemically or enzymatically using methods known in the art.

[0263] In some embodiments, extracellular vesicles contain chemically conjugated immunomodulatory components. Chemical conjugation can be achieved by covalently binding the immunomodulatory component to another molecule, with or without a linker. The formation of such conjugates is within the skill of those skilled in the art, and various techniques are known to be available for accomplishing the conjugation, with the choice of a particular technique guided by the material to be conjugated. In some embodiments, peptides are conjugated to extracellular vesicles. In some other embodiments, non-peptides (e.g., lipids, carbohydrates, nucleic acids, and small molecules) are conjugated to extracellular vesicles.

[0264] In some embodiments, immunomodulatory components are introduced into extracellular vesicles via hypotonic lysis. In some of these embodiments, the extracellular vesicles are exposed to a low-ionic-strength buffer solution, causing them to rupture and allowing loading of the immunomodulatory components. In some alternative embodiments, controlled dialysis against a hypotonic solution is used to swell the extracellular vesicles and create pores in the extracellular vesicle membrane. The extracellular vesicles are then exposed to conditions that allow for resealing of the membrane.

[0265] In some embodiments, immunomodulatory components are introduced into extracellular vesicles via detergent treatment. In some embodiments, the extracellular vesicles are treated with a mild detergent that temporarily disrupts the extracellular vesicle membrane by creating pores, thereby allowing loading of immunomodulatory components. After loading, the detergent is washed off, thereby resealing the membrane.

[0266] In some embodiments, immunomodulatory components are introduced into extracellular vesicles via receptor-mediated endocytosis. In some embodiments, the extracellular vesicles have surface receptors that, upon binding of the immunomodulatory components, induce the internalization of the receptors and the associated immunomodulatory components.

[0267] In some embodiments, immunomodulatory components are introduced into extracellular vesicles via mechanical firing. In some embodiments, 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, causing them to traverse the extracellular vesicle membrane.

[0268] In some embodiments, immunomodulatory components are introduced into extracellular vesicles via filtration. In some embodiments, extracellular vesicles and immunomodulatory components may be forced through a filter with 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.

[0269] In some implementations, extracellular vesicles are subjected to several freeze-thaw cycles, causing the extracellular vesicle membrane to rupture, thereby allowing the loading of immunomodulatory components.

[0270] Methods of isolating extracellular vesicles

[0271] Extracellular vesicles can be isolated from production cells. In some embodiments, extracellular vesicles are released from production cells into a cell culture medium. All known methods of extracellular vesicle isolation are considered applicable herein. For example, extracellular vesicles can be separated from media or other source materials using physical properties, including separation based on charge (e.g., electrophoretic separation), size (e.g., filtration, molecular sieving, etc.), density (e.g., conventional or gradient centrifugation), and Swedberg constant (e.g., sedimentation with or without external force). Alternatively or additionally, separation can be based on one or more biological properties and includes methods that can employ surface labeling (e.g., for precipitation, reversible binding to a solid phase, FACS separation, specific ligand binding, nonspecific ligand binding, affinity purification, etc.).

[0272] Separation and enrichment can be performed in a general and non-selective manner, typically involving continuous centrifugation. Alternatively, separation and enrichment can be performed in a more specific and selective manner, such as using extracellular vesicles or producing 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.

[0273] In some embodiments, size exclusion chromatography can be used to separate extracellular vesicles. Size exclusion chromatography techniques are 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. Furthermore, in some embodiments, as is generally known in the art, after chromatographic separation, extracellular vesicles can be further separated by centrifugation techniques (one or more chromatographic fractions). In some embodiments, for example, density gradient centrifugation can be used to further separate extracellular vesicles. In some embodiments, it may be desirable to further separate extracellular vesicles of production cell origin from those of other origins. For example, extracellular vesicles of production cell origin can be separated from those of non-production cell origin by immunoadsorption capture using antigens or antibodies specific to production cells.

[0274] In some implementations, the separation of extracellular vesicles may involve a combination of methods, including but not limited to differential centrifugation, size-based membrane filtration, immunoprecipitation, FACS sorting, and magnetic separation.

[0275] Methods for measuring extracellular vesicle size

[0276] In some embodiments, the methods described herein include measuring the size of extracellular vesicles and / or populations of extracellular vesicles. Typically, the extracellular vesicle size is measured as the longest measurable size. The longest measurable size of an extracellular vesicle is also commonly referred to as its diameter.

[0277] Extracellular vesicle size can be measured using dynamic light scattering (DLS) and / or multi-angle light scattering (MALS). Methods for measuring extracellular vesicle size using DLS and / or MALS are known to those skilled in the art and include nanoparticle tracking assays (NTA, e.g., using a Malvern NanoSight NS300 nanoparticle tracking device). In one specific embodiment, the extracellular vesicle size is determined using a Malvern NanoSight NS300. In some embodiments, the extracellular vesicles described herein have a longest dimension of approximately 20-300 nm as measured by NTA (e.g., using a Malvern NanoSight NS300). In other embodiments, the extracellular vesicles described herein have a longest dimension of approximately 40-200 nm as measured by NTA (e.g., using a Malvern NanoSight NS300). In other embodiments, the extracellular vesicle population described herein comprises a population in which 90% of the extracellular vesicles have a longest dimension of approximately 20-300 nm as measured by NTA (e.g., using a Malvern NanoSight NS300). In other embodiments, the extracellular vesicle population described herein comprises a population in which 95% of the extracellular vesicles have a longest dimension of approximately 20-300 nm as measured by an NTA (e.g., using a Malvern NanoSight NS300). In other embodiments, the extracellular vesicle population described herein comprises a population in which 99% of the extracellular vesicles have a longest dimension of approximately 20-300 nm as measured by an NTA (e.g., using a Malvern NanoSight NS300). In other embodiments, the extracellular vesicle population described herein comprises a population in which 90% of the extracellular vesicles have a longest dimension of approximately 40-200 nm as measured by an NTA (e.g., using a Malvern NanoSight NS300). In other embodiments, the extracellular vesicle population described herein comprises a population in which 95% of the extracellular vesicles have a longest dimension of approximately 40-200 nm as measured by an NTA (e.g., using a Malvern NanoSight NS300). In other embodiments, the extracellular vesicle populations described herein include populations in which 99% of the extracellular vesicles have the longest dimension of approximately 40–200 nm as measured by NTA (e.g., using a Malvern NanoSight NS300).

[0278] Extracellular vesicle size can be measured using tunable resistive pulse sensing (TRPS). In one 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 approximately 20-300 nm as measured by TRPS (e.g., using iZON qNano Gold). In other embodiments, the extracellular vesicles described herein have a longest dimension of approximately 40-200 nm as measured by TRPS (e.g., using iZON qNano Gold). In other embodiments, the extracellular vesicle population described herein comprises a population in which 90% of the extracellular vesicles have a longest dimension of approximately 20-300 nm as measured by TRPS (e.g., using iZON qNano Gold). In other embodiments, the extracellular vesicle population described herein comprises a population in which 95% of the extracellular vesicles have a longest dimension of approximately 20-300 nm as measured by TRPS (e.g., using iZON qNano Gold). In other embodiments, the extracellular vesicle population described herein comprises a population in which 99% of the extracellular vesicles have a longest dimension of approximately 20-300 nm as measured by TRPS (e.g., using iZON qNano Gold). In other embodiments, the extracellular vesicle population described herein comprises a population in which 90% of the extracellular vesicles have a longest dimension of approximately 40-200 nm as measured by TRPS (e.g., using iZON qNano Gold). In other embodiments, the extracellular vesicle population described herein comprises a population in which 95% of the extracellular vesicles have a longest dimension of approximately 40-200 nm as measured by TRPS (e.g., using iZON qNano Gold). In other embodiments, the extracellular vesicle population described herein comprises a population in which 99% of the extracellular vesicles have a longest dimension of approximately 40-200 nm as measured by TRPS (e.g., using iZON qNano Gold).

[0279] Extracellular vesicle size can be measured using electron microscopy. In some embodiments, the electron microscope used to measure extracellular vesicle size is a transmission electron microscope. In one specific embodiment, the transmission electron microscope used to measure extracellular vesicle size is a Tecnai. TM G 2 Spirit BioTWIN. Methods for measuring extracellular vesicle size using electron microscopy are well known to those skilled in the art, and any such method can be adapted to measure extracellular vesicle size. In some embodiments, the extracellular vesicles described herein have the ability to be measured using a scanning electron microscope (e.g., Tecnai). TM G2 The longest dimension is approximately 20-300 nm, as measured by a Spirit BioTWIN scanning electron microscope. In other embodiments, the extracellular vesicles described herein have a maximum size of approximately 20-300 nm as measured by a scanning electron microscope (e.g., Tecnai). TM G 2 The longest dimension is approximately 40-200 nm, as measured by a Spirit BioTWIN scanning electron microscope. In other embodiments, the extracellular vesicle populations described herein include those in which 90% of the extracellular vesicles have a longest dimension of approximately 40-200 nm, as measured by a scanning electron microscope (e.g., Tecnai). TM G 2 The longest dimension of the extracellular vesicle population, as measured by a Spirit BioTWIN scanning electron microscope, is approximately 20-300 nm. In other embodiments, the extracellular vesicle populations described herein comprise extracellular vesicles in which 95% have a longest dimension as measured by a scanning electron microscope (e.g., Tecnai). TM G 2 The longest dimension of the extracellular vesicle population, as measured by a Spirit BioTWIN scanning electron microscope, is approximately 20-300 nm. In other embodiments, the extracellular vesicle populations described herein comprise extracellular vesicles in which 99% have a longest dimension as measured by a scanning electron microscope (e.g., Tecnai). TM G 2 The longest dimension of the extracellular vesicle population, as measured by a Spirit BioTWIN scanning electron microscope, is approximately 20-300 nm. In other embodiments, the extracellular vesicle populations described herein comprise extracellular vesicles in which 90% have a longest dimension as measured by a scanning electron microscope (e.g., Tecnai). TM G 2 The longest dimension of the extracellular vesicle population, measured by a Spirit BioTWIN scanning electron microscope, is approximately 40-200 nm. In other embodiments, the extracellular vesicle populations described herein comprise extracellular vesicles in which 95% have a longest dimension as measured by a scanning electron microscope (e.g., Tecnai). TM G 2 The longest dimension of the extracellular vesicle population, measured by a Spirit BioTWIN scanning electron microscope, is approximately 40-200 nm. In other embodiments, the extracellular vesicle populations described herein comprise extracellular vesicles in which 99% have dimensions as measured by a scanning electron microscope (e.g., Tecnai). TM G 2 The longest-sized population, approximately 40–200 nm, was measured using a Spirit BioTWIN scanning electron microscope.

[0280] Treatment methods for cancer, GvHD, and autoimmune diseases

[0281] In addition, this article provides methods for treating subjects with cancer, graft-versus-host disease (GvHD), and autoimmune diseases.

[0282] In various embodiments, the composition is administered to a subject suffering from cancer. In some of these embodiments, the composition may upregulate the immune response and enhance the tumor-targeting ability of the subject's immune system. In some embodiments, the treated cancer is characterized by the infiltration of leukocytes (T cells, B cells, macrophages, dendritic cells, monocytes) into the tumor microenvironment, or a so-called "hot tumor" or "inflammatory tumor." In some embodiments, the treated cancer is characterized by low or undetectable levels of leukocyte infiltration into the tumor microenvironment, or a so-called "cold tumor" or "non-inflammatory tumor." In some embodiments, the composition is administered in an amount and for a time sufficient to convert a "cold tumor" into a "hot tumor," i.e., the administration results in the infiltration of leukocytes (e.g., T cells) into the tumor microenvironment.

[0283] In some embodiments, the composition comprising extracellular vesicles 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-1gp100, and TNF-associated apoptosis-inducing ligand. In some embodiments, the tumor antigen is derived from a reference genomic sequence. In some embodiments, the tumor antigen is derived from the genomic sequence of a subject receiving the composition.

[0284] Cancers that can be treated with the composition include, but are not limited to, the cancers listed in Table 5.

[0285] In some 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 some embodiments, the composition for treating GvHD comprises Fas ligand (FasL). In some of these embodiments, FasL is expressed on the surface of extracellular vesicles.

[0286] In various embodiments, the composition is administered to a subject suffering from an autoimmune disease. In some of these embodiments, the composition may downregulate the immune response and inhibit the subject's immune activity.

[0287] Autoimmune diseases include, but are not limited to, multiple sclerosis, peripheral neuritis, Sjögren's syndrome, rheumatoid arthritis, alopecia, autoimmune pancreatitis, Behcet's disease, bullous pemphigus, celiac disease, Dweck's disease (neuromyelitis optica), glomerulonephritis, IgA nephropathy, mixed vasculitis, scleroderma, diabetes, arteritis, vitiligo, ulcerative colitis, irritable bowel syndrome, psoriasis, uveitis, and systemic lupus erythematosus.

[0288] In some embodiments, the composition is administered intravenously to the subject's circulatory system. In some embodiments, the composition is infused into a suitable fluid and administered intravenously to the subject.

[0289] In some embodiments, the composition is administered intra-arterially to the subject's circulatory system. In some embodiments, the composition is injected into a suitable fluid and administered into the subject's arteries.

[0290] In some embodiments, the composition is administered to the subject via intrathecal administration. In some embodiments, the composition is administered by injection into the spinal canal or subarachnoid space to reach the cerebrospinal fluid (CSF).

[0291] In some embodiments, the composition is administered intratumorally to one or more tumors of the subject.

[0292] In some embodiments, the composition is administered to the subject via intranasal application. In some embodiments, the composition may be inhaled through the nose in the form of a topical or systemic application. In some embodiments, the composition is administered as a nasal spray.

[0293] In some embodiments, the composition is administered to the subject via intraperitoneal administration. In some embodiments, the composition is infused into a suitable fluid and injected into the peritoneum of the subject. In some embodiments, the intraperitoneal administration results in the distribution of the composition (e.g., extracellular vesicles in the composition) to lymphatic vessels. In some embodiments, the intraperitoneal administration results in the distribution of the composition (e.g., extracellular vesicles in the composition) to the thymus, spleen, and / or bone marrow. In some embodiments, the intraperitoneal administration results in the distribution of the composition (e.g., extracellular vesicles in the composition) to one or more lymph nodes. In some embodiments, the intraperitoneal administration results in the distribution of the composition (e.g., extracellular vesicles in the composition) to one or more of the following: cervical lymph nodes, inguinal lymph nodes, mediastinal lymph nodes, or sternal lymph nodes. In some embodiments, the intraperitoneal administration results in the distribution of the composition (e.g., extracellular vesicles in the composition) to the pancreas.

[0294] In some embodiments, the composition is administered to the subject via periocular application. In some embodiments, the composition is injected into the periocular tissue. Periocular drug administration includes subconjunctival, anterior subocular, posterior subocular, and retroocular routes.

[0295] 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, intra-arterial 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.

[0296] In some embodiments, the dosage 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.

[0297] The composition can be administered to the subject once. Alternatively, it can be administered multiple times over a period of time. For example, the subject may be given two, three, four, five, or more doses. In some embodiments, administration may be given as needed, for example, as long as symptoms associated with the disease, disorder, or condition persist. In some embodiments, repeated administration may be instructed for the remainder of the subject's life. The duration of treatment may vary and may, for example, not exceed one year, six months, three months, two months, one month, two weeks, one week, three days, two days, or not exceed one day.

[0298] In some implementations, the dose of extracellular vesicles is administered at certain intervals, such as once daily, every other day, once weekly, twice weekly, once monthly, or twice monthly.

[0299] In some embodiments, the pharmaceutical composition is administered at a frequency sufficient to effectively increase the concentration of the immunomodulatory component in target cells or tissues to levels above those associated with symptoms of the disease, disorder, or condition.

[0300] 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 to deliver a sufficient amount of the immunomodulatory component to the target cells or tissues during the treatment period. In some embodiments, the pharmaceutical composition is administered a sufficient number of times during the treatment period to deliver a sufficient amount of the immunomodulatory component to the target cells or tissues 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 the immunomodulatory component in the target cells or tissues includes increasing the peak concentration, while in other embodiments, it includes increasing the average concentration. In some embodiments, a significant increase during the treatment period can be determined by comparing subjects before or after treatment, or by comparing measurements taken in a treated population with a matched untreated control population.

[0301] 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 cells or tissue for at least 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 cells or tissue for a period at least as long as the treatment period.

[0302] In some embodiments, the time interval between repeated administrations during the treatment period is no greater than a period in which the number of extracellular vesicles in 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.

[0303] In some embodiments, the method further includes one or more doses of non-therapeutic extracellular vesicles prior to the injection of an appropriate therapeutic dose of the therapeutically carrying extracellular vesicles. In some embodiments, the non-therapeutic extracellular vesicles are administered separately from the therapeutic extracellular vesicles and at different doses. In some embodiments, the dose of the non-therapeutic extracellular vesicles is greater than the dose of the therapeutic extracellular vesicles. In some other embodiments, the dose of the non-therapeutic extracellular vesicles is less than the dose of the therapeutic extracellular vesicles. In some embodiments, the dose of the non-therapeutic extracellular vesicles is the same as the dose of the therapeutic extracellular vesicles. In various embodiments, the method of administering non-therapeutic extracellular vesicles prior to the injection of an appropriate dose of the therapeutic extracellular vesicles reduces the turnover of therapeutic extracellular vesicles in the liver, lungs, and / or spleen.

[0304] Effective amounts of the composition are provided at least in part based on the target tissue, target cell type, route of administration, physical characteristics of the extracellular vesicles (e.g., size, and, in some cases, the extent to which molecules are to be delivered), and other determinants. Typically, effective amounts of the composition provide an effective cellular response to the target cells. Increased efficiency can be demonstrated by increased cell transfection (i.e., the percentage of cells transfected with the extracellular vesicle component), increased cellular responses, or reduced innate immune responses in the host subject.

[0305] The dosage and frequency of administration of 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 timing of administration, and other clinical factors. In one instance, intravenous administration is initiated with the minimum effective dose and the dose is increased over a pre-selected period until a positive effect is observed. Subsequently, dose escalation is limited to the level that produces the corresponding increase in effect, taking into account any possible side effects.

[0306] Example

[0307] The following embodiments are provided to provide those skilled in the art with a complete disclosure and description of how to prepare and use the invention. These embodiments are not intended to limit the scope of the invention as perceived by the inventors, nor are they intended to represent all or only the experiments performed. Efforts have been made to ensure the accuracy of the figures used (e.g., amounts, temperatures, etc.), but some experimental errors and biases should be taken into account. Unless otherwise specified, parts are all parts by weight, molecular weights are all weight-average molecular weights, temperatures are all in degrees Celsius, and pressures are all at or near atmospheric pressure. Standard abbreviations may be used, such as bp, base pair; kb, kilobase; p1, picoliter; s or sec, second; min, minute; h or hr, hour; aa, amino acid; nt, nucleotide, etc.

[0308] Unless otherwise stated, the present invention will be practiced using conventional methods of protein chemistry, biochemistry, recombinant DNA techniques, and pharmacology, which are well explained in the literature. See, for example, Tetreighton, Proteins: Structures and Molecular Properties (WH Freeman and Company, 1993); Allehninger, 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).

[0309] Method

[0310] Exosome purification

[0311] Collect the conditioned medium and centrifuge at 300-800×g for 5 minutes at room temperature to remove cells and large debris. Then add 1000 U / L to the supernatant of the medium. 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 at 4°C for 3 hours to precipitate the foreign bodies. Discard the supernatant and aspirate any residual culture medium from the bottom of the tube. Resuspend the precipitate in 200–1000 μL of PBS (-Ca-Mg).

[0312] To further enrich the exogenous population, density gradient purification (sucrose or Optiprep) was performed. TM Precipitation was treated. For sucrose gradient purification, the foreign body precipitate was layered at the top of the sucrose gradient, as shown in Table 6 below:

[0313] Table 6, Sucrose density gradient:

[0314]

[0315]

[0316] The gradient was rotated at 200,000 × g in a 12 mL Ultra-Clear (344059) tube placed in an SW 41Ti rotor at 4 °C for 16 hours to separate the foreign fraction.

[0317] The exogenous 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. The purified exogenous material was then ultracentrifuged again at 133,900 × g for 3 hours at 4 °C to precipitate the precipitate. The resulting precipitate was resuspended in a minimal volume of PBS (approximately 200 μL) and stored at 4 °C.

[0318] For Optiprep TM A three-layer sterile gradient of equal volumes of 10%, 30%, and 45% Optiprep was prepared in 12 mL Ultra-Clear (344059) tubes for use with SW 41 Ti rotors. The precipitate was added to the Optiprep... TM The foreign body fraction was separated by gradient centrifugation at 200,000 × g for 16 hours at 4 °C. The foreign body layer was then gently collected from the top of the test tube, approximately 3 mL.

[0319] The exogenous fraction was diluted in approximately 32 mL of PBS in a 38.5 mL Ultra-Clear (344058) tube and purified by ultracentrifugation at 133,900 × g for 3 hours at 4 °C. The precipitated exogenous fraction was then resuspended in a minimum volume of PBS (approximately 200 μL) and stored at 4 °C.

[0320] Example 1: Engineering exosomes to display immune checkpoint modulator antibodies

[0321] Human embryonic kidney (HEK) cell lines were grown to high density, and exogenous bodies were isolated from the culture medium according to methods known to those skilled in the art (e.g., the methods described herein). Exogenous bodies engineered with a cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) antibody were prepared by chemical conjugation according to techniques known in the art. Exogenous bodies modified with the CTLA4 antibody were selected by flow cytometry. Simultaneously, unmodified exogenous bodies were isolated according to the same standard methods.

[0322] Two populations of exogenous organisms were labeled with a radioactive tracer, and 150 μg of each formulation was injected into live mice (e.g., a mouse model of melanoma). Mice receiving either the CTLA-4 antibody-labeled exogenous organism or the unmodified exogenous organism were continuously monitored for 30 minutes, followed by monitoring again at four-hour intervals via whole-animal PET / CT. Whole-animal imaging allowed for real-time, high-resolution tracking of labeled exogenous organisms across various tissues.

[0323] Without prior labeling with a radioactive tracer, 150 μg of each exogenous body population was intravenously injected into two mouse cohorts. Mice were euthanized five weeks after administration. Tumor samples were collected and analyzed by immunohistochemistry and real-time PCR.

[0324] Example 2: Engineering exosomes to display Fas ligand

[0325] Human antigen-presenting cells were transfected with a plasmid encoding a puromycin resistance-selective marker and the Fas ligand. Transfected cells were treated with puromycin, resistant colonies were selected, and the surface expression of the Fas ligand was determined by flow cytometry. Cells stably expressing the Fas ligand were grown to high concentrations, and the resulting exogenous cells were isolated from the culture medium according to methods known to those skilled in the art (e.g., the methods described herein). Simultaneously, untransfected production cells were cultured, and the resulting exogenous cells were isolated using the same standard methods.

[0326] Two populations of exogenous organisms were labeled with radioactive tracers, and 150 μg of each formulation was injected into live mice (e.g., a mouse model of GvHD). Mice receiving either exogenous organisms derived from unmodified cells or from Fas ligand-expressing cells were monitored for 30 minutes, and then again by whole-animal PET / CT at four-hour intervals. Whole-animal imaging allowed for real-time, high-resolution tracking of labeled exogenous organisms across various tissues.

[0327] According to the method described herein, purified exogenous populations from unmodified production cells and production cells engineered to express Fas ligand were purified. 150 μg of each exogenous population was injected into two mouse cohorts without prior labeling with a radiotracer. Animals in both cohorts were euthanized three to five weeks post-administration for immunohistochemical analysis and real-time PCR.

[0328] Example 3: Lymphatic uptake of exosomes following intraperitoneal administration

[0329] To determine the biodistribution of the purified exogenous organism in vivo, the following experiments were conducted:

[0330] Conditioned culture medium from 293T cells was collected and centrifuged at 300–800 × g for 5 minutes at room temperature to remove cells and large debris. Then, 1000 U / L of the culture supernatant was added. 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 precipitate the foreign bodies. Discard the supernatant and aspirate any remaining culture medium from the bottom of the tube. Resuspend the precipitate in 200–1000 μL of PBS (-Ca-Mg).

[0331] To further enrich the exogenous population, the precipitate was purified using a sucrose density gradient as defined in Table 6.

[0332] The gradient was rotated at 200,000 × g in a 12 mL Ultra-Clear (344059) tube placed in an SW 41Ti rotor at 4 °C for 16 hours to separate the foreign fraction.

[0333] The exogenous 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. The purified exogenous material was then ultracentrifuged again at 133,900 × g for 3 hours at 4 °C to precipitate the precipitate. The resulting precipitate was resuspended in a minimal volume of PBS (approximately 200 μL) and stored at 4 °C.

[0334] To radiolabel purified exogenous material for in vivo imaging, 100 μL of 1x10 11 The purified exogenous compound 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, followed by incubation at 4 °C overnight. The DFO-exogenous compound was 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 / 1x10⁻⁶ was obtained. 10 Total yield of exogenous exogenous material (0.4 mCi of 89Zr-DFO-exogenous material in up to 0.8 mL PBS). Quality control (HPLC) was performed prior to release to ensure >95% RCP.

[0335] In vitro stability

[0336] Exogenous material (20 μCi / 2 x 10) 10 Incubate at room temperature under the following conditions:

[0337] a. Prepare buffer solution

[0338] b. Mouse serum (if possible, serum containing 10% exogenous body solution).

[0339] Two hours after the start of incubation, the solution was injected into HPLC to determine the stability of the tracer.

[0340] In vivo imaging

[0341] Mice (SKH-1, n=8, 5-8 weeks old) were randomly divided into two groups, weighed, and injected (the second group was injected immediately after the dynamic scan of the first group) with 1x10 10 / g exogenous body, to obtain the minimum radiation dose of 100μCi / mouse. Group 1 was administered intravenously (IV), and Group 2 was administered intraperitoneally (IP).

[0342] Whole-body PET / CT scans of mice were performed in four mouse hotels using the following schedule: 1-hour 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 scans were performed after each imaging time point to provide anatomical reference.

[0343] After the last imaging time point, the 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.

[0344] If the count is very high, allow the organ to decompose for 2-3 days, then count again.

[0345] Table 7

[0346]

[0347] result

[0348] Imaging of the two treated mouse cohorts was performed at 4, 24, and 48 hours post-treatment. Whole-body PET / CT imaging showed stable liver delivery in all mice in group 1 treated with IV. FIG. 1A In group 2 of the IP treatment, mice showed a clear non-overlapping distribution. FIG. 1B Dissected organs were analyzed using a radiographic gamma counter, which showed significant liver and spleen uptake in IV-treated mice. FIG. 2 In contrast, in IP-treated mice, uptake was primarily observed in the pancreas, spleen, thymus, and lymph nodes, with additional uptake in the liver and ovaries. These results indicate that different routes of administration lead to 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 to reach immune cells.

[0349] Example 4: Engineering CD40L exosomes to activate B cells

[0350] CD40L is a member of the tumor necrosis factor (TNF) superfamily, primarily expressed on T cells. The CD40L receptor, CD40, is expressed on antigen-presenting cells, including macrophages, dendritic cells, and B cells. CD40 signaling activates B cells and induces antigen-specific responses. Therefore, activation of the CD40 pathway influences the development of antitumor immunity across various tumor types. To determine whether engineered exogens could be generated to induce specific immune effects, exogens were generated from HEK293SF cells transfected with a plasmid containing full-length human CD40L. Transfected cells were placed under puromycin selection, and resistant cell populations were grown to high density. The exogens obtained were collected from conditioned medium as described above and processed using Optiprep... TM Gradient purification was performed. Exogenous CD40L cells from unmodified HEK293SF cells were also isolated as controls. Human peripheral blood mononuclear cells (PBMCs) were seeded at 150,000 cells per well in 96-well plates and incubated overnight at 37°C with either purified or native CD40L exogenous cells. One PBMC sample was incubated with 1 μg / mL of soluble recombinant CD40L-Fc as a positive control. FIG. 3A and 3B As shown, the CD40L exogene activated B cells in a dose-dependent manner, as measured by CD69 expression in two different donor samples. The native exogene failed to induce B cell activation. Importantly, the level of B cell activation induced by the CD40L exogene was comparable to that induced by CD40L-Fc.

[0351] To determine whether the observed exosome-mediated B cell activation was due to direct B cell activation or mediated by trans-acting immune cells, a similar experiment was performed using purified human B cells. Fifty,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 subjected to freeze-thaw cycles (CD40L-EVs [F / T]) and its B cell activation was tested. FIG. 4A and 4B As shown, the CD40L exogene activated purified B cells from both donors to a similar degree as CD40L-Fc. The native exogene failed to activate B cells, while the CD40L exogene-treated freeze-thawed sample successfully activated B cells, indicating that the action of the CD40L exogene is directly mediated through B cells, and that the presence of CD40L is sufficient to activate B cells. Furthermore, the engineered exogene remained stable and active during at least one freeze-thaw cycle.

[0352] To further validate the CD40L exogenous organism, a reporter system was used to measure the activity of the engineered exogenous organism. Activation of the CD40 pathway leads to NF-κB activation. 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, cell activation was confirmed by incubation in the presence of either an agonist anti-CD40 antibody (BioLegend, Inc.) crosslinked with an anti-Fc antibody (Jackson ImmunoResearch, Inc.) or recombinant human CD40L (ACROBiosystems) crosslinked with an anti-IgG antibody (Jackson ImmunoResearch, Inc.). FIG. 5A and 5B Incubating CD40L-engineered exogenous cells with engineered cells resulted in a robust increase in luciferase activity comparable to anti-CD40+ and anti-Fc effects. Importantly, the engineered exogenous cells did not require cross-linking antibodies, indicating that CD40L on the surface of the exogenous cells can form functional CD40L trimers sufficient to activate CD40.

[0353] Example 5: Engineering CD80 exosomes to activate T cells.

[0354] 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) via CD28 and CTLA-4 activates T cells during the initiation of an immune response. To determine whether exogens could be engineered to activate T cells, CD80-containing exogens were generated by transfecting and selecting HEK293SF cells, as described in Example 4. To verify the activity of the CD80 exogens, human PBMCs were seeded at 150,000 cells per well in 96-well plates and incubated with (i) purified CD80 exogens and anti-CD3 antibody, (ii) native exogens and anti-CD3 antibody, (iii) anti-CD3 antibody alone, or (iv) a combination of anti-CD28 and anti-CD3 antibodies. The samples were incubated at 37°C for three days, and CD4+ was analyzed. + T cells ( FIG. 6A ) and CD8 + T cells ( FIG. 6B The T cell count was measured. The CD80 exogenous antibody activated T cells in a dose-dependent manner, to a degree comparable to the positive controls of CD3 and CD28 antibodies. In contrast, the native exogenous antibody had no effect on T cell proliferation.

[0355] To confirm that CD80 exosomes induce functional activation of T cells, IFNγ levels were measured by AlphaLISA in PBMCs incubated with both native and CD80 exosomes in the presence of additional anti-CD3 antibody. FIG. 7A As shown, for the CD80 exogenous form, there was a dose-dependent increase in IFNγ levels, while this was not observed for the natural exogenous form. FIG. 7B As shown, the highest concentration of the CD80 exogenous resulted in higher IFNγ levels than any other condition, including the positive control (anti-CD28 / anti-CD3). These results indicate that exogenous substances can be engineered to possess specific activities that lead to immune cell activation.

[0356] Example 6: Engineering CD27L and OX40L exosomes to produce proinflammatory cytokines

[0357] CD27L (CD70) and OX40L are members of the TNF superfamily and bind to their homologous receptors (CD27 and OX40, respectively) on T cells. CD27L is expressed by certain T cell and B cell populations, while OX40L is expressed by certain antigen-presenting cell populations. Therefore, signaling via CD27 or OX40 has implications in immuno-oncology, particularly as a method for activating unresponsive T cells. To determine whether exogens could be engineered to induce the production of pro-inflammatory cytokines in PBMCs, exogens containing CD27L and OX40L were generated by transfecting and selecting HEK293SF cells, as described in Example 4. To verify the activity of the CD27L exogens, human PBMCs were seeded in 96-well plates and incubated with purified CD27L exogens and anti-CD3 antibodies, native exogens 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 the production of interferon-γ (IFNγ) was measured in two different donors. FIG. 8A and 8B ) and the production of IL-2 ( FIG. 9A and 9B The CD27L exogene induced the production of IFNγ and IL-2 in a dose-dependent manner, to a degree comparable to or significantly higher than that of the positive controls of CD3 and CD28 antibodies (donor 1) (donor 2). Conversely, the native exogene had no effect on the production of IFNγ or IL-2. Similarly, the OX40L exogene was sufficient to induce the production of IFNγ and IL-2 to similar or greater extent in two different donors. FIG. 10A and 10B as well as FIG. 11A and 11B ).

[0358] To further validate the OX40L exogenous variant, a reporter system was used to measure the activity of the engineered exogenous variant. Activation of the OX40 pathway leads to NF-κB activation. Using modified Jurkat T cell lines (Promega Corporation) engineered to overexpress OX40 on their surface and containing a luciferase reporter gene downstream of the NF-κB promoter, cell activation was confirmed by incubation in the presence of an agonistic anti-OX40 antibody (Biolegend) crosslinked with an anti-Fc antibody (Jackson ImmunoResearch, Inc.) or recombinant human OX40L (ACROBiosystems) crosslinked with an anti-IgG antibody (Jackson ImmunoResearch, Inc.). FIG. 12A and 12B Anti-OX40L antibodies cross-linked with anti-IgG failed to activate reporter cells, while recombinant OX40L cross-linked with anti-Fc led to robust activation of the reporter gene. FIG. 12B Notably, the engineered OX40L exogenous organism induced a higher level of reporter gene expression compared to anti-OX40 antibodies or recombinant OX40L. FIG. 12C Importantly, the engineered exogenous organism does not require cross-linking antibodies, indicating that OX40L on the surface of the exogenous organism can form a functional OX40L trimer sufficient to activate OX40.

[0359] Example 7: IL-7 engineered exosomes activate T cells

[0360] IL-7 is a cytokine involved in the proliferation of B cells and T cells and has implications for immunotherapy. Specifically, IL-7 may activate T cells and induce tumor antigen responses in tumors where leukocytes have difficulty infiltrating or in tumor microenvironments that induce T cell anergy. IL-7 induces interferon-γ (IFNγ) signaling via signaling of the heterodimeric IL-7 receptor, which can enhance T cell tumor-specific antigen responses. To determine whether exogenous bodies could be engineered to induce T cell activation, pDisplay, which encodes a fusion of IL-7 and PDGF receptors, was used. TM ThermoFisher plasmid was transfected into HEK293SF cells to generate IL-7-containing exogenous antibodies. The engineered exogenous antibodies were purified as described in the methods section. To verify the activity of the IL-7 exogenous antibodies, human PBMCs were seeded in 96-well plates and incubated with purified IL-7 exogenous antibodies and anti-CD3 antibody, native exogenous antibodies and anti-CD3 antibody, anti-CD3 antibody alone, or a combination of anti-CD28 and anti-CD3 antibody. Samples were incubated at 37°C for two days, and IFNγ (IFNγ) was measured. FIG. 13A and 13BCompared to anti-CD3 alone, the combination of IL-7 exogenous antibody and anti-CD3 antibody induced a greater degree of peak IFNγ production. FIG. 13B Furthermore, the IL-7 exogenous variant induced IFNγ in a dose-dependent manner, to a degree comparable to the positive controls of CD3 and CD28 antibodies. Conversely, the native exogenous variant had no effect on IFNγ. FIG. 14A ).

[0361] The IL-7 receptor is a heterodimeric complex composed of IL-7R and IL-2RG. In the presence of IL-7, it forms a ternary complex and induces downstream signaling via the JAK / STAT pathway, leading to cell proliferation. Assays based on synthetic cells are used to measure IL-7 signaling through the IL-7 receptor to assess the functional activity of engineered IL-7 exogenous organisms (DiscoverX Corporation). FIG. 14B Recombinant human IL-7 (rhIL-7) is sufficient to increase signal transduction via the IL-7 receptor. FIG. 14C Engineered IL-7 exogenes can induce signal transduction through IL-7 receptors, while natural exogenes cannot. FIG. 15A These data demonstrate that exogenous IL-7 expression is sufficient to induce signal transduction via the IL-7 receptor in vitro.

[0362] To determine whether the effects of the IL-7 exogenous variant observed in vitro could be reproduced in an in vivo model, the IL-7 exogenous variant was administered to C57BL / 6 mice. A cohort of 20 mice was divided into the following groups: (1) PBS, (2) recombinant human IL-7 (rhIL-7), (3) engineered IL-7 exogenous variant, and (4) unmodified natural exogenous variant. Five mice in each group received an intraperitoneal (IP) injection of 1 mg EdU and PBS once daily, 1 x 10⁻⁶ mg / mL. 11 Administer one natural or exogenous IL-7, or 10 μg rhIL-7, for three consecutive days. Mice were sacrificed, spleens were isolated, and EdU levels in spleen cells were measured by flow cytometry. FIG. 15B As shown, the percentage of positive CD8+ T cells was significantly increased in IL-7 exogenous mice and rhIL-7 mice compared to the control cohort. Although the T cell count in IL-7 exogenous mice was lower than in the rhIL-7 cohort, it was estimated that the amount of IL-7 molecules administered in the IL-7 exogenous cohort was reduced fivefold (data not shown). A similar trend in memory CD8+ T cells was observed by measuring the level of the memory marker CD45RO. FIG. 15A ).

[0363] As an orthogonal method, the level of CD71 (transferrin receptor) was measured in spleen cells isolated from exogenously treated mice. CD71 is essential for proliferation, and its level is correlated with the number of T cells. FIG. 16A and 15B As shown, the number of CD8+ T cells and memory CD-8+ T cells follows the same pattern as... Example 8: Fusion of IL-7 to proprietary scaffolds enhances specific activity and 16B The same trend observed in [the study]. In summary, these data suggest that engineered exogenous agents can induce specific immune cell effects in vivo, and that this activation may be more effective on a per-molecule basis compared to recombinant agonists.

[0364] FIG. 17A

[0365] To enhance the activity of engineered IL-7 exogenes, the IL-7 sequence was fused with a truncated portion of PTGFRN, a novel transmembrane protein highly expressed on the surface of the HEK293SF exogene. IL-7 was expressed as a translational fusion upstream of the PTGFRN truncated fragment, comprising the region preceding the C-terminal IgV domain, transmembrane domain, and intracellular domain of PTGFRN, as well as the FLAG tag. A series of expression constructs were generated by introducing a series of four-amino acid deletions between IL-7 and PTGFRN. FIG. 17B The resulting constructs are numbered pX-1 to pX-4. FIG. 18A The complete pX-4 sequence is shown. As indicated by Western blot analysis using an anti-IL-7 antibody, constructs pX-3 and pX-4 showed the highest expression levels. IL-7 expression levels in the PTGFRN backbone were significantly higher than those of pDisplay-IL-7 used in Example 7. FIG. 18B Increased IL-7 expression suggests that these novel fusion proteins can induce higher levels of IL-7-mediated T cell activation. To determine the potency of the PTGFRN-IL-7 fusion, an in vitro model of T cell activation was established. Following IL-7-mediated T cell activation, IL-7 receptor (IL-7R) levels decreased in a dose-dependent manner over 24 hours (Ghawazi et al., Immunol CellBiol. 2013 Feb; 91(2):149-58). Therefore, IL-7R levels were monitored after PBMCs were incubated with various IL-7-engineered exogenous proteins. Example 9: Exosomes engineered with anti-CD3 antibody fragmentsAs shown, the natural exogenous compound failed to reduce IL-7R levels, while the pDisplay-IL-7 exogenous compound (IL-7-pD) only reduced IL-7R levels at high doses. Conversely, the PTGFRN-IL-7 exogenous compounds (IL-7-pX3 to pX4) completely reduced IL-7R levels at much lower doses, indicating enhanced potency of these engineered exogenous compounds. As a measure of IC50, the potency of the PTGFRN-IL-7 exogenous compound was 20 to 76 times higher than that of the IL-7-pD exogenous compound (Table 2), suggesting that the increased ligand density was sufficient to increase biopotency. Furthermore, these results suggest that specifically truncated PTGFRN may be an ideal scaffold for engineered therapeutic exogenous compounds.

[0366] Table 8

[0367]

[0368] FIG. 19A

[0369] As shown in the previous examples, exogenous bodies can be engineered to overexpress functional endogenous sequences of immunomodulatory proteins. To determine whether synthetic agonists can be engineered on the surface of exogenous bodies, 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 engineered to express anti-CD3 single-chain Fv (scFv) (… FIG. 20A and 19B ) or single-chain Fab (scFab) ( FIG. 19A and 20B The foreign organisms were incubated overnight together. As a positive control, PBMCs were incubated with ImmunoCult according to the manufacturer's protocol. TM CD3 / CD28 activators (Stem Cell Technologies) were incubated together. In the presence of anti-CD28 co-stimulation, all engineered exogenous-induced T cells ( FIG. 19B and 20A ) and B cells ( FIG. 21A and 20B The activation of the anti-CD3 exogenous antibody was comparable to that of the positive control, while it was absent in the unengineered exogenous control. To measure the effect of the anti-CD3 exogenous antibody on the immune cell population, the CD69 positivity of T cells and B cells was determined by flow cytometry. FIG. 21B As shown, PBMCs incubated with exogenous anti-CD3 scFv expressing a fusion with the CD80 transmembrane domain resulted in approximately 40% T cell activation. A similar effect was observed for B cell activation. FIG. 22A ).

[0370] To determine whether anti-CD3 exosome-mediated T cell activation was due to direct T cell activation or mediated by trans-acting immune cells, the activation of purified T cells was measured. 100,000 purified human T cells were seeded in 96-well arrays in wells pre-coated with non-targeting antibodies or anti-CD3 exosomes in the presence or absence of anti-CD28 antibodies, or incubated with soluble anti-CD3 exosomes in the presence or absence of anti-CD28 antibodies. FIG. 22B As shown, CD69 expression measurements revealed that both soluble and plate-coated anti-CD3 scFv exogens activated T cells in the presence of anti-CD28 antibody. Example 10: IL-12-PTGFRN exosomes have potent immunomodulatory activity in vitro and in vivo. As shown, in the presence of anti-CD28 antibody, the plate-coated anti-CD3 antibody activated T cells to the same extent as the plate-coated anti-CD3scFv in the presence of anti-CD28 antibody. Surprisingly, although soluble anti-CD3 antibody was sufficient to activate approximately 30% of T cells in the presence of anti-CD28 antibody, soluble anti-CD3scFv exogenes activated a significantly higher proportion of T cells in the presence of anti-CD28 antibody. This suggests that exogenes engineered to overexpress antibody fragments can induce higher levels of T cell activation compared to soluble antibodies. In summary, these results indicate that exogenes can be engineered to overexpress antibody fragments with functional activity against specific cell types.

[0371] FIG. 23A

[0372] IL-12 is a potent immunostimulatory cytokine produced by antigen-presenting cells in response to infection and other antigenic stimuli. IL-12 production by activated dendritic cells, macrophages, and neutrophils induces IFNγ production by CD8+ and CD4+ T cells and induces cytotoxicity in 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, myeloid-derived suppressor cell (MDSC) reprogramming, and anti-angiogenic effects. IL-12-mediated antitumor effects have generated durable T-cell responses and antitumor immunity in many animal models. IL-12 has previously been tested as an immunotherapeutic agent in humans, but despite the detectable robust IFNγ response induction, it has exhibited significant toxicity in patients with renal cell carcinoma (Leonard et al., Blood. 1 Oct 1, 1997; 90(7):2541-8). Therefore, due to the high local concentration of cytokines and the hypothetical pharmacological effect of tumor retention, exogenous bodies may represent an ideal delivery route for IL-12.

[0373] IL-12 consists of two domains, p35 and p40. The human IL-12 dimer is encoded by a domain similar to the full-length PTGFRN (…).FIG. 23B Construct 871, SEQ ID NO:3) or a shortened fragment of PTGFRN capable of achieving high-density surface display ( FIG. 24A A fusion protein (construction 873, SEQ ID NO:5) was prepared, and the construct was stably expressed in HEK293SF cells. Stable cell lines were grown in a chemically defined medium and expressed via Optiprep according to the method described. TM Exogens were purified from culture supernatant using a gradient purification process. The amount of IL-12 protein on the surface of the exogens was measured by ELISA and matched to rIL-12 concentration in all functional studies. Purified full-length and short hIL-12-PTGFRN exogens or recombinant hIL-12 (rhIL-12; BioLegend, catalog number 573004) were titrated in human PBMCs in the presence of suboptimal concentrations of anti-CD3 antibody to induce IFNγ expression. rhIL-12 resulted in robust IFNγ expression, EC50. 50 The concentration was 0.029 ng / ml, which is comparable to full-length IL12-PTGFRN, and both are approximately 10 times more potent than IL12-short-PTGFRN. FIG. 25-27 These results suggest that IL-12 displayed on a full-length PTGFRN scaffold may be a more effective immunomodulator than that of a short PTGFRN construct.

[0374] Mouse and human IL-12 proteins do not cross-react, and the data shown in Figure 24 in vitro indicate that mIL-12 fused with full-length PTGFRN is more effective than the short scaffold using PTGFRN. To determine the efficacy of the mIL-12-PTGFRN exogenous body in an in vivo cancer model, 1x10⁻¹ mIL-12 exogenous proteins were subcutaneously implanted in C57BL / 6 mice. 6 B16F10 mouse melanoma cells (n=5 mice per group). On days 5, 6, and 7 post-tumor inoculation, the tumors were injected with PBS, 0.2 μg of recombinant mouse IL-12 (mIL12; BioLegend, catalog number 577004), or 1 x 10⁻⁶ cells. 11 A full-length exogenous IL-12-PTGFRN was displayed (mIL12-exogenous; SEQ ID NO:4). Once the tumor volume reached 2,000 mmHg... 3 To kill animals. For example... FIG. 28As shown, tumors in the PBS group grew rapidly, while tumors in the rmIL12 and mIL12-Exo groups were significantly reduced (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 superior efficacy of IL-12 compared to soluble cytokines when displayed on exogenous surfaces. The IL-12-treated groups also showed a survival advantage compared to the PBS-treated groups. FIG. 29A ).

[0375] To understand the mechanistic advantage of the IL-12-PTGFRN exogenous form over rmIL12, Th1 gene expression was analyzed in tumors of both the control and treatment groups. Compared to the control group, IFNγ (…) was significantly higher in the IL-12-treated group. FIG. 29B ), T cell chemokine CXCL9 ( FIG. 29C ) and CXCL10 ( FIG. 29D ) and TGFβ ( FIG. 30 All of these increased. In most cases, cytokine signaling was higher in animals treated with mIL12-Exo compared to rmIL-12. IFNγ levels in splenic CD8+ T cells measured by flow cytometry showed significantly higher signaling in mice treated with Exo-mIL-12 compared to the PBS or rmIL-12 groups. Example 11: Exosomes displaying interferon gamma are potent immune cell activators In summary, these data demonstrate that IL-12 exhibited on the surface of exogenous tissue represents a novel and effective immunomodulatory strategy that promotes robust T cell activation in vitro and can be used to elicit potent antitumor effects in vivo in an aggressive mouse model of melanoma. Mechanistically, the IL-12 exogenous tissue exhibits superiority over rIL-12, thus representing a novel and differentiated approach to cancer immunotherapy.

[0376] FIG. 31A

[0377] Interferon-γ (IFNγ) is a cytokine involved in triggering innate and adaptive immune responses. It is expressed in response to a variety of signals (including IL-12) from multiple cell types 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. Exogenous IFNγ-expressing cells are produced by stable transfection of HEK293SF cells with full-length PTGFRN fused with either monomeric or dimeric human or mouse IFNγ (respectively...). FIG. 32 and 31BThe exogenous organisms from suspension cell cultures were purified as described above and analyzed by PAGE. Monomers (m) and tandem dimers (td) of the PTGFRN IFNγ exogenous organisms were expressed at comparable levels to the predicted molecular weights (arrows). Construct The purified exogens were analyzed by ELISA and compared with a standard curve using recombinant IFNγ (Biolegend, catalog number 570206) to calculate the number of IFNγ molecules for each exogen. The results in Table 9 show the number of IFNγ molecules for each of the four types of purified exogens. Notably, the tandem dimer IFNγPTGFRN exogen contained at least twice as many IFNγ molecules as the monomeric IFNγPTGFRN exogen, indicating that the tandem dimer exogen appropriately expressed the dimer IFNγ construct.

[0378] Table 9

[0379] IFN gamma molecule / exosome h-mIFN gamma-PTGFRN h-tdIFN gamma-PTGFRN 53 m-mIFN gamma-PTGFRN 173 m-tdIFN gamma-PTGFRN 47 FIG. 33 113

[0380] Human monomers and tandem dimers of PTGFRN-IFNγ exogenous proteins were incubated with human PBMCs at increasing concentrations for 24 hours. Monocyte activation was measured by PD-L1 expression, which is a downstream surface protein induced by IFNγ signaling. FIG. 33 As shown, the natural HEK293SF exogenous organism (WT) failed to induce PD-L1 expression, while both the monomeric and tandem dimer IFNγPTGFRN exogenous organisms induced PD-L1 in a dose-dependent manner, with the tandem dimer IFNγPTGFRN exogenous organism exhibiting greater activation. Exogenous organism-mediated PD-L1 activation was comparable to LPS-induced activation. Example 12: Exosomes expressing IL-15 induce NK cell activation These data demonstrate that soluble cytokines, in monomeric or dimeric form, can be functionally expressed on the surface of exogenous bodies and induce immune cell activation. The use of exogenous bodies expressing IFNγ in immuno-oncology could be to induce NK and T cell responses against tumor cells.

[0381] FIG. 34

[0382] 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 immunomodulatory therapies. Exogenous IL-15-expressing proteins are produced by stable transfection of HEK293SF cells with an expression plasmid encoding the transmembrane domain of PDGFR (pDisplay) fused with IL-β. The IL-15 / IL-15Rα fusion protein (…) FIG. 35As described above, the foreign body is processed via Optiprep. TM Density gradient ultracentrifugation purification was performed. The purified exogenous cells were incubated with human PBMCs for 24 hours, and NK cell activation was measured by flow cytometry as the percentage of CD69 positivity. pDisplay IL-15 exogenous cells were not found at a maximum concentration of 10-1 per cell in the PBMC culture. 5 A dose of exogenous organisms induces NK cell activation ( FIG. 34 The number of foreign constructs is as follows: FIG. 36A (As shown). To investigate whether higher density IL-15 display is needed to induce NK cell activation, HEK293SF cells were stably transfected with an expression plasmid encoding IL-15 fused with full-length PTGFRN. In addition, HEK293SF cells were stably transfected with an expression plasmid encoding a more potent IL-15 fused with full-length PTGFRN (IL-15N72D, e.g. J Immunol. 2009 Sep 15; 183(6):3598-607; ​ The expression of (as described above) was confirmed by anti-PTGFRN protein 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). The IL-15PTGFGN exogenous form was 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, measured by the percentage of CD69-positive NK cells. Furthermore, all constructs were comparable between the two donors, demonstrating meaningful comparative power. Figure 37 (The number of exogenous constructs is shown in Figure 36). These data indicate that IL-15 can be effectively and powerfully displayed on exogenous surfaces, but this requires high expression levels, such as those conferred by PTGFRN.

[0383] Example 13: Displaying exogenous anti-CD-3 antibody fragments on PTGFRN scaffolds to activate T cells

[0384] The results in Example 9 demonstrate that exogenous bodies displaying anti-CD3 antibody fragments can activate T cells. To determine whether the PTGFRN scaffold supports this activity, the anti-CD3 antibody fragment (OKT3 variant) was fused with 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 Exogenous binding is the use of RED96 (Pall) was confirmed using biological layer interferometry (BLI). The CD3 fragment was bound to the BLI probe (…). Figure 39 ii), washing ( Figure 39 iii), and add foreign body constructs ( Figure 39 (iv) Exogenous constructs from WTHEK293SF 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 display of foreign variants was tested. In vitro life Sex. T cell activation is measured by CD69 positivity on CD4+ T cells, such as by flow cytometry. Compared to the unmodified natural exosome, the anti-CD3 exosome fused with the PTGFRN fragment (exoCD3-short) is more effective in stimulating CD3 in vitro. Live CD4+ T cells (Fig. 40).

[0385] Example 14: Demonstrating that exogenous CD40L is an effective activator of B cells.

[0386] CD40 ligand (CD40L) is a ligand of the tumor necrosis superfamily (TNFSF) that binds to the costimulatory receptor CD40, which is highly expressed on B cells and other antigen-presenting cells. TNFSF ligand-mediated cell activation requires the formation of a trimer ligand complex, which forms on the cell surface and binds to its homologous receptor. To investigate whether exogenous CD40L exhibiting different conformations on its surface is 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 of the transmembrane domain of PDGFR, the 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 forced trimers (pCB-607 and pCB-527). Figure 41 (Bottom). To promote trimerization of monomer CD40L, constructs expressing fusions with multimerizing domains from TRAF2 (pCB-521 to pCB-523) or collagen XV (pCB-524 to pCB-526) were designed. In the monomer CD40L constructs, pCB-518 / 521 / 524 contain the full-length N-terminal stem sequence from endogenous CD40L; pCB-519 / 522 / 525 contain truncated N-terminal stem sequences from endogenous CD40L; and pCB-520 / 523 / 526 contain only the soluble portion of CD40L. Each engineered exogenous population was coupled with RosetteSep using... TMThe human B cell enrichment cocktail (Stemcell Technologies #15064) was used to incubate purified B cells isolated from human peripheral blood, and B cell activation was measured by detecting CD positivity of B cells using flow cytometry. ECs from each construct were then incubated together. 50 Calculate as a function of particle concentration in cell culture and plot on Figure 41 In the chart above, it's interesting to note that all the monomeric CD40L constructs exhibit moderate potency, while the trimer constructs are at least ten times more potent than the monomers. Figure 41 (See figure above). These results indicate that monomeric CD40L is a weak activator of B cells when present on the surface of exogenous organisms, but forced trimeric CD40L can induce robust B cell activation. Furthermore, PTGFRN has been shown to form dimer structures (PCT / US2018 / 048026), suggesting that higher-order multimeric structures may form on the surface of exogenous organisms to further promote target binding and immune cell activation.

[0387] Figure 41 All results shown used exogens containing luminal GFP fused to the C-terminus of PTGFRN. To generate tag-free CD40L exogens, a trimeric CD40L-PTGFRN construct identical to the leader construct pCB-527 but lacking C-terminal GFP was stably expressed in HEK293SF cells (pCB-766). The absolute concentration of CD40L on the surface of the engineered exogens was quantified using an ELISA (R&D Systems, catalog number DCDL40), as shown in Table 10 below.

[0388] Table 10

[0389] EC50 pCB-0766 pCB-0527 rhCD40L Particles / mL 6.63E+08 4.53E+08 N / A ng / mL 1.68 1.89 28.51

[0390] Purified CD40L-PTGFRN exogenous bodies were tested in B cell activation assays, as described above, compared to concentration-matched recombinant human CD40L (Biolegend, catalog number 591702). Both GFP-containing and tagless CD40L exogenous bodies were comparable B cell activators when measured as a function of particle number or CD40L concentration. Figure 42A Both exogenous formulations were more effective than concentration-matched CD40L. Figure 42B The native, unengineered exogenous material from HEK293SF cells failed to activate B cells, suggesting that the engineered CD40L trimer construct on the surface of the exogenous material was sufficient to effectively activate B cells.

[0391] Another way to stimulate CD40 and activate B cells is by using stimulating antibodies that are cross-linked with a secondary antibody. To compare the potency of exogenous antibodies expressing trimerized CD40L with those expressing stimulating CD40L, PBMC cultures were mixed with 2 μg / ml of an anti-CD40L antibody with a secondary cross-linked antibody (Jackson Immuno Research, catalog number 115-006-071). Clone 5C3) and incubate together. Figure 43A and 43B In the diagram, the dashed line indicates maximum B cell activation. This is observed in two independent donor PBMC libraries ( Figure 43A and 43B In this study, the pCB-527 exogenous antibody (PTGFRN-trimeric CD40L-GFP) induced greater maximal B cell activation than the cross-linked agonist antibody. Figure 43A and 43B This indicates that the trimeric CD40L exogenous body has superiority in activating immune cells.

[0392] Example 15: Simultaneous display of multiple immunotumor molecules on a single exogenous organism

[0393] Previous examples have demonstrated that a single immunomodulatory protein can be displayed on the surface of an exogenous body and induce functional alterations in one or more immune cell types. In some applications, it may be necessary to use combinatorially engineered exogenous bodies, i.e., exogenous bodies containing more than one molecule on their surface, each capable of transducing different immune cell pathways. HEK293SF cells were stably transfected with a plasmid expressing the PTGFRN-IL-12 and PTGFRN-CD40L fusion protein. The exogenous bodies were isolated and purified as described above. Exogenous bodies from unmodified HEK293SF cells were used as a negative control.

[0394] To demonstrate the simultaneous loading of different ligands, a pull-down co-staining assay was developed:

[0395] Reagents:

[0396] Dynabeads (Thermofisher Exogenous Streptavidin Separation / Detection Reagent, Catalog No. 10608D): 1x10 7 Beads / mL, 50% slurry

[0397] Separation buffer: 0.5% BSA / PBS (1:4 2% BSA)

[0398] Blocking buffer: 2% BSA / PBS (1gr / 50mL, filtered)

[0399] ● Wash 0.5 ml of beads with 0.5 ml of separation buffer and resuspend in 0.5 ml of separation buffer.

[0400] ●Add 1μg of biotinylated capture antibody (2.2ul 0.5ug / ul stock solution)

[0401] ● Rotate for 1 hour at room temperature

[0402] ●Wash with 500 μl of separation buffer

[0403] ● Resuspend in 500 μl of blocking buffer and rotate at room temperature for 10 minutes.

[0404] ●Incubate in 500 μl of separation buffer (1 x 10) 7 (beads / mL, 50% slurry)

[0405] ● Store under 4C conditions

[0406] A. Exogenous capture and flow

[0407] 1x10 5 Beads / sample (10 μl beads, 20 μl slurry)

[0408] 50,000 foreign objects / beads; 5x10 9 Foreign body / sample (1.2x10) 9 (Exogenous body / μL stock solution)

[0409] 5 μl of each fluorescently labeled detection antibody was flow-dried.

[0410] Mixed 5x10 9 Exogenous material + 20 μl Dynabeads slurry + 0.7 ml 0.1% BSA / PBS

[0411] program:

[0412] 1. Take 120 μl of slurry beads, remove the supernatant, add 0.7 ml of blocking buffer, mix, rotate at room temperature for 10 minutes, and remove the supernatant.

[0413] 2. Suspend the beads in 0.7 ml separation buffer + 25.2 μl of exogenous material and rotate at 4°C.

[0414] 3. Day 2: Quickly rotate the foreign object and the bead for 5 seconds.

[0415] 4. Place the tube on a magnet to remove the supernatant.

[0416] 5. Seal in 700 μl and rotate at room temperature for 10 minutes.

[0417] 6. Place the tube on a magnet to remove the supernatant.

[0418] 7. Resuspend in 600 μl of separation buffer: 6 x 100 μl per tube

[0419] 8. Add 1 μl of labeled detection antibody, mix, and incubate in the dark at 4°C for 30 minutes.

[0420] 9. Rotate at 500g for 2 minutes to remove the supernatant.

[0421] 10. Wash with 2x separation buffer

[0422] 11. Resuspend in 200 μl of separation buffer and run the flow.

[0423] Beads decorated with anti-CD40L and targeting IL-12 and CD40L ( Figure 44A ) or CD81 (an exogenous marker present on natural and engineered exogens) and CD40L ( Figure 44B Natural exogenous organisms were separated using fluorescent antibodies labeled with PTGFRN. Since no fluorescent signals of IL-12, CD40L, or CD81 were detected, CD40L beads did not pull down any natural exogenous organisms. Instead, exogenous organisms doubly engineered with PTGFRN-CD40L / IL-12 were incubated with anti-CD40L beads and separated as described above. All engineered exogenous organisms (greater than 97% of the counted beads) showed positive results for CD81. Figure 45A ), IL-12 or CD40L ( Figure 45B The staining of CD40L-mediated separation showed that IL-12 exogens could also be separated by CD40L-mediated separation. Similarly, anti-IL-12-decorated beads were incubated with IL-12 / CD40L-engineered exogens and stained for IL-12, CD40L, and CD81. More than 98% of all beads were positive for CD40L and either IL-12 or CD81. Figure 46A and 46B This indicates that the surface of the foreign body contains both IL-12 and CD40L.

[0424] The concentrations of IL-12 and CD40L were quantified by ELISA (Abcam catalog number ab119517) to test the in vitro potency of engineered exogens. Equal concentrations of recombinant IL-12, a mixture of recombinant IL-12 and recombinant CD40L, PTGFRN-IL-12 exogens, double-positive PTGFRN-CD40L / IL-12 exogens, or a mixture of PTGFRN-IL-12 and PTGFRN-CD40L exogens were added to human PBMCs (rhIL-12-BioLegend, catalog number 573004; rhCD40L-Biolegend, catalog number 591702) at increased concentrations. Cells were co-stimulated with anti-CD3 antibody, and IFNγ production was measured by (PerkinElmer, catalog number AL217C). Figure 47A and47B As shown, all exogenous formulations containing IL-12 elicited IFNγ responses comparable to those of recombinant cytokines. EC50 calculations under various conditions indicated that exogenous-associated IL-12 was more effective than concentration-matched IL-12, whether expressed alone or in combination on the exogenous surface. Figure 48 Under B cell activation, recombinant CD40L and single or double engineered CD40L exogenous bodies yielded similar results. Figure 49A And B). Similarly, CD40L-engineered exogenous compounds are more effective than soluble recombinant cytokines, and in this case, dual-engineered exogenous compounds are the most effective constructs tested in the assay. Figure 50 ).

[0425] To further explore the possibility of displaying exogenous proteins on combinatorial surfaces, HEK293SF cells were stably transfected with three separate constructs expressing PTGFRN-IL-12, PTGFRN-CD40L, or PTGFRN-FLT3L fusion proteins. Exogenous proteins were purified and isolated using the affinity bead method described above, but the presence of surface FLT3L was also queried using an anti-FLT3L-PE conjugated antibody. Exogenous proteins isolated using anti-IL-12 beads were incompatible with IL-12 and CD40L (…). Figure 51A ), IL-12 and FLT3L ( Figure 51B ), and CD40L and FLT3L ( Figure 51C The result was double positive. The exogenous antibody isolated using anti-CD40L beads tested positive for both IL-12 and CD40L. Figure 52A ), IL-12 and FLT3L ( Figure 52B ), and CD40L and FLT3L ( Figure 52C The presence of two positive results confirmed that each exogenous body expressed three immunomodulatory ligands. These results suggest that multiengineered immunomodulatory exogenous bodies are a viable therapeutic approach, and that they are comparable to or more effective than soluble cytokines in activating immune cells.

[0426] ***

[0427] Although the foregoing invention has been described in detail by way of illustration and examples for purposes of clarity, it will be apparent to those skilled in the art, based on the teachings of this disclosure, that certain changes and variations may be made therein without departing from the spirit or scope of the appended claims.

[0428] Therefore, the foregoing has merely illustrated the principles of the 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 invention and are included within its spirit and scope. Furthermore, all embodiments and conditional language described herein are primarily intended to aid the reader in understanding the principles of the invention without constituting a limitation on such specifically described embodiments and conditions. Moreover, all statements herein describing the principles, aspects, and embodiments of the invention, as well as specific embodiments thereof, are intended to cover their structural and functional equivalents. Furthermore, it is intended that such equivalents include currently known equivalents and those developed in the future, i.e., any element developed that performs the same function, regardless of its structure. Therefore, the scope of the invention is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of the invention are embodied in the appended claims.

[0429] sequence list

[0430] >SEQ ID NO:1

[0431]

[0432] >SEQ ID NO:2

[0433]

[0434] >hIL-12-PTGFRN;871(SEQ ID NO:3)

[0435]

[0436] >mIL-12-PTGFRN;872(SEQ ID NO:4)

[0437]

[0438] hIL-12-short PTGFRN; 873 (SEQ ID NO:5)

[0439]

[0440] mIL-12-short PTGFRN; 874 (SEQ ID NO:6)

[0441]

[0442] SEQ ID NO:7PTGFRN_IFN_γ monomer

[0443] MGRLASRPLLLALLSLALCRGQDPYVKEAENLKKYFNAGHSDVADNGTLFLGILKNWKEESDRKIMQSQIVSFYFKLFKNFKDDQSIQKSVETIKEDMNVKFFNSNKKKRDDFEKLTNYSVTDLNVQRKAIHELIQVMAELSPAAKTGSAGGGGSGGGGSRVVRVPTATLVRVVGTELVIPCNVSDYDGPSEQNFDWSFSSLGSSFVELASTWEVGFPAQLYQERLQRGEILLRRTANDAVELHIKNVQPSDQGHYKCSTPSTDATVQGNYEDTVQVKVLADSLHVGPSARPPPSLSLREGEPFELRCTAASASPLHTHLALLWEVHRGPARRSVLALTHEGRFHPGLGYEQRYHSGDVRLDTVGSDAYRLSVSRALSADQGSYRCIVSEWIAEQGNWQEIQEKAVEVATVVIQPSVLRAAVPKNVSVAEGKELDLTCNITTDRADDVRPEVTWSFSRMPDSTLPGSRVLARLDRDSLVHSSPHVALSHVDARSYHLLVRDVSKENSGYYYCHVSLWAPGHNRSWHKVAEAVSSPAGVGVTWLEPDYQVYLNASKVPGFADDPTELACRVVDTKSGEANVRFTVSWYYRMNRRSDNVVTSELLAVMDGDWTLKYGERSKQRAQDGDFIFSKEHTDTFNFRIQRTTEEDRGNYYCVVSAWTKQRNNSWVKSKDVFSKPVNIFWALEDSVLVVKARQPKPFFAAGNTFEMTCKVSSKNIKSPRYSVLIMAEKPVGDLSSPNETKYIISLDQDSVVKLENWTDASRVDGVVLEKVQEDEFRYRMYQTQVSDAGLYRCMVTAWSPVRGSLWREAATSLSNPIEIDFQTSGPIFNASVHSDTPSVIRGDLIKLFCIITVEGAALDPDDMAFDVSWFAVHSFGLDKAPVLLSSLDRKGIVTTSRRDWKSDLSLERVSVLEFLLQVHGSEDQDFGNYYCSVTPWVKSPTGSWQKEAEIHSKPVFITVKMDVLNAFKYPLLIGVGLSTVIGLLSCLIGYCSSHWCCKKEVQETRRERRRLMSMEMD

[0444] SEQ ID NO:8 PTGFRN_IFN_γ dimer

[0445]

[0446] MGRLASRPLLLALLSLALCRGRHGTVIESLESLNNYFNSSGIDVEEKSLFLDIWRNWQKDGDMKILQSQIISFYLRLFEVLKDNQAISNNISVIESHLITTFFSNSKAKKDAFMSIAKFEVNNPQVQRQAFNELIRVVHQLLPESSLRSAGGGGSGGGGSRVVRVPTATLVRVVGTELVIPCNVSDYDGPSEQNFDWSFSSLGSSFVELASTWEVGFPAQLYQERLQRGEILLRRTANDAVELHIKNVQPSDQGHYKCSTPSTDATVQGNYEDTVQVKVLADSLHVGPSARPPPSLSLREGEPFELRCTAASASPLHTHLALLWEVHRGPARRSVLALTHEGRFHPGLGYEQRYHSGDVRLDTVGSDAYRLSVSRALSADQGSYRCIVSEWIAEQGNWQEIQEKAVEVATVVIQPSVLRAAVPKNVSVAEGKELDLTCNITTDRADDVRPEVTWSFSRMPDSTLPGSRVLARLDRDSLVHSSPHVALSHVDARSYHLLVRDVSKENSGYYYCHVSLWAPGHNRSWHKVAEAVSSPAGVGVTWLEPDYQVYLNASKVPGFADDPTELACRVVDTKSGEANVRFTVSWYYRMNRRSDNVVTSELLAVMDGDWTLKYGERSKQRAQDGDFIFSKEHTDTFNFRIQRTTEEDRGNYYCVVSAWTKQRNNSWVKSKDVFSKPVNIFWALEDSVLVVKARQPKPFFAAGNTFEMTCKVSSKNIKSPRYSVLIMAEKPVGDLSSPNETKYIISLDQDSVVKLENWTDASRVDGVVLEKVQEDEFRYRMYQTQVSDAGLYRCMVTAWSPVRGSLWREAATSLSNPIEIDFQTSGPIFNASVHSDTPSVIRGDLIKLFCIITVEGAALDPDDMAFDVSWFAVHSFGLDKAPVLLSSLDRKGIVTTSRRDWKSDLSLERVSVLEFLLQVHGSEDQDFGNYYCSVTPWVKSPTGSWQKEAEIHSKPVFITVKMDVLNAFKYPLLIGVGLSTVIGLLSCLIGYCSSHWCCKKEVQETRRERRRLMSMEMD

[0447] SEQ ID NO:10PTGFRN_IFN_γ mouse dimer

[0448] MGRLASRPLLLALLSLALCRGRHGTVIESLESLNNYFNSSGIDVEEKSLFLDIWRNWQKDGDMKILQSQIISFYLRLFEVLKDNQAISNNISVIESHLITTFFSNSKAKKDAFMSIAKFEVNNPQVQRQAFNELIRVVHQLLPESSLRGSGGSGGSGGSGHGTVIESLESLNNYFNSSGIDVEEKSLFLDIWRNWQKDGDMKILQSQIISFYLRLFEVLKDNQAISNNISVIESHLITTFFSNSKAKKDAFMSIAKFEVNNPQVQRQAFNELIRVVHQLLPESSLRSAGGGGSGGGGSRVVRVPTATLVRVVGTELVIPCNVSDYDGPSEQNFDWSFSSLGSSFVELASTWEVGFPAQLYQERLQRGEILLRRTANDAVELHIKNVQPSDQGHYKCSTPSTDATVQGNYEDTVQVKVLADSLHVGPSARPPPSLSLREGEPFELRCTAASASPLHTHLALLWEVHRGPARRSVLALTHEGRFHPGLGYEQRYHSGDVRLDTVGSDAYRLSVSRALSADQGSYRCIVSEWIAEQGNWQEIQEKAVEVATVVIQPSVLRAAVPKNVSVAEGKELDLTCNITTDRADDVRPEVTWSFSRMPDSTLPGSRVLARLDRDSLVHSSPHVALSHVDARSYHLLVRDVSKENSGYYYCHVSLWAPGHNRSWHKVAEAVSSPAGVGVTWLEPDYQVYLNASKVPGFADDPTELACRVVDTKSGEANVRFTVSWYYRMNRRSDNVVTSELLAVMDGDWTLKYGERSKQRAQDGDFIFSKEHTDTFNFRIQRTTEEDRGNYYCVVSAWTKQRNNSWVKSKDVFSKPVNIFWALEDSVLVVKARQPKPFFAAGNTFEMTCKVSSKNIKSPRYSVLIMAEKPVGDLSSPNETKYIISLDQDSVVKLENWTDASRVDGVVLEKVQEDEFRYRMYQTQVSDAGLYRCMVTAWSPVRGSLWREAATSLSNPIEIDFQTSGPIFNASVHSDTPSVIRGDLIKLFCIITVEGAALDPDDMAFDVSWFAVHSFGLDKAPVLLSSLDRKGIVTTSRRDWKSDLSLERVSVLEFLLQVHGSEDQDFGNYYCSVTPWVKSPTGSWQKEAEIHSKPVFITVKMDVLNAFKYPLLIGVGLSTVIGLLSCLIGYCSSHWCCKKEVQETRRERRRLMSMEMD

[0449] SEQ ID NO:11 IL-15 441

[0450] MAPRRARGCRTLGLPALLLLLLLRPPATRGITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTTGGSGGGSGGGGSGGGGSGGGSGGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSSADYKDDDDKFEGGGGSGGGGSAVGQDTQEVIVVPHSLPFKVVVISAILALVVLTIISLIILIMLWQKKPRSGLLTGRT

[0451] SEQ ID NO:12 IL-15 442

[0452] MAPRRARGCRTLGLPALLLLLLLRPPATRGHHHHHHITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKC IRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTTGGSGGGSGGGGSTLDPRSFLLRNPNDKYEPFWEDEEKNESGGGGSGGGSGGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSSADYKDDDDKFEGGGGSGGGGSAVGQDTQEVIVVPHSLPFKVVVISAILALVVLTIISLIILIMLWQKKPRSGLLTGRT

[0453] SEQ ID NO:13 IL-15 443

[0454] METDTLLLWVLLLWVPGSTGNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSGGSGGGSGGGGSGGGGSGGGSGGSITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTTSADYKDDDDKFEGGGGSGGGGSAVGQDTQEVIVVPHSLPFKVVVISAILALVVLTIISLIILIMLWQKKPRSGLLTGRT

[0455] SEQ ID NO:14 IL-15 444

[0456] METDTLLLWVLLLWVPGSTGNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSDYKDDDDKGGSGGGSGGGGSTLDPRSFLLRNPNDKYEPFWEDEEKNESGGGGSGGGSGGSITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTTSAFEGGGGSGGGGSAVGQDTQEVIVVPHSLPFKVVVISAILALVVLTIISLIILIMLWQKKPRSGLLTGRTHHHHHH

[0457] SEQ ID NO:15 IL-15 1009

[0458]

[0459] SEQ ID NO:16 IL-15 1010

[0460]

[0461] SEQ ID NO:17 pDisplay - anti - CD3

[0462] MKIICLALVALLLTAQPAMAEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL SKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGSSGSGSGSTGTSSSGTGTSAGTTGTSASTSGSGSGGGGGSGGGGSAGGTATAGASSGSQVQLVESGGGVVQPGRSLRLSCAASGFKFSGYGMHWVRQAPGKGLEWVAVIWYDGSKKYYVDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARQMGYWHFDLWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTGGSGGGSGGGGSGGGGSGGGSGGSAVGQDTQEVIVVPHSLPFKVVVISAILALVVLTIISLIILIMLWQKKPRDYKDDDDK

[0463] SEQ ID NO:18 PTGFRN - anti - CD3

[0464]

[0465] SEQ ID NO:19PTGFRN_CD40L trimeric mouse

[0466]

[0467] SEQ ID NO:20PTGFRN_CD40L Trimer Human

[0468] METDTLLLWVLLLWVPGSTGMQKGDQNPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNRREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKLGSGGSGGSGMQKGDQNPQIAAHVISEASSKTTSVLQWAEKGYYTMS NNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKLSAGGGGGSGGGGSRVVRVPTATLVRVVGTELVIPCNVSDYDGPSEQNFDWSFSSLGSSFVELASTWEVGFPAQLYQERL QRGEILLRRTANDAVELHIKNVQPSDQGHYKCSTPSTDATVQGNYEDTVQVKVLADSLHVGPSARPPPSLSREGEPFELRCTAASASPLHTHLALLWEVHRGPARRSVLALTHEGRFHPGLGYEQRYHSGDVRLDTVGSDAYRLSVSRALSADQGSYRCIVSEWIAEQGNWQEIQEKAVEVATVVIQPSVLRAAVPKNVSVAEGKELDLTCNITTDRADDVRPEVTWSFSRMPDSTLPGSRVLARLDRDSLVHSSPHVALSHVDARSYHLLVRDVSKENSGYYYCHVSLWAPGHNRSWHKVAEAVSSPAGVGVTWLEPDYQVYLNASKVPGFADDPTELACRVVDTKSGEANVRFTVSWYYRMNRRSDNVVTSELLAVMDGDWTLKYGERSKQRAQDGDFIFSKEHTDTFNFRIQRTTEEDRGNYYCVVSAWTKQRNNSWVKSKDVFSKPVNIFWALEDSVLVVKARQPKPFFAAGNTFEMTCKVSSKNIKSPRYSVLIMAEKPVGDLSSPNETKYIISLDQDSVVKLENWTDASRVDGVVLEKVQEDEFRYRMYQTQVSDAGLYRCMVTAWSPVRGSLWREAATSLSNPIEIDFQTSGPIFNASVHSDTPSVIRGDLIKLFCIITVEGAALDPDDMAFDVSWFAVHSFGLDKAPVLLSSLDRKGIVTTSRRDWKSDLSLERVSVLEFLLQVHGSEDQDFGNYYCSVTPWVKSPTGSWQKEAEIHSKPVFITVKMDVLNAFKYPLLIGVGLSTVIGLLSCLIGYCSSHWCCKKEVQETRRERRRLMSMEMDSEQ ID NO:21 PTGFRN_Short - anti - CD3

[0469] MKIICLALVALLLTAQPAMAEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL SKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGSSGSGSGSTGTSSSGTGTSAGTTGTSASTSGSGSGGGGGSGGGGSAGGTATAGASSGSQVQLVESGGGGVQPGRSLRLSCAASGFKFSGYGMHWVRQAPGKGLEWVAVIWYDGSKYYVDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYCARQMGYWHFDLWG RGTLVTSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTGGSGGGSGGGSGGGSGGGSGGGSGGSGPIFNASVHSDTPSVIRGDLIKLFCIITVEGAALDPDDMAFDVSWFAVHSFGLDKAPVLLSSLDRKGIVTTSRRDWKSDLSLERVSVLEFLLQVHGSEDQDFGNYYCSVTPWVKSPTGSWQKEAEIHSKPVFITVKMDVLNAFKYPLLIGVGLSTVIGLLSCLIGYCSSHWCCKKEVQETRRERRRLMSMEMDTGGSGGSVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTL TYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKDYKDDDDK

[0470] SEQ ID NO:22FLT3L-PTGFRN

[0471]

[0472]

[0473]

[0474]

[0475]

[0476]

[0477]

[0478]

[0479]

[0480]

[0481]

[0482]

[0483]

[0484]

[0485]

[0486]

[0487]

[0488]

[0489]

[0490]

[0491]

[0492]

[0493]

Claims

1. A composition comprising: Extracellular vesicles comprising a cell membrane defining a closed volume, the cell membrane having an inner surface and an outer surface; and A first immunomodulatory component that is associated with or enclosed within the enclosed volume of the cell membrane.

2. The composition of claim 1, wherein the first immunomodulatory component is an inhibitor of a negative checkpoint modulator or an inhibitor of a binding partner of a negative checkpoint modulator.

3. The composition of claim 2, wherein the negative checkpoint modulator 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 immune receptor with Ig and ITIM domains (TIGIT), T-cell activation V-domain Ig inhibitor (VISTA), adenosine A2a receptor (A2aR), cytotoxic cell immunoglobulin-like receptor (KIR), indoleamine 2,3-dioxygenase (IDO), CD20, CD39, and CD73.

4. The composition according to claim 1, wherein the first immunomodulatory component is an activator of a positive costimulatory molecule or an activator of a binding partner of a positive costimulatory molecule.

5. The composition according to claim 4, wherein the positive co-stimulatory molecule is a member of the TNF receptor superfamily.

6. The composition of claim 5, wherein 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.

7. The composition according to claim 4, wherein the activator of the positive co-stimulatory molecule is a member of the TNF superfamily.

8. The composition of claim 7, wherein 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.

9. The composition of claim 8, wherein the TNF superfamily member is CD40L.

10. The composition of claim 8, wherein the TNF superfamily member is CD27L.

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