Recombinant polypeptides for programming extracellular vesicles

Abstract

Provided herein are recombinant tumor-selective viral particles comprising a nucleic acid encoding a recombinant polypeptide for directing extracellular vesicles (EVs) to at least one target cell, the recombinant polypeptide comprising at least one targeting moiety for directing the EVs to the at least one target molecule expressed by the at least one target cell; at least one EV anchor polypeptide; and at least one intravesicular polypeptide. The viral particle may be derived from an oncolytic virus. Recombinant polypeptides for programming EVs to target specific molecules are also provided. Therapeutic EVs for delivering payload polypeptides (and / or cargo molecules) to target cells, e.g., in vaccine or cell-free "CAR-T"-like applications, are also described, along with EVs for recruiting immune cells to target cells in EV-mediated BiTE-like applications. Furthermore, oncolytic viruses may be modified to provide additional therapeutic benefits by infecting tumor cells and administering the programmed EVs.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the benefit of priority of U.S. Provisional Patent Application No. 62 / 941,768, entitled "Recombinant Polypeptides for Programming Extracellular Vesicles", filed on November 28, 2019, which is incorporated herein by reference in its entirety. All publications, patents, and patent applications mentioned in this specification, as well as the attached documents, are incorporated herein by reference to the same extent as if each individual publication, patent, or patent application were specifically and individually indicated to be incorporated by reference.

[0002] Field The present disclosure generally relates to the delivery of molecules into cells. More particularly, the present disclosure relates to the targeted delivery of molecules into cells.

Background Art

[0003] Background The targeted delivery of therapeutic molecules into living systems is an important component of disease treatment. However, the targeted delivery of molecules to specific cell types remains problematic due to instability, off - target effects before reaching the target cells, issues arising from the toxicity of the molecules under other circumstances, and other problems.

Summary of the Invention

Problems to be Solved by the Invention

[0004] Therefore, it is desirable to provide a stable targeted therapeutic molecule that treats only the target cells.

Means for Solving the Problems

[0005] Summary It is an object of the present disclosure to eliminate or reduce at least one disadvantage of the prior art methods.

[0006] In a first aspect, the disclosure provides tumor-selective recombinant viral particles comprising a nucleic acid encoding a recombinant polypeptide for directing an extracellular vesicle (EV) to at least one target cell, wherein the recombinant polypeptide comprises at least one targeting moiety for directing the EV to at least one target molecule expressed by the at least one target cell, at least one EV anchor polypeptide, and at least one intravesicular polypeptide.

[0007] In another embodiment, a recombinant polypeptide is provided for directing an extracellular vesicle (EV) to at least one target cell, comprising at least one targeting moiety for directing the EV to at least one target molecule expressed by the at least one target cell, at least one EV anchor polypeptide, and at least one intravesicular polypeptide.

[0008] In one embodiment, a nucleic acid molecule encoding a recombinant polypeptide as described herein is provided.

[0009] In one embodiment, a vector comprising nucleic acid as defined herein is provided.

[0010] In one embodiment, a recombinant viral genome comprising nucleic acids as defined herein is provided.

[0011] In one embodiment, a virus particle containing nucleic acid as defined herein is provided.

[0012] In one embodiment, a host cell is provided comprising a nucleic acid, vector, recombinant viral genome, or viral particle as defined herein.

[0013] In one embodiment, a targeted extracellular vesicle (EV) comprising a recombinant polypeptide as defined herein is provided.

[0014] In one embodiment, a composition is provided comprising nucleic acids as defined herein, vectors as defined herein, recombinant viral genomes as defined herein, viral particles as defined herein, or targeted EVs as defined herein, together with a pharmaceutically acceptable excipient, diluent, or carrier.

[0015] One embodiment provides a method for binding a targeting portion to a target molecule of a target cell, the method comprising bringing the target cell into contact with a targeted EV as defined herein.

[0016] In one embodiment, a method is provided for delivering a payload molecule to a target cell, the method comprising bringing the target cell into contact with a targeted EV as defined herein.

[0017] In one embodiment, a method is provided for delivering cargo molecules to target cells, the method comprising bringing the target cells into contact with targeted EVs as defined herein.

[0018] In one embodiment, a method is provided for stimulating an immune response to an antigen, comprising administering a nucleic acid as defined herein, a vector as defined herein, a recombinant viral genome as defined herein, a viral particle as defined herein, or a targeted EV as defined herein, wherein the target cell includes an immune cell and the at least one EV therapeutic payload polypeptide includes an antigen.

[0019] In one embodiment, a method is provided for killing target cells, comprising administering nucleic acids as defined herein, vectors as defined herein, recombinant viral genomes as defined herein, viral particles as defined herein, or targeted EVs as defined herein, wherein at least one EV therapeutic payload polypeptide comprises a cytotoxic molecule.

[0020] In one aspect, a method of reprogramming immune cells, the method comprising contacting the immune cells with a nucleic acid as defined herein, a vector as defined herein, a recombinant viral genome as defined herein, a viral particle as defined herein, or a targeted EV as defined herein, wherein at least one EV therapeutic payload molecule comprises an immunomodulatory molecule, is provided.

[0021] In one aspect, a method of inducing an immune response against target disease cells, the method comprising administering to a subject a nucleic acid as defined herein, a vector as defined herein, a recombinant viral genome as defined herein, a viral particle as defined herein, or a targeted EV as defined herein, wherein the recombinant polypeptide comprises at least two targeting moieties each specifically binding to at least two different target molecules, and each of the at least two different target molecules is expressed by immune cells and disease cells, is provided.

[0022] In one aspect, a method of preparing therapeutically targeted EVs in a subject, the method comprising contacting cells obtained from the subject with a nucleic acid as defined herein, a vector as defined herein, a recombinant viral genome as defined herein, or a viral particle as defined herein, and collecting the targeted EVs, is provided.

[0023] In one embodiment, a method of producing targeted EVs, the method comprising expressing a nucleic acid as defined herein intracellularly or producing EVs by culturing a host cell as defined herein, and collecting the EVs, is provided.

[0024] Other aspects and features of the present disclosure will become apparent to those skilled in the art upon review of the following description of specific embodiments in conjunction with the attached drawings.

[0025] Embodiments of the present disclosure will be described herein by way of example only, with reference to the attached drawings.

Brief Description of the Drawings

[0026] [Figure 1] Schematic diagrams of different constructs considered by the present disclosure are shown. [Figure 2] Schematic diagram (Panel A) of a polypeptide in which a PD1 targeting moiety, a LAMP2B transmembrane domain, and a HA tag are embedded within extracellular vesicles according to the invention, and immunoblot (Panel B) showing successful expression of the polypeptide shown in Panel A from a vaccinia virus platform in whole cell lysates and isolated extracellular vesicles. [Figure 3] An immunoblot showing that the form of the PD1 targeting moiety in the polypeptide shown in FIG. 2 is accurate (outward-directed). [Figure 4] In Panel A, a schematic diagram of a competitive binding ELISA experimental design performed to obtain the data in Panel B is shown, demonstrating that the PD1 portion in the polypeptide as shown in FIG. 2 binds well to its PDL-1 receptor. [Figure 5] In Panel A, a timeline in the data represented in Panel B is shown, demonstrating that the construct of FIG. 2 activates T cells well when expressed by a virus. [Figure 6] In Panel A, a schematic diagram of a bispecific tetraspanin-based construct embedded in EVs targeting two different cell types (one cancer, one immune killer cell) is shown, and in Panel B, a brightfield microscopy image showing enhanced cell death when tumor cells are transfected with the construct of Panel A and then combined with immune cells is shown. [Figure 7] A Western blot showing the expression of six different constructs according to the invention during cell transfection is shown. [Figure 8] A Western blot showing the expression of five different constructs according to the invention during cell transfection is shown. [Figure 9] A Western blot is shown demonstrating the proper expression of six different constructs according to the present invention during cell transfection. [Figure 10] Western blots showing the proper expression of four different constructs according to the present invention during cell transfection are shown. [Figure 11] Western immunoblots of four different constructs targeted to DEC205 on dendritic cells are shown, demonstrating that small extracellular entities (EVs) with a CdaA payload, or targeted EVs, activate the STING pathway within dendritic cells with a DEC205 surface molecule. [Figure 12] Western immunoblots show that mouse fibroblasts lacking DEC205 on their cell surface do not possess the STING pathway activated when treated with small extravasation cells (EVs) having the construct Figure 11 that targets DEC205 with a CdaA payload, or with targeted EVs. [Figure 13] Western immunoblots are shown illustrating three constructs according to the present invention that target DEC205 on dendritic cells, demonstrating that exosomes with a CdaA payload, or targeted EVs, activate the STING pathway within dendritic cells. [Figure 14] Panel A shows Western blots demonstrating the proper expression of two constructs according to the present invention, while Panel B shows immunofluorescence images demonstrating the expression of a construct according to the present invention that targets dendritic cells. [Figure 15] This image shows an immunofluorescence image illustrating the cellular expression of a construct according to the present invention that targets dendritic cells containing a rotavirus antigen payload. [Figure 16] A schematic diagram illustrating the mechanism of action of programmed mono-targeting extracellular vesicles according to the present invention, which deliver a cytotoxic payload for immunotoxin-mediated cell death (apoptosis) and tumor regression (virus-mediated cell death) when the construct is expressed by a tumor-selective virus. [Figure 17]A schematic diagram of a chimeric fusion construct is shown, which has a VSVG transmembrane domain and an mGZMB payload, along with a single-strand variable fragment (scFv) targeting moiety that targets carcinoembryonic antigen (CEA) or carbonic anhydrase IX (CA9). [Figure 18] Western blots are shown demonstrating the appropriate expression of two constructs according to the present invention, expressed from plasmids or viruses and abundantly present in isolated extracellular vesicles or targeted extracellular vehicles, in three different cell types. [Figure 19A] The bar graph shows the reduced cell viability compared to a negative control for two constructs expressed from vaccinia virus according to the present invention, which have a cytotoxic payload. [Figure 19B] The image shows bright-field microscopy images illustrating enhanced cell death in cells that present target surface molecules on their cell surface and have been transfected with a construct that targets these cell surface molecules and delivers a cytotoxic payload according to the present invention. [Figure 19C] The bar graphs show the reduced cell viability compared to negative controls in two constructs carrying a cytotoxic payload according to the present invention, compared to untransfected cells, a construct lacking the targeting region, and a construct lacking the cytotoxic payload. [Figure 20] The bar graph shows reduced cell viability compared to a negative control for two constructs carrying a cytotoxic payload according to the present invention in two different cell types. [Figure 21] A schematic diagram illustrating the method of supernatant transplantation is shown. [Figure 22] The images show bright-field microscopy images of cells in a supernatant transplantation experiment, illustrating cell death limited to MC38 cells receiving supernatant (extracellular vesicle fraction) containing a construct that targets MC38 cells. [Figure 23] The images show bright-field microscopy images of cells in a supernatant transplantation experiment, illustrating cell death limited to cells that present targeted cell surface molecules when treated with supernatant from cells infected with a virus expressing a construct according to the present invention. [Figure 24]The images show immunofluorescence microscopy images of cells in a supernatant transplantation experiment demonstrating the expression of a construct according to the present invention that targets hCEA having an mCherry payload. The top panel shows that CEA-negative cells infected with a virus expressing the construct express the construct, while the two bottom panels show that only cells possessing a CEA target surface marker and undergoing supernatant transplantation take up the construct that targets this cell surface molecule. [Figure 25] A schematic diagram illustrating an outline of one aspect of the present invention for generating programmed extracellular vesicles from a producer cell line in vivo, in situ, or in vitro. [Figure 26] A schematic diagram illustrating five different constructs according to the present invention, each having a tetraspanin-based transmembrane domain and transporting a cytotoxic payload, is shown. [Figure 27] Figure 26 shows a Western blot illustrating the successful expression of the five constructs shown. [Figure 28] A schematic diagram illustrates seven different constructs according to the present invention, each having a transmembrane domain based on CD63 tetraspanin targeting CD19 and CD20, along with one payload for six of the illustrated constructs and two payloads and furin cleavage sites for one of the seven illustrated constructs. [Figure 29A] A Western blot is shown demonstrating that a construct according to the present invention, having a CTX-targeting moiety, a VSVG transmembrane domain, and a Nanoluc® payload, is well expressed from a plasmid transfected into HEK293T cells. [Figure 29B] The bar graph shows the luminescence observed in the supernatant transplantation experiment. Here, fluorescence is observed only in the supernatant of cells expressing the construct shown in Figure 29A. [Figure 29C] Figure 29B shows a bar graph illustrating the supernatant transferred to six different human glioblastoma cell lines, where luminescence in the cells corresponds exclusively to the proportion of target cell surface markers presented outside the treated cells in constructs containing the appropriate targeting region. [Figure 30]This graph shows that 786-O cancer cells infected with vaccinia virus (VacV) generate more extracellular organisms (EVs) than uninfected cells. [Figure 31] This Western blot shows that several cancer cell types infected with vaccinia virus generate more extracellular organisms (EVs) than uninfected cells. [Figure 32] A Western blot showing the expression of four different constructs according to the present invention in the isolated EV fraction during cell transfection is shown. [Figure 33] A Western blot showing the expression of the construct according to the present invention in the isolated EV fraction during cell transfection is shown. [Figure 34] A Western blot showing the expression of four different constructs according to the present invention in the isolated EV fraction during cell transfection is shown. [Figure 35A] This fluorescence microscopy image shows that a plasmid containing LDLRT (LDLR target)-VSV- fused to an RNA-binding motif can package mRNA encoding blue fluorescent protein (BFP) fused to Nanoluc® (Nluc), and deliver it to recipient cells positive for the LDLR target. [Figure 35B] Quantitative readout from the same experiment as 35A, in which Nanoluc™ activity in recipient cells was measured. [Figure 36] This graph shows the cell viability of fibroblast-activating protein (FAP)-positive pancreatic fibroblasts (PanFib), pancreatic cancer cells (BxPC3), and patient samples (P025, P032) treated with aFAP-VSVG-mGZMB EV. [Figure 37] This Western immunoblot shows that a PEV expressing aDEC205-mCD63-CdaA, which targets DEC205 on dendritic cells and has a CdaA payload, has the ability to activate the STING pathway within dendritic cells. [Figure 38]Western immunoblots of three different constructs targeted to MARCO on macrophages are shown, demonstrating that small EVs with a CdaA payload, or targeted EVs, activate the STING pathway in myeloid macrophages expressing MACRO on their cell surface. [Figure 39] This assay demonstrates the efficacy of an anti-MarcO-linked CdaA PEV construct in stimulating the interferon (IFN) signaling pathway. [Figure 40] The histogram shows that in the presence of EVs with the αCD3 construct, cell death of MC38 cells by mouse splenocytes is enhanced compared to mock controls (splenocyte:MC38 ratio of 10:1). [Figure 41] The bar graphs show the results of vaccination experiments using naive EVs or EVs decorated with either aDEC205-VSVGTM-OVA or both aDEC205-CD63D-CdaA-Flag and aDEC205-VSVGTM-OVA, demonstrating that both combinations of dendritic cell-targeting antigens [e.g., ovalbumin (OVA)] and immune adjuvants (e.g., CdaA enzyme) induce an immune response in vivo. [Figure 42] This assay demonstrates a dendritic cell-derived PEV construct that acts as an immune adjuvant by stimulating the interferon response. [Modes for carrying out the invention]

[0027] Detailed explanation Generally, the disclosure provides recombinant oncoselective viral particles comprising a nucleic acid encoding a recombinant polypeptide for directing an extracellular vesicle (EV) to at least one target cell, wherein the recombinant polypeptide comprises at least one targeting moiety for directing the EV to at least one target molecule expressed by the at least one target cell; at least one EV anchor polypeptide; and at least one intravesicular polypeptide. The viral particles may be derived from oncolytic viruses. Furthermore, the disclosure provides recombinant polypeptides for directing an extracellular vesicle (EV) to at least one target cell, comprising at least one targeting moiety for directing the EV to at least one target molecule expressed by the at least one target cell, at least one EV anchor polypeptide, and at least one intravesicular polypeptide.

[0028] Virus particles containing EV-programmed recombinant polypeptides In one embodiment, a recombinant oncoselective viral particle comprising a nucleic acid encoding a recombinant polypeptide for directing an extracellular vesicle (EV) to at least one target cell, wherein the recombinant polypeptide is - At least one targeting portion for guiding the EV to the at least one target molecule expressed by the at least one target cell, - At least one EV anchor polypeptide, and - At least one intravesicular polypeptide Virus particles containing [the specified element] are provided.

[0029] In one embodiment, recombinant tumor-selective virus particles belong to the category of oncolytic viruses.

[0030] Recombinant polypeptides In one embodiment, a recombinant polypeptide for guiding an extracellular vesicle (EV) to at least one target cell, - At least one targeting portion for guiding the EV to the at least one target molecule expressed by the at least one target cell, - At least one EV anchor polypeptide, and - At least one intravesicular polypeptide Recombinant polypeptides containing the above are provided.

[0031] "Single-specific" single-transmembrane (TM) domain constructs In one embodiment, the at least one EV anchor polypeptide comprises an EV-inducible transmembrane polypeptide linked to the at least one targeted portion.

[0032] In one embodiment, the EV-inducible transmembrane polypeptide comprises a transmembrane domain from LAMP2b, VSVG, CD81, CD82, or LAMP1.

[0033] In one embodiment, the EV-inducible transmembrane polypeptide includes a transmembrane domain from Junin virus glycoprotein, Lassa fever virus glycoprotein, LCMV (lymphocytic choriomeningitis virus) glycoprotein, SARS-CoV-2 glycoprotein, tamiamy virus glycoprotein, guanalithovirus glycoprotein, paranavirus glycoprotein, Machupovirus glycoprotein, Sabia virus glycoprotein, or CdaA.

[0034] In one embodiment, the EV-inducible transmembrane polypeptide includes a transmembrane domain derived from a rhabdoviral glycoprotein.

[0035] In one embodiment, the EV-inducible transmembrane polypeptide includes a transmembrane domain derived from an arenavirus glycoprotein.

[0036] In one embodiment, the at least one target cell includes a mammalian cell.

[0037] In one embodiment, the mammalian cell is a human cell.

[0038] In one embodiment, the at least one target cell is a tumor cell, a tumor stromal cell, or an immune cell.

[0039] In one embodiment, the tumor stromal cells include cancer-associated fibroblasts.

[0040] In one embodiment, the immune cells are T cells, B cells, natural killer (NK) cells, dendritic cells, macrophages, or neutrophils.

[0041] In one embodiment, the immune cell is a macrophage. In one embodiment, the at least one target molecule is a macrophage receptor (MARCO).

[0042] In one embodiment, the T cells are regulatory T cells or cytotoxic T cells.

[0043] In one embodiment, the at least one target molecule is a cell surface marker or a cell surface receptor.

[0044] In one embodiment, the at least one target molecule is a TNF-α family receptor, an integrin, a C-type lectin receptor, leptin, carcinoembryonic antigen, CD antigen, carbonic anhydrase, FAP, MMP2, DEC205, DC40, CLEC9, CD3, glycosaminoglycan, polysaccharide, or lipid.

[0045] In one embodiment, the at least one target molecule is - Expressed by diseased cells, not by healthy control cells, or -The diseased cells express more of this compared to the healthy control cells. Contains disease-specific cell surface molecules.

[0046] In one embodiment, the disease-specific cell surface molecule includes a tumor-associated antigen.

[0047] In one embodiment, the at least one target molecule includes DEC205, CLEC9A, CEACAM5, CTLA4, CD3, CD7, CD11c, CD19, CD20, CD22, CD40, CD44, CD206, EGFR, fibroblast-activating protein (FAP), CA9, MMP-2, PD-L1, SIRPa, chondroitin sulfate, αv-integrin, or folate receptor.

[0048] In one embodiment, the at least one targeting portion includes a receptor ligand, an antibody or a functional fragment thereof, an scFv, a single-domain antibody, or DARPin.

[0049] In one embodiment, the antibody is a single-domain antibody.

[0050] In one embodiment, the antibody is a humanized antibody.

[0051] In one embodiment, the functional fragment is Fab' or F(ab')2.

[0052] In one embodiment, the at least one targeted moiety includes anti-DEC205, anti-Clec9A, anti-FAP, anti-CEA, anti-CA9, anti-CTL4, anti-CD3, anti-CD206, anti-CD19, anti-CD20, anti-CD22, anti-CD44, anti-CD7, SIRPα external domain, GE11 peptide, CTX, VAR2Δ, CD40 ligand, CD40 targeted peptide, iRGD, and PD1.

[0053] In one embodiment, the intravesicular polypeptide may include a short amino acid tail for protruding into the intravesicular space. The intravesicular polypeptide may contain at least 9 amino acids. The intravesicular polypeptide may contain at least 10 amino acids. The intravesicular polypeptide may contain at least 11 amino acids. The intravesicular polypeptide may contain at least 12 amino acids. The intravesicular polypeptide may contain at least 13 amino acids. The intravesicular polypeptide may contain at least 14 amino acids. The intravesicular polypeptide may contain at least 15 amino acids. The intravesicular polypeptide may contain at least 9 amino acids. The intravesicular polypeptide may contain 9 to 15 amino acids.

[0054] In one embodiment, the intravesicular polypeptide comprises at least one EV payload polypeptide linked to the at least one targeting moiety via the EV anchor polypeptide. The EV payload polypeptide may include, for example, a therapeutic polypeptide, an imaging polypeptide, a diagnostic polypeptide, a suicide protein, or a receptor for a biomarker.

[0055] In one embodiment, at least one EV payload polypeptide comprises at least one EV therapeutic payload polypeptide.

[0056] "Single-specific" tetraspanin constructs In one embodiment, the EV anchor polypeptide and the intravesicular polypeptide together comprise an EV-inducible recombinant tetraspanin containing the at least one targeting moiety inserted between their two transmembrane domains.

[0057] In one embodiment, the recombinant tetraspanin contains or is derived from human CD63 or CD9.

[0058] In one embodiment, the at least one target cell includes a mammalian cell.

[0059] In one embodiment, the mammalian cell is a human cell.

[0060] In one embodiment, the at least one target cell is a tumor cell, a tumor stromal cell, or an immune cell.

[0061] In one embodiment, the tumor stromal cells include cancer-associated fibroblasts.

[0062] In one embodiment, the immune cells are T cells, B cells, natural killer (NK) cells, dendritic cells, macrophages, or neutrophils.

[0063] In one embodiment, the immune cell is a macrophage. In one embodiment, the at least one target molecule is a macrophage receptor (MARCO).

[0064] In one embodiment, the T cells are regulatory T cells or cytotoxic T cells.

[0065] In one embodiment, the at least one target molecule is a cell surface marker or a cell surface receptor.

[0066] In one embodiment, the at least one target molecule is a TNF-α family receptor, an integrin, a C-type lectin receptor, leptin, carcinoembryonic antigen, CD antigen, carbonic anhydrase, FAP, MMP2, DEC205, DC40, CLEC9, CD3, glycosaminoglycan, polysaccharide, or lipid.

[0067] In one embodiment, the at least one target molecule is - Expressed by diseased cells, not by healthy control cells, or -The diseased cells express more of this compared to the healthy control cells. Contains disease-specific cell surface molecules.

[0068] In one embodiment, the disease-specific cell surface molecule includes a tumor-associated antigen.

[0069] In one embodiment, the at least one target molecule includes DEC205, CLEC9A, CEACAM5, CTLA4, CD3, CD7, CD11c, CD19, CD20, CD22, CD40, CD44, CD206, EGFR, fibroblast-activating protein (FAP), CA9, MMP-2, PD-L1, SIRPa, chondroitin sulfate, αv-integrin, or folate receptor.

[0070] In one embodiment, the at least one targeting portion includes a receptor ligand, an antibody or a functional fragment thereof, an scFv, a single-domain antibody, or DARPin.

[0071] In one embodiment, the antibody is a single-domain antibody.

[0072] In one embodiment, the antibody is a humanized antibody.

[0073] In one embodiment, the functional fragment is Fab' or F(ab')2.

[0074] In one embodiment, the at least one targeted moiety includes anti-DEC205, anti-Clec9A, anti-FAP, anti-CEA, anti-CA9, anti-CTL4, anti-CD3, anti-CD206, anti-CD19, anti-CD20, anti-CD22, anti-CD44, anti-CD7, SIRPa external domain, GE11 peptide, CTX, VAR2Δ, CD40 ligand, CD40 targeted peptide, iRGD, and PD1.

[0075] In one embodiment, the recombinant polypeptide further comprises at least one EV payload polypeptide ligated to the N-terminus and / or C-terminus of the recombinant tetraspanin. The EV payload polypeptide may include, for example, a therapeutic polypeptide, an imaging polypeptide, a diagnostic polypeptide, a suicide protein, or a receptor for a biomarker.

[0076] In one embodiment, at least one EV payload polypeptide comprises at least one EV therapeutic polypeptide.

[0077] "Dual-specific" tetraspanin constructs In one embodiment, the at least one targeting portion comprises at least two targeting portions, where the EV anchor polypeptide and the intravesicular polypeptide together comprise an EV-inducible recombinant tetraspanin comprising four transmembrane domains numbered 1, 2, 3, and 4 from the N-terminus to the C-terminus, where the first of the two targeting portions is inserted between transmembrane domains 1 and 2, and the second of the two targeting portions is inserted between transmembrane domains 3 and 4.

[0078] In one embodiment, the EV-inducible recombinant tetraspanin is derived from human CD63 or CD9.

[0079] In one embodiment, the at least two targeting portions specifically bind to at least two different target molecules.

[0080] Targeting the same target cells In one embodiment, the at least two different target molecules are expressed by the same target cell.

[0081] In one embodiment, the target cells include mammalian cells.

[0082] In one embodiment, the mammalian cells include human cells.

[0083] In one embodiment, the target cells include tumor cells, tumor stromal cells, or immune cells.

[0084] In one embodiment, the tumor stromal cells include cancer-associated fibroblasts.

[0085] In one embodiment, the immune cells are T cells, B cells, natural killer (NK) cells, dendritic cells, macrophages, or neutrophils.

[0086] In one embodiment, the immune cell is a macrophage. In one embodiment, the at least one target molecule is a macrophage receptor (MARCO).

[0087] In one embodiment, the T cells are regulatory T cells or cytotoxic T cells.

[0088] In one embodiment, each of the at least two target molecules is a cell surface molecule.

[0089] In one embodiment, each of the at least two target molecules is a TNF-α family receptor, an integrin, a C-type lectin receptor, leptin, carcinoembryonic antigen, CD antigen, carbonic anhydrase, FAP, MMP2, DEC205, DC40, CLEC9, CD3, glycosaminoglycan, polysaccharide, or lipid.

[0090] In one embodiment, each of the at least two target molecules is - Expressed by diseased cells, not by healthy control cells, or -The diseased cells express more of this compared to the healthy control cells. Contains disease-specific cell surface molecules.

[0091] In one embodiment, the disease-specific cell surface molecule includes a tumor-associated antigen.

[0092] In one embodiment, each of the at least two target molecules independently includes DEC205, CLEC9A, CEACAM5, CTLA4, CD3, CD7, CD11c, CD19, CD20, CD22, CD40, CD44, CD206, EGFR, fibroblast-activating protein (FAP), CA9, MMP-2, PD-L1, SIRPa, chondroitin sulfate, αv-integrin, or folate receptor.

[0093] In one embodiment, each of the at least two targeting portions independently comprises a receptor ligand, an antibody or a functional fragment thereof, an scFv, a single-domain antibody, or DARPin.

[0094] In one embodiment, the antibody is a single-domain antibody.

[0095] In one embodiment, the antibody is a humanized antibody.

[0096] In one embodiment, the functional fragment is Fab' or F(ab')2.

[0097] In one embodiment, the at least one targeted moiety includes anti-DEC205, anti-Clec9A, anti-FAP, anti-CEA, anti-CA9, anti-CTL4, anti-CD3, anti-CD206, anti-CD19, anti-CD20, anti-CD22, anti-CD44, anti-CD7, SIRPα external domain, GE11 peptide, CTX, VAR2Δ, CD40 ligand, CD40 targeted peptide, iRGD, and PD1.

[0098] Targeting different target cells In one embodiment, the at least two different target molecules are expressed by different target cells.

[0099] In one embodiment, the different target cells include disease cells and immune cells, where the at least two targeting portions are each directed to disease cell surface molecules and immune cell surface molecules.

[0100] In one embodiment, the different target cells include tumor cells and immune cells, where the at least two targeting portions are each directed to tumor cell surface molecules and immune cell surface molecules.

[0101] In one embodiment, the immune cells are T cells, and the immune cell surface marker is a T cell surface molecule.

[0102] In one embodiment, the T cells are regulatory T cells or cytotoxic T cells.

[0103] In one embodiment, the immune cells are natural killer (NK) cells, and the immune cell surface marker is an NK cell surface molecule.

[0104] In one embodiment, the immune cells are B cells, and the immune cell surface marker is a B cell surface molecule.

[0105] In one embodiment, the immune cell is a macrophage, and the immune cell surface marker is a macrophage cell surface molecule. In one embodiment, the at least one target molecule is a macrophage receptor (MARCO).

[0106] In one embodiment, the immune cells are dendritic cells, and the immune cell surface markers are dendritic cell surface molecules.

[0107] In one embodiment, the immune cells are neutrophils, and the immune cell surface markers are neutrophil cell surface molecules.

[0108] In one embodiment, the tumor cell surface molecule includes a tumor-associated antigen.

[0109] In one embodiment, the tumor cell surface molecule includes one of the following: CEACAM5, CD19, CD20, CD22, EGFR, fibroblast-activating protein (FAP), CA9, MMP-2, PD-L1, SIRPα, chondroitin sulfate, αv-integrin, or folate receptor.

[0110] In one embodiment, the at least one targeting portion includes an antibody or a functional fragment thereof, an scFv, a single-domain antibody, or DARPin.

[0111] In one embodiment, the antibody is a single-domain antibody.

[0112] In one embodiment, the antibody is a humanized antibody.

[0113] In one embodiment, the functional fragment is Fab' or F(ab')2.

[0114] In one embodiment, the recombinant polypeptide further comprises at least one EV payload polypeptide. The EV payload polypeptide may include, for example, a therapeutic polypeptide, an imaging polypeptide, a diagnostic polypeptide, a suicide protein, or a receptor for a biomarker.

[0115] In one embodiment, the EV payload polypeptide comprises at least one EV therapeutic payload polypeptide.

[0116] In one embodiment, the EV therapeutic payload polypeptide is ligated to the N-terminus and / or C-terminus of the recombinant tetraspanin.

[0117] payload These payloads are intended for the embodiments described herein, as necessary.

[0118] In one embodiment, the at least one EV payload polypeptide is linked via a cleavage site for releasing the at least one EV payload polypeptide.

[0119] In one embodiment, the at least one EV therapeutic payload polypeptide is linked via a cleavage site for releasing the at least one EV therapeutic payload polypeptide.

[0120] In one embodiment, the cleavage site includes a self-cleaving peptide, a pH-dependent cleavage site, or a site for enzymatic cleavage.

[0121] In one embodiment, the EV therapeutic payload polypeptide includes an active pharmaceutical ingredient (API).

[0122] In one embodiment, the EV therapeutic payload polypeptide includes a cytotoxic molecule.

[0123] In one embodiment, the cytotoxic molecule includes human GZMB R201K, mouse GZMB, diphtheria toxin, the PE38 domain from Pseudomonas exotoxin A, or human TRAIL.

[0124] In one embodiment, the payload polypeptide includes an immunomodulatory molecule.

[0125] In one embodiment, the immunomodulatory molecule includes an enzyme that produces an immunogenic molecule.

[0126] In one embodiment, the immunomodulatory molecule includes STING or an ERAdP pathway activator.

[0127] In one embodiment, the STING or ERAdP pathway activator includes a bacterial dinucleotide cyclase.

[0128] In one embodiment, the bacterial dinucleotide cyclase contains CdaA.

[0129] In one embodiment, the payload polypeptide includes an enzyme.

[0130] In one embodiment, the payload polypeptide includes a nucleic acid-binding domain.

[0131] In one embodiment, the nucleic acid binding domain includes an RNA binding motif.

[0132] In one embodiment, the nucleic acid-binding domain includes an RNA-binding motif from a Cas13 family member protein. In one embodiment, the RNA-binding motif includes an RNA-binding motif from Cas13a. In one embodiment, the RNA-binding motif includes an RNA-binding motif from Cas13b. In one embodiment, the RNA-binding motif includes an RNA-binding motif from Cas13d.

[0133] In one embodiment, the RNA-binding motif includes an RNA-binding motif from Pum (Pumilio homology domain 1).

[0134] In one embodiment, the RNA-binding motif includes an RNA-binding motif from Stu1 (Staufen-1).

[0135] In one embodiment, the RNA-binding motif includes an RNA-binding motif from the alphavirus capsid protein L72AE.

[0136] In one embodiment, the nucleic acid binding includes an RNA-binding motif from the MS2 coat protein (hereinafter referred to as "MS2").

[0137] In one embodiment, the RNA-binding motif includes an RNA-binding motif from the VEEV capsid protein.

[0138] In one embodiment, the RNA-binding motif includes a nucleic acid ligand system.

[0139] In one embodiment, the payload polypeptide includes an antigen.

[0140] In one embodiment, the antigen is a tumor-associated antigen.

[0141] In one embodiment, the antigen is derived from a pathogen.

[0142] In one embodiment, the EV therapeutic payload polypeptide further comprises an adjuvant.

[0143] In one embodiment, the adjuvant includes STING or an ERAdP pathway activator.

[0144] In one embodiment, the STING or ERAdP pathway activator includes a bacterial dinucleotide cyclase.

[0145] In one embodiment, the bacterial dinucleotide cyclase contains CdaA.

[0146] In one embodiment, the at least one EV therapeutic payload polypeptide is linked to at least one further EV payload polypeptide.

[0147] In one embodiment, the at least one EV therapeutic payload polypeptide is linked to the at least one further EV payload polypeptide by a cleavage site.

[0148] In one embodiment, the at least one EV therapeutic payload polypeptide is separated from the at least one further EV payload polypeptide by at least two EV transmembrane domains.

[0149] nucleic acid molecule In one embodiment, a nucleic acid molecule encoding a recombinant polypeptide as described herein is provided.

[0150] In one embodiment, the nucleic acid further encodes separate EV cargo molecules.

[0151] In one embodiment, the EV cargo molecule includes nucleic acid.

[0152] In one embodiment, the nucleic acid includes RNA.

[0153] In one embodiment, the RNA comprises a target sequence from the sequences in Table 12. In one embodiment, the recombinant polypeptide comprises an RNA-binding motif from the sequence in Table 12 corresponding to the cargo target sequence.

[0154] In one embodiment, the RNA includes mRNA, miRNA, or shRNA.

[0155] In one embodiment, the EV cargo molecule includes a polypeptide.

[0156] In one embodiment, a nucleic acid molecule is provided that encodes a recombinant polypeptide containing an EV payload as defined herein.

[0157] In one embodiment, a nucleic acid molecule is provided that encodes a recombinant polypeptide containing an EV therapeutic payload as defined herein.

[0158] In one embodiment, the nucleic acid further encodes separate EV cargo molecules.

[0159] In one embodiment, the EV cargo molecule includes nucleic acid.

[0160] In one embodiment, the nucleic acid includes RNA.

[0161] In one embodiment, the RNA includes mRNA, miRNA, or shRNA.

[0162] In one embodiment, the EV cargo molecule includes a polypeptide.

[0163] vector In one embodiment, a vector comprising nucleic acid as defined herein is provided.

[0164] In one embodiment, a vector comprising nucleic acids as defined herein is provided, wherein the recombinant polypeptide comprises an EV therapeutic payload.

[0165] Recombinant viral genome In one embodiment, a recombinant viral genome comprising nucleic acids as defined herein is provided.

[0166] In one embodiment, the viral genome is derived from a lentivirus, the Tian Tan strain of vaccinia virus, or adeno-associated virus (AAV).

[0167] In one embodiment, the viral genome is derived from a tumor-selective virus.

[0168] In one embodiment, the viral genome is derived from an oncolytic virus.

[0169] In one embodiment, the oncolytic virus is vesicular stomatitis virus (VSV), vaccinia virus, herpes simplex virus type 1 (HSV-1), herpes virus type 2 (HSV-2), or adenovirus.

[0170] In one embodiment, a recombinant viral genome comprising nucleic acids as defined herein is provided, wherein the recombinant polypeptide comprises an EV therapeutic payload.

[0171] In one embodiment, the viral genome is derived from a tumor-selective virus.

[0172] In one embodiment, the viral genome is derived from an oncolytic virus.

[0173] In one embodiment, the oncolytic virus is vesicular stomatitis virus (VSV), vaccinia virus, herpes simplex virus type 1 (HSV-1), herpes virus type 2 (HSV-2), or adenovirus.

[0174] Virus particles In one embodiment, a virus particle containing nucleic acid as defined herein is provided.

[0175] In one embodiment, the virus particles belong to a lentivirus, a Tian Tan strain of vaccinia virus, or an adeno-associated virus (AAV).

[0176] In one embodiment, the virus particles belong to the category of tumor-selective viruses.

[0177] In one embodiment, the virus particles belong to the category of oncolytic viruses.

[0178] In one embodiment, the oncolytic virus is vesicular stomatitis virus (VSV), vaccinia virus, herpes simplex virus type 1 (HSV-1), herpes virus type 2 (HSV-2), or adenovirus.

[0179] In one embodiment, a viral particle comprising nucleic acid as defined herein is provided, wherein the recombinant polypeptide comprises an EV therapeutic payload.

[0180] In one embodiment, the virus particles belong to the category of tumor-selective viruses.

[0181] In one embodiment, the virus particles belong to the category of oncolytic viruses.

[0182] In one embodiment, the oncolytic virus is vesicular stomatitis virus (VSV), vaccinia virus, herpes simplex virus type 1 (HSV-1), herpes virus type 2 (HSV-2), or adenovirus.

[0183] host cell In one embodiment, a host cell is provided comprising a nucleic acid, vector, recombinant viral genome, or viral particle as defined herein.

[0184] In one embodiment, the host cell is a prokaryotic cell.

[0185] In one embodiment, the host cell is a eukaryotic cell.

[0186] In one embodiment, the host cell is a yeast cell or an insect cell.

[0187] In one embodiment, the host cell is a mammalian cell.

[0188] In one embodiment, the host cell is a human cell.

[0189] In one embodiment, the host cell is an immune cell.

[0190] In one embodiment, the host cell is a B cell, T cell, dendritic cell, macrophage, or neutrophil.

[0191] In one embodiment, the host cell is a regulatory T cell or a cytotoxic T cell.

[0192] In one embodiment, the host cell further encodes separate EV cargo molecules.

[0193] In one embodiment, the EV cargo molecule includes nucleic acid.

[0194] In one embodiment, the nucleic acid includes RNA.

[0195] In one embodiment, the nucleic acid-binding domain includes an RNA-binding motif from a Cas13 family member protein. In one embodiment, the RNA-binding motif includes an RNA-binding motif from Cas13a. In one embodiment, the RNA-binding motif includes an RNA-binding motif from Cas13b. In one embodiment, the RNA-binding motif includes an RNA-binding motif from Cas13d.

[0196] In one embodiment, the RNA-binding motif includes an RNA-binding motif from Pum(Pumilio homology domain 1).

[0197] In one embodiment, the RNA-binding motif includes an RNA-binding motif from Stu1 (Staufen-1).

[0198] In one embodiment, the RNA-binding motif includes an RNA-binding motif from the alphavirus capsid protein L72AE.

[0199] In one embodiment, the nucleic acid binding includes an RNA-binding motif from the MS2 coat protein (hereinafter referred to as "MS2").

[0200] In one embodiment, the RNA-binding motif includes an RNA-binding motif from the VEEV capsid protein.

[0201] In one embodiment, the recombinant polypeptide comprises one of the above-described RNA-binding motifs, and the EV cargo comprises a congeneral RNA target sequence in the RNA-binding motif. Examples of RNA-binding motifs and congeneral target sequences are shown in the sequences in Table 12.

[0202] In one embodiment, the RNA includes mRNA, miRNA, or shRNA.

[0203] In one embodiment, the EV cargo molecule includes a polypeptide.

[0204] In one embodiment, a host cell is provided comprising a nucleic acid, vector, recombinant viral genome, or viral particle as defined herein, wherein the recombinant polypeptide comprises an EV therapeutic payload.

[0205] In one embodiment, the host cell is a prokaryotic cell.

[0206] In one embodiment, the host cell is a eukaryotic cell.

[0207] In one embodiment, the host cell is a yeast cell or an insect cell.

[0208] In one embodiment, the host cell is a mammalian cell.

[0209] In one embodiment, the host cell is a human cell.

[0210] In one embodiment, the host cell is an immune cell.

[0211] In one embodiment, the host cell is a B cell, T cell, dendritic cell, macrophage, or neutrophil.

[0212] In one embodiment, the host cell is a regulatory T cell or a cytotoxic T cell.

[0213] In one embodiment, the host cell further encodes separate EV cargo molecules.

[0214] In one embodiment, the EV cargo molecule includes nucleic acid.

[0215] In one embodiment, the nucleic acid includes RNA.

[0216] In one embodiment, the RNA includes mRNA, miRNA, or shRNA.

[0217] In one embodiment, the EV cargo molecule includes a polypeptide.

[0218] Targeted extracellular vesicles (EVs) In one embodiment, a targeted extracellular vesicle (EV) comprising a recombinant polypeptide as defined herein is provided.

[0219] In one embodiment, the targeted EV further comprises separate EV cargo molecules.

[0220] In one embodiment, the EV cargo molecule includes nucleic acid.

[0221] In one embodiment, the nucleic acid includes RNA.

[0222] In one embodiment, the RNA comprises a target sequence from the sequences in Table 12. In one embodiment, the recombinant polypeptide comprises an RNA-binding motif from the sequence in Table 12 corresponding to the cargo target sequence.

[0223] In one embodiment, the RNA includes mRNA, miRNA, or shRNA.

[0224] In one embodiment, the EV cargo molecule includes a polypeptide.

[0225] In one embodiment, the EV cargo molecule includes an API.

[0226] In one embodiment, the targeted EV is an exosome.

[0227] In one embodiment, the targeted EV is a microvesicle.

[0228] In one embodiment, the targeted EV is an ectosome.

[0229] In one embodiment, the targeted extracellular matrix (EV) is an apoptotic body.

[0230] In one embodiment, the targeted EV is a virus-like particle.

[0231] In one embodiment, the targeted EV is a macrovesicle.

[0232] In one embodiment, the targeted extracellular matrix (EV) is an oncosome.

[0233] In one embodiment, the targeted EV is a gesicle.

[0234] In one embodiment, a targeted extracellular vesicle (EV) comprising a recombinant polypeptide as defined herein is provided, wherein the recombinant polypeptide comprises an EV therapeutic payload.

[0235] In one embodiment, the targeted EV further comprises separate EV cargo molecules.

[0236] In one embodiment, the EV cargo molecule includes nucleic acid.

[0237] In one embodiment, the nucleic acid includes RNA.

[0238] In one embodiment, the RNA includes mRNA, miRNA, or shRNA.

[0239] In one embodiment, the EV cargo molecule includes a polypeptide.

[0240] In one embodiment, the EV cargo molecule includes an API.

[0241] In one embodiment, the targeted EV is an exosome.

[0242] In one embodiment, the targeted EV is a microvesicle.

[0243] In one embodiment, the targeted EV is an ectosome.

[0244] In one embodiment, the targeted extracellular matrix (EV) is an apoptotic body.

[0245] Pharmaceutical composition In one aspect, there is provided a composition comprising a nucleic acid as defined herein, a vector as defined herein, a recombinant viral genome as defined herein, a viral particle as defined herein, or a targeted EV as defined herein, together with a pharmaceutically acceptable excipient, diluent, or carrier.

[0246] In one aspect, there is provided a composition comprising a nucleic acid as defined herein, a vector as defined herein, a recombinant viral genome as defined herein, a viral particle as defined herein, or a targeted EV as defined herein, together with a pharmaceutically acceptable excipient, diluent, or carrier, wherein the recombinant polypeptide comprises an EV therapeutic payload.

[0247] Methods and Uses Binding to a Target In one aspect, there is provided a method of binding a targeting moiety to a target molecule of a target cell, the method comprising contacting the target cell with a targeted EV as defined herein.

[0248] In one aspect, there is provided the use of a targeted EV as defined herein for binding a targeting moiety to a target molecule of a target cell.

[0249] In one aspect, there is provided a targeted EV as defined herein for use in binding a targeting moiety to a target molecule of a target cell.

[0250] Delivery of Payload In one aspect, there is provided a method of delivering a payload molecule to a target cell, the method comprising contacting the target cell with a targeted EV as defined herein.

[0251] In one aspect, there is provided the use of a targeted EV as defined herein for delivering a payload molecule to a target cell.

[0252] In one embodiment, an EV as defined herein is provided for use in the delivery of a payload molecule to target cells.

[0253] Cargo delivery In one embodiment, a method is provided for delivering cargo molecules to target cells, the method comprising bringing the target cells into contact with targeted EVs as defined herein.

[0254] In one embodiment, the use of targeted EVs as defined herein for delivering cargo molecules to target cells is provided.

[0255] In one embodiment, a targeted EV as defined herein is provided for use in the delivery of cargo molecules to target cells.

[0256] Stimulation of the immune response In one embodiment, a method is provided for stimulating an immune response to an antigen, comprising administering a nucleic acid as defined herein, a vector as defined herein, a recombinant viral genome as defined herein, a viral particle as defined herein, or a targeted EV as defined herein, wherein the target cell includes an immune cell and the at least one EV therapeutic payload polypeptide includes an antigen.

[0257] In one embodiment, the antigen includes a disease cell-specific antigen.

[0258] In one embodiment, the antigen includes a tumor-specific antigen.

[0259] In one embodiment, the antigen is derived from a pathogen.

[0260] In one embodiment, the at least one EV therapeutic payload polypeptide further comprises an adjuvant.

[0261] In one embodiment, a use is provided for stimulating an immune response to an antigen of a nucleic acid as defined herein, a vector as defined herein, a recombinant viral genome as defined herein, a viral particle as defined herein, or a targeted EV as defined herein, wherein the target cell comprises an immune cell and the at least one EV therapeutic payload polypeptide comprises an antigen.

[0262] In one embodiment, the antigen includes a disease cell-specific antigen.

[0263] In one embodiment, the antigen includes a tumor-specific antigen.

[0264] In one embodiment, the antigen is derived from a pathogen.

[0265] In one embodiment, the at least one EV therapeutic payload polypeptide further comprises an adjuvant.

[0266] In one embodiment, a nucleic acid as defined herein, a vector as defined herein, a recombinant viral genome as defined herein, a viral particle as defined herein, or a targeted EV as defined herein is provided for use in stimulating an immune response to an antigen, wherein the target cell comprises an immune cell and the at least one EV therapeutic payload polypeptide comprises an antigen.

[0267] In one embodiment, the antigen includes a disease cell-specific antigen.

[0268] In one embodiment, the antigen includes a tumor-specific antigen.

[0269] In one embodiment, the antigen is derived from a pathogen.

[0270] In one embodiment, the at least one EV therapeutic payload polypeptide further comprises an adjuvant.

[0271] Killing of target cells In one aspect, there is provided a method of killing target cells, comprising administering to a subject a nucleic acid as defined herein, a vector as defined herein, a recombinant viral genome as defined herein, a viral particle as defined herein, or a targeted EV as defined herein, wherein the at least one EV therapeutic payload polypeptide comprises a cytotoxic molecule.

[0272] In one embodiment, the cytotoxic molecule comprises human GZMB R201K, mouse GZMB, diphtheria toxin, the PE38 domain from Pseudomonas exotoxin A, or human TRAIL.

[0273] In one embodiment, the target cells comprise diseased cells.

[0274] In one embodiment, the diseased cells are tumor cells.

[0275] In one aspect, there is provided the use of a nucleic acid as defined herein, a vector as defined herein, a recombinant viral genome as defined herein, a viral particle as defined herein, or a targeted EV as defined herein for killing target cells, wherein the at least one EV therapeutic payload polypeptide comprises a cytotoxic molecule.

[0276] In one embodiment, the cytotoxic molecule comprises human GZMB R201K, mouse GZMB, diphtheria toxin, the PE38 domain from Pseudomonas exotoxin A, or human TRAIL.

[0277] In one embodiment, the target cells comprise diseased cells.

[0278] In one embodiment, the diseased cells are tumor cells.

[0279] In one embodiment, there exists a nucleic acid as defined herein, a vector as defined herein, a recombinant viral genome as defined herein, a viral particle as defined herein, or a targeted EV as defined herein for use in killing a target cell, wherein the at least one EV therapeutic payload polypeptide comprises a cytotoxic molecule.

[0280] In one embodiment, the cytotoxic molecule includes human GZMB R201K, mouse GZMB, diphtheria toxin, the PE38 domain from Pseudomonas exotoxin A, or human TRAIL.

[0281] In one embodiment, the target cells include diseased cells.

[0282] In one embodiment, the diseased cells are tumor cells.

[0283] Reprogramming of immune cells In one embodiment, a method is provided for reprogramming immune cells, comprising contacting the immune cells with nucleic acids as defined herein, vectors as defined herein, recombinant viral genomes as defined herein, viral particles as defined herein, or targeted EVs as defined herein, wherein at least one EV therapeutic payload molecule comprises an immunomodulatory molecule.

[0284] In one embodiment, the immunomodulatory molecule includes STING or an ERAdP pathway activator.

[0285] In one embodiment, the STING or ERAdP pathway activator includes a bacterial dinucleotide cyclase.

[0286] In one embodiment, the bacterial dinucleotide cyclase contains CdaA.

[0287] In one embodiment, the immune cells include B cells, T cells, NK cells, dendritic cells, macrophages, or neutrophils. In one embodiment, the immune cells include macrophages.

[0288] In one embodiment, the immunomodulatory molecule comprises a STING pathway activator, the immune cells comprise macrophages, and the at least one target molecule is a macrophage receptor (MARCO).

[0289] In one embodiment, a use is provided for reprogramming immune cells having nucleic acids as defined herein, vectors as defined herein, recombinant viral genomes as defined herein, viral particles as defined herein, or targeted EVs as defined herein, wherein at least one EV therapeutic payload molecule comprises an immunomodulatory molecule.

[0290] In one embodiment, the immunomodulatory molecule includes STING or an ERAdP pathway activator.

[0291] In one embodiment, the STING or ERAdP pathway activator includes a bacterial dinucleotide cyclase.

[0292] In one embodiment, the bacterial dinucleotide cyclase contains CdaA.

[0293] In one embodiment, the immune cells include B cells, T cells, NK cells, dendritic cells, macrophages, or neutrophils. In one embodiment, the immune cells include macrophages.

[0294] In one embodiment, the immunomodulatory molecule comprises a STING pathway activator, the immune cells comprise macrophages, and the at least one target molecule is a macrophage receptor (MARCO).

[0295] In one embodiment, an immune cell is provided for use in the reprogramming of immune cells, having a nucleic acid as defined herein, a vector as defined herein, a recombinant viral genome as defined herein, a viral particle as defined herein, or a targeted EV as defined herein, wherein at least one EV therapeutic payload molecule comprises an immunomodulatory molecule.

[0296] In one embodiment, the immunomodulatory molecule includes STING or an ERAdP pathway activator.

[0297] In one embodiment, the STING or ERAdP pathway activator includes a bacterial dinucleotide cyclase.

[0298] In one embodiment, the bacterial dinucleotide cyclase contains CdaA.

[0299] In one embodiment, the immune cells include B cells, T cells, NK cells, dendritic cells, macrophages, or neutrophils. In one embodiment, the immune cells include macrophages.

[0300] In one embodiment, the immunomodulatory molecule comprises a STING pathway activator, the immune cells comprise macrophages, and the at least one target molecule is a macrophage receptor (MARCO).

[0301] Induction of an immune response against a target In one embodiment, a method is provided for inducing an immune response against target disease cells, comprising administering a nucleic acid as defined herein, a vector as defined herein, a recombinant viral genome as defined herein, a viral particle as defined herein, or a targeted EV as defined herein, wherein the recombinant polypeptide comprises at least two targeting moieties, each specifically bound to at least two different target molecules, the at least two different target molecules, each expressed by immune cells and disease cells.

[0302] In one embodiment, the diseased cells include tumor cells.

[0303] In one embodiment, one of the at least two different target molecules includes a tumor-associated antigen.

[0304] In one embodiment, the immune cells include T cells, B cells, natural killer (NK) cells, dendritic cells, macrophages, or neutrophils.

[0305] In one embodiment, the immune cells are T cells.

[0306] In one embodiment, the immune cells are regulatory T cells or cytotoxic T cells.

[0307] In one embodiment, the immune cells are NK cells.

[0308] In one embodiment, a use is provided for inducing an immune response against a target disease cell of a vector as defined herein, a recombinant viral genome as defined herein, a viral particle as defined herein, or a targeted EV as defined herein, wherein the recombinant polypeptide comprises at least two targeting moieties, each specifically bound to at least two different target molecules, the at least two different target molecules, each expressed by an immune cell and a disease cell.

[0309] In one embodiment, the diseased cells include tumor cells.

[0310] In one embodiment, one of the at least two different target molecules includes a tumor-associated antigen.

[0311] In one embodiment, the immune cells include T cells, B cells, natural killer (NK) cells, dendritic cells, macrophages, or neutrophils.

[0312] In one embodiment, the immune cells are T cells.

[0313] In one embodiment, the immune cells are regulatory T cells or cytotoxic T cells.

[0314] In one embodiment, the immune cells are NK cells.

[0315] In one embodiment, a vector as defined herein, a recombinant viral genome as defined herein, a viral particle as defined herein, or a targeted EV as defined herein is provided for use in inducing an immune response against targeted disease cells, wherein the recombinant polypeptide comprises at least two targeting moieties, each specifically bound to at least two different target molecules, the at least two different target molecules, respectively, expressed by immune cells and disease cells.

[0316] In one embodiment, the diseased cells include tumor cells.

[0317] In one embodiment, one of the at least two different target molecules includes a tumor-associated antigen.

[0318] In one embodiment, the immune cells include T cells, B cells, natural killer (NK) cells, dendritic cells, macrophages, or neutrophils.

[0319] In one embodiment, the immune cells are T cells.

[0320] In one embodiment, the immune cells are regulatory T cells or cytotoxic T cells.

[0321] In one embodiment, the immune cells are NK cells.

[0322] Preparation of therapeutically targeted extracellular viable cells (EVs) In one embodiment, a method for preparing therapeutically targeted extravasation products (EVs) in a subject, - Contacting cells obtained from the subject with nucleic acids as defined herein, vectors as defined herein, recombinant viral genomes as defined herein, or viral particles as defined herein, - To recover targeted EVs, A method including this is provided.

[0323] In one embodiment, the cells are tumor cells.

[0324] In one embodiment, the cells are immune cells.

[0325] In one embodiment, the immune cells include T cells, B cells, natural killer (NK) cells, dendritic cells, macrophages, or neutrophils.

[0326] In one embodiment, the use of nucleic acids, vectors, recombinant viral genomes, or viral particles as defined herein is provided for preparing therapeutically targeted extravasations (EVs) in a target.

[0327] In one embodiment, the cells are tumor cells.

[0328] In one embodiment, the cells are immune cells.

[0329] In one embodiment, the immune cells include T cells, B cells, natural killer (NK) cells, dendritic cells, macrophages, or neutrophils.

[0330] In one embodiment, nucleic acids, vectors, recombinant viral genomes, or viral particles as defined herein are provided for use in the preparation of therapeutically targeted extravasation villi in a subject.

[0331] In one embodiment, the cells are tumor cells.

[0332] In one embodiment, the cells are immune cells.

[0333] In one embodiment, the immune cells include T cells, B cells, natural killer (NK) cells, dendritic cells, macrophages, or neutrophils.

[0334] Manufacturing method In one embodiment, a method is provided for producing a targeted extracellular matrix (EV), comprising: generating an EV by expressing a nucleic acid as defined herein in a cell or culturing a host cell as defined herein; and recovering the EV.

[0335] Definitions and Embodiments The following definitions are provided to facilitate understanding of the terms used herein.

[0336] A "tumor-selective" virus is a virus that preferentially proliferates or replicates within tumor cells.

[0337] "Oncolytic virus" refers to any one of many viruses that, when active, have been shown to replicate and kill tumor cells in vitro or in vivo. These viruses may be naturally oncolytic viruses or viruses that have been modified to produce or improve oncolytic activity. Oncolytic viruses include rhabdoviruses. Rhabdoviruses include Carajas virus, Chandipura virus, Cocal virus, Isfahan virus, Piry virus, Alagoas virus (vesicular stomatitis), BeAn157575 virus, Boteke virus, Calchaqui virus, Eelvirus American, Gray Lodge virus, Jurona virus, Klamath virus, Kwatta virus, La Joya virus, and Malpais Spring virus. Spring virus, Mount Elgon bat virus, Perinet virus, Tupaia virus, Farmington, Bahia Grande virus, Muir Springs virus, Reed Ranch virus, Hart Park virus, Flanders virus, Kamese virus, Mosqueiro virus, Mossuril virus, Barur virus, Fukuoka virus, Kern Canyon virus, Nkolbisson virus, Le Dantec virus, Keuralibha virus, Connecticut virus, New MintMinto virus, Sawgrass virus, Chaco virus, Sena Madureira virus, Timbo virus, Almpiwar virus, Aruac virus, Bangoran virus, Bimbo virus, Bivens Arm virus, Blue crab virus, Charleville virus, Coastal Plains virus, DakArK7292 virus, Entamoeba virus, Garba virus, Gossas virus, Humpty Doo virus, Joinjakaka virus, Kannamangalam virus, Kolongo virus, Koolpinyah virus, Kotonkon virus, Landjia virus, Manitoba virus, Marco virus, Nasoule virus, Navarro virus, Ngaingan virus, Oak-Vale virus, Obodhiang virus, Oita virus, Ouango virus, Parry Creek virus, RioGrandecichlid virus, Sandjimba virus, Sigma virus, Sripur virus, Sweetwater Branch virus, Tibrogargan virus, Xiburema virus, Yata virus, Rhode Island virus, Adelaide River virus, Berrimah virus, Kimberley virus, Maraba virus, Bovine ephemeral feverIncludes fever viruses or modified variants thereof.

[0338] Extracellular vesicles (EVs) are cell-derived membrane structures, including exosomes, microvesicles, virus-like particles, macrovesicles, oncosomes, gesicles, and apoptotic bodies. These extracellular vesicles are generally categorized based on their size, specific markers, cellular origin, and biosynthetic processes. Exosomes are endosomal vesicles, 30–160 nm in size, released from cells upon fusion of the multivesicular (MVB) membrane with the plasma membrane. Exosomes are produced by all cell types, and their release can be induced by various stimuli, including stress, hypoxia, cell death, and viral infection. Classical microvesicles (also known as microparticles) are 100 nm–1 μm vesicles released from cells upon shedding of the plasma membrane. Cancer cells may also secrete larger microvesicles (>1 μm), called oncosomes, which differ from classical microvesicles primarily in their size. Similar to exosomes, the release of microvesicles can be induced by stress and viral infection, and their contents are heterogeneous. Apoptotic bodies are larger EVs released from apoptotic cells by vesicle formation, ranging in size from 200 nm to 5 μm. These phosphatidylserine and annexin V-coated EVs contain cytoplasmic contents from dead cells. Traditionally, EVs pelleted at 100,000 g have been called exosomes, but in reality, this pellet contains a combination of microvesicles and exosomes. Although their biosynthetic pathways differ, exosomes and microvesicles share many similarities, and once released from cells, they are difficult to distinguish from each other. Recently, the International Society for Extracellular Vesicles proposed that the term small EV (sEV) should be used for particles smaller than 200 nm, while the term large EV (lEV) should be used for particles larger than 200 nm.

[0339] The terms “programmed EV” (PEV) and “targeted EV” are used herein synonymously to refer to EVs comprising recombinant polypeptides, as defined herein, and therefore possessing modified and acquired affinity (provided by the targeting moiety) for a target molecule.

[0340] "Recombinant" refers to nucleic acid or polypeptide molecules having segments of different origins, such as (but not limited to) products of genetic engineering using recombinant DNA technology.

[0341] "EV anchor polypeptide" refers to a polypeptide that anchors recombinant polypeptides to the EV membrane.

[0342] "EV-inducible transmembrane polypeptides" refer to a subset of transmembrane proteins that are present throughout the phospholipid bilayer membrane of EVs and that innately target (are transported to) the EV membrane.

[0343] Proteins having such EV-inducible transmembrane domains may be derived from viruses (e.g., VSVG) or cells (e.g., CD63 and Iamp2b). The membrane traversal across the domain may be a single pass or may be multiple passes, for example, four passes (4-pass or tetraspanin).

[0344] The single-pass and tetraspanin domains can be modified via the linker sequence to harbor one or more payloads, and the single-pass domain can similarly be modified to harbor one or more targeting portions in tandem.

[0345] Similarly, "EV-inducible tetraspanins" refers to a subset of tetraspanins that are transported to the EV membrane. Tetraspanins are a family of membrane proteins found in all multicellular eukaryotes and are also known as the transmembrane 4 superfamily (TM4SF) proteins. They have four transmembrane α-helices and two extracellular domains, one short extracellular domain or loop, and one longer extracellular domain / loop. While some protein families have four transmembrane α-helices, tetraspanins are defined by a conserved amino acid sequence that includes four or more cysteine ​​residues within the EC2 domain, in addition to two cysteine ​​residues within a highly conserved "CCG" motif.

[0346] Tetraspanin can be modified to directly possess up to two targeting segments and up to two payloads, or more when linked together.

[0347] Table 1 presents several examples of proteins that are specifically guided to and abundantly present within the EV membrane. These examples include single-pass and tetraspanin domains.

[0348] [Table 1]

[0349] [Table 2]

[0350] In relation to recombinant tetraspanins, "derived from ~" should be understood as meaning that native tetraspanins are modified to include exogenous sequences, such as a targeted portion inserted into one or both of the extracellular loops and / or a payload linked to the tetraspanin.

[0351] The "targeting moiety" refers to a molecule that has the ability to bind to a target molecule with sufficient affinity and specificity to target the extracellular gene (EV) to target cells expressing the target molecule. Non-limiting examples of targeting moieties include antibodies, their functional fragments, modified fragments, ligands (that target receptors), engineered ankyrin repeat proteins (DARPins) (that bind to target proteins), and domains that mediate specific protein-protein interactions. It will be understood that the targeting moiety of recombinant polypeptides is intended to be externally directed in relation to EVs.

[0352] Table 2 shows several examples of targeted areas.

[0353] [Table 3]

[0354] [Table 4]

[0355] [Table 5]

[0356] [Table 6]

[0357] [Table 7]

[0358] Single-domain antibodies (sdAbs), also known as nanobodies, are antibody fragments consisting of a single monomeric variable antibody domain. Like whole antibodies, they can selectively bind to specific antigens. With a molecular weight of only 12-15 kDa, single-domain antibodies are much smaller than common antibodies, which consist of two heavy chains and two light chains. sdABs can be produced by immunizing dromedary camels, camels, llamas, alpacas, or sharks, or they can be designed from common IgG, which has four chains.

[0359] A "functional fragment" refers to a portion of an antibody that maintains a paratope (including the complementarity-determining region, i.e., CDR) and has the ability to bind to the same target molecule as the parent antibody from which it originates. Examples include the Fab and F(ab')2 fragments.

[0360] A "modified fragment" refers to a recombinant polypeptide that originates from the parent antibody and retains the paratope, thus enabling it to bind to the same target molecule as the parent antibody. One example is a heavy chain of immunoglobulin (V) linked to a short linker peptide, typically 10 to about 25 amino acids. H ) and light chain (V L It is a single-stranded variable fragment (scFv), which is a fusion protein of the variable region of ).

[0361] DARPin is a repeat protein that typically contains several repeating structural domains (generally 4-6 repeats) of 33 amino acids. Because DARPin can bind to their target antigens with high affinity and specificity, it can be selected and used as an alternative scaffold in specific targeting. The main advantage of using DARPin compared to monoclonal antibodies is that DARPin generally has a low molecular weight and contains between 40 and 100 amino acid residues. For example, HER2 is frequently overexpressed in breast cancer cells. DARPin that binds to the extracellular domain of HER2 can be selected and used to direct therapeutic EVs toward malignant cells expressing HER2.

[0362] The term "target molecule" refers to the molecule to which the targeting moiety binds. Such a molecule may be a cell surface molecule that can be specifically bound by the targeting moiety, such as a polypeptide, lipid, or polysaccharide.

[0363] "Target cell" refers to a cell that expresses the target molecule, which is bound by the targeting moiety and to which the payload (if applicable) and / or cargo (if applicable) are directed.

[0364] "Intravesical polypeptide" refers to the polypeptide portion of a recombinant polypeptide that extends internally into the EV. It will be understood that the intravascular polypeptide may include a short polypeptide (e.g., at least 9 amino acids) that protrudes into the intravesical space. However, it will be understood that in other configurations described herein, the intravascular polypeptide may include an EV payload polypeptide. In yet another configuration, the EV-inducible transmembrane domain and the intravascular polypeptide may together include an EV-inducible recombinant tetraspanin, which may contain or may not contain at least one EV payload polypeptide ligated to the N-terminus and / or C-terminus.

[0365] "Single-targeted" indicates that a population of EVs is targeted to a target molecule. However, if "at least one" target is identified, it will be understood that this includes EVs that are directed to two or more target molecules, and therefore, it is also intended that the EVs are at least single-targeted.

[0366] Similarly, "bispecificity" means that the EV targets two target molecules. When "at least two" is specified, it will be understood that this also includes EVs directed to three or more target molecules, and that the EV is minimally bispecific.

[0367] "Cell surface molecules" means any molecule that is immobilized on the cell surface, or otherwise associates with it, enabling cell targeting by recombinant polypeptides via a targeting moiety. Such molecules may include, for example, polypeptides, polysaccharides, or lipids (including polysaccharide and lipid modifications of polypeptides). Examples include endometrial proteins, surface membrane proteins, and their modifications.

[0368] A "cell surface marker" is a cell surface molecule that is specific to (or abundantly present within) a particular cell type. A cell surface marker or combination of cell surface markers may be specific to a given cell type or cellular state (such as a disease state).

[0369] "Tumor stroma" refers to cells in the tumor environment other than cancer cells themselves, such as cancer-associated fibroblasts.

[0370] A "tumor-associated antigen" (TAA) refers to any immunogen that is associated with tumor cells and is absent or not very abundant in healthy cells or (as applicable and required) corresponding healthy cells. For example, a tumor-associated antigen may be specific to tumor cells in relation to the organism. A TAA may be, for example, a tumor-specific mutation, an abnormally spliced ​​protein, a carcinoembryonic antigen, or an endogenous retroviral protein. A TAA may be a neoantigen containing a neoepitope. A neoantigen is a newly formed (non-autologous) antigen that has not been previously recognized by the immune system and may arise, for example, from a tumor mutation.

[0371] The terms "payload" and "cargo" are used distinctly here. The former is intended to be part of a recombinant polypeptide, while the latter is intended to be another molecule to be transported within the EV.

[0372] "EV payload polypeptide" means any polypeptide that is part of the recombinant polypeptide itself and therefore co-encoded by the same nucleic acid molecule. EV payload polypeptides include any polypeptide for which EV loading or EV-mediated targeting or delivery is desired.

[0373] An "EV therapeutic payload polypeptide" refers to a therapeutic polypeptide that is part of the recombinant polypeptide itself and therefore co-encoded by the same nucleic acid molecule. Payload categories include (but are not limited to) cytotoxic molecules (e.g., GZMB variants, diphtheria toxin, pe38 (domain from Pseudomonas exotoxin A), or TRAIL), immune reprogramming molecules (e.g., STING or ERAdP pathway activators, e.g., bacterial cyclases), enzymes, nucleotide-binding domains, and antigens (tumor antigens, or infectious pathogens, e.g., antigens from dengue virus, malaria, or rotavirus). Non-limiting examples are shown in Table 3.

[0374] [Table 8]

[0375] [Table 9]

[0376] In contrast, "EV cargo" refers to a molecule that is transported within an EV but is different from the recombinant polypeptide that leads other EVs to their target. Therefore, cargo may be encoded by the same or different nucleic acids that encode the recombinant polypeptide (in the latter case, they would be understood to be expressed as separate polypeptides). For example, a host cell engineered to express a recombinant polypeptide is expected to be able to encode (or be modified to express) cargo separately (or vice versa). Cargo molecules do not need to be polypeptides because they are separate molecules from the recombinant polypeptide. For example, cargo molecules may be small molecules, such as small molecule drugs or imaging agents. Cargo may be nucleic acids, such as mRNA, miRNA, shRNA, or siRNA. For example, in embodiments where the payload includes a nucleic acid binding domain, nucleic acids may be preferentially loaded onto the vesicle. Such binding domains may be sequence-specific binding to sequence motifs within the nucleic acid molecule. Cargo molecules may also include polypeptides, such as cytotoxic molecules, immunoreprogramming molecules, enzymes, or antigens.

[0377] The term "linked" indicates that two parts are covalently linked, but such linkage does not need to be direct. For example, when "A" and "B" are "linked," it will be understood that the linkage may include additional amino acid residues or polypeptides. Similarly, when linked "via" a feature, such as a linker polypeptide or payload, it indicates that the feature is located between (and separates) "A" and "B" in relation to the recombinant polypeptide. However, when neither "A" nor "B" is linked, it must be directly linked to the intervening feature.

[0378] An "adjuvant" can be understood as a molecule that enhances and / or modulates the immune response to an antigen toward a desired immune response.

[0379] Where nucleic acids and amino acid molecules are referenced herein, it will be understood that embodiments including sequence variants thereof will also be explicitly considered. For example, such sequence variants may be intended to be polypeptides (or nucleic acid molecules encoding polypeptides) that have substantially the same function as, or retain the same function as, their parent molecules. Such sequence variants may be at least 70% identical to the parent molecule. They may be at least 80% identical to the parent molecule. They may be at least 90% identical to the parent molecule. They may be at least 95% identical to the parent molecule. They may be at least 96% identical to the parent molecule. They may be at least 97% identical to the parent molecule. They may be at least 98% identical to the parent molecule. They may be at least 99% identical to the parent molecule. The sequence variants considered herein may include conservative amino acid substitutions (or encoding nucleic acid sequence changes). The sequence variants considered herein may include silent mutations. [Examples]

[0380] Examples The following examples outline embodiments of the present invention and / or tests performed relating to the present invention. While these examples are illustrative, the present invention is by no means limited to the following exemplary embodiments.

[0381] Examples Attempts have been made to program extracellular vehicles (EVs) to deliver or transport therapeutic drugs using multiple molecules, mechanisms, and means, which are expensive, require ex vivo procedures, may be time-consuming (e.g., via electroporation), cannot be stored long-term, and are poorly validated.

[0382] Therefore, there is a need for means of delivering molecules to cells, for example, therapeutic agents and toxins, to targeted cells in vivo, or for preparing them simply, inexpensively, and continuously in a simple, stable manner in vivo, in vitro, or ex vivo.

[0383] Recombinant peptides are designed for targeted delivery of molecules to cells.

[0384] Figure 1 provides an overview of an example of the three-dimensional arrangement of structures discussed herein: Figure 1, Panel (a) - Single target - No payload Figure 1, Panel (b) - Two targets - No payload Figure 1, Panel (c) - Two targets - with payload Figure 1, Panel (d) - Single target - Payload Figure 1, Panel (e) - Control - No target, single payload Figure 1, Panel (f) - Control - No transmembrane domain

[0385] Example 1 Single targeting - no payload (2-part structure) Application: Blocks or activates the function of cell surface receptors.

[0386] The modes of operation of these platforms: They can act as competitive conjugates or blockers.

[0387] Advantages: Lack of stability and immunogenicity. If PEVs are produced from a viral platform, this has the potential for in-situ delivery. Coincidentally, the method is also more cost-effective thanks to these solutions.

[0388] Delivery / Production Method: Virus-based platforms (e.g., vaccinia virus, lentivirus, adeno-associated virus [AAV], VSV, HSV-1, etc.), plasmids (e.g., pcDNA3.1), and free PEVs.

[0389] Example 1(a): Programmed cell death protein 1 (PD1) is a surface protein preferentially expressed in immune cells such as T, B, NK cells, and bone marrow-derived dendritic cells. PD1 transmits an immunosuppressive signal upon association with its ligand, PD-L1. Molecules (monoclonal antibodies) that antagonize the PD1:PD-L1 interaction by binding to PD1 and PD-L1 have been shown to promote T cell-mediated killing of tumor cells. While these methods have shown positive results in several clinical applications, they tend to result in large amounts of consumed antigen that do not reach their final destination / use (e.g., exosomes expressing PD-L1). These factors reduce bioavailability and increase the toxicity risk of anti-PD1 and anti-PD-L1 monoclonal antibodies.

[0390] In this manner, we will examine a PEV that targets PD1 along with PD-L1 as the targeting part, thereby blocking its function. Targeting part: PD-L1 (which blocks the application / adjuvant in ICI).

[0391] Payload: Absent or optional.

[0392] Transmembrane domains (TD domains): All examples listed in Table 1 were usable.

[0393] Example 1(b): Similarly, T cells could be activated via CD3 surface proteins using a CD3-targeting moiety, e.g., an anti-CD3 antibody (application of a T cell activator / engager).

[0394] Payload: Absent or optional.

[0395] Transmembrane domains (TD domains): All examples listed in Table 1 were usable.

[0396] [Table 10]

[0397] Results: Data on PD1 targeting can be found in Figures 2-5.

[0398] Figure 2: Oncolytic vaccinia virus (VV or VacV) can program extracellular organisms (EVs) via a "chimeric blocking construct" encoding the external domain of PD1. A PEV construct was expressed that created a VV skeleton and programmed the EVs to present the mouse PD-1 external domain (mPD-1) on their surface. These PD-1-presenting EVs can specifically bind to tumor cells expressing PD-L1 on their surface in mice.

[0399] Figure 2A: A schematic diagram of a "chimeric blocking construct" in which mPD-1 is presented on the extracellular surface of a PEV and acts as a targeting site. mPD-1 is fused to a transmembrane LAMP2B domain, which facilitates the construct's round trip to the PEV. Notably, LAMP2B is generally abundant within EVs. This entire PEV construct is HA-labeled on the intracellular portion of the chimeric transgene construct, enabling PEV tracking and visualization.

[0400] Figure 2B: Human renal cell carcinoma 786-O cells were mock-infected or infected with VV-Exo control (this control construct expresses the LAMP2B transmembrane domain and HA tag at the C-terminus of the construct, but the EV targeting portion was replaced with a FLAG tag) or VV-Exo-PD1 (PEV construct shown in panel Figure 2A, MOI (multiple infections) of 1 for 48 hours). 48 hours after infection, cells were collected and lysed for immunoblot analysis. Similarly, extracellular vesicles were isolated from the culture medium by serial centrifugation. Next, Western blot analysis of the EV fraction and whole cell lysate (WCL) was performed using antibodies against mPD-1 and HA tags (to confirm appropriate chimeric transgene expression). The EV marker Alix and VV antigen A27L were also tested to appropriately indicate EV isolation and VV infection, respectively.

[0401] Figure 3: This shows that PEVs expressing the construct shown in Figure 2A can be isolated using an anti-PD1 antibody, and immunoblots show that the chimeric protein construct is incorporated into the EV, forming a PEV with an externally targeted region. Molecules bound to the anti-PD1 antibody express Alix and Flotirin (EV markers), and these are shown to be incorporated into the EV. Oncolytic vaccinia virus (VV) can program EVs from infected cells with "chimeric blocking constructs" that present PD1 on their surface. Using a VV platform expressing the above-mentioned chimeric PEV construct and its control in Figure 2, 786 cancer cells were infected at an MOI of 1 for 48 hours, and then cell lysates and EVs were isolated. The images in this figure show immunoblots of the EV fraction collected after immunoprecipitation (IP) pulldown of intact EVs using an anti-PD1 antibody. The resulting WCLs are also shown. EVs and WCLs were collected from mock or infected cells (at an MOI of 1 for 48 hours). Furthermore, the membrane was probed with the EV marker Alix and Flotilin 1. Detection of antibody heavy chains due to cross-reactivity was used as a loading control under each condition.

[0402] It is noteworthy that only EVs derived from cells infected with VV expressing the mPD-1-LAMP2B-HA tag construct were pulled down by the anti-PD1 antibody. This data indicates that the construct is not only expressed and incorporated into EVs, but that the "morphology or orientation" of the PEV construct within the EVs is as expected.

[0403] Figure 4 shows that VV can program EV with a "PEV blocking construct" to present PD1 that specifically binds to its binding partner PD-L1.

[0404] Figure 4A: Schematic diagram of the competitive binding ELISA setup performed to obtain the data shown in Panel B: SA-streptavidin; HRP-horseradish peroxidase. In this experimental setup, the biotin-conjugated mPD-L1 protein binds to mPD-1 from the Exo-PD1 construct along with B14R as a control, yielding a quantifiable fluorescence signal; therefore, samples lacking mPD-1 expression (due to the absence of the Exo-PD1 chimera) should not bind to mPD-L1 and yield little to no fluorescence signal. This shows a schematic diagram of the competitive binding enzyme-linked immunosorbent assay (ELISA) experiment used to obtain the data shown in Figure 4B. Here, cell lysates are washed across the entire well plate coated with anti-mPD1 antibody. Thus, constructs presenting mPD1 will bind to the plate. Washing the plate leaves only the bound constructs, which are then incubated with mPD-L1, the biotin-fused native binding ligand for PD1. Following this incubation, 2-SA-HRP (where 2 indicates this is a secondary antibody wash, with PD1 being the primary) is then washed across the entire plate, where it interacts with biotin and generates a quantifiable fluorescent signal. Thus, a well plate presenting functional PD-1 outside of cell lysate EV should exhibit fluorescence, as shown in Figure 4b.

[0405] Figure 4B: To demonstrate whether the Exo-PD1 chimera can bind to mPD-L1, a VV platform expressing the chimeric PEV construct and its control as described herein in Figure 2 was used. Mouse colorectal CT26 cancer cells were infected with a MOI of 10 for 48 hours, and then cell lysates were prepared. The cell lysates were used in a competitive binding ELISA adapted as shown in Panel A. In this binding ELISA, the samples under each condition were first subjected to a bicinchoninate protein assay (BCA) analysis to determine the protein concentration. Based on this analysis, known total input protein concentrations were used for each condition. Each viral treatment included an additional experimental condition (negative control) in which the lysate was pre-incubated for 2 hours before being added to the ELISA setup with an anti-mPD-1 antibody; this was done to block the interaction between mPD-1 and mPD-L1 (of the Exo-PD1 chimera) and to confirm that any fluorescence observed was attributable to this interaction. Absorbance readings relative to the negative control under each condition were plotted as a function of total input protein in the CT26 cell lysate sample, as specified. An additional condition was included in which the cell lysate sample was pre-incubated with anti-mPD-1 antibody for 2 hours. The results shown in Panel B indicate that the maximum observed fluorescence signal corresponds to the VV-Exo-PD1 condition. It is also important to note that the fluorescence signal under the VV-Exo-PD1 condition is lost when the lysate is pre-incubated with anti-mPD-1 antibody (negative control); therefore, it is important to confirm that the observed signal is due to the binding of mPD-1 to mPD-L1. This is further confirmed by the observation that there is little or no difference in absorbance under mock infection and VV control virus conditions (in the presence and absence of anti-PD-1 antibody). *** (P<0.001). This figure shows fluorescence with respect to input protein concentration, demonstrating that vaccinia virus expressing PD1 (without co-expression of anti-PD1) shows increased fluorescence with increasing protein concentration.

[0406] Figure 5 provides a schematic timeline of the experiment over time and data showing that PD-1-expressing PEVs successfully bind to PD-L1 on T cells, thereby activating various mRNA immune markers within these T cells.

[0407] (Panel a, Figure 5): Schematic diagram of the experimental time-course setup for qPCR analysis of T cell activation during PEV treatment, as shown in Panel B. In short, T cells were isolated from mouse spleens; then the cells were activated overnight with anti-CD3e and anti-CD28 antibodies. In parallel, EVs were collected from CT26 cells infected with either a mock virus, VV-Exo control virus, or VV-Exo-PD1 virus (described in Figure 2) at a MOI of 10 for 48 hours. The EVs were purified by fractional centrifugation under each condition and then transferred to activated T cells over 48 hours. RNA was then extracted from the T cells and subjected to qPCR analysis. Further experimental conditions included no transfer of EVs to activated T cells (T cells alone), as well as analysis of TGF-β mRNA levels (this marker serves as a non-targeted control as it is not one of the cytokines known to be modified by the PD1:PD-L1 inhibitory system). Before transferring the EVs, it is important to mention that by normalizing the EVs under each condition, we ensured that equal concentrations were used for each condition.

[0408] Panel B of Figure 5: Data demonstrating that Exo-PD1 EV can activate various immune markers at the mRNA level in mouse T cells. qPCR analysis of mRNA collected from mouse T cells was performed after treatment with equal concentrations of EV. EV was collected from treated CT26-WT cells (mock-infected cells or cells infected with control VV virus or VV-exo-PD1), as shown in the graph. The immune T cell activation markers used in this study were IL-2, IFN-γ, TNF-α, and IL-12. TGF-β was included as a non-targeted control. * P<0.05, ** P<0.01, *** P<0.001.

[0409] Example 2 Multiple targeting of two-part structures (EV-BiKe and EV-BiTe) (without payload) Background: Naturally, major histocompatibility complex (MHC) is necessary for T cells to recognize and kill tumor cells. However, most tumors downregulate MHC expression to evade immune attack. One existing method in the art to circumvent tumor evasion mechanisms involves bispecific antibodies modified to guide T cells and tumor cells to their vicinity. These bispecific antibodies are also called bispecific T-cell engagers or BiTEs.

[0410] These BiTEs can mediate the ability of T cells to recognize and kill tumor cells in an MHC-independent manner. BiTEs consist of variable-chain antibody fragments specifically linked to the T cell antigen CD3 and certain tumor-associated antigens (TAAs). Similarly, bispecific NK cell engagers, or BiKEs, can promote NK cell-dependent killing of tumor cells by mediating the simultaneous binding of activating receptors and surface tumor antigens on NK cells. While existing BiKE and BiTE technologies are promising, many currently in clinical development face challenges such as associated toxicity during systemic administration, drug stability issues (short half-lives), and the challenge of achieving sufficiently high local concentrations to be effective in most solid tumors.

[0411] Application: A PEV construct having two targeting moieties: one that recognizes a T (or NK) cell target, and the other that targets tumor cells (cancer cells or CAFs).

[0412] The mechanism of operation of these platforms: These PEVs facilitate the direct killing of tumor cells by these immune cell types by promoting the pairing between T cells and tumor cells or between NK cells and tumor cells.

[0413] Advantages: Presentation of BiTE and BiKE in PEV format is more stable than bispecific antibody constructs.

[0414] Special features: Generally, PEV constructs without payloads are stable, bispecific cell engagers that bring T or NK cells closer to cancer cells. These PEVs can be generated in vivo or ex vivo.

[0415] Delivery method: OV is used as the delivery medium in the patient, causing infected cancer cells to secrete BiTE and BiKE. In this way, PEV is delivered to the precise site where it is needed, and is therefore likely to be effective at picomolar concentrations, i.e., lower doses than current bispecific antibody methods. Virus-based platforms such as vaccinia virus (abbreviated as VacV or VV), lentivirus, adeno-associated virus [AAV], VSV, and HSV-1 have been available.

[0416] We will prepare the virus and infected cells, and also investigate plasmids (e.g., pcDNA3.1) for producing isolated PEVs.

[0417] Targeted portion: Single-stranded variable fragments or nanobodies as described above. These are, - Tumor cells: Surface tumor antigen targets (e.g., anti-CEA, anti-CA9, anti-FAP, etc.) -T cells: Molecules that bind to T cells (e.g., via CD3 targeting or anti-CD3 scFV targeting moieties), -NK cells: Molecules that bind to NK cell receptor targets (e.g., anti-CD16 or anti-NKG2D) It joins to one of the following.

[0418] Payload: None

[0419] Transmembrane domains: All examples listed in Table 1 were usable. An example included here is the tetraspanin protein, but "multimerization technology" may also be used for single-pass TM proteins (see Special Features for details).

[0420] [Table 11]

[0421] result: Figure 6: EXO-bite PEV constructs targeting CEA on the surface of cancer cells and CD3 on the surface of T cells enhance cancer cell death. HT-29 cells were either transfected or untransfected with the ExoBiTE construct (i.e., an anti-CEA + anti-CD3-CD63 construct presenting antibodies that recognize CEACAM5 and CD3 on the surface of cancer cells and T cells, respectively; a schematic diagram showing the Exo-bite construct in EV and its morphology is shown in the left panel). The cells were then co-incubated with mouse splenocytes (1:3 ratio) for 48 hours or unco-incubated. Note that HT-29 cells transfected with the Exo-bite construct and then co-cultured with splenocytes exhibit enhanced cell death (approximately 50%). See the large enclosed panel.

[0422] The left panel of Figure 6 shows a schematic diagram of an example of a PEV with a tetraspanin-based chimeric construct that targets T cells and cancer cells. The right panel of Figure 6 shows that cell death is enhanced when cells are transfected with a bispecific PEV that directs T cells to cancer cells.

[0423] Example 3 Single-targeting immune adjuvant payload Background / Context: Pharmacological stimulation of innate immune processes represents an interesting way to achieve multiple therapeutic outcomes, including inhibition of viral replication, boosting of antitumor immunity, and enhancement of vaccine immunogenicity. The platforms described herein may represent effective means for enhancing and prolonging cellular and oncological immune responses induced by infectious disease and cancer vaccines, respectively.

[0424] Application: Immunoadjuvant payloads (e.g., STING or ERAdP activators that generate immunogenic molecules to stimulate the immune system) can be specifically delivered to antigen-presenting cells (APCs), such as dendritic cells (DCs), by targeting specific DC surface molecules.

[0425] The mechanism of operation of these platforms: Antigen-presenting cells (APCs), e.g., DCs, often exhibit an immature or immunologically tolerant phenotype (not yet functionally ready to accept presented antigens or act to suppress immune responses). Delivery of immunoadjuvants (i.e., STING or ERAdP pathway activators, e.g., bacterial dinucleotide cyclases, e.g., c-di-AMP cyclases CdaA and MtbDisa, and c-di-GMP cyclase VCA0848, or mouse / human cGAS) to DCs via PEV may result in activation of STING and / or ERAdP, enhancing the antigen-presenting ability of DCs and increasing the expression of T cell co-stimulating molecules, thereby boosting APC activity. In some cases, these platforms can be used in combination with vaccine techniques.

[0426] Advantages: Stability, reduced off-target toxicity, and personalized delivery.

[0427] Targeting moiety: The target is antigen-presenting cell surface molecules including CD40, TNF-α family receptors, DEC-205, type C lectin receptors, and integrin receptor CD11c, via a targeting moiety containing specific monoclonal antibodies, scFv, single-domain antibodies, or nanobodies (i.e., anti-DEC205, anti-Clec9A, anti-CD11c, anti-lectin receptors). Peptides and ligands represent preferred alternatives to antibodies as active targeting agents (e.g., CD40 ligand or CD40-targeted peptide).

[0428] Payload: Bacterial dinucleotide cyclases (i.e., CdaA, etc.) (Note: Since these payloads are enzymes, these examples demonstrate that functionally active enzymes can also be delivered by PEV).

[0429] Transmembrane domains: All examples listed in Table 1 were available. To date, all of our examples have been constructed using VSV-G and CD63.

[0430] Delivery / Preparation Method: Platforms based on viruses such as vaccinia virus, lentivirus, adeno-associated virus [AAV], VSV, and HSV-1 were available. Plasmids for preparing recombinant viruses and transfected cells (e.g., pcDNA3.1), as well as the prepared and isolated PEVs, will also be investigated.

[0431] [Table 12]

[0432] result: Figures 7-10 present Western blots showing the expression of constructs from the pcDNA3.1 plasmid and in cells from VV and EV. Note that HEK293T (human embryonic kidney) cells were selected simply for their ease of transfection and represent several cell types. Note that these figures also represent constructs that act as cancer vaccines, as described below.

[0433] Figure 7: Various chimeric PEV constructs are appropriately expressed upon cell transfection. HEK293T cells were transfected either untransfected or transfected with a specified CD63 plasmid having an anti-DEC205 targeting portion and a different payload [inserted within the second loop of CD63] (all constructs were Flag-tagged at their C-terminus). After 24 hours, cell lysates were collected, immunoblotted against a specified antibody (anti-Flag, or anti-β-actin as a loading control), and probed. Red oval shapes indicate the desired band.

[0434] Figure 8: Appropriate expression of various chimeric PEV constructs during cell transfection. HEK293T cells were transfected either untransfected or transfected with a specified CD63 plasmid (all constructs were Flag-tagged at their C-terminus) having an anti-DEC205 targeting moiety and a different payload [inserted within the first loop of CD63]. After 24 hours, cell lysates were collected and immunoblotted against a specified antibody (anti-Flag, or anti-β-actin as a loading control). Red oval shapes indicate the desired band.

[0435] Figure 9: Various chimeric PEV constructs are appropriately expressed during cell transfection. HEK293T cells were transfected either untransfected or with designated pcDNA3.1 plasmids containing the VSV-G™ domain of the anti-DEC205 targeting region and different payloads (all constructs were Flag-tagged at their C-terminus). After 24 hours, cell lysates were collected, immunoblotted against designated antibodies (anti-Flag, or anti-β-actin as a loading control), and probed. Red oval shapes indicate the desired band.

[0436] Figure 10: Various chimeric PEV constructs are appropriately expressed during cell transfection. HEK293T cells were either untransfected or transfected with specified pcDNA3.1 plasmids (external domain negative and flag-tagged) containing an anti-DEC205 or anti-CLEC9A targeting moiety (VSV-G) and a different payload (CdaA or mCherry). After 24 hours, cell lysates were collected, immunoblotted against specified antibodies (anti-Flag, or anti-β-actin as a loading control), and probed. Red oval shapes indicate the desired band.

[0437] Figures 11–13 show three-panel Western immunoblots demonstrating that isolated PEVs with a chimeric construct targeting Dec205 successfully activate STING or ERAdP in a dose-dependent manner within dendritic cells, along with the VSVG transmembrane domain and CdaA payload (STING and / or ERAdP pathway activator) (Figure 11), but do not activate STING in non-targeted mouse fibroblasts (Figure 12, L929 cells with c-di-AMP and B-DNA positive controls). Delivery of this dinucleotide cyclase to DCs results in activation of the STING signaling pathway, as indicated by phosphorylation of TBK1 (Figure 13). These figures show that STING phosphorylation occurs within DCs (activation).

[0438] Isolated PEVs containing the anti-DEC205-VSVG-CdaA chimeric construct induce STING activation in a dose-dependent manner. The STING (interferon gene stimulant) pathway contributes to the activation of antigen-presenting cells, including DCs. STING activation is mediated by its phosphorylation. In DCs, STING activation is crucial for IFN-β expression and IL-12 production, as well as the surface expression of activation markers CD40 and CD86. The role of the cGAS-STING pathway is important in pathogen detection and cancer immunity. STING activation, and ERAdP activation, appear to be essential components in the recruitment of immune cells into the tumor microenvironment, which is optimal for tumor elimination. STING activation also contributes to adjuvant function during vaccination.

[0439] The data presented here show that only EVs decorated with the anti-DEC205-VSVG-CdaA construct can activate the STING-TBK1-IRF3 signaling pathway in primary mouse dendritic cells after 24-hour treatment with a specified amount of EVs isolated from HEK293T cells transfected with the specified PEV construct and control plasmid pcDNA3.1 (Figure 11), but not in mouse fibroblasts (Figure 12), indicating that while they do not express DEC205 on the cell surface, they are responsive to the classical STING agonist c-di-AMP and B-DNA. Note that STING activation in this specification is demonstrated using an antibody that recognizes STING phosphorylated at serine 365. The loading control includes total STING and β-actin. Figure 13: Activation of the STING-TBK1-IRF3 signaling pathway in primary mouse DCs after 24-hour treatment with EVs isolated from HEK293T cells transfected with the VSVG plasmid as specified. The activation of STING and TBK1 by phosphorylation is demonstrated by Western blotting using phosphorylation site-specific antibodies. Furthermore, all levels of STING and TBK1 are shown as loading controls. STING has been identified as a crucial signaling molecule required for the detection of cytoplasmic nucleic acids, particularly dsDNA derived from pathogens and viruses, as well as endogenous secondary messengers, such as cyclic di-GMP and -AMP. These events lead to the generation of innate immune response genes through the activation (phosphorylation) of the IRF3 and NF-κB pathways by cellular kinases, including TBK1. TBK1 activity is regulated by autophosphorylation of its Ser172. Note that in Panel C, only PEV carrying the anti-DEC205-VSVG-CdaA construct leads not only to the activation (phosphorylation) of STING in mouse dendritic cells, but also to the phosphorylation (=activation) of its downstream molecule (TBK1).

[0440] Example 4 Single-target cancer vaccine payload Background / Situation: Tumor-associated antigens and / or immune reprogramming moieties (e.g., STING or ERAdP pathway activators) can be specifically delivered via PEVs to surface molecules on APCs, such as dendritic cells. This construct would express a targeting moiety for targeting PEVs to DCs (dendritic cells) and could simultaneously deliver one or more payloads.

[0441] Application: These platforms represent an effective means of inducing robust tumor antigen-specific immunity.

[0442] The operation of these platforms: DCs often exhibit immature or tolerant phenotypes. Tumor antigen delivery via PEVs (as payload or cargo), combined with co-delivery of adjuvants (DC maturation stimulants such as anti-CD40mAb agonists, poly(I:C), cytosine-phosphate-guanine (CpG), lipopolysaccharide (LPS), or Toll-like receptor 7 / 8 (TLR7 / 8) agonists), or targeted co-delivery of PEVs with STING or ERAdP pathway activators as described above (e.g., bacterial dinucleotide cyclases, e.g., c-di-AMP cyclases CdaA and MtbDisa, and c-di-GMP cyclase VCA0848), results in enhanced tumor-associated antigen presentation ability and enhanced expression of T cell co-stimulating molecules.

[0443] Tumor-associated antigens, either alone, in combination with an adjuvant, or in combination with an immune reprogramming moiety (e.g., STING or ERAdP pathway activator), can be specifically delivered to surface molecules on dendritic cells via PEV.

[0444] Targeting moiety: Antigen-presenting cell surface molecules, including CD40, TNF-α family receptors, DEC205, type C lectin receptor (CLEC9), and integrin receptor CD11c, are targeted by a targeting moiety containing specific monoclonal antibodies, scFv, single-domain antibodies, nanobodies (i.e., anti-DEC205, anti-Clec9A, anti-CD11c), ligands, or targeted peptides (e.g., CD40 ligand or CD40 targeted peptide).

[0445] Payload: Specific tumor-associated antigens (for proof-of-concept in mouse tumor models: DCT and OVA are under consideration). Human tumor-associated antigens important for clinical trials are available (e.g., HPV-E6 and E7, NY-ESO-1, etc.). Cancer-specific neoantigens are also available.

[0446] The co-expression of specific disease cell antigens, such as tumor-related / specific antigens (e.g., OVA, DCT, mERKm9, etc.), is being investigated. Furthermore, adjuvant molecules such as STING or ERAdP activators can be delivered simultaneously with disease-specific antigens or tumor-related / specific antigens.

[0447] Transmembrane domains: All examples listed in Table 1 were available. To date, all of our examples are constructed with VSV-G.

[0448] Delivery / Preparation Method: Virus-based platforms such as vaccinia virus, lentivirus, adeno-associated virus [AAV], VSV, and HSV-1 were available. Plasmids for transfecting cells (e.g., pcDNA3.1), as well as prepared and isolated PEVs, will also be investigated.

[0449] [Table 13]

[0450] result: Figures 7-9 present Western blots showing the expression of constructs in cells and extracellular life (EVs) from pcDNA3.1 vector plasmids after transfection.

[0451] Figure 14: Panel A of Figure 14 is a Western blot showing the expression of a transmembrane domain based on a dendritic cell-targeting PEV (targeting portion - anti-Dec205) and VSVG, as well as mCherry (control) or OVA (cancer target). Panel B of Figure 14 is an immunofluorescence image showing the cellular expression of anti-DEC205-VSVG-OVA upon transfection of the plasmid encoding this construct. These figures demonstrate that the chimeric PEV construct, which targets DEC205 and delivers the ovalbumin (OVA) antigen, is properly expressed upon cell transfection.

[0452] Panel A of Figure 14: HEK293T cells were transfected with a specified pcDNA3.1 plasmid encoding either an anti-DEC205-VSVG-OVA or an anti-DEC205-VSVG-mCherry control construct. Note that both constructs were HIS-tagged at the N-terminus. Twenty-four hours after transfection, cell lysates were collected and prepared for immunoblotting with specific antibodies against the HIS tag and the loaded control GAPDH.

[0453] Panel B of Figure 14: HEK293T cells were transfected with a specified pcDNA3.1 plasmid encoding the anti-DEC205-VSVG-OVA construct. Note that both constructs were HIS-tagged at the N-terminus. Twenty-four hours after transfection, the cells were fixed and prepared for immunofluorescence staining with anti-HIS antibody (green). The nuclei were stained with DAPI (blue).

[0454] Example 5 Single-target infectious disease vaccine payload Background / Situation: Pathogen-specific antigens and / or immune reprogramming moieties (e.g., STING or ERAdP pathway activators) can be specifically delivered to surface molecules on dendritic cells via PEVs. This construct would express a targeting moiety for regulating PEVs to DCs and could also deliver multiple payloads simultaneously.

[0455] Application: These platforms represent an effective means of inducing robust pathogen-specific antigen-driven immunity.

[0456] The operation of these platforms: As above, DCs often exhibit an immature phenotype. Pathogen-specific antigen delivery via PEVs, combined with co-delivery of adjuvants (DC maturation stimulants such as anti-CD40mAb agonists, poly(I:C), cytosine-phosphate-guanine (CpG), lipopolysaccharide (LPS), or Toll-like receptor 7 / 8 (TLR7 / 8) agonists), or targeted co-delivery of PEVs with STING or ERAdP pathway activators (e.g., bacterial dinucleotide cyclases, e.g., c-di-AMP cyclases CdaA and MtbDisa, and c-di-GMP cyclase VCA0848), results in enhanced pathogen-specific antigen presentation ability and enhanced expression of T cell co-stimulating molecules.

[0457] Targeting moiety: The targets include antigen-presenting cell surface molecules, including CD40, TNF-o family receptors, DEC-205, type C lectin receptors, and integrin receptor CD11c, and are targeted by targeting moieties such as specific monoclonal antibodies, scFv, single-domain antibodies, nanobodies (i.e., anti-DEC205, anti-Clec9A, anti-CD11c), ligands, or targeted peptides (e.g., CD40 ligand or CD40 targeted peptide).

[0458] Payload: Pathogen-specific antigens (e.g., dengue PM and E antigens, malaria CS30, rotavirus VP6, etc.). To boost vaccine activity, we were able to explore the co-expression of specific infection-related antigens with adjuvant molecules such as STING or ERAdP activators.

[0459] Transmembrane domains: All examples listed in Table 1 were available. To date, all of the inventors' examples are constructed with VSV-G.

[0460] Delivery / Production Method: Virus-based platforms (e.g., vaccinia virus, lentivirus, adeno-associated virus [AAV], VSV, etc.), plasmids (e.g., pcDNA3.1), and free PEVs.

[0461] [Table 14]

[0462] result: Figure 15 is an immunofluorescence image showing cell expression of the rotavirus antigen, anti-DEC205-VSVG-VP6, upon transfection of the plasmid encoding this construct. Upon cell transfection, a chimeric PEV construct targeting DEC205 and containing the rotavirus antigen (VP6) is appropriately expressed. HEK293T cells were transfected with a specified pcDNA3.1 plasmid encoding the anti-DEC205-VSVG-VP6 construct. After 24 hours, the cells were fixed and prepared for immunofluorescence staining with anti-HIS antibody (green). The nuclei were stained with DAPI (blue). Note that both anti-DEC205-VSVG-VP6 constructs are HIS-tagged at the N-terminus.

[0463] Example 6 Single-targeting immune reprogramming payload Background / Situation: Immune reprogramming molecules (e.g., cytokines, miRNAs) can be specifically delivered via PEVs to surface molecular targets on specific immune cell populations as payloads or cargo. This construct would express a targeting portion for tailoring PEVs to specific immune cell populations and could also carry multiple payloads simultaneously.

[0464] Applications: These platforms perform specific functions by reprogramming or educating immune cells (e.g., activating them, altering their phenotype, etc.), thus representing an effective means of combating inflammatory diseases and cancer. Furthermore, these PEVs can be used to enhance the visibility (immunogenicity) of cancer cells to immune cells (e.g., by promoting immunogenic cell death).

[0465] Example 6(a): M1 / M2 imbalance The mechanism of operation of these platforms: Immunosuppressive M2 macrophages are transformed into immune-boosting M1 macrophages ready to phagocytose tumor cells. Furthermore, certain subsets of macrophages are important in causing inflammatory diseases such as asthma, atherosclerosis, rheumatoid arthritis, osteoarthritis, endometriosis, type 1 and type 2 diabetes, and obesity. Macrophage reprogramming can be performed by PEV.

[0466] Targeting portion: Single-stranded variable fragments or peptides bound to CD206 (mannose receptor) can be used because they specifically target M2 macrophages (also known as protumor-promoting macrophages).

[0467] Payload: Either without a cargo, or with a payload that specifically captures a cargo modifying macrophage polarization and is an RNA-binding motif for reducing inflammatory gene expression via RNAi, multiple genes can be simultaneously downregulated. Cargo targets may include inflammatory mediators, e.g., cytokines (e.g., TNF-α, IL-6, IL-1β), chemokines (e.g., CCL2, CCL3, CCL5), and transduction targets involved in promoting inflammation, e.g., members of the NF-κB signaling cascade. A miRNA cassette targeting siRNA specific to IκBα, mitogen-activated protein kinase kinase kinase 4 (Map4k4), reduced systemic inflammation by decreasing Tnf-α mRNA in macrophages.

[0468] Example 6(b): Reprogramming the Treq: The mechanism of operation of these platforms: Regulatory T cells (Tregs) are known to limit the function of effector T cells. In relation to cancer, Tregs are potent inhibitors of antitumor immunity, and the presence of these cells in the tumor microenvironment leads to tumor growth. Direct targeting of regulatory molecules in Tregs by PEV leads to the conversion of these cells into IFNg-secreting effector cells (anti-cancer cells).

[0469] Targeting region: A single-stranded variable fragment specific to CTL4 (cytotoxic T lymphocyte-associated antigen 4) on the surface of immunosuppressive T cells.

[0470] Payload: Either no payload with cargo, or a payload that is an RNA-binding motif for specifically capturing cargo that converts immunosuppressive regulatory T cells (Tregs) into anti-cancer T cells by downregulating CARMA1 and / or MALT1. For example, a miRNA cassette having shRNA for CARMA1 and / or MALT1. T cell activation. These miRNA cassettes may be EV-inducible miRNA cassettes in the presence or absence of an RNA sequence corresponding to the RNA-binding motif recognition site of the payload. Alternatively, these miRNA cassettes may be regular non-EV-inducible cassettes containing an RNA sequence corresponding to the RNA-binding motif recognition site of the payload.

[0471] Transmembrane domains: All examples listed in Table 1 were available.

[0472] Delivery / Production Method: Virus-based platforms (e.g., vaccinia virus, lentivirus, adeno-associated virus [AAV], VSV, etc.), plasmids (e.g., pcDNA3.1), and free PEVs.

[0473] Example 6(c): T cell activation The mechanisms of operation of these platforms: T cell dysfunction has been described in chronic viral, bacterial, and parasitic infections, as well as in cancer. CD3-targeted PEVs can be used to stimulate the activity of anti-disease T cells in the immune system.

[0474] Targeting portion: T cell activation: Single-chain variable fragment or single-domain antibody that targets CD3 on T cells.

[0475] Payload: CD3-targeted constructs have no payload. Association with CD3 in T cells may be sufficient to activate them, recruit them, and kill cancer cells. Anti-CD3 monoclonal antibodies (mAbs) initiate signals that lead to T lymphocyte activation via the T cell receptor (TCR), including the phosphatidylinositol pathway, PKC activation, and increased intracellular calcium (Cai2+).

[0476] Example 7 Single-targeting and multi-targeting - EV-CAR-like, cytotoxic payloads Background / Situation: These provide extracellular genes (EVs) that function like targeted cytotoxic T cells (similar to CAR-T therapy, but without the T cells). This allows our PEVs to kill highly immunosuppressive, immunologically "cold" tumors and MHC-I deficient cancers.

[0477] Application: A cytotoxic PEV used as a drug targeting specific tumor cell types, as described in the example below. Other cell types could also be considered.

[0478] Mechanism of Operation of These Platforms: Figure 16 illustrates the proposed mechanism of action of a single-targeted EV delivering a cytotoxic payload by a virus-based platform. Here, vaccinia virus is represented as the single-targeting portion, while this can be extended to other delivery / production modes such as other viruses (lentivirus, adeno-associated virus, vesicular stomatitis virus, etc.) via plasmid expression (i.e., pcDNA3.1) and free PEV. In short, a modified vaccinia virus would infect cancer cells. In infected cells, the viral genome would be transcribed and translated to produce further viral progeny, which would be released to infect adjacent tumor cells, leading to tumor regression or virus-mediated cell death. In parallel, any transgene encoded in the viral genome would be further translated by the infected cells. This transgene consists of a targeting region (purple) fused to a transmembrane linker domain (gray) that will carry a cytotoxic payload (green). The transmembrane domain, i.e., VSVG, preferentially transports the construct back and forth to the PEV, so that the targeting region resides on the extracellular surface of the PEV while the payload is sequestered inside the cell. These PEVs are then secreted by infected cells. The PEVs then bind to target antigens on adjacent cancer cells via the extracellular targeting region, leading to the uptake of these PEVs and the release of their cytotoxic payloads to recipient cells, resulting in the death of antigen-positive target cells.

[0479] Advantages: Non-autologous, stable, specifically targeted, can be manufactured for viral delivery or long-term storage.

[0480] Targeting portion: Single-chain variable fragment or single-domain antibody (i.e., anti-CD19, anti-CD20, anti-CD22, anti-EGFR, anti-FAP, anti-CEA, anti-CA9) or via targeted peptide [i.e., MMP2-targeted chlorotoxin (CTX), proteoglycan-targeted VAR2Δ (VAR2Δ is also called VAR2CSA and binds to different types of chondroitin sulfate (CS) that are exclusively expressed in the placenta and are also found at high rates on cancer cells), GE11 peptide that targets EGFR with high affinity].

[0481] Payload: Cytotoxic payloads such as mouse granzyme B (mGZMB), human granzyme B (hGZMB R201K) (note that the R201K mutation confers resistance to endogenous human granzyme B inhibitors), diphtheria toxin (DT), TRAIL (a cytokine that primarily causes cell death in tumor cells), and cleaved Pseudomonas exotoxin 38 (PE38).

[0482] Transmembrane domains: All examples listed in Table 1 were available.

[0483] Delivery / Production Method: Virus-based platforms (e.g., vaccinia virus, lentivirus, adeno-associated virus [AAV], VSV, etc.), plasmids (e.g., pcDNA3.1), and free PEVs.

[0484] [Table 15]

[0485] [Table 16]

[0486] [Table 17]

[0487] [Table 18]

[0488] result: Figure 17 shows a schematic diagram of a chimeric fusion construct having a single-stranded variable fragment (scFv) targeting moiety that targets carcinoembryonic antigen (CEA) or carbonic anhydrase IX (CA9), along with a VSVG transmembrane domain and an mGZMB payload. Purple: single-stranded variable fragment (scFv) consisting of heavy and light chains, or single-domain antibody, or nanobody, or targeting domain of the construct by other targeting modes; gray: single-pass transmembrane linker domain (VSV-G); green: granzyme B payload. Note that the targeting moiety may also consist of peptides other than scFv, single-domain antibodies, or nanobodies, such as MMP2-targeted chlorotoxin or proteoglycan-targeted VAR2Δ. His and Flag function as tags for visualizing and tracking the expression of the chimeric construct.

[0489] Figure 18 shows immunoblots demonstrating successful expression of the construct shown in Figure 17 in small extracellular viable cells (EVs) isolated from the pcDNA3.1 plasmid during transfection of HEK293T cells, from viral infection of human osteosarcoma U2OS cells, and from virally infected 786-O human kidney cell adenocarcinoma cells. The negative control used in these experiments was eGFP expressed at the site of the chimeric construct sequence. This demonstrates successful plasmid-mediated and viral-mediated PEV expression in transfected and infected cells, respectively, and their successful embedding in EVs derived from transfected and infected cells. All blots were probed with anti-granzyme B antibody. The blots show the expression of anti-CEA-VSVG-mGZMB and anti-CA9-VSVG-mGZMB in the following: Left blot - 293T cells transfected with the pcDNA3.1 plasmid encoding the construct. Intermediate blot - U2OS human osteosarcoma cells infected with vaccinia virus (VACV) encoding these constructs. Right blot - Small extracellular vesicles (sEVs) isolated from virus-infected 786-O human renal cell adenocarcinoma cells.

[0490] As expected, no signal was obtained in cells infected with VACV-eGFP, which does not express granzyme B protein at all.

[0491] These blots not only show chimeric granzyme B fusion constructs expressed by plasmids, but also demonstrate successful sorting and packaging in sEVs via transmembrane VSVG linkers.

[0492] Figures 19A, 19B, and 19C show that cancer cells displaying CEA or CA9, infected with vaccinia virus expressing the PEVs shown in Figures 17 and 18, enhance cell death mediated by cytotoxic payloads delivered by PEVs that target CEA or CA9 on recipient cells, respectively.

[0493] Figure 19A: Cytotoxicity of vaccinia virus encoding a single-target PEV derived from either human CEA or CA9 carrying a cytotoxic granzyme B payload in human colon cancer cell line (HT-29), known to express high levels of CEA and CA9 on its surface. In short, HT-29 cells were infected with the individual viruses at an MOI of 0.1. Cell viability was assessed 48 hours after infection using the Alamar Blue® viability assay (n=4 biological replicas). While the eGFP control virus certainly induced cell death, the degree of cell death was significantly greater in this cell line after infection with CEA-targeted and CA9-targeted viruses. This was as expected given the fact that HT-29 expresses both CEA and CA9 antigens at the cell surface level.

[0494] Figure 19B: HCT116 cells transfected with hCEA-VSVG-mGZMB produce PEVs in the supernatant upon transfection, but do not die because they do not express CEACAM5, which is required for EV uptake. In contrast, transfected HT-29 cells that express CEACAM5 produce EVs in the supernatant upon transfection, but the majority of the cells (approximately 60%) die because they take up EVs containing GZMB.

[0495] Figure 19C: Quantification of cell viability of cells overexpressing MDA-MB-231 and MDA-MB-231-CA9, which are killed upon taking up PEVs containing hCEA-VSVG-mGZMB and CA9-VSVG-mGZMB generated during plasmid transfection of a specified cell line. The cells do not die upon exposure to a PEV control lacking mGZMB or the target antibody.

[0496] Figure 20 shows that two cancer cell lines exhibit enhanced cell death upon exposure to PEVs that present VAR2Δ and transport mGZMB. Cells were transfected with a plasmid expressing a VAR2-VSVG-mGZMB chimeric construct. The cell viability of HT-29 (human colorectal cancer cell line) and BxPC3 (human pancreatic cancer cell line) cells, which die upon taking up PEVs containing VAR2-VSVG-mGZMB generated during plasmid transfection, was quantified.

[0497] Figure 21 is a schematic diagram showing the method for supernatant transplantation (see Figure 6 for data and results). In short, on day 0, 786-O cells are seeded to reach a concentration density by the following day. Next, the cells are infected with (1) VACV-eGFP, (2) VACV αCEA-VSVG-mGZMB, or (3) VACV αCA9-VSVG-mGZMB at MOI=0.1. These cells are considered producer cell lines for EVs. Next, 2 hours after infection, the medium on the cells is replaced with DMEM + 10% exosome-depleted FBS. Next, the plates are incubated at 37°C + 5% CO2 for 48 hours (at which point viral replication and the generation of chimeric granzyme B constructs and their subsequent packaging and secretion in EVs are allowed). After 48 hours, the supernatant is collected and centrifuged at 4°C, 2000×g for 20 minutes to remove cell debris and dead cells. Next, the supernatant is passed through a 0.2 μm filter, and vaccinia virus is removed by size exclusion, so that the filtered supernatant is virus-free and contains only extravasation germ cells (EVs). Then, the supernatant is transferred to the recipient cell line.

[0498] Figure 22 shows the results of the supernatant transplantation experiment described above with two PEV constructs (vaccinia virus expressing anti-CEA-mGZMB with a VSVG transmembrane domain, and vaccinia virus expressing anti-CA9-mGZMB with a VSVG transmembrane domain). The controls were uninfected cells and vaccinia virus expressing eGFP. This was done according to the method described in Figure 21. All PEV constructs express eGFP. There are three controls (uninfected, eGFP only, and anti-CA9 construct). Only MC38-CEA cells (a mouse colorectal cancer cell line genetically modified to express human CEA) exhibiting observable cell death received supernatant containing hCEA-VSVG-mGZMB PEV (third panel), which is compared to mocked control MC38 cells (uninfected and eGFP only) in the two left panels, and to CA9-targeted PEV (fourth panel from the left; note that MC38-CEA does not express human CA9). The green channels are shown to demonstrate that no infectious viral particles passed through during the filtration step (eGFP is expressed from all vectors) and that the observable results are from supernatant transplantation alone. The fourth panel further demonstrates that CA9-targeted PEV is ineffective, as the target cells (MC38-CEA) do not have CA9 expressed on their surface.

[0499] Figure 23 shows another supernatant transplantation experiment using supernatant from 786-O cells transfected with the anti-CEA-VSVG-mGZMB plasmid, or supernatant from untransfected 786-O cells as a negative control. The supernatant is transferred to wild-type (CEA-negative) or CEA-expressing MC38 cells and CEA-expressing HT-29 cells. 786-O cells are used as a PEV producer cell line and are transfected with the hCEA-VSVG-mGZMB plasmid. After 24 hours, the supernatant (i.e., containing PEV) is transferred to either a cell line expressing or not expressing the target antigen, in this case hCEA. MC38 WT cells were CEA-negative and showed no significant difference after receiving mock supernatant from untransfected 786-O cells versus supernatant from 786-O cells transfected with the hCEA-VSVG-mGZMB plasmid. Both MC38-hCEA and HT-29 cells are CEA-positive cell lines, and when they received supernatant from 786-O transfected with hCEA-VSVG-mGZMB, they showed significant cell death compared to mock supernatant, suggesting specificity of the targeting portion of PEV (in the supernatant) to target cells.

[0500] Figure 24 shows a supernatant transplantation experiment using mCherry as a payload in PEVs targeting CEA on cells that are either CEA-negative or CEA-positive. CEA-negative 293T cells were transfected with a plasmid encoding hCEA-VSVG-mCherry to generate hCEA-VSVG-mCherry PEVs (top panel). Supernatants from these cells were collected and used to treat new 293T (CEA-negative, middle panel) and HT-29 (CEA-positive, bottom panel) cells for 24 hours. CEA-positive cells showed significant mCherry signaling, suggesting that PEVs are incorporated only by target cells (CEA-positive).

[0501] Figure 25 shows a schematic diagram illustrating the EV-CAR (chimeric antigen receptor) platform when PEVs are generated from donor cells. A producer cell line, prepared to stably express the EV-CAR construct (e.g., through retroviral transduction), will generate EVs that carry the desired construct. In this example, the construct consists of a CD63 transmembrane domain scfold (tetraspanin), at least one single-stranded variable fragment (scFv) targeting moiety specific to tumor-associated antigens (TAAs) (e.g., CD19, CD20), and a cytotoxic payload (e.g., granzyme B). The PEVs generated by the producer cell line can be isolated and administered to patients. When the PEV (EV-CAR) reaches tumor cells, TAA recognition leads to receptor-mediated endocytosis, which phagocytoses the PEV. After uptake, the PEV releases the chimeric construct / EV contents and, consequently, the cytotoxic payload, resulting in the induction of apoptosis in tumor cells. The diagram provides a specific example of targeting cancer cells.

[0502] Figure 26 shows schematic diagrams of different constructs having a tetraspanin CD63 transmembrane domain, including one construct with two targeting regions and a single payload, two constructs with one targeting region and a single payload (where the targeting regions are located at different positions within the construct), one construct with two targeting regions and no payload, and a control construct without targeting regions and with a single payload.

[0503] Figure 27 shows a Western blot probed for granzyme B to demonstrate the successful protein expression of the full-length construct shown in Figure 26, expressed by the plasmid.

[0504] Figure 28 shows schematic diagrams of various bispecific tetraspanin-based chimeric constructs that have been prepared and are undergoing experimental validation. These contain six different payloads, one of which is a construct having a furin-cleavable site in a second payload on the opposite side of the construct.

[0505] Example 8 Single-targeting and multi-targeting - Tumor cell programming payloads Background / Situation: The reprogramming portion can be specifically delivered as a free cargo (therapeutic miRNA, mRNA) or by binding to an RNA-binding protein / domain payload (e.g., RNA-binding protein / domain MS2, CAS13, or others) linked via a PEV to a surface molecular target on a specific tumor (e.g., immune cell population, CAF, or cancer cells). This construct would express a targeting portion to adjust the PEV to the desired cell type, and could carry one or more payloads simultaneously, and / or these constructs could be combined with specific cargo having corresponding sequences.

[0506] Applications: These platforms perform specific functions by reprogramming or educating tumor-resident cells (e.g., activating them, altering their phenotype, etc.), and thus represent an effective means of combating cancer.

[0507] The operating modes of these platforms are as follows: For example, these PEVs can be used to enhance the visibility (immunogenicity) of cancer cells to immune cells (e.g., to promote immunogenic cell death), or to reprogram T cells as CAR-T cells in the tumor microenvironment, in situ.

[0508] Special features: A "nucleic acid ligand system" between an "RNA-binding payload (e.g., MS2, CAS13)" and a therapeutic RNA molecular cargo (i.e., mRNA, IncRNA, microRNA) having a "matching" RNA-binding motif (RNA ligand domain) bound by the RNA-binding payload.

[0509] Targeted portion: All examples listed above.

[0510] Payload: RNA-binding protein or its RNA-binding motif (e.g., Cas13, MS2 coat protein, Staufen-1, human Pumilio homology domain 1).

[0511] Transmembrane domains: All examples listed in Table 1 were available.

[0512] Delivery / Production Method: Virus-based platform (e.g., vaccinia virus, lentivirus, adeno-associated virus [AAV], VSV, etc.), plasmid (e.g., pcDNA3.1), and free PEV.

[0513] result: Figure 29C shows that PEVs containing CTX-VSVG-Nanoluc® specifically reprogram recipient cells and become "luminescent" during the enzymatic reaction with luciferin (substrate) (Nanoluc® is the enzyme). Note that Nanoluc® is an enzyme, and this data indicates not only targeted delivery of Nanoluc® via PEVs but also delivery of functional enzymes.

[0514] Figure 13 shows that the CdaA enzyme can be specifically delivered to DCs via PEV, and that the enzyme is functionally active if it is within the recipient cell.

[0515] Example 9 Simultaneous manifestation of cargo (can be combined with various PEV structures) Background / Context: The term "cargo" is defined herein as a molecule that is co-expressed but is not part of a chimeric protein construct. As such, a cargo may be included / co-expressed with or without the presence of a payload in the construct. A cargo may be a nucleic acid or protein that preferentially leads to an EV, and / or an RNA that may contain a specific sequence recognized by a payload that recognizes a particular RNA contained in the PEV construct.

[0516] Applications: Any application is possible where targeting of molecules to target cells may be suitable, especially when they cannot maintain activity / function within the PEV construct. Otherwise, specific delivery of EV cargo molecules may be facilitated.

[0517] Special features: The RNA may have either an RNA-binding motif recognition site for binding to an RNA-binding motif payload, or a motif that leads to an extracellular gene (EV). The protein may be preferentially led to the EV by specific sequences (for which targeting is known in the art).

[0518] Targeted area: Varies depending on the situation.

[0519] Payload: RNA-binding motif, for example, if the cargo has an RNA-binding motif recognition sequence.

[0520] To generate EVs that simultaneously transport a controlled, specific nucleic acid molecule of interest (cargo; e.g., microRNA and mRNA), a PEV can be designed to transport a payload having one or more RNA-binding domains (RBDs). The RNA cargo presents a binding motif recognized by the RBD (also called the RNA ligand domain), and therefore can specifically interact and be transported by the RBD-containing PEV ("RNA nucleic acid ligand system" system). For example, any well-characterized RNA-binding domain found in cellular RNA-binding proteins, such as RRM, KH, cold-stimulated domain (CSD), and Zn finger CCHC domain, can be used. Similarly, RNA-binding domains found in viral coat or capsid proteins (e.g., MS2 bacteriophage coat protein) or bacterial RNA-binding Cas proteins (e.g., Cas13) can be designed as part of the payload in a PEV construct that transports RNA or functions as a nucleic acid ligand system. The congeneral RNA-binding ligand will be included in the RNA cargo molecule.

[0521] Transmembrane domains (PEVs) may have any transmembrane domains that conform to the other categories outlined in this document, while cargo is not part of the chimeric constructs described herein.

[0522] [Table 19]

[0523] Example 10 Contrast structures These constructs are used as controls for various experiments across multiple categories. Some of these are independently proof-of-concept constructs, such as functional payload delivery (mCherry or Nanoluc®) or placement of target molecules within tetraspanin transmembrane domains for a single target.

[0524] [Table 20]

[0525] [Table 21]

[0526] [Table 22]

[0527] [Table 23]

[0528] result: Figures 29A, 29B, and 29C are proof-of-concept demonstrating that a PEV (PEV targeting portion: CTX) targeting MMP-2 on the surface of cancer cells can deliver a functional payload, in this case the enzyme Nanoluc®.

[0529] Figure 29A: HEK293T cells were either untransfected or transfected with a specified PEV plasmid (flag-tagged) in the presence or absence of a chlorotoxin (CTX) targeting moiety and reporter payload (Nanoluc®). All constructs had a VSVG transmembrane domain and a FLAG tag at the C-terminus. Cell lysates were collected 24 hours after transfection and immunoblotted against a specified antibody (Flag, or β-actin as a loading control). Data show the expression of the desired experimental construct (CTX-VSVG-Nanoluc®) and its respective negative control [VSVG-Nanoluc® (without targeting moiety) or CTX-VSVG (without payload)].

[0530] As shown in Figure 29B:(A), HEK293T cells were transfected and cultured in EV-depleted medium. After 24 and 48 hours, the supernatant was collected and the luminescence level was measured using a luminometer and a standard luciferase detection assay. Abbreviations: UT: untransfected; VC: CTX-VSVG construct; VN: VSVG-Nanoluc® construct; CVN: CTX-VSVG-Nanoluc® construct. Note that the Nanoluc® signal was detected only in the supernatant (containing EVs) derived from cells transfected with VSVG-Nanoluc® and CTX-VSVG-Nanoluc® constructs.

[0531] Figure 29C: 48 hours after transfection, various human glioblastoma cell lines were treated with supernatant collected from transfected HEK293T cells. Eight hours after transfer to conditioning medium, luminescence in cell lysates was measured using Nanoluc® substrate (luciferase assay) and a luminometer. Note that the uptake of EVs presenting CTX-VSVG-Nanoluc® was enhanced, particularly in U87MG cells. This cell line expresses high levels of MMP-2 (the protein to which CTX binds).

[0532] Example 10 Viral infection enhances EV secretion.

[0533] It has been demonstrated that viral infections (e.g., vaccinia virus infection) enhance the secretion of small extravasation cells (EVs).

[0534] Figure 30 shows a graph illustrating the analysis of the entirety of small extracellular organisms (EVs) generated from 786-O cancer cell lines that were either uninfected (mock) or infected with vaccinia virus (VacV), using a nanoparticle tracking analysis system (ZetaView® software). * P<0.05, ** P<0.01.

[0535] Figure 31 shows Western blot analysis of extracellular vesicles (EVs) and whole cell lysates (WCLs) from Vero, 786-O, and HT-29 cells 48 hours after infection, following mock infection (M) or CopWT infection (VACV) (V) at a MOI of 1. The membranes were probed for EV markers (Alix, TSG101, and Flotilin 1), cell / non-EV markers (GM130, Calreticulin, Tom20), and the A27L protein of VacV.

[0536] Example 11 Alternative viral glycoproteins as EV-inducible transmembrane polypeptides result: Figure 32 shows a Western blot illustrating the expression of four different constructs in the isolated EV fraction at the time of cell transfection, demonstrating that viral glycoproteins (G) derived from VSV, LCMV (lymphocytic choriomeningitis virus), Lassa (Lassa fever virus), and Junin virus (also known as Argentinian mammarenavirus) can be used as transmembrane domains to transport the payload (Flag tag) back and forth within the EV. In short, HEK293T cells were transfected with pCDNA3.1 plasmids expressing VSV-G, LCMV-G, Lassa virus-G, Junin virus-G, or an empty vector as a control (mock). After 48 hours, cell lysates were collected using RIPA buffer, and EVs were collected from the supernatant of the transfected cells using EXO-quick-TC reagent (System Biosciences) according to the manufacturing protocol. Next, cell lysates and purified extracellular proteins (EVs) were subjected to SDS-PAGE and Western blotting using an anti-Flag antibody. Notably, all glycoprotein constructs displayed Flag tags at their C-terminus, facilitating detection.

[0537] Figure 33 shows a Western blot illustrating the expression of the construct in the isolated EV fraction at the time of cell transfection, demonstrating that the viral glycoprotein derived from SARS-CoV-2 can be used as a transmembrane domain to transport the payload back and forth within the EV. In short, HEK293T cells were transfected with a pCDNA3.1 plasmid expressing either the SARS-CoV-2 Spike protein or an empty vector as a control (mock). After 48 hours, cell lysates were collected using RIPA buffer, and EVs were collected from the supernatant of the transfected cells using EXO-quick-TC reagent (System Biosciences) according to the manufacturing protocol. Next, the cell lysates and purified EVs were SDS-PAGE and Western blotted with an anti-Spike antibody.

[0538] Figure 34 shows a Western blot illustrating the expression of four different constructs in the isolated EV fraction at the time of cell transfection, demonstrating that viral glycoproteins derived from Tamiami, Guanarito, Parana, Machupo, and Sabia viruses can be used as transmembrane domains to transport the payload (Flag tag) back and forth within the EV. In short, HEK293T cells were transfected with pCDNA3.1 plasmids expressing Tamiami virus-G, Guanarito virus-G, Parana virus-G, Machupo virus-G, Sabia virus-G, or an empty vector as a control (mock). After 48 hours, cell lysates were collected using RIPA buffer, and EVs were collected from the supernatant of the transfected cells using EXO-quick-TC reagent (System Biosciences) according to the manufacturing protocol. Next, the cell lysates and purified EVs were SDS-PAGE and Western blotted with anti-HA antibody. Notably, all glycoprotein constructs present HA tags at their C-terminus, facilitating detection.

[0539] Example 12 Simultaneous expression of cargo As described above in Example 9.

[0540] Background / Situation: Cargo can be contained / concurrently expressed, regardless of whether or not there is a payload within the structure.

[0541] Applications: Any application is possible where targeting of molecules to target cells may be appropriate, especially when they cannot maintain activity / function within the PEV construct. Otherwise, specific delivery of EV cargo molecules to target cells may be facilitated.

[0542] Special features: The cargo RNA molecule may have either an RNA-binding motif recognition site for binding to an RNA-binding motif payload, or a motif that leads to an extracellular gene (EV). The protein may be preferentially directed to the EV by specific sequences (for which targeting is known in the art).

[0543] Targeted area: Varies depending on the application.

[0544] Payload: RNA-binding motif, for example, if the cargo has an RNA-binding motif recognition sequence.

[0545] To generate EVs that simultaneously transport a controlled, specific nucleic acid molecule of interest (cargo, e.g., microRNA and mRNA), a PEV can be designed to transport a payload having one or more RNA-binding domains (RBDs). The RNA cargo presents a binding motif recognized by the RBD (also referred to as the RNA ligand domain), and therefore can specifically interact with and be transported by the RBD-containing PEV ("RNA nucleic acid ligand system" system). For example, any well-characterized RNA-binding domain found in cellular RNA-binding proteins, such as RRM domains, K homology (KH) domains, cold-stimulated domains (CSDs), and Zn finger CCHC domains, can be used. Similarly, RNA-binding domains found in viral coat or capsid proteins (e.g., MS2 bacteriophage coat protein) or bacterial RNA-binding Cas proteins (e.g., Cas13) can be designed as part of the payload in a PEV construct that transports RNA or functions as a nucleic acid ligand system. The homologous RNA-binding ligand will be included in the RNA cargo molecule.

[0546] Transmembrane domains (PEVs) may have any transmembrane domains that conform to the other categories outlined in this document, while cargo is not part of the chimeric constructs described herein.

[0547] result: Table 12 presents amino acid motifs (RNA-binding motifs) experimentally shown to specifically bind to certain target RNA sequences in Figure 35. The RNA sequences are selectively recognized by each of these RNA-binding motifs and, as determined by bioinformatics analysis, are not present in the human genome. These RNA-binding motifs can be used as payloads, as described in the present invention and as shown in the figures below.

[0548] [Table 24]

[0549] Table 25

[0550] Table 26

[0551] Table 27

[0552] Table 28

[0553] Table 29

[0554] Table 30

[0555] Table 31

[0556] Table 32

[0557] Figure 35A. Quantitative readings of Nanoluc® activity in recipient cells educated with EVs loaded with different PEV constructs. Next, by quantifying Nanoluc® activity, we demonstrated that a plasmid containing an LDLRT (LDLR-targeted)-VSVG transmembrane domain (VSVG™) fused to an RNA-binding motif can package mRNA encoding blue fluorescent protein (BFP) fused to Nanoluc® (Nluc) and deliver it to LDLR-positive recipient cells on the cell surface. In short, these experiments used the Transwell® system, where HEK293T cells seeded in Transwell® inserts (represented as donor cells in the diagram) were given LDLR-VSVGTM-dCas13a+gRNA motif-BFP_NLuc or LDLR-VSVGTM-dCas13a+BFP_NLuc-3XCBD or LDLR-VSVGTM-dCas13d+gRNA motif-BFP_NLuc or LDLR-VSVGTM-dCas13d+BFP_NLuc-3XCBD or LDLR-VSVGTM-Pum+BFP_NLuc-3XPBD or LDLR-VSVGTM-Stuf+BFP_NLuc-3XSBD or LDLR-VSVGTM-SD-VEEV+BFP_NLuc-3XPS or LDLR-VSVGTM-L72AE+BFP_NLuc-3XC / D Plasmids expressing Box or LDLR-VSVGTM-MS2+BFP_NLuc-3XHAM were co-transfected. After transfection, Transwell® was incubated with recipient cells (seeded at the bottom of the Transwell® system) for 24 hours, and then cells (recipient cells) from the bottom compartment of the Transwell® system were collected, and Nanoluc® activity in the recipient cells was measured according to the manufacturer's proposed protocol (Promega).

[0558] Figure 35B. Representative fluorescence microscope image from the same experiment shown in Figure 35A. After transfection as shown in Figure 35A, the Transwell® was incubated with recipient cells (seed at the bottom of the Transwell® system) for 24 hours. Cells in the bottom and upper compartments of the Transwell® system were then imaged using an EVOS fluorescence microscope to detect BFP expression (shown as a light gray signal in the figure instead of actual blue fluorescence) not only in the transfected cells (donor) but also in the recipient cells. Notably, Figure 35B shows a bright-field image of the recipient cells, demonstrating that although the cells were under mock control conditions, there was no BFP expression.

[0559] Example 13 Single targeting with cytotoxic payloads As described above in Example 7.

[0560] Application: Any application is possible where targeting of cytotoxic molecules [e.g., granzyme B (GZMB)] to target cells may be appropriate. In this example, the targeted cells are cancer-associated fibroblasts or activated fibroblasts, cancer cells, and tumor samples from pancreatic cancer patients expressing fibroblast-activating protein (FAP).

[0561] result Figure 36 shows the cell viability of fibroblast-activating protein (FAP)-positive pancreatic fibroblasts (PanFib), pancreatic cancer cells (BxPC3), and samples from pancreatic cancer patients (P025, P032) treated with EV cells loaded with anti-FAP-VSVG-mGZMB. In short, HEK293T cells were either transfected with a plasmid expressing anti-FAP-VSVG-mGZMB or left untransfected as a mock-negative control. After 24 hours, the supernatant was collected from the transfected cells, and cell debris was pre-removed by centrifugation at 1000×g for 10 minutes. Next, the supernatant was transferred to various cell types known to express FAP, and cell viability was measured using Alamar Blue according to the manufacturer's instructions. The viability of cells exposed to supernatant derived from donor cells transfected with anti-FAP-VSVG-mGZMB was calculated as the change in viability (%) compared to cells exposed to negative control supernatant.

[0562] Example 14 Single-target immune adjuvant payload As described above in Example 3.

[0563] Figure 37 shows that EVs loaded with the anti-DEC205 targeting moiety and the CdaA payload [inserted into the first loop of CD63] (their expression is shown in Figure 8) are efficient in activating the STING pathway in isolated primary dendritic cells. HEK293T cells were transfected with the PEV construct aDEC205-CD63D-CdaA-Flag in the pcDNA3.1 vector (indicated as CD63 EV in the figure) or left untransfected. After 48 hours, the EVs were purified by serial ultracentrifugation and stored at -80°C until use. Bone marrow progenitor cells from the femoral and tibia of C57BL / 6 mice were cultured in RPMI with mouse GM-CSF (PeproTech#315-03, final concentration 40 ng / ml) for 6 days. On day 7, differentiated DCs were seeded and treated with either untransfected exosomes or the specified CD63 EV for 24 hours. Subsequently, STING phosphorylation (S365) in the treated DCs was evaluated by Western blotting using a STING phosphorylation state-specific antibody (Cell Signaling Technology #72971). Total STING protein levels were evaluated using a STING antibody (Cell Signaling Technology #13647). β-actin was used as a loading control.

[0564] Example 15 Single-targeting immune reprogramming payload As described above in Examples 3, 4, and 6a.

[0565] Application: Any application is possible where targeting of the “immune reprogramming” molecule to target immune cells may be appropriate. In this example, the targeted cells are primary macrophages. In this particular example, macrophages are treated with PEV construct-loaded EVs, which specifically target macrophages (via the anti-Marco targeting moiety) and simultaneously deliver the bacterial enzyme CdaA, a STING pathway activator. It is noteworthy that macrophages, in particular tumor-associated macrophages (TAMs) characterized by high levels of MARCO on their surface, are known in the literature to be reprogrammed (or polarized) into a pro-inflammatory phenotype upon STING activation.

[0566] result Figure 38. This figure shows a Western blot illustrating the activation of STING in activated primary macrophages treated with EVs loaded with three different PEV constructs, as described below. In short, anti-MARCO-VSVG™-CdaA, anti-MARCO-SARS2™-CdaA, or anti-MARCO-CdaATM-CdaA vectors were expressed in HEK293T cells. After 48 hours, the supernatant was collected and EVs were isolated by serial ultracentrifugation. The pelleted EVs were resuspended in PBS and stored at -80°C until use. Bone marrow progenitor cells from the femur and tibia of C57BL / 6 mice were cultured for 6 days in DMEM containing mouse M-CSF (PeproTech#315-02-25 ng / ml final concentration). On day 7, differentiated macrophages were seeded and either left untreated or pretreated with 5 EU units / ml of lipopolysaccharide (LPS) for 24 hours to induce MARCO expression on the surface of these cells. Subsequently, the pretreated macrophages were either left untreated or treated with various EV formulations for 24 hours. STING phosphorylation (S365) in the treated macrophages was then evaluated by Western blotting using a STING phosphorylation state-specific antibody (Cell Signaling Technology #72971). As a control, total STING protein levels were evaluated using a STING antibody (Cell Signaling Technology #13647). β-actin was used as a loading control.

[0567] Figure 39 shows experiments demonstrating the efficacy of various anti-Marco-linked CdaA PEV constructs (i.e., anti-MARCO-VSVG™-CdaA, anti-MARCO-SARS2™-CdaA, or anti-MARCO-CdaATM-CdaA vectors) in stimulating the interferon (IFN) signaling pathway. The functional efficacy of various MARCO-CdaA fusion proteins of the bacterial cyclase CdaA was evaluated for generating a c-di-AMP-STING signaling response. For this purpose, HEK293T cells were transfected for 24 hours with various MARCO plasmids, either mock or as specified. Subsequently, lysates containing c-di-AMP were prepared and applied to THP1-Blue-ISG IRF (IFN regulator) reporter cells (InvivoGen) to stimulate the STING-IRF signaling pathway for 24 hours, enabling the Quanti-Blue assay according to the production protocol.

[0568] Example 16 Multiplex targeting bipartite construct (EV-BiTE) As described above in Example 2.

[0569] Background: Major histocompatibility complexes (MHC) are necessary for T cells to recognize and kill tumor cells. However, most tumors evade immune attack by downregulating MHC expression. One existing method in the art to circumvent tumor evasion mechanisms involves bispecific antibodies modified to guide T cells and tumor cells to immediate proximity. These bispecific antibodies are also known as bispecific T-cell engagers or BiTEs.

[0570] BiTEs can mediate the ability of T cells to recognize and kill tumor cells in an MHC-independent manner. BiTEs consist of variable-chain antibody fragments specifically linked to the T cell antigen CD3 and certain tumor-associated antigens (TAAs). Similarly, bispecific NK cell engagers, or BiKEs, can promote NK cell-dependent killing of tumor cells by mediating the simultaneous binding of activating receptors and surface tumor antigens on NK cells. While existing BiKE and BiTE technologies are promising, many currently in clinical development face challenges such as associated toxicity during systemic administration, drug stability issues (short half-life), and the challenge of achieving sufficiently high local concentrations to be effective in most solid tumors.

[0571] Application: A PEV construct having two targeting moieties: one that recognizes a T cell target and the other that targets tumor cells (cancer cells or CAFs).

[0572] The mechanism of operation of these platforms: These PEVs facilitate the direct killing of tumor cells by these immune cell types by promoting the pairing of T cells and tumor cells.

[0573] Advantages: Presentation of BiTE and BiKE in PEV format is more stable than bispecific antibody constructs.

[0574] Special features: Generally, PEV constructs without payloads are stable, bispecific cell engagers that bring T or NK cells closer to cancer cells. These PEVs can be generated in vivo or ex vivo.

[0575] Delivery method: A tumor-selective virus is used as the delivery medium in the patient, causing infected cancer cells to secrete BiTE and BiKE. In this way, PEV is delivered to the precise site where it is needed, and is therefore likely to be effective at picomolar concentrations, i.e., lower doses than current bispecific antibody methods. Virus-based platforms such as vaccinia virus, lentivirus, adeno-associated virus [AAV], VSV, and HSV-1 have been available.

[0576] We will prepare the virus and infected cells, and also investigate plasmids (e.g., pcDNA3.1) for producing isolated PEVs.

[0577] Targeting portion: Single-stranded variable fragments or nanobodies as described above. These bind to tumor cells via surface tumor antigen targets (e.g., anti-CEA, anti-CA9, anti-FAP, etc.) and to T cells via molecules that bind to T cells (e.g., CD3 targets, via anti-CD3 scFV targeting portions).

[0578] Payload: None

[0579] Transmembrane domains: All examples listed in Table 1 were usable. Examples included here include tetraspanin proteins, but "multimerization techniques" may also be used for single-pass TM proteins (see Special Features for details).

[0580] result Figure 40 shows cell viability in MC38 (WT and CEA-expressing) cells (spleen:MC38 ratio 10:1) co-cultured with mouse splenocytes in the presence of naive EVs or EVs decorated with αCD3-VSVG, αCD3-αFAP-CD63, or αCD3-αCEA-CD63. Exo-BiTE constructs on EVs enhance cell death in splenocytes compared to naive EVs. In short, vectors encoding αCD3-VSVG, αCD3-αFAP-CD63, or αCD3-αCEA-CD63 were expressed in HEK293T cells. After 48 hours, the supernatant was collected and EVs were isolated by serial ultracentrifugation. The pelleted EVs were resuspended in PBS and stored at -80°C until use. Splenocytes were collected from homogenized mouse spleens and used fresh.

[0581] Example 17 Single-target cancer vaccine payload As described above in Examples 3 and 4.

[0582] Background / Situation: Tumor-associated antigens and / or immune reprogramming moieties (e.g., STING or ERAdP pathway activators) can be specifically delivered via PEVs to surface molecules on APCs, such as dendritic cells. This construct would express a targeting moiety for targeting PEVs to DCs (dendritic cells) and could simultaneously deliver one or more payloads.

[0583] Application: These platforms represent an effective means of inducing robust tumor antigen-specific immunity.

[0584] The operation of these platforms: DCs often exhibit immature or tolerant phenotypes. Tumor antigen delivery via PEVs (as payload or cargo), combined with co-delivery of adjuvants (DC maturation stimulants such as anti-CD40mAb agonists, poly(I:C), cytosine-phosphate-guanine (CpG), lipopolysaccharide (LPS), or Toll-like receptor 7 / 8 (TLR7 / 8) agonists), or targeted co-delivery of PEVs with STING or ERAdP pathway activators as described above (e.g., bacterial dinucleotide cyclases, e.g., c-di-AMP cyclases CdaA and MtbDisa, and c-di-GMP cyclase VCA0848), results in enhanced tumor-associated antigen presentation ability and enhanced expression of T cell co-stimulating molecules.

[0585] Tumor-associated antigens, either alone, in combination with an adjuvant, or in combination with an immune reprogramming moiety (e.g., STING or ERAdP pathway activator), can be specifically delivered to surface molecules on dendritic cells via PEV.

[0586] Targeting moiety: Antigen-presenting cell surface molecules, including CD40, TNF-α family receptors, DEC205, type C lectin receptor (CLEC9), and integrin receptor CD11c, are targeted by a targeting moiety containing specific monoclonal antibodies, scFv, single-domain antibodies, nanobodies (i.e., anti-DEC205, anti-Clec9A, anti-CD11c), ligands, or targeted peptides (e.g., CD40 ligand or CD40 targeted peptide).

[0587] Payload: Specific tumor-associated antigens (for proof-of-concept in mouse tumor models: DCT and OVA are under consideration). Human tumor-associated antigens important for clinical trials are available (e.g., HPV-E6 and E7, NY-ESO-1, etc.). Cancer-specific neoantigens are also available.

[0588] The co-expression of specific disease cell antigens, such as tumor-related / specific antigens (e.g., OVA, DCT, mERKm9, etc.), is being investigated. Furthermore, adjuvant molecules such as STING or ERAdP activators can be delivered simultaneously with disease-specific antigens or tumor-related / specific antigens.

[0589] Transmembrane domains: All examples listed in Table 1 were available.

[0590] Delivery / Preparation Method: Virus-based platforms such as vaccinia virus, lentivirus, adeno-associated virus [AAV], VSV, and HSV-1 were available. Plasmids for transfecting cells (e.g., pcDNA3.1), as well as prepared and isolated PEVs, will also be investigated.

[0591] result Figure 41. Vaccination experiments with naive EVs, or EVs decorated with aDEC205-VSVGTM-OVA (referred to as "OVA" in the figure), or both aDEC205-CD63D-CdaA-Flag and aDEC205-VSVGTM-OVA (referred to as OVA+cyclase in the figure) (note that these constructs were previously shown in Figures 8, 14, and 38) demonstrated that combinations of both dendritic cell-targeting antigens [e.g., ovalbumin (OVA)] and immune adjuvants (e.g., CdaA enzyme) induce an immune response in vivo. In short, vectors encoding aDEC205-CD63D-CdaA-Flag and / or anti-DEC205-VSVGTM-OVA were expressed in HEK293T cells. Transfection with empty vectors was included as a negative mock EV control. After 48 hours, the supernatant was collected, and extracellular viable cells (EVs) were isolated by serial ultracentrifugation, resuspended in 750 μl of PBS, and stored at -80°C until further use. Next, mice were vaccinated with 200 μl of each EV formulation per mouse IV (naive mice n=5, OVA only n=3, and oocyte + cyclase n=3). After 14 days, splenocytes were collected and stained with SIINFEKL tetramer. Data are expressed as tetramer + CD8+ T cells, representing the percentage of total CD8+ T cells relative to the background staining observed in naive mice.

[0592] Example 18 Single-target immune adjuvant payload As described above in Example 3.

[0593] Geobacter sulfurreducens' "response receiver-regulated diguanylate cyclase" [GsPCA] generates cyclic AMP-GMP (3',3'-cGAMP) in this common soil bacterium. Deletion of the REC (signal reception / dimerization) regulatory domain and expression of the diguanylate cyclase (DGC) or GGDEF domain resulted in a specific constitutively active form. When this active form was targeted to dendritic cells by anti-DEC205 scFV and expressed in PEV constructs loaded with EVs via the VSVG transmembrane domain, functional activation of the interferon response was observed.

[0594] Figure 42. This figure shows a dendritic cell-guided PEV construct that can act as an immunoadjuvant by stimulating the interferon response in reported cell lines. Functional characterization of wild-type and various mutant c-di-GMP and c-di-AMP bacterial cyclase enzymes in mammalian cell THP1-Blue-ISG cells using the Quanti-Blue colorimetric assay. Various codon-optimized transgene constructs, as detailed below, were transfected into HEK293T cells for 48 hours. Cell lysates were collected and transferred to IFN-β promoter-SEAP reporter cells [THP1-Blue-ISG IRF (IFN regulator) reporter cells (InvivoGen)], and the STING-IRF signaling pathway was stimulated for 24 hours to enable the Quanti-Blue assay according to the manufacturer's protocol. In Figure 42, GsPCA: anti-DEC205-VSVGTM-GsPCA-FLAG-pcDNA3.1(+) plasmid. The positive control includes (1) CdaA:CD63_anti-DEC205_CD63_CdaA_FLAG-pcDNA3.1(+) plasmid and (2) c-di-AMP:10pg / ml. The negative control includes mock-transfected cells.

[0595] When a feature is named herein, it will be understood that an example sequence corresponding to the feature (or a sequence containing it) may be found in Table 13.

[0596] Table 33

[0597] Table 34

[0598] Table 35

[0599] Table 36

[0600] Table 37

[0601] Table 38

[0602] Table 39

[0603] Table 40

[0604] Table 41

[0605] Table 42

[0606] Table 43

[0607] Table 44

[0608] Table 45

[0609] Table 46

[0610] Table 47

[0611] Table 48

[0612] Table 49

[0613] Table 50

[0614] Table 51

[0615] Table 52

[0616] Table 53

[0617] Table 54

[0618] Table 55

[0619] Table 56

[0620] Table 57

[0621] Table 58

[0622] Table 59

[0623] Table 60

[0624] Table 61

[0625] Table 62

[0626] Table 63

[0627] Table 64

[0628] Table 65

[0629] Table 66

[0630] Table 67

[0631] Table 68

[0632] Table 69

[0633] Table 70

[0634] Table 71

[0635] Table 72

[0636] Table 73

[0637] Table 74

[0638] It will be understood that functional variants are included in several embodiments. Functional variants may include sequences that are at least 80% identical to the sequence examples shown in Table 13, and such variants retain substantially the same function as their parent molecules from which they originate. Functional variants may be at least 90% identical to their respective parent molecules. Functional variants may be at least 95% identical to their respective parent molecules. Functional variants may be at least 96% identical to their respective parent molecules. Functional variants may be at least 97% identical to their respective parent molecules. Functional variants may be at least 98% identical to their respective parent molecules. Functional variants may be at least 99% identical to their respective parent molecules. Similarly, certain embodiments include functional fragments that retain substantially the same function as their full-length parent molecules from which they originate.

[0639] In the preceding paragraphs, every conceivable detail is provided for explanatory purposes and to enable a thorough understanding of the embodiments. However, it will be apparent to those skilled in the art that these specific details are unnecessary.

[0640] The embodiments described above are intended to be illustrative only. Those skilled in the art will be able to modify, modify, and vary specific embodiments. The scope of the claims should not be limited by the specific embodiments shown herein, but should be interpreted in a manner consistent with the entire specification. All references used herein are incorporated by reference in their entirety.

[0641] reference Stickney, Z., Losacco, J., McDevitt, S., Zhang, Z., and Lu, B. (2016) Development of exosome surface display technology in living human cells.Biochem Biophys Res Commun.472(1):53-9. Meyer,C.,Losacco,J.,Stickney,Z.,Li,L,Marriott,G.,and Lu,B.(2017) Pseudotyping exosomes for enhanced protein delivery in mammalian cells.Int J Nanomedicine.12:3153-3170. U.S. Patent No. 10,617,768 U.S. Patent No. 10,758,486 U.S. Patent Application No. 16 / 900,383

Claims

1. The use of a nucleic acid encoding a recombinant polypeptide or a vector containing the nucleic acid for directing an extracellular vesicle (EV) to at least one target cell, wherein the recombinant polypeptide is - At least one targeting portion for guiding the EV to at least one target molecule expressed by the at least one target cell, - At least one EV anchor polypeptide, and - At least one vesicular polypeptide Includes, The use described above is for the manufacture of a pharmaceutical product that induces in vivo secretion by producer cells of an EV expressing the recombinant polypeptide, wherein the producer cells are transfected or introduced with the nucleic acid, and the EV targets at least one target cell.

2. The use according to claim 1, wherein the at least one EV anchor polypeptide comprises an EV-inducible transmembrane polypeptide linked to the at least one targeted portion.

3. The use according to claim 2, wherein the EV-inducible transmembrane polypeptide comprises a transmembrane domain from LAMP2b, VSVG, CD81, CD82, LAMP1, human CD63, human CD9, Junin virus glycoprotein, Lassa fever virus glycoprotein, LCMV (lymphocytic choriomeningitis virus) glycoprotein, SARS-CoV-2 glycoprotein, tamiamy virus glycoprotein, guanalithovirus glycoprotein, paranavirus glycoprotein, Machupovirus glycoprotein, Sabia virus glycoprotein, or CdaA.

4. The use according to claim 1, wherein the at least one target cell includes a mammalian cell, which is optionally a tumor cell or an immune cell.

5. The use according to claim 1, wherein the at least one target molecule is a cell surface marker or cell surface receptor, optionally selected from TNF-α family receptors, integrins, C-type lectin receptors, leptins, carcinoembryonic antigens, CD antigens, carbonic anhydrase, FAP, MMP2, DEC205, DC40, CLEC9, CD3, glycosaminoglycans, polysaccharides, and lipids.

6. The use of claim 1, wherein the at least one targeting portion comprises a receptor ligand, an antibody, scFv, a single-domain antibody, or DARPin.

7. The use according to claim 1, wherein the intravesicular polypeptide is linked to the at least one targeted portion via the EV anchor polypeptide.

8. The use according to claim 1, wherein the at least one targeting portion comprises at least two targeting portions, and the at least two targeting portions specifically bind to at least two different target molecules.

9. The intravesicular polypeptide comprises at least one EV payload polypeptide linked to the at least one targeting portion via the anchor polypeptide, The aforementioned EV payload polypeptide is - Active pharmaceutical ingredient (API), - cytotoxic molecules, - Immunomodulatory molecules containing STING or ERAdP pathway activators, -enzyme, - Nucleic acid binding domain, or antigen, The use according to claim 7, including the use described in claim 7.

10. The use according to claim 9, wherein the EV payload polypeptide comprises a cytotoxic molecule including human GZMB R201K, mouse GZMB, diphtheria toxin, the PE38 domain from Pseudomonas exotoxin A, or human TRAIL.

11. The use according to claim 9, wherein the EV payload polypeptide comprises a STING pathway activator containing a bacterial dinucleotide cyclase.

12. The use according to claim 9, wherein the EV payload polypeptide comprises an antigen that is a tumor-associated antigen.

13. The use according to claim 9, wherein the nucleic acid binding domain includes an RNA binding motif such as the RNA binding motif of Staufen-1.

14. The use according to claim 1, wherein the EV includes a cargo separate from the recombinant polypeptide.

15. The use according to claim 14, wherein the cargo is nucleic acid.

16. The use according to claim 15, wherein the nucleic acid is RNA selected from mRNA, miRNA, or shRNA.

17. The use according to claim 1, wherein the vesicular polypeptide of the EV comprises an antigen and further comprises an adjuvant molecule such as STING or an ERAdP pathway activator.

18. The use according to claim 1, wherein the vector is a virus particle.

19. The use according to claim 18, wherein the virus particles are tumor-selective virus particles.

20. The use according to claim 1, wherein the producer cells are tumor cells.

21. An extracellular vesicle comprising a recombinant polypeptide containing an intravesicular polypeptide which is a bacterial dinucleotide cyclase.

22. The recombinant polypeptide guides an extracellular vesicle (EV) to at least one target cell, and the recombinant polypeptide further: - At least one targeting portion for guiding the EV to at least one target molecule expressed by the at least one target cell, and - At least one EV anchor polypeptide, The vesicle according to claim 21, comprising:

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