Methods of administering STING agonists

Abstract

Provided herein are methods of preventing or treating a tumor in a subject in need thereof, the method comprising administering to the subject a dose of an interferon gene protein stimulating factor (STING) agonist wherein the amount of the STING agonist in the dose is from about 0.01 [mu] g to about 100 [mu] g.

Description

Background Art

[0001] The stimulator of interferon genes (STING) is a cytoplasmic sensor of cyclic dinucleotides that are usually produced by bacteria. Upon activation, it causes the production of type I interferons and initiates an immune response. Activation of STING has been shown to be a promising method for generating immune responses against tumors preclinically. Unfortunately, given the wide expression spectrum of STING, systemic delivery of STING agonists can lead to systemic inflammation. This limits the dose that can be administered, which in turn limits the therapeutic effect. An alternative to systemic delivery is to inject STING agonists directly into the tumor. Intratumoral injection is quite effective; however, the intratumoral injection is limited to solid tumors that can be touched with a needle and cause tissue damage. Therefore, there is a need for improved methods of delivering STING agonists. Summary of the invention

[0002] Some aspects of the present disclosure relate to a method of preventing or treating a tumor in a subject in need thereof, the method comprising administering to the subject a dose of a stimulator of interferon genes (STING) agonist, wherein the amount of the STING agonist in the dose is about 0.01 μg to about 100 μg.

[0003] In some aspects, the STING agonist is delivered via an extracellular vesicle. In some aspects, the STING agonist is associated with an extracellular vesicle.

[0004] In some aspects, the dose comprises about 0.1 μg to about 1 μg, about 0.1 μg to about 10 μg, about 0.1 μg to about 20 μg, about 0.1 μg to about 30 μg, about 0.1 μg to about 40 μg, about 0.1 μg to about 50 μg, about 0.1 μg to about 60 μg, about 0.1 μg to about 70 μg, about 0.1 μg to about 80 μg, 0.1 μg to about 90 μg, about 0.1 to about 100 μg, about 1 μg to about 10 μg, about 1 μg to about 20 μg, about 1 μg to about 30 μg, about 1 μg to about 40 μg, about 1 μg to about 50 μg. g to about 50 μg, about 1 μg to about 60 μg, about 1 μg to about 70 μg, about 1 μg to about 80 μg, about 1 μg to about 90 μg, about 1 μg to about 100 μg, about 10 μg to about 20 μg, about 10 μg to about 30 μg, about 10 μg to about 40 μg, about 10 μg to about 50 μg, about 10 μg to about 60 μg, about 10 μg to about 70 μg, about 10 μg to about 80 μg, about 90 μg to about 10 μg; about 20 μg to about 30 μg, about 20 μg to about 40 μg, about 20 μg to about 50 μg, about 20 μg to about g to about 60 μg, about 20 μg to about 80 μg, about 20 μg to about 90 μg, about 20 μg to about 100 μg, about 30 μg to about 40 μg, about 30 μg to about 50 μg, about 30 μg to about 60 μg, about 30 μg to about 70 μg, about 30 μg to about 80 μg, about 30 μg to about 90 μg, about 30 μg to about 100 μg, about 40 μg to about 50 μg, about 40 μg to about 60 μg, about 40 μg to about 70 μg, about 40 μg to about 80 μg, about 40 μg to about 90 μg, about 40 μg to about 10 The STING agonist may be present in an amount of about 0 μg, about 50 μg to about 60 μg, about 50 μg to about 70 μg, about 50 μg to about 80 μg, about 50 μg to about 90 μg, about 50 μg to about 100 μg, about 60 μg to about 70 μg, about 60 μg to about 80 μg, about 60 μg to about 90 μg, about 60 μg to about 100 μg, about 70 μg to about 80 μg, about 70 μg to about 90 μg, about 70 μg to about 100 μg, about 80 μg to about 90 μg, about 80 μg to about 100 μg, or about 90 μg to about 100 μg of the STING agonist.

[0005] In some aspects, the dose comprises about 0.1 μg, about 0.2 μg, about 0.3 μg, about 0.4 μg, about 0.5 μg, about 0.6 μg, about 0.7 μg, about 0.8 μg, about 0.9 μg, about 1.0 μg, about 1.1 μg, about 1.2 μg, about 1.3 μg, about 1.4 μg, about 1.5 μg, about 1.6 μg, about 1.7 μg, about 1.8 μg, about 1.9 μg, about 2.0 μg, about 2.5 μg, about 3.0 μg, about In some aspects, the dosage comprises about 0.3 μg of the STING agonist. In some aspects, the dosage comprises about 1.0 μg of the STING agonist. In some aspects, the dosage comprises about 3.0 μg of the STING agonist. In some aspects, the dosage comprises about 6.0 μg of the STING agonist. In some aspects, the dose comprises about 12.0 μg of the STING agonist.

[0006] In some aspects, the tumor is a primary tumor, a secondary tumor, or both a primary tumor and a secondary tumor. In some aspects, the tumor is late or metastatic. In some aspects, the tumor is recurrent, recurrent or refractory.

[0007] In some aspects, the tumor is selected from the group consisting of a cancer selected from the group consisting of lung cancer, ovarian cancer, cervical cancer, endometrial cancer, breast cancer, brain cancer, colon cancer, prostate cancer, gastrointestinal cancer, head and neck cancer, non-small cell lung cancer, nervous system cancer, kidney cancer, retinal cancer, skin cancer, liver cancer, pancreatic cancer, genitourinary cancer and bladder cancer, melanoma, leukemia, brain cancer (e.g., glioma, astrocytoma, ependymoma, oligodendroglioma and tumors with a mixture of two or more cell types, known as mixed gliomas, acoustic neuroma (neurilemmoma, schwannoma, neurothecoma), adenoma, astrocytoma, low grade astrocytoma, giant cell astrocytoma, intermediate and high grade astrocytoma, recurrent tumors, brain stem glioma, chordoma, choroid plexus papilloma, CNS lymphoma (primary malignant lymphoma), cyst, dermoid cyst, epidermoid Cyst, craniopharyngioma, ependymoma anaplastic ependymoma, gangliocytoma (ganglioma), ganglioglioma, glioblastoma multiforme (GBM), malignant astrocytoma, glioma, hemangioblastoma, inoperable brain tumor, lymphoma, medulloblastoma (MDL), meningioma, metastatic brain tumor, mixed glioma, neurofibromatosis, oligodendroglioma, optic nerve glioma, pineal region tumor, pituitary adenoma, PNET (primitive neuroectodermal tumor), spinal tumor, subependymoma and tuberous sclerosis (Bourneville's Disease), head and neck squamous cell carcinoma, triple negative breast cancer, undifferentiated thyroid cancer and cutaneous squamous cell carcinoma. In some aspects, the composition is administered to prevent the metastasis of the tumor, reduce the growth rate of the tumor, reduce the size of the tumor or a combination thereof.

[0008] In some aspects, the subject has previously received at least two anti-cancer therapies.

[0009] In some aspects, the administration is parenteral, oral, intravenous, intramuscular, intratumoral, or intraperitoneal. In some aspects, the administration is intratumoral.

[0010] In some aspects, the method comprises administering a second, third, fourth, or fifth dose of the STING agonist. In some aspects, one or more of the second, third, fourth, or fifth dose is administered at the same dose as the initial dose. In some aspects, one or more of the second, third, fourth, or fifth dose is administered at a different dose than the initial dose.

[0011] In some aspects, the extracellular vesicle is selected from exosomes, nanovesicles, apoptotic bodies, microvesicles, lysosomes, endosomes, liposomes, lipid nanoparticles, micelles, multilamellar structures, reencapsulated vesicles, and extruded cells. In some aspects, the extracellular vesicle is an exosome. In some aspects, the STING agonist is encapsulated in the extracellular vesicle.

[0012] In some aspects, the STING agonist is attached to the lipid bilayer of the extracellular vesicle. In some aspects, the STING agonist is attached to the lipid bilayer of the extracellular vesicle via a linker. In some aspects, the linker is a cleavable linker.

[0013] In some aspects, the extracellular vesicles overexpress PTGFRN protein or a portion thereof. In some aspects, the STING agonist is linked to the PTGFRN protein or the portion thereof. In some aspects, the STING agonist is linked to the PTGFRN protein or the portion thereof via a linker. In some aspects, the extracellular vesicles are produced by cells that overexpress PTGFRN protein.

[0014] In some aspects, the STING agonist is a cyclic dinucleotide. In some aspects, the STING agonist is a non-cyclic dinucleotide. In some aspects, the STING agonist comprises a lipid binding tag. In some aspects, the STING agonist is physically and / or chemically modified. In some aspects, the modified STING agonist has a different polarity and / or charge than the corresponding unmodified STING agonist.

[0015] In some aspects, the STING agonist comprises:

[0016]

[0017] in:

[0018] X 1 is H, OH or F;

[0019] X 2 is H, OH or F;

[0020] Z is OH, OR 1 , SH or SR 1 ,in:

[0021] R 1 is Na or NH 4 ,or

[0022] R 1 It is an enzyme-labile group that donates OH or SH in vivo, such as pivaloyloxymethyl;

[0023] B1 and B2 are bases selected from the group consisting of:

[0024]

[0025] The conditions are:

[0026] In formula (I): X 1 and X 2 Not OH,

[0027] In formula (II): when X 1 and X 2 When it is OH, B 1 is not adenine, and B 2 is not guanine, and

[0028] In formula (III): when X 1 and X 2 When it is OH, B 1 Not adenine, B 2 is not guanine, and Z is not OH, or a pharmaceutically acceptable salt thereof.

[0029] In some aspects, the STING agonist is selected from the group consisting of:

[0030]

[0031]

[0032] and pharmaceutically acceptable salts thereof.

[0033] In some aspects, the STING agonist is

[0034] or a pharmaceutically acceptable salt thereof.

[0035] In some aspects, the method further comprises administering to the subject a second anticancer agent. In some aspects, the second anticancer agent comprises chemotherapy, an antibody or antigen-binding portion thereof, additional extracellular vesicles, or any combination thereof.

[0036] In some aspects, the PTGFRN protein comprises SEQ ID NO: 33. In some aspects, the PTGFRN protein comprises at least about 70%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO: 1. In some aspects, the PTGFRN protein comprises the amino acid sequence as shown in SEQ ID NO: 1.

[0037] Some aspects of the present disclosure relate to a method of treating a tumor in a subject, the method comprising: (a) first administering to the patient a first composition of extracellular vesicles comprising a first STING agonist, the first composition comprising about 0.3 micrograms of the first STING agonist; (b) secondly administering to the patient a second composition of extracellular vesicles comprising a second STING agonist, the second composition comprising about 1.0 micrograms of the second STING agonist; and (c) thirdly administering to the patient a third composition of extracellular vesicles comprising a third STING agonist, the third composition comprising about 2.0 micrograms of the third STING agonist; wherein the first STING agonist, the second STING agonist, and the third STING agonist are the same; wherein the composition is administered intratumorally; and wherein the tumor is selected from the group consisting of: head and neck squamous cell carcinoma, triple-negative breast cancer, anaplastic thyroid cancer, and cutaneous squamous cell carcinoma, wherein the patient has previously received at least two anti-cancer treatments. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 A schematic diagram showing the method of loading exosomes with STING agonists.

[0039] Figures 2A-2C is a representative chromatogram of exoSTING obtained by reverse phase ultra-high performance liquid chromatography (RP-UHPLC). DETAILED DESCRIPTION

[0040] Before describing the present invention in more detail, it should be understood that the present invention is not limited to the specific aspects described, as such aspects may of course vary. It should also be understood that the terms used herein are only for the purpose of describing specific aspects, and are not intended to be restrictive, as the scope of the present invention will only be limited by the appended claims.

[0041] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, representative illustrative methods and materials are now described.

[0042] All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent was specifically and individually indicated to be incorporated by reference and is incorporated herein by reference to disclose and describe the methods and / or materials in connection with the cited publication.

[0043] As will be apparent to those skilled in the art upon reading this disclosure, each of the individual aspects described and illustrated herein has discrete components and features that can be readily separated or combined with the features of any other several aspects without departing from the scope or spirit of the invention. Any recited method may be performed in the order of events recited or in any other order that is logically possible.

[0044] I. Definitions

[0045] It should be noted that, as used herein and in the appended claims, the singular forms "a / an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, the terms "a" (or "an"), "one or more", and "at least one" are used interchangeably herein. It should be further noted that the claims may be drafted to exclude any optional elements. Therefore, this statement is intended to serve as a precondition for using such exclusive terms as "solely," "only," or the use of a "negative" limitation in conjunction with the recitation of claim elements.

[0046] Furthermore, the use of "and / or" herein should be considered as explicitly disclosing each of the two specified features or components with or without the other. Thus, the term "and / or" as used herein in phrases such as "A and / or B" is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Likewise, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to cover each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0047] It should be understood that wherever herein aspects are described with the language "comprising," otherwise similar aspects described according to "consisting of" and / or "consisting essentially of" are also provided.

[0048] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art in the relevant field of the present disclosure. For example, the following provides a common dictionary of many terms used in the present disclosure for technical personnel: Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd edition, 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd edition, 1999, Academic Press; and Oxford Dictionary of Biochemistry and Molecular Biology, revised edition, 2000, Oxford University Press.

[0049] Units, prefixes and symbols are expressed in the form accepted by the International System of Units (SI). Numerical ranges include numbers defining ranges. In the case of a range of narrated values, it is to be understood that each intermediate integer value and each fraction thereof between the narrated upper and lower limits of the range are also specifically disclosed, as well as each subrange between such values. The upper and lower limits of any range may be independently included in the range or excluded from the range, and each range including any limit, not including two limits or including two limits is also encompassed in the present disclosure. Therefore, the range narrated herein should be understood as a shorthand for all values ​​within the range, including the endpoints narrated. For example, a range of 1 to 10 should be understood as including any number, combination of numbers or subranges from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10.

[0050] Where a value is explicitly stated, it is understood that values ​​of a quantity or amount approximately the same as the stated value are also within the scope of the present disclosure. Where a combination is disclosed, each subcombination of the elements of the combination is also specifically disclosed and is within the scope of the present disclosure. Conversely, where different elements or groups of elements are disclosed separately, combinations thereof are also disclosed. Where any element disclosed is disclosed as having multiple alternatives, examples of the disclosure in which each alternative is excluded individually or in any combination with other alternatives are also hereby disclosed; more than one element disclosed may have such exclusions, and all combinations of elements having such exclusions are hereby disclosed.

[0051] Nucleotide is represented by its generally accepted single letter code.Unless otherwise indicated, nucleotide sequence is written from left to right with 5' to 3' direction.Nucleotide is represented by the commonly known single letter symbol recommended by IUPAC-IUB Biochemical Nomenclature Commission in this article.Therefore, A represents adenine, C represents cytosine, G represents guanine, T represents thymine, and U represents uracil.

[0052] Amino acid sequences are written left to right in amino to carboxyl orientation.Amino acids are referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.

[0053] The terms "about" or "approximately" are used herein to mean approximately, roughly, around, or in the region thereof. When the term "about" is used in conjunction with a numerical range, the term modifies the range by extending the boundaries above and below the numerical values ​​set forth. The term table used herein means within 5% of the reference amount, for example, about 50% is understood to cover a range of values ​​from 47.5% to 52.5%.

[0054] As used herein, the term "extracellular vesicle" or "EV" refers to a cell-derived vesicle comprising a membrane that wraps an internal space. Extracellular vesicles include all membrane-bound vesicles (e.g., exosomes, nanovesicles) whose diameter is less than the diameter of the cell from which it is derived. Typically, the diameter of an extracellular vesicle is in the range of 20nm to 1000nm, and may include being in the internal space (i.e., lumen), displayed on the outer surface of the extracellular vesicle, and / or spanning various macromolecular payloads of the membrane. The payload may include nucleic acids, proteins, carbohydrates, lipids, small molecules, and / or combinations thereof. In some aspects, an extracellular vesicle includes a scaffold portion. For example, and not limitation, extracellular vesicles include apoptotic bodies, cell fragments, vesicles derived from cells by direct or indirect manipulation (e.g., by continuous extrusion or treatment with an alkaline solution), vesicles, vesicle organelles, and vesicles produced by living cells (e.g., by direct plasma membrane budding or late endosomes and plasma membrane fusion). Extracellular vesicles can be derived from living or dead organisms, explanted tissues or organs, prokaryotic or eukaryotic cells, and / or cultured cells. In some aspects, extracellular vesicles are produced by cells expressing one or more transgenic products.

[0055] As used herein, the term "exosome" refers to a cell-derived small (diameter between 20-300nm, for example, diameter between 40-200nm) vesicle, the cell-derived vesicle comprising a membrane that encapsulates the internal space (ie, lumen), and produced from the cell by direct plasma membrane budding or by late endosome fusion with the plasma membrane. In some aspects, EV (eg, exosome) is about 20nm to about 300nm. Exosomes are extracellular vesicles of a species. Exosomes contain lipids or fatty acids and polypeptides, and optionally contain a payload (eg, a therapeutic agent), an acceptor (eg, a targeting moiety), a polynucleotide (eg, nucleic acid, RNA or DNA), a sugar (eg, a monosaccharide, a polysaccharide or a polysaccharide) or other molecules. In some aspects, exosomes include a scaffold portion. Exosomes can be derived from producer cells and can be separated from producer cells based on their size, density, biochemical parameters or a combination thereof. In some aspects, the exosomes disclosed herein are produced by cells expressing one or more transgenic products.

[0056] As used herein, the term "nanovesicle" refers to a cell-derived small (diameter between 20-250nm, more preferably between 30-150nm) vesicle, the cell-derived vesicle comprising a membrane that wraps the inner space, and produced from the cell by direct or indirect manipulation, so that the producer cell does not produce the nanovesicle in the absence of the manipulation. Suitable manipulation of producer cells includes but is not limited to continuous extrusion, treatment with alkaline solution, ultrasonic treatment or its combination. In some cases, the generation of nanovesicles can cause the destruction of producer cells. Preferably, the nanovesicle colony is substantially free of vesicles derived from producer cells by direct budding from the plasma membrane or late endosomes and plasma membrane fusion. The nanovesicle comprises lipids or fatty acids and polypeptides, and optionally comprises a payload (e.g., a therapeutic agent), an acceptor (e.g., a targeting moiety), a polynucleotide (e.g., a nucleic acid, RNA or DNA), a sugar (e.g., a monosaccharide, a polysaccharide or a polysaccharide) or other molecules. In some aspects, the nanovesicle comprises a scaffold portion. When the nanovesicles are derived from producer cells according to the manipulations described, they can be isolated from the producer cells based on their size, density, biochemical parameters, or a combination thereof.

[0057] The term "modified" when used in the context of exosomes described herein refers to the alteration or engineering of EVs such that the modified EVs differ from naturally occurring EVs. In some aspects, the modified EVs described herein comprise a membrane that differs from the membrane of a naturally occurring EV in terms of the composition of proteins, lipids, small molecules, carbohydrates, etc. (e.g., the membrane comprises a higher density or amount of native EV proteins and / or the membrane comprises proteins that do not naturally occur in EVs. In certain aspects, such modifications to the membrane alter the outer surface of the EV. In certain aspects, such modifications to the membrane alter the lumen of the EV.

[0058] As used herein, the term "scaffold moiety" refers to a molecule that can be used to anchor a STING agonist disclosed herein and / or any other compound of interest (e.g., a payload) to an EV, the luminal surface or the external surface of an EV. In certain aspects, the scaffold moiety comprises a synthetic molecule. In some aspects, the scaffold moiety comprises a non-polypeptide moiety. In other aspects, the scaffold moiety comprises a lipid, carbohydrate, or protein that is naturally present in an EV. In some aspects, the scaffold moiety comprises a lipid, carbohydrate, or protein that is not naturally present in an exosome. In certain aspects, the scaffold moiety is Scaffold X. In some aspects, the scaffold moiety is Scaffold Y. In other aspects, the scaffold moiety comprises both Scaffold X and Scaffold Y. In certain aspects, the scaffold portion comprises Lamp-1, Lamp-2, CD13, CD86, Flotillin, Syntaxin-3, CD2, CD36, CD40, CD40L, CD41a, CD44, CD45, ICAM-1, Integrin α4, L1CAM, LFA-1, Mac-1 α and β, Vti-1A and B, CD3ε and ζ, CD9, CD18, CD37, CD53, CD63, CD81, CD82, CXCR4, FcR, GluR2 / 3, HLA-DM (MHCII), immunoglobulin, MHC-I or MHC-II component, TCRβ, a tetraspanin, or a combination thereof.

[0059] As used herein, the term "Scaffold X" refers to an exosomal protein recently identified on the surface of exosomes. See, e.g., U.S. Pat. No. 10,195,290, which is incorporated herein by reference in its entirety. Non-limiting examples of Scaffold X proteins include: prostaglandin F2 receptor negative regulator ("PTGFRN protein"); basic glycoprotein ("BSG protein"); immunoglobulin superfamily member 2 ("IGSF2 protein"); immunoglobulin superfamily member 3 ("IGSF3 protein"); immunoglobulin superfamily member 8 ("IGSF8 protein"); integrin beta-1 ("ITGB1 protein"); integrin alpha-4 ("ITGA4 protein"); 4F2 cell surface antigen heavy chain ("SLC3A2 protein"); and a class of ATP transporters ("ATP1A1 protein", "ATP1A2 protein", "ATP1A3 protein", "ATP1A4 protein", "ATP1B3 protein", "ATP2B1 protein", "ATP2B2 protein", "ATP2B3 protein", "ATP2B protein"). In some aspects, the Scaffold X protein can be a whole protein or a fragment thereof (e.g., a functional fragment, e.g., a minimal fragment capable of anchoring another portion to the outer surface or lumen surface of an EV (e.g., an exosome)). In some aspects, Scaffold X can anchor a moiety (e.g., a STING agonist) to the exterior surface or the luminal surface of an EV (e.g., an exosome).

[0060] As used herein, the term "scaffold Y" refers to an exosomal protein recently identified within the lumenal surface of an exosome. See, for example, International Publication No. WO / 2019 / 099942, which is incorporated herein by reference in its entirety. Non-limiting examples of scaffold Y proteins include: protein kinase C substrate rich in myristoylated alanine ("MARCKS protein"); protein kinase C substrate-like 1 rich in myristoylated alanine ("MARCKSL1 protein"); and brain acid soluble protein 1 ("BASP1 protein"). In some aspects, the scaffold Y protein can be a whole protein or a fragment thereof (e.g., a functional fragment, e.g., a minimal fragment capable of anchoring a portion on the lumenal surface of an EV (e.g., an exosome)). In some aspects, scaffold Y can anchor a portion (e.g., a STING agonist) to the lumen of an EV (e.g., an exosome).

[0061] As used herein, the term "fragment" of a protein (e.g., a therapeutic protein, scaffold X, or scaffold Y) refers to an amino acid sequence of a protein that is shorter than the naturally occurring sequence of the protein, the portion of the N-terminus and / or the C-terminus that is missing, or any portion thereof, as compared to the naturally occurring protein. As used herein, the term "functional fragment" refers to a protein fragment that retains the function of the protein. Thus, in some aspects, a functional fragment of a scaffold X protein retains the ability to anchor a moiety to the luminal surface and / or the outer surface of an EV. Similarly, in some aspects, a functional fragment of a scaffold Y protein retains the ability to anchor a moiety to the luminal surface of an EV. Whether a fragment is a functional fragment can be assessed by any known method for determining the protein content of an EV, including Western blot, FACS analysis, and fusion of a fragment with an autofluorescent protein, such as GFP. In some aspects, a functional fragment of a scaffold X protein retains the ability of a naturally occurring scaffold X protein, for example, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100% of the ability to anchor a moiety. In some aspects, a functional fragment of a Scaffold Y protein retains the ability of a naturally occurring Scaffold Y protein, e.g., at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100% of the ability to anchor another molecule.

[0062] As used herein, the term "variant" of a molecule (e.g., a functional molecule, an antigen, a scaffold X and / or a scaffold Y) refers to a molecule that shares certain structural and functional identities with another molecule after comparison by methods known in the art. For example, a variant of a protein may include a substitution, insertion, deletion, frameshift or rearrangement in another protein.

[0063] In some aspects, variants of Scaffold X comprise variants having at least about 70% identity to full-length, mature PTGFRN, BSG, IGSF2, IGSF3, IGSF8, ITGB1, ITGA4, SLC3A2 or ATP transporter or fragments (e.g., functional fragments) of PTGFRN, BSG, IGSF2, IGSF3, IGSF8, ITGB1, ITGA4, SLC3A2 or ATP transporter. In some aspects, variants of PTGFRN or variants of fragments share at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with PTGFRN according to SEQ ID NO: 1 or with a functional fragment thereof.

[0064] In some aspects, variants of Scaffold Y comprise variants having at least 70% identity with MARCKS, MARCKSL1, BASP1 or fragments of MARCKS, MARCKSL1 or BASP1. In some aspects, variants of MARCKS or variants of fragments share at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity with MARCKS according to SEQ ID NO: 401 or a functional fragment thereof. In some aspects, variants of MARCKSL1 or variants of fragments share at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity with MARCKSL1 according to SEQ ID NO: 402 or a functional fragment thereof. In some aspects, the variants of BASP1 or variants of fragments share at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with BASP1 according to SEQ ID NO: 403 or a functional fragment thereof. In some aspects, the variants of Scaffold Y protein or variants of fragments retain the ability to specifically target to the lumen of EVs. In some aspects, Scaffold Y comprises one or more mutations, e.g., conservative amino acid substitutions.

[0065] "Conservative amino acid substitution" is a substitution in which an amino acid residue is replaced by an amino acid residue with a similar side chain. Families of amino acid residues with similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, if an amino acid in a polypeptide is replaced by another amino acid from the same side chain family, the substitution is considered to be conservative. On the other hand, a string of amino acids can be conservatively replaced by a structurally similar string that differs in the order and / or composition of the side chain family members.

[0066] The term "percentage of sequence identity" or "percentage of identity" between two polynucleotide or polypeptide sequences refers to the number of identical matching positions shared by the sequences within the comparison window, taking into account additions or deletions (i.e., gaps) that must be introduced for optimal alignment of the two sequences. A matching position is any position where the same nucleotide or amino acid is present in both the target sequence and the reference sequence. Gaps present in the target sequence are not counted because gaps are not nucleotides or amino acids. Likewise, gaps present in the reference sequence are not counted because the target sequence nucleotides or amino acids are counted, not the nucleotides or amino acids from the reference sequence.

[0067] The percentage of sequence identity is calculated by determining the number of positions where the same amino acid residue or nucleic acid base occurs in the two sequences, dividing the number of matching positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percentage of sequence identity. The comparison of the sequence and the determination of the percentage of sequence identity between the two sequences can be completed using readily available software that can be used and downloaded online. Suitable software programs can be obtained from various sources, and can be used for the comparison of protein sequences and nucleotide sequences. A suitable program for determining the percentage of sequence identity is bl2seq, which is a part of the BLAST program suite that can be obtained from the National Center for Biotechnology Information (National Center for Biotechnology Information) BLAST website (blast.ncbi.nlm.nih.gov) of the U.S. government. Bl2seq uses BLASTN or BLASTP algorithms to compare between two sequences. BLASTN is used to compare nucleic acid sequences, and BLASTP is used to compare amino acid sequences. Other suitable programs are for example Needle, Stretcher, Water or Matcher, which are part of the EMBOSS suite of bioinformatics programs and are also available from the European Bioinformatics Institute (EBI) at www.ebi.ac.uk / Tools / psa.

[0068] Different regions within a single polynucleotide or polypeptide target sequence aligned to a polynucleotide or polypeptide reference sequence can each have their own percent sequence identity. It should be noted that percent sequence identity values ​​are rounded to the nearest tenth. For example, 80.11, 80.12, 80.13, and 80.14 are rounded down to 80.1, while 80.15, 80.16, 80.17, 80.18, and 80.19 are rounded up to 80.2. It should also be noted that length values ​​will always be integers.

[0069] Those skilled in the art will appreciate that the generation of sequence alignments for calculating sequence identity percentages is not limited to binary sequence-sequence comparisons driven completely by primary sequence data. Sequence alignments can be derived from multiple sequence alignments. A suitable program for generating multiple sequence alignments is ClustalW2, which can be obtained from www.clustal.org. Another suitable program is MUSCLE, which can be obtained from www.drive5.com / muscle / . ClustalW2 and MUSCLE can alternatively be obtained from, for example, EBI.

[0070] It should also be understood that sequence alignments can be generated by integrating sequence data with data from heterogeneous sources, such as structural data (e.g., crystal protein structures), functional data (e.g., the location of mutations), or phylogenetic data. A suitable program for integrating heterogeneous data to generate multiple sequence alignments is T-Coffee, which is available at www.tcoffee.org and alternatively available from, for example, EBI. It should also be understood that the final alignment for calculating percent sequence identity can be automatically or manually curated.

[0071] Polynucleotide variants can contain changes in coding region, noncoding region or both.On the one hand, polynucleotide variants contain silent substitutions, additions or deletions that do not change the characteristics or active changes of the encoded polypeptide.On the other hand, due to the degeneracy of the genetic code, nucleotide variants are produced by silent substitutions.In other aspects, wherein 5-10, 1-5 or 1-2 amino acids are substituted, lacked or added variants in any combination.Polynucleotide variants can be produced for a variety of reasons, for example, for optimizing the codon expression of a specific host (codons in human mRNA are changed to other codons, for example bacterial hosts, such as Escherichia coli (E.coli)).

[0072] Naturally occurring variants are called "allelic variants" and refer to one of several alternative forms of a gene occupying a given locus on a chromosome of an organism (Genes II, Lewin, B., ed., John Wiley & Sons, New York (1985)). These allelic variants may vary at the polynucleotide and / or polypeptide level and are included in the present disclosure. Alternatively, non-naturally occurring variants may be produced by mutagenesis techniques or direct synthesis.

[0073] Using known methods of protein engineering and recombinant DNA technology, variants can be produced to improve or change the properties of a polypeptide. For example, one or more amino acids can be deleted from the N-terminus or C-terminus of a secreted protein without significantly losing biological function. Ron et al., J. Biol. Chem. 268: 2984-2988 (1993), which is incorporated herein by reference in its entirety, reports variant KGF proteins with heparin binding activity even after the deletion of 3, 8 or 27 amino terminal amino acid residues. Similarly, interferon gamma exhibits up to ten times higher activity after the deletion of 8-10 amino acid residues from the carboxyl terminus of this protein. (Dobeli et al., J. Biotechnology 7: 199-216 (1988), which is incorporated herein by reference in its entirety.)

[0074] In addition, there is sufficient evidence to show that variants generally retain biological activities similar to those of naturally occurring proteins. For example, Gayle and colleagues (Journal of Biological Chemistry 268:22105-22111 (1993), the document is incorporated herein by reference in its entirety) have been subjected to extensive mutation analysis of human cytokine IL-1a. It uses random mutagenesis to produce more than 3,500 individual IL-1a mutants with an average of 2.5 amino acid changes per variant over the entire length of the molecule. Multiple mutations are examined at each possible amino acid position. The researchers found that "[most of] the molecule can be changed, with little effect on [combination or biological activity]. "(See Abstract.) In fact, in the more than 3,500 nucleotide sequences examined, only 23 unique amino acid sequences produced proteins significantly different in activity from the wild type.

[0075] As described above, polypeptide variants include, for example, modified polypeptides. Modifications include, for example, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, pegylation (Mei et al., Blood 116:270-79 (2010), which is incorporated herein by reference in its entirety), proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination. In some aspects, Scaffold X and / or Scaffold Y are modified at any convenient location.

[0076] As used herein, the term "producer cell" refers to a cell used to produce EVs. Producer cells can be cells cultured in vitro or cells cultured in vivo. Producer cells include, but are not limited to, cells known to be effective in producing EVs (e.g., exosomes), for example, HEK293 cells, Chinese hamster ovary (CHO) cells, mesenchymal stem cells (MSCs), BJ human foreskin fibroblasts, s9f cells, fHDF fibroblasts, Neuronal precursor cells, In some aspects, the producer cell is an antigen presenting cell. In some aspects, the producer cell is a bacterial cell. In some aspects, the producer cell is a dendritic cell, a B cell, a mast cell, a macrophage, a neutrophil, a Kupffer-Browicz cell or a cell derived from any of these cells or any combination thereof. In some aspects, the producer cell is not a bacterial cell. In other aspects, the producer cell is not an antigen presenting cell.

[0077] As used herein, the term "associated with" refers to encapsulating a first portion (e.g., a STING agonist) into a second portion (e.g., an extracellular vesicle), or refers to a covalent bond or non-covalent bond formed between a first portion (e.g., a STING agonist) and a second portion (e.g., an extracellular vesicle), respectively. For example, in some aspects, a scaffold portion, such as scaffold X (e.g., PTGFRN protein) is expressed in or on an extracellular vesicle, and the STING agonist is loaded in the lumen of the extracellular vesicle or on the outer surface. On the one hand, the term "associated with" means a covalent bond, a non-peptide bond, or a non-covalent bond. For example, the amino acid cysteine ​​contains a thiol group that can form a disulfide bond or bridge with a sulfhydryl group on a second cysteine ​​residue. Examples of covalent bonds include, but are not limited to, peptide bonds, metal bonds, hydrogen bonds, disulfide bonds, sigma bonds, pi bonds, delta bonds, glycosidic bonds, π hydrogen bonds, bent bonds, dipole bonds, pi reverse bonds, double bonds, triple bonds, quadruple bonds, pentapole bonds, sextuple bonds, conjugation, hyperconjugation, aromaticity, Haptor numbers, or antibonding. Non-limiting examples of non-covalent bonds include ionic bonds (e.g., cation-π bonds or salt bonds), metal bonds, hydrogen bonds (e.g., dihydrogen bonds, dihydrogen complexes, low-barrier hydrogen bonds, or symmetric hydrogen bonds), van der Walls forces, London dispersion forces, mechanical bonds, halogen bonds, gold affinity, intercalation, stacking, entropic forces, or chemical polarity. In other aspects, the term "associated with" means that the first portion (e.g., an extracellular vesicle) encapsulates the second portion (e.g., a STING agonist). In some aspects, the first portion and the second portion may be connected to each other. In other aspects, the first portion and the second portion are not physically and / or chemically connected to each other.

[0078] As used herein, the terms "linked to" or "conjugated to" are used interchangeably and refer to a covalent bond or non-covalent bond formed between a first portion and a second portion (e.g., a STING agonist and an extracellular vesicle, respectively). In some aspects, the scaffold portion is expressed in or on an extracellular vesicle, e.g., Scaffold X (e.g., PTGFRN protein), and the STING agonist is linked or conjugated to a portion of the Scaffold X protein (e.g., PTGFRN protein) exposed on the surface of the extracellular vesicle (e.g., "surface display of a STING agonist"). In some aspects, the scaffold portion is expressed in or on an extracellular vesicle, e.g., Scaffold X (e.g., PTGFRN protein), and the STING agonist is linked or conjugated to a portion of the Scaffold X protein (e.g., PTGFRN protein) exposed to the lumen of the extracellular vesicle.

[0079] As used herein, the term "loading" or grammatically different forms of the term (e.g., load or loaded) refers to the state or process of associating a first portion (e.g., a STING agonist) with a second portion (e.g., an EV, such as and an exosome). In some aspects, the first portion is chemically or physically linked to the second portion. In some aspects, the first portion is not chemically or physically linked to the second portion. In some aspects, the first portion is present within the second portion, such as within the lumen of an EV (e.g., an exosome), such as, "encapsulated". In some aspects, the first portion is associated with the outer surface of the second portion, such as, linked or conjugated to the surface of the EV (e.g., an exosome), such as, "surface display" of the second portion.

[0080] The term "encapsulated" or grammatically different forms of the term (e.g., encapsulation or encapsulating) refers to the state or process of having a first portion (e.g., a STING agonist) located inside a second portion (e.g., an EV, e.g., an exosome) without chemically or physically connecting the two portions. In some aspects, the term "encapsulated" can be used interchangeably with "in the lumen of..." Non-limiting examples of encapsulating a first portion (e.g., a STING agonist) into a second portion (e.g., an EV, e.g., an exosome) are disclosed elsewhere herein.

[0081] As used herein, the terms "isolate," "isolated," and "isolating," or "purify," "purified," and "purifying," and "extracted," and "extracting," are used interchangeably and refer to the state (e.g., multiple known or unknown amounts and / or concentrations) of a desired preparation of EVs that have been subjected to one or more purification processes, e.g., selection or enrichment of a desired EV preparation. In some aspects, as used herein, isolation or purification is the process of removing, partially removing (e.g., fractionating) EVs from a sample containing producer cells. In some aspects, the isolated EV composition has no detectable undesirable activity, or alternatively, the level or amount of the undesirable activity is at or below an acceptable level or amount. In other aspects, the isolated EV composition has an amount and / or concentration of the desired EVs that is at or above an acceptable amount and / or concentration. In other aspects, the isolated EV composition is enriched compared to the starting material (e.g., producer cell preparation) from which the composition is obtained. Compared to the starting material, this enrichment can be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99%, 99.999%, 99.9999% or greater than 99.9999%. In some aspects, the isolated EV preparation is substantially free of residual biological products. In some aspects, the isolated EV preparation is 100% free, 99% free, 98% free, 97% free, 96% free, 95% free, 94% free, 93% free, 92% free, 91% free or 90% free of any contaminating biological substances. Residual biological products may include non-biological materials (including chemicals) or undesirable nucleic acids, proteins, lipids, or metabolites. Substantially free of residual biological products may also mean that the EV composition contains no detectable producer cells, and only EVs are detectable.

[0082] As used herein, the term "agonist" refers to a molecule that binds to a receptor and activates the receptor to produce a biological response. Receptors can be activated by endogenous or exogenous agonists. Non-limiting examples of endogenous agonists include hormones, neurotransmitters, and cyclic dinucleotides. Non-limiting examples of exogenous agonists include drugs, small molecules, and cyclic dinucleotides. Agonists can be complete agonists, partial agonists, or inverse agonists.

[0083] As used herein, the term "antagonist" refers to a molecule that blocks or retards an agonist-mediated response rather than itself stimulating a biological response when bound to a receptor. Many antagonists achieve their effectiveness by competing with endogenous ligands or substrates at a structurally defined binding site on a receptor. Non-limiting examples of antagonists include alpha blockers, beta blockers, and calcium channel blockers. Antagonists can be competitive, noncompetitive, or noncompetitive antagonists.

[0084] As used herein, the term "free STING agonist" means a STING agonist that is not associated with an extracellular vesicle, but is otherwise identical to a STING agonist associated with an extracellular vesicle. In particular, the free STING agonist is the same STING agonist associated with an extracellular vesicle when compared to an extracellular vesicle associated with the STING agonist. In some aspects, when the free STING agonist is compared to an extracellular vesicle comprising the STING agonist in terms of its efficacy, toxicity, and / or any other properties, the amount of the free STING agonist is the same as the amount of the STING agonist associated with the EV when compared to the STING agonist associated with the extracellular vesicle.

[0085] As used herein, the term "exoSTING" refers to an exosome loaded with a STING agonist. In some aspects, the exosome comprises a STING agonist located in the lumen of the exosome. In some aspects, the STING agonist is associated with the lumen surface of the exosome, for example, with a scaffold protein, for example, Scaffold X (e.g., PTGFRN). In some aspects, the STING agonist is encapsulated in the lumen of the exosome and is not associated with the scaffold protein. In some aspects, the exosome comprises a STING agonist located on the surface of the exosome. In some aspects, the STING agonist is associated with the outer surface of the exosome. In some aspects, the STING agonist is connected or conjugated to the outer surface of the exosome. In some aspects, the STING agonist is connected or conjugated to the surface-exposed scaffold protein, for example, Scaffold X protein (e.g., PTGFRN protein). In some aspects, the STING agonist is connected or conjugated to the lipid bilayer of the exosome.

[0086] As used herein, the term "ligand" refers to a molecule that binds to a receptor and modulates the receptor to produce a biological response. Modulation can be activation, inactivation, blocking or retardation of a receptor-mediated biological response. Receptors can be modulated by endogenous ligands or exogenous ligands. Non-limiting examples of endogenous ligands include antibodies and peptides. Non-limiting examples of exogenous agonists include drugs, small molecules and cyclic dinucleotides. A ligand can be a complete ligand, a partial ligand or a reverse ligand.

[0087] As used herein, the term "antibody" encompasses immunoglobulins and fragments thereof that are produced naturally or partially or completely synthetically. The term also encompasses any protein having a binding domain that is homologous to an immunoglobulin binding domain. "Antibodies" further include polypeptides that specifically bind to and recognize an antigen and comprise framework regions from an immunoglobulin gene or a fragment thereof. The use of the term antibody is intended to include complete antibodies, polyclonal antibodies, monoclonal antibodies and recombinant antibodies, fragments thereof, and further include single-chain antibodies, humanized antibodies, murine antibodies, chimeric antibodies, mouse-human antibodies, mouse-primate antibodies, primate-human monoclonal antibodies, anti-idiotypic antibodies, antibody fragments, such as scFv, (scFv) 2 , Fab, Fab' and F(ab') 2 、F(ab1) 2 , Fv, dAb and Fd fragments, bifunctional antibodies and antibody-related polypeptides. Antibodies include bispecific antibodies and multispecific antibodies, as long as they exhibit the desired biological activity or function.

[0088] As used herein, the term "therapeutically effective amount" is an amount of an agent or pharmaceutical compound sufficient to produce a desired therapeutic, pharmacological and / or physiological effect on a subject in need thereof. A therapeutically effective amount can be a "prophylactically effective amount" because prevention and treatment can be considered therapy.

[0089] As used herein, the term "pharmaceutical composition" refers to one or more compounds, e.g., EVs, of the compounds described herein that are mixed or doped with one or more other chemical components, such as pharmaceutically acceptable carriers and excipients, or suspended in the one or more other chemical components. One purpose of a pharmaceutical composition is to facilitate the preparation of EVs for administration to a subject. The term "excipient" or "carrier" refers to an inert substance added to a pharmaceutical composition to further facilitate the administration of a compound. The term "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" and its grammatical variants cover any agent approved by a regulatory agency of the U.S. federal government or listed in the U.S. Pharmacopoeia for use in animals, including humans, and any carrier or diluent that does not produce an undesirable physiological effect to the extent that it inhibits the administration of the composition to a subject and does not eliminate the biological activity and properties of the administered compound. Excipients and carriers that can be used to prepare pharmaceutical compositions and are generally safe, non-toxic and desirable are included.

[0090] As used herein, the term "payload" refers to a therapeutic agent that acts on a target (e.g., a target cell) that contacts an EV. Payloads that can be introduced into EVs and / or producer cells include therapeutic agents such as nucleotides (e.g., nucleotides that contain a detectable moiety or a toxin or that disrupt transcription), nucleic acids (e.g., DNA or mRNA molecules that encode polypeptides such as enzymes, or RNA molecules with regulatory functions such as miRNA, dsDNA, lncRNA, and siRNA), amino acids (e.g., amino acids that contain a detectable moiety or a toxin or that disrupt translation), polypeptides (e.g., enzymes), lipids, carbohydrates, and small molecules (e.g., small molecule drugs and toxins).

[0091] The terms "administration," "administering," and variations thereof refer to the introduction of a composition (such as an EV) or an agent into a subject and include simultaneous and sequential introduction of a composition or agent. The composition or agent is introduced into a subject by any suitable route, including intratumorally, orally, pulmonary, intranasally, parenterally (intravenously, intraarterially, intramuscularly, intraperitoneally, or subcutaneously), rectally, intralymphatically, intrathecally, periocularly, or topically. Administration includes self-administration and administration by another person. A suitable route of administration allows the composition or agent to perform its intended function. For example, if the suitable route is intravenous, the composition is administered by introducing the composition or agent into a vein of the subject.

[0092] As used herein, the terms "treat," "treatment," or "treating" refer to, for example, reducing the severity of a disease or condition; reducing the duration of a disease process; ameliorating or eliminating one or more symptoms associated with a disease or condition; providing a beneficial effect to a subject with a disease or condition, but not necessarily curing the disease or condition. The terms also include preventing or preventing a disease or condition, or a symptom thereof. In one aspect, the terms "treating" or "treatment" means inducing an immune response to an antigen in a subject.

[0093] As used herein, the term "prevent" or "preventing" refers to reducing or decreasing the incidence or severity of a particular outcome. In some aspects, an outcome is prevented by prophylactic treatment.

[0094] As used herein, the terms "modulate", "modulate", "modify" and / or "modulator" generally refer to the ability to change by increasing or decreasing, for example, directly or indirectly promoting / stimulating / upregulating or interfering / inhibiting / downregulating a specific concentration, level, expression, function or behavior, such as acting as an antagonist or agonist. In some cases, a modulator can increase and / or decrease a certain concentration, level, activity or function relative to a control or relative to a generally expected average activity level or relative to a control activity level.

[0095] As used herein, "mammalian subjects" include all mammals, including but not limited to humans, domestic animals (e.g., dogs, cats, etc.), farm animals (e.g., cows, sheep, pigs, horses, etc.), and laboratory animals (e.g., monkeys, rats, mice, rabbits, guinea pigs, etc.).

[0096] The terms "individual," "subject," "host," and "patient" are used interchangeably herein and refer to any mammalian subject, particularly a human, for whom diagnosis, treatment, or therapy is desired. The methods described herein are applicable to both human therapy and veterinary applications. In some aspects, the subject is a mammal, and in other aspects, the subject is a human.

[0097] As used herein, the term "substantially free" means that the sample containing EVs contains less than 10% macromolecules by mass / volume (m / v) percentage concentration. Some fractions may contain less than 0.001%, less than 0.01%, less than 0.05%, less than 0.1%, less than 0.2%, less than 0.3%, less than 0.4%, less than 0.5%, less than 0.6%, less than 0.7%, less than 0.8%, less than 0.9%, less than 1%, less than 2%, less than 3%, less than 4%, less than 5%, less than 6%, less than 7%, less than 8%, less than 9% or less than 10% (m / v) macromolecules.

[0098] As used herein, the term "macromolecule" means nucleic acid, exogenous protein, lipid, carbohydrate, metabolite or a combination thereof.

[0099] As used herein, the terms "insignificant," "reduced," or "negligible" refer to the presence, level, or amount of an inflammatory response in a subject following administration of a sample comprising EVs encapsulating a STING agonist relative to the subject's baseline inflammatory response or compared to the inflammatory response following administration of a free STING agonist to the subject. For example, the presence, level, or amount of systemic inflammation that is negligible or insignificant relative to baseline inflammation in the subject or compared to the subject's immune response to administration of a free STING agonist may be less than 0.001%, less than 0.01%, less than 0.1%, less than 0.2%, less than 0.3%, less than 0.4%, less than 0.5%, less than 0.6%, less than 0.7%, less than 0.8%, less than 0.9%, less than 1%, less than 2%, less than 3%, less than 4%, less than 5%, less than 6%, less than 7%, less than 8%, less than 9%, less than 10%, less than 12%, less than 15%, less than 17%, less than 20%, or less than 25% of systemic inflammation. The level or amount of systemic inflammation can be 0.1 fold less, 0.5 fold less, 0.5 fold less, 1 fold less, 1.5 fold less, 2 fold less than baseline or the inflammatory response to administration of a free STING agonist.

[0100] As used herein, "primary tumor" refers to the original or first tumor in a subject's body where the tumor begins to grow. Primary tumors are used in contrast to "secondary tumors," which refer to tumors that arise at a location other than the location of the primary tumor after the primary tumor begins to grow, such as due to metastasis of cells in the primary tumor.

[0101] The ranges recited herein are to be understood as shorthand for all values ​​within the range, including the recited endpoints. For example, a range of 1 to 50 is to be understood to include any number, combination of numbers, or subrange from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50.

[0102] Unless otherwise stated, reference to a compound having one or more stereocenters is intended to refer to each stereoisomer thereof and all combinations of stereoisomers thereof.

[0103] II. Treatment Methods

[0104] Certain aspects of the present disclosure relate to a method of preventing or treating a tumor in a subject in need thereof, the method comprising administering to the subject a dose of a STING agonist, wherein the amount of the STING agonist in the dose is about 0.01 μg to about 100 μg. In some aspects, the STING agonist is delivered via extracellular vesicles (EVs). In some aspects, the STING agonist is associated with an EV.

[0105] In some aspects, the STING agonist is loaded in the lumen of the EV. In some aspects, the STING agonist is not associated with the EV. In some aspects, the STING agonist is loaded in nanoparticles. In some aspects, the STING agonist is loaded in nanoparticles selected from the group consisting of lipid nanoparticles, liposomes, polymer micelles, dendrimers, chitosan nanoparticles, alginate nanoparticles, xanthan gum-based nanoparticles, cellulose nanocrystals, inorganic nanoparticles (e.g., silver, gold, iron oxide and silica nanoparticles), nanocrystals, metal nanoparticles, quantum dots, and any combination thereof.

[0106] In some aspects, the tumor that can be treated by the method of the present invention is a primary tumor, a secondary tumor, or both a primary tumor and a secondary tumor. In some aspects, the volume of the tumor is reduced by administration. In some aspects, the volume of the primary tumor is reduced by administration. In some aspects, compared with the monotherapy after administration of the 14th day, the volume of the primary tumor can be reduced by at least about 1.5 times, at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times or at least about 10 times.

[0107] In some aspects, use reduces the tumor growth rate of the primary tumor. In some aspects, administration can reduce the growth rate of the primary tumor by at least about 1.5 times, at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, or at least about 10 times, compared to monotherapy after administration of the 14th day. In some aspects, administration can ablate or prevent primary tumor growth.

[0108] The method of any one of claims 4 to 8, wherein the administration reduces the volume of secondary tumors. In some aspects, the administration can reduce the volume of secondary tumors by at least about 1.5 times, at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, or at least about 10 times compared to monotherapy after day 14 of administration.

[0109] In some aspects, use reduces the tumor growth rate of secondary tumors. In some aspects, administration can reduce the growth rate of secondary tumors by at least about 1.5 times, at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, or at least about 10 times compared to monotherapy after administration of day 14. In some aspects, administration can ablate or prevent secondary tumor growth.

[0110] In some aspects, the method further comprises administering an anticancer agent. In some aspects, the anticancer agent comprises a checkpoint inhibitor. In some aspects, the checkpoint inhibitor comprises an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-CTLA-4 antibody, an anti-LAG-3 antibody, an anti-TIM-3 antibody, or any combination thereof. In some aspects, the checkpoint inhibitor is an anti-PD-1 antibody.

[0111] II.A. STING agonists

[0112] The innate immune system recognizes pathogen-associated molecular patterns (PAMPs) through pattern recognition receptors (PRRs) that induce immune responses. PRRs recognize a variety of pathogen molecules including single-stranded and double-stranded RNA and DNA. PRRs, such as retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs) and some toll-like receptors (TLRs), recognize RNA ligands. DNA ligands are recognized by cyclic GMP-AMP synthase (cGAS), AIM2, and other TLRs. TLRs, RLRs, and AIM2 directly interact with other signaling cascade adaptor proteins to activate transcription factors, while cGAS produces cGAMP, a cyclic dinucleotide molecule that activates the stimulator of interferon genes (STING) receptor. Both STING and RLRs activate the adaptor kinase TBK1, which induces the activation of transcription factors IRF3 and NF-κB, and causes the production of type I IFNs and proinflammatory cytokines.

[0113] Cyclic dinucleotides (CDNs) were first identified as bacterial signaling molecules characterized by two 3',5' phosphodiester bonds as in the molecule c-di-GMP. While STING can be activated by bacterial CDNs, innate immune responses in mammalian cells are also mediated by the CDN signaling molecule cGAMP produced by cGAS. cGAMP is characterized by mixed 2',5' and 3',5' phosphodiester bonds. Both bacterial and mammalian CDNs interact directly with STING to induce a pro-inflammatory signaling cascade, leading to the production of type I IFNs such as IFNα and IFNβ.

[0114] The STING agonists used in the present disclosure may be cyclic dinucleotides (CDNs) or non-cyclic dinucleotide agonists. It is known that cyclic purine dinucleotides such as, but not limited to, cGMP, cyclic diGMP (c-di-GMP), cAMP, cyclic diAMP (c-di-AMP), cyclic GMP-AMP (cGAMP), cyclic diIMP (c-di-IMP), cyclic AMP-IMP (cAIMP), and any analogs thereof, can stimulate or enhance an immune or inflammatory response in a patient. The CDNs may have 2'2', 2'3', 2'5', 3'3', or 3'5' bonds connecting the cyclic dinucleotides, or any combination thereof.

[0115] Cyclic purine dinucleotides can be modified by standard organic chemistry techniques to produce analogs of purine dinucleotides. Suitable purine dinucleotides include, but are not limited to, adenine, guanine, inosine, hypoxanthine, xanthine, isoguanine, or any other suitable purine dinucleotides known in the art. Cyclic dinucleotides can be modified analogs. Any suitable modification known in the art can be used, including but not limited to phosphorothioate modification, phosphorodithioate modification, fluoro ester modification, and difluoro ester modification.

[0116] Acyclic dinucleotide agonists may also be used, such as 5,6-dimethylxanthone-4-acetic acid (DMXAA), or any other acyclic dinucleotide agonist known in the art.

[0117] It is contemplated that any STING agonist may be used. wherein the STING agonist is DMXAA, STING agonist-1, ML RR-S2 CDA, ML RR-S2c-di-GMP, ML-RR-S2 cGAMP, 2'3'-c-di-AM(PS)2, 2'3'-cGAMP, 2'3'-cGAMPdFHS, 3'3'-cGAMP, 3'3'-cGAMPdFSH, cAIMP, cAIM(PS)2, 3'3'-cAIMP, 3'3'-cAIMPdFSH, 2'2'-cGAMP, 2'3'-cGAM(PS)2, 3'3'-cGAMP, c-di-AMP, 2'3'-c-di-AMP, 2'3'-c-di-AM(PS)2, c-di-GMP, 2'3'-c-di-GMP, c-di-IMP, c-di-UMP or any combination thereof. In a preferred aspect, the STING agonist is 3'3'-cAIMPdFSH, alternatively designated 3-3cAIMPdFSH. Additional STING agonists known in the art may also be used.

[0118] In some aspects, STING agonists useful in the present disclosure include compounds having the following formula:

[0119]

[0120] in:

[0121] X 1 is H, OH or F;

[0122] X 2 is H, OH or F;

[0123] Z is OH, OR 1 , SH or SR 1 ,in:

[0124] i)R 1 is Na or NH 4 ,or

[0125] ii) R 1 It is an enzyme-labile group that donates OH or SH in vivo, such as pivaloyloxymethyl;

[0126] Bi and B2 are bases selected from the group consisting of:

[0127]

[0128] The conditions are:

[0129] - In formula (I): X 1 and X 2 Not OH,

[0130] - In formula (II): when X 1 and X 2 When it is OH, B 1 is not adenine, and B 2 is not guanine, and

[0131] - In formula (III): when X 1 and X 2 When it is OH, B 1 Not adenine, B 2 is not guanine, and Z is not OH. See WO 2016 / 096174, the contents of which are incorporated herein by reference in their entirety.

[0132] In some aspects, STING agonists useful in the present disclosure include:

[0133]

[0134]

[0135] Its pharmaceutically acceptable salts. See WO 2016 / 096174A1.

[0136] In another aspect, STING agonists useful in the present disclosure include compounds having the formula:

[0137]

[0138]

[0139] or any pharmaceutically acceptable salt thereof.

[0140] In some aspects, STING agonists useful in the present disclosure include compounds having the following formula:

[0141]

[0142] Each of the symbols therein is defined in WO 2014 / 093936, the contents of which are incorporated herein by reference in their entirety.

[0143] In some aspects, STING agonists useful in the present disclosure include compounds having the following formula:

[0144]

[0145] Each of the symbols therein is defined in WO 2014 / 189805, the contents of which are incorporated herein by reference in their entirety.

[0146] In some aspects, STING agonists useful in the present disclosure include compounds having the following formula:

[0147]

[0148] Each symbol is defined in WO 2015 / 077354, the contents of which are incorporated herein by reference in their entirety. See also Cell Reports 11, 1018-1030 (2015).

[0149] In some aspects, STING agonists useful in the present disclosure include c-di-AMP, c-di-GMP, c-di-IMP, c-AMP-GMP, c-AMP-IMP, and c-GMP-IMP described in WO 2013 / 185052 and Sci. Transl. Med. 283, 283ra52 (2015), which are incorporated herein by reference in their entireties.

[0150] In some aspects, STING agonists useful in the present disclosure include compounds having the following formula:

[0151]

[0152] Each of the symbols therein is defined in WO 2014 / 189806, the contents of which are incorporated herein by reference in their entirety.

[0153] In some aspects, STING agonists useful in the present disclosure include compounds having the following formula:

[0154]

[0155] Each of the symbols therein is defined in WO 2015 / 185565, the contents of which are incorporated herein by reference in their entirety.

[0156] In some aspects, STING agonists useful in the present disclosure include compounds having the following formula:

[0157]

[0158] Each of the symbols therein is defined in WO 2014 / 179760, the contents of which are incorporated herein by reference in their entirety.

[0159] In some aspects, STING agonists useful in the present disclosure include compounds having the following formula:

[0160]

[0161]

[0162] Each of the symbols therein is defined in WO 2014 / 179335, the contents of which are incorporated herein by reference in their entirety.

[0163] In some aspects, STING agonists useful in the present disclosure include compounds having the following formula:

[0164]

[0165] It is described in WO 2015 / 017652, the contents of which are incorporated herein by reference in their entirety.

[0166] In some aspects, STING agonists useful in the present disclosure include compounds having the following formula:

[0167]

[0168] It is described in WO 2016 / 096577, the contents of which are incorporated herein by reference in their entirety.

[0169] In some aspects, STING agonists useful in the present disclosure include compounds having the following formula:

[0170]

[0171] Each of the symbols is defined in WO 2016 / 120305, the contents of which are incorporated herein by reference in their entirety.

[0172] In some aspects, STING agonists useful in the present disclosure include compounds having the following formula:

[0173]

[0174] Each of the symbols is defined in WO 2016 / 145102, the contents of which are incorporated herein by reference in their entirety.

[0175] In some aspects, STING agonists useful in the present disclosure include compounds having the following formula:

[0176]

[0177] Each of the symbols is defined in WO 2017 / 027646, the contents of which are incorporated herein by reference in their entirety.

[0178] In some aspects, STING agonists useful in the present disclosure include compounds having the following formula:

[0179]

[0180] Each of the symbols is defined in WO 2017 / 075477, the contents of which are incorporated herein by reference in their entirety.

[0181] In some aspects, STING agonists useful in the present disclosure include compounds having the following formula:

[0182]

[0183] Each of the symbols is defined in WO 2017 / 027645, the contents of which are incorporated herein by reference in their entirety.

[0184] In some aspects, STING agonists useful in the present disclosure include compounds having the following formula:

[0185]

[0186] Each of the symbols is defined in WO 2018 / 100558, the contents of which are incorporated herein by reference in their entirety.

[0187] In some aspects, STING agonists useful in the present disclosure include compounds having the following formula:

[0188]

[0189] Each of the symbols is defined in WO 2017 / 175147, the contents of which are incorporated herein by reference in their entirety.

[0190] In some aspects, STING agonists useful in the present disclosure include compounds having the following formula:

[0191]

[0192] Each of the symbols is defined in WO 2017 / 175156, the contents of which are incorporated herein by reference in their entirety.

[0193] In some aspects, the STING agonist that can be used in the present disclosure is CL606, CL611, CL602, CL655, CL604, CL609, CL614, CL656, CL647, CL626, CL629, CL603, CL632, CL633, CL659, or a pharmaceutically acceptable salt thereof. In some aspects, the STING agonist that can be used in the present disclosure is CL606 or a pharmaceutically acceptable salt thereof. In some aspects, the STING agonist that can be used in the present disclosure is CL611 or a pharmaceutically acceptable salt thereof. In some aspects, the STING agonist that can be used in the present disclosure is CL602 or a pharmaceutically acceptable salt thereof. In some aspects, the STING agonist that can be used in the present disclosure is CL655 or a pharmaceutically acceptable salt thereof. In some aspects, the STING agonist that can be used in the present disclosure is CL604 or a pharmaceutically acceptable salt thereof. In some aspects, the STING agonist that can be used in the present disclosure is CL609 or a pharmaceutically acceptable salt thereof. In some aspects, the STING agonist useful in the present disclosure is CL614 or a pharmaceutically acceptable salt thereof. In some aspects, the STING agonist useful in the present disclosure is CL656 or a pharmaceutically acceptable salt thereof. In some aspects, the STING agonist useful in the present disclosure is CL647 or a pharmaceutically acceptable salt thereof. In some aspects, the STING agonist useful in the present disclosure is CL626 or a pharmaceutically acceptable salt thereof. In some aspects, the STING agonist useful in the present disclosure is CL629 or a pharmaceutically acceptable salt thereof. In some aspects, the STING agonist useful in the present disclosure is CL603 or a pharmaceutically acceptable salt thereof. In some aspects, the STING agonist useful in the present disclosure is CL632 or a pharmaceutically acceptable salt thereof. In some aspects, the STING agonist useful in the present disclosure is CL633 or a pharmaceutically acceptable salt thereof. In some aspects, the STING agonist useful in the present disclosure is CL659 or a pharmaceutically acceptable salt thereof.

[0194] In some aspects, EVs (e.g., exosomes) comprise cyclic dinucleotide STING agonists and / or non-cyclic dinucleotide STING agonists. In some aspects, when several cyclic dinucleotide STING agonists are present on the EVs (e.g., exosomes) disclosed herein, such STING agonists may be the same or they may be different. In some aspects, when several non-cyclic dinucleotide STING agonists are present, such STING agonists may be the same or they may be different. In some aspects, the EV (e.g., exosome) compositions disclosed herein may comprise two or more EV populations (e.g., exosomes), wherein each EV population (e.g., exosomes) comprises a different STING agonist or a combination thereof.

[0195] STING agonists can also be modified to increase encapsulation of the agonist in extracellular vesicles or EVs (e.g., unbound in the lumen). In some aspects, the STING agonist is linked to a scaffold portion (e.g., scaffold Y). In certain aspects, the modification allows the STING agonist to be better expressed on the outer surface of an EV (e.g., exosome) (e.g., linked to a scaffold portion disclosed herein, such as scaffold X). This modification can include adding a lipid binding tag by treating the agonist with a chemical or enzyme, or by physically or chemically changing the polarity or charge of the STING agonist. The STING agonist can be modified by a single treatment or by a combination of treatments, for example, adding only a lipid binding tag, or adding a lipid binding tag and changing the polarity. The previous examples are meant to be non-limiting illustrative examples. It is contemplated that any combination of modifications can be practiced.

[0196] II.B. Administration

[0197] In some aspects, the method comprises administering a dose of a STING agonist to a subject, wherein the amount of the STING agonist in the dose is about 0.1 μg to about 100 μg. In some aspects, the amount of the STING agonist is measured using A260. In some aspects, the amount of the STING agonist is measured using mass spectrometry. In some aspects, the amount of the STING agonist is measured using reverse phase high performance liquid chromatography (RP-HPLC). In some aspects, the dose of the STING agonist administered is based on the weight of the STING agonist, excluding the weight of other linkers that can be used to associate the STING agonist with an extracellular vesicle (e.g., exosome).

[0198] In some aspects, the dose comprises about 0.1 μg to about 1 μg, about 0.1 μg to about 10 μg, about 0.1 μg to about 20 μg, about 0.1 μg to about 30 μg, about 0.1 μg to about 40 μg, about 0.1 μg to about 50 μg, about 0.1 μg to about 60 μg, about 0.1 μg to about 70 μg, about 0.1 μg to about 80 μg, 0.1 μg to about 90 μg, about 0.1 to about 100 μg, about 1 μg to about 10 μg, about 1 μg to about 20 μg, about 1 μg to about 30 μg, about 1 μg to about 40 μg, about 1 μg to about 50 μg. g to about 50 μg, about 1 μg to about 60 μg, about 1 μg to about 70 μg, about 1 μg to about 80 μg, about 1 μg to about 90 μg, about 1 μg to about 100 μg, about 10 μg to about 20 μg, about 10 μg to about 30 μg, about 10 μg to about 40 μg, about 10 μg to about 50 μg, about 10 μg to about 60 μg, about 10 μg to about 70 μg, about 10 μg to about 80 μg, about 90 μg to about 10 μg; about 20 μg to about 30 μg, about 20 μg to about 40 μg, about 20 μg to about 50 μg, about 20 μg to about g to about 60 μg, about 20 μg to about 80 μg, about 20 μg to about 90 μg, about 20 μg to about 100 μg, about 30 μg to about 40 μg, about 30 μg to about 50 μg, about 30 μg to about 60 μg, about 30 μg to about 70 μg, about 30 μg to about 80 μg, about 30 μg to about 90 μg, about 30 μg to about 100 μg, about 40 μg to about 50 μg, about 40 μg to about 60 μg, about 40 μg to about 70 μg, about 40 μg to about 80 μg, about 40 μg to about 90 μg, about 40 μg to about 10 In some aspects, the dosage comprises about 0.1 μg to about 12.0 μg of the STING agonist. In some aspects, the dosage comprises about 0.3 μg to about 12.0 μg of the STING agonist. In some aspects, the dose comprises about 0.3 μg to about 6.0 μg of the STING agonist. In some aspects, the dose comprises about 1.0 μg to about 12.0 μg of the STING agonist. In some aspects, the dose comprises about 1.0 μg to about 6.0 μg of the STING agonist.In some aspects, the dose comprises about 1.0 μg to about 3.0 μg of the STING agonist. In some aspects, the dose comprises about 3.0 μg to about 6.0 μg of the STING agonist.

[0199] In some aspects, the dose comprises at least about 0.1 μg, at least about 0.2 μg, at least about 0.3 μg, at least about 0.4 μg, at least about 0.5 μg, at least about 0.6 μg, at least about 0.7 μg, at least about 0.8 μg, at least about 0.9 μg, at least about 1.0 μg, at least about 1.1 μg, at least about 1.2 μg, at least about 1.3 μg, at least about 1.4 μg, at least about 1.5 μg, at least about 1.6 μg, at least about 1.7 μg, at least about 1.8 μg, at least about 1.9 μg, at least about 2.0 μg, at least about 2.1 μg, at least about 2.2 μg, At least about 2.3 μg, at least about 2.4 μg, at least about 2.5 μg, at least about 2.6 μg, at least about 2.7 μg, at least about 2.8 μg, at least about 2.9 μg, at least about 3.0 μg, at least about 3.1 μg, at least about 3.2 μg, at least about 3.3 μg, at least about 3.4 μg, at least about 3.5 μg, at least about 3.6 μg, at least about 3.7 μg, at least about 3.8 μg, at least about 3.9 μg, at least about 4.0 μg, at least about 4.1 μg, at least about 4.2 μg, at least about 4.3 μg, at least about 4.4 μg, at least about 4.5 μg, at least about 4.6 μg, at least about 4.7 μg, at least about 4.8 μg, at least about 4.9 μg, at least about 5.0 μg, at least about 5.1 μg, at least about 5.2 μg, at least about 5.3 μg, at least about 5.4 μg, at least about 5.5 μg, at least about 5.6 μg, at least about 5.7 μg, at least about 5.8 μg, at least about 5.9 μg, at least about 6.0 μg, at least about 6.5 μg, at least about 7.0 μg, at least about 7.5 μg, at least about 8.0 μg, at least about 8.5 μg, at least about 9.0 μg, at least about 9.5 μg, at least about 10.0 μg, at least about 10 .5 μg, at least about 11.0 μg, at least about 11.5 μg, at least about 12.0 μg, at least about 12.5 μg, at least about 13.0 μg, at least about 14 μg, at least about 15 μg, at least about 16 μg, at least about 17 μg, at least about 18 μg, at least about 19 μg, at least about 20 μg, at least about 25 μg, at least about 30 μg, at least about 35 μg, at least about 40 μg, at least about 45 μg, at least about 50 μg, at least about 60 μg, at least about 70 μg, at least about 80 μg, at least about 90 μg or at least about 100 μg of the STING agonist.

[0200] In some aspects, the dose comprises at least about 0.3 μg of the STING agonist. In some aspects, the dose comprises at least about 0.4 μg of the STING agonist. In some aspects, the dose comprises at least about 0.5 μg of the STING agonist. In some aspects, the dose comprises at least about 0.6 μg of the STING agonist. In some aspects, the dose comprises at least about 0.7 μg of the STING agonist. In some aspects, the dose comprises at least about 0.8 μg of the STING agonist. In some aspects, the dose comprises at least about 0.9 μg of the STING agonist. In some aspects, the dose comprises at least about 1.0 μg of the STING agonist. In some aspects, the dose comprises at least about 1.5 μg of the STING agonist. In some aspects, the dose comprises at least about 2.0 μg of the STING agonist. In some aspects, the dose comprises at least about 2.5 μg of the STING agonist. In some aspects, the dose comprises at least about 3.0 μg of the STING agonist. In some aspects, the dose comprises at least about 3.5 μg of the STING agonist. In some aspects, the dose comprises at least about 4.0 μg of the STING agonist. In some aspects, the dose comprises at least about 4.5 μg of the STING agonist. In some aspects, the dose comprises at least about 5.0 μg of the STING agonist. In some aspects, the dose comprises at least about 5.5 μg of the STING agonist. In some aspects, the dose comprises at least about 6.0 μg of the STING agonist. In some aspects, the dose comprises at least about 6.5 μg of the STING agonist. In some aspects, the dose comprises at least about 7.0 μg of the STING agonist. In some aspects, the dose comprises at least about 7.5 μg of the STING agonist. In some aspects, the dose comprises at least about 8.0 μg of the STING agonist. In some aspects, the dose comprises at least about 8.5 μg of the STING agonist. In some aspects, the dose comprises at least about 9.0 μg of the STING agonist. In some aspects, the dose comprises at least about 9.5 μg of the STING agonist. In some aspects, the dose comprises at least about 10.0 μg of the STING agonist. In some aspects, the dose comprises at least about 10.5 μg of the STING agonist. In some aspects, the dose comprises at least about 11.0 μg of the STING agonist. In some aspects, the dose comprises at least about 11.5 μg of the STING agonist. In some aspects, the dose comprises at least about 12.0 μg of the STING agonist. In some aspects, the dose comprises at least about 12.5 μg of the STING agonist.In some aspects, the dose comprises at least about 13.0 μg of the STING agonist. In some aspects, the dose comprises at least about 13.5 μg of the STING agonist. In some aspects, the dose comprises at least about 14.0 μg of the STING agonist. In some aspects, the dose comprises at least about 14.5 μg of the STING agonist. In some aspects, the dose comprises at least about 15 μg of the STING agonist. In some aspects, the dose comprises at least about 16 μg of the STING agonist. In some aspects, the dose comprises at least about 17 μg of the STING agonist. In some aspects, the dose comprises at least about 18 μg of the STING agonist. In some aspects, the dose comprises at least about 19 μg of the STING agonist. In some aspects, the dose comprises at least about 20 μg of the STING agonist. In some aspects, the dose comprises at least about 21 μg of the STING agonist. In some aspects, the dose comprises at least about 22 μg of the STING agonist. In some aspects, the dose comprises at least about 23 μg of the STING agonist. In some aspects, the dose comprises at least about 24 μg of the STING agonist. In some aspects, the dose comprises at least about 25 μg of the STING agonist. In some aspects, the dose comprises at least about 26 μg of the STING agonist. In some aspects, the dose comprises at least about 27 μg of the STING agonist. In some aspects, the dose comprises at least about 28 μg of the STING agonist. In some aspects, the dose comprises at least about 29 μg of the STING agonist. In some aspects, the dose comprises at least about 30 μg of the STING agonist.

[0201] In some aspects, the dose of the STING agonist is administered at least two times, at least three times, at least four times, at least five times, or at least six times. In some aspects, the method comprises administering a second dose, a third dose, a fourth dose, or a fifth dose of the STING agonist. In some aspects, one or more of the second dose, the third dose, the fourth dose, or the fifth dose is administered at the same dose as the initial dose. In some aspects, one or more of the second dose, the third dose, the fourth dose, or the fifth dose is administered at a different dose than the initial dose.

[0202] In some aspects, the dosage of the STING agonist is administered about once a week, about once every two weeks, about once every three weeks, about once every four weeks, about once every six weeks, or about once every eight weeks. In some aspects, the STING agonist is administered about once a week. In some aspects, the STING agonist is administered about once every two weeks. In some aspects, the STING agonist is administered about once every three weeks. In some aspects, the STING agonist is administered about once every four weeks.

[0203] In some aspects, the dose is administered on about day 1 and day 15 of the first 28-day cycle. In some aspects, the dose is administered on about day 1 and day 15 of the second 28-day cycle. In some aspects, the dose is administered on about day 1 of the third 28-day cycle. In some aspects, the dose is administered on day 1 of each subsequent 28-day cycle. In some aspects, the dose is administered (i) on about day 1 and day 15 of the first 28-day cycle, and (ii) on about day 1 and day 15 of the second 28-day cycle. In some aspects, the dose is administered (i) on about day 1 and day 15 of the first 28-day cycle, (ii) on about day 1 and day 15 of the second 28-day cycle, and (iii) on about day 1 of the third 28-day cycle. In certain aspects, the dose is administered (i) on about days 1 and 15 of a first 28-day cycle, (ii) on about days 1 and 15 of a second 28-day cycle, (iii) on about day 1 of a third 28-day cycle, and (iii) about every 28 days thereafter in the third 28-day cycle.

[0204] In some aspects, a dosage of about 0.3 μg is applied on about the 1st and 15th day of the first 28-day cycle. In some aspects, a dosage of about 0.3 μg is applied on about the 1st day of the second 28-day cycle. In some aspects, a dosage of about 0.3 μg is applied on about the 1st day of the third 28-day cycle. In some aspects, a dosage of about 0.3 μg is applied on the 1st day of each subsequent 28-day cycle. In some aspects, a dosage of about 0.3 μg is applied (i) on about the 1st and 15th day of the first 28-day cycle, and (ii) on about the 1st and 15th day of the second 28-day cycle. In some aspects, a dosage of about 0.3 μg is applied (i) on about the 1st and 15th day of the first 28-day cycle, (ii) on about the 1st and 15th day of the second 28-day cycle, and (iii) on about the 1st day of the third 28-day cycle. In certain aspects, a dose of about 0.3 μg is administered (i) on about days 1 and 15 of a first 28-day cycle, (ii) on about days 1 and 15 of a second 28-day cycle, (iii) on about day 1 of a third 28-day cycle, and (iii) about every 28 days thereafter in the third 28-day cycle.

[0205] In some aspects, a dosage of about 1.0 μg is applied on about the 1st and 15th day of the first 28-day cycle. In some aspects, a dosage of about 1.0 μg is applied on about the 1st day of the second 28-day cycle. In some aspects, a dosage of about 1.0 μg is applied on about the 1st day of the third 28-day cycle. In some aspects, a dosage of about 1.0 μg is applied on the 1st day of each subsequent 28-day cycle. In some aspects, a dosage of about 1.0 μg is applied (i) on about the 1st and 15th day of the first 28-day cycle, and (ii) on about the 1st and 15th day of the second 28-day cycle. In some aspects, a dosage of about 1.0 μg is applied (i) on about the 1st and 15th day of the first 28-day cycle, (ii) on about the 1st and 15th day of the second 28-day cycle, and (iii) on about the 1st day of the third 28-day cycle. In certain aspects, a dose of about 1.0 μg is administered (i) on about days 1 and 15 of a first 28-day cycle, (ii) on about days 1 and 15 of a second 28-day cycle, (iii) on about day 1 of a third 28-day cycle, and (iii) about every 28 days thereafter in the third 28-day cycle.

[0206] In some aspects, a dosage of about 3.0 μg is applied on about the 1st and 15th day of the first 28-day cycle. In some aspects, a dosage of about 3.0 μg is applied on about the 1st day of the second 28-day cycle. In some aspects, a dosage of about 3.0 μg is applied on about the 1st day of the third 28-day cycle. In some aspects, a dosage of about 3.0 μg is applied on the 1st day of each subsequent 28-day cycle. In some aspects, a dosage of about 3.0 μg is applied (i) on about the 1st and 15th day of the first 28-day cycle, and (ii) on about the 1st and 15th day of the second 28-day cycle. In some aspects, a dosage of about 3.0 μg is applied (i) on about the 1st and 15th day of the first 28-day cycle, (ii) on about the 1st and 15th day of the second 28-day cycle, and (iii) on about the 1st day of the third 28-day cycle. In certain aspects, a dose of about 3.0 μg is administered (i) on about days 1 and 15 of a first 28-day cycle, (ii) on about days 1 and 15 of a second 28-day cycle, (iii) on about day 1 of a third 28-day cycle, and (iii) about every 28 days thereafter in the third 28-day cycle.

[0207] In some aspects, a dosage of about 6.0 μg is applied on about the 1st and 15th day of the first 28-day cycle. In some aspects, a dosage of about 6.0 μg is applied on about the 1st day of the second 28-day cycle. In some aspects, a dosage of about 6.0 μg is applied on about the 1st day of the third 28-day cycle. In some aspects, a dosage of about 6.0 μg is applied on the 1st day of each subsequent 28-day cycle. In some aspects, a dosage of about 6.0 μg is applied (i) on about the 1st and 15th day of the first 28-day cycle, and (ii) on about the 1st and 15th day of the second 28-day cycle. In some aspects, a dosage of about 6.0 μg is applied (i) on about the 1st and 15th day of the first 28-day cycle, (ii) on about the 1st and 15th day of the second 28-day cycle, and (iii) on about the 1st day of the third 28-day cycle. In certain aspects, a dose of about 6.0 μg is administered (i) on about days 1 and 15 of a first 28-day cycle, (ii) on about days 1 and 15 of a second 28-day cycle, (iii) on about day 1 of a third 28-day cycle, and (iii) about every 28 days thereafter in the third 28-day cycle.

[0208] In some aspects, a dosage of about 12.0 μg is applied on about the 1st and 15th day of the first 28-day cycle. In some aspects, a dosage of about 12.0 μg is applied on about the 1st day of the second 28-day cycle. In some aspects, a dosage of about 12.0 μg is applied on about the 1st day of the third 28-day cycle. In some aspects, a dosage of about 12.0 μg is applied on the 1st day of each subsequent 28-day cycle. In some aspects, a dosage of about 12.0 μg is applied (i) on about the 1st and 15th day of the first 28-day cycle, and (ii) on about the 1st and 15th day of the second 28-day cycle. In some aspects, a dosage of about 12.0 μg is applied (i) on about the 1st and 15th day of the first 28-day cycle, (ii) on about the 1st and 15th day of the second 28-day cycle, and (iii) on about the 1st day of the third 28-day cycle. In certain aspects, a dose of about 12.0 μg is administered (i) on about days 1 and 15 of a first 28-day cycle, (ii) on about days 1 and 15 of a second 28-day cycle, (iii) on about day 1 of a third 28-day cycle, and (iii) about every 28 days thereafter in the third 28-day cycle.

[0209] In some aspects, administration of the IL-12 polypeptide continues until a clinical benefit is achieved.

[0210] II.C. Scaffold-engineered EVs (e.g., exosomes)

[0211] In some aspects, the EVs of the present disclosure comprise a membrane that is modified in its composition. For example, its membrane composition can be modified by changing the protein, lipid, or glycan content of the membrane.

[0212] In some aspects, surface engineered EVs are produced by chemical and / or physical methods (such as PEG-induced fusion and / or ultrasonic fusion). In other aspects, surface engineered EVs (e.g., exosomes) are produced by genetic engineering. EVs produced by genetically modified producer cells or progeny of genetically modified cells may contain modified membrane compositions. In some aspects, surface engineered EVs (e.g., exosomes) have a higher or lower density (e.g., a higher number) of scaffold portions (e.g., exosomal proteins, e.g., Scaffold X), or include variants or fragments of scaffold portions.

[0213] For example, surface engineered EVs (e.g., Scaffold X engineered or Scaffold Y engineered EVs) can be produced by cells (e.g., HEK293 cells) transformed with exogenous sequences encoding scaffold portions (e.g., exosomal proteins, e.g., Scaffold X and / or Scaffold Y) or variants or fragments thereof. EVs comprising scaffold portions expressed by exogenous sequences can include modified membrane compositions.

[0214] Various modifications or fragments of the scaffold portion can be used in various aspects of the present disclosure. For example, a scaffold portion modified to have an enhanced affinity for a binding agent can be used to produce a surface engineered EV that can be purified using a binding agent. Scaffold portions modified to more effectively target EVs (e.g., exosomes) and / or membranes can be used. Scaffold portions modified to contain the minimum fragments required for specific and effective targeting to EVs (e.g., exosomes), membranes can also be used.

[0215] In some aspects, a STING agonist disclosed herein is expressed on the surface of an EV (e.g., an exosome) as a fusion protein (e.g., a fusion protein of a STING agonist with Scaffold X and / or Scaffold Y). For example, the fusion protein can comprise a STING agonist disclosed herein linked to a scaffold portion (e.g., Scaffold X or Scaffold Y).

[0216] III.C.1. Scaffold X protein

[0217] In certain aspects, scaffold X comprises PTGFRN protein, BSG protein, IGSF2 protein, IGSF3 protein, IGSF8 protein, ITGB1 protein, ITGA4 protein, SLC3A2 protein, ATP transporter, Lamp-1 protein, Lamp-2 protein, CD13 protein, CD86 protein, floater protein, syntaxin-3 protein, CD2 protein, CD36 protein, CD40 protein, CD40L protein, CD41a protein, CD44 protein, CD45 protein, ICAM-1 protein, integrin α4 protein, L1CAM protein, LFA-1 protein, Mac-1α and β protein, Vti-1A and B protein, CD3ε and ζ protein, CD9 protein, CD18 protein, CD37 protein, CD53 protein, CD63 protein, CD81 protein, CD82 protein, CXCR4 protein, FcR protein, GluR2 / 3 protein, HLA-DM (MHC II) protein, immunoglobulin, MHC-I or MHC-II component protein, TCRβ protein, tetraspanin or fragments or variants thereof.

[0218] In some aspects, the surface-engineered EVs (e.g., exosomes (e.g., Scaffold X engineered EVs, e.g., exosomes) described herein exhibit superior properties compared to EVs (e.g., exosomes) known in the art. For example, surface-engineered EVs (e.g., Scaffold X) contain modified proteins that are more highly enriched on their surfaces compared to naturally occurring EVs (e.g., exosomes) or EVs (e.g., exosomes) produced using conventional exosomal proteins. In addition, surface-engineered EVs (e.g., exosomes) (e.g., Scaffold X engineered EVs, e.g., exosomes) of the present invention may have greater, more specific, or more controlled biological activity compared to naturally occurring EVs (e.g., exosomes) or EVs (e.g., exosomes) produced using conventional exosomal proteins.

[0219] In other aspects, the EVs (e.g., exosomes) of the present disclosure contain a STING agonist and a scaffold X, wherein the STING agonist is linked to the scaffold X. In some aspects, the EVs (e.g., exosomes) of the present disclosure contain a STING agonist and a scaffold X, wherein the STING agonist is not linked to the scaffold X.

[0220] In some aspects, the support X that can be used for the present disclosure comprises prostaglandin F2 receptor negative regulator (PTGFRN polypeptide).PTGFRN protein can also be referred to as CD9 partner 1 (CD9P-1), protein F (EWI-F) containing Glu-Trp-Ile EWI motif, prostaglandin F2-α receptor regulatory protein, prostaglandin F2-α receptor associated protein or CD315.The full-length amino acid sequence of human PTGFRN protein (Uniprot accession number Q9P2B2) is shown as SEQ ID NO:1 in Table 2.PTGFRN polypeptide contains signal peptide (SEQ ID NO:1 amino acid 1 to 25), extracellular domain (SEQ ID NO:1 amino acid 26 to 832), transmembrane domain (SEQ ID NO:1 amino acid 833 to 853) and cytoplasmic domain (SEQ ID NO:1 amino acid 854 to 879).Mature PTGFRN polypeptide is by SEQ ID NO:1 without signal peptide, i.e. SEQ ID NO:1 amino acid 26 to 879 composition. In some aspects, the PTGFRN polypeptide fragments useful in the present disclosure comprise the transmembrane domain of the PTGFRN polypeptide. In other aspects, the PTGFRN polypeptide fragments useful in the present disclosure comprise the transmembrane domain of the PTGFRN polypeptide and (i) at least five, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150 amino acids located at the N-terminus of the transmembrane domain, (ii) at least five, at least 10, at least 15, at least 20 or at least 25 amino acids located at the C-terminus of the transmembrane domain, or both (i) and (ii).

[0221] In some aspects, Scaffold X comprises an amino acid sequence that is at least about at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO: 33.

[0222] Table 2. Exemplary Scaffold X Protein Sequences

[0223]

[0224] In other aspects, Scaffold X comprises an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO: 2, 3, 4, 5, 6, 7, or 8.

[0225] Non-limiting examples of other Scaffold X proteins that can be used to link a STING agonist to the surface of an EV (e.g., an exosome) can be found in U.S. Pat. Nos. 10,195,290 Bl and 10,561,740 B2, each of which is incorporated herein by reference in its entirety.

[0226] In some aspects, the scaffold X described herein can also be used to simultaneously attach a STING agonist to the lumen surface and / or the outer surface of an EV (e.g., an exosome). For example, in addition to the surface of an EV (e.g., an exosome), a PTGFRN polypeptide can also be used to attach a STING agonist to the interior of the lumen. In some aspects, the scaffold X can be used to attach a STING agonist and an additional therapeutic agent to an EV (e.g., an exosome) (e.g., a payload). Thus, in some aspects, the scaffold X disclosed herein can be used for dual purposes.

[0227] In some aspects, the EVs (e.g., exosomes) of the present disclosure comprise an internal space (i.e., a lumen) that is different from the internal space of a naturally occurring EV (e.g., exosome). For example, an EV (e.g., exosome) can be altered such that the composition in the lumen side of the EV (e.g., exosome) has a protein, lipid, or glycan content that is different from the protein, lipid, or glycan content of a naturally occurring EV (e.g., exosome).

[0228] In some aspects, engineered EVs (e.g., exosomes) can be produced by cells transformed with exogenous sequences encoding a scaffold portion (e.g., an exosomal protein, e.g., Scaffold Y) or a modification or fragment of a scaffold portion that alters the composition or content of the lumenal side of the EV (e.g., exosome). Various modifications or fragments of exosomal proteins that can be expressed in the lumenal side of EVs (e.g., exosomes) can be used in various aspects of the present disclosure.

[0229] In some aspects, the STING agonists disclosed herein are located in the lumen of an EV (e.g., an exosome) (i.e., encapsulated). In some aspects, the STING agonist is attached to the lumen surface of an EV (e.g., an exosome). As used herein, when a molecule (e.g., a STING agonist) is described as being in the "lumen" of an EV (e.g., an exosome), this means that the molecule is located within (e.g., associated with) the EV (e.g., exosome), but is not attached to any molecule on the lumen surface of the EV. In other aspects, the STING agonist is expressed as a fusion molecule on the lumen surface of an EV (e.g., an exosome), e.g., a fusion molecule of a STING agonist and a scaffold portion (e.g., scaffold X or scaffold Y).

[0230] III.C.2. Scaffold Y protein

[0231] In some aspects, a STING agonist is expressed as a fusion molecule on the luminal surface of an EV (e.g., an exosome), e.g., a fusion molecule of a STING agonist and a scaffold Y portion. In some aspects, an engineered EV (e.g., an exosome) can be produced by cells transformed with an exogenous sequence encoding a scaffold portion (e.g., an exosomal protein, e.g., scaffold Y) or a modification or fragment of a scaffold portion that changes the composition or content of the luminal surface of an EV (e.g., an exosome). Various modifications or fragments of exosomal proteins that can be expressed on the luminal surface of an EV (e.g., an exosome) can be used in various aspects of the present disclosure.

[0232] In some aspects, exosomal proteins on the luminal surface of EVs (e.g., exosomes) can be altered, including but not limited to myristoylated alanine-rich protein kinase C substrate (MARCKS) protein, myristoylated alanine-rich protein kinase C substrate-like 1 (MARCKSL1) protein, brain acid soluble protein 1 (BASP1) protein, or any combination thereof.

[0233] Disclosed herein are non-limiting examples of scaffold Y proteins that can be used in the present disclosure. In some aspects, the scaffold Y protein comprises an amino acid sequence selected from SEQ ID NO: 411, 438, 446, and 455-567. In some aspects, the scaffold Y protein consists of an amino acid sequence selected from SEQ ID NO: 411, 438, 446, and 455-567. In some aspects, the scaffold Y protein comprises or consists of any scaffold Y protein disclosed in International Publication No. WO / 2019 / 099942 or No. WO 2020 / 101740, each of which is incorporated herein by reference in its entirety.

[0234] II.D. Connector

[0235] The EVs of the present disclosure may comprise one or more linkers that connect the STING agonist to the EV or to a scaffold portion (e.g., scaffold X on the outer surface of the EV). In some aspects, the STING agonist is connected to the EV directly or through a linker in a scaffold portion on the EV. In some aspects, the STING agonist is connected to the lipid bilayer of the EV, for example, through a linker. The linker can be any chemical moiety known in the art.

[0236] In some aspects, the term "joint" refers to a peptide or polypeptide sequence (e.g., a synthetic peptide or polypeptide sequence) or to a non-polypeptide. In some aspects, two or more joints can be connected in series. Typically, the joint provides flexibility or prevents / improves steric hindrance. The joint is usually not cut; however, in some aspects, such cutting may be desired. Therefore, in some aspects, the joint may include one or more protease cleavage sites, which may be located within the sequence of the joint or at either end of the joint sequence.

[0237] In some aspects, the joint is a peptide joint. In some aspects, the peptide joint can include at least about two, at least about three, at least about four, at least about five, at least about 10, at least about 15, at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least about 55, at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, at least about 90, at least about 95 or at least about 100 amino acids.

[0238] In some aspects, the peptide linker is synthetic, i.e., non-naturally occurring. In one aspect, the peptide linker comprises a peptide (or polypeptide) (e.g., a naturally or non-naturally occurring peptide) comprising an amino acid sequence that connects or genetically fuses a first linear sequence of amino acids to a second linear sequence of amino acids, the peptide not being naturally connected or genetically fused to the second linear sequence of amino acids in nature. For example, in one aspect, the peptide linker can comprise a non-naturally occurring polypeptide that is a modified form of a naturally occurring polypeptide (e.g., comprising a mutation, such as an addition, substitution, or deletion).

[0239] The linker may be susceptible to cleavage (a "cleavable linker"), thereby facilitating the release of the STING agonist or other payload. In some aspects, the linker is a "reduction-sensitive linker". In some aspects, the reduction-sensitive linker contains a disulfide bond. In some aspects, the linker is an "acid-labile linker". In some aspects, the acid-labile linker contains a hydrazone. Suitable acid-labile linkers also include, for example, a cis-aconitine linker, a hydrazide linker, a thiocarbamoyl linker, or any combination thereof. In some aspects, the linker comprises a non-cleavable linker.

[0240] In some aspects, the linker comprises a cholesterol moiety. In some aspects, the cholesterol moiety is embedded into the surface of the EV. In some aspects, the cholesterol moiety comprises cholesterol or a derivative thereof. In some aspects, the cholesterol moiety is linked to the STING agonist via a cleavable peptide linker (e.g., valine-alanine or valine-citrulline).

[0241] In some aspects, the cholesterol moiety comprises:

[0242]

[0243] In some aspects, the linker is conjugated to the phosphate backbone of a STING agonist (e.g., a cyclic dinucleotide (CDN)). In some aspects, the linker is conjugated to the base of a CDN (STING agonist).

[0244] In some aspects, the STING agonist comprises a linker and cholesterol according to:

[0245]

[0246] II.E. Producer Cells and Modifications

[0247] EVs (e.g., exosomes) can be produced by cells grown in vitro or by body fluids of a subject. When EVs (e.g., exosomes) are produced from in vitro cell culture, various producer cells (e.g., HEK293 cells) can be used. Additional cell types that can be used to produce the lumen-engineered EVs (e.g., exosomes) described herein include, but are not limited to, mesenchymal stem cells, T cells, B cells, dendritic cells, macrophages, and cancer cell lines. Additional examples include: Chinese hamster ovary (CHO) cells, mesenchymal stem cells (MSCs), BJ human foreskin fibroblasts, fHDF fibroblasts, Neuronal precursor cells, Amniotic cells, adipose-derived mesenchymal stem cells, and RPTEC / TERT1 cells. In certain aspects, the producer cell is not a dendritic cell, a macrophage, a B cell, a mast cell, a neutrophil, a Kupfer-Blauvich cell, a cell derived from any of these cells, or any combination thereof.

[0248] More specifically, the EV (e.g., exosome) of the present invention can be produced by cells transformed with sequences encoding one or more additional exogenous proteins, including but not limited to ligands, cytokines or antibodies or any combination thereof. These additional exogenous proteins can enable activation or regulation of additional immunostimulatory signals in combination with STING agonists. Exemplary additional exogenous proteins for use include proteins, ligands and other molecules described in detail in U.S. Patent Application 62 / 611,140, ​​International Publication No. WO / 2019 / 133934 and U.S. Patent Nos. 10,195,290B1 and 10,561,740B2, each of which is incorporated herein by reference in its entirety. In some aspects, EV (e.g., exosome) is further modified by a ligand comprising CD40L, OX40L or CD27L. In some aspects, EV (e.g., exosome) is further modified by a cytokine comprising IL-7, IL-12 or IL-15. Any of the one or more exosomal proteins described herein can be expressed from a plasmid, an exogenous sequence inserted into the genome, or other exogenous nucleic acid, such as a synthetic messenger RNA (mRNA).

[0249] In some aspects, EVs (e.g., exosomes) are further modified to display antagonistic antibodies or agonistic antibodies or fragments thereof on the surface of EVs (e.g., exosomes) to direct EV uptake, activate or block cellular pathways, thereby enhancing the combined effect of STING agonists. In some specific aspects, the antibody or fragment thereof is an antibody against DEC205, CLEC9A, CLEC6, DCIR, DC-SIGN, LOX-1 or Langerin. The producer cells can be modified to contain additional exogenous sequences encoding antagonistic antibodies or agonistic antibodies. Alternatively, the antagonistic antibody or agonistic antibody can be covalently linked or conjugated to EVs (e.g., exosomes) by any suitable linking chemistry known in the art. Non-limiting examples of suitable linking chemistries include amine reactive groups, carboxyl reactive groups, sulfhydryl reactive groups, aldehyde reactive groups, photoreactive groups, ClickIT chemistry, biotin-streptavidin or other avidin conjugates or any combination thereof.

[0250] II.F. Methods for treating tumors

[0251] Some aspects of the present disclosure relate to a method for preventing or treating a tumor in a subject in need thereof, the method comprising administering a dose of a STING agonist to the subject, wherein the amount of the STING agonist in the dose is about 0.01 μg to about 100 μg. In some aspects, the STING agonist is capable of upregulating a STING-mediated immune response in the subject, thereby enhancing tumor targeting of the subject's immune system. In some aspects, the STING agonist is administered intratumorally to the subject. In some aspects, the STING agonist is administered parenterally, orally, intravenously, intramuscularly, intraperitoneally, or by any other appropriate route of administration.

[0252] Also provided herein is a method for preventing cancer metastasis in a subject. The method comprises administering a dose of a STING agonist to a subject, wherein the amount of the STING agonist in the dose is about 0.01 μg to about 100 μg, wherein the STING agonist is capable of preventing one or more tumors at one location of the subject from promoting the growth of one or more tumors at another location of the subject. In some aspects, a STING agonist is administered intratumorally in a first tumor in one location, and a STING agonist is administered in the first tumor to prevent metastasis of one or more tumors at a second location.

[0253] In some aspects, administration of a STING agonist disclosed herein inhibits and / or reduces tumor growth in a subject. In some aspects, tumor growth (e.g., tumor volume or weight) is reduced by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% compared to a reference (e.g., tumor volume in a corresponding subject after administration of a free STING agonist).

[0254] In some aspects, the cancer being treated is characterized by infiltration of leukocytes (T cells, B cells, macrophages, dendritic cells, monocytes) into the tumor microenvironment, or so-called "hot tumors" or "inflammatory tumors". In some aspects, the cancer being treated is characterized by infiltration of leukocytes into the tumor microenvironment, or low or undetectable levels in so-called "cold tumors" or "non-inflammatory tumors". In some aspects, the amount and time of EV (e.g., exosomes) administered are sufficient to convert "cold tumors" into "hot tumors", that is, the administration causes leukocytes (such as T cells) to infiltrate into the tumor microenvironment. In some aspects, cancer comprises bladder cancer, cervical cancer, renal cell carcinoma, testicular cancer, colorectal cancer, lung cancer, head and neck cancer and ovarian, lymphoma, liver cancer, glioblastoma, melanoma, myeloma, leukemia, pancreatic cancer or a combination thereof. As used herein, the term "distant tumor", "distant tumor" or "secondary tumor" refers to a tumor that spreads from the original (or primary) tumor to a distant organ or distant tissue (e.g., lymph node). In some aspects, the EVs (e.g., exosomes) disclosed herein treat tumors after metastatic spread.

[0255] Non-limiting examples of cancers (or tumors) that can be treated with the methods disclosed herein include squamous cell carcinoma, small cell lung cancer (SCLC), non-small cell lung cancer, squamous non-small cell lung cancer (NSCLC), non-squamous NSCLC, gastrointestinal cancer, renal cancer (e.g., clear cell carcinoma), ovarian cancer, liver cancer (e.g., hepatocellular carcinoma), colorectal cancer, endometrial cancer, kidney cancer (e.g., renal cell carcinoma (RCC)), prostate cancer (e.g., hormone-refractory prostate cancer), thyroid cancer, pancreatic cancer, cervical cancer, gastric cancer, bladder cancer, liver cancer, breast cancer, colon cancer, and head and neck cancer (or carcinomas), gastric cancer, germ cell tumors, small cell carcinomas, and prostate cancer. sarcoma, paranasal sinus natural killer, melanoma (e.g., metastatic malignant melanoma, such as skin or intraocular malignant melanoma), bone cancer, skin cancer, uterine cancer, anal cancer, testicular cancer (e.g., choriocarcinoma and non-seminoma), fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, esophageal cancer (e.g., gastroesophageal junction cancer), small intestine cancer, endocrine system cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, solid tumors in children, ureteral cancer, renal pelvis cancer, tumor angiogenesis, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma malignancies, including those caused by leukemia, lymphoma, myeloid leukemia, and myeloma, including those caused by asbestos, malignant cell carcinoma, T-cell lymphoma, environmentally induced cancers (including those induced by asbestos), virus-related cancers or cancers of viral origin (e.g., human papillomavirus (HPV-related or HPV-derived tumors)), and hematological malignancies arising from one of the two major blood cell lineages, the myeloid cell lineage (which produces granulocytes, erythrocytes, platelets, macrophages, and mast cells) or the lymphoid cell lineage (which produces B, T, NK, and plasma cells), such as all types of leukemias, lymphomas, and myelomas, e.g., acute, chronic, lymphocytic, and / or myeloid leukemias, such as acute leukemia (ALL), acute myeloid Leukemia (AML), chronic lymphocytic leukemia (CLL) and chronic myeloid leukemia (CML), undifferentiated AML (MO), myeloblastic leukemia (M1), myeloblastic leukemia (M2; in which the cells are mature), promyelocytic leukemia (M3 or M3 variant [M3V]), myelomonocytic leukemia (M4 or M4 variant with eosinophilia [M4E]), monocytic leukemia (M5), erythroleukemia (M6), megakaryocytic leukemia (M7), isolated granulocytic sarcoma and chloroma; lymphomas, such as Hodgkin's lymphoma (Hodgkin's lymphoma (HL), non-Hodgkin's lymphoma (NHL), B-cell hematological malignancies, e.g., B-cell lymphoma, T-cell lymphoma, lymphoplasmacytoid lymphoma, monocytoid B-cell lymphoma, mucosa-associated lymphoid tissue (MALT) lymphoma, anaplastic (e.g., Ki1 +) large cell lymphoma, adult T-cell lymphoma / leukemia, mantle cell lymphoma, angioimmunoblastic T-cell lymphoma, angiocentric lymphoma, intestinal T-cell lymphoma, primary mediastinal B-cell lymphoma, precursor T-lymphoblastic lymphoma, T-lymphoblastic; and lymphoma / leukemia (T-Lbly / T-ALL), peripheral T-cell lymphoma, lymphoblastic lymphoma, post-transplant lymphoproliferative disorder, true histiocytic lymphoma, primary effusion lymphoma, B-cell lymphoma, lymphoblastic lymphoma (LBL), lymphoid lineage hematopoietic neoplasms, acute lymphoblastic leukemia, diffuse large B-cell lymphoma, Burkitt's lymphoma (Burkitt's lymphoma, follicular lymphoma, diffuse histiocytic lymphoma (DHL), immunoblastic large cell lymphoma, precursor B lymphoblastic lymphoma, cutaneous T-cell lymphoma (CTLC) (also called mycosis fungoides or Sezary syndrome), and lymphoplasmacytoid lymphoma (LPL) with Waldenstrom's macroglobulinemia; myelomas, such as IgG myeloma, light chain myeloma, nonsecretory myeloma, smoldering myeloma myeloma) (also known as indolent myeloma), solitary plasmacytoma and multiple myeloma, chronic lymphocytic leukemia (CLL), hairy cell lymphoma; myeloid hematopoietic tumors, tumors of mesenchymal origin, including fibrosarcoma and rhabdomyosarcoma; seminoma, teratoma, tumors of mesenchymal origin, including fibrosarcoma, rhabdomyosarcoma and osteosarcoma; and other tumors, including melanoma, xeroderma pigmentosum, keratoacanthoma, seminoma, thyroid follicular carcinoma and teratoma, hematopoietic tumors of the lymphoid lineage, such as T-cell and B-cell tumors, including but not limited to T-cell disorders, such as T-prolymphocytic leukemia (T-PLL), including small cell and cerebriform cell types; large granular lymphocytic leukemia (LGL) of the T-cell type; a / d T-NHL hepatosplenic lymphoma; peripheral / post-thymic T-cell lymphoma (pleomorphic and immunoblastic subtypes); angiocentric (nasal) T-cell lymphoma; head and neck cancer, kidney cancer, rectal cancer, thyroid cancer; acute myeloid lymphoma and any combination thereof.

[0256] In some aspects, the cancer (or tumor) that can be treated comprises breast cancer, head and neck cancer, uterine cancer, brain cancer, skin cancer, kidney cancer, lung cancer, colorectal cancer, prostate cancer, liver cancer, bladder cancer, kidney cancer, peritoneal cancer, pancreatic cancer, thyroid cancer, esophageal cancer, eye cancer, stomach (gastric) cancer, gastrointestinal cancer, carcinoma, sarcoma, leukemia, lymphoma, myeloma or a combination thereof. In certain aspects, the cancer that can be treated with the present disclosure is pancreatic cancer and / or peritoneal cancer.

[0257] In some aspects, the methods described herein can also be used to treat metastatic cancer, unresectable refractory cancer (e.g., cancer refractory to previous cancer therapy) and / or recurrent cancer. In some aspects, the subject has received at least one previous therapy for cancer treatment. In some aspects, at least one previous therapy comprises standard of care therapy. In some aspects, at least one previous therapy comprises immunotherapy, chemotherapy, radiotherapy, surgery or any combination thereof. In some aspects, at least one previous therapy comprises a checkpoint inhibitor. In some aspects, at least one previous therapy comprises platinum-based chemotherapy, such as platinum-based dual chemotherapy.

[0258] In some aspects, the EVs (e.g., exosomes) disclosed herein can be used in combination with one or more additional anticancer and / or immunomodulatory agents. Such agents can include, for example, chemotherapeutic drugs, small molecule drugs, or antibodies that stimulate an immune response to a given cancer. In some aspects, the methods described herein are used in combination with standard of care treatments (e.g., surgery, radiation, and chemotherapy).

[0259] In some aspects, the methods disclosed herein for treating cancer can comprise administering (i) EVs (e.g., exosomes) comprising a STING agonist (e.g., encapsulated or expressed on the lumen or outer surface) in combination with (ii) an anti-cancer agent, e.g., one or more immuno-oncology agents (e.g., checkpoint inhibitors), such that multiple elements of the immune pathway can be targeted.

[0260] Non-limiting examples of such combinations include: therapies that enhance tumor antigen presentation (e.g., dendritic cell vaccines, cellular vaccines that secrete GM-CSF, CpG oligonucleotides, imiquimod); therapies that inhibit negative immunomodulation, for example, by inhibiting CTLA-4 and / or PD1 / PD-L1 / PD-L2 pathways and / or depleting or blocking Tregs or other immunosuppressive cells (e.g., myeloid-derived suppressor cells); therapies that stimulate positive immunomodulation, for example, using agonists that stimulate the CD-137, OX-40 and / or CD40 or GITR pathways and / or stimulate T cell effector function; therapies that systemically increase the frequency of anti-tumor T cells; therapies that deplete or inhibit Tregs (e.g., Tr in tumors); therapies that affect suppressive myeloid cell function in the tumor; therapies that enhance the immunogenicity of tumor cells (e.g., anthracyclines); adoptive T cell or NK cell transfer comprising genetically modified cells, e.g., cells modified by chimeric antigen receptors (CAR-T therapy); therapies that inhibit metabolic enzymes such as indoleamine dioxygenase (IDO), dioxygenase, arginase, or nitric oxide synthase; therapies that reverse / prevent T cell anergy or exhaustion; therapies that trigger innate immune activation and / or inflammation at the tumor site; administration of immunostimulatory cytokines; or blockade of immunosuppressive cytokines.

[0261] In some aspects, the immuno-oncology agent that can be used in combination with the STING agonists disclosed herein further comprises a checkpoint inhibitor (ie, blocking signaling through a specific immune checkpoint pathway). Non-limiting examples of checkpoint inhibitors that can be used in the methods of the present invention include CTLA-4 antagonists (eg, anti-CTLA-4 antibodies), PD-1 antagonists (eg, anti-PD-1 antibodies, anti-PD-L1 antibodies), TIM-3 antagonists (eg, anti-TIM-3 antibodies), or a combination thereof.

[0262] In some aspects, the immuno-oncology agent comprises an immune checkpoint activator (ie, promoting signal transduction through a specific immune checkpoint pathway). In certain aspects, the immune checkpoint activator comprises an OX40 agonist (eg, an anti-OX40 antibody), a LAG-3 agonist (eg, an anti-LAG-3 antibody), a 4-1BB (CD137) agonist (eg, an anti-CD137 antibody), a GITR agonist (eg, an anti-GITR antibody), or any combination thereof.

[0263] In some aspects, the combination of a STING agonist disclosed herein and a second agent discussed herein (e.g., an immune checkpoint inhibitor) can be administered simultaneously as a single composition in a pharmaceutically acceptable carrier. In other aspects, the combination of EVs (e.g., exosomes) and a second agent discussed herein (e.g., an immune checkpoint inhibitor) can be administered simultaneously as a separate composition. In other aspects, the combination of EVs (e.g., exosomes) and a second agent discussed herein (e.g., an immune checkpoint inhibitor) can be administered sequentially. In some aspects, EVs (e.g., exosomes) are administered prior to administering a second agent (e.g., an immune checkpoint inhibitor).

[0264] III. Methods for generating EVs using STING agonists

[0265] The STING agonist can be encapsulated in EVs (e.g., exosomes) by any suitable technique known in the art. All known ways of loading biomolecules into EVs (e.g., exosomes) are considered suitable for use herein. Such techniques include passive diffusion, electroporation, chemical or polymer transfection, viral transduction, mechanical membrane disruption or mechanical shearing, or any combination thereof. During encapsulation, the STING agonist and EVs (e.g., exosomes) can be incubated in an appropriate buffer.

[0266] In one aspect, the STING agonist is encapsulated by EV (e.g., exosome) by passive diffusion. The STING agonist and EV (e.g., exosome) can be mixed together and incubated for a period of time sufficient to diffuse the STING agonist through the vesicle lipid bilayer, thereby being encapsulated in the EV (e.g., exosome). The STING agonist and EV (e.g., exosome) can be incubated together for about 1 to 30 hours, 2 to 24 hours, 4 to 18 hours, 6 to 16 hours, 8 to 14 hours, 10 to 12 hours, 6 to 12 hours, 12 to 20 hours, 14 to 18 hours, or 20 to 30 hours. The STING agonist and EV (e.g., exosome) can be incubated together for about 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, 26 hours, or 30 hours.

[0267] The buffer conditions of the EV (e.g., exosome) solution can also be varied to optimize encapsulation of the STING agonist. In one aspect, the buffer can be phosphate buffered saline (PBS) with sucrose. PBS is a buffer well known to those skilled in the art. Additional buffer modifications can also be used, such as shear protectants, viscosity modifiers, and / or solutes that affect the structural properties of the vesicles. Excipients can also be added to improve the efficiency of STING agonist encapsulation, such as membrane softening materials and molecular crowding agents. Other modifications to the buffer can include specific pH ranges and / or concentrations of salts, organic solvents, small molecules, detergents, zwitterions, amino acids, polymers, and / or any combination of the foregoing, including multiple concentrations.

[0268] The temperature of the solution of EVs (e.g., exosomes) and STING agonists during incubation can be varied to optimize encapsulation of the STING agonist. The temperature can be room temperature. The temperature can be between about 15°C and 90°C, 15-30°C, 30-50°C, 50-90°C. The temperature can be about 15°C, 20°C, 35°C, 30°C, 35°C, 37°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, 80°C, 85°C, or 90°C.

[0269] The concentration of the STING agonist can also be varied during incubation of the agonist with EVs (e.g., exosomes) to optimize encapsulation of the STING agonist. The concentration of the agonist can be between at least 0.01 mM and 100 mM STING agonist. The concentration of the agonist can be at least 0.01-1 mM, 1-10 mM, 10-50 mM, or 50-100 mM. The concentration of the agonist can be at least 0.01 mM, 0.02 mM, 0.03 mM, 0.04 mM, 0.05 mM, 0.06 mM, 0.07 mM, 0.08 mM, 0.09 mM, 0.1 mM, 0.2 mM, 0.3 mM, 0.4 mM, 0.5 mM, 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 15 mM, 20 mM 30mM, 35mM, 40mM, 45mM, 50mM, 55mM, 60mM, 65mM, 70mM, 75mM, 80mM, 85mM, 90mM, 95mM or 100mM.

[0270] The amount of extracellular particles incubated with the STING agonist can also be varied to optimize the encapsulation of the STING agonist. The amount of purified EV (e.g., exosome) particles can be at least about 10% of the total particles of the purified vesicles. 6 to at least about 10 20The number of purified particles can account for about 10% of the total particles of the purified vesicles. 8 to 10 18 10 10 to 10 16 10 8 to 10 14 or 10 10 to 10 12 The number of purified particles can account for at least about 10 of the total particles of the purified vesicles. 6 10 8 10 10 10 12 10 14 10 16 10 18 or 10 20 indivual.

[0271] In some aspects, one or more parts can be introduced into suitable producer cells using synthetic macromolecules (such as cationic lipids and polymers) (Papapetrou et al., Gene Therapy 12: S118-S130 (2005)). In some aspects, cationic lipids form complexes with one or more parts through charge interactions. In some of these aspects, the positively charged complex binds to the negatively charged cell surface and is absorbed by the cell through endocytosis. In some other aspects, cationic polymers can be used to transfect producer cells. In some of these aspects, the cationic polymer is polyethyleneimine (PEI). In some aspects, chemicals (such as calcium phosphate, cyclodextrin or polybrene) can be used to introduce one or more parts into producer cells. Physical methods (such as particle-mediated transfection, "gene gun", gene gun method or particle bombardment technology) can also be used to introduce one or more parts into producer cells (Papapetrou et al., Gene Therapy 12: S118-S130 (2005)). Reporter genes (eg, β-galactosidase, chloramphenicol acetyltransferase, luciferase, or green fluorescent protein) can be used to assess the transfection efficiency of producer cells.

[0272] In some aspects, one or more moieties are introduced into producer cells by viral transduction. Many viruses can be used as gene transfer vehicles, including Moloney murine leukemia virus (MMLV), adenovirus, adeno-associated virus (AAV), herpes simplex virus (HSV), slow virus, and foamy virus. Virus-mediated gene transfer vehicles include vectors based on DNA viruses (such as adenovirus, adeno-associated virus, and herpes virus), as well as retroviral-based vectors.

[0273] In some aspects, one or more parts are introduced into producer cells by electroporation. Electroporation produces transient holes in the cell membrane, thereby allowing various molecules to be introduced into cells. In some aspects, DNA and RNA and polypeptide and non-polypeptide therapeutic agents can be introduced into producer cells by electroporation.

[0274] In some aspects, one or more moieties are introduced into the producer cells by microinjection.In some aspects, a glass micropipette can be used to inject one or more moieties into the producer cells at a microscopic level.

[0275] In some aspects, one or more moieties are introduced into the producer cell by extrusion.

[0276] In some aspects, one or more moieties are introduced into the producer cells by sonication. In some aspects, the producer cells are exposed to high intensity sound waves, resulting in transient rupture of the cell membrane, thereby allowing loading of the one or more moieties.

[0277] In some aspects, one or more parts are introduced into producer cells by cell fusion. In some aspects, one or more parts are introduced by electrical cell fusion. In other aspects, polyethylene glycol (PEG) is used to fuse producer cells. In other aspects, Sendai virus (sendai virus) is used to fuse producer cells.

[0278] In some aspects, one or more parts are introduced into producer cells by hypotonic lysis. In such aspects, producer cells can be exposed to low ionic strength buffers to burst colonies (burst), thereby allowing loading of one or more parts. In other aspects, controlled dialysis for hypotonic solutions can be used to expand producer cells and produce holes in producer cell membranes. Subsequently, producer cells are exposed to conditions that allow the resealing of membranes.

[0279] In some aspects, one or more moieties are introduced into the producer cells by detergent treatment. In some aspects, the producer cells are treated with a mild detergent that transiently damages the producer cell membrane by creating pores that allow loading of the one or more moieties. After loading the producer cells, the detergent is washed away, thereby resealing the membrane.

[0280] In some aspects, one or more moieties are introduced into the producer cell by receptor-mediated endocytosis.In certain aspects, the producer cell has a surface receptor that, when bound to the one or more moieties, induces internalization of the receptor and the associated moiety.

[0281] In some aspects, one or more parts are introduced into the producer cell by filtration. In some aspects, the producer cell and one or more parts can be forced to pass through a filter with a pore size smaller than the producer cell, thereby causing a transient rupture of the producer cell membrane and allowing one or more parts to enter the producer cell.

[0282] In some aspects, producer cells are subjected to several freeze-thaw cycles, resulting in rupture of the cell membrane, thereby allowing loading of one or more fractions.

[0283] IV. EV Purification

[0284] EVs (e.g., exosomes) prepared for the present disclosure can be isolated from producer cells. It is considered that all known ways of isolating EVs (e.g., exosomes) are considered applicable to this article. For example, the physical properties of EVs (e.g., exosomes) can be used to separate the EVs from culture media or other source materials, including separations based on: charge (e.g., electrophoretic separation), size (e.g., filtration, molecular sieves, etc.), density (e.g., conventional or gradient centrifugation), Svedberg constant (e.g., sedimentation with or without external force, etc.). Alternatively or additionally, separation can be based on one or more biological properties, and include methods that can use surface markers (e.g., for precipitation, reversible binding to a solid phase, FACS separation, specific ligand binding, non-specific ligand binding, etc.). In yet another contemplated method, EVs (e.g., exosomes) can also be fused using chemical and / or physical methods, including PEG-induced fusion and / or ultrasonic fusion.

[0285] EVs (e.g., exosomes) can also be purified after incubation with a STING agonist to remove free, unencapsulated STING agonist from the composition. All methods previously disclosed are also considered applicable herein, including separation based on physical or biological properties of EVs (e.g., exosomes).

[0286] Separation, purification and enrichment can be performed in a general and non-selective manner (usually including continuous centrifugation). Alternatively, separation, purification and enrichment can be performed in a more specific and selective manner (e.g., using producer cell-specific surface markers). For example, specific surface markers can be used for immunoprecipitation, FACS sorting, affinity purification, bead-bound ligands for magnetic separation, etc.

[0287] In some aspects, size exclusion chromatography can be used to separate or purify EVs (e.g., exosomes). Size exclusion chromatography techniques are known in the art. Exemplary non-limiting techniques are provided herein. In some aspects, the void volume fraction is separated and contains the EVs of interest (e.g., exosomes). In some aspects, for example, density gradient centrifugation can be used to further separate EVs (e.g., exosomes). Still further, in some aspects, it may be desirable to further separate producer cell-derived EVs (e.g., exosomes) from EVs from other sources. For example, by using an antigen-antibody specific to producer cells for immunoabsorption capture, producer cell-derived EVs (e.g., exosomes) can be separated from non-producer cell-derived EVs (e.g., exosomes).

[0288] In some aspects, the separation of EVs (e.g., exosomes) may involve size exclusion chromatography or ion chromatography, such as anion exchange, cation exchange, or mixed mode chromatography. In some aspects, the separation of EVs (e.g., exosomes) may involve desalting, dialysis, tangential flow filtration, ultrafiltration, or diafiltration, or any combination thereof. In some aspects, the separation of EVs (e.g., exosomes) may involve a combination of methods, including but not limited to differential centrifugation, size-based membrane filtration, concentration, and / or rate zonal centrifugation. In some aspects, the separation of EVs (e.g., exosomes) may involve one or more centrifugation steps. Centrifugation may be performed at about 50,000 to 150,000 xg. Centrifugation may be performed at about 50,000 xg, 75,000 xg, 100,000 xg, 125,000 xg, or 150,000 xg.

[0289] Examples

[0290] Examples are provided for illustrative purposes only and should not be construed as limiting the scope or content of the present invention in any way. Unless otherwise indicated, the practice of the present invention will adopt conventional methods of protein chemistry, biochemistry, recombinant DNA technology and pharmacology within the technology of the art. Such technology is fully explained in the literature. See, e.g., TECreighton, Proteins: Structures and Molecular Properties (WH Freeman and Company, 1993); Green and Sambrook et al., Molecular Cloning: A Laboratory Manual, 4th ed. (Cold Spring Harbor Laboratory Press, 2012); Colowick and Kaplan, Methods in Enzymology (Academic Press); Remington: The Science and Practice of Pharmacy, 22nd ed. (Pharmaceutical Press, 2012); Sundberg and Carey, Advanced Organic Chemistry: Parts A and B, 5th ed. (Springer, 2007).

[0291] method

[0292] Exosome purification

[0293] HEK293SF cells were grown to high density in chemically defined medium for 7 days. Conditioned cell culture medium was collected and centrifuged at 300-800 x g for 5 minutes at room temperature to remove cells and large debris. The culture supernatant was supplemented and incubated at 37°C for 1 hour in a water bath. The supernatant was collected and centrifuged at 16,000xg for 30 minutes at 4°C to remove residual cell debris and other large contaminants. The supernatant was then ultracentrifuged at 133,900xg for 3 hours at 4°C to precipitate the exosomes. The supernatant was discarded and any remaining culture medium was aspirated from the bottom of the tube. The pellet was resuspended in 200-1000 μL PBS (-Ca-Mg).

[0294] To further enrich the exosome population, the pellet was purified by density gradient purification (sucrose or OPTIPREP TM ) treatment. For sucrose gradient purification, the exosome pellet was layered on a sucrose gradient as defined in Table 3 below.

[0295] Table 3:

[0296]

[0297] The gradient was spun at 200,000 x g for 16 hours at 4 °C in 12 mL Ultra Clear (344059) tubes placed in a SW 41Ti rotor to isolate the exosome fraction.

[0298] The exosome layer was gently removed from the top layer and diluted in approximately 32.5 mL PBS in a 38.5 mL ultra-clear (344058) tube and ultracentrifuged again at 133,900 x g for 3 hours at 4°C to pellet the purified exosomes. The resulting pellet was resuspended in a minimal volume of PBS (approximately 200 μL) and stored at 4°C.

[0299] For OPTIPREP TM Gradient, in 12 mL Ultra Clear (344059) tubes for SW 41Ti rotors, with equal volumes of 10%, 30%, and 45% OPTIPREP TM Prepare a 3-step sterile gradient. Add the precipitate to the OPTIPREP TM The exosome fraction was isolated by ultracentrifugation at 200,000 x g for 16 h at 4 °C. The exosome layer was then gently collected at approximately 3 mL from the top of the tube.

[0300] The exosome fraction was diluted in approximately 32 mL of PBS in a 38.5 mL ultra-clear (344058) tube and ultracentrifuged at 133,900 x g for 3 hours at 4°C to pellet the purified exosomes. The pelleted exosomes were then resuspended in a minimal volume of PBS (approximately 200 μL) and stored at 4°C.

[0301] use Conducting in vivo intratumoral microinjection studies

[0302] Tumor cell culture

[0303] A20 cells (ATCC batch number 70006082) were cultured at 37°C and 5% CO 2, cultured in RPMI1640 with L-glutamine (ThermoFisher), 10% fetal bovine serum (ThermoFisher) and 50 nanomolar BME. An IMPACT III test (IDEXX Bioresearch, USA) was performed to confirm mycoplasma-free and pathogen-free status. After obtaining the cells from the supplier, the cells were expanded and cryopreserved after 2-3 passages. After thawing, the cells were maintained for up to 8 weeks by subculturing 3 times per week and thereafter supplemented from fresh frozen stock solutions.

[0304] In vivo studies

[0305] All experiments in mice were approved by the IACUC Presage Biosciences Committee in Seattle, Washington (Protocol No. PR-001) and performed at Presage in accordance with relevant guidelines and regulations. All relevant procedures were performed under anesthesia, and every effort was made to minimize pain and suffering. Female BALB / cAnNHsd mice (Envigo) with an average body weight of 18 gm were used for experiments 5-7 weeks old. To generate A20 allografts, mice were inoculated with 1 million A20 cells in a 100 μl inoculation volume.

[0306] Intratumoral microinjection

[0307] CIVO intratumoral microinjections were performed as described in Klinghoffer et al., (2016) Science Translational Medicine. Briefly, mice (n=6 / time point, 4 hours and 24 hours) participated in microinjection studies when implanted tumors reached the following approximate sizes: 14 mm (length), 10 mm (width), and 7 mm (depth). The CIVO device was configured with 6 thirty-gauge injection needles, with a total delivery volume of 2.0 μl. Presage's fluorescent tracking marker (FTM, 5% by volume) was added to the injection contents for spatial targeting. The microinjected agents were as follows: control PTGFRN++GFP exosomes, ML RR-S2CDA-loaded PTGFRN++GFP exosomes, ML RR-S2 CDA-loaded PTGFRN++GFP desialylated exosomes, MLRR-S2 CDA-loaded native exosomes, all at 10 ng / μl of ML RR-S2CDA, resulting in a total delivery of 20 ng. Free ML RR-S2 CDA was microinjected at both 20 ng and 2 μg. At 4 and 24 h after CIVO microinjection, CO2 Inhalation euthanized mice for biomarker analysis.

[0308] Histology, immunohistochemistry, and in situ hybridization

[0309] The excised tumors were cut into 2 mm thick slices perpendicular to the injection column and fixed in 10% buffered formalin for 48 hours. Based on the signal from the FTM injected at each CIVO site, UV imaging was used to confirm CIVO microinjection. The 2 mm thick tissue sections were then processed for standard paraffin embedding. As described below, 4 μm thick sections were used for all histological determinations. Hematoxylin-eosin (H&E) staining was performed using standard methods.

[0310] Immunohistochemistry

[0311] Formalin-fixed paraffin-embedded tumors were cut onto slides with a thickness of 4 μm. The slides were baked at 60°C for 1 hour, dewaxed in xylene, and rehydrated through graded alcohols.

[0312] Slides were subjected to incubation of the target repair solution at 100°C for 20 minutes, followed by cooling to room temperature for 20 minutes. Serum was blocked (TBST containing 5% normal goat serum) for 1 hour at room temperature. Primary antibody staining was performed overnight with appropriate primary antibodies in 5% NGS TBS diluent at room temperature. The corresponding isotype control was included in each batch. Secondary antibody staining was performed overnight with appropriate secondary antibodies in 5% NGS TBS diluent at room temperature. Slides were counterstained with DAPI for 10 minutes and coverslipped with Prolong Gold mounting medium (Invitrogen). The stained slides were imaged using a digital, automated, high-resolution scanner.

[0313] In situ hybridization was performed using RNAscope Multiplex Fluorescence Reagent Kit v2 (Advanced Cell Diagnostics). Formalin-fixed paraffin-embedded tumors were cut onto slides with a thickness of 4 μm. The slides were baked at 60°C for 1 hour, dewaxed in xylene, and rehydrated through graded alcohols. Hydrogen peroxide was added for 10 minutes to quench endogenous peroxidase activity. The slides were subjected to a 15-minute incubation of the target repair solution at 100°C, followed by a 15-minute protease digestion at 40°C. RNAscope ISH assays were performed using a mouse Ifnb1 probe (Advanced Cell Diagnostics) and TSA plus cyanine 5 detection (Perkin Elmer). The slides were counterstained with DAPI for 10 minutes and covered with Prolong Gold mounting media (Invitrogen). The stained slides were imaged using a digital, automated, high-resolution scanner.

[0314] Whole slide scanning and image analysis

[0315] By digitization, automation, high-resolution whole tissue scanning (3D Histech Panoramic 250Flash), images of each cell from each tissue section stained are captured. Using Presage's custom CIVO analyzer image analysis platform, tumor responses are quantitatively measured from image files from each tissue section. The whole tissue section images captured by the slide scanner are automatically processed by the CIVO analyzer. Each cell from each tissue section is segmented based on the nuclear (DAPI) signal, and each cell is classified as biomarker negative or biomarker positive using a cell profiler (Broad Institute). After cell segmentation and classification, in each image around the FTM at each position, a circular region of interest (ROI) is positioned around each microinjection site, wherein the radius of the maximum ROI is not greater than 2000 μm. In order to mitigate the impact of pre-existing necrosis on biomarker measurement, before quantitative analysis, the injection site falling into most of the cell-free tumor area is excluded.

[0316] Example 1: Exosome-encapsulated STING agonist

[0317] Encapsulation of STING agonists

[0318] 1 mM STING agonists including ML RR-S2 CDA ammonium salt (MedChem Express, catalog number HY-12885B) and (3-3cAIMPdFSH; InvivoGen, catalog number tlrl-nacairs) were incubated with purified exosomes (1E12 total particles) in 300 ul of PBS at 37°C overnight. The mixture was then washed twice in PBS and purified by ultracentrifugation at 100,000 x g ( Figure 1 ).

[0319] Quantification of cyclic dinucleotide STING agonists

[0320] Sample preparation for LC-MS analysis

[0321] All samples were received in phosphate buffered saline (PBS) buffer or PBS and 5% sucrose. Particle concentration (P / mL) was measured by nanoparticle tracking analysis (NTA) on the NanoSight NS300 prior to analysis. All standards and samples were prepared so that each injection contained virtually the same number of particles. This was achieved by a combination of diluted samples and exosomes spiked into standards to achieve a final concentration of 1.0-4.0E+11 P / mL, depending on the initial particle concentration of the sample.

[0322] A standard curve was prepared by spiking known concentrations of STING agonist into PBS buffer and then preparing additional standards by serial dilution. Individual standards were typically prepared so that the final concentrations (after all sample preparation steps) were 25, 50, 250, 500, 1250, 2500, and 5000 nM STING agonist. First, 75.0 μL of each appropriately diluted sample and each matrix-matched standard was prepared in a separate 1.5 mL microcentrifuge tube. Next, 25.0 μL of exosome lysis buffer (60 mM Tris, 400 mM GdmCl, 100 mM EDTA, 20 mM TCEP, 1.0% Triton X-100) was added to each tube, and all tubes were then vortexed to mix and briefly centrifuged to settle. Finally, 1.0 μL of concentrated proteinase K enzyme solution (Dako, reference S3004) was added to each tube, and all tubes were vortexed again and then briefly centrifuged before incubation for 60 minutes at 55° C. Prior to injection on the LC-MS, samples were cooled to room temperature and transferred to HPLC sample vials.

[0323] LC-MS analysis

[0324] 20.0 μL of standards and samples were injected directly into an UltiMate 3000RSCLnano (Thermo Fisher Scientific) low flow chromatography system without cleanup. A Phenomenex Kinetex EVO C18 core-shell analytical column (50 x 2.1 mm, 2.6 μm particle size, pore size) and a loading pump that delivered a gradient of mobile phase A (MPA: water, 0.1% formic acid) and mobile phase B (MPB: acetonitrile, 0.1% formic acid) at a flow rate of 500 microliters / minute for separation of analytes. The gradient started with 2% MPB and was held for 2 minutes to load and desalt the STING agonist analytes. The MPB percentage was then increased from 2-30% over 3 minutes to elute the STING agonist analytes. The MPB percentage was then increased from 30-95% over 1 minute, held at 95% for 3 minutes, decreased from 95-2% over 1 minute, and then held at 2% for another 3 minutes to re-equilibrate the column. The total run time for the method was 13 minutes, and the LC flow was only introduced into the MS between 2.5-4.5 minutes. Typical carryover was less than 0.05% of the peak area of ​​the previous injection, so blank injections were not performed between analytical injections.

[0325] Mass analysis was performed using a Q Exactive Basic (Thermo Fisher Scientific) mass spectrometer with an Ion Max source and a HESI-II probe operating in negative ion mode, and mass spectra were collected from 500-800 Da using a full MS-SIM mode scan with an AGC target of 1E+6 ions, a maximum injection time of 200 milliseconds, and a resolution of 35,000. STING agonist quantification was performed using the monoisotopic-1 STING agonist peak by selectively extracting all ions in the m / z range of 688.97-689.13 Da and then integrating the resulting peaks within a retention time between 3.80-3.90 minutes. The concentration of STING agonist in a given sample was determined by comparing the STING agonist peak area in the sample with the STING agonist peak area generated by the standard, which is a typical relative quantification.

[0326] Example 2: In vivo administration of exo-STING

[0327] A Phase 1 / 2 study is ongoing in patients enrolled in a Phase 1 / 2 study, to whom five escalating dose cohorts (0.3mcg, 1.0μg, 3.0μg, 6.0μg, and 12.0μg of STING agonist) are administered. Trial participants (n=23) are administered exoSTING (a composition comprising a STING agonist and exosomes) intratumorally, and all subjects have received at least two prior therapies prior to study entry, with the majority of subjects (73%) having progressed on checkpoint inhibitors.

[0328] Plasma pharmacokinetic (PK) measurements of patients receiving exoSTING showed no systemic exposure to the agonist. Further, analysis of available plasma biomarkers demonstrated a lack of detectable systemic inflammatory cytokines in the blood following exoSTING administration.

[0329] Across all dose cohorts, exoSTING was generally well tolerated, and no dose-limiting toxicities or treatment-emergent adverse events of grade 3 or higher were observed. Treatment-related serious adverse events were observed in three patients.

[0330] Blood biomarker assessments after dosing demonstrated dose-dependent activation of the STING pathway. Paired tumor biopsies from cohorts 1-4 showed evidence of an adaptive immune response, including a sustained increase in CD-8 effector T cells and PD-L1 in the tumor microenvironment.

[0331] In 11 of the 17 tumor types, signs of antitumor activity were observed in both injected and distant tumor shrinkage.

[0332] Example 3: Measurement of STING agonists

[0333] Pure Payload

[0334] The identity, determination, purity and related substances of CP-201 intermediates were evaluated using a reversed phase UHPLC (RP-UHPLC) method. The method uses a gradient of mobile phases A and B, wherein UV detection is performed at 254nm. Samples and reference standard solutions with a concentration of 1.0 mg / mL were prepared in a sample solvent (9:1 water: methanol). The identification standard solutions of CP-201 isomers (CP-230, CP-231 and CP-232) were prepared in the range of 0.25 to 2.0 mg / mL to detect isomers. The method was performed according to the conditions described in Table 4.

[0335] The identity of the CP-201 intermediate is confirmed by inspection of the chromatogram; both the sample and the reference standard must contain a single major peak with similar peak shape and retention time (11.0 ± 1.5 minutes). The assay (as is, w / w %) is determined by comparing the peak response of the sample with the peak response of the reference standard. The purity and content of related substances and impurities are calculated as a percentage of the total peak area. All peaks below 0.05% and peaks present in the matrix are excluded from the sum of the total peak areas. Purity is calculated as: (100% - the sum of all individual impurities ≥ 0.05 area %). Related substances are reported relative to the retention time of the parent peak (RRT). Purity, assay and related substance results are reported as the average of four assay results (double injections of two discrete sample preparations). Representative chromatograms are shown in Figure 2A As shown in the figure, the main peak of the CP-201 intermediate migrates at a retention time of about 11 minutes. The retention times of the migration of the CP-201 isomers are 8.8 minutes (CP-230), 12.1 minutes (CP-231), and 17.2 minutes (CP-232), respectively. Figure 2B It is a chromatogram showing the peaks of the CP-201 isomer identification standard.

[0336] Table 4. RP-UHPLC method conditions

[0337] column Luna Omega 1.6μm Polar C18, 50mm x 2.1mm Mobile phase A 20 mM triethylamine and acetic acid in water, pH 8.0 Mobile phase B Methanol Detection UV: λ = 254nm Column temperature 30℃ Injection volume 1.0μL Flow rate 0.4 ml / min Collection time 30 minutes Autosampler temperature 5℃

[0338] With exosome payload

[0339] The RP-UHPLC method was used to assess the content, identity, and purity of the CP-201 STING agonist component in the CDK-002 drug product. This method is similar to the RP-UHPLC method described in Section 3.2.S.4.2.3 (CP-201 Intermediate), with modifications to account for the presence of exosomes in the drug product.

[0340] The CP-201 intermediate was reconstituted in a formulation buffer and used to prepare a standard curve. The CP-201 standard and CDK-002 drug product samples were mixed with exosome lysis buffer (21mM tris(2-carboxyethyl)phosphine hydrochloride, 90mM EDTA, 462mM guanidine hydrochloride, 85mM tromethamine, reduced 14.5% (w / v) Triton X-100) and incubated at 55°C for 1 hour to dissolve the exosomes. After incubation, the samples were diluted and mixed with a buffer consisting of a 3:1 ratio of formulation buffer and exosome lysis buffer. The samples were then analyzed by RP-UHPLC using the parameters shown in Table 5.

[0341] The chromatography method uses an aqueous mobile phase containing 0.1M triethylammonium acetate (pH 7) as an ion pairing agent, an acetonitrile gradient, and data is collected from the absorbance at 254nm. The collected data are processed and reported using automated chromatography software. Identity is determined by comparing the retention time of the test sample with the known CP-201STING agonist reference standard. The content is determined relative to the standard curve of the CP-201STING agonist reference standard. Product purity is reported as a percentage of the main peak area relative to the total peak area. Product-related designated impurities (CP-201STING agonist product-related isomers and degradation products) and process-related designated impurities (N-methylbenzamide) are reported in the results as area percentages (area %) of all reported peaks. Related substances are identified relative to the retention time of the parent peak (RRT).

[0342] Representative chromatograms of CDK-002 drug product are shown in Figure 2C As shown in the figure, the retention time of the CP-201 intermediate main peak migration is about 13.2 minutes. The retention time of the CP-201 isomer, CP-230 migration is 9.4 minutes, and the retention time of N-methylbenzamide migration is 7.1 minutes. The retention time of CP-231 migration is 14.3 minutes, but it was not detected in this sample. Other unspecified impurities exist at low levels, wherein the retention times are shown in the figure.

[0343] Table 1: RP-UHPLC method conditions

[0344]

[0345] Incorporated by Reference

[0346] All publications, patents, patent applications, and other documents cited in this application are hereby incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, or other document was individually indicated to be incorporated by reference for all purposes.

[0347] Equivalent

[0348] The present disclosure provides, among other things, compositions of exosomes encapsulating STING agonists for use as therapeutic agents. The present disclosure also provides methods of producing exosomes encapsulating STING agonists and methods of administering such exosomes as therapeutic agents. Although various specific aspects have been illustrated and described, the above description is not limiting. It should be understood that various changes can be made without departing from the spirit and scope of the present invention. Many changes will become apparent to those skilled in the art after reading this specification.

Claims

1. A method for preventing or treating a tumor in a subject in need thereof, the method comprising administering to the subject a dose of a stimulator of interferon genes (STING) agonist, wherein the amount of the STING agonist in the dose is about 0.01 μg to about 100 μg.

2. The method of claim 1, wherein the STING agonist is delivered via extracellular vesicles.

3. The method of claim 1 or 2, wherein the STING agonist is associated with extracellular vesicles.

4. The method of any one of claims 1 to 3, wherein the dose comprises about 0.1 μg to about 1 μg, about 0.1 μg to about 10 μg, about 0.1 μg to about 20 μg, about 0.1 μg to about 30 μg, about 0.1 μg to about 40 μg, about 0.1 μg to about 50 μg, about 0.1 μg to about 60 μg, about 0.1 μg to about 70 μg, about 0.1 μg to about 80 μg, 0.1 μg to about 90 μg, about 0.1 to about 100 μg, about 1 μg to about 10 μg, about 1 μg to about 20 μg, about 1 μg to about 30 μg, about 0.1 μg to about 40 μg, about 0.1 μg to about 50 μg, about 0.1 μg to about 60 μg, about 0.1 μg to about 70 μg, about 0.1 μg to about 80 μg, about 0.1 μg to about 90 μg, about 0.1 to about 100 μg, about 1 μg to about 10 μg, about 1 μg to about 20 μg, about 1 μg to about 30 μg, about 1 μg to about 40 μg, about 1 μg to about 50 μg, about 1 μg to about 60 μg, about 1 μg to about 70 μg, about 1 μg to about 80 μg, about 1 μg to about 90 μg, about 1 μg to about 100 μg, about 10 μg to about 20 μg, about 10 μg to about 30 μg, about 10 μg to about 40 μg, about 10 μg to about 50 μg, about 10 μg to about 60 μg, about 10 μg to about 70 μg, about 10 μg to about 80 μg, about 90 μg to about 10 μg; about 20 μg to about 30 μg, about 20 μg to about 40 μg, about 20 μg to about 50μg, about 20μg to about 60μg, about 20μg to about 80μg, about 20μg to about 90μg, about 20μg to about 100μg, about 30μg to about 40μg, about 30μg to about 50μg, about 30μg to about 60μg, about 30μg to about 70μg, about 30μg to about 80μg, about 30μg to about 90μg, about 30μg to about 100μg, about 40μg to about 50μg, about 40μg to about 60μg, about 40μg to about 70μg, about 40μg to about 80μg, about 40μg to about 90μg, about 40μg to about to about 100 μg, about 50 μg to about 60 μg, about 50 μg to about 70 μg, about 50 μg to about 80 μg, about 50 μg to about 90 μg, about 50 μg to about 100 μg, about 60 μg to about 70 μg, about 60 μg to about 80 μg, about 60 μg to about 90 μg, about 60 μg to about 100 μg, about 70 μg to about 80 μg, about 70 μg to about 90 μg, about 70 μg to about 100 μg, about 80 μg to about 90 μg, about 80 μg to about 100 μg, or about 90 μg to about 100 μg of the STING agonist.

5. The method of any one of claims 1 to 4, wherein the dose comprises about 0.1 μg, about 0.2 μg, about 0.3 μg, about 0.4 μg, about 0.5 μg, about 0.6 μg, about 0.7 μg, about 0.8 μg, about 0.9 μg, about 1.0 μg, about 1.1 μg, about 1.2 μg, about 1.3 μg, about 1.4 μg, about 1.5 μg, about 1.6 μg, about 1.7 μg, about 1.8 μg, about 1.9 μg, about 2.0 μg, about 2.5 μg. , about 3.0 μg, about 3.5 μg, about 4.0 μg, about 4.5 μg, about 5.0 μg, about 5.5 μg, about 6.0 μg, about 6.5 μg, about 7.0 μg, about 7.5 μg, about 8.0 μg, about 8.5 μg, about 9.0 μg, about 9.5 μg, about 10 μg, about 11 μg, about 12 μg, about 13 μg, about 14 μg, about 15 μg, about 16 μg, about 17 μg, about 18 μg, about 19 μg or about 20 μg of the STING agonist.

6. The method of any one of claims 1 to 5, wherein the dose comprises about 0.3 μg of the STING agonist.

7. The method of any one of claims 1 to 5, wherein the dose comprises about 1.0 μg of the STING agonist.

8. The method of any one of claims 1 to 5, wherein the dose comprises about 3.0 μg of the STING agonist.

9. The method of any one of claims 1 to 5, wherein the dose comprises about 6.0 μg of the STING agonist.

10. The method of any one of claims 1 to 5, wherein the dose comprises about 12.0 μg of the STING agonist.

11. The method of any one of claims 1 to 10, wherein the tumor is a primary tumor, a secondary tumor, or both a primary tumor and a secondary tumor.

12. The method of any one of claims 1 to 11, wherein the tumor is advanced or metastatic.

13. The method of any one of claims 1 to 12, wherein the tumor is recurrent, relapsed or refractory.

14. The method according to any one of claims 1 to 13, wherein the tumor is selected from the group consisting of a cancer selected from the group consisting of lung cancer, ovarian cancer, cervical cancer, endometrial cancer, breast cancer, brain cancer, colon cancer, prostate cancer, gastrointestinal cancer, head and neck cancer, non-small cell lung cancer, nervous system cancer, kidney cancer, retinal cancer, skin cancer, liver cancer, pancreatic cancer, genitourinary cancer and bladder cancer, melanoma, leukemia, brain cancer (e.g., gliomas, astrocytomas, ependymomas, oligodendrogliomas and tumors with a mixture of two or more cell types, known as mixed gliomas, acoustic neuromas (neurilemmomas, schwannomas, neurothecomas), adenomas, astrocytomas, low-grade astrocytomas, giant cell astrocytomas, intermediate and high-grade astrocytomas, Recurrent tumors, brain stem gliomas, chordomas, choroid plexus papilloma, CNS lymphomas (primary malignant lymphomas), cysts, dermoid cysts, epidermoid cysts, craniopharyngiomas, ependymomas, anaplastic ependymomas, gangliocytomas (gangliomas), gangliogliomas, glioblastoma multiforme (GBM), malignant astrocytomas, gliomas, hemangioblastomas, inoperable brain tumors, lymphomas, medulloblastomas (MDL), meningiomas, metastatic brain tumors, mixed gliomas, neurofibromatosis, oligodendrogliomas, optic nerve gliomas, pineal region tumors, pituitary adenomas, PNETs (primitive neuroectodermal tumors), spinal tumors, subependymomas and tuberous sclerosis (Bourneville's Disease), head and neck squamous cell carcinoma, triple-negative breast cancer, anaplastic thyroid cancer, and skin squamous cell carcinoma.

15. The method of any one of claims 1 to 14, wherein administering the composition prevents metastasis of the tumor, reduces the growth rate of the tumor, reduces the size of the tumor, or a combination thereof.

16. The method according to any one of claims 1 to 15, wherein the subject has previously received at least two anti-cancer treatments.

17. The method of any one of claims 1 to 16, wherein the administration is parenteral, oral, intravenous, intramuscular, intratumoral, or intraperitoneal.

18. The method of any one of claims 1 to 17, wherein the administration is intratumoral.

19. The method of any one of claims 1 to 18, comprising administering a second, third, fourth or fifth dose of the STING agonist.

20. The method of any one of claims 1 to 19, wherein one or more of the second, third, fourth, or fifth doses is administered at the same dose as the initial dose.

21. The method of any one of claims 1 to 20, wherein one or more of the second, third, fourth, or fifth doses is administered at a different dose than the initial dose.

22. The method according to any one of claims 2 to 21, wherein the extracellular vesicle is selected from exosomes, nanovesicles, apoptotic bodies, microvesicles, lysosomes, endosomes, liposomes, lipid nanoparticles, micelles, multilamellar structures, reencapsulated vesicles and extruded cells.

23. The method of any one of claims 2 to 22, wherein the extracellular vesicles are exosomes.

24. The method of any one of claims 2 to 23, wherein the STING agonist is encapsulated within the extracellular vesicle.

25. The method of any one of claims 2 to 24, wherein the STING agonist is associated with the lipid bilayer of the extracellular vesicle.

26. The method of claim 25, wherein the STING agonist is attached to the lipid bilayer of the extracellular vesicle via a linker.

27. The method of claim 26, wherein the linker is a cleavable linker.

28. The method according to any one of claims 2 to 27, wherein the extracellular vesicles overexpress PTGFRN protein or a portion thereof.

29. The method of claim 28, wherein the STING agonist is linked to the PTGFRN protein or the portion thereof.

30. The method of claim 27 or 28, wherein the STING agonist is linked to the PTGFRN protein or the portion thereof via a linker.

31. The method of any one of claims 2 to 30, wherein the extracellular vesicles are produced by cells that overexpress PTGFRN protein.

32. The method of any one of claims 1 to 31, wherein the STING agonist is a cyclic dinucleotide.

33. The method of any one of claims 1 to 32, wherein the STING agonist is a non-cyclic dinucleotide.

34. The method of any one of claims 1 to 33, wherein the STING agonist comprises a lipid binding tag.

35. The method of any one of claims 1 to 34, wherein the STING agonist is physically and / or chemically modified.

36. The method, EV or composition of claim 35, wherein the modified STING agonist has a different polarity and / or charge than the corresponding unmodified STING agonist.

37. The method of any one of claims 1 to 36, wherein the STING agonist comprises: in: X1 is H, OH or F; X2 is H, OH or F; Z is OH, OR1, SH or SR1, where: i) R1 is Na or NH4, or ii) R1 is an enzyme-labile group that donates OH or SH in vivo, such as pivaloyloxymethyl; B1 and B2 are bases selected from the group consisting of: The conditions are: - In formula (I): X1 and X2 are not OH, - In formula (II): when X1 and X2 are OH, B1 is not adenine, and B2 is not guanine, and - In formula (III): when X1 and X2 are OH, B1 is not adenine, B2 is not guanine, and Z is not OH, or a pharmaceutically acceptable salt thereof.

38. The method of any one of claims 1 to 36, wherein the STING agonist is selected from the group consisting of: and pharmaceutically acceptable salts thereof.

39. The method of any one of claims 1 to 38, wherein the STING agonist is or a pharmaceutically acceptable salt thereof.

40. The method of any one of claims 1 to 39, further comprising administering to the subject a second anticancer agent.

41. The method of claim 40, wherein the second anti-cancer agent comprises a chemotherapy, an antibody or antigen-binding portion thereof, an additional extracellular vesicle, or any combination thereof.

42. The method of any one of claims 29 to 41, wherein the PTGFRN protein comprises SEQ ID NO:

33.

43. according to the method described in any one of claims 29 to 42, wherein the PTGFRN protein comprises at least about 70%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical to SEQ ID NO:

1.

44. A method according to any one of claims 29 to 43, wherein the PTGFRN protein comprises the amino acid sequence shown in SEQ ID NO:

1.

45. A method of treating a tumor in a subject, the method comprising: a. first administering to a patient a first composition of extracellular vesicles comprising a first STING agonist, wherein the first composition comprises about 0.3 micrograms of the first STING agonist; b. secondly administering to the patient a second composition of extracellular vesicles comprising a second STING agonist, The second composition comprises about 1.0 microgram of the second STING agonist; and c. administering to the patient a third time a third composition comprising extracellular vesicles of a third STING agonist, wherein the third composition comprises about 2.0 micrograms of the third STING agonist; wherein the first STING agonist, the second STING agonist, and the third STING agonist are the same; wherein the composition is administered intratumorally; and wherein the tumor is selected from the group consisting of head and neck squamous cell carcinoma, triple-negative breast cancer, anaplastic thyroid cancer, and cutaneous squamous cell carcinoma, The patient has previously received at least two anti-cancer treatments.

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