Cholesterol-oligonucleotide tethers with aptamers for ligand display and extracellular vesicle functionalization

Abstract

Provided herein are Treg-derived extracellular vesicles (EVs) modified with an aptamer that binds an affinity-tagged protein. The affinity-tagged protein may be CTLA4, such that the Treg EVs target B7-dislaying cells, such as T-cells, B-cells, APCs, dendritic cells, or macrophage. The CTLA4-modified Treg EVs are useful in reducing an immune response, such as in transplantation patients, cancer patients, autoimmune disease patients, or inflammatory disease patients.

Description

CHOLESTEROL-OLIGONUCLEOTIDE TETHERS WITH APTAMERS FOR LIGAND DISPLAY AND EXTRACELLULAR VESICLE FUNCTIONALIZATIONCROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of United States Provisional Patent Application No. 63 / 531 ,335, filed August 08, 2023, the disclosure of which is hereby incorporated by reference in its entirety.REFERENCE TO A SEQUENCE LISTING

[0002] The Sequence Listing associated with this application is filed in electronic format and is hereby incorporated by reference into the specification in its entirety. The name of the XML file containing the Sequence Listing is 06526-2404467.xmL The size of the XML file is 2,768 bytes and the XML file was created on August 08, 2024.

[0003] Regulatory T (Treg) cells are a subpopulation of CD4+ T cells that regulates immunological self-tolerance and immune homeostasis. Treg cells operate to regulate immune response, and perturbation of the action of Treg cells can result in in autoimmune disease, inflammatory disease, transplant rejection, and tumors. Action of Tregs include contact-dependent and contact-independent mechanisms. As part of their action, Treg cells secrete extracellular vesicles (EVs) to regulate other cell types without direct contact. Extracellular vesicles (EVs) are secreted by cells to the extracellular environment and can play a role in intercellular communication by serving as vehicles for the transport of proteins, lipids, and nucleic acids (See, e.g., Lin C, et al. Roles of Regulatory T Cell-Derived Extracellular Vesicles in Human Diseases. IntJ Mol Sci. 2022 Sep 23;23(19):11206). Treg cells-derived extracellular vesicles (Treg- EVs) maintain self-immune tolerance and regulate immune responses by expressing specific molecules and delivering cargoes as a novel contact-independent mechanism of Treg cells.

[0004] Engineered EV’s that that can be obtained from Treg cells, and that can be targeted easily, inexpensively, and in high quantity are desirable, for instance for use in targeting immune cells in order to promote tolerance, for example in transplant rejection, cancer, autoimmune disease, and inflammatory disease.SUMMARY

[0005] A particle is provided, comprising: a Treg extracellular vesicle (EV); and an anti-affinity tag (anti-TAG) aptamer linked to a lipophilic anchor embedded in a phospholipid bilayer of the EV, wherein the anti-TAG aptamer binds a functional protein linked to an affinity tag binding partner to the anti-TAG aptamer.

[0006] A method of preparing a modified Treg EV particle is provided. The method comprising contacting a Treg EV particle with an anti-affinity tag (anti-TAG) aptamer linked to a lipophilic membrane anchor to produce an anti-TAG aptamer-modified Treg EV particle, and contacting the anti-TAG-modified Treg EV particle with a functional protein linked to an affinity tag binding partner to the anti-TAG aptamer, thereby binding the functional protein via the affinity tag to the anti-TAG aptamer of the anti-TAG aptamer-modified Treg EV particle. A particle also is provided that is a product of the preceding method.

[0007] A composition is provided comprising a particle comprising a Treg extracellular vesicle (EV); and an anti-affinity tag (anti-TAG) aptamer linked to a lipophilic anchor embedded in a phospholipid bilayer of the EV, wherein the anti-TAG aptamer binds a functional protein linked to an affinity tag binding partner to the anti-TAG aptamer, and a pharmaceutically-acceptable excipient.

[0008] A method of reducing an immune response in a patient, comprising contacting a cell expressing membrane B7 protein, such as an immune cell, e.g., a T-cell, a 13- cell, an APC, a dendritic cell, or a macrophage, of the patient with a particle comprising a Treg extracellular vesicle (EV); and an anti-affinity tag (anti-TAG) aptamer linked to a lipophilic anchor embedded in a phospholipid bilayer of the EV, wherein the anti-TAG aptamer binds a functional protein linked to an affinity tag binding partner to the anti- TAG aptamer, such that the EV binds a B7 membrane protein of the cell, e.g., a T-cell, a B-cell, an APC, a dendritic cell, or a macrophage of the patient to reduce an immune response in the patient.

[0009] A method of delivering a Treg EV particle to a cell expressing membrane B7 protein, such as an immune cell, e.g., a T-cell, a B-cell, an APC, a dendritic cell, or a macrophage, comprising contacting the cell with a particle comprising a Treg extracellular vesicle (EV); and an anti-affinity tag (anti-TAG) aptamer linked to a lipophilic anchor embedded in a phospholipid bilayer of the EV, wherein the anti-TAG aptamer binds a functional protein linked to an affinity tag binding partner to the anti-TAG aptamer, such that the cell internalizes the Treg EV particle e.g., by endocytosis or trogocytosis).

[0010] The following numbered clauses outline various non-limiting aspects, embodiments, or examples of the present invention.

[0011] Clause 1. A particle comprising: a Treg extracellular vesicle (EV); and an anti-affinity tag (anti-TAG) aptamer linked to a lipophilic anchor embedded in a phospholipid bilayer of the EV, wherein the anti-TAG aptamer binds a functional protein linked to an affinity tag binding partner to the anti-TAG aptamer.

[0012] Clause 2. The particle of clause 1 , wherein the anti-TAG aptamer comprises an anti-Fc aptamer, e.g., SEQ ID NO: 1 , or an anti-HIS aptamer, e.g., SEQ ID NO: 2.

[0013] Clause 3. The particle of clause 1 or 2, wherein the particle further comprises a functional protein linked to the affinity tag binding partner to the anti-TAG aptamer, bound to the anti-TAG aptamer.

[0014] Clause 4. The particle of clause 3, wherein the functional protein is a ligand for binding a membrane-bound protein of a target cell.

[0015] Clause 5. The particle of clause 4, wherein the functional protein linked to the affinity tag is a CTLA4 protein that binds cell membrane-bound B7 protein.

[0016] Clause 6. The particle of clause 4 or 5, wherein the target cell is a human cell.

[0017] Clause 7. The particle of any one of clauses 1 -6, bound to a B7 membrane protein of a target cell via the CTLA4 protein.

[0018] Clause 8. The particle of clause 7, wherein the target cell is a T-cell, a B- cell, an APC, a dendritic cell, or a macrophage.

[0019] Clause 9. The particle of any one of clauses 1 -8, wherein the Treg EV is obtained from a human Treg cell.

[0020] Clause 10. The particle of any one of clauses 1 -9, wherein the lipophilic anchor comprises a cholesterol moiety.

[0021] Clause 11. A composition comprising the particle of any one of clauses 1 -10 and a pharmaceutically-acceptable excipient.

[0022] Clause 12. A method of preparing a modified Treg EV particle, comprising contacting a Treg EV particle with an anti-affinity tag (anti-TAG) aptamer linked to a lipophilic membrane anchor to produce an anti-TAG aptamer-modified Treg EVparticle, and contacting the anti-TAG-modified Treg EV particle with a functional protein linked to an affinity tag binding partner to the anti-TAG aptamer, thereby binding the functional protein via the affinity tag to the anti-TAG aptamer of the anti-TAG aptamer-modified Treg EV particle.

[0023] Clause 13. The method of clause 12, wherein the anti-TAG aptamer comprises an anti-Fc aptamer or an anti-HIS aptamer.

[0024] Clause 14. The method of clause 12 or 13, wherein the functional protein is a ligand for binding a membrane-bound protein of a target cell.

[0025] Clause 15. The method of clause 14, wherein the functional protein linked to the affinity tag is a CTLA4 protein that binds cell membrane-bound B7 protein (e.g., B7-1 (CD80) and / or B7-2 (CD86)).

[0026] Clause 16. The method of clause 14 or 15, wherein the target cell is a human cell.

[0027] Clause 17. The method of any one of clauses 12-16, wherein the Treg EV is obtained from a human Treg cell.

[0028] Clause 18. The method of any one of clauses 12-17, wherein the lipophilic anchor comprises a cholesterol moiety.

[0029] Clause 19. The method of any one of clauses 12-18, further comprising mixing the modified Treg EV particle with a pharmaceutically-acceptable excipient.

[0030] Clause 20. A method of reducing an immune response in a patient, comprising contacting a cell expressing membrane B7 protein, such as an immune cell, e.g., a T-cell, a B-cell, an APC, a dendritic cell, or a macrophage, of the patient with the particle of clause 5, such that the EV binds a B7 membrane protein of the cell, such as a B-cell, an APC, a dendritic cell, or a macrophage, of the patient to reduce the immune response in the patient.

[0031] Clause 21 . The method of clause 20, wherein the patient is a transplantation patient, a cancer patient, an autoimmune disease patient, or an inflammatory disease patient.

[0032] Clause 22. A method of delivering a Treg EV particle to a cell expressing membrane B7 protein, such as an immune cell, e.g., a T-cell, a B-cell, an APC, a dendritic cell, or a macrophage, comprising contacting the cell with the particle of clause 5, such that the cell internalizes the Treg EV particle (e.g., by endocytosis or trogocytosis).

[0033] Clause 23. The method of clause 22, wherein the cell is a cell of a transplantation patient, a cancer patient, an autoimmune disease patient, or an inflammatory disease patient.BRIEF DESCRIPTION OF THE DRAWINGS

[0034] FIG. 1. Rational for engineering CTLA-4 onto Treg EVs to increase targeting to immune cells. CTLA-4 on engineered EVs binds to B7 on APCs and prevents T cell activation via CD28 (Indirect Inhibition) and binds to B7 on Teff cells to trigger anergy (Direct Inhibition). EVs are internalized by APCs or macrophages (via endocytosis), unactivated T cells or Teff cells (via trogocytosis). Engineered EVs interfere with the formation of the immunological synapse and reduce enforced T cell-APC contact times. Additional intraluminal cargo can then be delivered directly to the cytosol during internalization.

[0035] FIGS. 2A and 2B. Histogram representations and graph of fluorescence from flow cytometry experiments evaluating ssDNA (Chol-DNA-Cy3) tethering to Treg EVs. Increasing concentration of Chol-DNA-Cy3 in the EV membrane resulted in corresponding increase in the Cy3 fluorescence intensity from anti-CD63 conjugated beads.

[0036] FIG. 3A provides representative confocal images of CTLA4 bound to Treg EVs captured with anti-CD63 conjugated beads. Chol-ssDNA-His Aptamer or Chol- ssDNA-Fc Aptamer were prebound to EVs with corresponding His-CTLA4 or Fc- CTLA4 (both fluorescently labeled with Dylight 650. Tethered EVs were then captured with anti-C63 magnetic beads. Captured EVs were pipetted onto a standard glass slide, allowed to dry, and imaged using ZEISS LSM 880 confocal microscope under constant settings. Increasing concentration of Chole-ssDNA-His aptamer bound to EVs resulted in a corresponding increase in fluorescence from the beads. FIG. 3B provides a graph showing specificity of CTLA4 binding to EVs via aptamer and with an aptamer free Chol-DNA tether.

[0037] FIG 4. A. Representative confocal images showing internalization of DID- labeled Treg EVs modified with Chol-DNA tethers with and without CTLA4 in murine J774 macrophage cells. B. Quantification of confocal images. C. Cell up take of CTLA4 requires aptamer bound CTLA4.

[0038] FIG. 5A. Representative confocal images in right showing internalization of DID-labeled Treg EVs modified with Chol-DNA-HisApt with and without CTLA4 in human Jurkat T cells. FIG. 5B. Quantification of imaging data.

[0039] FIG. 6. Schematic of Chol-DNA with aptamer sequence for protein display on exosome surface.DETAILED DESCRIPTION

[0040] Other than in the operating examples, or where otherwise indicated, the use of numerical values in the various ranges specified in this application are stated as approximations as though the minimum and maximum values within the stated ranges are both preceded by the word “about”. In this manner, slight variations above and below the stated ranges can be used to achieve substantially the same results as values within the ranges. Also, unless indicated otherwise, the disclosure of ranges is intended as a continuous range including every value between the minimum and maximum values. Further, as used herein, all numbers expressing dimensions, physical characteristics, processing parameters, quantities of ingredients, reaction conditions, and the like, used in the specification and claims are to be understood as being modified in all instances by the term “about”. Moreover, unless otherwise specified, all ranges disclosed herein are to be understood to encompass the beginning and ending range values and any and all subranges subsumed therein. For example, a stated range of “1 to 10” should be considered to include any and all subranges between (and inclusive of) the minimum value of 1 and the maximum value of 10; that is, all subranges beginning with a minimum value of 1 or more and ending with a maximum value of 10 or less, e.g., 1 to 3.3, 4.7 to 7.5, 5.5 to 10, and the like.

[0041] As used herein “a” and “an” refer to one or more. The term “comprising” is open-ended and may be synonymous with “including”, “containing”, or “characterized by”. The term “consisting essentially of” limits the scope of a claim to the specified materials or steps and those that do not materially affect the basic and novel characteristic(s) of the claimed invention.

[0042] As used herein, spatial or directional terms, such as “left”, “right”, “inner”, “outer”, “above”, “below”, “over”, “under”, and the like, relate to the invention as it is shown in the drawing figures are provided solely for ease of description and illustration, and do not imply directionality, unless specifically required for operation of the described aspect of the invention. It is to be understood that the invention can assume various alternative orientations and, accordingly, such terms are not to be considered as limiting.

[0043] As used herein, a "patient" is an animal, such as a mammal, including a primate (such as a human, a non-human primate, e.g., a monkey, and a chimpanzee), a nonprimate (such as a cow, a pig, a camel, a llama, a horse, a goat, a rabbit, a sheep, a hamster, a guinea pig, a cat, a dog, a rat, a mouse, a horse, and a whale), or a bird (e.g., a duck or a goose). As used herein, the terms "treating” or "treatment" refer to a beneficial or desired result, such as improving one of more functions, or symptoms of a disease.

[0044] "Therapeutically effective amount," as used herein, is intended to include the amount of a recognition reagent as described herein that, when administered to a subject having a disease, is sufficient to effect treatment of the disease (e.g., by diminishing, ameliorating, or maintaining the existing disease or one or more symptoms of disease). The "therapeutically effective amount" may vary depending on compound or composition, how it is administered, the disease and its severity and the history, age, weight, family history, genetic makeup, the types of preceding or concomitant treatments, if any, and other individual characteristics of the subject to be treated.

[0045] A "therapeutically-effective amount" also includes an amount of an agent that produces some desired local or systemic effect at a reasonable benefit / risk ratio applicable to any treatment. Compounds and compositions described herein may be administered in a sufficient amount to produce a reasonable benefit / risk ratio applicable to such treatment.

[0046] The phrase "pharmaceutically-acceptable carrier" as used herein means a pharmaceutically-acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc magnesium, calcium or zinc stearate, or steric acid), or solvent encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject being treated. Some examples of materials which can serve as pharmaceutically- acceptable carriers include: (1 ) sugars, such as lactose, glucose and sucrose; (2) starches, such as com starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such as magnesium state, sodium lauryl sulfate and talc; (8) excipients, such as cocoa butter andsuppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil and soybean oil; (10) glycols, such as propylene glycol; (11 ) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21 ) polyesters, polycarbonates and / or polyanhydrides; (22) bulking agents, such as polypeptides and amino acids (23) serum component, such as serum albumin, HDL and LDL; and (22) other non-toxic compatible substances employed in pharmaceutical formulations.

[0047] As used herein, the term “nucleic acid” refers to deoxyribonucleic acids (DNA) and ribonucleic acids (RNA). Nucleic acid analogs include, for example and without limitation: 2’-O-methyl-substituted RNA, locked nucleic acids, unlocked nucleic acids, triazole-linked DNA, peptide nucleic acids, morpholino oligomers, dideoxynucleotide oligomers, glycol nucleic acids, threose nucleic acids and combinations thereof including, optionally ribonucleotide or deoxyribonucleotide residue(s). Herein, “oligonucleotide” is a short, single-stranded structure made of up nucleotides, includes nucleic acids, nucleic acid analogs, or a chimera thereof, as oligonucleotides may include a combination of both standard nucleotide monomer residues and synthetic nucleotide monomer residues. An oligonucleotide may be referred to by the length (i.e., number of nucleotides) of the strand, through the nomenclature “-mer”. For example, an oligonucleotide of 22 nucleotides would be referred to as a 22-mer. An oligonucleotide comprises a sequence of nucleobases (“has a sequence of bases”, or simply “has a sequence”) that is able to hybridize to a complementary sequence on an oligonucleotide, a nucleic acid, or a nucleic acid analog by cooperative base pairing, e.g., Watson-Crick base pairing or Watson-Crick-like base pairing.

[0048] A “nucleic acid analog” is a composition comprising a sequence of nucleobases arranged on a substrate, such as a polymeric backbone, and can bind DNA and / or RNA by hybridization by Watson-Crick, or Watson-Crick-like hydrogen bond base pairing. Non-limiting examples of common nucleic acid analogs include peptide nucleic acids (PNAs), such as yPNA, morpholino nucleic acids, phosphorothioates, locked nucleic acid (2’-O-4’-C-methylene bridge, including oxy, thio or amino versions thereof), unlocked nucleic acid (the C2’-C3’ bond is cleaved), a, [3-con strained nucleic acid, 2’-fluoro RNA, phosphorodiamidate morpholino, 2’-O-methyl-substituted RNA,threose nucleic acid, glycol nucleic acid, 2',4'-constrained ethyl nucleic acid, 2’, 4’ bridged nucleic acid NC (N-H), 2’, 4’ bridged nucleic acid NC (N-methyl), ((S)-5’-C- methyl DNA (RNA)), and 5’-E-vinylphosphonate nucleic acid, among others. A "peptide nucleic acid" (PNA) refers to a nucleic acid analog, or DNA or RNA mimic, in which the sugar phosphodiester backbone of the DNA or RNA is replaced by an N- (2-aminoethyl)glycine unit. A gamma PNA (yPNA) is an oligomer or polymer of gamma-modified N-(2-aminoethyl)glycine monomers to produce a chiral center.

[0049] In the context of the present disclosure, a “nucleotide” refers to a monomer comprising at least one nucleobase and a backbone element (backbone moiety), which in a nucleic acid, such as RNA or DNA, is ribose or deoxyribose. “Nucleotides” also typically comprise reactive groups that permit polymerization under specific conditions. In natural DNA and RNA, those reactive groups are the 5’ phosphate and 3’ hydroxyl groups. For chemical synthesis of nucleic acids and analogs thereof, the bases and backbone monomers may contain modified groups, such as blocked amines, as are known in the art. A “nucleotide residue” refers to a single nucleotide that is incorporated into an oligonucleotide or polynucleotide. The backbone monomer can be any suitable nucleic acid backbone monomer, such as a ribose triphosphate or deoxyribose triphosphate, or a monomer of a nucleic acid analog, such as peptide nucleic acid (PNA), such as a gamma PNA (yPNA). The backbone monomer may be a ribose mono-, di-, or tri-phosphate or a deoxyribose mono-, di-, or tri-phosphate, such as a 5’ monophosphate, diphosphate, or triphosphate of ribose or deoxyribose. The backbone monomer includes both the structural “residue” component, such as the ribose in RNA, and any active groups that are modified in linking monomers together, such as the 5’ triphosphate and 3’ hydroxyl groups of a ribonucleotide, which are modified when polymerized into RNA to leave a phosphodiester linkage. Likewise, for PNA, the C-terminal carboxyl and N-terminal amine active groups of the N-(2- aminoethyl)glycine backbone monomer are condensed during polymerization to leave a peptide (amide) bond.

[0050] Complementary refers to the ability of polynucleotides (nucleic acids) to hybridize to one another, forming inter-strand base pairs. Base pairs are formed by hydrogen bonding between nucleotide units in polynucleotide or polynucleotide analog strands that are typically in antiparallel orientation. Complementary polynucleotide strands can base pair (hybridize) in the Watson-Crick manner (e.g., A to T, A to U, C to G), or in any other manner that allows for the formation of duplexes. In RNA asopposed to DNA, uracil rather than thymine is the base that is complementary to adenosine. Two sequences comprising complementary sequences can hybridize if they form duplexes under specified conditions, such as in water, saline (e.g., normal saline, or 0.9% w / v saline) or phosphate-buffered saline), or under other stringency conditions, such as, for example and without limitation, 0.1 X SSC (saline sodium citrate) to 10X SSC, where 1 X SSC is 0.15M NaCI and 0.015M sodium citrate in water. Hybridization of complementary sequences is dictated, e.g., by the nucleobase content of the strands, the presence of mismatches, the length of complementary sequences, salt concentration, temperature, with the melting temperature (Tm) lowering with shorter complementary sequences, increased mismatches, and increased stringency. Perfectly matched sequences are said to be “fully complementary”, though one sequence (e.g., a target sequence in an mRNA) may be longer than the other.

[0051] Although described in terms of nucleobase, backbone, and complementarity, in the context of the present disclosure nucleic acids and nucleic acid analogs, are used as aptamers. Aptamers are short, single-stranded DNA or RNA (ssDNA or ssRNA) molecules that can selectively bind to a specific target, including proteins, peptides, carbohydrates, small molecules, toxins, and even live cells, akin to the way an antibody might bind to the same. Rather than primary sequence complementarity as in Watson-Crick-like binding by complementarity, aptamer binding is determined by its tertiary structure. Target recognition and binding involve three-dimensional, shapedependent interactions as well as hydrophobic interactions, base-stacking, and intercalation. Aptamers may be developed, for example, by a process called Sequential Evolution of Ligands by Exponential Enrichment (SELEX). The aptamer molecules described herein, with Fc-specific or His-tag-specific binding may be developed using SELEX, but are commercially available via various manufacturers, and can be readily modified (e.g., end-modified) with a lipophilic moiety or other membrane anchor moiety, such as a cholesterol moiety.

[0052] An “extracellular vesicle” (EV) is a double-layer phospholipid membrane vesicle known to be released by most cells. EVs may carry biologically active molecules that can traffic to local or distant targets and execute defined biological functions. EVs typically have a diameter of 10 nm and above. However, EVs may be classified by size, biogenetic pathways, and function. Common classification includes endosomal sorting complexes required for transport (ESCRT) protein-based formation ofintraluminal vesicles within multivesicular bodies (MVBs) (“exosomes”), a pathway that is shared by viruses; formation by pinching off from the plasma membrane (“microvesicles”); and membrane disintegration (“apoptotic bodies”). (See, e.g., Margolis L, Sadovsky Y (2019) The biology of extracellular vesicles: The known unknowns. PLOS Biology 17(7): e3000363; Li, S., Lin, Z., Jiang, X. et al. Exosomal cargo-loading and synthetic exosome-mimics as potential therapeutic tools. Acta Pharmacol Sin 39, 542-551 (2018)). Although many compounds, compositions, and methods described herein may be described in the context of exosomes, unless specifically indicated to the contrary, those compounds, compositions, and methods may be considered applicable to other EV types. Extracellular vesicles include but are not limited to exomeres, exosomes, outer-membrane vesicles, matrix vesicles, microvesicles or apoptotic bodies. For purposes herein, unless otherwise indicated, to the contrary, extracellular vesicles, e.g., exosomes, may be prepared or obtained from any biological source, such as, without limitation, from any living organism that produces extracellular vesicles, from cells, tissue, or organ cultures, e.g., from mammals of mammalian cell culture. Non-limiting examples of sources for extracellular vesicles include primary cell culture, stem cell culture, progenitor cell culture, recombinant cell culture, dendritic cell culture, among others. In the context of the present disclosure, the EVs may be Treg EVs, for example, as described in Lin C, et al., Int J Mol Sci. 2022 Sep 23;23(19):11206, and which can be prepared by any useful method, such as the methods described herein and / or, for example and without limitation, as described and / or referenced in: Thome AD, etal. Extracellular Vesicles Derived From Ex Vivo Expanded Regulatory T Cells Modulate In Vitro and In Vivo Inflammation. Front Immunol. 2022 Jun 22; 13:875825; Tung SL, etal. Regulatory T Cell Extracellular Vesicles Modify T-Effector Cell Cytokine Production and Protect Against Human Skin Allograft Damage. Front Cell Dev Biol. 2020 May 20;8:317; and Heydari, Z., etal. EVs vs. EVs: MSCs and Tregs as a source of invisible possibilities. J Mol Med 101 , 51-63 (2023).

[0053] United States Patent No. 10,513,710, incorporated herein by reference for its exemplary technical disclosure, describing exosomes, methods of preparing exosomes, and methods and reagents useful for producing exosomes decorated with hydrophobically modified nucleic acids that are RNA interference reagents. The oligonucleotides are hydrophobically modified by linking a hydrophobic (e.g., lipophilic) moiety to the oligonucleotide as described therein. The hydrophobic moieties may bea sterol such as cholesterol, GM1 , a lipid, a vitamin, a small molecule, or a peptide, or a combination thereof. The hydrophobically modified nucleic acids (hydrophobically modified nucleic acid cargo) are associated with the exosome, for example and without intent to be bound by this theory, by insertion of the hydrophobic moiety of the hydrophobically modified nucleic acid in the lipid bilayer of the exosome. Other publications describe anchoring hydrophobically-modified oligonucleotides in exosomes (Pi etal. “Nanoparticle orientation to control RNA loading and ligand display on extracellular vesicles for cancer regression”, Nature Nanotechnology, 2018, 13:82- 89). Cholesterol TEG (triethylene glycol spacer), for linking to oligonucleotides, is commercially available, e.g., as Cholesterol-TEG phosphoramidite (Glen Research, Sterling, VA).

[0054] A “moiety” (pl. “moieties”) is a part of a chemical compound, and includes groups, such as functional groups but can include any portion of a compound. As such, a nucleobase moiety is a nucleobase that is modified by attachment to another compound moiety, such as a polymer monomer, e.g., the nucleic acid or nucleic acid analog monomers described herein, or a polymer, such as a nucleic acid or nucleic acid analog as described herein.

[0055] Various linking reactions may be utilized to link or conjugate a first molecule to a second molecule, such as in linking a biologically active agent, such as a therapeutic agent, a polymer, or a member of a binding pair to an oligonucleotide. In the context of the present disclosure, conjugates may be prepared by reacting a functional group on an oligonucleotide with a functional group or groups on the item to be conjugated to the oligonucleotide, such as a lipophilic or hydrophobic moiety-containing molecule, a member of a binding pair such as an antibody, or a therapeutic agent. The reaction of the functional groups may be a “click” reaction, such as, for example and without limitation, a Staudinger ligation, an azide-alkyne cycloaddition, a reaction of tetrazine with a trans-cyclooctene, a disulfide linking reaction, a thiol-ene reaction, a hydrazinealdehyde reaction, a hydrazine-ketone reaction, a hydroxyl amine-aldehyde reaction, a hydroxyl amine-ketone reaction or a Diels-Alder reaction. Affinity tags may be attached to the C-terminus or N-terminus of a protein, such as the functional proteins described herein. The amino acid sequence of the tag, e.g., (His)e-9 or Fc, may be encoded in-frame by a gene for expression of the protein such that the protein is translated with the tag already incorporated at one end.

[0056] A “polymer composition” is a composition comprising one or more polymers. As a class, “polymers” includes, without limitation, homopolymers, heteropolymers, copolymers, block polymers, block co-polymers and can be both natural and / or synthetic. Homopolymers contain one type of building block, or monomer, whereas copolymers contain more than one type of monomer. The term “(co)polymer” and like terms refer to either homopolymers or copolymers. A polymer may have any shape for the chain making up the backbone of the polymer, including, without limitation: linear, branched, networked, star, brush, comb, or dendritic shapes. Amino acids and nucleic acids are polymers.

[0057] A polymer “comprises” or is “derived from” a stated monomer if that monomer is incorporated into the polymer. Thus, the incorporated monomer (monomer residue) that the polymer comprises is not the same as the monomer prior to incorporation into a polymer, in that at the very least, certain groups / moieties are missing and / or modified when incorporated into the polymer backbone. A polymer is said to comprise a specific type of linkage if that linkage is present in the polymer, such as, without limitation: ester, amide, carbonyl, ether, thioester, thioether, disulfide, sulfonyl, amine, carbonyl, or carbamate bonds. The polymer may be a homopolymer, a copolymer, and / or a polymeric blend. Polypeptides (e.g., proteins) comprise amino acid residues, while nucleic acids comprise ribonucleotide or deoxyribonucleotide residues.

[0058] Association of one molecule with another may be covalent or non-covalent. By attaching or linking one moiety to another, it is meant the linkage is covalent, as in, for example, polymerization, cross-linking, click chemistry reactions, or linking reactions using linkers. Complexing two molecules refers to a non-covalent association, such as by Van der Waals forces, hydrogen bonding, pi stacking, or ionic interactions. Hybridization of two complementary oligonucleotides, nucleic acids, and / or nucleic acid analogs, is a form of complexing, as used herein.

[0059] In the context of recognition reagents, the term “ligand” refers to a binding moiety for a specific target, its binding partner. Collectively the ligand and its binding partner are termed a “binding pair”. A binding partner can be a cognate receptor, a protein, a small molecule, a hapten, or any other relevant molecule, such as an affibody or a paratope-containing molecule. One common, and non-limiting example of a binding pair is streptavidin / avidin and biotin. A classic binding pair is an antibody and its corresponding antigen, where a paratope of the antibody binds an epitope of the antigen. Likewise, in the context of the present disclosure, an aptamer forms abinding pair with its target to which it binds specifically. “Specific binding” refers to binding of members of a binding pair to the substantial exclusion of cross-reactivity with other molecules in the context of a therapeutic approach, diagnostic assay, or production method in which the specific binding is to be employed. Sufficiency of the specificity of binding may be evaluated during development of a therapeutic drug product or diagnostic assay and may be evaluated according to any medically- or pharmaceutically-relevant standard, such as governmental regulatory standards.

[0060] Antigen binding molecules include for example and without limitation, antibodies, antibody fragments, engineered paratope-containing molecules, and aptamers. The term “antibody” typically refers to an immunoglobulin, derivatives thereof which maintain specific binding ability, and proteins having a binding domain which is homologous or largely homologous to an immunoglobulin binding domain. As such, the antibody operates as a ligand for its cognate antigen, which can be virtually any molecule. Other antigen binding molecules include, for example, and without limitation: an affibody, an aptamer, an affilin, an affimer, an affitin, an alphabody, an aticalin, an avimer, a DARPin, a funomer, a Kunitz domain peptide, a monobody, a nanoclamp, or other engineered protein ligands, e.g., comprising a paratope targeting any suitable epitope present in a sample. Further examples of antigen binding molecules include, but are not limited to, Fab, Fab', F(ab')2, Fv, Fd, dsFv, scFv, diabody, triabody, tetrabody, di-scFv (dimeric single-chain variable fragment), bispecific T-cell engager (BiTE), single-domain antibody (sdAb), or antibody binding domain fragments. In the context of targeting ligands, the antigen binding molecule may be a single chain antibody fragment. An antigen binding molecule may comprise multiple chains that are linked together, for instance, by disulfide linkages. The antigen binding molecule may also optionally be a multimolecular complex. A functional antibody fragment will typically comprise at least about 50 amino acids and more typically will comprise at least about 200 amino acids.

[0061] Ligands also include other engineered binding reagents, such as affibodies and designed ankyrin repeat proteins (DARPins), that exploit the modular nature of repeat proteins (Forrer T, Stumpp MT, Binz HK, Pluckthun A: A novel strategy to design binding molecules harnessing the modular nature of repeat proteins, FEBS Lett 2003, 539: 2-6; Gebauer A, Skerra A: Engineered protein scaffolds as next-generation antibody therapeutics, Curr Opin Chem Biol 2009, 13:245-255), comprising, often as a single chain, one or more antigen-binding or epitope-binding sequences and at aminimum any other amino acid sequences needed to ensure appropriate specificity, delivery, and stability of the composition (see also, e.g., Nelson, AL, “Antibody Fragments Hope and Hype” (2010) MAbs 2(1 ):77-83).

[0062] As used herein, the terms “cell” and “cells” refer to any types of cells from or in any animal, such as, without limitation, rat, mice, monkey, and human. For example and without limitation, cells can be progenitor cells, such as stem cells, or differentiated cells, such as endothelial cells, smooth muscle cells. In certain embodiments, cells for medical procedures can be obtained from the patient for autologous procedures or from other donors for allogeneic procedures.

[0063] Extracellular vesicles may be obtained from Treg cells and used as-is, or optionally are loaded with any compatible biologically-active agent, such as a therapeutic agent by any useful method. Non-limiting examples of drug loading include passive or active absorption or adsorption, electroporation, and membraneassociation with hydrophobic agents or agents comprising hydrophobic moieties (see, e.g., Olivier G. de Jong, et al. Drug Delivery with Extracellular Vesicles: From Imagination to Innovation. Accounts of Chemical Research 2019 52 (7), 1761 -1770 doi:10.1021 / acs. accounts.9b00109 and Lamichhane TN, Jay SM. Production of Extracellular Vesicles Loaded with Therapeutic Cargo. Methods Mol Biol. 2018; 1831 :37-47. doi :10.1007 / 978-1 -4939-8661 -3_4).

[0064] The biologically active agent or therapeutic agent may, for example, be a partially or fully complementary strand of RNA, DNA, PNA or chimera. In a number of embodiments, the biologically active agent is a partially or fully complementary strand of guide RNA, siRNA, or any useful reagent for RNA interference or antisense methods. The biologically-active agent may be a recombinant genetic construct for expression of a gene and / or for introduction into, or modification of the genome of the target cell.

[0065] One or more therapeutic agents that may be complexed with the Treg EV compositions described herein include, without limitation, anti-inflammatories, such as, without limitation, NSAIDs (non-steroidal anti-inflammatory drugs) such as salicylic acid, indomethacin, sodium indomethacin trihydrate, salicylamide, naproxen, colchicine, fenoprofen, sulindac, diflunisal, diclofenac, indoprofen sodium salicylamide, anti-inflammatory cytokines, and anti-inflammatory proteins or steroidal anti-inflammatory agents); antibiotics; anticlotting factors such as heparin, Pebac, enoxaprin, aspirin, hirudin, plavix, bivalirudin, prasugrel, idraparinux, warfarin,coumadin, clopidogrel, PPACK, GGACK, tissue plasminogen activator, urokinase, and streptokinase; growth factors. Therapeutic agents include, without limitation: (1 ) immunosuppressants; glucocorticoids such as hydrocortisone, betamethisone, dexamethasone, flumethasone, isoflupredone, methylpred-nisolone, prednisone, prednisolone, and triamcinolone acetonide; (2) antiangiogenics such as fluorouracil, paclitaxel, doxorubicin, cisplatin, methotrexate, cyclophosphamide, etoposide, pegaptanib, lucentis, tryptophanyl-tRNA synthetase, retaane, CA4P, AdPEDF, VEGF- TRAP-EYE, AG-103958, Avastin, JSM6427, TG100801 , ATG3, OT-551 , endostatin, thalidomide, becacizumab, neovastat; (3) antiproliferatives such as sirolimus, paclitaxel, perillyl alcohol, farnesyl transferase inhibitors, FPTIII, L744, antiproliferative factor, Van 10 / 4, 5-FU, Daunomycin, Mitomycin, dexamethasone, azathioprine, chlorambucil, methotrexate, mofetil, vasoactive intestinal polypeptide, and PACAP; (4) antibodies; (5) drugs acting on immunophilins, such as cyclosporine, zotarolimus, everolimus, tacrolimus and sirolimus (rapamycin), interferons, TNF binding proteins; (6) taxanes, such as docetaxel; statins, such as atorvastatin, lovastatin, simvastatin, pravastatin, fluvastatin and rosuvastatin; (7) nitric oxide donors or precursors, such as, without limitation, Angeli's Salt, L-Arginine, Free Base, Diethylamine NONOate, Diethylamine NONOate / AM, Glyco-SNAP-1 , Glyco-SNAP-2, S-Nitroso-N- acetylpenicillamine, S-Nitrosoglutathione, NOC-5, NOC-7, NOC-9, NOC-12, NOC-18, NOR-1 , NOR-3, SIN-1 , Sodium Nitroprusside, Dihydrate, Spermine NONOate, Streptozotocin; and (8) antibiotics, such as, without limitation: acyclovir, afloxacin, ampicillin, amphotericin B, atovaquone, azithromycin, ciprofloxacin, clarithromycin, clindamycin, clofazimine, dapsone, diclazaril, doxycycline, erythromycin, ethambutol, fluconazole, fluoroquinolones, foscarnet, ganciclovir, gentamicin, iatroconazole, isoniazid, ketoconazole, levofloxacin, lincomycin, miconazole, neomycin, norfloxacin, ofloxacin, paromomycin, penicillin, pentamidine, polymixin B, pyrazinamide, pyrimethamine, rifabutin, rifampin, sparfloxacin, streptomycin, sulfadiazine, tetracycline, tobramycin, trifluorouridine, trimethoprim sulphate, Zn-pyrithione, ciprofloxacin, norfloxacin, afloxacin, levofloxacin, gentamicin, tobramycin, neomycin, erythromycin, trimethoprim sulphate, polymixin B and silver salts such as chloride, bromide, iodide and periodate.

[0066] Any useful cytokine or chemoattractant can be associated with any composition as described herein. For example and without limitation, useful components include growth factors, interferons, interleukins, chemokines, monokines, hormones, andangiogenic factors. In certain non-limiting aspects, the therapeutic agent is a growth factor, such as a neurotrophic or angiogenic factor, which optionally may be prepared using recombinant techniques. Non-limiting examples of growth factors include basic fibroblast growth factor (bFGF), acidic fibroblast growth factor (aFGF), vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), insulin-like growth factors 1 and 2 (IGF-1 and IGF-2), platelet derived growth factor (PDGF), stromal derived factor 1 alpha (SDF-1 alpha), nerve growth factor (NGF), ciliary neurotrophic factor (CNTF), neurotrophin-3, neurotrophin-4, neurotrophin-5, pleiotrophin protein (neurite growth-promoting factor 1 ), midkine protein (neurite growth-promoting factor 2), brain-derived neurotrophic factor (BDNF), tumor angiogenesis factor (TAF),corticotrophin releasing factor (CRF), transforming growth factors a and [3 (TGF- a and TGF-[3), interleukin-8 (IL-8), granulocyte-macrophage colony stimulating factor (GM-CSF), interleukins, and interferons. Commercial preparations of various growth factors, including neurotrophic and angiogenic factors, are available from R & D Systems, Minneapolis, Minnesota; Biovision, Inc., Mountain View, California; ProSpec-Tany TechnoGene Ltd., Rehovot, Israel; and Cell Sciences®, Canton, Massachusetts.

[0067] The therapeutic agent may be an angiogenic therapeutic agent, such as: erythropoietin (EPO), basic fibroblast growth factor (bFGF), acidic fibroblast growth factor (aFGF), fibroblast growth factor-2 (FGF-2), granulocyte colony stimulating factor (G-CSF), granulocyte macrophage colony stimulating factor (GM-CSF), hepatocyte growth factor (HGF), insulin-like growth factors 1 and 2 (IGF-1 and IGF-2), placental growth factor (PIGF), platelet derived growth factor (PDGF), stromal derived factor 1 alpha (SDF-1 alpha), vascular endothelial growth factor (VEGF), angiopoietins (Ang 1 and Ang 2), matrix metalloproteinase (MMP), delta-like ligand 4 (DIM), and class 3 semaphorins (SEMA3s), all of which are broadly-known, and are available from commercial sources.

[0068] The therapeutic agent may be an antimicrobial agent, such as, without limitation, isoniazid, ethambutol, pyrazinamide, streptomycin, clofazimine, rifabutin, fluoroquinolones, ofloxacin, sparfloxacin, rifampin, azithromycin, clarithromycin, dapsone, tetracycline, erythromycin, ciprofloxacin, doxycycline, ampicillin, amphotericin B, ketoconazole, fluconazole, pyrimethamine, sulfadiazine, clindamycin, lincomycin, pentamidine, atovaquone, paromomycin, diclazaril, acyclovir, trifluorouridine, foscarnet, penicillin, gentamicin, ganciclovir, iatroconazole,miconazole, Zn-pyrithione, and silver salts such as chloride, bromide, iodide and periodate.

[0069] The therapeutic agent may be an anti-inflammatory agent, such as, without limitation, an NSAID, such as salicylic acid, indomethacin, sodium indomethacin trihydrate, salicylamide, naproxen, colchicine, fenoprofen, sulindac, diflunisal, diclofenac, indoprofen, sodium salicylamide; an anti-inflammatory cytokine; an antiinflammatory protein; a steroidal anti-inflammatory agent; or an anti-clotting agents, such as heparin.

[0070] Extracellular vesicles may be loaded with an appropriate, e.g., and effective amount of a therapeutic agent in any manner, such as by absorption or adsorption. Protein or nucleic acid therapeutic agents, such as biological drugs, therapeutic RNAs, or genetic constructs containing a gene for expression in a target cell or organism, may be produced in cell culture, e.g., in recombinant cells expressing a gene or producing an mRNA, or a recombinant viral genome, and extracellular vesicles produced by the cells including the therapeutic agent may be used in the methods and tethered extracellular vesicles as described herein.

[0071] Provided herein is a particle comprising: a Treg extracellular vesicle (EV); and an anti-TAG aptamer linked (covalently) to a lipophilic anchor embedded in a phospholipid bilayer of the EV, wherein the anti-TAG aptamer binds a functional protein linked to an affinity tag (“TAG”, as used herein). The lipophilic anchor may be a cholesterol anchor. An anti-TAG aptamer is an aptamer that is a binding partner with a protein tag such as a His tag (polyhistidine tag), which is a contiguous sequence of His amino acid residues, typically attached to a C-terminus or N-terminus of a protein. His tags often have from 6-9 contiguous His residues. An Fc-tag comprises an immunoglobulin Fc sequence (e.g., lgG1 ) typically attached to a C-terminus of a protein. The Fc tag may have any sequence and length so long as the tag can be recognized by an aptamer binding partner, but for example may comprise the constant region (domain 3 and 4) of immunoglobulin heavy-chain. Other affinity tags that may be attached to a functional protein, e.g. CTLA4, may include streptavidin (see, e.g., Srisawat C, et al. Streptavidin aptamers: affinity tags for the study of RNAs and ribonucleoproteins. RNA. 2001 Apr;7(4):632-41 ), Flag tag peptide (see, e.g., Einhauer A., et al. (2001 ). The FLAG peptide, a versatile fusion tag for the purification of recombinant proteins. J. Biochem. Biophys. Methods 49 455-65), Glutathione-S- transferase (see, e.g., Smith D. B., Johnson K. S. (1988). Single-step purification ofpolypeptides expressed in Escherichia coli as fusions with glutathione-S-transferase. Gene 6731-40) or other tags (see, e.g., Mishra V. Affinity Tags for Protein Purification. Curr Protein Pept Sci. 2020;21 (8):821 -830). In one embodiment, the anti-TAG aptamer comprises an anti-Fc aptamer or an anti-HIS aptamer which are well- described and are also commercially-available.

[0072] An exemplary Anti-Fc aptamer sequence, as used in the EXAMPLES below, is provided below (see, e.g., Ge YANG, et al. (2020) Screening of Aptamer for Human IgG Fc Fragment by Capillary Electrophoresis-Systematic Evolution of Ligands by Exponential Enrichment, Chinese Journal of Analytical Chemistry, 48(5): 601 -607; ISSN 1872-2040):5’-GCCACATTAGTCTCACCACTACCTGCGTACCTACCGCCGCTTT-3’ (SEQ ID NO: 1 )

[0073] An exemplary Anti-HIS aptamer sequence, as used in the EXAMPLES below, is provided below (see, e.g., Filip Bartnicki, et al. (2015) Imidazole-free purification of His3-tagged recombinant proteins using ssDNA aptamer-based affinity chromatography, Journal of Chromatography A, Volume 1418:130-139; ISSN 0021 - 9673):5’-GTTTGCCGGTGGGCAGGTTTAGGGTCTGCTCGGGATTGCGGAGGAAC ATGCGTCGCAAACTTT-3’ (SEQ ID NO: 2).

[0074] The particle may comprise a functional protein linked (covalently) to the affinity tag binding partner of the anti-TAG aptamer, bound to the anti-TAG aptamer. The functional protein may be a ligand for binding a membrane-bound protein of a target cell. In the context of the particles provided herein, the functional protein linked to the affinity tag may be a CTLA4 protein that binds cell membrane-bound B7 protein (e.g., B7-1 (CD80) and / or B7-2 (CD86)), for example in a human cell. The particle may be bound to a B7 membrane protein of a target cell via the CTLA4 protein. The target cell may be a T-cell, a B-cell, an APC, a dendritic cell, or a macrophage. In examples, the Treg EV may be obtained from a human Treg cell. For use in patients, such as human patients, the particle may be formulated into a composition comprising the Treg EV particle and a pharmaceutically-acceptable excipient.

[0075] A method of preparing a modified Treg EV particle also is provided. The constituents of the particle may be as described in the context of the particle described above. The method comprises contacting a Treg EV particle with an anti-TAG aptamer linked to a lipophilic membrane anchor to produce an anti-TAG aptamer-modified TregEV particle, essentially as described above. The anti-TAG-modified Treg EV particle is contacted with a functional protein linked to a tag binding partner for binding to the anti-TAG aptamer of the anti-TAG aptamer-modified Treg EV particle.

[0076] In use, the described modified Treg EV particle comprising affinity tag-bound CTLA4 protein may be used to modify, e.g., reduce, an immune response, and is therefore useful in circumstances where immunosuppression is desirable, including, without limitation in treatment of transplantation patients for prevention or treatment of graft rejection, in patients with inflammation or an inflammatory disease (including diseases that have an inflammation component), in patients with autoimmune disease, and in cancer patients. In an immunosuppression method, a composition comprising the described Treg EV particle comprising affinity tag-bound CTLA4 protein and a pharmaceutically-acceptable excipient is administered to a patient in an amount and by a route effective to treat the disease in the patient, e.g., to reduce an immune response in a patient. Similarly, a method of down-regulating an immune response, comprising contacting a T-cell, a B-cell, an APC, a dendritic cell, or a macrophage with the described Treg EV particle comprising affinity tag-bound CTLA4 protein is provided. The method may be conducted in vitro or in vivo. A method also is provided for delivering a Treg EV particle to an immune cell expressing membrane B7 protein, comprising contacting the cell with the described Treg EV particle comprising affinity tag-bound CTLA4 protein, such that the cell internalizes the Treg EV particle (e.g., by endocytosis or trogocytosis).EXAMPLES

[0077] The research goal was to translate an oligonucleotide tethering approach to functionalize the surface of Treg extracellular vesicles (EVs). First, representative experiments were conducted to demonstrate successful integration of oligonucleotide tethers into Treg EVs (essentially based on Yemeni SS, et al. Rapid On-Demand Extracellular Vesicle Augmentation with Versatile Oligonucleotide Tethers. ACS Nano. 2019 Sep 24; 13(9): 10555-10565 and US 2022 / 0145291 A1 ). Tethering of DNA via a conjugated cholesterol moiety to the EV lipid bilayer was demonstrated. The distal end of the tether was functionalized with either a fluorescent dye (Cy3) or an oligonucleotide aptamer. The Chol-DNA-Cy3 tether demonstrated that we could load DNA tethers in a concentration dependent manner with these new EV. For the aptamer tethers, two types of aptamers were used, one to His, with binding affinity to His tag toimmobilize recombinant His-CTLA4, and the second with binding affinity to Fc tag to immobilize recombinant Fc-CTLA4. This approach preserves the native cell-EV binding interactions as assessed using on-bead flow cytometry and fluorescence microscopy-based cell uptake studies. This method is shown to be useful in readily functionalizing Treg EV membranes to carry bioactive cargo and demonstrates how this method can be used controllably immobilize CTLA4, a membrane surface active protein, onto the Treg EV surface. Better immobilization was demonstrated using the His aptamer rather than the Fc aptamer, therefore efforts were primarily focused on the His aptamer. Treg EVs with His-CTLA4 exhibited far better cell uptake then nonmodified Treg EVs in representative immune cells, macrophages (J7774 murine cell line) and T-cells (human Jurket cell line). Rationale for engineering CTLA4 onto Treg EVs is presented in FIG. 1.

[0078] DNA tethers for EVs: A 22-mer DNA tether with a 5’- tetra(ethylene glycol) (TEG) spacer and cholesterol modification (Chol-DNA) was used at varied concentrations to treat Treg EVs. This was accomplished by simply vortexing the EVs with Chol-DNA in buffered solution (PBS) at ambient room temperature for five minutes. Analysis by flow cytometry using a tether consisting of a Cy3 labeled DNA complimentary to the DNA-cholesterol showed a proportional increase in Cy3 signal with increasing tether concentration from 1 pm to 10 pm (Representative data presented in FIG. 2).

[0079] Functionalization EVs with aptamers specific for CTLA4: Chol-DNA tethers were altered using aptamers to either His or Fc with the intent of binding to recombinant His tagged or Fc tagged recombinant proteins, respectively. The aptamer sequence was directly displayed on the EVs using a ‘tail’ sequence on the Chol-DNA strand. These aptamer sequences were based on literature reports on sequences that were selected to bind with high affinity (Kd in the nM range) to either His-tags or human IgFc ( Chromatography A 2015; 1218:130-139 and the Chinese J Anal Chem 2020; 48:601 -607, respectively). In order to test the functionality of the aptamer tails on the EVs, His-CTLA4 or Fc-CTLA4 protein were fluorescently labeled (Dylight 650), then incubated with EVs with surface immobilized either Chol-DNA-His Aptamer or Chol- DNA-Fc Aptamer. After a 100 kD filtration to wash out unbound CTLA4, florescent readings were taken to assess the relative protein binding. Anti-CD63 magnetic beads were then added to the EV and CTLA4 incubation mixtures and fluorescence wasseparately measured through flow cytometry. As concentration of EV immobilized Chol-DNA-His aptamer increased there was a corresponding increase in the fluorescence on the anti-CD63 beads, indicating the ability to alter EV surface CTLA4 concentration (representative data presented in FIG. 3A). His-CTLA4 was observed binding to EVs indirectly without the His Aptamer (FIG. 3B) suggesting that CTLA4 can bind directly to Treg EVs. It is unclear at this time whether there are B7 receptors on available on the EV surface to mediate binding or if the binding is non-specific. Lastly, due to the higher specificity of the His aptamer for His-CTLA4 then the Fc aptamer for Fc-CTLA4, primary efforts were focused on going forward on the His aptamer.

[0080] Functional assessment of CTLA4 modified Treg EVs. Internalization into macrophages: Cell uptake of EVs as determined by exogenous surface CTLA4 was considered for both His-CTLA4 and Fc-CTLA4. Either Chol-DNA-HisApt-CTLA4, Chol- DNA-FcApt-CTLA4 or Chol-DNA-Cy3 were loaded onto DiD fluorescently-labeled Treg EVs. These EVs were then incubated with murine J774 macrophage cells for 2 hr at 37 °C. Cells were then rinsed 2 times with ice cold PBS, then incubated with acid stripping buffer (500 mM NaCI and 0.5% acetic acid in DI water, pH 3) for 1 min, then cells fixed in 4% paraformaldehyde at room temperature for 20 min. Cells were permeabilized with 0.1 % Triton-X for 1 min then nuclei stained with 1 :1000 DAPI in PBS for 5 min. Following rinsing imaging of internalized EVs was performed using EVOS fluorescence microscope. Representative data is presented in FIG 4 (A). FIG. 4 (B and C) demonstrate that the His Aptamer is required for CTLA4 internalization although CTLA4 can bind to EVs directly as shown in FIG. 3B. Additional internalization experiments were performed demonstrating dose dependent increase in EV internalization as a function of amount of surface CTLA4.

[0081] Functional assessment of CTLA4 modified Treg EVs. Internalization into T cells: Similar to macrophages the effect of immobilized EV surface CTLA4 on the internalization of Treg EVs into human Jurkat T cells was determined. Cell uptake Treg EVs were assessed as described for macrophages detailed immediately above. FIG. 5A represents two separate experiments evaluating the dose dependency of EV surface CTLA4 to impact EV internalization. FIG. 5B represents the quantification of the imaging data using Image J. As the concentration of Chol-DNA-HisApt tethers increased, resulting in increased His-CTLA4 immobilized on the EVs surface, there was an increase in internalized EVs. However, at the highest concentration, 2 pM ofChol-DNA-HisApt integrated into the EV surface, there was a drastic decrease in internalization. This could have been caused by an EV surface saturation of CTLA4- His protein. A crowding of protein displayed by the EVs may lead to increased difficulty for any single protein to interact with the Treg cell.

[0082] In FIG. 6, providing a schematic, cholesterol-oligonucleotide (Chol-DNA) tethers are used to display CTLA4 protein through an aptamer - either a His-Tag aptamer or Fc-tag aptamer sequence on the DNA, on the surface of Treg extracellular vesicles (EVs) as an example. The CTLA4 protein that is displayed on the Treg EV surface is bioactive and bio-functional.

[0083] Data is provided demonstrating successful Chol-DNA tether immobilization on the Treg EV surface (FIG. 2B), the ability of complementary DNA with a terminal His binding aptamer to both localize His-CTLA4 to the Treg EV surface (FIG. 4C) but also demonstrates that CTLA4-Treg EVs have an -350% increase in Treg EV uptake into T cells in comparison to Treg EVs alone (FIGS. 5Aand 5B). In sum, it is demonstrated that one can successfully incorporate oligonucleotide tethers containing aptamers for His or Fc tagged proteins. It was determined that tethers consisting of Chol-DNA-His Aptamers had the highest affinity for His-CTLA4, and that Treg EVs with aptamer bound CTLA4 resulted in cell binding and internalization into both macrophages and T cells. These data suggest that the tethering of Treg EV surface CTLA4 has the potential to bind to immune cells and to deliver associated EV cargo to the target cell cytoplasm. Future work may include biodistribution and blood cell targeting studies of CTLA4 engineered onto Treg EVs.

[0084] While the present invention is described with reference to several distinct embodiments, those skilled in the art may make modifications and alterations without departing from the scope and spirit. Accordingly, the above detailed description is intended to be illustrative rather than restrictive.

Claims

CLAIMS:1 . A particle comprising: a Treg extracellular vesicle (EV); and an anti-affinity tag (anti-TAG) aptamer linked to a lipophilic anchor embedded in a phospholipid bilayer of the EV, wherein the anti- TAG aptamer binds a functional protein linked to an affinity tag binding partner to the anti-TAG aptamer.

2. The particle of claim 1 , wherein the anti-TAG aptamer comprises an anti-Fc aptamer, e.g., SEQ ID NO: 1 , or an anti-HIS aptamer, e.g., SEQ ID NO: 2.

3. The particle of claim 1 or claim 2, wherein the particle further comprises a functional protein linked to the affinity tag binding partner to the anti-TAG aptamer, bound to the anti-TAG aptamer.

4. The particle of claim 3, wherein the functional protein is a ligand for binding a membrane-bound protein of a target cell.

5. The particle of claim 4, wherein the functional protein linked to the affinity tag is a CTLA4 protein that binds cell membrane-bound B7 protein.

6. The particle of claim 4 or 5, wherein the target cell is a human cell.

7. The particle of any one of claims 1 -6, bound to a B7 membrane protein of a target cell via the CTLA4 protein.

8. The particle of claim 7, wherein the target cell is a T-cell, a B-cell, an APC, a dendritic cell, or a macrophage.

9. The particle of any one of claims 1 -8, wherein the Treg EV is obtained from a human Treg cell.

10. The particle of any one of claims 1 -9, wherein the lipophilic anchor comprises a cholesterol moiety.

11. A composition comprising the particle of any one of claims 1 -10 and a pharmaceutically-acceptable excipient.

12. A method of preparing a modified Treg EV particle, comprising contacting a Treg EV particle with an anti-affinity tag (anti-TAG) aptamer linked to a lipophilic membrane anchor to produce an anti-TAG aptamer-modified Treg EV particle, and contacting the anti-TAG-modified Treg EV particle with a functional protein linked to an affinity tag binding partner to the anti-TAG aptamer, thereby binding the functional protein via the affinity tag to the anti-TAG aptamer of the anti-TAG aptamer-modified Treg EV particle.

13. The method of claim 12, wherein the anti-TAG aptamer comprises an anti-Fc aptamer or an anti-HIS aptamer.

14. The method of claim 12 or 13, wherein the functional protein is a ligand for binding a membrane-bound protein of a target cell.

15. The method of claim 14, wherein the functional protein linked to the affinity tag is a CTLA4 protein that binds cell membrane-bound B7 protein (e.g., B7-1 (CD80) and / or B7-2 (CD86)).

16. The method of claim 14 or 15, wherein the target cell is a human cell.

17. The method of any one of claims 12-16, wherein the Treg EV is obtained from a human Treg cell.

18. The method of any one of claims 12-17, wherein the lipophilic anchor comprises a cholesterol moiety.

19. The method of any one of claims 12-18, further comprising mixing the modified Treg EV particle with a pharmaceutically-acceptable excipient.

20. A method of reducing an immune response in a patient, comprising contacting a cell expressing membrane B7 protein, such as an immune cell, e.g., a T-cell, a B-cell, an APC, a dendritic cell, or a macrophage, of the patient with the particle of claim 5, such that the EV binds a B7 membrane protein of the cell of the patient to reduce the immune response in the patient.21 . The method of claim 20, wherein the patient is a transplantation patient, a cancer patient, an autoimmune disease patient, or an inflammatory disease patient.

22. A method of delivering a Treg EV particle to a cell expressing membrane B7 protein, such as an immune cell, e.g., a T-cell, a B-cell, an APC, a dendritic cell, or a macrophage, comprising contacting the cell with the particle of claim 5, such that the cell internalizes the Treg EV particle (e.g., by endocytosis or trogocytosis).

23. The method of claim 22, wherein the cell is a cell of a transplantation patient, a cancer patient, an autoimmune disease patient, or an inflammatory disease patient.

Images