Extracellular vesicles for delivering payloads to eukaryotic cells

Abstract

This invention relates to the production, modification, and use of extracellular vesicles (EVs), particularly for the delivery of nucleic acid payloads. EVs contain retroviral capsids, which can be coated with polymers. EVs can be treated with proteases to remove proteins from their outer surface, thereby producing shaved EVs. Shaved EVs can then be coated with polymers and used to deliver nucleic acid payloads to cells.

Description

[Background technology]

[0001] Extracellular vesicles (EVs) are vesicles secreted by many different types of cells. See, for example, Du et al., Pharmaceuticals 2022, 14:2236. EVs are phospholipid-based bilayer particles. EVs can be endosome-derived ("exosomes") and typically have a diameter of about 30 nm to 150 nm. Alternatively, EVs can be generated by budding outside the membrane ("exosomes" or "microvesicles") and typically have a diameter of about 50 nm to 1000 nm. See, for example, Teng and Fussenegger, Advanced Science 2021, 8:2003505.

[0002] Cellular vein cells (EVs) function as natural media for transmitting information from one cell to another. Studies have shown that receptor cells can be modified by loading EVs with nucleic acids, proteins, lipids, and small molecules. See, for example, Joshi and Zuhorn, Materials Today Nano 2021, 100148. Therefore, EVs can be used as therapeutic tools for the clinical application of nucleic acid drugs. See, for example, Rezaie et al., Cell Communication and Signaling 2022, 20:145.

[0003] Similarly, retroviruses are phospholipid-based bilayer particles. Retroviruses acquire host-derived lipid membranes when passing through the cell membrane. See, for example, Rheinemann et al., Encyclopedia of Virology, 4th Edition, 2021 Elsevier Ltd., Volume 1, 519-528. All retroviruses, including lentiviruses, express Gag polyproteins that regulate virion assembly. See, for example, Coffin et al., Retroviruses, Cold Spring Harbor Laboratory Press, 1997. When expressed alone, Gag polyproteins are released into virus-like particles. This indicates that Gag polyproteins are sufficient for virion assembly, envelope formation, and budding. See, for example, Bell and Lever, Trends in Microbiology 2013, 21:136-44.

[0004] Retroviral vectors, particularly lentiviral vectors, have been used to deliver payloads containing therapeutic molecules that produce therapeutic effects directly or indirectly to mammalian cells. See, for example, Dautzenberg et al., Gene Therapy 2021, 28:89-104. For example, they have been used to generate chimeric antigen receptor (CAR) T cells. See, for example, U.S. Patent No. 9,102,760. Virus-like particles (VLPs), formed by structural viral proteins that mimic the form of the original virus, are non-infectious, non-replicating, and lack genetic material, and have also been used as vectors. See, Rheinemann et al., Encyclopedia of Virology, 4th Edition, 2021 Elsevier Ltd., Volume 1, 519-528.

[0005] Delivering therapeutic molecules to cells holds great potential in treating diseases such as cancer. Novel compositions and methods are needed to increase the delivery of therapeutic molecules to cells. The applicant's invention meets this need. [Overview of the Initiative]

[0006] This invention encompasses extracellular vesicles (EVs) and methods for producing, modifying, and using them.

[0007] This invention includes extracellular proteins (EVs) that are treated with proteases to remove proteins from the outer surface of the EV.

[0008] In one embodiment, the EV does not have any full-length viral envelope protein on its surface. In one embodiment, the EV does not have any full-length CAR, non-retroviral protein, or non-lentiviral protein on its surface.

[0009] In one embodiment, the EV comprises a retroviral capsid containing a pair of RNAs encoding a non-retroviral protein or a non-lentiviral protein. In one embodiment, the RNAs encode a chimeric antigen receptor. In one embodiment, the capsid is an HIV-1 capsid. In one embodiment, the capsid contains a reverse transcriptase. In one embodiment, the capsid contains an integrase.

[0010] Polymeric nanoparticles (PNPs) can be formed by coating EVs with a polymer. In one embodiment, EVs are coated with a cationic polymer. In another embodiment, EVs are coated with an anionic polymer. In yet another embodiment, EVs are coated with a zwitterionic polymer. The polymers can be selected from polyarginine, polylysine, polyetherimide (PEI), chitosan, poly(β-aminoester) (PBAE), PEI-co-polyhistidine copolymer, polylysine dendrimer / dendrimer / branched polymer, star-shaped polyamino acid-based polymer, chondroitin sulfate, heparosan, polyglutamic acid (PGA), hyaluronic acid, dextran sulfate, polysarcosine, polyethylene glycol (PEG), polycarboxybetaine, polysulfobetaine, or polyphosphorylcholine.

[0011] In one embodiment, the protease is proteinase K.

[0012] In one embodiment, EV comprises a CD3 or NKG2D targeting agent.

[0013] In one embodiment, EVs are produced in a mammalian cell line. The present invention encompasses cells containing the EVs of the present invention.

[0014] The present invention encompasses a method comprising contacting the EV / PNP of the present invention with mammalian cells. In one embodiment, the cells are human cells. In one embodiment, the cells are T cells or NK cells.

[0015] The present invention encompasses methods for modifying the outer surface of extracellular proteins (EVs). In one embodiment, the method includes purifying EVs from cells, treating the EVs with a protease to remove proteins from the outer surface of the EVs, inactivating the protease, and purifying the treated EVs from the protease and the removed extracellular protein fragments. In one embodiment, the cells express retroviral Gag protein, Rev protein, and Pol protein. In one embodiment, the EVs contain RNA encoding a non-retroviral protein or a lentiviral protein. In one embodiment, the RNA encodes a chimeric antigen receptor. In one embodiment, the method includes contacting the modified EVs / PNPs with mammalian cells. [Brief explanation of the drawing]

[0016] [Figure 1] A-C show the results of direct ELISA detection of human CD9 (A), human CD63 (B), and human CD81 (C) surface receptors on two unshaved EV batches produced in HEK293-derived cells. [Figure 2] Figures A-C show the results of simultaneous detection of human CD81 / CD9 (A), human CD63 / CD9 (B), and human CD63 / CD81 (C) surface receptors on an unshaved EV batch produced in HEK293-derived cells using the sandwich ELISA method. [Figure 3] Figures A-C show the results of sandwich ELISA performed on untreated EV and LV batches produced in HEK293-derived cells by A) titrating EV / LV using a CD81 / CD9 antibody pair, B) titrating EV / LV using a CD63 / CD9 antibody pair, and C) titrating EV / LV using a CD63 / CD81 antibody pair. [Figure 4]This shows the expression levels of human receptors expressed on the surface of unshaved EV and LV batches produced in HEK293-derived cells. Expression levels were measured by flow cytometry using the MACSplex exosome kit. [Figure 5] The results of measuring the zeta potentials of unshaved EV and LV batches produced in HEK293-derived cells using MADLS (Multi-Angle Dynamic Light Scattering) are shown. [Figure 6] This shows the particle size (Dh), measured by MADLS (Multi-Angle Dynamic Light Scattering), and the average hydrodynamic diameter of aggregates detected above 200 nm (Dh aggregates) for unshaved LV batches produced in HEK293-derived cells. [Figure 7] The mean hydrodynamic diameter (an indicator of particle size) and polydispersion index (PdI, an indicator of size distribution) measured by MADLS (Multi-Angle Dynamic Light Scattering) for an unshaved EV batch produced in HEK293-derived cells are shown. [Figure 8] A and B show the size distribution of unshaved LV(A) and EV(B) batches produced in HEK293-derived cells, measured by MADLS (Multi-Angle Dynamic Light Scattering). [Figure 9] A-D show the time course (up to 14 days) of GFP expression in cell lines transduced from untreated LV at MOI 1-30. Quantification was performed by flow cytometry, and A) shows the GFP expression rate (%) expressed by HEK293 cells, B) the GFP expression rate (%) expressed by Jurkat cells, C) the average fluorescence intensity of GFP on transduced HEK293 cells, and D) the average fluorescence intensity of GFP on transduced Jurkat cells. [Figure 10] This document outlines general workflows for the upstream and downstream processing of extracellular viable cells (EVs) and pulmonary leukocytes (LVs) produced within HEK293-derived cells. [Figure 11] Figures A and B show the solubility of cationic polymers in water, 100 mM acetate buffer (pH 5), or acetic acid when dissolved at a concentration of 5 mg / mL and analyzed by dynamic light scattering. Figure A) shows the number of particles in the solution, reflecting the number of nanoparticles resulting from polymer self-assembly, while Figure B) shows the measured size of the polymer self-assemblies. The gray areas correspond to the assumed sizes of polymer-coated or uncoated EVs or LVs, and therefore to exclusion areas for polymers that readily form self-assemblies in solution. [Figure 12] The size difference of 3.6 × 10⁹ EV particles produced in HEK293-derived cells was measured by dynamic light scattering after being treated with proteinase K (PK) at an elevated PK concentration for 10 minutes at 25°C. [Figure 13] Figures A-C show the detection of VSV-G protein on unshaved LV and EV batches produced in HEK293-derived cells by direct ELISA. Titration was performed using A) anti-VSVG neutralizing monoclonal antibody 8G5F11, B) anti-VSVG monoclonal antibody 1E9F9, and C) anti-VSVG polyclonal antibody. [Figure 14] A-D show the detection of tetraspanin and VSV-G proteins on unshaved LV and EV batches produced in HEK293-derived cells using sandwich ELISA. A) Captured with anti-VSVG 8G5F11 and detected with anti-human CD9, B) Captured with anti-VSVG 8G5F11 and detected with anti-human CD81, C) Captured with anti-VSVG 1E9F9 and detected with anti-human CD9, and D) Captured with anti-VSVG 1E9F9 and detected with anti-human CD9. [Figure 15]This report presents the characterization of shaved and unshaved LV produced in HEK293-derived cells using a sandwich ELISA method. Shaving was performed by 15 minutes of proteinase K at 10, 500, 1000, and 5000 nM, followed by 25 minutes of treatment with 20 mM AEBSF. Data are normalized to the signal under unshaved conditions (dotted line and 0 nMPK conditions). A) Captured with anti-human CD63 and detected with anti-human CD81, B) Captured with anti-human CD81 and detected with anti-human CD9, C) Captured with anti-VSVG 8G5F11 and detected with anti-human CD9, D) Captured with anti-VSVG 1E9F9 and detected with anti-human CD9, E) Captured with anti-VSVG 8G5F11 and detected with anti-human CD81, and F) Captured with anti-VSVG 1E9F11 and detected with anti-human CD81. [Figure 16] A and B show the transduction efficiency of shaved and unshaved LV in Jurkat cells. Shaving was performed with 10, 500, 1000, and 5000 nM proteinase K for 15 minutes, followed by treatment with proteinase K in 20 m of MAEBSF for 25 minutes. Shaved and unshaved LV samples were transduced into Jurkat cells at various particle concentrations, i.e., 1.6 × 10⁹ particles corresponding to MOI 5 (top panel) and 6.1 × 10⁹ particles corresponding to MOI 20 (bottom panel). GFP expression (A) was monitored by flow cytometry from day 3 to day 20 using the percentage of cells expressing GFP (%) and the mean fluorescence intensity (MFI) of GFP. Vector copy number (B) was quantified by qPCR on DNA extracted from transduced cells from day 3 to day 20. [Figure 17]A and B show the transduction efficiency of shaved and unshaved LV in activated primary T cells. Shaving was performed with 500 and 5000 nM proteinase K for 15 minutes, followed by treatment with 20 m MAEBSF for 25 minutes. Transduction was performed into activated human primary T cells using shaved and unshaved LV samples at various particle concentrations, i.e., 1.9 × 10⁸ particles (upper panel) corresponding to MOI 5, and 7.7 × 10⁸ particles (lower panel) corresponding to MOI 20. GFP expression was monitored from day 4 to day 14 post-transduction by flow cytometry, using the percentage of cells expressing GFP (%) (A) and the mean fluorescence intensity (MFI) of GFP. Vector copy number was quantified by qPCR on DNA extracted from cells transduced at MOI 5 on days 4 and 7 (upper panel, asterisks). [Figure 18] Figures A and B show the effect of formulation buffer on Jurkat cell viability and LV transduction. (A) is the percentage of viable Jurkat cells after 2 hours of incubation with formulation medium at pH 5 or pH 7.4 at 25%, 50%, 75%, 87.5%, or 100%. (B) is the GFP expression rate (%) after transduction of VSVG LV with 0%, 1%, 50%, and 75% formulation buffer. P: phosphate, C: citrate, T: tris, A: acetate, S: succinate, H: histidine, OS: sucrose and NaCl. [Figure 19] The size difference of 5.9 × 10⁹ LV particles produced in HEK293-derived cells was measured by dynamic light scattering after treatment with increasing concentrations of proteinase K (PK) at 25°C for 5 minutes. Proteinase K inhibition with a final concentration of 20 mM AEBSF was performed for 25 minutes. [Figure 20]Panels A-D show the size distribution of unshaved LV, shaved LV, or shaved and filtered LV produced in HEK293-derived cells, measured by MADLS (Multi-Angle Dynamic Light Scattering). Four independent shaving reactions (Batch 1: Panel A, Batch 2: Panel B, Batch 3: Panel C, Batch 4: Panel D) were performed on 1.43 × 10¹¹ LV particles, followed by a 15-minute incubation step with 5 μM proteinase K, and then inhibition with 20 mM AEBSF for 25 minutes. The reaction products were filtered sequentially through 0.45 μm and 0.22 μm PVDF membranes, or not filtered. [Figure 21] Panels A and B show the changes in zeta potential (Panel A) as a function of pH and (z-mean as a size scale - Panel B) for unshaved LV particles or shaved and filtered LV particles produced in HEK293-derived cells. After adjusting the pH of the virus particles by diluting them 10-fold in DPBS-sucrose, measurements were performed by MADLS (Multi-Angle Dynamic Light Scattering). [Figure 22] Panels A and B show the changes in hydrodynamic diameter (z-mean, a measure of size) and zeta potential of polymeric nanoparticles as a function of virus particle concentration (Panel A) or cationic polymer concentration (Panel B) for shaved and filtered LV particles produced in HEK293-derived cells. Virus particles and cationic polymers were diluted in 20 mM histidine, 50 mM sucrose, and 127 mM NaCl (pH 5) buffer. LV was added to the polymer, and a coating reaction was carried out at 25°C for 30 minutes, after which measurements were performed by MADLS (Multi-Angle Dynamic Light Scattering). [Figure 23]Panels A and B show the changes in hydrodynamic diameter (z-mean as a measure of size) (Panel A) and size distribution (PdI as a polydispersity index) (Panel B) of polymeric nanoparticles obtained from two different batches of shaved (using 5000 nM proteinase K) and filtered LV particles produced in HEK293-derived cells. The virus particles and cationic polymers were diluted in 20 mM histidine, 50 mM sucrose, and 127 mM NaCl (pH 5) buffer. LV was added to the polymer, and a coating reaction was carried out at 25°C for 30 minutes, after which measurements were performed by MADLS (Multi-Angle Dynamic Light Scattering). [Figure 24] Panels A and B show the size distribution (Panel A) and zeta potential changes (Panel B) of polymeric nanoparticles relative to shaved and filtered LV particles produced in HEK293-derived cells. Virus particles, H-PBAE (cationic polymer), and HA (anionic polymer) polymers were diluted in 20 mM M histidine, 50 mM sucrose, and 127 mM NaCl (pH 5) buffer. LV was added to the polymers and a coating reaction was carried out at 25°C for 30 minutes, after which measurements were performed using MADLS (Multi-Angle Dynamic Light Scattering). Nanoparticles obtained in the first coating step were added to the anionic HA polymer and a coating reaction was carried out at 25°C for 30 minutes, after which measurements were performed using MADLS. [Figure 25]Panels A-D show the changes in hydrodynamic diameter (z-mean, a measure of size) (Panels A and C) and size distribution (Panels B and D) of polymeric nanoparticles relative to shaved and filtered LV particles produced in HEK293-derived cells. Virus particles, cationic polymers (PBAE-447, H-PBAE, and R-PBAE), and anionic polymers (heparosan and HA) were diluted in 20 mM histidine, 50 mM sucrose, and 127 mM NaCl (pH 5) buffer. LV was added to the polymers and a coating reaction was carried out at 25°C for 30 minutes, after which measurements were performed by MADLS (Multi-Angle Dynamic Light Scattering). Nanoparticles obtained after the first coating step were added to the heparosan or HA anionic polymers and a coating reaction was carried out at 25°C for 30 minutes, after which measurements were performed by MADLS. [Figure 26] Figures A and B show the detection of CD19-CAR and tetraspanin proteins by sandwich ELISA in unshaved LV (Batch 1) cells (Vitamins) encoding CD19-CAR or GFP produced in HEK293-derived cells. A) Captured with anti-human CD63 or CD81 and detected with recombinant CD19 protein or anti-FMC63 idiotype antibody; B) Captured with recombinant CD19 protein and detected with anti-human CD81 or CD9 antibody. [Modes for carrying out the invention]

[0017] This invention encompasses electric vehicles (EVs) and methods for manufacturing, modifying, and using them. Before disclosing and describing the compositions, methods, and other embodiments of this invention, it should be understood that the terms used herein are intended solely to describe specific embodiments and are not intended to limit them.

[0018] Note that, as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural nouns unless the context clearly indicates otherwise.

[0019] As used herein, the term “comprising” is synonymous with “including” or “containing,” and is comprehensive or open-ended and does not exclude additional, undescribed aggregate elements, elements, or method steps.

[0020] As used herein, the term "in vitro" refers to events that occur in an artificial environment, such as a test tube, reaction vessel, or cell culture medium, rather than in a living organism.

[0021] As used herein, the term "in vivo" refers to events that occur within a living organism.

[0022] As used herein, the term "exvivo" refers to an event that occurs outside of a living organism using material directly taken from a living organism.

[0023] As used herein, the terms “contact” or “bring into contact” refer to placing two compounds or compositions (e.g., cells and extracellular vesicles) together without a physical barrier between them (e.g., in a solution in the same tube).

[0024] As used herein, the term "purified" refers to PNP (polymeric nanoparticles), EV, LV, capsid, or other compositions or molecules (e.g., proteins) that are separated from some of the other constituent substances related in the mixture. For example, "purified" EV can be one separated from the cells producing EV or one separated from the protease that has processed EV. Similarly, "purified" capsid can be one separated from cell lysates or one separated from the lipid membrane components outside retrovirus particles or lentivirus particles. The degree of purification is based on the degree of removal of other components. Thus, the purity can range from 50% or less to 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or more. For example, 50% purified EV can refer to one from which 50% of the processed protease has been removed.

[0025] Extracellular vesicles (EV) In various embodiments, the present invention includes EV and methods for producing and using EV.

[0026] The present invention includes 1, 10, 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , 10 14 , 10 15 , etc. It includes EV or PNP, or any range between these amounts (e.g., 10 5 ~ 10 8 EV or PNP).

[0027] The present invention includes 10 2 / ml, 10 3 / ml, 10 4 / ml, 10 5 / ml, 10 6 / ml, 107 / ml, 10 8 / ml, 10 9 / ml, 10 10 / ml, 10 11 / ml, 10 12 / ml, 10 13 / ml, 10 14 / ml, 10 15 The concentration of EV or PNP in units such as / ml, or any range between these amounts (e.g., 10 9 / ml~10 12 Includes / ml)

[0028] In some embodiments, the extracellular matrix (EV) is endosome-derived ("exosome") having a diameter of about 30 nm to about 150 nm. Preferably, the EV is generated by budding outside the membrane ("exosome" or "microvesicle") and has a diameter of about 50 nm to about 1000 nm.

[0029] In some embodiments, EVs are exosomes with a size of approximately 100 nm to approximately 300 nm. In one embodiment, EVs are exosomes with a size of approximately 150 nm.

[0030] Preferably, the EV batch contains less than 600 ng / mL of residual DNA and less than 15 μg / mL of host cell protein.

[0031] Extracellular vesicles (EVs) can be generated from most types of extracellular cells. Preferably, EVs are generated from eukaryotic cells. Examples of eukaryotic cells include those from yeast, birds, and mammals such as chickens, mice, primates, monkeys, humans, hamsters, and rats.

[0032] In some embodiments, the eukaryotic cells are JURKAT, VERO, HELA, COS-7, CHO, HEK293, BHK-21, 293T, MDCK, BSC-1, NSO, PER.C6, VERO, CRL-7030, HS578BST, NIH 3T3, HEPG2, A549, DAUDI, R1.1, BW-5147, LM, BSC-40, YB / 20, P3X63Ag8.653, SP2 / 0-Ag14, or BMT10 cell lines. Many of these cell lines are readily available, for example, from the American Type Culture Collection (ATCC).

[0033] In some embodiments, the eukaryotic cells are of animal origin, such as from birds or mammals like chickens, mice, primates, monkeys, humans, hamsters, and rats.

[0034] In some embodiments, eukaryotic cells are stem cells, including fibroblasts, immune cells, or iPSCs.

[0035] In some embodiments, eukaryotic cells express viral proteins such as vesicular stomatitis virus (VSV), retroviruses, and measles virus proteins. These proteins include viral envelope proteins, particularly those used for pseudotyping vectors. These include the envelope protein of mouse leukemia virus (MULV), gibbon leukemia virus (GALV), feline endogenous RD114 retrovirus, Moloney's MULV 4070A, Moloney's MULV strain 10A1, the envelope glycoprotein of vesicular stomatitis virus (VSV-G protein), the glycoprotein of rabies virus, and measles virus hemagglutinin and fusion glycoproteins.

[0036] In some embodiments, eukaryotic cells express retroviral proteins. Examples of retroviral proteins include Gag protein, Pol protein, Rev protein, and Env protein. Further examples of retroviral proteins include capsid proteins, matrix proteins, nucleocapsid proteins, protease proteins, reverse transcriptases, and integrase proteins. Retroviral accessory proteins include Tat, Nef, Vif, Vpr, Vpu, and Vpx.

[0037] Cells can express one or more retroviral proteins transiently or stably. Preferably, retroviral Gag and Pol proteins are expressed by transient transfection of cells. Preferably, RNA containing a retroviral packaging sequence is expressed by transient transfection of cells.

[0038] In some embodiments, the EV contains retroviral envelope (e.g., lentiviral) proteins on its outer surface. In some embodiments, the EV contains CAR on its outer surface.

[0039] In some embodiments, the retrovirus is avian leukemia virus, Rous sarcoma virus (RSV), mouse mammary tumor virus (MMTV), mouse leukemia virus (MuLV), feline leukemia virus (FLV), bovine leukemia virus (BLV), or human T lymphotropic virus (HTLV).

[0040] In some embodiments, the retrovirus is a lentivirus (LV), such as human immunodeficiency virus type 1 (HIV-1), human immunodeficiency virus type 2 (HIV-2), simian immunodeficiency virus (SIV), or feline immunodeficiency virus (FIV).

[0041] In some embodiments, the EV of the present invention includes a payload for delivery to another cell. This payload may be a protein, nucleic acid, or small molecule. The protein or nucleic acid can be expressed in the cell from which the EV is generated, or introduced into the cell from which the EV is generated. In this way, the payload is associated with the EV at the time it is generated from the cell. Alternatively, the payload may be loaded onto the EV after purification. The payload may include one or more therapeutic proteins and nucleic acids encoding one or more therapeutic proteins.

[0042] In some embodiments, therapeutic proteins are, for example, interferons, interleukins, lymphokines, and tumor necrosis factors, such as cytokines or growth factors like IL-1, IL-2, IL-4, IL-6, IL-10, IL-11, IL-13, IL-17, IL-35, TNFα, IFN-α, IFN-β, IFN-γ, TGF-β, GM-CSF, M-CSF, G-CSF, EPO, BMP, EGF, FGF, and VEGF.

[0043] In some embodiments, the therapeutic protein is an antibody or other inactivating molecule, which includes programmed death 1 (PD-1), cytotoxic T lymphocyte antigen 4 (CTLA-4), LAG3, TIM3, BTLA, BY55, TIGIT, LAIR1, SIGLEC10, 2B4, PPP2CA, PPP2CB, PTPN6, PTPN22, CD96, CRTAM, SIGLEC7, SIGLEC9, TNFRSF10B, TNFRSF10A, The targets are CASP8, CASP10, CASP3, CASP6, CASP7, FADD, FAS, TGFBRII, TGFRBRI, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA, IL10RB, HMOX2, IL6R, IL6ST, EIF2AK4, CSK, PAG1, SIT1, FOXP3, PRDM1, BATF, GUCY1A2, GUCY1A3, GUCY1B2, and GUCY1B3.

[0044] Examples of such therapeutic proteins include chimeric antigen receptors and costimulatory receptors (CCRs).

[0045] Chimeric antigen receptors (CARs) include carbonic anhydrase IX (CAIX), carcinoembryonic antigen (CEA), CD8, CD7, CD10, CD19, CD20, CD22, CD30, CD33, CLL1, CD34, CD38, CD41, CD44, CD49f, CD56, CD74, CD117, CD133, CD138, CD123, CD44V6, antigens of cytomegalovirus (CMV) infected cells (e.g., cell surface antigens), epithelial glycoprotein-2 (EGP-2), epithelial glycoprotein-40 (EGP-40), epithelial cell adhesion molecule (EpCAM), and receptor tyrosine protein kinases erb-B2, 3, 4 (erb-B B2, 3, 4), folate-binding protein (FBP), fetal acetylcholine receptor (ACHR), folate receptor-a, FLT3, ganglioside G2 (GD2), ganglioside G3 (GD3), human epidermal growth factor receptor 2 (HER-2), human telomerase reverse transcriptase (HTERT), interleukin-13 receptor subunit alpha-2 (IL-13Ra2), K-light chain kinase insertion domain receptor (KDR), Lewis Y (LeY), L1 cell adhesion molecule (LICAM), melanoma antigen family A1 (MAGE-A1), mucin 16 (MUC16), mucin 1 (MUC1), mesothelin (MSLN), ERBB2, MAGE-A3, MAGE-A4, p53, MART1, GP100, Proteinase 3 (PR1), Tyrosinase, Sulbibin, hTERT, EphA2, NKG2D ligand, Cancer-testicular antigen NY-ESO-1, Fetal carcinoma antigen (h5T4), Prostate stem cell antigen (PSCA), Prostate-specific membrane antigen (PSMA), ROR1, Tumor-associated glycoprotein 72 (TAG-72), Vascular endothelial growth factor R2 (VEGF-R2), Wilms tumor protein (WT-1), BCMA, NKCS1, EGFIR, EGFR-VIII, CD99, CD70, ADGRE2, CCR1, LILRB2, PRAMEExamples include receptors for CCR4, CD5, CD3, TRBC1, TRBC2, TIM-3, integrin B7, ICAM-1, CD70, Tim3, CLEC12A, ERBB, B-cell activator receptor (BAFF-R), thymic stromal lymphocyte generating factor (TSLPR), SLAM family member 7 (SLAMF7), and G protein-coupled receptor family C group 5 member D (GPRC5D).

[0046] Examples of chimeric antigen receptors (CARs) include those for CD19, CD20, CD22, CD30, CD33, CD38, CD123, CD133, CD147, CD317, FLT3, BCMA, DR4, CS-1, CLL-1, ADGRE2, HER2, HSP70, VEGF, GD2, GD3, MUC1, MUC16, NKR2, NKG2D ligand, CYP1B1, SP17, PRAME, and WT1. The chimeric antigen receptor (CAR) is preferably CD19 (present in B-cell derived cancers such as acute lymphoblastic leukemia (ALL) and diffuse large B-cell lymphoma (DLBCL)), CD30 (present in refractory Hodgkin lymphoma), CD33, CD123, ADGRE2 and FLT3 (present in acute myeloid leukemia (AML)), and BCMA (present in multiple myeloma).

[0047] Preferably, the CAR includes an extracellular antigen-binding domain, a transmembrane domain, a hinge domain, a costimulatory signaling region, and an intracellular domain. Examples of chimeric antigen receptors (CARs) include first-generation, second-generation, and third-generation CARs. See US2020 / 0317777, which is incorporated herein by reference in its entirety.

[0048] First-generation CARs typically consist of an extracellular antigen-binding domain (e.g., scFv), which is fused to a transmembrane domain, which is further fused to a cytoplasmic / intracellular signaling domain. Second-generation CARs provide additional signaling to T cells by adding intracellular signaling domains derived from various co-stimulatory molecules (e.g., CD28, 4-1BB, ICOS, OX40, CD27, CD40 / My88, and NKG2D) to the cytoplasmic tail of the CAR. Third-generation CARs include those possessing multiple co-stimulatory (e.g., CD28 and 4-1BB) domains as well as an activation (e.g., CD3ζ) domain.

[0049] In some embodiments, the extracellular antigen-binding domain of the CAR includes human scFv, humanized scFv, mouse scFv, F(ab)2, VHH (camelid heavy chain), or a portion of the innate ligands of cell surface receptors.

[0050] In some embodiments, the transmembrane domain of CAR includes native or modified CD8, CD28, CD3ζ, CD40, 4-1BB, OX40, CD84, CD166, CD8α, CD8β, ICOS, ICAM-1, CTLA-4, CD27, CD40 / My88, NKG2D, synthetic polypeptides, or combinations thereof.

[0051] In some embodiments, the CAR includes a hinge region. In some embodiments, the hinge domain of the CAR includes CD8 polypeptide, CD28 polypeptide, CD3ζ polypeptide, CD40 polypeptide, 4-1BB polypeptide, OX40 polypeptide, CD84 polypeptide, CD166 polypeptide, CD8α polypeptide, CD8β polypeptide, ICOS polypeptide, ICAM-1 polypeptide, CTLA-4 polypeptide, CD27 polypeptide, CD40 / My88 peptide, NKG2D peptide synthetic polypeptide (not based on a protein related to the immune response), or a combination thereof. The hinge spacer region may be a hinge region derived from IgG1, or the CH2 and CH3 regions of an immunoglobulin, as well as a portion of CD3, a portion of the CD28 polypeptide.

[0052] In some embodiments, the CAR comprises one or more co-stimulatory signaling regions. In some embodiments, the co-stimulatory signaling region comprises at least one native or modified CD8, CD3ζ, CD27, CD28, CD40, 4-1BB, OX40, CD84, CD166, CD8α, CD8β, ICOS, ICAM-1, CTLA-4, CD40 / My88, NKG2D, synthetic polypeptide, or a combination thereof. Examples of co-stimulatory ligands include CD80, CD86, CD70, OX40L, and 4-1BB ligand.

[0053] In some embodiments, the intracellular domain of CAR includes a modified or unmodified CD3ζ polypeptide on the ITAM motif.

[0054] In some embodiments, the therapeutic protein is a combination of different types of therapeutic proteins.

[0055] In some embodiments, the payload is a gene editing system, such as a TALEN, ZFN, or CRISPR-based gene editing system.

[0056] Production method The present invention encompasses methods for producing extracellular viable cells (EVs). In some embodiments, EVs are prepared by isolating EVs from EV-producing cells ("producing cells") using techniques well known in the art. If the producing cells are adherent cells, the supernatant can be collected from the cells, or the cells can be removed from the plate or flask (e.g., using trypsin) and the producing cells removed by centrifugation or filtration.

[0057] If the producing cells are non-adherent cells, they can be removed by centrifugation or filtration. The supernatant can be subjected to centrifugation and / or filtration to remove all cells and / or other contaminants.

[0058] To increase EV production, specialized cell culture methods, including specially prepared culture flasks or 3D culture, can be used. See Du et al., Pharmaceuticals 2022, 14:2236, which is incorporated herein by reference. Ca2+-dependent regulatory induction, stimulation by stress culture conditions (hypoxia, low pH, electrical stimulation, liposome stimulation, acoustic treatment, and drug stimulation), and specialized EV separation methods (tangential flow filtration) can also be used to increase production.

[0059] EV can be purified by numerous techniques well known in the art. See Chen et al., Front. Bioeng. Biotechnol. 2022, 9:811971, 1-18, which is incorporated herein by reference. Separation techniques include ultrafiltration, multimodal chromatography, immunoaffinity, ultracentrifugation, acoustic devices, and microfluidic devices.

[0060] For example, these techniques can be used to remove proteins, lipids, and nucleic acids, as well as other molecules and structures smaller than 50 nm, 100 nm, or 150 nm. Similarly, these techniques can be used to remove proteins, lipids, and nucleic acids, as well as other molecules and structures larger than 200 nm, 300 nm, 400 nm, or 500 nm.

[0061] In some embodiments, this technique removes at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 97%, or 99% of molecules and structures smaller than 150 nm.

[0062] In some embodiments, this technique removes at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 97%, or 99% of molecules and structures larger than 300 nm.

[0063] Shaving of extracellular vesicles This invention encompasses methods for modifying the surface of extracellular proteins (EVs). EVs can be treated with enzymes to remove parts of their outer surface ("shaving"). For example, EVs can be treated with proteases to cleave accessible proteins on the EV surface.

[0064] The present invention encompasses a method for modifying the surface of extracellular proteins (EVs), comprising: a) treating the EVs with a protease to remove all or part of the proteins on the EV outer surface; b) inactivating the protease; and c) purifying the treated EVs from the protease. In some embodiments, EVs are treated with a protease in the presence of EV-producing cells. The producing cells can be removed by centrifugation and / or filtration.

[0065] The present invention encompasses a method for modifying the surface of purified extracellular proteins (EVs), comprising: a) purifying EVs from cells; b) treating the EVs with a protease to remove proteins from the outer surface of the EVs; c) inactivating the protease; and d) purifying the treated EVs from the residue of the protease and proteins.

[0066] Preferably, the EV comprises a retroviral capsid. The retroviral capsid may contain retroviral proteins necessary for reverse transcription, which may or may not have the ability to integrate. More preferably, the capsid comprises an RNA molecule encoding a protein, preferably a therapeutic protein (e.g., CAR or CCR). In some embodiments, the capsid comprises an RNA molecule encoding a gene editing system, such as a TALEN, ZFN, or CRISPR-based gene editing system.

[0067] The method further includes coating the EV with one or more polymers to generate polymeric nanoparticles (PNPs). Preferably, the polymeric nanoparticles include a targeting moiety, most preferably a targeting moiety against T cells (e.g., CD3 targeting agents such as anti-CD3 antibodies or aptamers) or natural killer (NK) cells (e.g., CD56 or NKG2D targeting agents such as anti-CD56 antibodies or NKG2D antibodies or aptamers). See Menon et al., Cancers 2023, 15:1189 and Lian et al., Int. J. Mol. Sci. 2022, 23, 164, which are incorporated herein by reference.

[0068] This method may further involve contacting coated EV / PNPs with target cells, in which case the target drug may or may not be included. In one embodiment, the target cells are T cells. In some embodiments, the target cells are NK cells or hematopoietic stem cells. The coated EV / PNPs and cells can be contacted in vitro, ex vivo, or in vivo.

[0069] The protease may be a mixture of proteases or a purified protease. In one embodiment, the protease is an endopeptidase. In one embodiment, the protease is an exopeptidase. In some embodiments, the protease is derived from a serine protease, asparagine protease, metalloprotease, or cysteine ​​protease.

[0070] Examples of proteases include proteinase K, trypsin, chymotrypsin, endoprotease Asp-N, endoprotease Arg-C, endoprotease Glu-C, endoprotease Lys-C, pepsin, thermolysin, elastase, papain, subtilisin, and endopeptidases such as clostripine.

[0071] Examples of proteases include carboxypeptidase A, carboxypeptidase B, carboxypeptidase P, carboxypeptidase Y, cathepsin C, acyl amino acid-releasing enzymes, and endopeptidases such as pyroglutamate aminopeptidase.

[0072] Proteases can be selected based on whether the protein to be cleaved is desirable or whether it is desirable to leave it uncleaved. For example, VSV-pseudotyped lentiviral vectors are less susceptible to trypsin inactivation than other envelope-pseudotyped lentiviral vectors. See Dautzenberg et al., Gene Therapy 2021, 28:89-104, which is incorporated herein by reference.

[0073] The protease is preferably mixed with EV after isolation and incubated at an appropriate temperature and concentration for a certain period of time, thereby enabling the cleavage of the protein of interest (multiple proteins).

[0074] The extracellular portion of the vector surface protein can be digested on purified or unpurified EVs with proteinase K or other broad-spectrum proteases.

[0075] In one embodiment, the protease is proteinase K, which cleaves peptide bonds adjacent to the carboxyl groups of aliphatic and aromatic amino acids. Enzymatic treatment involves adding recombinant proteinase K (e.g., Invitrogen 25530015) to the vector sample at a final concentration of approximately 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.1, 0.15, 0.2, 0.25, 0.3, 0.4, 0.5, 1, 2, 5, 10, 20, 50, 100, 200, or 500 μg / ml, or any range between these amounts (e.g., 0.03-0.3 μg / ml), and then 10 6 , 10 7 , 10 8 , 10 9 , 10 10 Or 10 11 This can be done by directly adding to EV for at least 1, 2, 5, 10, 15, 30, 60 minutes to 2, 4, 6, 12, or 24 hours, or any range between these amounts (e.g., 60 to 180 minutes), at 20, 25, 30, 35, 37, or 40°C, and at a pH of 6.5 to 9.5, with or without stirring. In one embodiment, 3.6 × 10 9 The EV of the particles is treated with 0.03-10 μg / ml of proteinase K at 25°C for 10 minutes.

[0076] After incubation, proteolysis can be halted using 10 mM phenylmethylsulfonyl fluoride (PMSF, Thermo Scientific, 36978), 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF, Thermo Scientific, 78431), or diisopropyl fluorophosphate (DFP). Excess PMSF, AEBSF, or DFP should be used to ensure complete inactivation of the enzyme before proceeding with purification and encapsulation.

[0077] Preferably, the protease removes cellular proteins and / or viral proteins from the outer surface of the EV. Preferably, the protease removes all or part of one or more cellular proteins, namely MHC, CD24, CD29, CD44, CD55, CD146, CD326, cadherins, ZO-1, occludins, tetraspanins (CD9, CD63, CD81, CD82), EGFR, or integrins.

[0078] Preferably, the protease removes all or part of one or more viral proteins, namely retroviral envelope protein / lentiviral envelope protein, measles virus envelope protein, or varicella stomatitis virus (VSV) envelope protein.

[0079] Preferably, the protease removes all or part of one or more extracellular therapeutic proteins, such as chimeric antigen receptors (CARs) or chimeric costimulatory receptors (CCRs).

[0080] In some embodiments, the protease effectively removes cellular and / or viral proteins from the outer surface of the EV, rendering the LV inoperable. In some embodiments, the protease removes at least 10%, 20%, 30%, 40%, 50%, 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the detectable cellular and / or viral proteins from the outer surface of the EV.

[0081] In some embodiments, the protease removes at least 50%, 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the extracellular components of the CAR and CCR from the outer surface of the EV.

[0082] Removal of cellular proteins, viral proteins, and therapeutic proteins from the outer surface of EVs can be measured by assessing the levels of these proteins before and after protease treatment, for example, by measuring the binding of antibodies to these proteins, as described in the examples herein.

[0083] In some embodiments, the protease removes at least 5%, 10%, 20%, 30%, 40%, 50%, 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the detectable chimeric antigen and / or costimulatory receptor on the outer surface of the EV.

[0084] In some embodiments, fragments of cellular proteins and / or viral proteins and / or therapeutic proteins remain within or inside the EV membrane. In some embodiments, fragments of vesicular stomatitis virus (VSV) envelope protein remain within or inside the EV membrane. In some embodiments, fragments and / or domains of chimeric antigen receptors and chimeric costimulatory receptors remain within or inside the EV membrane.

[0085] In some embodiments, a protease inhibitor is added after protease treatment to inactivate the protease. Examples of protease inhibitors include a cocktail of protease inhibitors or individual protease inhibitors. Preferred protease inhibitors are phenylmethanesulfonyl fluoride (PMSF, Thermo Scientific, 36978), 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF, Thermo Scientific, 78431), and diisopropyl fluorophosphate (DFP).

[0086] The present invention encompasses methods for removing proteases from treated extracellular viable (EVs). Removal methods include tangential flow filtration (TFF) and filtration methods such as size exclusion. In some embodiments, one of the following is used: EXOEASY Maxi kit (Qiagen, 76064), EXOQUICK-TC ULTRA (System Biosciences, ECULTRA-20TC-1), AMICON Ultra-15 100 kDa (Merck, UFC910008), or MACROSEP Advance 100K (Pall, MAP100C36).

[0087] Refinement after shaving Prior to polymer coating, removal of impurities generated during the proteolytic treatment is essential. The purification strategy used is based on removing impurities from the shaved vector using molecular weight / molecular size-based methods.

[0088] For vectors with a volume of less than 10 mL after shaving, ultrafiltration is used, employing polyvinylidene fluoride (PVDF), polyethersulfone (PES), modified polyethersulfone (mPES), or a cellulose centrifugal filter (Millipore, AMICON ultra filter) with a molecular weight cutoff (MWCO) of 100-500 kD. After the vector concentration step, diafiltration is performed using at least three 1 / 10e dilution steps to ensure appropriate buffer exchange to the final polymer coating buffer.

[0089] For higher volumes and greater purity, purification, concentration, and formulation are performed in a specific polymer-coated buffer using size exclusion chromatography (SEC), multimodal chromatography, followed by tangential flow filtration (TFF, hollow fiber, or cassette), or the TFF step alone.

[0090] Evaluation of the shaving process Multiple detection of surface proteins Using the MACSPLEX Exosome Kit (Miltenyi Biotec, 30-108-813), surface epitopes detected by EV before and after shaving are compared. This method allows for the quantification of expression of up to 37 human membrane epitopes using specific antibodies. The overnight protocol is followed according to the manufacturer's instructions for 10 minutes. 7 , 10 8 or 10 9 The procedure is performed using individual EV particles, and detection is carried out using anti-CD9 antibody, anti-CD63 antibody, or anti-CD81 antibody, or using all three antibodies simultaneously.

[0091] Direct ELISA method and sandwich ELISA method EVs produced in HEK293T, or EVs derived therefrom, are expected to express the human tetraspanins CD9, CD63, and CD81 on their surface, as well as the membrane proteins CD24, CD29, CD44, CD146, and CD346. In addition, lentiviral vectors may also express the VSV-G protein, as well as transmembrane genes such as CAR and CCR. Therefore, the shaving efficiency is determined by either direct ELISA or sandwich ELISA on the EV surface, targeting anti-CD9 (Biolegend, 312102, clone HI9a, Invitrogen, MA5-33125, clone 3A2), CD24 ((Biolegend, 311102, clone ML5), CD29 / integrin beta 1 (Invitrogen, MA5-17103, clone 3B6), CD44 (Invitrogen, MA5-15462, clone 8E2F3), CD63 ((Invitrogen, MA5-301) 87 clone 142, Biolegend, 353039, clone H5C6, CD81 (Biolegend, 349502, clone 5A6, Invitrogen, MA5-33123, clone 9F7), CD146 (Invitrogen, MA5-29414, clone 12), CD326 (Invitrogen, 14-9326-82, clone 1B7), VSVG (Sigma-Aldrich, MABF2337-100UG, clone 8G5F11, Sigma-Aldrich, MABF2321-100UL, 1E9F9), CAR (FMC63 For scFv, it can be evaluated by measuring the absence or reduction of binding of a CCR antibody (Miltenyi Biotec, 130-127-983, clone REA1297; Miltenyi Biotec, 130-127-984, clone REA1298), or an antigen for CAR and CCR such as CD19 (Miltenyi Biotec, 130-129-550, ACROBiosystem, CD9-H52H2).

[0092] For the direct ELISA method, 10 samples were taken per well in a MAXISORP 96-well plate (Thermo Scientific, NUNC, 439454). 8 Individual EVs can be coated overnight in DPBS 1X (Gibco, 14190094). After saturating with 2% BSA (Thermo Scientific, 37525) for 2 hours, incubate with a primary antibody or antigen (listed above) within the dilution range for 1.5 hours. After washing with a solution of DPBS 1X and 0.05% Tween (Sigma, P9416), incubate with a secondary antibody or streptavidin / avidin conjugated to HRP for 30 minutes. The HRP conjugate reagents used were 1 / 5,000 of goat anti-mouse IgG(H+L)HRP complex (Invitrogen, 31430), 1 / 10,000 of goat anti-mouse IgG(H+L)HRP complex (Invitrogen, 31460), 1 / 10,000 of streptavidin-HRP (Thermo Scientific, 21130), or 1 / 5,000 of avidin-HRP (Biolegend, 405103). After washing the wells, 1X TMB substrate solution (Invitrogen, 00-4201-56) was added for 10 minutes, and the mixture was stopped with stop solution (Thermofisher, N600). Absorbance was measured at 450 nm using GLOMAX DISCOVER (Promega, GM3000).

[0093] Antibody pairs were selected to simultaneously detect two different protein surface epitopes (tetraspanin, VSVG, and / or CAR). After coating the primary antibody overnight, a saturation step was performed, followed by incubation of various EVs at room temperature for 2 hours using the sandwich ELISA method. After incubating the biotinylated antibody for 1.5 hours, it was washed and incubated with streptavidin-HRP (Thermo Scientific, Pierce, 21130) at a 1 / 10,000e dilution for 30 minutes. Absorbance was measured according to the instructions for the direct ELISA method.

[0094] Western blotting for detection of cytoplasmic domains Tetraspanin, VSVG, CAR, and other proteins expressed on the EV surface are transmembrane proteins. Even after removal of the surface domain, their cytoplasmic domains remain intact and are not altered by the shaving procedure. Therefore, the integrity of EVs after shaving can be assessed by Western blotting using specific antibodies targeting the cytoplasmic domains of the following proteins: CD9 (Invitrogen, MA5-33125, clone 3A2), CD63, CD81 (AbboMax, 630-910), CD326 (Abcam, ab233919, clone EGP40-1110), VSVG (Abcam, ab50549, clone P5D4), CD3 zeta (Sigma-Aldrich, MABF3032, clone 6B10.2), and CD28 (Invitrogen, MA5-36070, clone RM404, Invitrogen, PA5-116751). First, the EV sample is dissolved in RIPA buffer (Thermo Scientific, 89900) or an equivalent lysis buffer containing NP-40 or Triton, and the protein is extracted from the EV sample. The extracted and denatured EV sample is loaded onto a NUPAGE 4-12% Bis-Tris gel (Thermofisher, NP0335BOX) and electrophoresis is performed in MES SDS running buffer (Thermofisher, NP0002) according to the manufacturer's instructions. Next, the blot is transferred onto a nitrocellulose membrane (Invitrogen, IB23002) using an IBLOT 2 dry blotting system (Thermofisher, IB21001). This blot is blocked for 1 hour in a solution of PBS 1X, 0.1% Tween 20 (PBST), and 3% BSA, and washed three times in PBST. Next, the primary antibody is incubated in PBST containing 0.2% BSA according to the manufacturer's instructions, followed by three washes, and then incubation of the HRP conjugate reagent. Detection is performed using SUPERSIGNAL WEST PICO plus chemiluminescent substrate (Thermofisher, 34580), and then acquired on an IBRIGHT FL1500 imaging system (Thermofisher, A44241).

[0095] Transduction efficiency Titration of shaved retroviral (e.g., lentivirus) vectors can be performed by flow cytometry or qPCR, and loss of infectivity after shaving can be confirmed on two different cell lines: HEK293T (ATCC, CRL-3216) cells and Jurkat (ATCC, TIB-152) cells.

[0096] Flow cytometry Four hours before transduction, HEK293T cells were transferred in DMEM (Gibco, 61965026) containing 10% FBS (Gibco, 10091148) and 1% penicillin / streptomycin (PS) (Gibco, 15140122) in 8x10⁶ cells. 5 Seed cells / well into a 48-well plate (Corning, 3548) and incubated at 37°C and 5% CO2. Immediately before transduction, Jurkat cells were divided into 8x10⁶ 5Cells are seeded in RPMI (Gibco, A1049101) containing 10% FBS and 1% PS within each well. Unshaved and shaved vectors are incubated on cells at 37°C in 5% CO2 using serial dilution (with the same physical titer). After 2 hours, fresh culture medium is added to each well, and the culture is extended to 3 days. On day 3, cells are harvested (using a trypsin treatment step with TrypLE, Gibco, 2563011 for HEK293T), washed with DPBS 1X, stained with Zombie NIR (Biolegend, 423106) diluted 1 / 1000 with DPBS 1X for viability determination, and then fixed with fixation buffer (Biolegend, 420801) for identification of the GFP-encoding vector (SEQ ID NO: 3). For the CD19-CAR encoding vector (SEQ ID NO: 4), first, incubate with CD19 CAR detection reagent (Miltenyi Biotec, 130-129-550) for 10 minutes, then wash, and incubate with biotin antibody-VB515 (Miltenyi Biotec, 130-110-957, clone REA746) to perform an additional staining step before fixation.

[0097] The number of viable fluorescent cells after transduction using shaved or unshaved vectors is measured using a flow cytometer (ATTUNE NXT, Thermofisher), and the infectivity titer is quantified using cells expressing 5–25% GFP or CD19 CAR.

[0098] qPCR reaction for measuring infectivity titer Four hours before transduction, HEK293T cells were transferred in DMEM (Gibco, 61965026) containing 10% FBS (Gibco, 10091148) and 1% penicillin / streptomycin (PS) (Gibco, 15140122) in 8x10⁶ cells. 4 Seed cells / well in 24-well plates (Corning, 3513) and incubated at 37°C and 5% CO2. Immediately before transduction, 8x10⁶ Jurkat cells were placed in a single well.4 Cells are seeded in RPMI (Gibco, A1049101) containing 10% FBS and 1% PS per well. Cells are transduced using shaved or unshaved extracellular viable cells that have undergone at least three serial dilutions, and cultured at 37°C and 5% CO2 for 72 hours. After 72 hours, cells are harvested and the pellet is collected. Whole genomic DNA is extracted from the transduced cell pellet using the MAXWELL RSC Blood DNA Kit (Promega, AS1400) according to the manufacturer's instructions. Proviral DNA is quantified by qPCR using PERFECTA MULTIPLEX TOUGHMIX (Quantabio, 95147-250). The integrated sequence is amplified using LTR (long terminal repeat) specific probes and primers.

[0099] Physical titer Particle concentration measurements of shaved retroviral (e.g., lentiviral) vectors can be performed to ensure that shaving does not affect the integrity of vectors produced or derived within HEK293T by using three orthogonal methods based on particle count (NTA), quantification of capsid P24 protein, or quantification of viral RNA copies (RT-qPCR).

[0100] The particle concentration of vectors produced in or derived from HEK293T can be determined by nanoparticle tracking analysis using a ZETAVIEW instrument (Particle Metrix GmbH) or by interferometry using a VIDEODROP instrument (Myriade). Newly prepared or freeze-thawed vectors produced in or derived from HEK293T can be homogenized by diluting 1000-fold in DPBS-50 mM sucrose and gently vortexing before measurement.

[0101] Detection and quantification of the p24 capsid lentivirus vector protein can be performed using the HIV-1 p24 antigen ELISA 2.0 kit (Zeptometrix, 0801002) according to the manufacturer's instructions.

[0102] Proviral RNA copy quantification can be performed by isolating and purifying viral RNA using the MAXWELL RSC TNA virus kit (Promega, AS1330) according to the manufacturer's instructions, followed by the use of the LENTI-X QRT-PCR TITRATION kit (Takara, 631235).

[0103] membrane integrity By using multi-angle dynamic light scattering (MALS), the total charge (zeta potential), size (mean hydrodynamic diameter), and size distribution (polydispersion index) of vectors produced in or derived from HEK293T can be compared before and after shaving, thereby confirming the integrity of the treated film. Newly prepared or freeze-thawed EVs can be homogenized by diluting them 16-fold (zeta potential and polydispersion index) or 20-fold (mean hydrodynamic diameter) in DPBS-50mM sucrose and gently vortexing them before analysis on a ZETASIZER ULTRA instrument (Malvern Instruments).

[0104] Shaved extracellular vesicles (shaved EVs) The present invention encompasses extracellular vesicles (EVs) whose outer surface portion has been removed by enzymatic treatment ("shaved extracellular vesicles" or "shaved EVs").

[0105] The present invention is 1, 10, 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10, 10 11 , 10 12 , 10 13 , 10 14 , 10 15 Shaved EVs such as 10, or any range between these amounts (e.g., 10) 5 ~10 8 It includes individual EVs.

[0106] The present invention is 10 2 / ml, 10 3 / ml, 10 4 / ml, 10 5 / ml, 10 6 / ml, 10 7 / ml, 10 8 / ml, 10 9 / ml, 10 10 / ml, 10 11 / ml, 10 12 / ml, 10 13 / ml, 10 14 / ml, 10 15 The concentration of shaved EV such as / ml, or any range between these amounts (e.g., 10 9 / ml~10 12 Includes / ml)

[0107] In some embodiments, the shaved EVs are endosome-derived ("exosomes") having a diameter of approximately 30 nm to 150 nm. Preferably, the shaved EVs are generated by budding ("ectosomes") having a diameter of approximately 50 nm to 300 nm outside the membrane.

[0108] In some embodiments, the shaved EV contains less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% of the original levels of detectable cells and / or viral proteins on the outer surface of the EV before shaving.

[0109] In some embodiments, the shaved EV contains less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% of the original levels of detectable CD9, CD29, CD63, CD81, CD82, CD146, or VSV proteins on the outer surface of the EV before shaving.

[0110] The removal of cellular or viral proteins from the outer surface of EVs can be measured by measuring the levels of these proteins before and after protease treatment using techniques such as those described in the examples of this specification.

[0111] In some embodiments, the shaved EV includes less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% of the original level of detectable chimeric antigen receptors on the outer surface of the EV before shaving.

[0112] Preferably, the shaved EVs are further purified to remove the proteases used to shave them. Removal methods include tangential flow filtration (TFF) and other filtration methods such as size exclusion or multimodal chromatography.

[0113] Retrovirus capsid The present invention encompasses extracellular viable tissue (EV), preferably shaved EV, containing a retroviral capsid. The retroviral capsid may be from any retroviral. Preferably, the retroviral capsid is a lentiviral capsid.

[0114] Retroviral capsids can be introduced into extracellular viable cells (EVs) by transfection or infection of cells, or by using stably transformed cell lines, by expressing retroviral proteins within the producing cells. Cells can express a variety of retroviral proteins from RNA or DNA encoding retroviral proteins, as illustrated, for example, in Dautzenberg et al., Gene Therapy 2021, 28:89-104 (incorporated herein by reference).

[0115] Extraviral viruses (EVs) containing retroviral capsids can be produced in well-known ways using 3-4 plasmid production approaches, for example, as described in Zufferey et al., Nature biotechnol 1997, 15:871-875.

[0116] The present invention encompasses a method comprising: a) producing a capsid in cells by expressing a retroviral Gag protein; b) purifying extracellular viable cells (EVs) containing the capsid from the cells; c) removing proteins from the outer surface of the EVs by treating them with a protease; and d) purifying the EVs treated with the protease.

[0117] This method may further include generating polymeric nanoparticles by coating the EV with one or more polymers. Preferably, the polymeric nanoparticles include a targeting moiety, most preferably a targeting moiety against T cells (e.g., CD3 targeting agents such as anti-CD3 antibodies or aptamers) or natural killer (NK) cells (e.g., CD56 or NKG2D targeting agents such as anti-CD56 antibodies or NKG2D antibodies or aptamers).

[0118] This method may further include contacting coated EV / PNPs with target cells, in which case the target drug may or may not be included. In one embodiment, the target cells are T cells. The coated EV / PNPs and cells can be contacted in vitro, ex vivo, or in vivo.

[0119] The retroviral Gag protein can be expressed in the producing cell to form a capsid that is released from the cell during EV. In some embodiments, the capsid comprises the retroviral Gag protein and Pol protein. Preferably, the capsid comprises a capsid protein, a matrix protein, a nucleocapsid protein, a protease, a reverse transcriptase, and an integrase protein.

[0120] In some embodiments, the retrovirus is avian leukemia virus, Rous sarcoma virus, mouse mammary tumor virus, mouse leukemia virus, feline leukemia virus, bovine leukemia virus, or human T lymphotropic virus. In some embodiments, the retrovirus is lentivirus such as human immunodeficiency virus type 1 (HIV-1), human immunodeficiency virus type 2 (HIV-2), monkey immunodeficiency virus, or feline immunodeficiency virus.

[0121] Preferably, the capsid contains retroviral Gag and Pol proteins. Most preferably, the capsid contains a capsid protein, a matrix protein, a nucleocapsid protein, a protease, a reverse transcriptase, and an integrase protein.

[0122] In some embodiments, the capsid contains a pair of RNA molecules. The RNA molecules preferably contain a retroviral packaging signal. The RNA molecules also preferably contain a retroviral signal for reverse transcription.

[0123] In some embodiments, the RNA encodes a protein, preferably a non-retroviral protein. In some embodiments, the protein is a marker protein such as β-galactosidase, chloramphenicol acetyltransferase, green fluorescent protein, red fluorescent protein, and luciferase enzyme.

[0124] In some embodiments, the protein is a cytokine or growth factor, such as interferon, interleukin, lymphokine, and tumor necrosis factor, for example, IL-1, IL-2, IL-4, IL-6, IL-10, IL-11, IL-13, IL-17, IL-35, TNFα, IFN-α, IFN-β, IFN-γ, TGF-β, GM-CSF, M-CSF, G-CSF, EPO, BMP, EGF, FGF, and VEGF.

[0125] Examples of such therapeutic proteins include chimeric antigen receptors and costimulatory receptors (CCRs). CCRs are chimeric receptors containing an antigen-binding extracellular domain, a transmembrane domain, and an intracellular signaling domain (Sadelain et al., Cancer Discov. 2013, 3(4):388-398), but lacking a T cell activation domain and instead possessing a costimulatory domain such as CD28, 4-1BB, OX40, ICOS, DAPIO, 2B4, or CD70. CCRs can be used in combination with T cell receptors or CARs to enhance the responsiveness of T cells to T cells expressing dual antigens. CCRs can also be used to enhance selective tumor targeting (Kloss et al., Nat Biotechnol, 2013, 31(1):71-5). CCRs are antigen-specific costimulatory receptors that, upon binding to their binding partner, i.e., the target antigen, mimic 4-1BB, OX40, ICOS, or CD70 (depending on the costimulatory domain of the CCR).

[0126] Chimeric antigen receptors (CARs) include carbonic anhydrase IX (CAIX), carcinoembryonic antigen (CEA), CD8, CD7, CD10, CD19, CD20, CD22, CD30, CD33, CLL1, CD34, CD38, CD41, CD44, CD49f, CD56, CD74, CD117, CD133, CD138, CD123, CD44V6, antigens of cytomegalovirus (CMV) infected cells (e.g., cell surface antigens), epithelial glycoprotein-2 (EGP-2), epithelial glycoprotein-40 (EGP-40), epithelial cell adhesion molecule (EpCAM), and receptor tyrosine protein kinases erb-B2, 3, 4 (erb-B B2, 3, 4), folate-binding protein (FBP), fetal acetylcholine receptor (ACHR), folate receptor-a, FLT3, ganglioside G2 (GD2), ganglioside G3 (GD3), human epidermal growth factor receptor 2 (HER-2), human telomerase reverse transcriptase (HTERT), interleukin-13 receptor subunit alpha-2 (IL-13Ra2), K-light chain kinase insertion domain receptor (KDR), Lewis Y (LeY), L1 cell adhesion molecule (LICAM), melanoma antigen family A1 (MAGE-A1), mucin 16 (MUC16), mucin 1 (MUC1), mesothelin (MSLN), ERBB2, MAGE-A3, MAGE-A4, p53, MART1, GP100, Proteinase 3 (PR1), Tyrosinase, Sulbibin, hTERT, EphA2, NKG2D ligand, Cancer-testicular antigen NY-ESO-1, Fetal carcinoma antigen (h5T4), Prostate stem cell antigen (PSCA), Prostate-specific membrane antigen (PSMA), ROR1, Tumor-associated glycoprotein 72 (TAG-72), Vascular endothelial growth factor R2 (VEGF-R2), Wilms tumor protein (WT-1), BCMA, NKCS1, EGFIR, EGFR-VIII, CD99, CD70, ADGRE2, CCR1, LILRB2, PRAMEExamples include receptors for CCR4, CD5, CD3, TRBC1, TRBC2, TIM-3, integrin B7, ICAM-1, CD70, Tim3, CLEC12A, ERBB, B-cell activator receptor (BAFF-R), thymic stromal lymphocyte generating factor (TSLPR), SLAM family member 7 (SLAMF7), and G protein-coupled receptor family C group 5 member D (GPRC5D).

[0127] Preferably, the chimeric antigen receptor (CAR) is for CD19, CD20, CD22, CD30, CD33, CD38, CD123, CD133, CD147, CD317, FLT3, BCMA, DR4, CS-1, CLL-1, ADGRE2, HER2, HSP70, VEGF, GD2, GD3, MUC1, MUC16, NKR2, NKG2D ligand, CYP1B1, SP17, PRAME, and WT1. The chimeric antigen receptor (CAR) is preferably CD19 (present in B-cell derived cancers such as acute lymphoblastic leukemia (ALL) and diffuse large B-cell lymphoma (DLBCL)), CD30 (present in refractory Hodgkin lymphoma), CD33, CD123, ADGRE2 and FLT3 (present in acute myeloid leukemia (AML)), and BCMA (present in multiple myeloma).

[0128] polymer The present invention encompasses polymer-coated EVs, preferably shaved EVs. EVs can be coated with at least one polymer to form polymeric nanoparticles.

[0129] The present invention encompasses a method comprising: a) providing EVs; b) treating EVs with a protease to remove proteins from the outer surface of EVs; c) purifying the treated (shaved) EVs from the protease; d) coating the treated (shaved) EVs with at least one polymer to form polymeric nanoparticles; and e) purifying the treated (shaved) EVs from the uncoated polymers.

[0130] The present invention encompasses a method comprising: a) providing an EV comprising a capsid; b) treating the EV with a protease to remove proteins on the outer surface of the EV; c) purifying the treated (shaved) EV from the protease; d) coating the treated (shaved) EV with at least one polymer to form polymeric nanoparticles; and e) purifying the treated (shaved) EV from the polymer that has not been coated.

[0131] Preferably, the polymeric nanoparticles comprise a targeting moiety, and most preferably those directed against T cells or NK cells (e.g., CD3 or NKG2D targeting agents such as anti-CD3 antibodies or anti-NKG2D antibodies or aptamers). The method can further comprise contacting the coated EV / PNP with target cells, where the target agent may or may not be included. In one embodiment, the target cells are T cells or NK cells. The coated EV / PNP and the cells can be contacted in vitro, ex vivo, or in vivo.

[0132] In various embodiments, the number of polymer molecules per vector particle is at least about 10 3 、10 4 、10 5 、10 6 、10 7 、10 8 、10 9 、10 10 、10 11 、10 12 、10 13 、10 14 、10 15 、10 16 、10 17 、10 18 、10 19 、10 20 or is within any range between these amounts (e.g., about 10 8 ~10 10 ).

[0133] The polymer can be added to the EV by mixing it with the EV to bind them together. Mixing can be facilitated using a vortex treatment. In some embodiments, the mixture can be bound for at least 1 minute, 2 minutes, 5 minutes, 10 minutes, 20 minutes, 30 minutes, 45 minutes, or 60 minutes. In some embodiments, the mixture can be bound for at least 1 hour, 2 hours, 3 hours, 6 hours, 12 hours, 18 hours, or 24 hours.

[0134] In some embodiments, the mixture can be mixed at approximately 1°C, approximately 2°C, approximately 4°C, approximately 10°C, approximately 15°C, approximately 20°C, approximately 25°C, approximately 30°C, approximately 35°C, approximately 37°C, approximately 40°C, approximately 45°C, or approximately 50°C, or any range between these temperatures (e.g., approximately 4°C to approximately 20°C).

[0135] In some embodiments, the mixture can be mixed in pharmacopoeia-approved buffers suitable for systemic administration, such as histidine (20 mM), sodium citrate phosphate (10 mM), sodium succinate phosphate (10 mM), sodium phosphate (10 mM), tris citrate (10 mM), sodium acetate phosphate (10 mM), tris acetate (10 mM), and tris succinate (10 mM), all of which have a pH in the range of 5 to 7.4, in which case the preparation may include or exclude sucrose (50 mM) and / or sodium chloride (175 mM).

[0136] Shaved EV coating Single layer coating Polymer coating of shaved EVs can be performed as follows: The polymer is diluted in the same pharmacopoeia-compliant formulation buffer used for the purified shaved EVs and homogenized by short-time vortexing. The polymer solution is added to the shaved vectors in an appropriate mass ratio, altering the overall charge of the particles, and homogenization is performed by liquid-phase mixing (vortexing or microfluidics, etc.). After incubation of this complex at room temperature for 30 minutes, it can be used for analysis or transduction.

[0137] A polymer coating can be constructed from a mixture of free polymers and polymers covalently bonded to a target site.

[0138] Alternating lamination coating The alternating lamination method involves adding a shielding polymer to coated and shaved EVs prepared by following a "single-layer coating" method using a cationic polymer. To this end, excess polymer is removed from the formed polymeric nanoparticles (PNPs) using a hollow fiber tangential flow filtration step. This step allows for the recombination of the coated nanoparticles in a pharmacopoeia-compliant buffer used to dilute the shielding polymer. The shielding polymer solution consists of a mixture of free polymer and polymer covalently bonded to the target moiety. After a short mixing (e.g., vortexing) for homogenization, the shielding polymer solution is added to the coated nanoparticles in an appropriate mass ratio to alter the global charge of the particles, and homogenization is performed by liquid-phase mixing (e.g., vortexing or microfluidics). After incubation of this complex at room temperature for 30 minutes, it is used for analysis or transduction. Again, excess polymer is removed by the hollow fiber tangential flow filtration step.

[0139] Characterization Method Using multi-angle dynamic light scattering (MADLS), the total charge (zeta potential), size (mean hydrodynamic diameter), and size distribution (polydispersion index) of shaved EVs can be compared before and after polymer addition to confirm coating efficiency. Newly prepared coated EVs / PNPs can be diluted 16-fold (zeta potential and polydispersion index) or 20-fold (mean hydrodynamic diameter) in the final formulation buffer, homogenized by gentle vortexing, and then analyzed on a ZETASIZER ULTRA instrument (Malvern Instruments).

[0140] Transduction efficiency by flow cytometry By performing PNP titration on two cell lines (HEK293T cells and Jurkat cells) as described below, it is possible to confirm that infectivity is restored after single-layer coating or alternating layer coating.

[0141] Four hours before transduction, HEK293T cells were transferred in DMEM containing 10% FBS and 1% penicillin / streptomycin (PS) in 8x10⁻¹⁴⁻¹ 5 Seed cells / well in 24-well plates and incubated at 37°C and 5% CO2. Immediately before transduction, Jurkat cells were placed in RPMI containing 10% FBS and 1% PS in 8x10⁶ cells. 5Seed cells per well. Incubate cells with serial dilutions of shaved retrovirus particles and PNP (of the same physical titer) at 37°C and 5% CO2. After 2 hours, add fresh culture medium to each well and extend the culture to 3 days. On day 3, harvest the cells (using a trypsinization step for HEK293T), wash in DPBS 1X, and fix directly with fixation buffer (Biolegend, 420801) for GFP-encoding vectors (SEQ ID NO: 3) or with Zombie NIR for viability discrimination. For CD19-CAR-encoding vectors (SEQ ID NO: 4), perform an additional staining step before fixation: first, incubate with CD19 CAR detection reagent (Miltenyi Biotec, 30-129-550) for 10 minutes, wash, and then incubate with biotin antibody-VB515 (Miltenyi Biotec, 130-110-957).

[0142] The number of viable fluorescent cells after transduction of shaved retroviral particles or PNPs is measured using a flow cytometer (ATTUNE NXT, Thermofisher).

[0143] qPCR reaction for measuring infectivity titer Four hours before transduction, HEK293T cells were transferred in DMEM (Gibco, 61965026) containing 10% FBS (Gibco, 10091148) and 1% penicillin / streptomycin (PS) (Gibco, 15140122) in 8x10⁶ cells. 4 Seed cells / well in 24-well plates (Corning, 3513) and incubated at 37°C and 5% CO2. Immediately before transduction, 8x10⁶ Jurkat cells were placed in a single well. 4Cells are seeded in RPMI (Gibco, A1049101) containing 10% FBS and 1% PS per well. Cells are transduced with shaved retroviral particles or PNPs diluted at least three times in series, and cultured at 37°C and 5% CO2 for 72 hours. After 72 hours, cells are harvested and the pellet is collected. Whole genomic DNA is extracted from the transduced cell pellet using the Maxwell RSC Blood DNA Kit (Promega, AS1400) according to the manufacturer's instructions. Proviral DNA is quantified by qPCR using PERFECTA MultiPlex ToughMix (Quantabio, 95147-250). The integrated sequence is amplified using the following LTR (long terminal repeat) probe (5′-6FAM-AACCATTAGGAGTAGCACCCACCAAGG-BHQ1-3′ (SEQ ID NO: 5)) and primers (forward 5′-TGGAGGAGGAGATATGAGGG-3′ (SEQ ID NO: 6) and reverse 5′-CTGCTGCACTATACCAGACA-3′ (SEQ ID NO: 7)). Analysis is performed using QUANTSTUDIO 7 Pro (Applied Biosystems) to quantify the number of proviral copies per cell.

[0144] Preferably, the EV is coated with one or two layers of polymer. In some embodiments, the polymer is cationic, neutral, or zwitterionic, in which case the targeting agent may or may not be included. In some embodiments, the first polymer layer is a cationic polymer and the second polymer layer is anionic, neutral, or zwitterionic polymer, in which case the targeting agent may or may not be included.

[0145] Examples of polymers include polyarginine, polylysine, PEI, chitosan, PBAE, PEI-co-polyhistidine, polylysine dendrimer / dendrimer / branched polymers, star-shaped polyamino acid-based polymers, chondroitin sulfate, heparosan, polyglutamic acid, hyaluronic acid, or dextran sulfate, polysarcosine, PEG, polycarboxybetaine, polysulfobetaine, or polyphosphorylcholine.

[0146] The present invention encompasses EVs containing cationic polymers. Examples of cationic polymers include polyamino acids, polyarginine, polylysine, polyetherimine (PEI), chitosan, poly(β-aminoester) (PBAE), polyethyleneimine / polytetrahydrofuran (PEI / THF), OM-PBAE, lysine dendrigrafts (DGL), and their variants.

[0147] Examples of anionic polymers include chondroitin sulfate, sodium hyaluronate, heparosan, polyglutamic acid (PGA), alginic acid, and dextran sulfate. Cationic and anionic polymers can be used with shielding agents, such as polysarcosine, polyfwitterions, polyethylene glycol (PEG), polycarboxybetaine, polysulfobetaine, or polyphosphorylcholine.

[0148] The zwitterionic polymer contains a positively charged nitrogen atom and a negatively charged oxygen atom, separated from each other by at least one carbon atom, preferably two to three carbon atoms. Examples of zwitterionic polymers include polycarboxybetaine, polysulfobetaine, and polyphosphorylcholine.

[0149] In some embodiments, the zwitterion is a fusion of an anionic or cationic compound and a zwitterion.

[0150] Targeting agent This invention encompasses extracellular vehicles (EVs) that include a targeting agent. The targeting agent can guide the EV to a specific cellular target.

[0151] In various embodiments, the targeting agent includes human, mouse, camel, and shark antibodies, single-chain antibodies, nanobodies, dibodies, and nucleic acids such as antigen-binding fragments of antibodies, peptides, aptamers, or oligonucleotides, as well as ligands that bind to cell surface receptors or extracellular matrix components.

[0152] In one embodiment, the targeting agent is a CD3 targeting agent, such as an anti-CD3 antibody or aptamer. In various embodiments, the targeting agent is an anti-NKG2D antibody or aptamer.

[0153] The aptamer may be any aptamer disclosed in Molecules 2020, 25, 5227, 1-34, which are incorporated herein by reference.

[0154] The present invention encompasses a method for adding a targeting agent to EVs. In various embodiments, the targeting agent (e.g., an aptamer) is a charged molecule (e.g., negatively charged) that interacts with and can be incorporated into polymeric nanoparticles.

[0155] The targeting agent can be covalently grafted onto the polymer (the second layer or the layer in contact with the extracellular medium). A mixture of the target polymer and the non-target polymer can be used for coating the EV.

[0156] How to deliver a payload The EV of the present invention can be used as a delivery means for payloads such as therapeutic proteins and nucleic acids that encode them.

[0157] In various embodiments, the present invention includes methods comprising contacting EVs with eukaryotic cells in vitro, ex vivo, or in vivo.

[0158] The present invention comprises a) providing polymeric nanoparticles including shaved EVs containing a payload, and b) a method for contacting polymeric nanoparticles (PNPs) with eukaryotic cells.

[0159] The present invention comprises a method for a) providing a shaved EV containing a payload, b) coating the treated EV with at least one polymer to form polymeric nanoparticles, and c) contacting the polymeric nanoparticles (PNPs) with eukaryotic cells.

[0160] The present invention encompasses a method comprising: a) providing an EV containing a payload; b) treating the EV with a protease to remove proteins from the outer surface of the EV; c) purifying the treated EV from the protease; d) coating the treated EV with at least one polymer to form polymeric nanoparticles (PNPs); and e) contacting the PNPs with eukaryotic cells. In some embodiments, the PNPs are purified before contacting eukaryotic cells.

[0161] Preferably, the EV comprises a retroviral capsid. The retroviral capsid may contain retroviral proteins necessary for reverse transcription, which may or may not have the ability to integrate. More preferably, the capsid comprises an RNA molecule encoding a protein, preferably a therapeutic protein (e.g., CAR).

[0162] Preferably, the polymeric nanoparticles include a targeting moiety, most preferably a moiety targeting T cells or NK cells (e.g., CD3 or NKG2D targeting agents, e.g., anti-CD3, anti-NKG2D antibodies or aptamers). The method may further include contacting the coated EV / PNPs with target cells, in which case the target agent may or may not be included. In one embodiment, the target cells are T cells or NK cells. The coated EV / PNPs and cells can be contacted in vitro, ex vivo, or in vivo.

[0163] In one embodiment, the cells are T cells or NK cells, and the payload is a pair of RNAs encoding CARs.

[0164] In various embodiments, the ratio of EVs (or PNPs) to eukaryotic cells is approximately 1:100, 1:10, 1:1, 10:1, 100:1, 10 3 :1, 10 4 :1, 10 5 :1, or 10 6 :1, or any range between these ratios (e.g., about 100:1 to 10 4 :1) It can be.

[0165] In various embodiments, 10 4 , 10 5 , 10 6 , 10 7 , 108, 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , 10 14 , 10 15 , etc. EV (or PNP), preferably shaved EV, 10 4 pieces, 10 5 pieces, 10 6 pieces, 10 7 pieces, 10 8 pieces, 10 9 pieces, 10 10 pieces, 10 11 pieces, 10 12 pieces, 10 13 pieces, 10 14 pieces, 10 15 Cells such as 10, preferably T cells, or any range between these amounts (for example, 10 7 ~10 pieces 10 10 EVs 5 ~10 pieces 8 It can be brought into contact with individual cells.

[0166] In one embodiment, the method is 10 4 pieces, 10 5 pieces, 10 6pieces, 10 7 pieces, 10 8 pieces, 10 9 pieces, 10 10 pieces, 10 11 pieces, 10 12 pieces, 10 13 pieces, 10 14 pieces, 10 15 This includes contact with individual items, etc. 10 4 pieces, 10 5 pieces, 10 6 pieces, 10 7 pieces, 10 8 pieces, 10 9 pieces, 10 10 pieces, 10 11 pieces, 10 12 pieces, 10 13 pieces, 10 14 pieces, 10 15 This is ex vivo EV (or PNP) using individual cells, etc. T cells or NK cells are collected from the patient, and these T cells or NK cells are reintroduced into the patient.

[0167] In some embodiments, the eukaryotic cells are of animal origin, such as from birds or mammals like chickens, mice, primates, monkeys, humans, hamsters, and rats.

[0168] Examples of cells include immune cells and fibroblasts. In some embodiments, the cells are peripheral blood mononuclear cells, B cells, NK cells, T cells, macrophages, dendritic cells, stem cells, neurons, or iPSCs.

[0169] Isolated T cells and NK cells can be prepared using well-known techniques, for example, using paramagnetic polystyrene beads coated with anti-CD3 and anti-CD28 monoclonal antibodies, as described in Laport et al., Blood 2003, 102:2004-2013. Contact between cells and EVs containing capsids can be carried out as described in Levine et al., PNAS 2006, 103:17372-17377.

[0170] The present invention further encompasses cells comprising the EV, capsid, or payload of the present invention. [Examples]

[0171] Example 1 - Production of EV A. Capsid-free Extracellular vesicles were produced in HEK293-derived cells (Gibco Invitrogen) in suspension. HEK293-derived cells were raised in a glass bioreactor to 3.5 x 10⁻⁶ 5 Cells were seeded at a concentration of cells / mL in 1000 mL of LV-MAX® production medium (Gibco Invitrogen). After incubation for 5 days at 37°C with constant magnetic stirring under 8% CO2 humidity, the mixture was collected in its entirety by centrifugation at 2000 g for 15 minutes, and cells and cellular debris in the supernatant were removed. The mixture was then clarified by filtration through 0.45 μm and 0.22 μm PVDF membranes (STERICUP, Merk Millipore).

[0172] B. Those containing capsids Capsid-containing extracellular viable cells (EVs) were produced in HEK293-derived cells (Gibco Invitrogen) in suspension, and then transiently transfected with LV-MAX® reagent containing third-generation Gag-Pol, Rev packaging, and VSV-G envelope plasmids, as well as transfer plasmids encoding GFP- or CD19CAR. See, for example, Perry et al., Viruses 2021, 13:268.

[0173] HEK293-derived cells were placed in 204 mL of LV-MAX® production medium (Gibco Invitrogen) in an 8 x 1000 mL Erlenmeyer flask (Corning) at a rate of 3.5 x 10⁶ cells. 6 Cells were seeded at cells / mL. These Erlenmeyer flasks were cultured at 37°C, 120 rpm, and under 8% CO2 humidity. The cell density was 4x10 the day after seeding. 6Transient transfection was performed at a cell / mL level. LV-MAX® transfection reagent (Gibco Invitrogen) was mixed with GFP or CD19 CAR transfer plasmid, Gag-Pol packaging plasmid, Rev packaging plasmid, and VSV-G envelope plasmid. After incubation at room temperature for 10 minutes, the transfection mix / plasmid complex was slowly added to the cells and incubated at 37°C, 120 rpm, and 8% CO2 humidity. Six hours post-transfection, EV production including the capsid was stimulated by adding LV-MAX® enhancer (Gibco Invitrogen). The entire bulk mixture was cultured for 48 hours at 37°C, 120 rpm, and 8% CO2 humidity. One hour before clarification, 5 U / mL of DENARASE (c-LEcta GmbH) in 2 mM MgCl2 was added, and DNAse treatment was performed at 37°C, 120 rpm, and 8% CO2 humidity. The entire mixture was collected by centrifugation at 1300 g for 15 minutes, cells and cellular debris were removed from the supernatant, and the mixture was filtered through a 0.45 μm PVDF membrane (STERICUP, Merk Millipore) for clarification.

[0174] Example 2 - Production of EV A. Capsid-free EVs were purified by multimodal chromatography on a CAPTO Core 700 membrane (Cytiva). Tangential flow filtration was performed on a 300 kDa MIDIKROS MPES membrane (Repligen) to reduce the volume, allowing for preparation in pharmacopoeia-compliant formulations. After sterile filtration (Millipore) at 0.22 μm, the final product was packed into 2 mL cryotubes with aliquots of less than 1 mL, then labeled, frozen, and stored at below -70°C.

[0175] Table 1: Characteristics of EV batches produced from HEK293-derived cells. Physical titer was measured by NTA (Nano Tracking Analyzer) and MADLS. [Table 1]

[0176] B. Those containing capsids The extravasation vesicles (EVs) containing the capsid were purified by anion exchange chromatography using a Q Mustang membrane (Pall Corporation) and stepwise elution with NaCl. Tangential flow filtration was performed on a 500 kDa MIDIKROS MPES membrane (Repligen) to reduce the volume, allowing for preparation in pharmacopoeia-compliant formulations. The final product was packed into 2 mL cryotubes with aliquots of less than 1 mL, then labeled, frozen, and stored at below -70°C.

[0177] Table 2: Characteristics of LV batches produced from HEK293-derived cells. [Table 2]

[0178] In HEK293-derived cells, similar biomanufacturing processes were performed for both capsid-containing and capsid-free cells, yielding EVs with comparable biophysical and biochemical properties. These steps, which ensure batch-to-batch consistency, are summarized in Figure 10.

[0179] Example 3 - Assay of surface proteins on EV Multiple detection of surface proteins The MACSPLEX exosome kit (Miltenyi Biotec) allows for the comparison of surface epitopes detected on vectors before and after shaving. This method enables the quantification of expression of up to 37 human membrane epitopes using specific antibodies. Follow the overnight protocol according to the manufacturer's instructions for 10 minutes. 7 , 10 8 or 10 9The study was conducted using individual EV particles, and detection was performed using anti-CD9 antibody, anti-CD63 antibody, or anti-CD81 antibody, or all three antibodies. As shown in Figure 4, the surface expression profiles of capsid-free EVs (EVs) and capsid-containing EVs (LVs) produced in HEK293T-derived cells are similar. The results confirmed that CD9, CD63, and CD81 are interesting markers present on both types of EVs.

[0180] Direct ELISA method and sandwich ELISA method EVs produced in HEK293T, or EVs derived therefrom, are expected to express the human tetraspanins CD9, CD63, and CD81 on their surface, as well as the membrane proteins CD24, CD29, CD44, CD146, and CD346. In addition, LVs can possess a pseudo-envelope such as the VSV-G protein on their surface, as well as transmembrane transgenes such as CAR and CCR.

[0181] Figures 13-14 show the results of experiments analyzing unshaved EV and LV samples using ELISA to determine the presence of VSVG. Similar results were obtained when titrated with anti-VSVG neutralizing monoclonal antibody 8G5F11, anti-VSVG monoclonal antibody 1E9F9, and anti-VSVG polyclonal antibody.

[0182] Furthermore, Figure 26 shows the results of an experiment in which unshaved LVs were analyzed by sandwich ELISA for the presence of CD19-CAR using CD19 recombinant protein or anti-FMC63 idiotype antibody. CD19-CAR was detected on LV expressing CAR, but, as expected, was not detected on LV expressing GFP.

[0183] Therefore, the efficiency of the shaving process is determined by the use of anti-CD9 antibody (Biolegend, 312102, clone HI9a, Invitrogen, MA5-33125, clone 3A2), anti-CD24 antibody (Biolegend, 311102, clone ML5), anti-CD29 / integrin beta 1 antibody (Invitrogen, MA5-17103, clone 3B6), anti-CD44 antibody (Invitrogen, MA5-15462, clone 8E2F3), anti-CD63 antibody (Invitrogen, MA5-30187, clone 142, Biolegend, 353039, clone 3A2) on the EV surface. Clone H5C6), anti-CD81 antibody (Biolegend, 349502, clone 5A6, Invitrogen, MA5-33123, clone 9F7), anti-CD146 antibody (Invitrogen, MA5-29414, clone 12), anti-CD326 antibody (Invitrogen, 14-9326-82, clone 1B7), anti-VSVG antibody (Sigma-Aldrich, MABF2337-100UG, clone 8G5F11, Sigma-Aldrich, MABF2321-100UL, clone 1E9F9), CAR antibody (For FMC63scFv, see Miltenyi Biotec, 130-127-983, clone REA1297; Miltenyi Biotec, 130-127-984, clone REA1298); or CCR antibodies; or, for CAR and CCR, the absence or reduction of binding to an antigen such as CD19 antigen (Miltenyi, 130-129-550, ACROBiosystem, CD9-H52H2) by either direct ELISA or sandwich ELISA.

[0184] In the direct ELISA method, 10 samples are taken per well in a MAXISORP 96-well plate (Thermo Scientific, NUNC, 439454). 8Each EV was coated overnight in DPBS 1X (Gibco, 14190094). After saturating in 2% BSA (Thermo Scientific, 37525) for 2 hours, a primary antibody or antigen (listed above) within the dilution range was incubated for 1.5 hours. After washing with DPBS 1X and 0.05% Tween's solution (Sigma, P9416), a secondary antibody or streptavidin / avidin conjugated to HRP was incubated for 30 minutes. The HRP conjugates used were 1 / 5,000 of goat anti-mouse IgG(H+L)HRP conjugate (Invitrogen, 31430), 1 / 10,000 of goat anti-mouse IgG(H+L)HRP conjugate (Invitrogen, 31460), 1 / 10,000 of streptavidin-HRP (Thermo Scientific, 21130), or 1 / 5,000 of avidin-HRP (Biolegend, 405103). After washing the wells, 1X TMB substrate solution (Invitrogen, 00-4201-56) was added for 10 minutes, and the wells were stopped with stop solution (Thermofisher, N600). Absorbance was measured at 450 nm using GLOMAX Discover (Promega, GM3000). Figures 1A-C show titration curves obtained by direct ELISA using anti-CD9, anti-CD63, and CD81 antibodies for two independent batches of capsid-free EV.

[0185] Antibody pairs were selected to simultaneously detect two different protein surface epitopes (tetraspanin, VSVG, and / or CAR). After coating the primary antibody overnight, a saturation step was performed, followed by incubation of various EVs at room temperature for 2 hours using the sandwich ELISA method. After incubating the biotinylated antibody for 1.5 hours, it was washed and incubated with streptavidin-HRP (Thermo Scientific, Pierce, 21130) at a 1 / 10,000e dilution for 30 minutes. Absorbance was measured as described for the direct ELISA method. As shown in Figures 2A-C, the integrity of capsid-free EVs can be assessed using pairs of anti-CD9, anti-CD63, and CD81 antibodies in the sandwich ELISA method. As shown in Figures 3A-C, the titration profile and detection sensitivity of the sandwich ELISA method are equivalent whether the method is applied to capsid-containing EVs or capsid-free EVs.

[0186] Example 4 - Shaving process of EV The extracellular portion of the vector surface protein is digested by proteinase K or other broad-spectrum proteases, whether on a purified or unpurified vector.

[0187] Enzymatic treatment is performed by directly adding recombinant proteinase K (Invitrogen, 25530015) to the vector sample at a final concentration of 0.01–100 μg / mL. The mixture is incubated at room temperature for up to 2 hours. Proteolysis is then stopped with PMSF (Thermo Scientific) or AEBSF (Thermo Scientific). Excess PMSF or AEBSF must be used to completely inactivate the enzyme before proceeding with purification and encapsulation.

[0188] 5.9 × 10⁶ cells produced in HEK293-derived cells 9Each LV particle was treated with proteinase K (PK) at 25°C for 5 minutes, increasing the concentration of proteinase K. After inhibiting proteinase K with 20 mM AEBSF for 25 minutes, the size difference was evaluated by dynamic light scattering. The results are shown in Figure 19.

[0189] Example 5 - Purification of shaved EV Prior to polymer coating, it is essential to remove impurities after proteolysis. The purification strategy used is based on removing impurities from shaved EV using a method based on the amount / size of molecules.

[0190] If the volume of shaved EV is less than 10 mL, ultrafiltration is used with a PVDF or cellulose centrifugal filter (Millipore, AMICON ultra filter) with a molecular weight cutoff (MWCO) of 100-500 kD. After the vector concentration step, diafiltration is performed using at least three 1 / 10e dilution steps to ensure proper buffer exchange to the final polymer coating buffer.

[0191] For higher volumes and greater purity, purification, concentration, and formulation can be performed in a specific polymer-coated buffer using size exclusion chromatography (SEC), multimodal chromatography followed by tangential flow filtration (TFF, hollow fiber, or cassette), or the TFF step alone.

[0192] Four independent shaving treatment reactions (Batch 1: Panel A, Batch 2: Panel B, Batch 3: Panel C, Batch 4: Panel D) were performed at a rate of 1.43 × 10⁻⁶ 11The reaction was performed on individual LV particles, followed by a 15-minute incubation step with 5 μM proteinase K, and then inhibition with 20 mM AEBSF for 25 minutes. The reaction products were filtered either by successively passing through 0.45 μm and 0.22 μm PVDF membranes or without filtration. The size distribution of unshaved LV, shaved LV, or shaved and filtered LV produced in HEK293-derived cells was measured by MADLS (Multi-Angle Dynamic Light Scattering). The results are shown in Figures 20A-D.

[0193] For unshaved, shaved, and filtered LV particles, the change in zeta potential and the hydrodynamic diameter (z-mean), a size index as a function of pH, were measured by MADLS (Multi-Angle Dynamic Light Scattering). The results are shown in Figures 21A-B.

[0194] Example 6 - Evaluation of the shaving process Multiple detection of surface proteins Using the MACSPLEX exosome kit (Miltenyi Biotec, 130-108-813), surface epitopes detected on the EV are compared before and after shaving. This method allows for the quantification of expression of up to 37 human membrane epitopes using specific antibodies. The overnight protocol is followed according to the manufacturer's instructions.

[0195] Direct ELISA method and sandwich ELISA method EVs produced in or derived from HEK293T express human tetraspanins CD9, CD63, and CD81 on their surface. In addition, lentiviral vector particles may harbor VSV-G and possibly CAR on their surface. Therefore, the efficiency of the shaving process can be measured by either direct ELISA or sandwich ELISA by the absence or reduction of binding of anti-CD9 antibodies, anti-CD63 antibodies, anti-CD81 antibodies, anti-VSVG antibodies, and anti-CAR antibodies and recombinant antigens to the vector.

[0196] For the direct ELISA method, 10 per well 8 Each EV was coated overnight in 1X DPBS in a MAXISORP 96-well plate (Thermofisher, NUNC, 439454). After saturating in 2% BSA for 2 hours, a primary antibody in the dilution range was incubated for 1.5 hours, and after washing, an HRP-conjugated secondary antibody was incubated for 30 minutes. After washing the wells, 1X TMB substrate solution (eBiosciences, 00-4201-56) was added for 10 minutes, and the mixture was stopped in stop solution (Thermofisher N600). Absorbance was measured at 450 nm using GLOMAX DISCOVER (Promega, GM3000).

[0197] The antibody pairs are selected to simultaneously detect two different protein surface epitopes (tetraspanin, VSVG, and / or transgenes). After coating the primary antibody overnight, a saturation step is performed, followed by incubation of various vectors at room temperature for 2 hours to perform the sandwich ELISA method. After incubating the biotinylated antibody or biotinylated antigen for 1.5 hours, wash, and incubate with streptavidin-HRP (Thermo Scientific, Pierce, 21130) at a 1 / 10,000e dilution for 30 minutes. Measure the absorbance according to the instructions for the direct ELISA method.

[0198] Various LV batches were shaved with 10 nM, 500 nM, 1000 nM, and 5000 nM proteinase K for 15 minutes, followed by treatment with 20 mM AEBSF for 25 minutes. The resulting shaved EVs were then analyzed by capturing with anti-human CD63 and detecting with anti-human CD81, capturing with anti-human CD81 and detecting with anti-human CD9, capturing with anti-VSVG 8G5F11 and detecting with anti-human CD9, D) capturing with anti-VSVG 1E9F9 and detecting with anti-human CD9, capturing with anti-VSVG 8G5F11 and detecting with anti-human CD81, or capturing with anti-VSVG 1E9F11 and detecting with anti-human CD81, and compared with unshaved LVs. The results are shown in Figure 15. All markers used demonstrated that almost all of the tested surface markers could be removed by shaving. Shaving with 500–5000 nM proteinase K was sufficient to remove almost all surface proteins.

[0199] Western blotting for detection of intracellular and extracellular domains Tetraspanin, VSVG, CAR, and other proteins expressed on the eV surface are transmembrane proteins. Even after removal of the surface domain, their cytoplasmic domains remain intact and are not altered by the shaving procedure. Therefore, the integrity of EVs after shaving can be assessed by Western blotting using specific antibodies targeting the cytoplasmic domains of the following proteins: CD9 (Invitrogen, MA5-33125, clone 3A2), CD63, CD81 (AbboMax, 630-910), CD326 (Abcam, ab233919, clone EGP40-1110), VSVG (Abcam, ab50549, clone P5D4), CD3 zeta (Sigma-Aldrich, MABF3032, clone 6B10.2), and CD28 (Invitrogen, MA5-36070, clone RM404, Invitrogen, PA5-116751). First, the EV sample is dissolved in RIPA buffer (Thermo Scientific, 89900) or an equivalent lysis buffer containing NP-40 or Triton, and the protein is extracted from the EV sample. The extracted and denatured EV sample is loaded onto a NUPAGE 4-12% Bis-Tris gel (Thermofisher, NP0335BOX) and electrophoresis is performed in MES SDS running buffer (Thermofisher, NP0002) according to the manufacturer's instructions. Then, the blot is transferred onto a nitrocellulose membrane (Invitrogen, IB23002) using the iBlot 2 dry blotting system (Thermofisher). This blot is blocked for 1 hour in a solution of PBS 1X, 0.1% Tween 20 (PBST), and 3% BSA, and washed three times in PBST. Next, the primary antibody is incubated in PBST containing 0.2% BSA according to the manufacturer's instructions, then washed three times, and the HRP conjugate reagent is incubated. Detection is performed using the SUPERSIGNAL West Pico plus chemiluminescent substrate (Thermofisher, 34580), and then the images are acquired on the IBRIGHT FL1500 imaging system (Thermofisher, A44241).

[0200] Transduction efficiency The shaved vectors were titrated as follows, and the loss of infectivity after shaving was confirmed in two different cell lines, HEK293T cells and Jurkat cells.

[0201] Four hours before transduction, 8x10 in DMEM containing 10% FBS and 1% PS. 5 HEK293T cells were seeded in 24-well plates at 10 cells / well and incubated at 37°C and 5% CO2. Immediately before transduction, Jurkat cells were transferred to RPMI containing 10% FBS and 1% PS in 8x10⁶ cells. 5 Seed cells per well. Unshaved and shaved vectors are incubated on cells at 37°C in 5% CO2 using serial dilution (with the same physical titer). After 2 hours, fresh culture medium is added to each well, and the culture is extended to 3 days. On day 3, cells are harvested (with a trypsination step in the case of HEK293T), washed in DPBS 1X, and fixed directly with fixation buffer for GFP coding vectors (Biolegend, 420801). For CD19-CAR coding vectors, an additional staining step is performed by first incubating with CD19 CAR detection reagent (Miltenyi Biotec, 130-129-550) for 10 minutes before fixation, followed by washing and incubation with biotin antibody-VB515 (Miltenyi Biotec, 130-110-957).

[0202] The number of fluorescent positive cells after transfection with shaved vectors or unshaved vectors is measured using a flow cytometer (ATTUNE NXT, Thermofisher). As shown in Figure 9, the transfection efficiency of GFP-encoding EVs containing capsids is dose-dependent and equivalent in both cell models (regardless of mean fluorescence intensity or percentage of GFP cell readout values). The fact that the transgene reaches maximum expression on the third day after transfection and remains constant over 14 days supports the stable integration of the delivered transgene.

[0203] Genomic integration of shaved EVs, unshaved EVs, or transgenes delivered by PNP is quantified by qPCR. HEK293T cells or Jurkat cells are seeded at 8x10 4 cells / well in a 24-well plate. Shaved EVs, unshaved EVs, or PNP are serially diluted at least three times and transfected into the cells, which are then incubated at 37°C and 5% CO2 for 72 hours. After 72 hours, the cells are harvested and the pellets are recovered. Total genomic DNA is extracted from the transfected cell pellets using the MAXWELL RSC BLOOD DNA kit (Promega). Quantification of proviral DNA is performed by qPCR using PERFECTA MULTIPLEX TOUGHMIX (Quantabio).

[0204] Amplification of the integrated sequences is performed using probes and primers specific for the LTR (long terminal repeat sequence).

[0205] Using shaved LV samples and unshaved LV, Jurkat cells are transfected with various particles, namely 1.6×10 9 particles corresponding to MOI 5 and 6.1×10 9Transfection was performed with individual particles. From 3 days to 20 days after transfection, GFP expression was monitored by flow cytometry using the percentage of GFP-expressing cells (%) and the mean fluorescence intensity (MFI) of GFP. The copy number of the vector was quantified by qPCR on DNA extracted from the transfected cells from 3 days to 20 days. The results are shown in Figure 16. Shaving treatment with 500 - 5000 nM proteinase K was sufficient to reduce both GFP expression and proviral DNA integration, and at 5000 nM proteinase K, expression and integration were at undetectable levels.

[0206] In the case of proteinase K, 20 mg / mL = 692 μM. Therefore, 500 nM = 14.45 μg / mL, 1000 nM = 28.9 μg / mL, and 5000 nM = 144.5 μg / mL.

[0207] The transfection efficiency of shaved LV and unshaved LV was similarly evaluated in primary activated human T cells. The results are shown in Figure 17. Shaving treatment with 500 - 5000 nM proteinase K was sufficient to reduce both GFP expression and proviral DNA integration, and at 5000 nM proteinase K, expression and integration were at undetectable levels.

[0208] The effect of formulation buffer on the viability of Jurkat cells and LV introduction efficiency was evaluated in different formulation media at pH 5 or pH 7.4. The results are shown in Figure 18.

[0209] Physical titer Quantification of the physical titer enables titration of non-infectious shaved lentiviral vectors. Three orthogonal methods based on quantification of particle number (NTA), quantification of capsid P24 protein, or quantification of viral RNA copies (RT-qPCR) are used.

[0210] The particle concentration of shaved EV can be measured by nanoparticle tracking analysis using a ZETAVIEW instrument (Particle Metrix). Before measurement, newly prepared or frozen shaved EV can be diluted 1000-fold in DPBS-50 mM sucrose and homogenized by gentle vortexing.

[0211] The p24 capsid vector protein was detected and quantified using the HIV-1 p24 antigen ELISA 2.0 kit (Zeptometrix, 0801002) according to the manufacturer's instructions.

[0212] After isolating and purifying viral RNA using the MAXWELL RSC TNA virus kit (Promega, AS1330), the proviral RNA copies were quantified using the LENTI-X QRT-PCR titration kit (Takara, 631235) according to the manufacturer's instructions.

[0213] Example 7 - Coating of EVs using polymers Single layer coating Polymer coating of shaved vectors can be performed as follows: Dilute the polymer in the same pharmacopoeia-compliant formulation buffer used for the purified shaved vector and vortex briefly for homogenization. Add the polymer solution to the shaved vector in an appropriate mass ratio to alter the overall charge of the particles and homogenize by liquid-phase mixing (vortex or microfluidics, etc.). Incubate this complex at room temperature for 30 minutes before use for analysis or transduction.

[0214] A polymer coating can be constructed from a mixture of free polymers and polymers covalently bonded to a target site.

[0215] Virus particles and cationic polymers were diluted in a buffer of 20 mM histidine, 50 mM sucrose, and 127 mM pH 5 NaCl. LVs were added to the polymer, and a coating reaction was carried out at 25°C for 30 minutes. Measurements were then performed by multi-angle dynamic light scattering (MADLS). For LVs particles produced from HEK293-derived cells that were shaved and filtered, the hydrodynamic diameter (z-mean as a size measure) and zeta potential of polymeric nanoparticles were measured as a function of virus particle concentration or cationic polymer concentration. The results are shown in Figures 22A-B.

[0216] Virus particles and cationic polymers were diluted in a buffer of 20 mM histidine, 50 mM sucrose, and 127 mM pH 5 NaCl. LVs were added to the polymer, and a coating reaction was carried out at 25°C for 30 minutes. Measurements were then performed by multi-angle dynamic light scattering (MADLS). Changes in hydrodynamic diameter (z-mean, a measure of size) and size distribution (PdI, representing the polydispersity index) of polymeric nanoparticles obtained from two different batches of shaved (with 5000 nM proteinase K added) and filtered LVs particles produced in HEK293-derived cells were measured. The results are shown in Figures 23A-B.

[0217] Alternating lamination coating The alternating lamination method adds a shielding polymer to coated and shaved vectors prepared according to the "single-layer coating" method using a cationic polymer. To this end, excess polymer is removed from the formed polymeric nanoparticles using a hollow fiber tangential flow filtration step. This step allows for the recombination of the coated nanoparticles in a pharmacopoeia-compliant buffer used to dilute the shielding polymer. The shielding polymer solution consists of a mixture of free polymer and polymer covalently bonded to the targeting moiety. After a short mixing (e.g., vortexing) for homogenization, the shielding polymer solution is added to the coated nanoparticles in an appropriate mass ratio to alter the global charge of the particles, and homogenization is performed by liquid-phase mixing (e.g., vortexing or microfluidics). After incubation of this complex at room temperature for 30 minutes, it is used for analysis or transduction. Again, excess polymer is removed by the hollow fiber tangential flow filtration step.

[0218] Virus particles, H-PBAE (cationic polymer), and HA (anionic polymer) were diluted in a buffer of 20 mM histidine, 50 mM sucrose, and 127 mM NaCl (pH 5). LV was added to the polymers, and a coating reaction was carried out at 25°C for 30 minutes. Measurements were then performed by multi-angle dynamic light scattering (MADLS). The nanoparticles obtained after the first coating step were added to the anionic HA polymer, and a coating reaction was carried out at 25°C for 30 minutes. Measurements were then performed by MADLS. The results are shown in Figures 24A-B.

[0219] Virus particles, cationic polymers (PBAE-447, H-PBAE, and R-PBAE), and anionic polymers (heparosan and HA) were diluted in a buffer of 20 mM histidine, 50 mM sucrose, and 127 mM NaCl (pH 5). LV was added to the polymers, and a coating reaction was carried out at 25°C for 30 minutes, after which measurements were performed by multi-angle dynamic light scattering (MADLS). Nanoparticles obtained after the first coating step were added to heparosan or HA anionic polymers, and a coating reaction was carried out at 25°C for 30 minutes, after which measurements were performed by MADLS. The results are shown in Figures 25A-D.

[0220] Characterization Method Using multi-angle dynamic light scattering (MADLS), the total charge (zeta potential), size (mean hydrodynamic diameter), and size distribution (polydispersion index) of shaved vectors are compared before and after polymer addition to confirm coating efficiency. Newly prepared coated vectors can be diluted 16-fold (zeta potential and polydispersion index) or 20-fold (mean hydrodynamic diameter) in the final formulation buffer, homogenized by gentle vortexing, and then analyzed on a ZETASIZER ULTRA instrument (Malvern Instruments).

[0221] Both EVs containing and without capsids exhibit similar overall charge (see Figure 5), size (see Figures 6 and 7), and size distribution (see Figures 7 and 8). These biophysical properties are maintained even across batches, providing further evidence of the robustness of the biomanufacturing process performed using HEK293-derived cells.

[0222] As shown in Fig. 12, when protease K-treated EVs without capsids are used, the extracellular portion of the proteins expressed on the surface of the EVs is removed, resulting in a 10% decrease in the size of the vector. Surprisingly, this modification occurs only at a limited range of protease concentrations (0.03 - 0.3 μg / mL). Excess protease induces the formation of EVs of larger sizes, probably due to the formation of protein / particle aggregates.

[0223] Since the shaving treatment process modifies the biophysical properties of the vector, it is extremely important to precisely control the coating reaction and the behavior of the polymer in solution. As shown in Fig. 11, when a cationic polymer is exposed to a pharmacopoeial-compliant formulation buffer, it can form self-assemblies. Therefore, the coating conditions for the shaving-treated EVs must generate nanoparticles with a size, size distribution, and zeta potential that can be easily distinguished from excess polymer, and the excess polymer must be easily removable from the formulated product.

[0224] Transduction efficiency The titration of polymeric nanoparticles can be performed as follows to confirm the recovery of infectivity after a single-layer coating method or an alternating layer-by-layer coating method on two different cell lines, HEK293T cells and Jurkat cells.

[0225] Four hours before transduction, HEK293T cells are seeded in a 24-well plate at 8 x 10 5 cells / well in DMEM containing 10% FBS and 1% penicillin / streptomycin (PS) and incubated at 37°C and 5% CO2. Immediately before transduction, Jurkat cells are seeded at 8 x 10 5Seed cells per well. Incubate shaved vectors and serial dilutions of shaved and shaved vectors (with the same physical titer) on the cells at 37°C and 5% CO2. After 2 hours, add fresh culture medium to each well and extend the culture to 3 days. On day 3, harvest the cells (with a trypsination step in the case of HEK293T), wash with DPBS 1X, fix directly with fixation buffer (Biolegend, 420801) in the case of GFP coding vectors, and identify viability by Zombie NIR. For CD19-CAR coding vectors, perform an additional staining step by first incubating with CD19 CAR detection reagent (Miltenyi Biotec, 130-129-550) for 10 minutes before fixation, washing, and then incubating with biotin antibody-VB515 (Miltenyi Biotec, 130-110-957).

[0226] The number of surviving fluorescent cells after transduction with shaved vectors, or coated and shaved vectors, is measured using a flow cytometer (ATTUNE NXT, Thermofisher).

[0227] Genomic integration of transgenes delivered via shaved EVs, unshaved EVs, or PNPs is quantified by qPCR. HEK293T cells or Jurkat cells are placed in 24-well plates in 8x10⁶ cells. 4Seed cells per well. Transduction was performed by sequentially diluting shaved or unshaved extracellular viable cells (EVs) at least three times, and the cells were incubated at 37°C and 5% CO2 for 72 hours. After 72 hours, the cells were harvested and the pellet was collected. Whole genomic DNA was extracted from the transduced cell pellet using the MAXWELL RSC Blood DNA Kit (Promega). Proviral DNA was quantified by qPCR using PERFECTA MultiPlex ToughMix (Quantabio). The integrated sequence was amplified using LTR (long terminal repeat) specific probes and primers.

[0228] Example 8 - Coating treatment of EV using a targeting agent The coating treatment of EVs with a targeting agent is generally carried out as described in Example 7, except that a mixture of free polymers and polymers covalently bonded to the targeting moiety is used. The buffer composition is adapted so as not to affect the solubility of the selected polymer bonded to the targeting agent.

[0229] Table 3 summarizes the characteristics of aptamers used as specific targeting agents for T lymphocytes or NK cells. [Table 3]

[0230] Example 9 - Contact between PNP and cells The ability to introduce a target gene by contacting primary cells with PNPs containing or without a targeting agent, thereby restoring transduction, or potentially by transfection, will be evaluated.

[0231] Contact with the cell lines was performed on Jurkat cells and HEK293T cells as described in Example 7.

[0232] Contact between PNPs and primary cells is performed on peripheral blood mononuclear cells (PBMCs) or on isolated human T cells and NK cells. Human buffy coats are collected from Etablissement Francais du Sang (Rungis, France). PBMCs are purified by FICOLL (Cytiva, FICOLL-PAQUE PLUS) gradient separation. Next, T cells or NK cells are purified from the PBMCs by magnetic separation using a T cell or NK cell negative or positive separation kit (Miltenyi Biotec), or directly from the buffy coat using a STRAIGHTFROM kit (Miltenyi Biotec). The cells are then cultured in TEXMACS medium (Miltenyi Biotec, 130-097-196), with or without T cell Transact (Miltenyi Biotec, 130-128-758), for polyclonal stimulation. Polymer-coated EV / PNPs are cultured in PBMCs, isolated T cells, or NK cells at different MOIs (MOI 1, 2, 5, 10, 20, 50, or 100) at 37°C and 5% CO2. GFP or CAR expression is evaluated by flow cytometry (ATTUNE NXT, Thermofisher) at days 3, 7, 10, and 14 post-transduction to assess the stability of transgene expression.

[0233] Simultaneously, cell pellets are collected, and integration into the cell genome is quantified from the extracted genomic DNA and by qPCR. Overall, flow cytometry-based expression of the transgene and time-dependent quantification by qPCR allow for the characterization of the stability of gene transfer (e.g., stable transduction or transient transfection).

[0234] Furthermore, detailed cellular phenotypic analysis is performed using flow cytometry at each point in time after contact with the coated and shaved EVs.

[0235] Among PBMCs, viable CD3+ T lymphocytes, CD19+ B cells, CD56+ NK cells, CD3+CD56+ NKT cells, and CD16+ / SSCs are available. high Neutrophils and CD16- / SSC high Eosinophils and CD14+ monocytes (including classical, intermediate, and non-classical subsets) are characterized by their proportion and GFP or CD19CAR expression levels. The contact specificity of coated EV / PNPs with targeting agents was compared with coated EV / PNPs without targeting agents.

[0236] The phenotype of isolated T cells expressing the transgene will be studied by detecting CD4+ T cells, CD8+ T cells, Treg cells, and TCR gamma / delta T lymphocytes. Among the CD4+ and CD8+ T cell subpopulations, naive cells, memory cells, effector memory cells, and central memory cells will be characterized using antibodies specific to CD45RA, CD62L, CCR7, CD27, CD45RO, and CD127. Activation markers such as CD25, CD30, CD69, CD137, and CD154 will be detected, and fatigue profiles after contact with coated EV / PNPs will be evaluated using antibodies specific to PD1, CTLA4, TIM3, LAG3, and TIGIT.

[0237] Isolated NK cells expressing GFP or CD19 CAR are detected using CD56+ expression and CD3+ expression absence. NK phenotyping is performed to evaluate NK activation and fatigue profiles using a range of markers including, but not limited to, NKG2A, NKG2D, NKp30, NKp44, NKp46, CD11a, CD11c, CD16, CD25, CD28, CD54 (ICAM-1), CD69, CD226, DNAM-1, TIM3, and PD1.

[0238] Finally, human cytokines secreted by PBMCs, isolated T cells, or NK cells at 3, 7, 10, and 14 days post-culturing were quantified on the cell culture supernatant using Biolegend's LEGENDPLEX kit.

[0239] References Du R., Wang C., Zhu L., and Yang Y. Extracellular Vesicles as Delivery Vehicles for Therapeutic Nucleic Acids in Cancer Gene Therapy: Progress and Challenges, Pharmaceutics, 2022, 14:2236 Teng F. and Fussenegger M. Shedding Light on Extracellular Vesicle Biogenesis and Bioengineering, Advanced Science, 2021, 8:2003505 Joshi B.S., Ortiz D., and Zuhorn I.S. Converting extracellular vesicles into nanomedicine: loading and unloading of cargo, Materials Today Nano, 2021, 16:100148 Rezaie J., Feghhi M., & Etemadi T. A review on exosomes application in clinical trials: perspective, questions, and challenges, Cell Communication and Signaling, 2022, 20:145 Rheinemann L. and Sundquist W.I. Virus Budding, Encyclopedia of Virology, 4 th Edition, 2021, 519 - 528 Coffin J.M., Hughes S.H, and Varmus H.E. Retroviruses, Cold Spring Harbor Laboratory Press, 1997 Bell N.M.and LeverA.M.L.HIV Gag polyprotein:processing and early viral particle assembly,Trends in Microbiology,2013,21:136-44 Dautzenberg I.J.C ,Martijn J.W.E.and Hoeben R.C.The stability of envelope-pseudotyped lentiviral vectors,Gene Therapy,2021,28:89-104 Chen J.,Li P.,Zhang T.,Xu Z.,Huang X.,Wang R and Du.L.Review on Strategies and Technologies for Exosome Isolation and Purification,Frontiers in Bioengineering and Biotechnology,2022,9:811971,1-18 Menon A.P.,Moreno B.,Meraviglia-Crivelli D.,Nonatelli F.,Villanueva H.,Barainka M.,Zheleva A.,van Santen H.M.and Pastor F.Modulating T Cell Responses by Targeting CD3,Cancers,2023,15:1189 Lian G.,Mak T.S.K.,Yu X.and Lan H.Y.Challenges and Recent Advances in NK Cell-Targeted Immunotherapies in Solid Tumors,International Journal of Molecular Sciences,2022,23:164 Zufferey,R.,Nagy D.,Mandel R.J.,Naldini L.and Trono D.Multiply attenuated lentiviral vector achieves efficient gene delivery in vivo,Nature Biotechnology,1997,15:871-875 Sadelain M.,Brentjens R.and Riviere I.The basic principles of chimeric antigen receptor design,Cancer Discovery,2013,3:388-398 Kloss C.C.,Condomines M.,Cartellieri M.,Bachmann M.and Sadelain M.Combinatorial antigen recognition with balanced signaling promotes selective tumor eradication by engineered T cells,Nature Biotechnology,2013,31:71-75 Riccardi C.,Napolitano E.,Musumeci D.and Montesarchio D.Dimeric and Multimeric DNA Aptamers for Highly Effective Protein Recognition,Molecules,2020,25:5227 Levine B.L,Humeau L.M.,Boyer J.,MacGregor R.R.,Rebello T,Lu X.,Binder G.K.,Slepushkin V.,Lemiale F.,Mascola J.R.,Bushman F.D.,Dropulic B.and June C.H.Gene transfer in humans using a conditionally replicating lentiviral vector,PNAS,2006,103:17372-17377 Perry C.and Rayat A.C.M.E.Lentiviral Vector Bioprocessing,Viruses,2021,13:268

Claims

1. Extracellular vesicles (EVs), which are treated with a protease to remove proteins from the outer surface of the EVs.

2. The EV according to claim 1, wherein the EV does not have any full-length viral envelope protein on its surface.

3. The EV according to claim 1 or 2, comprising a retroviral capsid containing a pair of RNAs encoding non-lentiviral proteins.

4. The EV according to claim 3, wherein the RNA encodes a chimeric antigen receptor.

5. The EV according to any one of claims 1 to 4, wherein the EV does not have any full-length CARs or non-lentiviral proteins on its surface.

6. The EV according to any one of claims 3 to 5, wherein the capsid is an HIV-1 capsid.

7. The EV according to any one of claims 1 to 6, wherein the EV is produced in a mammalian cell line.

8. The EV according to any one of claims 1 to 7, wherein the protease is proteinase K.

9. An EV according to any one of claims 1 to 8, which is coated with a cationic polymer.

10. An EV according to any one of claims 1 to 8, which is coated with an anionic polymer.

11. An EV according to any one of claims 1 to 8, which is coated with a zwitterionic polymer.

12. The EV according to any one of claims 1 to 11, further comprising a CD3 or NKG2D targeting agent.

13. An EV according to any one of claims 1 to 12, comprising a polymer selected from polyarginine, polylysine, polyethyleneimine (PEI), chitosan, poly(β-aminoester) (PBAE), PEI-co-polyhistidine copolymer, polylysine denzigraft / dendrimer / branched polymer, star-shaped polyamino acid-based polymer, chondroitin sulfate, heparosan, polyglutamic acid (PGA), hyaluronic acid, dextran sulfate, polysarcosine, polyethylene glycol (PEG), polycarboxybetaine, polysulfobetaine, or polyphosphorylcholine.

14. The EV according to any one of claims 3 to 13, wherein the capsid comprises reverse transcriptase.

15. The EV according to any one of claims 3 to 14, wherein the capsid comprises integrase.

16. A cell containing EV according to any one of claims 1 to 13.

17. A method comprising bringing an EV according to any one of claims 1 to 15 into contact with a mammalian cell.

18. The method according to claim 17, wherein the cells are human cells.

19. The method according to claim 18, wherein the cells are T cells or NK cells.

20. A method for modifying the outer surface of extracellular vesicles (EVs), a) Purifying EV from cells, b) Treating the EV with a protease to remove the protein on the outer surface of the EV, c) Purifying the processed EV from the protease, Methods that include...

21. The method according to claim 20, wherein the cells express retroviral Gag protein and Pol protein.

22. The method according to claim 20 or 21, wherein the EV comprises RNA encoding a non-lentiviral protein.

23. The method according to claim 23, wherein the RNA encodes a chimeric antigen receptor.

24. The method according to any one of claims 20 to 23, further comprising bringing the EV into contact with mammalian cells.

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