Controlled Expression of Therapeutically Relevant Biomolecules for Luminal-Localized Payloads in Biomimetic Nanovesicles and Exosomes

Abstract

Disclosed herein are methods for generating therapeutic biomimetic nanovesicles (BioNVs) or therapeutic exosomes filled with treatment-related biomolecules derived from hypoimmunogenic cells in the lumen, compositions of the therapeutic BioNVs or therapeutic exosomes, and methods of using them for treating or preventing a disease or disorder.

Description

Technical Field

[0001] The present disclosure provides, in part, methods for preparing lumen-filled biomimetic nanovesicles and exosomes, as well as compositions thereof and methods of using them, for the treatment of diseases in mammalian subjects such as humans.

[0002] Cross - Reference to Related Applications This application claims the benefit and priority of U.S. Provisional Patent Application No. 63 / 345,659, filed May 25, 2022, and U.S. Provisional Patent Application No. 63 / 424,978, filed November 14, 2022, the contents of each of which are hereby incorporated by reference in their entirety.

Background Art

[0003] Cell - derived vesicles (CDV) such as biomimetic nanovesicles (BioNV) and exosomes are powerful and novel tools for treating a number of diseases, including cancer, genetic diseases, tissue damage, endocrine diseases, and infectious diseases. BioNV can be obtained from cells through various cell disruption processes. In contrast, exosomes are naturally released from whole cells via the endocytosis pathway. The diameter and volume of both BioNV and exosomes (the latter being defined by the lumen compartment within the lipid bilayer) can be pre - determined depending on the method used to disrupt the cells from which they are derived (or, in the case of exosomes, to process the cells).

[0004] The lumen of BioNV can be filled with therapeutically relevant molecules mainly in three ways: (1) by spontaneously encapsulating biomolecules (nucleic acids, proteins / peptides) expressed intracellularly from which CDV is derived into the lumen of CDV; (2) by adding (bio)molecules (nucleic acids, proteins / peptides, small molecules, or biological agents) to the concentration chambers divided during BioNV formation to ensure controlled filling; or (3) a combination of (1) and (2), where essential cell-derived biomolecules are encapsulated within the lumen of CDV while desired biomolecules are added for a specific purpose (e.g., addition of doxorubicin for delivery to targeted cancer cells).

[0005] Cells express numerous biomolecules (cytokines, chemokines, regulatory nucleic acids, etc.) that generally assist in cell regeneration, tissue repair, and cytotoxicity through complex pathways governed by external stimuli. These biomolecules typically begin with surface receptors that sense environmental stimuli, followed by triggering the intracellular domain of the receptor, resulting in signal transduction events that may involve complex cross-talk between signal transduction pathways propagating intracellularly through tightly regulated cellular pathways. The endpoints of these signal transduction pathways are generally systems of transcriptional activators and repressors that regulate the gene function and expression of biomolecules. Within these complex and organized pathways, there are multiple types of migrating effector molecules and biomolecules such as phosphate-containing compounds (cAMP, ATP, GTP, etc.), metal ions (calcium, iron, zinc, etc.), signal-regulating enzymes (phosphatases, kinases, phosphorylases, etc.), transcription factors and initiators, as well as translation factors, forming layers.

[0006] Each cell type within an organism has a set of distinct surface receptors, each with different downstream functions, ultimately resulting in a specific biomolecular repertoire. The expression of these biomolecules from healthy cells generally contains effector functions designed for the protection and maintenance of homeostasis of the cell or the tissue in which they reside, as well as for the overall survival of the organism. For example, T cells contain a T cell receptor (TCR) or a chimeric antigen receptor (when engineered), which, when engaged with an infected or transformed cell, triggers the T cell to initiate the rapid expression of inflammatory cytokines and apoptotic cytokines, including interferon, perforin, granzyme, and granulysin. These cytokines form lytic granules within the cytoplasm and then localize at the interface (called the immunological synapse (IS)) between the TCR and the ligand of the target cell. In another example, M1 macrophages are activated through the CD38 surface receptor and resist bacterial invasion of cells via pro-inflammatory cytokines, activating the phagocytosis and digestion of necrotic cells. Also, M2 macrophages are activated through the EGR2 surface receptor and shift the cells into a repair mode by (partially) producing anti-inflammatory cytokines. In another example, natural killer cells contain multiple surface and transmembrane receptors linked to a complex intercellular signaling network, which gives the cells excellent environmental sensing properties, allowing them to precisely recognize and destroy the difference between healthy cells and infected or transformed cells. Because this complex network is sensitive, natural killer cells have adapted multiple intercellular signaling pathways and specific receptors that often cooperate in a synergistic manner to regulate their cytotoxic phenotype against specific diseases caused by viral or bacterial infections, genetic abnormalities, or cancer. This level of synergy between receptor-signaling pathways is necessary for natural killer cells because they are primed / preloaded with cytotoxic secretory lysosomes containing perforin, granzyme, granulysin, and other cytotoxins that can cause chaos if not tightly regulated.Some subsets of CD4+ and CD8+ T cells (excluding CD8+ killer T cells primed with low levels of lytic granules) are not primed with lytic granules and thus do not require such a stringent biochemical regulatory network.

[0007] Recent reports have shown that, in addition to immune regulatory cells, there is a strong correlation of cytokine signaling between leukocytes and cardiomyocytes, and their interaction plays an important role in the regulation of inflammation after heart injury. In patients with heart injury, the pro-inflammatory cytokines tumor necrosis factor TNF-α, interleukin-6 (IL-6), and IL-1β are observed at high levels. These cytokines may be induced under pathological conditions of heart tissue, and the runaway of their pro-inflammatory effects is associated with the high mortality rate of heart failure patients. In cardiomyocytes, low levels of IL-6 expression may have the advantage of repairing heart tissue, but high levels can lead to the runaway of inflammation and heart failure. IL-6 is regulated by the level of IL-1β, suggesting that by directly regulating IL-1β, the level of IL-6 can be manipulated and adjusted in a positive and controlled manner.

[0008] To encapsulate a therapeutically beneficial desired biomolecule into a delivery system such as AAV, an anellovirus, or a lipid nanoparticle (LNP), a complex encapsulation process is required. Currently, the concentration of biomolecule(s) that can be encapsulated into these systems and the types of biomolecule(s) are very limited and depend on the engineered promoter gene construct and encapsulation size limitations. Further, naturally secreted exosomes have the ability to combine multiple biomolecules from the cells from which they are secreted, but typically have a low, undesired, and not "controllable" concentration of the desired biomolecule(s) (compared to BioNV). This is due to the standard pathways involved in the types of these extracellular vesicles (EVs). Thus, an additional system is needed to control the filling of the lumen. Further, cell-derived vesicles (CDVs) from resting cells in a "natural somatic state" have limited therapeutic sources because they have low (or no / suppressed) expression levels of naturally occurring biomolecules with therapeutic properties. Therefore, there is still a need for a method to control and regulate the gene expression of therapeutically desirable / related biomolecules within the genotype repertoire of a cell and encapsulate them into the lumen of BioNV (or exosomes) to enable treatment of diseases by the concentration and types of therapeutically desirable biomolecules. SUMMARY OF THE INVENTION

[0009] In various embodiments, a method of generating a therapeutic biomimetic nanovesicle (BioNV) includes (a) obtaining low-immunogenicity cells, (b) activating the low-immunogenicity cells to express one or more therapeutically relevant biomolecules, and (c) treating the activated low-immunogenicity cells to generate a therapeutic BioNV, wherein the therapeutic BioNV comprises the plasma membrane of the low-immunogenicity cells and encapsulates one or more therapeutically relevant biomolecules.

[0010] In various embodiments, a method of generating therapeutic extracellular vesicles (EVs), such as exosomes and microsomes, comprises: (a) obtaining hypoimmunogenic cells; (b) activating the hypoimmunogenic cells to express one or more therapeutically relevant biomolecules; and (c) harvesting therapeutic exosomes naturally secreted from the activated hypoimmunogenic cells, wherein the therapeutic exosomes comprise the plasma membrane of the hypoimmunogenic cells and encapsulate the one or more therapeutically relevant biomolecules.

[0011] In various embodiments, a method of treating or preventing a disease or disorder comprises administering a therapeutically effective amount of a therapeutic biomimetic nanovesicle (BioNV) generated according to claim 1 to a subject in need thereof.

[0012] In various embodiments, a method of treating or preventing a disease or disorder comprises administering a therapeutically effective amount of a therapeutic exosome generated according to claim 2 to a subject in need thereof.

[0013] In various embodiments, the hypoimmunogenic cells are stem cells, induced pluripotent stem cells (iPSCs), reprogrammed totipotent or pluripotent cells, embryonic stem cells, mesenchymal stem cells, or differentiated cells derived from any of these stem cells. In various embodiments, the hypoimmunogenic cells are T cells, helper T cells, memory T cells, or NK cells. In various embodiments, the hypoimmunogenic cells are macrophages. In various embodiments, the hypoimmunogenic cells are monocytes. In various embodiments, the hypoimmunogenic cells are hepatocytes, cardiomyocytes, neurons, endothelial cells, pancreatic cells, or retinal pigment epithelial (RPE) cells.

[0014] In various embodiments, the low immunogenicity cells substantially lack one or more MHC class I proteins, MHC class II proteins, T cell receptor (TCR) proteins, and / or cytokine release syndrome (CRS) proteins. In various embodiments, the low immunogenicity cells have a disruption of the β2-microglobulin (B2M) gene and / or a disruption that reduces or ablates the expression and / or activity of MHC class I proteins. In various embodiments, the low immunogenicity cells have a disruption of the CIITA gene and / or a disruption that reduces or ablates the expression and / or activity of MHC class II proteins.

[0015] In various embodiments, the low immunogenicity cells have a disruption of the HLA-A gene and / or a disruption that reduces or ablates the expression and / or activity of HLA-A proteins. In various embodiments, the low immunogenicity cells have a disruption of the HLA-B gene and / or a disruption that reduces or ablates the expression and / or activity of HLA-B proteins. In various embodiments, the low immunogenicity cells have a disruption of the HLA-C gene and / or a disruption that reduces or ablates the expression and / or activity of HLA-C proteins. In various embodiments, the low immunogenicity cells have a disruption of the HLA-E gene or a disruption of the HLA-G gene and / or a disruption that reduces or ablates the expression and / or activity of HLA-E or HLA-G proteins. In various embodiments, the low immunogenicity cells have a disruption of the HLA-F gene and / or a disruption that reduces or ablates the expression and / or activity of HLA-F proteins.

[0016] In various embodiments, the low immunogenicity cells have a disruption of the T cell alpha constant (TRAC) gene and / or a disruption that reduces or ablates the expression and / or activity of TRAC proteins. In various embodiments, the low immunogenicity cells have a disruption of the T cell beta constant (TRBC) gene and / or a disruption that reduces or ablates the expression and / or activity of TRBC proteins.

[0017] In various embodiments, the hypoimmunogenic cells have reduced or absent expression of the PD-1 gene and / or reduced or absent expression and / or activity of the PD-1 protein, where the hypoimmunogenic cells are activated; or the hypoimmunogenic cells express the PD-1 gene and / or gene product or have increased expression thereof, where the hypoimmunogenic cells are not activated. In various embodiments, the hypoimmunogenic cells have a disruption of the IL-4 gene and / or a disruption that reduces or ablates the expression and / or activity of the IL-4 protein. In various embodiments, the hypoimmunogenic cells have a disruption of the IL-6 gene and / or a disruption that reduces or ablates the expression and / or activity of the IL-6 protein. In various embodiments, the hypoimmunogenic cells have a disruption of the IL-10 gene and / or a disruption that reduces or ablates the expression and / or activity of the IL-10 protein. In various embodiments, the hypoimmunogenic cells have a disruption of the IL-16 gene and / or a disruption that reduces or ablates the expression and / or activity of the IL-16 protein.

[0018] In various embodiments, the hypoimmunogenic cells have a disruption of the SerpinB9 gene and / or a disruption that reduces or ablates the expression and / or activity of the SerpinB9 protein. In various embodiments, the hypoimmunogenic cells express the SerpinB9 gene and / or gene product or have increased expression and / or activity thereof.

[0019] In various embodiments, the hypoimmunogenic cells express the CCL2 gene and / or gene product or have increased expression and / or activity thereof. In various embodiments, the hypoimmunogenic cells express the PD-L1 gene and / or gene product or have increased expression and / or activity thereof, where the hypoimmunogenic cells are not activated. In various embodiments, the hypoimmunogenic cells express the H2-M3 gene and / or gene product or have increased expression and / or activity thereof.

[0020] In various embodiments, the low immunogenicity cells express the CD47 gene and / or gene product, or have increased expression and / or activity thereof. In various embodiments, the low immunogenicity cells express the CD24 gene and / or gene product, or have increased expression and / or activity thereof. In various embodiments, the low immunogenicity cells express the chimeric CD24 / CD47 gene and / or gene product, or have increased expression and / or activity thereof.

[0021] In various embodiments, the low immunogenicity cells express the CTLA-4 gene and / or gene product, or have increased expression and / or activity thereof. In various embodiments, the low immunogenicity cells express the CD200 gene and / or gene product, or have increased expression and / or activity thereof. In various embodiments, the low immunogenicity cells express the chimeric CD24 / CD200 gene and / or gene product, or the chimeric CD47 / CD200 gene and / or gene product, or have increased expression and / or activity thereof. In various embodiments, the low immunogenicity cells express the MFG-E8 gene and / or gene product, or have increased expression and / or activity thereof. In various embodiments, the low immunogenicity cells express the NCAM gene and / or gene product, or have increased expression and / or activity thereof. In various embodiments, the low immunogenicity cells express the α-phagocyte integrin gene and / or gene product, or have increased expression and / or activity thereof. In various embodiments, the low immunogenicity cells express an antibody or antibody format molecule (anti-IL-6R) that targets the IL-6 surface receptor, or have increased expression and / or activity thereof.

[0022] In various embodiments, the low immunogenicity cells express the FasL gene and / or gene product. In various embodiments, the low immunogenicity cells do not overexpress the FasL gene and / or gene product.

[0023] In various embodiments, the hypoimmunogenic cells substantially lack the expression and / or activity of one or more immunogenic proteins and express or have increased expression of one or more immunoprotective proteins.

[0024] In various embodiments, the hypoimmunogenic cells have reduced or absent expression and / or activity of three or more immunogenic proteins, four or more immunogenic proteins, five or more immunogenic proteins, six or more immunogenic proteins, seven or more immunogenic proteins, eight or more immunogenic proteins, nine or more immunogenic proteins, ten or more immunogenic proteins, eleven or more immunogenic proteins, or twelve or more immunogenic proteins. In various embodiments, the hypoimmunogenic cells express or have increased expression of three or more immunoprotective proteins, four or more immunoprotective proteins, five or more immunoprotective proteins, six or more immunoprotective proteins, seven or more immunoprotective proteins, eight or more immunoprotective proteins, nine or more immunoprotective proteins, or ten or more immunoprotective proteins.

[0025] In various embodiments, the hypoimmunogenic cells have reduced or absent expression and / or activity of any one gene and / or gene product of HLA-A, HLA-B, HLA-C, HLA-F, CIITA, IL-6, TRAC, TRBC, and either HLA-E or HLA-G.

[0026] In various embodiments, the hypoimmunogenic cells have reduced or absent expression and / or activity of any one gene and / or gene product of HLA-A, HLA-B, HLA-C, HLA-F, CIITA, IL-6, TRAC, TRBC, SerpinB9, and either HLA-E or HLA-G.

[0027] In various embodiments, the hypoimmunogenic cells have reduced or absent expression and / or activity of any one gene and / or gene product of HLA-A, HLA-B, HLA-C, HLA-F, CIITA, IL-6, TRAC, TRBC, SerpinB9, and either HLA-E or HLA-G.

[0028] In various embodiments, the hypoimmunogenic cells include hypoimmunogenic cells in which the expression and / or activity of any one of HLA-A, HLA-B, HLA-C, HLA-F, CIITA, IL-6, TRAC, TRBC, SerpinB9, HLA-E, or HLA-G, and one or more genes and / or gene products among IL-4, IL-10, and IL-16 are reduced or eliminated.

[0029] In various embodiments, the hypoimmunogenic cells express α-phagocyte integrin, CCL2, H2-M3, FasL, MFEG8, and / or express or have increased expression and / or activity of PD-L1 and / or CTLA-4, where the hypoimmunogenic cells do not overexpress FasL, the hypoimmunogenic cells are not activated by the expression of PD-L1, and express any one of CD24, CD47, CD200, chimeric CD24 / CD47, chimeric CD24 / CD200, and chimeric CD47 / CD200, or express or have increased expression and / or activity of any two of CD24, CD47, and CD200.

[0030] In various embodiments, the hypoimmunogenic cells express α-phagocyte integrin, CCL2, H2-M3, FasL, MFEG8, SerpinB9, and / or express or have increased expression and / or activity of PD-L1 and / or CTLA-4, where the hypoimmunogenic cells do not overexpress FasL, the hypoimmunogenic cells are not activated by the expression of PD-L1, and express any one of CD24, CD47, CD200, chimeric CD24 / CD47, chimeric CD24 / CD200, and chimeric CD47 / CD200, or express or have increased expression and / or activity of any two of CD24, CD47, and CD200.

[0031] In various embodiments, the low immunogenicity cells express the CD200 gene and / or gene product, or have increased expression and / or activity thereof, and do not express and / or are substantially deleted of either the CD24 or CD47 gene and / or gene product. In various embodiments, the low immunogenicity cells do not have the expression and / or activity of the SerpinB9 gene and / or gene product and the CD200 gene and / or gene product.

[0032] In various embodiments, the low immunogenicity cells are allogeneic. In various embodiments, the low immunogenicity cells do not cause an immune response in the patient to whom the cells or BioNV derived therefrom are administered.

[0033] In various embodiments, the low immunogenicity cells comprise one or more targeting agents. In various embodiments, the one or more targeting agents comprise a chimeric antigen receptor (CAR). In various embodiments, the CAR is bispecific. In various embodiments, the CAR lacks an intracellular portion. In various embodiments, the CAR is a targeting agent, a transmembrane domain, and an intracellular domain comprising an intracellular domain comprising a costimulatory domain and / or a signaling domain. In various embodiments, the transmembrane domain is derived from CD28, CD3ζ, CD4, CD8α, or ICOS, or fragments thereof. In various embodiments, the intracellular domain comprises the intracellular signaling domain of the CD3ζ chain and / or one or more costimulatory molecules, optionally selected from CD28, 4-1BB, ICOS, CD27, and OX40. In various embodiments, the one or more targeting agents comprise an antibody or antibody format. In various embodiments, the antibody or antibody format is a monoclonal antibody, a polyclonal antibody, an antibody fragment, Fab, Fab’, Fab’-SH, F(ab’)2, Fv, single-chain Fv (scFv), V NAR 、V HIt is selected from one or more of H, affilin, diabody, nanobody, linear antibody, bispecific antibody, multispecific antibody, chimeric antibody, humanized antibody, human antibody, and a fusion protein containing an antigen-binding portion of an antibody. In some embodiments, the antibody format is scFv. In some embodiments, one or more targeting agents include a viral epitope recognition receptor (VERR) or a viral ligand. In some embodiments, one or more targeting agents include a ligand for a receptor. In some embodiments, one or more targeting agents include a receptor for a ligand.

[0034] In some embodiments, the hypoimmunogenic cell is a T cell, but those skilled in the art will understand that, given the overall advantages of the present disclosure, the hypoimmunogenic cell can be any cell type, and activation can occur via any cell receptor present on the cell. In some embodiments, activation of the hypoimmunogenic cell includes activation of the TCR / CD3 receptor complex by a protein antigen. In some embodiments, activation of the hypoimmunogenic cell includes activation of the TCR / CD3 receptor complex by a small molecule. In some embodiments, the hypoimmunogenic cell includes activation of the TCR / CD3 receptor complex by a viral antigen.

[0035] In some embodiments, activation of the hypoimmunogenic cell includes activation of a calcium-dependent channel. In some embodiments, activation of the hypoimmunogenic cell includes activation of the LFA-1 integrin receptor. In some embodiments, activation of the hypoimmunogenic cell includes activation of the CD28 receptor. In some embodiments, activation of the hypoimmunogenic cell includes activation of the IL-2 receptor.

[0036] In some embodiments, activation of the hypoimmunogenic cell includes activation of the SLAMF1 (CD150) receptor. In some embodiments, activation of the SLAMF1 (CD150) receptor is by the measles virus. In some embodiments, activation of the SLAMF1 (CD150) receptor is by Gram-negative bacteria.

[0037] In various embodiments, the activation of the low immunogenicity cells includes the activation of the IFNγ receptor. In various embodiments, the activation of the low immunogenicity cells includes the activation of the CD4 receptor by one or more viruses.

[0038] In various embodiments, activation of low immunogenic cells such as natural killer cells (or T cells if the receptor(s) listed below are associated with T cell activation) includes activation of the receptor(s) (alone or in a combination that enables a synergistic activation response), and the receptor(s) include, for example, CD2, CD3, CD16, CD28, CD314 (NKG2D), CD335, B7-H6, CD158d, IL-2R, IL-12R, DNAM-1, CD2, CD44, CD137, CX3CR1, CD27, CD160, 2B4, etc., but are not limited to these sensory receptors. CD16 can be activated via binding to the Fc region of an antibody (anti-S2 IgG). CD134, CD335, B7-H6 can be activated in combination with ICAM-1 combined with the Fc region of an antibody. CD314 can be activated by ULBP1, MICA, MICB, or H60. CD158d can be activated to induce pro-inflammatory molecules via HLA-G. IL-2R can be activated by the molecule IL-2. IL-12R can be activated by the molecule IL-12. DNAM-1 can be activated by CD155 or CD112 or NKp30. CD2 can be activated by LFA3. CD44 can be activated by hyaluronic acid, hyaluronan, osteopontin, collagen, or matrix metalloprotease. The CD28 homolog can be activated by the B7H7 protein ligand. CD137 can be activated by itself and by members of the tumor necrosis factor receptor family of proteins. CX3CR1 can be activated by CX3CL1. CD27 can be activated by CD70. CD160 can be activated by HLA-C. 2B4 can be activated by CD48. The levels of cytotoxic biomolecules can vary depending on each activation type, and each of these can be therapeutically beneficial when encapsulated in BioNV.Activation of each (or any) of the above combinations can occur by adding each of the following ligands (or any other relevant ligand) either in soluble form (partial peptide, domain or whole protein), immobilized on a membrane (partial peptide, domain or whole protein), as a target cell membrane component (partial peptide, domain or whole protein), or cross-linked to a solid phase including but not limited to Sephadex or magnetic beads (as a partial peptide, domain or whole protein).

[0039] In some embodiments, activation of some natural killer receptors (or T cells if the receptors listed below are relevant for T cell activation) does not result in degranulation or polarization of granules to the IS. These receptors include, but are not limited to: i) 2B4 alone, ii) CD134 alone, iii) CD134 combined with either 2B4 and the inhibitory receptor KIR or CD94, iv) LFA-1 combined with either inhibitory receptor KIR or CD94, v) CD16 combined with either inhibitory receptor KIR or CD94. Activation of each (or any) of the above combinations can occur by adding each of the following ligands (or any other relevant ligand): i) CD48, ii) ULBP1, iii) HLA-E, iv) HLA-C combined with HLA-E, or v) HLA-E either in soluble form (partial peptide, domain or whole protein), immobilized on a membrane (partial peptide, domain or whole protein), as a target cell membrane component (partial peptide, domain or whole protein), or cross-linked to a solid phase including but not limited to Sephadex or magnetic beads (as a partial peptide, domain or whole protein).

[0040] In some embodiments, activation of some natural killer receptors (or T cells if the receptor(s) listed below are associated with T cell activation) results in degranulation. Immediately prior to degranulation, perforin and granzymes may (or may not) undergo post-translational modifications that enhance their activity, such as calcium binding to perforin to render the protein active. To achieve the maximum effect of the cytotoxic proteins, it may be preferable to activate the cells along the degranulation pathway. These receptors include, but are not limited to, i) CD16 in combination with either LFA-1 and the inhibitory receptor KIR or CD94, ii) CD134 in combination with 2B4, or iii) CD16 alone. Activation of each (or any) of the above combinations can occur by adding each of the following ligands (or any other relevant ligand): i) HLA-C in combination with HLA-E, ii) ULBP1 in combination with CD48, or iii) anti-S2 IgG, in either a soluble form (partial peptide, domain or full protein), membrane-fixed (partial peptide, domain or full protein), target cell membrane component (partial peptide, domain or full protein), or cross-linked to a solid phase including but not limited to Sephadex or magnetic beads (as a partial peptide, domain or full protein).

[0041] In some embodiments, receptors that, when activated, cause polarization of soluble granules may be less than optimal (or may be preferred) because their localization to the cell membrane may minimize encapsulation into BioNV but maximize encapsulation into secreted EVs (exosomes and microsomes). This is due to the difference between extrusion production (BioNV) and cellular exocytosis processing (EV). Some receptors that induce polarization of soluble granules into IS include, but are not limited to: i) CD16 in combination with LFA-1 (which also causes degranulation), ii) LFA-1 alone, or iii) CD134 in combination with LFA-1 and 2B4 (which also results in degranulation). Activation of each (or any) of the above combinations can occur by adding each of the following ligands (or any other relevant ligand): i) anti-S2 IgG in combination with ICAM-1, ii) ICAM-1, or iii) ULBP1 in combination with CD48 and ICAM-1, in either soluble form (partial peptide, domain or full protein), immobilized on a membrane (partial peptide, domain or full protein), a target cell membrane component (partial peptide, domain or full protein), or cross-linked to a solid phase including but not limited to Sephadex or magnetic beads (as partial peptide, domain or full protein).

[0042] In some embodiments, activation of low-immunogenicity cells includes activation by engineered non-natural biomolecules selected from one or more of soluble peptides, chimeric antigen receptors, small molecule decoys, small molecule ligands, designer nucleic acid ligands, carbohydrate ligands, viral ligands, chimeric biomolecule ligands, fusion proteins, antibodies, and antibody format molecules. In some embodiments, activation of low-immunogenicity cells includes activation by inorganic compounds. In some embodiments, activation of low-immunogenicity cells includes activation by increased expression, overexpression, or activity of transcription factors.

[0043] In various embodiments, the activation of low immunogenicity cells includes activation at the DNA level by one or more of a transposase-based method, a Cre / Lox-based method, an endonuclease-based method, a homologous recombination (HR)-based method, a non-homologous end joining (NEHJ)-based method, a microhomology-mediated end joining (MMEJ)-based method, a homology-mediated end joining (HMEJ)-based method, small molecule RNAs, or combinations thereof. In various embodiments, the small molecule RNAs include one or more of guide RNA (gRNA), tracer RNA (tracrRNA), microRNA (miRNA), RNA interference (RNAi), small interfering RNA (siRNA), double-stranded RNA, Piwi-interacting RNA (piRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), antisense oligonucleotide (ASO), locked nucleic acid (LNA), splice-switching oligonucleotide (SSO), tRNA, complementary messenger RNA, repeat-associated small interfering RNA (rasiRNA), endonuclease, and small non-coding RNA.

[0044] In various embodiments, the activation of low immunogenicity cells includes activation at the RNA level by one or more of guide RNA (gRNA), tracer RNA (tracrRNA), microRNA (miRNA), RNA interference (RNAi), small interfering RNA (siRNA), double-stranded RNA, Piwi-interacting RNA (piRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), antisense oligonucleotide (ASO), locked nucleic acid (LNA), splice-switching oligonucleotide (SSO), tRNA, complementary messenger RNA, repeat-associated small interfering RNA (rasiRNA), endonuclease, small non-coding RNA, IRES element, or combinations thereof. In various embodiments, the activation includes one or more RNA-guided endonucleases.

[0045] In various embodiments, activation of the hypoimmunogenic cells includes activation by an endogenous promoter region and / or enhancer region. In various embodiments, activation of the hypoimmunogenic cells includes activation by stably integrating a genetic element. In various embodiments, activation of the hypoimmunogenic cells includes activation by transient expression of a genetic element.

[0046] In various embodiments, activation of the hypoimmunogenic cells results in a metabolically altered state of the hypoimmunogenic cells.

[0047] In various embodiments, one or more therapeutically relevant biomolecules are chemokines, interferons, interleukins, alarmins, lymphokines, perforin, granzyme, granulysin, tumor necrosis factor (TNF), colony stimulating factor, bone morphogenetic protein (BMP), erythropoietin (EPO), granulocyte stimulating factor (G-CSF), granulocyte macrophage colony stimulating factor (GM-CSF), gene editing payloads, fusion proteins, antibodies or antibody formats, or combinations thereof. In various embodiments, the cytokine is a pro-inflammatory cytokine. In various embodiments, the cytokine is an anti-inflammatory cytokine. In various embodiments, the granzyme is granzyme A, B, H, K, or M.

[0048] In various embodiments, the gene editing payload includes one or more gene editor nucleic acids and / or proteins, or one or more nucleic acids encoding one or more gene editors. In various embodiments, one or more gene editors are site-specific endonucleases, TALENs, ZFNs, RNase P RNA, CRISPR / Cas nucleases, C2c1, C2c2, C2c3, Cas9, Cpf1, TevCas9, archaeal Cas9, CasY.1, CasY.2, CasY.3, CasY.4, CasY.5, CasY.6, CasX, Cas omega, transposases, and / or any orthologs or homologs thereof. In various embodiments, the gene editing payload includes a trans-activation response region (TAR) loop system.

[0049] In some embodiments, the therapeutic BioNV comprises (i) one or more membrane-embedded targeting agents targeted to one or more cell biomarkers, (ii) one or more membrane-embedded proteins of alpha-phagocyte integrin, CCL2, H2-M3, FasL, MFEG8, PD-L1 and / or CTLA-4, SerpinB9, and an anti-IL-6R antibody or antibody format, (iii) any one of CD24, CD47, CD200, chimeric CD24 / CD47, chimeric CD24 / CD200, and chimeric CD47 / CD200, or any two of CD24, CD47, and CD200 as membrane-embedded proteins, and (iv) any one of HLA-A, HLA-B, HLA-C, HLA-F, CIITA, IL-6, TRAC, TRBC, HLA-E or HLA-G, and one or more proteins of IL-4, IL-10 and IL-16, and the membrane substantially lacks one or more of the following therapeutic-related biomolecules, which are chemokines, interferons, interleukins, alarmins, lymphokines, perforin, granzymes, granulysin, tumor necrosis factor (TNF), colony stimulating factors, bone morphogenetic proteins (BMP), erythropoietin (EPO), granulocyte stimulating factor (G-CSF), granulocyte macrophage colony stimulating factor (GM-CSF), gene editing payloads, fusion proteins, antibodies or antibody formats, or combinations thereof.

[0050] In various embodiments, the therapeutic BioNVs comprise (i) one or more membrane-embedded targeting agents targeted to one or more cell biomarkers, (ii) one or more membrane-embedded proteins of α-phagocyte integrin, CCL2, H2-M3, FasL, MFEG8, PD-L1 and / or CTLA-4, SerpinB9, and an anti-IL-6R antibody or antibody format, (iii) a membrane-embedded protein of either CD24 and CD47, or chimeric CD24 / CD47, and (iv) one or more of HLA-A, HLA-B, HLA-C, HLA-F, CIITA, IL-6, TRAC, TRBC, HLA-E or HLA-G, SerpinB9, and CD200, and one or more of IL-4, IL-10, and IL-16, and the membrane substantially lacks one or more of the following therapeutic-related biomolecules: chemokines, interferons, interleukins, alarmins, lymphokines, perforin, granzyme, granulysin, tumor necrosis factor (TNF), colony-stimulating factors, bone morphogenetic proteins (BMP), erythropoietin (EPO), granulocyte-stimulating factor (G-CSF), granulocyte macrophage colony-stimulating factor (GM-CSF), gene editing payloads, fusion proteins, antibodies or antibody formats, or combinations thereof.

[0051] In various embodiments, the one or more targeting agents are antibodies or antibody formats. In various embodiments, the one or more targeting agents are CARs. In various embodiments, treating the activated hypoimmunogenic cells is by one or more of sonication, adaptive focused acoustics technology, French press, extrusion, continuous extrusion, enzymatic lysis, cell lysis by surfactants, and / or electroporation. In various embodiments, treating the activated hypoimmunogenic cells is by continuous extrusion.

[0052] In various embodiments, the therapeutic BioNVs are sized from about 10 nm to about 1200 nm. In various embodiments, the therapeutic BioNVs are sized from about 10 nm to about 100 nm. In various embodiments, the therapeutic BioNVs are sized from about 100 nm to about 200 nm. In various embodiments, the therapeutic BioNVs are sized from about 200 nm to about 500 nm. In various embodiments, the therapeutic BioNVs are sized from about 500 nm to about 1200 nm.

[0053] In various embodiments, the therapeutic exosomes comprise a membrane that is substantially lacking in one or more of (i) one or more membrane-embedded targeting agents targeted to one or more cell biomarkers, (ii) one or more membrane-embedded proteins of alpha-phagocyte integrin, CCL2, H2-M3, FasL, MFEG8, PD-L1 and / or CTLA-4, SerpinB9, and an anti-IL-6R antibody or antibody format, (iii) any one of CD24, CD47, CD200, chimeric CD24 / CD47, chimeric CD24 / CD200, and chimeric CD47 / CD200, or any two of CD24, CD47, and CD200, and (iv) any one of HLA-A, HLA-B, HLA-C, HLA-F, CIITA, IL-6, TRAC, TRBC, HLA-E or HLA-G, and one or more of IL-4, IL-10, and IL-16, wherein the one or more therapeutically relevant biomolecules are chemokines, interferons, interleukins, alarmins, lymphokines, perforin, tumor necrosis factor (TNF), colony stimulating factors, bone morphogenetic proteins (BMP), erythropoietin (EPO), granulocyte stimulating factor (G-CSF), granulocyte macrophage colony stimulating factor (GM-CSF), gene editing payloads, fusion proteins, antibodies or antibody formats, or combinations thereof.

[0054] In various embodiments, the therapeutic exosomes comprise (i) one or more membrane-embedded targeting agents targeted to one or more cellular biomarkers, (ii) one or more membrane-embedded proteins of alpha-phagocyte integrin, CCL2, H2-M3, FasL, MFEG8, PD-L1 and / or CTLA-4, SerpinB9, and an anti-IL-6R antibody or antibody format, (iii) a membrane-embedded protein of either CD24 and CD47, or chimeric CD24 / CD47, and (iv) one or more of HLA-A, HLA-B, HLA-C, HLA-F, CIITA, IL-6, TRAC, TRBC, HLA-E or HLA-G, SerpinB9, and CD200, and one or more of IL-4, IL-10 and IL-16, and a membrane substantially lacking one or more of the following therapeutic-related biomolecules: chemokines, interferons, interleukins, alarmins, lymphokines, perforin, granzyme, granulysin, tumor necrosis factor (TNF), colony-stimulating factors, bone morphogenetic proteins (BMP), erythropoietin (EPO), granulocyte-stimulating factor (G-CSF), granulocyte macrophage colony-stimulating factor (GM-CSF), gene editing payloads, fusion proteins, antibodies or antibody formats, or combinations thereof.

[0055] In some embodiments, one or more targeting agents are antibodies or antibody formats. In some embodiments, one or more targeting agents are CARs. In some embodiments, harvesting therapeutic exosomes from activated hypoimmunogenic cells includes inducing hypoxia. In some embodiments, harvesting therapeutic exosomes from activated hypoimmunogenic cells includes expressing or increasing the expression of one or more cytokines. In some embodiments, harvesting therapeutic exosomes from activated hypoimmunogenic cells includes supplying one or more small molecule exosome modulators. In some embodiments, harvesting therapeutic exosomes from activated hypoimmunogenic cells includes supplementing the medium with one or more exosome factors. In some embodiments, the therapeutic exosomes are about 10 nm to about 200 nm in size. In some embodiments, the therapeutic exosomes are about 10 nm to about 100 nm in size. In some embodiments, the therapeutic exosomes are about 100 nm to about 200 nm in size.

[0056] In some embodiments, the mammalian disease is cancer, an infectious disease, a genetic disorder, or a rare disease.

[0057] In some aspects, described herein are methods of treating and / or preventing a disease or disorder using co - administration of BioNV and whole cells. In some embodiments, the method includes administering a therapeutically effective amount of BioNV comprising one or more biomimetic nanovesicle (BioNV) antigen - binding constructs and administering a therapeutically effective amount of whole cells comprising one or more whole - cell antigen - binding constructs.

[0058] In some embodiments, the disease or disorder is cancer. In some embodiments, the cancer is one or more of carcinoma, sarcoma, myeloma, leukemia, lymphoma, mixed cancer, and / or metastatic cancer.

[0059] In some embodiments, BioNV is administered first and whole cells are administered second, or whole cells are administered first and BioNV is administered second, or BioNV and whole cells are administered simultaneously.

[0060] In some embodiments, one or more BioNV antigen-binding constructs comprise a CAR. In some embodiments, one or more whole cell antigen-binding constructs comprise a CAR. In some embodiments, one or more whole cell antigen-binding constructs bind to cancer cells. In some embodiments, one or more BioNV antigen-binding constructs bind to cancer cells. In some embodiments, one or more whole cell antigen-binding constructs bind to BioNV. In some embodiments, one or more BioNV antigen-binding constructs bind to whole cells. In some embodiments, the whole cells comprise a CAR that binds to an antigen, ligand, or receptor present on both cancer cells and BioNV.

[0061] In some embodiments, BioNV comprises a CAR that binds to an antigen, ligand, or receptor present on cancer cells and an antigen, ligand, and / or receptor that binds to a CAR on whole cells. In some embodiments, the whole cells comprise a first CAR that binds to an antigen, ligand, or receptor present on cancer cells and a second CAR that binds to an antigen, ligand, and / or receptor present on BioNV.

[0062] In some embodiments, the antigen, ligand, and / or receptor on the BioNV is, comprises, or is similar to an antigen, ligand, or receptor present on a cancer cell; or the antigen, ligand, and / or receptor on the BioNV is different from an antigen, ligand, or receptor present on a cancer cell. In some embodiments, the first CAR can signal through a pathway that results in cell-mediated cytotoxicity or comprises one or more intracellular signaling domains that can signal through a pathway that results in cell-mediated cytotoxicity. In some embodiments, the second CAR can signal through a pathway that results in cell-mediated cytotoxicity by all cells or comprises one or more intracellular signaling domains that can signal through a pathway that results in cell-mediated cytotoxicity. In some embodiments, the second CAR cannot signal through a pathway that results in cell-mediated cytotoxicity by all cells or lacks one or more intracellular signaling domains that can signal through a pathway that results in cell-mediated cytotoxicity. In some embodiments, the second CAR can signal through a pathway that results in the persistence, survival, and / or proliferation of all cells or comprises one or more intracellular signaling domains that can signal through a pathway that results in the persistence, survival, and / or proliferation of all cells.

[0063] In some embodiments, administering the BioNV improves the function of all cells. In some embodiments, the function of all cells includes one or more of cell-mediated cytotoxicity, cytokine release, killing of tumor cells or cancer cells, honing to tumor cells or cancer tissue, tissue infiltration, proliferation, persistence, and / or survival. In some embodiments, administering the BioNV reduces one or more toxicities of all cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0064]

Figure 1

Figure 2

Figure 3

Figure 4A

Figure 4B

Figure 4C

Figure 4D

Figure 5

[0065] The present disclosure relates, in part, to methods of generating therapeutic biomimetic nanovesicles (BioNVs), or extracellular vesicles (EVs) such as naturally secreted therapeutic exosomes or microsomes, which are utilized in the treatment of diseases. Although reference is made below to exosomes, it should be understood that any other EVs can also be used. Therapeutic BioNVs and / or therapeutic exosomes lack the expression and / or activity of one or more immunogenic molecules (e.g., MHC class I / II, HLA, T cell receptor (TCR), cytokine release syndrome (CRS) molecules, etc.) and are derived from hypoimmunogenic cells that are modified to express and / or have increased expression of one or more immunoprotective molecules (e.g., CD47, CD24, CD200, α-macrophage integrin, etc.). The hypoimmunogenic cells are subjected to genetic regulation and activation to control the expression of therapeutic-related biomolecules that can be filled or encapsulated within the lumen of the BioNVs and exosomes. Therapeutic BioNVs and therapeutic exosomes can be derived from any hypoimmunogenic cell type, including a subset of stem cells or cells differentiated therefrom.

[0066] The present disclosure relates, in part, to compositions of BioNVs and / or exosomes comprising surface-directed targeting agents (e.g., CARs) that recognize one or more cell biomarkers. This results in cell-targeted (or disease-specific) therapeutic BioNVs / exosomes that are much smaller in size than conventional cell-based CAR-T / NK cell therapies, on the order of 20 - 1200 nm. BioNVs / exosomes can be derived from cell types such as stem cells, iPSCs, reprogrammed totipotent or pluripotent cells, embryonic stem cells, mesenchymal stem cells, or cells differentiated from any stem cell. Plasma membrane-derived BioNVs or naturally secreted exosomes retain low immunogenicity from low-immunogenic cells. These properties can be obtained from low-immunogenic cells by gene manipulation focusing on the knockout of specific immunogenic cell surface markers or immunogenic molecules (e.g., MHC class I / II, T cell receptor (TCR), cytokine release syndrome (CRS), etc.) and / or the expression or increased expression of immunoprotective cell surface markers (e.g., CD47, CD34, CD24, CD200, alpha-phagocytes, etc.). The targeting agent can include any variant of an antibody construct, e.g., Fab, Fab’, Fab’-SH, F(ab’)2, scFv, diabody, nanobody, linear antibody, bispecific antibody, multispecific antibody, chimeric antibody, humanized antibody, human antibody, and fusion proteins containing the antigen-binding portion of an antibody, V H H nanobody, V NARS and other antibody formats, enabling BioNVs / exosomes to target any desired cell (targeting any type of cell surface biomarker). BioNVs / exosomes can include targeting agents that are virus epitope recognition receptors (VERRs) or virus ligands, ligands for receptors, or receptors for ligands. BioNVs / exosomes can also encapsulate and deliver to a cell target intended for treating cancer any selected small molecule, biological agent, nucleic acid, gene editing therapeutic payload, etc.

[0067] Furthermore, the present disclosure relates, in part, to methods of treating or preventing a disease or disorder by administering a therapeutically effective amount of a therapeutic BioNV or therapeutic exosome to a subject in need thereof.

[0068] Method for producing biomimetic nanovesicles (BioNV) and / or exosomes In various aspects, the present disclosure provides a method for producing a therapeutic biomimetic nanovesicle (BioNV), comprising: (a) obtaining hypoimmunogenic cells; (b) activating the hypoimmunogenic cells to express one or more therapeutically relevant biomolecules; and (c) treating the activated hypoimmunogenic cells to produce the therapeutic BioNV, wherein the therapeutic BioNV comprises the plasma membrane of the hypoimmunogenic cells and encapsulates one or more therapeutically relevant biomolecules.

[0069] In various aspects, the present disclosure provides a method for producing a therapeutic exosome, comprising: (a) obtaining hypoimmunogenic cells; (b) activating the hypoimmunogenic cells to express one or more therapeutically relevant biomolecules; and (c) harvesting the therapeutic exosomes spontaneously released from the activated hypoimmunogenic cells, wherein the therapeutic exosomes comprise the plasma membrane of the hypoimmunogenic cells and encapsulate the one or more therapeutically relevant biomolecules.

[0070] In various embodiments, the therapeutic BioNVs and / or exosomes are derived from hypoimmunogenic cells, which can be stem cells (e.g., iPSCs, mesenchymal, etc.), induced pluripotent stem cells (iPSCs), reprogrammed totipotent or pluripotent cells, embryonic stem cells, mesenchymal stem cells, or differentiated cells derived from any of these modified cells. In various embodiments, the hypoimmunogenic cells from which the BioNVs and / or exosomes are derived are T cells, helper T cells, memory T cells, or NK cells. In various embodiments, the hypoimmunogenic cells are macrophages. In various embodiments, the cells are monocytes. In various embodiments, the hypoimmunogenic cells are hepatocytes, cardiomyocytes, neurons, endothelial cells, pancreatic cells, or retinal pigment epithelial (RPE) cells. In various embodiments, the hypoimmunogenic cells can be any terminally differentiated cells, such as, but not limited to, muscle cells (satellite cells), adipocytes, osteocytes, cardiomyocytes, hepatocytes, blood cells (including erythrocytes, platelets, and all immune cell types), glial cells (such as other neuron cell types), epithelial cells, epidermal cells, stromal cells (e.g., respiratory stromal cells), fibroblasts (e.g., skin fibroblasts), endothelial cells (e.g., bronchial endothelial cells), oral cells, stromal cells, or germ cells. In various embodiments, the hypoimmunogenic cells can be any functionally specific cell type, such as, but not limited to, exocrine gland secretory epithelial cells, hormone-secreting cells (e.g., enteroendocrine cells, thyroid cells, pancreatic islet cells, etc.), sensory transducer cells, autonomic nerve cells, sensory organ cells (e.g., pillar cells, olfactory cells, Schwann cells, satellite glial cells, etc.), barrier cells (e.g., lung cells, duct cells, kidney cells, podocytes, etc.), extracellular matrix cells (e.g., tendon fibroblasts, osteoblasts, connective tissue cells, etc.), or contractile cells (e.g., skeletal muscle cells, cardiomyocytes, myoepithelial cells, etc.).

[0071] In various embodiments, the modified cells are iPSCs. In various embodiments, the iPSCs differentiate into specific cells. In various embodiments, the modified cells express or do not express various cell surface markers upon differentiation. In various embodiments, the method includes BioNVs derived from hypoimmunogenic iPSCs.

[0072] In various embodiments, the therapeutic BioNV / exosomes are derived from iPSCs engineered to be hypoimmunogenic. In various embodiments, the iPSCs are reverted from a somatic cell state using microRNA technology instead of small molecule transactivators. The use of microRNA provides a more stringent differentiation system, which results in higher quality iPSCs. Without wishing to be bound by theory, such high quality iPSCs are less likely to undergo expression suppression (of proteins such as CD47 after genetic manipulation) and genetic drift, and have excellent quality / quantity of culture splitting (can be split more times than other methods before problems arise with cell integrity).

[0073] In various embodiments, the hypoimmunogenic cells from which the BioNV and / or exosomes are derived retain the functions of hypoimmunogenic cells, such as, but not limited to, the ability to cross the blood-brain barrier in the case of macrophages / monocytes, or tissue-specific factors in the case of cardiomyocytes, hepatocytes, etc.

[0074] In various embodiments, activated cells having allogeneic and hypoimmunogenic properties (e.g., derived from iPSCs) are created by knocking out, silencing, inactivating, blocking, or otherwise invalidating the transcriptional efficiency of immunogenic molecules. In various embodiments, hypoimmunogenic cells substantially lack one or more MHC class I proteins, MHC class II proteins, T cell receptor (TCR) proteins, and / or cytokine release syndrome (CRS) proteins. Inactivation of immunogenic molecules can include, for example, disruption of the MHC class I and β2-microglobulin (B2M) genes (e.g., in the case of the CD8+ T cell lineage), as well as disruption of the MHC class II and MHC II transactivator (CIITA) genes (e.g., in the case of the CD4+ T cell lineage). Without wishing to be bound by theory, these proteins contribute to the immunogenicity of human leukocyte antigen (HLA) and require HLA allele matching in donor-recipient for treatment by cell-based therapies. In various embodiments, allogeneic and hypoimmunogenic properties are achieved by disrupting the genes encoding TCR proteins, e.g., the α and β chains (in the case of αβ T cells) or γ and δ chains (in the case of γδ T cells) that form the ligand-binding site, as well as the signaling modules CD3δ, CD3γ, CD3ε, and CD3ζ. By doing so, irrelevant TCR types other than the TCR of the CAR cassette can be reduced, the uniformity of the desired CAR can be further improved, and off-target effects in BioNV / exosome formation can be reduced.

[0075] In some embodiments, the hypoimmunogenic cells comprise a knockout or disruption of the expression of immunogenic cell surface proteins and / or intracellular or secreted proteins. In some embodiments, the hypoimmunogenic cells have a disruption of the β2-microglobulin (B2M) gene and / or a disruption that reduces or abolishes the expression and / or activity of MHC class I proteins. In embodiments, knocking out the B2M gene risks preventing the long-term acceptance of BioNV by the recipient as observed in the whole-cell-based approach described above, reducing the number of doses that can potentially be administered. To overcome this problem, in some embodiments, the HLA-E or HLA-G gene is left intact to allow the immune system to adapt to the resulting BioNV. In some embodiments, HLA-A, HLA-B, HLA-C, HLA-F, and HLA-E or HLA-G (but not both HLA-E and HLA-G) are successively knocked out.

[0076] In some embodiments, the hypoimmunogenic cells have a disruption of the HLA-A gene and / or a disruption that reduces or abolishes the expression and / or activity of HLA-A protein. In some embodiments, the hypoimmunogenic cells have a disruption of the HLA-B gene and / or a disruption that reduces or abolishes the expression and / or activity of HLA-B protein. In some embodiments, the hypoimmunogenic cells have a disruption of the HLA-C gene and / or a disruption that reduces or abolishes the expression and / or activity of HLA-C protein. In some embodiments, the hypoimmunogenic cells have a disruption of the HLA-E gene or a disruption of the HLA-G gene and / or a disruption that reduces or abolishes the expression and / or activity of HLA-E or HLA-G protein. In some embodiments, the hypoimmunogenic cells have a disruption of the HLA-F gene and / or a disruption that reduces or abolishes the expression and / or activity of HLA-F protein.

[0077] In some embodiments, the hypoimmunogenic cells have a disruption of the CIITA gene and / or a disruption that reduces or abolishes the expression and / or activity of MHC class II proteins. In some embodiments, in allogeneic iPSCs, the MHC class I and MHC class II complexes are disrupted by knocking out B2M, a serum protein found associated with the MHC class I heavy chain on the surface of almost all nucleated cells and involved in peptide antigen presentation to the immune system. In some embodiments, in allogeneic iPSCs, the CIITA gene is disrupted, such that the resulting differentiated cell lines (e.g., DCs, monocytes, endothelial cells, thymic epithelial cells, B cells, etc.) do not express MHC class II proteins.

[0078] In some embodiments, the hypoimmunogenic cells have a disruption of the T cell alpha constant (TRAC) gene and / or a disruption that reduces or abolishes the expression and / or activity of the TRAC protein. In some embodiments, the hypoimmunogenic cells have a disruption of the T cell beta constant (TRBC) gene and / or a disruption that reduces or abolishes the expression and / or activity of the TRBC protein. In some embodiments, the hypoimmunogenic cells have reduced or abolished expression of the PD-1 gene and / or reduced or abolished expression and / or activity of the PD-1 protein, where the hypoimmunogenic cells are activated; or the hypoimmunogenic cells express the PD-1 gene and / or gene product or have increased expression thereof, where the hypoimmunogenic cells are not activated.

[0079] In various embodiments, as used herein, "increased expression and / or activity" refers to an increase in expression and / or activity in hypoimmunogenic cells as compared to their native or wild-type cognate cells. For example, in various embodiments, an increase in the expression and / or activity of one or more biomolecules described herein can confer hypoimmunogenic properties to iPSCs as compared to iPSCs with different protein expression patterns or levels of expression. In various embodiments, the "increase in expression and / or activity" is due to genetic modification such as knock-in.

[0080] CRS is a major concern in all cell therapies, and despite being engineered to be hypoimmunogenic, there is a risk that effector functions and other consequences of the interaction of the cells after injection can result in the release of biomolecules, which in turn can cause a systemic inflammatory syndrome characterized by fever, multiple organ dysfunction, etc. In various embodiments, hypoimmunogenic cells are engineered to disrupt the expression and / or activity of one or more proteins that contribute to CRS. In various embodiments, hypoimmunogenic cells have reduced or absent expression and / or activity of CRS-related cytokines (e.g., knockout or silencing).

[0081] In various embodiments, the hypoimmunogenic cells have a disruption of the IL-4 gene and / or a disruption that reduces or eliminates the expression and / or activity of the IL-4 protein. In various embodiments, the hypoimmunogenic cells have a disruption of the IL-6 gene and / or a disruption that reduces or eliminates the expression and / or activity of the IL-6 protein. In various embodiments, the hypoimmunogenic cells have a disruption of the IL-10 gene and / or a disruption that reduces or eliminates the expression and / or activity of the IL-10 protein. In various embodiments, the hypoimmunogenic cells have a disruption of the IL-16 gene and / or a disruption that reduces or eliminates the expression and / or activity of the IL-16 protein. In various embodiments, the modified cells include gene disruptions (e.g., knockouts) in interleukins associated with other cytokine release syndromes (CRS). In various embodiments, the reduction or elimination of the interleukin that causes CRS reduces the likelihood of CRS.

[0082] Serine protease inhibitor B9 (SerpinB9) is a member of the serine protease inhibitor superfamily. SerpinB9 has been reported to protect cells from the immune killing action of granzyme B. In various embodiments, the hypoimmunogenic cells have a disruption of the SerpinB9 gene and / or a disruption that reduces or eliminates the expression and / or activity of the SerpinB9 protein. In various embodiments, the hypoimmunogenic cells have SerpinB9 knocked out and / or silenced. Alternatively, in various embodiments, the hypoimmunogenic cells express the SerpinB9 gene and / or gene product, or the expression and / or activity thereof is increased.

[0083] In various embodiments, overexpression of SerpinB9 suppresses the function of granzyme B, which is associated with immune activation responses such as apoptosis of target cells and / or diseased cells. In various embodiments, granzyme B is inhibited in cell types such as activated lymphocytes, NK cells, macrophages, and follicular DCs. In various embodiments, for example, in the case of BioNV, which is intended to deliver a non-granzyme payload, such as a gene editing payload, the low immunogenic cells may express and / or overexpress SerpinB9.

[0084] In various embodiments, the low immunogenic cells express the CCL2 gene and / or gene product, or the expression and / or activity thereof is increased. In various embodiments, the low immunogenic cells express the PD-L1 gene and / or gene product, or the expression thereof is increased, where the low immunogenic cells are not activated; or the modified cells have reduced or abolished expression of the PD-L1 gene and / or gene product, where the low immunogenic cells are activated. In various embodiments, the low immunogenic cells express the H2-M3 gene and / or gene product, or the expression and / or activity thereof is increased. In various embodiments, the low immunogenic cells express the CTLA-4 gene and / or gene product, or the expression and / or activity thereof is increased.

[0085] In various embodiments, the low immunogenic cells express the CD47 gene and / or gene product, or the expression and / or activity thereof is increased. In various embodiments, preventing a potential inhibitory phenotype of CD47 expression across the cell is accomplished by interfering with the inhibitory mechanism of action of a series of microRNAs against the 3’UTR of the CD47 gene. This is done by deleting this region in a stable construct or by eliminating / inhibiting the expression of the microRNAs. In various embodiments, this may solve the problem of inhibition caused by microRNAs.

[0086] In various embodiments, the low immunogenicity cells express the CD24 gene and / or gene product, or have increased expression and / or activity thereof. CD24 is a sialoglycoprotein expressed on mature granulocytes and B cells and is also an anti-phagocytosis protein. CD24 prevents phagocytosis through interaction with Siglec-G / 10 on macrophages. In various embodiments, the low immunogenicity cells express or overexpress the CD24 protein and / or gene product. In various embodiments, the low immunogenicity cells express the chimeric CD24 / CD47 gene and / or gene product, or have increased expression and / or activity thereof. In various embodiments, the low immunogenicity cells comprise CD24 / CD47 with a transmembrane domain linked thereto. In various embodiments, the domains of CD47 isoform 2 and CD24 can be expressed separately or linked to form a two-leaf chimeric protein. In various embodiments, the low immunogenicity cells are iPSCs derived from fibroblasts rather than ABO cells.

[0087] In various embodiments, the hypoimmunogenic cells express the CD200 gene and / or gene product, or have increased expression and / or activity thereof. In various embodiments, the CD200 tag minimizes phagocytosis by macrophages and also prevents granulocyte activation. In various embodiments, when it is not desirable to suppress granulocytes, e.g., in a solid tumor microenvironment (TME), the hypoimmunogenic cells do not express CD200. This is to complement the mechanism of action of BioNV designed such that granulocyte activation releases granzymes and perforins. However, in various embodiments, when the CD47 or CD24 tag is used, or when the CD24 / CD47 chimeric bivalent protein tag (each preventing phagocytosis) is used in combination with overexpressed H2-M3 (attenuating the NK response), clearance of BioNV is enabled while achieving stability without CD200. In various embodiments, when granzymes and perforins are not selected as therapeutic biomolecules, CD200 can be expressed to prevent granulocyte activation, while on the other hand, the CD47 tag or the CD24 tag (but not both tags) can be removed. In various embodiments, the hypoimmunogenic cells express the chimeric CD24 / CD200 gene and / or gene product, or the chimeric CD47 / CD200 gene and / or gene product, or have increased expression and / or activity thereof. In various embodiments, these CD200 strategies are hypoimmunogenic cell lines for generating BioNV for targeting non-cancer cells, such as targeting liver, kidney, cardiomyocytes, and / or tissue regeneration pathways.

[0088] In various embodiments, the resulting hypoimmunogenic cells (or cells differentiated therefrom) do not express and / or do not overexpress all three of CD47, CD24, and CD200. In various embodiments, the hypoimmunogenic cells are engineered to stabilize the BioNV or exosomes obtained from the hypoimmunogenic cell line, but not to prevent the BioNV or exosomes from being cleared from the body. BioNV / exosomes that are too stable may ultimately cause a humoral response, resulting in limited dosing or treatment frequency.

[0089] In various embodiments, the hypoimmunogenic cells express the MFG-E8 gene and / or gene product, or have increased expression and / or activity thereof. In various embodiments, the hypoimmunogenic cells express the NCAM gene and / or gene product, or have increased expression and / or activity thereof. In various embodiments, the hypoimmunogenic cells express the α-phagocyte integrin gene and / or gene product, or have increased expression and / or activity thereof.

[0090] In various embodiments, the hypoimmunogenic cells express an antibody or antibody format molecule (anti-IL-6R) that targets the IL-6 surface receptor, or have increased expression and / or activity thereof. In various embodiments, the hypoimmunogenic cells are iPSC cell lines in which anti-IL-6R antibodies are incorporated into the iPSC cell lines. In various embodiments, BioNV / exosomes carry α-IL-6R, thereby blocking the activation of signaling pathways on localized immune cells in the tumor environment.

[0091]

[0092] In various embodiments, the hypoimmunogenic cells express the FasL gene and / or gene product. In various embodiments, the hypoimmunogenic cells do not overexpress the FasL gene and / or gene product. In various embodiments, the hypoimmunogenic cells do not overexpress FasL. This is because, in the membrane of BioNV, for example, after treatment by continuous extrusion, enrichment of native expression levels of FasL is observed. If the concentration of FasL is too high, it can have a reverse effect and may prevent the recruitment of T cells to solid tumors and / or may cause premature death of T cells.In various embodiments, the hypoimmunogenic cells can express one or more fusion proteins of one or more portions of any of the immunoprotective proteins herein. For example, in various embodiments, the construct can be made when an appropriate portion of a selected ligand is linked to a transmembrane domain. In various embodiments, the construct can be made when biologically relevant portions of two or more proteins are joined together and / or linked to a transmembrane domain.

[0093] In various embodiments, the hypoimmunogenic cells are substantially lacking in the expression and / or activity of one or more immunogenic proteins and express or have increased expression of one or more immunoprotective proteins.

[0094] In various embodiments, the hypoimmunogenic cells have reduced or absent expression and / or activity of one or more immunogenic proteins, such as proteins that give rise to an immune response, donor-recipient mismatch, HLA alloimmunity, inflammation, CRS, etc. in a subject, such as MHC class I proteins, MHC class II proteins, HLA proteins, TCR proteins, CRS proteins, etc. In various embodiments, the hypoimmunogenic cells have reduced or absent expression and / or activity of 3 or more immunogenic proteins, 4 or more immunogenic proteins, 5 or more immunogenic proteins, 6 or more immunogenic proteins, 7 or more immunogenic proteins, 8 or more immunogenic proteins, 9 or more immunogenic proteins, 10 or more immunogenic proteins, 11 or more immunogenic proteins, or 12 or more immunogenic proteins.

[0095] In some embodiments, the modified cells have an increased expression and / or activity of one or more immune-protective proteins, such as proteins that result in preventing or reducing an immune response in a subject, preventing or reducing early clearance of BioNV in a subject, preventing or reducing phagocytosis, conferring barrier passage function, etc., such as CD47, CD24, CD200, CD34, CCL2, H2-M3, MFEG8, PD-L1 (non-activated cell source), CTLA-4, etc. In some embodiments, the low-immunogenicity cells express or have an increased expression of 3 or more immune-protective proteins, 4 or more immune-protective proteins, 5 or more immune-protective proteins, 6 or more immune-protective proteins, 7 or more immune-protective proteins, 8 or more immune-protective proteins, 9 or more immune-protective proteins, or 10 or more immune-protective proteins.

[0096] In some embodiments, the low-immunogenicity cells have a reduced or abolished expression and / or activity of any one gene and / or gene product of HLA-A, HLA-B, HLA-C, HLA-F, CIITA, IL-6, TRAC, TRBC, and HLA-E or HLA-G.

[0097] In some embodiments, the low-immunogenicity cells have a reduced or abolished expression and / or activity of any one gene and / or gene product of HLA-A, HLA-B, HLA-C, HLA-F, CIITA, IL-6, TRAC, TRBC, SerpinB9, and HLA-E or HLA-G.

[0098] In some embodiments, the low-immunogenicity cells include low-immunogenicity cells having a reduced or abolished expression and / or activity of any one of HLA-A, HLA-B, HLA-C, HLA-F, CIITA, IL-6, TRAC, TRBC, SerpinB9, HLA-E or HLA-G, and one or more genes and / or gene products of IL-4, IL-10, and IL-16.

[0099] In various embodiments, the hypoimmunogenic cells express, or have increased expression and / or activity of, alpha - macrophage integrin, CCL2, H2 - M3, FasL, MFEG8, and PD - L1 and / or CTLA - 4, where the hypoimmunogenic cells do not overexpress FasL, the hypoimmunogenic cells are not activated by the expression of PD - L1, and express any one of CD24, CD47, CD200, chimeric CD24 / CD47, chimeric CD24 / CD200, and chimeric CD47 / CD200, or any two of CD24, CD47, and CD200, or have increased expression and / or activity thereof.

[0100] In various embodiments, the hypoimmunogenic cells express, or have increased expression and / or activity of, alpha - macrophage integrin, CCL2, H2 - M3, FasL, MFEG8, SerpinB9, and PD - L1 and / or CTLA - 4, where the hypoimmunogenic cells do not overexpress FasL, the hypoimmunogenic cells are not activated by the expression of PD - L1, and express any one of CD24, CD47, CD200, chimeric CD24 / CD47, chimeric CD24 / CD200, and chimeric CD47 / CD200, or any two of CD24, CD47, and CD200, or have increased expression and / or activity thereof.

[0101] In various embodiments, the hypoimmunogenic cells express, or have increased expression and / or activity of, the CD200 gene and / or gene product, do not express either the CD24 or CD47 gene and / or gene product, and / or are substantially deleted thereof. In various embodiments, the hypoimmunogenic cells do not have the expression and / or activity of the SerpinB9 gene and / or gene product and the CD200 gene and / or gene product.

[0102] In some embodiments, the low immunogenicity cells express one or more antigens, ligands, and / or receptors of tumor cells or cancer tissues, or their expression and / or activity is increased. In some embodiments, one or more antigens, ligands, and / or receptors of tumor cells or cancer tissues are listed in Table 4, Table 5, and / or Table 6 below. In some embodiments, one or more antigens, ligands, and / or receptors of tumor cells or cancer tissues are neoantigens such as variant variants or peptides specific to cancer or disease states that are not normally present in the subject to be treated. In some embodiments, the low immunogenicity cells express co-stimulatory molecules for immune cells, or their expression and / or activity is increased. In some embodiments, the co-stimulatory molecule for immune cells is IL-15.

[0103] In some embodiments, the low immunogenicity cells are allogeneic. In some embodiments, the low immunogenicity cells do not cause an immune response in the patient to whom the cell or BioNV derived therefrom is administered.

[0104] In some embodiments, the low immunogenicity cells contain one or more targeting agents. In some embodiments, the one or more targeting agents include a chimeric antigen receptor (CAR). In some embodiments, the CAR is bispecific. In some embodiments, the CAR lacks an intracellular portion. In some embodiments, the CAR is a targeting agent, a transmembrane domain, and an intracellular domain, and the intracellular domain includes an intracellular domain including a co-stimulatory domain and / or a signaling domain. In some embodiments, the transmembrane domain is derived from CD28, CD3ζ, CD4, CD8α, or ICOS, or fragments thereof. In some embodiments, the intracellular domain includes an intracellular signaling domain of the CD3ζ chain and / or one or more co-stimulatory molecules, optionally selected from CD28, 4-1BB, ICOS, CD27, and OX40.

[0105] In some embodiments, one or more targeting agents include an antibody or an antibody format. In some embodiments, the antibody or antibody format is selected from one or more of monoclonal antibodies, polyclonal antibodies, antibody fragments, Fab, Fab’, Fab’-SH, F(ab’)2, Fv, single-chain Fv (scFv), V NAR , V H H, affilins, diabodies, nanobodies, linear antibodies, bispecific antibodies, multispecific antibodies, chimeric antibodies, humanized antibodies, human antibodies, and fusion proteins comprising the antigen-binding portion of an antibody. In some embodiments, the antibody format is scFv. In some embodiments, one or more targeting agents include a viral epitope recognition receptor (VERR) or a viral ligand. In some embodiments, one or more targeting agents include a ligand for a receptor. In some embodiments, one or more targeting agents include a receptor for a ligand.

[0106] In some embodiments, the resulting hypoimmunogenic cells can have B2M knockout (KO), CIITA KO, CD47tg knock-in (KI), IL-6 KO integrated into the iPSCs, and the TRAC and TRBC genes knocked out. In some embodiments, only one gene of each is knocked out, rather than both on separate alleles. The TRAC and TRBC genes can be knocked out as described herein. The purpose of knocking out the TRAC and TRBC genes is to eliminate the T cell receptor. In some embodiments, the modified cells differentiate into a subset of T cells lacking a T cell receptor for inducing BioNV / exosomes. By genetically modifying the cells to substantially lack the TCR, the possibility of competing ligands that can non-specifically target another tissue against the CAR construct can be reduced. Thus, in some embodiments, knocking out the TCR gene is a strategy for reducing the off-target effects of BioNV / exosomes. In some embodiments, TRAC / TRBC knockout generally reduces the likelihood of CRS and the toxicity of BioNV / exosomes.

[0107] In various embodiments, the generated BioNV / exosomes express CD47. Exosomes and vesicles are readily removed from the body by macrophages through phagocytosis. Phagocytosis has a great impact on the therapeutic benefit and effectiveness of exosomes. Without wishing to be bound by theory, in order to prevent macrophage depletion of BioNV / exosomes, in various embodiments, BioNV / exosomes have a CD47 tag added to their surface. The CD47tg (tag) provides a "do not eat me" signal, which in various embodiments increases the half-life and serum stability of BioNV / exosomes in a subject. In various embodiments, the molecular CD47 isoform 2 (the isoform that interacts with the SIRPα receptor on macrophages) is incorporated into modified cells (e.g., iPSC cell lines). Without CD47tg, the half-life of BioNV / exosomes is shortened by phagocytosis inhibition, resulting in the need for higher doses and / or more frequent administrations.

[0108] In various embodiments, the obtained low-immunogenic cells have B2M gene disruption (or other genes capable of achieving MHC I suppression), CIITA gene disruption (or other genes capable of achieving MHC II suppression), CD47tg knock-in, and knockout of major interleukins (ILs) associated with the cause of cytokine release syndrome. In various embodiments, the modified cells include disruption of the IL-6 gene. Interleukin 6 (IL-6) is a major interleukin that causes CRS. In various embodiments, IL-6 knockout prevents unwanted encapsulation of IL-6 into BioNV / exosomes and reduces the contribution of BioNV (or exosomes) to localized (and concentrated by biomarker targeting) and / or potential systemic CRS events.

[0109] In various embodiments, the modified cells are expanded in culture after genetic manipulation. Any small-scale expansion culture method or large-scale feeder system expansion culture method known in the art can be used.

[0110] In various embodiments, the low-immunogenicity cells for generating BioNV / exosomes express one or more of the above proteins by a regulatable expression element and can be regulated throughout the BioNV / exosome manufacturing process. In various embodiments, the regulatable expression element includes a regulatable promoter (such as a Tet-on / off promoter) or a regulatable expression element such as a CRISPRa / i regulatory system. In various embodiments, CAR expression is controlled by a regulatable expression element. Simple overexpression of a construct from a CMV (or other type of) promoter may result in too high a surface density of the CAR, which may cause many problems such as "overactivation", leading to in vitro exhaustion and cell death after activation. Cell death results in the loss of the cell line for generating BioNV / exosomes. In various embodiments, the surface density of the CAR protein construct is regulated. For example, in various embodiments, the typical concentration range of the CAR protein per microgram of T cells is between 0.20 ng and 0.70 ng. If the expression of the CAR is too low, the targeting of biomarkers will be insufficient. However, the density cannot exceed the limit of the cells. If the CAR density is too high, the parent cells may become exhausted in the in vitro activation process before the induction of BioNV / exosomes, and the quality of BioNV / exosomes may also decrease due to the high protein concentration in the plasma membrane. By setting an upper limit, the targeting efficiency for low biomarker expression on cancer cells can be increased. The density limit of cell therapeutics is listed in published US Non-Provisional Application 20220040106 A1, which is incorporated herein by reference.

[0111] In various embodiments, the low immunogenic cells for generating BioNV / exosomes can include stable integration of any of the gene elements described herein into the cells (e.g., iPSCs) (safe harbor gene loci), which can be controlled by implementing a Tet-regulated CRISPRa and targeted 3× transcription factor-targeted gRNA system. The CRISPR activation system for the three upstream transcription factors can trigger signal cascade events that promote the production of CARs in which the ORF of the endogenous antibody is replaced at the designated locus (loci). This system can be made "tunable" by including a Tet-regulated promoter and can vary the concentration of CARs on the surface of the cells. Next, stable cell replacement with the CAR cassette of the CDR as well as the heavy and light chain antibody regions can be achieved by Cpf-1-induced homologous recombination repair (HDR). Finally, the stably integrated CAR cassette can include adjacent gRNA binding sites, thereby allowing for repeated exchange or modification of scFVs (such as other antibody formats) or VERR / viral ligands, enabling rapid and consistent insertion of the desired sequences.

[0112] In various embodiments, the concentration of CARs on the surface of iPSC-based cell lines or any downstream differentiated cells (and the resulting BioNV / exosomes) can be regulated using various transcriptional control elements such as a tetracycline on / off promoter (or a similar drug-regulated promoter) to drive the expression of the CRISPR activation / gRNA (CRISPRa) system. The CRISPRa system can then activate antibody regulatory transcription factors, such as Drm2, Fr5, and Bxp2, which regulate the expression of the engineered CAR cassette integrated at the site of the antibody locus (where the antibody gene has been replaced). Additionally, similar transcriptional control elements can be provided at defined manufacturing stages for purposes such as controlling overexpression of genes (e.g., CD47), driving genes that control differentiation, and the like.

[0113] In various embodiments, the hypoimmunogenic cells have B2M KO, CIITA KO, CD47tg KI, IL-6 KO, and an IL-2p GFP reporter is constructed in these cells, and the CAR construct can be incorporated / engineered into the modified cells. In various embodiments, the CAR construct can be knocked into the TRAC / TRBC genes and the remaining TRAC / TRBC genes can be knocked out simultaneously to obtain cells that are CAR+ and TRAC / TRBC− / −. In various embodiments, the CAR construct can be knocked into the TRAC / TRBC locus simultaneously at both loci to obtain cells that are CAR+ / + and TRAC / TRBC− / −.

[0114] In various embodiments, the hypoimmunogenic cells have engineered B2M KO, CIITA KO, CD47tg KI, IL-6 KO, IL-2p GFP, and CAR-modified cells (e.g., iPSCs), and the quality of the immune synapse (IS) between the CAR recognition domain and the biomarker is measured during the BioNV / exosome generation process. In various embodiments, the quality of the IS of the BioNV / exosomes can be directly related to the effectiveness in whole cell therapy.

[0115] In various embodiments, the therapeutic BioNV / exosomes, or modified / activated cells derived therefrom, contain nucleic acids encoding green fluorescent protein (GFP). In various embodiments, when B2M KO, CIITA KO, CD47tg KI, IL-6 KO, TRAC / TRBC single KO are incorporated into iPSCs, GFP molecules can be incorporated into the modified cell line. In various embodiments, this functions as a control cell line. In various embodiments, the non-control cell line (therapeutic cell line) does not have GFP. In various embodiments, the nucleic acid encoding GFP is operably linked to a promoter derived from one or more of IL-2, perforin, granzyme, granulysin, alarmin, TNF, INF, combinations thereof, and / or any other cell-specific gene or reporter gene. When lymphocytes are widely / globally activated from various stimuli, the IL-2 promoter is constitutively activated. In various embodiments, more focused activation / suppression (regulation) is used. In various embodiments, the IL-2p GFP reporter gene functions as an indicator of the degree of widespread / global activation of cells (part of the BioNV / exosome induction process). In various embodiments, by combining the GFP signal with immunoblot analysis of cytokine levels (such as perforin, granzyme, alarmin, TNF, and INF), the degree of widespread / global activation of lymphocytes upon exposure to activating antigens can be efficiently regulated. In various embodiments, GFP is used to compare the degree of activation between manufacturing lots and ensure consistency in therapeutic drug development.

[0116] In some embodiments, BioNV / exosomes are formed by obtaining induced pluripotent stem cells (hypo-iPSCs). In some embodiments, hypo-iPSCs are characterized by a plasma membrane profile of B2M− / −, CIITA− / −, CD47+ / +, PD1− / − and can be used for the production of the present BioNV / exosomes. Hypo-BioNV / exosomes can be produced from parental iPSC cell lines by methods such as sonication, adaptive focused acoustics technology, French press, extrusion, continuous extrusion, cell lysis with surfactants, and electroporation. In some embodiments, continuous extrusion is the method used to produce Hypo-BioNV / exosomes. Continuous extrusion of iPSCs can produce BioNV / exosomes that are tgCD47+ and HLA1 / HLA2 negative (low immunogenicity) and show elimination of PD1 resistance.

[0117] In some embodiments, the obtained low-immunogenic cells are CD34+ or are derived from CD34+ cells such as human CD34+ cord blood. In some embodiments, CD34+ cord blood-derived cell lines can function as base cell lines for the development, production, and manufacture of therapeutic BioNV / exosomes for the delivery of gene editing therapeutics. In some embodiments, CD34+ cord blood-derived low-immunogenic cell lines have been experimentally confirmed for low expression of HLA1 / 2 and overexpression of CD47 (Deuse T. et al. “Hypoimmunogenic derivatives of induced pluripotent stem cells evade immune rejection in fully immunocompetent allogeneic recipients.” Nat Biotechnol. 2019;37(3):252-258).

[0118] In various embodiments, the hypoimmunogenic cells can include cells engineered using a plurality of hypoimmunogenic engineering techniques described, for example, by Deuse et al., Han et al., Xu et al., Harding et al., as well as Deuse's published U.S. Patent Application Nos. 20190376045, 20190376045, 20210308183, and 20210292715, Nagy's US20210161971, Strominger's US20180141992, and Poirot's published European Patent Application No. 3693384, each of which is incorporated herein by reference in its entirety (Han X, et al. “Generation of hypoimmunogenic human pluripotent stem cells.” PNAS. Vol. 116, No. 21 2019: pp. 10441 - 10446. doi:10.1073 / pnas.1902566116.) and (Xu H, et al. “Targeted Disruption of HLA Genes via CRISPR - Cas9 Generates iPSCs with Enhanced Immune Compatibility.” Cell Stem Cell. Vol. 24, No. 4, 2019: pp. 566 - 578. doi:10.1016 / j.stem.2019.02.005.).

[0119] In various embodiments, the BioNV / exosomes are derived from cells in which the HLA genes encoding MHC membrane glycoproteins that give rise to immune responses associated with GVHD rejection have been eliminated. The HLA gene cluster can be classified into three categories: 1) the MHC class I pathway, 2) the MHC class II pathway, and 3) the MHC class III pathway. In GVHD, only the MHC class I and II pathways express protein complexes that trigger an immune response, and the MHC class III complex is not involved in immune activity.

[0120] When the MHC class protein complex is removed, natural killer cells and macrophages may be induced into an active clearance mode, after which the cells are destroyed. To avoid this killing mechanism, in various embodiments, by incorporating an addition of a transmembrane molecular protein tag of CD47 isoform 2 into the cell membrane of the modified cells, the natural killer and macrophage-mediated killing response can be avoided. For example, it is described in Willingham et al., Deuse et al., and Han et al. (Willingham SB, et al. “The CD47-signal regulatory protein alpha (SIRPa) interaction is a therapeutic target for human solid tumors.” PNAS. Vol. 109, No. 17, 2012: pp. 6662-7. doi: 10.1073 / pnas.1121623109.).

[0121] In various embodiments, cells can be engineered to prevent these responses using additional mechanisms such as those described below: 1) a CD24 transmembrane protein tag (e.g., as implemented in Zhao et al.) (Zhao W, et al. “Strategies for Genetically Engineering Hypoimmunogenic Universal Pluripotent Stem Cells.” iScience. Vol. 23, No. 6, 2020:101162. doi:10.1016 / j.isci.2020.101162.), 2) membrane-bound surfactant protein-D (SP-D) (e.g., as implemented in Jiaravuthisan et al.) (Jiaravuthisan P, et al. “A membrane-type surfactant protein D(SP-D) suppresses macrophage-mediated cytotoxicity in swine endothelial cells.” Transpl Immunol. Vol. 47, 2018: pp. 44-48. doi:10.1016 / j.trim.2018.02.003.), and 3) a molecular PD-L1 tag for preventing T cell responses. In various embodiments, BioNVs derived from “activated” cells encapsulate and / or release perforin and / or granzyme, resulting in targeted cell death. In various embodiments, activated cells produce perforin and / or granzyme encapsulated within the BioNVs. In various embodiments, hypoimmunogenic cells to be activated do not express PD-L1 to avoid the resulting BioNVs targeting PD-1 on T cells. In various embodiments, this reduces the likelihood that perforin and / or granzyme are released and unwanted T cell death occurs. In various embodiments, PD-L1 is overexpressed in BioNVs derived from cells that are not activated and not filled with apoptotic cytokines. In various embodiments, hypoimmunogenic cells to be activated have PD-L1 downregulated, knocked out, or otherwise silenced.In various embodiments, for non-activated, low immunogenic cells, i.e., BioNVs used for gene editor delivery, PD-L1 is upregulated. In various embodiments, CD47 isoform 2 can be incorporated into cells to prevent both macrophage- and natural killer-mediated cytotoxicity. This is because CD47 isoform 2 functions as a “don’t eat me” tag through the SIRP-α receptor expressed on cells such as these cells. In various embodiments, CD47 can be utilized for immune tolerance to innate immune cells in genetically engineered iPSCs, for example, as in Chhabra et al., Han et al., and Jaiswal et al. (Chhabra A, et al. “Hematopoietic stem cell transplantation in immunocompetent hosts without radiation or chemotherapy.” Sci Transl Med. Vol. 8, No. 351, 2016:351ra105. doi:10.1126 / scitranslmed.aae0501.) and (Jaiswal S, et al. “CD47 is upregulated on circulating hematopoietic stem cells and leukemia cells to avoid phagocytosis.” Cell. Vol. 138, No. 2, 2009:pp. 271-85. doi:10.1016 / j.cell.2009.05.046.). In various embodiments, the cells can be modified as described in Jaiswal et al.’s U.S. Patent No. 8,562,997, which is hereby incorporated by reference in its entirety.

[0122] In some embodiments, for example, as done in Xu et al. and Han et al., instead of completely knocking out all HLA genes, only HLA genes with high relevance to the immune response are knocked out, and HLA genes that attenuate macrophage or NK responses (e.g., HLA-E, HLA-F, and HLA-G) are left intact. An approach can also be used in which this approach does not require the addition of a CD47 tag, and the modified cells can be engineered to produce BioNV / exosomes with or without CD47.

[0123] In some embodiments, the method improves the low immunogenicity approach of Table 1. Table 1: Three modification methods of cells using HLA knockout combined with CD47 isoform 2 tag and PD-L1 transmembrane tag (Zhao, et al.) and (Gornalusse GG, et al. “HLA-E-expressing pluripotent stem cells escape allogeneic responses and lysis by NK cells.” Nat Biotechnol. Vol. 35, No. 8, 2017: pp. 765-772. doi: 10.1038 / nbt.3860.).

Table 1

[0124] In various embodiments, the development of allogeneic off-the-shelf modified cells involves the removal of MHC class I and MHC class II protein complexes by disruption of a specific HLA gene or B2M knockout followed by knockout of the CIITA gene. In various embodiments, due to the rapid mechanism of action, knockout can be performed using a CRISPR gene editing approach. In various embodiments, the knockout is performed using zinc finger nucleases (ZFNs) and / or TALENs. In various embodiments, a Cre / Lox recombinase system is used to generate modified cells. In various embodiments, RNA silencing (RNAi, shRNA, microRNA, CRISPR Cas13a-d, etc.) is used to generate modified cells.

[0125] In some embodiments, the development of allogeneically modified cells includes the method of Harding et al. that creates a different type of allogenicity than the methods described above (Harding et al., “Induction of long-term allogeneic cell acceptance and formation of immune privileged tissue in immunocompetent hosts.” BioRxiv 716571 [Preprint], July 30, 2019. doi:10.1101 / 716571.). In some embodiments, rather than deleting MHC class I / II genes and risking preventing long-term acceptance by the recipient, the method of Harding et al. includes an alternative approach based on immune evasion mechanisms that occur in nature. In some embodiments, the method relies on the biomimicry of Harding et al. based on the devil facial tumor disease (DFTD) type 2 of horizontally transmissible cancer that is common in the Tasmanian devil. In some embodiments, the development of allogeneically modified cells includes overexpression of the immunomodulatory proteins CCL21, PD-L1, FasL, SerpinB9, H2-M3, CD47, CD200, and / or MFG-E8 to protect cell derivatives from long-term immune rejection responses in mice (and humans) without deleting MHC class I / II proteins. In some embodiments, the modified cells express one or more of the proteins shown in Table 2 (including any splice variants and / or isoforms of any of the proteins shown, e.g., CD200 splice variants). In some embodiments, this system can be used to inhibit the activity of antigen-presenting cells (APCs), macrophages, natural killer cells, and T lymphocytes.In various embodiments, the modified cell line can also include the safe cell system developed by Liang et al. 2018, in which the cell division gene is linked to a suicide gene to prevent the runaway of teratoma leading to cancer (Liang Q, et al. “Linking a cell-division gene and a suicide gene to define and improve cell therapy safety.” Nature. Vol. 563, No. 7733, 2018: pp. 701-704. doi: 10.1038 / s41586-018-0733-7.).

[0126] In various embodiments, the method improves the low immunogenicity approach of Table 2. Table 2: Expression or increased expression of exemplary proteins for generating allogeneic modified cells.

Table 2

[0127] In various embodiments, obtaining cells from which BioNV / exosomes are derived includes cells engineered to have one or more knockouts of HLA-A, HLA-B, HLA-C, HLA-E, HLA-G, HLA-F, CIITA, IL-6, IL-4, IL-10, IL-16, TRAC, TRBC, and / or any combination thereof, and one or more knockins of CCL2, PD-L1 (in BioNV derived from non-activated cell sources), CTLA-4, H2-M3, CD24, CD47 (removing the 3’UTR region or having an alternative 3’UTR region that does not contain the binding site for inhibitory microRNA), MFG-E8, CD200, and / or any combination thereof.

[0128] In various embodiments, BioNV is generated from modified cells having one or more of the modifications of Table 3. Table 3: Exemplary engineered cell expression profiles for BioNV formation for human use (Fife BT and Bluestone JA. “Control of peripheral T-cell tolerance and autoimmunity via the CTLA-4 and PD-1 pathways.” Immunol Rev. Vol. 224, 2008: pp. 166-82. doi: 10.1111 / j.1600-065X.2008.00662.x.) and (Rong Z, et al. “An effective approach to prevent immune rejection of human ESC-derived allografts.” Cell Stem Cell. Vol. 14, No. 1 2014: pp. 121-30. doi: 10.1016 / j.stem.2013.11.014.). [Table 3]

[0129] In some embodiments, gene inactivation / activation is controlled by an inducible promoter throughout the BioNV / exosome differentiation, activation, and manufacturing processes. In some embodiments, disruption of the MHC, TCR, and CRS genes generates allogeneic iPSCs that are - / - CRS and - / - TCR and that, when injected into a subject, come to have a plasma membrane that exhibits low immunogenicity. CRS genes involved in the etiology of CRS include cytokines such as IL-6, IL-10, IFN-γ, monocyte chemoattractant protein 1 (MCP-1), granulocyte macrophage colony-stimulating factor (GM-CSF), and also tumor necrosis factor (TNF), IL-1, IL-2, IL-2-receptor-α, and IL-8. In some embodiments, one or more of these genes are inactivated, for example, in the cells from which the BioNV / exosomes are derived.

[0130] In various embodiments, the hypoimmunogenic cells obtained to generate BioNV / exosomes are metabolically adjusted (e.g., to a specific differentiated cell lineage) to produce one or more desired therapeutically relevant biomolecules or to increase (or adjust to a desired concentration) the concentration of therapeutically relevant biomolecules, and then the adjusted cells can be processed into BioNV / exosomes. In various embodiments, the cells

[0131] In various embodiments, broad or global regulation of cells results in the expression of multiple genes to produce therapeutically relevant biomolecules that "activate" the cells, such as cytokines, chemokines, regulatory nucleic acids, etc., whereby the cells are enhanced to achieve a metabolically defined goal / phenotype. In various embodiments, the controlled expression of one or more therapeutically relevant biomolecules in cells can reproduce, for example, the activated state that occurs when a T cell receptor (TCR) engages an antigen-presenting cell (APC), such that the T lymphocyte enters an activated state (Figure 1). In various embodiments, the controlled expression of one or more therapeutically relevant biomolecules in cells can reproduce the interaction(s) between antigenic peptides presented by the major histocompatibility complex (MHC) and the TCR of a T cell, thereby initiating a conformational change in the TCR and triggering an intercellular signaling cascade and massive gene expression of cytokines, including perforin, granzyme, alarmin, interleukin, and interferon, etc. (Figure 1). Typically, cytokines enable cells to help eliminate antigens from the host and mobilize immune cells to the site of infection by "activating" the cells in a specific manner, thereby assisting in the elimination of antigen / infected cells and the repair of surrounding tissues. In various embodiments, these cellular processes occur in cell lines without the supply of exogenous cytokines that can have detrimental effects, such as exhaustion, competing signaling pathways, etc. In various embodiments, for example, the activated state of T cells can be utilized in BioNV / exosomes through a method of treating cytokines or transmembrane ligands in a form that entraps them in the lumen and / or membrane of BioNV / exosomes, respectively (Figure 2).

[0132] In some embodiments, broad or global activation of cells (e.g., T cells, natural killer cells) can be achieved outside the host in a culture medium (in vitro). In some embodiments, the activated cells can proliferate and be processed into BioNV / exosomes that function like miniature T cells without the complex genetic baggage of the whole cell. Broad regulation of T cells in vitro can be achieved by a number of methods described herein.

[0133] In some embodiments, activation of low-immunogenicity cells includes activation of the TCR / CD3 receptor complex by protein antigens (in the case of T cells). In some embodiments, activation of low-immunogenicity cells includes activation of the TCR / CD3 receptor complex by small molecules. In some embodiments, the small molecule stimuli used for cell activation by the TCR / CD3 receptor may include phorbol esters. In some embodiments, activation of low-immunogenicity cells includes activation of the TCR / CD3 receptor complex by viral antigens. In some embodiments, the viral antigen can be a recombinant viral protein, such as an extracellular viral antigen (e.g., glycoprotein or spike protein) or an intracellular viral antigen, etc.

[0134] In some embodiments, activation of low-immunogenicity cells includes activation of calcium-dependent channels. In some embodiments, the calcium-dependent channels include, for example, calcium release-activated channels (CRAC) that are activated by an increase in Ca2+ concentration by supply to the culture medium. In some embodiments, potassium channels (K channels) or calcium channels (Ca channels) can be used or overexpressed in cells, and the cells can be activated by supplying the corresponding inorganic components to the medium. In some embodiments, a long-term increase in intracellular calcium or potassium is a signal for mimicking the activation of lymphocytes by antigens or mitogens.

[0135] In various embodiments, activation of the hypoimmunogenic cells includes activation of the LFA-1 integrin receptor. In various embodiments, activation of the LFA-1 integrin receptor can include supplying an activating ligand peptide including ICAM1, ICAM2 (CD102), ICAM3 (CD50), ICAM4, ICAM5, JAM-A, and / or a portion thereof.

[0136] In various embodiments, activation of the hypoimmunogenic cells includes activation of the CD28 receptor. In various embodiments, activation by CD28 can be effected by supplying one or more ligand peptides for CD28 including CD80, CD86, GRAP2, and Grb2. In various embodiments, activation by CD28 is effected in a cell subset that is not memory T cells.

[0137] In various embodiments, activation by IL-12 can be effected by supplying one or more recombinant human IL-12. More specifically, NK cells constitutively express perforin and granzyme. Secretory lysosomes (about 500 nm in diameter) containing perforin and granzyme are retained at a constant concentration intracellularly, enabling natural killer cells to be primed / readied for cytotoxic activity without delay. CD8+ killer T cells (and their cell subsets) also contain low to medium concentrations of perforin and granzyme in their lytic granules. However, in other T cells (e.g., CD4+ / CD8+ memory and helper T cells), perforin and granzyme in the lytic granules are usually low or absent, resulting in a delay in expression and cytotoxic action, and it may take several days to a week to accumulate sufficient lytic granules to initiate a cytotoxic phenotype. This delay is a regulated and buffered function between different cell types to prevent a runaway reaction. By way of example, NK cells and killer T cells are like a garden hose filled with water. When someone turns on the faucet, water comes out immediately. On the other hand, other T cells are like an empty garden hose. It takes time for water to come out even when the faucet is turned on. That is, the reaction is slow. From the perspective of activation for production, it is ideal to use iPSC-derived NK cells and / or killer T cells. This is because these cells are already primed with secretory lysosomes and lytic granules respectively and can be stimulated / activated to rapidly produce additional perforin and granzyme as needed. Other T cells (or other cells such as neutrophils) can also be used as a source of cytotoxic proteins, but the time to express them may be extended. The former cell type reduces costs.

[0138] In some embodiments, activating low-immunogenicity cells includes activating the IL-2 receptor and the IL-12 receptor. In some embodiments, activation by IL-2 can be effected by supplying one or more recombinant human IL-2s. In some embodiments, activation by IL-12 can be effected by supplying one or more recombinant human IL-12s. IL-2 and IL-12 increase the accumulation of perforin and granzyme B mRNA in NK cells and killer T cells. The mRNA transcripts of perforin and granzyme(s) form a low-level "primed sustainable pool" (water in a garden hose) in the cytoplasm and are likely to function as a source for rapid deployment during activation. IL-2 and / or IL-12 are likely to be better candidates for in vitro cell activation than other sources or modes of action such as antigen engagement in other cells or antigen stimulation of surface receptors. This is because these pathways have been shown to only cause an increase in granzyme(s). Thus, the production of both perforin and granzyme is desirable, which can be achieved by IL-2 and IL-12 stimulation through their respective receptors. Additionally, IL-2 has been shown to stabilize the stability of perforin and granzyme mRNA in the cytoplasm. Thus, IL-2 is likely to be a preferred activating cytokine over IL-12 as it helps maintain these cytoplasmic mRNA reservoirs (IL-2 holds water in the hose) (REF-PMID:8103068).

[0139] In some embodiments, the cytokine INF-α can also be used to activate the JAK1 / TYK2-STAT pathway via the INFAR1 and / or INFAR2 receptor, at least in T cells, to produce perforin and granzyme(s).

[0140] In various embodiments, the activation of low immunogenic cells such as natural killer cells (or T cells if the receptor(s) listed below are related to the activation of T cells) includes the activation of receptors (alone or in a combination that allows for a synergistic activation response), and the receptors include, for example, CD2, CD3, CD16, CD28, CD314 (NKG2D), CD335, B7-H6, CD158d, IL-2R, IL-12R, DNAM-1, CD2, CD44, CD137, CX3CR1, CD27, CD160, 2B4, etc., but are not limited to these sensory receptors. CD16 can be activated through binding to the Fc region of an antibody (anti-S2 IgG). CD134, CD335, B7-H6 can be activated in combination with ICAM-1 combined with the Fc region of an antibody. CD314 can be activated by ULBP1, MICA, MICB, or H60. CD158d can be activated to induce pro-inflammatory molecules via HLA-G. IL-2R can be activated by the molecule IL-2. IL-12R can be activated by the molecule IL-12. DNAM-1 can be activated by CD155 or CD112 or NKp30. CD2 can be activated by LFA3. CD44 can be activated by hyaluronic acid, hyaluronan, osteopontin, collagen, or matrix metalloproteinase. The CD28 homolog can be activated by the B7H7 protein ligand. CD137 can be activated by itself and by members of the tumor necrosis factor receptor family of proteins. CX3CR1 can be activated by CX3CL1. CD27 can be activated by CD70. CD160 can be activated by HLA-C. 2B4 can be activated by CD48. Each activation type can result in different levels of cytotoxic biomolecules.Activation of each (or any) of the above combinations can occur by adding each of the following ligands (or any other relevant ligand) either in soluble form (partial peptide, domain or whole protein), immobilized on a membrane (partial peptide, domain or whole protein), as a target cell membrane component (partial peptide, domain or whole protein), or cross-linked to a solid phase including but not limited to Sephadex or magnetic beads (as a partial peptide, domain or whole protein).

[0141] In some embodiments, activation of some natural killer receptors (or T cells if the receptor(s) listed below are relevant for T cell activation) does not result in degranulation or polarization of granules to the IS. These receptors include, but are not limited to: i) 2B4 alone, ii) CD134 alone, iii) CD134 combined with either 2B4 and the inhibitory receptor KIR or CD94, iv) LFA-1 combined with either the inhibitory receptor KIR or CD94, v) CD16 combined with either the inhibitory receptor KIR or CD94. Activation of each (or any) of the above combinations can occur by adding each of the following ligands (or any other relevant ligand): i) CD48, ii) ULBP1, iii) HLA-E, iv) HLA-C in combination with HLA-E, or v) HLA-E, either in soluble form (partial peptide, domain or whole protein), immobilized on a membrane (partial peptide, domain or whole protein), as a target cell membrane component (partial peptide, domain or whole protein), or cross-linked to a solid phase including but not limited to Sephadex or magnetic beads (as a partial peptide, domain or whole protein).

[0142] In some embodiments, activation of some natural killer receptors (or T cells if the receptor(s) listed below are associated with T cell activation) results in degranulation. Immediately prior to degranulation, perforin and granzymes may (or may not) undergo post-translational modifications that enhance their activity. To achieve the maximum effect of the cytotoxic proteins, it may be preferable to activate the cells along the degranulation pathway. These receptors include, but are not limited to, i) CD16 in combination with either LFA-1 and the inhibitory receptor KIR or CD94, ii) CD134 in combination with 2B4, iii) CD16 alone. Activation of each (or any) of the above combinations can occur by adding each of the following ligands (or any other relevant ligand): i) HLA-C in combination with HLA-E, ii) ULBP1 in combination with CD48, iii) anti-S2 IgG, in either a soluble form (partial peptide, domain or full protein), membrane-fixed (partial peptide, domain or full protein), target cell membrane component (partial peptide, domain or full protein), or cross-linked to a solid phase including but not limited to sephadex or magnetic beads (as a partial peptide, domain or full protein).

[0143] In some embodiments, receptors that, when activated, cause polarization of soluble granules may be less than ideal (or may be preferred) because localization to the cell membrane can minimize encapsulation into BioNV but maximize encapsulation into secreted EVs (exosomes and microsomes). This is due to the differences between extrusion manufacturing (BioNV) and cellular exocytosis processing (EV). Some receptors that induce polarization of soluble granules into IS include, but are not limited to, i) CD16 in combination with LFA-1 (which also causes degranulation), ii) LFA-1 alone, iii) CD134 in combination with LFA-1 and 2B4 (which also results in degranulation). Activation of each (or any) of the above combinations can occur by adding each of the following ligands (or any other relevant ligand): i) anti-S2 IgG in combination with ICAM-1, ii) ICAM-1, iii) ULBP1 in combination with CD48 and ICAM-1, in either soluble form (partial peptide, domain or full protein), membrane-fixed (partial peptide, domain or full protein), target cell membrane component (partial peptide, domain or full protein), or cross-linked to a solid phase including but not limited to Sephadex or magnetic beads (as partial peptide, domain or full protein).

[0144] In some embodiments, activation of low immunogenic cells includes activation of the SLAMF1 (CD150) receptor. In some embodiments, activation of cells via the SLAMF1 (CD150) receptor can be performed using one or more SLAM-associated proteins. In some embodiments, the above activation by the SLAMF1 (CD150) receptor is due to interaction with measles virus. In some embodiments, the above activation by the SLAMF1 (CD150) receptor is due to interaction with Gram-negative bacteria.

[0145] In various embodiments, the activation of hypoimmunogenic cells includes the activation of the IFNγ receptor. In various embodiments, the activation by the IFNγ receptor can include any Toll-like receptor (TLR) superfamily member and can be effected by supplying one or more TLR receptor agonists such as IFN-γ, lipoteichoic acid (LTA), lipopolysaccharide (LPS), gardiquimod, R848, CpG, Pam3CSK4, and the like.

[0146] In various embodiments, the activation of hypoimmunogenic cells includes the activation of the CD4 receptor by one or more viruses. In various embodiments, the activating viruses include, but are not limited to, two distantly related arenaviruses, Pichinde virus and lymphocytic choriomeningitis virus. Pichinde virus and lymphocytic choriomeningitis virus have been shown to induce up to 50% of the tumor-specific CTL response in the circulating CD8+ T cell pool. In various embodiments, the ex vivo activation of CD8+ T cells can increase the levels of alarmins (e.g., but not limited to, IL-1α, IL-33, and IL-17), which, after being derived from virally activated cells, are encapsulated into BioNV / exosomes (along with other anti-cancer biomolecules such as perforin and granzyme). In various embodiments, alarmins can increase the induction of a potent cytotoxic effector T lymphocyte (CTLeff) response in the treatment of disease. In various embodiments, the response resulting from the administration of the BioNV / exosomes herein is localized to the site of the disease specifically targeted by the BioNV / exosomes carrying the alarmins (e.g., tumor, infected cells, etc.).

[0147] In various embodiments, activation of the expression of cytotoxic biomolecules such as perforin and granzyme(s) can be achieved by targeting cytoplasmic signaling molecules (e.g., kinases, phosphatases, GTPases, ATPases, etc.) that are part of the signal transduction pathway downstream of membrane-bound receptors and factors. In various embodiments, examples of such approaches in T cells can involve small molecules that activate STAT, Blimp1, Tbet, Runx3, thPOK (by blocking Runx3 or eome inhibition).

[0148] In similar embodiments, PKC (PKCtheta), a serine and threonine specific protein kinase(s) and a transduction signaling molecule central to the activation of T cells and NK cells, can be activated by calcium and the second messenger diacylglycerol. Small molecules that pass through the cell membrane to activate PKC and cause the production of perforin and granzyme can also be added to the cells. There are several commercially available small molecules that can activate PKC. These include, but are not limited to, the following.

[0149] Bryostatin 1: A potent agonist with binding affinity for classical and novel PKC isozymes subgroups (REF-PMID: 16834754).

[0150] Ingenol-3-angelate (I3A, PEP005): A broad-spectrum PKC activator. It has apoptosis-promoting and immunostimulatory effects and may promote the degranulation process. (REF-PMID: 22069553).

[0151] Phorbol 12-myristate 13-acetate: A reversible PKC activator. It activates Ca2+-ATPase (REF-PMID: 3478199).

[0152] Prostratin: A PKC and NF-κB activator. It synergizes with the calcium / calcineurin signaling pathway. A potent synergistic activator of perforin and granzyme(s). (REF-PMID: 24204950).

[0153] SC-9, SC-10: PKC activators that induce PKC phosphorylation. PKC inhibition can be restored using the SC-10 dual system that activates cells and then reduces the activation state when perforin and granzyme(s) reach optimal levels. (REF-PMID: 1414485).

[0154] Phorbol 12,13-dibutyrate (PDBu): Increases kinase phosphorylation. Induces contraction of vascular smooth muscle. The release of mobilized Ca2+ may lead to the activation of perforin in the preload state (REF-PMID: 19632318)

[0155] Other PKC activators are available from Santa Cruz.

[0156] In similar embodiments, Crk, an adapter protein that binds to several tyrosine-phosphorylated proteins and acts as an important regulator of activation in NK cells, can be the target of activation or release of secondary inhibitory-related functions to achieve the expression of perforin and granzyme. MHC-I inhibitory receptors block activation signals, leading to phosphorylation of Crk and subsequent association with c-Abl (REF-PMID: 18835194). HLA-E induces phosphorylation of Crk in NKG2A+ NK cells (REF-PMID: 22464172). Since Crk is required for the activation of CD16 (as described above), prevention of the inhibitory mechanism by interference with small molecules in combination with a CD16 receptor activator can be an alternative means of activating NK cells that produce perforin and granzyme within secretory lysosomes. Also, a small molecule blocker of Crk phosphorylation (1-(2,6-dichlorophenyl)-1,5-dihydro-6-((4-(2-hydroxyethoxy)phenyl)methyl)-3-(1-methylethyl)-4H-pyrazolo[3,4-d]pyrimidin-4-one) is an example of a small molecule that inhibits Crk phosphorylation in combination with a small molecule activator and can be an alternative method for activating NK cells. In various embodiments, the Crk phosphorylation site can be mutated to prevent inhibition. The mutation can be transient or stable within the cell. In such mutants, small molecule activating molecules can be used to activate the cells through Crk. Similar strategies can be used at other points in the signaling pathways that lead to NK activation and the production of perforin and granzyme. As described above, some of the activation points (targeting proteins in the signaling pathway(s)) can cause crosstalk between signaling pathways, which can result in changes in the degree of calcium-dependent perforin activation and / or polarization of perforin and granzyme within the cell. Therefore, depending on the desired state of each protein before the BioNV manufacturing process, the type of activating molecule needs to be selected to suit their purpose. For example, folded perforin in a Ca2+-unbound state may be more desirable than Ca2+-bound perforin.The latter, when encapsulated in BioNV, may cause unintended off-target effects on healthy tissues. Therefore, small molecules that activate the expression of native perforin without inducing calcium release are desired.

[0157] In various embodiments, activation of transcription factors leading to the activation of perforin and granzyme may also be used. In this approach, if it is intended to increase the amount of perforin to a higher level than granzyme, a transcription factor that targets only the perforin promoter for increased expression may be desirable. In another approach, if it is intended to increase the amount of granzyme to a higher level than perforin, a transcription factor that targets only the granzyme promoter for increased expression may be desirable. In another approach, by activating each transcription factor simultaneously, equal levels of each protein can be produced. Small molecules can be used to activate transcription factors. Proteins (transiently or stably expressed) designed / engineered to interact with a transcription factor or transcription factor gene can also be used to activate the transcription factor. Examples of transcription factors that positively regulate the expression of perforin and granzyme include, but are not limited to, eome and NF-κB. Eome can be activated by a CRISPR activation plasmid (REF-EOMES CRISPR). NF-κB can be activated by the small molecule(s) 4-hydroxyquinazoline; 4-kinazolinol.

[0158] In various embodiments, a CRISPR activation and inhibition system (CRISPRa / i) can be used to directly activate or suppress the gene expression functions of perforin and / or granzyme, independent of upstream signaling. In such a system, CRISPRa / i can be transiently (episomally) engineered into the desired iPSC cells or stably integrated, and those cells can differentiate into the desired cell type (e.g., T cells or NK). After differentiation, in the stably integrated cell line, CRISPRa can be activated using a drug-inducible promoter such as tetracycline (but not limited to tetracycline or tetracycline-based promoters). When induced, CRISPRa and each of its gRNAs targeting the promoter regions of perforin and granzyme are expressed. The CRISPRa gRNAs bind to perforin and granzyme and express them. The advantage of this system compared to the above activation approach is to control the expression of perforin and granzyme without activating the cells to an active state. This system can be used to control the expression of other cytotoxic proteins or proteins for therapeutically beneficial purposes that can be encapsulated within the lumen of BioNV. Subsequently, the expression of the desired protein can be turned off using CRISPRi and a gRNA targeting the same promoter region.

[0159] In various embodiments, the perforin and granzyme genes can be incorporated into iPSCs and include regulatory properties embedded within promoters, enabling direct and controlled expression, for example, using drug-inducible or CRISPRa / i or microRNA control systems. In this approach, iPSCs containing engineered cytotoxic genes can be differentiated into any desired cell type, such as monocytes, macrophages, fibroblasts, kidney cells, liver cells, etc. This approach allows cells to express perforin and granzyme (and potentially other engineered cytotoxic proteins if needed or applicable) that would not normally be expressed naturally (and cannot be activated to express these proteins). The purpose of this approach is to enable the expression of such proteins while leveraging cell properties different from those of soluble granule-producing cells (such as natural killer cells, T cells, neutrophils, etc.). Such available cell properties include tissue tropism, barrier-passing phenotypes, and surface markers that improve the high precision of targeting cells or tissues that may be lacking in natural killer cells, T cells, neutrophils, etc.

[0160] In various embodiments, activation of hypoimmunogenic cells includes activation by engineered non-natural biomolecules selected from one or more of soluble peptides, chimeric antigen receptors, small molecule decoys, small molecule ligands, designer nucleic acid ligands, carbohydrate ligands, viral ligands, chimeric biomolecule ligands, fusion proteins, antibodies, and antibody format molecules. In various embodiments, T cells (or desired cells) can be engineered to contain a CAR, and the intracellular domain of the CAR construct can be developed to broadly activate the cells through its signaling domain. For example, in various embodiments, by incorporating 4-1BB into a conventional CD3 intercellular activation domain(s), activation of T cells can be expanded and maintained through activation of non-canonical NF-kB pathways (and subsequent pathways). In various embodiments, the cells are activated by a biomarker antigen engineered such that the extracellular domain of the CAR construct recognizes it through its scFv recognition domain(s). In various embodiments, this broad activation can occur through different types of extracellular CAR domains such as scFv, V H H nanobodies, V NAR , affilins (or other affinity-targeted chemical ligands), or CER, or through biomarker recognition via TCR ligands and components.

[0161] In various embodiments, activation of hypoimmunogenic cells includes activation by inorganic compounds (e.g., iron, calcium, potassium, tin, mercury, arsenic, pyrophosphate, etc.).

[0162] In various embodiments, activation of hypoimmunogenic cells includes activation by expression, overexpression, or increased activity of transcription factors (e.g., signal transducer and activator of transcription (STAT), T-bet, Blimp1, NF-kB, MAPK, etc.). In various embodiments, the cells are activated by expression and / or overexpression of a constitutively active form of an intracellular signaling pathway such as a constitutively active STAT5 molecule to drive signaling.

[0163] In various embodiments, the CAR is activated prior to BioNV / exosome formation. In various embodiments, the CAR is activated via its target, through another receptor and / or virus. In various embodiments, the CAR construct typically activates T / NK / macrophage cells (such cell types) into an active mode (metabolically regulated) to result in the expression of various biomolecules such as alarmins, interferons, granzymes, perforins, etc., including first-generation, second-generation, third-generation, or fourth-generation intracellular signaling modalities. However, in various embodiments, the degree of activation is regulated through different receptors and can result in a lower level of activation of more directed or targeted cytokines for a particular purpose. In various embodiments, the modified cells are activated by IL-15 alone, and by packing this cytokine into BioNV / exosomes, the naturally occurring lymphocytes can be mobilized to the tumor site.

[0164] In various embodiments, the CAR construct (extracellular region) is activated by its antigen (e.g., the soluble form of the antigen biomarker targeted by the CAR construct). In various embodiments, the antigen can be attached to magnetic beads, added to the cell culture, and the magnetic beads can be removed from the culture after activation, or the cells can be passed through a chromatography column containing the bound antigen and be activated as they flow through the column.

[0165] In various embodiments, the CAR construct can be activated via an appropriate biomarker "antigen" recognized by the CAR or a portion thereof, which includes scFv, VERR / viral ligand, V H H nanobody, V NAR, an affinity (or other affinity target biochemical ligand), CER, or a binding moiety, epitope, or peptide of a TCR ligand or component may be included, but is not limited thereto. In various embodiments, the antigen can be a target biomarker or an activated virus, or a protein(s) of the activated virus. In various embodiments, activation of the antigen occurs by binding the antigen on a chromatography column and then passing the modified cells over the column such that they are captured by the antigen via CAR-antigen interaction. In various embodiments, the CAR-modified cells are lymphocytes, which are then eluted from the column to obtain purified activated lymphocytes. In various embodiments, activation by the antigen includes addition of a low level of the antigen to the medium of the modified cells. In various embodiments, after activation (through antigen-second generation or antigen-third generation CAR interaction), the cells are processed by the methods described herein to produce BioNV / exosomes. In various embodiments, the BioNV / exosomes include a second generation (or third generation) CAR on their surface and important lymphocyte activation proteins (lymphocyte anti-cancer cells or anti-deficient cell cytokine repertoire), which may include, but are not limited to, perforin and granzyme B.

[0166] Generally, activation by the antigen can give rise to considerations due to problems in separating the biomarker antigen from the CAR receptor of the modified cells. In various embodiments, some viruses can be added to or exposed to the modified cells (e.g., T cells) to activate them outside the CAR receptor. In various embodiments, the antibody binds to an iron piece or magnetic nanoparticles (iron oxide), and using magnetism, the virus can be separated from the activated cells and removed.

[0167] In various embodiments, each “natural” activation mode (e.g., calcium / potassium-dependent channels, LFA-1 integrin receptor, IL-2 receptor, SLAMF1 (CD150) receptor, IFNγ receptor, TLR receptor, CD4 receptor, virus, etc.) can be complemented by an engineered version of an activated soluble peptide, CAR, small molecule decoy, small molecule ligand, designer nucleic acid ligand, carbohydrate ligand, viral ligand, chimeric biomolecule ligand, or fusion protein, antibody, antibody format molecule, and / or combinations thereof. In various embodiments, activation is performed simultaneously by at least one, at least two, at least three, or at least four or more different activation modes. In various embodiments, any one or more of the activation modes results in a broad and / or global activation of T cells sufficient to produce a minimal level of a therapeutically relevant biomolecule (such as a cytokine described herein), and the therapeutically relevant biomolecule that can be captured from the activated and proliferated T cells can be processed into the lumen of BioNV / exosomes.

[0168] In various embodiments, in addition to the activation pathway, a method of broad or global cell modulation can include conjugating an activating molecule such as an antigen(s) to magnetic beads or streptavidin-biotin beads to ensure separation of the antigen from the cell activation receptor (FIG. 3). For example, in various embodiments, a biomarker antigen that forms an immune synapse (IS) with a CAR construct is conjugated to magnetic beads or streptavidin-biotin beads and then added to CAR-containing cells in vitro to activate the cells. In various embodiments, the biomarker antigen(s) can be mechanically removed from the cell suspension upon activation.

[0169] In various embodiments, activation of low immunogenicity cells includes activation at the DNA level by one or more of a transposase-based method, a Cre / Lox-based method, an endonuclease-based method, a homologous recombination (HR)-based method, a non-homologous end joining (NEHJ)-based method, a microhomology-mediated end joining (MMEJ)-based method, a homology-mediated end joining (HMEJ)-based method, small RNAs, or combinations thereof. In various embodiments, small RNAs include one or more of guide RNA (gRNA), tracer RNA (tracrRNA), microRNA (miRNA), RNA interference (RNAi), small interfering RNA (siRNA), double-stranded RNA, Piwi-interacting RNA (piRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), antisense oligonucleotide (ASO), locked nucleic acid (LNA), splice-switching oligonucleotide (SSO), tRNA, complementary messenger RNA, repeat-associated small interfering RNA (rasiRNA), and small non-coding RNA.

[0170] In various embodiments, activation of low immunogenicity cells includes activation at the RNA level by one or more of guide RNA (gRNA), tracer RNA (tracrRNA), microRNA (miRNA), RNA interference (RNAi), small interfering RNA (siRNA), double-stranded RNA, Piwi-interacting RNA (piRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), antisense oligonucleotide (ASO), locked nucleic acid (LNA), splice-switching oligonucleotide (SSO), tRNA, complementary messenger RNA, repeat-associated small interfering RNA (rasiRNA), endonuclease, small non-coding RNA, IRES element, or combinations thereof.

[0171] In various embodiments, the cells are activated by one or more RNA-guided endonucleases. In various embodiments, the RNA-guided endonucleases are used to inactivate (e.g., cause knockout mutations) and / or activate (e.g., knockout gene repressors) gene expression for the purpose of metabolically engineering the cells or activating signaling pathways that otherwise result in therapeutically relevant biomolecules. In various embodiments, activation of the hypoimmunogenic cells includes activation by an endogenous promoter region and / or enhancer region, e.g., by knock-in of a constitutively active promoter / enhancer element upstream of the gene of interest. In various embodiments, activation of the hypoimmunogenic cells includes activation by stable integration of gene elements. In various embodiments, activation of the hypoimmunogenic cells includes activation by transient expression of gene elements.

[0172] In various embodiments, the above examples of activation (e.g., calcium / calcium-dependent channels, LFA-1 integrin receptor, IL-2 receptor, SLAMF1 (CD150) receptor, IFNγ receptor, TLR receptor, CD4 receptor, and viruses) relate to T cells and subsets of T cells (e.g., helper T cells, memory T cells, etc.) as well as NK cells. However, in various embodiments, activation of cells through surface receptors for the purpose of producing therapeutic-related biomolecules encapsulated within the lumen of BioNV / exosomes can occur in any other cell type that may be applicable to the target disease. For example, in various embodiments, through broad or overall regulation of macrophages, different inflammatory cytokines and / or anti-antigen-directed cytokines can be produced at different concentrations in response to activation of surface receptors. In various embodiments, by using alternative cell types, phenotypes associated with cell membrane proteins can be utilized, which has been shown to transfer to the properties of BioNV / exosomes. For example, in various embodiments, macrophage cell membrane proteins involved in overall cell organization and barrier passage events can be captured in the resulting BioNV / exosomes. The resulting BioNV / exosomes may be able to pass through similar barriers such as the blood-brain barrier.

[0173] In various embodiments, hepatocytes can be regulated to produce therapeutic-related biomolecules that can be used in the context of human diseases such as liver diseases. In various embodiments, hepatocytes can be subject to broad / overall regulation to alter cell surface receptors responsible for downstream cytokine-driven growth and repair at each stage of the cell cycle (e.g., regulation of repair pathways can be divided into different stages of repair and growth: G0, G1, S phase). In various embodiments, hepatocytes can be engaged (activated and / or genetically engineered) in the manner described above in a T cell control scenario.

[0174] In various embodiments, the activation of hypoimmunogenic cells results in a metabolically altered state of the hypoimmunogenic cells. In various embodiments, the activation and / or manipulation methods may be applied to other cell types involved in any single or multifunctional pathway that regulates cells from a resting state to a metabolically altered state, and in the metabolically altered state, newly expressed biomolecules have therapeutic relevance and can be utilized in BioNV / exosomes through the BioNV / exosome processing method.

[0175] In various embodiments, one or more therapeutically relevant biomolecules are chemokines, interferons, interleukins, alarmins, lymphokines, perforin, granzymes, granulysin, tumor necrosis factor (TNF), colony stimulating factors, bone morphogenetic proteins (BMP), erythropoietin (EPO), granulocyte stimulating factor (G-CSF), granulocyte macrophage colony stimulating factor (GM-CSF), gene editing payloads, fusion proteins, antibodies or antibody formats, or combinations thereof.

[0176] In various embodiments, one or more therapeutically relevant biomolecules include cytokines. In various embodiments, cytokines refer to a broad category of small proteins in the range of about 5 kDa to about 50 kDa that have important functions in cell signaling; cytokines typically cannot pass through the lipid bilayer of cells and enter the cytoplasm. In various embodiments, cytokines can include chemokines, interferons (IFNα / β / γ), interleukins (IL), alarmins, lymphokines, and tumor necrosis factor (TNF), colony stimulating factors, bone morphogenetic proteins (BMP), erythropoietin (EPO), granulocyte stimulating factor (G-CSF), granulocyte macrophage colony stimulating factor (GM-CSF), or combinations thereof. In various embodiments, one or more therapeutically relevant biomolecules are pro-inflammatory cytokines. In various embodiments, one or more therapeutically relevant biomolecules are anti-inflammatory cytokines. In various embodiments, one or more therapeutically relevant biomolecules are perforin. In various embodiments, one or more therapeutically relevant biomolecules are granzymes (e.g., granzyme A, B, H, K, and M). Table 4: Exemplary interleukins that can be encapsulated in BioNV / exosomes. [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4]

[0177] In various embodiments, when a cell is activated, differences in surface markers, cytoplasmic proteins, mRNA and / or DNA expression profiles that indicate that the cell has been activated occur. By analyzing the surface markers by flow cytometry, the degree of activation can be accurately measured. Furthermore, the degree of cytotoxic protein production in the cytoplasm can also be analyzed using flow cytometry, immunoblotting, mass spectrometry, ELISA, etc. alone or in combination to measure the degree of activation. Furthermore, the mRNA level can be measured using standard mRNA detection and amplification procedures such as (but not limited to) nested or qPCR methods. Furthermore, by analyzing the differences in gene profiling between quiescent cells and activated cells, the degree of activation can be determined. This can be achieved by DNA expression arrays or similar techniques.

[0178] In various embodiments, the same measurements as above (excluding gene measurements since BioNV does not contain cellular nucleic acids) can be used to detect and monitor the degree of cell activation and can be similarly applied to BioNV. By measuring the same markers of activation, an indirect measurement is achieved.

[0179] In various embodiments, the therapeutic exosomes encapsulate a payload, such as a "lumen-loading" (i.e., the exosome can fill the lumen (the space inside the biomimetic nanovesicle) with the payload) payload. In various embodiments, the payload is one or more of a biological agent, a nucleic acid, a fusion protein, a fluorescent protein, a tracer dye, a radionuclide, and / or a small molecule. In various embodiments, the payload is a therapeutic payload for the disease type targeted by the CAR. In various embodiments, the payload comprises one or more of an alkylating agent, an anthracycline, an antimetabolite, an antitumor antibiotic, an antibody or antibody format, a corticosteroid, a plant alkaloid, a topoisomerase inhibitor, a checkpoint inhibitor, an anti-infective agent, and / or a growth factor.

[0180] In various embodiments, the nucleic acid payload encodes one or more of a CRISPR / Cas component, a guide RNA (gRNA), a tracer RNA (tracrRNA), a microRNA (miRNA), RNA interference (RNAi), small interfering RNA (siRNA), double-stranded RNA, Piwi-interacting RNA (piRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), an antisense oligonucleotide (ASO), a locked nucleic acid (LNA), a splice-switching oligonucleotide (SSO), a tRNA, a ribosomal RNA (rRNA), a short hairpin (shRNA) complementary messenger RNA, a repeat-associated small interfering RNA (rasiRNA), and a small non-coding RNA.

[0181] In various embodiments, one or more gene editors are site-specific endonucleases, TALENs, ZFNs, RNase P RNAs, CRISPR / Cas nucleases, C2c1, C2c2, C2c3, Cas9, Cpf1, TevCas9, archaeal Cas9, CasY.1, CasY.2, CasY.3, CasY.4, CasY.5, CasY.6, CasX, Cas omega, transposases, and / or any orthologs or homologs thereof. In various embodiments, the gene editor may also include a gRNA, which refers to a guide RNA as used herein. In various embodiments, the gRNA may be a sequence complementary to a coding or non-coding sequence and can be tailored to a specific sequence to be targeted. In various embodiments, the gRNA may be a sequence complementary to a protein-coding sequence, such as a sequence encoding one or more viral structural proteins (e.g., gag, pol, env, and tat). In various embodiments, the gRNA sequence may be a sense or antisense sequence. In various embodiments, when the gene editor composition is administered herein, preferably, but not limited to, it contains two or more gRNAs. However, a single gRNA can also be used.

[0182] In various embodiments, the gene editing payload includes a trans-activation response region (TAR) loop system. In various embodiments, the therapeutic BioNV / exosome expresses a gene editor and encapsulates a plasmid containing a TAR loop sequence between the 5' end of the promoter and the gene editor / guide cassette, and this TAR loop sequence functions as a barrier to block transcription. In various embodiments, transcription is induced only in cells that are infected and contain the HIV Tat protein. In various embodiments, the Tat protein binds to the TAR loop, relaxes it, liberates the promoter for transcription, thereby expressing the editor and its guide.

[0183] In various embodiments, BioNV / exosomes can encapsulate disease-specific therapeutic payloads, for example, encapsulating an increase in perforin or granzyme from activated lymphocytes into the resulting BioNV / exosomes to be able to pierce cancer cells. In various embodiments, an increase in cellular alarmin production can be encapsulated into the resulting BioNV / exosomes to enhance the recruitment of additional lymphocytes to the solid tumor site. In various embodiments, cells are modified to increase the production of gene editing payloads, such that BioNV / exosomes can incorporate gene editing materials for tissue-specific targeting.

[0184] In various embodiments, when a single type of cytokine with therapeutic relevance value, or a small subset of cytokines, encapsulated within the lumen of BioNV / exosomes is desired, rather than a broad / global expression method that often involves a large series of regulatory scenarios, a focused cellular control may be required.

[0185] In various embodiments, a therapeutically relevant biomolecule (e.g., a cytokine) can be selectively expressed from any given cell by manipulating regulatory points within the signaling points. In various embodiments, for example, activation of a kinase, (de)activation of a phosphorylase, activation of a hydrolase (e.g., GTPase), introduction of an inhibitor (to branch or block signals from one pathway to another), or activation of an activator / agonist to accelerate, one point (or multiple points) within a single signaling pathway (or multiple pathways) can be activated. In various embodiments, these signaling pathways can be selectively activated by supplying cells with proteins, peptides, small molecules, nucleic acids, carbohydrates, chimeric molecules, viral ligands, inorganic elements / compounds (e.g., calcium), etc. alone or in combination, concentrating intracellular pathways to drive the expression of therapeutically relevant biomolecules within the cell, and then encapsulating those biomolecules into the lumen of BioNV / exosomes (Figure 3). In various embodiments, the regulatory points can include, but are not limited to, one or more cell surface receptors that are targeted alone or in combination to concentrate signals within the cell to express the desired biomolecule. In various embodiments, the cells can be genetically modified to be particularly sensitive to the activation of these pathways, resulting in metabolically / phenotypically adjusted cells (of any type) from which BioNV / exosomes can be made. In various embodiments, the cells can be made "particularly sensitive" to activation by specific stimuli by overexpression of cell surface receptors, overexpression of intracellular signaling molecules (e.g., STAT, NF-κB, MAPK / ERK / ATM kinase, etc.), and / or their constitutively active variants.

[0186] In various embodiments, regulatory points within the cell can be manipulated to express a particular therapeutically relevant protein as a stable cell line (e.g., stable integration for constitutive expression).

[0187] In some embodiments, the transcription factor can be activated intracellularly by supplying small molecules, (de)phosphorylation events, nucleic acids, etc. In some embodiments, the transcription factor can be transiently expressed by a plasmid. In some embodiments, the transcription factor can be stably expressed from integration (e.g., using a constitutively active promoter) to generate a stable cell line having constitutive signaling of the signaling pathway related to the therapeutic biomolecule.

[0188] In some embodiments, the promoter region or enhancer region of a gene can be regulated by overexpression of a specific transcription factor, can be regulated by microRNA, can be regulated by tRNA, and can be activated or inhibited using a (g)RNA-guided endonuclease linked to an activation domain or an inhibitory domain (e.g., CRIPSRa / CRISPRi).

[0189] In some embodiments, the therapeutic-related gene of interest can be (stably or transiently) integrated into the target cells (based on desired properties such as membrane proteins suggesting barrier passage). In some embodiments, the stably integrated gene can be activated by any one of the methods described herein.

[0190] In some embodiments, background (unwanted) mRNA can be silenced by interfering RNAs (e.g., siRNA, RNAi, etc.) to enhance the presence / expression of the desired mRNA for the protein of interest. In some embodiments, the mRNA can also be regulated through an IRES element. In some embodiments, specific splicing variants can be enhanced by the addition of biomolecules that enhance and / or suppress the IRES, and produce the desired therapeutic-related peptide / protein that can be encapsulated within BioNV / exosomes during post-activation cell processing.

[0191] In various embodiments, unwanted genes can be knocked out to enhance the expression of a desired therapeutically relevant gene. Those skilled in the art will understand methods available for transiently and stably knocking out unwanted genes and / or entire signaling pathways during the processing of BioNV / exosomes.

[0192] In various embodiments, focused activation through a CAR construct can be performed. In various embodiments, the intracellular domain of the CAR construct can be designed to include an activation domain that specifically focuses on a single (or multiple) signaling pathway. In various embodiments, these constructs can be activated through the extracellular binding domain of the CRA construct (e.g., supplying a specific stimulus to the cell).

[0193] In various embodiments, to enhance the presence of a therapeutically relevant biomolecule within a desired cell (metabolically engineered to produce the desired therapeutically relevant biomolecule), methods for regulating the genes described herein are used to increase (or adjust to a desired concentration) the concentration of the therapeutically relevant biomolecule, and then the regulated cells are processed to become BioNVs or naturally secreted exosomes containing the therapeutically relevant biomolecule at the desired concentration within the lumen, which can treat mammalian diseases.

[0194] Therapeutic BioNV In various aspects, the present disclosure provides a membrane that substantially lacks one or more of (i) one or more membrane-embedded targeting agents targeted to one or more cell biomarkers, (ii) one or more membrane-embedded proteins of alpha-phagocyte integrin, CCL2, H2-M3, FasL, MFEG8, PD-L1 (in BioNV derived from non-activated cell sources) and / or CTLA-4, SerpinB9, and an anti-IL-6R antibody or antibody format, (iii) any one of CD24, CD47, CD200, chimeric CD24 / CD47, chimeric CD24 / CD200, and chimeric CD47 / CD200, or any two of CD24, CD47, and CD200, and (iv) any one of HLA-A, HLA-B, HLA-C, HLA-F, CIITA, IL-6, TRAC, TRBC, HLA-E, or HLA-G, and one or more of IL-4, IL-10, and IL-16, wherein the one or more therapeutically relevant biomolecules include chemokines, interferons, interleukins, alarmins, lymphokines, perforin, tumor necrosis factor (TNF), colony stimulating factors, bone morphogenetic proteins (BMP), erythropoietin (EPO), granulocyte stimulating factor (G-CSF), granulocyte macrophage colony stimulating factor (GM-CSF), gene editing payloads, fusion proteins, antibodies or antibody formats, or combinations thereof, and the membrane is included in a therapeutic BioNV.

[0195] In various aspects, the present disclosure includes a membrane-embedded targeting agent targeted to one or more cell biomarkers, (ii) one or more membrane-embedded proteins of α-phagocyte integrin, CCL2, H2-M3, FasL, MFEG8, PD-L1 (in BioNV derived from non-activated cell sources) and / or CTLA-4, SerpinB9, and an anti-IL-6R antibody or antibody format, (iii) a membrane-embedded protein of either CD24 and CD47, or chimeric CD24 / CD47, and (iv) one or more of HLA-A, HLA-B, HLA-C, HLA-F, CIITA, IL-6, TRAC, TRBC, HLA-E or HLA-G, SerpinB9, and CD200, and one or more of IL-4, IL-10 and IL-16, and a membrane substantially lacking one or more of the following therapeutic-related biomolecules, wherein the one or more therapeutic-related biomolecules include chemokines, interferons, interleukins, alarmins, lymphokines, perforin, granzyme, granulysin, tumor necrosis factor (TNF), colony-stimulating factors, bone morphogenetic proteins (BMP), erythropoietin (EPO), granulocyte stimulating factor (G-CSF), granulocyte macrophage colony-stimulating factor (GM-CSF), gene editing payloads, fusion proteins, antibodies or antibody formats, or combinations thereof, and includes a therapeutic BioNV.

[0196] In embodiments, the BioNV expressing PD-L1 is derived from non-activated cells, and the BioNV is substantially lacking perforin and / or granzyme.

[0197] In various embodiments, BioNV expresses and / or has the activity of one or more antigens, ligands, and / or receptors of tumor cells or cancer tissues. In various embodiments, one or more antigens, ligands, and / or receptors of tumor cells or cancer tissues are listed in Table 4, Table 5, and / or Table 6. In various embodiments, one or more antigens, ligands, and / or receptors of tumor cells or cancer tissues are neoantigens such as variant variants or peptides specific to a cancer or disease state that are not normally present in the subject being treated. In various embodiments, BioNV expresses and / or has the activity of a costimulatory molecule for immune cells. In various embodiments, the costimulatory molecule for immune cells is IL-15.

[0198] In various embodiments, BioNV contains one or more outer-facing, membrane-embedded targeting agents (e.g., CARs) that can bind to one or more target molecules. In various embodiments, BioNV contains an outer-facing, membrane-embedded CAR that can bind to a target molecule. In various embodiments, therapeutic BioNV is biomimetic due to the nanovesicle composition derived from the plasma membrane of allogeneic hypoimmunogenic modified cells. In various embodiments, therapeutic BioNV contains a lipid bilayer derived from the plasma membrane and completely encapsulates an aqueous core that can accommodate various cell-derived molecules including perforin, granzyme, cytokines, gene editing payloads, etc. In various embodiments, the aqueous core of therapeutic BioNV can further encapsulate therapeutic agents such as exogenous biological agents, fluorescent proteins, tracer dyes, radionuclides, and small molecules.

[0199] In various embodiments, the CAR construct can include various structural molecules. The structure-function of a prototype CAR includes an extracellular (or outward-facing) binding moiety (e.g., scFv) connected by a hinge peptide (e.g., CH2 / CH3 domain from the IgG Fc region, Gly-Gly-Ser peptide bond, CD28 peptide, CD8α peptide, etc.) to a transmembrane domain (e.g., CD28, CD3ζ, CD4, CD8α, ICOS, etc.), followed by various intracellular signaling domains (e.g., 4-1BB, CD3ζ, CD28, 4-1BB, ICOS, CD27, OX40, etc.) in the form of a fusion protein. In various embodiments, since BioNV lacks the intracellular machinery of whole cells, it does not require intracellular signaling molecules in CAR design (primary CAR construct). In various embodiments, the CAR construct includes an extracellular scFV binding moiety fused to the transmembrane domain of CD28 with an IgG CH2 / CH3 linker and substantially lacks an intracellular domain or functionality. In various embodiments, the CAR construct replaces the intracellular domain of the prototype or otherwise fuses with an anchor protein, such as the PLA2 domain from AAV, a fusion protein, a radionuclide binding domain, a cytoskeletal element, a small molecule transport domain, etc., which can assist in fusion to target cells and / or encapsulation and release of therapeutic payloads.

[0200] In various embodiments, the CAR antigen-binding molecule includes various binding moieties including antibody-based or antibody-format binding domains. In various embodiments, the therapeutic BioNV includes an antibody or antibody-format binding moiety selected from one or more of monoclonal antibodies, polyclonal antibodies, antibody fragments, Fab, Fab’, Fab’-SH, F(ab’)2, Fv, single-chain Fv (scFv), diabody, nanobody, linear antibody, bispecific antibody, multispecific antibody, chimeric antibody, humanized antibody, human antibody, and fusion proteins containing the antigen-binding portion of an antibody. In various embodiments, the CAR construct includes a bispecific T cell engager (BiTE), a viral epitope recognition receptor (VERR), a variable heavy chain IgG fragment VHH, VNAR, or a binding moiety via an engineered T cell receptor (TCR).

[0201] In order to ensure the proper orientation of the CAR and eliminate BioNV lacking the CAR, in some embodiments, HPLC-based affinity chromatography techniques can be used to select and concentrate only those BioNV having a sufficient surface concentration of CAR exposed to the solvent. Using HPLC-based affinity chromatography techniques, the concentration of NV having contaminating cellular material and immunogenic cell surface markers can be reduced by either positive or negative selection.

[0202] In some embodiments, the therapeutic BioNV can include NV having only the outer plasma membrane leaflet, only the inner plasma membrane leaflet, and / or both leaflets of an intact plasma membrane lipid bilayer. In some embodiments, iPSC-derived NV is added with additional lipid additives (e.g., phosphatidylethanolamine, phosphatidylcholine, phosphatidylinositol, ceramide, lecithin, etc.), nonionic surfactants (e.g., sorbitan monostearate, octadecylamine, etc.), sterols (e.g., cholesterol, bile salt derivatives, etc.), polyols (e.g., maltodextrin, sorbitol, sucrose, mannitol, etc.), and proteins (e.g., serum albumin, etc.) to improve physicochemical properties such as thermal stability and encapsulation / release of therapeutic payloads. The amount of cholesterol and the length and saturation of the hydrocarbon chains of phospholipids can affect the rigidity and stability of the bilayer, and thus can affect the ability of NV to receive and release drugs, biomolecules, and other therapeutic payloads. In some embodiments, the therapeutic BioNV also incorporates zwitterionic lipids and methods of using zwitterionic lipids as described, for example, in U.S. Patent Publication US20130216607, the content of which is hereby incorporated by reference in its entirety. Correspondingly, by functionalizing the hydrophilic head of the lipid with a polymer or biomolecule, new features can be provided on the vesicle surface, and interactions with blood components, tissues, and the immune system can be formed in vivo.

[0203] In various embodiments, the therapeutic BioNV encapsulates a payload, such as a "lumen-loaded" payload (i.e., a payload that the BioNV can load into the lumen, the space within the biomimetic nanovesicle). In various embodiments, the payload is one or more of a biological agent, a nucleic acid, a fusion protein, a fluorescent protein, a tracer dye, a radionuclide, and / or a small molecule. In various embodiments, the payload is a therapeutic payload for the disease type targeted by the CAR. In various embodiments, the payload comprises one or more of an alkylating agent, an anthracycline, an antimetabolite, an antitumor antibiotic, an antibody or antibody format, a corticosteroid, a plant alkaloid, a topoisomerase inhibitor, a checkpoint inhibitor, an anti-infective agent, and / or a growth factor.

[0204] In various embodiments, the nucleic acid payload encodes one or more of a CRISPR / Cas component, a guide RNA (gRNA), a tracer RNA (tracrRNA), a microRNA (miRNA), RNA interference (RNAi), small interfering RNA (siRNA), double-stranded RNA, Piwi-interacting RNA (piRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), an antisense oligonucleotide (ASO), a locked nucleic acid (LNA), a splice-switching oligonucleotide (SSO), a tRNA, a ribosomal RNA (rRNA), a short hairpin (shRNA) complementary messenger RNA, a repeat-associated small interfering RNA (rasiRNA), and a small non-coding RNA.

[0205] In various embodiments, the payload includes gene editing nucleic acids and / or proteins, such as TALENs, ZFNs, RNase P RNAs, C2c1, C2c2, C2c3, Cas9, Cpf1, TevCas9, archaeal Cas9, CasY.1, CasY.2, CasY.3, CasY.4, CasY.5, CasY.6, CasX, Cas omega, transposases, and / or any orthologs or homologs of any of these editors. In various embodiments, the gene editor may also include a gRNA, which as used herein refers to a guide RNA. In various embodiments, the gRNA can be a sequence complementary to a coding or non-coding sequence and can be tailored to a specific sequence to be targeted. In various embodiments, the gRNA can be a sequence complementary to a protein-coding sequence, such as a sequence encoding one or more viral structural proteins (e.g., gag, pol, env, and tat). In various embodiments, the gRNA sequence can be a sense or antisense sequence. In various embodiments, when the gene editor composition is administered herein, preferably but not limited to, it includes two or more gRNAs. However, a single gRNA can also be used.

[0206] In various embodiments, the therapeutic BioNV includes one or more perforin molecules and / or one or more granzyme molecules derived from the cells from which the BioNV is derived. In various embodiments, the therapeutic BioNV includes one or more perforin molecules and one or more granzyme molecules added externally to the BioNV. In various embodiments, BioNVs designed to carry granzyme lack CD200 and SerpinB9.

[0207] In various embodiments, the therapeutic BioNV can deliver a gene editing payload that includes a trans-activation response region (TAR) loop system. In various embodiments, the therapeutic BioNV expresses a gene editor and encapsulates a plasmid that includes a TAR loop sequence between the 5' end of a promoter and the gene editor / guide cassette, and this TAR loop sequence functions as a barrier to block transcription. In various embodiments, transcription is induced only in cells that are infected and contain the HIV Tat protein. In various embodiments, the Tat protein binds to the TAR loop, relaxes it, liberates the promoter for transcription, thereby expressing the editor and its guide.

[0208] In various embodiments, the primary targeting therapeutic BioNV can be used to deliver a small molecule therapeutic payload. In various embodiments, a second-generation (or third-generation or fourth-generation) CAR-containing therapeutic BioNV derived from activated lymphocytes can contain cytokines and other cytotoxic peptides. In various embodiments, the therapeutic BioNV can be formatted to encapsulate and deliver plasmid DNA, for example, to express a gene editing nuclease and gRNA in target cells. Alternatively, or additionally, in various embodiments, the therapeutic BioNV can encapsulate a nuclease and gRNA. In various embodiments, the targeted second-generation (or third-generation or fourth-generation) therapeutic BioNV can be designed to encapsulate and deliver an additional therapeutic protein or peptide of interest.

[0209] In various embodiments, the therapeutic BioNV is allogeneic and / or hypo-immunogenic by substantially lacking the above. In various embodiments, when injected into a subject to treat a disease, the therapeutic BioNV does not cause a harmful immune response.

[0210] In various embodiments, the therapeutic BioNVs are formed by treating hypoimmunogenic cells activated by one or more of sonication, adaptive focused acoustics technology, French press, extrusion, continuous extrusion, enzymatic lysis of cells (e.g., trypsinization), cell lysis by surfactants, and / or electroporation. In various embodiments, the cell disruption is by continuous extrusion; for example, when the BioNVs are derived from whole cells using a continuous extrusion process, the decrease in the pore size of the polycarbonate filter matches the diameter and volume of the BioNVs.

[0211] In various embodiments, therapeutic BioNVs can be analyzed for size uniformity by methods of determining particle size such as dynamic light scattering (DLS), flow cytometry, mass photometry, etc. In various embodiments, therapeutic BioNVs can be filtered by particle size or particle size range to optimize renal clearance and other clinically relevant NV properties. In various embodiments, therapeutic BioNVs are sized from about 20 nm to 1200 nm. In various embodiments, therapeutic BioNVs are sized about 10 nm, about 20 nm, about 30 nm, about 40 nm, about 50 nm, about 60 nm, about 70 nm, about 80 nm, about 90 nm, about 100 nm, about 120 nm, about 140 nm, about 160 nm, about 180 nm, about 200 nm, about 300 nm, about 400 nm, about 500 nm, about 600 nm, about 700 nm, about 800 nm, about 900 nm, about 1000 nm, about 1100 nm, or about 1200 nm. In various embodiments, the size of therapeutic BioNVs ranges from about 10 nm to 20 nm, about 20 nm to 30 nm, about 30 nm to 40 nm, about 40 nm to 50 nm, about 50 nm to 60 nm, about 60 nm to 70 nm, about 70 nm to 80 nm, about 80 nm to 90 nm, about 90 nm to 100 nm, about 10 nm to 100 nm, about 100 nm to 200 nm, about 200 nm to 400 nm, about 400 nm to 600 nm, about 600 nm to 800 nm, about 800 nm to 1000 nm, or about 1000 nm to 1200 nm.

[0212] Therapeutic exosomes In various aspects, the present disclosure includes a membrane-embedded targeting agent targeted to one or more cell biomarkers, (ii) one or more membrane-embedded proteins of α-phagocyte integrin, CCL2, H2-M3, FasL, MFEG8, PD-L1 (in BioNV derived from non-activated cell sources) and / or CTLA-4, SerpinB9, and an anti-IL-6R antibody or antibody format, (iii) any one of CD24, CD47, CD200, chimeric CD24 / CD47, chimeric CD24 / CD200, and chimeric CD47 / CD200, or any two of CD24, CD47, and CD200 as membrane-embedded proteins, (iv) any one of HLA-A, HLA-B, HLA-C, HLA-F, CIITA, IL-6, TRAC, TRBC, HLA-E or HLA-G, and one or more proteins selected from IL-4, IL-10, and IL-16, and a membrane substantially lacking one or more of the above-mentioned treatment-related biomolecules, wherein the one or more treatment-related biomolecules include chemokines, interferons, interleukins, alarmins, lymphokines, perforin, tumor necrosis factor (TNF), colony stimulating factors, bone morphogenetic proteins (BMP), erythropoietin (EPO), granulocyte stimulating factor (G-CSF), granulocyte macrophage colony stimulating factor (GM-CSF), gene editing payloads, fusion proteins, antibodies or antibody formats, or combinations thereof, and include therapeutic exosomes.

[0213] In various aspects, the present disclosure includes a membrane-embedded targeting agent targeted to one or more cell biomarkers, (ii) one or more membrane-embedded proteins of α-phagocyte integrin, CCL2, H2-M3, FasL, MFEG8, PD-L1 (in BioNV derived from non-activated cell sources) and / or CTLA-4, SerpinB9, and an anti-IL-6R antibody or antibody format, (iii) a membrane-embedded protein of either CD24 and CD47, or chimeric CD24 / CD47, and (iv) one or more of HLA-A, HLA-B, HLA-C, HLA-F, CIITA, IL-6, TRAC, TRBC, HLA-E or HLA-G, SerpinB9, and CD200, and one or more of IL-4, IL-10 and IL-16, and a membrane substantially lacking one or more of the above proteins, wherein the one or more treatment-related biomolecules include chemokines, interferons, interleukins, alarmins, lymphokines, perforin, granzyme, granulysin, tumor necrosis factor (TNF), colony stimulating factor, bone morphogenetic protein (BMP), erythropoietin (EPO), granulocyte stimulating factor (G-CSF), granulocyte macrophage colony stimulating factor (GM-CSF), gene editing payload, fusion protein, antibody or antibody format, or combinations thereof, and includes therapeutic exosomes.

[0214] In embodiments, exosomes expressing PD-L1 are derived from non-activated cells and the exosomes are substantially lacking perforin and / or granzyme.

[0215] In some embodiments, the exosomes express and / or have the activity of one or more antigens, ligands, and / or receptors of tumor cells or cancer tissues. In some embodiments, one or more antigens, ligands, and / or receptors of tumor cells or cancer tissues are listed in Table 4, Table 5, and / or Table 6. In some embodiments, one or more antigens, ligands, and / or receptors of tumor cells or cancer tissues are neoantigens such as mutant variants or peptides specific to a cancer or disease state that are not normally present in the subject being treated. In some embodiments, the exosomes express and / or have the activity of co-stimulatory molecules for immune cells. In some embodiments, the co-stimulatory molecule for immune cells is IL-15. In some embodiments, exosomes expressing and / or having the activity of one or more neoantigens and / or co-stimulatory molecules can be used, for example, in the activation / stimulation targeting neoantigens, in all cells described herein, and / or in the production of BioNV.

[0216] In some embodiments, the exosomes contain one or more outer-facing, membrane-embedded targeting agents (e.g., CARs) that can bind to one or more target molecules. In some embodiments, the exosomes contain an outer-facing, membrane-embedded CAR that can bind to a target molecule. In some embodiments, the therapeutic exosomes are biomimetic due to the nanovesicle composition derived from the plasma membrane of allogeneic low-immunogenic modified cells. In some embodiments, the therapeutic exosomes contain a lipid bilayer derived from the plasma membrane and completely enclose an aqueous core that can accommodate various cell-derived molecules including perforin, granzyme, cytokines, gene editing payloads, etc. In some embodiments, the aqueous core of the therapeutic exosomes can further encapsulate therapeutic agents such as exogenous biological agents, fluorescent proteins, tracer dyes, radionuclides, and small molecules.

[0217] In various embodiments, the CAR construct can include various structural molecules. The structure-function of a prototype CAR includes an extracellular (or outward-facing) binding moiety (e.g., scFv) connected by a hinge peptide (e.g., CH2 / CH3 domain from the IgG Fc region, Gly-Gly-Ser peptide bond, CD28 peptide, CD8α peptide, etc.) to a transmembrane domain (e.g., CD28, CD3ζ, CD4, CD8α, ICOS, etc.), followed by various intracellular signaling domains (e.g., 4-1BB, CD3ζ, CD28, 4-1BB, ICOS, CD27, OX40, etc.), in the form of a fusion protein. In various embodiments, therapeutic exosomes lack the intracellular machinery of whole cells and thus do not require intracellular signaling molecules in the CAR design (primary CAR construct). In various embodiments, the CAR construct includes an extracellular scFV binding moiety fused to the transmembrane domain of CD28 with an IgG CH2 / CH3 linker and substantially lacks an intracellular domain or functionality. In various embodiments, the CAR construct replaces the intracellular domain of the prototype or otherwise fuses with an anchor protein, such as the PLA2 domain from AAV, a fusion protein, a radionuclide binding domain, a cytoskeletal element, a small molecule transport domain, etc., which can assist in fusion to target cells and / or encapsulation and release of the therapeutic payload.

[0218] In various embodiments, the CAR antigen-binding molecule includes various binding moieties including antibody-based or antibody-format binding domains. In various embodiments, the therapeutic exosome includes an antibody or antibody-format binding moiety selected from one or more of monoclonal antibodies, polyclonal antibodies, antibody fragments, Fab, Fab’, Fab’-SH, F(ab’)2, Fv, single-chain Fv (scFv), diabody, nanobody, linear antibody, bispecific antibody, multispecific antibody, chimeric antibody, humanized antibody, human antibody, and fusion proteins containing the antigen-binding portion of an antibody. In various embodiments, the CAR construct includes a bispecific T cell engager (BiTE), a viral epitope recognition receptor (VERR), a variable heavy chain IgG fragment VHH, VNAR, or a binding moiety via an engineered T cell receptor (TCR).

[0219] In various embodiments, to ensure the proper orientation of the CAR and to exclude exosomes lacking the CAR, HPLC-based affinity chromatography techniques can be used to select and concentrate only those exosomes with a sufficient surface concentration of CAR exposed to the solvent. The HPLC-based affinity chromatography techniques can be used to reduce the concentration of NVs with contaminating cellular material and immunogenic cell surface markers by either positive or negative selection.

[0220] In various embodiments, therapeutic exosomes are naturally discharged from cells via the standard vesicle pathway, i.e., exosomes can be collected from cells without irreversibly mechanically disrupting (e.g., rupturing, shearing, etc.) the plasma membrane of the cells from which the exosomes are derived, which is different from therapeutic BioNVs. In various embodiments, exosomes can be collected from the cell culture medium, for example, by differential ultracentrifugation of the culture medium.

[0221] In various embodiments, collecting therapeutic exosomes from activated low-immunogenic cells includes inducing hypoxia. In various embodiments, therapeutic exosome formation can be increased from cells using many strategies such as induction of cellular stress.

[0222] In various embodiments, collecting therapeutic exosomes from activated low-immunogenic cells includes expressing or increasing the expression of one or more cytokines. In various embodiments, overexpression of cytokines such as tetraspanin CD9 or hypoxia-inducible factor-1α induces exosome formation and enables collection.

[0223] In various embodiments, harvesting therapeutic exosomes from activated low-immunogenic cells involves supplying one or more small molecule exosome modulators. In various embodiments, the small molecule modulators supplied may include phenoterol, norepinephrine, N-methyl dopamine, and / or mephenesin.

[0224] In various embodiments, harvesting therapeutic exosomes from activated low-immunogenic cells involves supplementing the medium with one or more exosome factors. In various embodiments, exosome factors may include nutrient supplementation of the medium, such as by forskolin. In various embodiments, exosome factors can include providing specific cell type stimulation to increase the production of therapeutic exosomes. For example, cardiac fibroblasts can be stimulated with TGF-β to increase collagen expression, which can lead to exosome formation.

[0225] In various embodiments, therapeutic exosomes encapsulate a payload, such as a "loaded lumen" (i.e., the exosome can fill the lumen (the space within the biomimetic nanovesicle) with the payload). In various embodiments, the payload is one or more of a biological agent, nucleic acid, fusion protein, fluorescent protein, tracer dye, radionuclide, and / or small molecule. In various embodiments, the payload is a therapeutic payload for the disease type targeted by the CAR. In various embodiments, the payload includes one or more of an alkylating agent, anthracycline, antimetabolite, antitumor antibiotic, antibody or antibody format, corticosteroid, plant alkaloid, topoisomerase inhibitor, checkpoint inhibitor, anti-infective agent, and / or growth factor.

[0226] In various embodiments, the nucleic acid payload encodes one or more of a CRISPR / Cas component, guide RNA (gRNA), tracer RNA (tracrRNA), microRNA (miRNA), RNA interference (RNAi), small interfering RNA (siRNA), double-stranded RNA, Piwi-interacting RNA (piRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), antisense oligonucleotide (ASO), locked nucleic acid (LNA), splice-switching oligonucleotide (SSO), tRNA, ribosomal RNA (rRNA), short hairpin (shRNA) complementary messenger RNA, repeat-associated small interfering RNA (rasiRNA), and small non-coding RNA.

[0227] In various embodiments, the payload includes gene editing nucleic acids and / or proteins such as TALEN, ZFN, RNase P RNA, C2c1, C2c2, C2c3, Cas9, Cpf1, TevCas9, archaeal Cas9, CasY.1, CasY.2, CasY.3, CasY.4, CasY.5, CasY.6, CasX Cas omega, transposase, and / or any ortholog or homolog of any of these editors. In various embodiments, the gene editor may also include a gRNA, which as used herein refers to a guide RNA. In various embodiments, the gRNA can be a sequence complementary to a coding or non-coding sequence and can be tailored to a specific sequence of interest. In various embodiments, the gRNA can be a sequence complementary to a protein-coding sequence, such as a sequence encoding one or more viral structural proteins (e.g., gag, pol, env, and tat). In various embodiments, the gRNA sequence can be a sense or antisense sequence. In various embodiments, when a gene editor composition is administered herein, it preferably includes, but is not limited to, two or more gRNAs. However, a single gRNA can also be used.

[0228] In some embodiments, therapeutic exosomes can deliver a gene editing payload that includes a trans-activation response region (TAR) loop system. In some embodiments, a therapeutic BioNV expresses a gene editor and encapsulates a plasmid that includes a TAR loop sequence between the 5’ end of a promoter and the gene editor / guide cassette, and this TAR loop sequence functions as a barrier to block transcription. In some embodiments, transcription is induced only in cells that are infected and contain the HIV Tat protein. In some embodiments, the Tat protein binds to the TAR loop, relaxes it, liberates the promoter for transcription, thereby expressing the editor and its guide.

[0229] In some embodiments, primary targeted therapeutic exosomes can be used to deliver a small molecule therapeutic payload. In some embodiments, second-generation (or third-generation or fourth-generation) CAR-containing therapeutic exosomes derived from activated lymphocytes can contain cytokines and other cytotoxic peptides. In some embodiments, therapeutic exosomes can be formatted to encapsulate and deliver plasmid DNA, for example, to express a gene editing nuclease and gRNA in target cells. Alternatively, or additionally, in some embodiments, therapeutic exosomes can encapsulate a nuclease and gRNA. In some embodiments, targeted second-generation (or third-generation or fourth-generation) therapeutic exosomes can be designed to encapsulate and deliver an additional therapeutic protein or peptide of interest.

[0230] In various embodiments, therapeutic exosomes can be analyzed for size uniformity by methods for determining particle size such as dynamic light scattering (DLS), flow cytometry, and molecular weight photometry. In various embodiments, therapeutic exosomes can be filtered by particle size or particle size range to optimize renal clearance and other clinically relevant NV properties. In various embodiments, therapeutic exosomes are in the size range of about 10 nm to 200 nm. In various embodiments, therapeutic exosomes are about 10 nm in size, about 20 nm in size, about 30 nm in size, about 40 nm in size, about 50 nm in size, about 60 nm in size, about 70 nm in size, about 80 nm in size, about 90 nm in size, about 100 nm in size, about 120 nm in size, about 140 nm in size, about 160 nm in size, about 180 nm in size, about 200 nm in size. In various embodiments, the size of therapeutic exosomes is in the range of about 10 nm to 20 nm, about 20 nm to 30 nm, about 30 nm to 40 nm, about 40 nm to 50 nm, about 50 nm to 60 nm, about 60 nm to 70 nm, about 70 nm to 80 nm, about 80 nm to 90 nm, about 90 nm to 100 nm, about 10 nm to 100 nm, about 100 nm to 200 nm.

[0231] Method for treating a disease In various embodiments, therapeutic BioNV / exosomes can be used to treat and / or prevent a disease or disorder. In various embodiments, the disease or disorder is cancer, an infectious disease, a genetic disorder, or a rare disease.

[0232] In various embodiments, the cancer is one or more of carcinoma, sarcoma, myeloma, leukemia, lymphoma, mixed cancer, and / or metastatic cancer.

[0233] In various embodiments, the cancer is acute biphenotypic leukemia, acute eosinophilic leukemia, acute lymphoblastic leukemia, acute lymphocytic leukemia, acute myeloid dendritic cell leukemia, acute myeloid leukemia, lung adenocarcinoma, adrenal tumor, adrenocortical carcinoma, AIDS-related cancer, AIDS-related lymphoma, alveolar soft part sarcoma and cardiac sarcoma, amyloidosis, anal cancer, anaplastic large cell lymphoma, angioimmunoblastic T cell lymphoma, appendiceal cancer, astrocytoma, ataxia telangiectasia, attenuated familial adenomatous polyposis, B-cell prolymphocytic leukemia, basal cell carcinoma, Beckwith-Wiedemann syndrome, bile duct cancer, Birt-Hogg-Dubé syndrome, bladder cancer, bone cancer, central nervous system cancer, brain stem glioma, brainstem glioma, brain tumor, breast cancer, bronchial adenoma / carcinoid, Burkitt lymphoma, carcinoid tumor, carcinoid tumors, Carney complex, central nervous system tumor, cerebellar astrocytoma, cerebral astrocytoma, cervical cancer, childhood desmoplastic ganglioglioma, cholangiocarcinoma, chondrosarcoma, chronic lymphocytic leukemia, chronic myelogenous leukemia, chronic myeloid leukemia, chronic T-cell lymphocytic leukemia, colon cancer, colorectal cancer, Cowden syndrome, craniopharyngioma, cutaneous T-cell lymphoma, dermatofibrosarcoma protuberans, desmoplastic small round cell tumor, diffuse gastric cancer, diffuse large B-cell lymphoma, endocrine cancer, endocrine tumor, endometrial cancer, eosinophilic leukemia, epithelioma, epitheloid hemangioendothelioma (EHE), esophageal cancer, Ewing sarcoma, extragonadal germ cell tumor, extrahepatic bile duct cancer, eye cancer, eyelid cancer, fallopian tube cancer, familial adenomatous polyposis, familial malignant melanoma, familial clear cell renal cell carcinoma (RCC), follicular lymphoma, gallbladder cancer, Gardner syndrome, gastric cancer, gastrointestinal cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, gastrointestinal stromal tumor (GIST), genitourinary cancer and gynecologic cancer, germ cell tumor, gestational trophoblastic disease, gestational trophoblastic tumor, glioblastoma, glioma, hairy cell leukemia, head and neck cancer, hematologic cancer, hepatocellular carcinoma, hepatosplenic T-cell lymphoma, HIV-related cancer, Hodgkin lymphoma, hypopharyngeal cancer, inflammatory breast cancer, intravascular large B-cell lymphoma, invasive cribriform carcinoma, invasive lobular carcinoma, islet cell carcinoma (pancreatic endocrine),Pancreatic cell tumors, juvenile polyposis syndrome, Kaposi sarcoma, keratoacanthoma, renal cancer, lacrimal gland tumors, large granular lymphocyte leukemia, laryngeal cancer and hypopharyngeal cancer, leiomyomatosis and renal cell carcinoma, leiomyosarcoma, Li-Fraumeni syndrome (LFS), liposarcoma, liver cancer, lung cancer, primary cutaneous lymphoma, lymphomatoid granulomatosis, lymphoplasmacytic lymphoma, Lynch syndrome, malignant fibrous histiocytoma of bone, mantle cell lymphoma, marginal zone B-cell lymphoma, mast cell leukemia, mastocytosis, mediastinal large cell type B-cell lymphoma, medullary carcinoma, medulloblastoma, melanoma, meningioma, Merkel cell carcinoma, mesothelioma, mixed polyposis syndrome, mucosa-associated lymphoid tissue lymphoma, Muir-Torre syndrome (MTS), multiple endocrine neoplasia syndrome, multiple endocrine neoplasia type 1, multiple endocrine neoplasia type 2, multiple myeloma, multiple myeloma / plasma cell tumor, fungating polypoid tumor, myelodysplastic syndrome, MYH-related polyposis, myxosarcoma, nasal and paranasal cavity cancer, nephroblastoma, nasopharyngeal cancer, nasopharyngeal carcinoma, neuroblastoma, neuroendocrine tumor, neurofibromatosis type 1, neurofibromatosis type 2, nevoid basal cell carcinoma syndrome, nodal marginal zone B-cell lymphoma, non-Hodgkin lymphoma, non-small cell lung cancer, non-small cell lung carcinoma, anaplastic glioma, optic nerve glioma, oral cancer and / or oropharyngeal cancer, osteosarcoma, ovarian cancer, ovarian epithelial cancer, ovarian germ cell tumor, pancreatic cancer, papillary renal cell carcinoma, paranasal cavity and nasal cavity cancer, parathyroid cancer, pelvic cancer, penile cancer, Peutz-Jeghers syndrome, pharyngeal cancer, pheochromocytoma, phyllodes tumor, pilocytic astrocytoma, pineal astrocytoma, pituitary adenoma, pituitary tumor, plasmablastic lymphoma, pleuropulmonary blastoma, precursor B-lymphoblastic leukemia, primary central nervous system lymphoma, primary cutaneous follicular lymphoma, primary cutaneous immunocytoma, primary effusion lymphoma, primary neuroectodermal tumor, prostate cancer, rectal cancer, renal cancer, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, primary cutaneous sarcoma, sebaceous adenocarcinoma, Sézary syndrome, skin appendage tumor, skin cancer, small bowel cancer, small cell lung cancer, small intestine cancer, splenic marginal zone lymphoma, squamous cell carcinoma of the lung, squamous cell carcinoma, squamous cell skin cancer, stomach cancer, superficial epithelial stromal tumor, T-cell prolymphocytic leukemia, testicular cancer, thoracic and respiratory cancer, thymoma, thymic carcinoma, thyroid cancer, transitional cell carcinoma, transitional cell carcinoma adenoid cystic carcinoma, tuberous sclerosis syndrome, tubular carcinoma, Turcot syndrome, carcinoma of unknown primary, unclassified carcinoma, ureteral cancer, urethral cancer, uterine cancer, uterine sarcoma, uveal melanomaIt is selected from vaginal cancer, optic chiasma hypothalamic glioma, von Hippel-Lindau (VHL) syndrome, vulvar cancer, Wilms tumor, and xeroderma pigmentosum.,

[0234] In various embodiments, BioNV can target cancer cells associated with adenoid cystic carcinoma, adrenal tumor, amyloidosis, anal cancer, appendiceal cancer, astrocytoma, ataxia telangiectasia, attenuated familial adenomatous polyposis, Beckwith-Wiedemann syndrome, bile duct cancer, Birt-Hogg-Dubé syndrome, bladder cancer, bone cancer, brainstem glioma, brain tumor, breast cancer, carcinoid tumor, Carney complex, central nervous system tumor, cervical cancer, colorectal cancer, Cowden syndrome, craniopharyngioma, desmoplastic infantile ganglioglioma, endocrine tumor, epithelioma, esophageal cancer, Ewing sarcoma, eye cancer, eyelid cancer, fallopian tube cancer, familial adenomatous polyposis, familial malignant melanoma, familial non-VHL clear cell renal cell carcinoma, gallbladder cancer, Gardner syndrome, gastrointestinal stromal tumor, germ cell tumor, gestational trophoblastic disease, head and neck cancer, diffuse gastric cancer, leiomyomatosis and renal cell carcinoma, mixed polyposis syndrome, pancreatitis, papillary renal cell carcinoma, HIV and AIDS-related cancer, islet cell tumor, juvenile polyposis syndrome, kidney cancer, lacrimal gland tumor, laryngeal cancer and hypopharyngeal cancer, acute lymphoblastic leukemia, acute lymphocytic leukemia, acute myeloid leukemia, B-cell prolymphocytic leukemia, hairy cell leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, chronic T-cell lymphocytic leukemia, eosinophilic leukemia, Li-Fraumeni syndrome, liver cancer, lung cancer, Hodgkin lymphoma, non-Hodgkin lymphoma, Lynch syndrome, mastocytosis, medulloblastoma, melanoma, meningioma, mesothelioma, Muir-Torre syndrome, multiple endocrine neoplasia type 1, multiple endocrine neoplasia type 2, multiple myeloma, myelodysplastic syndrome, MYH-related polyposis, nasal and paranasal cancer, nasopharyngeal cancer, neuroblastoma, neuroendocrine tumor, neurofibromatosis type 1, neurofibromatosis type 2, nevoid basal cell carcinoma syndrome, oral and oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, parathyroid cancer, penile cancer, Peutz-Jeghers syndrome, pituitary tumor, pleuropulmonary blastoma, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, alveolar soft part sarcoma and cardiac sarcoma, Kaposi sarcoma, skin cancer, small intestine cancer, stomach cancer, testicular cancer, thymoma, thyroid cancer, tuberous sclerosis syndrome, Turcot syndrome, cancer of unknown primary origin, uterine cancer, vaginal cancer, von Hippel-Lindau syndrome, Wilms tumor, or xeroderma pigmentosum.

[0235] In various embodiments, the therapeutic BioNV / exosomes used to treat and / or prevent a disease or disorder are targeted to one or more biomarkers in Table 5 and / or Table 6. Table 5: Exemplary checkpoint biomarkers and ligands of checkpoint biomarkers. [Table 5] Table 6: Exemplary cancer cell biomarkers. [Table 6-1] [Table 6-2] [Table 6-3]

[0236] In various embodiments, the infectious disease includes a lytic viral infection, a lysogenic viral infection, and / or both.

[0237] Co-administration of CAR-T BioNV In various embodiments, the methods for treating and / or preventing the diseases or disorders herein include co-administering a whole cell therapy (e.g., T cells, NK cells, TIL, macrophage therapy) with BioNV. In various embodiments, by supplementing the whole cell therapy with the therapeutic BioNV / exosomes, the required effective amount of the whole cell therapy can be reduced, and the CRS, off-target effects, the possibility of teratoma, etc. can be reduced. In various embodiments, the methods for treating and / or preventing a disease or disorder include treating cancer including delaying the growth of a tumor or cancer tissue, reducing the tumor volume, suppressing metastasis, increasing tumor infiltrating lymphocytes (TIL), and / or improving symptoms.

[0238] In various embodiments, the methods for treating and / or preventing a disease or disorder herein include administering one or more BioNVs in combination with one or more whole cell therapies (e.g., as shown in FIGS. 4A - 4C). In various embodiments, the combination is selected according to the disease to be treated. In various embodiments, the combination is selected according to the desired BioNV - whole cell interaction. In various embodiments, the combination is selected according to both the disease to be treated and the nature of the desired interaction between the BioNV and the whole cell therapy.

[0239] In various embodiments, the methods for treating and / or preventing a disease or disorder include co - administering a BioNV having at least one cancer antigen - binding ligand / receptor with a whole cell therapy having at least one cancer antigen - binding ligand / receptor. In various embodiments, the BioNV and the whole cell therapy target the same cancer antigen. In various embodiments, the BioNV and the whole cell therapy target different cancer antigens. Without wishing to be bound by theory, targeting different antigens (i.e., two or more) can target tumors presenting two or more biomarkers and / or prevent or reduce the likelihood of antigen escape in tumors or cancerous tissues. For example, without wishing to be bound by theory, the limitation of cancer treatment is that cancer cells can lose the expression of the antigen / biomarker targeted by the treatment.

[0240] In some embodiments, the BioNV for co - administration comprises, on its surface, an antigen - binding construct (e.g., a CAR) and one or more cancer antigens / biomarkers, or neo - antigens derived therefrom. In some embodiments, the cancer antigen / biomarker, or neo - antigen derived therefrom, is or comprises one or more of the proteins listed in Table 4, Table 5, and / or Table 6. In some embodiments, the BioNV has one or more co - stimulatory molecules (e.g., IL - 15) present on its surface to stimulate co - administered whole cells (e.g., T cells, NK cells, or macrophages). In some embodiments, the BioNV antigen - binding construct does not substantially bind to or recognize the antigen / biomarker, or neo - antigen derived therefrom, also present on the BioNV (e.g., to prevent cross - reactivity).

[0241] In some embodiments, the BioNV - presented cancer antigen / biomarker, or neo - antigen derived therefrom, is administered as a monotherapy. Without wishing to be bound by theory, in some embodiments, the neo - antigen - presenting BioNV stimulates a natural T - cell response in a subject being treated via T - cell receptor (TCR) engagement, wherein natural T cells (activated in the bloodstream) show improved homing to the site of the tumor or cancer tissue and improved cell - mediated cytotoxicity capacity that would not normally be present.

[0242] In various embodiments, the BioNV-presented cancer antigen / biomarker, or neoantigen derived therefrom, is co-administered with one or more whole cell therapies. Without wishing to be bound by theory, in various embodiments, BioNV, CAR, and the antigen enable the activation / stimulation of the whole cell therapy (e.g., T cells, NK cells, macrophages, etc.) co-administered. Without wishing to be bound by theory, in various embodiments, the whole cells (e.g., CAR-T cells) administered to the subject can be activated by BioNV circulating in the blood, and once activated, instead of waiting for the whole cells to be activated in the tumor microenvironment, the homing of the whole cells to the tumor or cancer tissue can be improved. Without wishing to be bound by theory, in various embodiments, co-administering the cancer antigen / biomarker (or neoantigen) present on BioNV with the whole cell counterpart can improve the kinetics (e.g., pharmacokinetics / pharmacodynamics) of tumor cell killing / removal by the whole cell therapy or BioNV alone.

[0243] In various embodiments, the methods for treating and / or preventing a disease or disorder herein include administering a first antigen-binding protein, such as a CAR that recognizes a cancer antigen (e.g., CAR1 shown in FIG. 4A), and a BioNV having a second ligand, receptor, and / or antigen recognized by whole cells (e.g., shown in FIG. 4A). In various embodiments, the second ligand, receptor, and / or antigen is the same as, includes, or is similar to a ligand, receptor, and / or antigen expressed by the cancer cells to be targeted (e.g., CAR 2R shown in FIG. 4A). In various embodiments, the second ligand, receptor, and / or antigen on the BioNV is a cancer neoantigen. In various embodiments, this BioNV is co-administered with whole cells containing an antigen-binding protein, such as a CAR (e.g., CAR 2 shown in FIG. 4A), wherein this antigen-binding protein recognizes both an antigen, ligand, or receptor present on the cancer cells and an antigen, ligand, or receptor present on the BioNV (e.g., shown in FIG. 4A). Without wishing to be bound by theory, this treatment strategy improves the targeting of whole cells to tumor cells or cancer tissue by 1) increasing the local concentration of the target antigen, ligand, or receptor, and enhances the activation / stimulation of whole cells via antigen-specific constructs by 2) higher-order binding through the combination of interactions with both tumor cells or cancer tissue and the BioNV, thereby improving the application of conventional whole cell therapies.

[0244] In various embodiments, the methods of treating and / or preventing a disease or disorder herein include administering a BioNV having a first antigen-binding protein, such as a CAR that recognizes a cancer antigen (e.g., CAR1 shown in FIG. 4B), and a second ligand, receptor, and / or antigen recognized by whole cells (e.g., shown in FIG. 4B). In various embodiments, the second ligand, receptor, and / or antigen is the same as, includes, or is similar to a ligand, receptor, and / or antigen expressed by the cancer cells to be targeted (e.g., CAR 2R shown in FIG. 4B). In various embodiments, the second ligand, receptor, and / or antigen on the BioNV is a cancer neoantigen. In various embodiments, the BioNV is co-administered with whole cells comprising two or more antigen-binding protein constructs, such as CARs (e.g., CAR 2 and CAR 2A shown in FIG. 4B). In various embodiments, the whole cells include a first antigen-binding construct (e.g., a CAR) that binds to an antigen, ligand, or receptor present on tumor cells or cancer tissue on the cancer cells, and a second antigen-binding construct (e.g., a CAR) that binds to an antigen, ligand, and / or receptor present on the BioNV (e.g., as shown in FIG. 4B).

[0245] In some embodiments, two or more antigen-binding constructs present on the whole cell have different intracellular domains such as co-stimulatory domains and / or intracellular signaling domains described herein. In some embodiments, two or more antigen-binding constructs have different signaling functions. For example, in some embodiments, two or more antigen-binding constructs have intracellular domains that signal through different non-competing signaling pathways, such as a sustained promotion / survival promotion pathway and a cell-mediated cytotoxicity pathway. In some embodiments, two or more antigen-binding constructs signal through redundant or overlapping signaling pathways. Without wishing to be bound by theory, this therapeutic strategy, without limitation, 1) improves the targeting of whole cells to tumor cells or cancer tissue by increasing the local concentration of the target antigen, ligand, or receptor; 2) enhances the activation / stimulation of whole cells via antigen-specific constructs by higher-order binding through the combination of the interaction between tumor cells or cancer tissue and BioNV; and 3) enhances the activation / stimulation of whole cells through two or more different antigen-specific constructs having different intracellular domains, thereby improving the administration of conventional whole cell therapies.

[0246] In various embodiments, the methods of treating and / or preventing a disease or disorder herein include administering a BioNV having a first antigen-binding protein, such as a CAR (e.g., CAR1 shown in FIG. 4C), and a second ligand, receptor, and / or antigen (e.g., as shown in FIG. 4C) recognized by whole cells. In various embodiments, the second ligand, receptor, and / or antigen is not the same as, does not include, or is not similar to a ligand, receptor, and / or antigen expressed by the targeted cancer cells (e.g., CAR 2R shown in FIG. 4C). In various embodiments, the second ligand, receptor, and / or antigen (recognized by whole cells) present on the BioNV is selected for a specific cell-BioNV interaction that is intended to occur rarely, if at all, at other sites in the subject being treated. In various embodiments, the BioNV is co-administered with whole cells comprising at least two different antigen-binding constructs, e.g., a CAR (e.g., CAR 2 shown in FIG. 4C) that binds to an antigen, ligand, or receptor present on cancer cells, and a CAR (e.g., CAR 2A shown in FIG. 4C) that binds to the BioNV. In various embodiments, the whole cells comprise a first antigen-binding construct that binds to an antigen, ligand, or receptor present on tumor cells or cancer tissue, and a second antigen-binding construct that binds to a ligand, receptor, and / or antigen present on the BioNV (e.g., as shown in FIG. 4C), wherein the ligand, receptor, and / or antigen on the BioNV is different from the antigen, ligand, or receptor present on the tumor cells or cancer tissue.

[0247] In some embodiments, the first antigen-binding construct and the second antigen-binding construct present on all cells differ in intracellular domains such as the co-stimulatory domain and / or intracellular signaling domain described herein. In some embodiments, the two antigen-binding constructs have different signaling functions. For example, in some embodiments, all cells have a first antigen-binding construct (e.g., CAR 2-CAR 2R shown in FIG. 4C) that binds to tumor cells or cancer tissue, and this antigen-binding construct contains an intracellular domain that signals through a cell-mediated cytotoxic signaling pathway (e.g., for the cellular release of anti-tumor cytokines such as granzyme, perforin, etc.). For example, in some embodiments, all cells have a second antigen-binding construct (e.g., CAR 2A shown in FIG. 4C) that binds to BioNV, and this antigen-binding construct contains an intracellular domain that signals through a different non-cell-mediated cytotoxic signaling pathway (e.g., a pathway that promotes persistence, survival, and / or proliferation). Without wishing to be bound by theory, this treatment strategy, but not limited to, 1) improves the targeting of all cells to tumor cells or cancer tissue by increasing the local concentration of the target antigen, ligand, or receptor, 2) enhances the activation / stimulation of all cells via antigen-specific constructs by higher-order binding due to the combination of the interaction between tumor cells or cancer tissue and BioNV, 3) enhances the activation / stimulation of all cells via two or more different intracellular domains, and 4) reduces the peripheral or off-target activation / stimulation of all cells by requiring both tumor cell or cancer tissue interaction and BioNV interaction for the full functionality of all cells, thereby improving the application of conventional all-cell therapies.

[0248] In some embodiments, a T cell receptor (TCR) can be used instead of or in addition to the CAR constructs described herein (e.g., as shown in FIG. 4D). In some embodiments, the TCR is a native TCR derived from the subject intended to be treated. In some embodiments, the TCR is an engineered T cell receptor.

[0249] In various embodiments, a method for treating and / or preventing a disease or disorder includes co - administration that first administers an effective amount (e.g., once or multiple times) of BioNV and then administers an effective amount (e.g., once or multiple times) of whole - cell therapy. In various embodiments, BioNV is first administered about 2 hours before or at least about 2 hours before whole - cell therapy, about 4 hours before or at least about 4 hours before whole - cell therapy, about 6 hours before or at least about 6 hours before whole - cell therapy, about 8 hours before or at least about 8 hours before whole - cell therapy, about 12 hours before or at least about 12 hours before whole - cell therapy, about 24 hours before or at least about 24 hours before whole - cell therapy, about 2 days before or at least about 2 days before whole - cell therapy, about 3 days before or at least about 3 days before whole - cell therapy, about 4 days before or at least about 4 days before whole - cell therapy, about 5 days before or at least about 5 days before whole - cell therapy, about 6 days before or at least about 6 days before whole - cell therapy, or more than about 1 week before or at least about 1 week before whole - cell therapy. In various embodiments, BioNV is first administered to modify cancer cells before administering whole - cell therapy.

[0250] In various embodiments, a method for treating and / or preventing a disease or disorder includes co - administering BioNV and whole - cell therapy simultaneously.

[0251] In various embodiments, a method for treating and / or preventing a disease or disorder includes co - administration that includes administering an effective amount (e.g., one or more times) of a whole - cell therapy first, and then administering an effective amount (e.g., one or more times) of BioNV. In various embodiments, the whole - cell is first administered about 2 hours or at least about 2 hours before BioNV, about 4 hours or at least about 4 hours before BioNV, about 6 hours or at least about 6 hours before BioNV, about 8 hours or at least about 8 hours before BioNV, about 12 hours or at least about 12 hours before BioNV, about 24 hours or at least about 24 hours before BioNV, about 2 days or at least about 2 days before BioNV, about 3 days or at least about 3 days before BioNV, about 4 days or at least about 4 days before BioNV, about 5 days or at least about 5 days before BioNV, about 6 days or at least about 6 days before BioNV, or more than about 1 week or at least about 1 week before BioNV. In various embodiments, because BioNV has relatively easy penetration into tissues / tumors, the whole - cell therapy is administered first.

[0252] In some embodiments, the whole cells are whole cell therapies comprising one or more of CAR-T cell, CAR-NK cell, and / or CAR-macrophage cell therapies.In various embodiments, the all-cell therapy is one or more of ABECMA (idecabtagene vicleucel, Celgene Corporation, Bristol-Myers Squibb), ADSTILADRIN (nadofaragene firadenovec, Ferring Pharmaceuticals A / S), BREYANZI (lisocabtagene maraleucel, Juno Therapeutics, Inc., Bristol-Myers Squibb), CARVYKTI (cilta-cabtagene autoleucel, Janssen Biotech, Inc.), GINTUIT (allogeneic cultured keratinocytes and fibroblasts in bovine collagen, Organogenesis Inc.), HEMGENIX (etranacogene dezaparvovec, CSL Behring LLC), HPC (umbilical cord blood, multiple sources), IMLYGIC (talimogene laherparepvec, BioVex, Inc., Amgen Inc.), KYMRIAH (tisagenlecleucel, Novartis Pharmaceuticals Corporation), LAVIV (azficel-T, Fibrocell Technologies), LUXTURNA (voretigene neparvovec, Spark Therapeutics, Inc.), MACI (autologous cultured chondrocytes on porcine collagen membrane, Vericel Corp.), PROVENGE (sipuleucel-T, Dendreon Corp.), RETHYMIC (allogeneic processed thymus tissue, Enzyvant Therapeutics GmbH), SKYSONA (elivaldogene autotemcel, bluebird bio, Inc.), STRATAGRAFT (allogeneic cultured keratinocytes and dermal fibroblasts in murine collagen, Stratatech Corporation), TECARTUS (brexucabtagene autoleucel, Kite Pharma, Inc.), YESCARTA (axicabtagene ciloleucel, Kite Pharma, Inc.), ZYNTEGLO (betibeglogene autotemcel, Bluebird Bio, Inc.), ZOLGENSMA (onasemnogene abeparvovec-xioi; Novartis Gene Therapies, Inc.).In some embodiments, the whole cell therapy is one or more of the whole cell therapies described herein that have been engineered to include one or more BioNV-compatible cell surface proteins as described herein. In some embodiments, the whole cell therapy is one or more of the whole cell therapies described herein that have been engineered to be hypoimmunogenic cells as described herein.

[0253] In some embodiments, by administering BioNV in addition to the whole cells, the function of the whole cells is improved as compared to administration of the whole cells alone. In some embodiments, the function of the whole cells includes one or more of cell-mediated cytotoxicity, cytokine release, killing of tumor cells or cancer cells, homing to tumor cells or cancer tissue, tissue infiltration, proliferation, persistence, and / or survival. In some embodiments, BioNV reduces one or more toxicities of the whole cells. In some embodiments, the toxicity of the whole cell therapy includes the tendency for off-target binding (e.g., binding in peripheral tissues, non-specific activation, etc.) and related adverse events.

[0254] In some embodiments, a method of treating and / or preventing a disease or disorder includes administering an additional therapeutic agent. In some embodiments, the additional therapeutic agent can be any additional anti-cancer agent, anti-infective agent, analgesic, and / or non-steroidal anti-inflammatory agent (NSAID).

[0255] In some embodiments, the therapeutic BioNV / exosomes can be frozen at -80°C or can be suitable for storage at about -80°C and / or can be lyophilized (e.g., for reconstitution in buffer). In some embodiments, the therapeutic BioNV / exosomes can be stable at approximately ambient temperature, about -20°C, about 4°C, about 25°C, or about 37°C for at least about 1 hour, at least about 2 hours, at least about 4 hours, at least about 6 hours, at least about 12 hours, at least about 24 hours, at least about 2 days, at least about 1 week, or at least about 1 month or more.

[0256] In various embodiments, treating and / or preventing a disease or disorder can be achieved within about 2 weeks, within about 4 weeks, within about 6 weeks, within about 12 weeks, within about 18 weeks, within about 24 weeks, within about 6 months, within about 1 year, or within about 2 years or less from the administration of the composition and methods using such compositions.

[0257] Dosage and Administration The dosages and administration schedules of the therapeutic BioNV / exosomes disclosed herein can depend on a variety of parameters and factors including, but not limited to, the particular therapeutic BioNV / exosome, the disease being treated, the severity of the condition, whether the condition is being treated or prevented, the age, weight, and overall health of the subject, and the discretion of the administering physician. Further, pharmacogenomic (the effect of genotype on the pharmacokinetics, pharmacodynamics, or efficacy profile of a therapeutic agent) information regarding a particular subject can affect the dosage used. Additionally, the exact individual dosage can be somewhat adjusted according to various factors such as the particular combination of agents being administered, the duration of administration, the route of administration, the nature of the formulation, the rate of excretion, the disease being treated, the severity of the disorder, and the anatomical location of the disorder. Some variation in dosage can be expected.

[0258] In various embodiments, delivery of the therapeutic BioNV / exosomes can be similar to the delivery of vesicles, particularly liposomes (see Langer, 1990, Science 249:1527 - 1533; Treat et al., in Liposomes in Therapy of Infectious Disease and Cancer, Lopez - Berestein and Fidler (eds.), Liss, New York, pp. 353 - 365 (1989)).

[0259] Methods of treating and / or preventing a disease or disorder using the therapeutic BioNV / exosomes described herein can, in various embodiments, include dosage ranges at a concentration of the number of therapeutic BioNV / exosomes per kilogram (kg) of the subject's body weight. In various embodiments, an appropriate dosage range for the methods described herein is about 10 3BioNV / kg (or exosomes / kg) to about 10 12 may contain BioNV / kg (or exosomes / kg). In some embodiments, the therapeutic BioNV / exosomes are about 10 3 BioNV / mL (or exosomes / mL) to about 10 14 present in the composition at a concentration of BioNV / mL (or exosomes / mL). Alternatively, in some embodiments, the therapeutic BioNV / exosome composition is present in the composition as a weight / volume in the range of about 5 ng / mL to about 500 mg / mL. In some embodiments, the dosage of the therapeutic BioNV / exosomes is based on the size of the therapeutic BioNV / exosomes used for treatment. For example, a 1000 nm therapeutic BioNV / exosome is provided in an amount about 5 to 10 times less than a 100 nm BioNV / exosome to obtain an equivalent dose.

[0260] In various embodiments, the therapeutic BioNV / exosomes disclosed herein are administered by controlled or sustained release means or by delivery of a device well known to those of skill in the art. Examples include, but are not limited to, those described in U.S. Patent Nos. 3,845,770; 3,916,899; 3,536,809; 3,598,123; 4,008,719; 5,674,533; 5,059,595; 5,591,767; 5,120,548; 5,073,543; 5,639,476; 5,354,556; and 5,733,556 (each of which is incorporated herein by reference in its entirety). Such dosage forms can be useful for providing controlled or sustained release of one or more active ingredients and a desired release profile at various rates, for example, using hydroxypropyl methylcellulose, other polymeric matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, microspheres, or combinations thereof. The controlled or sustained release of the active ingredient can be stimulated by various conditions including, but not limited to, changes in pH, changes in temperature, stimulation by light of an appropriate wavelength, enzyme concentration or availability, water concentration or availability, or other physiological conditions or compounds.

[0261] In various embodiments, polymeric materials are used (see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Florida (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, 1983, J. Macromol. Sci. Rev. Macromol. Chem. 23:61; Levy et al., 1985, Science 228:190; During et al., 1989, Ann. Neurol. 25:351; Howard et al., 1989, J. Neurosurg. 71:105).

[0262] In various embodiments, the controlled release system is placed near the target area to be treated and thus requires only a fraction of the systemic dose (see, for example, Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)). Other controlled release systems discussed in the review by Langer, 1990, Science 249:1527-1533 may be used.

[0263] In various embodiments, methods of using therapeutic BioNV / exosomes include applying the therapeutic BioNV / exosomes to the surface of a device (e.g., a catheter) or housing them within a pump, patch, or other drug delivery device. The excipient or carrier can be selected based on the mode of administration and route of administration. Suitable pharmaceutical carriers and essential ingredients for pharmaceutical formulations are described in Remington’s Pharmaceutical Sciences (E.W. Martin) and USP / NF (United States Pharmacopeia and the National Formulary), well-known references in the art.

[0264] In various embodiments, therapeutic BioNV / exosomes can be administered at a dose adapted to the dose of whole cells, for example, based on the CAR concentration. In various embodiments, the typical concentration range of CAR protein per microgram of T cells is 0.20 ng to 0.70 ng, while a single BioNV may have a total number of CARs that is 1 / 5 to 1 / 10,000 of that of whole cells. As a result, when converting the mass of BioNV to CAR concentration, it can be assumed that the CAR concentration is equivalent to (such as in the case of exosomes) or increased (such as in the case of BioNV) to that of the cells from which it is derived (e.g., T cells). In various embodiments, the concentration and / or surface density of a targeting agent (e.g., CAR) increases on BioNV compared to the whole cells from which it is derived. In various embodiments, the concentration and / or surface density of a targeting agent (e.g., CAR) is concentrated by continuous extrusion treatment of whole cells. In various embodiments, the concentration and / or surface density of cell surface molecules such as a targeting agent (e.g., CAR) on BioNV increases 2-fold to 100-fold compared to whole cells. In various embodiments, since exosomes are naturally secreted, the concentration and / or surface density of cell surface molecules such as a targeting agent (e.g., CAR) is substantially the same as that of whole cells.

[0265] Any dosing regimen utilizing a therapeutic BioNV / exosome disclosed herein can be selected according to various factors such as the type, species, age, weight, gender, and medical condition of the subject's cancer; the severity of the condition being treated; the route of administration; the subject's renal or hepatic function; the individual pharmacogenomic makeup; and the specific composition of the present disclosure being used. Any therapeutic BioNV / exosome disclosed herein may be administered once a day or the total daily dose may be administered in divided doses of 2, 3, or 4 times a day. Further, any therapeutic BioNV / exosome disclosed herein may be administered continuously rather than intermittently throughout the dosing regimen.

[0266] In embodiments, the therapeutic BioNV / exosome is administered in continuous dosing at about once per hour, about once every 2 hours, about once every 6 hours, about once every 12 hours, about once every 24 hours, about once every 2 days, about once every 4 days, about once every 7 days, about once every 2 weeks, about once every 4 weeks, or about once per month.

[0267] Additional therapeutic agents In embodiments, the compositions or methods described herein further comprise a therapeutically effective amount of one or more additional therapeutic agents. In embodiments, the therapeutically effective amount of one or more additional therapeutic agents may be in a solution containing the therapeutic BioNV / exosome, adsorbed on the surface of the NV, or a payload encapsulated within the therapeutic BioNV / exosome. In embodiments, the additional therapeutic agent is one or more of a checkpoint inhibitor, an analgesic, and / or an anti-infective agent.

[0268] In various embodiments, the composition or method contemplates other additional therapeutic agents, such as, for example, an analgesic agent to assist in treating inflammation or pain at the administration site, or an anti-infective agent to prevent infection at the treatment site by the composition.Non-limiting examples of additional therapeutic agents include analgesics such as non-steroidal anti-inflammatory drugs, opioid agonists, and salicylates; anti-infective agents such as anthelmintics, anti-anaerobics, antibiotics, aminoglycoside antibiotics, antifungal antibiotics, cephalosporin antibiotics, macrolide antibiotics, other β-lactam antibiotics, penicillin antibiotics, quinolone antibiotics, sulfonamide antibiotics, tetracycline antibiotics, anti-mycobacterial drugs, anti-tuberculosis mycobacterial drugs, anti-protozoal drugs, anti-malaria protozoal drugs, antiviral agents, anti-retroviral agents, anti-scabies drugs, anti-inflammatory agents, corticosteroid anti-inflammatory agents, antipruritics / local anesthetics, topical anti-infective agents, antifungal topical anti-infective agents, antiviral topical anti-infective agents; electrolytes and renal agents such as acidifying agents, alkalinizing agents, diuretics, carbonic anhydrase inhibitor diuretics, loop diuretics, osmotic diuretics, potassium-sparing diuretics, thiazide diuretics, electrolyte replenishers, and uric acid excretants; enzymes such as pancreatic enzymes and thrombolytic enzymes; gastrointestinal drugs such as antidiarrheals, antiemetics, gastrointestinal anti-inflammatory agents, salicylic acid gastrointestinal anti-inflammatory agents, antacid anti-ulcer agents, gastric acid pump inhibitor anti-ulcer agents, gastric mucosal anti-ulcer agents, H2 blocker anti-ulcer agents, gallstone dissolving agents, digestive agents, emetics, laxatives and stool softeners, and gastrointestinal motility promoters; general anesthetics such as inhaled anesthetics, halogenated inhaled anesthetics, intravenous anesthetics, barbiturate intravenous anesthetics, benzodiazepine intravenous anesthetics, and opioid agonist intravenous anesthetics; hormone regulators such as abortifacients, adrenocortical agents, corticosteroid agents, androgens, anti-androgens; immunobiological agents such as immunoglobulins, immunosuppressants, toxoids, and vaccines; local anesthetics such as amide-type local anesthetics and ester-type local anesthetics; musculoskeletal agents such as anti-gout anti-inflammatory agents, corticosteroid anti-inflammatory agents, gold compound anti-inflammatory agents, immunosuppressive anti-inflammatory agents, non-steroidal anti-inflammatory drugs (NSAIDs), salicylic acid anti-inflammatory agents; minerals; vitamins such as water-soluble or fat-soluble vitamins, vitamin A, vitamin B, vitamin C, vitamin D, vitamin E, and / or vitamin K; and radionuclides such as yttrium 90, iodine 131, samarium 153, lutetium 177, astatine 211, lead 212 / bismuth 212, radium 223, actinium 225, and thorium 227.

[0269] Additional non-limiting examples of useful therapeutic agents of the above categories include: (1) analgesics in general such as lidocaine or its derivatives, and NSAID analgesics such as diclofenac, ibuprofen, ketoprofen, and naproxen; (2) opioid-acting analgesics such as codeine, fentanyl, hydromorphone, and morphine; (3) salicylic acid analgesics such as aspirin (ASA) (enteric-coated ASA); (4) Hi-blocker antihistamines such as clemastine and terfenadine; (5) anti-infective agents such as mupirocin; (6) anti-anaerobic anti-infective agents such as chloramphenicol and clindamycin; (7) antifungal antibiotic anti-infective agents such as amphotericin b, clotrimazole, fluconazole, and ketoconazole; (8) macrolide antibiotic anti-infective agents such as azithromycin and erythromycin; (9) other β-lactam antibiotic anti-infective agents such as aztreonam and imipenem; (10) penicillin antibiotic anti-infective agents such as nafcillin, oxacillin, penicillin G, and penicillin V; (11) quinolone antibiotic anti-infective agents such as ciprofloxacin and norfloxacin; (12) tetracycline antibiotic anti-infective agents such as doxycycline, minocycline, and tetracycline; (13) anti-tuberculosis mycobacterium anti-infective agents such as isoniazid (INH) and rifampin; (14) anti-protozoal anti-infective agents such as atovaquone and dapsone; (15) anti-malaria protozoal anti-infective agents such as chloroquine and pyrimethamine; (16) anti-retroviral anti-infective agents such as ritonavir and zidovudine; (17) anti-viral anti-infective agents such as acyclovir, ganciclovir, interferon alpha, remdesivir, and rimantadine; (18) antifungal topical anti-infective agents such as amphotericin B, clotrimazole, miconazole, and nystatin; (19) anti-viral topical anti-infective agents such as acyclovir; (20) electrolytes and renal agents such as lactulose; (21) loop diuretics such as furosemide; (22) potassium-sparing diuretics such as triamterene; (23) thiazide diuretics such as hydrochlorothiazide (HCTZ); (24) uric acid excretants such as probenecid; (25) enzymes such as RNase and DNase; (26) anti-emetics such as prochlorperazine;(27) Salicylic acid gastrointestinal anti-inflammatory agents such as sulfasalazine; (28) Gastric acid pump inhibitor anti-ulcer agents such as omeprazole; (29) H2 blocker anti-ulcer agents such as cimetidine, famotidine, nizatidine, and ranitidine; (30) Digestants such as pancreatin; (31) Gastrointestinal motility promoters such as erythromycin; (32) Ester-type local anesthetics such as benzocaine and procaine; (33) Skeletal system corticosteroid anti-inflammatory agents such as beclomethasone, betamethasone, cortisone, dexamethasone, hydrocortisone, and prednisone; (34) Skeletal system anti-inflammatory immunosuppressive agents such as azathioprine, cyclophosphamide, and methotrexate; (35) Skeletal system non-steroidal anti-inflammatory drugs (NSAIDs) such as diclofenac, ibuprofen, ketoprofen, ketorolac, and naproxen; (36) Minerals such as iron, calcium, and magnesium; (37) Vitamin B compounds such as cyanocobalamin (vitamin B12) and niacin (vitamin B3); (38) Vitamin C compounds such as ascorbic acid; and (39) Vitamin D compounds such as calcitriol are included.;

[0270] Composition of Therapeutic BioNV / Exosomes In various aspects, the present disclosure relates to a composition that can be used for the treatment of mammalian diseases, including allogeneic low-immunogenic therapeutic BioNV / exosomes containing one or more treatment-related biomolecules as described herein.;

[0271] In various embodiments, the composition includes therapeutic BioNV / exosomes. In various embodiments, the composition may include therapeutic BioNV / exosomes and at least one or more of an anti-cancer therapeutic agent, an anti-infective therapeutic agent, or a gene editing payload. In various embodiments, the composition includes therapeutic BioNV / exosomes that can adsorb a therapeutic molecule on the surface of the NV and / or encapsulate a therapeutic payload within the aqueous compartment of the NV. In various embodiments, the composition includes a therapeutically effective amount of BioNV / exosomes.;

[0272] In various embodiments, the composition is allogeneic and / or hypoimmunogenic. In various embodiments, the composition is derived from iPSCs (or other cell types such as) that have been modified to reduce the expression of immunogenic molecules and / or increase the expression of immunoprotective molecules.

[0273] In various embodiments, the therapeutic BioNV / exosome composition is hypoimmunogenic. For example, in various embodiments, the composition does not elicit an inflammatory response and / or an immune response when administered. In various embodiments, the therapeutic BioNV / exosomes are hypoimmunogenic. In various embodiments, when administered to a subject, the composition, optionally the therapeutic BioNV / exosomes within the composition, elicits, for example, compared to the counterpart of an allogeneic whole cell therapy, an inflammation or immune response of about 50%, about 45%, about 40%, about 35%, about 30%, about 25%, about 24%, about 23%, about 22%, about 21%, about 20%, about 19%, about 18%, about 17%, about 16%, about 15%, about 14%, about 13%, about 12%, about 11%, about 10%, about 9%, about 8, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, or less than about 1% as measured according to cytokine, chemokine, or immunomodulatory enzyme concentrations such as IL-1, IL-2, IL-3, IL-4, IL-6, IL-7, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-20, IFN-α / β / γ, TNFα / β, IDO, HLA-G, HGF, PGE2, or any combination thereof.

[0274] In various embodiments, the therapeutic BioNV / exosomes are about 10 3 BioNV / mL (or exosomes / mL) to about 10 14 BioNV / mL (or exosomes / mL) and are present in the composition at a concentration. Alternatively, in various embodiments, the therapeutic BioNV / exosome composition is present in the composition as a weight / volume in the range of about 5 ng / mL to about 500 mg / mL.

[0275] In various embodiments, the composition is substantially free of one or more bacteria, viruses, fungi, spores, mycoplasma, pyrogens, and in more particular embodiments, is substantially free of all of the foregoing. In various embodiments, the composition is substantially free of whole cells and intracellular cell components including cell organelles such as nuclei, mitochondria, Golgi, and / or is substantially free of non-CAR expressing NVs, and / or is substantially free of ruptured and damaged NVs. In various embodiments, the composition is substantially free of cell chromatin, nucleosomes, as well as other genetic materials and non-therapeutic nucleic acids. In various embodiments, the BioNV / exosome and BioNV / exosome compositions are substantially free of cellular genomic DNA.

[0276] In various embodiments, the therapeutic BioNV / exosomes are modular and allogeneic (off-the-shelf) due to the lack of immunogenicity derived from engineered iPSCs. In various embodiments, due to the lack of whole cell signaling components, the BioNV / exosomes are readily adjustable with respect to target specificity and resistance to immunosuppressive signals. In various embodiments, the therapeutic BioNV / exosomes lack gene elements that contribute to cytokine storm rampage, minimizing the risk of cytokine release syndrome (CRS) in patients. In various embodiments, the amounts of active cytokines, perforin, granzyme, granulysin, interferon, interleukin, etc. encapsulated within the therapeutic BioNV / exosomes are regulated during upstream (prior to BioNV / exosome induction) cell processes. In various embodiments, the therapeutic BioNV / exosomes are derived from cells that can pass through biological barriers and / or are known to facilitate passage of viral receptors.

[0277] While not wishing to be bound by theory, therapeutically relevant BioNV / exosomes generated from iPSCs derived from engineered allogeneic-based cell lines are immunologically invisible. That is, therapeutically relevant BioNV / exosomes have the potential for multiple administrations, with antibody-mediated neutralization of therapeutically relevant BioNV / exosomes minimized and immune cell-mediated clearance (T cells and macrophages) avoided. In various embodiments, therapeutically relevant BioNV / exosomes do not contain viable genetic material from cells that cause CRS or teratomas. In various embodiments, the mobilization of natural T cells can be achieved by increasing the expression of certain cytokines encapsulated within therapeutically relevant BioNV / exosomes. In various embodiments, BioNV / exosomes can be derived from modified cell types with or without barrier-permeable ligands to further control activity after injection.

[0278] Pharmaceutical Compositions and Formulations of Therapeutically Relevant BioNV / Exosomes In various aspects, the composition is a pharmaceutical composition. In various embodiments, the pharmaceutical compositions of the present disclosure are formulated to provide a therapeutically effective amount of therapeutically relevant BioNV / exosomes as an active ingredient. In various embodiments, the pharmaceutical compositions of the present disclosure are formulated to provide a therapeutically effective amount of one or more anti-cancer therapeutics as a payload within therapeutically relevant BioNV / exosomes as an active ingredient. Typically, the pharmaceutical composition also includes one or more pharmaceutically acceptable excipients, carriers such as inert solid diluents and fillers, diluents such as sterile aqueous solutions and various organic solvents, penetration enhancers, solubilizers, and adjuvants.

[0279] Pharmaceutical excipients can be liquids such as water and oil, including, for example, those derived from petroleum, animals, plants, or synthesis, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Pharmaceutical excipients can be, for example, physiological saline, acacia gum, gelatin, starch paste, talc, keratin, colloidal silica, urea, etc. In addition, adjuvants, stabilizers, thickeners, lubricants, and coloring agents may be used. Pharmaceutically acceptable excipients are generally sterile when administered to a subject. Water is a useful excipient when any of the drugs disclosed herein are administered intravenously. Physiological saline and aqueous solutions of dextrose and glycerol can also be used as liquid excipients, particularly for injection solutions. Suitable pharmaceutical excipients also include starch, glucose, lactose, sucrose, gelatin, malt, rice, wheat, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol, etc. Any composition disclosed herein can also be formulated with a wetting agent or emulsifier, or a pH buffer, if desired. Other examples of suitable pharmaceutical excipients are described in Remington’s Pharmaceutical Sciences 1447-1676 (Alfonso R. Gennaro eds., 19th ed. 1995), which is incorporated herein by reference.

[0280] In some embodiments, the composition includes an excipient or carrier. In some embodiments, the diluent is a pharmaceutically acceptable excipient or carrier.

[0281] In various embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable diluent. Non-limiting examples of diluents include liquid diluents such as water, ethanol, propylene glycol, glycerin, and various combinations thereof, as well as inert solid diluents such as calcium carbonate, calcium phosphate, or kaolin. In various embodiments, the diluent comprises one or more of physiological saline, phosphate buffered saline, Dulbecco's Modified Eagle Medium (DMEM), Alpha Modified Minimal Essential Medium (Alpha MEM), Roswell Park Memorial Institute Medium 1640 (RPMI Media 1640), HBSS, human albumin, Ringer's solution, or any combination thereof.

[0282] In various embodiments, the active ingredient is typically mixed with, diluted by, or encapsulated within such a carrier in the form of, for example, capsules, tablets, sachets, papers, or other containers. When the excipient functions as a diluent, the excipient can be a solid, semi-solid, or liquid material (e.g., physiological saline) and acts as a vehicle, carrier, or medium for the active ingredient. In various embodiments, the composition can be in the form of tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as a solid or in a liquid medium), lotions, creams, ointments, gels, soft and hard gelatin capsules, suppositories, sterile injectable solutions, and sterile packaged powders. As is known in the art, the type of diluent can vary depending on the intended route of administration. In various embodiments, the resulting composition can contain additional agents such as preservatives, cryopreservatives (e.g., DMSO), and / or lyoprotectants (e.g., polyols, salts). In various embodiments, the carrier can be or can include a lipid-based or polymer-based colloid. In various embodiments, the carrier material can be formulated as a colloid in the form of liposomes, hydrogels, microparticles, nanoparticles, or block copolymer micelles. In various embodiments, the carrier material can form a capsule, and the material can be a polymer-based colloid.

[0283] In various embodiments, a pharmaceutical composition comprising therapeutic BioNV / exosomes comprises a solubilizing agent. In various embodiments, a pharmaceutical composition comprising therapeutic BioNV / exosomes comprises a cryoprotectant such as DMSO or glycerol, or an agent that improves thermal stability. In various embodiments, the pharmaceutical composition can be delivered using a suitable vehicle or delivery device known in the art.

[0284] In various embodiments, the composition comprises a scaffold of a biomaterial. In a non-limiting example, the three-dimensional biomaterial comprises therapeutic BioNV / exosomes attached to the scaffold, or dispersed within the scaffold, or embedded in an extracellular matrix captured within the scaffold. In various embodiments, the biomaterial is biodegradable and / or synthetic.

[0285] In various embodiments, the scaffold comprises a biodegradable biomaterial. Non-limiting examples of biodegradable biomaterials include fibrin, collagen, elastin, gelatin, vitronectin, fibronectin, laminin, reconstituted basement membrane matrix, starch, dextran, alginate, hyaluronic acid, chitin, chitosan, agarose, saccharides, hyaluronic acid, poly(lactic acid), poly(glycolic acid), polyethylene glycol, decellularized tissue, self-assembling peptides, polypeptides, glycosaminoglycans, their derivatives and mixtures. Other useful biodegradable polymers or polymer species include polydioxanone, polycarbonate, polyoxalate, poly(α-ester), polyanhydride, polyacetate, polycaprolactone, poly(orthoester), polyamino acid, polyamide, and their mixtures and copolymers, L-lactic acid and D-lactic acid stereopolymers, copolymers of bis(p- carboxyphenoxy)propionic acid and sebacic acid, sebacic acid copolymers, caprolactone copolymers, poly(lactic acid) / poly(glycolic acid) / polyethylene glycol copolymers, polyurethanes and poly(lactic acid) copolymers, polyurethanes and poly(lactic acid) copolymers, α-amino acid copolymers, α-amino acid and caproic acid copolymers, Α-benzylglutamic acid and polyethylene glycol copolymers, succinate and poly(glycol) copolymers, polyphosphazene, polyhydroxyalkanoates and mixtures thereof, but are not limited thereto. Binary and ternary systems are also contemplated. In various embodiments, the scaffold comprises one or more of collagen, various proteoglycans, alginate-based substrates, and chitosan. In various embodiments, the scaffold comprises one or more of hydrogels, silk, Matrigel, acellular and / or decellularized scaffolds, poly-ε-caprolactone scaffolds, absorbable scaffolds, and nanofiber hydrogel composites.

[0286] In various embodiments, the scaffold comprises a synthetic biomaterial. Non-limiting examples of synthetic biomaterials include lactone-based polyesters or copolyesters, such as polylactic acid, polycaprolactone glycolide, polyorthoesters, polyanhydrides, polyamino acids, polysaccharides, polyphosphazenes, poly(ether-ester) copolymers (e.g., PEO-PLLA); polydimethylsiloxane, poly(ethylene vinyl acetate), acrylate-based polymers or copolymers (e.g., polyhydroxyethyl methyl methacrylate, polyvinylpyrrolidone), fluorinated polymers, such as polytetrafluoroethylene and cellulose esters.

[0287] In various embodiments, the composition can be prepared by any method well known in the pharmaceutical art and can be administered by various routes (e.g., subcutaneous, intravenous, etc.) depending on whether local or systemic treatment is desired and the area being treated. In various embodiments, administration can be local (including ophthalmic, as well as delivery to mucous membranes including intranasal, intravaginal, and rectal delivery), pulmonary (e.g., by inhalation or insufflation of powders or aerosols including those by nebulizer; intratracheal, intranasal, epithelial, and transdermal), ocular, oral, or parenteral. In various embodiments, the method can include intravitreal delivery, topical administration (eye drops), subconjunctival, periocular or intravitreal injection, or introduction by balloon catheter, or an ophthalmic insert surgically placed in the conjunctival sac. In various embodiments, parenteral administration includes intravenous, arterial, subcutaneous, intraperitoneal, or intramuscular injection or infusion, or intracranial, e.g., intrathecal or intraventricular administration. In various embodiments, parenteral administration can be in the form of a single bolus dose or, e.g., by continuous infusion pump.

[0288] In various embodiments, pharmaceutical compositions and formulations for local administration can include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, solutions, powders, and the like. In various embodiments, methods of treating and / or preventing cancer include the use of a pharmaceutical carrier, an aqueous, powder or oily base, a thickening agent, and the like.

[0289] In various embodiments, the pharmaceutical composition contains, as an active ingredient, the nucleic acids and vectors described herein, in combination with one or more pharmaceutically acceptable carriers. In various embodiments, the term "pharmaceutically acceptable" (or "pharmacologically acceptable") refers to molecular entities and compositions that do not cause adverse reactions, allergic reactions, or other undesirable reactions when administered, as necessary, to animals or humans. The methods and compositions disclosed herein can be applied to a wide range of species, such as, for example, humans, non-human primates (e.g., monkeys), horses, or other livestock, dogs, cats, ferrets, or other mammals raised as pets, rats, mice, or other laboratory animals. In various embodiments, the term "pharmaceutically acceptable carrier" includes any solvent, dispersion medium, coating agent, antibacterial agent, isotonic agent and absorption delaying agent, buffer, excipient, binder, lubricant, gel, surfactant, etc. that can potentially be used as a vehicle for pharmaceutically acceptable substances.

[0290] In various embodiments, the composition can be applied to the surface of a device (e.g., a catheter) or can be contained within a pump, patch, or other drug delivery device. In various embodiments, the composition can be administered alone or as a mixture in the presence of a pharmaceutically acceptable excipient or carrier (e.g., physiological saline). The excipient or carrier is selected based on the mode of administration and the route of administration. Suitable pharmaceutical carriers and essential ingredients for pharmaceutical formulations are described in Remington’s Pharmaceutical Sciences (E.W. Martin) and USP / NF (United States Pharmacopeia and the National Formulary), well-known references in the art.

[0291] In various embodiments, the compositions disclosed herein, such as pharmaceutical compositions, are resuspended in a physiological saline buffer (including but not limited to TBS, PBS, etc.).

[0292] The present technology includes therapeutic BioNV / exosomes disclosed in the formulations of various pharmaceutical compositions. In various embodiments, the therapeutic BioNV / exosomes disclosed herein can take the form of solutions, suspensions, emulsions, drops, tablets, pills, pellets, capsules, liquid-containing capsules, powders, sustained-release formulations, emulsions, aerosols, sprays, suspensions, or any other form suitable for use.

[0293] The pharmaceutical compositions containing the therapeutic BioNV / exosomes described herein can be conveniently provided in unit dosage forms and can be prepared by any of the methods well known in the pharmaceutical art. Such methods generally include the step of associating the therapeutic agent with a carrier that constitutes one or more accessory ingredients. Typically, the pharmaceutical composition is prepared by uniformly and intimately associating the therapeutic agent with a liquid carrier, a finely divided solid carrier, or both, and then shaping the product into the dosage form of the desired formulation, if necessary (e.g., wet or dry granulation, powder blending, etc., followed by tableting using conventional methods known in the art).

[0294] In various embodiments, any of the therapeutic BioNV / exosomes disclosed herein are formulated according to conventional methods as a pharmaceutical composition adapted to the mode of administration disclosed herein.

[0295] Subject and / or animal In various embodiments, the subject and / or animal intended for use with the therapeutic BioNV / exosomes is a mammal, such as a human, mouse, rat, guinea pig, dog, cat, horse, cow, pig, rabbit, sheep, or non-human primate. In various embodiments, the subject and / or animal is a non-mammal, such as a zebrafish, for example. In various embodiments, the subject and / or animal is a transgenic animal that includes fluorescent cells, such as RPE cells and / or immune cells having GFP, for example. In various embodiments, the subject and / or animal is a human. In various embodiments, the therapeutic BioNV / exosomes are derived from fluorescent-tagged cells and / or encapsulated with a fluorescent-tagged protein or tag (e.g., with GFP). In various embodiments, the human is a pediatric human, adult, elderly, infant or child. In other embodiments, the human may be referred to as a patient.

[0296] In various embodiments, the treatment method includes administering to a human of an age in the range of from about 0 months to about 6 months, from about 6 months to about 12 months, from about 12 months to about 18 months, from about 18 months to about 36 months, from about 1 year to about 5 years, from about 5 years to about 10 years, from about 10 years to about 15 years, from about 15 years to about 20 years, from about 20 years to about 25 years, from about 25 years to about 30 years, from about 30 years to about 35 years, from about 35 years to about 40 years, from about 40 years to about 45 years, from about 45 years to about 50 years, from about 50 years to about 55 years, from about 55 years to about 60 years, from about 60 years to about 65 years, from about 65 years to about 70 years, from about 70 years to about 75 years, from about 75 years to about 80 years, from about 80 years to about 85 years, from about 85 years to about 90 years, from about 90 years to about 95 years, or from about 95 years to about 100 years.

[0297] In various embodiments, the subject is a non-human animal, and thus the present disclosure relates to veterinary use. In various embodiments, the non-human animal is a household pet. In various embodiments, the non-human animal is a livestock animal.

[0298] In various embodiments, serum and / or immune cells and / or tumor cells are evaluated and / or affected. In various embodiments, immune cells include cells of the innate immune system of a subject and / or an animal. In various embodiments, such cells include, but are not limited to, NK cells, monocytes, DCs, B cells, macrophages, CD4+ T cells, and CD8+ T cells. In various embodiments, the present disclosure provides for detecting the presence, absence, or amount of tumor volume, tumor cells, metastases, cDNA, or RNA in a sample derived from a subject.

[0299] Kit In various embodiments, the present disclosure provides a kit that can simplify the administration of any of the agents described herein. Exemplary kits of the present disclosure include any of the agents described herein in unit dosage form. In various embodiments, the unit dosage form is a container such as a prefilled syringe that can be sterile and contains any of the agents described herein and a pharmaceutically acceptable carrier, diluent, excipient, or vehicle. In various embodiments, the kit further includes a label or printed instructions that direct the use of any of the agents described herein. In various embodiments, the kit also includes an eyelid retractor, a local anesthetic, and a cleanser for the injection site. In various embodiments, the kit further includes one or more additional agents described herein.

[0300] In various aspects, the present disclosure includes a syringe containing one or more of the compositions of the present disclosure. In various embodiments, the syringe is prefilled with a certain amount of the composition. In various embodiments, the syringe is prefilled with a volume of from about 1 mL to about 10 mL. In various embodiments, the syringe is prefilled with a volume of the composition of about 10 mL, about 9 mL, about 8 mL, about 7 mL, about 6 mL, about 5 mL, about 4 mL, about 3 mL, about 2 mL, about 1.9 mL, about 1.8 mL, about 1.7 mL, about 1.6 mL, about 1.5 mL, about 1.4 mL, about 1.3 mL, about 1.2 mL, about 1.1 mL, or about 1.0 mL or less.

[0301] In various embodiments, the syringe contains a composition having a storage stability in the range of about 1 hour to about 1 week. In various embodiments, the syringe contains a composition having a storage stability of at least about 12 hours, about 24 hours, about 36 hours, about 48 hours, or about 72 hours when stored at a temperature in the range of about -85°C to about 25°C. In various embodiments, the syringe contains a composition having a storage stability of at least about 12 hours, about 24 hours, about 36 hours, about 48 hours, or about 72 hours when stored at a temperature in the range of about 15°C to about 25°C.

[0302] In various embodiments, the storage temperature is about -80°C. In various embodiments, the storage temperature is about -20°C. In various embodiments, the storage temperature is about 4°C. In various embodiments, the storage temperature is about 21°C. In various embodiments, the kit contains lyophilized BioNV / exosomes.

[0303] In one embodiment, the kit contains a container including a composition containing the BioNV / exosomes of the present disclosure, a therapeutically effective amount of an additional therapeutic agent as described herein, and instructions for use.

Example

[0304] Example 1: Regulated expression of therapeutically relevant biomolecules in cells for lumen-localized payloads in BioNV and exosomes Isolated CAR NK cells are grown until a consistent desired logarithmic phase (which is defined by the amount of BioNV obtained and / or size limitations associated with the growth media flask, container, or bioreactor) is obtained. The cells are then stimulated for activation. Several activation mechanisms can be used, including i) irradiated antigen-presenting cells, ii) anti-CD3 / CD28 coated beads, iii) IL-2, iv) stimulatory molecule(s) such as antigen-coated beads recognized by the CAR construct, v) chemicals such as phorbol esters, or vi) other methods known to those skilled in the art.

[0305] The cells can be washed or separated from the stimulatory substances for activation, such as by filtration, centrifugation, affinity column, and / or magnetism (e.g., when using magnetic beads), to remove them from anti-CD3 / CD28 coated beads (or beads conjugated with another activating antigen). Alternatively, the cells may be washed from antigen-presenting cells by flow cytometry or affinity column chromatography, or a combination of these, or both.

[0306] Next, the cells are returned to a resting state. The cell size is observed over a period of 1 - 14 days to identify the resting state. After the cells have returned to the resting state, a second activation process is performed. During this period, the cells are examined for one or more of CAR density, CD57 / CD16 expression, and intracellular therapeutic protein concentration using analytical methods such as enzyme-linked immunosorbent assay (ELISA), flow cytometry, iodixanol density gradient centrifugation, immunoblotting, and / or PCR analysis. The cells are restimulated by irradiated antigen-presenting cells, anti-CD3 and / or anti-CD28 coated beads (or beads conjugated with another activating antigen), chemicals such as phorbol esters, or any of the other methods described in the art. This restimulation step reduces activation-induced cell death of NK cells.

[0307] Thereafter, the beads are separated and the cells are grown according to known protocols outlined, for example, as described in WO2020172328, WO2017037083, WO2011080740, and WO2014028453 (each document is incorporated herein by reference in its entirety). Cell growth can be from more than 1-fold expansion to the desired fold depending on the amount of cells required to obtain an appropriate number of BioNVs.

[0308] The final doubly-activated cell population is resampled and examined for one or more of CAR density, CD57 / CD16 expression, and intracellular therapeutic protein concentration using a combination of analytical methods such as enzyme-linked immunosorbent assay (ELISA), flow cytometry, immunoblotting, and / or PCR analysis.

[0309] The protocol is carried out using appropriate controls that vary depending on the purpose of the CAR construct and the CAR-containing BioNV. One of the controls includes non-activated cells, i.e., non-activated CAR NK, processed in parallel using the above protocol. Similar activation protocols can be used in other cell types, and the verification of cell markers (in this case, CD57 / CD16 for NK cells) varies depending on the cell type used.

[0310] Example 2: Generation of BioNV by continuous extrusion Biomimetic nanovesicles (BioNV) can be substantially produced as shown in the scheme illustrated in Figure 5.

[0311] The level of CAR expression can be measured in low-immunogenic cell lines using a combination of flow cytometry and iodixanol density gradient (e.g., step 1 in Figure 5).

[0312] The differentiation of iPSC-expressing surface CAR into CAR lymphocytes can be analyzed by the identification of lymphocyte markers such as cell surface markers such as CD4 / CD8 (T cells) or CD56 / CD16 (natural killer cells) (e.g., step 2 in Figure 5). The expression profile can be determined by flow cytometry, RT-PCR, and / or CRISPR-based analysis.

[0313] Next, the activation of CAR lymphocytes can be achieved in two steps over a period of two weeks at a predetermined low concentration using biomarker antigen-coated beads (e.g., step 3 of FIG. 5). This process can also measure i) the quantification of F-actin accumulation at the synapse formation site, ii) the distribution of pZeta at the synapse, iii) the clustering of antigens through the IS position, and / or iv) the polarization of soluble granules (LG) containing perforin and granzyme, using a well-established protocol to analyze the quality of the immune synapse (IS) between the CAR and the antigen-coated beads.

[0314] After lymphocyte activation, the cells are expanded using an established protocol (e.g., step 4 of FIG. 5). After expansion, the levels of perforin and granzyme (or other luminal payloads where applicable) are analyzed for each cell population to ensure consistent concentration levels for each batch. This is accomplished using a series of qPCR, immunoblotting, flow cytometry, and / or mass spectrometry. If a cell population of sufficient size can be achieved in step 3, the expansion step may not be necessary.

[0315] Once the cells are activated and the desired therapeutic protein(s) is / are produced, the cells are expanded, harvested, washed several times, and then placed into a buffered extrusion medium. The cells are then completely processed by continuous extrusion through each step of a polycarbonate filter system with decreasing pore sizes (e.g., step 5 of FIG. 5). In the first extrusion step of the continuous extrusion process, nuclei (along with nuclear components including nuclear pores, genomic material, and transcription factors) and mitochondria are removed. The sample is then treated with an endonuclease, e.g., BENZONASE. BENZONASE is a non-specific recombinant endonuclease that cleaves all types of DNA and RNA variants into non-functional fragments less than 8 soluble base pairs. This leads to maximum reduction of nucleic acid loading per sample and on a scalable basis without interfering with the chemical properties of the BioNV membrane. This cleavage process also removes the viscosity of the nucleic acids, allowing for subsequent filling of the material and passage through the next extrusion filter set.

[0316] The continuous extrusion process avoids the removal of other organelles such as the Golgi apparatus or the ER. The membrane systems of these organelles are highly evolved such that vesicles transport (release and uptake) between the folded membranes. For example, the cis and trans faces of the Golgi apparatus have unique lipid compositions that facilitate the absorption and release of low energy barriers in vesicle transport. These components are relatively less abundant in the plasma membrane. Thus, isolating the plasma membrane to obtain BioNV is not very advantageous. BioNV is disrupted based on pore size and spontaneously formed when passing through a polycarbonate filter in the continuous extrusion process. This process yields BioNV containing membranes with a homogeneously mixed lipid content and protein components of the cytoplasm, Golgi, and ER, which can significantly increase the affinity for delivery and uptake into cells and tissues compared to BioNV that has been processed to remove these organelles. These characteristics can potentially result in better and more consistent uptake of BioNV into target cells at much lower doses than systems that do not incorporate these properties.

[0317] After the extrusion step, BioNV passes through an α-CD3 HPLC (FPLC at scale-up) column to remove a low percentage (about 0.05%) of inverted BioNV that forms naturally during the continuous extrusion process (e.g., step 6 in Figure 5). This is done to ensure that the resulting BioNV has a uniform orientation with respect to the membrane. Since this step is a flow-through process that captures impurities, the yield loss during this step is minimal. When BioNV is collected after the HPLC / FPLC step, it is examined through a standardization process.

[0318] The standardization process includes one or more of the following assays.

[0319] Homogeneity of BioNV: By using nanoparticle flow cytometry (NanoFCM), the BioNV concentration, size uniformity, density of BioNV, and / or homogeneity of the lumen components of BioNV can be confirmed.

[0320] Concentration of lumen payload: By using NanoFM technology, the type and concentration of nucleic acid / protein encapsulated in the lumen of BioNV can be determined. These data can be confirmed in parallel with one or more methods including immunoblot, mass spectrometry, and BCA analysis to determine the nucleic acid and protein content of BioNV.

[0321] Stability of BioNV: One or more of nanoparticle tracking analysis (NTA), dynamic light scattering (DLS), and electron microscopy (EM) can be used in combination with immunoblot and / or mass spectrometry to determine the physical and biochemical characteristics of BioNV over 8 - 10 months. Data from these assays may include protein expression profiles, the degree of intact BioNV membrane / enclosure, and / or the degree of aggregation.

[0322] Integrity of the membrane: The integrity of the BioNV membrane is evaluated using a calcein release assay combined with NanoFCM to assess membrane permeability. These results may provide insights into the leakage characteristics of BioNV relative to a standardized BioNV panel.

[0323] Quality of the lumen payload: The quality of the payload encapsulated in the lumen can be determined using multiple analytical assays depending on the nature of the payload. If the delivery is nucleic acid, qPCR and / or sequencing over 8 - 10 months can be used to confirm the integrity and amount of the nucleic acid payload. In the case of protein, the protein payload can be analyzed using analysis of BioNV components using one or more of NanoFCM, mass spectrometry, and immunoblot analysis.

[0324] Quality and surface density of CAR: The CAR surface density can be determined using NanoFCM, mass spectrometry, and / or immunoblot analysis. In BioNV, the CAR surface density is expected to be at least about 5-fold to at least about 10-fold higher compared to the total cell surface density. This may significantly enhance the targeting to the antigen compared to whole cells. The CAR quality can be determined at the cell stage as described above (e.g., as in step 3). Mathematical models can be used to extrapolate cell quality data and apply it to BioNV by associating it with the data results of the efficacy test.

[0325] Functionality of BioNV: BioNV can be tested for basic functionality in a plurality of defined standardized assays, such as in vitro cell uptake into target cells when the antigen is expressed and when it is not expressed, and the ability to pass through high-density tissues such as the human retina model. Following these basic functionality assays that can be performed immediately after the continuous extrusion process, in preclinical trials, the remaining quality and functionality characteristics of BioNV are addressed.

[0326] Definitions The following definitions are used in connection with the present disclosure. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0327] "Effective amount" or "therapeutically effective amount" is an amount effective to treat, prevent, or ameliorate a disease in a mammal.

[0328] As used herein, "a", "an", or "the" may mean one or more than one.

[0329] As used herein, the term "comprising" and variations thereof are intended to be non-limiting, such that the recitation of items in a list is not to be construed as excluding other similar items that may be useful in the materials, compositions, devices, and methods of this technology. Similarly, the terms "can" and "may" and variations thereof are intended to be non-limiting, such that the recitation that a particular embodiment can or may include a particular element or feature does not exclude other embodiments of this technology that do not include those elements or features.

[0330] As used herein, for purposes of describing and claiming the present disclosure, the open-ended term "comprising" is used as a synonym for terms such as "including," "containing," or "having," although the present disclosure or embodiments thereof may alternatively be described using alternative terms, such as "consisting of" or "consisting essentially of."

[0331] In various embodiments, "BioNV", "exosome", "therapeutic BioNV", or "therapeutic exosome" refers to a biomimetic nanovesicle (NV) that encapsulates an aqueous fraction containing at least one therapeutic biomolecule. In various embodiments, the therapeutic BioNV / exosome is allogeneic and / or hypoimmunogenic. In various embodiments, the therapeutic BioNV / exosome comprises at least one surface-directed membrane-embedded CAR. In various embodiments, the "nanovesicle (NV)" referred to herein is a lipid-bound vesicle sized from about 10 nm to about 1200 nm that encapsulates an aqueous core. In various embodiments, the lipid-bound NV can be formed using a lipid monolayer, a lipid bilayer, or can maintain a multilamellar morphology. In various embodiments, the therapeutic BioNV / exosome refers to a bio-derived nanosized vesicle that can have a designed biological functionalization. In various embodiments, the therapeutic BioNV / exosome is "biomimetic" in that it is derived from endogenous cellular material, and more specifically, substantially reproduces the plasma membrane material found within cells. In various embodiments, the cells from which the therapeutic BioNV / exosome is derived can include any type of stem cell, including cell types differentiated from the stem cells. In various embodiments, the therapeutic BioNV / exosome is substantially free of encapsulated cellular debris, including nucleic acids, organelles, or organelle parts. In various embodiments, the therapeutic BioNV / exosome is characterized by having one or more, two or more, three or more, four or more, five or more, or six or more of the following. a. having a size from about 10 nm to about 1200 nm; b. having a total volume of about 500 nm 3 to about 5 μm 3 assuming a spherical shape); c. having a content of at least one phospholipid and cholesterol; d. The surface membrane has one or more of CD34, CCL21, PD-L1 (in BioNV / exosomes derived from non-activated cell sources), FasL, SerpinB9, H2-M3, CD47, CTLA-4, CD24, CD200, MFG-E8, NCAM, α-phagocyte integrin, and / or an anti-6R antibody or antibody format, or a chimera of any one or more of them; the surface membrane is substantially lacking in T cell receptor components (TRAC and / or TRBC), MHC class I components, and / or MHC class II components, lacking one or more proteins of HLA-A, HLA-B, HLA-C, HLA-E or HLA-G (but not both HLA-E and HLA-G), HLA-F, and / or CIITA, SerpinB9, and substantially lacking one or more proteins of IL-4, IL-6, IL-10, and / or IL-16 inside the vesicles; e. Encapsulating one or more therapeutically relevant biomolecules, such as cytokines, such as chemokines, interferons (IFNα / β / γ), interleukins, alarmins, lymphokines, tumor necrosis factor (TNF), colony stimulating factors, bone morphogenetic proteins (BMP), erythropoietin (EPO), granulocyte stimulating factor (G-CSF), granulocyte macrophage colony stimulating factor (GM-CSF), pro-inflammatory cytokines, anti-inflammatory cytokines, perforin granzymes (such as granzyme A, B, H, K, and M), gene editing payloads, fusion proteins, antibody or antibody format constructs, or combinations thereof; f. CAR, monoclonal antibody, polyclonal antibody, antibody fragment, Fab, Fab’, Fab’-SH, F(ab’)2, Fv, single-chain Fv (scFv), diabody, nanobody, linear antibody, bispecific antibody, multispecific antibody, chimeric antibody, humanized antibody, human antibody, fusion protein containing the antigen-binding portion of an antibody, bispecific T cell engager (BiTE), virus epitope recognition receptor (VERR) or virus ligand, variable heavy chain IgG fragment V H H or V NARA membrane-embedded targeting agent comprising an antibody or antibody format selected from one or more thereof, or a targeting moiety via a T cell receptor (TCR), wherein the targeting agent can target a single biomarker or multiple biomarkers, or multiple portions of a single biomarker; g. being able to adsorb and / or encapsulate one or more therapeutic payloads such as perforin, granzyme, cytokine, cytotoxic protein, cell-acting agent of non-natural origin, checkpoint inhibitor, recombinant gene editing payload, antibody or antibody fragment, small molecule inhibitor, biological agent, radionuclide, tracer, dye, fluorescent protein, and / or any combination thereof; and h. being capable of not causing a harmful immune reaction in a subject.

[0332] In various embodiments, "induced pluripotent stem cell" or "iPSC" refers to a stem cell that can be generated directly from an adult cell. An iPSC can arise from a differentiated cell that is reprogrammed back to an embryonic-like pluripotent state. An iPSC can generally proliferate indefinitely and become any cell type of the organism from which it is derived.

[0333] In various embodiments, as used herein, "allogeneic" refers to biological materials, tissues, or cells that are not genetically similar and are originally immunologically incompatible, even though they are derived from the same species. For example, allogeneic BioNV / exosomes are materials derived from a first subject (iPSC donor) and can be provided to any number of different subjects that are not genetically identical.

[0334] In various embodiments, "low immunogenicity" or "low immune" as used herein with respect to modified cells and / or BioNV / exosomes refers to a reduction in the ability to generate an immune response. In various embodiments, iPSCs and BioNV / exosomes can be hypoimmunogenic because they have reduced or absent expression and / or activity of one or more specific cell surface proteins and / or secreted proteins, such as T cell receptor (TCR) proteins, cytokine response syndrome proteins, MHC class I or II proteins, and the like. In various embodiments, iPSCs and BioNV / exosomes can be hypoimmunogenic due to increased expression of immune-protective cell surface proteins such as CD47, CD34, CD24, CD200, α-macrophage integrin. In various embodiments, BioNV / exosomes can be hypoimmunogenic by not inducing CRS in a subject and / or not inducing HLA incompatibility.

[0335] In various embodiments, "knockout," "silencing," "inactivation," "disruption," or "blockade," and their equivalents, with respect to transcription, gene expression, or protein expression, refer to a decrease in the amount of transcription, gene, or protein expression in a particular cell subset from a normal state or less than the wild-type state. This decrease can be significant such that gene expression does not occur at all or the amount of expression is very low.

[0336] In various embodiments, "overexpression" as used herein refers to an increase in the amount of transcription, gene, or protein expression in a particular cell subset from a normal state or more than the wild-type state.

[0337] One of ordinary skill in the art will be able to recognize or confirm many equivalents of the specific embodiments specifically described herein using only routine experimentation. Such equivalents are intended to be encompassed within the scope of the following claims.

[0338] As used herein, all headings are for organizational purposes only and are not intended to limit the disclosure in any way. The content of any individual section may be equally applicable to all sections.

[0339] All patents and publications referred to herein are hereby incorporated by reference in their entirety, including the published PCT application WO2020 / 227369 entitled "Tailored Hypoimmune Nanovesicle Delivery Systems for Cancer Tumors", filed on May 6, 2020, and the published US non-provisional application 20220040106A1 entitled "Tailored Hypoimmune Nanovesicular Delivery Systems for Cancer Tumors, Hereditary and Infectious Diseases.", filed on August 3, 2021.

Claims

**Claim 1** A method for generating a therapeutic biomimetic nanovesicle (BioNV), comprising: (a) obtaining hypoimmunogenic cells; (b) activating the hypoimmunogenic cells to express one or more therapy-related biomolecules; (c) treating the activated hypoimmunogenic cells to generate the therapeutic BioNV, wherein the therapeutic BioNV comprises the plasma membrane of the hypoimmunogenic cells and encapsulates the one or more therapy-related biomolecules. The method as described above. **Claim 2** A method for generating a therapeutic exosome, comprising: (a) obtaining hypoimmunogenic cells; (b) activating the hypoimmunogenic cells to express one or more therapy-related biomolecules; (c) collecting the therapeutic exosomes naturally secreted from the activated hypoimmunogenic cells, wherein the therapeutic exosomes comprise the plasma membrane of the hypoimmunogenic cells and encapsulate the one or more therapy-related biomolecules. The method as described above. **Claim 3** A method for treating or preventing a disease or disorder, comprising administering a therapeutically effective amount of the therapeutic biomimetic nanovesicle (BioNV) generated according to Claim 1 to a subject in need thereof. **Claim 4** A method for treating or preventing a disease or disorder, comprising administering a therapeutically effective amount of the therapeutic exosomes generated according to Claim 2 to a subject in need thereof. **Claim 5** The method according to any one of Claims 1 to 4, wherein the hypoimmunogenic cells are stem cells, induced pluripotent stem cells (iPSCs), reprogrammed totipotent or pluripotent cells, embryonic stem cells, mesenchymal stem cells, or differentiated cells derived from any of these stem cells. **Claim 6** The method according to any one of Claims 1 to 4, wherein the hypoimmunogenic cells are T cells, helper T cells, memory T cells, or NK cells. **Claim 7** The method according to any one of Claims 1 to 4, wherein the hypoimmunogenic cells are macrophages. **Claim 8** The method according to any one of Claims 1 to 4, wherein the hypoimmunogenic cells are monocytes. **Claim 9** The method according to any one of Claims 1 to 4, wherein the hypoimmunogenic cells are hepatocytes, cardiomyocytes, neurons, endothelial cells, pancreatic cells, or retinal pigment epithelial (RPE) cells. **Claim 10** The method according to any one of the preceding claims, wherein the hypoimmunogenic cells are substantially lacking one or more MHC class I proteins, MHC class II proteins, T cell receptor (TCR) proteins, and / or cytokine release syndrome (CRS) proteins.

11. The method according to any one of the preceding claims, wherein the hypoimmunogenic cells have a disruption of the β2-microglobulin (B2M) gene and / or a disruption that reduces or abolishes the expression and / or activity of MHC class I proteins.

12. The method according to any one of the preceding claims, wherein the hypoimmunogenic cells have a disruption of the CIITA gene and / or a disruption that reduces or abolishes the expression and / or activity of MHC class II proteins.

13. The method according to any one of the preceding claims, wherein the hypoimmunogenic cells have a disruption of the HLA-A gene and / or a disruption that reduces or abolishes the expression and / or activity of HLA-A proteins.

14. The method according to any one of the preceding claims, wherein the hypoimmunogenic cells have a disruption of the HLA-B gene and / or a disruption that reduces or abolishes the expression and / or activity of HLA-B proteins.

15. The method according to any one of the preceding claims, wherein the hypoimmunogenic cells have a disruption of the HLA-C gene and / or a disruption that reduces or abolishes the expression and / or activity of HLA-C proteins.

16. The method according to any one of the preceding claims, wherein the hypoimmunogenic cells have a disruption of the HLA-E gene or a disruption of the HLA-G gene and / or a disruption that reduces or abolishes the expression and / or activity of HLA-E or HLA-G proteins.

17. The method according to any one of the preceding claims, wherein the hypoimmunogenic cells have a disruption of the HLA-F gene and / or a disruption that reduces or abolishes the expression and / or activity of HLA-F proteins.

18. The method according to any one of the preceding claims, wherein the hypoimmunogenic cells have a disruption of the T cell alpha constant (TRAC) gene and / or a disruption that reduces or abolishes the expression and / or activity of TRAC proteins.

19. The method according to any one of the preceding claims, wherein the hypoimmunogenic cells have a disruption of the T cell beta constant (TRBC) gene and / or a disruption that reduces or abolishes the expression and / or activity of the TRBC protein.

20. The method according to any one of the preceding claims, wherein the hypoimmunogenic cells have reduced or abolished expression of the PD-1 gene and / or reduced or abolished expression and / or activity of the PD-1 protein, where the hypoimmunogenic cells are activated; or the hypoimmunogenic cells express the PD-1 gene and / or gene product or have increased expression thereof, where the hypoimmunogenic cells are not activated.

21. The method according to any one of the preceding claims, wherein the hypoimmunogenic cells have a disruption of the IL-4 gene and / or a disruption that reduces or abolishes the expression and / or activity of the IL-4 protein.

22. The method according to any one of the preceding claims, wherein the hypoimmunogenic cells have a disruption of the IL-6 gene and / or a disruption that reduces or abolishes the expression and / or activity of the IL-6 protein.

23. The method according to any one of the preceding claims, wherein the hypoimmunogenic cells have a disruption of the IL-10 gene and / or a disruption that reduces or abolishes the expression and / or activity of the IL-10 protein.

24. The method according to any one of the preceding claims, wherein the hypoimmunogenic cells have a disruption of the IL-16 gene and / or a disruption that reduces or abolishes the expression and / or activity of the IL-16 protein.

25. The method according to any one of the preceding claims, wherein the hypoimmunogenic cells have a disruption of the SerpinB9 gene and / or a disruption that reduces or abolishes the expression and / or activity of the SerpinB9 protein.

26. The method according to any one of claims 1 to 24, wherein the hypoimmunogenic cells express the SerpinB9 gene and / or gene product or have increased expression and / or activity thereof.

27. The method according to any one of the preceding claims, wherein the hypoimmunogenic cells express the CCL2 gene and / or gene product or have increased expression and / or activity thereof.

28. The method according to any one of the preceding claims, wherein the hypoimmunogenic cell expresses the PD-L1 gene and / or gene product, or its expression is increased, and wherein the hypoimmunogenic cell is not activated; or the modified cell has reduced or abolished expression of the PD-L1 gene and / or gene product, and wherein the hypoimmunogenic cell is activated. Claim 29 The method according to any one of the preceding claims, wherein the hypoimmunogenic cell expresses the H2-M3 gene and / or gene product, or its expression and / or activity is increased. Claim 30 The method according to any one of the preceding claims, wherein the hypoimmunogenic cell expresses the CD47 gene and / or gene product, or its expression and / or activity is increased. Claim 31 The method according to any one of the preceding claims, wherein the hypoimmunogenic cell expresses the CD24 gene and / or gene product, or its expression and / or activity is increased. Claim 32 The method according to any one of the preceding claims, wherein the hypoimmunogenic cell expresses the chimeric CD24 / CD47 gene and / or gene product, or its expression and / or activity is increased. Claim 33 The method according to any one of the preceding claims, wherein the hypoimmunogenic cell expresses the CTLA-4 gene and / or gene product, or its expression and / or activity is increased. Claim 34 The method according to any one of the preceding claims, wherein the hypoimmunogenic cell expresses the CD200 gene and / or gene product, or its expression and / or activity is increased. Claim 35 The method according to any one of the preceding claims, wherein the hypoimmunogenic cell expresses the chimeric CD24 / CD200 gene and / or gene product or the chimeric CD47 / CD200 gene and / or gene product, or its expression and / or activity is increased. Claim 36 The method according to any one of the preceding claims, wherein the hypoimmunogenic cell expresses the MFG-E8 gene and / or gene product, or its expression and / or activity is increased. Claim 37 The method according to any one of the preceding claims, wherein the hypoimmunogenic cell expresses the NCAM gene and / or gene product, or its expression and / or activity is increased.

38. The method according to any one of the preceding claims, wherein the hypoimmunogenic cell expresses an α-phagocyte integrin gene and / or gene product, or the expression and / or activity thereof is increased.

39. The method according to any one of the preceding claims, wherein the hypoimmunogenic cell expresses an antibody or antibody format molecule (anti-IL-6R) targeting the IL-6 surface receptor, or the expression and / or activity thereof is increased.

40. The method according to any one of the preceding claims, wherein the hypoimmunogenic cell expresses a FasL gene and / or gene product.

41. The method according to any one of the preceding claims, wherein the hypoimmunogenic cell does not overexpress a FasL gene and / or gene product.

42. The method according to any one of the preceding claims, wherein the hypoimmunogenic cell substantially lacks the expression and / or activity of one or more immunogenic proteins and expresses one or more immunoprotective proteins, or the expression thereof is increased.

43. The method according to any one of the preceding claims, wherein the hypoimmunogenic cell has reduced or abolished the expression and / or activity of 3 or more immunogenic proteins, 4 or more immunogenic proteins, 5 or more immunogenic proteins, 6 or more immunogenic proteins, 7 or more immunogenic proteins, 8 or more immunogenic proteins, 9 or more immunogenic proteins, 10 or more immunogenic proteins, 11 or more immunogenic proteins, or 12 or more immunogenic proteins.

44. The method according to any one of the preceding claims, wherein the hypoimmunogenic cell expresses 3 or more immunoprotective proteins, 4 or more immunoprotective proteins, 5 or more immunoprotective proteins, 6 or more immunoprotective proteins, 7 or more immunoprotective proteins, 8 or more immunoprotective proteins, 9 or more immunoprotective proteins, or 10 or more immunoprotective proteins, or the expression thereof is increased.

45. The method according to any one of the preceding claims, wherein the hypoimmunogenic cell has reduced or abolished the expression and / or activity of any one gene and / or gene product of HLA-A, HLA-B, HLA-C, HLA-F, CIITA, IL-6, TRAC, TRBC, and HLA-E or HLA-G.

46. The method according to any one of claims 1 to 44, wherein the hypoimmunogenic cells have reduced or abolished expression and / or activity of any one gene and / or gene product of HLA-A, HLA-B, HLA-C, HLA-F, CII TA, IL-6, TRAC, TRBC, SerpinB9, and HLA-E or HLA-G.

47. The method according to any one of claims 1 to 44, wherein the hypoimmunogenic cells include hypoimmunogenic cells having reduced or abolished expression and / or activity of any one of HLA-A, HLA-B, HLA-C, HLA-F, CII TA, IL-6, TRAC, TRBC, SerpinB9, HLA-E or HLA-G, and one or more genes and / or gene products of IL-4, IL-10, and IL-16.

48. The hypoimmunogenic cells express α-phagocyte integrin, CCL2, H2-M3, FasL, MFEG8, and PD-L1 and / or CTLA-4, or their expression and / or activity is increased, where the hypoimmunogenic cells do not overexpress FasL and the hypoimmunogenic cells are not activated by the expression of PD-L1; also The method according to any one of claims 1 to 47, wherein any one of CD24, CD47, CD200, chimeric CD24 / CD47, chimeric CD24 / CD200, and chimeric CD47 / CD200, or any two of CD24, CD47, and CD200 are expressed, or their expression and / or activity is increased.

49. The hypoimmunogenic cells express α-phagocyte integrin, CCL2, H2-M3, FasL, MFEG8, SerpinB9, and PD-L1 and / or CTLA-4, or their expression and / or activity is increased, where the hypoimmunogenic cells do not overexpress FasL and the hypoimmunogenic cells are not activated by the expression of PD-L1; also The method according to any one of claims 1 to 47, wherein any one of CD24, CD47, CD200, chimeric CD24 / CD47, chimeric CD24 / CD200, and chimeric CD47 / CD200, or any two of CD24, CD47, and CD200 are expressed, or their expression and / or activity is increased.

50. The method according to any one of claims 1 to 47, wherein the hypoimmunogenic cells express the CD200 gene and / or gene product, or have increased expression and / or activity thereof, and do not express and / or are substantially deleted of either the CD24 or CD47 gene and / or gene product.

51. The method according to any one of claims 1 to 47, wherein the hypoimmunogenic cells do not have the expression and / or activity of the SerpinB9 gene and / or gene product and the CD200 gene and / or gene product.

52. The method according to any one of the preceding claims, wherein the hypoimmunogenic cells are allogeneic.

53. The method according to any one of the preceding claims, wherein the hypoimmunogenic cells do not cause an immune response in a patient to whom the cells or BioNV or exosomes derived therefrom have been administered.

54. The method according to any one of the preceding claims, wherein the hypoimmunogenic cells comprise one or more targeting agents.

55. The method according to claim 54, wherein the one or more targeting agents comprise a chimeric antigen receptor (CAR).

56. The method according to claim 55, wherein the CAR is bispecific.

57. The method according to claim 55, wherein the CAR lacks an intracellular portion.

58. The method according to claim 55, wherein the CAR is a targeting agent, a transmembrane domain, and an intracellular domain, and the intracellular domain comprises a costimulatory domain and / or a signaling domain.

59. The method according to claim 58, wherein the transmembrane domain is derived from CD28, CD3ζ, CD4, CD8α, or ICOS, or a fragment thereof.

60. The method according to claim 58, wherein the intracellular domain is the intracellular signaling domain of the CD3ζ chain and / or one or more costimulatory molecules, optionally including one or more of the costimulatory molecules selected from CD28, 4-1BB, ICOS, CD27, and OX40.

61. The method according to claim 54, wherein the one or more targeting agents comprise an antibody or an antibody format.

62. The method according to claim 61, wherein the antibody or antibody format is selected from one or more of monoclonal antibodies, polyclonal antibodies, antibody fragments, Fab, Fab’, Fab’-SH, F(ab’)2, Fv, single-chain Fv (scFv), V ＮＡＲ , V Ｈ H, affilins, diabodies, nanobodies, linear antibodies, bispecific antibodies, multispecific antibodies, chimeric antibodies, humanized antibodies, human antibodies, and fusion proteins comprising the antigen-binding portion of an antibody

63. The method according to claim 62, wherein the antibody format is scFv.

64. The method according to claim 54, wherein the one or more targeting agents comprise a viral epitope recognition receptor (VERR) and / or a viral ligand.

65. The method according to claim 54, wherein the one or more targeting agents comprise a ligand for a receptor and / or a receptor for a ligand.

66. The method according to any one of the preceding claims, wherein the activation of the hypoimmunogenic cells comprises activation of the TCR / CD3 receptor complex by a protein antigen.

67. The method according to any one of claims 1 to 65, wherein the activation of the hypoimmunogenic cells comprises activation of the TCR / CD3 receptor complex by a small molecule.

68. The method according to any one of claims 1 to 65, wherein the activation of the hypoimmunogenic cells comprises activation of the TCR / CD3 receptor complex by a viral antigen.

69. The method according to any one of claims 1 to 65, wherein the activation of the hypoimmunogenic cells comprises activation of a calcium-dependent channel.

70. The method according to any one of claims 1 to 65, wherein the activation of the hypoimmunogenic cells comprises activation of the LFA-1 integrin receptor.

71. The method according to any one of claims 1 to 65, wherein the activation of the hypoimmunogenic cells comprises activation of the CD28 receptor.

72. The method according to any one of claims 1 to 65, wherein the activation of the hypoimmunogenic cells comprises activation of the IL-2 receptor.

73. The method according to any one of claims 1 to 65, wherein the activation of the hypoimmunogenic cells comprises activation of the SLAMF1 (CD150) receptor.

74. The method according to claim 73, wherein the activation of the SLAMF1 (CD150) receptor is by a measles virus.

75. The method according to claim 73, wherein the activation of the SLAMF1 (CD150) receptor is by a gram-negative bacterium.

76. The method according to any one of claims 1 to 65, wherein the activation of the hypoimmunogenic cells comprises activation of the IFNγ receptor.

77. The method according to any one of claims 1 to 65, wherein the activation of the hypoimmunogenic cells comprises activation of the CD4 receptor by one or more viruses.

78. The method according to any one of claims 1 to 65, wherein the activation of the hypoimmunogenic cells comprises activation by an engineered non-natural biomolecule selected from one or more of a soluble peptide, a chimeric antigen receptor, a small decoy, a small ligand, a designer nucleic acid ligand, a carbohydrate ligand, a viral ligand, a chimeric biomolecule ligand, a fusion protein, an antibody, and an antibody format molecule.

79. The method according to any one of claims 1 to 65, wherein the activation of the hypoimmunogenic cells comprises activation by an inorganic compound.

80. The method according to any one of claims 1 to 65, wherein the activation of the hypoimmunogenic cells comprises activation by expression, overexpression, or increased activity of a transcription factor.

81. The method according to any one of the preceding claims, wherein the activation of the hypoimmunogenic cells comprises activation at the DNA level by one or more of a transposase-based method, a Cre / Lox-based method, an endonuclease-based method, a homologous recombination (HR)-based method, a non-homologous end joining (NEHJ)-based method, a microhomology-mediated end joining (MMEJ)-based method, a homology-mediated end joining (HMEJ)-based method, a small RNA, or a combination thereof.

82. The method according to claim 81, wherein the small RNA comprises one or more of a guide RNA (gRNA), a tracer RNA (tracrRNA), a microRNA (miRNA), RNA interference (RNAi), a small interfering RNA (siRNA), a double-stranded RNA, a Piwi-interacting RNA (piRNA), a small nuclear RNA (snRNA), a small nucleolar RNA (snoRNA), an antisense oligonucleotide (ASO), a locked nucleic acid (LNA), a splice-switching oligonucleotide (SSO), a tRNA, a complementary messenger RNA, a repeat-associated small interfering RNA (rasiRNA), an endonuclease, and a small non-coding RNA.

83. The activation of the hypoimmunogenic cells comprises activation at the RNA level by one or more of guide RNA (gRNA), tracer RNA (tracrRNA), microRNA (miRNA), RNA interference (RNAi), small interfering RNA (siRNA), double-stranded RNA, Piwi-interacting RNA (piRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), antisense oligonucleotide (ASO), locked nucleic acid (LNA), splice-switching oligonucleotide (SSO), tRNA, complementary messenger RNA, repeat-associated small interfering RNA (rasiRNA), endonuclease, small non-coding RNA, IRES element, or a combination thereof, according to any one of claims 1 to 80.

84. The method according to any one of claims 1 to 80, wherein the activation comprises one or more RNA-induced endonucleases.

85. The method according to any one of claims 1 to 80, wherein the activation of the hypoimmunogenic cells comprises activation by an endogenous promoter region and / or enhancer region.

86. The method according to any one of claims 1 to 80, wherein the activation of the hypoimmunogenic cells comprises activation by stably integrating a gene element.

87. The method according to any one of claims 1 to 80, wherein the activation of the hypoimmunogenic cells comprises activation by transient expression of a gene element.

88. The method according to any one of the preceding claims, wherein the activation of the hypoimmunogenic cells results in a metabolically altered state of the hypoimmunogenic cells.

89. The method according to any one of the preceding claims, wherein the one or more treatment-related biomolecules are chemokines, interferons, interleukins, alarmins, lymphokines, perforin, granzyme, granulysin, tumor necrosis factor (TNF), colony-stimulating factors, bone morphogenetic proteins (BMP), erythropoietin (EPO), granulocyte colony-stimulating factor (G-CSF), granulocyte macrophage colony-stimulating factor (GM-CSF), gene editing payloads, fusion proteins, antibodies or antibody formats, or a combination thereof.

90. The method according to claim 89, wherein the cytokine is a pro-inflammatory cytokine.

91. The method according to claim 89, wherein the cytokine is an anti-inflammatory cytokine.

92. The method according to claim 89, wherein the granzyme is granzyme A, B, H, K, or M.

93. The method according to claim 89, wherein the gene editing payload comprises one or more gene editor nucleic acids and / or proteins, or one or more nucleic acids encoding one or more gene editors.

94. The method according to claim 93, wherein the one or more gene editors are site-specific endonucleases, TALENs, ZFNs, RNase P RNA, CRISPR / Cas nucleases, C2c1, C2c2, C2c3, Cas9, Cpf1, TevCas9, archaeal Cas9, CasY.1, CasY.2, CasY.3, CasY.4, CasY.5, CasY.6, CasX, Cas omega, transposases, and / or any orthologs or homologs thereof.

95. The method according to claim 89, wherein the gene editing payload comprises a trans-activation response region (TAR) loop system.

96. The therapeutic BioNV is (i) one or more membrane-embedded targeting agents targeted to one or more cell biomarkers, (ii) one or more membrane-embedded proteins of α-phagocyte integrin, CCL2, H2-M3, FasL, MFEG8, PD-L1 and / or CTLA-4, SerpinB9, and anti-IL-6R antibody or antibody format, (iii) any one of CD24, CD47, CD200, chimeric CD24 / CD47, chimeric CD24 / CD200, and chimeric CD47 / CD200, or any two of CD24, CD47, and CD200 membrane-embedded proteins, and (iv) any one of HLA-A, HLA-B, HLA-C, HLA-F, CIIITA, IL-6, TRAC, TRBC, HLA-E or HLA-G, and one or more proteins of IL-4, IL-10 and IL-16 substantially lacking, a membrane containing. The method according to any one of claims 1, 3, and 5 to 95, wherein the one or more treatment-related biomolecules are chemokines, interferons, interleukins, alarmins, lymphokines, perforin, tumor necrosis factor (TNF), colony-stimulating factors, bone morphogenetic proteins (BMP), erythropoietin (EPO), granulocyte-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), gene editing payloads, fusion proteins, antibodies or antibody formats, or combinations thereof.

97. The therapeutic BioNV is (i) one or more membrane-embedded targeting agents targeted to one or more cell biomarkers, (ii) one or more membrane-embedded proteins of α-phagocyte integrin, CCL2, H2-M3, FasL, MFEG8, PD-L1 and / or CTLA-4, and an anti-IL-6R antibody or antibody format, (iii) a membrane-embedded protein of either CD24 and CD47, or chimeric CD24 / CD47, and (iv) one or more of HLA-A, HLA-B, HLA-C, HLA-F, CII TA, IL-6, TRAC, TRBC, HLA-E or HLA-G, SerpinB9, and CD200, and a membrane substantially lacking one or more of IL-4, IL-10 and IL-16 proteins. The method according to any one of claims 1, 3, and 5 to 95, wherein the one or more treatment-related biomolecules are chemokines, interferons, interleukins, alarmins, lymphokines, perforin, granzyme, granulysin, tumor necrosis factor (TNF), colony-stimulating factors, bone morphogenetic proteins (BMP), erythropoietin (EPO), granulocyte-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), gene editing payloads, fusion proteins, antibodies or antibody formats, or combinations thereof.

98. The method according to claim 96 or 97, wherein the one or more targeting agents are antibodies or antibody formats.

99. The method according to claim 98, wherein the one or more targeting agents are CARs.

100. Processing the activated hypoimmunogenic cells is by one or more of sonication, adaptive focused acoustics technology, French press, extrusion, continuous extrusion, enzymatic disruption, cell lysis by surfactant, and / or electroporation, the method according to any one of claims 1 and 96 to 99.

101. The method according to claim 100, wherein processing the activated hypoimmunogenic cells is by continuous extrusion.

102. The method according to any one of claims 1 and 96 to 101, wherein the therapeutic BioNV is sized from about 10 nm to about 1200 nm.

103. The method according to claim 102, wherein the therapeutic BioNV is sized from about 10 nm to about 100 nm.

104. The method according to claim 102, wherein the therapeutic BioNV is sized from about 100 nm to about 200 nm.

105. The method according to claim 102, wherein the therapeutic BioNV is sized from about 200 nm to about 500 nm.

106. The method according to claim 102, wherein the therapeutic BioNV is sized from about 500 nm to about 1200 nm.

107. The therapeutic exosome is (i) one or more membrane-embedded targeting agents targeted to one or more cell biomarkers, (ii) one or more membrane-embedded proteins of α-phagocyte integrin, CCL2, H2-M3, FasL, MFEG8, PD-L1 and / or CTLA-4, SerpinB9, and anti-IL-6R antibody or antibody format, (iii) any one of CD24, CD47, CD200, chimeric CD24 / CD47, chimeric CD24 / CD200, and chimeric CD47 / CD200, or any two of CD24, CD47, and CD200 membrane-embedded proteins, and (iv) any one of HLA-A, HLA-B, HLA-C, HLA-F, CII TA, IL-6, TRAC, TRBC, HLA-E or HLA-G, and one or more proteins of IL-4, IL-10 and IL-16 substantially lacking membranes. The method according to any one of claims 2 and 4 to 95, wherein the one or more treatment-related biomolecules are chemokines, interferons, interleukins, alarmins, lymphokines, perforin, tumor necrosis factor (TNF), colony-stimulating factors, bone morphogenetic proteins (BMP), erythropoietin (EPO), granulocyte-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), gene editing payloads, fusion proteins, antibodies or antibody formats, or combinations thereof.

108. The therapeutic exosome is (i) one or more membrane-embedded targeting agents targeted to one or more cell biomarkers, (ii) one or more membrane-embedded proteins of α-phagocyte integrin, CCL2, H2-M3, FasL, MFEG8, PD-L1 and / or CTLA-4, SerpinB9, and anti-IL-6R antibody or antibody format, (iii) a membrane-embedded protein of either CD24 and CD47, or chimeric CD24 / CD47, and (iv) a membrane substantially lacking one or more of HLA-A, HLA-B, HLA-C, HLA-F, CII TA, IL-6, TRAC, TRBC, HLA-E or HLA-G, SerpinB9, and CD200, and one or more of IL-4, IL-10 and IL-16. The method according to any one of claims 2 and 4 to 95, wherein the one or more treatment-related biomolecules are chemokines, interferons, interleukins, alarmins, lymphokines, perforin, granzyme, granulysin, tumor necrosis factor (TNF), colony-stimulating factors, bone morphogenetic proteins (BMP), erythropoietin (EPO), granulocyte-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), gene editing payloads, fusion proteins, antibodies or antibody formats, or combinations thereof.

109. The method according to claim 107 or 108, wherein the one or more targeting agents are antibodies or antibody formats.

110. The method according to claim 109, wherein the one or more targeting agents are CARs.

111. The method according to any one of claims 2 and 107 to 110, wherein collecting the therapeutic exosomes from the activated hypoimmunogenic cells includes inducing hypoxia.

112. The method according to any one of claims 2 and 107 to 111, wherein collecting the therapeutic exosomes from the activated hypoimmunogenic cells includes expressing or increasing the expression of one or more cytokines.

113. The method according to any one of claims 2 and 107 to 112, wherein collecting the therapeutic exosomes from the activated hypoimmunogenic cells includes supplying one or more small molecule exosome modulators.

114. The method according to any one of claims 2 and 107 to 113, wherein collecting the therapeutic exosomes from the activated hypoimmunogenic cells includes supplementing the medium with one or more exosome factors.

115. The method according to any one of claims 2 and 110 to 114, wherein the therapeutic exosomes are sized from about 10 nm to about 200 nm.

116. The method according to claim 115, wherein the therapeutic exosomes are sized from about 10 nm to about 100 nm.

117. The method according to claim 115, wherein the therapeutic exosomes are sized from about 100 nm to about 200 nm.

118. The method according to claim 3 or 4, wherein the mammalian disease is cancer, an infectious disease, a genetic disorder, or a rare disease.

119. The method according to claim 1 or 2, wherein activation of the hypoimmunogenic cells includes activating the JAK1 / TYK2-STAT pathway via the INFAR1 and / or INFAR2 receptor using INF-α.

120. The method according to any one of claims 1 to 2 and 119, wherein the hypoimmunogenic cells are natural killer cells, and the activation includes activating a receptor selected from the group consisting of CD2, CD3, CD16, CD314 (NKG2D), CD335, B7-H6, CD158d, IL-2R, IL-12R, DNAM-1, CD2, CD44, CD137, CX3CR1, CD27, CD160, 2B4, and combinations thereof.

121. The method according to any one of claims 1 to 2 and 119 to 120, wherein the hypoimmunogenic cells are natural killer cells, killer T cells, T cells (all subsets), and the activation does not result in degranulation or polarization of granules to the immunological synapse.

122. The method according to any one of claims 1 to 2 and 119 to 121, wherein the hypoimmunogenic cells are natural killer cells, killer T cells, T cells (all subsets), and the activation results in granulation.

123. The method according to any one of claims 1 to 2 and 119 to 122, wherein the hypoimmunogenic cells are natural killer cells, killer T cells, T cells (all subsets), and the activation results in polarization of soluble granules.

124. The method according to any one of claims 1 to 2 and 119 to 123, wherein activating comprises activating the expression of cytotoxic biomolecules by targeting cytoplasmic signaling molecules that are part of a signaling pathway.

125. The method according to any one of claims 1 to 2 and 119 to 124, wherein activating comprises activating PKC with a small molecule or a biological agent.

126. The method according to any one of claims 1 to 2 and 119 to 125, wherein activating comprises activating PKC with calcium and diacylglycerol.

127. The method according to any one of claims 1 to 2 and 119 to 126, wherein the activation step comprises activating PKC by adding a molecule selected from the group consisting of bryostatin 1, ingenol-3-angelate, phorbol 12-myristate 13-acetate, prostratin, SC-9, SC-10, and phorbol 12,13-dibutyrate to the hypoimmunogenic cells.

128. The method according to any one of claims 1 to 2 and 119 to 120, wherein activating comprises targeting Crk for perforin and granzyme expression using a small molecule or a biological agent.

129. The method according to claim 128, wherein the Crk targeting step comprises blocking or mutating the phosphorylation of Crk.

130. The method according to any one of claims 1 to 2 and 119 to 129, wherein said activating comprises activating a transcription factor that leads to activating a compound selected from the group consisting of perforin, granzyme, other cytotoxic proteins, and combinations thereof.

131. The method according to any one of claims 1 to 2 and 119 to 130, wherein said activating further comprises activating or suppressing a gene expression function by a CRISPR activation and inhibition system (CRISPRa / i) of a compound selected from the group consisting of perforin, granzyme, other cytotoxic proteins, and combinations thereof.

132. The method according to any one of claims 1 to 2 and 119 to 131, wherein said low immunogenic cells are induced pluripotent stem cells (iPSCs) engineered to contain at least one perforin and / or granzyme gene, and said iPSCs contain regulatory properties or regulatory elements embedded within one or more of its promoters that enable direct and regulated expression of said at least one perforin and / or granzyme gene.

133. The method according to any one of claims 1 to 2 and 119 to 132, further comprising said analyzing, wherein said analyzing is selected from the group consisting of surface markers, cytoplasmic proteins, differences in mRNA expression profiles, differences in DNA expression profiles, and combinations thereof.

134. The method according to any one of claims 1 to 2 and 119 to 133, further comprising measuring the degree of activation of said activated low immunogenic cells.

135. A method for treating a disease or disorder, comprising: administering a therapeutically effective amount of BioNVs comprising one or more biomimetic nanovesicle (BioNV) antigen-binding constructs; and administering a therapeutically effective amount of whole cells comprising one or more whole cell antigen-binding constructs. The method as described above.

136. The method according to claim 135, wherein said disease or disorder is cancer.

137. The method according to claim 136, wherein said cancer is one or more of carcinoma, sarcoma, myeloma, leukemia, lymphoma, mixed cancer, and / or metastatic cancer.

138. The method according to any one of claims 135 to 137, wherein the BioNV is administered first and the whole cells are administered second, or the whole cells are administered first and the BioNV is administered second, or the BioNV and the whole cells are administered simultaneously.

139. The method according to any one of claims 135 to 138, wherein the one or more BioNV antigen-binding constructs comprise a CAR.

140. The method according to any one of claims 135 to 139, wherein the one or more whole cell antigen-binding moieties comprise a CAR.

141. The method according to any one of claims 135 to 140, wherein the one or more whole cell antigen-binding constructs bind to cancer cells.

142. The method according to any one of claims 135 to 141, wherein the one or more BioNV antigen-binding constructs bind to cancer cells.

143. The method according to any one of claims 135 to 142, wherein the one or more whole cell antigen-binding constructs bind to the BioNV.

144. The method according to any one of claims 135 to 143, wherein the one or more BioNV antigen-binding constructs bind to the whole cells.

145. The method according to any one of claims 135 to 144, wherein the whole cells comprise a CAR that binds to an antigen, ligand, or receptor present on both cancer cells and the BioNV.

146. The BioNV is (i) a CAR that binds to an antigen, ligand, or receptor present on cancer cells, and (ii) an antigen, ligand, and / or receptor that binds to the CAR on the whole cells, The method according to any one of claims 135 to 145, comprising.

147. The whole cells are (i) a first CAR that binds to an antigen, ligand, or receptor present on cancer cells, and (ii) a second CAR that binds to an antigen, ligand, and / or receptor present on the BioNV, The method according to any one of claims 135 to 146, comprising.

148. The antigen, ligand, and / or receptor on the BioNV is, comprises, or is similar to the antigen, ligand, or receptor present on the cancer cell; or the antigen, ligand, and / or receptor on the BioNV is different from the antigen, ligand, or receptor present on the cancer cell, the method according to claim 147.

149. The method according to claim 147 or 148, wherein the first CAR is capable of signaling via a pathway that results in cell-mediated cytotoxicity or comprises one or more intracellular signaling domains capable of signaling via a pathway that results in cell-mediated cytotoxicity.

150. The method according to claim 147 or 148, wherein the second CAR is capable of signaling via a pathway that results in cell-mediated cytotoxicity by the whole cell or comprises one or more intracellular signaling domains capable of signaling via a pathway that results in cell-mediated cytotoxicity.

151. The method according to claim 147 or 148, wherein the second CAR is incapable of signaling via a pathway that results in cell-mediated cytotoxicity by the whole cell or lacks one or more intracellular signaling domains capable of signaling via a pathway that results in cell-mediated cytotoxicity.

152. The method according to claim 147 or 148, wherein the second CAR is capable of signaling via a pathway that results in the persistence, survival, and / or proliferation of the whole cell or comprises one or more intracellular signaling domains capable of signaling via a pathway that results in the persistence, survival, and / or proliferation of the whole cell.

153. The method according to any one of claims 135 to 152, wherein the function of the whole cell is improved by administering the BioNV.

154. The method according to claim 153, wherein the function of the whole cell comprises one or more of cell-mediated cytotoxicity, cytokine release, killing of tumor cells or cancer cells, homing to tumor cells or cancer tissues, tissue invasion, proliferation, persistence, and / or survival.

155. The method according to any one of claims 135 to 154, wherein administering the BioNV reduces the toxicity of one or more of the whole cells.

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