Controlled expression of therapy-related biomolecules for localized vessels and exosomes by

Abstract

Disclosed herein are methods of generating therapeutic biomimetic nanovesicles (BioNVs) or therapeutic exosomes with lumen-loaded therapy-related biomolecules from low immunogen cells, compositions of therapeutic BioNVs or therapeutic exosomes, and methods of treating or preventing diseases or conditions using the same.

Description

Technical Field

[0001] The present disclosure provides, in part, methods and compositions for preparing lumen-loaded biomimetic nanovesicles and exosomes, and methods of using the same to treat diseases in, for example, mammalian subjects, such as humans.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of and priority to U.S. Provisional Application No. 63 / 345,659, filed May 25, 2022, and U.S. Provisional Application No. 63 / 424,978, filed November 14, 2022, the contents of which are incorporated herein by reference in their entirety. Background Art

[0004] Cell-derived vesicles (CDVs), including biomimetic nanovesicles (BioNVs) and exosomes, are powerful and novel tools for treating a wide range of diseases, including cancer, genetic disorders, tissue damage, endocrine disorders, and infectious diseases. BioNVs can be obtained from cells through various cell disruption procedures. In contrast, exosomes are naturally shed from whole cells via endocytic pathways. The diameter and volume of BioNVs and exosomes (the latter defined by the luminal compartment within the lipid bilayer) can be predetermined by the method used to disrupt the cells from which they are derived (or to treat the cells to obtain the exosomes).

[0005] The lumen of a BioNV can be loaded with therapeutically relevant molecules by three main approaches: (1) spontaneous inclusion of biomolecules (nucleic acids, proteins / peptides) expressed within the cells from which the CDV is derived into the lumen of the CDV; (2) addition of (bio)molecules (nucleic acids, proteins / peptides, small molecules or biologics) into the segmented concentrating compartments during the BioNV formation process to ensure controlled loading; or (3) a combination of (1) and (2), where the necessary cell-derived biomolecules are packaged in the lumen of the CDV and the desired biomolecules are added for a specific purpose, e.g., doxorubicin is added for delivery to targeted cancer cells.

[0006] Cells express a variety of biomolecules (cytokines, chemokines, regulatory nucleic acids, etc.), which help cell regeneration, tissue repair, and cytotoxicity through complex pathways that are usually controlled by external stimuli. These biomolecules are strictly regulated by cellular pathways, which usually start from surface receptors that sense environmental stimuli and then trigger the intracellular domain of the receptor, resulting in signal transduction events that propagate within the cell, which may involve complex crosstalk between signal pathways. The endpoints of these signal transduction pathways are usually systems of transcriptional activators and inhibitors that regulate gene function and biomolecule expression. Within these complex and orderly pathways, there are various types of conversion effector molecules and biomolecules, such as phosphate-containing compounds (cAMP, ATP, GTP, etc.), metal ions (calcium, iron, zinc, etc.), signal regulatory enzymes (phosphatases, kinases, phosphorylases, etc.), transcription factors and promoters, and translation factors, etc.

[0007] Each cell in an organism possesses a distinct set of surface receptors, each with distinct downstream functions, ultimately resulting in a specialized repertoire of biomolecules. The expression of these biomolecules in healthy cells typically incorporates effector functions designed to protect and maintain homeostasis of the cell or tissue in which it resides, as well as the overall survival of the organism. For example, T cells contain T cell receptors (TCRs) or, if engineered, chimeric antigen receptors. Upon contact with pathogen-infected or transformed cells, these cells are triggered to initiate the rapid expression of inflammatory and apoptotic cytokines, including interferons, perforins, granzymes, and granulysins. These lytic granules form in the cytoplasm and subsequently localize within sight of the interface between the TCR and target cell ligand, known as the immunological synapse (IS). In another example, M1 macrophages are activated via the CD38 surface receptor, enabling the cell to resist bacterial invasion and activate the phagocytosis and digestion of necrotic cells through proinflammatory cytokines. Furthermore, M2 macrophages, activated via the EGR2 surface receptor, put the cell into a repair mode by producing (partially) anti-inflammatory cytokines. In another example, natural killer cells contain multiple surface and transmembrane receptors that are connected to a complex intercellular signaling network, endowing the cells with unique environmental sensing properties, allowing them to discern the difference between healthy cells and infected or transformed cells with high precision for destruction. This complex network is so sensitive that natural killer cells have adapted specific receptors that often coordinate with multiple intercellular signaling pathways 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 essential for natural killer cells because they are primed / preloaded with cytotoxic secretory lysosomes containing perforins, granzymes, granulysins, and other cytotoxins, which can cause severe damage if not strictly controlled. CD4+ and some CD8+ T cell subsets (except for CD8+ killer T cells that are primed with low levels of lytic granules) are not primed with lytic granules and therefore do not require such a strict biochemical regulatory network.

[0008] In addition to immunoregulatory cells, recent reports indicate a strong correlation between cytokine signaling between leukocytes and cardiomyocytes, and that this interaction plays an important role in controlling inflammation following cardiac injury. Elevated levels of the proinflammatory cytokines tumor necrosis factor (TNF)-α, interleukin-6 (IL-6), and IL-1β have been observed in patients with cardiac injury. These cytokines can be triggered under pathological conditions in cardiac tissue, and uncontrolled proinflammatory effects are associated with high mortality in patients with heart failure. Low levels of IL-6 expression in cardiomyocytes can have reparative benefits for cardiac tissue, but high levels can lead to uncontrolled inflammation and heart failure. IL-6 is regulated by IL-1β levels, suggesting that direct regulation of IL-1β can be manipulated to adjust IL-6 levels in a proactive and controlled manner.

[0009] Packaging desired biomolecules of therapeutic value into delivery systems such as AAV, anelloviridae, or lipid nanoparticles (LNPs) requires complex packaging processes. Currently, the concentration and variety of biomolecules that can be packaged in these systems are very limited and depend on the engineered promoter gene construct and packaging size constraints. Furthermore, although naturally shed exosomes can bind a wide range of biomolecules from the cells they shed, due to the typical pathways involved in these types of extracellular vesicles (EVs), exosomes typically have lower, less ideal, and less 'controllable' concentrations of desired biomolecules (compared to BioNVs). Therefore, additional systems are needed to control the loading into the lumen. Furthermore, CDVs derived from quiescent cells in their 'natural somatic state' are limited as therapeutic sources due to the low expression levels (or absence / suppression) of naturally occurring biomolecules with therapeutic properties. Therefore, there remains a need for a method to control and regulate the gene expression of therapeutically desired / relevant biomolecules in a genotypic repertoire of cells so that they can be packaged into the lumen of BioNVs (or exosomes) at concentrations and diversity that make disease treatment feasible. Summary of the Invention

[0010] In embodiments, a method of producing therapeutic biomimetic nanovesicles (BioNVs) comprises (a) obtaining low immunogenic cells, (b) activating the low immunogenic cells to express one or more therapeutically relevant biomolecules, and (c) processing the activated low immunogenic cells to produce therapeutic BioNVs, wherein the therapeutic BioNVs comprise a low immunogenic cell plasma membrane and encapsulate the one or more therapeutically relevant biomolecules.

[0011] In embodiments, a method of producing therapeutic extracellular vesicles (EVs), such as exosomes and microsomes, comprises (a) obtaining low immunogenic cells, (b) activating the low immunogenic cells to express one or more therapeutically relevant biomolecules, and (c) harvesting naturally shed therapeutic exosomes from the activated low immunogenic cells, wherein the therapeutic exosomes comprise a low immunogenic cell plasma membrane and encapsulate one or more therapeutically relevant biomolecules.

[0012] In an embodiment, a method for treating or preventing a disease or condition comprises administering a therapeutically effective amount of the therapeutic biomimetic nanovesicles (BioNV) produced by Technical Solution 1 to a subject in need thereof.

[0013] In an embodiment, a method for treating or preventing a disease or disorder comprises administering a therapeutically effective amount of therapeutic exosomes produced by Technical Solution 2 to a subject in need thereof.

[0014] In embodiments, low immunogenic cells are stem cells, induced pluripotent stem cells (iPSC), the multipotent or multipotent cells of reprogramming, embryonic stem cells, mesenchymal stem cells or the differentiated cells derived from any stem cell thereof. In embodiments, low immunogenic cells are T cells, helper T cells, T memory cells or NK cells. In embodiments, low immunogenic cells are macrophages. In embodiments, low immunogenic cells are monocytes. In embodiments, low immunogenic cells are hepatocytes, cardiomyocytes, neurons, endothelial cells, pancreatic cells or retinal pigment epithelial (RPE) cells.

[0015] In embodiments, low immunogenic 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 embodiments, low immunogenic cells have β2-macroglobulin (B2M) gene disruption and / or reduce or eliminate MHC class I protein expression and / or active destruction. In embodiments, low immunogenic cells have CIITA gene disruption and / or reduce or eliminate MHC class II protein expression and / or active destruction.

[0016] In embodiments, the low immunogenic cells have an HLA-A gene disruption and / or a disruption that reduces or eliminates HLA-A protein expression and / or activity. In embodiments, the low immunogenic cells have an HLA-B gene disruption and / or a disruption that reduces or eliminates HLA-B protein expression and / or activity. In embodiments, the low immunogenic cells have an HLA-C gene disruption and / or a disruption that reduces or eliminates HLA-C protein expression and / or activity. In embodiments, the low immunogenic cells have an HLA-E gene disruption or an HLA-G gene disruption and / or a disruption that reduces or eliminates HLA-E or HLA-G protein expression and / or activity. In embodiments, the low immunogenic cells have an HLA-F gene disruption and / or a disruption that reduces or eliminates HLA-F protein expression and / or activity.

[0017] In embodiments, the low immunogenic cells have a T cell alpha constant (TRAC) gene disruption and / or a disruption that reduces or eliminates TRAC protein expression and / or activity. In embodiments, the low immunogenic cells have a T cell beta constant (TRBC) gene disruption and / or a disruption that reduces or eliminates TRBC protein expression and / or activity.

[0018] In embodiments, low immunogenic cells have reduced or eliminated PD-1 gene expression and / or reduced or eliminated PD-1 protein expression and / or activity, wherein low immunogenic cells are activated; or low immunogenic cells have expression of PD-1 genes and / or gene products or increase in expression thereof, wherein low immunogenic cells are not activated. In embodiments, low immunogenic cells have IL-4 gene disruption and / or disruption that reduces or eliminates IL-4 protein expression and / or activity. In embodiments, low immunogenic cells have IL-6 gene disruption and / or disruption that reduces or eliminates IL-6 protein expression and / or activity. In embodiments, low immunogenic cells have IL-10 gene disruption and / or disruption that reduces or eliminates IL-10 protein expression and / or activity. In embodiments, low immunogenic cells have IL-16 gene disruption and / or disruption that reduces or eliminates IL-16 protein expression and / or activity.

[0019] In embodiments, the low immunogenic cells have a SerpinB9 gene disruption and / or a disruption that reduces or eliminates SerpinB9 protein expression and / or activity. In embodiments, the low immunogenic cells express the SerpinB9 gene and / or gene product, or have increased expression and / or activity.

[0020] In embodiments, the low immunogenic cells express CCL2 genes and / or gene products, or their expression and / or activity is increased. In embodiments, the low immunogenic cells express PD-L1 genes and / or gene products, or their expression and / or activity is increased, wherein the low immunogenic cells are not activated. In embodiments, the low immunogenic cells express H2-M3 genes and / or gene products, or their expression and / or activity is increased.

[0021] In one embodiment, the low immunogenic cells express the CD47 gene and / or gene product, or the expression and / or activity thereof is increased. In one embodiment, the low immunogenic cells express the CD24 gene and / or gene product, or the expression and / or activity thereof is increased. In one embodiment, the low immunogenic cells express the chimeric CD24 / CD47 gene and / or gene product, or the expression and / or activity thereof is increased.

[0022] In embodiments, low immunogenic cells express CTLA-4 genes and / or gene products, or their expression and / or activity increase. In embodiments, low immunogenic cells express CD200 genes and / or gene products, or their expression and / or activity increase. In embodiments, low immunogenic cells express chimeric CD24 / CD200 genes and / or gene products or chimeric CD47 / CD200 genes and / or gene products, or their expression and / or activity increase. In embodiments, low immunogenic cells express MFG-E8 genes and / or gene products, or their expression and / or activity increase. In embodiments, low immunogenic cells express NCAM genes and / or gene products, or their expression and / or activity increase. In embodiments, low immunogenic cells express α-phagocytic integrin genes and / or gene products, or their expression and / or activity increase. In embodiments, low immunogenic cells express antibodies or antibody-like molecules targeting IL-6 surface receptors (anti-IL-6R), or their expression and / or activity increase.

[0023] In embodiments, the low immunogenic cells express the FasL gene and / or gene product. In embodiments, the low immunogenic cells do not express the FasL gene and / or gene product.

[0024] In embodiments, the low immunogenic cells substantially lack expression and / or activity of one or more immunogenic proteins and express, or have increased expression of, one or more immunoprotective proteins.

[0025] In embodiments, 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 of the low immunogenic cells are reduced or eliminated. In embodiments, the low immunogenic cells express 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 their expression is increased.

[0026] In embodiments, the expression and / or activity of genes and / or gene products of HLA-A, HLA-B, HLA-C, HLA-F, CIITA, IL-6, TRAC, TRBC, and one of HLA-E or HLA-G are reduced or eliminated in the low immunogenic cells.

[0027] In an embodiment, the expression and / or activity of genes and / or gene products of HLA-A, HLA-B, HLA-C, HLA-F, CIITA, IL-6, TRAC, TRBC, SerpinB9, and one of HLA-E or HLA-G are reduced or eliminated in the low immunogenic cells.

[0028] In an embodiment, the expression and / or activity of genes and / or gene products of HLA-A, HLA-B, HLA-C, HLA-F, CIITA, IL-6, TRAC, TRBC, SerpinB9, and one of HLA-E or HLA-G are reduced or eliminated in the low immunogenic cells.

[0029] In an embodiment, the low immunogenic cells include low immunogenic cells in which the expression and / or activity of genes and / or gene products of 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 of IL-4, IL-10 and IL-16 are reduced or eliminated.

[0030] In embodiments, the low immunogenic cells express, or have increased expression and / or activity of, alpha-phagocytic integrin, CCL2, H2-M3, FasL, MFEG8, and PD-L1 and / or CTLA-4, wherein the low immunogenic cells do not overexpress FasL, and wherein the low immunogenic cells are not activated by expression of PD-L1, and express, or have increased expression and / or activity of, CD24, CD47, CD200, chimeric CD24 / CD47, chimeric CD24 / CD200, and chimeric CD47 / CD200, or both CD24, CD47, and CD200.

[0031] In embodiments, the low immunogenic cells express, or have increased expression and / or activity of, alpha-phagocytic integrin, CCL2, H2-M3, FasL, MFEG8, SerpinB9, and PD-L1 and / or CTLA-4, wherein the low immunogenic cells do not overexpress FasL, and wherein the low immunogenic cells are not activated by expression of PD-L1, and express one of CD24, CD47, CD200, chimeric CD24 / CD47, chimeric CD24 / CD200, and chimeric CD47 / CD200, or both of CD24, CD47, and CD200, or have increased expression and / or activity.

[0032] In an embodiment, the low immunogenic cells express the CD200 gene and / or gene product, or have increased expression and / or activity, and do not express and / or substantially lack the CD24 or CD47 genes and / or gene products. In an embodiment, the low immunogenic 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.

[0033] In embodiments, the low immunogenic cells are allogeneic. In embodiments, the low immunogenic cells do not elicit an immune response in a patient to whom they or a BioNV derived therefrom is administered.

[0034] In embodiments, low immunogenic cells include one or more targeting agents. In embodiments, one or more targeting agents include chimeric antigen receptors (CAR). In embodiments, CAR is bispecific. In embodiments, CAR lacks an intracellular portion. In embodiments, CAR includes a targeting agent, a transmembrane domain and an intracellular domain, which include a costimulatory domain and / or a signaling domain. In embodiments, the transmembrane domain is derived from CD28, CD3ζ, CD4, CD8α or ICOS, or a fragment thereof. In embodiments, the intracellular domain includes an intracellular signaling domain and / or one or more costimulatory molecules of the CD3ζ chain, optionally selected from CD28, 4-1BB, ICOS, CD27 and OX40. In embodiments, one or more targeting agents include antibodies or antibody forms. In embodiments, antibodies or antibody forms are selected from one or more of the following: monoclonal antibodies, polyclonal antibodies, antibody fragments, Fab, Fab', Fab'-SH, F(ab')2, Fv, single-chain Fv (scFv), V NAR 、V H In some embodiments, the present invention relates to a fusion protein comprising an antigen-binding portion of an antibody. In some embodiments, the antibody is a scFv. In some embodiments, the one or more targeting agents include a viral epitope recognition receptor (VERR) or a viral ligand. In some embodiments, the one or more targeting agents include a ligand for a receptor. In some embodiments, the one or more targeting agents include a receptor for a ligand.

[0035] In embodiments, the low immunogenic cells are T cells; however, those skilled in the art, having the benefit of the entire disclosure, will appreciate that the low immunogenic cells can be any cell type and can be activated by any cell receptor present on the cell. In embodiments, activating the low immunogenic cells comprises activating the TCR / CD3 receptor complex by a protein antigen. In embodiments, activating the low immunogenic cells comprises activating the TCR / CD3 receptor complex by a small molecule. In embodiments, the low immunogenic cells comprises activating the TCR / CD3 receptor complex by a viral antigen.

[0036] In embodiments, activating low immunogenic cells comprises activating calcium-dependent channels. In embodiments, activating low immunogenic cells comprises activating LFA-1 integrin receptors. In embodiments, activating low immunogenic cells comprises activating CD28 receptors. In embodiments, activating low immunogenic cells comprises activating IL-2 receptors.

[0037] In embodiments, activating low immunogenic cells comprises activating a SLAMF1 (CD150) receptor. In embodiments, activation of the SLAMF1 (CD150) receptor is by measles virus. In embodiments, activation of the SLAMF1 (CD150) receptor is by gram-negative bacteria.

[0038] In embodiments, activating the poorly immunogenic cells comprises activating an IFNy receptor. In embodiments, activating the poorly immunogenic cells comprises activating a CD4 receptor by one or more viruses.

[0039] In embodiments, activation of low immunogenic cells, such as natural killer cells (or T cells, if the following receptors are associated with T cell activation) includes activating receptors (alone or in combination to allow for a coordinated activation reaction), such as CD2, CD3, CD16, CD28, CD314 (NKG2D), CD335, B7-H6, CD158d, IL-2R, IL-12R, DNAM-1, CD2, CD44, CD137, CX3CR1, CD27, CD160, 2B4, but not limited to these sensory receptors. CD16 can be activated by binding to the Fc region of an antibody (anti-S2 IgG). CD134, CD335, B7-H6 can be activated in combination with ICAM-1, which is combined with the Fc region of an antibody. CD314 can be activated by ULBP1, MICA, MICB, or H60. CD158d can be activated by HLA-G to trigger proinflammatory molecules. 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, CD112, or NKp30. CD2 can be activated by LFA3. CD44 can be activated by hyaluronic acid, hyaluronan, osteopontin, collagen, or matrix metalloproteinases. CD28 homologs can be activated by the B7H7 protein ligand. CD137 can be activated by itself and by members of the tumor necrosis factor receptor protein family. 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 type of activation can produce different levels of cytotoxic biomolecules, each of which may have therapeutic value when packaged into BioNV. Activation of each (or any) of the above combinations can be achieved by adding each of the following ligands (or any other relevant ligands) separately, wherein the ligand is in soluble form (partial peptide, domain or whole protein), anchored to the membrane (partial peptide, domain or whole protein), is a component of the target cell membrane (partial peptide, domain or whole protein) or cross-linked to a solid phase, such as but not limited to cross-linked dextran or magnetic beads (as partial peptide, domain or whole protein).

[0040] In embodiments, activation of some natural killer receptors (or T cells, if the following receptors are involved in T cell activation) does not result in degranulation or granule polarization to IS. These receptors include, but are not limited to: i) 2B4 alone, ii) CD134 alone, iii) CD134 in combination with 2B4 and one of the inhibitory receptors KIR or CD94, iv) LFA-1 in combination with one of the inhibitory receptors KIR or CD94, v) CD16 in combination with one of the inhibitory receptors KIR or CD94. Activation of each (or any) of the above combinations can be achieved by adding each of the following ligands (or any other relevant ligands) separately, wherein the ligand is in soluble form (partial peptide, domain or whole protein), anchored to the membrane (partial peptide, domain or whole protein), a component of the target cell membrane (partial peptide, domain or whole protein) or cross-linked to a solid phase, such as but not limited to cross-linked dextran or magnetic beads (as partial peptide, domain or whole protein): i) CD48, ii) ULBP1, iii) HLA-E, iv) a combination of HLA-C and HLA-E, or v) HLA-E.

[0041] In embodiments, activation of certain natural killer receptors (or T cells, if the following receptors are associated with T cell activation) leads to degranulation. Immediately prior to degranulation, perforin and granzymes may or may not undergo post-translational modifications to enhance their activity, such as calcium binding to perforin, which activates the protein. Activating cells along the degranulation pathway may be beneficial for achieving maximum efficacy of cytotoxic proteins. These receptors include, but are not limited to: i) CD16 in combination with LFA-1 and one of the inhibitory receptors KIR or CD94, ii) CD134 in combination with 2B4, or iii) CD16 alone. Activation of each (or any) of the above combinations can be achieved by adding each of the following ligands (or any other relevant ligands) separately, wherein the ligand is in soluble form (partial peptide, domain or whole protein), anchored to the membrane (partial peptide, domain or whole protein), a target cell membrane component (partial peptide, domain or whole protein) or cross-linked to a solid phase, such as but not limited to cross-linked dextran or magnetic beads (as partial peptide, domain or whole protein): i) a combination of HLA-C and HLA-E, ii) a combination of ULBP1 and CD48, or iii) anti-S2 IgG.

[0042] In embodiments, receptors that cause polarization of lytic granules when activated may not (or may be) advantageous because their localization to the cell membrane may minimize their packaging in BioNV, but may maximize their packaging in secreted EVs (exosomes and microsomes) due to differences in extrusion manufacturing (BioNV) versus cellular exocytosis processing (EV). Some receptors that induce polarization of lytic granules to IS include, but are not limited to, i) CD16 in combination with LFA-1 (also leading to degranulation), ii) LFA-1 alone, or iii) CD134 in combination with LFA-1 and 2B4 (also leading to degranulation). Activation of each (or any) of the above combinations can be achieved by adding each of the following ligands (or any other relevant ligands) separately, wherein the ligand is in soluble form (partial peptide, domain or whole protein), anchored to the membrane (partial peptide, domain or whole protein), a component of the target cell membrane (partial peptide, domain or whole protein) or cross-linked to a solid phase, such as but not limited to cross-linked dextran or magnetic beads (as partial peptide, domain or whole protein): i) anti-S2 IgG in combination with ICAM-1, ii) ICAM-1, or iii) ULBP1 in combination with CD48 and ICAM-1.

[0043] In embodiments, activating the low immunogenicity cells comprises activating by an engineered non-natural biomolecule selected from one or more of the following: a soluble peptide, a chimeric antigen receptor, a small molecule decoy, a small molecule ligand, a designed nucleic acid ligand, a carbohydrate ligand, a viral ligand, a chimeric biomolecule ligand, a fusion protein, an antibody, and an antibody-formulated molecule. In embodiments, activating the low immunogenicity cells comprises activating by an inorganic compound. In embodiments, activating the low immunogenicity cells comprises activating by expression, overexpression, or increased activity of a transcription factor.

[0044] In embodiments, activating low immunogenic cells comprises activating at the DNA level by one or more of the following: a transposase-based approach, a Cre / Lox-based approach, an endonuclease-based approach, a homologous recombination (HR)-based approach, a non-homologous end joining (NEHJ)-based approach, a microhomology-mediated end joining (MMEJ)-based approach, a homology-mediated end joining (HMEJ)-based approach, a small RNA, or a combination thereof. In embodiments, the small RNA comprises one or more of the following: 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.

[0045] In embodiments, activation of the low immunogenic 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. In embodiments, activation comprises one or more RNA-guided endonucleases.

[0046] In embodiments, activating low immunogenic cells comprises activating by endogenous promoter regions and / or enhancer regions. In embodiments, activating low immunogenic cells comprises activating by stable integration of genetic elements. In embodiments, activating low immunogenic cells comprises activating by transient expression of genetic elements.

[0047] In embodiments, activation of the poorly immunogenic cells results in a metabolically altered state of the poorly immunogenic cells.

[0048] In embodiments, one or more therapeutically relevant biomolecules are chemokines, interferons, interleukins, alarmins, lymphokines, perforins, granzymes, granulysins, 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 form, or a combination thereof. In embodiments, cytokines are proinflammatory cytokines. In embodiments, cytokines are anti-inflammatory cytokines. In embodiments, granzymes are granzymes A, B, H, K or M.

[0049] In embodiments, the gene editing payload comprises one or more gene editing agent nucleic acids and / or proteins, or one or more nucleic acids encoding one or more gene editing agents. In embodiments, the one or more gene editing agents are site-directed endonucleases, TALENs, ZFNs, ribonuclease P RNAs, CRISPR / Cas nucleases, C2c1, C2c2, C2c3, Cas9, Cpf1, TevCas9, Archaea Cas9, CasY.1, CasY.2, CasY.3, CasY.4, CasY.5, CasY.6, CasX, Cas omega, transposases, and / or any orthologs thereof. In embodiments, the gene editing payload comprises a transactivation response region (TAR) loop system.

[0050] In embodiments, the therapeutic BioNV comprises (i) one or more membrane-embedded targeting agents that target one or more cellular biomarkers, (ii) one or more α-phagocytic integrin, CCL2, H2-M3, FasL, MFEG8, PD-L1 and / or CTLA-4, SerpinB9 and anti-IL-6R antibodies or membrane-embedded proteins in the form of antibodies, (iii) one of CD24, CD47, CD200, chimeric CD24 / CD47, chimeric CD24 / CD200 and chimeric CD47 / CD200 or a membrane-embedded protein of CD24, CD47 and CD200, and (iv) a cell-free cell line substantially lacking HLA-A, HLA-B, HLA-C, or HLA-D. -C, HLA-F, CIITA, IL-6, TRAC, TRBC, one of 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, perforins, granzymes, granulysins, 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.

[0051] In embodiments, the therapeutic BioNV comprises (i) one or more membrane-embedded targeting agents that target one or more cellular biomarkers, (ii) one or more of α-phagocytic integrin, CCL2, H2-M3, FasL, MFEG8, PD-L1 and / or CTLA-4, SerpinB9 and anti-IL-6R antibodies or membrane-embedded proteins in the form of antibodies, (iii) membrane-embedded proteins of CD24 and CD47 or chimeric CD24 / CD47, and (iv) substantially lacking 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, wherein the one or more therapeutically relevant biomolecules are chemokines, interferons, interleukins, alarmins, lymphokines, perforins, granzymes, granulysins, tumor necrosis factor (TNF), colony stimulating factors, bone morphogenetic proteins (BMPs), 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.

[0052] In embodiments, one or more targeting agents are antibodies or antibody forms. In embodiments, one or more targeting agents are CARs. In embodiments, processing activated low immunogenic cells is performed by one or more of the following: sonication, adaptive focused acoustic technology, French press, extrusion, continuous extrusion, enzymatic rupture, detergent cell lysis and / or electroporation. In embodiments, processing activated low immunogenic cells is performed by continuous extrusion.

[0053] In embodiments, the therapeutic BioNV has a size of about 10 nm to about 1200 nm. In embodiments, the therapeutic BioNV has a size of about 10 nm to about 100 nm. In embodiments, the therapeutic BioNV has a size of about 100 nm to about 200 nm. In embodiments, the therapeutic BioNV has a size of about 200 nm to about 500 nm. In embodiments, the therapeutic BioNV has a size of about 500 nm to about 1200 nm.

[0054] In embodiments, the therapeutic exosomes include (i) one or more membrane-embedded targeting agents that target one or more cellular biomarkers, (ii) one or more α-phagocytic integrin, CCL2, H2-M3, FasL, MFEG8, PD-L1 and / or CTLA-4, SerpinB9 and anti-IL-6R antibodies or membrane-embedded proteins in the form of antibodies, (iii) one of CD24, CD47, CD200, chimeric CD24 / CD47, chimeric CD24 / CD200 and chimeric CD47 / CD200 or a membrane-embedded protein of CD24, CD47 and CD200, and (iv) a membrane-embedded protein that is substantially devoid of HLA-A, HLA-B, Membranes of proteins comprising one or more of HLA-C, HLA-F, CIITA, IL-6, TRAC, TRBC, one of 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, perforins, tumor necrosis factor (TNF), colony stimulating factors, bone morphogenetic proteins (BMPs), 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 embodiments, the therapeutic exosomes include (i) one or more membrane-embedded targeting agents that target one or more cellular biomarkers, (ii) one or more α-phagocytic integrins, CCL2, H2-M3, FasL, MFEG8, PD-L1 and / or CTLA-4, SerpinB9, and anti-IL-6R antibodies or membrane-embedded proteins in the form of antibodies, (iii) membrane-embedded proteins of CD24 and CD47 or chimeric CD24 / CD47, and (iv) substantially lack one or more of HLA-A, HLA-B, HLA-C, HLA-F, CIITA, IL-6, TRAC, TRBC, A membrane comprising a protein comprising one of HLA-E or HLA-G, SerpinB9 and CD200, and one or more of IL-4, IL-10, and IL-16, wherein the one or more therapeutically relevant biomolecules is a chemokine, an interferon, an interleukin, an alarmin, a lymphokine, a perforin, a granzyme, a granulysin, a tumor necrosis factor (TNF), a colony stimulating factor, a bone morphogenetic protein (BMP), erythropoietin (EPO), granulocyte-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), a gene editing payload, a fusion protein, an antibody or an antibody format, or a combination thereof.

[0056] In embodiments, one or more targeting agents are antibodies or antibody forms. In embodiments, one or more targeting agents are CARs. In embodiments, harvesting therapeutic exosomes from activated low immunogenic cells includes inducing hypoxia. In embodiments, harvesting therapeutic exosomes from activated low immunogenic cells includes expressing one or more cytokines or increasing their expression. In embodiments, harvesting therapeutic exosomes from activated low immunogenic cells includes providing one or more small molecule exosome regulators. In embodiments, harvesting therapeutic exosomes from activated low immunogenic cells includes supplementing the culture medium with one or more exosomal factors. In embodiments, the size of the therapeutic exosomes is about 10 nm to about 200 nm. In embodiments, the size of the therapeutic exosomes is about 10 nm to about 100 nm. In embodiments, the size of the therapeutic exosomes is about 100 nm to about 200 nm.

[0057] In embodiments, the mammalian disease is cancer, an infectious disease, a genetic disorder, or an orphan disease.

[0058] In certain aspects, methods are described herein for treating and / or preventing a disease or condition using co-administration of BioNV and whole cells. In embodiments, the methods comprise 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.

[0059] In embodiments, the disease or disorder is cancer. In embodiments, the cancer is one or more of carcinoma, sarcoma, myeloma, leukemia, lymphoma, mixed cancer, and / or metastatic cancer.

[0060] In embodiments, BioNV is administered first and then whole cells, whole cells are administered first and then BioNV, or BioNV and whole cells are administered simultaneously.

[0061] In embodiments, one or more BioNV antigen-binding constructs comprise a CAR. In embodiments, one or more whole cell antigen-binding constructs comprise a CAR. In embodiments, one or more whole cell antigen-binding constructs bind to a cancer cell. In embodiments, one or more BioNV antigen-binding constructs bind to a cancer cell. In embodiments, one or more whole cell antigen-binding constructs bind to BioNV. In embodiments, one or more BioNV antigen-binding constructs bind to a whole cell. In embodiments, a whole cell comprises a CAR that binds to an antigen, ligand, or receptor present on both a cancer cell and a BioNV.

[0062] In embodiments, BioNV comprises a CAR that binds to an antigen, ligand, or receptor present on a cancer cell, and an antigen, ligand, and / or receptor that binds to a CAR on a whole cell. In embodiments, the whole cell comprises a first CAR that binds to an antigen, ligand, or receptor present on a cancer cell, and a second CAR that binds to an antigen, ligand, and / or receptor present on the BioNV.

[0063] In embodiments, the antigen, ligand and / or receptor on 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 BioNV is different from an antigen, ligand or receptor present on a cancer cell. In embodiments, the first CAR is capable of signaling via a pathway that leads to cell-mediated cytotoxicity, or comprises one or more intracellular signaling domains that can signal via a pathway that leads to cell-mediated cytotoxicity. In embodiments, the second CAR is capable of signaling via a pathway that leads to cell-mediated cytotoxicity of the whole cell, or comprises one or more intracellular signaling domains that can signal via a pathway that leads to cell-mediated cytotoxicity. In embodiments, the second CAR is not capable of signaling via a pathway that leads to cell-mediated cytotoxicity of the whole cell, or lacks one or more intracellular signaling domains that can signal via a pathway that leads to cell-mediated cytotoxicity. In embodiments, the second CAR is capable of signaling by a pathway that leads to persistence, survival and / or proliferation of the whole cell, or comprises one or more intracellular signaling domains that can signal by a pathway that leads to persistence, survival and / or proliferation of the whole cell.

[0064] In embodiments, administration of BioNV improves the function of whole cells. In embodiments, the function of whole cells includes one or more of the following: cell-mediated cytotoxicity, cytokine release, tumor cell or cancer cell killing, integration into tumor cell or cancer tissue, tissue infiltration, proliferation, persistence, and / or survival. In embodiments, administration of BioNV reduces one or more toxicities of whole cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0065] Figure 1 A non-limiting schematic diagram depicts T cell activation through interactions between T cell receptors (TCRs) and MHC class I synapses. Target peptides displayed by MHC class I complexes on virus-infected cells, cancer cells, etc. are recognized by the hypervariable regions of TCR α / β on lymphocytes, leading to conformational changes in the intracellular domains, thereby triggering signaling and the production of cytotoxic cytokines.

[0066] Figure 2 A non-limiting schematic depicts activated immune cells containing anti-cytokines engineered into biomimetic nanovesicles (BioNVs). Anti-cytokines (and various cytotoxic peptides) can be loaded into BioNVs for robust delivery.

[0067] Figure 3A non-limiting diagram of the process of activating cells with biomarker antigens through their CAR / TCR receptors is depicted. The biomarker antigen can be bound to magnetic beads or streptavidin beads (or other) and then added to a cell suspension to cause activation. After activation, the antigen can be separated from the cell suspension, leaving the activated cells with unbound CAR / TCR complexes.

[0068] Figures 4A-4D Depicted is a non-limiting schematic representation of a BioNV whole cell therapy co-administration strategy for treating cancer. Figure 4A A BioNV+whole-cell cancer cell targeting strategy is shown, in which BioNV and whole cells (e.g., CAR-T cells) target tumor cells through different antigen recognition constructs (e.g., CAR 1-CAR 1R and CAR 2-CAR 2R), and whole cells recognize tumor cells and BioNV through the same construct to improve cancer cell targeting. Figure 4B A BioNV+ whole cell cancer cell targeting strategy is shown, in which BioNV and whole cells (e.g., CAR-T cells) target tumor cells through different antigen recognition constructs (e.g., CAR 1-CAR 1R and CAR 2-CAR 2R), and whole cells recognize tumor cells and BioNV through the same antigen binding domain but different intracellular signaling domains (e.g., CAR 2 A ) to improve cancer cell targeting and whole-cell activation / stimulation through potentially redundant, overlapping, or distinct pathways. Figure 4C A BioNV+ whole cell cancer cell targeting strategy is shown, in which BioNV and whole cells (e.g., CAR-T cells) target tumor cells through different antigen recognition constructs (e.g., CAR 1-CAR 1R and CAR2-CAR 2R), and whole cells recognize tumor cells and BioNV through different antigen binding domains, and the intracellular signaling domain is separated between CAR 2 and CAR 2R. A different between them) to improve cancer cell targeting, whole-cell activation / stimulation, and reduce off-target binding through potentially redundant, overlapping, or distinct pathways. Figure 4D Shows the Figure 4C Similar strategies, where T cell receptors (TCRs) can replace (or supplement) CARs to target whole cells (e.g., TCR 2) to tumor cells, target whole cells to BioNV (e.g., via TCR 2) A ), and targets BioNV to tumor cells (e.g., TCR 1) to the cognate TCR ligand (e.g., TCR 1L) on the tumor cells.

[0069] Figure 5 A non-limiting schematic diagram of continuous extrusion to produce BioNV is depicted. DETAILED DESCRIPTION

[0070] The present disclosure relates in part to methods for producing therapeutic biomimetic nanovesicles (BioNVs) or extracellular vesicles (EVs), such as naturally shed therapeutic exosomes or microsomes, that can be used to treat diseases. It should be understood that although exosomes are mentioned below, any other EVs can be used. Therapeutic BioNVs and / or therapeutic exosomes are derived from low immunogenic cells that have been modified to 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 express one or more immunoprotective molecules (e.g., CD47, CD24, CD200, α-phagocytic integrin, etc.) and / or increase their expression. Low immunogenic cells are genetically regulated and activated to control the expression of therapeutically relevant biomolecules, which can be lumenally loaded or encapsulated in BioNVs and exosomes. Therapeutic BioNVs and therapeutic exosomes can be derived from any low immunogenic cell type, including stem cell subsets or cells differentiated therefrom.

[0071] This disclosure relates in part to a composition of BioNV and / or exosomes and a surface-directed targeting agent (e.g., CAR) that recognizes one or more cell biomarkers. This produces therapeutic BioNV / exosomes with cell-targeted (or disease-specific) properties of about 20-1200 nm in size, which is much smaller than traditional cell-based CAR-T / NK cell therapies. BioNV / exosomes can be derived from cells, such as stem cells, iPSCs, reprogrammed multipotent or pluripotent cells, embryonic stem cells, mesenchymal stem cells, or differentiated cells from any stem cells, as well as other cell types. Plasma membrane-derived BioNV, or naturally shed exosomes, retain the low immunogenicity of low-immunogenic cells. These properties can be obtained from low immunogenic cells by genetic engineering that focuses on knocking out 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 expressing immunoprotective cell surface markers (e.g., CD47, CD34, CD24, CD200, α-phagocytes, etc.) or increasing their expression. Targeting agents may include all variants of antibody constructs, including, for example, Fab, Fab′, Fab′-SH, F(ab′)2, scFv, 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, V H H nanobody, V NARSAs well as other antibody formats, allowing BioNV / exosomes to be targeted to any cell of interest (targeting any type of cell surface biomarker). BioNV / exosomes can contain targeting agents that are viral epitope recognition receptors (VERRs) or viral ligands, ligands of receptors, or receptors of ligands. BioNV / exosomes can also encapsulate any selected small molecules, biologics, nucleic acids, gene editing therapeutic payloads, etc., and deliver them to the intended cellular target for treating cancer.

[0072] Furthermore, the present disclosure relates in part to methods of treating or preventing a disease or disorder by administering a therapeutically effective amount of therapeutic BioNVs or therapeutic exosomes to a subject in need thereof.

[0073] Methods for producing biomimetic nanovesicles (BioNV) and / or exosomes

[0074] In certain aspects, the present disclosure includes methods of producing therapeutic biomimetic nanovesicles (BioNVs), the methods comprising (a) obtaining low immunogenic cells, (b) activating the low immunogenic cells to express one or more therapeutically relevant biomolecules, and (c) processing the activated low immunogenic cells to produce therapeutic BioNVs, wherein the therapeutic BioNVs comprise a low immunogenic cell plasma membrane and encapsulate the one or more therapeutically relevant biomolecules.

[0075] In certain aspects, the present disclosure includes a method of producing therapeutic exosomes, the method comprising (a) obtaining low immunogenic cells, (b) activating the low immunogenic cells to express one or more therapeutically relevant biomolecules, and (c) harvesting naturally shed therapeutic exosomes from the activated low immunogenic cells, wherein the therapeutic exosomes comprise a low immunogenic cell plasma membrane and encapsulate the one or more therapeutically relevant biomolecules.

[0076] In embodiments, therapeutic BioNV and / or exosomes are derived from low immunogenic cells, wherein the low immunogenic cells can be stem cells (e.g., iPSC, mesenchymal cells, etc.), induced pluripotent stem cells (iPSC), reprogrammed pluripotent or multipotent cells, embryonic stem cells, mesenchymal stem cells, or differentiated cells derived from any modified cells thereof. In embodiments, the low immunogenic cells from which BioNV and / or exosomes are derived are T cells, helper T cells, T memory cells, or NK cells. In embodiments, the low immunogenic cells are macrophages. In embodiments, the cells are monocytes. In embodiments, the low immunogenic cells are hepatocytes, cardiomyocytes, neurons, endothelial cells, pancreatic cells, or retinal pigment epithelial (RPE) cells. In embodiments, the poorly immunogenic cells can be any terminally differentiated cells, such as, but not limited to, muscle cells (satellite cells), adipocytes, bone cells, cardiomyocytes, hepatocytes, blood cells (including erythrocytes, thrombocytes, and all immune cell types), glial cells (and other neuronal cell types), epithelial cells, epidermal cells, mesenchymal cells (e.g., respiratory mesenchymal cells), fibroblasts (e.g., dermal fibroblasts), endothelial cells (e.g., bronchial endothelial cells), oral cells, stromal cells, or germ cells. In an embodiment, the low immunogenic cells can be any function-specific cell type, such as, but not limited to, exocrine secretory epithelial cells, hormone-secreting cells (e.g., enteroendocrine cells, thyroid cells, pancreatic islet cells, etc.), sensory transduction cells, autonomic nerve cells, sensory organ cells (e.g., columnar cells, olfactory cells, Schwann cells, satellite glial cells, etc.), barrier cells (e.g., lung cells, ductal 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.).

[0077] In embodiments, the modified cells are iPSCs. In embodiments, the iPSCs are differentiated into specific cells. In embodiments, the modified cells are differentiated to express or not express multiple cell surface markers. In embodiments, the method comprises a BioNV derived from a low immunogenic iPSC.

[0078] In the embodiment, therapeutic BioNV / exosomes are derived from iPSCs that have been engineered to have low immunogenicity. In the embodiment, microRNA technology is used instead of small molecule transactivators to restore iPSCs from the somatic state. The use of microRNAs can provide a tighter differentiation system, thereby producing higher quality iPSCs. Without wishing to be bound by theory, these high-quality iPSCs are less prone to expression suppression (post-engineering proteins, such as CD47) and genetic drift, and have a higher quality / quantity of culture divisions (cultures can be divided more times than other methods before cell integrity issues arise).

[0079] In embodiments, the low immunogenic cells from which BioNVs and / or exosomes are derived retain the functions of low immunogenic cells, such as, but not limited to, the ability to cross the blood-brain barrier, such as in the case of macrophages / monocytes, or tissue-specific factors, such as in the case of cardiomyocytes, hepatocytes, etc.

[0080] In embodiments, the allogeneic and low immunogenic characteristics of activated cells (for example, derived from iPSC) are produced by knocking out, silencing, inactivating, blocking or otherwise denying the transcription efficiency of immunogenic molecules. In embodiments, low immunogenic cells substantially lack one or more MHC I class proteins, MHC II class proteins, T cell receptor (TCR) protein and / or cytokine release syndrome (CRS) protein. The inactivation of immunogenic molecules can include, for example, destroying MHC I class and beta-2-microglobulin (B2M) gene, such as the situation of CD8+T cell lineage, and destroying MHC II class and MHC II transcription activator (CIITA) gene, such as the situation of CD4+T cell lineage. Not wishing to be bound by theory, these proteins contribute to human leukocyte antigen (HLA) immunogenicity, and it needs the HLA allele matching in donor-recipient to treat by cell-based therapy. In embodiments, allogeneic and low immunogenic properties are achieved by disrupting genes encoding T cell receptor (TCR) proteins, including, for example, the α and β chains (as in the case of αβ T cells) or the γ and δ chains (as in the case of γδ T cells) that form the ligand binding site, and the signaling modules CD3δ, CD3γ, CD3ε, and CD3ζ. This can be done to reduce foreign T cell receptor types other than those of the CAR cassette, and further improve the homogeneity of the CAR of interest and reduce off-target effects in BioNV / exosome formation.

[0081] In embodiments, low immunogenic cells include knockout or destruction of expression of immunogenic cell surface proteins and / or intracellular or secretory proteins. In embodiments, low immunogenic cells have β2-macroglobulin (B2M) gene destruction and / or reduction or elimination of MHC class I protein expression and / or activity destruction. In embodiments, knockout B2M gene reduction needs to administer potential dosage quantity, because there is the risk of preventing the recipient from receiving BioNV for a long time, such as the situation observed in the above-mentioned whole cell-based method. To overcome this problem, in embodiments, HLA-E or HLA-G genes remain intact, so that the immune system can adapt to the BioNV produced. In embodiments, HLA-A, HLA-B, HLA-C, HLA-F and HLA-E or HLA-G (but not both HLA-E and HLA-G) are sequentially knocked out.

[0082] In embodiments, the low immunogenic cells have an HLA-A gene disruption and / or a disruption that reduces or eliminates HLA-A protein expression and / or activity. In embodiments, the low immunogenic cells have an HLA-B gene disruption and / or a disruption that reduces or eliminates HLA-B protein expression and / or activity. In embodiments, the low immunogenic cells have an HLA-C gene disruption and / or a disruption that reduces or eliminates HLA-C protein expression and / or activity. In embodiments, the low immunogenic cells have an HLA-E gene disruption or an HLA-G gene disruption and / or a disruption that reduces or eliminates HLA-E or HLA-G protein expression and / or activity. In embodiments, the low immunogenic cells have an HLA-F gene disruption and / or a disruption that reduces or eliminates HLA-F protein expression and / or activity.

[0083] In embodiments, low immunogenic cells have CIITA gene destruction and / or reduction or elimination of MHC II class protein expression and / or activity destruction. In embodiments, the MHC class I and MHC II class complexes of allogeneic iPSC are destroyed by knocking out the key proteins involved in their expression, such as B2M, a serum protein found to be associated with almost all MHC class I heavy chains on the surface of nucleated cells, involved in presenting peptide antigens to the immune system. In embodiments, the CIITA gene of allogeneic iPSC is destroyed so that the resulting differentiated cell lines (e.g., DC, mononuclear phagocytes, endothelial cells, thymic epithelial cells, B cells, etc.) do not express MHC II class proteins.

[0084] In embodiments, low immunogenic cells have T cell alpha constant (TRAC) gene disruption and / or reduction or elimination of TRAC protein expression and / or disruption of activity. In embodiments, low immunogenic cells have T cell beta constant (TRBC) gene disruption and / or reduction or elimination of TRBC protein expression and / or disruption of activity. In embodiments, low immunogenic cells have reduced or eliminated PD-1 gene expression and / or reduced or eliminated PD-1 protein expression and / or activity, wherein low immunogenic cells are activated; or low immunogenic cells have expression of PD-1 genes and / or gene products or increase in their expression, wherein low immunogenic cells are not activated.

[0085] In embodiments, "increased expression and / or activity" as used herein refers to an increase in expression and / or activity in a low immunogenic cell compared to a native or wild-type homologous cell. For example, in embodiments, increased expression and / or activity of one or more biomolecules described herein can confer low immunogenicity to an iPSC relative to an iPSC that does not have the same protein expression pattern or expression level. In embodiments, the "increased expression and / or activity" is due to a genetic modification, such as a knock-in.

[0086] CRS is a major problem with whole cell therapies, and despite the presence of engineered low immunogenicity, the effector functions and other consequences of interacting with cells after infusion may lead to the release of biomolecules, resulting in systemic inflammatory syndrome characterized by fever, multiple organ dysfunction, etc. In embodiments, low immunogenic cells are engineered to destroy the expression and / or activity of one or more proteins that contribute to CRS. In embodiments, low immunogenic cells have reduced or eliminated expression and / or activity (e.g., knockout or silencing) of CRS-associated cytokines.

[0087] In embodiments, low immunogenic cells have IL-4 gene destruction and / or reduce or eliminate IL-4 protein expression and / or active destruction. In embodiments, low immunogenic cells have IL-6 gene destruction and / or reduce or eliminate IL-6 protein expression and / or active destruction. In embodiments, low immunogenic cells have IL-10 gene destruction and / or reduce or eliminate IL-10 protein expression and / or active destruction. In embodiments, low immunogenic cells have IL-16 gene destruction and / or reduce or eliminate IL-16 protein expression and / or active destruction. In embodiments, modified cells contain gene destruction (for example, knockout) of other cytokine release syndrome (CRS) related interleukins. In embodiments, reducing or eliminating the interleukins causing CRS can reduce the possibility of CRS.

[0088] SerpinB9 is a member of the serine protease inhibitor superfamily. SerpinB9 is reported to protect cells from the immune killing effects of granzyme B. In embodiments, the low immunogenic cells have a disruption of the SerpinB9 gene and / or a disruption that reduces or eliminates SerpinB9 protein expression and / or activity. In embodiments, SerpinB9 in the low immunogenic cells is knocked out and / or silenced. Alternatively, in embodiments, the low immunogenic cells express the SerpinB9 gene and / or gene product, or their expression and / or activity is increased.

[0089] In embodiments, overexpression of SerpinB9 sequesters the function of granzyme B associated with immune stimulatory responses (e.g., apoptosis of target and / or diseased cells). In embodiments, granzyme B is inhibited in activated lymphocytes, NK cells, macrophages, and follicular DCs, among other cell types. In embodiments, for example, when BioNV is intended to deliver a non-granzyme payload (e.g., a gene editing payload), low immunogenic cells may express and / or overexpress SerpinB9.

[0090] In embodiments, low immunogenic cells express CCL2 genes and / or gene products, or their expression and / or activity is increased. In embodiments, low immunogenic cells express PD-L1 genes and / or gene products, or their expression is increased, and wherein the low immunogenic cells are not activated; or wherein the expression of the PD-L1 genes and / or gene products of the modified cells is reduced or eliminated, and wherein the low immunogenic cells are activated. In embodiments, low immunogenic cells express H2-M3 genes and / or gene products, or their expression and / or activity is increased. In embodiments, low immunogenic cells express CTLA-4 genes and / or gene products, or their expression and / or activity is increased.

[0091] In embodiments, low immunogenic cells express the CD47 gene and / or gene product, or their expression and / or activity is increased. In embodiments, the potential suppressive phenotype of CD47 expression across cells is prevented by interfering with the inhibitory action mechanism of a series of microRNAs on the 3'UTR of the CD47 gene, by deleting this region in a stable construct or by eliminating / inhibiting the expression of microRNAs. In embodiments, this can address the problem of inhibition caused by microRNAs.

[0092] In the embodiments, the low immunogenic cells express the CD24 gene and / or gene product, or their expression and / or activity are increased. CD24 is a sialoglycoprotein expressed on mature granulocytes and B cells and is also an anti-phagocytic protein. CD24 prevents phagocytosis by interacting with Siglec-G / 10 on macrophages. In the embodiments, the low immunogenic cells express or overexpress the CD24 protein and / or gene product. In the embodiments, the low immunogenic cells express chimeric CD24 / CD47 genes and / or gene products, or their expression and / or activity are increased. In the embodiments, the low immunogenic cells include CD24 / CD47 with a tethered transmembrane domain. In the embodiments, the domains of CD47 isoform 2 and CD24 can be expressed separately or tethered to form a bilobed chimeric protein. In the embodiments, the low immunogenic cells are iPSCs from fibroblasts rather than ABO cells.

[0093] In embodiments, low immunogenic cells express the CD200 gene and / or gene product, or their expression and / or activity is increased. In embodiments, the CD200 tag minimizes phagocytosis of macrophages and also prevents the activation of granulocytes. In embodiments, when there is no need to prevent granulocytes, such as in a solid tumor microenvironment (TME), low immunogenic cells do not express CD200, because the activation of granulocytes will complement the mechanism of action of BioNV, which is intended to release granzymes and perforins. However, in embodiments, if CD47 or CD24 tags are used, or a combination of CD24 / CD47 chimeric bilobed protein tags (each preventing phagocytosis) and overexpressed H2-M3 (inhibiting NK responses) is used, stability can be achieved in the absence of CD200 while allowing BioNV to be cleared. In embodiments where granzymes and perforins are not preferred therapeutic biomolecules, CD200 can be expressed to prevent the activation of granulocytes while eliminating the CD47 tag or the CD24 tag, but not both tags. In embodiments, the low immunogenic cells express chimeric CD24 / CD200 genes and / or gene products or chimeric CD47 / CD200 genes and / or gene products, or have increased expression and / or activity. In embodiments, these CD200 strategies represent a method for generating low immunogenic cell lines for targeting non-cancerous cells, e.g., targeting liver, kidney, heart cells and / or tissue regeneration pathways.

[0094] In embodiments, the obtained low immunogenic cells (or cells differentiated therefrom) do not express and / or overexpress all three of CD47, CD24, and CD200. In embodiments, the low immunogenic cells are engineered so that the BioNV or exosomes produced by the low immunogenic cell line are stable, but not to the extent that the BioNV or exosomes resist clearance from the body. Excessively stable BioNV / exosomes may ultimately trigger a humoral response, thereby limiting the dose that can be administered or the number of treatments.

[0095] In one embodiment, the low immunogenic cells express the MFG-E8 gene and / or gene product, or its expression and / or activity is increased. In one embodiment, the low immunogenic cells express the NCAM gene and / or gene product, or its expression and / or activity is increased. In one embodiment, the low immunogenic cells express the α-phagocytic integrin gene and / or gene product, or its expression and / or activity is increased.

[0096] In an embodiment, the low immunogenic cells express an antibody or antibody-like molecule targeting an IL-6 surface receptor (anti-IL-6R), or their expression and / or activity is increased. In an embodiment, the low immunogenic cells are iPSC cell lines with anti-IL-6R antibodies engineered into the cell line. In an embodiment, BioNV / exosomes carry α-IL-6R, thereby preventing the activation of signaling pathways on local immune cells in the tumor environment.

[0097] In embodiments, the low immunogenic cells express the FasL gene and / or gene product. In embodiments, the low immunogenic cells do not express the FasL gene and / or gene product. In embodiments, the low immunogenic cells do not overexpress FasL because, after processing (e.g., by continuous extrusion), enrichment of the native expression level of FasL is observed in the BioNV membrane. Excessive concentrations of FasL may be counterproductive and prevent T cell recruitment to solid tumors and / or lead to premature T cell death.

[0098] In embodiments, low immunogenic cells may express one or more fusion proteins of one or more portions of any of the immunoprotective proteins described herein. For example, in embodiments, constructs may be prepared in which the appropriate portion of a selected ligand is tethered to a transmembrane domain. In embodiments, constructs may be prepared in which biologically relevant portions of two or more proteins are tethered together and / or to a transmembrane domain.

[0099] In embodiments, the low immunogenic cells substantially lack expression and / or activity of one or more immunogenic proteins and express, or have increased expression of, one or more immunoprotective proteins.

[0100] In embodiments, the low immunogenic cells have reduced or eliminated expression and / or activity of one or more immunogenic proteins, e.g., proteins that lead to an immune response, donor-recipient mismatch, HLA alloimmunization, inflammation, CRS, etc. in a subject, e.g., MHC class I proteins, MHC class II proteins, HLA proteins, TCR proteins, CRS proteins, etc. In embodiments, the low immunogenic cells have reduced or eliminated 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.

[0101] In embodiments, the modified cells have expression or increased expression and / or activity of one or more immunoprotective proteins, e.g., proteins that prevent or reduce an immune response in a subject, prevent or reduce premature clearance of BioNV in a subject, prevent or reduce phagocytosis, confer barrier function, etc., e.g., CD47, CD24, CD200, CD34, CCL2, H2-M3, MFEG8, PD-L1 (from non-activated cells), CTLA-4, etc. In embodiments, the low immunogenic cells express 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 have increased expression thereof.

[0102] In embodiments, the expression and / or activity of genes and / or gene products of HLA-A, HLA-B, HLA-C, HLA-F, CIITA, IL-6, TRAC, TRBC, and one of HLA-E or HLA-G are reduced or eliminated in the low immunogenic cells.

[0103] In an embodiment, the expression and / or activity of genes and / or gene products of HLA-A, HLA-B, HLA-C, HLA-F, CIITA, IL-6, TRAC, TRBC, SerpinB9, and one of HLA-E or HLA-G are reduced or eliminated in the low immunogenic cells.

[0104] In an embodiment, the low immunogenic cells include low immunogenic cells in which the expression and / or activity of genes and / or gene products of 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 of IL-4, IL-10 and IL-16 are reduced or eliminated.

[0105] In embodiments, the low immunogenic cells express, or have increased expression and / or activity of, alpha-phagocytic integrin, CCL2, H2-M3, FasL, MFEG8, and PD-L1 and / or CTLA-4, wherein the low immunogenic cells do not overexpress FasL, and wherein the low immunogenic cells are not activated by expression of PD-L1, and express, or have increased expression and / or activity of, CD24, CD47, CD200, chimeric CD24 / CD47, chimeric CD24 / CD200, and chimeric CD47 / CD200, or both CD24, CD47, and CD200.

[0106] In embodiments, the low immunogenic cells express, or have increased expression and / or activity of, alpha-phagocytic integrin, CCL2, H2-M3, FasL, MFEG8, SerpinB9, and PD-L1 and / or CTLA-4, wherein the low immunogenic cells do not overexpress FasL, and wherein the low immunogenic cells are not activated by expression of PD-L1, and express one of CD24, CD47, CD200, chimeric CD24 / CD47, chimeric CD24 / CD200, and chimeric CD47 / CD200, or both of CD24, CD47, and CD200, or have increased expression and / or activity.

[0107] In an embodiment, the low immunogenic cells express the CD200 gene and / or gene product, or have increased expression and / or activity, and do not express and / or substantially lack the CD24 or CD47 genes and / or gene products. In an embodiment, the low immunogenic 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.

[0108] In embodiments, low immunogenic cells express one or more antigens, ligands and / or receptors of tumor cells or cancerous tissues, or their expression and / or activity increase. In embodiments, one or more antigens, ligands and / or receptors of tumor cells or cancerous tissues are listed in Table 4, Table 5 and / or Table 6 below. In embodiments, one or more antigens, ligands and / or receptors of tumor cells or cancerous tissues are neoantigens, such as mutant variants or peptides that are specific for cancer or disease state, and the mutant variants or peptides are not present in the subject to be treated in other cases. In embodiments, low immunogenic cells express co-stimulatory molecules of immune cells, or their expression and / or activity increase. In embodiments, the co-stimulatory molecule of immune cells is IL-15.

[0109] In embodiments, the low immunogenic cells are allogeneic. In embodiments, the low immunogenic cells do not elicit an immune response in a patient to whom they or a BioNV derived therefrom is administered.

[0110] In embodiments, low immunogenic cells include one or more targeting agents. In embodiments, one or more targeting agents include chimeric antigen receptors (CAR). In embodiments, CAR is bispecific. In embodiments, CAR lacks an intracellular portion. In embodiments, CAR includes a targeting agent, a transmembrane domain and an intracellular domain, which include costimulatory domains and / or signaling domains. In embodiments, the transmembrane domain is derived from CD28, CD3 ζ, CD4, CD8 α or ICOS, or a fragment thereof. In embodiments, the intracellular domain includes an intracellular signaling domain and / or one or more costimulatory molecules of a CD3 ζ chain, optionally selected from CD28, 4-1BB, ICOS, CD27 and OX40.

[0111] In embodiments, the one or more targeting agents comprise an antibody or antibody form. In embodiments, the antibody or antibody form is selected from one or more of the following: a monoclonal antibody, a polyclonal antibody, an antibody fragment, Fab, Fab', Fab'-SH, F(ab')2, Fv, single chain Fv (scFv), V NAR 、V H In one embodiment, the one or more targeting agents include a viral epitope recognition receptor (VERR) or a viral ligand. In one embodiment, the one or more targeting agents include a ligand for a receptor. In one embodiment, the one or more targeting agents include a receptor for a ligand.

[0112] In the embodiments, the low immunogenic cells obtained have B2M knockout (KO), CIITAKO, CD47tg knock-in (KI), IL-6KO, are engineered into iPSC, and TRAC and TRBC genes can be knocked out. In the embodiments, only one gene of each is knocked out, rather than two genes on a single allele. TRAC and TRBC genes can be knocked out as described herein. The purpose of knocking out TRAC and TRBC genes is to eliminate T cell receptors. In the embodiments, modified cells are differentiated into T cell subsets lacking T cell receptors to derive BioNV / exosomes. Cells are genetically modified so that they are substantially lacking TCR, reducing the chance of competing ligands for the CAR constructs that can non-specifically target alternative tissues. Therefore, in the embodiments, TCR genes are knocked out as a strategy to reduce the off-target effects of BioNV / exosomes. In the embodiments, TRAC / TRBC knockouts typically reduce the possibility of CRS and BioNV / exosome toxicity.

[0113] In an embodiment, the produced BioNV / exosomes express CD47. Exosomes and vesicles are easily cleared from the body by macrophages through phagocytosis. Phagocytosis greatly affects the therapeutic value and efficacy of exosomes. In order to prevent macrophage consumption of BioNV / exosomes, but not wishing to be bound by theory, in an embodiment, BioNV / exosomes carry a CD47 tag on the surface. CD47tg (tag) sends a "don't eat me" signal, which in an embodiment increases the half-life and serum stability of BioNV / exosomes in the subject. In an embodiment, the molecule CD47 isoform 2 (an isoform that interacts with the SIRPα receptor on macrophages) is engineered into modified cells (e.g., iPSC cell lines). Without CD47tg, the half-life of BioNV / exosomes would be shortened due to inhibition of phagocytosis, resulting in the need for higher and / or more frequent doses.

[0114] In embodiments, the low immunogenic cells obtained have B2M gene disruption (or other genes that can achieve MHC I suppression), CIITA gene disruption (or other genes that achieve MHC II suppression), CD47tg knock-in and knockout of key interleukins (IL) related to the cause of cytokine release syndrome. In embodiments, modified cells include IL-6 gene disruption. Interleukin 6 (IL-6) is the main interleukin that causes CRS. In embodiments, IL-6 knockout prevents undesirable IL-6 from being packaged into BioNV / exosomes, and reduces the contribution of BioNV (or exosomes) to local (and concentrated due to biomarker targeting) and / or potential systemic CRS events.

[0115] In embodiments, the modified cells are expanded after engineering; any small-scale expansion or large-scale feeder system expansion method known in the art can be used.

[0116] In an embodiment, the low immunogenic cells used to produce BioNV / exosomes express one or more of the above-mentioned proteins through an adjustable expression element, which can be adjusted throughout the BioNV / exosome manufacturing process. In an embodiment, the adjustable expression element includes an adjustable promoter (e.g., a Tet on / off promoter) or a CRISPRa / i regulatory system, as well as other adjustable expression elements. In an embodiment, CAR expression is controlled by an adjustable expression element. Simple overexpression of a construct from a CMV (or other type) promoter may result in too high a surface density of CAR, which may cause many problems, such as 'overactivation', leading to in vitro exhaustion and cell death after activation. Cell death leads to loss of the cell line that produces BioNV / exosomes. In an embodiment, the surface density of the CAR protein construct is adjusted. For example, in an embodiment, the typical concentration range of CAR protein per microgram of T cell is between 0.20ng-0.70ng. Too little CAR expression results in poor biomarker targeting. However, the density cannot exceed the cell limit. Too high a CAR density can lead to depletion of the mother cells during in vitro activation prior to BioNV / exosome derivation and can also result in poor BioNV / exosome quality due to high protein concentrations in the plasma membrane. An upper limit allows for improved targeting efficiency in cancer cells with low biomarker expression. Density limits for cell therapy are outlined in published U.S. non-provisional application US20220040106A1, which is hereby incorporated by reference.

[0117] In embodiments, the low immunogenic cells for producing BioNV / exosomes may comprise stable cell integration (safe harbor genetic position) of any genetic element described herein in cells (e.g., iPSC), which can be controlled by implementing a Tet-regulated CRISPRa+ targeting 3x transcription factor targeting gRNA system. Activation of three upstream transcription factors by the CRISPR activation system can trigger a signal cascade event, enhancing the production of CAR that replaces endogenous antibody ORFs at designated sites. This system can be 'tuned' by including a Tet-regulated promoter, thereby being able to change the concentration of cell surface CAR. Next, Cpf-1 directed homology-directed repair (HDR) can be used to achieve stable cell replacement of CDRs and heavy and light chain antibody regions with CAR cassettes. Finally, the stably integrated CAR cassette may contain flanking gRNA binding sites, which allow scFV (and other antibody forms) or VERR / viral ligands to be repeatedly exchanged or changed to quickly and consistently insert the desired sequence.

[0118] In embodiments, a variety of transcriptional control elements, such as tetracycline on / off promoters (or similar drug-regulated promoters) can be used to regulate the CAR concentration on the surface of the iPSC base cell line or any downstream differentiated cells (and the BioNV / exosomes produced therefrom) 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 engineered CAR boxes integrated into the antibody gene locus (the position where the antibody gene has been replaced). In addition, similar transcriptional control elements can be provided to control the overexpression of genes (e.g., CD47) at defined manufacturing stages, drive genes that control differentiation, and the like.

[0119] In the embodiment, the low immunogenic cells have B2M KO, CIITA KO, CD47tgKI, IL-6KO, wherein an IL-2p GFP reporter gene is constructed, and the CAR construct can be integrated / engineered into the modified cells. In the embodiment, the CAR construct can be knocked into the TRAC / TRBC gene while knocking out the remaining TRAC / TRBC genes, thereby generating CAR+ and TRAC / TRBC- / - cells. In the embodiment, the CAR construct can simultaneously knock into the TRAC / TRBC gene position on two loci, thereby generating CAR+ / + and TRAC / TRBC- / - cells.

[0120] In embodiments, low immunogenic cells have engineered B2M KO, CIITA KO, CD47tg KI, IL-6 KO, IL-2pGFP, and CAR cells (e.g., iPSCs), and the quality of the immune synapse (IS) is measured between the CAR recognition domain and the biomarker during the method for producing BioNV / exosomes. In embodiments, the IS quality of BioNV / exosomes can be directly correlated with the efficacy of whole cell therapy.

[0121] In embodiments, therapeutic BioNV / exosomes, or modified / activated cells derived therefrom, comprise nucleic acids encoding green fluorescent protein (GFP). In embodiments, once B2M KO, CIITAKO, CD47tg KI, IL-6KO, TRAC / TRBC single KO are engineered into iPSC, GFP molecules can be engineered into modified cell lines. In embodiments, this serves as a control cell line. In embodiments, non-control cell lines (therapeutic cell lines) do not have GFP. In embodiments, nucleic acids encoding GFP are operably linked to promoters from one or more of the following: IL-2, perforin, granzyme, granulysin, alarmin, TNF, INF, combinations thereof, and / or any other cell-specific genes or reporter genes. When lymphocytes are widely / overall activated due to various stimuli, the IL-2 promoter is constitutively activated. In embodiments, more centralized activation / inhibition (regulation) is used. In embodiments, the IL-2p GFP reporter gene serves as an indicator (part of the BioNV / exosome-derived process) of the degree of extensive / overall activation of cells. In embodiments, GFP signaling is combined with immunoblot analysis of cytokine levels (e.g., perforin, granzymes, alarmins, TNF, and INF) to effectively modulate the extent of widespread / global activation of lymphocytes upon exposure to activating antigens. In embodiments, GFP is used to compare activation levels between manufacturing batches and ensure consistency in therapeutic development.

[0122] In an embodiment, BioNV / exosomes are formed by obtaining iPSCs (hypo-iPSCs). In an embodiment, hypo-iPSCs are characterized by B2M- / -, CIITA- / -, CD47+ / +, PD1- / - plasma membrane profiles and can be used to produce the BioNV / exosomes of the present invention. Hypo-BioNV / exosomes can be produced from parental iPSC cell lines by methods such as ultrasonic treatment, adaptive focused acoustic technology, French press, extrusion, continuous extrusion, detergent cell lysis, and electroporation. In an embodiment, continuous extrusion is a method for producing Hypo-BioNV / exosomes. Continuous extrusion of iPSCs can produce HLA1 / HLA2 negative (low immunogenicity) and tgCD47+ BioNV / exosomes that exhibit PD1 resistance elimination.

[0123] In the embodiment, the low immunogenic cells obtained are CD34+, or derived from CD34+ cells, such as human CD34+ umbilical cord blood. In the embodiment, CD34+ umbilical cord blood-derived cell lines can be used as the basis cell lines for the development, production and manufacture of therapeutic BioNV / exosomes for the delivery of gene editing therapeutics. In the embodiment, the low immunogenic cell lines derived from CD34+ umbilical cord blood have been experimentally confirmed to have low expression of HLA 1 / 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).

[0124] In embodiments, the hypoimmunogenic cells may include cells engineered using a variety of hypoimmunogenic engineering techniques, for example, as described in Deuse et al., Han et al., Xu et al., Harding et al., and in published U.S. patent applications Nos. 20190376045, 20190376045, 20210308183, and 20210292715 to Deuse, 20210161971 to Nagy, 20180141992 to Strominger, and published European patent application No. 3693384 to Poirot, each of which is incorporated herein by reference in its entirety (Han X et al., "Generation of hypoimmunogenic human pluripotent stem cells"). 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.).

[0125] In embodiments, BioNV / exosomes are derived from cells that have been depleted of HLA genes, which encode MHC membrane glycoproteins that confer immune responses associated with GVHD rejection. HLA gene clusters can be divided into three categories: 1) MHC class I pathway, 2) MHC class II pathway, and 3) MHC class III pathway. Only the MHC class I and class II pathways express protein complexes that trigger immune responses in GVHD, while MHC class III complexes are not involved in immune activity.

[0126] Elimination of MHC class protein complexes can trigger natural killer cells and macrophages to enter active clearance mode, followed by cell destruction. To avoid this killing mechanism, in embodiments, a CD47 isoform 2 transmembrane molecule protein tag can be added to the cell membrane of the modified cells to avoid natural killer and macrophage-mediated killing reactions, for example, as described in Willingham et al., Deuse et al., and Han et al. (Willingham SB et al. "The CD47-signalregulatory protein alpha (SIRPa) interaction is atherapeutic target for humansolid tumors." PNAS. Vol. 109, No. 17, 2012: p. 6662-7. doi: 10.1073 / pnas.1121623109.).

[0127] In embodiments, cells can be engineered to use additional mechanisms to prevent these reactions, such as those described in the following literature: 1) CD24 transmembrane molecule protein tag (e.g., as performed 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 performed in Jiaravuthisan et al.) (Jiaravuthisan P et al. "Amembrane-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 embodiments, BioNV derived from 'activated' cells will encapsulate and / or release perforin and / or granzymes, resulting in targeted cell death. In embodiments, activated cells will produce perforin and / or granzymes to be packaged into BioNV. In embodiments, low immunogenic cells to be activated do not express PD-L1 to avoid the resulting BioNV being targeted to PD-1 on T cells. In embodiments, this reduces the possibility of releasing perforin and / or granzymes, thereby causing unwanted T cell death. In embodiments, PD-L1 is overexpressed in BioNV derived from cells that have not yet been activated and are not loaded with apoptotic cytokines. In embodiments, PD-L1 of low immunogenic cells to be activated is downregulated, knocked out, or otherwise silenced. In embodiments, non-activated low immunogenic cells have upregulated PD-L1, i.e., for BioNV for gene editing agent delivery. In embodiments, CD47 isoform 2 can be engineered into cells to prevent macrophage and natural killer mediated cytotoxicity because it acts as a "don't eat me" tag through the SIRP-α receptor expressed on these and other cells.In embodiments, CD47 can be used in genetically engineered iPSCs to achieve immune tolerance to innate immune cells, such as in studies by Chhabra et al., Han et al., and Jaiswal et al. (Chhabra A et al. “Hematopoietic stem cell transplantation inimmunocompetent 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 embodiments, cells may be modified as described in US Patent No. 8,562,997 to Jaiswal et al., which is incorporated herein by reference in its entirety.

[0128] In embodiments, some methods that do not completely knock out all HLA genes can be used, for example, as done by Xu et al. and Han et al., only HLA genes highly associated with immune response are knocked out, while HLA genes that inhibit macrophage or NK response remain intact (e.g., HLA-E, HLA-F, and HLA-G). In embodiments, this method does not require the addition of a CD47 tag; modified cells can be engineered to produce BioNV / exosomes with or without CD47.

[0129] In an embodiment, the method improves upon the low immunogenicity method of Table 1.

[0130] Table 1: Three methods for modifying cells using HLA knockout combined with a CD47 isoform 2 tag and a 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." NatBiotechnol. Vol. 35, No. 8, 2017: Pages 765–772. doi:10.1038 / nbt.3860.).

[0131]

[0132]

[0133] In embodiments, development allogeneic modified cells involve removing MHC class I and MHC class II protein complexes by destroying certain HLA genes or B2M knockouts, and then knocking out the CIITA gene. In embodiments, due to its rapid mechanism of action, CRISPR gene editing methods can be used to knock out. In embodiments, zinc finger nucleases (ZFNs) and / or TALENS are used to knock out. In embodiments, Cre / Lox recombinase systems are used to produce modified cells. In embodiments, RNA silencing (RNAi, shRNA, microRNA, CRISPR Cas13a-d, etc.) is used to produce modified cells.

[0134] In embodiments, developing allogeneic modified cells includes a method of creating allogeneicity by Harding et al., which is different from the above-mentioned method (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 embodiments, the method of Harding et al. includes an alternative method based on immune escape mechanisms in nature, rather than deleting MHC class I / II genes and risking preventing long-term acceptance by the recipient. In embodiments, the method relies on biomimetic work by Harding et al. based on the horizontally transmitted cancer Tasmanian devil facial tumor disease (DFTD) type 2 (dominant in Tasmanian devils). In embodiments, development of allogeneic modified cells includes overexpression of immunomodulatory proteins CCL21, PD-L1, FasL, SerpinB9, H2-M3, CD47, CD200 and / or MFG-E8 to protect cell derivatives from long-term immune rejection in mice (and humans) without deleting MHC class I / II proteins. In embodiments, the modified cells express one or more proteins shown in Table 2, including any splice variants and / or isoforms of any of the shown proteins (e.g., CD200 splice variants). In embodiments, this system can be used to interfere with the activity of antigen presenting cells (APCs), macrophages, natural killer cells, and T lymphocytes. In an embodiment, the modified cell line may also contain a safe cell system developed by Liang et al. in 2018, in which a cell division gene is linked to a suicide gene to prevent uncontrolled teratomas from leading to cancer (Liang Q et al. "Linking a cell-division gene and a suicide gene to define and improve celltherapy safety." Nature. Vol. 563, No. 7733, 2018: pp. 701-704. doi: 10.1038 / s41586-018-0733-7.).

[0135] In an embodiment, the method improves upon the low immunogenicity method of Table 2.

[0136] Table 2: Expression or increased expression of illustrative proteins used to generate allogeneic modified cells.

[0137]

[0138] In embodiments, cells from which BioNVs / exosomes are derived include cells engineered to knock out one or more of the following: 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 to knock in one or more of the following: CCL2, PD-L1 (in BioNVs from non-activated cell sources), CTLA-4, H2-M3, CD24, CD47 (minus the 3'UTR region or an alternative 3'UTR region that does not contain inhibitory microRNA binding sites), MFG-E8, CD200, and / or any combination thereof.

[0139] In embodiments, BioNV is produced by a modified cell having one or more modifications of Table 3.

[0140] Table 3: Illustrative engineered cell expression profiles for BioNV formation for human use (Fife BT and BluestoneJA. "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 immunerejection of human ESC-derived allografts.” Cell Stem Cell. Volume 14, Issue 1, 2014: Pages 121–30. doi:10.1016 / j.stem.2013.11.014.).

[0141]

[0142] In embodiments, the inactivation / activation of genes is controlled by an inducible promoter throughout the differentiation, activation and manufacturing process of BioNV / exosomes. In embodiments, the destruction of MHC, TCR and CRS genes will produce allogeneic iPSCs of - / -CRS and - / -TCR, resulting in a plasma membrane that exhibits low immunogenicity when infused into a subject. The CRS genes related to the pathogenesis of CRS include IL-6, IL-10, IFN-γ, monocyte chemoattractant protein 1 (MCP-1), granulocyte macrophage colony stimulating factor (GM-CSF), and other cytokines, including tumor necrosis factor (TNF), IL-1, IL-2, IL-2-receptor-α and IL-8. In embodiments, one or more of these genes are inactivated, for example, in cells derived from BioNV / exosomes.

[0143] In embodiments, the low immunogenic cells obtained for production of BioNV / exosomes are metabolically regulated (e.g., specialized and differentiated cell lineages) to produce one or more desired therapeutically relevant biomolecules, or to increase the concentration of a therapeutically relevant biomolecule (or to a desired concentration), such that the regulated cells can then be processed into BioNV / exosomes. In some embodiments, the cells

[0144] In embodiments, broad or global regulation of a cell results in the expression of multiple genes to produce cytokines, chemokines, regulatory nucleic acids, and other therapeutically relevant biomolecules that cause the cell to enter an 'activated' state, thereby enhancing the cell's ability to achieve its metabolically predetermined purpose / phenotype. In embodiments, the controlled expression of one or more therapeutically relevant biomolecules in a cell can reproducibly, for example, cause a T lymphocyte to enter an activated state, wherein binding of the T cell receptor (TCR) to an antigen presenting cell (APC) occurs ( Figure 1 In embodiments, the controlled expression of one or more therapeutically relevant biomolecules in cells can recapitulate the interaction between antigenic peptides presented in the major histocompatibility complex (MHC) and the TCR of T cells, which triggers conformational changes in the TCR, thereby triggering intercellular signaling cascades and the expression of a large number of genes for cytokines, including perforins, granzymes, alarmins, interleukins, and interferons, etc. ( Figure 1 Typically, cytokines 'activate' cells into a specialized mode that allows them to clear host antigens and recruit immune cells to the site of infection to help clear antigens / infected cells and repair surrounding tissues. In embodiments, these cellular processes occur without the provision of exogenous cytokines, which can lead to deleterious effects in cell lines, such as exhaustion, competing signaling pathways, etc. In embodiments, the activation state of T cells can be exploited in BioNV / exosomes, for example, by processing methods that capture cytokines or transmembrane ligands in their lumen and / or membrane, respectively. Figure 2 ).

[0145] In embodiments, extensive or global activation of cells (e.g., T cells, natural killer cells) can be achieved in culture outside a host (in vitro). In embodiments, the activated cells can be expanded and processed into BioNV / exosomes, which act similarly to miniature T cells without the complex genetic baggage of whole cells. Extensive regulation of T cells in vitro can be achieved by the various methods described herein.

[0146] In embodiments, activating low immunogenic cells comprises activating TCR / CD3 receptor complexes by protein antigens (in the case of T cells). In embodiments, activating low immunogenic cells comprises activating TCR / CD3 receptor complexes by small molecules. In embodiments, small molecule stimulation for activating cells by TCR / CD3 receptors may comprise phorbol esters. In embodiments, activating low immunogenic cells comprises activating TCR / CD3 receptor complexes by viral antigens. In embodiments, viral antigens may be recombinant viral proteins, such as extracellular viral antigens (e.g., glycoproteins or spike proteins) or intracellular viral antigens, etc.

[0147] In embodiments, activation of low immunogenic cells includes activation of calcium-dependent channels. In embodiments, calcium-dependent channels include calcium release activated channels (CRAC), which are activated by an increase in Ca2+ concentration, for example, by being supplied to a culture medium. In embodiments, potassium channels (K channels) or calcium channels (Ca channels) can be used, or overexpressed in cells, and homologous inorganic components are provided in the culture medium to activate cells. In embodiments, the long-term rise in intracellular calcium or potassium is a signal that simulates antigens or mitogens to activate lymphocytes.

[0148] In embodiments, activating low immunogenic cells comprises activating the LFA-1 integrin receptor. In embodiments, LFA-1 integrin receptor activation comprises providing an activating ligand peptide comprising ICAM1, ICAM2 (CD102), ICAM3 (CD50), ICAM4, ICAM5, JAM-A, and / or portions thereof.

[0149] In embodiments, activating low immunogenic cells comprises activating the CD28 receptor. In embodiments, activation of CD28 can be achieved by providing one or more ligand peptides for CD28, including CD80, CD86, GRAP2, and Grb2. In embodiments, activation of CD28 is performed in a subpopulation of non-memory T cells.

[0150] In embodiments, activation of IL-12 can be performed by providing one or more recombinant human IL-12s. In more detail, NK cells constitutively express perforin and granzymes. Secretory lysosomes (about 500 nm in diameter) containing perforin and granzymes maintain a constant concentration within the cell, enabling natural killer cells to immediately initiate / prepare for cytotoxic activity. CD8+ killer T cells (and their cell subsets) also contain low to moderate concentrations of perforin and granzymes in their lytic granules. However, in other T cells (such as CD4+ / CD8+ memory and helper T cells), perforin and granzymes within the lytic granules are typically low or absent, resulting in delayed expression and cytotoxic effects. It may take several days to a week to accumulate enough lytic granules to initiate their cytotoxic phenotype. This delay is a regulatory and buffering feature between different cell types to prevent runaway reactions. To give an analogy, NK cells and killer T cells are like garden hoses filled with water. When someone turns on the faucet, water flows out immediately. On the other hand, other T cells are like empty garden hoses. After turning on a tap, it takes a while for the water to flow—it's a delayed reaction. From a manufacturing activation perspective, using iPSC-derived NK and / or killer T cells is ideal because they are already primed with secretory lysosomes and lytic granules, respectively, and can be stimulated / activated in a way that rapidly produces additional perforins and granzymes. Other T cells (or other cells, such as neutrophils) can still be used as a source of cytotoxic proteins if needed, but their expression may be prolonged. The former cell type will reduce costs.

[0151] In embodiments, activating low immunogenic cells includes activating IL-2 receptors and IL-12 receptors. In embodiments, IL-2 activation can be performed by providing one or more recombinant human IL-2s. In embodiments, IL-12 activation can be performed by providing one or more recombinant human IL-12s. IL-2 and IL-12 increase the accumulation of perforin and granzyme B mRNA in NK and killer T cells. Perforin and granzyme mRNA transcripts are likely to form low-level 'start-up and sustainable pools' (water in a garden hose) in the cytoplasm and serve as a source for rapid deployment during activation. IL-2 and / or IL-12 are likely to be more suitable for in vitro cell activation than other sources or modes of action (e.g., antigen receptor engagement on other cells, or antigen stimulation on surface receptors) because these pathways have been shown to only cause an increase in granzymes. Therefore, the production of perforin and granzymes is ideal and can be achieved by stimulating IL-2 and IL-12 with their respective receptors. In addition, IL-2 has been shown to stabilize the stability of perforin and granzyme mRNA in the cytoplasm. Therefore, IL-2 may be a preferable activating cytokine over IL-12 because it helps maintain these cytoplasmic mRNA reservoirs (IL-2 keeps water in the water pipes) (REF-PMID: 8103068).

[0152] In embodiments, the cytokine INF-α can also be used to activate the JAK1 / TYK2 to STAT pathway, at least in T cells, via the INFAR1 and / or INFAR2 receptors, to produce perforin and granzymes.

[0153] In embodiments, activation of low immunogenic cells, such as natural killer cells (or T cells, if the following receptors are associated with T cell activation) includes activating receptors (alone or in combination to allow for a coordinated activation reaction), such as CD2, CD3, CD16, CD28, CD314 (NKG2D), CD335, B7-H6, CD158d, IL-2R, IL-12R, DNAM-1, CD2, CD44, CD137, CX3CR1, CD27, CD160, 2B4, but not limited to these sensory receptors. CD16 can be activated by binding to the Fc region of an antibody (anti-S2 IgG). CD134, CD335, B7-H6 can be activated in combination with ICAM-1, which is combined with the Fc region of an antibody. CD314 can be activated by ULBP1, MICA, MICB, or H60. CD158d can be activated by HLA-G to trigger proinflammatory molecules. 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, CD112, or NKp30. CD2 can be activated by LFA3. CD44 can be activated by hyaluronic acid, hyaluronan, osteopontin, collagen, or matrix metalloproteinases. CD28 homologs can be activated by the B7H7 protein ligand. CD137 can be activated by itself and by members of the tumor necrosis factor receptor protein family. 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 type of activation results in different levels of cytotoxic biomolecules. Activation of each (or any) of the above combinations can be achieved by adding each of the following ligands (or any other relevant ligands) separately, wherein the ligand is in soluble form (partial peptide, domain or whole protein), anchored to the membrane (partial peptide, domain or whole protein), is a component of the target cell membrane (partial peptide, domain or whole protein) or cross-linked to a solid phase, such as but not limited to cross-linked dextran or magnetic beads (as partial peptide, domain or whole protein).

[0154] In embodiments, activation of some natural killer receptors (or T cells, if the following receptors are involved in T cell activation) does not result in degranulation or granule polarization to IS. These receptors include, but are not limited to: i) 2B4 alone, ii) CD134 alone, iii) CD134 in combination with 2B4 and one of the inhibitory receptors KIR or CD94, iv) LFA-1 in combination with one of the inhibitory receptors KIR or CD94, v) CD16 in combination with one of the inhibitory receptors KIR or CD94. Activation of each (or any) of the above combinations can be achieved by adding each of the following ligands (or any other relevant ligands) separately, wherein the ligand is in soluble form (partial peptide, domain or whole protein), anchored to the membrane (partial peptide, domain or whole protein), a component of the target cell membrane (partial peptide, domain or whole protein) or cross-linked to a solid phase, such as but not limited to cross-linked dextran or magnetic beads (as partial peptide, domain or whole protein): i) CD48, ii) ULBP1, iii) HLA-E, iv) a combination of HLA-C and HLA-E, or v) HLA-E.

[0155] In embodiments, activation of certain natural killer receptors (or T cells, if the following receptors are associated with T cell activation) leads to degranulation. Prior to degranulation, perforin and granzymes may or may not undergo post-translational modifications to enhance their activity. Activating cells along the degranulation pathway may be beneficial for achieving maximum efficacy of cytotoxic proteins. These receptors include, but are not limited to: i) CD16 in combination with LFA-1 and one of the inhibitory receptors KIR or CD94, ii) CD134 in combination with 2B4, iii) CD16 alone. Activation of each (or any) of the above combinations can be achieved by adding each of the following ligands (or any other relevant ligands) separately, wherein the ligand is in soluble form (partial peptide, domain or whole protein), anchored to the membrane (partial peptide, domain or whole protein), a target cell membrane component (partial peptide, domain or whole protein) or cross-linked to a solid phase, such as but not limited to cross-linked dextran or magnetic beads (as partial peptide, domain or whole protein): i) a combination of HLA-C and HLA-E, ii) a combination of ULBP1 and CD48, iii) anti-S2 IgG.

[0156] In embodiments, receptors that cause polarization of lytic granules when activated may not (or may be) advantageous because their localization to the cell membrane may minimize their packaging in BioNV, but may maximize their packaging in secreted EVs (exosomes and microsomes) due to differences in extrusion manufacturing (BioNV) versus cellular exocytosis processing (EV). Some receptors that induce polarization of lytic granules to IS include, but are not limited to, i) CD16 in combination with LFA-1 (also leading to degranulation), ii) LFA-1 alone, iii) CD134 in combination with LFA-1 and 2B4 (also leading to degranulation). Activation of each (or any) of the above combinations can be achieved by adding each of the following ligands (or any other relevant ligands) separately, wherein the ligand is in soluble form (partial peptide, domain or whole protein), anchored to the membrane (partial peptide, domain or whole protein), a component of the target cell membrane (partial peptide, domain or whole protein) or cross-linked to a solid phase, such as but not limited to cross-linked dextran or magnetic beads (as partial peptide, domain or whole protein): i) anti-S2 IgG in combination with ICAM-1, ii) ICAM-1, iii) ULBP1 in combination with CD48 and ICAM-1.

[0157] In embodiments, activating low immunogenic cells comprises activating a SLAMF1 (CD150) receptor. In embodiments, activation of cells via the SLAMF1 (CD150) receptor can be performed using one or more SLAM-associated proteins. In embodiments, said activation of the SLAMF1 (CD150) receptor is achieved through interaction with measles virus. In embodiments, said activation of the SLAMF1 (CD150) receptor is achieved through interaction with gram-negative bacteria.

[0158] In embodiments, activation of low immunogenic cells comprises activation of IFNγ receptors. In embodiments, activation of IFNγ receptors can include any Toll-like receptor (TLR) superfamily member and can be performed by providing one or more TLR receptor agonists, such as IFN-γ, lipoteichoic acid (LTA), lipopolysaccharide (LPS), gardiquimod, R848, CpG, Pam3CSK4, etc.

[0159] In embodiments, activation of low-immunogenic cells comprises activation of the CD4 receptor via one or more viruses. In 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 tumor-specific CTL responses in up to 50% of the circulating CD8+ T cell pool. In embodiments, ex vivo activation of CD8+ T cells can increase levels of alarmins (e.g., but not limited to, IL-1α, IL-33, and IL-17), which are then packaged (along with other anti-cancer biomolecules, such as perforin and granzymes) into BioNV / exosomes after they are derived from virally activated cells. In embodiments, alarmins can increase the potent cytotoxic effector T lymphocyte (CTLeff) responses elicited when treating a disease. In embodiments, the responses generated by administration of the BioNV / exosomes herein will be localized to the disease site (e.g., tumor, infected cells, etc.) specifically targeted by the alarmin-carrying BioNV / exosomes.

[0160] In embodiments, the expression of cytotoxic biomolecules, such as perforin and granzymes, can be activated by targeting cytoplasmic signaling molecules (e.g., kinases, phosphatases, GTPases, ATPases, etc.) that are part of the signaling pathway and are located downstream of membrane-associated receptors and factors. In embodiments, an example of this approach in T cells can involve small molecules that activate STAT, Blimp1, Tbet, Runx3, thPOK (by blocking Runx3 or eome inhibition).

[0161] In a similar embodiment, PKC (PKCtheta), a serine- and threonine-specific protein kinase and signaling molecule crucial for T cell and NK cell activation, can be activated by calcium and the second messenger diacylglycerol. Small molecules can also be added to cells, cross the cell membrane, and activate PKC, thereby producing perforin and granzymes. There are many commercially available small molecules that can activate PKC. These include (but are not limited to):

[0162] Bryostatin 1: a potent agonist with binding affinity for subsets of classical and novel PKC isozymes (REF-PMID: 16834754).

[0163] Ingenol-3-angelate (I3A, PEP005): A broad PKC activator. Its pro-apoptotic and immunostimulatory effects may promote degranulation. (REF-PMID: 22069553).

[0164] Phorbol 12-myristate 13-acetate: A reversible PKC activator that activates Ca2+-ATPase (REF-PMID: 3478199).

[0165] Prostratin: Activator of PKC and NF-κB. Synergistic with calcium / calcineurin signaling. Potent coactivator of perforin and granzymes. (REF-PMID: 24204950).

[0166] SC-9 and SC-10: PKC activators that induce PKC phosphorylation. PKC inhibition can be reversed by the SC-10 dual system, which activates cells and then reduces activation after perforin and granzyme levels reach optimal levels. (REF-PMID: 1414485).

[0167] Phorbol-12,13-dibutyrate (PDBu): Increases kinase phosphorylation. Induces vascular smooth muscle contraction. Release of mobilized Ca2+ may lead to perforin activation in preloaded states (REF-PMID: 19632318)

[0168] Other PKC activators can be found in Santa Cruz.

[0169] In a similar embodiment, Crk, an adaptor protein that binds to some tyrosine-phosphorylated proteins and acts as a key regulator of activation in NK cells, can be targeted to activate or release secondary inhibitory-related functions to achieve the expression of perforin and granzymes. MHC-I inhibitory receptors block the activation signal, leading to Crk phosphorylation and its subsequent binding to c-Abl (REF-PMID: 18835194). HLA-E induces Crk phosphorylation in NKG2A+ NK cells (REF-PMID: 22464172). Since Crk is required for CD16 activation (as described above), preventing the inhibitory mechanism by small molecule blockade in conjunction with CD16 receptor activators can be an alternative approach to activating NK cells to produce perforin and granzymes in secretory lysosomes. In addition, 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, which can be combined with a small molecule activator to activate NK cells. In an embodiment, Crk phosphorylation sites can be mutated to prevent inhibition. Mutations can be transient or stable in cells. In such mutants, small activating molecules can be used to activate cells through Crk. Similar strategies can be used for other points in the signal transduction pathway that lead to NK activation to produce perforin and granzymes. As described above, some activation points (target proteins in the signaling pathway) may lead to crosstalk between signaling pathways, which may cause different degrees of calcium-dependent perforin activation and / or perforin and granzyme polarization 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 these purposes. For example, perforin in a folded and unbound state may be more desirable than perforin bound to Ca2+. The latter may lead to unintended off-target effects in healthy tissues in the context of BioNV packaging. Therefore, small molecules that activate native perforin expression without inducing calcium release are needed.

[0170] In an embodiment, the activation of transcription factors that cause perforin and granzyme activation can also be used. In this method, if the purpose is to increase the amount of perforin to a level higher than that of granzymes, it may be necessary to target only the perforin promoter to increase the transcription factor expressed. In another method, if the purpose is to increase the amount of granzymes to a level higher than that of perforin, it may be necessary to target only the granzyme promoter to increase the transcription factor expressed. In another method, each transcription factor can be activated simultaneously to produce each protein at an equivalent level. Small molecules can be used to activate transcription factors. Proteins that are designed / engineered (transient or stably expressed) to interact with transcription factors or transcription factor genes can also be used to activate transcription factors. Examples of transcription factors that positively regulate the expression of perforin and granzymes include eomes and NF-κB (but are not limited to these). Eomes can be activated by CRISPR activation plasmid (REF-EOMES CRISPR). NF-κB can be activated by small molecules 4-hydroxyquinazoline; 4-quinazolinol.

[0171] In embodiments, a CRISPR activation and inhibition system (CRISPRa / i) can be used to directly activate or inhibit perforin and / or granzyme gene expression function, independent of upstream signaling. In such a system, CRISPRa / i can be transiently (freely) or stably integrated into desired iPSC cells, which can then differentiate into desired cell types (e.g., T cells or NK cells). After differentiation, in stably integrated cell lines, CRISPRa can be activated using a drug-inducible promoter such as tetracycline (but not limited to tetracycline or tetracycline-based promoters). Once induced, CRISPRa and its respective gRNAs targeting the perforin and granzyme promoter regions are expressed. CRISPRa gRNAs bind to and express perforin and granzymes. This system has the advantage over the aforementioned activation methods that perforin and granzyme expression can be controlled without placing the cells in an activated state. This system can be used to control the expression of other cytotoxic proteins or proteins of interest with therapeutic value, which can be packaged into the BioNV lumen. CRISPRi can then be used with a gRNA targeting the same region of the protomer to shut down expression of the desired protein.

[0172] In an embodiment, perforin and granzyme genes can be engineered into iPSC and contain regulatory properties embedded in promoters to allow direct and controlled expression using, for example, drug induction or CRISPRa / i or microRNA regulatory systems. In this method, iPSC containing engineered cytotoxic genes can be differentiated into any desired cell type, such as monocytes, macrophages, fibroblasts, kidney cells, hepatocytes, etc. This method will allow cells to express perforin and granzymes (and potential other engineered cytotoxic proteins, if necessary or applicable), otherwise they cannot be naturally expressed (nor can they be activated to express these proteins). The purpose of this method is to allow the expression of such proteins while utilizing cell properties that are different from cells that produce lytic granules (such as natural killer cells, T cells, neutrophils, etc.). This available cell property includes surface markers that enhance tissue tropism, phenotypes that cross barriers, and more accurate cell or tissue targeting, which may be lacking in natural killer cells, T cells, neutrophils, etc.

[0173] In embodiments, activation of low immunogenic cells includes activation by one or more engineered non-natural biomolecules selected from the group consisting of soluble peptides, chimeric antigen receptors, small molecule decoys, small molecule ligands, designed nucleic acid ligands, carbohydrate ligands, viral ligands, chimeric biomolecule ligands, fusion proteins, antibodies, and antibody form molecules. In embodiments, T cells (or desired cells) can be engineered to include CAR, wherein the intercellular domain of the CAR construct can be developed to activate cells extensively through its signal transduction domain. For example, in embodiments, 4-1BB can be engineered into traditional CD3 intercellular activation domains to expand and maintain T cell activation by activating non-classical NF-kB pathways (and their subsequent pathways). In embodiments, cells are activated by biomarker antigens, which are engineered to be recognized by their scFv recognition domains by the extracellular domains of the CAR construct. In embodiments, this extensive activation can be achieved by different types of extracellular CAR domains (e.g., scFv, V H H nanobody, V NAR , afilin (or other affinity targeting biochemical ligand) or CER) biomarker recognition or occurs through TCR ligands and components.

[0174] In embodiments, activating the low immunogenic cells comprises activation by an inorganic compound (eg, iron, calcium, potassium, tin, mercury, arsenic, pyrophosphate, etc.).

[0175] In embodiments, activating low immunogenic cells comprises activating 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 embodiments, cells are activated by expression and / or overexpression of a constitutively active form of an intracellular signaling pathway (e.g., a constitutively active STAT5 molecule) to drive signal transduction.

[0176] In the embodiments, CAR is activated before BioNV / exosomes are formed. In the embodiments, CAR is activated via its target, by another receptor and / or virus. In the embodiments, the CAR construct generally comprises the 1st, 2nd, 3rd or 4th generation intracellular signaling mode, which is designed to activate T / NK / macrophages (and other cell types) into active mode (metabolic regulation), thereby resulting in the expression of various biomolecules (such as alarmins, interferons, granzymes, perforins, etc.). However, in the embodiments, the degree of activation is regulated by different receptors, which can result in low-level activation of cytokines that are more targeted or more targeted for a specific purpose. In the embodiments, the modified cells can be activated by IL-15 alone to package BioNV / exosomes with the cytokines, thereby recruiting naturally occurring lymphocytes to the tumor site.

[0177] In embodiments, the CAR construct (extracellular region) is activated by its antigen (e.g., a soluble form of an antigen biomarker targeted thereto). In embodiments, the antigen can be attached to a magnetic bead and added to a cell culture, which can then be removed from the culture after activation, or the cells can be passed through a chromatographic column containing a bound antigen so that they are activated as they flow through the chromatographic column.

[0178] In embodiments, the CAR construct can be activated by an appropriate biomarker "antigen" or portion thereof recognized by the CAR, which may include but is not limited to scFv, VERR / viral ligand, V H H nanobody, V NAR, Afilin (or other affinity targeting biochemical ligands), CER or TCR ligand or component binding portion, epitope or peptide. In embodiments, the antigen can be a targeted biomarker or an activation virus, or a protein that activates the virus. In embodiments, antigen activation occurs by binding the antigen to a chromatographic column and then passing the modified cell through the column to be captured by the antigen through CAR-antigen interaction. In embodiments, the CAR-modified cells are lymphocytes, which can then be eluted from the column to obtain purified, activated lymphocytes. In embodiments, antigen activation involves adding low levels of antigen to the culture medium of the modified cells. In embodiments, after activation (by antigen-second generation or antigen-third generation CAR interactions), cells are processed by the methods described herein to produce BioNV / exosomes. In embodiments, BioNV / exosomes contain second generation (or third generation) CAR on their surface, as well as necessary lymphocyte activation proteins (lymphocyte anti-cancer cell or anti-defective cell cytokine libraries), which may include but are not limited to perforin and granzyme B.

[0179] In general, due to problems separating biomarker antigens from modified cell CAR receptors, activation with antigens may cause warnings. In embodiments, several viruses can be added or exposed to modified cells (e.g., T cells) to activate them outside of the CAR receptor. In embodiments, antibodies can be attached to a piece of iron or magnetic nanoparticles (iron oxide), and magnetism can be used to separate the virus from the cell after activation to remove the virus.

[0180] In embodiments, each "natural" activation mode (e.g., calcium / potassium-dependent channel, LFA-1 integrin receptor, IL-2 receptor, SLAMF1 (CD150) receptor, IFNγ receptor, TLR receptor, CD4 receptor, virus, etc.) can be supplemented by activating soluble peptides, CARs, small molecule decoys, small molecule ligands, designed nucleic acid ligands, carbohydrate ligands, viral ligands, chimeric biomolecule ligands or fusion proteins, antibodies, antibody-form molecules and / or combinations thereof. In 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 embodiments, any one or more activation modes will result in extensive and / or overall T cell activation sufficient to produce minimal levels of therapeutically relevant biomolecules (e.g., cytokines described herein) that can be captured from activated and expanded T cells for processing into the lumen of BioNV / exosomes.

[0181] In embodiments, methods of broad or global cell modulation may also include binding of the antigen (in addition to other activating molecules) to magnetic or streptavidin-biotin beads to ensure separation of the antigen from the cell activating receptors ( Figure 3 ). For example, in embodiments, the biomarker antigen that forms an immune synapse (IS) with the CAR construct can be conjugated to magnetic or streptavidin-biotin beads and then added to cells containing CAR in vitro to activate the cells. In embodiments, once activated, the biomarker antigen can be mechanically removed from the cell suspension.

[0182] In embodiments, activating low immunogenic cells comprises activating at the DNA level by one or more of the following: a transposase-based approach, a Cre / Lox-based approach, an endonuclease-based approach, a homologous recombination (HR)-based approach, a non-homologous end joining (NEHJ)-based approach, a microhomology-mediated end joining (MMEJ)-based approach, a homology-mediated end joining (HMEJ)-based approach, a small RNA, or a combination thereof. In embodiments, the small RNA comprises one or more of the following: 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), and a small non-coding RNA.

[0183] In embodiments, activating the low immunogenic 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 oligonucleotides (ASO), locked nucleic acid (LNA), splice-switching oligonucleotides (SSO), tRNA, complementary messenger RNA, repeat-associated small interfering RNA (rasiRNA), endonuclease, small non-coding RNA, IRES element, or a combination thereof.

[0184] In embodiments, cell is activated by the endonuclease guided by one or more RNA.In embodiments, the endonuclease guided by RNA can be used for inactivation (for example, causing knockout mutation) and / or activation gene expression (for example, knockout gene repressor), for metabolic regulation cell or otherwise activate the signal transduction pathway of treatment-related biomolecule.In embodiments, activating low immunogenic cells includes activating by endogenous promoter region and / or enhancer region, for example, by knocking in constitutively active promoter / enhancer element upstream of the gene of interest.In embodiments, activating low immunogenic cells includes activating by stable integration genetic elements.In embodiments, activating low immunogenic cells includes activating by transient expression of genetic elements.

[0185] In embodiments, the aforementioned examples of activation (e.g., calcium / potassium-dependent channels, LFA-1 integrin receptors, IL-2 receptors, SLAMF1 (CD150) receptors, IFNγ receptors, TLR receptors, CD4 receptors, and viruses) involve T cells and T cell subsets (e.g., T helper cells, T memory cells, etc.) and NK cells. However, in embodiments, activation of cells via surface receptors with the goal of producing therapeutically relevant biomolecules packaged within the lumen of the BioNV / exosomes can occur in any other cell type applicable to the target disease. For example, in embodiments, broad or global modulation of macrophages can produce varying concentrations of different inflammatory and / or anti-antigen-directed cytokines based on surface receptor activation. In embodiments, the use of alternative cell types can exploit phenotypes associated with cell membrane proteins, properties that have been demonstrated to transfer to BioNV / exosomes. For example, in embodiments, macrophage membrane proteins involved in whole cell organization and barrier-crossing events can be captured in the resulting BioNV / exosomes; the resulting BioNV / exosomes can then cross similar barriers, such as the blood-brain barrier.

[0186] In embodiments, hepatocytes can be regulated to produce therapeutically relevant biomolecules that can be used to treat human diseases (e.g., liver disease). In embodiments, hepatocytes can be broadly / globally regulated 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 segmented into different stages of repair and growth: G0, G1, S phase). In embodiments, hepatocytes can be engaged (activated and / or genetically engineered) by methods similar to those described above for T cell regulation scenarios.

[0187] In embodiments, activation of low immunogenic cells results in a metabolically altered state of the low immunogenic cells. In embodiments, the activation and / or engineering methods can be applied to other cell types involving monofunctional or multifunctional pathways that require regulation of cells from a quiescent state to a metabolically altered state, wherein the newly expressed biomolecules have therapeutically relevant value and can be utilized in BioNV / exosomes via the BioNV / exosome processing methods.

[0188] In embodiments, the one or more therapeutically relevant biomolecules are chemokines, interferons, interleukins, alarmins, lymphokines, perforins, granzymes, granulysins, tumor necrosis factor (TNF), colony stimulating factors, bone morphogenetic proteins (BMPs), 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.

[0189] In embodiments, one or more treatment-related biomolecules include cytokines. In embodiments, cytokines refer to small proteins of about 5kDa to about 50kDa that play an important role in cell signaling; cytokines are generally unable to pass through the lipid bilayer of the cell into the cytoplasm. In embodiments, cytokines may include chemokines, interferons (IFNα / β / γ), interleukins (IL), alarmins, lymphokines and tumor necrosis factors (TNF), colony stimulating factors, bone morphogenetic proteins (BMPs), erythropoietin (EPO), granulocyte stimulating factor (G-CSF), granulocyte macrophage colony stimulating factor (GM-CSF) or a combination thereof. In embodiments, one or more treatment-related biomolecules are proinflammatory cytokines. In embodiments, one or more treatment-related biomolecules are anti-inflammatory cytokines. In embodiments, one or more treatment-related biomolecules are perforins. In embodiments, one or more treatment-related biomolecules are granzymes (e.g., granzymes A, B, H, K and M).

[0190] Table 4: Illustrative interleukins that can be packaged into BioNV / exosomes.

[0191]

[0192]

[0193]

[0194]

[0195] In embodiments, once a cell is activated, it will produce differences in surface markers, cytoplasmic proteins, mRNA and / or DNA expression profiles indicating that the cell has been activated. Surface markers can be analyzed by flow cytometry to accurately measure the degree of activation. In addition, any of the following can be used alone or in combination to analyze the degree of cytoplasmic cytotoxic protein production by flow cytometry, immunoblotting, mass spectrometry, ELISA, etc. to measure the degree of activation. In addition, standard mRNA detection and amplification procedures (such as nested or qPCR, but not limited to these) can be used to measure mRNA levels. In addition, the gene spectrum differences between resting cells and activated cells can be analyzed to determine the degree of activation. This can be achieved by DNA expression arrays or similar technologies.

[0196] In embodiments, the same measurements described above (minus genetic measurements, as BioNV does not contain cellular nucleic acids) can be used to detect and monitor the extent of cellular activation and can also be applied to BioNV. Indirect measurements can be achieved by measuring the same activation markers.

[0197] In embodiments, therapeutic exosomes encapsulate a payload; For example, "lumen load", or the ability of exosomes to load a payload into the lumen (the space in the biomimetic nanovesicle). In embodiments, the payload is one or more of a biologic, a nucleic acid, a fusion protein, a fluorescent protein, a tracer dye, a radionuclide, and / or a small molecule. In embodiments, the payload is a therapeutic payload for the type of disease targeted by CAR. In embodiments, the payload includes one or more of: an alkylating agent, an anthracycline, an antimetabolite, an antitumor antibiotic, an antibody or an antibody form, a corticosteroid, a plant alkaloid, a topoisomerase inhibitor, a checkpoint inhibitor, an anti-infective agent, and / or a growth factor.

[0198] In embodiments, the nucleic acid payload encodes one or more of the following: a CRISPR / Cas component, 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 ribosomal RNA (rRNA), a short hairpin (shRNA) complementary messenger RNA, a repeat-associated small interfering RNA (rasiRNA), and a small non-coding RNA.

[0199] In an embodiment, one or more gene editing agents are site-directed endonucleases, TALENs, ZFNs, ribonuclease P RNAs, CRISPR / Cas nucleases, C2c1, C2c2, C2c3, Cas9, Cpf1, TevCas9, Archaea 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 an embodiment, the gene editing agent may further include gRNA, which as used herein refers to guide RNA. In an embodiment, the gRNA may be a sequence complementary to a coding or non-coding sequence and may be customized according to the specific sequence to be targeted. In an embodiment, 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 an embodiment, the gRNA sequence may be a sense sequence or an antisense sequence. In embodiments, when a gene editing agent composition is administered herein, it is preferably, but not limited to, including two or more gRNAs; however, a single gRNA may also be used.

[0200] In embodiments, the gene editing payload includes a transactivation response region (TAR) loop system. In embodiments, therapeutic BioNV / exosomes encapsulate a plasmid expressing a gene editing agent and contain a TAR loop sequence between the 5' end of the promoter and the gene editing agent / guide cassette, acting as a barrier to block transcription. In embodiments, transcription will only be triggered in infected cells containing the HIV Tat protein. In embodiments, the Tat protein binds to the TAR loop, relaxing it and releasing the promoter for transcription, thereby expressing the editing agent and its guide.

[0201] In embodiments, BioNV / exosomes can encapsulate disease-specific therapeutic payloads. For example, increased levels of perforin or granzymes from activated lymphocytes can be packaged into the resulting BioNV / exosomes to penetrate cancer cells. In embodiments, increased cellular production of alarmins can be packaged into the resulting BioNV / exosomes to enhance recruitment of additional lymphocytes to solid tumor sites. In embodiments, cells can be modified to increase production of gene-editing payloads, allowing BioNV / exosomes to incorporate gene-editing materials for tissue-specific targeting.

[0202] In embodiments, if a single type of cytokine or a small subset of cytokines with therapeutically relevant value needs to be encapsulated within the lumen of the BioNV / exosome, focused cellular regulation may be warranted, rather than a broad / global expression approach that primarily accompanies a multitude of regulatory scenarios.

[0203] In embodiments, by manipulating regulatory points within signal transduction sites, therapeutically relevant biomolecules (e.g., cytokines) can be selectively expressed from within any given cell. In embodiments, one point (or multiple points) within a single signal transduction pathway (or pathways) can be activated, for example, activating a kinase, (de)activating a phosphorylase, activating a hydrolase (e.g., a GTPase), introducing an inhibitor (to diffuse or block a signal from one pathway to another), or accelerating an activator / agonist. In embodiments, these signal transduction pathways can be selectively activated by providing proteins, peptides, small molecules, nucleic acids, carbohydrates, chimeric molecules, viral ligands, inorganic elements / compounds (e.g., calcium), etc., to cells, alone or in combination, to focus cellular pathways to drive the expression of therapeutically relevant biomolecules within the cell, which can then be packaged into the lumen of the BioNV / exosomes ( Figure 3 In embodiments, regulatory points may include, but are not limited to, one or more cell surface receptors that are targeted individually or in combination to focus intracellular signals to express biomolecules of interest. In embodiments, cells may be genetically modified to be particularly sensitive to activation of these pathways, thereby generating metabolic / phenotype regulatory cells (of any type) that can produce BioNV / exosomes. In embodiments, cells may be made "particularly sensitive" to activation by certain stimuli by overexpressing cell surface receptors, overexpressing intracellular signaling molecules (e.g., STAT, NF-κB, MAPK / ERK / ATM kinases, etc.), and / or constitutively active mutants thereof.

[0204] In embodiments, regulatory points within cells can be engineered to express a particular therapeutically relevant protein of interest into a stable cell line (e.g., stable integration for constitutive expression).

[0205] In embodiments, transcription factors can be activated intracellularly by providing small molecules, (de)phosphorylation events, or nucleic acids, etc. In embodiments, transcription factors can be transiently expressed via plasmids. In embodiments, transcription factors can be stably expressed by integration (e.g., with a constitutively active promoter) to generate stable cell lines with constitutive signaling of signaling pathways associated with therapeutic biomolecules.

[0206] In an embodiment, the promoter region or enhancer region of a gene can be regulated by overexpression of a specific transcription factor, by microRNA, by tRNA, or by activation or repression using a (g)RNA-guided endonuclease with an activation or repression domain associated therewith (e.g., CRIPSRa / CRISPRi).

[0207] In embodiments, therapeutically relevant genes of interest can be integrated (stably or transiently) into cells of interest (based on desired properties, such as membrane proteins that are inferred to cross a barrier). In embodiments, stably integrated genes can be activated by any of the methods described herein.

[0208] In embodiments, background (unwanted) mRNA can be silenced using interfering RNA (e.g., siRNA, RNAi, etc.) to enhance the presence / expression of the desired mRNA for the protein of interest. In embodiments, mRNA can also be regulated by an IRES element. In embodiments, specific splice variants can be enhanced by adding IRES enhancing and / or inhibiting biomolecules to produce desired therapeutically relevant peptides / proteins that can be packaged into BioNV / exosomes during post-activation cellular processing.

[0209] In embodiments, unwanted genes can be knocked out to enhance expression of desired therapeutically relevant genes. One skilled in the art will appreciate methods that can be used to transiently and stably knock out unwanted genes and / or entire signaling pathways during BioNV / exosome processing.

[0210] In embodiments, it is possible to centrally activate by CAR constructs. In embodiments, the intercellular domain of CAR constructs can be designed to include an activation domain specifically focused on a single (or multiple) signal transduction pathway. In embodiments, these constructs can be activated (for example, providing specific stimulation to cells) by the extracellular binding domain of CRA constructs.

[0211] In embodiments, the gene modulation methods described herein for enhancing the presence of therapeutically relevant biomolecules within desired cells whose metabolism is adapted to produce the desired therapeutically relevant biomolecules can be used to increase the concentration of the therapeutically relevant biomolecules (or to modulate to a desired concentration) such that the modulated cells can then be processed into BioNVs or naturally shed exosomes containing the desired concentration of the therapeutically relevant biomolecule within their lumen to treat a mammalian disease.

[0212] Therapeutic BioNV

[0213] In some aspects, the present disclosure includes therapeutic BioNVs comprising (i) one or more membrane-embedded targeting agents that target one or more cellular biomarkers, (ii) one or more α-phagocytic integrin, CCL2, H2-M3, FasL, MFEG8, PD-L1 (in BioNVs derived from unactivated cell sources) and / or CTLA-4, SerpinB9, and anti-IL-6R antibodies or antibody-based membrane-embedded proteins, (iii) one of CD24, CD47, CD200, chimeric CD24 / CD47, chimeric CD24 / CD200, and chimeric CD47 / CD200, or both of CD24, CD47, and CD200, and (iv) substantially A membrane lacking a protein of one or more of HLA-A, HLA-B, HLA-C, HLA-F, CIITA, IL-6, TRAC, TRBC, one of 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 is a chemokine, an interferon, an interleukin, an alarmin, a lymphokine, a perforin, a tumor necrosis factor (TNF), a colony stimulating factor, a bone morphogenetic protein (BMP), erythropoietin (EPO), granulocyte-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), a gene editing payload, a fusion protein, an antibody or an antibody format, or a combination thereof.

[0214] In some aspects, the present disclosure includes therapeutic BioNVs comprising (i) one or more membrane-embedded targeting agents that target one or more cellular biomarkers, (ii) one or more α-phagocytic integrin, CCL2, H2-M3, FasL, MFEG8, PD-L1 (in BioNVs derived from unactivated cell sources) and / or CTLA-4, SerpinB9, and anti-IL-6R antibodies or antibody-form membrane-embedded proteins, (iii) membrane-embedded proteins of CD24 and CD47 or chimeric CD24 / CD47, and (iv) a cell-free cell that is substantially free of HLA-A, HLA-B, HLA-C, HLA-F, CIITA, IL-6, TRA C, a membrane of a protein comprising one or more of TRBC, HLA-E or HLA-G, one of SerpinB9 and CD200, and one or more of IL-4, IL-10, and IL-16, wherein the one or more therapeutically relevant biomolecules are a chemokine, an interferon, an interleukin, an alarmin, a lymphokine, a perforin, a granzyme, a granulysin, a tumor necrosis factor (TNF), a colony stimulating factor, a bone morphogenetic protein (BMP), erythropoietin (EPO), granulocyte-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), a gene editing payload, a fusion protein, an antibody or an antibody form, or a combination thereof.

[0215] In embodiments, the PD-L1 expressing BioNV is derived from unactivated cells and the BioNV is substantially devoid of perforin and / or granzymes.

[0216] In an embodiment, BioNV expresses and / or has activity on one or more antigens, ligands and / or receptors of a tumor cell or cancer tissue. In an embodiment, the one or more antigens, ligands and / or receptors of a tumor cell or cancer tissue are listed in Table 4, Table 5 and / or Table 6. In an embodiment, the one or more antigens, ligands and / or receptors of a tumor cell or cancer tissue are neoantigens, such as mutant variants or peptides specific for a cancer or disease state that are not otherwise present in the subject to be treated. In an embodiment, BioNV expresses and / or has activity on an immune cell co-stimulatory molecule. In an embodiment, the co-stimulatory molecule of an immune cell is IL-15.

[0217] In an embodiment, BioNV comprises one or more outward-facing, membrane-embedded targeting agents (e.g., CARs) capable of binding to one or more target molecules. In an embodiment, BioNV comprises an outward-facing, membrane-embedded CAR capable of binding to a target molecule. In an embodiment, therapeutic BioNV is biomimetic due to a nanovesicle composition derived from the plasma membrane of an allogeneic, low-immunogenic modified cell. In an embodiment, therapeutic BioNV comprises a plasma membrane-derived lipid bilayer that fully encapsulates an aqueous core that can accommodate a variety of cell-derived molecules, including perforins, granzymes, cytokines, gene editing payloads, and the like. In an embodiment, the aqueous core of therapeutic BioNV can further encapsulate exogenous biologics, fluorescent proteins, tracer dyes, radionuclides, and small molecules, as well as other therapeutic agents.

[0218] In embodiments, CAR constructs can include a variety of structural molecules. The structural function of prototype CAR includes fusion protein, and the fusion protein includes extracellular (or outward-facing) binding moiety (for example, scFv), which is connected to a transmembrane domain (for example, CD28, CD3ζ, CD4, CD8α, ICOS, etc.) by a hinge peptide (for example, CH2 / CH3 domains, Gly-Gly-Ser peptide bonds, CD28 peptides, CD8α peptides, etc. from IgG Fc areas), followed by various intracellular signaling domains (for example, 4-1BB, CD3ζ, CD28, 4-1BB, ICOS, CD27, OX40, etc.). In embodiments, BioNV lacks the intracellular mechanism of whole cells, so CAR design does not require any intracellular signaling molecules (primary CAR constructs). In embodiments, CAR constructs include the extracellular scFV binding moiety fused to CD28 transmembrane domains with IgG CH2 / CH3 linkers, and substantially lack any intracellular domain or function. In embodiments, the CAR construct has a prototypical intracellular domain, such as a PLA2 domain from AAV, a fusion protein, a radionuclide binding domain, a cytoskeletal element, a small molecule transport domain, etc., exchanged or otherwise fused with an anchor protein, which may facilitate fusion with the target cell and / or packaging and release of the therapeutic payload.

[0219] In embodiments, CAR antigen binding molecules include a variety of binding moieties, including binding domains based on antibodies or antibody forms. In embodiments, therapeutic BioNV includes an antibody or antibody form binding moiety selected from one or more of the following: monoclonal antibody, polyclonal antibody, antibody fragment, Fab, Fab', Fab'-SH, F(ab')2, Fv, single-chain Fv (scFv), double antibody, nano antibody, linear antibody, bispecific antibody, multispecific antibody, chimeric antibody, humanized antibody, human antibody and a fusion protein comprising the antigen binding portion of an antibody. In embodiments, CAR constructs include binding moieties with bispecific T cell engagers (BiTE), viral epitope recognition receptors (VERR), variable heavy chain IgG fragment VHH, VNAR or by engineering T cell receptors (TCR).

[0220] To ensure the correct directionality of the CAR and eliminate BioNVs lacking the CAR, in embodiments, HPLC-based affinity chromatography techniques can be used to select and concentrate only BioNVs exposed to the solvent with sufficient surface concentrations of CARs. HPLC-based affinity chromatography techniques can be used to reduce the concentration of contaminating cellular material and NVs containing immunogenic cell surface markers by positive or negative selection.

[0221] In embodiments, therapeutic BioNVs may include NVs with only the outer plasma membrane leaflet, only the inner plasma membrane leaflet, and / or both leaflets of the plasma membrane lipid bilayer intact. In embodiments, iPSC-derived NVs include additional lipid additives (e.g., phosphatidylethanolamine, phosphatidylcholine, phosphatidylinositol, ceramide, phosphatidylcholine, etc.), non-ionic surfactants (e.g., sorbitol 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, Fc, etc.) to improve physicochemical properties such as thermal stability, clearance, and therapeutic payload packaging / release. The cholesterol content and the length and saturation of the hydrocarbon chains of the phospholipids affect the rigidity and stability of the bilayer, which in turn affects the ability of the NV to load and release drugs, biomolecules, and other therapeutic payloads. In embodiments, therapeutic BioNVs also incorporate zwitterionic lipids and methods of using zwitterionic lipids, as described, for example, in U.S. Patent Publication No. US20130216607, the contents of which are incorporated herein by reference in their entirety. Accordingly, functionalizing the hydrophilic head of the lipid with polymers or biomolecules can provide additional features to the vesicle surface, thereby shaping its interactions with blood components, tissues, and the immune system in the body.

[0222] In embodiments, therapeutic BioNV encapsulates a payload; for example, "lumen load", or the ability of BioNV to load a payload into the lumen (the space in the biomimetic nanovesicle). In embodiments, the payload is one or more of a biologic, a nucleic acid, a fusion protein, a fluorescent protein, a tracer dye, a radionuclide, and / or a small molecule. In embodiments, the payload is a therapeutic payload for the type of disease targeted by CAR. In embodiments, the payload includes one or more of: an alkylating agent, an anthracycline, an antimetabolite, an antitumor antibiotic, an antibody or an antibody form, a corticosteroid, a plant alkaloid, a topoisomerase inhibitor, a checkpoint inhibitor, an anti-infective agent, and / or a growth factor.

[0223] In embodiments, the nucleic acid payload encodes one or more of the following: a CRISPR / Cas component, 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 ribosomal RNA (rRNA), a short hairpin (shRNA) complementary messenger RNA, a repeat-associated small interfering RNA (rasiRNA), and a small non-coding RNA.

[0224] In embodiments, the payload includes gene editing nucleic acids and / or proteins, such as TALEN, ZFN, ribonuclease P RNA, C2c1, C2c2, C2c3, Cas9, Cpf1, TevCas9, Archaea 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 editing agents. In embodiments, the gene editing agent may also include gRNA, which refers to guide RNA as used herein. In embodiments, gRNA can be a sequence complementary to a coding or non-coding sequence and can be customized according to the specific sequence to be targeted. In embodiments, 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 embodiments, the gRNA sequence can be a sense sequence or an antisense sequence. In embodiments, when a gene editing agent composition is administered herein, it is preferably, but not limited to, including two or more gRNAs; however, a single gRNA may also be used.

[0225] In embodiments, the therapeutic BioNV comprises one or more perforin molecules and / or one or more granzyme molecules derived from the cell from which the BioNV was derived. In embodiments, the therapeutic BioNV comprises one or more perforin molecules and one and / or more granzyme molecules exogenously added to the BioNV. In embodiments, the BioNV designed to carry granzymes lacks CD200 and SerpinB9.

[0226] In embodiments, Therapeutic BioNV can deliver a gene-editing payload containing a transcriptional activation response region (TAR) loop system. In embodiments, Therapeutic BioNV encapsulates a plasmid expressing a gene-editing agent and contains a TAR loop sequence between the 5' end of the promoter and the gene-editing agent / guide cassette, acting as a barrier to block transcription. In embodiments, transcription will only be triggered in cells that are infected and contain the HIV Tat protein. In embodiments, the Tat protein binds to the TAR loop, causing it to relax and releasing the promoter for transcription, thereby expressing the editing agent and its guide.

[0227] In an embodiment, the primary targeted therapeutic BioNV can be used to deliver small molecule therapeutic payloads. In an embodiment, the second generation (or third or fourth generation) of therapeutic BioNV containing CAR derived from activated lymphocytes may contain cytokines and other cytotoxic peptides. In an embodiment, the therapeutic BioNV can be formatted to encapsulate and deliver plasmid DNA, for example, to express gene editing nucleases and gRNA in target cells. Alternatively or additionally, in an embodiment, the therapeutic BioNV can encapsulate nucleases and gRNA. In an embodiment, the targeted second generation (or third or fourth generation) therapeutic BioNV can be designed to encapsulate and deliver additional therapeutic proteins or peptides of interest.

[0228] In embodiments, the therapeutic BioNV is substantially devoid of the above, and is thus allogeneic and / or hypoimmunogenic. In embodiments, the therapeutic BioNV does not produce an adverse immune response when infused into a subject to treat a disease.

[0229] In embodiments, therapeutic BioNVs are formed by processing activated low immunogenic cells by one or more of the following: sonication, adaptive focused acoustic technology, French press, extrusion, continuous extrusion, enzymatic disruption of cells (e.g., trypsinization), detergent cell lysis, and / or electroporation. In embodiments, cell disruption is performed by continuous extrusion, e.g., if the BioNVs are derived from whole cells using a continuous extrusion process, the gradually decreasing pore size of the polycarbonate filter is consistent with the diameter and volume of the BioNVs.

[0230] In embodiments, the size uniformity of therapeutic BioNVs can be analyzed by dynamic light scattering (DLS), flow cytometry, mass spectrometry, and other methods for determining particle size. In embodiments, therapeutic BioNVs can be filtered based on particle size or size range to optimize renal clearance and other clinically relevant NV properties. In embodiments, the size of therapeutic BioNVs is about 20 nm to 1200 nm. In embodiments, the size of therapeutic BioNVs is 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 embodiments, the therapeutic BioNVs have a size 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, 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.

[0231] Therapeutic exosomes

[0232] In some aspects, the present disclosure includes therapeutic exosomes comprising (i) one or more membrane-embedded targeting agents that target one or more cellular biomarkers, (ii) one or more α-phagocytic integrin, CCL2, H2-M3, FasL, MFEG8, PD-L1 (in BioNV derived from an unactivated cell source) and / or CTLA-4, SerpinB9 and anti-IL-6R antibodies or antibody-based membrane-embedded proteins, (iii) one of CD24, CD47, CD200, chimeric CD24 / CD47, chimeric CD24 / CD200 and chimeric CD47 / CD200, or both of CD24, CD47 and CD200, (iv) a membrane-embedded protein substantially lacking Membranes of proteins comprising one or more of HLA-A, HLA-B, HLA-C, HLA-F, CIITA, IL-6, TRAC, TRBC, one of 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, perforins, tumor necrosis factor (TNF), colony stimulating factors, bone morphogenetic proteins (BMPs), 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.

[0233] In some aspects, the present disclosure includes therapeutic exosomes comprising (i) one or more membrane-embedded targeting agents that target one or more cellular biomarkers, (ii) one or more α-phagocytic integrin, CCL2, H2-M3, FasL, MFEG8, PD-L1 (in BioNV derived from unactivated cell sources) and / or CTLA-4, SerpinB9 and anti-IL-6R antibodies or antibody-form membrane-embedded proteins, (iii) membrane-embedded proteins of CD24 and CD47 or chimeric CD24 / CD47, and (iv) substantially lacking HLA-A, HLA-B, HLA-C, HLA-F, CIITA, IL-6, TRAC , one or more of TRBC, HLA-E or HLA-G, one of SerpinB9 and CD200, 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, perforins, granzymes, granulysins, 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 forms, or combinations thereof.

[0234] In embodiments, the PD-L1 expressing exosomes are derived from unactivated cells and the exosomes are substantially devoid of perforin and / or granzymes.

[0235] In embodiments, exosomes express one or more antigens, ligands and / or receptors of tumor cells or cancer tissues and / or have their activity. In 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 embodiments, one or more antigens, ligands and / or receptors of tumor cells or cancer tissues are neoantigens, such as mutant variants or peptides that are specific to cancer or disease states, and the mutant variants or peptides are not present in the subject to be treated under other circumstances. In embodiments, exosomes express immune cell co-stimulatory molecules and / or have their activity. In embodiments, the co-stimulatory molecule of immune cells is IL-15. In embodiments, exosomes expressing one or more neoantigens and-or co-stimulatory molecules and / or having their activity can be used to produce whole cells and / or BioNVs described herein, for example, by neoantigen targeted activation / stimulation.

[0236] In embodiments, exosomes include one or more outward-facing, membrane-embedded targeting agents (e.g., CARs) capable of binding to one or more target molecules. In embodiments, exosomes include outward-facing, membrane-embedded CARs capable of binding to target molecules. In embodiments, therapeutic exosomes are biomimetic due to the nanovesicle compositions of the plasma membrane of allogeneic, low immunogenic modified cells. In embodiments, therapeutic exosomes include a lipid bilayer derived from the plasma membrane, which fully encapsulates an aqueous core that can accommodate a variety of cell-derived molecules, including perforins, granzymes, cytokines, gene editing payloads, etc. In embodiments, the aqueous core of therapeutic exosomes can further encapsulate exogenous biological agents, fluorescent proteins, tracer dyes, radionuclides, small molecules, and other therapeutic agents.

[0237] In embodiments, CAR constructs may include a variety of structural molecules. The structural function of prototype CAR includes a fusion protein, which includes an extracellular (or outward-facing) binding portion (e.g., scFv), which is connected to a transmembrane domain (e.g., CD28, CD3ζ, CD4, CD8α, ICOS, etc.) by a hinge peptide (e.g., CH2 / CH3 domains, Gly-Gly-Ser peptide bonds, CD28 peptides, CD8α peptides, etc. from IgG Fc regions), followed by various intracellular signaling domains (e.g., 4-1BB, CD3ζ, CD28, 4-1BB, ICOS, CD27, OX40, etc.). In embodiments, therapeutic exosomes lack the intracellular mechanism of whole cells, so CAR design does not require any intracellular signaling molecules (primary CAR constructs). In embodiments, CAR constructs include an extracellular scFV binding portion fused to a CD28 transmembrane domain with an IgG CH2 / CH3 linker, and substantially lack any intracellular domain or function. In embodiments, the CAR construct has a prototypical intracellular domain, such as a PLA2 domain from AAV, a fusion protein, a radionuclide binding domain, a cytoskeletal element, a small molecule transport domain, etc., exchanged or otherwise fused with an anchor protein, which may facilitate fusion with the target cell and / or packaging and release of the therapeutic payload.

[0238] In embodiments, CAR antigen binding molecules include a variety of binding moieties, including binding domains based on antibodies or antibody forms. In embodiments, therapeutic exosomes include antibodies or antibody forms binding moieties selected from one or more of the following: monoclonal antibodies, polyclonal antibodies, antibody fragments, Fab, Fab', Fab'-SH, F(ab')2, Fv, single-chain Fv (scFv), double antibodies, nano antibodies, linear antibodies, bispecific antibodies, multispecific antibodies, chimeric antibodies, humanized antibodies, human antibodies and fusion proteins comprising the antigen binding portion of antibodies. In embodiments, CAR constructs include binding moieties with bispecific T cell engagers (BiTE), viral epitope recognition receptors (VERR), variable heavy chain IgG fragments VHH, VNAR or by engineering T cell receptors (TCR).

[0239] To ensure the correct directionality of CAR and eliminate CAR-deficient exosomes, in embodiments, HPLC-based affinity chromatography can be used to select and concentrate only exosomes with sufficient surface concentrations of solvent-exposed CAR. HPLC-based affinity chromatography can be used to reduce the concentration of contaminating cellular material and NV containing immunogenic cell surface markers by positive or negative selection.

[0240] In embodiments, therapeutic exosomes differ from therapeutic BioNVs in that they are naturally shed from cells via a canonical vesicular pathway, i.e., exosomes can be harvested from cells without irreversibly mechanically disrupting (e.g., rupturing, shearing, etc.) the plasma membrane of the cells from which they are derived. In embodiments, exosomes can be harvested from cell culture medium, e.g., by differential ultracentrifugation of the culture medium.

[0241] In embodiments, harvesting therapeutic exosomes from activated low immunogenic cells comprises inducing hypoxia. In embodiments, various strategies (e.g., inducing cellular stress) can be used to increase therapeutic exosome formation by cells.

[0242] In embodiments, harvesting therapeutic exosomes from activated low immunogenic cells comprises expressing or increasing the expression of one or more cytokines. In embodiments, overexpression of cytokines such as tetraspanin CD9 or hypoxia-inducible factor-1α can induce exosome formation for harvesting.

[0243] In embodiments, harvesting therapeutic exosomes from activated low immunogenic cells comprises providing one or more small molecule exosome modulators. In embodiments, the provided small molecule modulators may include fenoterol, norepinephrine, N-methyldopamine, and / or mephenesin.

[0244] In embodiments, harvesting therapeutic exosomes from activated low immunogenic cells comprises supplementing the culture medium with one or more exosomal factors. In embodiments, the exosomal factors may comprise nutritional supplements to the culture medium, such as forskolin. In embodiments, the exosomal factors may comprise providing stimulation of specific cell types to increase the production of therapeutic exosomes, for example, cardiac fibroblasts may be stimulated with TGF-β to increase collagen expression, thereby leading to exosome formation.

[0245] In embodiments, therapeutic exosomes encapsulate a payload; For example, "lumen load", or the ability of exosomes to load a payload into the lumen (the space in the biomimetic nanovesicle). In embodiments, the payload is one or more of a biologic, a nucleic acid, a fusion protein, a fluorescent protein, a tracer dye, a radionuclide, and / or a small molecule. In embodiments, the payload is a therapeutic payload for the type of disease targeted by CAR. In embodiments, the payload includes one or more of: an alkylating agent, an anthracycline, an antimetabolite, an antitumor antibiotic, an antibody or an antibody form, a corticosteroid, a plant alkaloid, a topoisomerase inhibitor, a checkpoint inhibitor, an anti-infective agent, and / or a growth factor.

[0246] In embodiments, the nucleic acid payload encodes one or more of the following: a CRISPR / Cas component, 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 ribosomal RNA (rRNA), a short hairpin (shRNA) complementary messenger RNA, a repeat-associated small interfering RNA (rasiRNA), and a small non-coding RNA.

[0247] In embodiments, the payload includes gene editing nucleic acids and / or proteins, such as TALEN, ZFN, ribonuclease P RNA, C2c1, C2c2, C2c3, Cas9, Cpf1, TevCas9, Archaea 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 editing agents. In embodiments, the gene editing agent may also include gRNA, which refers to guide RNA as used herein. In embodiments, gRNA can be a sequence complementary to a coding or non-coding sequence and can be customized according to the specific sequence to be targeted. In embodiments, 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 embodiments, the gRNA sequence can be a sense sequence or an antisense sequence. In embodiments, when a gene editing agent composition is administered herein, it is preferably, but not limited to, including two or more gRNAs; however, a single gRNA may also be used.

[0248] In embodiments, therapeutic exosomes can deliver gene-editing payloads containing a transcriptional activation response region (TAR) loop system. In embodiments, therapeutic BioNV encapsulates a plasmid expressing a gene-editing agent and contains a TAR loop sequence between the 5' end of the promoter and the gene-editing agent / guide cassette, acting as a barrier to block transcription. In embodiments, transcription will only be triggered in infected cells containing the HIV Tat protein. In embodiments, the Tat protein binds to the TAR loop, relaxing it and releasing the promoter for transcription, thereby expressing the editing agent and its guide.

[0249] In embodiments, primary targeted therapeutic exosomes can be used to deliver small molecule therapeutic payloads. In embodiments, second-generation (or third or fourth generation) therapeutic exosomes derived from activated lymphocytes containing CAR may contain cytokines and other cytotoxic peptides. In embodiments, therapeutic exosomes may be formatted to encapsulate and deliver plasmid DNA, for example, to express gene editing nucleases and gRNA in target cells. Alternatively or additionally, in embodiments, therapeutic exosomes may encapsulate nucleases and gRNA. In embodiments, targeted second-generation (or third or fourth generation) therapeutic exosomes may be designed to encapsulate and deliver additional therapeutic proteins or peptides of interest.

[0250] In embodiments, the size uniformity of therapeutic exosomes can be analyzed by dynamic light scattering (DLS), flow cytometry, mass spectrometry, and other methods for determining particle size. In embodiments, therapeutic exosomes can be filtered according to particle size or size range to optimize renal clearance and other clinically relevant NV characteristics. In embodiments, the large size of therapeutic exosomes is about 10 nm to 200 nm. In embodiments, the size of therapeutic exosomes may be 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. In embodiments, the therapeutic exosomes have a size 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.

[0251] Methods of treating diseases

[0252] In embodiments, therapeutic BioNV / exosomes can be used to treat and / or prevent a disease or disorder. In embodiments, the disease or disorder is cancer, an infectious disease, a genetic disorder, or an orphan disease.

[0253] In embodiments, the cancer is one or more of carcinoma, sarcoma, myeloma, leukemia, lymphoma, mixed cancer, and / or metastatic cancer.

[0254] In embodiments, the cancer is selected from acute biphenotypic leukemia, acute eosinophilic leukemia, acute lymphoblastic leukemia, acute lymphocytic leukemia, acute myeloid dendritic cell leukemia, acute myeloid leukemia, lung adenocarcinoma, adrenal tumors, adrenocortical carcinoma, AIDS-related cancers, AIDS-related lymphomas, alveolar soft tissue and cardiac sarcomas, amyloidosis, anal cancer, anaplastic large cell lymphoma, angioimmunoblastic T-cell lymphoma, appendix cancer, astrocytoma, ataxia telangiectasia, attenuating familial adenomatous polyposis, B-cell prolymphocytic leukemia, basal cell carcinoma, Beckwith-Wiedemann Syndrome, bile duct cancer, Birt-Hogg-Dubé Syndrome, bladder cancer, bone cancer, brain and nervous system cancers, brain stem glioma, brainstem glioma, Glioma, brain tumor, breast cancer, bronchial adenoma / carcinoid, Burkitt's Lymphoma, carcinoid tumor, Carney Complex, central nervous system tumor, cerebellar astrocytoma, cerebral astrocytoma, cervical cancer, fibroproliferative ganglioglioma in children, bile duct cancer, chondrosarcoma, chronic lymphocytic leukemia, chronic myeloid leukemia, chronic T-cell lymphocytic leukemia, colon cancer, colorectal cancer, Cowden Syndrome, craniopharyngioma, cutaneous T-cell lymphoma, dermatofibrosarcoma protuberans, fibroproliferative small round cell tumor, diffuse gastric cancer, diffuse large B-cell lymphoma, endocrine system cancer, endocrine tumor, endometrial cancer, eosinophilic leukemia, ependymoma, epithelioid hemangioendothelioma (EHE), esophageal cancer, Ewing's sarcoma. Sarcoma), extragonadal germ cell tumors, 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 and gynecological cancers, germ cell tumors, gestational trophoblastic disease, gestational trophoblastic tumor, glioblastoma, glioma, hairy cell leukemia, head and neck cancer, hematopoietic cancer, hepatocellular carcinoma, hepatosplenic T-cell lymphoma, HIV-related cancers, Hodgkin's lymphoma Lymphoma), hypopharyngeal cancer, inflammatory breast cancer, intravascular large B-cell lymphoma, invasive cribriform carcinoma, invasive lobular carcinoma, islet cell carcinoma (endocrine pancreas), islet cell tumors, juvenile polyposis syndrome, Kaposi sarcomaSarcoma), keratoacanthomas, renal cancer, lacrimal gland tumors, large granular lymphocytic leukemia, laryngeal and hypopharyngeal cancer, leiomyomatosis and renal cell carcinoma, leiomyosarcoma, Li-Fraumeni syndrome (LFS), liposarcoma, liver cancer, lung cancer, primary cutaneous lymphoma, lymphomatoid granuloma, lymphoplasmacytic lymphoma, Lynch syndrome, malignant fibrous histiocytoma of bone, mantle cell lymphoma, marginal zone B-cell lymphoma, mast cell leukemia, mastocytosis, mediastinal large B-cell lymphoma, medullary carcinoma, medulloblastoma, melanoma, meningioma, Merkel cell carcinoma, mesothelioma, mixed polyposis syndrome, mucosa-associated lymphoid tissue lymphoma, Muir-Torre syndrome Syndrome; MTS), Multiple Endocrine Neoplasia Syndrome, Multiple Endocrine Neoplasia Type 1, Multiple Endocrine Neoplasia Type 2, Multiple Myeloma, Multiple Myeloma / Plasma Cell Neoplasm, Mycosis Fungoides, Myelodysplastic Syndrome, MYH-Associated Polyposis, Myxosarcoma, Nasal and Paranasal Sinus Cancer, Wilms Tumor, Nasopharyngeal Carcinoma, Nasopharyngeal Carcinoma, Neuroblastoma, Neuroendocrine Tumors, 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 Cancer, Oligodendroglioma, Optic Pathway Glioma, Oral and / or Oropharyngeal Cancer, Osteosarcoma, Ovarian Cancer, Ovarian Epithelial Cancer, Ovarian Germ Cell Tumor, Pancreatic Cancer, Papillary Renal Cell Carcinoma, Paranasal Sinus and Nasal Cavity Cancer, Parathyroid Cancer, Pelvic Cancer, Penile Cancer, Peutz-Jeghers Syndrome 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, primitive neuroectodermal tumor, prostate cancer, rectal cancer, kidney cancer, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, primary cutaneous sarcoma, sebaceous carcinoma, Sézary Syndrome, skin appendage tumor, skin cancer, small bowel cancer, small cell lung cancer, small intestinal cancerCancer), splenic marginal zone lymphoma, squamous cell carcinoma of the lung, squamous cell carcinoma, squamous cell skin cancer, gastric cancer, surface epithelial-stromal tumors, T-cell prolymphocytic leukemia, testicular cancer, thoracic and respiratory tract cancer, thymoma, thymic carcinoma, thyroid cancer, transitional cell carcinoma, transitional cell carcinoma, adenoid cystic carcinoma, tuberous sclerosis complex, tubular carcinoma, Turcot syndrome, carcinoma of unknown primary, unclassified carcinoma, ureteral cancer, urethral cancer, uterine cancer, uterine sarcoma, uveal melanoma, vaginal cancer, optic pathway and hypothalamic gliomas, von Hippel-Lindau (VHL) syndrome, vulvar cancer, Wilms tumor, and xeroderma pigmentosum.

[0255] In an embodiment, BioNV can target cancer cells associated with adenoid cystic carcinoma, adrenal tumors, amyloidosis, anal cancer, appendix cancer, astrocytoma, ataxia telangiectasia, familial adenomatous polyposis, Beckwith-Wiedemann syndrome, bile duct cancer, Burt-Hoag-Dub syndrome, bladder cancer, bone cancer, brainstem glioma, brain tumor, breast cancer, carcinoid tumor, Carney syndrome, central nervous system tumor, cervical cancer, colorectal cancer, Cowden syndrome, craniopharyngioma, desmoplastic infantile ganglioglioma, endocrine tumors, ependymoma, esophageal cancer, Ewing's sarcoma, tumors, 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 tumors, germ cell tumors, 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 cancers, islet cell tumors, juvenile polyposis syndrome, kidney cancer, lacrimal gland tumors, laryngeal and hypopharyngeal cancers, acute lymphoblastic leukemia, acute lymphocytic leukemia, acute myeloid leukemia, B cell Proliferative lymphocytic 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-Tore syndrome, multiple endocrine neoplasia type 1, multiple endocrine neoplasia type 2, multiple myeloma, myelodysplastic syndrome, MYH-associated polyposis, nasal and paranasal sinus cancer, nasopharyngeal carcinoma, neuroblastoma, neuroendocrine tumors, Neurofibromatosis type 1, neurofibromatosis type 2, nevoid basal cell carcinoma syndrome, oral cavity and oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, parathyroid cancer, penile cancer, Peutz-Jeghers syndrome, pituitary tumors, pleuropulmonary blastoma, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, alveolar soft tissue and cardiac sarcomas, Kaposi sarcoma, skin cancer, small intestine cancer, stomach cancer, testicular cancer, thymoma, thyroid cancer, tuberous sclerosis complex, Tucker's syndrome, macular degeneration of unknown primary, uterine cancer, vaginal cancer, von Hippel-Lindau syndrome, Wilms tumor, and xeroderma pigmentosum.

[0256] In embodiments, therapeutic BioNVs / exosomes for treating and / or preventing a disease or condition target one or more biomarkers in Table 5 and / or Table 6.

[0257] Table 5: Illustrative checkpoint biomarkers and checkpoint biomarker ligands.

[0258] Biomarkers Descriptive Features CTLA-4 Immune checkpoints PD-L1 Suppresses the adaptive immune system PD-L2 Immune checkpoint receptor ligands B7-H3 Immune checkpoints B7-H4 Immune co-suppressive molecules BTLA Immunomodulatory receptors HVEM TNF receptor-associated factor molecule TIM3 immune response modifiers GAL9 Induce T cell death LAG3 Immune checkpoints VISTA Immunomodulatory receptors KIR Regulating immune tolerance 2B4 Regulate NK cells CD160 Checkpoint inhibitors CGEN-15049 Immune checkpoints CHK1 Initiate DNA damage response CHK2 Regulate cell division A2A Signal adenosine B-7 family ligands Regulating immune responses

[0259] Table 6: Illustrative cancer cell biomarkers.

[0260]

[0261]

[0262]

[0263] In embodiments, the infectious disease comprises a lytic viral infection, a lysogenic viral infection, and / or both.

[0264] CAR-T BioNV co-administration

[0265] In embodiments, methods for treating and / or preventing a disease or condition include co-administering whole cell therapy (e.g., T cell, NK cell, TIL, macrophage therapy) with BioNV. In embodiments, supplementing whole cell therapy with therapeutic BioNV / exosomes can be used to reduce the effective dose of whole cell therapy required, reduce CRS, off-target effects, teratoma potential, etc. In embodiments, methods for treating and / or preventing a disease or condition include treating cancer, including slowing the growth of tumors or cancer tissue, reducing tumor volume, reducing metastasis, increasing tumor infiltrating lymphocytes (TIL), and / or improving symptoms.

[0266] In embodiments, the method of treating and / or preventing a disease or condition comprises administering one or more BioNVs (e.g., Figures 4A-4C In embodiments, the pairing is selected based on the disease to be treated. In embodiments, the pairing is selected based on the desired BioNV-whole cell interaction. In embodiments, the pairing is selected based on the disease to be treated and the nature of the desired interaction between the BioNV and the whole cell therapy.

[0267] In embodiments, methods of treating and / or preventing a disease or condition comprise 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 embodiments, the BioNV and whole cell therapy target the same cancer antigen. In embodiments, the BioNV and whole cell therapy target different cancer antigens. Without wishing to be bound by theory, targeting different antigens (i.e., two or more) allows for targeting tumors that present two or more biomarkers and / or prevents or reduces the likelihood of antigen escape from tumors or cancerous tissue. For example, without wishing to be bound by theory, one limitation of treating cancer is that cancer cells may lose expression of the antigen / biomarker targeted by the therapy.

[0268] In embodiments, the BioNV for co-administration includes an antigen binding construct (e.g., CAR) and one or more cancer antigens / biomarkers, or new antigens derived therefrom, on its surface. In embodiments, the cancer antigens / biomarkers or new antigens derived therefrom are or include one or more proteins listed in Table 4, Table 5, and / or Table 6. In embodiments, BioNV has one or more co-stimulatory molecules (e.g., IL-15) present on the surface for stimulating co-administered whole cells (e.g., T cells, NK cells, or macrophages). In embodiments, the BioNV antigen binding construct does not substantially bind to or recognize antigens / biomarkers also present on BioNV or new antigens derived therefrom (e.g., to prevent cross-reactions).

[0269] In embodiments, BioNVs presenting cancer antigens / biomarkers or neoantigens derived therefrom are administered as a monotherapy. Without wishing to be bound by theory, in embodiments, the neoantigen-presenting BioNVs stimulate a natural T cell response in the subject to be treated via T cell receptor (TCR) engagement, wherein the natural T cells (now activated in circulation) exhibit improved attribution to the tumor or cancer tissue site and exhibit improved cell-mediated cytotoxicity capabilities that would otherwise not be present.

[0270] In embodiments, BioNV presenting cancer antigens / biomarkers or neoantigens derived therefrom are co-administered with one or more whole cell therapies. Without wishing to be bound by theory, in embodiments, BioNV with CAR and antigen is capable of activating / stimulating co-administered whole cell therapies (e.g., T cells, NK cells, macrophages, etc.). Without wishing to be bound by theory, in embodiments, whole cells (e.g., CAR-T cells) administered to a subject can be activated by BioNV circulating in the blood, which, after activation, can improve the running-in of whole cells to tumors or cancer tissues, rather than waiting for whole cells to be activated in the tumor microenvironment. Without wishing to be bound by theory, in embodiments, cancer antigens / biomarkers (or neoantigens) present on BioNV co-administered with whole cell counterparts can increase the kinetics (e.g., pharmacokinetics / pharmacodynamics) of killing / clearing tumor cells by whole cell therapy or a single BioNV.

[0271] In embodiments, the method of treating and / or preventing a disease or condition herein comprises administering a BioNV having a first antigen binding protein, such as a CAR that recognizes a cancer antigen (e.g., Figure 4A CAR 1 as shown), and a second ligand, receptor and / or antigen recognized by the whole cell (e.g., Figure 4AIn embodiments, the second ligand, receptor and / or antigen is identical to, comprises or is similar to the ligand, receptor and / or antigen expressed by the cancer cell to be targeted (e.g., Figure 4A In embodiments, the second ligand, receptor and / or antigen on the BioNV is a cancer neoantigen. In embodiments, the BioNV is bound to a protein comprising an antigen binding protein, such as a CAR (e.g., Figure 4A CAR 2) shown in the figure, wherein the antigen binding protein recognizes an antigen, ligand or receptor present on the cancer cell and an antigen, ligand or receptor present on the BioNV (e.g., Figure 4A Without wishing to be bound by theory, this therapeutic strategy improves upon traditional applications of whole-cell therapy by: 1) improving targeting of whole cells to tumor cells or cancer tissues by increasing the local concentration of target antigens, ligands, or receptors; and 2) enhancing whole-cell activation / stimulation by antigen-specific constructs due to higher-order binding resulting from combined interactions with tumor cells or cancer tissues and BioNV.

[0272] In embodiments, the method of treating and / or preventing a disease or condition herein comprises administering a BioNV having a first antigen binding protein, such as a CAR that recognizes a cancer antigen (e.g., Figure 4B CAR 1 as shown), and a second ligand, receptor and / or antigen recognized by the whole cell (e.g., Figure 4B In embodiments, the second ligand, receptor and / or antigen is identical to, comprises or is similar to the ligand, receptor and / or antigen expressed by the cancer cell to be targeted (e.g., Figure 4B In embodiments, the second ligand, receptor and / or antigen on the BioNV is a cancer neoantigen. In embodiments, the BioNV is combined with a construct comprising two or more antigen binding proteins, such as a CAR (e.g., Figure 4B In embodiments, the whole cell comprises a first antigen binding construct (e.g., CAR) that binds to an antigen, ligand, or receptor present on a tumor cell or cancerous tissue on a cancer cell, and a second antigen binding construct (e.g., CAR) that binds to an antigen, ligand, and / or receptor present on BioNV (e.g., Figure 4B shown).

[0273] In embodiments, two or more antigen-binding constructs present on a whole cell are different in their intracellular domains (including costimulatory domains and / or intracellular signaling domains), as described herein. In embodiments, two or more antigen-binding constructs are different in their signaling functions. For example, in embodiments, two or more antigen-binding constructs have intracellular domains that signal through different, non-competitive signaling pathways, such as pro-persistent / pro-survival pathways and cell-mediated cytotoxicity pathways. In embodiments, two or more antigen-binding constructs signal through redundant or overlapping signaling pathways. Without wishing to be bound by theory, this treatment strategy improves upon the traditional administration of whole-cell therapy by increasing the local concentration of target antigens, ligands, or receptors. 2) whole-cell activation / stimulation is enhanced by antigen-specific constructs due to a higher-order binding produced by the combined interaction of tumor cells or cancer tissues and BioNV. And 3) whole-cell activation / stimulation is enhanced by two or more different antigen-specific constructs with different intracellular domains.

[0274] In embodiments, the method of treating and / or preventing a disease or condition herein comprises administering a BioNV having a first antigen binding protein, such as a CAR (e.g., Figure 4C CAR 1 as shown), and a second ligand, receptor and / or antigen recognized by the whole cell (e.g., Figure 4C In embodiments, the second ligand, receptor and / or antigen is not identical to, does not contain, or is not similar to the ligand, receptor and / or antigen expressed by the cancer cell to be targeted (e.g., Figure 4C CAR 2R as shown). In embodiments, the second ligand, receptor and / or antigen present on BioNV (which will be recognized by the whole cell) is selected for a specific cell-BioNV interaction that is not intended to occur significantly elsewhere in the subject to be treated. In embodiments, BioNV is co-administered with whole cells comprising at least two different antigen binding constructs, such as CARs that bind to antigens, ligands or receptors present on cancer cells (e.g., Figure 4C CAR 2) and a CAR that binds BioNV (e.g., Figure 4C In embodiments, the whole cell has a first antigen binding construct that binds to an antigen, ligand, or receptor present on a tumor cell or cancer tissue, and a second antigen binding construct that binds to a ligand, receptor, and / or antigen present on the BioNV (e.g., Figure 4C ), wherein the ligand, receptor and / or antigen on the BioNV is different from the antigen, ligand or receptor present on the tumor cell or cancer tissue.

[0275] In embodiments, the first antigen binding construct and the second antigen binding construct present on the whole cell differ in their intracellular domains (including costimulatory domains and / or intracellular signaling domains), as described herein. In embodiments, the two antigen binding constructs differ in their signaling function. For example, in embodiments, the whole cell has a first antigen binding construct that binds to a tumor cell or cancer tissue (e.g., Figure 4C CAR 2-CAR 2R) shown in FIG, which comprises an intracellular domain that signals through a cell-mediated cytotoxic signaling pathway (e.g., for the cell to release granzymes, perforins, and other anti-tumor cytokines). For example, in embodiments, the whole cell has a second antigen-binding construct that binds to BioNV (e.g., Figure 4C CAR 2A shown in ), which comprises intracellular domains that signal through different non-cell-mediated cytotoxic signaling pathways, such as pro-persistence, pro-survival and / or pro-proliferation pathways. Without wishing to be bound by theory, this therapeutic strategy improves upon the traditional application of whole-cell therapy by, but not limited to, the following means: 1) improving the targeting of whole cells to tumor cells or cancer tissues by increasing the local concentration of target antigens, ligands or receptors; 2) enhancing whole-cell activation / stimulation by antigen-specific constructs due to higher-order binding resulting from combined interactions with tumor cells or cancer tissues and BioNV; 3) enhancing whole-cell activation / stimulation by two or more different intracellular domains; and 4) reducing peripheral or off-target activation / stimulation of whole cells by requiring tumor cell or cancer tissue interactions as well as BioNV interactions to achieve full function of whole cells.

[0276] In embodiments, a T cell receptor (TCR) may be used in place of or in addition to a CAR construct described herein (e.g., Figure 4D In embodiments, the TCR is a natural TCR derived from the subject to be treated. In embodiments, the TCR is an engineered T cell receptor.

[0277] In embodiments, methods of treating and / or preventing a disease or condition include co-administration, including first administering an effective amount (e.g., one or more doses) of BioNV prior to administering an effective amount (e.g., one or more doses) of whole cell therapy. In embodiments, BioNV is first administered about or at least about 2 hours prior to whole cell therapy, about or at least about 4 hours prior to whole cell therapy, about or at least about 6 hours prior to whole cell therapy, about or at least about 8 hours prior to whole cell therapy, about or at least about 12 hours prior to whole cell therapy, about or at least about 24 hours prior to whole cell therapy, about or at least about 2 days prior to whole cell therapy, about or at least about 3 days prior to whole cell therapy, about or at least about 4 days prior to whole cell therapy, about or at least about 5 days prior to whole cell therapy, about or at least about 6 days prior to whole cell therapy, or about or at least about 1 week or more prior to whole cell therapy. In embodiments, BioNV is first administered to decorate cancer cells prior to administering whole cell therapy.

[0278] In embodiments, the method of treating and / or preventing a disease or condition comprises concurrently administering BioNV and whole cell therapy.

[0279] In embodiments, methods of treating and / or preventing a disease or condition include co-administration, including first administering an effective amount (e.g., one or more doses) of whole cells prior to administering an effective amount (e.g., one or more doses) of BioNV. In embodiments, the whole cell therapy is administered first about or at least about 2 hours prior to BioNV, about or at least about 4 hours prior to BioNV, about or at least about 6 hours prior to BioNV, about or at least about 8 hours prior to BioNV, about or at least about 12 hours prior to BioNV, about or at least about 24 hours prior to BioNV, about or at least about 2 days prior to BioNV, about or at least about 3 days prior to BioNV, about or at least about 4 days prior to BioNV, about or at least about 5 days prior to BioNV, about or at least about 6 days prior to BioNV, or about or at least about 1 week or more prior to BioNV. In embodiments, the whole cell therapy is administered first because BioNV relatively easily penetrates tissue / tumors.

[0280] In embodiments, the whole cell is a whole cell therapy comprising one or more of a CAR-T cell, a CAR-NK cell, and / or a CAR-macrophage cell therapy. In embodiments, the whole 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 (ciltacabtagene 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, various 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 membranes, Vericel Corp.), PROVENGE (sipuleucel-T, Dendreon Corp.), RETHYMIC (allogeneic processed thymic 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 embodiments, the whole cell therapy is one or more whole cell therapies described herein that have been engineered to include one or more BioNV-compatible cell surface proteins, as described herein. In embodiments, the whole cell therapy is one or more whole cell therapies described herein that have been engineered to be hypoimmunogenic, as described herein.

[0281] In embodiments, administering BioNV in addition to whole cells improves whole cell function relative to administering whole cells alone. In embodiments, whole cell function includes one or more of the following: cell-mediated cytotoxicity, cytokine release, tumor cell or cancer cell killing, integration into tumor cell or cancer tissue, tissue infiltration, proliferation, persistence, and / or survival. In embodiments, BioNV reduces one or more toxicities of whole cells. In embodiments, toxicities of whole cell therapy include propensity for off-target binding (e.g., binding in peripheral tissues, nonspecific activation, etc.) and related adverse events.

[0282] In embodiments, the method of treating and / or preventing a disease or condition comprises administering an additional therapeutic agent. In embodiments, the additional therapeutic agent may be any additional anti-cancer agent, anti-infective agent, analgesic agent, and / or nonsteroidal anti-inflammatory agent (NSAID).

[0283] In embodiments, the therapeutic BioNV / exosomes may be frozen at -80°C or suitable for storage at about -80°C and / or lyophilized (e.g., for reconstitution in a buffer). In embodiments, the therapeutic BioNV / exosomes may be stable at about 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 a week, or at least about a month or longer.

[0284] In embodiments, treatment and / or prevention of a disease or condition 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 more following administration of the compositions and methods employing such compositions.

[0285] Dosage and administration

[0286] The dosage and dosing schedule of the therapeutic BioNV / exosomes disclosed herein may depend on various parameters and factors, including, but not limited to, the specific therapeutic BioNV / exosome, the disease being treated, the severity of the condition, whether the condition is being treated or prevented, the age, weight, and general health of the subject, and the discretion of the attending physician. Additionally, pharmacogenomic information (the influence of genotype on the pharmacokinetics, pharmacodynamics, or efficacy profile of a therapeutic agent) about a particular subject may influence the dosage used. Furthermore, the exact individual dosage may be adjusted to some extent based on a variety of factors, including the specific combination of drugs being administered, the time of administration, the route of administration, the nature of the formulation, the rate of excretion, the disease being treated, the severity of the condition, and the anatomical location of the condition. Some variation in dosage is to be expected.

[0287] In embodiments, delivery of therapeutic BioNVs / exosomes can be similar to delivery of vesicles, particularly liposomes (see Langer, 1990, Science 249: 1527-1533; Treat et al., Liposomes in Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989)).

[0288] In embodiments, the methods of treating and / or preventing a disease or condition using the therapeutic BioNV / exosomes described herein include a dosage range in units of therapeutic BioNV / exosome number concentration per kilogram (kg) of subject body weight. In embodiments, a suitable dosage range for the methods described herein may include about 10 3 BioNV / kg (or exosomes / kg) to about 10 12 In one embodiment, the therapeutic BioNV / exosomes are present in the composition at a concentration of about 10 3 BioNV / mL (or exosomes / mL) to about 10 14BioNV / exosomes / mL (or exosomes / mL). Alternatively, in embodiments, the therapeutic BioNV / exosome composition is present in the composition at a weight / volume range of about 5 ng / mL to about 500 mg / mL. In embodiments, the therapeutic BioNV / exosome dosage is based on the size of the therapeutic BioNV / exosomes used in treatment, for example, 1000 nm therapeutic BioNV / exosomes are provided in an amount that is approximately 5-fold to 10-fold less than the amount of 100 nm therapeutic BioNV / exosomes at the same dose.

[0289] In embodiments, the therapeutic BioNV / exosomes disclosed herein are administered by controlled or sustained release means or delivery via devices well known to those of ordinary 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 adapted to provide controlled or sustained release of one or more active ingredients using, for example, hydroxypropylcellulose, other polymer matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, microspheres, or combinations thereof, to provide a desired release profile in varying proportions. Controlled or sustained release of an 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, concentration or availability of an enzyme, concentration or availability of water, or other physiological conditions or compounds.

[0290] In an embodiment, polymeric materials are used (see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Press., 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).

[0291] In an embodiment, a controlled release system is placed adjacent to the target area to be treated, thereby requiring only a fraction of the systemic dose (see, e.g., Goodson, 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.

[0292] In an embodiment, methods of using therapeutic BioNV / exosomes include applying the therapeutic BioNV / exosomes to the surface of a device (e.g., a catheter) or containing them in a pump, patch, or other drug delivery device. The excipient or carrier can be selected based on the mode and route of administration. Suitable pharmaceutical carriers and pharmaceutical essentials for pharmaceutical formulations are described in the well-known reference books in the field, Remington's Pharmaceutical Sciences (EW Martin) and USP / NF (United States Pharmacopoeia and National Formulary).

[0293] In embodiments, therapeutic BioNV / exosomes can be administered at a dose consistent with that of whole cells, for example, based on CAR concentration. In embodiments, the typical concentration range of CAR protein per microgram of T cell is between 0.20ng-0.70ng, while the total number of CARs in a single BioNV may be 5 to 10,000 times less than that of a whole cell, resulting in a conversion of BioNV mass to CAR concentration, wherein it can be assumed that the CAR concentration is equal to the cell (e.g., T cell) from which it is derived (e.g., the case in exosomes) or increases (e.g., the case in BioNV). In embodiments, the concentration and / or surface density of the targeting agent (e.g., CAR) on BioNV is increased compared to the whole cell from which it is derived. In embodiments, the concentration and / or surface density of the targeting agent (e.g., CAR) is enriched by continuous extrusion processing of the whole cell. In embodiments, the concentration and / or surface density of the targeting agent (e.g., CAR) and other cell surface molecules on BioNV are increased by 2 to 100 times relative to the whole cell. In embodiments, because exosomes are naturally shed, their concentration and / or surface density of targeting agents (e.g., CARs) and other cell surface molecules are substantially the same as whole cells.

[0294] The dosage regimen for any of the therapeutic BioNV / exosomes disclosed herein can be selected based on a variety of factors, including the subject's cancer type, species, age, weight, sex, and medical condition; the severity of the condition being treated; the route of administration; the subject's renal or hepatic function; the individual's pharmacogenomic makeup; and the specific composition employed in the present disclosure. Any of the therapeutic BioNV / exosomes disclosed herein can be administered in a single daily dose, or the total daily dose can be administered in divided doses of two, three, or four times daily. Furthermore, any of the therapeutic BioNV / exosomes disclosed herein can be administered continuously rather than intermittently throughout the dosing regimen.

[0295] In embodiments, therapeutic BioNV / exosomes are administered in continuous doses 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 a month.

[0296] Additional therapeutic agents

[0297] 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 solution with the therapeutic BioNV / exosomes, adsorbed onto the surface of the NVs, or encapsulated as a payload within the therapeutic BioNV / exosomes. In embodiments, the additional therapeutic agent is one or more of a checkpoint inhibitor, an analgesic, and / or an anti-infective agent.

[0298] In embodiments, the compositions or methods of the present invention contemplate other additional therapeutic agents, such as analgesics to help treat inflammation or pain at the site of administration, or anti-infectives to prevent infection at the site treated with the composition. Non-limiting examples of additional therapeutic agents include analgesics, such as nonsteroidal anti-inflammatory drugs, opioid agonists, and salicylates; anti-infectives, such as anthelmintics, antianaerobics, antibiotics, aminoglycoside antibiotics, antifungal antibiotics, cephalosporin antibiotics, macrolide antibiotics, other beta-lactam antibiotics, penicillins, quinolones, sulfonamides, tetracyclines, antimycobacterials, antituberculosis antimycobacterials, antiprotozoals, antimalarial antiprotozoals, antivirals, antiretrovirals, scabicides, anti-inflammatory drugs, corticosteroids, Steroidal anti-inflammatory drugs, antipruritic / local anesthetics, topical anti-infectives, antifungal topical anti-infectives, antiviral topical anti-infectives; electrolyte and renal agents, such as acidifiers, alkalinizers, diuretics, carbonic anhydrase inhibitor diuretics, loop diuretics, osmotic diuretics, potassium-sparing diuretics, thiazide diuretics, electrolyte substitutes, and uricosuric agents; enzymes, such as pancreatic enzymes and thrombolytic enzymes; gastrointestinal agents, such as antidiarrheals, antiemetics, gastrointestinal anti-inflammatory agents, salicylate gastrointestinal anti-inflammatory agents, antacids and anti-ulcers, acid pump inhibitors and anti-ulcers, and gastric mucosal anti-ulcers , H2-blockers, antiulcers, gallstone dissolving agents, digestives, emetics, laxatives and stool softeners and prokinetics; general anesthetics, such as inhalation anesthetics, halogenated inhalation anesthetics, intravenous anesthetics, intravenous barbiturates, intravenous benzodiazepines, and intravenous opioid agonists; hormones and hormone modulators, such as abortifacients, adrenal medications, corticosteroid adrenal medications, androgens, antiandrogens; immunobiological agents, such as immunoglobulins, immunosuppressants, toxoids, and vaccines; local anesthetics, such as amide local anesthetics and ester local anesthetics Anesthetics; musculoskeletal agents, such as antigout anti-inflammatory agents, corticosteroid anti-inflammatory agents, gold compound anti-inflammatory agents, immunosuppressive anti-inflammatory agents, nonsteroidal anti-inflammatory drugs (NSAIDs), salicylate 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.

[0299] Additional non-limiting examples of useful therapeutic agents in the above categories include: (1) general analgesics, such as lidocaine or its derivatives, and NSAID analgesics, including diclofenac, ibuprofen, ketoprofen, and naproxen; (2) opioid agonist analgesics, such as codeine, fentanyl, hydromorphone, and morphine; (3) salicylate analgesics, such as aspirin (ASA) (enteric-coated ASA); (4) Hi-blocker antihistamines, such as clemastine and terfenadine; (5) narcotic analgesics, such as dapoxetine and fentanyl; (6) narcotic analgesics, such as dapoxetine and fentanyl; (7) narcotic analgesics, such as dapoxetine and fentanyl; (8) narcotic analgesics, such as dapoxetine and fentanyl; (9) narcotic analgesics, such as dapoxetine and fentanyl; (10) narcotic analgesics, such as dapoxetine and fentanyl; (11) narcotic analgesics, such as dapoxetine and fentanyl; (12) narcotic analgesics, such as dapoxetine and fentanyl; (13) narcotic analgesics, such as dapoxetine and fentanyl; (14) narcotic analgesics, such as dapoxetine and fentanyl; (15) narcotic analgesics, such as dapoxetine and fentanyl; (16) narcotic analgesics, such as dapoxetine and fentanyl; (17) narcotic analgesics, such as (5) Anti-infectives, such as mupirocin; (6) Anti-anaerobic anti-infectives, such as chloramphenicol and clindamycin; (7) Antifungal antibiotic anti-infectives, such as amphotericin B, clotrimazole, fluconazole and ketoconazole; (8) Macrolide antibiotic anti-infectives, such as azithromycin and erythromycin; (9) Other β-lactam antibiotic anti-infectives, such as aztreonam and imipenem; (10) Penicillin antibiotic anti-infectives, such as nafcillin, oxacillin, penicillin G and penicillin V; (11) Quinolone antibiotic anti-infectives, such as ciprofloxacin ) and norfloxacin; (12) tetracycline antibiotics, such as doxycycline, minocycline, and tetracycline; (13) antituberculosis and antimycobacterial anti-infectives, such as isoniazid (INH) and rifampin; (14) antiprotozoal anti-infectives, such as atovaquone and dapsone; (15) antimalarial antiprotozoal anti-infectives, such as chloroquine and pyrimethamine; (16) antiretroviral anti-infectives, such as ritonavir and zidovudine; (17) antiviral anti-infectives, such as acyclovir, ganciclovir, interferon α , remdesivir and rimantadine; (18) antifungal topical anti-infective drugs, such as amphotericin B, clotrimazole, miconazole and nystatin; (19) antiviral topical anti-infective drugs, such as acyclovir; (20) electrolyte and renal drugs, such as lactulose; (21) loop diuretics, such as furosemide; (22) potassium-sparing diuretics, such as triamterene; (23) thiazide diuretics, such as hydrochlorothiazide (HCTZ); (24) uricosuric drugs, such as probenecid; (25) enzymes, such as RNAse and DNAse; (26) antiemetics, such as prochlorperazine; (27) salicylic acid gastrointestinal anti-inflammatory drugs, such as sulfasalazine; (28) gastric acid pump inhibitor anti-ulcer drugs, such as omeprazole;(29) H2-receptor blocker antiulcer drugs, such as cimetidine, famotidine, nizatidine, and ranitidine; (30) digestive agents, such as pancreatic lipase; (31) prokinetic drugs, such as erythromycin; (32) ester local anesthetics, such as benzocaine and procaine; (33) musculoskeletal corticosteroid anti-inflammatory drugs, such as beclomethasone, betamethasone, cortisone, etc. sone), dexamethasone, hydrocortisone, and prednisone; (34) musculoskeletal anti-inflammatory immunosuppressants, such as azathioprine, cyclophosphamide, and methotrexate; (35) musculoskeletal nonsteroidal anti-inflammatory drugs (NSAIDs), such as diclofenac, ibuprofen, ketoprofen, ketoprofen, 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.

[0300] Therapeutic BioNV / exosome compositions

[0301] In some aspects, the present disclosure relates to compositions useful for treating mammalian disease, comprising allogeneic, hypoimmunogenic therapeutic BioNV / exosomes comprising one or more therapeutically relevant biomolecules, as described herein.

[0302] In embodiments, the composition comprises therapeutic BioNV / exosomes. In embodiments, the composition may comprise therapeutic BioNV / exosomes and at least one or more of an anti-cancer therapeutic, an anti-infective therapeutic, or a gene editing payload. In embodiments, the composition comprises therapeutic BioNV / exosomes that can adsorb therapeutic molecules onto the surface of the NV and / or encapsulate the therapeutic payload within the aqueous compartment of the NV. In embodiments, the composition comprises a therapeutically effective amount of BioNV / exosomes.

[0303] In embodiments, the compositions are allogeneic and / or hypoimmunogenic.In embodiments, the compositions are derived from iPSCs (and other cell types) that have been modified to reduce expression of immunogenic molecules and / or increase expression of immunoprotective molecules.

[0304] In embodiments, the composition of therapeutic BioNV / exosomes is low immunogenic. For example, in embodiments, the composition does not cause an inflammatory response and / or an immune response when administered. In embodiments, the therapeutic BioNV / exosomes are low immunogenic. In embodiments, upon administration to a subject, the composition (optionally the therapeutic BioNV / exosomes therein) elicits less than 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 about 1% of inflammatory responses.

[00135] In some embodiments, the present invention provides a method for producing an immunomodulatory effect or immune response, as measured as a function of the concentration of a cytokine, chemokine, or immunomodulatory enzyme, 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 the like, or any combination thereof, for example, compared to a homologous whole cell therapy counterpart.

[0305] In an embodiment, the therapeutic BioNV / exosomes are present in the composition at a concentration of about 10 3 BioNV / mL (or exosomes / mL) to about 10 14 Alternatively, in embodiments, the therapeutic BioNV / exosome composition is present in the composition at a weight / volume range of about 5 ng / mL to about 500 mg / mL.

[0306] In embodiments, the composition is substantially free of one or more bacteria, viruses, fungi, spores, mycoplasmas, pyrogens, and in more specific embodiments, substantially free of all of the foregoing. In embodiments, the composition is substantially free of whole cells and intracellular cellular components, including organelles such as the nucleus, mitochondria, Golgi apparatus, etc., and / or substantially free of non-CAR-expressing NV and / or substantially free of ruptured, damaged NV. In embodiments, the composition is substantially free of cellular chromatin, nucleosomes, and other genetic material and non-therapeutic nucleic acids. In embodiments, BioNV / exosomes and BioNV / exosome compositions are substantially free of cellular genomic DNA.

[0307] In embodiments, due to the lack of immunogenicity of engineered iPSCs, therapeutic BioNV / exosomes are modular and allogeneic (off-the-shelf). In embodiments, the lack of whole-cell signaling components enables BioNV / exosomes to easily modulate target specificity and resistance to immunosuppressive signals. In embodiments, therapeutic BioNV / exosomes lack genetic elements that cause uncontrolled cytokine storms, thereby minimizing the risk of cytokine release syndrome (CRS) in patients. In embodiments, the amount of active cytokines, perforins, granzymes, granulysins, interferons, interleukins, etc. encapsulated in therapeutic BioNV / exosomes is regulated in the process upstream of the cell (before BioNV / exosome derivation). In embodiments, therapeutic BioNV / exosomes are derived from cells that can cross biological barriers and / or viral receptors known to facilitate crossing.

[0308] Without wishing to be bound by theory, therapeutic BioNV / exosomes produced by iPSC-engineered allogeneic basal cell lines represent immune stealth, meaning that therapeutic BioNV / exosomes have the potential for multiple dosing, antibody-mediated neutralization of therapeutic BioNV / exosomes is minimized, and immune cell-mediated clearance is evaded (T cells and macrophages). In embodiments, therapeutic BioNV / exosomes do not contain viable genetic material from the cells from which they are derived to cause CRS or teratomas. In embodiments, increased expression of certain cytokines encapsulated within therapeutic BioNV / exosomes can recruit naive T cells. In embodiments, BioNV / exosomes can be derived from modified cell types with or without barrier-penetrating ligands to further control activity after infusion.

[0309] Pharmaceutical compositions and formulations of therapeutic BioNV / exosomes

[0310] In some aspects, the composition is a pharmaceutical composition. In embodiments, the pharmaceutical composition of the present disclosure is formulated to provide a therapeutically effective amount of therapeutic BioNV / exosomes as an active ingredient. In embodiments, the pharmaceutical composition of the present disclosure is formulated to provide a therapeutically effective amount of one or more anti-cancer therapeutic agents as a payload within the therapeutic BioNV / exosomes as an active ingredient. Typically, the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients, carriers (including inert solid diluents and fillers), diluents (including sterile aqueous solutions and various organic solvents), penetration enhancers, solubilizers, and adjuvants.

[0311] Pharmaceutical excipients can be liquids, such as water and oils, including those of petroleum, animal, plant or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Pharmaceutical excipients can be, for example, normal saline, gum arabic, gelatin, starch paste, talc, keratin, colloidal silicon dioxide, urea, etc. In addition, adjuvants, stabilizers, thickeners, lubricants and colorants can be used. When applied to a subject, pharmaceutically acceptable excipients are generally sterile. When any agent disclosed herein is administered intravenously, water is a useful excipient. Saline solutions and aqueous dextrose solutions and glycerol solutions can also be used as liquid excipients, particularly for injectable solutions. Suitable pharmaceutical excipients also include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene, ethylene glycol, water, ethanol, etc. If desired, any composition disclosed herein can also be formulated with a wetting agent or emulsifier or pH buffer. Other examples of suitable pharmaceutical excipients are described in Remington's Pharmaceutical Sciences 1447-1676 (Alfonso R. Gennaro ed., 19th ed. 1995), which is incorporated herein by reference.

[0312] In an embodiment, the composition comprises an excipient or carrier. In an embodiment, the diluent is a pharmaceutically acceptable excipient or carrier.

[0313] In an embodiment, the pharmaceutical composition comprises a pharmaceutically acceptable diluent. Non-limiting examples of diluents include liquid diluents such as water, ethanol, propylene glycol, glycerol, and various combinations thereof, and inert solid diluents such as calcium carbonate, calcium phosphate, or kaolin. In an embodiment, the diluent comprises one or more of the following: saline, phosphate-buffered saline, Dulbecco's modified Eagle's medium (DMEM), alpha-modified minimum essential medium (αMEM), Roswell Park Memorial Institute medium 1640 (RPMI medium 1640), HBSS, human albumin, Ringer's solution, etc., or any combination thereof.

[0314] In embodiments, the active ingredient is typically mixed with an excipient, diluted by an excipient, or encapsulated in a carrier such as a capsule, tablet, pouch, paper, or other container. When an excipient is used as a diluent, it can be a solid, semisolid, or liquid substance (e.g., normal saline) as a vehicle, carrier, or medium for the active ingredient. In embodiments, the composition can be in the form of tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as solids or in liquid media), lotions, creams, ointments, gels, soft and hard gelatin capsules, suppositories, sterile injectable solutions, and sterile packaged powders. As known in the art, the type of diluent can vary depending on the intended route of administration. In embodiments, the resulting composition can include additional agents, such as preservatives, cryopreservatives (e.g., DMSO), and / or lyoprotectants (e.g., polyols, salts). In embodiments, the carrier can be or can include a lipid-based or polymer-based colloid. In embodiments, the carrier material may be a colloid formulated as a liposome, a hydrogel, a microparticle, a nanoparticle, or a block copolymer micelle. In embodiments, the carrier material may form a capsule, and the material may be a polymer-based colloid.

[0315] In embodiments, the pharmaceutical composition comprising therapeutic BioNV / exosomes includes a solubilizing agent. In embodiments, the pharmaceutical composition comprising therapeutic BioNV / exosomes includes a cryoprotectant or an agent that improves thermal stability, such as DMSO or glycerol. In embodiments, the pharmaceutical composition can be delivered using a suitable vehicle or delivery device known in the art.

[0316] In embodiments, the composition comprises a scaffold of a biomaterial. In a non-limiting example, a three-dimensional biomaterial comprises therapeutic BioNV / exosomes embedded in an extracellular matrix that is attached to, dispersed within, or entrapped within the scaffold. In embodiments, the biomaterial is biodegradable and / or synthetic.

[0317] In embodiments, the scaffold comprises a biodegradable biomaterial. Non-limiting examples of biodegradable biomaterials include fibrin, collagen, elastin, gelatin, vitronectin, fibronectin, laminin, reconstructed basement membrane matrix, starch, dextran, alginate, hyaluronic acid, chitin, chitosan, agarose, sugar, hyaluronic acid, poly (lactic acid), poly (glycolic acid), polyethylene glycol, decellularized tissue, self-assembling peptides, polypeptides, glycosaminoglycans, derivatives thereof, and mixtures thereof. Other useful biodegradable polymers or polymer types include, but are not limited to, polydioxanone, polycarbonate, polyoxalate, poly(α-ester), polyanhydride, polyacetate, polycaprolactone, poly(orthoester), polyamino acid, polyamide and mixtures and copolymers thereof, 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, polyurethane and poly(lactic acid) copolymers, polyurethane and poly(lactic acid) copolymers, α-amino acid copolymers, α-amino acid and caproic acid copolymers, A-glutamic acid benzyl ester and polyethylene glycol copolymers, succinate and poly(ethylene glycol) copolymers, polyphosphazenes, polyhydroxyalkanoates and mixtures thereof. Binary and ternary systems are also contemplated. In embodiments, the scaffold includes one or more of collagen, various proteoglycans, alginate-based matrix and chitosan. In embodiments, the scaffold comprises one or more of a hydrogel, silk, matrigel, acellular and / or decellularized scaffolds, poly-ε-caprolactone scaffolds, absorbable scaffolds, and nanofiber-hydrogel composites.

[0318] In embodiments, the scaffold comprises a synthetic biomaterial. Non-limiting examples of synthetic biomaterials include lactone-based polyesters or copolyesters, such as polylactide, polycaprolactone, 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, polyvinyl pyrrolidone), fluorinated polymers (e.g., polytetrafluoroethylene), and cellulose esters.

[0319] In embodiments, the composition can be prepared in any manner well known in the pharmaceutical art and can be administered by a variety of routes (e.g., subcutaneous, intravenous, etc.), depending on whether local or systemic treatment is required and the area to be treated. In embodiments, administration can be topical (including ophthalmic and mucosal, including intranasal, vaginal and rectal delivery), pulmonary (e.g., by inhalation or insufflation of powder or aerosol, including by a nebulizer; intratracheal, intranasal, epidermal and transdermal), ocular, oral or parenteral. In embodiments, methods may include ocular delivery, topical administration (eye drops), subconjunctival, periocular or intravitreal injection or introduction by surgically placed balloon catheter or ophthalmic insert in the conjunctival sac. In embodiments, parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial administration, such as intrathecal or intraventricular administration. In embodiments, parenteral administration may be in the form of a single bolus dose, or may be, for example, by a continuous infusion pump.

[0320] In embodiments, pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, powders, etc. In embodiments, methods of treating and / or preventing cancer include the use of pharmaceutical carriers; aqueous, powder or oily bases; thickeners, etc.

[0321] In an embodiment, the pharmaceutical composition contains as active ingredients the nucleic acids and vectors described herein and one or more pharmaceutically acceptable carriers. In an embodiment, the term "pharmaceutically acceptable" (or "pharmacologically acceptable") refers to molecular entities and compositions that do not produce side effects, allergies or other adverse reactions when administered to animals or humans, as the case may be. The methods and compositions disclosed herein can be applied to a variety of species, such as humans, non-human primates (e.g., monkeys), horses or other livestock, dogs, cats, ferrets, or other mammals kept as pets, rats, mice or other experimental animals. In an embodiment, the term "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antimicrobial agents, isotonic and absorption delaying agents, buffers, excipients, adhesives, lubricants, gels, surfactants, etc. that can be used as a medium for a pharmaceutically acceptable substance.

[0322] In an embodiment, the composition can be applied to the surface of a device (e.g., a catheter) or be contained in a pump, patch or other drug delivery device. In an embodiment, the composition can be applied alone or in the presence of a pharmaceutically acceptable excipient or carrier (e.g., normal saline) as a mixture. Excipient or carrier is selected based on mode of administration and approach. Suitable pharmaceutical carriers and pharmaceutical necessities for pharmaceutical formulations are described in the well-known reference books Remington's Pharmaceutical Sciences (EW Martin) and USP / NF (United States Pharmacopoeia and National Formulary) in this area.

[0323] In embodiments, a composition disclosed herein, e.g., a pharmaceutical composition, is suspended in a saline buffer (including but not limited to TBS, PBS, etc.).

[0324] The present technology includes the disclosed therapeutic BioNV / exosomes in various formulations of pharmaceutical compositions. In embodiments, the therapeutic BioNV / exosomes disclosed herein can be in the form of solutions, suspensions, emulsions, drops, tablets, pills, pellets, capsules, capsules containing liquids, powders, sustained-release formulations, emulsions, aerosols, sprays, suspensions, or any other form suitable for use.

[0325] Pharmaceutical compositions comprising the therapeutic BioNV / exosomes described herein can be conveniently presented in unit dosage form and can be prepared by any method well known in the pharmaceutical art. Such methods generally include the step of combining the therapeutic agent with a carrier consisting of one or more additional ingredients. Typically, pharmaceutical compositions are prepared by uniformly and intimately combining the therapeutic agent with a liquid carrier, a finely divided solid carrier, or both, and then, if desired, forming the product into a dosage form of the desired formulation (e.g., wet or dry granulation, powder blends, etc., followed by tableting using conventional methods known in the art).

[0326] In an embodiment, any of the therapeutic BioNV / exosomes disclosed herein are formulated according to routine procedures into pharmaceutical compositions suitable for the modes of administration disclosed herein.

[0327] Subjects and / or animals

[0328] In embodiments, the subject and / or animal intended for use with 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 embodiments, the subject and / or animal is a non-mammal, such as a zebrafish. In embodiments, the subject and / or animal is a transgenic animal comprising fluorescent cells, such as RPE cells and / or immune cells with GFP. In embodiments, the subject and / or animal is a human. In embodiments, therapeutic BioNV / exosomes are derived from fluorescently labeled cells and / or packaged with fluorescently labeled proteins or tags (e.g., GFP). In embodiments, the human is a pediatric human, adult, elderly, infant or child. In other embodiments, the human refers to a patient.

[0329] In an embodiment, the methods of treatment comprise administering to a human whose age range is from about 0 months to about 6 months, about 6 months to about 12 months, about 12 months to about 18 months, about 18 months to about 36 months, about 1 year to about 5 years, about 5 years to about 10 years, about 10 years to about 15 years, about 15 years to about 20 years, about 20 years to about 25 years, about 25 years to about 30 years, about 30 years to about 35 years, about 35 years to about 40 years, about 40 years to about 45 years, about 45 years to about 50 years, about 50 years to about 55 years, about 55 years to about 60 years, about 60 years to about 65 years, about 65 years to about 70 years, about 70 years to about 75 years, about 75 years to about 80 years, about 80 years to about 85 years, about 85 years to about 90 years, about 90 years to about 95 years, or about 95 years to about 100 years.

[0330] In embodiments, the subject is a non-human animal, and thus the disclosure relates to veterinary uses. In embodiments, the non-human animal is a domestic pet. In embodiments, the non-human animal is a livestock animal.

[0331] In embodiments, serum and / or immune cells and / or tumor cells are assessed and / or affected. In embodiments, immune cells include cells of the innate immune system of a subject and / or animal. In 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 disclosure provides for detecting the presence, absence, or amount of tumor volume, tumor cells, metastasis, cDNA, or RNA in a sample derived from a subject.

[0332] Reagent test kit

[0333] In an embodiment, the present disclosure provides a kit that can simplify the administration of any agent described herein. An exemplary kit of the present disclosure includes any agent described herein in unit dosage form. In an embodiment, the unit dosage form is a container, such as a prefilled syringe, which can be sterile, containing any agent described herein and a pharmaceutically acceptable carrier, diluent, excipient, or vehicle. In an embodiment, the kit also includes a label or printed instructions indicating the use of any agent described herein. In an embodiment, the kit also includes an eyelid speculum, a local anesthetic, and a cleanser for the injection surface. In an embodiment, the kit further includes one or more additional agents described herein.

[0334] In some aspects, the present disclosure includes syringes comprising one or more compositions of the present disclosure. In embodiments, the syringe is pre-filled with a certain volume of composition. In embodiments, the volume of the syringe pre-filled is about 1mL to about 10mL. In embodiments, the syringe is pre-filled with a volume of about 10mL, about 9mL, about 8mL, about 7mL, about 6mL, about 5mL, about 4mL, about 3mL, about 2mL, about 1.9mL, about 1.8mL, about 1.7mL, about 1.6mL, about 1.5mL, about 1.4mL, about 1.3mL, about 1.2mL, about 1.1mL or about 1.0mL or less composition.

[0335] In an embodiment, the syringe comprises a composition having a shelf life in the range of about 1 hour to about 1 week. In an embodiment, the syringe comprises a composition having a shelf life 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 an embodiment, the syringe comprises a composition having a shelf life 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.

[0336] In embodiments, the storage temperature is about -80°C. In embodiments, the storage temperature is about -20°C. In embodiments, the storage temperature is about 4°C. In embodiments, the storage temperature is about 21°C. In embodiments, the kit comprises lyophilized BioNV / exosomes.

[0337] In one embodiment, the kit includes a container containing a composition comprising the BioNV / exosomes of the present disclosure, a therapeutically effective amount of an additional therapeutic agent (eg, a therapeutic agent described herein), and instructions for use.

[0338] Example

[0339] Example 1: Luminal-localized payloads of therapeutically relevant biomolecules in cells for BioNV and exosomes controlled expression

[0340] The isolated CAR NK cells will be grown to the desired logarithmic phase consistency (defined by the amount of BioNV to be derived and / or the size limitations associated with the growth medium flask, container, or bioreactor). The cells will then be stimulated and activated. Several activation mechanisms can be used, including i) irradiated antigen presenting cells, ii) anti-CD3 / CD28 coated beads, iii) IL-2, iv) stimulatory molecules, such as beads coated with antigens recognized by the CAR construct, v) chemicals, such as phorbol esters, or vi) by other methods known to those skilled in the art.

[0341] The cells can be cleaned or separated from the stimuli used for activation, for example, by removal from anti-CD3 / CD28 coated beads (or beads with an alternative activating antigen bound thereto) by filtration, centrifugation, affinity columns, and / or magnetism (e.g., if magnetic beads are used). Antigen-presenting cells can also be removed from the cells by flow cytometry or affinity column chromatography, or a combination of both.

[0342] Next cells will be allowed to return to resting state. The size of cells will be observed within 1-14 days to determine their resting state. After cells return to resting state, a second activation process will be implemented. During this period, the analytical method of enzyme-linked immunosorbent assay (ELISA), flow cytometry, iodixanol density gradient centrifugation, immunoblotting and / or PCR analysis will be used to test one or more of CAR density, CD57 / CD16 expression and intracellular therapeutic protein concentration. Cells will be restimulated by irradiated antigen-presenting cells, anti-CD3 and / or anti-CD28 coated beads (or beads with alternative activation antigens combined therewith), chemicals (such as phorbol esters) or other methods described in the art. This restimulation step reduces the cell death of activation-induced in NK cells.

[0343] The beads are then separated and the cells are expanded according to known protocols, for example, as described in WO2020172328, WO2017037083, WO2011080740, and WO2014028453, each of which is hereby incorporated by reference in its entirety. Cell expansion can range from 1-fold expansion to higher fold increases, depending on the number of cells required to obtain an appropriate number of BioNVs.

[0344] The final dual-activated cell population will be sampled again and tested for one or more of CAR density, CD57 / CD16 expression, and intracellular therapeutic protein concentration using a combination of enzyme-linked immunosorbent assay (ELISA), flow cytometry, immunoblotting, and / or PCR analysis.

[0345] The protocol will be performed under appropriate controls, which will vary depending on the use of the CAR construct and the BioNV containing CAR. A control will include parallel processing and use of cells that have not yet been activated using the above protocol - i.e., unactivated CARNK. Similar activation protocols can be used for other cell types, and cell marker verification (in this case, CD57 / CD16 of NK cells) varies depending on the cell type used.

[0346] Example 2: Production of BioNV by continuous extrusion

[0347] Biomimetic nanovesicles (BioNV) can be basically Figure 5 The scheme described in the description is produced as described.

[0348] CAR expression levels in poorly immunogenic cell lines can be measured using a combination of flow cytometry and iodixanol density gradients (e.g., Figure 5 Step 1).

[0349] Differentiation of surface CAR-expressing iPSCs into CAR lymphocytes can be analyzed by identification of lymphocyte markers, including, for example, CD4 / CD8 (T cells) or CD56 / CD16 (natural killer cells) and other cell surface markers (e.g., Figure 5 Step 2). The expression profile can be determined by flow cytometry, RT-PCR and / or CRISPR-based analysis.

[0350] Next, activation of CAR lymphocytes can be achieved using low predetermined concentrations of biomarker antigen-coated beads in two phases over two weeks (e.g., Figure 5 This process also allows analysis of the quality of the immunological synapse (IS) between the CAR and antigen-coated beads, using well-established protocols to measure i) quantification of F-actin accumulation at the site of synapse formation, ii) distribution of pZeta at the synapse, iii) antigen recruitment across the IS, and / or iv) polarization of lytic granules (LGs) containing perforin and granzymes.

[0351] After lymphocyte activation, cells are expanded using a defined protocol (e.g. Figure 5 After expansion, each cell population is analyzed for perforin and granzyme (or other luminal payload, if applicable) levels to ensure consistent concentration levels across batches. This is accomplished using a series of qPCR, immunoblotting, flow cytometry, and / or mass spectrometry analyses. If a sufficiently large cell population is available in step 3, the expansion step may not be necessary.

[0352] Once the cells are activated and producing the desired therapeutic protein, they are expanded, harvested, washed several times, and then placed into a buffered extrusion medium. The cells are then fully processed (e.g., Figure 5 Step 5). In the initial extrusion step of the continuous extrusion process, the nucleus (along with nuclear components, including nuclear pores, genomic material and transcription factors) and mitochondria are eliminated. The sample is then treated with an endonuclease, such as BENZONASE. BENZONASE is a non-specific recombinant endonuclease that cuts all types of DNA and RNA variants into non-functional fragments of <8 soluble base pairs. This minimizes the amount of nucleic acid loaded per sample and on a scalable basis without interfering with the BioNV membrane chemistry. The lysis process also eliminates nucleic acid viscosity, allowing the material to be subsequently loaded and passed through the next set of extrusion filters.

[0353] The continuous extrusion process avoids the elimination of other organelles, such as the Golgi apparatus or the endoplasmic reticulum. The membrane systems of these organelles are highly evolved to transport vesicles between folded membranes (release and uptake). For example, the cis and trans faces of the Golgi apparatus contain unique lipid compositions that facilitate low-energy barrier uptake and release in vesicle transport. These components are relatively low in the cytoplasmic membrane. Therefore, isolating the cytoplasmic membrane for BioNV derivatization is not as advantageous. When BioNVs pass through polycarbonate filters during the continuous extrusion process, they are disrupted and spontaneously formed depending on the pore size. The BioNVs produced by this process contain membranes with a homogeneous mixture of cytoplasmic, Golgi, and ER lipid content and protein components, which significantly improves their affinity for cell and tissue delivery and uptake compared to BioNVs that have been treated to eliminate these organelles. These characteristics can translate into better and more consistent uptake of BioNVs into target cells at much lower doses than systems without these properties.

[0354] After the extrusion step, the BioNV was passed through an α-CD3 HPLC (FPLC under scale-up) column to remove a low percentage (approximately 0.05%) of reversed BioNV (e.g., Figure 5 This is done to ensure that the resulting BioNV has homogeneous orientation relative to the membrane. Yield loss is low in this step because it serves as a flow-through process to capture impurities. After collecting the BioNV following the HPLC / FPLC step, it will be tested using a standardized process.

[0355] The standardization process involves determining one or more of the following:

[0356] BioNV homogeneityNanoparticle flow cytometry (NanoFCM) can be used to confirm the homogeneity of BioNV concentration, size, density, and / or luminal composition of BioNV.

[0357] Luminal payload concentration: NanoFM technology can be used to determine the type and concentration of nucleic acids / proteins packaged into the lumen of BioNV. These data can be confirmed in parallel by one or more methods including immunoblotting, mass spectrometry, and BCA analysis to determine the nucleic acid and protein content of BioNV.

[0358] BioNV stability One or more of nanoparticle tracking analysis (NTA), dynamic light scattering (DLS), and electron microscopy (EM) can be used in combination with immunoblotting and / or mass spectrometry to determine the physical and biochemical characteristics of BioNV over a period of 8 to 10 months. Data from these assays can include protein expression profiles, the extent of intact BioNV membrane / packaging, and / or the extent of aggregation.

[0359] Membrane integrity: BioNV membrane integrity was assessed using a calcein release assay combined with NanoFCM to evaluate membrane permeability. The results provide insight into the leakiness properties of BioNV relative to a standardized set of BioNVs.

[0360] Mass of lumen payload: Depending on the nature of the payload, a variety of analytical assays can be used to determine the quality of the lumen-encapsulated payload. If the delivery is a nucleic acid, the integrity and quantity of the nucleic acid payload can be checked using 8-10 month qPCR and / or sequencing. For proteins, BioNV components can be analyzed using one or more of NanoFCM, mass spectrometry, and immunoblot analysis to analyze protein payloads.

[0361] CAR mass and surface density: NanoFCM, mass spectrometry and / or immunoblotting analysis can be used to determine the CAR surface density. Compared to the whole cell surface density, the CAR surface density in BioNV is expected to be at least about 5 times to at least about 10 times higher. Compared to whole cells, this can significantly enhance the targeting of antigens. CAR quality can be determined at the cell stage, as described above (e.g., as described in step 3). Mathematical models can be used to infer cell quality data and applied to BioNV related to efficacy study data results.

[0362] BioNV features:BioNVs can be tested for basic functionality, including a variety of well-defined, standardized assays, such as in vitro cellular uptake into target cells with and without antigen expression, and their ability to penetrate dense tissues, such as those in human retinal models. These basic functional assays can be performed immediately following the continuous extrusion process, after which preclinical studies will address the remaining quality and functional properties of BioNVs.

[0363] definition

[0364] The following definitions are used in conjunction with the disclosure herein. Unless defined otherwise, 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 belongs.

[0365] An "effective amount" or "therapeutically effective amount" is an amount effective to treat, prevent, or ameliorate a disease in a mammal.

[0366] As used herein, "a," "an," or "the" may mean one or more than one.

[0367] As used herein, the word "include" and variations thereof are intended to be non-limiting, such that recitation of items in a list does not exclude other similar items that may also be applicable to materials, compositions, devices, and methods of the present technology. Likewise, the terms "can" and "may" and variations thereof are intended to be non-limiting, such that recitation of an embodiment that may or may include certain elements or features does not exclude other embodiments of the present technology that do not include those elements or features.

[0368] Although the open-ended term "comprising," as a synonym for terms such as including, containing, or having, is used herein to describe and claim the present disclosure, alternative terms such as "consisting of" or "consisting essentially of" may alternatively be used to describe the present disclosure or its embodiments.

[0369] In embodiments, "BioNV," "exosome," "therapeutic BioNV," or "therapeutic exosome" refers to a biomimetic nanovesicle (NV) that encapsulates an aqueous compartment containing at least one therapeutic biomolecule. In embodiments, the therapeutic BioNV / exosome is allogeneic and / or hypoimmune. In embodiments, the therapeutic BioNV / exosome comprises at least one surface-oriented, membrane-embedded CAR. In embodiments, the "nanovesicle (NV)" referred to herein is a lipid-bound vesicle having a size of about 10 nm to about 1200 nm that encapsulates an aqueous core. In embodiments, the lipid-bound NV can be formed using a lipid monolayer, a lipid bilayer, or maintained in a multilayer form. In embodiments, the therapeutic BioNV / exosome refers to a biologically derived nanosized vesicle that can have designed biological functionalizations. In embodiments, the therapeutic BioNV / exosome is "biomimetic" because it is derived from endogenous cellular material, more specifically, it substantially recreates the plasma membrane material found in cells. In embodiments, the cells from which the therapeutic BioNV / exosomes are derived may include any type of stem cell, including cell types differentiated from such stem cells. In embodiments, the therapeutic BioNV / exosomes are substantially free of encapsulated cellular debris, including nucleic acids, organelles, or organelle portions. In embodiments, the therapeutic BioNV / exosomes are 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:

[0370] a. A size of about 10 nm to about 1200 nm;

[0371] b. The total volume is about 500nm 3 to about 5 μm 3 (assuming a spherical shape);

[0372] c. having a content of at least one phospholipid and cholesterol;

[0373] d. having a surface membrane having one or more of CD34, CCL21, PD-L1 (in therapeutic BioNV / exosomes derived from non-activated cell sources), FasL, SerpinB9, H2-M3, CD47, CTLA-4, CD24, CD200, MFG-E8, NCAM, α-phagocytic integrin, and / or anti-6R antibody or antibody form, or a chimera of one or more thereof; having a surface membrane that substantially lacks T cell receptor components (TRAC and / or TRBC), MHC class I components and / or MHC Class II components lack proteins from one or more 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 are substantially lacking proteins from one or more of IL-4, IL-6, IL-10, and / or IL-16 within the vesicles;

[0374] e. Encapsulation of one or more therapeutically relevant biomolecules, including, for example, cytokines, including chemokines, interferons (IFNα / β / γ), interleukins, alarmins, lymphokines, tumor necrosis factor (TNF), colony stimulating factors, bone morphogenetic proteins (BMPs), erythropoietin (EPO), granulocyte stimulating factor (G-CSF), granulocyte macrophage colony stimulating factor (GM-CSF), proinflammatory cytokines, anti-inflammatory cytokines, perforin granzymes (e.g., granzymes A, B, H, K, and M), gene editing payloads, fusion proteins, antibodies, or antibody-format constructs, or combinations thereof.

[0375] f. having a membrane embedded targeting agent comprising a target binding portion, wherein the target binding portion may include an antibody or antibody format selected from one or more of the following: 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 comprising the antigen binding portion of an antibody, bispecific T cell engager (BiTE), viral epitope recognition receptor (VERR) or viral ligand, variable heavy chain IgG fragment V H H or V NAR or through the T cell receptor (TCR), where the targeting agent can target a single biomarker or multiple biomarkers, or multiple portions of a single biomarker;

[0376] g. payloads capable of adsorbing and / or encapsulating one or more perforins, granzymes, cytokines, cytotoxic proteins, non-naturally occurring cellular agents, checkpoint inhibitors, recombinant gene editing payloads, antibodies or antibody fragments, small molecule inhibitors, biologics, radionuclides, tracers, dyes, fluorescent proteins, and other therapeutic payloads and / or any combination thereof; and

[0377] h. be capable of not eliciting a harmful immune response in the subject.

[0378] In the embodiments, "induced pluripotent stem cells" or "iPSCs" refer to stem cells that can be generated directly from adult cells. iPSCs can be derived from differentiated cells that have been reprogrammed back to an embryonic-like pluripotent state. iPSCs can generally propagate indefinitely and become any cell type of the organism from which they originated.

[0379] In embodiments, "allogeneic" as used herein refers to biological material, tissue, or cells that, while derived from the same species, are genetically different and initially immunologically incompatible. For example, allogeneic BioNV / exosomes are material derived from a first subject (iPSC donor) and can be provided to any number of different, genetically non-identical subjects.

[0380] In embodiments, "low immunogenicity" or "low immunity" as used herein for modified cells and / or BioNV / exosomes refers to a reduced ability to generate an immune response. In embodiments, iPSCs and BioNV / exosomes may have low immunogenicity due to reduced or eliminated expression and / or activity of one or more specific cell surface proteins and / or secreted proteins (e.g., T cell receptor (TCR) proteins, cytokine response syndrome proteins, MHC class I or class II proteins, etc.). In embodiments, iPSCs and BioNV / exosomes may have low immunogenicity due to increased expression of immune protective cell surface proteins (e.g., CD47, CD34, CD24, CD200, α-phagocytic integrin, etc.). In embodiments, BioNV / exosomes may have low immunogenicity due to not triggering CRS in a subject and / or not inducing HLA incompatibility.

[0381] In embodiments, "knockout," "silencing," "inactivation," "disruption," or "blocking," and equivalents thereof, with respect to transcription, gene expression, or protein expression, refers to a reduction in the amount of transcription, gene, or protein expression from a normal state or below the wild-type state in a particular cell subpopulation. The reduction may be significant such that no gene expression occurs, or only a negligible amount of expression occurs.

[0382] In embodiments, "overexpression" as used herein refers to an increase in the amount of transcription, gene or protein expression from a normal state or exceeding the wild-type state in a specific cell subpopulation.

[0383] Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation many equivalents to the specific embodiments described specifically herein. Such equivalents are intended to be encompassed by the scope of the appended claims.

[0384] As used herein, all headings are for organizational purposes only and are not intended to limit the disclosure in any way. The contents of any individual section may apply equally to all sections.

[0385] All patents and publications cited herein are hereby incorporated by reference in their entirety, including published PCT application WO 2020 / 227369, filed on May 6, 2020, entitled “Tailored Hypoimmune Nanovesicle Delivery Systems for Cancer Tumors,” and published U.S. non-provisional application US 20220040106A1, filed on August 3, 2021, entitled “Tailored Hypoimmune Nanovesicular Delivery Systems for Cancer Tumors, Hereditary and Infectious Diseases.”

Claims

1. A method for producing a therapeutic biomimetic nanovesicle (BioNV), the method comprising: (a) obtaining low immunogenic cells; (b) activating the low immunogenic cells to express one or more therapeutically relevant biomolecules; and (c) processing the activated low immunogenic cells to produce a therapeutic BioNV, wherein the therapeutic BioNV comprises a low immunogenic cell plasma membrane and encapsulates the one or more therapeutically relevant biomolecules.

2. A method for producing therapeutic exosomes, the method comprising: (a) obtaining low immunogenic cells; (b) activating the low immunogenic cells to express one or more therapeutically relevant biomolecules; and (c) harvesting naturally shed therapeutic exosomes from the activated low immunogenic cells, wherein the therapeutic exosomes comprise low immunogenic cell plasma membranes and encapsulate the one or more therapeutically relevant biomolecules.

3. A method of treating or preventing a disease or condition, the method comprising administering to a subject in need thereof a therapeutically effective amount of the therapeutic biomimetic nanovesicle (BioNV) produced by claim 1.

4. A method of treating or preventing a disease or condition, the method comprising administering a therapeutically effective amount of the therapeutic exosomes produced by claim 2 to a subject in need thereof.

5. The method of any one of claims 1 to 4, wherein the low immunogenicity cell is a stem cell, an induced pluripotent stem cell (iPSC), a reprogrammed pluripotent or multipotent cell, an embryonic stem cell, a mesenchymal stem cell, or a differentiated cell derived from any stem cell thereof.

6. The method of any one of claims 1 to 4, wherein the low immunogenic cells are T cells, helper T cells, T memory cells or NK cells.

7. The method of any one of claims 1 to 4, wherein the low immunogenic cells are macrophages.

8. The method of any one of claims 1 to 4, wherein the low immunogenic cells are monocytes.

9. The method of any one of claims 1 to 4, wherein the low immunogenicity cell is a hepatocyte, a cardiomyocyte, a neuron, an endothelial cell, a pancreatic cell or a retinal pigment epithelial (RPE) cell.

10. The method of any one of the preceding claims, wherein the low immunogenic 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.

11. The method of any of the preceding claims, wherein the low immunogenic cells have a disruption of the β2-macroglobulin (B2M) gene and / or a disruption that reduces or eliminates the expression and / or activity of MHC class I proteins.

12. The method of any of the preceding claims, wherein the low immunogenic cells have a disruption of the CIITA gene and / or a disruption that reduces or eliminates expression and / or activity of MHC class II proteins.

13. The method of any of the preceding claims, wherein the low immunogenic cells have a disruption of the HLA-A gene and / or a disruption that reduces or eliminates HLA-A protein expression and / or activity.

14. The method of any of the preceding claims, wherein the low immunogenic cells have a disruption of an HLA-B gene and / or a disruption that reduces or eliminates HLA-B protein expression and / or activity.

15. The method of any of the preceding claims, wherein the low immunogenic cells have a disruption of the HLA-C gene and / or a disruption that reduces or eliminates HLA-C protein expression and / or activity.

16. The method of any of the preceding claims, wherein the low immunogenic cells have an HLA-E gene disruption or an HLA-G gene disruption and / or a disruption that reduces or eliminates HLA-E or HLA-G protein expression and / or activity.

17. The method of any of the preceding claims, wherein the low immunogenic cells have a disruption of the HLA-F gene and / or a disruption that reduces or eliminates HLA-F protein expression and / or activity.

18. The method of any of the preceding claims, wherein the low immunogenic cells have a T cell alpha constant (TRAC) gene disruption and / or a disruption that reduces or eliminates TRAC protein expression and / or activity.

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

20. The method of any of the preceding claims, wherein the low immunogenic cells have reduced or eliminated PD-1 gene expression and / or reduced or eliminated PD-1 protein expression and / or activity, wherein the low immunogenic cells are activated; or the low immunogenic cells have expression of the PD-1 gene and / or gene product or increased expression thereof, wherein the low immunogenic cells are not activated.

21. The method of any of the preceding claims, wherein the low immunogenic cells have an IL-4 gene disruption and / or a disruption that reduces or eliminates IL-4 protein expression and / or activity.

22. The method of any of the preceding claims, wherein the low immunogenic cells have a disruption of an IL-6 gene and / or a disruption that reduces or eliminates IL-6 protein expression and / or activity.

23. The method of any one of the preceding claims, wherein the low immunogenic cells have a disruption of an IL-10 gene and / or a disruption that reduces or eliminates IL-10 protein expression and / or activity.

24. The method of any of the preceding claims, wherein the low immunogenic cells have a disruption of an IL-16 gene and / or a disruption that reduces or eliminates IL-16 protein expression and / or activity.

25. The method of any of the preceding claims, wherein the low immunogenic cells have a SerpinB9 gene disruption and / or a disruption that reduces or eliminates SerpinB9 protein expression and / or activity.

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

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

28. The method of any one of the preceding claims, wherein the low immunogenic cells express the PD-L1 gene and / or gene product, or the expression thereof is increased, and wherein the low immunogenic cells are not activated; or wherein the expression of the PD-L1 gene and / or gene product of the modified cells is reduced or eliminated, and wherein the low immunogenic cells are activated.

29. The method of any one of the preceding claims, wherein the low immunogenic cells express the H2-M3 gene and / or gene product, or have increased expression and / or activity.

30. The method of any one of the preceding claims, wherein the low immunogenic cells express the CD47 gene and / or gene product, or have increased expression and / or activity.

31. The method of any one of the preceding claims, wherein the low immunogenic cells express the CD24 gene and / or gene product, or have increased expression and / or activity.

32. The method of any of the preceding claims, wherein the low immunogenic cells express chimeric CD24 / CD47 genes and / or gene products, or have increased expression and / or activity thereof.

33. The method of any of the preceding claims, wherein the low immunogenic cells express the CTLA-4 gene and / or gene product, or have increased expression and / or activity.

34. The method of any one of the preceding claims, wherein the low immunogenic cells express the CD200 gene and / or gene product, or have increased expression and / or activity.

35. The method of any of the preceding claims, wherein the low immunogenic cells express chimeric CD24 / CD200 genes and / or gene products or chimeric CD47 / CD200 genes and / or gene products, or have increased expression and / or activity.

36. The method of any of the preceding claims, wherein the low immunogenic cells express the MFG-E8 gene and / or gene product, or have increased expression and / or activity.

37. The method of any of the preceding claims, wherein the low immunogenic cells express the NCAM gene and / or gene product, or have increased expression and / or activity.

38. The method of any of the preceding claims, wherein the low immunogenic cells express an alpha-phagocytic integrin gene and / or gene product, or have increased expression and / or activity thereof.

39. The method of any of the preceding claims, wherein the low immunogenic cells express an antibody or antibody-like molecule targeting an IL-6 surface receptor (anti-IL-6R), or have increased expression and / or activity.

40. The method of any one of the preceding claims, wherein the low immunogenic cells express the FasL gene and / or gene product.

41. The method of any one of the preceding claims, wherein the low immunogenic cells do not overexpress the FasL gene and / or gene product.

42. methods as described in any one of the preceding claims, wherein the low immunogenicity cells substantially lack expression and / or activity of one or more immunogenic proteins, and express one or more immunoprotective proteins, or their expression is increased.

43. methods as described in any one of the preceding claims, expression and / or the 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 of wherein said low immunogenic cells are reduced or eliminated.

44. methods as described in any one of the preceding claims, wherein the low immunogenicity cells express 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 increase their expression.

45. The method of any of the preceding claims, wherein the expression and / or activity of genes and / or gene products of one of HLA-A, HLA-B, HLA-C, HLA-F, CIITA, IL-6, TRAC, TRBC, and HLA-E or HLA-G of the low immunogenic cells is reduced or eliminated.

46. ​​The method of any one of claims 1 to 44, wherein the expression and / or activity of genes and / or gene products of one of HLA-A, HLA-B, HLA-C, HLA-F, CIITA, IL-6, TRAC, TRBC, SerpinB9, and HLA-E or HLA-G of the low immunogenic cells is reduced or eliminated.

47. A method as described in any one of claims 1 to 44, wherein the low immunogenic cells include low immunogenic cells in which the expression and / or activity of genes and / or gene products of one or more of HLA-A, HLA-B, HLA-C, HLA-F, CIITA, IL-6, TRAC, TRBC, SerpinB9, HLA-E or HLA-G and IL-4, IL-10 and IL-16 are reduced or eliminated.

48. The method of any one of claims 1 to 47, wherein the low immunogenic cells express or increase the expression and / or activity of α-phagocytic integrin, CCL2, H2-M3, FasL, MFEG8, and PD-L1 and / or CTLA-4, wherein the low immunogenic cells do not overexpress FasL; and wherein the low immunogenic cells are not activated by expression of PD-L1; and Expressing one of CD24, CD47, CD200, chimeric CD24 / CD47, chimeric CD24 / CD200 and chimeric CD47 / CD200, or two of CD24, CD47 and CD200, or increasing their expression and / or activity.

49. The method of any one of claims 1 to 47, wherein the low immunogenic cells express or increase the expression and / or activity of α-phagocytic integrin, CCL2, H2-M3, FasL, MFEG8, SerpinB9, and PD-L1 and / or CTLA-4, wherein the low immunogenic cells do not overexpress FasL; and wherein the low immunogenic cells are not activated by the expression of PD-L1; and Expressing one of CD24, CD47, CD200, chimeric CD24 / CD47, chimeric CD24 / CD200 and chimeric CD47 / CD200, or two of CD24, CD47 and CD200, or increasing their expression and / or activity.

50. The method of any one of claims 1 to 47, wherein the low immunogenic cells express or increase the expression and / or activity of the CD200 gene and / or gene product, and do not express and / or substantially lack CD24 or CD47 genes and / or gene products.

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

52. The method of any one of the preceding claims, wherein the low immunogenic cells are allogeneic.

53. The method of any of the preceding claims, wherein the low immunogenic cells do not elicit an immune response in a patient to whom the cells or BioNVs or exosomes derived therefrom are administered.

54. The method of any one of the preceding claims, wherein the low immunogenicity cells comprise one or more targeting agents.

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

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

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

58. A method as claimed in claim 55, wherein the CAR comprises a targeting agent, a transmembrane domain and an intracellular domain, which comprises a co-stimulatory domain and / or a signaling domain.

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

60. A method as claimed in claim 58, wherein the intracellular domain comprises the 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.

61. The method of claim 54, wherein the one or more targeting agents comprise an antibody or an antibody form.

62. The method of claim 61, wherein the antibody or antibody form is selected from one or more of the following: a monoclonal antibody, a polyclonal antibody, an antibody fragment, Fab, Fab', Fab'-SH, F(ab')2, Fv, single chain Fv (scFv), V NAR 、V H H, afilin, 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 of claim 62, wherein the antibody format is a scFv.

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

65. The method of 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 of any of the preceding claims, wherein activating the low immunogenic cells comprises activating the TCR / CD3 receptor complex by a protein antigen.

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

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

69. The method of any one of claims 1 to 65, wherein activating the low immunogenicity cells comprises activating calcium-dependent channels.

70. The method of any one of claims 1 to 65, wherein activating the low immunogenic cells comprises activating the LFA-1 integrin receptor.

71. The method of any one of claims 1 to 65, wherein activating the low immunogenic cells comprises activating the CD28 receptor.

72. The method of any one of claims 1 to 65, wherein activating the low immunogenic cells comprises activating an IL-2 receptor.

73. The method of any one of claims 1 to 65, wherein activating the low immunogenic cells comprises activating the SLAMF1 (CD150) receptor.

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

75. The method of claim 73, wherein the activation of the SLAMF1 (CD150) receptor is by Gram-negative bacteria.

76. The method of any one of claims 1 to 65, wherein activating the low immunogenic cells comprises activating an IFNy receptor.

77. The method of any one of claims 1 to 65, wherein activating the low immunogenic cells comprises activating the CD4 receptor by one or more viruses.

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

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

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

81. The method of any of the preceding claims, wherein activating the low immunogenic cells comprises activating 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 of claim 81, wherein the small RNA comprises one or more of the following: guide RNA (gRNA), tracer RNA (tracrRNA), micro RNA (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.

83. The method of any one of claims 1 to 80, wherein activating the low immunogenic cells comprises activating at the RNA level by one or more of: guide RNA (gRNA), tracer RNA (tracrRNA), micro RNA (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.

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

85. The method of any one of claims 1 to 80, wherein activating the low immunogenicity cells comprises activating through an endogenous promoter region and / or enhancer region.

86. The method of any one of claims 1 to 80, wherein activating the low immunogenicity cells comprises activating by stably integrated genetic elements.

87. The method of any one of claims 1 to 80, wherein activating the low immunogenicity cells comprises activating by transient expression of a genetic element.

88. The method of any one of the preceding claims, wherein activating the low immunogenic cells results in a metabolically altered state of the low immunogenic cells.

89. The method of any of the preceding claims, wherein the one or more therapeutically relevant biomolecules are chemokines, interferons, interleukins, alarmins, lymphokines, perforins, granzymes, granulysins, tumor necrosis factor (TNF), colony stimulating factors, bone morphogenetic proteins (BMPs), erythropoietin (EPO), granulocyte stimulating factor (G-CSF), granulocyte macrophage colony stimulating factor (GM-CSF), gene editing payloads, fusion proteins, antibodies or antibody forms, or combinations thereof.

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

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

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

93. The method of claim 89, wherein the gene editing payload comprises one or more gene editing agent nucleic acids and / or proteins, or one or more nucleic acids encoding one or more gene editing agents.

94. The method of claim 93, wherein the one or more gene editing agents are site-directed endonucleases, TALENs, ZFNs, ribonuclease P RNAs, CRISPR / Cas nucleases, C2c1, C2c2, C2c3, Cas9, Cpf1, TevCas9, Archaea 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 of claim 89, wherein the gene editing payload comprises a transactivation response region (TAR) loop system.

96. The method of any one of claims 1, 3, and 5 to 95, wherein the therapeutic BioNV comprises: (i) one or more membrane-embedded targeting agents that target one or more cellular biomarkers; (ii) one or more of α-phagocytic integrin, CCL2, H2-M3, FasL, MFEG8, PD-L1 and / or CTLA-4, SerpinB9 and an anti-IL-6R antibody or a membrane embedded protein in antibody form; (iii) a membrane embedded protein of one of CD24, CD47, CD200, chimeric CD24 / CD47, chimeric CD24 / CD200 and chimeric CD47 / CD200, or two of CD24, CD47 and CD200; and (iv) a membrane substantially lacking proteins of one or more of HLA-A, HLA-B, HLA-C, HLA-F, CIITA, IL-6, TRAC, TRBC, one or more of HLA-E or HLA-G, and one or more of IL-4, IL-10, and IL-16, and The one or more therapeutically relevant biomolecules are chemokines, interferons, interleukins, alarmins, lymphokines, perforins, tumor necrosis factor (TNF), colony stimulating factors, bone morphogenetic proteins (BMPs), erythropoietin (EPO), granulocyte stimulating factor (G-CSF), granulocyte macrophage colony stimulating factor (GM-CSF), gene editing payloads, fusion proteins, antibodies or antibody forms, or combinations thereof.

97. The method of any one of claims 1, 3, and 5 to 95, wherein the therapeutic BioNV comprises: (i) one or more membrane-embedded targeting agents that target one or more cellular biomarkers; (ii) one or more of α-phagocytic integrin, CCL2, H2-M3, FasL, MFEG8, PD-L1 and / or CTLA-4 and an anti-IL-6R antibody or a membrane embedded protein in antibody form; (iii) a membrane chimeric protein of CD24 and CD47, or a chimeric CD24 / CD47; and (iv) a membrane substantially lacking proteins of one or more of HLA-A, HLA-B, HLA-C, HLA-F, CIITA, IL-6, TRAC, TRBC, HLA-E or HLA-G, one or more of SerpinB9 and CD200, and one or more of IL-4, IL-10, and IL-16, and The one or more therapeutically relevant biomolecules are chemokines, interferons, interleukins, alarmins, lymphokines, perforins, granzymes, granulysins, tumor necrosis factor (TNF), colony stimulating factors, bone morphogenetic proteins (BMPs), 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 of claim 96 or 97, wherein the one or more targeting agents are antibodies or antibody forms.

99. The method of claim 98, wherein the one or more targeting agents is a CAR.

100. The method of any one of claims 1 and 96 to 99, wherein processing the activated low immunogenicity cells is performed by one or more of: sonication, adaptive focused acoustic technology, French press, extrusion, continuous extrusion, enzymatic disruption, detergent cell lysis and / or electroporation.

101. The method of claim 100, wherein processing the activated reduced immunogenicity cells is performed by continuous extrusion.

102. The method of any one of claims 1 and 96 to 101, wherein the therapeutic BioNV has a size of about 10 nm to about 1200 nm.

103. The method of claim 102, wherein the therapeutic BioNV has a size of about 10 nm to about 100 nm.

104. The method of claim 102, wherein the therapeutic BioNV has a size of about 100 nm to about 200 nm.

105. The method of claim 102, wherein the therapeutic BioNV has a size of about 200 nm to about 500 nm.

106. The method of claim 102, wherein the therapeutic BioNV has a size of about 500 nm to about 1200 nm.

107. The method of any one of claims 2 and 4 to 95, wherein the therapeutic exosomes comprise: (i) one or more membrane-embedded targeting agents that target one or more cellular biomarkers; (ii) one or more of α-phagocytic integrin, CCL2, H2-M3, FasL, MFEG8, PD-L1 and / or CTLA-4, SerpinB9 and an anti-IL-6R antibody or a membrane embedded protein in antibody form; (iii) a membrane embedded protein of one of CD24, CD47, CD200, chimeric CD24 / CD47, chimeric CD24 / CD200 and chimeric CD47 / CD200, or two of CD24, CD47 and CD200; and (iv) a membrane substantially lacking proteins of one or more of HLA-A, HLA-B, HLA-C, HLA-F, CIITA, IL-6, TRAC, TRBC, one or more of HLA-E or HLA-G, and one or more of IL-4, IL-10, and IL-16, and The one or more therapeutically relevant biomolecules are chemokines, interferons, interleukins, alarmins, lymphokines, perforins, tumor necrosis factor (TNF), colony stimulating factors, bone morphogenetic proteins (BMPs), erythropoietin (EPO), granulocyte stimulating factor (G-CSF), granulocyte macrophage colony stimulating factor (GM-CSF), gene editing payloads, fusion proteins, antibodies or antibody forms, or combinations thereof.

108. The method of any one of claims 2 and 4 to 95, wherein the therapeutic exosomes comprise: (i) one or more membrane-embedded targeting agents that target one or more cellular biomarkers; (ii) one or more of α-phagocytic integrin, CCL2, H2-M3, FasL, MFEG8, PD-L1 and / or CTLA-4, SerpinB9 and an anti-IL-6R antibody or a membrane embedded protein in antibody form; (iii) a membrane chimeric protein of CD24 and CD47, or a chimeric CD24 / CD47; and (iv) a membrane substantially lacking proteins of one or more of HLA-A, HLA-B, HLA-C, HLA-F, CIITA, IL-6, TRAC, TRBC, HLA-E or HLA-G, one or more of SerpinB9 and CD200, and one or more of IL-4, IL-10, and IL-16, and The one or more therapeutically relevant biomolecules are chemokines, interferons, interleukins, alarmins, lymphokines, perforins, granzymes, granulysins, tumor necrosis factor (TNF), colony stimulating factors, bone morphogenetic proteins (BMPs), 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 of claim 107 or 108, wherein the one or more targeting agents are antibodies or antibody forms.

110. The method of claim 109, wherein the one or more targeting agents is a CAR.

111. The method of any one of claims 2 and 107 to 110, wherein harvesting the therapeutic exosomes from the activated low immunogenic cells comprises inducing hypoxia.

112. The method of any one of claims 2 and 107 to 111, wherein harvesting the therapeutic exosomes from the activated low immunogenic cells comprises expressing or increasing expression of one or more cytokines.

113. The method of any one of claims 2 and 107 to 112, wherein harvesting the therapeutic exosomes from the activated low immunogenic cells comprises providing one or more small molecule exosome modulators.

114. The method of any one of claims 2 and 107 to 113, wherein harvesting the therapeutic exosomes from the activated low immunogenic cells comprises supplementing culture medium with one or more exosomal factors.

115. The method of any one of claims 2 and 110 to 114, wherein the therapeutic exosomes have a size of about 10 nm to about 200 nm.

116. The method of claim 115, wherein the therapeutic exosomes have a size of about 10 nm to about 100 nm.

117. The method of claim 115, wherein the therapeutic exosomes have a size of about 100 nm to about 200 nm.

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

119. The method of claim 1 or 2, wherein activating the low immunogenic cells comprises activating the JAK1 / TYK2 to STAT pathway via the INFAR1 and / or INFAR2 receptors using INF-α.

120. The method of any one of claims 1 to 2 and 119, wherein the low immunogenic cells are natural killer cells, and wherein the activation comprises 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 of any one of claims 1 to 2 and 119 to 120, wherein the low immunogenic cells are natural killer cells, killer T cells, T cells (all subsets), and wherein the activation does not result in degranulation or granule polarization of the immune synapse.

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

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

124. The method of any one of claims 1 to 2 and 119 to 123, wherein the activation comprises activating expression of a cytotoxic biomolecule by targeting a cytoplasmic signaling molecule that is part of a signaling pathway.

125. The method of any one of claims 1 to 2 and 119 to 124, wherein the activation comprises activating PKC by a small molecule or a biologic.

126. The method of any one of claims 1 to 2 and 119 to 125, wherein the activation comprises activation of PKC by calcium and diacylglycerol.

127. The method of any one of claims 1 to 2 and 119 to 126, wherein the activating step comprises activating PKC by adding to the low immunogenic cells 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.

128. The method of any one of claims 1 to 2 and 119 to 120, wherein the activation comprises targeting Crk with small molecules or biologics to express perforin and granzymes.

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

130. The method of any one of claims 1-2 and 119-129, wherein the activation comprises activating a transcription factor that results in activation of a compound selected from the group consisting of perforin, granzymes, other cytotoxic proteins, and combinations thereof.

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

132. A method as described in any one of claims 1 to 2 and 119 to 131, wherein the low immunogenic cells are induced pluripotent stem cells (iPSCs) engineered to contain at least one perforin and / or granzyme gene, and wherein the iPSCs contain regulatory properties or elements embedded in one or more of their promoters, thereby allowing direct and controlled expression of at least one perforin and / or granzyme gene.

133. The method of any one of claims 1 to 2 and 119 to 132, further comprising analyzing the activated low immunogenic cells for a substance 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 of any one of claims 1 to 2 and 119 to 133, further comprising measuring the degree of activation of the activated low immunogenic cells.

135. A method of treating a disease or condition, the method comprising: administering a therapeutically effective amount of a biomimetic nanovesicle (BioNV) comprising one or more BioNV antigen-binding constructs; as well as A therapeutically effective amount of whole cells comprising one or more whole cell antigen binding constructs is administered.

136. The method of claim 135, wherein the disease or condition is cancer.

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

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

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

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

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

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

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

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

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

146. The method of any one of claims 135 to 145, wherein the BioNV comprises: (i) CARs that bind to antigens, ligands, or receptors present on cancer cells; as well as (ii) an antigen, ligand and / or receptor that binds to the CAR on the whole cell.

147. The method of any one of claims 135 to 146, wherein the whole cell comprises: (i) a first CAR that binds to an antigen, ligand or receptor present on a cancer cell; as well as (ii) a second CAR that binds to an antigen, ligand and / or receptor present on the BioNV.

148. The method of claim 147, wherein 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 wherein the antigen, ligand and / or receptor on the BioNV is different from the antigen, ligand or receptor present on the cancer cell.

149. A method as described in claim 147 or 148, wherein the first CAR is capable of signaling via a pathway that leads to cell-mediated cytotoxicity, or comprises one or more intracellular signaling domains that are capable of signaling via a pathway that leads to cell-mediated cytotoxicity.

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

151. A method as described in claim 147 or 148, wherein the second CAR is unable to signal via a pathway that causes cell-mediated cytotoxicity of the whole cell, or lacks one or more intracellular signaling domains that are capable of signaling via a pathway that causes cell-mediated cytotoxicity.

152. A method as described in claim 147 or 148, wherein the second CAR is capable of signaling through a pathway that leads to the persistence, survival and / or proliferation of the whole cell, or comprises one or more intracellular signaling domains that are capable of signaling through a pathway that leads to the persistence, survival and / or proliferation of the whole cell.

153. The method of any one of claims 135 to 152, wherein administering the BioNV improves the function of the whole cell.

154. The method of claim 153, wherein the function of the whole cell comprises one or more of: cell-mediated cytotoxicity, cytokine release, tumor cell or cancer cell killing, integration into tumor cell or cancer tissue, tissue infiltration, proliferation, persistence and / or survival.

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

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