Low-immunogenic cells for producing biomimetic nanovesicles

Abstract

Disclosed herein are methods for producing hypoimmunogenic cells, methods for using hypoimmunogenic cells for treating mammalian diseases, and methods for preparing biomimetic nanovesicles (BioNVs).

Description

Technical Field

[0001] The present disclosure provides, in part, a method for producing hypoimmunogenic cells for producing biomimetic nanovesicles (BioNVs), as well as compositions and uses thereof.

[0002] Cross - reference to related applications This application claims the benefit and priority of U.S. Provisional Application No. 63 / 345,669, filed May 25, 2022, and U.S. Provisional Application No. 63 / 387,516, filed November 14, 2022, the contents of which are hereby incorporated by reference in their entirety.

[0003] Description of electronically submitted text file The content of the text file submitted electronically with this application, namely, a copy of the computer - readable XML - formatted array listing (file name: "CAR - 006PC_Sequence_Listing.xml", recording date: May 24, 2023, file size: 117,233 bytes) is hereby incorporated by reference in its entirety.

Background Art

[0004] Whole - cell therapy is becoming an increasingly effective treatment. Since cell therapy is performed by transferring whole cells from a provider to a patient, it is necessary to closely match the immunogenicity of the provider and the recipient. Multiple low - immunogenic engineering methods for induced pluripotent stem cells (iPSCs) have been reported. All of these methods aim to remove the human leukocyte antigen (HLA) genes that encode multi - tissue compatibility complex (MHC) membrane glycoproteins, which cause immune reactions related to graft - versus - host disease (GVHD) rejection. The HLA gene group is divided into three categories. That is, 1) the MHC class I pathway, 2) the MHC class II pathway, and 3) the MHC class III pathway. Only the MHC class I and II pathways express protein complexes that cause immune reactions in GVHD, while the MHC class III complex is not involved in immune activities.

[0005] Removing or suppressing these complexes provides some protection against harmful immune responses in patients, but there are additional surface proteins and mechanisms that need to be removed / suppressed to enhance the immunosuppressive properties of the cells. For example, removal of MHC class I / II protein complexes leads NK cells and macrophages into an "active clearance mode", where the cells are destroyed.

[0006] Some systems are promising, but they still express accessory non-MHC antigens and may have genetic polymorphisms that contribute to immunological rejection, have a high potential for teratomas, and may ultimately lead to the development of humoral immunity against the cells. In addition, even low-immunogenicity engineering approaches for whole-cell therapy have the potential for cytokine release syndrome (CRS), cell exhaustion, problems with tissue and tumor penetration, problems crossing biological barriers, difficulty in payload encapsulation, targeting problems (e.g., off-target effects), etc. Furthermore, low-immunogenicity cells may require additional engineering manipulations to avoid persistence and initiate controlled cell death (e.g., a "suicide switch"). With each manipulation, the manufacturing cost of whole-cell therapeutics increases. There remains a need to combine the low-immunogenicity properties and disease-specific targeting of engineered whole-cell therapeutics while avoiding the problems and manufacturing costs to enable clinically viable treatment options. SUMMARY OF THE INVENTION

[0007] In embodiments, described herein is a method of generating low-immunogenicity cells, comprising reducing or eliminating the expression and / or activity of one or more immunogenic proteins in the cells, and expressing one or more immunoprotective proteins in the cells or increasing their expression and / or activity, thereby generating low-immunogenicity cells. In embodiments, described herein are low-immunogenicity cells produced using the method of the invention.

[0008] In an embodiment, the 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 of these stem cells. In an embodiment, the differentiated cell is a T cell, a helper T cell, a T memory cell, or an NK cell. In an embodiment, the differentiated cell is a macrophage. In an embodiment, the differentiated cell is a monocyte. In an embodiment, the differentiated cell is a hepatocyte, a cardiomyocyte, a neuron, an endothelial cell, a pancreatic cell, or a retinal pigment epithelial (RPE) cell.

[0009] In an embodiment, the hypoimmunogenic cell substantially lacks one or more of the MHC class I protein complex, the MHC class II complex, the T cell receptor (TCR) complex, and / or the cytokine release syndrome (CRS) protein. In an embodiment, reducing or eliminating the expression and / or activity of one or more immunogenic proteins includes disrupting the β2-microglobulin (B2M) gene and / or disrupting to reduce or eliminate the expression and / or activity of the MHC class I protein. In an embodiment, reducing or eliminating the expression and / or activity of one or more immunogenic proteins includes disrupting the CIITA gene and / or disrupting to reduce or eliminate the expression and / or activity of the MHC class II protein.

[0010] In embodiments, reducing or eliminating the expression and / or activity of one or more immunogenic proteins includes disrupting the HLA-A gene and / or disrupting to reduce or eliminate the expression and / or activity of the HLA-A protein. In embodiments, reducing or eliminating the expression and / or activity of one or more immunogenic proteins includes disrupting the HLA-B gene and / or disrupting to reduce or eliminate the expression and / or activity of the HLA-B protein. In embodiments, reducing or eliminating the expression and / or activity of one or more immunogenic proteins includes disrupting the HLA-C gene and / or disrupting to reduce or eliminate the expression and / or activity of the HLA-C protein. In embodiments, reducing or eliminating the expression and / or activity of one or more immunogenic proteins includes disrupting the HLA-E gene or the HLA-G gene and / or disrupting to reduce or eliminate the expression and / or activity of the HLA-E or HLA-G protein. In embodiments, reducing or eliminating the expression and / or activity of one or more immunogenic proteins includes disrupting the HLA-F gene and / or disrupting to reduce or eliminate the expression and / or activity of the HLA-F protein.

[0011] In embodiments, reducing or eliminating the expression and / or activity of one or more immunogenic proteins includes disrupting the T cell alpha constant (TRAC) gene and / or disrupting to reduce or eliminate the expression and / or activity of the TRAC protein. In embodiments, reducing or eliminating the expression and / or activity of one or more immunogenic proteins includes disrupting the T cell beta constant (TRBC) gene and / or disrupting to reduce or eliminate the expression and / or activity of the TRBC protein.

[0012] In an embodiment, reducing or eliminating the expression and / or activity of one or more immunogenic proteins includes disrupting the PD-1 gene and / or reducing or eliminating the expression and / or activity of the PD-1 protein. In an embodiment, reducing or eliminating the expression and / or activity of one or more immunogenic proteins includes disrupting the IL-4 gene and / or reducing or eliminating the expression and / or activity of the IL-4 protein. In an embodiment, reducing or eliminating the expression and / or activity of one or more immunogenic proteins includes disrupting the IL-6 gene and / or reducing or eliminating the expression and / or activity of the IL-6 protein. In an embodiment, reducing or eliminating the expression and / or activity of one or more immunogenic proteins includes disrupting the IL-10 gene and / or reducing or eliminating the expression and / or activity of the IL-10 protein. In an embodiment, reducing or eliminating the expression and / or activity of one or more immunogenic proteins includes disrupting the IL-16 gene and / or reducing or eliminating the expression and / or activity of the IL-16 protein. In an embodiment, reducing or eliminating the expression and / or activity of one or more immunogenic proteins includes disrupting the SerpinB9 gene and / or reducing or eliminating the SerpinB9 protein expression activity.

[0013] In embodiments, the reduction or elimination of the expression and / or activity of one or more immunogenic proteins is at the DNA level by one or more of a transferase-based method, a Cre / Lox-based method, an endonuclease-based method, a homologous recombination (HR)-based method, a non-homologous end joining (NEHJ)-based method, a microhomology-mediated end joining (MMEJ)-based method, a homology-mediated end joining (HMEJ)-based method, small RNAs, or combinations thereof. In embodiments, the small RNAs are guide RNAs (gRNAs), tracer RNAs (tracrRNAs), microRNAs (miRNAs), RNA interference (RNAi), small interfering RNAs (siRNAs), double-stranded RNAs, Piwi-interacting RNAs (piRNAs), small nuclear RNAs (snRNAs), small nucleolar RNAs (snoRNAs), antisense oligonucleotides (ASOs), locked nucleic acids (LNAs), splice-switching oligonucleotides (SSOs), tRNAs, complementary messenger RNAs, repeat-associated small interfering RNAs (rasiRNAs), endonucleases, and small non-coding RNAs, or include one or more of them. In embodiments, reducing or eliminating the expression and / or activity of one or more immunogenic proteins is at the RNA level by one or more guide RNAs (gRNAs), tracer RNAs (tracrRNAs), microRNAs (miRNAs), RNA interference (RNAi), small interfering RNAs (siRNAs), double-stranded RNAs, Piwi-interacting RNAs (piRNAs), small nuclear RNAs (snRNAs), small nucleolar RNAs (snoRNAs), antisense oligonucleotides (ASOs), locked nucleic acids (LNAs), splice-switching oligonucleotides (SSOs), tRNAs, complementary messenger RNAs, repeat-associated small interfering RNAs (rasiRNAs), endonucleases, and small non-coding RNAs.

[0014] In embodiments, expressing or increasing the expression and / or activity of one or more immune defense proteins comprises expressing or increasing the expression of a CD34 gene and / or gene product. In embodiments, expressing or increasing the expression and / or activity of one or more immune defense proteins comprises expressing or increasing the expression of a CCL2 gene and / or gene product. In embodiments, expressing or increasing the expression and / or activity of one or more immune defense proteins comprises expressing or increasing the expression of a PD-L1 gene and / or gene product, wherein the cells are not activated. In embodiments, expressing or increasing the expression and / or activity of one or more immune defense proteins comprises expressing or increasing the expression of a H2-M3 gene and / or gene product.

[0015] In embodiments, expressing or increasing the expression and / or activity of one or more immune defense proteins comprises expressing or increasing the expression of a CD47 gene and / or gene product. In embodiments, expressing or increasing the expression and / or activity of one or more immune defense proteins comprises expressing or increasing the expression of a CD24 gene and / or gene product. In embodiments, expressing or increasing the expression and / or activity of one or more immune defense proteins comprises expressing or increasing the expression of a chimeric CD24 / CD47 gene and / or gene product.

[0016] In embodiments, expressing or increasing the expression and / or activity of one or more immune defense proteins comprises expressing or increasing the expression of the CTLA-4 gene and / or gene product.

[0017] In embodiments, expressing one or more immune defense proteins or increasing their expression and / or activity includes expressing the CD200 gene and / or gene product or increasing its expression. In embodiments, expressing one or more immune defense proteins or increasing their expression and / or activity includes expressing the chimeric CD24 / CD200 gene and / or gene product, or the chimeric CD47 / CD200 gene and / or gene product, or increasing their expression.

[0018] In embodiments, expressing one or more immune defense proteins or increasing their expression and / or activity includes expressing the MFG-E8 gene and / or gene product or increasing its expression. In embodiments, expressing one or more immune defense proteins or increasing their expression and / or activity includes expressing the NCAM gene and / or gene product or increasing its expression. In embodiments, expressing one or more immune defense proteins or increasing their expression and / or activity includes expressing the α-phagocytic integrin gene and / or gene product or increasing its expression. In embodiments, expressing one or more immune defense proteins or increasing their expression and / or activity includes expressing an antibody or antibody format molecule (anti-IL-6R) that targets the IL-6 surface receptor or increasing its expression.

[0019] In embodiments, expressing one or more immune defense proteins or increasing their expression and / or activity includes expressing the FasL gene and / or gene product. In embodiments, expressing one or more immune defense proteins or increasing their expression and / or activity does not include overexpressing the FasL gene and / or gene product.

[0020] In an embodiment, the hypoimmunogenic cell has 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. In an embodiment, the hypoimmunogenic cell has expressed 3 or more immune defense proteins, 4 or more immune defense proteins, 5 or more immune defense proteins, 6 or more immune defense proteins, 7 or more immune defense proteins, 8 or more immune defense proteins, 9 or more immune defense proteins, or 10 or more immune defense proteins, or has increased expression and / or activity thereof.

[0021] In an embodiment, the hypoimmunogenic cell has reduced or eliminated one or more genes and / or gene products of HLA-A, HLA-B, HLA-C, HLA-F, CIITA, IL-6, TRAC, TRBC, and either HLA-E or HLA-G. In an embodiment, the hypoimmunogenic cell has reduced or eliminated one or more genes and / or gene products of HLA-A, HLA-B, HLA-C, HLA-F, CIITA, IL-6, TRAC, TRBC, SerpinB9, and either HLA-E or HLA-G. In an embodiment, the hypoimmunogenic cell has reduced or eliminated expression and / or activity of one of HLA-A, HLA-B, HLA-C, HLA-F, CIITA, IL-6, TRAC, TRBC, SerpinB9, either HLA-E or HLA-G, and one or more genes and / or gene products of IL-4, IL-10, and IL-16.

[0022] In an embodiment, the hypoimmunogenic cell expresses or has increased expression of α-phagocytic integrin, CCL2, H2-M3, FasL, MFG-E8, and PD-L1 and / or CTLA-4, where the hypoimmunogenic cell does not overexpress FasL, and the hypoimmunogenic cell is not activated by the expression of any one of PD-L1, and CD24, CD47, CD200, chimeric CD24 / CD47, chimeric CD24 / CD200, and chimeric CD47 / CD200, or any two of CD24, CD47, and CD200. In an embodiment, the hypoimmunogenic cell expresses or has increased expression of α-phagocytic integrin, CCL2, H2-M3, FasL, MFG-E8, SerpinB9, and PD-L1 and / or CTLA-4, where the hypoimmunogenic cell does not overexpress FasL, and the hypoimmunogenic cell is not activated by the expression of any one of PD-L1, and CD24, CD47, CD200, chimeric CD24 / CD47, chimeric CD24 / CD200, and chimeric CD47 / CD200, or any two of CD24, CD47, and CD200.

[0023] In an embodiment, the hypoimmunogenic cell expresses or has increased expression of the CD200 gene and / or gene product and does not express or is substantially lacking in any one of the CD24 gene or CD47 gene and / or gene product. In an embodiment, the hypoimmunogenic cell does not have the expression and / or activity of the SerpinB9 gene and / or gene product and the CD200 gene and / or gene product.

[0024] In an embodiment, the expression of one or more immune defense proteins, or the increase in expression, is by the introduction of an exogenous genetic element. In an embodiment, the introduction of the exogenous gene element is by stable integration into the cell genome. In an embodiment, the stable integration is 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, or a combination thereof. In an embodiment, the stable integration is by a viral vector. In an embodiment, the introduction of the exogenous genetic element is by transient transfection. In an embodiment, the expression of one or more immune defense proteins or the increase in their expression is by an exogenous promoter and / or enhancer, and / or an endogenous promoter and / or enhancer, or a combination thereof. In an embodiment, the expression of one or more immune defense proteins or the increase in their expression is under the control of a constitutively active promoter.

[0025] In embodiments, expressing or increasing the expression of one or more immune defense proteins is at the DNA 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, and small non-coding RNA. In embodiments, expressing or increasing the expression of one or more immune defense proteins is 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, and small non-coding RNA. In embodiments, expressing or overexpressing one or more immune defense proteins is by one or more of small regulatory RNA, miRNA, IRES element, transcription factor, or combinations thereof.

[0026] In embodiments, the hypoimmunogenic cells are allogeneic. In embodiments, the hypoimmunogenic cells do not elicit an immune response in the patient to whom the cells or biological compositions derived therefrom are administered.

[0027] In embodiments, the hypoimmunogenic cells comprise one or more targeting agents. In embodiments, the one or more targeting agents comprise a chimeric antigen receptor (CAR). In embodiments, the CAR is bispecific. In embodiments, the CAR lacks an intracellular portion. In embodiments, the CAR comprises a targeting agent, a transmembrane region, and an intracellular region comprising a co-stimulatory region and / or a signaling region. In embodiments, the transmembrane region is derived from CD28, CD3ζ, CD4, CD8α, or ICOS, or fragments thereof. In embodiments, the intracellular region comprises the intracellular signaling region of the CD3ζ chain and / or one or more co-stimulatory molecules optionally selected from CD28, 4-1BB, ICOS, CD27, and OX40.

[0028] In embodiments, the one or more targeting agents comprise an antibody or antibody format. In embodiments, the antibody or antibody format is selected from one or more of monoclonal antibodies, polyclonal antibodies, antibody fragments, Fab, Fab’, Fab’-SH, F(ab’)2, Fv, single-chain Fv (scFv), VNAR, VHH, affilins, diabodies, nanobodies, linear antibodies, bispecific antibodies, multispecific antibodies, chimeric antibodies, humanized antibodies, human antibodies, and fusion proteins comprising the antigen-binding portion of an antibody. In embodiments, the antibody format is scFv. In embodiments, the one or more targeting agents comprise a virus epitope recognition receptor (VERR) or a viral ligand. In embodiments, the one or more targeting agents comprise a ligand for a receptor. In embodiments, the one or more targeting agents comprise a receptor for a ligand.

[0029] In embodiments, the one or more targeting agents are operably linked to a regulatable expression element.

[0030] In embodiments, described herein are hypoimmunogenic cells produced by any of the methods described herein. In embodiments, described herein is a pharmaceutical composition comprising hypoimmunogenic cells produced by any of the methods described herein and one or more excipients.

[0031] In an embodiment, described herein is a hypoimmunogenic cell comprising (a) one or more membrane-embedded targeting agents targeted to one or more cell biomarkers, (b) (i) alpha-phagocytic integrin, CCL2, H2-M3, MFG-E8, and FasL (where FasL is not overexpressed), (ii) PD-L1 and / or CTLA-4 with expression of PD-L1 (where hypoimmunogenic cells are not activated), and (iii) expression or increased expression of an immune defense protein comprising any one of CD24, CD47, CD200, chimeric CD24 / CD47, chimeric CD24 / CD200, and chimeric CD47 / CD200, or any two of CD24, CD47, and CD200, and (c) (i) substantially lacking expression and / or activity of immunogenic proteins including HLA-A, HLA-B, HLA-C, HLA-F, CIITA, PD-1, IL-6, T cell alpha chain (TRAC), and T cell beta chain (TRBC), and (ii) HLA-E or HLA-G.

[0032] In an embodiment, described herein is a hypoimmunogenic cell comprising (a) one or more membrane-embedded targeting agents targeted to one or more cell biomarkers, (b) (i) alpha-phagocytic integrin, CCL2, H2-M3, MFG-E8, and FasL (where FasL is not overexpressed), (ii) PD-L1 and / or CTLA-4 with expression of PD-L1 (where hypoimmunogenic cells are not activated), (iii) either CD24 and CD47, or chimeric CD24 / CD47, and (c) (i) substantially lacking expression and / or activity of immunogenic proteins including HLA-A, HLA-B, HLA-C, HLA-F, CIITA, PD-1, SerpinB9, IL-6, T cell alpha chain (TRAC) and / or T cell beta chain (TRBC), and (ii) HLA-E or HLA-G.

[0033] In embodiments, the hypoimmunogenic cells substantially lack the expression and / or activity of one or more of IL-4, IL-10, and / or IL-16. In embodiments, the hypoimmunogenic cells further comprise the expression or increased expression of an antibody or antibody format molecule (anti-IL-6R) that targets the IL-6 surface receptor. In embodiments, the hypoimmunogenic cells further comprise the expression or increased expression of NCAM.

[0034] In embodiments, one or more targeting agents comprise a chimeric antigen receptor (CAR). In embodiments, one or more targeting agents are an antibody or antibody format. In embodiments, one or more targeting agents are an antibody or antibody format. In embodiments, the antibody or antibody format is selected from one or more of monoclonal antibodies, polyclonal antibodies, antibody fragments, Fab, Fab’, Fab’-SH, F(ab’)2, Fv, single-chain Fv (scFv), VNAR, VHH, affilins, diabodies, nanobodies, linear antibodies, bispecific antibodies, multispecific antibodies, chimeric antibodies, humanized antibodies, human antibodies, and fusion proteins comprising the antigen-binding portion of an antibody. In embodiments, one or more targeting agents are a viral epitope recognition receptor (VERR) or a viral ligand. In embodiments, one or more targeting agents are a ligand for a receptor or a receptor for a ligand.

[0035] In embodiments, the hypoimmunogenic cells are allogeneic. In embodiments, the hypoimmunogenic cells do not cause an immune response in the patient to whom the cells or a biological composition derived therefrom is administered.

Brief Description of the Drawings

[0036]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Figure 11

Figure 12

Figure 13

Figure 14

Figure 15

Figure 16

Figure 17

Figure 18

Figure 19

Figure 20

Figure 21

Figure 22

Figure 23

Figure 24

Figure 25

Figure 26

Figure 27

Modes for Carrying Out the Invention

[0037] The present disclosure relates, in part, to a method for producing hypoimmunogenic cells for use in producing cell-derived biomimetic nanovesicles (BioNVs). BioNVs retain cell-derived functionality while avoiding the concerns of whole cell therapy. Nanovesicles (NVs) have a lower manufacturing cost compared to whole cell therapy, but NV therapeutics can suffer from half-life and excretion problems, such as being cleared by immune cells or the kidneys. The methods of Harding et al., Deuse et al., and Zhao et al. are directly related to whole cell therapy and, by achieving hypoimmunogenicity in part, enable the basis for truly “off-the-shelf” therapeutics but are not useful for BioNV formation (Harding et al., “Induction of long-term allogeneic cell acceptance and formation of immune privileged tissue in immunocompetent hosts.” BioRxiv 716571 [Patent], July 30, 2019 doi:10.1101 / 716571.), (Deuse et al. “Hypoimmunogenic derivatives of induced pluripotent stem cells evade immune rejection in fully immunocompetent allogeneic recipients.” Nat. Biotechnol. Vol. 37, No. 3, 2019: pp. 252-258. doi:10.1038 / s41587-019-0016-3), and (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.).

[0038] In an embodiment, a method of generating low immunogenic cells used to produce BioNV that overcomes the drawbacks of other cells used in BioNV is, for example: 1) The expression of SerpinB9 inhibits granzyme function, thereby limiting the functionality of the mechanism of action of BioNV; 2) The overexpression of FasL is not necessary due to the increased density of naturally expressed FasL on the surface of BioNV that occurs during cell processing (e.g., after extrusion); 3) The overexpression of CD200 may inhibit granulocyte function necessary for efficacy in the solid tumor microenvironment (when used for cancer treatment purposes); 4) The overexpression of too many anti-phagocytosis tags may contribute to cell products that are too stable after the therapeutic effect and difficult to excrete from the body.

[0039] In an embodiment, the method of producing hypoimmunogenic cells improves other methods of producing hypoimmunogenic cells, which focuses on reducing or eliminating (e.g., knocking out) the expression and / or activity of MHC class I / II genes and / or HLA genes and then overexpressing CD47. For example, overexpression of CD47 can inhibit the function of BioNV for several reasons. (1) Knockout of the B2M gene and the CIITA gene in combination with overexpression of the CD47 tag may weaken the desired NK cell response at the target site of BioNV. This requirement is generally not required for all cell therapies, but BioNV lacks the genetic material that contributes to the trigger of the NK cell response against cells lacking all HLA genes. (2) Overexpression of CD47 in some subsets of differentiated cells may be suppressible, whereby the dependence on CD47 limits the types of cells that can be used for BioNV. Since CD47 is important for cell stability, it is a highly regulated surface protein. If the expression of CD47 is too low, cells (and BioNVs) are at risk of premature immune clearance. Conversely, if the expression of CD47 is too high (e.g., as occurs in cancer cells), the cells may not be eliminated from the immune system in a timely manner. The expression of the CD47 isoform 2 gene is tightly regulated at the levels of transcription (through several transcription factors including STAT3, NF-kβ, Hif-1, Myc), RNA translation by microRNAs (miR-708, miR-192, miR-222, miR-133a, miR-155, miR-200a, and miR-340), and post-translational protein modification (formation of N-terminal pyroglutamic acid that aids in interaction with SIRPα). The tight control of CD47 in the cellular environment may cause the purpose of overexpression as an anti-phagocytic defense tag to be inhibited in cells differentiated from engineered iPSC sources, even if the iPSC is derived from syncytiotrophoblast cells in which CD47 is naturally overexpressed (i.e., control may be reduced by lineage differentiation). (3) The TCR gene remains intact. In iPSCs differentiated into the T cell (naive) lineage, the presence of TCRs can contribute to GVHD in recipient patients.Numerous studies have reported on the occurrence of non-MHC antigen rejection reactions via TCR in patients.

[0040] In an embodiment, a method of producing a low immunogenic cell line that is allogeneic and engineered not to cause a harmful immune reaction in a patient is provided. The low immunogenic cell can be engineered to express a chimeric antigen receptor (CAR) among other targeting agent formats (e.g., VERR, V NAR , V H H, scFv, bispecific T cell engager (BiTE), artificial T cell receptor (TCR), dual affinity retargeting (DART) antibody, etc.). The low immunogenic cell can retain the ability to be activated to express various intracellular therapeutic biomolecules (e.g., cytokines, perforin, granzyme, interferon (INF), tumor necrosis factor (TNF), interleukin (IL), etc.) that can be encapsulated in a BioNV. The low immunogenic cell can be engineered to produce a gene editing payload, including nucleic acids (e.g., tracer RNA (trRNA), guide RNA (gRNA), miRNA, tRNA, RNAi, small RNA, and plasmids and gene cassettes that express endonucleases such as CRISPR, TALEN, ZFN, etc.) among other gene editing formats that can be encapsulated in the resulting BioNV. The low immunogenic cell can be engineered to produce and encapsulate therapeutic fusion proteins, antibodies and antibody fragments, nucleic acids, etc. that can be encapsulated in a BioNV or naturally secreted as exosomes. The low immunogenic cell can be differentiated into, for example, T cells, macrophages, cardiomyocytes, etc. while retaining its characteristics, and they can then endow the cells with new characteristics such as the ability to cross biological barriers and cell mobilization ability. The BioNV derived from the low immunogenic cells described herein can retain these characteristics and payloads of the cells and represents an effective alternative to whole cell therapy.

[0041] In an embodiment, what is described herein is a low immunogenic cell. In an embodiment, what is described herein is a low immunogenic cell produced using the method of the present invention.

[0042] Low immunogenic cells can be modified to express one or more targeting agents (e.g., CAR) targeted to one or more cell biomarkers. Low immunogenic cells can be modified to express or have increased expression of one or more membrane-embedded immune defense surface markers, including α-gulphagocytic integrin, CCL2, PD-1, CTLA-4, H2-M3, SerpinB9, CD24, CD47, a chimeric form of CD24 / CD47, CD200, a CD200 chimera with either CD24 or CD47, MFG-E8, anti-IL-6R, NCAM, and / or FasL. Low immunogenic cells can be modified to substantially lack the expression and / or activity of one or more immunogenic proteins, including MHC class I, MHC class II, HLA-A, HLA-B, HLA-C, HLA-E or HLA-G, HLA-F, CIITA, PD-1, SerpinB9, IL-4, IL-6, IL-10, IL-16, T cell alpha chain (TRAC), and / or T cell beta chain (TRBC). Depending on the desired functionality of the BioNV to be derived therefrom, low immunogenic cells can either express or not express CD200 and / or SerpinB9, for example, for the delivery of granzyme.

[0043] In embodiments, as used herein, "increased expression and / or activity" refers to an increase in the expression and / or activity in low immunogenic cells as compared to their native or wild-type cognate cells. For example, in embodiments, the increased expression and / or activity of one or more biomolecules described herein can confer immune-lowering properties to iPSCs as compared to iPSCs that do not have the same expression pattern or level of protein expression. In embodiments, "increased expression and / or activity" is due to genetic modification such as knock-in.

[0044] In embodiments, described herein are compositions of hypoimmunogenic cells and kits containing hypoimmunogenic cells. The compositions can include whole cells suspended in a solution compatible with cryopreservation of the cells or administration of the cells to a subject (e.g., intravenously, intraperitoneally, intramuscularly, etc.). The hypoimmunogenic cell compositions can include additional therapeutic agents for use as whole cell therapies. The kits can include, for example, any of the hypoimmunogenic cell compositions described herein packaged in syringes, IV bags, and can also include instructions for use and diagnostic materials.

[0045] Method for producing hypoimmunogenic cell line In aspects, the disclosure includes methods for producing hypoimmunogenic cells, including reducing or eliminating the expression and / or activity of one or more immunogenic proteins in the cells and expressing or increasing the expression and / or activity of one or more immune defense proteins in the cells, thereby producing hypoimmunogenic cells.

[0046] In embodiments, the hypoimmunogenic cells can be derived from any cell that can differentiate into a specific cell type. In embodiments, the cells are stem cells, induced pluripotent stem cells (iPSCs), reprogrammed pluripotent or multipotent cells, embryonic stem cells, mesenchymal stem cells, or differentiated cells derived from any of these stem cells. In embodiments, the differentiated cells are lymphoid cells such as T cells, helper T cells, T memory cells, NK cells. In embodiments, the differentiated cells are myeloid cells such as macrophages, monocytes, neutrophils. In embodiments, the differentiated cells are tissue-specific cells such as hepatocytes, cardiomyocytes, neurons, endothelial cells, pancreatic cells, retinal pigment epithelial (RPE) cells. In embodiments, the hypoimmunogenic cells are, for example, but not limited to, muscle cells (satellite cells), adipocytes, osteocytes, cardiomyocytes, hepatocytes, blood cells (including erythrocytes, platelets, and all immune cell types), glial cells (among other neuron cell types), epithelial cells, epidermal cells, stromal cells (such as epithelial cells, epidermal cells, stromal cells (e.g., respiratory stromal cells), fibroblasts (e.g., dermal fibroblasts), endothelial cells (e.g., bronchial endothelial cells), oral cells, stromal cells, or germ cells), or any terminally differentiated cell. In embodiments, the hypoimmunogenic cells are, for example, but not limited to, exocrine epithelial cells, hormone-secreting cells (e.g., enteroendocrine cells, thyroid cells, pancreatic islet cells, etc.), sensory transducer cells, autonomic nerve cells, sensory organ cells (e.g., pillar cells, olfactory cells, Schwann cells, satellite glial cells, etc.), barrier cells (lung cells, duct cells, kidney cells, podocytes, etc.), extracellular matrix cells (tendon fibroblasts, osteoblasts, connective tissue cells, etc.), or contractile cells (skeletal muscle cells, cardiomyocytes, myoepithelial cells, etc.), or any functionally specific cell type.

[0047] In embodiments, the hypoimmunogenic cells (or the cells used to generate hypoimmunogenic cells) are human female cells (e.g., from a female donor or source). In embodiments, the hypoimmunogenic cells (or the cells used to generate hypoimmunogenic cells) are human male cells (e.g., from a male donor or source). In embodiments, the hypoimmunogenic cells (or the cells used to generate hypoimmunogenic cells) are human female fibroblast iPSCs. In embodiments, the hypoimmunogenic cells (or the cells used to generate hypoimmunogenic cells) are human male fibroblast iPSCs.

[0048] In embodiments, the allogeneic and hypoimmunogenic properties of cells (e.g., iPSC-derived) are created by knocking out, suppressing, inactivating, blocking, or otherwise rendering ineffective the expression, transcriptional efficiency, and / or activity of one or more immunogenic molecules. In embodiments, reducing or eliminating the expression and / or activity of one or more immunogenic proteins includes disrupting the β2-microglobulin (B2M) gene and / or reducing or eliminating the expression and / or activity of MHC class I proteins, as in the case of the CD8+ T cell lineage. In embodiments, reducing or eliminating the expression and / or activity of one or more immunogenic proteins includes disrupting the CIITA gene and / or reducing or eliminating the expression and / or activity of MHC class II proteins, as in the case of the CD4+ T cell lineage. Without wishing to be bound by theory, these proteins contribute to human leukocyte antigen (HLA) immunogenicity, which requires HLA allele matching in provider-recipients for treatment by cell-based therapies. In embodiments, the allogeneic and / or hypoimmunogenic properties are achieved by reducing or eliminating the expression and / or activity of genes encoding T cell receptor (TCR) proteins, which include, for example, the alpha and beta chains (as in the case of αβ T cells) or the gamma and delta chains (as in the case of γδ T cells) that form the ligand-binding site, as well as the signal transduction modules CD3δ, CD3γ, CD3ε, and CD3ζ. In embodiments, this is performed to reduce the types of foreign T cell receptors other than the receptor of the CAR cassette, further improve the uniformity of the CAR of interest, and reduce non-specific effects in BioNV formation.

[0049] In embodiments, the hypoimmunogenic cells substantially lack one or more of the MHC class I protein complex, the MHC class II complex, the T cell receptor (TCR) complex, and / or the cytokine release syndrome (CRS) protein. In embodiments, reducing or eliminating the expression and / or activity of one or more immunogenic proteins includes β2-microglobulin (B2M) gene disruption and / or disruption that reduces or eliminates the expression and / or activity of the MHC class I protein. In embodiments, knocking out the B2M gene may reduce the expected number of administrations to be performed due to the risk of preventing the long-term acceptance of BioNV by the recipient, as observed in the above-described all-cell-based approaches. To overcome this problem, in embodiments, the HLA-E or HLA-G gene remains intact, allowing the immune system to adapt to the resulting BioNV. 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.

[0050] In embodiments, reducing or eliminating the expression and / or activity of one or more immunogenic proteins includes disrupting the HLA-A gene and / or disrupting to reduce or eliminate the expression and / or activity of the HLA-A protein. In embodiments, reducing or eliminating the expression and / or activity of one or more immunogenic proteins includes HLA-B gene disruption and / or disrupting to reduce or eliminate the expression and / or activity of the HLA-B protein. In embodiments, reducing or eliminating the expression and / or activity of one or more immunogenic proteins includes HLA-C gene disruption and / or disrupting to reduce or eliminate the expression and / or activity of the HLA-C protein. In embodiments, reducing or eliminating the expression and / or activity of one or more immunogenic proteins includes HLA-E gene disruption or HLA-G gene disruption and / or disrupting to reduce or eliminate the expression and / or activity of the HLA-E or HLA-G protein. In embodiments, reducing or eliminating the expression and / or activity of one or more immunogenic proteins includes HLA-F gene disruption and / or disrupting to reduce or eliminate the expression and / or activity of the HLA-F protein.

[0051] In embodiments, hypoimmunogenic cells include CIITA gene disruption and / or disruption that inhibits MHC class II protein expression. In embodiments, allogeneic iPSCs have their CIITA genes disrupted, and the resulting differentiated cell lines (e.g., DCs, monocytes, endothelial cells, thymic epithelial cells, B cells, etc.) do not express or have reduced expression of MHC class II proteins.

[0052] In embodiments, reducing or eliminating the expression and / or activity of one or more immunogenic proteins includes disrupting the T cell alpha constant (TRAC) gene and / or disrupting to reduce or eliminate the expression and / or activity of the TRAC protein. In embodiments, reducing or eliminating the expression and / or activity of one or more immunogenic proteins includes disrupting the T cell beta constant (TRBC) gene and / or disrupting to reduce or eliminate the expression and / or activity of the TRBC protein. In embodiments, reducing or eliminating the expression and / or activity of one or more immunogenic proteins includes disrupting the PD-1 gene and / or disrupting to reduce or eliminate the expression and / or activity of the PD-1 protein.

[0053] CRS is a major concern in whole cell therapy, and despite engineered hypoimmunogenicity, effector functions and other interactions with cells post-injection as causal relationships can lead to the release of biomolecules that cause a systemic inflammatory syndrome characterized by fever, multi-organ dysfunction, etc. In embodiments, hypoimmunogenic cells are engineered to disrupt one or more proteins contributing to CRS. In embodiments, hypoimmunogenic cells reduce or eliminate the expression and / or activity (e.g., knockout or suppression) of CRS-related cytokines.

[0054] In embodiments, reducing or eliminating the expression and / or activity of one or more immunogenic proteins includes IL-4 gene disruption and / or disruption that reduces or eliminates the expression and / or activity of the IL-4 protein. In embodiments, reducing or eliminating the expression and / or activity of one or more immunogenic proteins includes IL-6 gene disruption and / or disruption that reduces or eliminates the expression and / or activity of the IL-6 protein. In embodiments, IL-6 knockout prevents the unwanted packaging of IL-6 into BioNV and reduces the contribution of BioNV to local (and focused by biomarker targeting) and / or potentially systemic CRS events. In embodiments, reducing or eliminating the expression and / or activity of one or more immunogenic proteins includes IL-10 gene disruption and / or disruption that reduces or eliminates the expression and / or activity of the IL-10 protein. In embodiments, reducing or eliminating the expression and / or activity of one or more immunogenic proteins includes IL-16 gene disruption and / or disruption that reduces or eliminates the expression and / or activity of the IL-16 protein. In embodiments, reducing or eliminating the interleukin reduces the likelihood of CRS.

[0055] Serine protease inhibitor B9 (SerpinB9) is a member of the serine protease inhibitor superfamily. SerpinB9 has been reported to protect cells from the immune killing action of granzyme B. In embodiments, the low immunogenic cells express SerpinB9 or have increased expression. In embodiments, the low immunogenic cells have SerpinB9 knocked out and / or suppressed. In embodiments, reducing or eliminating the expression and / or activity of one or more immunogenic proteins includes SerpinB9 gene disruption and / or disruption that reduces or eliminates SerpinB9 protein expression activity.

[0056] In embodiments, overexpression of SerpinB9 blocks the function of granzyme B, which is associated with immune-stimulatory responses such as 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, in the case of BioNV intended to deliver a non-granzyme payload, such as a gene editing payload, low immunogenic cells may express and / or overexpress SerpinB9.

[0057] In embodiments, reducing or eliminating the expression and / or activity of one or more immunogenic proteins is at the DNA level by one or more of a transferase-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 molecule RNA, or combinations thereof. In embodiments, the small molecule RNA includes one or more of a guide RNA (gRNA), a tracer RNA (tracrRNA), a microRNA (miRNA), RNA interference (RNAi), a small interfering RNA (siRNA), 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. In embodiments, reducing or eliminating the expression and / or activity of one or more immunogenic proteins is at the RNA level by one or more of a guide RNA (gRNA), a tracer RNA (tracrRNA), a microRNA (miRNA), RNA interference (RNAi), a small interfering RNA (siRNA), 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.

[0058] In embodiments, the site-specific nuclease used for any gene modification herein may include a CRISPR / Cas endonuclease, TALENs, ZFNs, or any other site-specific nuclease system for gene suppression and / or knockout. In embodiments, gene disruption is, for example, by a gene editing system that includes one or more proteins and / or nucleic acids that cooperate as found in TALEN, ZFN, ribonuclease P RNA, C2c1, C2c2, C2c3, Cas9, Cpf1, TevCas9, ArchaeaCas9, CasY.1, CasY.2, CasY.3, CasY.4, CasY.5, CasY.6, CasX Cas omega, transposase, and / or any ortholog or homolog of any of these editors.

[0059] In embodiments, the gRNA sequence compatible with the CRISPR / Cas9 system can be designed (e.g., as described in Table 8) to be targeted to at least a portion of the B2M gene (including coding and non-coding sequences that control gene expression). For example, in non-limiting embodiments, SEQ ID NOs: 22-23 enumerate exemplary B2M gene sequences for designing gRNAs for knockout purposes. In non-limiting embodiments, a series of exemplary gRNA sequences (described in DNA form) are described in SEQ ID NOs: 21, 24, 26-29, and 133-134 that are targeted to the sequences of SEQ ID NOs: 22-23. In non-limiting embodiments, such gRNAs can create B2M allele knockouts (including, for example, bi-allelic knockouts), resulting in, for example, cloned DNA sequences as described in SEQ ID NOs: 43-49. In embodiments, the genomic DNA sequence obtained from the CRISPR / Cas-modified B2M can be used to insert one or more genes, coding sequences, DNA sequences, etc. (e.g., within the space(s) flanked by SEQ ID NOs: 43-49 for B2M knockout).

[0060] In an embodiment, a gRNA sequence compatible with the CRISPR / Cas9 system can be designed (e.g., as described in Table 8) to be targeted to at least a portion of the CIITA gene, including coding and non-coding sequences that control gene expression. For example, in a non-limiting embodiment, SEQ ID NOs: 32-33 enumerate exemplary CIITA gene sequences for designing gRNAs for knockout purposes. In a non-limiting embodiment, a series of exemplary gRNA sequences (described in DNA format) are described in SEQ ID NOs: 31, 34, and 36-42, which are targeted to the sequences of SEQ ID NOs: 32-33. In a non-limiting embodiment, such gRNAs can create CIITA allele knockouts (e.g., including bi-allelic knockouts), resulting in, for example, clone DNA sequences such as those described in SEQ ID NOs: 50-55. In an embodiment, the genomic DNA sequence obtained from the CRISPR / Cas modification of CIITA can be used to insert one or more genes, coding sequences, DNA sequences, etc. (e.g., within the space(s) flanked by SEQ ID NOs: 50-55 for CIITA knockout).

[0061] In embodiments, a gRNA sequence compatible with the CRISPR / Cas9 system can be designed (e.g., as described in Table 8) to target at least a portion of the TRAC gene, including the coding and non-coding sequences that control gene expression. For example, in a non-limiting embodiment, SEQ ID NOs: 56-57 enumerate exemplary TRAC gene sequences for designing gRNAs for knockout purposes. In a non-limiting embodiment, a series of exemplary gRNA sequences (described in DNA format) are described in SEQ ID NOs: 60-75 that target the sequences of SEQ ID NOs: 56-57. In a non-limiting embodiment, such gRNAs can create TRAC allele knockouts (e.g., including bi-allelic knockouts), resulting in, for example, cloned DNA sequences such as those described in SEQ ID NOs: 102-108. In embodiments, genomic DNA sequences obtained from CRISPR / Cas modification of TRAC can be used to insert one or more genes, coding sequences, DNA sequences, etc. (e.g., within one or more spaces (s) flanked by SEQ ID NOs: 102-108 for TRAC knockout).

[0062] In embodiments, the gRNA sequence compatible with the CRISPR / Cas9 system can be designed to target at least a portion of the TRBC1 gene (including the coding and non-coding sequences that control gene expression) (e.g., as described in Table 8). For example, in a non-limiting embodiment, SEQ ID NOs: 76-77 enumerate exemplary TRBC1 gene sequences for designing gRNAs for knockout purposes. In non-limiting embodiments, a series of exemplary gRNA sequences (described in DNA format) are described in SEQ ID NOs: 79-101 that are targeted to the sequences of SEQ ID NOs: 76-77. In non-limiting embodiments, such gRNAs can create allelic knockouts of TRBC1 (including, for example, bi-allelic knockouts), resulting in, for example, cloned DNA sequences such as those described in SEQ ID NOs: 109-110 and 112-116. In embodiments, the genomic DNA sequences obtained from the CRISPR / Cas modification of TRBC1 can be used to insert one or more genes, coding sequences, DNA sequences, etc. (e.g., within one or more spaces (s) flanked by SEQ ID NOs: 109-110 and 112-116 for TRBC1 knockout).

[0063] In embodiments, the gRNA sequence compatible with the CRISPR / Cas9 system can be designed to target at least a portion of the IL-6 gene (including the coding and non-coding sequences that control gene expression) (e.g., as described in Table 8). For example, in a non-limiting embodiment, SEQ ID NOs: 117-118 enumerate exemplary IL-6 gene sequences for designing gRNAs for knockout purposes. In non-limiting embodiments, a series of exemplary gRNA sequences (described in DNA format) are described in SEQ ID NOs: 119-130 that are targeted to the sequences of SEQ ID NOs: 117-118. In non-limiting embodiments, such gRNAs can create allelic knockouts of IL-6 (including, for example, bi-allelic knockouts), resulting in cloned DNA sequences. In embodiments, the genomic DNA sequences obtained from the CRISPR / Cas modification of IL-6 can be used to insert one or more genes, coding sequences, DNA sequences, etc.

[0064] In embodiments, low immunogenic cells can be generated by knocking out one or more genes described herein using one or more gRNAs described herein. For example, in embodiments, the gRNA has about or at least about 85% sequence identity, about or at least about 86% sequence identity, about or at least about 87% sequence identity, about or at least about 89% sequence identity, about or at least about 90% sequence identity, about or at least about 91% sequence identity, about or at least about 92% sequence identity, about or at least about 93% sequence identity, about or at least about 94% sequence identity, about or at least about 95% sequence identity, about or at least about 96% sequence identity, about or at least about 97% sequence identity, about or at least about 98% sequence identity, or about or at least about 99% sequence identity to one or more of SEQ ID NOs: 31, 24, 26 - 29, 31, 34, 36 - 42, 60 - 75, 79 - 101, 119 - 130, and 133 - 134.

[0065] In embodiments, low immunogenic cells can be generated by targeting one or more genes described herein for knockout and / or for insertion of one or more proteins for increased expression and / or expression and / or activity, as described herein, and / or by targeting one or more DNA sequences. For example, in embodiments, the DNA sequence includes one or more of SEQ ID NOs: 22 - 23, 32 - 33, 56 - 57, 76 - 77, and 117 - 118, which include sequences having one or more substitutions, deletions, insertions, variants, and / or nucleotide polymorphisms (e.g., single nucleotide polymorphism SNPs) therein.

[0066] In embodiments, the hypoimmunogenic cells (and BioNVs derived therefrom) express or have increased expression of one or more immune defense proteins. In embodiments, expressing one or more immune defense proteins or increasing their expression and / or activity includes expressing or increasing the expression of the CD34 gene and / or gene product. In embodiments, expressing one or more immune defense proteins or increasing their expression and / or activity includes expressing or increasing the expression of the CCL2 gene and / or gene product. In embodiments, expressing one or more immune defense proteins or increasing their expression and / or activity includes expressing or increasing the expression of the PD-L1 gene and / or gene product, where the hypoimmunogenic cells are not activated by the expression of PD-L1. In embodiments, expressing one or more immune defense proteins or increasing their expression and / or activity includes expressing or increasing the expression of the H2-M3 gene and / or gene product.

[0067] In embodiments, expressing one or more immune defense proteins or increasing their expression and / or activity includes expressing or increasing the expression of the CD47 gene and / or gene product. In embodiments, preventing the potentially inhibitory phenotype of CD47 expression across the cell is accomplished through deletion of this region in a stable construct or by eliminating / inhibiting the expression of microRNAs, through interference with the inhibitory mechanism of action of a set of microRNAs against the 3’UTR of the CD47 gene. In embodiments, this can address the problem of inhibition caused by microRNAs.

[0068] In an embodiment, expressing one or more immune defense proteins or increasing their expression and / or activity includes expressing the CD24 gene and / or gene product or increasing its expression. CD24 is a sialoglycoprotein expressed on mature granulocytes and B cells and is also an anti-phagocytic protein. CD24 blocks phagocytosis through interaction with Siglec-G / 10 on macrophages. In an embodiment, the hypoimmunogenic cells express or overexpress the CD24 protein and / or gene product. In an embodiment, expressing one or more immune defense proteins or increasing their expression and / or activity includes expressing the chimeric CD24 / CD47 gene and / or gene product or increasing its expression. In an embodiment, the hypoimmunogenic cells comprise CD24 / CD47 having a tethered transmembrane region. In an embodiment, the CD47 isoform 2 and the CD24 region may be expressed separately or tethered to form a double-membrane chimeric protein. In an embodiment, the hypoimmunogenic cells are fibroblast-derived iPSCs and not ABO cell-derived.

[0069] In embodiments, expressing one or more immune defense proteins or increasing their expression and / or activity includes expressing the CD200 gene and / or gene product or increasing its expression. In embodiments, the CD200 tag minimizes phagocytosis by macrophages and also prevents granulocyte activation. In embodiments, since granulocyte activation would complement the mechanism of action of BioNV designed to release granzyme and perforin, for example, when it is not desirable to block granulocytes in the solid tumor microenvironment (TME), low immunogenic cells do not express CD200. However, in embodiments, when the CD47 or CD24 tag is used, or a CD24 / CD47 chimeric bilayer protein tag (each preventing phagocytosis) is used in combination with overexpressed H2-M3 (attenuating the NK response), clearance of BioNV can be enabled while achieving stability without CD200. In embodiments, when granzyme and perforin are not selected as therapeutic biomolecules, CD200 can be expressed to prevent granulocyte activation, while the CD47 tag or CD24 tag can be excluded, but both tags cannot be excluded. In embodiments, expressing one or more immune defense proteins or increasing their expression and / or activity includes expressing or increasing the expression of a chimeric CD24 / CD200 gene and / or gene product, or a chimeric CD47 / CD200 gene and / or gene product. In embodiments, these CD200 strategies correspond to low immunogenic cell lines for creating BioNVs for targeting non-cancer cells, such as those targeting liver, kidney, heart cells, and / or tissue regeneration pathways.

[0070] In an embodiment, the low immunogenicity cells (or cells differentiated therefrom) do not express and / or overexpress all three of CD47, CD24, and CD200. In an embodiment, the low immunogenicity cells are engineered such that BioNV derived from the low immunogenicity cell line is stabilized, but not engineered to the extent that BioNV is resistant to elimination from the body. A BioNV that is too stable may ultimately trigger a humoral response, which may limit the number of administrations or treatments that can be administered.

[0071] In an embodiment, expressing or increasing the expression and / or activity of one or more immune defense proteins includes expressing or increasing the expression of the CTLA-4 gene and / or gene product. In an embodiment, expressing or increasing the expression and / or activity of one or more immune defense proteins includes expressing or increasing the expression of the MFG-E8 gene and / or gene product. In an embodiment, expressing or increasing the expression and / or activity of one or more immune defense proteins includes expressing or increasing the expression of the NCAM gene and / or gene product. In an embodiment, expressing or increasing the expression and / or activity of one or more immune defense proteins includes expressing or increasing the expression of the α-phagocytic integrin gene and / or gene product. In an embodiment, expressing or increasing the expression and / or activity of one or more immune defense proteins includes expressing or increasing the expression of an antibody or antibody format molecule (anti-IL-6R) that targets the IL-6 surface receptor.

[0072] In embodiments, expressing one or more immune defense proteins or increasing their expression and / or activity includes the expression of the FasL gene and / or gene product. In embodiments, expressing one or more immune defense proteins or increasing their expression and / or activity does not include overexpression of the FasL gene and / or gene product. In embodiments, overexpression of FasL is avoided because enrichment of the naturally expressed level of FasL is observed, for example, in the membrane of BioNV after processing via continuous extrusion. If the concentration of FasL is too high, it can have a reverse effect, preventing the recruitment of T cells to solid tumors and / or causing premature T cell death.

[0073] In embodiments, low immunogenic cells can express one or more fusion proteins of one or more portions of any of the immune defense proteins herein. For example, in embodiments, constructs can be made in which an appropriate portion of a selected ligand is tethered to a transmembrane region. In embodiments, constructs can be made such that biologically relevant portions of two or more proteins are joined together and / or tethered to a transmembrane region.

[0074] In embodiments, low immunogenic cells have reduced or eliminated expression and / or activity of one or more immunogenic proteins that cause an immune response, provider-recipient mismatch, HLA allogeneic immunity, inflammation, CRS, etc. in a subject, such as MHC class I proteins, MHC class II proteins, HLA proteins, TCR proteins, CRS proteins, etc. In embodiments, 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.

[0075] In embodiments, the modified cells have the expression or increased expression and / or activity of one or more immune defense proteins, such as proteins that prevent or reduce the immune response in a subject, prevent or reduce the premature clearance of BioNV in a subject, prevent or reduce phagocytosis, confer barrier-crossing functionality, etc., such as CD47, CD24, CD200, CD34, CCL2, H2-M3, MFG-E8, PD-L1, CTLA-4, etc. In embodiments, the hypoimmunogenic cells are not activated or are in an activated state by the expression of PD-L1. In embodiments, the hypoimmunogenic cells have the expression or increased expression of 3 or more immune defense proteins, 4 or more immune defense proteins, 5 or more immune defense proteins, 6 or more immune defense proteins, 7 or more immune defense proteins, 8 or more immune defense proteins, 9 or more immune defense proteins, or 10 or more immune defense proteins.

[0076] In embodiments, the hypoimmunogenic cells have a reduction or removal of the gene and / or gene product of any one of HLA-A, HLA-B, HLA-C, HLA-F, CIITA, IL-6, TRAC, TRBC, and HLA-E or HLA-G.

[0077] In embodiments, the hypoimmunogenic cells have a reduction or removal of the gene and / or gene product of any one of HLA-A, HLA-B, HLA-C, HLA-F, CIITA, IL-6, TRAC, TRBC, SerpinB9, and HLA-E or HLA-G.

[0078] In embodiments, the hypoimmunogenic cells have a reduction or removal of the expression and / or activity of the gene and / or gene product of any one of HLA-A, HLA-B, HLA-C, HLA-F, CIITA, IL-6, TRAC, TRBC, SerpinB9, HLA-E or HLA-G, and one or more of IL-4, IL-10, and IL-16.

[0079] In an embodiment, the hypoimmunogenic cell expresses or has increased expression of α-phagocytic integrin, CCL2, H2-M3, FasL, MFG-E8, and PD-L1 and / or CTLA-4, where the hypoimmunogenic cell does not overexpress FasL, and where the hypoimmunogenic cell is not activated by the expression of any one of PD-L1, CD24, CD47, CD200, chimeric CD24 / CD47, chimeric CD24 / CD200, chimeric CD47 / CD200, or any two of CD24, CD47, CD200.

[0080] In an embodiment, the hypoimmunogenic cell expresses or has increased expression of α-phagocytic integrin, CCL2, H2-M3, FasL, MFG-E8, SerpinB9, and PD-L1 and / or CTLA-4, where the hypoimmunogenic cell does not overexpress FasL, and the hypoimmunogenic cell is not activated by the expression of any one of PD-L1, CD24, CD47, CD200, chimeric CD24 / CD47, chimeric CD24 / CD200, chimeric CD47 / CD200, or any two of CD24, CD47, CD200.

[0081] In an embodiment, the hypoimmunogenic cell expresses or has increased expression of the CD200 gene and / or gene product and does not express or is substantially lacking in either the CD24 gene or the CD47 gene and / or gene product. In an embodiment, the hypoimmunogenic cell does not have the expression and / or activity of the SerpinB9 gene and / or gene product, and the CD200 gene and / or gene product.

[0082] Immunodeficient HLA complex For the production of BioNV, the low immunogenic cells, in embodiments, express one or more immune deficiency complexes or have increased expression. In embodiments, BioNV has one or more immune deficiency complexes. In embodiments, the one or more immune deficiency complexes include one or more human leukocyte antigens (HLAs) such that when the complex interacts with one or more T cells, the induction of a T cell response is inhibited. In embodiments, the immune deficiency complex includes a fusion protein that is inhibited from activating T cells but maintains structural integrity to allow for some effector function.

[0083] For the production of BioNV, the low immunogenic cells, in embodiments, express or have increased expression of one or more T cell receptors (TCRs) instead of or in addition to the CAR construct as described herein.

[0084] The immune-deficient HLA construct, in embodiments, includes one or more nucleic acids encoding the immune-deficient HLA. In embodiments, the complex optionally includes, in order from N-terminus to C-terminus, an amino acid sequence, or a nucleic acid sequence encoding an amino acid sequence including at least a portion of a peptide and a human HLA class 1 heavy chain sequence. In embodiments, the immune-deficient HLA is a fusion protein having a peptide that binds to the HLA and interferes with one or more interactions that the HLA molecule has with cell surface complexes on immune cells. In embodiments, when the peptide interacts with one or more T cells, the peptide does not elicit a substantial T cell response. In embodiments, the peptide is unable to activate one or more T cells (e.g., CD4+ T cells, CD8+ T cells, etc.). In embodiments, the peptide is capable of binding to the receptor of one or more T cells, and the binding is insufficient to activate one or more T cells. In embodiments, the peptide binds to one or more HLA-binding groove region residues of the human HLA class 1 heavy chain sequence. In embodiments, the peptide modulates the conformation of the human HLA class 1 heavy chain sequence. This conformation, in embodiments, prevents one or more T cells from binding to the human HLA class 1 heavy chain sequence.

[0085] MHC class I molecules typically bind peptides that are about 8 - 10 amino acids in length, but can also bind non-standard long peptides (e.g., 13 amino acids or longer). Peptides, in embodiments, are about or at least about 7 amino acids, 8 amino acids, 9 amino acids, 10 amino acids, 11 amino acids, 12 amino acids, 13 amino acids, 14 amino acids, or 15 amino acids or longer in length. In some embodiments, the human HLA class 1 heavy chain sequence comprises one or more class 1 HLAs.

[0086] The human HLA class 1 heavy chain sequence, in embodiments, is HLA - A, HLA - B, HLA - C, or any combination thereof. In embodiments, the human HLA class 1 heavy chain sequence comprises multiple versions of HLA - A, HLA - B, HLA - C, or any combination thereof. In embodiments, the human HLA class 1 heavy chain sequence comprises HLA - A, where HLA - A is positioned between HLA - B and HLA - C.

[0087] The complex, in embodiments, has one or more linkers between the peptide and the human HLA class 1 heavy chain sequence. The one or more linkers, in embodiments, are configured to resist proteolytic cleavage or reduce cleavage. In embodiments, the peptide is bound to the complex by a disulfide bond. In embodiments, the one or more linkers are configured to form a conformation that does not block one or more killer cell immunoglobulin - like receptor (KIR) binding sites on the human HLA class 1 heavy chain sequence.

[0088] The complex, in embodiments, is a fusion protein with one or more immune checkpoint agonists. In embodiments, the one or more immune checkpoint agonists include CD47, PD - L1, PD - L2, A2AR, B7 - H3, B7 - H4, BTLA, CTLA - 4, IDO, KIR, LAG3, NOX2, PD - 1, TIM - 3, VISTA, SIGLEC7, or combinations thereof.

[0089] The human HLA class I heavy chain sequence, in embodiments, includes HLA-E or a fragment thereof, HLA-F or a fragment thereof, HLA-G or a fragment thereof, or any combination thereof. In embodiments, at least one of HLA-E or a fragment thereof, HLA-F or a fragment thereof, HLA-G or a fragment thereof, or any combination thereof is inhibited from inducing a T cell response when the complex interacts with one or more T cells.

[0090] The complex, in embodiments, has a regulatory peptide. The regulatory peptide, in embodiments, is an apoptosis-inducing peptide, for example, acting as a "kill switch" for controlling the complex. BioNV, in embodiments, does not require a regulatory peptide in its complex, whereas low immunogenic cells intended for use as cell therapy use a regulatory peptide in their complex.

[0091] The complex, in embodiments, has an epitope configured to enable detection of the complex. In embodiments, the epitope is 3,5-dinitrosalicylic acid or includes 3,5-dinitrosalicylic acid.

[0092] The complex, in embodiments, includes a human β2M amino acid sequence. In embodiments, the complex has one or more linkers between the peptide sequence and the human β2M sequence, or between the human β2M sequence and the human HLA class I heavy chain sequence, or has one or more linkers between both.

[0093] In embodiments, the complex has one or more linkers. In embodiments, the one or more linkers are disposed between the peptide and the HLA class I sequence, or alternatively, the one or more linkers are disposed between the HLA class I sequence and one or more of β2M, which is a checkpoint agonist sequence, and / or a second HLA sequence. In non-limiting embodiments, the one or more linkers have a sequence with at least about 70%, 80%, 90%, 95%, or 99% sequence identity to an influenza A virus M1 peptide or a histone M3 peptide. In non-limiting embodiments, the one or more linkers have a sequence consisting essentially of glycine and / or serine residues, such as, for example, Gly-Gly-Ser, Gly-Gly-Gly-Ser, Gly-Gly-Gly-Gly-Ser, Gly-Ser-Ser, and the like.

[0094] In embodiments, the complex comprises a fusion construct from the N-terminus to the C-terminus, a peptide (e.g., for binding to the HLA groove), one or more linkers, a human β2M sequence, one or more linkers, and a human HLA class 1 heavy chain sequence. In embodiments, the complex comprises one or more HLAs, where the one or more HLAs are inhibited from causing a T cell response when the complex interacts with one or more T cells. In embodiments, the HLA fusion has one or more linkers that adopt a conformation that allows (e.g., does not inhibit) interaction with one or more killer cell immunoglobulin-like receptor (KIR) binding sites on the human HLA class 1 heavy chain sequence.

[0095] In embodiments, the one or more HLAs have one or more mutations that reduce or inhibit the HLA from causing a T cell response when the complex interacts with one or more T cells, such as cytotoxic T lymphocytes (e.g., CD4+ and / or CD8+ T cells).

[0096] In embodiments, the complex has one or more proteins or fragments thereof that reduce or inhibit the immune response by the complement system. In embodiments, the one or more proteins or fragments thereof include CD48, CD59, or a combination thereof.

[0097] In embodiments, the immunodeficient HLA takes the form of a nucleic acid molecule that optionally functions to replace one or more sequences encoding the native HLA gene in hypoimmunogenic cells. In embodiments, the hypoimmunogenic cells have a genetic element configured to receive one or more sequences for deletion / disruption at the HLA locus. In embodiments, the one or more sequences encode the human HLA class 1 heavy chain sequence fusion complex described herein.

[0098] BioNV for neoantigen presentation In aspects, the disclosure includes BioNV having one or more surface-exposed neoantigens. In embodiments, the neoantigens are the proteins (or peptides derived therefrom) of one or more biomarkers in Table 5 and / or Table 6 including their isoforms or mutants, or include them. In embodiments, the neoantigens are one or more peptides from Table 1. Without wishing to be bound by theory, the one or more biomarkers stimulate whole cells expressing cognate CARs in co-therapeutic applications while also retaining the CAR-directed killing power of BioNV. TIFF2025518125000002.tif69159

[0099] In embodiments, BioNV (and the cells from which BioNV is derived) express MHC class I / II receptors that display neoantigens. In embodiments, the MHC class I / II receptors are immunoincompatible HLA molecules as described herein. In embodiments, the MHC class I / II receptors display neoantigens by direct binding interactions. In embodiments, the MHC class I / II receptors are fused to neoantigens (e.g., via flexible peptide linkages). In embodiments, the fusion includes one or more amino acid linker sequences as described herein.

[0100] In embodiments, "expression or increased expression" of "gene and / or gene product" encompasses increased expression at the DNA level (e.g., stable expression from gene knock-in, insertion of a transgene cassette having a gene and an enhancer, internal ribosome entry site (IRES) element, etc.), or changes outside the gene itself that result in an increase in its gene product (e.g., increased transcription factor concentration, higher activity of a promoter inserted / replaced upstream of the gene, etc.). In embodiments, expressing or overexpressing one or more immune defense proteins is at the RNA level using one or more of small regulatory RNAs, IRES elements, regulation of the concentration of activated transcription factors in the cell, or combinations thereof.

[0101] In embodiments, expressing or increasing the expression of one or more immune defense proteins is by introduction of exogenous genetic elements. In embodiments, the introduction of exogenous gene elements is by stable integration into the cell genome. In embodiments, stable integration is 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, or combinations thereof. In embodiments, stable integration is by a viral vector. In embodiments, the introduction of exogenous genetic elements is by transient transfection. In embodiments, expressing or increasing the expression of one or more immune defense proteins is by an exogenous promoter and / or enhancer, and / or an endogenous promoter and / or enhancer, or combinations thereof. In embodiments, expressing or increasing the expression of one or more immune defense proteins is under the control of a constitutively active promoter.

[0102] In embodiments, expressing or increasing the expression of one or more immune defense proteins is at the DNA 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, and small non-coding RNA. In embodiments, expressing or increasing the expression of one or more immune defense proteins is 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, and small non-coding RNA. In embodiments, the expression or overexpression of one or more immune defense proteins is by one or more of small regulatory RNAs, miRNAs, IRES elements, transcription factors, or combinations thereof.

[0103] The expression or overexpression of one or more immune defense proteins is by knock-in of genetic elements. In embodiments, the expression or overexpression of one or more immune defense proteins is by the Cre / Lox recombinase system, a transposase-based system, or an endonuclease system, as described herein. In embodiments, "knock-in" of "genetic elements" can refer to engineering the introduction of a promoter / enhancer element upstream of an existing gene, or inserting a new transgene cassette into a gene copy within a framework having one or more promoters, enhancers, introns, IRES sequences, or any other element that can be used to increase the expression of the gene and / or gene product. In embodiments, the expression or overexpression is by an exogenous promoter or enhancer (e.g., from a plasmid, or knock-in of a promoter or newly added cis-acting DNA element). In embodiments, the expression or overexpression is by an endogenous promoter or enhancer, e.g., an increase in the concentration of a transcription factor acting on the endogenous promoter (e.g., without genomic manipulation). In embodiments, the expression or overexpression is under the control of a constitutively active promoter (e.g., for mammalian systems, SV40, CMV, UBC, EF1A, PGK CAGG, etc.) and / or an inducible promoter (e.g., tetracycline control, etc.).

[0104] In embodiments, expression or overexpression is by stably integrating the gene into the cell. In embodiments, stable integration is by a viral vector. In embodiments, iPSCs (among other cells) are genetically engineered for the integration of gene cassettes (e.g., CARs or any other optional elements described herein). In embodiments, integration of gene cassettes can include both integrative and non-integrative transgene insertions. Non-limiting examples of non-integrative transgene insertions include the mRNA method, the non-integrative lentiviral method, and the endonuclease targeting method. Integrative gene cassette insertion methods include stable retroviral vector insertion systems and integrase-based integration systems. Stable gene cassette introduction can be achieved, for example, using a retroviral vector such that the iPSC maintains the gene elements encoding the gene through differentiation, expansion, and activation. In embodiments, clinical-level stable introduction of the CAR cassette into T cells has been achieved for brexucabtagene autoleucel (Tecartus®, Kite Pharma Inc.) and axicabtagene ciloleucel (Yescarta®, Kite Pharma Inc.) using a GRV vector, while tisagenlecleucel (Kymriah®, Novartis International AG) has been introduced using a lentiviral vector (Labbe, R.P., et al. “Lentiviral Vectors for T Cell Engineering: Clinical Applications, Bioprocessing and Future Perspectives.” Viruses 13 (2021); 1528. doi:10.3390 / v13081528).

[0105] In embodiments, the low immunogenicity cells can express one or more immune defense proteins by transient transfection (e.g., electroporation, lipid reagents, etc.). In embodiments, the low immunogenicity cells can be genetically modified to include one or more knockouts of the immunogenic proteins, and the expression or overexpression of the one or more immune defense proteins can be controlled by transient expression in a later step of the manufacturing pipeline.

[0106] In embodiments, the low immunogenicity cells (and BioNVs derived therefrom) are allogeneic. In embodiments, allogeneic means the quality of an "off-the-shelf" of cells from a single source that should be used to create a clonal cell population that is suitable for the treatment of multiple diseases and can be administered to multiple patients regardless of the immunological profile of the subject to be treated.

[0107] In embodiments, the hypoimmunogenic cells, or biological compositions derived therefrom, do not cause an immune response in the patient to whom they are administered. For example, in embodiments, the hypoimmunogenic cells do not result in an inflammatory response and / or an immune response upon administration. In embodiments, upon administration to a subject, the hypoimmunogenic cells cause less than about 50%, less than about 45%, less than about 40%, less than about 35%, less than about 30%, less than about 25%, less than about 24%, less than about 23%, less than about 22%, less than about 21%, less than about 20%, less than about 19%, less than about 18%, less than about 17%, less than about 16%, less than about 15%, less than about 14%, less than about 13%, less than about 12%, less than about 11%, less than about 10%, less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% of an inflammatory or immune response, as measured as a function of cytokine, chemokine, and immunomodulatory enzyme concentrations, such as, for example, but not limited to, 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-alpha / beta / gamma, TNF alpha / beta, IDO, HLA-G, HGF, PGE2, or any combination thereof, compared to allogeneic whole cell therapeutics.

[0108] In embodiments, the hypoimmunogenic cells comprise one or more targeting agents. In embodiments, the cells comprise one or more targeting agents. In embodiments, the one or more targeting agents comprise a chimeric antigen receptor (CAR). In embodiments, the CAR is bispecific. In embodiments, the CAR lacks an intracellular portion. In embodiments, the CAR comprises a targeting agent, a transmembrane region, and an intracellular region comprising a co-stimulatory region and / or a signaling region. In embodiments, the transmembrane region is derived from CD28, CD3ζ, CD4, CD8α, or ICOS, or a fragment thereof. In embodiments, the intracellular region comprises the intracellular signaling region of the CD3ζ chain and / or one or more co-stimulatory molecules optionally selected from CD28, 4-1BB, ICOS, CD27, and OX40. In embodiments, the one or more targeting agents comprise an antibody or an antibody format. In embodiments, the antibody or antibody format is selected from one or more of a monoclonal antibody, a polyclonal antibody, an antibody fragment, Fab, Fab’, Fab’-SH, F(ab’)2, Fv, single-chain Fv (scFv), VNAR, VHH, affilin, diabody, nanobody, 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, the antibody format is scFv. In embodiments, the one or more targeting agents comprise a viral epitope recognition receptor (VERR) or a viral ligand. In embodiments, the one or more targeting agents comprise a ligand for a receptor. In embodiments, the one or more targeting agents comprise a receptor for a ligand.

[0109] In embodiments, the low immunogenicity cells express one or more targeting agents by an adjustable expression element. In embodiments, the adjustable expression element includes, among other adjustable expression elements, an adjustable promoter (e.g., Tet on / off promoter), or a CRISPRa / i regulatory system. In embodiments, CAR expression is controlled by an adjustable expression element. Simple overexpression of constructs from CMV (or other types) promoters can lead to too high a surface density of CAR, which can result in many problems such as "overactivation" leading to in vitro exhaustion and cell death after activation. Cell death leads to the loss of cell lines for generating BioNV. In embodiments, the surface density of the CAR protein construct is regulated. For example, in embodiments, the typical concentration range of CAR protein per microgram of T cells is 0.20 ng to 0.70 ng. If the expression level of CAR is too low, biomarker targeting will be insufficient. However, it cannot exceed the density limit of the cells. If the CAR density is too high, the parent cells may become exhausted during the in vitro activation process before BioNV induction, and the protein concentration in the cell membrane will increase, which may reduce the quality of BioNV. By setting an upper limit, the targeting efficiency can be increased when the biomarker expression on cancer cells is low. The density limit of cell therapeutics is described, for example, in the published US non-provisional application US20220040106, which is incorporated herein by reference.

[0110] In embodiments, stable cellular integration (safe harbor genomic location) of any of the genetic elements described herein in cells (e.g., iPSCs) can be controlled by implementing a Tet-regulated CRISPRa + targeted 3× transcription factor-targeted gRNA system. The CRISPR activation system for three upstream transcription factors can trigger signal cascade events that enhance the production of CARs that replace the endogenous antibody ORF at the designated locus (loci). This system is "tunable" by including a Tet control promoter and can vary the concentration of CARs on the cell surface. Next, stable replacement by the CDR and the CAR cassette of the heavy and light antibody regions can be achieved by Cpf-1-directed homology-directed repair (HDR). Finally, the stably integrated CAR cassette can include adjacent gRNA binding sites that allow for rapid and consistent insertion of the desired sequence by repeatedly swapping or varying scFVs (among other antibody formats) or VERR / viral ligands.

[0111] In embodiments, the concentration of CARs on the surface of iPSC-based cell lines, or any downstream differentiated cells (and resulting BioNVs), can be regulated using various transcriptional control elements, such as a tetracycline on / off promoter (or similar, drug-regulated promoter) to drive the expression of the CRISPR activation / gRNA (CRISPRa) system. The CRISPRa system can then activate antibody regulatory transcription factors, such as Drm2, Fr5, Bxp2, etc., that control the expression of the engineered CAR cassette integrated at the site of the antibody locus (where the antibody gene was replaced). Additionally, similar transcriptional control elements can be provided to control the overexpression of genes (e.g., CD47), drive genes that control differentiation, etc., at defined manufacturing stages.

[0112] In embodiments, the regulatory points within the cell can be engineered to express specific therapeutically relevant proteins of interest as stable cell lines (e.g., stable integration for constitutive expression).

[0113] In embodiments, the transcription factor can be activated intracellularly by supplying a small molecule, (de)phosphorylation event, nucleic acid, etc. In embodiments, the transcription factor can be transiently expressed by a plasmid. In embodiments, the transcription factor can be stably expressed from integration (e.g., using a constitutively active promoter) to create a stable cell line having constitutive signaling of a signaling pathway related to a therapeutic biomolecule. In embodiments, therapeutic biomolecules expressed in hypoimmunogenic cells include cytokines (e.g., for functions such as pro-inflammatory, anti-antigen, mobilization, etc.), perforin, granzyme, chemokine, interferon (IFNα / β / γ), interleukin, alarmin, lymphokine, 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), or combinations thereof.

[0114] In embodiments, the promoter region or enhancer region of a gene can be regulated by overexpression of a specific transcription factor, controlled by microRNA, tRNA, or activated or inhibited using a (g)RNA-guided endonuclease (e.g., CRIPSRa / CRISPRi) with an activation region or inhibitory region linked thereto.

[0115] In embodiments, a therapeutically relevant gene of interest can be integrated (stably or transiently) into a cell of interest (based on desirable properties such as a membrane protein that implies barrier crossing). In embodiments, a stably integrated gene can be activated by any one of the methods described herein.

[0116] In embodiments, background (unwanted) mRNA can be suppressed by interfering RNAs (e.g., siRNA, RNAi, etc.) to enhance the presence / expression of the desired mRNA for the target protein. In embodiments, mRNA can also be regulated through an IRES element. In embodiments, certain conjugated variants can be enhanced by the addition of IRES-enhancing and / or suppressing biomolecules to produce the desired therapeutically relevant peptides / proteins that can be encapsulated in BioNV during cell processing after activation.

[0117] In embodiments, unwanted genes can be knocked out to enhance the expression of the desired therapeutically relevant genes. Those skilled in the art will understand the methods available for transiently and stably knocking out unwanted genes and / or entire signaling pathways during the processing of BioNV.

[0118] In embodiments, low immunogenic cells are derived from iPSCs engineered as described herein. In embodiments, iPSCs are reverted from a somatic cell state using microRNA technology instead of small molecule transactivators. The use of microRNA provides a more stringent differentiation lineage and results in higher quality iPSCs. Without wishing to be bound by theory, such high quality iPSCs are less likely to undergo expression attenuation (such as of modified proteins like CD47) or genetic drift, and also have a high quality / quantity of culture splitting (able to split the culture more times than other methods before problems occur with cell integrity).

[0119] In embodiments, BioNVs derived from iPSC-derived low immunogenic cells retain the functionality of the low immunogenic cells, e.g., the ability to cross the blood-brain barrier as in the case of macrophages / monocytes, or tissue-specific factors as in the case of cardiomyocytes, hepatocytes, etc.

[0120] In an embodiment, allogeneic iPSC cells have their MHC class I and MHC class II complexes disrupted by knocking out B2M, a serum protein found in association with MHC class I heavy chains on the surface of almost all nucleated cells involved in peptide antigen presentation to the immune system, which is an important protein involved in their expression.

[0121] In an embodiment, once B2M knockout (KO), CIITA KO, IL-6 KO, and CD47tg knock-in (KI) are engineered into iPSCs, the TRAC and TRBC genes can be knocked out. In an embodiment, only one gene of each, rather than both alleles separately, is knocked out. In an embodiment, the TRAC gene and the TRBC gene can be knocked out as described herein. The purpose of knocking out the TRAC gene and the TRBC gene is to remove the T cell receptor. In an embodiment, the modified cells are differentiated into a subset of T cells lacking the T cell receptor to induce BioNV. Genetically modifying the cells to substantially lack the TCR reduces the potential for competing ligands for CAR constructs that can be non-specifically targeted to alternative tissues. Thus, in an embodiment, the TCR gene is knocked out as a strategy to reduce the non-specific effects of BioNV. In an embodiment, the TRAC / TRBC knockout not only reduces the likelihood of CRS but also generally reduces the toxicity of BioNV.

[0122] In an embodiment, the modified cells are expanded after engineering. Small-scale expansion methods known in the art, or large-scale feeder system expansion methods, can be used.

[0123] In an embodiment, after constructing B2M KO, CIITA KO, IL-6 KO, CD47tg KI, and an IL-2 promoter-driven green fluorescent protein (GFP) (IL-2p GFP) reporter, the CAR construct can be incorporated / gene-introduced into cells. In an embodiment, the CAR construct can be knocked into the TRAC / TRBC gene, while simultaneously knocking out the remaining TRAC / TRBC gene, resulting in cells that are CAR+ and TRAC / TRBC− / −. In an embodiment, the CAR construct can be simultaneously knocked into the positions of the TRAC / TRBC gene on both loci, resulting in cells that are CAR+ / + and TRAC / TRBC− / −.

[0124] In an embodiment, once B2M KO, CIITA KO, IL-6 KO, CD47tg KI, IL-2p GFP KI, and CAR-modified cells (e.g., iPSCs) are designed, the quality of the immunological synapse (IS) between the CAR recognition region and the biomarker is measured. In an embodiment, the quality of the BioNV's IS can be directly related to the effectiveness in whole cell therapy.

[0125] In embodiments, the BioNVs, or hypoimmunogenic cells derived therefrom, contain nucleic acids encoding GFP (among other fluorescent proteins). In embodiments, once B2M KO, CIITA KO, CD47tg KI, IL-6 KO, TRAC / TRBC single KO are engineered into iPSCs, GFP molecules are engineered into the modified cell line. In embodiments, this functions as a control cell line. In embodiments, non-control cell lines (therapeutic cell lines) do not have GFP. In embodiments, the nucleic acid encoding GFP is operably linked to a promoter from one or more of IL-2, perforin, granzyme, alarmin, TNF, INF, combinations thereof, and / or any other cell-specific or reporter gene. The IL-2 promoter is constitutively activated when lymphocytes are broadly / globally activated from various stimuli. In embodiments, more focused activation / suppression (regulation) is used. In embodiments, the IL-2p GFP reporter gene functions as an indicator of the degree of broad / global activation of cells (as part of the BioNV induction process). In embodiments, the GFP signal, in combination with immunoblot analysis of cytokine levels (such as perforin, granzyme, alarmin, TNF, INF, etc.), enables efficient regulation of the degree of broad / global activation of lymphocytes when exposed to activation antigens. In embodiments, GFP is used to compare the degree of activation between manufacturing lots and ensure consistency in therapeutic development.

[0126] In embodiments, broad or global regulation of cells results in the expression of multiple genes for producing cytokines, chemokines, regulatory nucleic acids, among other therapeutically relevant biomolecules that adapt the cells to an "activated" state, whereby the cells are enhanced to fulfill a metabolically ordained purpose / phenotype. In embodiments, the controlled expression of one or more therapeutically relevant biomolecules in cells can recapitulate, for example, activated T lymphocytes entering an activated state in which the T cell receptor (TCR) engages an antigen-presenting cell (APC) (Figure 1). In embodiments, the controlled expression of one or more therapeutically relevant biomolecules in cells can mimic the interaction between the antigen peptide presented in the major histocompatibility complex (MHC) and the TCR of a T cell that initiates a conformational change in the TCR, inducing massive gene expression of intercellular signaling cascades and cytokines (e.g., perforin, granzyme, alarmin, interleukin, and interferon) (Figure 1). Typically, cytokines "activate" cells in a specific mode such that they remove antigens from the host, recruit immune cells to the site of infection to assist in the removal of antigens / infected cells and the repair of surrounding tissues. In embodiments, these cellular processes occur in cell lines without the provision of exogenous cytokines that can have deleterious effects such as exhaustion, competing signaling pathways, etc. In embodiments, the activated state of cells (e.g., T cells) is utilized by BioNV / exosomes through a processing method in a form that captures cytokines or transmembrane ligands in their lumen and / or membrane, as shown in Figure 2.

[0127] In an embodiment, the hypoimmunogenic cells are activated and / or purified as shown in FIG. 3. In an embodiment, a broad or global cell modulation method includes, in addition to the activation pathway, among other activation molecules, binding an antigen(s) to magnetic beads or streptavidin-biotin beads to ensure separation of the antigen from the cell activation receptor (FIG. 3). For example, in an embodiment, a biomarker antigen that forms an immunological synapse (IS) with a CAR construct can be bound to magnetic beads or streptavidin-biotin beads and then added to CAR-containing cells in vitro to activate the cells. In an embodiment, once activated, the biomarker antigen is mechanically removed from the cell suspension.

[0128] In embodiments, therapeutically relevant biomolecules (e.g., cytokines) can be selectively expressed from any cell by manipulating regulatory points within signaling points. In embodiments, for example, activation of kinases, (de)activation of phosphorylases, activation of hydrolases (such as GTPases), introduction of inhibitors (to branch or block signals from one pathway to another), or introduction of promoting activators / agonists, one or more points within one signaling pathway (or multiple pathways) can be activated. In embodiments, these signaling pathways can be selectively activated by supplying the cell with proteins, peptides, small molecules, nucleic acids, carbohydrates, chimeric molecules, viral ligands, inorganic elements / compounds (e.g., calcium), etc., alone or in combination, to concentrate cellular pathways to promote the expression of therapeutically relevant biomolecules intracellularly, which can then be encapsulated within the lumen of BioNV / exosomes (Figure 3). In embodiments, the regulatory points include, but are not limited to, one or more cell surface receptors that are targeted alone or in combination to concentrate intracellular signals to express the biomolecule of interest. In embodiments, cells can be genetically modified to be particularly sensitive to the activation of these pathways to obtain metabolically / phenotypically adjusted cells (regardless of type). In embodiments, cells can be made "particularly sensitive" to activation by specific stimuli by overexpression of cell surface receptors, overexpression of intracellular signaling molecules (e.g., STAT, NF-κB, MAPK / ERK / ATM kinases, etc.), and / or their constitutively active mutants.

[0129] In an embodiment, the hypoimmunogenic cells are CD34+ or derived from CD34+ cells such as human CD34+ cord blood. In an embodiment, the CD34+ cord blood-derived cell line can function as a base cell line for the development, production, and manufacturing of BioNV for the delivery of gene editing therapeutics. In an embodiment, the hypoimmunogenic cell line derived from CD34+ cord blood has been experimentally confirmed for low expression of HLA1 / 2 and overexpression of CD47 (Deuse T. et al. “Hypoimmunogenic derivatives of induced pluripotent stem cells evade immune rejection in fully immunocompetent allogeneic recipients.” Nat Biotechnol. 2019;37(3):252-258).

[0130] In embodiments, hypoimmunogenic cells can be engineered using multiple hypoimmunogenic engineering techniques, such as those described in Deuse et al., Han et al., Xu et al., Harding et al., and also as described in the published patent applications US20190376045, US20190376045, US20210308183, and US20210292715 to Deuse, US20210161971 to Nagy, US20180141992 to Strominger, and published European patent application 3693384 to Poirot. Each of these is incorporated herein by reference in its entirety (Han X, et al. “Generation of hypoimmunogenic human pluripotent stem cells.” PNAS. Vol. 116, No. 21 2019: pp. 10441 - 10446. doi:10.1073 / pnas.1902566116. and Xu H, et al. “Targeted Disruption of HLA Genes via CRISPR - Cas9 Generates iPSCs with Enhanced Immune Compatibility.” Cell Stem Cell. Vol. 24, No. 4, 2019: pp. 566 - 578. doi:10.1016 / j.stem.2019.02.005.).

[0131] In embodiments, BioNV is derived from cells in which the HLA genes encoding MHC membrane glycoproteins, which give rise to immune responses associated with GVHD rejection, have been eliminated. The HLA gene complex is divided into three categories. Namely, 1) the MHC class I pathway, 2) the MHC class II pathway, and 3) the MHC class III pathway. Only the MHC class I and II pathways express protein complexes that induce immune responses in GVHD, while the MHC class III complex is not involved in immune activity.

[0132] When the MHC class protein complex is removed, NK cells and macrophages shift to an active clearance mode, after which the cells are destroyed. To avoid this killing mechanism, in embodiments, the addition of a CD47 isoform 2 transmembrane molecular protein tag can be engineered into the cell membrane of the modified cells to avoid the killing response mediated by NK and macrophages, as described, for example, in Willingham et al., Deuse et al., and Han et al. (Willingham SB, et al. “The CD47-signal regulatory protein alpha (SIRPa) interaction is a therapeutic target for human solid tumors.”. PNAS. Vol. 109, No. 17, 2012: pp. 6662-7. doi: 10.1073 / pnas.1121623109.).

[0133] In embodiments, the cells can be engineered to use additional mechanisms to prevent these killing responses, such as, for example, those described below: 1) a CD24 transmembrane protein tag (such as that performed by Zhao et al.), 2) membrane-bound surfactant protein D (SP-D) (such as that performed by Jiaravuthisan et al.) (Jiaravuthisan P, et al. “A membrane-type surfactant protein D (SP-D) suppresses macrophage-mediated cytotoxicity in swine endothelial cells.” Transpl Immunol. Vol. 47, 2018: pp. 44-48. doi:10.1016 / j.trim.2018.02.003.), and 3) a molecular PD-L1 tag to prevent T cell responses. In embodiments, BioNVs derived from “activated” cells will encapsulate and / or release perforin and / or granzyme, resulting in targeted cell death. In embodiments, activated cells will produce the perforin and / or granzyme to be encapsulated in the BioNVs. In embodiments, the hypoimmunogenic cells to be activated will not express PD-L1, in order to avoid the resulting BioNVs targeting PD-1 on T cells. In embodiments, this reduces the likelihood of releasing perforin and / or granzyme, resulting in unwanted T cell death. In embodiments, PD-L1 is overexpressed in BioNVs derived from non-activated cells and is not loaded with apoptotic cytokines. In embodiments, the hypoimmunogenic cells to be activated have PD-L1 downregulated, knocked out, or otherwise suppressed. In embodiments, the hypoimmunogenic cells not to be activated have PD-L1 upregulated. That is, for the BioNVs used for gene editor delivery.In an embodiment, among other cells, in particular, CD47 isoform 2 acts as a "do not eat" tag via the SIRP-α receptor expressed on these cells, and thus can be engineered into cells to block both macrophage- and NK cell-mediated cytotoxicity. In an embodiment, CD47 can be utilized in genetically engineered iPSCs for immune tolerance to innate immune cells, as, for example, in Chhabra et al., Han et al., and Jaiswal, et al. (Chhabra A, et al. “Hematopoietic stem cell transplantation in immunocompetent hosts without radiation or chemotherapy.” Sci Transl Med. Vol. 8, No. 351, 2016: 351ra105. doi:10.1126 / scitranslmed.aae0501. and Jaiswal S, et al. “CD47 is upregulated on circulating hematopoietic stem cells and leukemia cells to avoid phagocytosis.” Cell. Vol. 138, No. 2, 2009: pp. 271-85. doi:10.1016 / j.cell.2009.05.046.). In an embodiment, cells can be modified as described in U.S. Patent No. 8,562,997 to Jaiswal, et al.

[0134] In an embodiment, for example, in Xu et al. and Han et al., a method is also used that does not completely knockout all HLA genes, but rather only knocks out HLA genes highly associated with the immune response, leaving HLA genes (e.g., HLA-E, HLA-F, HLA-G) that attenuate macrophage and NK responses intact, as was carried out. In an embodiment, this method does not require the addition of a CD47 tag. The modified cells can be engineered to create BioNV regardless of the presence or absence of CD47.

[0135] In an embodiment, the method improves the low immunogenicity approach of Table 2. TIFF2025518125000003.tif82159

[0136] In an embodiment, the development of allogeneically modified cells requires the removal of MHC class I and MHC class II protein complexes by disruption of specific HLA genes, or B2M knockout, followed by knockout of the CIITA gene. In an embodiment, the knockout can be performed using the CRISPR gene editing technique due to its rapid mechanism of action. In an embodiment, the knockout is performed using zinc finger nucleases (ZFNs) and / or TALENs. In an embodiment, modified cells are produced using the Cre / Lox recombinase system. In an embodiment, modified cells are produced using RNA suppression (RNAi, shRNA, microRNA, CRISPR Cas13a-d, etc.).

[0137] In embodiments, the method of creating allogeneic hypoimmunogenic modified cells is different from the allogeneic creation method of Harding et al. Instead of deleting MHC class I / II genes and risking long-term rejection by the recipient, the method of Harding et al. includes alternative approaches based on naturally occurring immune evasion mechanisms. This method relies on the biomimicry of Harding et al. based on the prominent horizontally transmitted cancer DFTD2 type in the Tasmanian devil. In embodiments, creating allogeneic modified cells involves expressing or increasing the expression of the immunomodulatory proteins CCL21, PD-L1, FasL, SerpinB9, H2-M3, CD47, CD200, and / or MFG-E8 without deleting MHC class I / II proteins to protect cell derivatives from long-term immune rejection in mice (and humans). In embodiments, the modified cells express one or more of the proteins shown in Table 3 (including any splice variants and / or isoforms of any of the indicated proteins, e.g., CD200 splice variants). In embodiments, this system can be used to interfere with the activity of APCs, macrophages, NK cells, and T lymphocytes. In embodiments, the modified cell line can also include the safe cell line developed by Liang et al. (2018), where the cell division gene is linked to a suicide gene to prevent the runaway of teratomas leading to cancer (Liang Q, et al. “Linking a cell-division gene and a suicide gene to define and improve cell therapy safety.” Nature. Vol. 563, No. 7733, 2018: pp. 701-704. doi:10.1038 / s41586-018-0733-7.).

[0138] In embodiments, the method improves the hypoimmunogenic approach of Table 3. TIFF2025518125000004.tif93159

[0139] In embodiments, the low immunogenic cells derived from BioNVs are engineered to have one or more knockouts of HLA-A, HLA-B, HLA-C, HLA-E, HLA-G, HLA-F, CIITA, IL-6, IL-4, IL-10, IL-16, TRAC, TRBC, SerpinB9, and / or any combination thereof, and one or more knockins of CCL2, PD-L1 (in BioNVs derived from non-activated cell sources), CTLA-4, H2-M3, CD24, CD47 (excluding the 3'UTR region or an alternative 3'UTR region without the binding site for inhibitory microRNAs), MFG-E8, CD200, and / or any combination thereof.

[0140] In embodiments, BioNVs are produced from low immunogenic cells that have undergone one or more of the modifications in Table 4. TIFF2025518125000005.tif153159

[0141] In embodiments, gene inactivation / activation is controlled by an inducible promoter throughout the differentiation, activation, and manufacturing processes for BioNVs. In embodiments, disruption of MHC, TCR, and CRS genes produces allogeneic iPSCs that are - / -CRS and - / -TCR and have cell membranes that exhibit low immunogenicity upon injection into a subject. CRS genes involved in the etiology of CRS include, among other cytokines, tumor necrosis factor (TNF), IL-1, IL-2, IL-2-receptor-α, and IL-8, including IL-6, IL-10, IFN-γ, monocyte chemoattractant protein 1 (MCP-1), granulocyte macrophage colony-stimulating factor (GM-CSF). In embodiments, one or more of these genes are inactivated, for example, in cells from which BioNVs are derived.

[0142] In an embodiment, BioNV is formed by disrupting the cell membrane of artificial iPSCs. In an embodiment, low iPSCs are characterized by a B2M− / −, CIITA− / −, CD47+ / +, PD1− / − plasma membrane profile and can be used to generate BioNV. Low immunogenic BioNV can be produced from parental iPSC cell lines via, among other methods, sonication, adaptive focused acoustics technology, French press, extrusion, continuous extrusion, detergent-based cell lysis, electroporation. In an embodiment, continuous extrusion is the method used to produce low immunogenic BioNV. In an embodiment, continuous extrusion of iPSCs can produce BioNV that is HLA1 / HLA2 negative (low immunogenic) with tgCD47+ and exhibits PD1-resistant elimination.

[0143] BioNV produced from low immunogenic cells In aspects, the disclosure includes BioNV that is between about 20 and 1200 nm in size and, in embodiments, can include an extracellular, membrane-embedded targeting agent (e.g., CAR) that can bind to one or more target molecules. In an embodiment, BioNV is biomimetic due to its nanovesicle composition derived from the cell membrane of allogeneic low immunogenic cell-modified cells. In an embodiment, BioNV includes a lipid bilayer membrane derived from the cell membrane and completely encapsulates an aqueous core that can accommodate various cell-derived molecules such as perforin, granzyme, cytokines, gene editing payloads. In an embodiment, the aqueous core of BioNV can further encapsulate, among other therapeutic agents that can be synthesized intracellularly prior to disruption or added during the cell processing step, biologics, fluorescent proteins, tracking dyes, radionuclides, small molecule compounds.

[0144] In embodiments, BioNV can inherit a CAR construct from hypoimmunogenic cells that may contain various structural molecules. The typical structure - function of a CAR includes an extracellular (or outward - facing) binding portion (such as scFv) connected by a hinge peptide (e.g., the CH2 / CH3 region from IgG Fc region, Gly - Gly - Ser peptide bond, CD28 peptide, CD8α peptide, etc.) to a transmembrane region (such as CD28, CD3ζ, CD4, CD8α, ICOS, etc.), followed by various intracellular signaling regions (such as 4 - 1BB, CD3ζ, CD28, 4 - 1BB, ICOS, CD27, OX40, etc.). In embodiments, since BioNV lacks the intracellular machinery of whole cells, the CAR design does not require any intracellular signaling molecules (primary CAR construct). In embodiments, the CAR construct includes an extracellular scFV binding portion fused to the CD28 transmembrane region by an IgG CH2 / CH3 linker and is substantially lacking in an intracellular region or functionality.

[0145] In embodiments, the CAR construct has a prototypical intracellular region that is either replaced or fused to an anchor protein, such as the PLA2 region from AAV, a fusion protein, a radionuclide - binding region, a cytoskeletal element, a small - molecule transport region, etc., that can assist in fusion to target cells and / or packaging and release of a therapeutic payload. In embodiments, BioNV expresses one or more factors that increase uptake, including, by way of non - limiting example, one or more membrane - embedded proteins, surface functionalizations and / or anionic or cationic conjugated lipids, and / or viral ligands / receptors that improve or facilitate the uptake of BioNV.

[0146] In embodiments, the CAR antigen-binding molecule comprises various binding moieties, including an antibody-based or antibody-formatted binding region. In embodiments, BioNV comprises an antibody or antibody-formatted binding site selected from one or more of monoclonal antibodies, polyclonal antibodies, antibody fragments, Fab, Fab’, Fab’-SH, F(ab’)2, Fv, single-chain Fv (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. In embodiments, the CAR construct comprises a bispecific T cell engager (BiTE), a viral epitope recognition receptor (VERR) or viral ligand, variable heavy chain IgG fragment V H H, V NAR , or a binding moiety having an artificial T cell receptor (TCR). In embodiments, BioNV comprises, as a target agent, a ligand for a receptor or a receptor for a ligand.

[0147] To ensure proper targeting of the CAR and eliminate BioNV lacking the CAR, in embodiments, HPLC-based affinity chromatography techniques can be used to select and concentrate only BioNV having a CAR exposed to a solvent with sufficient surface concentration. HPLC-based affinity chromatography techniques can be used to reduce the concentration of contaminating cell material and NV bearing immunogenic cell surface markers, either by positive or negative selection.

[0148] In embodiments, BioNVs can include NVs having only the outer layer of the cell membrane, only the inner layer of the cell membrane, and / or both intact layers of the cell membrane lipid bilayer. In embodiments, iPSC-derived NVs are added with additional lipid additives (e.g., phosphatidylethanolamine, phosphatidylcholine, phosphatidylinositol, ceramide, lecithin, etc.), nonionic surfactants (e.g., sorbitan monostearate, octadecylamine, etc.), sterols (e.g., cholesterol, bile acid derivatives, etc.), polyols (e.g., maltodextrin, sorbitol, sucrose, mannitol, etc.), proteins (e.g., serum albumin, Fc, etc.) to improve physicochemical properties such as thermal stability, clearance, packaging / release of therapeutic payloads. The amount of cholesterol, as well as the length and saturation of the hydrocarbon chains of phospholipids, can affect the rigidity and stability of the bilayer and, consequently, the ability of NVs to load and release drugs, biomolecules, and other therapeutic payloads. In embodiments, BioNVs also include zwitterionic lipids and methods of using zwitterionic lipids as described in U.S. Patent Publication No. 20130216607, the entire content of which is incorporated herein by reference. Correspondingly, by functionalizing the hydrophilic head of the lipid with a polymer or biomolecule, additional features can be imparted to the vesicle surface, resulting in the formation of interactions with blood components, tissues, and the immune system in vivo.

[0149] In embodiments, low immunogenic cells can synthesize one or more therapeutically relevant biomolecules for encapsulation in BioNVs. In embodiments, the one or more therapeutically relevant biomolecules are cytokines, inflammatory cytokines, anti-antigenic cytokines, perforin, granzyme, chemokines, interferons (IFNα / β / γ), interleukins, alarmins, lymphokines, and tumor necrosis factor (TNF), colony-stimulating factors, bone morphogenetic proteins (BMP), erythropoietin (EPO), granulocyte-stimulating factor (G-CSF), granulocyte macrophage colony-stimulating factor (GM-CSF), or combinations thereof.

[0150] In embodiments, the low immunogenicity cells can be used to encapsulate a payload; for example, "lumen-loading", or the ability of BioNV derived from cells to fill the lumen (the space within the biomimetic nanovesicle) with the payload. In embodiments, the payload is one or more of a biological agent, nucleic acid, fusion protein, fluorescent protein, tracking dye, radionuclide, and / or small molecule. In embodiments, the payload is a therapeutic payload for the disease type targeted by the CAR. In embodiments, the payload comprises one or more of an alkylating agent, anthracycline, antimetabolite, antitumor antibiotic, antibody or antibody format, corticosteroid, plant alkaloid, topoisomerase inhibitor, checkpoint inhibitor, anti-infective agent, and / or growth factor.

[0151] In embodiments, the nucleic acid payload encodes one or more of CRISPR / Cas components, guide RNA (gRNA), tracer RNA (tracrRNA), microRNA (miRNA), RNA interference (RNAi), small interfering RNA (siRNA), double-stranded RNA, Piwi-interacting RNA (piRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), antisense oligonucleotide (ASO), locked nucleic acid (LNA), splice-switching oligonucleotide (SSO), tRNA, complementary messenger RNA, repeat-associated small interfering RNA (rasiRNA), endonuclease, and small non-coding RNA.

[0152] In embodiments, the payload comprises gene editing nucleic acids and / or proteins, such as, for example, 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, transferases, and / or any ortholog or homolog of any of these editors. In embodiments, the gene editor may also include a gRNA (as used herein, refers to guide RNA). In embodiments, the gRNA may be a sequence complementary to a coding or non-coding sequence and may be tailored to a specific sequence to be targeted. In embodiments, the gRNA may be a sequence complementary to a protein coding sequence, such as, for example, a sequence encoding one or more viral structural proteins (e.g., gag, pol, env, and tat). In embodiments, the gRNA sequence may be a sense or antisense sequence. In embodiments, when a gene editor composition is administered herein, preferably, without limitation, it includes two or more gRNAs. However, a single gRNA may also be used.

[0153] In embodiments, BioNV delivers a gene editing payload that includes a transactivation response region (TAR) loop system. In embodiments, BioNV expresses a gene editor and encapsulates a plasmid that contains a TAR loop sequence between the 5' end of the promoter and the gene editor / guide cassette and serves as a barrier to block transcription. In embodiments, transcription is induced only in cells that are infected and contain the HIV Tat protein. In embodiments, the Tat protein binds to the TAR loop, releases it, liberates the promoter for transcription, thereby expressing the editor and its guide.

[0154] In embodiments, the primary targeting BioNV is used to deliver small molecule therapeutic payloads. In embodiments, the second generation (or third or fourth generation) CAR-containing BioNV derived from activated lymphocytes may include cytokines and other cytotoxic peptides. In embodiments, the BioNV can be engineered to encapsulate and deliver plasmid DNA, for example, to express gene editing nucleases and gRNAs in target cells. Alternatively or additionally, in embodiments, the BioNV can encapsulate nucleases and gRNAs. In embodiments, the targeted second generation (or third or fourth generation) BioNV can be designed to encapsulate and deliver additional therapeutic proteins or peptides of interest.

[0155] In embodiments, the BioNV includes one or more of CD34, CCL21, PD-L1 (from non-activated cells) and / or CTLA-4, FasL, SerpinB9, H2-M3, any one of CD24, CD47, CD200, chimeric CD24 / CD47, chimeric CD24 / CD200, and chimeric CD47 / CD200, or any two of CD24, CD47, CD200, MFG-E8, NCAM, α-phagocytic integrin, an anti-IL-6R antibody or antibody format, and / or fusions or portions thereof. In embodiments, the BioNV can have NCAM, or any other protein that promotes fusion of the BioNV to the cell membrane of the target cell. In embodiments, the BioNV encapsulating at least one granzyme may be lacking SerpinB9 and / or CD200. In embodiments, the BioNV expressing PD-L1 is from non-activated cells and the BioNV is substantially lacking perforin and / or granzyme.

[0156] In embodiments, BioNV is substantially deficient in one or more of MHC class I complex, MHC class II complex, HLA-A complex, HLA-B complex, HLA-C complex, HLA-E or HLA-G complex, HLA-F complex, T cell receptor alpha chain (TRAC) protein, T cell receptor beta chain (TRBC) protein, PD-1, SerpinB9, IL-4, IL-6, IL-10, and / or IL-16. In embodiments, BioNV is substantially deficient in one or more of the above because it is allogeneic and / or hypoimmunogenic. In embodiments, BioNV does not produce a harmful immune response upon injection into a subject for treating a disease.

[0157] In embodiments, BioNV comprises one or more targeting agents. In embodiments, the cells from which BioNV is derived can be modified to express one or more targeting agents. In embodiments, one or more targeting agents are CARs. In embodiments, one or more targeting agents can be any antibody or antibody format described herein.

[0158] In embodiments, BioNV is targeted to one or more biomarkers of Table 5 and / or Table 6. TIFF2025518125000006.tif130159TIFF2025518125000007.tif233159TIFF2025518125000008.tif242154TIFF2025518125000009.tif87159

[0159] In embodiments, BioNV is formed by disrupting activated cells by one or more of sonication, adaptive focused acoustics technology, French press, extrusion, continuous extrusion, enzymatic lysis of cells (e.g., trypsinization), cell lysis with detergents, and / or electroporation. In embodiments, cell disruption is by continuous extrusion.

[0160] In embodiments, BioNV can be analyzed for particle size uniformity by, among other methods for determining particle size, dynamic light scattering (DLS), flow cytometry, and mass photometry. In embodiments, BioNV can be filtered for a particle size or range of particle sizes to optimize renal clearance and other clinically relevant NV properties. In embodiments, BioNV is on the order of about 20 nm to 1200 nm in size. In embodiments, BioNV is on the order of about 10 nm in size, about 20 nm in size, about 30 nm in size, about 40 nm in size, about 50 nm in size, about 60 nm in size, about 70 nm in size, about 80 nm in size, about 90 nm in size, about 100 nm in size, about 120 nm in size, about 140 nm in size, about 160 nm in size, about 180 nm in size, about 200 nm in size, about 300 nm in size, about 400 nm in size, about 500 nm in size, about 600 nm in size, about 700 nm in size, about 800 nm in size, about 900 nm in size, about 1000 nm in size, about 1100 nm in size, or about 1200 nm in size. In embodiments, BioNV is sized in the range of about 10 nm to about 20 nm, about 20 nm to about 30 nm, about 30 nm to about 40 nm, about 40 nm to about 50 nm, about 50 nm to about 60 nm, about 60 nm to about 70 nm, about 70 nm to about 80 nm, about 80 nm to about 90 nm, about 90 nm to about 100 nm, about 10 nm to about 100 nm, about 100 nm to about 200 nm, about 200 nm to about 400 nm, about 400 nm to about 600 nm, about 600 nm to about 800 nm, about 800 nm to about 1000 nm, or about 1000 nm to about 1200 nm.

[0161] Method for treating mammalian diseases In embodiments, hypoimmunogenic cells can be used to treat mammalian diseases. In embodiments, BioNV derived from hypoimmunogenic cells can be used to treat mammalian diseases. In embodiments, mammalian diseases are cancer, infectious diseases, genetic diseases, and / or rare diseases.

[0162] In embodiments, methods of treating mammalian diseases herein include co-administering a second whole cell therapy (e.g., T cell, NK cell, TIL, macrophage therapy). In embodiments, supplementing whole cell therapy with hypoimmunogenic cells and / or BioNV can be used to reduce the effective amount of whole cell therapy required and to reduce CRS, non-specific effects, the potential for teratomas, etc.

[0163] In embodiments, methods of treating mammalian diseases include administering an additional therapeutic agent. In embodiments, the additional therapeutic agent can be any additional anti-cancer agent, anti-infective agent, analgesic, and / or non-steroidal anti-inflammatory agent (NSAID).

[0164] In embodiments, hypoimmunogenic cells can be frozen at about -80°C or are suitable for storage at -80°C. In embodiments, BioNV can be frozen at about -80°C or is suitable for storage at -80°C and / or can be lyophilized (e.g., for reconstitution in buffer). In embodiments, hypoimmunogenic cells and / or BioNV can be stable at ambient temperature, about -20°C, about 4°C, about 25°C, or about 37°C for at least about 1 hour, at least about 2 hours, at least about 4 hours, at least about 6 hours, at least about 12 hours, at least about 24 hours, at least about 2 days, at least about 1 week, or at least about 1 month or more.

[0165] In embodiments, treatment of mammalian diseases 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 from administration of the compositions and methods with such compositions.

[0166] Dosage and Administration The dosage and administration schedule of any of the low immunogenic cells (or BioNVs) disclosed herein may depend on a variety of parameters and factors including, but not limited to, the particular low immunogenic cell, the disease being treated, the severity of the condition, whether the condition is being treated or prevented, the age, weight, general health of the subject, and the discretion of the administering physician. Further, pharmacogenetic information (the effect of genotype on the pharmacokinetic, pharmacodynamic or efficacy profile of a therapeutic agent) regarding a particular subject may affect the dosage used. Additionally, the exact individual dosage may be somewhat adjusted by various factors such as the particular combination of agents 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 disorder, the anatomical site of the disorder, etc. Some variation in dosage is to be expected.

[0167] In embodiments, delivery of BioNVs can be effected, for example, by 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)).

[0168] The methods of treating mammalian diseases using low immunogenic cells described herein include, in embodiments, dosage ranges in terms of the concentration of the number of low immunogenic cells per kilogram (kg) of subject body weight. In embodiments, suitable dosage ranges for the methods described herein are from about 10 3 cells / kg (or BioNVs / kg) to about 10 9 cells / kg (or BioNVs / kg). In embodiments, the low immunogenic cells are from about 10 3 cells / mL (or BioNVs / mL) to about 10 9It is present in the composition at a concentration of cells / mL (or BioNVs / mL). Alternatively, in embodiments, the low immunogenic cell (or BioNV) composition is present in the composition in the range of about 5 ng / mL to about 500 mg / mL as weight / volume. In embodiments, the dosage of BioNV is based on the size of the BioNV used for treatment. For example, a 1000 nm BioNV is provided in an amount about 5 to 10 times less than that of a 100 nm BioNV for an equivalent dosage.

[0169] In embodiments, the BioNVs (or low immunogenic cells) disclosed herein are administered by delivery with controlled release or sustained release means, or means well known to those skilled in the art. Examples include, but are not limited to, those described in U.S. Patent Nos. 3,845,770, 3,916,899, 3,536,809, 3,598,123, 4,008,719, 5,674,533, 5,059,595, 5,591,767, 5,120,548, 5,073,543, 5,639,476, 5,354,556, and 5,733,556 (each of which is incorporated herein by reference in its entirety). Such dosage forms can be useful for controlled or sustained release of one or more active ingredients by providing the desired release profile at various rates using, for example, hydroxypropylmethylcellulose, other polymeric matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, microspheres, or combinations thereof. The controlled or sustained release of the active ingredient can be stimulated by various conditions including, but not limited to, changes in pH, changes in temperature, stimulation by light of an appropriate wavelength, enzyme concentration or efficiency, water concentration or efficiency, and other physiological conditions or compounds.

[0170] In an embodiment, a polymeric material is used (see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Florida (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, 1983, J. Macromol. Sci. Rev. Macromol. Chem. 23:61; Levy et al., 1985, Science 228:190; During et al., 1989, Ann. Neurol. 25:351; Howard et al., 1989, J. Neurosurg. 71:105).

[0171] In an embodiment, the controlled release system is placed in the vicinity of the target area to be treated and thus requires only a fraction of the systemic dose (see, for example, Goodson, Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)). Other controlled release systems discussed in a review by Langer, 1990, Science 249:1527-1533 may be used.

[0172] In an embodiment, methods of using low immunogenic cells (or BioNV) include applying the low immunogenic cells to the surface of a device (e.g., a catheter) or including them within a pump, patch, or other drug delivery device. Excipients or carriers can be selected based on the mode of administration and route of administration. Suitable pharmaceutical carriers and pharmaceutical necessities for use in pharmaceutical formulations are described in the well-known reference books Remington’s Pharmaceutical Sciences (E.W. Martin) and USP / NF (United States Pharmacopeia and the National Formulary) in this field.

[0173] In embodiments, the low immunogenicity cells (or BioNVs) can be administered at a dose that matches the dose of the whole cells, for example, based on the CAR concentration. In embodiments, the typical concentration range of the CAR protein per microgram of T cells is 0.20 ng to 0.70 ng. On the other hand, a single BioNV may have 5 to 10,000 times fewer total CARs than the whole cells. As a result, when converting the mass of the BioNV to the CAR concentration, it can be assumed that the CAR concentration is equivalent (e.g., in the case of exosomes) to or increased (e.g., in the case of BioNVs) compared to the cells from which it is derived (e.g., T cells). In embodiments, the concentration and / or surface density of the targeting agent (e.g., CAR) increases on the BioNV compared to the whole cells from which it is derived. In embodiments, the concentration and / or surface density of the targeting agent (e.g., CAR) is concentrated by continuous extrusion treatment of the whole cells. In embodiments, among other cell surface molecules, the concentration and / or surface density of the targeting agent (e.g., CAR) on the BioNV is increased 2 to 100 times compared to the whole cells. In embodiments, since exosomes are naturally secreted, the concentration and / or surface density of the targeting agent (e.g., CAR) among other cell surface molecules is substantially the same as that of the whole cells.

[0174] The dosing regimen using any of the low immunogenicity cells (or BioNVs) disclosed herein can be selected according to various factors including the cancer type, species, age, weight, gender, and medical condition of the subject, the severity of the condition being treated, the route of administration, the renal or hepatic function of the subject, the pharmacogenomic makeup of the individual, and the specific composition of the disclosure employed. Any of the low immunogenicity cells (or BioNVs) disclosed herein can be administered as a once-daily dose or the total daily dose can be divided and administered 2, 3, or 4 times a day. Further, any of the low immunogenicity cells (or BioNVs) disclosed herein can be administered continuously rather than intermittently throughout the dosing regimen.

[0175] In embodiments, the hypoimmunogenic cells (or BioNVs) are administered continuously at about hourly, about every two hours, about every six hours, about every twelve hours, about every twenty-four hours, about every two days, about every four days, about every seven days, about every two weeks, about every four weeks, or about once a month.

[0176] Additional therapeutic agent 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 can be in solution with the BioNV, adsorbed on the surface of the NV, or a payload encapsulated within the BioNV. In embodiments, the additional therapeutic agent is one or more of a checkpoint inhibitor, an analgesic, and / or an anti-infective agent.

[0177] In an embodiment, the present composition or method contemplates other additional therapeutic agents, such as analgesics to assist in treating inflammation or pain at the administration site, or anti-infective agents to prevent infection at the treatment site by the composition.Non-limiting examples of additional therapeutic agents include analgesics such as non-steroidal anti-inflammatory drugs, opiates, and salicylates; anthelmintics, anti-anaerobic agents, antibiotics, aminoglycoside antibiotics, antifungal antibiotics, cephalosporin antibiotics, macrolide antibiotics, miscellaneous B-lactam antibiotics, penicillin antibiotics, quinolone antibiotics, sulfonamide antibiotics, tetracycline antibiotics, anti-mycobacterial agents, anti-tuberculosis anti-mycobacterial agents, anti-protozoal agents, anti-malaria anti-protozoal agents, antiviral agents, anti-retroviral agents, scabicides, anti-inflammatory agents, corticosteroid anti-inflammatory agents, antipruritics / local anesthetics, anti-infective agents, anti-fungal anti-infective agents, anti-viral anti-infective agents, etc.; acidifying agents, alkalinizing agents, diuretics, carbonic anhydrase inhibitor diuretics, loop diuretics, osmotic diuretics, potassium-sparing diuretics, thiazide diuretics, electrolyte replenishers, and uremic agents, etc., electrolytic and renal agents; enzymes such as pancreatic enzymes and thrombolytic enzymes; antidiarrheals, antiemetics, gastrointestinal anti-inflammatory drugs, salicylic gastrointestinal anti-inflammatory drugs, antacid anti-ulcer drugs, gastric acid pump inhibitor anti-ulcer drugs, gastric mucosal anti-ulcer drugs, H2-blocker anti-ulcer drugs, gallstone dissolving agents, digestive agents, emetics, laxatives and stool softeners, and prokinetic agents, etc., gastrointestinal drugs; inhalation anesthetics, halogenated inhalation anesthetics, intravenous anesthetics, barbiturate intravenous anesthetics, benzodiazepine intravenous anesthetics, and opiate agonist intravenous anesthetics, etc., general anesthetics; single-dose drugs, adrenal cortical hormone agents, corticosteroid adrenal cortical hormone agents, androgens, anti-androgen agents, etc., hormones and hormone regulators; immunobiological agents such as immunoglobulins, immunosuppressants, toxoids, and vaccines; local anesthetics such as amide local anesthetics and ester local anesthetics; skeletal muscle agents such as anti-gout anti-inflammatory agents, corticosteroid anti-inflammatory agents, gold compound anti-inflammatory agents, immunosuppressive anti-inflammatory agents, non-steroidal anti-inflammatory drugs (NSAIDs), salicylate anti-inflammatory agents, etc.; minerals; water-soluble or fat-soluble vitamins, vitamins such as 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, thorium-227, etc.

[0178] Additional non-limiting examples of useful therapeutic agents from the above categories include the following: (1) analgesics in general such as lidocaine or its derivatives, and NSAID analgesics such as diclofenac, ibuprofen, ketoprofen, and naproxen; (2) opioid 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) anti-infective agents such as mupirocin; (6) anti-anaerobic anti-infective agents such as chloramphenicol and clindamycin; (7) antifungal antibiotic anti-infective agents such as amphotericin b, clotrimazole, fluconazole, and ketoconazole; (8) macrolide antibiotic anti-infective agents such as azithromycin and erythromycin; (9) miscellaneous β-lactam antibiotic anti-infective agents such as aztreonam and imipenem; (10) penicillin antibiotic anti-infective agents such as nafcillin, oxacillin, penicillin G, and penicillin V; (11) quinolone antibiotic anti-infective agents such as ciprofloxacin and norfloxacin; (12) tetracycline antibiotic anti-infective agents such as doxycycline, minocycline, and tetracycline; (13) anti-tuberculosis anti-mycobacterial anti-infective agents such as isoniazid (INH) and rifampin; (14) anti-protozoal agents such as atovaquone and dapsone; (15) anti-malarial anti-protozoal agents such as chloroquine and pyrimethamine; (16) anti-retroviral anti-infective agents such as ritonavir and zidovudine; (17) anti-viral anti-infective agents such as acyclovir, ganciclovir, interferon alpha, remdesivir, and rimantadine; (18) antifungal topical anti-infective agents such as amphotericin B, clotrimazole, miconazole, and nystatin; (19) anti-viral topical anti-infective agents such as acyclovir; (20) electrolyte and renal agents such as lactulose; (21) loop diuretics such as furosemide; (22) potassium-sparing diuretics such as triamterene; (23) thiazide diuretics such as hydrochlorothiazide (HCTZ); (24) uremia agents such as probenecid; (25) enzymes such as ribonuclease and deoxyribonuclease.(26) Antiemetics such as prochlorperazine, (27) Salicylic acid gastrointestinal anti-inflammatory drugs such as sulfasalazine, (28) Anti-ulcer agents of gastric acid pump inhibitors such as omeprazole, (29) H2 blocker anti-ulcer agents such as cimetidine, famotidine, nizatidine, and ranitidine, (30) Digestants such as pancreatin, (31) Promotility agents such as erythromycin, (32) Ester local anesthetics such as benzocaine and procaine, (33) Skeletal corticosteroid anti-inflammatory agents such as beclomethasone, betamethasone, cortisone, dexamethasone, hydrocortisone, and prednisone, (34) Skeletal anti-inflammatory immunosuppressive agents such as azathioprine, cyclophosphamide, and methotrexate, (35) Skeletal non-steroidal anti-inflammatory drugs (NSAIDs) such as diclofenac, ibuprofen, ketoprofen, ketorolac, and naproxen, (36) Minerals such as iron, calcium, and magnesium, (37) Vitamin B compounds such as cyanocobalamin (vitamin B12) and niacin (vitamin B3), (38) Vitamin C compounds such as ascorbic acid, and (39) Vitamin D compounds such as calcitriol.,

[0179] Low immunogenic cells and / or compositions of BioNV In aspects, the present disclosure relates to a composition comprising allogeneic low immunogenic cells and / or BioNV that can be used for the treatment of mammalian diseases. In embodiments, the low immunogenic cells and / or BioNV comprise one or more therapeutically relevant biomolecules.

[0180] In embodiments, the composition comprises low immunogenic cells. In embodiments, the composition may comprise BioNV. In embodiments, the composition comprises low immunogenic cells and BioNV. In embodiments, the composition comprises at least one of an anti-cancer therapeutic agent, an anti-infection therapeutic agent, or a gene editing payload. In embodiments, the composition comprises low immunogenic cells (or BioNV) that can adsorb therapeutic molecules on the surface of the therapeutic payload and / or encapsulate the therapeutic payload. In embodiments, the composition comprises a therapeutically effective amount of low immunogenic cells and / or BioNV.

[0181] In embodiments, the composition is allogeneic and / or hypoimmunogenic. In embodiments, the composition is derived from iPSCs that have been modified to reduce the expression of immunogenic molecules and / or increase the expression of immune defense molecules (among other cell types).

[0182] In embodiments, the hypoimmunogenic cells and / or compositions of BioNVs are hypoimmunogenic. For example, in embodiments, the composition does not elicit an inflammatory and / or immune response upon administration. In embodiments, upon administration to a subject, the composition, optionally the hypoimmunogenic cells therein, elicit, for example, compared to allogeneic whole cells, less than about 50%, less than about 45%, less than about 40%, less than about 35%, less than about 30%, less than about 25%, less than about 24%, less than about 23%, less than about 22%, less than about 21%, less than about 20%, less than about 19%, less than about 18%, less than about 17%, less than about 16%, less than about 15%, less than about 14%, less than about 13%, less than about 12%, less than about 11%, less than about 10%, less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% of an inflammatory or immune response as measured as a function of cytokine, chemokine, or immunomodulatory enzyme concentration, 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 combinations thereof.

[0183] In embodiments, the hypoimmunogenic cells are about 10 3 cells / mL (or BioNVs / mL) to about 10 9 cells / mL (or BioNVs / mL) and are present in the composition at a concentration. Alternatively, in embodiments, the cell and / or BioNV composition is present in the composition at a weight / volume in the range of about 5 ng / mL to about 500 mg / mL.

[0184] In embodiments, the composition is substantially free of one or more bacteria, viruses, fungi, spores, mycoplasma, and pyrogens, and in more particular embodiments, is substantially free of all of the foregoing. In embodiments, the BioNV composition is substantially free of whole cells and intracellular components including organelles such as nuclei, mitochondria, and Golgi bodies, and / or is substantially free of non-CAR-expressing NVs, and / or is substantially free of ruptured and damaged NVs. In embodiments, the composition is substantially free of extracellular chromatin, nucleosomes, other genetic material, and non-therapeutic nucleic acids. In embodiments, the BioNV composition is substantially free of cellular genomic DNA.

[0185] In embodiments, the low-immunogenicity cells are modular and allogeneic (off-the-shelf) because they lack immunogenicity from engineered iPSCs. In embodiments, BioNV lacks the cell-wide signaling components, so the target specificity and resistance to immunosuppressive signals can be easily adjusted. In embodiments, the low-immunogenicity cells lack genetic elements that contribute to cytokine storm rampage, thus minimizing the patient's CRS risk. In embodiments, the amounts of active cytokines, perforin, granzyme, interferon, interleukin, etc. encapsulated within BioNV are regulated during upstream (prior to BioNV induction) cell processing. In embodiments, the low-immunogenicity cells are derived from cells that can pass through biological barriers and / or from viral receptors known to facilitate passage.

[0186] While not wishing to be bound by theory, hypoimmunogenic cells generated from allogeneic-based cell lines engineered with iPSCs correspond to immune-invisible cells, which means that hypoimmunogenic cells have the potential for multiple administrations, and antibody-mediated neutralization is minimized, and immune cell-mediated clearance is avoided (T cells and macrophages). In embodiments, BioNVs derived from hypoimmunogenic cells do not contain viable genetic material such as to cause CRS or teratoma from the cells from which they were derived. In embodiments, increased expression of certain cytokines in hypoimmunogenic cells is encapsulated within BioNVs that can mobilize native T cells. In embodiments, BioNVs may be derived from modified cell types with or without barrier-penetrating ligands to further control activity after injection.

[0187] Pharmaceutical Compositions and Formulations of Hypoimmunogenic Cells and / or BioNVs In aspects, the composition is a pharmaceutical composition. In embodiments, the pharmaceutical compositions of the present disclosure are formulated to provide a therapeutically effective amount of hypoimmunogenic cells as an active ingredient. In embodiments, the pharmaceutical compositions of the present disclosure are formulated to provide a therapeutically effective amount of one or more anti-cancer agents as a payload within BioNVs as an active ingredient. Typically, the pharmaceutical compositions also include one or more pharmaceutically acceptable excipients, carriers including inert solid diluents and fillers, diluents including sterile aqueous solutions and various organic solvents, penetration enhancers, solubilizing agents, and adjuvants.

[0188] Pharmaceutical excipients can be liquids such as water and oil, which include those of petroleum, animal, plant, or synthetic origin, for example, peanut oil, soybean oil, mineral oil, sesame oil, etc. Pharmaceutical excipients can be, for example, physiological saline, acacia gum, gelatin, gelatinized starch, talc, keratin, colloidal silica, urea, etc. Further, adjuvants, stabilizers, thickeners, lubricants, colorants, etc. can be used. Pharmaceutically acceptable excipients are generally sterile when administered to a subject. Water is a useful excipient when the drugs disclosed herein are administered intravenously. Aqueous solutions of physiological saline, dextrose, and glycerol can also be employed, especially as liquid excipients for injection solutions. Suitable pharmaceutical excipients also include starch, glucose, lactose, sucrose, gelatin, malt, rice, wheat flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol, etc. The compositions disclosed herein can be formulated, if desired, with wetting agents or emulsifiers, or pH buffering agents. Examples of other suitable pharmaceutical excipients are described in Remington’s Pharmaceutical Sciences 1447-1676 (Alfonso R. Gennaro eds., 19th ed. 1995), which is incorporated herein by reference.

[0189] In embodiments, the composition includes an excipient or a carrier. In embodiments, the diluent can be a pharmaceutically acceptable excipient or carrier.

[0190] In embodiments, the pharmaceutical composition includes a pharmaceutically acceptable diluent. Non-limiting examples of diluents include liquid diluents such as water, ethanol, propylene glycol, glycerin, and various combinations thereof, as well as inert solid diluents such as calcium carbonate, calcium phosphate, or kaolin. In embodiments, the diluent includes one or more of physiological saline, phosphate buffered saline, Dulbecco's modified Eagle's medium (DMEM), alpha-modified minimal essential medium (alpha-MEM), Roswell Park Memorial Institute medium 1640 (RPMI medium 1640), HBSS, human albumin, Ringer's solution, etc., or any combination thereof.

[0191] In embodiments, the active ingredient is usually mixed with, diluted by, or encapsulated in a carrier such as, for example, a capsule, tablet, sachet, paper, or other container form. When the excipient functions as a diluent, it can be a solid, semi-solid or liquid material (e.g., normal saline), and functions as a vehicle, carrier or medium for the active ingredient. In embodiments, the composition can be in the form of tablets, pills, powders, troches, sachets, oblongs, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as a solid or in a liquid medium), lotions, creams, ointments, gels, soft and hard gelatin capsules, suppositories, sterile injectable solutions, and sterile packaged powders. As is known in the art, the type of diluent can vary depending on the intended route of administration. In embodiments, the resulting composition can include additional agents such as preservatives, cryopreservatives (e.g., DMSO), and / or anti-precipitants (e.g., polyols, salts). In embodiments, the carrier can be or can include lipid-based or polymer-based colloids. In embodiments, the carrier material can be formulated as liposomes, hydrogels, microparticles, nanoparticles, or block copolymer micelles. In embodiments, the carrier material can form capsules, and the material can be a polymer-based colloid.

[0192] In an embodiment, a pharmaceutical composition containing hypoimmunogenic cells includes a solubilizing agent. In an embodiment, a pharmaceutical composition containing hypoimmunogenic cells includes a cryoprotectant or an agent for improving thermal stability, such as DMSO or glycerol. The pharmaceutical composition can be delivered, in an embodiment, using a suitable vehicle or delivery device as known in the art.

[0193] In an embodiment, the composition includes a scaffold. In an embodiment, the scaffold includes a biomaterial. In a non-limiting example, the three-dimensional biomaterial includes hypoimmunogenic cells that adhere to the scaffold, disperse within the scaffold, or are embedded in an extracellular matrix trapped within the scaffold. In an embodiment, the biomaterial is biodegradable and / or synthetic.

[0194] In embodiments, the scaffold comprises a biodegradable biomaterial. Non-limiting examples of biodegradable biomaterials include fibrin, collagen, elastin, gelatin, vitronectin, fibronectin, laminin, reconstituted basement membrane matrix, starch, dextran, alginate, hyaluronic acid, chitin, chitosan, agarose, sugars, hyaluronic acid, polylactic acid, polyglycolic acid, polyethylene glycol, decellularized tissue, self-assembling peptides, polypeptides, glycosaminoglycans, their derivatives and mixtures. Other useful biodegradable polymers or polymer species include polydioxanone, polycarbonate, polyoxalate, poly-α-ester, polyanhydride, polyacetate, polycaprolactone, polyorthoester, polyamino acid, polyamide, and their mixtures and copolymers, stereopolymers of L-lactic acid and D-lactic acid, copolymers of bis(paracarboxyphenoxy)propanoic acid and sebacic acid, sebacic acid copolymers, caprolactone copolymers, polylactic acid / polyglycolic acid / polyethylene glycol copolymers, copolymers of polyurethane and polylactic acid, copolymers of polyurethane and polylactic acid, α-amino acid copolymers, copolymers of α-amino acid and caproic acid, copolymers of α-benzyl glutamate and polyethylene glycol, copolymers of succinate and polyglycol, polyphosphazene, polyhydroxyalkanoate, and their mixtures, but are not limited thereto. Binary and ternary systems are also contemplated. In embodiments, the scaffold comprises one or more of collagen, various proteoglycans, an alginate-based substrate, and chitosan. In embodiments, the scaffold comprises one or more of a hydrogel, silk, Matrigel, cellular and / or decellularized scaffolds, poly-ε-caprolactone scaffolds, resorbable scaffolds, and nanofiber-hydrogel composites.

[0195] In an embodiment, the scaffold comprises a synthetic biomaterial. Non-limiting examples of synthetic biomaterials include lactone-based polyesters or copolyesters such as polylactide, polycaprolactone glycolide, polyorthoesters, polyanhydrides, polyamino acids, polysaccharides, polyphosphazenes, poly(ether-ester) copolymers (e.g., PEO-PLLA); fluorinated polymers such as polydimethylsiloxane, poly(ethylene-vinyl acetate), acrylate-based polymers or copolymers (e.g., polyhydroxyethyl methyl methacrylate, polyvinylpyrrolidone), polytetrafluoroethylene, and cellulose esters.

[0196] In an embodiment, the composition can be prepared by any pharmaceutically well-known method and can be administered by various routes (e.g., subcutaneous, intravenous, etc.) depending on whether local or systemic treatment is desired and the site to be treated. In an embodiment, administration can be local (including ophthalmic and administration to mucous membranes including intranasal, intravaginal, and rectal delivery), intrapulmonary (e.g., including by nebulizer, inhalation of powder or aerosol or by insufflation; intratracheal, intranasal, epidermal and transdermal), intraocular, oral, or parenteral. In an embodiment, the method can include intraocular delivery, topical administration (eye drops), subconjunctival, periocular or intravitreal injection, or introduction by a balloon catheter or ophthalmic insert surgically placed in the conjunctival sac. In an embodiment, parenteral administration includes intravenous, arterial, subcutaneous, intraperitoneal or intramuscular injection or infusion, or intracranial, e.g., intrathecal or intraventricular administration. In an embodiment, parenteral administration can be in the form of a single bolus dose or, for example, by a continuous perfusion pump.

[0197] In an embodiment, pharmaceutical compositions and formulations for topical administration can include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, powders, etc. In an embodiment, methods of treating and / or preventing cancer include the use of aqueous, powder or oil-based pharmaceutical carriers, thickeners, etc.

[0198] In embodiments, the pharmaceutical composition comprises, as an active ingredient, the nucleic acids and vectors described herein in combination with one or more pharmaceutically acceptable carriers. In embodiments, the term "pharmaceutically acceptable" (or "pharmacologically acceptable") refers to molecular entities and compositions that do not produce adverse side effects, allergic reactions, or other harmful reactions when administered to animals or humans as necessary. The methods and compositions disclosed herein can be applied to a wide range 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 laboratory animals. In embodiments, the term "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial agents, isotonic agents and absorption delaying agents, buffers, excipients, binders, lubricants, gels, surfactants, etc. that can be used as a vehicle for pharmaceutically acceptable substances.

[0199] In embodiments, the composition can be applied to the surface of a device (e.g., a catheter) or can be included within a pump, patch, or other drug delivery device. In embodiments, the composition can be administered alone or in a mixture in the presence of a pharmaceutically acceptable excipient or carrier (e.g., physiological saline). The excipient or carrier is selected based on the mode of administration and the route of administration. Suitable pharmaceutical carriers and pharmaceutical necessities for use in pharmaceutical formulations are described in the well-known reference books Remington’s Pharmaceutical Sciences (E.W. Martin) and USP / NF (United States Pharmacopeia and the National Formulary) in this field.

[0200] In embodiments, the compositions disclosed herein, such as pharmaceutical compositions, are suspended in a physiological saline buffer (including, but not limited to, TBS, PBS, etc.).

[0201] The present technology includes the disclosed low-immunogenicity cells in various formulations of pharmaceutical compositions. The low-immunogenicity cells (or BioNVs) disclosed herein can, in embodiments, be in the form of a solution, suspension, emulsion, drops, tablets, pills, pellets, capsules, capsules containing a liquid, powders, sustained-release formulations, emulsions, aerosols, sprays, suspensions, or any other suitable form for use.

[0202] The pharmaceutical compositions containing the low-immunogenicity cells described herein can be conveniently presented in unit dosage form and can be prepared by any method well known in the art of pharmacy. Such methods generally include the step of associating the therapeutic agent with a carrier that constitutes one or more accessory ingredients. Typically, the pharmaceutical composition uniformly and closely associates the therapeutic agent with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shapes the product into the dosage form of the desired formulation (e.g., by wet or dry granulation, powder blending, etc., and then tableting using conventional methods known in the art).

[0203] In embodiments, any of the low-immunogenicity cells disclosed herein are formulated according to routine procedures as pharmaceutical compositions compatible with the modes of administration disclosed herein.

[0204] Subject and / or animal In embodiments, the subject and / or animal intended for use with hypoimmunogenic cells and / or BioNV are mammals, such as humans, mice, rats, guinea pigs, dogs, cats, horses, cows, pigs, rabbits, sheep, or non-human primates. In embodiments, the subject and / or animal is a non-mammal, such as a zebrafish. In embodiments, the subject and / or animal is a genetically modified animal comprising fluorescent cells, such as, for example, RPE cells and / or immune cells having GFP. In embodiments, the subject and / or animal is a human. In embodiments, the hypoimmunogenic cells are derived from fluorescent-tagged cells and / or are encapsulated with a fluorescent-tagged protein or tag (such as having GFP). In embodiments, the human is a pediatric human, human adult, elderly human, infant or child. In other embodiments, the human is referred to as a patient or subject.

[0205] In embodiments, the treatment method comprises administering to a human having an age in the range of from about 0 months to about 6 months, from about 6 months to about 12 months, from about 12 months to about 18 months, from about 18 months to about 36 months, from about 1 year to about 5 years, from about 5 years to about 10 years, from about 10 years to about 15 years, from about 15 years to about 20 years, from about 20 years to about 25 years, from about 25 years to about 30 years, from about 30 years to about 35 years, from about 35 years to about 40 years, from about 40 years to about 45 years, from about 45 years to about 50 years, from about 50 years to about 55 years, from about 55 years to about 60 years, from about 60 years to about 65 years, from about 65 years to about 70 years, from about 70 years to about 75 years, from about 75 years to about 80 years, from about 80 years to about 85 years, from about 85 years to about 90 years, from about 90 years to about 95 years, or from about 95 years to about 100 years.

[0206] In embodiments, the subject is a non-human animal and thus the present disclosure relates to veterinary use. In embodiments, the non-human animal is a household pet. In embodiments, the non-human animal is a farm animal.

[0207] In embodiments, serum and / or immune cells, and / or tumor cells are evaluated and / or affected. In embodiments, immune cells include cells of the innate immune system of a subject and / or an 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 present disclosure provides for detecting the presence, absence, or amount of tumor burden, tumor cells, metastases, cDNA, or RNA in a sample derived from a subject.

[0208] Kit In embodiments, the present disclosure provides a kit that can simplify the administration of any of the agents described herein. Exemplary kits of the present disclosure include any of the agents / compositions described herein in unit dosage form. In embodiments, the unit dosage form is a container, such as a pre-filled syringe that can be sterile, containing any of the agents described herein and a pharmaceutically acceptable carrier, diluent, excipient, or vehicle. In embodiments, the kit further includes a label or printed instructions that direct the use of any of the agents described herein. In embodiments, the kit also includes a coverslip, a local anesthetic, and a cleanser for the injection surface. In embodiments, the kit further includes one or more additional agents described herein.

[0209] In aspects, the present disclosure includes a syringe containing one or more compositions of the present disclosure. In embodiments, the syringe is pre-filled with the volume of the composition. In embodiments, the syringe is pre-filled with a volume of from about 1 mL to about 10 mL. In embodiments, the syringe is pre-filled with a volume of the composition of about 10 mL, about 9 mL, about 8 mL, about 7 mL, about 6 mL, about 5 mL, about 4 mL, about 3 mL, about 2 mL, about 1.9 mL, about 1.8 mL, about 1.7 mL, about 1.6 mL, about 1.5 mL, about 1.4 mL, about 1.3 mL, about 1.2 mL, about 1.1 mL, or about 1.0 mL or less.

[0210] In an embodiment, the syringe contains a composition having a storage stability in the range of about 1 hour to about 1 week. In an embodiment, the syringe contains a composition having a storage stability of at least about 12 hours, about 24 hours, about 36 hours, about 48 hours, or about 72 hours when stored at a temperature in the range of about -85°C to about 25°C. In an embodiment, the syringe contains a composition having a storage stability of at least about 12 hours, about 24 hours, about 36 hours, about 48 hours, or about 72 hours when stored at a temperature in the range of about 15°C to about 25°C.

[0211] In an embodiment, the storage temperature is about -80°C. In an embodiment, the storage temperature is about -20°C. In an embodiment, the storage temperature is about 4°C. In an embodiment, the storage temperature is about 21°C. In an embodiment, the kit contains lyophilized BioNV.

[0212] In one embodiment, the kit contains a container containing a composition comprising the CDV / exosome of the present disclosure, a therapeutically effective amount of an additional therapeutic agent as described herein, and instructions for use.

Examples

[0213] Example 1: Construction of a low-immunogenic cell line for producing BioNV The low-immunogenic cells are made from human fibroblast-induced pluripotent stem cells (iPSCs). Genetic modification of human fibroblast iPSC cells is performed using lentivirus introduction by the CRISPR Cas9 system. For each target locus, the design of each gRNA that minimizes non-specific effects is performed. The lentiviral method is suitable for the production of BioNV because the product is not a cell-based therapy.

[0214] The following knockouts (KOs) are sequentially performed:

[0215] Option 1:

[0216] HLA-A, HLA-B, HLA-C, HLA-E / G (select the gene corresponding to the gRNA most suitable by design for CRISPR Cas9), and HLA-F;

[0217] Option 2:

[0218] Each of HLA A, HLA B, HLA C, HLA E / G, and HLA F was knocked out simultaneously (or in two steps) using the current CRISPR Cas9 shotgun-based approach. Simultaneously knocking out HLA to ensure permanent suppression of the B2M locus has no lethal or phenotypic effects (other than on the expression of MHC I);

[0219] MHC class II NO:

[0220] CIITA, and

[0221] CRS-related cytokines:

[0222] IL-6, IL-4, IL-10, and / or IL-16. The cytokines are knocked out sequentially starting from IL-6. The CRS effect is evaluated upon KO of IL-6, and if CRS or a phenotype related to CRS is obtained upon KO of IL-6, further cytokines are removed.

[0223] The following knock-ins (KIs) are performed sequentially:

[0224] CCL2, CTLA-4, H2-M3, MFG-E8;

[0225] CD24, CD200, and / or the CD47 anti-phagocytosis transmembrane region; and

[0226] IL-2p GFP construct (as a reporter for diagnosis and preclinical experiments). A single integration with a drug / reporter selection marker is used for each KI.

[0227] Table 7 below details the steps in which the cell line bank and engineering sequence are carried out. TIFF2025518125000010.tif114159

[0228] All cell lines are examined during operation for mycoplasma, HBV, HIV, and other standardized contaminant screenings. Clonal evaluation of cell lines suitable for gene editing is carried out during gene modification, including conditions such as the type of growth medium, splitting time, and fitness such as cell viability.

[0229] The design, assembly, and verification of CRISPR and / or TUNR (AMSBIO) are first determined to evaluate whether either KO of the HLA and / or CIITA gene is lethal. TUNR is a product that incorporates a DNA portion into a gene of interest and reduces its expression to a near-lethal level when necessary for cell line development.

[0230] Transfection / nucleofection by the lentiviral system is used for knock-in and knock-out operations, respectively. KO and KI are carried out in pools at each step, followed by single-cell dilution, cloning, drug selection / gene testing, and conservation of clones derived from single cells. Clones at each step are then expanded for subsequent steps.

[0231] Pools of single-cell-derived clones are screened by deep sequencing to prepare a gene library that enables analysis for continuous deep sequencing operation and additional clone analysis. Data are collected and provided at each stage. In the case of KO using CRISPR, cells with the lowest indel occurrence rate are selected for further steps in the engineering process.

[0232] Perform final positive clone expansion (after KO and KI, before the CAR platform cross-line), and confirm the final genotype and cryopreserve the clones.

[0233] Examples of gene modifications for generating human fibroblast - reprogrammed iPSC cell lines are shown in Figures 4 - 26, which summarize the CRISPR / Cas9 gRNA design, knockout efficiency, and off - target analysis for sequential B2M and CIITA KO, human CD47 (hCD47) isoform 2 knock - in in human fibroblast - reprogrammed iPSCs, and KO of TRAC and TRBC1. In summary, clone sequencing data demonstrate that each of B2M, CIITA, TRAC, and TRBC1 (one of the TRBC genes) was successfully targeted by the gRNA, and CD47 was successfully knocked in as evident from RNA extraction.

[0234] The B2M knockout strategy is shown in Figure 4, for example, which illustrates the B2M gene knockout strategy for the development of low - immunogenic cell lines from human fibroblast - reprogrammed iPSCs. β2 - microglobulin (B2M) is a serum protein found in association with the major histocompatibility complex (MHC) class I heavy chain on the surface of almost all nucleated cells, which is involved in peptide antigen presentation to the immune system. Figure 5 is an illustration of the B2M gRNA design for the development of low - immunogenic cell lines. This strategy requires, in an embodiment, using gRNAs specifically designed to target CRISPR / Cas9 and the relevant regions of the B2M gene (e.g., SEQ ID NOs: 22 - 23), such as the gRNAs of SEQ ID NOs: 21, 24, 26 - 29, and 133 - 134. Figure 6 presents the B2M gRNA off - target analysis for the development of low - immunogenic cell lines in tabular form.

[0235] The CIITA knockout strategy is illustrated in FIG. 7, which depicts, for example, the B2M gene knockout strategy for the development of low-immunogenic cell lines from human fibroblast reprogrammed iPSCs. The master control factor CIITA is the MHC class II transcriptional activator that is involved in the transcriptional regulation of all MHC II genes. FIG. 8 is an illustration of the CIITA gRNA design for the development of low-immunogenic cell lines. This strategy requires, in embodiments, the use of CRISPR / Cas9 and gRNAs specifically designed to target the relevant regions of the CIITA gene (e.g., SEQ ID NOs: 32-33), such as the gRNAs of SEQ ID NOs: 31, 34, and 36-42. FIG. 9 presents in tabular form the CIITA gRNA non-specific analysis for the development of low-immunogenic cell lines.

[0236] FIG. 10 is an illustration of the B2M knockout clone sequence analysis for the development of low-immunogenic cell lines from human fibroblast reprogrammed iPSCs. The results of two selected B2M / CIITA double knockout clones are shown. The sequence data indicate that B2M KO was successful in the double KO clones and the genomic sequences of SEQ ID NOs: 43-44 were obtained. FIG. 11 is an illustration of the CIITA knockout clone sequence analysis for the development of low-immunogenic cell lines from human fibroblast reprogrammed iPSCs. The results of two selected B2M / CIITA double knockout clones are shown. The sequence data indicate that CIITA KO was successful in the double KO clones and the genomic sequences of SEQ ID NOs: 50-55 were obtained.

[0237] Morphological analysis by optical microscopy (e.g., Figures 12 and 23) shows that the clonal cell population appears to have a normal morphology and is capable of replication. Figure 12 is an overview of the B2M / CIITA double knockout clone sequence and an illustration of the clonal morphology in in vitro human fibroblast reprogrammed iPSC low immunogenicity cells. This data indicates that the clonal population resulted in different overall knockouts. Figure 23 is an illustration of low immunogenicity cells of B2M / CIITA double knockout, TRAC / TRBC1 double knockout, hCD47 KI human fibroblast reprogrammed iPSCs. This data shows the morphological analysis of the clones in vitro.

[0238] Figure 13 is an illustration of human CD47 (hCD47) isoform 2 knock-in for the development of low immunogenic cell lines from human fibroblast reprogrammed iPSCs. The pcDNA3.1(+)XCC92 mammalian vector (length 6271 bp) is used together with a selection marker. Deletion of the 3’UTR of CD47 is performed for stable clonal surface expression of CD47. The 3’UTR has at least six microRNA binding sites that suppress the expression of CD47, and instead, the bGH polyA tail is used. Figure 14 is an illustration of the selection of hCD47 isoform 2 knock-in clones for the development of low immunogenic cell lines in B2M / CIITA double KO human fibroblast reprogrammed iPSCs. The data indicates that hCD47 KI was successful in the double KO cell line. The data shows that hCD47 increases by approximately two-fold at the exon 1-2 and 3-4 junctions.

[0239] Figure 15 is a diagram of a TRAC gene knockout strategy for the development of a low-immunogenic cell line from human fibroblast reprogrammed iPSCs. In an embodiment, the elimination of the T cell receptor alpha chain (TRAC) is performed to prevent CAR targeting and interference with non-specific effects. Figure 16 is a diagram of a TRAC gRNA design for the development of a low-immunogenic cell line. This strategy requires, in an embodiment, the use of CRISPR / Cas9 and gRNAs specifically designed to target the relevant regions of the TRAC gene (e.g., SEQ ID NOs: 56-57), such as the gRNAs of SEQ ID NOs: 60-75. Figure 17 is a tabular representation of a TRAC gRNA non-specific analysis for the development of a low-immunogenic cell line.

[0240] Figure 18 is a diagram of a TRBC1 gene knockout strategy for the development of a low-immunogenic cell line from human fibroblast reprogrammed iPSCs. In an embodiment, the elimination of the T cell receptor beta chain 1 (TRBC1) is performed to prevent CAR targeting and interference with non-specific effects. Figure 19 is a diagram of a TRAC gRNA design for the development of a low-immunogenic cell line. This strategy requires, in an embodiment, the use of CRISPR / Cas9 and gRNAs specifically designed to target the relevant regions of the TRAC gene (e.g., SEQ ID NOs: 76-77), such as the gRNAs of SEQ ID NOs: 79-101. Figure 20 is a tabular representation of a TRAC gRNA non-specific analysis for the development of a low-immunogenic cell line.

[0241] Figure 21 is a schematic diagram of the analysis of the TRAC knockout clone sequence for the development of a low immunogenic cell line for B2M / CIITA double knockout and hCD47 knock-in in human fibroblast reprogrammed iPSCs. The results of two clones are shown together with the resulting sequences, such as SEQ ID NOs: 102 to 108. The sequence data indicates the success of TRAC knockout in the double KO / CD47 KI clones. Figure 22 is a schematic diagram of the analysis of the TRBC1 knockout clone sequence for the development of a low immunogenic cell line for B2M / CIITA double knockout, hCD47 knock-in, and TRAC knockout in human fibroblast reprogrammed iPSCs. The results of two clones are shown together with the resulting sequences, such as SEQ ID NOs: 109 to 110, and 112 to 116.

[0242] Figure 23 shows that the disruption of two genes, B2M, CIITA, TRAC, and TRBC1, has been successful as revealed by the sequencing data. Furthermore, the normal cell morphology of the clones is maintained.

[0243] Figure 24 is a schematic diagram of the IL-6 gene knockout strategy for the development of a low immunogenic cell line from human fibroblast reprogrammed iPSCs. The elimination of interleukin-6 (IL-6) is for the prevention of cytokine release syndrome (CRS). Figure 25 is a schematic diagram of the IL-6 gRNA design for the development of a low immunogenic cell line. This strategy requires, in an embodiment, the use of gRNAs, such as SEQ ID NOs: 119 to 120 and 122 to 130, which are specifically designed to target the CRISPR / Cas9 and the related regions of the IL-6 gene (e.g., SEQ ID NOs: 117 to 118). Figure 26 presents the IL-6 gRNA non-specific analysis for the development of a low immunogenic cell line in tabular form TIFF2025518125000011.tif221159TIFF2025518125000012.tif233159TIFF2025518125000013.tif230159TIFF2025518125000014.tif190159

[0244] Example 2: Preparation of BioNV by Continuous Extrusion Biomimetic nanovesicles (BioNVs) can be produced from the above low-immunogenic cell lines as shown in the scheme of Figure 27.

[0245] The expression level of the CAR can be measured in the low-immunogenic cell line by combining flow cytometry and iodixanol density gradient (e.g., step 1 in Figure 27).

[0246] Differentiation of iPSC-expressing surface CAR into CAR lymphocytes can be analyzed by lymphocyte marker identification, including, among other cell surface markers, for example, CD4 / CD8 (T cells) or CD56 / CD16 (natural killer cells) (e.g., step 2 in Figure 27). The expression profile can be determined by flow cytometry, RT-PCR, and / or CRISPR-based analysis.

[0247] Next, activation of the CAR lymphocytes can be carried out in two steps over a period of two weeks using beads coated with a predetermined low concentration of biomarker antigen (e.g., step 3 in Figure 27). In this step, the quality of the immunological synapse (IS) between the CAR and the antigen-coated beads can also be analyzed using established protocols for measuring i) quantification of F-actin accumulation at the synapse formation site, ii) distribution of p-zeta at the synapse, iii) clustering of the antigen via the IS site, and / or iv) polarization of the lytic granules (LG) containing perforin and granzyme.

[0248] After lymphocyte activation, the cells are expanded using an established protocol (e.g., step 4 in Figure 27). After expansion, the levels of perforin and granzyme (or other luminal payloads where applicable) are analyzed for each cell population to ensure consistent concentration levels for each batch. This is achieved using a series of qPCR, immunoblotting, flow cytometry, and / or mass spectrometry. If the cell population obtained in step 3 is of sufficient size, the expansion step may not be necessary.

[0249] Once the cells are activated and the desired therapeutic protein is produced, the cells are expanded, harvested, washed several times, and then placed in a buffered extrusion medium. The cells are then processed globally by continuous extrusion through each step of a polycarbonate filter system with a gradually decreasing pore size (e.g., step 5 in FIG. 27). In the first extrusion step of the continuous extrusion process, the nuclei (including nuclear components such as nuclear pores, genomic material, and transcription factors) and mitochondria are removed. Next, the sample is treated with an endonuclease, such as BENZONASE. BENZONASE is a non-specific recombinant endonuclease that cleaves any type of DNA and RNA variant into non-functional fragments <8 soluble base pairs. This leads to the greatest reduction in nucleic acid load, per sample and in a measurable form, without interfering with the BioNV membrane chemistry. Also, the cleavage treatment eliminates the viscosity of the nucleic acids, enabling subsequent loading and passage through the next extrusion filter. There are FDA regulatory guidelines regarding the use of BENZONASE in vaccine manufacturing, which are also applicable to the extrusion process of BioNV.

[0250] The continuous extrusion process avoids the exclusion of other organelles such as the Golgi apparatus or the ER. The membrane systems of these organelles are highly evolved such that vesicles shuttle (release and uptake) between the folded membranes. For example, the cis and trans faces of the Golgi apparatus contain a unique lipid composition that facilitates the absorption and release of vesicles with low energy barriers during vesicle transport. These components are relatively scarce in the plasma membrane. Therefore, it is not very preferable to isolate the plasma membrane to induce BioNV. When passing through a polycarbonate filter in the continuous extrusion process, BioNV experiences disruption and spontaneous formation according to the pore size. This process results in BioNV containing membranes in which protein components that can significantly enhance the affinity for uptake into cells and tissues are homogeneously mixed, compared to BioNV treated to remove the cytoplasm, Golgi apparatus, and ER lipid contents, as well as these organelles. These characteristics may lead to better and more stable uptake of BioNV into target cells and the ability to perform this at low doses compared to systems without such properties.

[0251] After the extrusion process, BioNV is passed through an α-CD3 HPLC (FPLC on a large scale) column to remove the low percentage (about 0.05%) of inverted BioNV that forms spontaneously during the continuous extrusion process (e.g., step 6 in Figure 27). This is done to ensure that the resulting BioNV has a uniform orientation with respect to the membrane. Since this step is a flow-through process for capturing impurities, there is little reduction in yield. After the HPLC / FPLC step, once the BioNV is recovered, they are tested through a standardization process.

[0252] The standardization process includes one or more of the following assays:

[0253] Homogeneity of BioNV: By using nanoparticle flow cytometry (NanoFCM), the concentration, size homogeneity, density of BioNV, and / or the homogeneity of the lumen components of BioNV can be confirmed.

[0254] Concentration of the lumen payload: The NanoFM technology can be used to determine the type and concentration of nucleic acids / proteins encapsulated in the lumen of BioNV. These data can be confirmed to determine the nucleic acid and protein content of BioNV in parallel with one or more methods such as immunoblotting, mass spectrometry, and BCA analysis.

[0255] Stability of BioNV: Nanoparticle tracking analysis (NTA), dynamic light scattering (DLS), and electron microscopy (EM) can be used in combination with immunoblot analysis and mass spectrometry to determine the physical and biochemical characteristics of BioNV over 8 - 10 months. The data obtained from these assays can include protein expression profiles, the degree of intact BioNV membrane / packaging, and / or the degree of aggregation.

[0256] Integrity of the membrane: The integrity of the BioNV membrane is evaluated using a calcein release assay combined with NanoFCM to assess membrane permeability. From this result, the leakage characteristics of BioNV with respect to a standardized BioNV panel can be known.

[0257] Quality of the lumen payload: The quality of the payload encapsulated in the lumen can be determined using multiple analytical assays depending on the nature of the payload. If the delivery is a nucleic acid, qPCR and / or sequencing over 8 - 10 months can be used to check the integrity and amount of the nucleic acid payload. For proteins, analysis of BioNV components using one or more of NanoFCM, mass spectrometry, and immunoblot analysis can be used for the analysis of the protein payload.

[0258] Quality and surface density of CAR: The surface density of CAR can be determined using NanoFCM, mass spectrometry, and / or immunoblot analysis. The surface density of CAR is expected to be at least about 5-fold to at least about 10-fold higher in BioNV compared to the surface density of whole cells. This can significantly enhance the targeting to antigens compared to whole cells. The quality of CAR can be determined at the cell stage as described above (e.g., as in step 3). Mathematical models can be used to extrapolate cell quality data and apply it to BioNV in relation to the results of efficacy test data.

[0259] Functionality of BioNV: BioNV can be tested for basic functionality including multiple defined standardized assays such as in vitro cell uptake into target cells when the antigen is expressed and when it is not expressed, as well as their ability to cross dense tissues such as in a human retinal model. Following these basic functionality assays that can be performed immediately after the continuous extrusion process, preclinical trials will address the remaining aspects of the quality and functionality characteristics of BioNV.

[0260] Definitions The following definitions are used in connection with the disclosure herein. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0261] "Effective amount" or "therapeutically effective amount" means an amount effective for the treatment, prevention, or amelioration of a disease in a mammal.

[0262] As used herein, "a", "an", or "the" may mean one or more.

[0263] As used herein, the term "include" and variations thereof are intended to be non-limiting such that the recitation of items in a list is not to be construed as excluding other like items that may be useful in the materials, compositions, devices, and methods of this technology. Similarly, the terms "can" and "may" and variations thereof are intended to be non-limiting such that the recitation that an embodiment can or may include a particular element or feature is not to be construed as excluding other embodiments of this technology that do not include those elements or features.

[0264] As used herein, the open-ended term "comprising", as a synonym for terms such as "including", "containing", or "having", is used to describe and claim the disclosed methods and compositions, but the present disclosure, or embodiments thereof, may alternatively be described using alternative terms such as "consisting of" or "consisting essentially of".

[0265] In an embodiment, "BioNV" refers to a biomimetic nanovesicle (NV) that encapsulates an aqueous portion. In an embodiment, BioNV is allogeneic and / or hypoimmunogenic. In an embodiment, BioNV may refer to an NV (e.g., a cell-derived vesicle) created by rupturing / destroying cells, or a nanovesicle (e.g., an exosome) naturally secreted from cells. In an embodiment, BioNVs include at least one surface-directed membrane-embedded CAR. In an embodiment, as used herein, "nanovesicle (NV)" is a lipid-bound vesicle having a size of about 10 nm to about 1200 nm that encapsulates an aqueous core. In an embodiment, the lipid-bound NV can be formed using a lipid monolayer, a lipid bilayer, or a multi-layer morphology. In an embodiment, BioNV refers to a biologically induced nanoscale-sized vesicle that can have a programmed biological functionality. In an embodiment, BioNV is "biomimetic" in that it is derived from endogenous cellular materials, and more specifically, in that it substantially reproduces the cell membrane materials found within cells. In an embodiment, the cells from which BioNV is derived can include any type of stem cell, including cell types differentiated from stem cells. In an embodiment, BioNV substantially does not contain encapsulated cell debris, including nucleic acids, organelles, or parts of organelles. In an embodiment, BioNV is characterized by having one or more, two or more, three or more, four or more, five or more, or six or more of the following: a. having a size of about 10 nm to about 1200 nm, b. having a total volume of about 500 nm 3 to about 5 μm 3 (assuming a spherical shape), c. having a content of at least one phospholipid and cholesterol, d. Having a surface membrane having any one or more of CD34, CCL21, PD-L1 (in BioNV derived from inactivated cells), FasL, SerpinB9, H2-M3, CD47, CTLA-4, CD24, CD200, MFG-E8, NCAM, α-phagocytic integrin, and / or an anti-6R antibody or antibody form, or a chimera thereof; having a surface membrane substantially lacking T cell receptor components (TRAC and / or TRBC), MHC class I components, and / or MHC class II components, lacking one or more proteins of HLA-A, HLA-B, HLA-C, HLA-E or HLA-G (but not both HLA-E and HLA-G), HLA-F, and / or CIITA, SerpinB9, and substantially lacking one or more intracellular proteins of IL-4, IL-6, IL-10, and / or IL-16, e. Encapsulating one or more therapeutically relevant biomolecules, including, for example, cytokines containing chemokines, interferons (IFNα / β / γ), interleukins, alarmins, lymphokines, tumor necrosis factor (TNF), colony stimulating factors, bone morphogenetic proteins (BMP), erythropoietin (EPO), granulocyte stimulating factor (G-CSF), granulocyte macrophage colony stimulating factor (GM-CSF), inflammatory cytokines, anti-antigenic cytokines, perforin granzymes (e.g., granzyme A, B, H, K, and M), gene editing payloads, fusion proteins, antibody or antibody form constructs, or combinations thereof. f. CAR, monoclonal antibody, polyclonal antibody, antibody fragment, Fab, Fab’, Fab’-SH, F(ab’)2, Fv, single-chain Fv (scFv), diabody, nanobody, linear antibody, bispecific antibody, multispecific antibody, chimeric antibody, humanized antibody, human antibody, fusion protein containing the antigen-binding portion of an antibody, bispecific T cell engager (BiTE), virus epitope recognition receptor (VERR) or virus ligand, variable heavy chain IgG fragment V H H or V NARcomprising a membrane-embedded targeting agent comprising an antibody or antibody format selected from one or more of a fusion protein comprising the antigen-binding portion of a T cell receptor (TCR), wherein the CAR can target a single biomarker, multiple biomarkers, or multiple portions of a single biomarker, and one or more targeting agents can comprise a ligand for a receptor or a receptor for a ligand g. Among other therapeutic payloads, one or more perforin, granzyme, cytokine, cytotoxic protein, non-naturally occurring cell agent, checkpoint inhibitor, recombinant gene editing payload, antibody or antibody fragment, small molecule inhibitor, biologic, radionuclide, tracer, dye, fluorescent protein, and / or any combination thereof can be adsorbed and / or encapsulated, and h. not causing a harmful immune reaction in a subject.

[0266] "Induced pluripotent stem cell", or "iPSC", refers to a stem cell that can be directly generated from an adult cell. An iPSC can arise from a differentiated cell that has been reprogrammed to return to an embryonic-like pluripotent state. An iPSC can generally proliferate indefinitely and become any type of cell of the organism from which it is derived.

[0267] In embodiments, as used herein, "allogeneic" refers to biological materials, tissues, or cells that are genetically heterogeneous and originally immunologically incompatible, even though they are derived from the same species. For example, an allogeneic CDV / exosome is a material derived from a first subject (iPSC provider) and can be provided to any number of different subjects that are not genetically identical.

[0268] In embodiments, when used herein in connection with cells and / or BioNVs, "low immunogenic" or "low immunogenicity" refers to a reduced ability to generate an immunological response. In embodiments, cells and BioNVs can be low immunogenic due to reduced or eliminated expression of one or more specific cell surface proteins and / or secreted proteins, such as, for example, T cell receptor (TCR) proteins, cytokine response syndrome proteins, MHC class I or II proteins. In embodiments, cells and BioNVs can be low immunogenic due to increased expression of immune defense cell surface proteins such as CD47, CD34, CD24, CD200, α - eating integrin. In embodiments, BioNVs and / or cells can be low immunogenic because they do not induce CRS in a subject and / or do not induce HLA incompatibility.

[0269] In embodiments, "knockout," "suppression," "inactivation," "disruption," or "blockade," and their equivalents, with respect to transcription, gene expression, or protein expression, refer to a reduced amount of transcription, gene, or protein expression in a particular cell subset from a normal state or less than wild type. This reduction can be significant such that no gene expression occurs or an amount of expression occurs that is negligible.

[0270] In embodiments, "overexpression" as used herein refers to an increased amount of transcription, gene, or protein expression in a particular cell subset from a normal state or greater than wild type.

[0271] equivalents One of ordinary skill in the art will recognize, or be able to ascertain, numerous equivalents to the specific embodiments specifically described herein without undue experimentation. Such equivalents are intended to be encompassed by the following claims.

[0272] As used herein, all headings are for organizational purposes only and are not intended to limit the disclosure in any way. The content of any individual section may apply equally to all sections.

[0273] Incorporation by reference All patents and publications referred to herein are hereby incorporated by reference in their entirety. They include the published PCT application WO2020 / 227369, filed on May 6, 2020, entitled "Tailored Hypoimmune Nanovesicle Delivery Systems for Cancer Tumors", and the published US non-provisional application US20220040106A1, filed on August 3, 2021, entitled "Tailored Hypoimmune Nanovesicular Delivery Systems for Cancer Tumors, Hereditary and Infectious Diseases".

[0274] Throughout this application, various publications, including U.S. patents, are referenced by author and year, and patents and applications are referenced by number. Complete citations of the publications are shown below. The entire disclosures of these publications and patents are hereby incorporated by reference into this application to more fully describe the state of the art relevant to this disclosure.

[0275] It should be understood that the present disclosure is illustrated by way of example and that the terms used are not limiting and are intended as words of description.

[0276] Obviously, many modifications and variations of the present disclosure are possible in light of the above teachings. Accordingly, it is to be understood that within the scope of the appended claims, the present disclosure may be practiced otherwise than as specifically described.

Claims

**Claim 1** A method for producing hypoimmunogenic cells, comprising: (a) reducing or eliminating the expression and / or activity of one or more immunogenic proteins in the cells; and (b) expressing one or more immunoprotective proteins in the cells, or increasing the expression and / or activity thereof, thereby producing the hypoimmunogenic cells. **Claim 2** The method according to claim 1, wherein the cells are stem cells, induced pluripotent stem cells (iPSCs), reprogrammed pluripotent or multipotent cells, embryonic stem cells, mesenchymal stem cells, or differentiated cells derived from any of these stem cells. **Claim 3** The method according to claim 2, wherein the differentiated cells are T cells, helper T cells, T-memory cells, or NK cells. **Claim 4** The method according to claim 2, wherein the differentiated cells are macrophages. **Claim 5** The method according to claim 2, wherein the differentiated cells are monocytes. **Claim 6** The method according to claim 2, wherein the differentiated cells are hepatocytes, cardiomyocytes, neurons, endothelial cells, pancreatic cells, or retinal pigment epithelial (RPE) cells. **Claim 7** The method according to any one of the preceding claims, wherein the hypoimmunogenic cells are substantially lacking in one or more MHC class I proteins, MHC class II proteins, T cell receptor (TCR) proteins, and / or cytokine release syndrome (CRS) proteins. **Claim 8** The method according to any one of the preceding claims, wherein reducing or eliminating the expression and / or activity of one or more immunogenic proteins comprises disrupting the β2-microglobulin (B2M) gene and / or disrupting to reduce or eliminate the expression and / or activity of MHC class I proteins. **Claim 9** The method according to any one of the preceding claims, wherein reducing or eliminating the expression and / or activity of one or more immunogenic proteins comprises disrupting the CIITA gene and / or disrupting to reduce or eliminate the expression and / or activity of MHC class II proteins. **Claim 10** The method according to any one of the preceding claims, wherein reducing or eliminating the expression and / or activity of one or more immunogenic proteins comprises disrupting the HLA-A gene and / or disrupting to reduce or eliminate the expression and / or activity of HLA-A proteins. **Claim 11** The method according to any one of the preceding claims, wherein reducing and / or eliminating the expression and / or activity of one or more immunogenic proteins comprises disrupting the HLA-B gene and / or disrupting to reduce and / or eliminate the expression and / or activity of the HLA-B protein.

12. The method according to any one of the preceding claims, wherein reducing and / or eliminating the expression and / or activity of one or more immunogenic proteins comprises disrupting the HLA-C gene and / or disrupting to reduce and / or eliminate the expression and / or activity of the HLA-C protein.

13. The method according to any one of the preceding claims, wherein reducing and / or eliminating the expression and / or activity of one or more immunogenic proteins comprises disrupting the HLA-E gene or the HLA-G gene and / or disrupting to reduce and / or eliminate the expression and / or activity of the HLA-E or HLA-G protein.

14. The method according to any one of the preceding claims, wherein reducing and / or eliminating the expression and / or activity of one or more immunogenic proteins comprises disrupting the HLA-F gene and / or disrupting to reduce and / or eliminate the expression and / or activity of the HLA-F protein.

15. The method according to any one of the preceding claims, wherein reducing and / or eliminating the expression and / or activity of one or more immunogenic proteins comprises disrupting the T cell alpha constant (TRAC) gene and / or disrupting to reduce and / or eliminate the expression and / or activity of the TRAC protein.

16. The method according to any one of the preceding claims, wherein reducing and / or eliminating the expression and / or activity of one or more immunogenic proteins comprises disrupting the T cell beta constant (TRBC) gene and / or disrupting to reduce and / or eliminate the expression and / or activity of the TRBC protein.

17. The method according to any one of the preceding claims, wherein reducing and / or eliminating the expression and / or activity of one or more immunogenic proteins comprises disrupting the PD-1 gene and / or disrupting to reduce and / or eliminate the expression and / or activity of the PD-1 protein.

18. The method according to any one of the preceding claims, wherein reducing and / or eliminating the expression and / or activity of one or more immunogenic proteins comprises disrupting the IL-4 gene and / or disrupting to reduce and / or eliminate the expression and / or activity of the IL-4 protein.

19. Reducing and / or removing the expression and / or activity of one or more immunogenic proteins, which comprises disrupting the IL-6 gene and / or disrupting to reduce and / or remove the expression and / or activity of the IL-6 protein, the method according to any one of the preceding claims.

20. Reducing and / or removing the expression and / or activity of one or more immunogenic proteins, which comprises disrupting the IL-10 gene and / or disrupting to reduce and / or remove the expression and / or activity of the IL-10 protein, the method according to any one of the preceding claims.

21. Reducing and / or removing the expression and / or activity of one or more immunogenic proteins, which comprises disrupting the IL-16 gene and / or disrupting to reduce and / or remove the expression and / or activity of the IL-16 protein, the method according to any one of the preceding claims.

22. Reducing and / or removing the expression and / or activity of one or more immunogenic proteins, which comprises disrupting the SerpinB9 gene and / or disrupting to reduce and / or remove the expression activity of the SerpinB9 protein, the method according to any one of the preceding claims.

23. Reducing and / or removing the expression and / or activity of one or more immunogenic proteins, which is by one or more of a transferase-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, at the DNA level, the method according to any one of the preceding claims.

24. The method according to claim 23, wherein the small RNA is a guide RNA (gRNA), a tracer RNA (tracrRNA), a microRNA (miRNA), RNA interference (RNAi), a small interfering RNA (siRNA), 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, or comprises one or more of these.

25. Reducing or eliminating the expression and / or activity of one or more immunogenic proteins 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, and small non-coding RNA, at the RNA level, the method according to any one of the preceding claims.

26. The method according to any one of the preceding claims, comprising expressing one or more immune defense proteins or increasing their expression and / or activity by expressing the CD34 gene and / or gene product or increasing its expression.

27. The method according to any one of the preceding claims, comprising expressing one or more immune defense proteins or increasing their expression and / or activity by expressing the CCL2 gene and / or gene product or increasing its expression.

28. The method according to any one of the preceding claims, comprising expressing one or more immune defense proteins or increasing their expression and / or activity by expressing the PD-L1 gene and / or gene product or increasing its expression, wherein the cell is not activated.

29. The method according to any one of the preceding claims, comprising expressing one or more immune defense proteins or increasing their expression and / or activity by expressing the H2-M3 gene and / or gene product or increasing its expression.

30. The method according to any one of the preceding claims, comprising expressing one or more immune defense proteins or increasing their expression and / or activity by expressing the CD47 gene and / or gene product or increasing its expression.

31. The method according to any one of the preceding claims, comprising expressing one or more immune defense proteins or increasing their expression and / or activity, which includes expressing the CD24 gene and / or gene product or increasing its expression.

32. The method according to any one of the preceding claims, comprising expressing one or more immune defense proteins or increasing their expression and / or activity, which includes expressing the chimeric CD24 / CD47 gene and / or gene product or increasing its expression.

33. The method according to any one of the preceding claims, comprising expressing one or more immune defense proteins or increasing their expression and / or activity, which includes expressing the CTLA-4 gene and / or gene product or increasing its expression.

34. The method according to any one of the preceding claims, comprising expressing one or more immune defense proteins or increasing their expression and / or activity, which includes expressing the CD200 gene and / or gene product or increasing its expression.

35. The method according to any one of the preceding claims, comprising expressing one or more immune defense proteins or increasing their expression and / or activity, which includes expressing the chimeric CD24 / CD200 gene and / or gene product or the chimeric CD47 / CD200 gene and / or gene product or increasing their expression.

36. The method according to any one of the preceding claims, comprising expressing one or more immune defense proteins or increasing their expression and / or activity, which includes expressing the MFG-E8 gene and / or gene product or increasing its expression.

37. The method according to any one of the preceding claims, comprising expressing one or more immune defense proteins or increasing their expression and / or activity, which includes expressing the NCAM gene and / or gene product or increasing its expression.

38. The method according to any one of the preceding claims, comprising expressing one or more immune defense proteins or increasing their expression and / or activity, which includes expressing the α-phagocytic integrin gene and / or gene product or increasing its expression.

39. The method according to any one of the preceding claims, comprising expressing one or more immune defense proteins or increasing their expression and / or activity, which includes expressing an antibody or antibody format molecule targeting the IL-6 surface receptor (anti-IL-6R) or increasing its expression.

40. The method according to any one of the preceding claims, comprising expressing one or more immune defense proteins or increasing their expression and / or activity, which includes the expression of the FasL gene and / or gene product.

41. The method according to any one of the preceding claims, comprising expressing one or more immune defense proteins or increasing their expression and / or activity, which does not include overexpression of the FasL gene and / or gene product.

42. The method according to any one of the preceding claims, wherein 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 in the low immunogenic cells is reduced or eliminated.

43. The method according to any one of claims 1 to 41, wherein the low immunogenic cells have the expression of 3 or more immune defense proteins, 4 or more immune defense proteins, 5 or more immune defense proteins, 6 or more immune defense proteins, 7 or more immune defense proteins, 8 or more immune defense proteins, 9 or more immune defense proteins, or 10 or more immune defense proteins, or have increased expression.

44. The method according to any one of claims 1 to 41, wherein the expression and / or activity of the gene and / or gene product of any one of HLA-A, HLA-B, HLA-C, HLA-F, CII TA, IL-6, TRAC, TRBC, and HLA-E or HLA-G in the hypoimmunogenic cells is reduced or eliminated.

45. The method according to any one of claims 1 to 41, wherein the expression and / or activity of the gene and / or gene product of any one of HLA-A, HLA-B, HLA-C, HLA-F, CII TA, IL-6, TRAC, TRBC, SerpinB9, and HLA-E or HLA-G in the hypoimmunogenic cells is reduced or eliminated.

46. The method according to any one of claims 1 to 41, wherein the expression and / or activity of the gene and / or gene product of any one of HLA-A, HLA-B, HLA-C, HLA-F, CII TA, IL-6, TRAC, TRBC, SerpinB9, HLA-E or HLA-G, and one or more of IL-4, IL-10, and IL-16 in the hypoimmunogenic cells is reduced or eliminated.

47. The hypoimmunogenic cells express or have increased expression of α-phagocytic integrin, CCL2, H2-M3, FasL, MFG-E8, and PD-L1 and / or CTLA-4, wherein the hypoimmunogenic cells do not overexpress FasL, and wherein the hypoimmunogenic cells are not activated by the expression of PD-L1, and The method according to any one of claims 1 to 41, wherein the hypoimmunogenic cells express or have increased expression of any one of CD24, CD47, CD200, chimeric CD24 / CD47, chimeric CD24 / CD200, and chimeric CD47 / CD200, or any two of CD24, CD47, and CD200.

48. The hypoimmunogenic cells express or have increased expression of α-phagocytic integrin, CCL2, H2-M3, FasL, MFG-E8, SerpinB9, and PD-L1 and / or CTLA-4, wherein the hypoimmunogenic cells do not overexpress FasL, and wherein the hypoimmunogenic cells are not activated by the expression of PD-L1, and The method according to any one of claims 1 to 41, wherein the hypoimmunogenic cells express any one of CD24, CD47, CD200, chimeric CD24 / CD47, chimeric CD24 / CD200, and chimeric CD47 / CD200, or any two of CD24, CD47, and CD200, or have increased expression thereof.

49. The method according to any one of claims 1 to 41, wherein the hypoimmunogenic cells express the CD200 gene and / or gene product, or have increased expression thereof, and do not express or are substantially lacking in any one of the CD24 or CD47 gene and / or gene product. Do not express or are substantially lacking.

50. The method according to any one of claims 1 to 41, wherein the hypoimmunogenic cells do not have the expression and / or activity of the SerpinB9 gene and / or gene product, and the CD200 gene and / or gene product.

51. The method according to any one of claims 26 to 40, wherein the expression of one or more immune defense proteins or the increase in expression is by the introduction of exogenous genetic elements.

52. The method according to claim 51, wherein the introduction of the exogenous genetic element is by stable integration into the cell genome.

53. The method according to claim 52, wherein the stable integration is by one or more of a method based on transposase, a method based on Cre / Lox, a method based on endonuclease, a method based on homologous recombination (HR), a method based on non-homologous end joining (NEHJ), a method based on microhomology-mediated end joining (MMEJ), a method based on homology-mediated end joining (HMEJ), or a combination thereof.

54. The method according to claim 53, wherein the stable integration is by a viral vector.

55. The method according to claim 51, wherein the introduction of the exogenous genetic element is by transient transfection.

56. The method according to any one of claims 26 to 40, wherein the expression of one or more immune defense proteins or the increase in expression is by an exogenous promoter and / or enhancer, and / or an endogenous promoter and / or enhancer, or a combination thereof.

57. The method according to any one of claims 26 to 40, wherein expressing or increasing the expression of one or more immune defense proteins is under the control of a constitutively active promoter.

58. The method according to any one of claims 26 to 40, wherein expressing or increasing the expression of one or more immune defense proteins is at the DNA 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, and small non-coding RNA.

59. The method according to any one of the preceding claims, wherein expressing or increasing the expression of one or more immune defense proteins is 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, and small non-coding RNA.

60. The method according to any one of claims 26 to 40, wherein expressing or overexpressing one or more immune defense proteins is by one or more of small regulatory RNA, miRNA, IRES element, transcription factor, or a combination thereof.

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

62. The method according to any one of the preceding claims, wherein the hypoimmunogenic cells or a biological composition derived therefrom do not elicit an immune response in the patient to whom they are administered.

63. The method according to any one of the preceding claims, wherein the low immunogenic cells comprise one or more targeting agents.

64. The method according to claim 63, wherein the one or more targeting agents comprise a chimeric antigen receptor (CAR), an immunodeficient HLA complex, and / or a T cell receptor (TCR).

65. The method according to claim 64, wherein the CAR is bispecific.

66. The method according to claim 64, wherein the CAR lacks an intracellular portion.

67. The method according to claim 64, wherein the CAR comprises a targeting agent, a transmembrane region, and an intracellular region comprising a co-stimulatory region and / or a signaling region.

68. The method according to claim 67, wherein the transmembrane region is derived from CD28, CD3ζ, CD4, CD8α or ICOS, or a fragment thereof.

69. The method according to claim 67, wherein the intracellular region comprises an intracellular signaling region of the CD3ζ chain and / or one or more co-stimulatory molecules optionally selected from CD28, 4-1BB, ICOS, CD27, and OX40.

70. The method according to claim 63, wherein the one or more targeting agents are an antibody or an antibody format.

71. The antibody or antibody format is selected from one or more of monoclonal antibodies, polyclonal antibodies, antibody fragments, Fab, Fab', Fab'-SH, F(ab')2, Fv, single-chain Fv (scFv), VERR, V ＮＡＲ , V Ｈ H, affilins, diabodies, nanobodies, linear antibodies, bispecific antibodies, multispecific antibodies, chimeric antibodies, humanized antibodies, human antibodies, and fusion proteins containing the antigen-binding portion of an antibody, the method according to claim 70.

72. The method according to claim 71, wherein the antibody format is scFv.

73. The method according to claim 63, wherein the one or more targeting agents comprise a virus epitope recognition receptor (VERR) or a virus ligand.

74. The method according to claim 63, wherein the one or more targeting agents comprise a ligand for a receptor.

75. The method according to claim 63, wherein the one or more targeting agents comprise a receptor for a ligand.

76. The method according to claims 63 to 75, wherein the one or more targeting agents are operably linked to a regulatable expression element.

77. Low immunogenic cells produced by the method according to any one of claims 1 to 76.

78. A pharmaceutical composition comprising the low immunogenic cells according to claim 77 and one or more excipients.

79. Low immunogenic cells, comprising: (a) one or more membrane-embedded targeting agents targeted to one or more cell biomarkers; (b) (i) α - phagocytic integrin, CCL2, H2 - M3, MFG - E8, and FasL (where FasL is not overexpressed); (ii) PD-L1 and / or CTLA-4 (wherein the low immunogenicity cells are not activated by the expression of PD-L1), and (iii) any one of CD24, CD47, CD200, chimeric CD24 / CD47, chimeric CD24 / CD200, and chimeric CD47 / CD200, or any two of CD24, CD47, and CD200, expression of, or increased expression of, one or more immune defense proteins selected from (c) (i) HLA-A, HLA-B, HLA-C, HLA-F, CII TA, PD-1, IL-6, T cell alpha chain (TRAC), and T cell beta chain (TRBC), and (ii) HLA-E or HLA-G, substantially lacking the expression and / or activity of one or more immunogenic proteins selected from Low immunogenicity cells comprising **Claim 80** Low immunogenicity cells, comprising (a) one or more membrane-embedded targeting agents targeted to one or more cell biomarkers, (b) (i) α - phagocytic integrin, CCL2, H2-M3, MFG-E8, and FasL (wherein FasL is not overexpressed), (ii) PD-L1 and / or CTLA-4 (wherein the low immunogenicity cells are not activated by the expression of PD-L1), and (iii) either CD24 and CD47, or chimeric CD24 / CD47, expression of, or increased expression of, one or more immune defense proteins selected from (c) (i) HLA-A, HLA-B, HLA-C, HLA-F, CII TA, PD-1, SerpinB9, IL-6, T cell alpha chain (TRAC), and / or T cell beta chain (TRBC), and (ii) HLA-E or HLA-G, substantially lacking the expression and / or activity of one or more immunogenic proteins selected from Low immunogenicity cells comprising **Claim 81** The low immunogenicity cells according to claim 79 or 80, wherein the low immunogenicity cells substantially lack the expression and / or activity of one or more of IL-4, IL-10, and / or IL-16. **Claim 82** The low immunogenicity cells according to any one of claims 79 to 81, further comprising the expression or increased expression of an antibody or antibody format molecule (anti-IL-6R) targeting the IL-6 surface receptor. **Claim 83** The low immunogenicity cell according to any one of claims 79 to 82, further comprising the expression of NCAM or increased expression.

84. The low immunogenicity cell according to any one of claims 79 to 83, wherein the one or more targeting agents comprise a chimeric antigen receptor (CAR).

85. The low immunogenicity cell according to any one of claims 79 to 84, wherein the one or more targeting agents are an antibody or an antibody format.

86. The antibody or antibody format is a monoclonal antibody, polyclonal antibody, antibody fragment, Fab, Fab', Fab'-SH, F(ab')2, Fv, single-chain Fv (scFv), V ＮＡＲ , V Ｈ H, affilin, diabody, nanobody, linear antibody, bispecific antibody, multispecific antibody, chimeric antibody, humanized antibody, human antibody, and a fusion protein comprising an antigen-binding portion of an antibody, the low immunogenic cell according to claim 85.

87. The low immunogenicity cell according to any one of claims 79 to 83, wherein the one or more targeting agents are a virus epitope recognition receptor (VERR) or a virus ligand.

88. The low immunogenicity cell according to any one of claims 79 to 83, wherein the one or more targeting agents are a ligand for a receptor or a receptor for a ligand.

89. The low immunogenicity cell according to any one of claims 79 to 88, wherein the low immunogenicity cell is allogeneic.

90. The low immunogenicity cell according to any one of claims 79 to 89, wherein the low immunogenicity cell or a biological composition derived therefrom does not cause an immune response in a patient to whom it is administered.

91. A pharmaceutical composition comprising the low immunogenicity cell according to any one of claims 79 to 90 and one or more excipients.

Images