Engineered cells and methods of use

Abstract

The present disclosure provides methods and compositions for providing insertion of transgenes in stem or progenitor cells by in vitro T cell differentiation methods without detrimental effects on T cell differentiation. To achieve this, the inventors have found that the expression of the transgene under the control of the promoter region (e.g. Granzyme A) allows for a coordinated expression pattern that provides both 1) high expression of the transgene in mature T cells, and 2) coordinated expression levels of the transgene throughout the in vitro differentiation method, which allows for the production of a population of mature T cells.

Description

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 315,579 filed on March 2, 2022, U.S. Provisional Patent Application No. 63 / 315,581 filed on March 2, 2022, and U.S. Provisional Patent Application No. 63 / 442,966 filed on February 2, 2023, which are hereby incorporated by reference in their entirety.

[0002] Sequence Listing

[0003] This application contains a sequence listing prepared in accordance with the ST.26 format, and is hereby incorporated by reference in its entirety. The sequence listing created on March 1, 2023 is named UCLAP0162WO and is 61,982 bytes in size. Background Art 1. Technical Field

[0004] The present invention relates generally to the fields of cell culture and development.

[0005] 2. Description of Related Technology

[0006] Using stem cells or progenitor cells as T cell sources for adoptive cell therapy provides the possibility of alternatives for currently relying on the method for operating mature peripheral blood T cells. The T cells produced from PSC and HSPC provide advantages over the primary mature T cells harvested from blood, because stem cells have the ability of unlimited self-renewal in vivo (in the case of HSPC) or in vitro (in the case of PSC). Inserting biologically active genes in the genome of stem cells or progenitor cells can differentiate them into T cells with changed functional characteristics in vitro or in vivo. However, when some biologically active genes are expressed in the developmental stage prior to mature T cells, T cell development can be harmful. Therefore, this area needs to transform stem cells or progenitor cells so that they can carry transgenics and successfully differentiate into mature T cells. Summary of the invention

[0007] The present disclosure provides methods and compositions that provide for insertion of transgenes in stem or progenitor cells without deleterious effects on differentiation of T cells constitutively expressing the transgene during T cell differentiation in vitro or in vivo. To achieve this, the inventors have discovered that expression of a transgene under the control of a promoter region (e.g., granzyme A) allows for a coordinated expression pattern that provides both: 1) high expression of the transgene in mature T cells, and 2) a coordinated expression level of the transgene throughout an in vitro differentiation process, which allows for the generation of a population of mature T cells.

[0008] The present disclosure describes such a nucleic acid, which comprises a promoter region and a transgene located downstream of the transcription start site of the promoter region; wherein the promoter region comprises a promoter region from a gene of Table 1. Cells comprising the nucleic acid of the present disclosure are also described. The cell of the present disclosure may comprise a plurality of nucleic acids of the present disclosure. The cell may comprise a first nucleic acid and a second nucleic acid, wherein the first nucleic acid comprises a first promoter region and a first transgene located downstream of the transcription start site of the promoter region; wherein the promoter region comprises a promoter region from a gene of Table 1, and the second nucleic acid comprises a second promoter region and a second transgene located downstream of the transcription start site of the promoter region; wherein the promoter region comprises a promoter region selected from a gene of Table 1. The cell may comprise a first promoter region and a first transgene located downstream of the transcription start site of the promoter region; wherein the promoter region comprises a promoter region from the gene RAG1 or RAG2; and a second nucleic acid comprising a second promoter region and a second transgene located downstream of the transcription start site of the promoter region; wherein the promoter region comprises a promoter region selected from the following genes: GZMA, GZMB, GZMH, GZMK, GZMM, TNFSF14, XCL2, PTGDS, SAMD3, NCALD or XCL1. Also described are methods for preparing cells, comprising culturing cells of the present disclosure under conditions suitable for the in vitro differentiation of cells into mature cells. Also described are methods for treating subjects, comprising administering cells of the present disclosure.

[0009] Table 1 - Promoter Region

[0010]

[0011]

[0012] One or more genes and / or promoter regions thereof from Table 1 may not be included in the methods, nucleic acids, cells or compositions of the present disclosure. The promoter region, the first promoter region or the second promoter region may be a promoter region from a granzyme gene. The promoter region, the first promoter region or the second promoter region may be from a granzyme A, B, H, K or M gene. The promoter region, the first promoter region or the second promoter region may not include a promoter region from one or more of the granzyme A, B, H, K or M genes. The promoter, the first promoter region or the second promoter region may be a promoter region from a granzyme A gene. The promoter region, the first promoter region or the second promoter region may be a promoter region from a granzyme B gene. The promoter region, the first promoter region or the second promoter region may be a promoter region from a granzyme H gene. The promoter region, the first promoter region or the second promoter region may be a promoter region from a granzyme K gene. The promoter region, the first promoter region or the second promoter region may be a promoter region from a granzyme M gene. The promoter region, the first promoter region, or the second promoter region may comprise a RAG1 promoter region or a RAG2 promoter region. The RAG1 promoter region or the RAG2 promoter region may not be included in the embodiments of the present disclosure. The genomic DNA may lack genes encoding one or both of the functional RAG1 protein and / or the RAG2 protein. The genomic DNA may encode one or both of the functional RAG1 protein and / or the RAG2 protein. The transgenic, the first transgenic, or the second transgenic may be inserted into the RAG1 coding region or the RAG2 coding region, and / or wherein the transgenic, the first transgenic, or the second transgenic may at least partially replace the RAG1 coding region or the RAG2 coding region. The promoter region, the first promoter region, or the second promoter region may comprise an endogenous promoter region. An endogenous promoter region refers to a situation in which the promoter region is in its endogenous genomic environment, such that the promoter upstream sequences (i.e., the 5' region) are substantially the same as those in wild-type cells. Substantially identical can refer to a region having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with the wild-type upstream region. An endogenous promoter region also refers to a situation where the promoter is located at the same genomic location as the wild-type promoter. An endogenous promoter can refer to an unmodified promoter in a cell, or it has 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98 or 99% identity with the wild-type promoter or a promoter having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with the wild-type promoter.

[0013] The transgenic, the first transgenic or the second transgenic may comprise a chimeric antigen receptor (CAR) or a T-cell receptor (TCR). The transgenic, the first transgenic or the second transgenic may comprise FOXP3. The transgenic, the first transgenic or the second transgenic may comprise BCL11b. The transgenic, the first transgenic or the second transgenic may comprise a T cell receptor (TCR). The TCR may be a tumor antigen-specific TCR, a virus-specific TCR, a heterologous specific TCR, a cancer cell-specific TCR, a bacteria-specific TCR or a cancer-testis antigen-specific TCR. The TCR may be a tumor antigen-specific TCR (i.e., a TCR that recognizes a tumor antigen). The TCR may be a viral antigen-specific TCR (i.e., a TCR that recognizes a viral antigen). The CAR may be, for example, a tumor antigen-specific CAR, a virus-specific CAR, a heterologous specific CAR or a bacteria-specific CAR. The CAR may be a tumor antigen-specific CAR (i.e., a CAR that recognizes a tumor antigen). The transgenic, the first transgenic or the second transgenic may be a transcription factor.

[0014] Transgenic may be a cytokine receptor or a cytokine. Transgenic, the first transgenic or the second transgenic may not include a chimeric antigen receptor (CAR) or a T cell receptor (TCR). Transgenic, the first transgenic or the second transgenic may not include FOXP3. Transgenic, the first transgenic or the second transgenic may not include BCL11b. Transgenic, the first transgenic or the second transgenic may not include a T cell receptor (TCR). TCR may not include a tumor antigen-specific TCR, a virus-specific TCR, a heterologous specific TCR, a cancer cell-specific TCR, a bacteria-specific TCR or a cancer-testis antigen-specific TCR. TCR may not include a tumor antigen-specific TCR (i.e., a TCR that recognizes a tumor antigen). TCR may not include a viral antigen-specific TCR (i.e., a TCR that recognizes a viral antigen). CAR may not include, for example, a tumor antigen-specific CAR, a virus-specific CAR, a heterologous specific CAR or a bacteria-specific CAR. CAR may not include a tumor antigen-specific CAR (i.e., a CAR that recognizes a tumor antigen). Transgenic, the first transgenic or the second transgenic may not include a transcription factor. Transgenic may not include a cytokine receptor or a cytokine.

[0015] The size of the transgene, the first transgene or the second transgene may be, at least, or at most, and / or the size of the encoded protein may be, at least, or at most,

[0016] 500, 505, 510, 515, 520, 525, 530, 535, 540, 545, 550, 555, 560, 565, 570, 575, 580, 585, 590, 595, 600, 605, 610, 615, 620, 625, 630, 635, 640, 645, 650, 655, 660, 665, 670, 675, 680, 685 5, 690, 695, 700, 705, 710, 715, 720, 725, 730, 735, 740, 745, 750, 755, 760, 765, 770, 775, 780, 785, 790, 795, 800, 805, 810, 815, 820, 825, 830, 835, 840, 845, 850, 855, 860, 865, 870, 8 75, 880, 885, 890, 895, 900, 905, 910, 915, 920, 925, 930, 935, 940, 945, 950, 955, 960, 965, 970, 975, 980, 985, 990, 995, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 20 00, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4500, 5000, 5500, 6000, or 7000 amino acids or nucleic acids in length (or any range derivable therein).

[0017] The nucleic acid, the first nucleic acid or the second nucleic acid may comprise genomic DNA. The nucleic acid, the first nucleic acid or the second nucleic acid may comprise a coding region of a gene of Table 1 downstream of a transcription start site of a promoter region, a first promoter region or a second promoter region. Both transcription of the transgenic and the gene of Table 1 may be regulated by the promoter of the gene of Table 1. The transgenic, the first transgenic or the second transgenic and the gene of Table 1 are expressed on the same mRNA. The nucleic acid, the first nucleic acid or the second nucleic acid may comprise or further comprise a self-cleaving peptide site or an IRES. The nucleic acid may not comprise a self-cleaving peptide or an IRES. The transgenic, the first transgenic or the second transgenic may be located at the 3'-proximal end of the gene of Table 1. The transgenic, the first transgenic or the second transgenic may be located at the 5'-proximal end of the gene of Table 1. When the first region is connected to the 3' nucleic acid terminus of the second region, the first region may be located at the 3'-proximal end of the second region. There may be other intervening amino acid residues between the first region and the second region. It may also not comprise an intervening amino acid residue between the first region and the second region. Therefore, unless specifically indicated that there is no intervening amino acid residue, these regions do not need to be adjacent, which is also considered. The term "5'-proximal" is similarly defined as when the first region is connected to the 5' nucleic acid terminus of the second region, the first region is located at the 5'-proximal end of the second region. Similarly, unless otherwise indicated, there may be other intervening amino acid residues between the first region and the second region. The nucleic acid, the first nucleic acid or the second nucleic acid may include or further include a 3'UTR region downstream of the coding region gene and / or transgene, the first transgene or the second transgene of Table 1. The 3'UTR may include an endogenous 3'UTR. The promoter region, the first promoter region, the second promoter region, the 3'UTR, the transgene, the first transgene, the second transgene and / or the gene of the coding region of Table 1 are located in the genomic DNA at the endogenous locus of the gene of Table 1.

[0018] Nucleic acid, first nucleic acid, second nucleic acid genomic DNA and / or cell may lack genes encoding one or more of functional endogenous TCR-α (TCR-a) protein, functional endogenous TCR-β (TCR-b) protein, functional endogenous TCR-γ (TCR-g) protein or functional endogenous TCR-δ (TCR-d) protein. Nucleic acid, first nucleic acid, second nucleic acid genomic DNA and / or cell may lack genes encoding one or more of functional CD3γ protein, functional CD3δ protein, functional CD3ε protein or functional CD3ζ protein.

[0019] The nucleic acid, the first nucleic acid, the second nucleic acid genomic DNA and / or the cell may be effective for one or more genes encoding a functional endogenous TCR-α (TCR-a) protein, a functional endogenous TCR-β (TCR-b) protein, a functional endogenous TCR-γ (TCR-g) protein, or a functional endogenous TCR-δ (TCR-d) protein. The nucleic acid, the first nucleic acid, the second nucleic acid genomic DNA and / or the cell may be effective for one or more genes encoding a functional CD3γ protein, a functional CD3δ protein, a functional CD3ε protein, or a functional CD3ζ protein.

[0020] The cell may comprise a pluripotent stem cell (PSC). The cell may comprise an embryonic stem cell or an induced pluripotent stem cell (iPSC). The cell may comprise a stem cell, a hematopoietic stem or progenitor cell (HSPC), an embryonic stem cell, an induced pluripotent stem cell (iPSC), a human embryonic mesoderm progenitor cell or a pluripotent stem cell (PSC). The cell may be separated from or derived from cord blood, peripheral blood, bone marrow, peripheral blood, umbilical cord blood, placenta, adipose tissue or umbilical cord tissue. The cell may comprise a T cell. Mature cells may comprise mature T cells, mature regulatory T cells (regulatory Tcell, Treg), iNKT cells, innate lymphocytes or NK cells. The cell may comprise a CD4-single positive (SP) cell or a CD8 SP cell. The cell may comprise CD4+CD8- or CD8ab+CD4-T cells. The cell may comprise cells differentiated in vitro. In vitro differentiated cells can be T cells, mature Treg cells, iNKT cells, innate lymphocytes or NK cells. The cells can be in vivo and / or have been administered to a subject. The cells can be in vitro. The cells can be cells that have undergone differentiation in vitro. The cells can be cells that have undergone differentiation in vivo (i.e., in a human subject).

[0021] The cells of the present disclosure may not include PSC, iPSC, HSPC, embryonic stem cells, iPSC, human embryonic mesoderm progenitor cells, or cells separated from or derived from cord blood, peripheral blood, bone marrow, peripheral blood, cord blood, placenta, adipose tissue or umbilical cord tissue. Mature cells may not include one or more of mature T cells, mature regulatory T cells (Treg), iNKT cells, innate lymphocytes or NK cells. Cells may include CD4-single positive (SP) cells or CD8 SP cells. Cells may include CD4+CD8- or CD8ab+CD4-T cells. Cells may not include cells and / or mature Treg cells, iNKT cells, innate lymphocytes or NK cells differentiated in vitro.

[0022] The method may also include or further include culturing cells in three-dimensional (3D) cell aggregates, wherein the cell aggregates also include a selected matrix cell population expressing Notch ligands, and wherein the 3D cell aggregates are cultured in a serum-free medium containing insulin, biotin, transferrin and albumin for a period of time sufficient to differentiate stem cells or progenitor cells in vitro into mature cells, such as mature T cells. The matrix cells may include MS5 matrix cells. The matrix cells may be human matrix cells. The Notch ligands may include exogenous Notch ligands. The matrix cells may include transgenics expressing Notch ligands. The method may include or further include centrifuging stem cells or progenitor cells and matrix cells to form 3D cell aggregates. The cell culture medium may include or further include one or more of the following: externally added FLT3 ligand (FLT3L), interleukin 7 (interleukin 7, IL-7), stem cell factor (stem cell factor, SCF), thrombopoietin (thrombopoietin, TPO), thrombopoietin (TPO), IL-2, IL-4, IL-6, IL-15, IL-21, TNF-α, TGF-β, interferon-γ, interferon-λ, TSLP, thymopentin, pleiotrophin, pleiotrophin, midkine, or a combination thereof. The stromal cells may have an exogenous nucleotide sequence encoding a complete, partial or modified Notch ligand, and wherein the Notch ligand is DLL4, DLL1, JAG1, JAG2 or a combination thereof. The cell culture medium may not include one or more of the following: externally added FLT3 ligand (FLT3L), interleukin 7 (IL-7), stem cell factor (SCF), thrombopoietin (TPO), thrombopoietin (TPO), IL-2, IL-4, IL-6, IL-15, IL-21, TNF-α, TGF-β, interferon-γ, interferon-λ, TSLP, thymopentin, pleiotrophic factor, pleiotrophic growth factor, midkine, or a combination thereof. The stromal cells may not include one or more exogenous nucleotide sequences encoding complete, partial or modified Notch ligands, and wherein the Notch ligand is DLL4, DLL1, JAG1, JAG2, or a combination thereof.

[0023] The cell can be allogeneic. The cell can also be further limited to autologous. The subject can be a subject suffering from cancer. The method can be used to treat cancer. The method can be used to treat autoimmune diseases. The cancer or autoimmune disease can be one of those listed herein. The cancer can be selected from lung cancer, prostate cancer, ovarian cancer, testicular cancer, brain cancer, skin cancer, melanoma, colon cancer, rectal cancer, stomach cancer, esophageal cancer, tracheal cancer, head and neck cancer, pancreatic cancer, liver cancer, breast cancer, ovarian cancer, lymphoma including lymphoma and multiple myeloma, leukemia, osteosarcoma or soft tissue sarcoma, cervical cancer, and vulvar cancer. The cancer may not include one or more of the following: lung cancer, prostate cancer, ovarian cancer, testicular cancer, brain cancer, skin cancer, melanoma, colon cancer, rectal cancer, stomach cancer, esophageal cancer, tracheal cancer, head and neck cancer, pancreatic cancer, liver cancer, breast cancer, ovarian cancer, lymphoma including lymphoma and multiple myeloma, leukemia, osteosarcoma or soft tissue sarcoma, cervical cancer, and vulvar cancer.

[0024] One or more cells, particularly stromal cells, may express exogenous Notch ligands. The culture medium may contain externally added Notch ligands. Externally added Notch ligands may be connected to a solid support or immobilized. Some non-limiting examples of Notch ligands include complete (full length), partial (truncated form) or modified (comprising one or more mutations, such as conservative mutations) DLL4, DLL1, JAG1, JAG2 or a combination thereof. For example, stromal cells have an exogenous nucleotide sequence encoding a Notch ligand, which can be introduced into cells by transfection or transduction. The culture composition may not contain or may not contain a Notch ligand in other ways, or may not contain an externally added Notch ligand. The Notch ligand may be a human Notch ligand. The Notch ligand may be a human DLL1 or DLL4 Notch ligand.

[0025] The stromal cell can be a mouse stromal cell line, a human stromal cell line, a selected primary stromal cell group, a selected stromal cell group differentiated in vitro by pluripotent stem cells, or a combination thereof. The stromal cell can be a MS5, OP9, S17, HS-5 or HS-27a cell. The stromal cell line (e.g., MS5, OP9, S17, HS-5 or HS-27a cell) may not be included in the methods and compositions of the present disclosure. The stromal cell can be differentiated by human cells. The stromal cell can be differentiated by human pluripotent stem cells. The stromal cell can be differentiated by human or non-human HSPC or PSC cells.

[0026] The culture medium can be prepared using a culture medium for culturing animal cells as its basic culture medium, such as any of the following: AIM V, X-VIVO-15, NeuroBasal, EGM2, TeSR, BME, BGJb, CMRL1066, Glasgow MEM, Improved MEM Zinc Option, IMDM, Medium 199, Eagle MEM, αMEM, DMEM, Ham, RPMI-1640 and Fischer medium, and any combination thereof, but the culture medium may not be particularly limited thereto as long as it can be used for culturing animal cells. In particular, the culture medium may be xeno-free or chemically defined.

[0027] The culture medium can be a serum-containing culture medium or a serum-free culture medium, or a xeno-free culture medium. From the perspective of preventing contamination by xeno-animal-derived components, the serum can be derived from the same animal as the stem cells. A serum-free culture medium refers to a culture medium without unprocessed or unpurified serum, and therefore can include a culture medium with purified blood-derived components or animal tissue-derived components (e.g., growth factors).

[0028] The culture medium may or may not include any substitute for serum. The substitute for serum may include such a substance: it suitably includes albumin (e.g., lipid-rich albumin, bovine albumin, albumin substitutes such as recombinant albumin or humanized albumin, plant starch, dextran and protein hydrolysate), transferrin (or other iron transporters), fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thioglycerol (3'-thiolgiycerol), or its equivalent. The substitute for serum may be prepared by the method disclosed in, for example, International Publication No. 98 / 30679 (which is incorporated herein in its entirety). Alternatively, any commercially available substance may be used to obtain more convenience. Commercially available substances include knockout serum substitutes (Knockout Serum Replacement, KSR), chemically defined lipid concentrates (Gibco) and Glutamax (Gibco).

[0029] The culture medium can be a serum-free medium suitable for neural cell development. For example, the culture medium can contain a concentration effective to generate mature cells from 3D cell aggregates. Supplements, Xeno-Free supplement (available on the World Wide Web at http: / / www.thermofisher.com / us / en / home / technical-resources / media-formulation.250.html), NS21 supplement (Chen et al., J Neurosci Methods, 2008 Jun 30; 171(2):239–247, which is incorporated herein in its entirety), GS21 TM supplement (available on the World Wide Web at amsbio.com / B-27.aspx), or a combination thereof.

[0030] The culture medium may include one, two, three, four, five, six, seven, eight, nine, ten, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more of the following B-27 supplement ingredients: vitamins, such as biotin; DL alpha tocopheryl acetate; DL alpha-tocopherol; vitamin A (acetate); proteins, such as BSA (bovine serum albumin) or human albumin; fatty acid free fraction V; catalase; human recombinant insulin; human transferrin; superoxide dismutase; other components such as corticosterone; D-galactose; ethanolamine HCl; glutathione (reduced); L-carnitine HCl; linoleic acid; linolenic acid; progesterone; putrescine 2HCl; sodium selenite; and T3 (triodo-I-thyronine).

[0031] The culture medium may include externally added ascorbic acid. The culture medium may also include externally added fatty acids or lipids, amino acids (e.g., non-essential amino acids), monosaccharides, vitamins, growth factors, cytokines, antioxidant substances, 2-mercaptoethanol, pyruvic acid, buffers, inorganic ions and inorganic salts. Vitamins may include biotin, DLα tocopherol acetate, DLα tocopherol, vitamin A, choline chloride, calcium pantothenate, pantothenic acid, folic acid nicotinamide, pyridoxine, riboflavin, thiamine, inositol, vitamin B12 or a combination thereof or a salt thereof. Amino acids may include arginine, cysteine, isoleucine, leucine, lysine, methionine, glutamine, phenylalanine, threonine, tryptophan, histidine, tyrosine or valine or a combination thereof. Inorganic ions may include sodium, potassium, calcium, magnesium, nitrogen or phosphorus, or a combination thereof or a salt thereof. The culture medium may also include molybdenum, vanadium, iron, zinc, selenium, copper or manganese, or a combination thereof.

[0032] The culture medium may not contain one, two, three, four, five, six, seven, eight, nine, ten, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more of the following: B-27 supplement, biotin, DL alpha tocopheryl acetate, DL alpha tocopherol, vitamin A (acetate), BSA (bovine serum albumin), human albumin, fatty acid free fraction V, catalase, human recombinant insulin, human transferrin, superoxide dismutase, corticosterone, D-galactose, ethanolamine HCl, glutathione (reduced), L-carnitine HCl, linoleic acid, linolenic acid , progesterone, putrescine 2HCl, sodium selenite, T3 (triiodo-I-thyronine), ascorbic acid, fatty acids, lipids, amino acids, monosaccharides, vitamins, growth factors, cytokines, antioxidant substances, 2-mercaptoethanol, pyruvic acid, buffers, inorganic ions, inorganic salts, choline chloride, calcium pantothenate, pantothenic acid, folic acid niacinamide, pyridoxine, riboflavin, thiamine, inositol, vitamin B12, arginine, cysteine, isoleucine, leucine, lysine, methionine, glutamine, phenylalanine, threonine, tryptophan, histidine, tyrosine, valine, sodium, potassium, calcium, magnesium, nitrogen, phosphorus, molybdenum, vanadium, iron, zinc, selenium, copper and / or manganese.

[0033] One or more media components can be added at a concentration of at least, at most, or about 0.1, 0.5, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, or 1000 ng / L, ng / ml, μg / ml, mg / ml, or any range derivable therein.

[0034] The culture medium used can be supplemented with at least one externally added growth factor or cytokine at a concentration of about 0.1 ng / mL to about 500 ng / mL, more particularly 1 ng / mL to 100 ng / mL, or at least, at most, or about 0.1, 0.5, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900 or 1000 ng / L, ng / ml, μg / ml, mg / ml, or any range derivable therein. Suitable cytokines include, but are not limited to, FLT3 ligand (FLT3L), interleukin 7 (IL-7), stem cell factor (SCF), thrombopoietin (TPO), IL-2, IL-4, IL-6, IL-15, IL-21, TNF-α, TGF-β, interferon-γ, interferon-λ, TSLP, thymopentin, pleiotropic trophic factor and midkine. In particular, the culture medium may include at least one of FLT3L and IL-7. More particularly, the culture may include both FLT3L and IL-7. One or more of the following may not be included: FLT3 ligand (FLT3L), interleukin 7 (IL-7), stem cell factor (SCF), thrombopoietin (TPO), IL-2, IL-4, IL-6, IL-15, IL-21, TNF-α, TGF-β, interferon-γ, interferon-λ, TSLP, thymopentin, pleiotropic trophic factor and midkine.

[0035] Other culture conditions may be appropriately defined. For example, the culture temperature may be about 20°C to 40°C, for example, at least, at most, or about 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C (or any range derivable therein), but is not particularly limited thereto. CO2 concentration may be about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10% (or any range derivable therein), for example, about 2% to 10%, for example, about 2% to 5%, or any range derivable therein. Oxygen tension may be at least or about 1%, 5%, 8%, 10%, 20% or any range derivable therein.

[0036] The stromal cells and PSCs induced to differentiate into mature cells can be present in any ratio, for example, about 20:1, 10:1, 5:1, 1:1, 1:5, 1:10, 1:20, or any range derivable therein.

[0037] Any cell population, such as stromal cells, mature cells, stem cells, progenitor cells, or PSCs induced to differentiate into mature cells, may contain at least, about, or at most

[0038] 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,50,60,70,80,90,100,200,300,400,500,1x10 3 , 2x10 3 , 3x10 3 , 4x10 3 , 5x10 3 , 6x10 3 , 7x10 3 , 8x10 3 , 9x10 3 , 1x10 4 , 2x10 4 , 3x10 4 , 4x10 3 , 5x10 4 , 6x10 4 , 7x10 4 , 8x10 4 , 9x10 4 , 1x10 5 , 2x10 5 , 3x10 5 , 4x10 5 , 5x10 5 , 6x10 5 , 7x10 5 , 8x10 5 , 9x10 5 , 1x10 6 , or 2x10 6 cells.

[0039] The culture may continue for any length of time, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 hours, days or weeks, or any range derivable therein. The culture may or may not involve passaging of the cells.

[0040] The subject may comprise a laboratory test animal, such as a mouse, rat, rabbit, dog, cat, horse or pig. The subject may be a human.

[0041] Any genetic modification composition or method (e.g., gene editing, homologous recombination or non-homologous recombination, RNA-mediated genetic delivery, or any conventional nucleic acid delivery method) can be used to introduce exogenous nucleic acid into cells. Some non-limiting examples of genetic modification methods can include, for example, gene editing methods by CRISPR / CAS9, zinc finger nucleases, or TALEN technology.

[0042] The compositions and methods described herein can be modified so that the methods are used to prepare cells having a certain phenotype. The methods are used to prepare T cells having the following phenotypes:

[0043] CD4 + CD8 - T cells, CD4 - CD8 + T cells, CD34 + CD7 + CD1a + Cells, CD3+ TCRab+, CD3+TCRgd+, CD3+ TCRab+ CD4+ CD8-, CD3+ TCRab+ CD8+ CD4-, CD3+ TCRab+ CD4+ CD8-CD45RO- CD45RA+, CD3+ TCRab+ CD8+ CD4- CD45RO- CD45RA+, CD3+ TCRab+ CD4+ CD8-CD45RO- CD45RA+ CCR7+, CD3+ TCRab+ CD8+ CD4- CD45RO- CD45RA+ CCR7+, CD3+ TCRab+CD4+ CD8- CD45RO- CD45RA+ CD27+, CD3+ TCRab+ CD8+ CD4- CD45RO- CD45RA+ CD27+, CD34 + CD7 + CD1a + Cells, CD34+CD5+CD7+, CD34+CD5+CD7-,

[0044] Natural killer T cells, regulatory T cells, antigen-specific T cells, intraepithelial lymphocyte T cells, or the following cells: CD45+, CD11b+, CD11b-,

[0045] CD15+, CD15-, CD24+, CD24-, CD114+, CD114-, CD182+, CD182-, CD4+, CD4-, CD14+, CD14 -, CD11a+, CD11a-, CD91+, CD91-, CD16+, CD16-, CD3+, CD3-, CD25+, CD25-, Foxp3+, Fox3 p-, CD8+, CD8-, CD19+, CD19-, CD20+, CD20-, CD24+, CD24, CD38+, CD38-, CD22+, CD22-, C D61+, CD61-, CD16+, CD16-, CD56+, CD56-, CD31+, CD31-, CD30+, CD30-, CD38+, and / or CD38-

[0046] Or a combination thereof. For example, intraepithelial lymphocytes (IEL) can be prepared by expressing the cognate antigen in stromal cells.

[0047] Cells can express CD4, CD8, CD34, CD7,

[0048] One or more of CD1a, CD3, TCRab, TCRgd, CD45RO, CD45RA, CCR7, CD27, CD45, CD11b, CD15, CD24, CD114, CD182, CD91, CD99, CD16, CD25, Foxp3, CD20, CD38, CD22, CD61, CD56, CD31, and CD30 are positive or negative, and / or alternatively or in combination may be positive or negative for

[0049] One or more of CD4, CD8, CD34, CD7, CD1a, CD3, TCRab, TCRgd, CD45RO, CD45RA, CCR7, CD27, CD45, CD11b, CD15, CD24, CD114, CD182, CD91, CD99, CD16, CD25, Foxp3, CD20, CD38, CD22, CD61, CD56, CD31, and CD30 are negative.

[0050] The methods, cells and compositions may not include T cells having the following phenotypes:

[0051] CD4 + CD8 - T cells, CD4 - CD8 + T cells, CD34 + CD7 +CD1a + Cells, CD3+ TCRab+, CD3+TCRgd+, CD3+ TCRab+ CD4+ CD8-, CD3+ TCRab+ CD8+ CD4-, CD3+ TCRab+ CD4+ CD8-CD45RO- CD45RA+, CD3+ TCRab+ CD8+ CD4- CD45RO- CD45RA+, CD3+ TCRab+ CD4+ CD8-CD45RO- CD45RA+ CCR7+, CD3+ TCRab+ CD8+ CD4- CD45RO- CD45RA+ CCR7+, CD3+ TCRab+CD4+ CD8- CD45RO- CD45RA+ CD27+, CD3+ TCRab+ CD8+ CD4- CD45RO- CD45RA+ CD27+, CD34 + CD7 + CD1a + Cells, CD34+CD5+CD7+, CD34+CD5+CD7-,

[0052] Natural killer T cells, regulatory T cells, antigen-specific T cells, intraepithelial lymphocyte T cells, or the following:

[0053] CD45+, CD11b+, CD11b-, CD15+, CD15-, CD24+, CD24-, CD114+, CD114-, CD182+, CD182-, CD4+, CD4-, CD14+, CD14-, CD11a+, CD11a-, CD91+, CD91-, CD16+, CD16-, CD3+, CD3-, CD25+, CD25-, F oxp3+, Fox3p-, CD8+, CD8-, CD19+, CD19-, CD20+, CD20-, CD24+, CD24, CD38+, CD38-, CD22+, CD22-, CD61+, CD61-, CD16+, CD16-, CD56+, CD56-, CD31+, CD31-, CD30+, CD30-, CD38+, and / or CD38-

[0054] The cell can be a mature, conventional cell, such as a mature, conventional T cell having a CD4+CD8-(SP4) or CD4-CD8ab phenotype. The cell can also be a NK cell, an iNKT cell, a Treg cell, a TCRgd T cell, an innate immune cell, or a cell expressing CD8aa.

[0055] Genetic modification can also include the introduction of selectable markers or screenable markers that facilitate in vitro or in vivo selection or screening or imaging. In particular, in vivo imaging agents or suicide genes can be exogenously expressed or added to starting cells or daughter cells. The method can involve image-guided adoptive cell therapy.

[0056] The terms "subject" and "patient" are used interchangeably and may refer to a human subject. A subject may be limited to a mammalian subject. A subject may also be a mouse, rat, pig, horse, non-human primate, cat, dog, cow, etc.

[0057] Throughout this application, the term "about" is used according to its plain and ordinary meaning in the arts of cellular and molecular biology to indicate that a value includes the standard deviation of error for the device or method employed to determine the value.

[0058] When used in conjunction with the term "comprising", the use of a noun without a quantifier may mean "one", but it also complies with the meaning of "one or more", "at least one", and "one or more than one".

[0059] As used herein, the terms "or / or" and "and / or / or" are used to describe multiple components that are combined or exclusive of each other. For example, "x, y, and / or z" may refer to "x" alone, "y" alone, "z" alone, "x, y, and z", "(x and y) or z", "x or (y and z)", or "x or y or z". It is specifically contemplated that x, y, or z may be specifically excluded from an embodiment or aspect.

[0060] The words "comprising" (and any variations thereof), "having" (and any variations thereof), "including" (and any variations thereof), "characterized by" (and any variations thereof), or "containing" (and any variations thereof) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0061] The compositions and methods of use thereof may "comprise", "consist essentially of", or "consist of" any ingredient or step disclosed throughout this specification. The phrase "consisting of" excludes any unspecified element, step, or ingredient. The phrase "consisting essentially of" limits the scope of the subject matter to the specified substances or steps and those that do not materially affect its basic and novel characteristics. It is contemplated that some embodiments and aspects described in the context of the term "comprising", may also be implemented in the context of the term "consisting of" or "consisting essentially of".

[0062] Any method in the context of a therapeutic, diagnostic, or physiological purpose or effect may also be described in "use" claim language, such as "the use of any compound, composition, or agent discussed herein to achieve or carry out said therapeutic, diagnostic, or physiological purpose or effect."

[0063] The purposes of one or more sequences or compositions can be used based on any method described herein. Other embodiments and aspects are discussed throughout the application. Any embodiment discussed about an aspect of the present disclosure is equally applicable to other aspects of the present disclosure, and vice versa. In addition, any composition of the present invention can be used for any method of the present invention, and any method of the present invention can be used to produce or utilize any composition of the present invention. Some aspects of the embodiment set forth in the examples are also embodiments that can be implemented in the context of some embodiments discussed in other places in different embodiments or in other places in the application (e.g., in summary of the invention, specific embodiments, claims and accompanying drawings).

[0064] Other objects, features and advantages of the present invention will become apparent from the following detailed description. However, it should be understood that although the detailed description and specific examples indicate some specific embodiments of the present invention, they are given by way of example only, because various changes and modifications within the spirit and scope of the present invention will become apparent to those skilled in the art based on the detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0065] The following drawings constitute part of this specification and are included to further demonstrate certain aspects of the present disclosure. The present disclosure may be better understood by referring to one or more of these drawings in combination with the detailed description of specific embodiments shown herein.

[0066] Figure 1A to B: An example of stage-specific expression after the overall transgenic knock-in method is used to achieve endogenous gene expression. (A) Using CRISPR / Cas9-mediated homology-directed repair, the biologically active transgene (i.e., reporter, chimeric antigen receptor) is inserted into the framework of the 3'untranslated region of the endogenous gene. In this process, the stop codon is replaced by a serine-arginine linker and a self-cleaving peptide sequence (P2A). This method allows the expression of the inserted transgene to be regulated by an endogenous promoter, and the translation of the endogenous gene remains unchanged. In addition, the insertion of a puromycin resistance gene cassette helps to enrich the edited pluripotent stem cells. (B) After CRE-TAT treatment at the iPSC stage, the puromycin resistance cassette can be removed, leaving a single loxp3 sequence.

[0067] Figure 2ATo B: High constitutive lentiviral vector-mediated CAR19 expression blocks normal differentiation of pluripotent stem cells. H1 hESCs were transduced with a lentiviral vector co-expressing CAR19LH and the reporter eGFP. Cells with high eGFP expression were selected and cloned. Clone 2 was differentiated in artificial thymic organoids (ATO) and analyzed at weeks 2, 3, 4, and 5. Abnormal differentiation was manifested as low frequencies of (A) CD3+TCRab+ cells and SP4 and SP8, and (B) major populations of CD4-CD8-"DN" cells.

[0068] Figure 3 : Illustration of stage-specific expression of exogenous genes inserted downstream of GZMA. CRISPR / Cas9 editing was performed in the H1 hESC line to generate a GZMA reporter cell line. In undifferentiated H1 hESCs, mCitrine was knocked in (KI) downstream of the endogenous GZMA gene, and the cells were cloned: The clones were then differentiated in ATO and analyzed at weeks 1, 3, and 5 to assess the expression of the reporter in specific T cell populations. Shown is the analysis of heterozygous clones. Mcitrine is expressed in 23% of SP8 and 2% of SP4, but not in the precursor stages ISP4 or DP.

[0069] Figure 4A To B: It shows that delaying the initiation of CAR19 expression allows normal T cell differentiation. The expression of the exogenous gene CAR19LH in mature T cells is achieved by inserting downstream of the endogenous GZMA. Homozygous clones are generated by knocking CAR19LH into undifferentiated cells from the T-iPSC line "n11" at the GZMA locus. The edited T-iPSC clones are then differentiated in the ATO system. The figure shows (3a) DP (week 1) and normal differentiation of SP8 cells and SP4 cells (weeks 3 and 4). (3b) At week 4, 67% of SP8 cells and 6% of SP4 cells expressed CAR.

[0070] Figure 5A to D. (A) Schematic diagram of the scheme for producing mature T cells from hPSC. All genetic modifications using lentiviral transduction or CRISPR / Cas9 are performed at the pluripotent stage. (B) Schematic diagram of a lentiviral vector expressing a second-generation anti-CD19CAR (CAR19) under a constitutive ubiquitin promoter. (C) Representative flow cytometry diagrams of hPSC ATOs transduced with WT and CAR19 lentiviruses. Transduced ATOs show a significant loss of DP T cells and enrichment of DNs. (D) Further analysis of DNs in ATOs transduced with CAR19 lentiviruses shows an upregulation of type 2 innate lymphocyte (ILC2) markers. All FACS graphs are gated on CD45+CD56-.

[0071] Fig. 6A To B. (A) Table summarizing the candidate genes selected and expression levels (RPKM) at each stage of T cell differentiation. This analysis identified candidate genes that showed zero to low expression from PSC to late DP, but high expression in both SP8 and SP4. (B) T cell differentiation kinetics of GZMA-mCitrine reporter PSC. The expression of the reporter was driven using the endogenous GZMA promoter. Expression of the reporter mCitrine was only present in mature SPs, not in the early T cell stage.

[0072] Figure 7A to D. (A) Schematic diagram of donor template design for achieving specific CRISPR / Cas9-mediated HDR to knock in CAR19 into the 3' untranslated region (UTR) of the GZMA locus. (B) Representative flow cytometry of T cell differentiation of GZMA-CAR19LHT-iPSC. (C) Flow cytometry of ATO-derived T cells at week 5. (D) Representative flow cytometry of in vitro co-culture assays using GZMA-CAR19LH T-iPSC-derived T cells.

[0073] 8A-B: RAG1 / RAG2 double knockout (DKO) arrests T cell development at the double-positive stage of T cell development.

[0074] 9A-B: Constitutive exogenous TCR expression rescues RAG1 / 2DKO hPSC T cell differentiation.

[0075] Fig.10 : RNA sequencing data.

[0076] Fig.11ATo B. (A) FACS analysis of GZMA-CAR19LH T-iPSC after TRAC destruction compared to the analyzed WT T-iPSC and the control ATO with only TRAC destruction at weeks 2, 4, and 5. Throughout the differentiation of T-iPSC, TRAC destruction prevented the surface expression of TCRαβ and the expression of CD3. (B) FACS analysis of T cell development of GZMA-CAR19LH T-iPSC ATO with TRAC destruction compared to the analyzed wild type and the control with only TRAC destruction at weeks 2, 4, and 5. Due to the lack of TCRαβ and CD3 expression, T cells cannot be positively selected, and development stops at the DP stage. (C) Intracellular staining of GZMA showed that at week 5, there was no GZMA expression in both TRAC-destroyed DP T cells. Mature SP8 T cells generated from wildtype (WT) controls were used to generate isotypes and positive controls. (D) FACS analysis of CD19 CAR expression at week 5. Because DP T cells do not express GZMA, they do not express CAR.

[0077] Fig. 12A To C. (A) FACS analysis of RAG1-mCitrine T-iPSC ATOs at each stage of T cell development. The mCitrine fluorescent reporter protein was knocked into the 3'UTR of RAG1. Reporter expression was analyzed at weeks 1, 2, and 7 to capture the DN, ISP4, DP, and SP T cell stages. Before T cell commitment (i.e., T-iPSC, hEMP), there was no mCitrine expression (data not shown), but mCitrine expression increased from the DN to DP stage. After positive selection, there was no reporter expression at all in mature, initial SP8 and SP4 T cells. (B) Histogram and quantification of mCitrine reporter expression from RAG1-mCitrine T-iPSC ATOs. Reporter expression is only present in the DN to DP stage, but not before T cell commitment (i.e., T-iSPC and hEMP) and after positive selection (i.e., SP8 and SP4 T cells). (C) Analysis of RAG1-mCitrine SP8 and SP4 T cells after ex vivo culture and artificial stimulation. RAG1-mCitrine T-iPSC ATO disaggregated at week 7. SP8 and SP4 T cells were cultured with IL-2 and IL-7, and subsets were maximally stimulated with PMA / PTI to measure any possible aberrant RAG1 expression after activation. Under both conditions, no expression of mCitrine was detected in SP8 and SP4.

[0078] Fig.13Ato D. FACS analysis of TCRαβ and CD3 of TRAC-disrupted RAG1-NYESO TCR T-iPSC ATOs compared to analyzed wild-type and TRAC-disrupted controls at weeks 2, 4, and 6. The exogenous NYESO (1G4) TCR was knocked into the 3'UTR of RAG1. As a result, the exogenous TCR expression accurately reflected the expression profile of RAG1. Over the course of 6 weeks, as developing T cells achieve positive selection, the NYESO TCR is downregulated, resulting in the generation of SP8 and SP4 T cells that do not express TCRαβ or CD3 (see also Fig. 13B ). (B) FACS analysis of T cell development derived from the analyzed TRAC-disrupted RAG1-NYESO TCR T-iPSC ATOs at weeks 2, 4, and 6. At week 6, approximately 40% of T cells achieved positive selection and subsequently downregulated TCRαβ and CD3. Mature SP8 and SP4 T cells were only present in the TCRαβ-CD3- population, while the remaining TCRαβ+CD3+ cells were arrested at the DP stage. (C) Analysis of TCRαβ expression of the analyzed TRAC-disrupted RAG1-NYESO TCR T-iPSC ATOs at week 6. During development, TCRαβ expression is lost as cells achieve positive selection. (D) Characterization of TCR-SP8 T cells generated from TRAC-disrupted RAG1-NYESO TCR T-iPSC ATOs at week 6 compared to wild-type controls. The resulting TCRαβ-CD3-SP8 T cells robustly expressed CD45RA+ and CD62L+, and were CD8α+ / CD8β+.

[0079] Fig.14ATo E. (A) FACS analysis of TRAC-disrupted RAG1-NYESO TCR, GZMA-CAR19LH T-iPSC ATO analyzed at weeks 2 and 6. These three genetic modifications achieve the following: (1) TRAC disruption prevents endogenous TCR expression, (2) RAG1-NYESO TCR provides transient TCR expression to achieve positive selection and subsequent loss, (3) the resulting mature SP8 T cells then express CAR through the GZMA promoter. The combination of these strategies ultimately led to the robust generation of TCRαβ-CD3-SP8CAR T cells. (B) Characterization of TCRαβ-CD3-SP CAR T cells generated by ATO at week 6. Mature T cells robustly express mature, conventional and naive T cell markers as well as CD19 CAR. (C) Functional characterization of TCRαβ-CD3-SP8CAR T cells. T cells were isolated from ATO at week 6 and co-cultured with antigen-positive (CD19+K562) or negative (K562 WT) aAPC to evaluate antigen-specific responses. In response to antigens, CART cells upregulate activation markers CD25 and 4-1BB (CD137). (D) In ​​addition, T cells express a variety of cytokines and degranulation markers CD107a. All figures are gated on CD45+CD7+CD8+. (E) Characterization of TCRαβ-CD3-SP8 CAR T cells, which are produced using the same triple gene knock-in strategy but express different CAR constructs. The first T-iPSC line expresses CD19 CAR via GZMA, which includes a long hinge spacer (LH) and a CD28 costimulatory domain (CD28CS). The second T-iPSC line expresses CD19 CAR via GZMA without a long spacer (SH=short hinge) and a 4-1BB costimulatory domain (41BB). Both lines were able to generate TCRαβ-CD3-SP8 CART cells that expressed similar levels of mature, conventional, and naive T cell markers.

[0080] Description of Illustrative Embodiments

[0081] I. Introduction

[0082] The present disclosure provides a solution to the problem of expressing transgenes (such as FOXP3 and CAR) that are harmful to normal T cell differentiation by limiting exogenous gene expression to mature T cells. The inventors identified Granzyme A (GZMA) as an endogenous gene with tightly regulated stage-specific expression during T cell differentiation in vitro and in the normal thymus. They inserted a reporter gene downstream of GZMA into pluripotent stem cells (PSCs) to show that expression during differentiation is highly restricted to mature CD8+CD3+TCRab+CD4-(SP8)T cells and mature CD4+CD3+TCRab+CD8-(SP4)T cells ( Figure 3 ). The inventors also inserted the CAR19 construct into the GZMA locus of PSC and demonstrated normal differentiation of mature T cells expressing high levels of CAR constructs (Fig. 4). Therefore, the method can be applied to in vitro differentiation techniques using any type of pluripotent or multipotent cells or lymphoid cells as precursors of mature T cells described herein and known in the art.

[0083] II. Definitions

[0084] The term "transgene" refers to a gene that is transferred into a cell in vitro (i.e., by gene transfer / transduction / transfection technology). A transgene can be a gene that is not naturally expressed in a cell, such as a CAR or an engineered TCR.

[0085] The term "chimeric antigen receptor" or "CAR" refers to a modified receptor that is grafted to any specific immune effector cell. These receptors are used to graft the specificity of monoclonal antibodies to T cells; wherein the transfer of its coding sequence is promoted by retroviral or lentiviral vectors. The receptor is called a chimera because it is composed of parts from different sources. The most common form of these molecules is a fusion of a single-chain variable fragment (scFv) derived from a monoclonal antibody fused to a CD3-ζ transmembrane and internal domain, a CD28 or 41BB intracellular domain, or a combination thereof. Such a molecule allows the signal to be transmitted in response to scFv recognition of its target. An example of such a construct is 14g2a-ζ, which is a fusion of scFv derived from a hybridoma 14g2a (which recognizes disialoganglioside GD2). When T cells express the molecule (usually achieved by oncogenic retroviral vector transduction), they recognize and kill target cells (e.g., neuroblastoma cells) expressing GD2. To target malignant B cells, researchers use chimeric immune receptors specific for the B lineage molecule CD19 to redirect the specificity of T cells. The variable parts of the immunoglobulin heavy and light chains are fused through a flexible linker to form an scFv. The scFv is preceded by a signal peptide to guide the nascent protein to the endoplasmic reticulum and subsequently surface expressed (which is cleaved). The flexible spacer allows the scFv to be oriented in different directions to achieve antigen binding. The transmembrane domain is a typical hydrophobic alpha helix, which is usually derived from the original molecule of the signal transduction endodomain that protrudes into the cell and transmits the desired signal.

[0086] The term "antigen" refers to any substance to which the immune system produces antibodies or T cells to which it responds. An antigen can be a peptide of 5 to 50 amino acids in length, or at least, at most, or exactly 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 250 or 300 amino acids, or any range derivable therein.

[0087] The term "allogeneic to the recipient" is intended to refer to a cell that is not isolated from the recipient. The cell may be a cell that is not isolated from a patient. The cell may be a cell that is not isolated from a genetically matched individual (e.g., a relative with a compatible genotype).

[0088] The term "xeno-free (XF)" or "animal component-free (ACF)" or "animal-free" when used in relation to culture medium, extracellular matrix or culture conditions refers to a culture medium, extracellular matrix or culture conditions that are substantially free of foreign animal-derived components. For culturing human cells, any protein from a non-human animal (e.g., mouse) is a foreign component. A xeno-free matrix can be substantially free of any non-human animal-derived components, thus excluding mouse feeder cells or Matrigel. TM Matrigel TM It is a solubilized basement membrane preparation extracted from the Engelbreth-Holm-Swarm (EHS) mouse sarcoma, a tumor rich in extracellular matrix proteins including laminin (the major component), collagen IV, heparin sulfate proteoglycans, and entactin (nidogen).

[0089] The term "defined" when used in reference to a culture medium, extracellular matrix or culture conditions refers to a culture medium, extracellular matrix or culture conditions in which the identity and amounts of substantially all components are known.

[0090] "Chemically defined medium" refers to a medium in which the chemical nature and amounts of substantially all components are known. These media are also referred to as synthetic media. Some examples of chemically defined media include TeSR TM .

[0091] As used herein, a cell is "substantially free" of certain agents or components, such as serum, signal transduction inhibitors, animal components or feeder cells, exogenous genetic elements, or vector elements, when the cell has less than 10% of the component, and is "essentially free" of certain agents or components when the cell has less than 1% of the component. However, even more desirable are cell populations in which less than 0.5% or less than 0.1% of the total cell population contain exogenous genetic elements or vector elements.

[0092] A culture, matrix, or medium is "substantially free" of certain agents or components, such as serum, signal transduction inhibitors, animal components, or feeder cells, when the culture, matrix, or medium, respectively, has levels of the agent below detectable levels using conventional detection methods known to those of ordinary skill in the art, or when no such agent has been added exogenously to the culture, matrix, or medium. A serum-free medium can be substantially free of serum.

[0093] "Peripheral blood cells" refer to the cellular components of blood, including red blood cells, white blood cells, and platelets, which are found in the circulating pool of blood.

[0094] "Hematopoietic stem cells and progenitor cells" or "hematopoietic precursor cells" refer to cells that are phenotyped in the hematopoietic lineage but are capable of further hematopoietic differentiation and include hematopoietic stem cells, multipotent hematopoietic stem cells (hematoblasts), myeloid progenitor cells, megakaryocyte progenitor cells, erythroid progenitor cells, and lymphoid progenitor cells."Hematopoietic stem cells (HSCs)" are multipotent stem cells that produce all blood cell types, including myeloid lineages (monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes / platelets, dendritic cells) and lymphoid lineages (T cells, B cells, NK cells).

[0095] "Vector" or "construct" (sometimes referred to as a gene delivery or gene transfer "vehicle") refers to a macromolecule, molecular complex, or viral particle that contains a polynucleotide to be delivered to a host cell in vitro or in vivo. A polynucleotide can be a linear or circular molecule.

[0096] A "plasmid," a common type of vector, is an extrachromosomal DNA molecule separate from the chromosomal DNA that is capable of replicating independently of the chromosomal DNA. In certain cases, it is circular and double-stranded.

[0097] "Expression construct" or "expression cassette" means a nucleic acid molecule capable of directing transcription. An expression construct comprises at least a promoter or a structure functionally equivalent to a promoter. Additional elements may also be included, such as enhancers and / or transcription termination signals.

[0098] The term "corresponding to" is used herein to mean that a polynucleotide sequence is homologous (i.e., identical, not strictly evolutionarily related) to all or a portion of a reference polynucleotide sequence, or that a polypeptide sequence is identical to a reference polypeptide sequence. In contrast, the term "complementary to" is used herein to mean that a complementary sequence is homologous to all or a portion of a reference polynucleotide sequence. For illustration, the nucleotide sequence "TATAC" corresponds to the reference sequence "TATAC" and is complementary to the reference sequence "GTATA".

[0099] A "gene," "polynucleotide," "coding region," "sequence," "segment," "fragment," or "transgene" that "encodes" a specific protein is a nucleic acid molecule that is transcribed in vitro or in vivo and optionally also translated into a gene product, such as a polypeptide, when placed under the control of appropriate regulatory sequences. The coding region may be present in the form of cDNA, genomic DNA, or RNA. When present in the form of DNA, the nucleic acid molecule may be single-stranded (i.e., the sense strand) or double-stranded. The boundaries of the coding region are determined by the start codon at the 5' (amino) terminus and the translation stop codon at the 3' (carboxyl) terminus. A gene may include, but is not limited to, cDNA from prokaryotic or eukaryotic mRNA, genomic DNA sequences from prokaryotic or eukaryotic DNA, and synthetic DNA sequences. The transcription termination sequence will generally be located 3' to the gene sequence.

[0100] The term "cell" is used herein in its broadest sense in the art, and refers to a living organism that is a structural unit of organization of a multicellular organism, is surrounded by a membrane structure that isolates it from the outside, has the ability to self-replicate, and has genetic information and a mechanism for expressing it. The cell used herein may be a naturally occurring cell or an artificially modified cell (e.g., a fused cell, a genetically modified cell, etc.).

[0101] As used herein, the term "stem cell" refers to a cell that is capable of self-replication and has pluripotency or multipotency. Generally speaking, stem cells can regenerate injured tissue. The stem cell herein can be, but is not limited to, an embryonic stem (ES) cell, an induced pluripotent stem cell, or a tissue stem cell (also referred to as a tissue-specific stem cell or a somatic stem cell).

[0102] "Embryonic stem (ES) cells" are pluripotent stem cells derived from early embryos. ES cells were first established in 1981, which have also been used for the generation of knockout mice since 1989. In 1998, human ES cells were established, which are currently becoming useful for regenerative medicine.

[0103] Unlike ES cells, tissue stem cells have limited differentiation potential. Tissue stem cells are present in specific locations in tissues and have undifferentiated intracellular structures. Therefore, the pluripotency of tissue stem cells is generally low. Tissue stem cells have a higher nuclear / cytoplasmic ratio and have fewer intracellular organelles. Most tissue stem cells have low pluripotency, long cell cycles, and proliferation capabilities that exceed individual life spans. Based on the location from which the cells originate, such as the skin system, digestive system, bone marrow system, nervous system, etc., tissue stem cells are divided into several categories. Tissue stem cells in the skin system include epidermal stem cells, hair follicle stem cells, etc. Tissue stem cells in the digestive system include pancreatic (common) stem cells, liver stem cells, etc. Tissue stem cells in the bone marrow system include hematopoietic stem cells, mesenchymal stem cells, etc. Tissue stem cells in the nervous system include neural stem cells, retinal stem cells, etc.

[0104] "Induced pluripotent stem cells" (often abbreviated as iPS cells or iPSCs) refer to a type of pluripotent stem cells artificially prepared from non-pluripotent cells (usually somatic cells) or terminally differentiated cells (such as fibroblasts, hematopoietic cells, muscle cells, neurons, epidermal cells, etc.) by introducing certain factors called reprogramming factors.

[0105] "Pluripotency" refers to stem cells that have the potential to differentiate into all cells that constitute one or more tissues or organs, or in particular any of the three germ layers: endoderm (inner stomach lining, gastrointestinal tract, lungs), mesoderm (muscle, bone, blood, urogenital) or ectoderm (epidermal tissue and nervous system). "Pluripotent stem cells" as used herein refers to cells that can differentiate into cells derived from any of the three germ layers, such as totipotent cells or direct descendants of induced pluripotent cells.

[0106] As used herein, "embryonic mesodermal progenitor cells" or "EMP cells" refer to pluripotent stem cells (PSCs), which may include hematopoietic stem cells and progenitor cells that have been committed to the mesoderm, embryonic stem cells (ESCs), and induced pluripotent stem cells (iPSCs). Commitment to the mesoderm is initiated by epithelial-to-mesenchymal transition (EMT) and mutual changes in the expression of cell surface proteins EpCAM / CD326 and NCAM / CD56. EMP cells can be further defined as EMP CD326-CD56+. EMP CD326 - CD56 + The cells can be generated by culturing PSCs in the presence of components including, but not limited to, one or more of the following: Activin A, BMP4, VEGF, FGF2, and combinations thereof.

[0107] "Operably linked" with respect to nucleic acid molecules means that two or more nucleic acid molecules (e.g., nucleic acid molecules to be transcribed, promoters, and enhancer elements) are linked in such a way as to allow transcription of the nucleic acid molecules. "Operably linked" with respect to peptide and / or polypeptide molecules means that two or more peptide and / or polypeptide molecules are linked in such a way as to produce a single polypeptide chain having at least one property of each peptide and / or polypeptide component of the fusion, i.e., a fusion polypeptide. Fusion polypeptides are particularly chimeric, i.e., consist of heterologous molecules.

[0108] III. Transgenic

[0109] The transgene, the first transgene, or the second transgene may comprise an antigen targeting molecule, such as a CAR or a TCR. Some non-limiting examples of tumor antigens that can be targeted by the CAR and / or TCR of the present disclosure include at least the following: differentiation antigens such as tyrosinase TRP-1, TRP-2, and tumor-specific multi-lineage antigens such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, pi5; overexpressed embryonic antigens such as CEA; overexpressed oncogenes and mutated tumor suppressor genes such as p53, Ras, HER-2 / neu; unique tumor antigens generated by chromosomal translocations, such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR; and viral antigens such as Epstein Barr virus antigen (EBVA) and human papillomavirus (HPV) antigens E6 and E7. Other large protein-based antigens include TSP-180, MAGE-4,

[0110] MAGE-5, MAGE-6, RAGE, NY-ESO, p185erbB2, pl80erbB-3, c-met, nm-23H1, PSA, CA19-9, CA72-4, CAM17.1, NuMa, K-ras, β-catenin, CDK4, Mum-1, p15, p16, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein β-HCG, BCA225, BTAA, CA 125, CA 15-3\CA 27.29\BCAA, CA 195, CA242, CA-50, CAM43, CD68\P1, CO-029, FGF-5, G250, Ga733\EpCAM, HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB / 70K, NY-CO-1, RCASI, SDCCAG16, TA-90\Mac-2 binding protein\cyclophilin C-associated protein, TAAL6, TAG72, TLP, TPS, GPC3, MUC16, MUC18, LMP1, EBMA-1, BARF-1, CS1, CD319, HER1, B7H6, L1CAM, IL6, and MET.

[0111] Tumor antigens also include, for example, glioma-associated antigens, carcinoembryonic antigens, antigen, CEA), EGFRvlll, IL-IIRa, IL-13Ra, EGFR, FAP, B7H3, Kit, CALX, CS-1, MUC1, BCMA, bcr-abl, HER2, b-human chorionic gonadotropin, alpha-fetoprotein (AFP), ALK, CD19, cyclin B1, lectin-reactive AFP, Fos-related antigen 1, ADRB3, thyroglobulin, EphA2, RAGE-1, RUI, RU2, SSX2, AKAP-4, LCK, OY-TESI, PAX5, SART3, CLL-1, fucosyl GM1, GloboH, MN-CAIX, EPCAM, EVT6-AML, TGS5, human telomerase reverse transcriptase, plysialic acid, PLAC1, RUI, RU2 (AS), intestinal carboxylesterase, lewisY, sLe, LY6K, mut hsp70-2, M-CSF, MYCN, RhoC, TRP-2, CYPIBI, BORIS, prostate enzymes, prostate-specific antigen (PSA), PAX3, PAP, NY-ESO-1, LAGE-la, LMP2, NCAM, p53, p53 mutants, Ras mutants, gpl00, prostein, OR51 E2, PANX3, PSMA, PSCA, Her2 / neu, hTERT, HMWMAA, HAVCR1, VEGFR2, PDGFR-β, survival protein and telomerase, legumain, HPVE6, E7, sperm protein 17, SSEA-4, tyrosinase, TARP, WT1, prostate-carcinoma tumor antigen-1 (PCTA-1), ML-IAP, MAGE, MAGE-A1.MAD-CT-1, MAD-CT-2, MelanA / MART 1, XAGE1, ELF2M, ERG (TMPRSS2 ETS fusion gene), NA17, neutrophil elastase, sarcoma translocation breakpoints, NY-BR-1, ephnnB2, CD20, CD22, CD24, CD30, CD33, CD38, CD44v6, CD97, CD171, CD179a, androgen receptor, FAP, insulin growth factor (IGF)-I, IGFII, IGF-I receptor, GD2, o-acetyl-GD2, GD3, GM3, GPRC5D, GPR20, CXORF61, folate receptor (FRa), folate receptor beta, ROR1, Flt3, TAG72, TN Ag, Tie 2, TEM1, TEM7R, CLDN6, TSHR, UPK2, mesothelin, and any combination thereof. .

[0112] Other examples of tumor cell antigens that CAR and / or TCR can target include, for example, at least 5T4, 8H9, α vβ6 integrin, BCMA, B7-H3, B7-H6, CAIX, CA9, CD19, CD20, CD22, CD30, CD33, CD38, CD44, CD44v6, CD44v7 / 8, CD70, CD123, CD138, CD171, CD133, CEA, c-Met, CSPG4, EGFR, EGFR family including ErbB2 (HER2), EGFRvIII , EGP2, EGP40, ERBB3, ERBB4, ErbB3 / 4, EPCAM, EphA2, EpCAM, folate receptor-a, FAP, FBP, fetal AchR, FRα, GD2, G250 / CAIX, GD3, GPC3, GUCY2C, HER1, HER2, ICAM-1, IL-13Rα2, IL-11Rα, Kras, Kras G12D, L1CAM, λ(Lambda), Lewis-Y, κ(Kappa), KDR, MAGE, MCSP, MET, Mesothelin, Muc1, Muc16, MUC18, NCAM, NKG2D ligand, NY-ESO-1, PRAME, PSC1, PSCA, PSMA, ROR1, SP17, Survivin, TAG72, TEM, Carcinoembryonic antigen, HMW-MAA, AFP, CA-125, ETA, Tyrosinase, MAGE, Laminin receptor, HPV E6, E7, BING-4, calcium-activated chloride channel 2, cyclin B1, 9D7, EphA3, telomerase, SAP-1, BAGE family, CAGE family, GAGE ​​family, MAGE family, SAGE family, XAGE family, NY-ESO-1 / LAGE-1, PAME, SSX-2, Melan-A / MART-1, GP100 / pmel17, TRP-1 / -2, P. polypeptide, MC1R, prostate-specific antigen, β-catenin, BRCA1 / 2, CML66, fibronectin, MART-2, TGF-βRII, WT-1 or VEGF receptor (e.g., VEGFR2). CAR can be a first generation, second generation, third generation or more generation CAR. CAR can have bispecificity to any two non-identical antigens, or it can have specificity to more than two non-identical antigens.

[0113] Some non-limiting examples of bacterial antigens that can be targeted by the CAR and / or TCR of the present disclosure include at least the following: Aeromonas aminopeptidase; Arthrobacter globiformis chimeric protein; Arthrobacter globiformis DMGO protein; A. globiformis dimethylglycine oxidase; Bacillus intein; Bacillus thermolysin; Bacillus anthracis lethal factor; Bacillus anthracis protective antigen PA63; Bacillus anthracis protective antigen; Bacillus circulans intein; B. polymyxin B peptide; Bacillus anthracis edema factor; Bacillus anthracis edema factor (S447N mutant); Bacillus anthracis protective antigen PA20; Bacillus anthracis protective antigen PA 63 (activated); B. thuringiensis Cry1Ab toxin; Borrelia OspA; Borrelia Burgdorferi NapA; Borrelia BmpA; Borrelia P41; Borrelia p45; B. burgdorferi OspC; Borrelia OspA; Borrelia P41; Borrelia P41; Borrelia B31; Borrelia P100; Borrelia DbpA; Borrelia BmpA; Borrelia DbpB; Borrelia garinii; Borrelia garinii P58; Borrelia sparmanii OspC of B. spielmanii; Borrelia burgdorferi grade 2; Brucella bovis; B. pertussis toxin; B. pertussis; B. pertussis FHA; B. pertussis 165 LPS; B. pertussis adenylate cyclase antigen; B. pertussis pertussis toxin subunit (a protomer); B. pertussis pertussis adhesin; B. pertussis fimbriae 2 / 3; B. pertussis adenylate cyclase toxin; Bordetella pertussis whole cell (Tohama I strain); B. pertussis lipopolysaccharide; Campylobacter jejuni; Candida albicans enolase; Candida albicans; Chlamydia pneumoniae (C.Pneumoniae) outer membrane protein VD3; Chlamydia pneumoniae; Chlamydia trachomatis MOMP; Chlamydia trachomatis antigen; Chlamydia trachomatis HSP70 protein; Chlamydia trachomatis outer membrane protein; Chlamydia trachomatis HSP70; Chlamydia trachomatis; Chlamydia trachomatis MOMP; Chlamydia trachomatis LGV II; Chlamydia trachomatis PGP-3D; Chlamydia trachomatis active MOMP; Chlamydia trachomatis MOMP protein; Chlamydia trachomatis active MOMP; Chlamydia trachomatis active HSP70; Chlamydia trachomatis W4 MOMP; Chlamydia trachomatis W5 MOMP; Chlamydia trachomatis MOMP, E78; Clostridium difficile difficile toxin A; Clostridium difficile binary toxin A subunit; Clostridium difficile glutamate dehydrogenase; Clostridium difficile toxin B; Clostridium botulinum BoNT-A light chain; Clostridium botulinum BoNT-B light chain; Clostridium botulinum BoNT-E light chain; Clostridium difficile toxin B, TcdB; Clostridium botulinum BoNT-F light chain; Clostridium difficile toxin B; Paraputrificum chitinase; Clostridium botulinum C3 toxin; Clostridium difficile toxin A; Clostridium difficile toxin A ( diphtheriae toxin A (RNA type 078); Clostridium difficile toxin B (RNA type 027); Clostridium difficile toxin B (RNA type 078); Corynebacterium diphtheriae diphtheria toxin; Corynebacterium diphtheriae toxin (mutation CRM197); Corynebacterium diphtheriae toxin; Escherichia coli (E.Coli) chaperone protein fimC; E. coli GroEL, HSP60; E. coli GroES, HSP10; E. coli chaperone SURA; E. coli O 158; E. coli DnaJ protein; E. coli HSP60; E. coli heat shock protein 1; E. coli Eco; E. coli AmpC; E. coli CoaA; E. coli GroL; E. coli GroS protein; E. coli DnaK; E. coli DsbA protein; HIV OmpP2; Helicobacter pylori; Helicobacter pylori outer membrane protein; Helicobacter pylori Cag antigen; Helicobacter pylori flagellin A antigen; Helicobacter pylori urease small subunit antigen; Helicobacter pylori Vac (toxin) antigen; Helicobacter pylori protein; Helicobacter pylori urease large subunit protein; Legionella pneumophila; LipL32 lipoprotein of Leptospira interrogans; Leptospira biflexa; Listeria monocytogenesmonocytogenes internalization protein; Listeria monocytogenes flagellar protein; Active listeriolysin; Mycobacterium; Mycobacterium heat shock protein 65; Mycobacterium heat shock protein 70; Mycobacterium PstS1; Mycobacterium secretory protein; Mycobacterium DESAT6; Mycobacterium heat shock protein DnaK; Mycobacterium TB Ag85A; Mycobacterium tuberculosis tuberculosis early secretory target 6 protein; Mycobacterium tuberculosis ESAT-6; Mycobacterium tuberculosis (M. tuberculosis) FbpA; Mycobacterium tuberculosis; Mycobacterium tuberculosis PPD protein; Mycobacterium tuberculosis Old tuberculin; Mycobacterium bovis (M. bovis) HSP65; Mycobacterium tuberculosis heat shock protein 70; Mycobacterium tuberculosis heat shock protein 65; Mycobacterium tuberculosis MTB heat shock protein; Mycobacterium tuberculosis MTB lipoprotein; Mycobacterium tuberculosis MTB fragment and esat6 fragment; Pseudomonas aeruginosa (P. aeruginosa) exotoxin A; Urealyticum (Urealyticum) (serovar 3); Salmonella typhi OMP; Salmonella Typhimurium; Salmonella Paratyphi paratyphi) A antigen; Salmonella paratyphi B antigen; Salmonella typhimurium antigen; Salmonella beta-lactamase; Salmonella typhi pagC antigen; Salmonella typhi flag; Salmonella typhimurium uridine phosphorylase; Salmonella typhimurium flagellin protein; Salmonella minnesota (R595) lipid A monophosphoryl; Salmonella typhimurium LPS; Salmonella enterica (S. enterica) flagellin; Salmonella minnesota (S. minnesota) LPS; Staphylococcus aureus (Staphylococcus aureus δ-hemolysin; Staphylococcus aureus protease protein; Staphylococcus protein A; Staphylococcus aureus subsp. penicillin-binding protein 2; Staphylococcus aureus staphylokinase; Staphylococcus aureus protein A; Staphylococcus aureus; Staphylococcus aureus enterotoxin type B toxoid; Staphylococcus aureus HLA; Staphylococcus aureus Hlg B; Staphylococcus aureus leukocidin-D; Staphylococcus aureus leukocidin-E; Staphylococcus aureus LukF-PV; Staphylococcus aureus LukS-PV; Staphylococcus aureus phenolic modulator peptide α3; Staphylococcus aureus toxic shock syndrome toxin-1; Streptococcus pneumoniae (S.pneumoniae cell wall polysaccharide antigen; Streptococcus pneumoniae cell wall polysaccharide antigen; Streptococcus pneumoniae β(1-4)-galactosidase; Streptomyces avidinii streptavidin; Clostridium tetani (C. tetani) tetanus toxoid antigen; Clostridium tetani TeNT light chain; Clostridium tetani toxin C fragment; Treponema pallidum (T. pallidum) treponemal membrane protein A; Treponema pallidum active p41; Treponema pallidum Treponema active p47; Treponema pallidum P17; Treponema membrane protein A; Treponema pallidum active p15; Treponema pallidum antigen; Treponema pallidum Tp0453 antigen; Treponema pallidum tpp17; Yersinia enterocolitica (O subtype: 3); Yersinia enterocolitica (serotype O: 8); Yersinia enterocolitica (serotype O: 9); Arthroderma benhamiae MEP4; Arthroderma gypseum MEP5; Arthroderma gypseum MEP3; Arthroderma gypseum VPS10; Arthroderma gypseum AMPP; Arthroderma gypseum DAPB; Arthroderma otae CHO2; Arthroderma otae SEY1; Arthroderma otae VPS10; Spirulina maxima ferredoxin; Arthrospira platensis desA; Ascaris suum ATP6; Ascaris suum ND5; Ascaris suum ND4L; Ascaris suum V-type proton ATPase; Ashbya gossypii PET8; Ashbya gossypii TIM21; and Ashbya gossypii ATG22.

[0114] Some non-limiting examples of viral antigens that can be targeted by the CAR and / or TCR of the present disclosure include at least the following: Adenovirus (ADV); ADV type 3; ADV type 5; ADV type 2 hexon; ADV type 5 hexon; ADV type 40; ADV class 2; Cytomegalovirus (CMV); CMV AD169; CMV class 2; CMV Pp28; CMV; CMVPp38; CMV Pp150; CMV glycoprotein B; CMV UL80a; CMV P38, P65, P150, P52; Coronavirus spike glycoprotein; Human SARS coronavirus nucleoprotein; Coronavirus nucleocapsid 229E protein; SARS coronavirus spike glycoprotein 2; Canine coronavirus (strain 1-71) antigen; Feline coronavirus (strain WSU 79-1146) antigen; SARS coronavirus mosaic spike glycoprotein 2; SARS M protein; SARS nucleocapsid; SARS spike protein; SARS envelope protein; SARS S1; SARS S2; SARS core protein; SARS matrix protein; Coxsackievirus (B5); Coxsackievirus (B1); Coxsackievirus (B6); Coxsackievirus B1 (Tucson strain) VP1; Coxsackievirus B6 protein (Schmidt strain); Dengue virus (DENV); dengue virus subtype-2 envelope protein; DENV; DENV type 2 premembrane; DENV type 2; DENV type 1 capsid; DENV type 2 capsid; DENV type 3 capsid; DENV type 4 capsid; DENV type 4 PreM / M; DENV type 1 PreM / M; DENV type 2 PreM / M; DENV type 3 PreM / M; DENV envelope protein 1; DENV type 1 envelope protein; DENV type 2 envelope protein; DENV type 3 envelope protein; DENV Type 4 envelope protein; Epstein-Barr Virus (EBV); EBV NA1; EBV mosaic EBNA1 protein; EBV; EBV type 1; EBVpg350 / 220; EBV p18; EBV P23; EBV early antigen D; EBV type 1 nuclear antigen; EBV BFRF3; EBV BMRF1; EBVEBNA1; EBV early antigen; Enterovirus 71 (EV71); EV71 VP1; EV71 VP0; Enterovirus A CoxA16 protein; Enterovirus D EV70 protein; Ebola Zaire nucleoprotein; Ebola-Z nucleoprotein; Ebola-S nucleoprotein; Ebola-Z glycoprotein; Ebola-Z VP40; Ebola virus-like particle; Ebola virus glycoprotein; Ebola virus VP40 matrix protein;Reston Ebolavirus glycoprotein; Ebola NP antigen; Ebola GP (Sudan-Nakisamata); Ebola GP (Zaire); Hepatitis A (HAV); HAV antigen; HAV P3C; HAV P2C-P3A; HAV VP2-VP4; HAV VP3; HAV VP1-P2A; HAV P2C; HAV P2C-P3B; HAV P2C-P3A; HAV P3C; HAV VP1; HAV VP3; HAV VP4; Hepatitis B (HBV); HBV surface antigen; HBV core antigen; HBV PreS1 / S2; HBV E antigen; HBV type 3 PreS1; HBV type 2 PreS2; HBV type 3 PreS2; HBV X protein; HBV surface antigen preS2; HBV PreS1; HBV PreS2; Human Cytomegalovirus (HCMV); HCMV interleukin 10; Hepatitis C (HCV); HCV multiple antigens; HCV nonstructural protein 3-3; HCV type 1 core + NS3 antigen (Cap); HCV type 1 core + NS3 antigen (Det); HCV core, nonstructural protein 3, nonstructural protein 4, nonstructural protein 5; HCV type 2 nonstructural protein 5; HCV type 1 core antigen; HCV type 3 core antigen; HCV type 5 core antigen; HCV type 6 core antigen; HCV type 1a nonstructural protein 5; HCV type 1a nonstructural protein 3; HCV type 1a nonstructural protein 5; HCV nucleocapsid, nonstructural protein 3, nonstructural protein 4, nonstructural protein 5; Hepatitis D (HDV); Hepatitis E (HEV); HEV ORF3; HEV ORF2, ORF3; HEV mosaic ORF2, ORF3; HEV mosaic-S ORF2, ORF3; HEV mosaic protein; HEV mosaic-S protein; HEV ORF2; Human immunodeficiency virus (HIV); HIV-O antigen; HIV-1IIIB gp120 protein; HIV-1IIIBgp160 protein; HIV-1 451gp120 protein; HIV-1 451gp160 protein; HIV-1IIIB p24 protein; HIV-1gp120 and gp41 chimeric antigen; HIV-1Gp160 protein; HIV-1Gag polyprotein Pr55; HIV-1p17 protein; HIV-1p24 protein; Human papillomavirus (HPV);HPV type 16; HPV16 E6 protein; HPV16 E7 protein; HPV18 E6 protein; HPV18 E7 protein; HPV type 18 L1 protein; HPV type 2, HPV type 3; HPV type 18; HPV type 11; HPV type 16 L1 protein; HPV type 33 L1 protein; HPV type 6 L1 protein; HPV type 16 L1 protein; Herpes simplex virus (HSV); HSV type 1; HSV-2 gG peptide; HSV8 ORF8 and ORF65 proteins; HSV type 1 glycoprotein; HSV type 2 glycoprotein; HSV mosaic protein; HSV type 2; HSV type 1 glycoprotein D; HSV type 1 glycoprotein G; HSV type 2 glycoprotein G; HSV glycoprotein D; HSV type 1 glycoprotein G1; HSV type 1 MacIntyre; HSV Type 1 Class III antigen; Human T-lymphotropic virus (HTLV); HTLV type 1, 2; HTLV gp21 antigen; HTLV type 1 glycoprotein 21; HTLV type 1 envelope protein; HTLV type 1 glycoprotein 46; HTLV type 1 mosaic; HTLV type 1 p24 protein; HTLV glycoprotein 46 mosaic protein; HTLV mosaic protein; Human T-lymphotropic virus type I gp21+gp46 chimeric antigen; Human T-lymphotropic virus type II gp21+gp46 chimeric antigen; Human T-lymphotropic virus type I and II chimeric antigen; Influenza A (IAV); IAV H2N2 nucleoprotein protein; Recombinant IAV H2N2 hemagglutinin protein; IAV H3N2 hemagglutinin protein; Recombinant IAV H3N2 neuraminidase; IAV HIN1 Class II antigen; IAV H3N2 matrix protein 1; IAV H4N4 hemagglutinin protein; IAV H4N6 hemagglutinin protein; recombinant IAV H4N8 hemagglutinin protein; influenza B virus (IBV); influenza B virus antigen; IBV hemagglutinin; IBV hemagglutinin 1; IBV matrix protein 1; IBV nucleoprotein; Japanese encephalitis (JEV); JEV core protein; JEV PreM / M; MMLV leukemia virus reverse transcriptase; Measles virus (MeV); Measles virus C protein; Measles virus large polymerase; Measles virus mosaic protein; MeV hemagglutinin mosaic; MeV large polymerase; MeV polymerase; MeV hemagglutinin fusion protein; MeV nucleocapsid; Measles grade 2; Molluscum contagiosum virus (MCV); MCV MC148 protein; MCV type 2 chemokine-like protein; Orf virus VEGFE; Parvovirus VP2 VLP; Parvovirus VP2; Parvovirus VP1; Parvovirus PepD; Parvovirus Pep C;Parvovirus Pep B; Parvovirus Pep A; Parvovirus NS1; Parvovirus B19 VLP VP2; Parvovirus B19 VP2; Parvovirus B19 nonstructural protein; Parvovirus B19, VP1, VP2; Parvovirus B19; Parvovirus B19 VLP VP1 / VP2 cocapsid protein; Parvovirus B19 VP1 / 2; Parvovirus B19 VP1; Rabies Virus (RV); Rabies Virus Glycoprotein; RV M2; RV (strain MRV) nucleoprotein; RV nucleoprotein; Respiratory Syncytial Virus (RSV); Respiratory Syncytial Virus (long strain) antigen; Yersinia enterocolitica G protein; RSV Src; RSV Src kinase; RSV glycoprotein; RSV glycoprotein G; RSV nucleoprotein; Rift Valley Fever Virus (Rift Valley Fever Virus) Virus, RVFV); RVFV glycoprotein; RVFV nucleoprotein; RVFV nonstructural protein; Rubella virus (RuV); RuV IV grade; RuV envelope protein 1; RuV 2 grade; RuV K1S; RuV K2S; RuV nucleocapsid; RuV capsid; RuV E1 protein; RuV E2 protein; RuV E1 mosaic; RuV capsid C; Rotavirus 3 grade; Rotavirus; Tick-Borne Encephalitis Virus (TBEV); TBEV gE, TBEV NE protein; TBEV gE, TBEVNE, TBEV GE, TBEV CE proteins; TBEV glycoprotein E; TBEV PreM; TBEV core antigen; TBEV nonstructural protein 3; TBEV E peptide; Tick-Borne Encephalitis Virus gE protein; Simian Immunodeficiency Virus (SIV); SIV glycoprotein 120; SIV Gag; SIV P27; SIV Pol protein; SIV type 1 Nef protein; Tobacco Etch Virus (TEV); TEV N1a protein; TEV core protein; TEV PreM / M; TEV protease protein; EN-TEV active EN-TEV protease; Varicella Zoster Virus (VZV); VZV glycoprotein E; VZV ORF9; VZV ORF26; VZV glycoprotein; VZV grade 2; Smallpox virus variola CRMB; West Nile Virus (WNV); West Nile virus antigen; West Nile virus preM protein; West Nile virus NS3 protease protein; WNV envelope protein; WNV PreM; WNV E antigen;WNV nonstructural protein 1; WNV E protein peptide; WNV E protein blocking peptide; WNV M protein blocking peptide; WNV preM blocking peptide; Zika virus; Zika virus envelope protein; Zika virus NS1 protein; Zika virus antigen; Zika virus H / PF / 2013; Zika capsid; Zika E (domain III); Zika Pre-M; Chikungunya virus (CHIKV); Chikungunya virus gp E1; Chikungunya virus gp E2; Chikungunya virus gp VLP; CHIKV E1 protein; Chikungunya E1 antigen; Chikungunya E2 antigen; Chikungunya E1 E2 complex antigen; CHIKV capsid protein; CHIKV envelope antigen 2; HTNV N protein; FIV core antigen; Astrovirus type 1; hMPV; Mumps virus; HRV 3C protease; CDV HA; CDV matrix protein; Huaiyangshan virus nucleocapsid; Schmallenberg virus nucleocapsid; CDV nucleocapsid; MMTV Pr77; virus MIP MIP-I; virus MIP MIP-II; VACV Fc chimera; CCI Fc chimera; Phytolacca acinosa antiviral protein; Echovirus type 9; MuV; African Swine Fever Virus (ASFV); African Swine Fever Virus p30 protein; and African Swine Fever Virus p54 protein. ;

[0115] IV. Cells of the Disclosure

[0116] The cell of the present disclosure includes the following cells, such as pluripotent stem cells or hematopoietic stem cells or progenitor cells, and mature cells, such as mature T cells. The pluripotent stem cells comprising the transgenic, first transgenic or second transgenic located downstream of the transcription start site of the promoter region, the first promoter region or the second promoter region (such as the promoter region of the gene in Table 1) can be differentiated into mature cell types in vitro. The system and method for promoting cell differentiation into mature cells can use the stromal cells co-cultured with stem cells or progenitor cells. The cell of the present disclosure is further described below.

[0117] A. Stromal cells

[0118] Stromal cells are connective tissue cells in any organ such as the bone marrow, thymus, uterine mucosa (endometrium), prostate, and ovaries. They are cells that support the function of the parenchymal cells of that organ. Fibroblasts (also called mesenchymal stromal cells / MSCs) and pericytes are the most common types of stromal cells.

[0119] The interaction between stromal cells and tumor cells is known to play a major role in cancer growth and progression. Furthermore, the involvement of bone marrow stromal cells in human hematopoietic and inflammatory processes has been described by regulating local cytokine networks (e.g. M-CSF, LIF).

[0120] Stromal cells in the bone marrow, thymus, and other hematopoietic organs regulate the development of hematopoietic and immune cells through cell-cell ligand-receptor interactions and by releasing soluble factors including cytokines and chemokines. Stromal cells in these tissues form niches that regulate stem cell maintenance, lineage specification and commitment, and differentiation into effector cell types.

[0121] The stroma is composed of non-malignant host cells. Stromal cells also provide the extracellular matrix, on which tissue-specific cell types and, in some cases, tumors can grow.

[0122] B. Hematopoietic stem and progenitor cells

[0123] Due to the significant medical potential of hematopoietic stem cells and progenitor cells, a lot of work has been done to try to improve the method of differentiating hematopoietic progenitor cells from embryonic stem cells. In adults, the hematopoietic stem cells mainly present in the bone marrow produce a heterogeneous population of hematopoietic (CD34+) progenitor cells that differentiate into all cells of the blood system. In adults, hematopoietic progenitor cells proliferate and differentiate, causing hundreds of billions of mature blood cells to be produced every day. Hematopoietic progenitor cells are also present in umbilical cord blood. In vitro, human embryonic stem cells can be differentiated into hematopoietic progenitor cells. As described below, hematopoietic progenitor cells can also be increased or enriched from peripheral blood samples. Hematopoietic cells can be human, mouse or any other mammalian species.

[0124] Isolation of hematopoietic progenitor cells includes any selection method, including cell sorters, magnetic separation using antibody-coated magnetic beads, packed columns; affinity chromatography; cytotoxic agents linked to or used in conjunction with monoclonal antibodies, including but not limited to complement and cytotoxins; and "panning" with antibodies attached to a solid matrix such as a plate or any other convenient technique.

[0125] The use of separation or isolation techniques includes, but is not limited to, those based on differences in physics (density gradient centrifugation and counterflow centrifugal elutriation), cell surface (lectin and antibody affinity), and vital staining properties (mitochondrial binding dye rho123 and DNA binding dye Hoechst 33342). Techniques that provide precise separation include, but are not limited to, FACS (fluorescence activated cell sorting) or MACS (magnetic activated cell sorting), which can have varying degrees of complexity, such as multiple color channels, low-angle and obtuse light scattering detection channels, impedance channels, etc.

[0126] The antibodies used in the aforementioned techniques or techniques for assessing the purity of cell types (e.g., flow cytometry) can be conjugated to identifiable reagents, including but not limited to enzymes, magnetic beads, colloidal magnetic beads, haptens, fluorescent dyes, metal compounds, radioactive compounds, drugs, or haptens. Enzymes that can be conjugated to antibodies include but are not limited to alkaline phosphatase, peroxidase, urease, and beta-galactosidase. Fluorescent dyes that can be conjugated to antibodies include but are not limited to fluorescein isothiocyanate, tetramethylrhodamine isothiocyanate, phycoerythrin, allophycocyanin, and Texas Red. For other fluorescent dyes that can be conjugated to antibodies, see Handbook of Fluorescent Probes and Research Chemicals (1992-1994). Metal compounds that can be conjugated to antibodies include but are not limited to ferritin, colloidal gold, and particularly colloidal superparamagnetic beads. Haptens that can be conjugated to antibodies include but are not limited to biotin, digoxin, oxazolidinone, and nitrophenol. Radioactive compounds that can be conjugated to or incorporated into the antibody are known in the art and include, but are not limited to, technetium 99m (99TC), 125I, and amino acids containing any radionuclide including, but not limited to 14C, 3H, and 35S.

[0127] Other techniques for positive selection that allow accurate separation may be employed, such as affinity columns, etc. The method should allow the removal of non-target cell populations to a residual amount of less than about 20%, preferably less than about 5%.

[0128] Cells can be selected based on light scattering properties and the expression of their various cell surface antigens. By FACS analysis, the purified stem cells have low side scatter and low to moderate forward scatter spectra. Cytospin preparations show that the enriched stem cells have a size between mature lymphocytes and mature granulocytes.

[0129] It is also possible to enrich for CD34 before culture + A seeding population of cells may be prepared using, for example, the methods of Sutherland et al. (1992) and the methods described in U.S. Pat. No. 4,714,680. For example, the cells may be negatively selected to remove those cells expressing lineage-specific markers. The cell population may be subjected to negative selection to deplete non-CD34 +Hematopoietic cells and / or specific hematopoietic cell subsets. Negative selection can be performed based on the cell surface expression of a variety of molecules, including T cell markers such as CD2, CD4 and CD8; B cell markers such as CD10, CD19 and CD20; monocyte marker CD14; NK cell markers CD2, CD16 and CD56 or any lineage-specific markers. Negative selection can be performed based on the cell surface expression of a variety of molecules, such as a mixture of antibodies (e.g., CD2, CD3, CD11b, CD14, CD15, CD16, CD19, CD56, CD123 and CD235a) that can be used to separate other cell types (e.g., by MACS or column separation).

[0130] As used herein, lineage negative (LIN-) refers to cells lacking at least one marker associated with lineage committed cells, such as markers associated with T cells (such as CD2, 3, 4, and 8), B cells (such as CD10, 19, and 20), myeloid cells (such as CD14, 15, 16, and 33), natural killer ("NK") cells (such as CD2, 16, and 56), RBCs (such as glycophorin A), megakaryocytes (CD41), mast cells, eosinophils or basophils, or other markers such as CD38, CD71, and HLA-DR. Preferably, lineage-specific markers include, but are not limited to, at least one of CD2, CD14, CD15, CD16, CD19, CD20, CD33, CD38, HLA-DR, and CD71. More preferably, LIN- will include at least CD14 and CD15. Further purification can be achieved by positive selection, such as c-kit+ or Thy-1+. Further enrichment can be obtained by using the mitochondrial binding dye Rhodamine 123 and selecting for Rhodamine+ cells by methods known in the art. + , preferably CD34 + LIN - , most preferably CD34 + Thy-1 + LIN - A highly enriched composition can be obtained by using cells of the invention. Highly enriched populations in stem cells and methods for obtaining them are well known to those skilled in the art, see, for example, methods described in PCT / US94 / 09760, PCT / US94 / 08574, and PCT / US94 / 10501.

[0131] A variety of techniques can be used to separate cells by first removing cells of a dedicated lineage. Monoclonal antibodies are particularly useful for identifying markers associated with a specific cell lineage and / or differentiation stage. The antibodies can be attached to a solid support to allow for a rough separation. The separation technique employed should retain the viability of the fraction to be collected to the greatest extent possible. A variety of techniques of varying effectiveness can be used to obtain a "relatively rough" separation. This separation is where up to 10%, usually no more than about 5%, and preferably no more than about 1% of the total cells present are undesirable cells retained with the cell population to be retained. The specific technique employed will depend on the separation efficiency, the associated cytotoxicity, the ease and speed of operation, and the necessity of precision equipment and / or technical skills.

[0132] Selection of hematopoietic progenitor cells need not be achieved using only cell-specific markers. By using a combination of negative and positive selections, enriched cell populations can be obtained.

[0133] C. Source of blood cells

[0134] Hematopoietic stem cells (HSCs) normally reside in the bone marrow but can be forced into the blood, a process known as mobilization that is used clinically to harvest large numbers of HSCs in the peripheral blood. One mobilizing agent of choice is granulocyte colony-stimulating factor (G-CSF).

[0135] CD34 circulating in peripheral blood + Hematopoietic stem or progenitor cells can be collected by apheresis techniques in an undisturbed state or after mobilization following external administration of hematopoietic growth factors such as G-CSF. The number of stem or progenitor cells collected after mobilization is greater than the number obtained after apheresis in an undisturbed state. The source of the cell population can be an object whose cells have not been mobilized by externally applied factors, since there is no need to enrich hematopoietic stem or progenitor cells in vivo.

[0136] The cell population used for the methods described herein can be a mammalian cell, such as a human cell, a non-human primate cell, a rodent cell (e.g., a mouse or rat), a bovine cell, a sheep cell, a pig cell, a horse cell, a sheep cell, a dog cell, and a cat cell, or a mixture thereof. Non-human primate cells include rhesus monkey cells. Cells can be obtained from animals such as human patients, or they can be from cell lines. If cells are obtained from animals, they can be used as is, for example, as unseparated cells (i.e., mixed groups); they can have been first established in culture, for example, by transformation; or they can have been subjected to a preliminary purification method. For example, a cell population can be operated as follows: by positive or negative selection based on the expression of cell surface markers; by stimulation with one or more antigens in vitro or in vivo; by treatment with one or more biological regulators in vitro or in vivo; or any or all combinations of these.

[0137] Cell populations include peripheral blood mononuclear cells (PBMCs), whole blood or fractions thereof containing mixed populations, spleen cells, bone marrow cells, tumor infiltrating lymphocytes, cells obtained by leukapheresis, biopsy tissue, lymph nodes, such as lymph nodes draining from a tumor. Suitable donors include immunized donors, non-immunized (naive) donors, treated or untreated donors. A "treated" donor is a donor that has been exposed to one or more biological modulators. An untreated donor has not been exposed to one or more biological modulators.

[0138] For example, peripheral blood mononuclear cells (PBMC) can be obtained as described in accordance with methods known in the art. Some examples of such methods are discussed in Kim et al. (1992), Biswas et al. (1990), Biswas et al. (1991).

[0139] Methods for obtaining hematopoietic progenitor cells from a cell population are also well known in the art. Hematopoietic progenitor cells can be expanded using a variety of cytokines, such as hSCF, hFLT3 and / or IL-3 (Akkina et al., 1996), or CD34 can be enriched using MACS or FACS. + As described above, negative selection techniques can also be used to enrich for CD34 + cell.

[0140] Cell samples can also be obtained from a subject and subsequently enriched for desired cell types. For example, PBMC and / or CD34 +Hematopoietic cells can be isolated from blood as described herein. Cells can also be separated from other cells using a variety of techniques, such as separation and / or activation with antibodies that bind to epitopes on the cell surface of the desired cell type. Another method that can be used includes negative selection using antibodies to cell surface markers to selectively enrich for specific cell types without activating cells by receptor engagement.

[0141] Bone marrow cells can be obtained from the iliac crest, femur, tibia, spine, ribs or other medullary spaces. Bone marrow can be removed from a patient and isolated by a variety of separation and washing procedures. An exemplary procedure for isolating bone marrow cells includes the following steps: a) centrifuging a bone marrow suspension into three fractions and collecting the middle fraction or buffycoat; b) centrifuging the buffycoat fraction from step (a) in a separation fluid (usually Ficoll (trademark of Pharmacia Fine Chemicals AB)) again and collecting the middle fraction containing the bone marrow cells; and c) washing the collected fractions from step (b) to recover re-infusible bone marrow cells.

[0142] D. Pluripotent stem cells

[0143] Hematopoietic stem cells and progenitor cells can also be prepared by in vitro differentiation of pluripotent stem cells. The cells used in the methods described herein can be pluripotent stem cells (hematopoietic stem cells and progenitor cells, embryonic stem cells or induced pluripotent stem cells) directly inoculated into a cell differentiation system, which differentiates the cells into mature cells, such as artificial thymic organoids (ATOs). The cells used in the methods and compositions described herein can be derivatives or progeny of PSCs, such as, but not limited to, mesodermal progenitor cells, hematopoietic-endothelial progenitor cells or hematopoietic progenitor cells.

[0144] Pluripotent stem cells may be embryonic stem (ES) cells derived from the inner cell mass of a blastocyst. Pluripotent stem cells may be induced pluripotent stem cells obtained by reprogramming somatic cells. Pluripotent stem cells may be embryonic stem cells obtained by somatic cell nuclear transplantation.

[0145] Embryonic stem (ES) cells are pluripotent cells derived from the inner cell mass of the blastocyst. ES cells can be isolated by removing the trophectoderm of the developing embryo and then culturing the inner mass cells on a feeder layer of non-growing cells. Under appropriate conditions, colonies of proliferating, undifferentiated ES cells are produced. The colonies can be taken out, dissociated into single cells, and then re-plated on a fresh feeder layer. The re-plated cells can continue to proliferate and produce new undifferentiated ES cell colonies. Then the new colonies can be taken out, dissociated, re-plated again and allowed to grow. This process of "subculture" or "passage" of undifferentiated ES cells can be repeated many times to produce cell lines containing undifferentiated ES cells (U.S. Patent Nos. 5,843,780; 6,200,806; 7,029,913). "Primary cell culture" is a culture of cells directly obtained from the inner cell mass of a tissue such as a blastocyst. "Subculture" is any culture derived from a primary cell culture.

[0146] The method for obtaining mouse ES cells is well known. In one method, the preimplantation blastocyst from 129 strain mice is treated with mouse antiserum to remove the trophectoderm, and the inner cell mass is cultured on a feeder cell layer of chemically inactivated mouse embryonic fibroblasts in a culture medium containing fetal bovine serum. The colonies of the undifferentiated ES cells that appear are subcultured on a mouse embryonic fibroblast feeder layer in the presence of fetal bovine serum to produce ES cell groups. In some methods, mouse ES cells can be grown in the absence of a feeder layer by adding cytokine leukemia inhibitory factor (LIF) to a serum-containing culture medium (Smith, 2000). In other methods, mouse ES cells can be grown in a serum-free culture medium in the presence of bone morphogenetic protein and LIF (Ying et al., 2003).

[0147] Human ES cells can be obtained from blastocysts using previously described methods (Thomson et al., 1995; Thomson et al., 1998; Thomson and Marshall, 1998; Reubinoff et al., 2000). In one method, human blastocysts on day 5 are exposed to rabbit anti-human spleen cell antiserum and then to a 1:5 dilution of guinea pig complement to lyse trophectoderm cells. After removing the lysed trophectoderm cells from the intact inner cell mass, the inner cell mass is cultured on a feeder layer of γ-inactivated mouse embryonic fibroblasts and in the presence of fetal bovine serum. After 9 to 15 days, cell masses derived from the inner cell mass can be chemically (i.e., exposed to trypsin) or mechanically dissociated and re-plated in fresh culture medium containing fetal bovine serum and a feeder layer of mouse embryonic fibroblasts. After further proliferation, colonies with undifferentiated morphology are selected by micropipette, mechanically dissociated into clumps and re-plated (see U.S. Patent No. 6,833,269). The ES-like morphology is characterized by dense colonies with a significantly high ratio of nuclei to cytoplasm and prominent nucleoli. The resulting ES cells can be routinely passaged by simple trypsin digestion or by selecting single colonies with a micropipette. In some methods, human ES cells can be cultured in the absence of serum by culturing ES cells on a feeder layer of fibroblasts in the presence of basic fibroblast growth factor (Amit et al., 2000). In other methods, cells are cultured in a medium such as Matrigel in the presence of a "conditioned" medium containing basic fibroblast growth factor. TM Human ES cells can be cultured without a feeder cell layer by culturing them on a protein matrix containing either fibroblasts or laminin (Xu et al., 2001). The medium is previously conditioned by co-culturing with fibroblasts.

[0148] Methods for isolating rhesus monkey and common marmoset ES cells are also known (Thomson, and Marshall, 1998; Thomson et al., 1995; Thomson and Odorico, 2000).

[0149] Another source of ES cells is the ES cell line established. Various mouse cell lines and human ES cell lines are known, and their growth and proliferation conditions have been determined. For example, the mouse CGR8 cell line is established from the inner cell mass of the mouse strain 129 embryo, and the culture of CGR8 cells can be grown in the presence of LIF without a feeder layer. As another example, Thompson et al. have established human ES cell lines H1, H7, H9, H13 and H14. In addition, subclones H9.1 and H9.2 of the H9 system have been developed.

[0150] The source of ES cells can be blastocysts, cells derived from the inner cell mass of cultured blastocysts, or cells obtained from cultures of established cell lines. Therefore, as used herein, the term "ES cell" can refer to inner cell mass cells of blastocysts, ES cells obtained from inner mass cell cultures, and ES cells obtained from cultures of ES cell lines.

[0151] Induced pluripotent stem (iPS) cells are cells with the characteristics of ES cells but obtained by the reprogramming of differentiated somatic cells. Induced pluripotent stem cells have been obtained by a variety of methods. In one method, retroviral transduction is used to transfect adult skin fibroblasts (Takahashi et al., 2007) with transcription factors Oct4, Sox2, c-Myc and Klf4. The transfected cells are plated on SNL feeder cells (mouse cell fibroblast line producing LIF) in a culture medium supplemented with basic fibroblast growth factor (bFGF). After about 25 days, colonies similar to human ES cell colonies appear in the culture. Pick ES cell-like colonies and expand on feeder cells in the presence of bFGF.

[0152] Based on cell characteristics, the cells of ES cell-like colonies are induced pluripotent stem cells. Induced pluripotent stem cells are morphologically similar to human ES cells and express a variety of human ES cell markers. Moreover, when grown under conditions known to cause human ES cell differentiation, induced pluripotent stem cells differentiate accordingly. For example, induced pluripotent stem cells can differentiate into cells with neuronal structures and neuronal markers.

[0153] In another method, human fetal or neonatal fibroblasts are transfected with four genes, Oct4, Sox2, Nanog and Lin28, using lentiviral transduction (Yu et al., 2007). Colonies with human ES cell morphology become visible on days 12 to 20 after infection. The colonies are picked and expanded. The induced pluripotent stem cells constituting the colonies are morphologically similar to human ES cells, express a variety of human ES cell markers, and form teratomas with neural tissue, cartilage and intestinal epithelium after injection into mice.

[0154] Methods for preparing induced pluripotent stem cells from mice are also known (Takahashi and Yamanaka, 2006). Inducing iPS cells generally requires expression or exposure to at least one member from the Sox family and at least one member from the Oct family. Sox and Oct are considered to be the core of the transcriptional regulatory hierarchy that specifies ES cell identity. For example, Sox can be Sox-1, Sox-2, Sox-3, Sox-15 or Sox-18; Oct can be Oct-4. Other factors can improve reprogramming efficiency, such as Nanog, Lin28, Klf4 or c-Myc; a specific group of reprogramming factors can be a group comprising Sox-2, Oct-4, Nanog and optionally Lin-28; or a group comprising Sox-2, Oct4, Klf and optionally c-Myc.

[0155] Like ES cells, IPS cells have typical antigens that can be identified or confirmed by immunohistochemistry or flow cytometry using antibodies to SSEA-1, SSEA-3, and SSEA-4 (Developmental Studies Hybridoma Bank, National Institute of Child Health and Human Development, Bethesda Md.) and antibodies to TRA-1-60 and TRA-1-81 (Andrews et al., 1987). The pluripotency of embryonic stem cells can be confirmed by adding approximately 0.5 to 10 × 10 6 This was confirmed by injecting 10 cells into the hind leg muscle of 8- to 12-week-old male SCID mice. Teratomas developed that demonstrated at least one cell type from each of the three germ layers.

[0156] E. Embryonic mesodermal progenitor cells

[0157] Embryonic mesodermal progenitor (EMP) cells are cells derived from PSC. EMP cells can be prepared by culturing PSC (e.g., ESC) in a basal medium supplemented with optimal concentrations of human VEGF, bFGF, BMP4, and optionally activin A. EMP cells are a cell population that downregulates CD326 and obtains CD56 cell surface expression on the 3rd and 5th day of culture, i.e., CD326 that has undergone the EMT process. - CD56 +EMP. A marked upregulation of key transcriptional regulators of EMT, Snail-1, Snail-2, and Twist, was consistent with the downregulation of E-cadherin and CD326, as well as tight junction-related genes such as claudin, syndecan, and occludin. Based on the upregulation of vimentin, a clear reorganization of cytoskeletal proteins could be recorded. An upregulation of fibronectin expression was also seen, consistent with the role of this extracellular matrix protein in promoting cell migration. In addition, CD326 was upregulated, as demonstrated by the downregulation of CD9 and SSEA-4 (cell surface markers commonly used to identify undifferentiated cells), as well as Nanog, Sox-2, and Oct-4 / Pou5f1 (three key transcription factors associated with ESC pluripotency). - CD56 + EMPs lose their pluripotency. - CD56 + EMP expression of known mesoderm markers KDR, PDGFR-α, and CD34 continued to increase 2 weeks after mesoderm induction. - CD56 + EMPs represent a primitive population of cells that arise from PSCs through the process of epithelial to mesenchymal transition. + Cells appear before more lineage-restricted mesodermal populations, at a time when mesodermal potential is still present.

[0158] After mesoderm induction, CD326 - CD56 + Cells can be isolated by fluorescence activated cell sorting (FACS) at day 3.5 and optionally further differentiated to the mesodermal lineage under conditions of hemogenic endothelial cells, cardiac cells, or mesenchymal stem cells.

[0159] V. Transgenic

[0160] A. Antigen-targeting transgenes

[0161] The transgene, the first transgene, or the second transgene may comprise a molecule that targets an antigen, such as a CAR or TCR. Some non-limiting examples of tumor antigens that can be targeted by the CAR and / or TCR of the present disclosure include at least the following: differentiation antigens such as tyrosinase TRP-1, TRP-2, and tumor-specific multi-lineage antigens such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, pi 5; overexpressed embryonic antigens such as CEA; overexpressed oncogenes and mutated tumor suppressor genes such as p53, Ras, HER-2 / neu; unique tumor antigens generated by chromosomal translocations, such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR; and viral antigens such as Epstein Barr virus antigen (EBVA) and human papillomavirus (HPV) antigens E6 and E7. Other large protein-based antigens include TSP-180, MAGE-4,

[0162] MAGE-5, MAGE-6, RAGE, NY-ESO, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, CA19-9, CA 72-4, CAM17.1, NuMa, K-ras, β-catenin, CDK4, Mum-1, p15, p16, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein β-HCG, BCA225, BTAA, CA 125, CA 15-3\CA27.29\BCAA, CA 195, CA 242, CA-50, CAM43, CD68\P1, CO-029, FGF-5, G250, Ga733\EpCAM, HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB / 70K, NY-CO-1, RCASI SDCCAG16, TA-90\Mac-2 binding protein\cyclophilin C-associated protein, TAAL6, TAG72, TLP, TPS, GPC3, MUC16, MUC18, LMP1, EBMA-1, BARF-1, CS1, CD319, HER1, B7H6, L1CAM, IL6, and MET.

[0163] Tumor antigens also include, for example, glioma-associated antigen, carcinoembryonic antigen (CEA), EGFRvlll, IL-IIRa, IL-13Ra, EGFR, FAP, B7H3, Kit, CALX, CS-1, MUC1, BCMA, bcr-abl, HER2, b-human chorionic gonadotropin, alpha-fetoprotein (AFP), ALK, CD19, cyclin B1, lectin-reactive AFP, Fos-related antigen 1, ADRB3, thyroid Adenoglobulin, EphA2, RAGE-1, RUI, RU2, SSX2, AKAP-4, LCK, OY-TESI, PAX5, SART3, CLL-1, fucosyl GM1, GloboH, MN-CAIX, EPCAM, EVT6-AML, TGS5, human telomerase reverse transcriptase, plysialic acid, PLAC1, RUI, RU2 (AS), intestinal carboxylesterase, lewisY, sLe, LY6K, mut hsp70-2, M-CSF, MYCN, RhoC, TRP-2, CYPIBI, BORIS, prostate enzymes, prostate-specific antigen (PSA), PAX3, PAP, NY-ESO-1, LAGE-la, LMP2, NCAM, p53, p53 mutants, Ras mutants, gp100, prostein, OR51 E2, PANX3, PSMA, PSCA, Her2 / neu, hTERT, HMWMAA, HAVCR1, VEGFR2, PDGFR-β, survivin and telomerase, podocyte protein, HPV E6, E7, sperm protein 17, SSEA-4, tyrosinase, TARP, WT1, prostate cancer tumor antigen-1 (PCTA-1), ML-IAP, MAGE, MAGE-A1.MAD-CT-1, MAD-CT-2, MelanA / MART 1, XAGE1, ELF2M, ERG(TMPRSS2 ETS fusion gene), NA17, neutrophil elastase, sarcoma translocation breakpoints, NY-BR-1, ephnnB2, CD20, CD22, CD24, CD30, CD33, CD38, CD44v6, CD97, CD171, CD179a, androgen receptor, FAP, insulin growth factor (IGF)-I, IGFII, IGF-I receptor, GD2, o-acetyl-GD2, GD3, GM3, GPRC5D, GPR20, CXORF61, folate receptor (FRa), folate receptor beta, ROR1, Flt3, TAG72, TN Ag, Tie 2, TEM1, TEM7R, CLDN6, TSHR, UPK2, mesothelin, and any combination thereof.

[0164] Other examples of tumor cell antigens that CAR and / or TCR can target include, for example, at least 5T4, 8H9, α v β6 integrin, BCMA, B7-H3, B7-H6, CAIX, CA9, CD19, CD20, CD22, CD30, CD33, CD38, CD44, CD44v6, CD44v7 / 8, CD70, CD123, CD138, CD171, CD133, CEA, c-Met, CSPG4, EGFR, EGFR family including ErbB2 (HER2), EGFRvIII , EGP2, EGP40, ERBB3, ERBB4, ErbB3 / 4, EPCAM, EphA2, EpCAM, folate receptor-a, FAP, FBP, fetal AchR, FRα, GD2, G250 / CAIX, GD3, GPC3, GUCY2C, HER1, HER2, ICAM-1, IL-13Rα2, IL-11Rα, Kras, Kras G12D, L1CAM, λ(Lambda), Lewis-Y, κ(Kappa), KDR, MAGE, MCSP, MET, Mesothelin, Muc1, Muc16, MUC18, NCAM, NKG2D ligand, NY-ESO-1, PRAME, PSC1, PSCA, PSMA, ROR1, SP17, Survivin, TAG72, TEM, Carcinoembryonic antigen, HMW-MAA, AFP, CA-125, ETA, Tyrosinase, MAGE, Laminin receptor, HPV E6, E7, BING-4, calcium-activated chloride channel 2, cyclin B1, 9D7, EphA3, telomerase, SAP-1, BAGE family, CAGE family, GAGE ​​family, MAGE family, SAGE family, XAGE family, NY-ESO-1 / LAGE-1, PAME, SSX-2, Melan-A / MART-1, GP100 / pmel17, TRP-1 / -2, P. polypeptide, MC1R, prostate-specific antigen, β-catenin, BRCA1 / 2, CML66, fibronectin, MART-2, TGF-βRII, WT-1 or VEGF receptor (e.g., VEGFR2). CAR can be a first generation, second generation, third generation or more generation CAR. CAR can have bispecificity to any two non-identical antigens, or it can have specificity to more than two non-identical antigens.

[0165] Some non-limiting examples of bacterial antigens that can be targeted by the CAR and / or TCR of the present disclosure include at least the following: Aeromonas aminopeptidase; Arthrobacter sphaericus chimeric protein; Arthrobacter sphaericus DMGO protein; Arthrobacter sphaericus dimethylglycine oxidase; Bacillus intein; Bacillus thermolysin; Bacillus anthracis lethal factor; Bacillus anthracis protective antigen PA63; Bacillus anthracis protective antigen; Bacillus circulans intein; B. polymyxin B peptide; Bacillus anthracis edema factor; Bacillus anthracis edema factor (S447N mutant); Bacillus anthracis protective antigen PA20; Bacillus anthracis protective antigen PA63 (activated); Bacillus thuringiensis Cry1Ab toxin; Borrelia OspA; Borrelia burgdorferi NapA; Borrelia BmpA; Borrelia P41; Borrelia p45; Borrelia burgdorferi OspC; Borrelia burgdorferi OspA; Borrelia burgdorferi P41; Borrelia burgdorferi P41; Borrelia burgdorferi B31; Borrelia burgdorferi P100; Borrelia burgdorferi DbpA; Borrelia burgdorferi BmpA; Borrelia burgdorferi DbpB; Borrelia garinii; Borrelia garinii P58; Borrelia sparmannii OspC; Borrelia burgdorferi grade 2; Brucella bovis; Borrelia pertussis toxin; Borrelia pertussis; Borrelia pertussis FHA; Borrelia pertussis 165 LPS; Borrelia pertussis adenylate cyclase antigen; Borrelia pertussis pertussis toxin subunit (a protomer); Borrelia pertussis pertussis adhesin; Borrelia pertussis fimbriae 2 / 3; Borrelia pertussis adenylate cyclase toxin; Borrelia pertussis whole cell (Tohama I strain); Bordetella pertussis lipopolysaccharide; Campylobacter jejuni; Candida albicans enolase; Candida albicans; Chlamydia pneumoniae outer membrane protein VD3; Chlamydia pneumoniae; Chlamydia trachomatis MOMP; Chlamydia trachomatis antigen; Chlamydia trachomatis HSP70 protein; Chlamydia trachomatis outer membrane protein; Chlamydia trachomatis HSP70; Chlamydia trachomatis; Chlamydia trachomatis MOMP; Chlamydia trachomatis LGV II; Chlamydia trachomatis PGP-3D; Chlamydia trachomatis active MOMP; Chlamydia trachomatis MOMP protein; Chlamydia trachomatis active MOMP; Chlamydia trachomatis active HSP70; Chlamydia trachomatis W4 MOMP; Chlamydia trachomatis W5 MOMP; Chlamydia trachomatis MOMP, E78; Clostridium difficile toxin A; Clostridium difficile binary toxin A subunit; Clostridium difficile glutamate dehydrogenase; Clostridium difficile toxin B; Clostridium botulinum BoNT-A light chain; Clostridium botulinum BoNT-B light chain; Clostridium botulinum BoNT-E light chain; Clostridium difficile toxin B, TcdB; Clostridium botulinum BoNT-F light chain; Clostridium difficile toxin B; Clostridium putrefaciens chitinase; Clostridium botulinum C3 toxinoid; Clostridium difficile toxin A; Clostridium difficile toxin A (RNA type 027); Clostridium difficile toxin A (RNA type 078); Clostridium difficile toxin B (RNA type 027);Clostridium difficile toxin B (ribotype 078); Corynebacterium diphtheriae diphtheria toxinoid; Corynebacterium diphtheriae toxin (mutation CRM197); Corynebacterium diphtheriae toxin; Escherichia coli chaperone protein fimC; Escherichia coli GroEL, HSP60; Escherichia coli GroES, HSP10; Escherichia coli chaperone SURA; Escherichia coli O158; Escherichia coli DnaJ protein; Escherichia coli HSP60; Escherichia coli heat shock protein 1; Escherichia coli Eco; Escherichia coli AmpC; Escherichia coli CoaA; Escherichia coli GroL; Escherichia coli GroS protein; Escherichia coli DnaK; Escherichia coli DsbA protein; HIV OmpP2; Helicobacter pylori; Helicobacter pylori outer membrane protein; Helicobacter pylori Cag antigen; Helicobacter pylori flagellin A antigen; Helicobacter pylori urease small subunit antigen; Helicobacter pylori Vac (toxin) antigen; Helicobacter pylori protein; Helicobacter pylori urease large subunit protein; Legionella pneumophila; Leptospira interrogans LipL32 lipoprotein; Leptospira hyperbolicus; Listeria monocytogenes internalization protein; Listeria monocytogenes flagellin; active listeriolysin; Mycobacterium; Mycobacterium fever Shock protein 65; Mycobacterium heat shock protein 70; Mycobacterium PstS1; Mycobacterium secretory protein; Mycobacterium DESAT6; Mycobacterium heat shock protein DnaK; Mycobacterium TBAg85A; Mycobacterium tuberculosis early secretory target 6 protein; Mycobacterium tuberculosis ESAT-6; Mycobacterium tuberculosis FbpA; Mycobacterium tuberculosis; Mycobacterium tuberculosis PPD protein; Mycobacterium tuberculosis Old tuberculin; Mycobacterium bovis HSP65; Mycobacterium tuberculosis heat shock protein 70; Mycobacterium tuberculosis heat shock protein 65; Mycobacterium tuberculosis MTB heat shock protein; Mycobacterium tuberculosis MTB lipoprotein; Mycobacterium tuberculosis MTB fragment and esat6 fragment; Pseudomonas aeruginosa exotoxin A; Ureaplasma urealyticum (serovar 3); Salmonella typhi OMP; Salmonella typhimurium; Salmonella paratyphi A antigen; Salmonella paratyphi B antigen; Salmonella typhimurium antigen; Salmonella β-lactamase; Salmonella typhi pagC antigen; Salmonella typhi flag; Salmonella typhimurium uridine phosphorylase; Salmonella typhimurium Flagellin protein; Salmonella minnesota (R595) lipid A monophosphoryl; Salmonella typhimurium LPS; Salmonella enterica flagellin; Salmonella minnesota LPS; Staphylococcus aureus delta hemolysin; Staphylococcus aureus protease protein; Staphylococcus protein A; Staphylococcus aureus subsp. penicillin binding protein 2; Staphylococcus aureus staphylokinase; Staphylococcus aureus protein A; Staphylococcus aureus; Staphylococcus aureus enterotoxin type B toxoid; Staphylococcus aureus HLA; Staphylococcus aureus Hlg B; Staphylococcus aureus leukocidin-D; Staphylococcus aureus leukocidin-E; Staphylococcus aureus LukF-PV;Staphylococcus aureus LukS-PV; Staphylococcus aureus phenolic modulated peptide α3; Staphylococcus aureus toxic shock syndrome toxin-1; Streptococcus pneumoniae cell wall polysaccharide antigen; Streptococcus pneumoniae cell wall polysaccharide antigen; Streptococcus pneumoniae β(1-4)-galactosidase; Streptococcus avidin streptavidin; Clostridium tetani tetanus toxoid antigen; Clostridium tetani TeNT light chain; Clostridium tetani toxin C fragment; Treponema pallidum treponemal membrane protein A; Treponema pallidum active p41; Treponema pallidum active p47; Treponema pallidum P17; Treponema pallidum membrane protein A; Treponema pallidum active p15; Treponema pallidum antigen; Treponema pallidum Tp0453 antigen; Treponema pallidum tpp17; small intestine Yersinia enterocolitica (O subtype: 3); Yersinia enterocolitica (serotype O: 8); Yersinia enterocolitica (serotype O: 9); A. benzonitida MEP4; A. benzonitida MEP5; A. benzonitida MEP3; A. gypseum VPS10; A. gypseum AMPP; A. gypseum DAPB; A. ohta CHO2; A. ohta SEY1; A. ohta VPS10; Spirulina maxima ferredoxin; A. platensis desA; Ascaris suum ATP6; Ascaris suum ND5; Ascaris suum ND4L; Ascaris suum V-type proton ATPase; Ashbya gossypii PET8; Ashbya gossypii TIM21; and Ashbya gossypii ATG22. ;

[0166] Some non-limiting examples of viral antigens that can be targeted by the CAR and / or TCR of the present disclosure include at least the following: adenovirus (ADV); ADV type 3; ADV type 5; ADV type 2 hexon; ADV type 5 hexon; ADV type 40; ADV class 2; cytomegalovirus (CMV); CMV AD169; CMV class 2; CMV Pp28; CMV; CMV Pp38; CMV Pp150; CMV glycoprotein B; CMV UL80a; CMV P38, P65, P150, P52; coronavirus spike glycoprotein; human SARS coronavirus nucleoprotein; coronavirus nucleocapsid 229E protein; SARS coronavirus spike glycoprotein 2; canine coronavirus (strain 1-71) antigen; feline coronavirus (strain WSU 79-1146) antigen; SARS coronavirus mosaic spike glycoprotein 2; SARS M protein; SARS nucleocapsid; SARS spike protein; SARS envelope protein; SARS S1; SARS S2; SARS core protein; SARS matrix protein; Coxsackievirus (B5); Coxsackievirus (B1); Coxsackievirus (B6); Coxsackievirus B1 (Tucson strain) VP1; Coxsackievirus B6 protein (Schmidt strain); Dengue virus (DENV); Dengue virus subtype-2 envelope protein; DENV; DENV type 2 premembrane; DENV type 2; DENV type 1 capsid; DENV type 2 capsid; DENV type 3 capsid; DENV type 4 capsid; DENV type 4 PreM / M; DENV type 1 PreM / M; DENV type 2 PreM / M; DENV type 3 PreM / M; DENV envelope protein 1; DENV type 1 envelope protein; DENV type 2 envelope protein; DENV type 3 envelope protein; DENV type 4 envelope protein; Epstein-Barr virus (EBV); EBV NA1; EBV mosaic EBNA1 protein; EBV; EBV type 1; EBV pg350 / 220; EBV p18; EBV P23; EBV early antigen D; EBV type 1 nuclear antigen; EBVBFRF3; EBV BMRF1; EBV EBNA1; EBV early antigen; enterovirus 71 (EV71); EV71 VP1; EV71 VP0; enterovirus ACox A16 protein; enterovirus D EV70 protein; Ebola Zaire nucleoprotein; Ebola-Z nucleoprotein; Ebola-S nucleoprotein; Ebola-Z glycoprotein; Ebola-Z VP40; Ebola virus-like particle; Ebola virus glycoprotein; Ebola virus VP40 matrix protein; Reston Ebola virus glycoprotein; Ebola NP antigen; Ebola GP (Sudan-Nakisamata); Ebola GP (Zaire); hepatitis A (HAV); HAV antigen; HAV P3C; HAV P2C-P3A;HAV VP2-VP4; HAV VP3; HAV VP1-P2A; HAV P2C; HAV P2C-P3B; HAV P2C-P3A; HAV P3C; HAV VP1; HAV VP3; HAV VP4; Hepatitis B (HBV); HBV surface antigen; HBV core antigen; HBV PreS1 / S2; HBV E antigen; HBV type 3 PreS1; HBV type 2 PreS2; HBV type 3 PreS2; HBV X protein; HBV surface antigen preS2; HBV PreS1; HBVPreS2; Human cytomegalovirus (HCMV); HCMV interleukin 10; Hepatitis C (HCV); HCV multiple antigens; HCV nonstructural protein 3-3; HCV type 1 core + NS3 antigen (Cap); HCV Type 1 core + NS3 antigen (Det); HCV core, nonstructural protein 3, nonstructural protein 4, nonstructural protein 5; HCV type 2 nonstructural protein 5; HCV type 1 core antigen; HCV type 3 core antigen; HCV type 5 core antigen; HCV type 6 core antigen; HCV type 1a nonstructural protein 5; HCV type 1a nonstructural protein 3; HCV type 1a nonstructural protein 5; HCV nucleocapsid, nonstructural protein 3, nonstructural protein 4, nonstructural protein 5; Hepatitis D (HDV); Hepatitis E (HEV); HEV ORF3; HEV ORF2, ORF3; HEV mosaic ORF2, ORF3; HEV mosaic-S ORF2, ORF3; HEV mosaic protein; HEV mosaic-S protein; HEV ORF2; Human immunodeficiency virus (HIV); HIV-O antigen; HIV-1IIIB gp120 protein; HIV-1IIIB gp160 protein; HIV-1 451gp120 protein; HIV-1 451gp160 protein; HIV-1IIIB p24 protein; HIV-1gp120 and gp41 chimeric antigen; HIV-1Gp160 protein; HIV-1Gag polyprotein Pr55; HIV-1p17 protein; HIV-1p24 protein; human papillomavirus (HPV); HPV type 16; HPV16 E6 protein; HPV16 E7 protein; HPV18 E6 protein; HPV18 E7 protein; HPV type 18 L1 protein; HPV type 2, HPV type 3; HPV type 18; HPV type 11; HPV type 16 L1 protein; HPV type 33 L1 protein; HPV type 6 L1 protein; HPV type 16 L1 protein; herpes simplex virus (HSV); HSV type 1; HSV-2 gG peptide; HSV8 ORF8 and ORF65 proteins; HSV type 1 glycoprotein; HSV type 2 glycoprotein; HSV mosaic protein; HSV type 2; HSV type 1 glycoprotein D; HSV type 1 glycoprotein G;HSV type 2 glycoprotein G; HSV glycoprotein D; HSV type 1 glycoprotein G1; HSV type 1 MacIntyre; HSV type 1 class III antigen; human T-lymphotropic virus (HTLV); HTLV types 1 and 2; HTLV gp21 antigen; HTLV type 1 glycoprotein 21; HTLV type 1 envelope protein; HTLV type 1 glycoprotein 46; HTLV type 1 mosaic; HTLV type 1 p24 protein; HTLV glycoprotein 46 mosaic protein; HTLV mosaic protein; human T-lymphotropic virus type I gp21+gp46 chimeric antigen; human T-lymphotropic virus type II gp21+gp46 chimeric antigen; human T-lymphotropic virus type I and II chimeric antigens; influenza A (IAV); IAV H2N2 nucleoprotein protein; recombinant IAV H2N2 hemagglutinin protein; IAV H3N2 hemagglutinin protein; recombinant IAV H3N2 neuraminidase; IAV HIN1 grade 2 antigen; IAV H3N2 matrix protein 1; IAV H4N4 hemagglutinin protein; IAV H4N6 hemagglutinin protein; recombinant IAV H4N8 hemagglutinin protein; influenza B (IBV); influenza B virus antigen; IBV hemagglutinin; IBV hemagglutinin 1; IBV matrix protein 1; IBV nucleoprotein; Japanese encephalitis (JEV); JEV core protein; JEV PreM / M; MMLV leukemia virus reverse transcriptase; measles virus (MeV); measles virus C protein; measles virus large polymerase; measles virus mosaic protein; MeV hemagglutinin mosaic; MeV large polymerase; MeV polymerase; MeV hemagglutinin fusion protein; MeV nucleocapsid; measles grade 2; molluscum contagiosum virus (MCV); MCV MC148 protein; MCV type 2 chemokine-like protein; Orf virus VEGFE; parvovirus VP2 VLP; Parvovirus VP2; Parvovirus VP1; Parvovirus Pep D; Parvovirus Pep C; Parvovirus PepB; Parvovirus Pep A; Parvovirus NS1; Parvovirus B19 VLP VP2; Parvovirus B19 VP2; Parvovirus B19 nonstructural protein; Parvovirus B19, VP1, VP2; Parvovirus B19; Parvovirus B19 VLP VP1 / VP2 cocapsid protein; Parvovirus B19 VP1 / 2; Parvovirus B19 VP1; Rabies virus (RV); Rabies virus glycoprotein; RV M2; RV (strain MRV) nucleoprotein; RV nucleoprotein; Respiratory syncytial virus (RSV); Respiratory syncytial virus (long strain) antigen; Yersinia enterocolitica G protein; RSV Src; RSV Src kinase; RSV glycoprotein; RSV glycoprotein G; RSV nucleoprotein; Rift Valley fever virus (RVFV); RVFV glycoprotein; RVFV nucleoprotein; RVFV nonstructural protein; Rubella virus (RuV); RuV class IV;RuV envelope protein 1; RuV class 2; RuV K1S; RuV K2S; RuV nucleocapsid; RuV capsid; RuV E1 protein; RuV E2 protein; RuV E1 mosaic; RuV capsid C; rotavirus class 3; rotavirus; tick-borne encephalitis virus (TBEV); TBEV gE, TBEV NE protein; TBEVgE, TBEV NE, TBEV GE, TBEV CE protein; TBEV glycoprotein E; TBEV PreM; TBEV core antigen; TBEV nonstructural protein 3; TBEV E peptide; tick-borne encephalitis virus gE protein; simian immunodeficiency virus (SIV); SIV glycoprotein 120; SIV Gag; SIV P27; SIV Pol protein; SIV type 1 Nef protein; tobacco etch virus (TEV); TEV N1a protein; TEV core protein; TEV PreM / M; TEV protease protein; EN-TEV active EN-TEV protease; varicella zoster virus (VZV); VZV glycoprotein E; VZV ORF9; VZV ORF26; VZV glycoprotein; VZV grade 2; variola virus variola CRMB; West Nile virus (WNV); West Nile virus antigen; West Nile virus preM protein; West Nile virus NS3 protease protein; WNV envelope protein; WNV PreM; WNV E antigen; WNV nonstructural protein 1; WNV E protein peptide; WNV E protein blocking peptide; WNV M protein blocking peptide; WNV preM blocking peptide; Zika virus; Zika virus envelope protein; Zika virus NS1 protein; Zika virus antigen; Zika virus H / PF / 2013; Zika capsid; Zika E (domain III); Zika Pre-M; Chikungunya virus (CHIKV); Chikungunya virus gpE1; Chikungunya virus gp E2; Chikungunya virus gp VLP; CHIKV E1 protein; Chikungunya E1 antigen; Chikungunya E2 antigen; Chikungunya E1 E2 composite antigen; CHIKV capsid protein; CHIKV envelope antigen 2; HTNV N protein; FIV core antigen; type 1 astrovirus; hMPV; mumps virus; HRV 3C protease; CDV HA; CDV matrix protein; Huaiyangshan virus nucleocapsid; Schmallenberg virus nucleocapsid; CDV nucleocapsid; MMTV Pr77; viral MIP MIP-I; viral MIP MIP-II; VACV Fc chimera; CCI Fc chimera; Phytolacca acinosa pokeweed antiviral protein; Echovirus type 9; MuV; African swine fever virus (ASFV); African swine fever virus p30 protein; and African swine fever virus p54 protein. ;

[0167] The transgene, first transgene, or second transgene disclosed herein may be comprised in a nucleotide sequence encoding a polypeptide. As used herein, "protein" or "polypeptide" refers to a molecule comprising at least five amino acid residues. As used herein, the term "wild type" refers to the endogenous form of a molecule naturally occurring in an organism. The wild type form of a protein or polypeptide is employed, whereas a modified protein or polypeptide is employed to generate an immune response. The above terms are used interchangeably. "Modified protein" or "modified polypeptide" or "variant" refers to a protein or polypeptide whose chemical structure, in particular, its amino acid sequence, has been altered relative to a wild type protein or polypeptide. A modified / variant protein or polypeptide may have at least one modified activity or function (recognizing that a protein or polypeptide may have multiple activities or functions). It is particularly contemplated that a modified / variant protein or polypeptide may be altered in one activity or function, but retains a wild type activity or function in other aspects (e.g., immunogenicity).

[0168] The size of the transgene (wild type or modified) may include, but is not limited to

[0169] 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52 ,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, 1000, 1100, 1200, 1300, 1400, 1500, 1750, 2000, 2250, 2500 amino acid residues or more, and any range derivable therein, or derivatives of the corresponding amino acid sequences described or referred to herein.

[0170] BT cell receptor (TCR) and methods for generating engineered TCRs.

[0171] T cell receptor or TCR is a molecule found on the surface of T lymphocytes (T cells), which is responsible for identifying antigen fragments of peptides bound to major histocompatibility complex (MHC) molecules. TCR is composed of two different protein chains (i.e., it is a heterodimer). In 95% of T cells in humans, TCR is composed of α chain (α, also referred to as "a" herein) and β (β, also referred to as "b" herein) chain, while in 5% of T cells, TCR is composed of γ and δ (γ / δ) chain. This ratio changes in individual development and in disease states and in different species.

[0172] When TCR is engaged with antigenic peptide and MHC (peptide / MHC), T lymphocytes are activated by signal transduction, i.e., a series of biochemical events mediated by related enzymes, co-receptors, specialized adapter molecules and activated or released transcription factors. TCR is a membrane-anchored heterodimeric protein connected by disulfide bonds, which is usually composed of highly variable α (α) and β (β) chains expressed as part of a complex with constant CD3 chain molecules. The T cells expressing this receptor are referred to as α:β (or αβ or ab) T cells, but a few T cells express alternative receptors formed by variable γ chains (γ, also referred to as "g" in this article) and δ chains (δ, also referred to as "d" in this article), referred to as γδ (or gd) T cells.

[0173] Each chain consists of two extracellular domains: a variable (V) region and a constant (C) region, both of which are immunoglobulin superfamily (IgSF) domains that form antiparallel β-folds. The constant region is close to the cell membrane, followed by a transmembrane region and a short cytoplasmic tail, while the variable region binds to the peptide / MHC complex.

[0174] The variable domains of both the TCR α and β chains each have three hypervariable regions or complementarity determining regions (CDRs), while the variable region of the β chain has an additional hypervariable region (HV4) that generally does not contact antigen and is therefore not considered a CDR.

[0175] The residues are located in two regions of the TCR: at the interface of the α and β chains and in the framework region of the β chain which is thought to be close to the CD3 signaling complex. CDR3 is the primary CDR responsible for recognizing processed antigens, but CDR1 of the α chain has also been shown to interact with the N-terminal portion of antigenic peptides, while CDR1 of the β chain interacts with the C-terminal portion of the peptide. CDR2 is thought to recognize MHC. CDR4 of the β chain is not thought to be involved in antigen recognition, but has been shown to interact with superantigens. The constant domain of the TCR domain consists of a short linker sequence in which cysteine ​​residues form disulfide bonds that form the connection between the two chains.

[0176] TCR is a member of IgSF protein, which means that it can be compared with antibodies and BCR. In terms of similarity, TCR is similar to half of an antibody with heavy and light chains, except that the heavy chain does not have its crystallizable part (Fc) (note: TCRα undergoes VJ recombination in terms of individual development, so it is similar to light chain; TCRβ undergoes VDJ recombination, so it is similar to heavy chain). Therefore, TCR is similar to one of the antibody binding fragments of antibodies in terms of ontological development. The two subunits of TCR are twisted together. In view of the fact that antibodies use their Fc regions to bind to Fc receptors on natural leukocytes, TCR has docked on the cell membrane. However, due to its short cytoplasmic tail, it is not able to mediate signal transduction itself, so TCR still needs CD3 and ζ to perform signal transduction at its position, just as antibodies need to be combined with FcR to start signal transduction. In this way, the MHC-TCR-CD3 interaction of T cells is functionally similar to the Ag-Ig-FcR interaction of myeloid leukocytes and the Ag-Ig-CD79 interaction of B cells.

[0177] Exogenous TCR includes proteins that can be expressed by TCR-α and TCR-β genes. Exogenous TCR may include proteins expressed by TCR-γ and TCR-δ genes. Exogenous TCR may include proteins expressed by TCR-α and TCR-β genes, and antigen recognition receptors include proteins expressed by TCR-γ and TCR-δ genes. Exogenous TCR may include proteins expressed by TCR-γ and TCR-δ genes, and antigen recognition receptors include proteins expressed by TCR-α and TCR-β genes.

[0178] Methods for producing antigen-specific TCRs are known in the art. Methods may include, for example: 1) synthesizing known or predicted HLA-restricted peptide epitopes derived from target proteins (e.g., tumor antigens, new antigens from sequencing data, etc.); 2) presenting these to a T cell library (e.g., tumor infiltrating lymphocytes in the case of tumor ag-specific T cells) from which TCR sequences are extracted by antigen presenting cells (for amplification) or tetramers (for direct sorting); 3) selecting or screening antigen-specific T cells (e.g., FACS sorting antigen-specific T cells based on tetramer binding); 4) cloning (by RT-PCR) and sequencing TCR genes (i.e., α and β chains or γ and δ chains of TCR); cloning and sequencing can be performed at the population or single cell level; and 5) determining and analyzing TCR specificity by, for example, transducing peripheral blood T cells with these sequences and assessing their reactivity to target cells expressing associated peptide-MHC complexes to test the function of TCR clones. Reactivity is usually measured based on cytokine production (e.g., interferon γ).

[0179] C. Chimeric antigen receptors (CARs) and methods of producing CARs.

[0180] The term "chimeric antigen receptor" or "CAR" refers to an engineered receptor that has any specificity grafted onto an immune effector cell. These receptors are used to graft the specificity of a monoclonal antibody onto immune cells; transfer of their coding sequences is facilitated by retroviral or lentiviral vectors. The receptor is called chimeric because it is composed of parts from different sources.

[0181] The most common form of these molecules is a fusion of a single-chain variable fragment (scFv) derived from a monoclonal antibody fused to a CD3-ζ transmembrane and endodomain, CD28 or 41BB intracellular domain, or a combination thereof. Such a molecule allows the signal to be transmitted in response to scFv recognition of its target. An example of such a construct is 14g2a-ζ, which is a fusion of scFv (which recognizes disialoganglioside GD2) derived from hybridoma 14g2a. When immune cells express the molecule (e.g., by oncoretroviral vector transduction), it identifies and kills target cells (e.g., neuroblastoma cells) expressing GD2.

[0182] The variable parts of the immunoglobulin heavy and light chains are fused by flexible linkers to form scFv. The scFv is preceded by a signal peptide to direct the nascent protein to the endoplasmic reticulum and subsequently surface expressed (which is cleaved). The flexible spacer allows the scFv to be oriented in different directions to enable antigen binding. The transmembrane domain is a typical hydrophobic alpha helix of the original molecule that is usually derived from the signal transduction endodomain, which protrudes into the cell and transmits the desired signal.

[0183] CAR may include at least one extracellular domain and at least one intracellular domain. The extracellular domain may include a target-specific binding element (in other cases referred to as an antigen binding portion or a ligand binding portion) that specifically binds to any particular antigen of interest.

[0184] The intracellular domain or in other cases the cytoplasmic domain may comprise one or more co-stimulatory signaling regions and a zeta chain portion. The co-stimulatory signaling region refers to the portion of the CAR that comprises the intracellular domain of a co-stimulatory molecule. The co-stimulatory molecule may be a cell surface molecule other than an antigen receptor or its ligand that is required for an immune cell to effectively respond to an antigen.

[0185] 1. Signal peptide

[0186] The polypeptides of the present disclosure may include a signal peptide. A "signal peptide" refers to a peptide sequence that directs protein transport and localization within a cell (e.g., to a cell organelle such as the endoplasmic reticulum and / or the cell surface). The signal peptide can direct the nascent protein to the endoplasmic reticulum. This is essential if the receptor is to be glycosylated and anchored in the cell membrane. Typically, a signal peptide that is naturally attached to the most amino-terminal component is used (e.g., in a scFv with a light chain-linker-heavy chain orientation, the natural signal of the light chain is used).

[0187] The signal peptide may be cleaved after passing through the endoplasmic reticulum (ER), ie, is a cleavable signal peptide. A restriction site may be located at the carboxyl terminus of the signal peptide to facilitate cleavage.

[0188] 2. Antigen binding domain

[0189] The polypeptides of the present disclosure may comprise one or more antigen binding domains. An "antigen binding domain" describes a region of a polypeptide that is capable of binding to an antigen under appropriate conditions. The antigen binding domain may be based on a single chain variable fragment (scFv) of one or more antibodies (e.g., a CD20 antibody). The antigen binding domain may comprise a heavy variable (VH) region and a light variable (VL) region, wherein the VH and VL regions are located on the same polypeptide. The antigen binding domain may comprise a linker between the VH and VL regions. The linker may enable the antigen binding domain to form a desired structure for antigen binding.

[0190] The variable region of the antigen-binding domain of the polypeptide of the present disclosure can be modified by mutating the amino acid residues in the VH and / or VL CDR 1, CDR 2 and / or CDR 3 regions to improve one or more binding properties (e.g., affinity) of the antibody. The term "CDR" refers to a complementary determining region, which is based on a portion of a variable chain in an immunoglobulin (antibody) and a T cell receptor produced by a B cell and a T cell, respectively, where these molecules bind to their specific antigens. Since most sequence variations associated with immunoglobulins and T cell receptors are present in the CDRs, these regions are sometimes referred to as hypervariable regions. Mutations can be introduced by site-directed mutagenesis or PCR-mediated mutagenesis, and the effects on antibody binding or other functional properties of interest can be evaluated in appropriate in vitro or in vivo assays. Preferably, conservative modifications are introduced, and typically no more than 1, 2, 3, 4 or 5 residues in the CDR regions are changed. Mutations can be amino acid substitutions, additions or deletions.

[0191] Framework modifications may be made to antibodies to reduce immunogenicity, for example, by "backmutating" one or more framework residues to the corresponding germline sequence.

[0192] It is also contemplated that the antigen binding domains can be multispecific or multivalent by multimerizing the antigen binding domains with pairs of VH and VL regions that bind the same antigen (multivalent) or different antigens (multispecific).

[0193] The binding affinity of the antigen binding region, such as the variable region (heavy chain and / or light chain variable region) or CDR can be at least 10 - 5 M, 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 M or 10 -13 In some embodiments, the KD of an antigen binding region, such as a variable region (heavy chain and / or light chain variable region) or a CDR, may be at least 10 -5 M, 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 M or 10 -13 M (or any range derivable therein).

[0194] Binding affinity, KA or KD can be determined by methods known in the art, for example by a biosensor based on surface plasmon resonance (SRP), by a kinetic exclusion assay (KinExA), by an optical scanner based on polarization-modulated oblique-incidence reflectivity difference (OI-RD) for microarray detection, or by ELISA.

[0195] A polypeptide comprising a humanized binding region may have the same, better, or at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 104%, 106%, 106%, 108%, 109%, 110%, 115%, or 120% binding affinity and / or expression level in a host cell as compared to a polypeptide comprising a non-humanized binding region (e.g., a binding region from a mouse).

[0196] Relative to the mouse framework, the framework regions (e.g., FR1, FR2, FR3, and / or FR4) of the human framework can each or collectively have at least, at most, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 ,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99,100,101,102,103,104,105,106,107,108,109,110,111,112 2,113,114,115,116,117,118,119,120,121,122,123,124,125,126,127,128,129,130,131,132,133,134,135,136,137,138,139,140,141,142,143,144,145,146,147,148,149,150,151,152,153,154,155,156,157,158,159,160,1 , 194, 195, 196, 197, 198, 199, or 200 (or any range derivable therein) amino acid substitutions, consecutive amino acid additions, or consecutive amino acid deletions.

[0197] Relative to the human framework, the framework regions (e.g., FR1, FR2, FR3, and / or FR4) of the mouse framework can each or collectively have at least, at most, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 ,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99,100,101,102,103,104,105,106,107,108,109,110,111,112 2,113,114,115,116,117,118,119,120,121,122,123,124,125,126,127,128,129,130,131,132,133,134,135,136,137,138,139,140,141,142,143,144,145,146,147,148,149,150,151,152,153,154,155,156,157,158,159,160,1 , 194, 195, 196, 197, 198, 199, or 200 (or any range derivable therein) amino acid substitutions, consecutive amino acid additions, or consecutive amino acid deletions.

[0198] The substitution may be in the heavy or light chain variable region FR1, FR2, FR3 or FR4

[0199] 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 digits.

[0200] 3. Extracellular peptide spacer

[0201] Between the extracellular domain and the transmembrane domain of CAR, and / or between the cytoplasmic domain and the transmembrane domain of CAR, a spacer domain may be incorporated. The term "spacer domain" as used herein generally refers to any oligopeptide or polypeptide that functions to connect the transmembrane domain in a polypeptide chain to the extracellular domain or cytoplasmic domain. The extracellular spacer may connect the antigen binding domain to the transmembrane domain. The peptide spacer may be flexible enough to orient the antigen binding domain in different directions to promote antigen binding.

[0202] The spacer may comprise a hinge region from an IgG. The spacer may comprise or further comprise a CH2CH3 region and a CD3 portion of an immunoglobulin. The CH2CH3 region may have L235E / N297Q or L235D / N297Q modifications, or have at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% amino acid sequence identity with the CH2CH3 region. The spacer may be from IgG4. The extracellular spacer may comprise a hinge region.

[0203] The term "hinge" as used herein refers to a flexible polypeptide connection region (also referred to herein as a "hinge region" or "spacer region") that provides structural flexibility and separates flanking polypeptide regions, and may be composed of natural or synthetic polypeptides. A "hinge" or "spacer region" derived from an immunoglobulin (e.g., IgG1) is generally defined as extending from Glu216 of human IgG1 to Pro230, for example (Burton (1985) Molec. Immunol., 22: 161-206). By placing the first and last cysteine ​​residues that form inter-heavy chain disulfide bonds (SS) in the same position, the hinge region of other IgG isotypes can be compared with the IgG1 sequence. The hinge region may be naturally occurring or non-naturally occurring, including but not limited to the hinge region of the changes described in U.S. Patent No. 5,677,425. The hinge region may include a complete hinge region derived from an antibody of a different class or subclass from the antibody of the CH1 domain. The term "hinge" may also include other receptors derived from CD8 and providing similar functions in providing flexibility and separating flanking regions. Other alternatives include the CH2CH3 region and CD3 portion of an immunoglobulin.

[0204] The length of the extracellular spacer may be at least, at most, or exactly 4, 5, 6, 7, 8, 9, 10, 12, 15, 16, 17, 18, 19, 20, 20, 25, 30, 35, 40, 45, 50, 75, 100, 110, 119, 120, 130, 140, 150, 160, 170, 180, 190, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221 12, 213, 214, 215, 216, 217, 218, 219, 220, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 260, 270, 280, 290, 300, 325, 350, or 400

[0205] The extracellular spacer may consist of or comprise a hinge region from an immunoglobulin (e.g., IgG). Immunoglobulin hinge region amino acid sequences are known in the art; see, e.g., Tan et al. (1990) Proc. Natl. Acad. Sci. USA 87:162; and Huck et al. (1986) Nucl. Acids Res.

[0206] The length of the extracellular spacer can have an effect on the signaling activity of the CAR and / or the expansion characteristics of the CAR-T cells of the CAR signaling in response to antigen stimulation. Shorter spacers can be used, for example, less than 50, 45, 40, 30, 35, 30, 25, 20, 15, 14, 13, 12, 11 or 10 amino acids. Longer spacers, for example, at least 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 225, 226 , 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 260, 270, 280, or 290 amino acids may have the advantage of increased in vivo or in vitro amplification.

[0207] As a non-limiting example, an immunoglobulin hinge region may comprise one of the following amino acid sequences:

[0208] Table: Exemplary hinge regions

[0209]

[0210] The extracellular spacer may comprise the amino acid sequence of a human IgG1, IgG2, IgG3 or IgG4 hinge region. The extracellular spacer may also comprise one or more amino acid substitutions and / or insertions and / or deletions compared to a wild-type (naturally occurring) hinge region. For example, His229 of a human IgG1 hinge may be replaced by Tyr such that the hinge region comprises the sequence

[0211] The extracellular spacer may comprise an amino acid sequence derived from human CD8; for example, the hinge region may comprise the amino acid sequence:

[0212] or variations thereof.

[0213] The extracellular spacer may include or further include a CH2 region. An exemplary CH2 region is

[0214] The extracellular spacer may include or further include a CH3 region. An exemplary CH3 region is

[0215] When the extracellular spacer comprises multiple parts, there may be anywhere between 0 to 50 amino acids between the multiple parts. For example, between the hinge and the CH2 or CH3 region, or between the CH2 and CH3 regions (when both are present), there may be at least, at most or exactly 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45 or 50 amino acids (or any derivable range therein). The extracellular spacer may consist essentially of the hinge, CH2 and / or CH3 regions, meaning that the hinge, CH2 and / or CH3 regions are the only identifiable regions present, and all other domains or regions are excluded, but amino acids that are not part of the identifiable regions may also be present.

[0216] 4. Transmembrane domain

[0217] Regarding the transmembrane domain, CAR can be designed to include a transmembrane domain fused to the extracellular domain of CAR. The transmembrane domain can be a hydrophobic alpha helix that spans the membrane. Different transmembrane domains can lead to different receptor stabilities.

[0218] A transmembrane domain that is naturally associated with one of the domains in the CAR can be used. In some cases, the transmembrane domain can be selected or modified by amino acid substitution to avoid binding of such a domain to a transmembrane domain of the same or different surface membrane protein, thereby minimizing interactions with other members of the receptor complex.

[0219] Transmembrane domains can be derived from natural or synthetic sources. In the case where the source is natural, the domain can be derived from any membrane-bound protein or transmembrane protein. Some illustrative but non-limiting examples of the transmembrane region particularly used in the CAR construct considered here can be derived from the following (i.e., comprising at least the following transmembrane region): α, β or ζ chain of T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154. Alternatively, the transmembrane domain can be synthetic, in which case it can mainly include hydrophobic residues such as leucine and valine. The triplets of phenylalanine, tryptophan and valine can be present at each end of the synthetic transmembrane domain. Optionally, a short oligopeptide linker or polypeptide linker (e.g., a length of 2 to about 10 amino acids) can form a bond between the transmembrane domain and the cytoplasmic signaling domain of CAR. Glycine-serine doublets provide particularly suitable linkers.

[0220] The transmembrane domain is inserted between the extracellular spacer and the cytoplasmic region. The transmembrane domain can be inserted between the extracellular spacer and one or more costimulatory regions. A linker can be located between the transmembrane domain and one or more costimulatory regions.

[0221] Any transmembrane domain that provides for insertion of a polypeptide into the cell membrane of a eukaryotic (eg, mammalian) cell may be suitable for use. The transmembrane domain may be derived from CD28, CD8, CD4, CD3-ζ, CD134 or CD7.

[0222] Exemplary transmembrane domains useful in any of the compositions and methods of the present disclosure include those in the following table:

[0223] Table: Exemplary transmembrane domain sequences

[0224]

[0225] 5. Cytoplasmic domain

[0226] The cytoplasmic domain of CAR or another intracellular signaling domain is responsible for activating at least one normal effector function of the immune cell in which CAR has been placed. After antigen and / or ligand recognition, receptors aggregate and signals are transmitted to cells through the cytoplasmic region. The cytoplasmic region may include an intracellular signaling domain. The intracellular signaling domain may include a primary signaling domain and one or more costimulatory domains. The costimulatory domain described herein may be part of the cytoplasmic region.

[0227] The term "effector function" refers to a specialized function of a cell. For example, the effector function of a T cell can be a cell lytic activity or an auxiliary activity, including the secretion of cytokines. Therefore, the term "intracellular signaling domain" refers to a portion of a protein that transduces an effector function signal and directs the cell to perform a specialized function. Although the entire intracellular signaling domain can generally be used, in many cases it is not necessary to use the entire chain. Insofar as a truncated portion of an intracellular signaling domain is used, such a truncated portion can be used in place of the complete chain as long as it transduces an effector function signal. The term intracellular signaling domain therefore means any truncated portion that contains an intracellular signaling domain that is sufficient to transduce an effector function signal.

[0228] The cytoplasmic region and / or co-stimulatory region of the CAR suitable for the present disclosure include any desired signaling domain, which responds to activation by binding of an antigen to an antigen binding domain and provides different and detectable signals (e.g., increased production of one or more cytokines by cells; changes in target gene transcription; changes in protein activity; changes in cell behavior, such as cell death; cell proliferation; cell differentiation; cell survival; regulation of cell signaling responses, etc.). The cytoplasmic region may include at least one (e.g., one, two, three, four, five, six, etc.) ITAM motif as described herein. The cytoplasmic region may include a DAP10 / CD28 type signaling chain. The cytoplasmic region may include CD3-ζ, DAP10, CD28, 2B4, DNAM-1, 4-1BB, OX40, CD30, CD40, ICOS, lymphocyte function-associated antigen-1 (lymphocyte function-associated antigen-1, LFA-1), CD2, CD7, LIGHT, and NKG2C type signaling chains.

[0229] The cytoplasmic region suitable for the polypeptide of the present disclosure comprises an intracellular signaling polypeptide containing an activation motif (ITAM) based on immunoreceptor tyrosine. The ITAM motif is YX1X2(L / I), wherein X1 and X2 are independently any amino acid. In some cases, the cytoplasmic region comprises 1, 2, 3, 4 or 5 ITAM motifs. In some cases, the ITAM motif is repeated twice in the intracellular domain, wherein the first and second instances of the ITAM motif are separated from each other by 6 to 8 amino acids, such as (YX1X2(L / I))(X3)m(YX1X2(L / I)), wherein n is an integer from 6 to 8, and 6 to 8 X3 can each be any amino acid.

[0230] A suitable cytoplasmic region may be a ΓΓΑΜ motif-containing portion derived from a polypeptide comprising an ITAM motif. For example, a suitable cytoplasmic region may be an ITAM motif-containing domain from any ITAM motif-containing protein. Therefore, a suitable intracellular domain need not contain the entire sequence of the entire protein from which it is derived. Some examples of suitable ITAM motif-containing polypeptides include, but are not limited to: DAP12, DAP10, FCER1G (Fcε receptor Iγ chain); CD3D (CD3δ); CD3E (CD3ε); CD3G (CD3γ); CD3-ζ; and CD79A (antigen receptor complex-associated protein α chain).

[0231] Exemplary cytoplasmic regions are known in the art. The cytoplasmic regions shown below also provide some examples of regions that can be incorporated into the CAR of the present disclosure:

[0232] In some cases, the cytoplasmic region is derived from DAP12 (also known as TYROBP; TYRO protein tyrosine kinase binding protein; KARAP; PLOSL; DN AX-activating protein 12; KAR-associated protein; TYRO protein tyrosine kinase binding protein; killer activated receptor-associated protein; killer activated receptor-associated protein, etc.). Suitable cytoplasmic regions may include a portion of the full-length DAP12 amino acid sequence containing an ITAM motif.

[0233] The cytoplasmic region may be derived from FCER1G (also known as FCRG; Fcε receptor I gamma chain; Fc receptor gamma chain; fc-εRl-γ; fcRγ; fceRIγ; high affinity immunoglobulin epsilon receptor subunit gamma; immunoglobulin E receptor, high affinity, gamma chain, etc.). A suitable cytoplasmic region may comprise a portion of the full-length FCER1G amino acid sequence containing an ITAM motif.

[0234] The cytoplasmic region may be derived from the T cell surface glycoprotein CD3 δ chain (also known as CD3D; CD3-δ; T3D; CD3 antigen, δ subunit; CD3δ; CD3d antigen, δ polypeptide (TiT3 complex); OKT3, δ chain; T cell receptor T3 δ chain; T cell surface glycoprotein CD3 δ chain; etc.). A suitable cytoplasmic region may comprise a portion of the full-length CD3 δ amino acid sequence containing an ITAM motif.

[0235] The cytoplasmic region can be derived from the T cell surface glycoprotein CD3ε chain (also known as CD3e, T cell surface antigen T3 / Leu-4ε chain, T cell surface glycoprotein CD3ε chain, AI504783, CD3, CD3ε, T3e, etc.). Suitable cytoplasmic regions can include a portion of the ITAM motif containing the full-length CD3ε amino acid sequence.

[0236] The cytoplasmic region may be derived from the T cell surface glycoprotein CD3γ chain (also known as CD3G, T cell receptor T3γ chain, CD3-γ, T3G, γ polypeptide (TiT3 complex), etc.). A suitable cytoplasmic region may comprise a portion of the full-length CD3γ amino acid sequence containing an ITAM motif.

[0237] The cytoplasmic region can be derived from the T cell surface glycoprotein CD3ζ chain (also known as CD3Z, T cell receptor T3ζ chain, CD247, CD3-ζ, CD3H, CD3Q, T3Z, TCRZ, etc.). Suitable cytoplasmic regions can include a portion of the ITAM motif containing the full-length CD3ζ amino acid sequence.

[0238] The cytoplasmic region may be derived from CD79A (also known as B cell antigen receptor complex-associated protein α chain; CD79a antigen (immunoglobulin-associated α); MB-1 membrane glycoprotein; ig-α; membrane-bound immunoglobulin-associated protein; surface IgM-associated protein; etc.). A suitable cytoplasmic region may include a portion of the full-length CD79A amino acid sequence containing an ITAM motif.

[0239] Suitable cytoplasmic regions may comprise a CD28-type signaling chain. Other cytoplasmic regions suitable for use in CARs of the present disclosure comprise a ZAP70 polypeptide.

[0240] Specific exemplary cytoplasmic regions are known in the art and are further shown in the table below.

[0241] Table: Cytoplasmic region

[0242]

[0243]

[0244] 6. Co-stimulatory Zone

[0245] The term "costimulatory ligand" used herein includes molecules on antigen presenting cells (e.g., APC, dendritic cells, B cells, etc.), which specifically bind to associated costimulatory molecules or domains on immune effector cells, thereby providing such a signal: the signal includes but is not limited to proliferation, activation, differentiation, etc. in addition to the main signal that mediates the response of immune effector cells. Costimulatory ligands also encompass antibodies specifically bound to costimulatory molecules present in particular on immune effector cells." costimulatory molecule" refers to an associated binding partner on immune effector cells, which specifically binds to costimulatory ligands, thereby mediating a costimulatory response by immune effectors, such as but not limited to proliferation and / or activation. "Costimulatory signal" used herein refers to a signal that causes immune cell activation, proliferation, and / or key molecule upregulation or downregulation in combination with a main signal.

[0246] The term "stimulation" means a primary response induced by the binding of a stimulatory molecule to its associated ligand, thereby mediating a signal transduction event (such as but not limited to signal transduction). Stimulation can mediate changes in the expression of certain molecules. The term "stimulatory molecule" used herein means a molecule on an immune effector cell that specifically binds to an associated stimulatory ligand present on an antigen presenting cell. "Stimulatory ligand" used herein means a ligand that, when present on an antigen presenting cell (such as an APC, a dendritic cell, a B cell, etc.), can specifically bind to an associated binding partner (referred to herein as a "stimulatory molecule") on an immune effector cell, thereby mediating a primary response by an immune effector cell, including but not limited to activation, the initiation of an immune response, proliferation, etc.

[0247] Some non-limiting examples of suitable co-stimulatory regions, such as those contained in the cytoplasmic region, include, but are not limited to, polypeptides from: 4-1BB (CD137), CD28, ICOS, OX-40, BTLA, CD27, CD30, CD40, GITR, 2B4, DNAM-1, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, and HVEM.

[0248] The co-stimulatory region can have a length of at least, at most, or exactly 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, or 300 amino acids, or any range derivable therein.

[0249] The co-stimulatory region may be derived from DAP10 (also known as HCST, DAP10, KAP10, PIK3AP, hematopoietic cell signaling molecules, etc.). The co-stimulatory region may be derived from the intracellular portion of the transmembrane protein 4-1BB (also known as tumor necrosis factor receptor superfamily member 9, TNFRSF9 (tumor necrosis factor receptor superfamily member 9); CD137; CDwl37; ILA, etc.). The co-stimulatory region may be derived from the intracellular portion of the transmembrane protein CD28 (also known as Tp44). The co-stimulatory region may be derived from the intracellular portion of the transmembrane protein ICOS (also known as induced T cell co-stimulation, AILIM, CD278 and CVID1). The co-stimulatory region may be derived from the intracellular portion of the transmembrane protein OX-40 (also known as tumor necrosis factor receptor superfamily member 4, TNFRSF4, RP5-902P8.3, ACT35, CD134, OX40, TXGP1L). The co-stimulatory region may be derived from the intracellular portion of the transmembrane protein BTLA (also known as B and T lymphocyte-associated protein, BTLA1, and CD272). The co-stimulatory region may be derived from the intracellular portion of the transmembrane protein CD27 (also known as S152, T14, tumor necrosis factor receptor superfamily member 7, TNFRSF7, and Tp55). The co-stimulatory region may be derived from the intracellular portion of the transmembrane protein CD30 (also known as tumor necrosis factor receptor superfamily member 8, TNFRSF8, D1S166E, and Ki-1). The co-stimulatory region may be derived from the intracellular portion of the transmembrane protein GITR (also known as tumor necrosis factor receptor superfamily member 18, TNFRSF18, RP5-902P8.2, AITR, CD357, ENERGEN, and GITR-D). The co-stimulatory region may be derived from the intracellular portion of the transmembrane protein HVEM (also known as tumor necrosis factor receptor superfamily member 14, TNFRSF14, RP3-395M20.6, ATAR, CD270, HVEA, LIGHTR and TR2). The co-stimulatory region may be derived from 2B4 (also known as CD244, NAIL, NKR2B4, Nmrk, SLAMF4, CD244 molecules, etc.). The co-stimulatory region may be derived from DNAM-1 (also known as CD226, DNAM1, PTA1, TLiSA1, CD226 molecules, etc.). The co-stimulatory region may be derived from CD40 (also known as Bp50, CDW40, TNFRSF5, p50, CD40 (protein), CD40 molecules, etc.). The co-stimulatory region may be derived from LFA-1 (also known as lymphocyte function-associated antigen 1, integrin αL, ITGAL, CD11A, LFA1A, integrin subunit αL, etc.). The co-stimulatory region may be derived from CD2 (also known as lymphocyte function antigen-2, LFA-2, SRBC, T11, CD2 molecule, etc.).The co-stimulatory region may be derived from CD7 (also known as GP40, LEU-9, TP41, Tp40, CD7 molecule, etc.). The co-stimulatory region may be derived from LIGHT (also known as TNFSF14, CD258, HVEML, LIGHT, LTg, TR2, TNLG1D, tumor necrosis factor superfamily member 14, etc.). The co-stimulatory region may be derived from NKG2C (also known as KLRC2, CD159c, NKG2-C, killer cell lectin-like receptor C2, etc.).

[0250] A specific exemplary costimulatory domain is represented by the following amino acid sequence:

[0251] Table: Co-stimulatory domain

[0252]

[0253] 7. Detection of Peptides

[0254] CAR described herein may also include a detection peptide or molecule. Suitable detection peptides include hemagglutinin (HA; e.g., YPYDVPDYA; SEQ ID NO: 56); FLAG (e.g., DYKDDDDK; SEQ ID ON: 57); c-myc (e.g., EQKLISEEDL; SEQ ID NO: 58), etc. Other suitable detection peptides are known in the art.

[0255] 8. Peptide linkers

[0256] In some embodiments, the polypeptide of the present disclosure includes a peptide linker (sometimes referred to as a linker). Peptide linkers can be used to separate any peptide domain / region described herein. For example, a linker can be between a signal peptide and an antigen binding domain, between the VH and VL of an antigen binding domain, between an antigen binding domain and a peptide spacer, between a peptide spacer and a transmembrane domain, a flanking costimulatory region or on the N- or C- region of a costimulatory region, and / or between a transmembrane domain and an intracellular domain. A peptide linker can have any of a variety of amino acid sequences. Domains and regions can be connected by peptide linkers that generally have flexible properties, but other chemical bonds are not excluded. A linker can be a peptide having a length of about 6 to about 40 amino acids or a length of about 6 to about 25 amino acids. These linkers can be produced by coupling proteins using synthetic oligonucleotides encoding linkers.

[0257] Peptide linkers with a certain degree of flexibility can be used. Peptide linkers can have virtually any amino acid sequence, keeping in mind that suitable peptide linkers will have sequences that result in generally flexible peptides. The use of small amino acids (e.g., glycine and alanine) can be used to produce flexible peptides. The generation of such sequences is routine for those skilled in the art.

[0258] Suitable linkers can be readily selected and can be of any suitable length, for example, from 1 amino acid (e.g., Gly) to 20 amino acids, from 2 amino acids to 15 amino acids, from 3 amino acids to 12 amino acids, including from 4 amino acids to 10 amino acids, from 5 amino acids to 9 amino acids, from 6 amino acids to 8 amino acids, or from 7 amino acids to 8 amino acids, and can be 1, 2, 3, 4, 5, 6 or 7 amino acids.

[0259] Suitable linkers can be readily selected and can be of any suitable different lengths, for example, from 1 amino acid (e.g., Gly) to 20 amino acids, from 2 amino acids to 15 amino acids, from 3 amino acids to 12 amino acids, including from 4 amino acids to 10 amino acids, from 5 amino acids to 9 amino acids, from 6 amino acids to 8 amino acids, or from 7 amino acids to 8 amino acids, and can be 1, 2, 3, 4, 5, 6 or 7 amino acids.

[0260] Exemplary flexible linkers include glycine polymers (G)n, glycine-serine polymers (including, for example, (GS)n, (GSGGS)n (SEQ ID NO: 59), (G4S)n, and (GGGS)n (SEQ ID NO: 60), where n is an integer of at least 1. n can be at least, at most, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (or any range derivable therein). Glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers can be used; both Gly and Ser are relatively unstructured and can therefore act as a neutral tether between components. Glycine polymers can be used; glycine has significantly more phi-psi space available than even alanine and is much less constrained than residues with longer side chains. Exemplary spacers can comprise amino acid sequences including, but not limited to, GGSG (SEQ ID NO: 61), GGSGG (SEQ ID NO: 62), GSGSG (SEQ ID NO: 63), GSGGG (SEQ ID NO: 64), GGGSG (SEQ ID NO: 65), GSSSG (SEQ ID NO: 66), etc. The linker may comprise repeats, such as consecutive repeats of one or more of SEQ ID NOs: 61 to 66, such as a linker comprising an amino acid sequence corresponding to one of SEQ ID NOs: 61 to 66, repeated at least, at most or exactly 2, 3, 4, 5, 6, 7, 8, 9 or 10 times, or any range derivable therein.

[0261] The linker may comprise (EAAAK)n (SEQ ID NO: 67), wherein n is an integer of at least one. n may be at least, at most, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (or any range derivable therein).

[0262] B. Therapeutic Control

[0263] The transgenic, the first transgenic or the second transgenic can be co-expressed with a therapeutic control. The therapeutic control regulates cell proliferation, promotes cell selection (e.g., selects cells expressing a chimeric antigen receptor of the present invention) or a combination thereof. Regulating cell proliferation can include upregulating cell proliferation to promote cell proliferation. Regulating cell proliferation can include downregulating cell proliferation to reduce or inhibit cell proliferation. The agent used as a therapeutic control can promote the enrichment of cells that express a chimeric antigen receptor that can lead to a therapeutic advantage. The agent used as a therapeutic control can interact biochemically with another composition to regulate the function of the therapeutic control. For example, EGFRt (therapeutic control) can interact biochemically with cetuximab to regulate the function of EGFRt in selection, tracking, cell ablation, or a combination thereof.

[0264] Exemplary therapeutic controls include truncated epidermal growth factor receptor (EGFRt), chimeric cytokine receptor (CCR) and / or dihydroxyfolate receptor (DHFR) (e.g., mutant DHFR). The polynucleotide encoding CAR and therapeutic controls can be connected by an IRES sequence or by a polynucleotide sequence encoding a cleavable linker. The CAR of the present invention is constructed so that it can be expressed in cells, which then proliferate in response to the presence of at least one molecule (e.g., antigen) that interacts with at least one antigen-specific targeting region. The therapeutic control may include a cell surface protein, wherein the protein lacks an intracellular signaling domain. It is contemplated that any cell surface protein lacking intracellular signaling or modified (e.g., by truncation) to lack intracellular signaling can be used. Other examples of therapeutic controls include truncated LNGFR, truncated CD19, etc., wherein the truncated protein lacks an intracellular signaling domain.

[0265] "Coexpression" as used herein refers to the simultaneous expression of two or more genes. A gene can be a nucleic acid encoding, for example, a single protein or a chimeric protein as a single polypeptide chain. For example, the CAR of the present disclosure can be coexpressed with a therapeutic control (e.g., truncated epidermal growth factor (EGFRt)), wherein the CAR is encoded by a first polynucleotide chain and the therapeutic control is encoded by a second polynucleotide chain. The first and second polynucleotide chains can be connected by a nucleic acid sequence encoding a cleavable joint. The polynucleotides encoding CAR and a therapeutic control system can be connected by an IRES sequence. Alternatively, CAR and a therapeutic control are encoded by two different polynucleotides that are not connected by a joint but are encoded by, for example, two different vectors. In addition, the CAR of the present disclosure can be coexpressed with a therapeutic control and CCR, a therapeutic control and DHFR (e.g., a mutant DHFR) or a therapeutic control and CCR and DHFR (e.g., a mutant DHFR). CAR, a therapeutic control, and CCR can be coexpressed and encoded by the first, second, and third polynucleotide sequences, respectively, wherein the first, second, and third polynucleotide sequences are connected by an IRES sequence or a sequence encoding a cleavable joint (e.g., T2A). Alternatively, these sequences are not connected by a joint, but are encoded by, for example, different vectors. CAR, therapeutic control and DHFR (e.g., mutant DHFR) can be co-expressed and encoded by the first, second and fourth polynucleotide sequences, respectively, wherein the first, second and fourth polynucleotide sequences are connected by an IRES sequence or by a sequence encoding a cleavable joint. Alternatively, these sequences are not connected by a joint, but are encoded by, for example, different vectors. CAR, therapeutic control, CCR and DHFR (e.g., mutant DHFR) can be co-expressed and encoded by the first, second, third and fourth polynucleotide sequences, respectively, wherein the first, second, third and fourth polynucleotide sequences are connected by an IRES sequence or a sequence encoding a cleavable joint. Alternatively, these sequences are not connected by a joint, but are encoded by, for example, different vectors. If the aforementioned sequences are encoded by different vectors, these vectors can be transfected simultaneously or sequentially.

[0266] Additional aspects of therapeutic control of the compositions of the present disclosure, CAR molecules, and methods of use can be found in U.S. Patent No.: 9,447,194, which is incorporated herein by reference for all purposes. VI. Gene Transfer Technology

[0267] A. Gene Editing

[0268] Transformed nuclease can be used to introduce transgene into cells, such as progenitor cells, stem cells, HSPC, ES cells, iPSC and human embryonic mesodermal progenitor cells. In the case where cells are genetically modified, such as adding or reducing one or more features, genetic modification can be carried out by any suitable method. For example, any genetic modification composition or method can be used to introduce exogenous nucleic acid into cells or edit genomic DNA, such as gene editing, homologous recombination or non-homologous recombination, RNA-mediated genetic delivery or any conventional nucleic acid delivery method. Gene transfer technology can include targeted integration into the endogenous locus of the genome of the cell, such as the locus of the gene in Table 1. Some non-limiting examples of genetic modification methods can include, for example, gene editing methods by CRISPR / CAS9, zinc finger nucleases or TALEN technology.

[0269] Genome editing or genome editing with engineered nuclease (GEEN) is a type of genetic engineering in which artificially engineered nucleases or "molecular scissors" are used to insert, replace or remove DNA from the genome. The nuclease generates a specific double-stranded break (DSB) at the desired location in the genome and prevents the cell's endogenous mechanisms from repairing the induced breaks through the natural processes of homologous recombination (HR) and nonhomologous end-joining (NHEJ).

[0270] Non-limiting engineered nucleases include: zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), CRISPR / Cas9 system and engineered meganuclease re-engineered homing endonuclease. Any engineered nuclease known in the art can be used in the present methods and compositions.

[0271] The general practice in genetic analysis is to interfere with gene or protein function in a sequence-specific manner and monitor its effect on the organism in order to understand the function of the gene or protein function. However, in some organisms, it is difficult or impossible to perform site-specific mutagenesis, so more indirect methods must be used, such as silencing the target gene by short RNA interference (siRNA). However, the gene disruption of siRNA may be variable and incomplete. Genome editing with nucleases such as ZFN is different from siRNA, because the modified nuclease can change the DNA binding specificity, so in principle any target position in the genome can be cut, and modification is introduced for the endogenous sequence of the gene that is not specifically targeted by conventional RNAi. In addition, the specificity of ZFN and TALEN is enhanced because two ZFNs are required in the recognition of its target part and then point to adjacent sequences.

[0272] Meganucleases commonly found in microbial species have the unique property of having very long recognition sequences (>14 bp), thus making them naturally very specific. This can be exploited to perform site-specific DSBs in genome editing; however, the challenge is that not enough meganucleases are known or may ever be known to cover all possible target sequences. To overcome this challenge, mutagenesis and high-throughput screening methods have been used to generate meganuclease variants that recognize unique sequences. Others have been able to fuse multiple meganucleases and generate hybrid enzymes that recognize new sequences. Still others have attempted to alter the DNA-interacting amino acids of meganucleases to design sequence-specific meganucleases in a method known as rationally designed meganucleases (U.S. Patent 8,021,867B2, which is incorporated herein by reference).

[0273] Meganucleases have the benefit of causing less toxicity in cells than approaches such as ZFNs, probably because of more stringent DNA sequence recognition; however, constructing sequence-specific enzymes for all possible sequences is expensive and time-consuming, as one cannot benefit from the combinatorial possibilities exploited by approaches such as ZFNs and TALENs. Thus, there are both advantages and disadvantages.

[0274] In contrast to meganucleases, the concepts behind ZFNs and TALENs are more based on non-specific DNA cleavage enzymes that are then linked to specific DNA sequence recognition peptides such as zinc fingers and transcription activator-like effectors (TALEs). One approach is to find an endonuclease in which the DNA recognition site and the cleavage site are separated from each other, which is an uncommon situation in restriction enzymes. Once such an enzyme is found, its cleavage portion can be separated, which will be very non-specific because it does not have recognition capabilities. This portion can then be linked to a sequence recognition peptide, which will result in very high specificity. An example of a restriction enzyme with this property is FokI. In addition, the advantage of FokI is that dimerization is required to have nuclease activity, which means that specificity is significantly improved because each nuclease partner recognizes a unique DNA sequence. In order to enhance this effect, the FokI nuclease has been modified to act only as a heterodimer and has improved catalytic activity. Heterodimer functional nucleases will avoid the possibility of unwanted homodimer activity, thereby improving the specificity of DSBs.

[0275] Although the nuclease parts of both ZFN and TALEN have similar properties, the difference between these modified nucleases lies in their DNA recognition peptides. ZFN relies on Cys2-His2 zinc fingers, and TALEN relies on TALE. The characteristics of these two DNA recognition peptide domains are that they occur naturally in combination in their proteins. Cys2-His2 zinc fingers are usually present in repetitions of 3bp apart, and exist in different combinations in various nucleic acid interaction proteins such as transcription factors. On the other hand, TALE exists in a repetition of a one-to-one recognition ratio between amino acids and recognized nucleotide pairs. Since both zinc fingers and TALE exist in a repetitive pattern, different combinations can be tried to produce a variety of sequence specificities. Zinc fingers are more mature in these terms and methods, such as modular assembly (wherein zinc fingers associated with triplet sequences are connected in a row to cover the desired sequence), OPEN (low stringency selection of peptide domains and triplet nucleotides, followed by high stringency selection of peptide combinations and final targets in bacterial systems) and bacterial single hybrid screening of zinc finger libraries have been used to prepare site-specific nucleases.

[0276] B. Transgene Delivery

[0277] The vector can be constructed to contain a transgenic nucleic acid sequence for genetically modifying any cell used herein, particularly a starting cell, such as a stem cell or progenitor cell induced to differentiate into a mature cell. Details of the components and delivery methods of these vectors are disclosed below.

[0278] Cells can contain one or more genetic changes by genetically modifying cells before or after differentiation (US2002 / 0168766). When exogenous nucleic acids or polynucleotides are transferred into cells by any suitable artificial manipulation means, or when cells are descendants of cells that have inherited the initial changes of polynucleotides, cells are referred to as "genetically altered", "genetically modified" or "transgenic". For example, cells can be treated with cells to increase their replication potential by genetically modifying cells to express telomerase reverse transcriptase before or after cells progress to restricted developmental lineage cells or terminally differentiated cells (US2003 / 0022367).

[0279] C. Carrier

[0280] Those skilled in the art will be well able to construct vectors by standard recombinant techniques (see, e.g., Maniatis et al., 1988 and Ausubel et al., 1994, both of which are incorporated herein by reference).

[0281] The vector may also contain other components or functions that further regulate gene delivery and / or gene expression, or otherwise provide beneficial properties to the target cell. Such other components include, for example, components that affect binding or targeting to cells (including components that mediate cell type or tissue specific binding); components that affect cell uptake of vector nucleic acid; components that affect the localization of polynucleotides in cells after uptake (e.g., agents that mediate nuclear localization); and components that affect the expression of polynucleotides.

[0282] Such components may also include markers, such as detectable markers and / or selection markers that can be used to detect or select cells that have taken in and expressed the nucleic acid delivered by the vector. Such components may be provided as natural features of the vector (e.g., using certain viral vectors with components or functions that mediate binding and uptake), or the vector may be modified to provide such functions. A large number of such vectors are known in the art and are generally available. When the vector is maintained in a host cell, the vector may be stably replicated by the cell as an autonomous structure during mitosis, incorporated into the genome of the host cell, or maintained in the nucleus or cytoplasm of the host cell.

[0283] D. Regulatory elements

[0284] The eukaryotic expression cassette contained in the vector contains, inter alia, a eukaryotic transcriptional promoter operably linked (in 5' to 3' direction) to the protein coding sequence, splicing signals containing intervening sequences, and transcription termination / polyadenylation sequences.

[0285] 1. Promoter / enhancer

[0286] "Promoter" is a control sequence, which is a region of a nucleic acid sequence that controls the initiation and rate of transcription. A promoter or enhancer may include a promoter or enhancer from a gene in Table 1. It may include genetic elements that regulatory proteins and molecules (e.g., RNA polymerase and other transcription factors) can bind to, to initiate specific transcription of a nucleic acid sequence. The phrases "effectively positioned," "effectively connected," "under control," and "under transcriptional control" mean that a promoter is in the correct functional position and / or orientation relative to a nucleic acid sequence to control transcription initiation and / or expression of the sequence.

[0287] The promoter region of the present disclosure may be an endogenous promoter region. An endogenous promoter region refers to a situation where the promoter region is in its endogenous genomic environment, so that the sequence upstream of the promoter (i.e., 5' region) is substantially the same as that in wild-type cells. Substantially the same may refer to a region with at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the upstream region of the wild type. An endogenous promoter region also refers to a situation where the promoter is located at the same genomic location as the wild-type promoter. An endogenous promoter may refer to a promoter in an unmodified cell, or a promoter with at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the wild-type promoter.

[0288] Promoters usually include sequences whose function is to locate the start site of RNA synthesis. The best-known example is the TATA box, but in some promoters lacking the TATA box (e.g., the promoter of the mammalian terminal deoxynucleotidyl transferase gene and the promoter of the SV40 late gene), the discrete elements covering the start site themselves help to determine the start position. Other promoter elements regulate the frequency of transcription initiation. Typically, these are located in the region 30 to 110 bp upstream of the start site, although it has been shown that many promoters also include functional elements downstream of the start site. In order to make the coding sequence "under the control" of the promoter, the 5' end of the transcription start site of the transcription reading frame is positioned "downstream" (i.e., 3') of the selected promoter. "Upstream" promoters stimulate the transcription of DNA and promote the expression of the encoded RNA.

[0289] The spacing between promoter elements is usually flexible so that promoter function is maintained when the elements are inverted or moved relative to each other. In the tk promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline. Depending on the promoter, it is shown that each element can function cooperatively or independently to activate transcription. Promoters may or may not be used in conjunction with "enhancers", which refer to cis-acting regulatory sequences that participate in the transcriptional activation of nucleic acid sequences.

[0290] 2. Protease cleavage site / self-cleaving peptide and internal ribosome binding site

[0291] Suitable protease cleavage sites and self-cleaving peptides are known to the skilled person (see for example Ryan et al., 1997; Scymczak et al., 2004). Some examples of protease cleavage sites are the following cleavage sites: potyvirus Nla protease (e.g., tobacco etch virus protease), potyvirus HC protease, potyvirus P1 (P35) protease, byovirus Nla protease, byovirus RNA-2 encoded protease, foot-and-mouth disease virus L protease, enterovirus 2A protease, rhinovirus 2A protease, picornavirus 3C protease, comovirus 24K protease, nepovirus 24K protease, RTSV (rice tungro spherical virus) 3C-like protease, PY\IF (parsnipyellow fleck virus) 3C-like protease, thrombin, factor Xa, and enterokinase. Due to its high cleavage stringency, the TEV (tobacco etch virus) protease cleavage site can be used.

[0292] Exemplary self-cleaving peptides (also called "cis-acting hydrolysis elements", CHYSEL; see deFelipe (2002)) are derived from potato virus Y and cardiovirus 2A peptides. Specific self-cleaving peptides can be selected from 2A peptides derived from FMDV (foot-and-mouth disease virus), equine rhinitis A virus, Thoseàasigna virus and porcine teschovirus.

[0293] Specific start signals can also be used to effectively translate the coding sequence in the polycistronic message. These signals include the ATG start codon or adjacent sequences. It may be necessary to provide exogenous translation control signals, including the ATG start codon. One of ordinary skill in the art will be able to easily determine this and provide the necessary signals. It is known that the start codon must be "in frame" with the reading frame of the desired coding sequence to ensure translation of the entire insert. Exogenous translation control signals and start codons can be natural or synthetic. Expression efficiency can be enhanced by including appropriate transcription enhancer elements.

[0294] The use of internal ribosome entry site (IRES) elements can be used to generate polygenic or polycistronic information. IRES elements can bypass the ribosome scanning model of 5' methylated cap-dependent translation and start translation at the internal site (Pelletier and Sonenberg, 1988). IRES elements from two members of the Picornaviridae (poliomyelitis and encephalomyocarditis) have been described (Pelletier and Sonenberg, 1988), as well as IRES from mammalian information (Macejak and Sarnow, 1991). IRES elements can be connected to heterologous open reading frames. Multiple open reading frames can be transcribed together, each separated by IRES, thereby generating polycistronic information. With IRES elements, each open reading frame can utilize ribosomes for efficient translation. Using a single promoter / enhancer to transcribe a single message, multiple genes can be effectively expressed (see U.S. Patent Nos. 5,925,565 and 5,935,819, each of which is incorporated herein by reference).

[0295] 3. Multiple cloning site

[0296] The vector may contain a multiple cloning site (MCS), which is a nucleic acid region containing multiple restriction enzyme sites, any of which can be used in conjunction with standard recombinant techniques to digest the vector (see, for example, Carbonelli et al., 1999, Levenson et al., 1998 and Cocea, 1997, which are incorporated herein by reference). "Restriction enzyme digestion" refers to the catalytic cleavage of a nucleic acid molecule with an enzyme that functions only at a specific position in the nucleic acid molecule. Many of these restriction enzymes are commercially available. The use of such enzymes is widely understood by those skilled in the art. Typically, a restriction enzyme that cuts within the MCS is used to linearize or fragment the vector so that an exogenous sequence can be connected to the vector. "Connection" refers to the process of forming a phosphodiester bond between two nucleic acid fragments, which may or may not be continuous with each other. Techniques involving restriction enzymes and ligation reactions are well known to those skilled in the art of recombinant technology.

[0297] 4. Splice Sites

[0298] Most transcribed eukaryotic RNA molecules will undergo RNA splicing to remove introns from the primary transcript. Vectors comprising genomic eukaryotic sequences may require donor and / or acceptor splice sites to ensure correct processing of transcripts for protein expression (see, e.g., Chandler et al., 1997, which is incorporated herein by reference).

[0299] 5. Termination Signal

[0300] The vector or construct may comprise at least one termination signal. A "termination signal" or "terminator" consists of a DNA sequence that participates in the specific termination of an RNA transcript by an RNA polymerase. Thus, termination signals that terminate the production of RNA transcripts are contemplated. Terminators may be necessary in vivo to achieve a desired message level.

[0301] In eukaryotic systems, the terminator region may also include a specific DNA sequence that allows site-specific cutting of new transcripts to expose polyadenylation sites. It signals a specialized endogenous polymerase to add a section of approximately 200 A residues (polyA) to the 3' end of the transcript. RNA molecules modified with this polyA tail show more stable and more effective translation. Therefore, the terminator may include a signal for RNA cutting, and the terminator signal promotes the polyadenylation of information. Terminators and / or polyadenylation site elements can be used to enhance information levels and minimize reading from the box to other sequences.

[0302] It is contemplated that terminators include any known transcription terminators described herein or known to those of ordinary skill in the art, including but not limited to, for example, termination sequences of genes, such as, for example, bovine growth hormone terminators, or viral termination sequences, such as, for example, SV40 terminators. Termination signals may lack transcribable or translatable sequences, such as, for example, due to sequence truncation.

[0303] 6. Polyadenylation signal

[0304] In expression, particularly eukaryotic expression, a polyadenylation signal is often included to achieve proper polyadenylation of the transcript. The nature of the polyadenylation signal is not believed to be critical to successful practice, and any such sequence may be used. Some exemplary embodiments include the SV40 polyadenylation signal or the bovine growth hormone polyadenylation signal, which are convenient and known to function well in a variety of target cells. Polyadenylation may increase the stability of the transcript or may promote cytoplasmic transport.

[0305] E. Vector Delivery

[0306] The genetic modification in the cells of the present disclosure or the introduction of transgenic nucleic acids can use any suitable method for nucleic acid delivery to transform cells, as described herein or as known to those of ordinary skill in the art. Methods for introducing and expressing genes into cells are known in the art. In the context of expression vectors, vectors can be easily introduced into host cells, such as mammals, bacteria, yeast or insect cells, by any method in the art. For example, expression vectors can be transferred to host cells by physical, chemical or biological means.

[0307] Such methods include, but are not limited to, direct delivery of DNA, such as by ex vivo transfection (Wilson et al., 1989, Nabel et al., 1989); by injection (U.S. Pat. Nos. 5,994,624, 5,981,274, 5,945,100, 5,780,448, 5,736,524, 5,702,932, 5,656,610, 5,589,466 and 5,580,859, each of which is incorporated herein by reference), including microinjection (Harland and Weintraub, 1985; U.S. Pat. No. 5,789,215, which is incorporated herein by reference); by electroporation (U.S. Pat. No. 5,384,253, which is incorporated herein by reference; Tur-Kaspa et al., 1986; Potter et al., 1984); by calcium phosphate precipitation (Graham and Van Der Eb, 1973; Chen and Okayama, 1987; Rippe et al., 1990); by using DEAE-dextran followed by polyethylene glycol (Gopal, 1985); by direct sonication (Fechheimer et al., 1987); by liposome-mediated transfection (Nicolau and Sene, 1982; Fraley et al., 1979; Nicolau et al., 1987; Wong et al., 1980; Kaneda et al., 1989; Kato et al., 1991) and receptor-mediated transfection (Wu and Wu, 1987; Wu and Wu, 1988); by microparticle bombardment (PCT Application Nos. WO 94 / 09699 and 95 / 06128; U.S. Pat. Nos. 5,610,042; 5,322,783 5,563,055, 5,550,318, 5,538,877 and 5,538,880, each of which is incorporated herein by reference); by agitation with silicon carbide fibers (Kaeppler et al., 1990; U.S. Pat. Nos. 5,302,523 and 5,464,765, each of which is incorporated herein by reference); by Agrobacterium-mediated transformation (U.S. Pat. Nos. 5,591,616 and 5,563,055, each of which is incorporated herein by reference); by PEG-mediated transformation of protoplasts (Omirulleh et al., 1993; U.S. Pat. Nos. 4,684,611 and 4,952,500, each of which is incorporated herein by reference); by desiccation / inhibition-mediated DNA uptake (Potrykus et al., 1994; U.S. Pat. Nos. 5 ...302,523 and 5,464,765, each of which is incorporated herein by reference); by Agrobacterium-mediated transformation (U.S. Pat. Nos. 5,591,616 and 5,563,055, each of which is incorporated herein by reference); by PEG-mediated transformation of protoplasts (Omirulleh et al, 1985), and any combination of such methods. By applying techniques such as these, organelles, cells, tissues or organisms can be transformed stably or transiently. .

[0308] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, liposome transfection, particle bombardment, microinjection, electroporation, etc. Methods for generating cells containing vectors and / or exogenous nucleic acids are well known in the art (see, e.g., Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). An illustrative but non-limiting method for introducing polynucleotides into host cells is calcium phosphate transfection.

[0309] Biological methods for introducing a polynucleotide of interest into a host cell may include the use of DNA and RNA vectors. Viral vectors and especially retroviral vectors have become the most widely used methods for inserting genes into mammalian (e.g., human) cells. Other viral vectors may be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, and adeno-associated viruses, etc. (see, e.g., U.S. Patent Nos. 5,350,674 and 5,585,362, etc.).

[0310] Chemical methods for introducing polynucleotides into host cells include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An illustrative colloidal system used as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).

[0311] 1. Liposome-mediated transfection

[0312] In the case of using a non-viral delivery system, an illustrative delivery vehicle is a lipid and / or liposome. Consider using lipid formulations to introduce nucleic acids into host cells (in vitro, in vitro or in vivo). Nucleic acids can be associated with lipids. Nucleic acids associated with lipids can be encapsulated in the aqueous interior of liposomes, dispersed in the lipid bilayer of liposomes, connected to liposomes by connecting molecules associated with liposomes and oligonucleotides, embedded in liposomes, compounded with liposomes, dispersed in a solution containing lipids, mixed with lipids, combined with lipids, included in lipids as a suspending agent, included or compounded with micelles, or otherwise associated with lipids. Lipids, lipid / DNA or lipid / expression vector-related compositions are not limited to any specific structure in solution. For example, they can exist as a double-layer structure, such as micelles, or have a "collapsed" structure. They may also be simply dispersed in solution, and may form aggregates of uneven size or shape. Lipid is a fatty substance, which can be a naturally occurring or synthetic lipid. For example, lipids include fat droplets naturally present in the cytoplasm as well as a class of compounds containing long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

[0313] Nucleic acids can be embedded in lipid complexes such as liposomes. Liposomes are vesicular structures characterized by a phospholipid bilayer membrane and an internal aqueous medium. Multilamellar liposomes have multiple lipid layers separated by an aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the closed structure is formed, and water and dissolved solutes are embedded between the lipid bilayers (Ghosh and Bachhawat, 1991). Nucleic acids complexed with Lipofectamine (Gibco BRL) or Superfect (Qiagen) are also contemplated. The amount of liposomes used can vary depending on the properties of the liposomes and the cells used, for example, about 5 to about 20 μg of vector DNA per 1 to 10 million cells can be considered.

[0314] Liposome-mediated nucleic acid delivery and expression of foreign DNA in vitro has been very successful (Nicolau and Sene, 1982; Fraley et al., 1979; Nicolau et al., 1987). The feasibility of liposome-mediated delivery and expression of foreign DNA in cultured chick embryo, HeLa and hepatoma cells has also been demonstrated (Wong et al., 1980).

[0315] Liposomes can be complexed with hemagglutinating virus (HVJ). This has been shown to facilitate fusion with cell membranes and promote entry of liposome-encapsulated DNA into cells (Kaneda et al., 1989). Liposomes can be complexed or used in combination with nuclear non-histone chromosomal proteins (HMG-1) (Kato et al., 1991). Liposomes can be complexed or used in combination with both HVJ and HMG-1. The delivery vehicle can include a ligand and a liposome.

[0316] Lipids suitable for use can be obtained from commercial sources. For example, dimyristoylphosphatidylcholine ("DMPC (dimyristyl phosphatidylcholine)") can be obtained from Sigma, St. Louis, Mo.; dicetyl phosphate ("DCP (dicetyl phosphate)") can be obtained from K & K Laboratories (Plainview, NY); cholesterol ("Choi (cholesterol)") can be obtained from Calbiochem-Behring; dimyristoylphosphatidylglycerol ("DMPG (dimyristyl phosphatidylglycerol)") and other lipids can be obtained from Avanti Polar Lipids, Inc. (Birmingham, Ala.). Stock solutions of lipids in chloroform or chloroform / methanol can be stored at about -20°C. Chloroform can be used as the only solvent because it evaporates more easily than methanol. "Liposome" is a general term that covers a variety of single and multi-layer lipid carriers formed by producing closed lipid bilayers or lipid aggregates. Liposomes can be characterized as having a vesicle structure with a phospholipid bilayer membrane and an internal aqueous medium. Multilamellar liposomes have multiple lipid layers separated by an aqueous medium. When phospholipids are suspended in an excess of aqueous solution, they form spontaneously. Before the closed structure forms, the lipid components undergo self-rearrangement, and water and dissolved solutes are embedded between the lipid bilayers (Ghosh et al. (1991) Glycobiology 5: 505-510). However, compositions having structures different from normal vesicle structures in solution are also contemplated. For example, lipids can assume micellar structures, or exist only as heterogeneous aggregates of lipid molecules. Lipofectamine-nucleic acid complexes are also contemplated.

[0317] 2. Electroporation

[0318] The nucleic acid is introduced into the cell by electroporation. Electroporation involves exposing a suspension of cells and DNA to a high voltage discharge. The recipient cells can be made more susceptible to transformation by mechanical injury. The amount of vector used can also vary depending on the nature of the cells used, for example, about 5 to about 20 μg of vector DNA per 1 to 10 million cells can be considered.

[0319] Transfection of eukaryotic cells using electroporation has been quite successful. Mouse pre-B lymphocytes have been transfected with human kappa immunoglobulin genes in this manner (Potter et al., 1984), and rat hepatocytes have been transfected with the chloramphenicol acetyltransferase gene (Tur-Kaspa et al., 1986).

[0320] 3. Calcium phosphate

[0321] Nucleic acids can be introduced into cells using calcium phosphate precipitation. This technique has been used to transfect human KB cells with adenovirus 5 DNA (Graham and Van Der Eb, 1973). In the same way, mouse L (A9), mouse C127, CHO, CV-1, BHK, NIH3T3 and HeLa cells were transfected with the neomycin marker gene (Chen and Okayama, 1987), and rat hepatocytes were transfected with multiple marker genes (Rippe et al., 1990).

[0322] 4.DEAE-dextran

[0323] Nucleic acids can be delivered to cells using DEAE-dextran followed by polyethylene glycol. In this manner, reporter plasmids have been introduced into mouse myeloma and erythroleukemia cells (Gopal, 1985). VII. Selectable or screenable markers

[0324] Regardless of the method used to introduce exogenous nucleic acid into the host cell or otherwise expose the cell to the inhibitor of the present disclosure, a variety of assays can be performed to confirm the presence of the recombinant DNA sequence in the host cell. Such assays include, for example, "molecular biology" assays known to those skilled in the art, such as Southern and Northern blots, RT-PCR and PCR; "biochemical" assays, such as by immunological means (ELISA and Western blots) or by assays described herein to detect the presence or absence of specific peptides to identify agents falling within the scope of the present disclosure.

[0325] Cells comprising exogenous nucleic acids can be identified in vitro or in vivo by including markers in expression vectors or exogenous nucleic acids. Such markers can confer identifiable changes to cells, thereby allowing easy identification of cells comprising expression vectors. In general, selection markers can be markers that confer properties that allow selection. Positive selection markers can be markers in which the presence of the marker allows its selection, while negative selection markers are markers in which its presence prevents its selection. An example of a positive selection marker is a drug resistance marker.

[0326] Drug selection markers are usually included to help clone and identify transformants, for example, genes that confer resistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin and histidinol are useful selection markers. In addition to conferring markers that allow the phenotype of transformants to be distinguished based on conditional implementation, other types of markers (including screenable markers, such as GFP, which is based on colorimetric analysis) are also considered. Alternatively, screenable enzymes can be used as negative selection markers, such as herpes simplex virus thymidine kinase (thymidine kinase, tk) or chloramphenicol acetyltransferase (chloramphenicol acetyltransferase, CAT). Those skilled in the art also know how to use immunological markers, which can be used in conjunction with FACS analysis. The marker used is not considered to be important, as long as it can be expressed simultaneously with the nucleic acid encoding the gene product. Other examples of selection markers and screenable markers are well known to those skilled in the art.

[0327] Selectable markers may include reporter gene types used in laboratory microbiology, molecular biology, and genetic engineering to indicate the success of transfection or other operations designed to introduce foreign DNA into cells. Selectable markers are usually antibiotic resistance genes; cells that have been manipulated to introduce foreign DNA are grown on a medium containing antibiotics, and these cells that can grow have successfully taken up and expressed the introduced genetic material. Some examples of selectable markers include: the Abicr gene or the Neo gene from Tn5, which confers antibiotic resistance to geneticin.

[0328] Screenable markers may include reporter genes that allow researchers to distinguish desired cells from undesirable cells. Reporter genes may be used to indicate specific cell lineages. For example, reporter genes may be located within expression elements and are generally expressed simultaneously under the control of ventricular or atrial selective regulatory elements associated with the coding region of ventricular or atrial selective genes. Reporters allow the separation of cells of specific lineages without placing them under drugs or other selective pressures or otherwise compromising cell viability.

[0329] Some examples of such reporters include genes encoding cell surface proteins (e.g., CD4, HA epitopes), fluorescent proteins, antigenic determinants, and enzymes (e.g., β-galactosidase). Cells containing the vector can be isolated by, for example, FACS using fluorescently tagged antibodies against cell surface proteins or substrates that can be converted into fluorescent products by vector-encoded enzymes.

[0330] The reporter gene can be a fluorescent protein. A wide range of fluorescent protein genetic variants have been developed, characterized in that the fluorescence emission spectrum is distributed across almost the entire visible spectrum. Mutagenesis efforts in the original Victoria multi-tube luminous jellyfish (Aequoreavictoria) jellyfish green fluorescent protein have produced new fluorescent probes, ranging from blue to yellow, and are some of the most widely used in vivo reporter molecules in biological research. Fluorescent proteins with longer wavelengths have been developed from sea anemones (marine anemone), Discosoma striata and coral reefs belonging to the coral animal class (Anthozoa) category, which emit in orange and red spectral regions. Other species have also been developed to produce similar proteins with cyan, green, yellow, orange and crimson fluorescence emission. Developmental research efforts are currently being carried out to improve the brightness and stability of fluorescent proteins, thereby improving their overall usefulness.

[0331] VIII. Cell Culture Conditions

[0332] Cell culture conditions can be provided for the culture of 3D cell aggregates described herein and the production of mature cells comprising transgenes. The starting cells of the selected population can comprise at least or about 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 The seeding density of the starting cell population can be at least or about 10, 10 1 , 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 cells / ml, or any range derivable therein.

[0333] A. Culture medium

[0334] The cells may be cultured in a specific culture medium at any stage in the process of producing mature cells expressing a transgene. The cells may be formulated in such a manner as to be suitable for delivery to a recipient without deleterious effects.

[0335] The culture medium can be prepared using a culture medium for culturing animal cells as its basic culture medium, such as any of the following: AIM V, X-VIVO-15, NeuroBasal, EGM2, TeSR, BME, BGJb, CMRL 1066, Glasgow MEM, modified MEM zinc selection, IMDM, medium 199, Eagle MEM, αMEM, DMEM, Ham, RPMI-1640 and Fischer medium, and any combination thereof, but the culture medium may not be particularly limited thereto as long as it can be used for culturing animal cells. In particular, the culture medium may be xeno-free or chemically defined.

[0336] The culture medium can be a serum-containing culture medium or a serum-free culture medium or a xeno-free culture medium. From the perspective of preventing contamination by xeno-animal-derived components, the serum can be derived from the same animal as the stem cells. A serum-free culture medium refers to a culture medium without unprocessed or unpurified serum, and therefore can include a culture medium with purified blood-derived components or animal tissue-derived components (e.g., growth factors).

[0337] The culture medium may or may not include any substitute for serum. The substitute for serum may include such a substance: it suitably includes albumin (e.g., lipid-rich albumin, bovine albumin, albumin substitutes such as recombinant albumin or humanized albumin, plant starch, dextran and protein hydrolysate), transferrin (or other iron transporters), fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thioglycerol (3'-thiolgiycerol), or its equivalent. The substitute for serum may be prepared by the method disclosed in, for example, International Publication No. 98 / 30679 (which is incorporated herein in its entirety). Alternatively, any commercially available substance may be used to obtain more convenience. Commercially available substances include knockout serum substitutes (knockout Serum Replacement, KSR), chemically defined lipid concentrates (Gibco) and Glutamax (Gibco).

[0338] The culture medium can be a serum-free medium suitable for cell development. For example, the culture medium can contain a concentration effective to generate T cells from the 3D cell aggregates. Supplements, Xeno-Free supplement (available on the World Wide Web at thermofisher.com / us / en / home / technical-resources / media-formulation.250.html), NS21 supplement (Chen et al., J Neurosci Methods, 2008 Jun 30; 171(2):239-247, which is incorporated herein in its entirety), GS21™ supplement (available on the World Wide Web at amsbio.com / B-27.aspx), or a combination thereof.

[0339] The culture medium may contain one, two, three, four, five, six, seven, eight, nine, ten, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more of the following: vitamins such as biotin; DL alpha tocopherol acetate; DL alpha-tocopherol; vitamin A (acetate); proteins such as BSA (bovine serum albumin) or human albumin; fatty acid free fraction V; catalase; human recombinant insulin; human transferrin; superoxide dismutase; other components such as corticosterone, D-galactose, ethanolamine HCl, glutathione (reduced form), L-carnitine HCl, linoleic acid, linolenic acid, progesterone, putrescine 2HCl, sodium selenite, and / or T3 (triiodo-1-thyronine).

[0340] The culture medium may also include vitamins. The culture medium may include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 of the following (and any range derivable therein): biotin, DLα-tocopherol acetate, DLα-tocopherol, vitamin A, choline chloride, calcium pantothenate, pantothenic acid, nicotinamide folic acid, pyridoxine, riboflavin, thiamine, inositol, vitamin B12, or the culture medium includes a combination thereof or a salt thereof. The culture medium may include or consist essentially of biotin, DLα-tocopherol acetate, DLα-tocopherol, vitamin A, choline chloride, calcium pantothenate, pantothenic acid, nicotinamide folic acid, pyridoxine, riboflavin, thiamine, inositol, and vitamin B12. The vitamins may include or consist essentially of biotin, DLα-tocopherol acetate, DLα-tocopherol, vitamin A, or a combination thereof or a salt thereof. The culture medium may also include protein. The protein may comprise albumin or bovine serum albumin, fractions of human albumin, catalase, insulin, transferrin, superoxide dismutase, or a combination thereof. The culture medium may also comprise one or more of the following: corticosterone, D-galactose, ethanolamine, glutathione, L-carnitine, linoleic acid, linolenic acid, progesterone, putrescine, sodium selenite or triiodo-1-thyronine, or a combination thereof. The culture medium may comprise one or more of the following: Supplements, Xeno-Free Supplement, GS21TM supplement, or a combination thereof. The culture medium may contain or further contain amino acids, monosaccharides, inorganic ions. The amino acids may contain arginine, cysteine, isoleucine, leucine, lysine, methionine, glutamine, phenylalanine, threonine, tryptophan, histidine, tyrosine or valine, or a combination thereof. The inorganic ions may contain sodium, potassium, calcium, magnesium, nitrogen or phosphorus, or a combination thereof or a salt thereof. The culture medium may also contain one or more of the following: molybdenum, vanadium, iron, zinc, selenium, copper or manganese, or a combination thereof. The culture medium may contain one or more of the vitamins discussed herein and / or one or more of the proteins discussed herein and / or one or more of the following, or may be substantially composed thereof: corticosterone, D-galactose, ethanolamine, glutathione, L-carnitine, linoleic acid, linolenic acid, progesterone, putrescine, sodium selenite, or triiodo-I-thyronine, Supplements, Xeno-Free supplements, GS21TM supplements, amino acids (e.g., arginine, cysteine, isoleucine, leucine, lysine, methionine, glutamine, phenylalanine, threonine, tryptophan, histidine, tyrosine or valine), monosaccharides, inorganic ions (e.g., sodium, potassium, calcium, magnesium, nitrogen and / or phosphorus) or their salts, and / or molybdenum, vanadium, iron, zinc, selenium, copper or manganese.

[0341] The culture medium may comprise externally added ascorbic acid. The culture medium may also comprise one or more externally added fatty acids or lipids, amino acids (e.g., non-essential amino acids), vitamins, growth factors, cytokines, antioxidant substances, 2-mercaptoethanol, pyruvic acid, buffers, and / or inorganic salts.

[0342] One or more media components can be added at a concentration of at least, at most, or about 0.1, 0.5, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 180, 200, 250 ng / L, ng / ml, μg / ml, mg / ml, or any range derivable therein.

[0343] The culture medium used can be supplemented with at least one exogenously added cytokine at a concentration of about 0.1 ng / mL to about 500 ng / mL, more particularly 1 ng / mL to 100 ng / mL, or at least, at most, or about 0.1, 0.5, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 180, 200, 250 ng / L, ng / ml, μg / ml, mg / ml, or any range derived therefrom. Suitable cytokines include, but are not limited to, FLT3 ligand (FLT3L), interleukin 7 (IL-7), stem cell factor (SCF), thrombopoietin (TPO), IL-2, IL-4, IL-6, IL-15, IL-21, TNF-α, TGF-β, interferon γ, interferon-λ, TSLP, thymopentin, pleiotropic factor and / or midkine. In particular, the culture medium may contain at least one of FLT3L and IL-7. More particularly, the culture may contain both FLT3L and IL-7.

[0344] Other culture conditions can be appropriately defined.For example, the culture temperature can be about 20°C to 40°C, such as at least, at most or about 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C (or any range from which it can be derived), but the temperature can be higher or lower than these values.CO2 concentration can be about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10% (or any range from which it can be derived), such as about 2% to 10%, such as about 2% to 5%, or any range from which it can be derived.Oxygen tension can be at least or about 1%, 5%, 8%, 10%, 20% or any range from which it can be derived.

[0345] B. Culture Container

[0346] The culture container for culturing cells and / or 3D cell aggregates or progeny cells thereof may include, but is not specifically limited to, bottles, bottles for tissue culture, dishes, culture dishes, dishes for tissue culture, multi-dishes, microplates, microwell plates, multi-plates, multi-well plates, microslides, chamber slides, tubes, trays, Chambers, culture bags and roller bottles, as long as they can culture stem cells therein. Stem cells can be cultured in the following volumes: at least or about 0.2, 0.5, 1, 2, 5, 10, 20, 30, 40, 50 ml, 100 ml, 150 ml, 200 ml, 250 ml, 300 ml, 350 ml, 400 ml, 450 ml, 500 ml, 550 ml, 600 ml, 800 ml, 1000 ml, 1500 ml, or any range from which it can be derived, depending on the needs of the culture. The culture vessel can be a bioreactor, which can refer to any device or system that supports a biologically active environment. The volume of the bioreactor can be at least or about 2, 4, 5, 6, 8, 10, 15, 20, 25, 50, 75, 100, 150, 200, 500 liters, 1, 2, 4, 6, 8, 10, 15 cubic meters, or any range that can be derived therefrom.

[0347] The culture container may be cell-adhesive or non-cell-adhesive and is selected according to the purpose. The cell-adhesive culture container may be coated with any substance for cell adhesion, such as an extracellular matrix (ECM) to improve the adhesion of the vascular surface to the cells. The substrate for cell adhesion may be any material intended for attaching stem cells or feeder cells (if used). Substrates for cell adhesion include collagen, gelatin, poly-L-lysine, poly-D-lysine, laminin and fibronectin and mixtures thereof, such as Matrigel. TM , and lysed cell membrane preparations.

[0348] C. Matrix components

[0349] A variety of defined matrix components can be used in the culture method or composition. For example, a combination of recombinant collagen IV, fibronectin, laminin, and vitronectin can be used to coat the culture surface as a means of providing a solid support for the growth of pluripotent cells, as described in Ludwig et al. (2006a; 2006b), which is incorporated by reference in its entirety.

[0350] The matrix composition can be fixed on the surface to provide support for cells. The matrix composition can include one or more extracellular matrix (ECM) proteins and aqueous solvents. The term "extracellular matrix" is recognized in the art. Its components include one or more of the following proteins: fibronectin, laminin, vitronectin, tenascin, haptospondin, thrombospondin, elastin, gelatin, collagen, fibrillin, partition protein, anchor protein, cartilage adhesion protein, connexin, bone sialoprotein, osteocalcin, osteopontin, epinectin, hyaluronectin, undulin, epidermal integrin and kalinin. Other extracellular matrix proteins are described in Kleinman et al, (1993), which is incorporated herein by reference. It is expected that the term "extracellular matrix" covers currently unknown extracellular matrices that may be found in the future, because its characterization as an extracellular matrix will be easily determined by those skilled in the art.

[0351] The total protein concentration in the matrix composition may be about 1 ng / mL to about 1 mg / mL. The total protein concentration in the matrix composition may be about 1 μg / mL to about 300 μg / mL. The total protein concentration in the matrix composition may be about 5 μg / mL to about 200 μg / mL.

[0352] Extracellular matrix (ECM) protein can be natural origin and purified from human tissue or animal tissue. Alternatively, ECM protein can be a genetically engineered recombinant protein or naturally synthesized. ECM protein can be a complete protein or in the form of a natural or modified peptide fragment. Some examples of ECM proteins that can be used for the matrix of cell culture include laminin, collagen I, collagen IV, fibronectin and vitronectin. Matrix composition can include synthetically produced fibronectin or peptide fragments of recombinant fibronectin.

[0353] The matrix composition may comprise a mixture of at least fibronectin and vitronectin.The matrix composition may comprise laminin.

[0354] The matrix composition preferably comprises a single type of extracellular matrix protein. The matrix composition may comprise fibronectin, particularly for culturing hematopoietic stem cells or progenitor cells. For example, a suitable matrix composition can be prepared by diluting human fibronectin in Dulbecco's phosphate buffered saline (DPBS) to a protein concentration of 5 μg / mL to about 200 μg / mL, such as human fibronectin sold by Becton, Dickinson & Co. of Franklin Lakes, NJ (BD) (Cat. No. 354008). In a specific example, the matrix composition comprises a fibronectin fragment, such as It is a protein of approximately 63 kDa (574 amino acids) that contains a central cell-binding domain (type III repeats, 8, 9, 10), a high-affinity heparin-binding domain II (type III repeats, 12, 13, 14), and a CS1 site within the alternatively spliced ​​IIICS region of human fibronectin.

[0355] The matrix composition may include laminin. For example, a suitable matrix composition can be prepared by diluting laminin (Sigma-Aldrich (St. Louis, Mo.); Catalog Nos. L6274 and L2020) in Dulbecco's phosphate buffered saline (DPBS) to a protein concentration of 5 μg / ml to about 200 μg / ml.

[0356] The matrix composition can be xeno-free, i.e., the matrix or its component proteins are of human origin only. This may be desirable for certain research applications. For example, in a xeno-free matrix for culturing human cells, human-derived matrix components can be used, wherein any non-human animal components can be excluded. TM Can be excluded from culture compositions as a substrate. TM It is a gelatinous protein mixture secreted by mouse tumor cells and is commercially available from BD Biosciences (New Jersey, USA). This mixture resembles the complex extracellular environment found in many tissues and is often used by cell biologists as a substrate for cell culture, but it may introduce undesirable foreign antigens or contaminants.

[0357] IX. Cell Culture Compositions and Methods

[0358] 3D culture compositions such as artificial thymic organoids (ATO) are optimized, efficient and highly reproducible off-the-shelf solutions for producing mature cells in vitro from pluripotent starting cell sources. 3D culture compositions can use serum-free conditions, avoid the use of human thymic tissue or proprietary scaffold materials, and are conducive to the positive selection and robust production of fully functional mature human cells (e.g., mature human T cells and NK cells) from stem cells. As a potential commercial platform for in vitro cell development, 3D culture compositions provide efficiency, repeatability, scalability, and reduced costs and labor compared to competing technologies. Non-restrictive commercial applications may include in vitro experimental modeling of human cell development, and in vitro production of cells and / or transgenic expression cells of modified targeted antigens from a variety of stem cell sources, which can be used for cell therapy, such as immunotherapy for the treatment of cancer and autoimmune disorders.

[0359] An optimized three-dimensional (3D) culture system for generating functional mature cells in vitro is also provided. The resulting cellular 3D structure can be referred to as an artificial thymic organoid (ATO).

[0360] The system may comprise aggregating PSCs with stromal cells expressing Notch ligands in a 3D structure in the presence of an optimized culture medium comprising FLT3 ligand (FLT3L), interleukin 7 (IL-7), B27, and ascorbic acid. Conditions that allow culture at an air-liquid interface may also be present.

[0361] Also provided is a method for 3D culture composition as developed (e.g., ATO production), which involves incubating MS-5 murine stromal cell line transduced with human DLL4 (hereinafter referred to as MS5-hDLL4) with stem cells or progenitor cells and / or CD34 isolated from human umbilical cord blood, bone marrow, whole blood samples or peripheral blood mononuclear cells (PBMCs). + PSC aggregation. As many as 1×10 6 stem or progenitor cells and / or CD34 + PSCs were mixed with MS5-hDLL4 cells at an optimized ratio (HSPC:stromal cells typically 1:10).

[0362] For example, aggregation is achieved by centrifuging a mixed cell suspension ("packed aggregation") followed by aspiration of the cell-free supernatant. The cell pellet can then be aspirated as a slurry in 5 to 10 μl of differentiation medium and transferred as a droplet onto a 0.4 μm nylon transwell culture insert that is floated in a well of differentiation medium such that the bottom of the insert is in contact with the medium and the top is in contact with air.

[0363] For example, differentiation medium is made up of RPMI-1640, 5ng / ml human FLT3L, 5ng / ml human IL-7, 4% serum-free B27 supplement and 30μM L-ascorbic acid. Culture inserts can be completely replaced with culture medium every 3 to 4 days. The change of scheme allows the use of alternative components with different effects on efficacy, specifically: basal medium RPMI can be replaced with several commercially available substitutes (such as IMDM). The stromal cell line used is the previously described mouse bone marrow cell line MS-5 (Itoh et al, 1989), but MS-5 can be replaced with similar mouse stromal cell lines (such as OP9, S17), human stromal cell lines (such as HS-5, HS-27a), primary human stromal cells or stromal cells derived from human pluripotent stem cells.

[0364] The stromal cell line is transduced with a lentivirus encoding human DLL4 cDNA; however, the method of gene delivery and the Notch ligand gene can vary. Alternative Notch ligand genes include DLL1, JAG1, JAG2, etc. Notch ligands also include those described in U.S. Patents 7795404 and 8377886, which are incorporated herein by reference. Notch ligands also include δ1, 3 and 4 and Jagged1, 2. Notch ligands also include functional fragments of Notch ligands. The types and sources of PSCs may include bone marrow, whole blood, umbilical cord blood, peripheral blood, peripheral blood mononuclear cells, or thymus, or PSCs or hematopoietic stem cells or progenitor cells may be differentiated from embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) in vitro. PSCs or hematopoietic stem cells or progenitor cells from primary tissues or ESCs or iPSCs may be derived from humans or non-human animals (e.g., mice).

[0365] X. Disease Treatment

[0366] The methods can be used for individuals who test positive for such diseases or are considered to be at risk for developing such conditions or related conditions.The compositions and methods described herein can be used to treat inflammatory or autoimmune components of the diseases listed herein and / or known in the art.

[0367] The present disclosure relates to the treatment of cancer and / or the use of cancer antigens. The cancer or antigen to be treated can be an antigen associated with any cancer known in the art, or for example: epithelial cancer (e.g., breast cancer, gastrointestinal cancer, lung cancer), prostate cancer, bladder cancer, lung (e.g., small cell lung) cancer, colon cancer, ovarian cancer, brain cancer, gastric cancer, renal cell carcinoma, pancreatic cancer, liver cancer, esophageal cancer, head and neck cancer, or colorectal cancer. The cancer or antigen to be treated may be from one of the following cancers: adenocortical carcinoma, myeloid metaplasia of unknown cause, AIDS-related cancers (e.g., AIDS-related lymphoma), anal cancer, appendix cancer, astrocytoma (e.g., cerebellar and cerebral), basal cell carcinoma, bile duct cancer (e.g., extrahepatic), bladder cancer, bone cancer, (osteosarcoma and malignant fibrous histiocytoma), brain tumor (e.g., glioma, brain stem glioma, cerebellar or cerebral astrocytoma (e.g., pilocytic astrocytoma, diffuse astrocytoma, anaplastic (malignant) astrocytoma), malignant glioma, ependymoma, oligodenglioma ), meningiomas, meningiosarcoma, craniopharyngiomas, haemangioblastomas, medulloblastomas, supratentorial primitive neuroectodermal tumors, optic pathway and hypothalamic gliomas and glioblastomas), breast cancer, bronchial adenocarcinoma / carcinoid, carcinoid tumors (e.g., gastrointestinal carcinoid tumors), cancer of unknown primary, central nervous system lymphoma, cervical cancer, colon cancer, colorectal cancer, chronic myeloproliferative disorders, endometrial cancer (e.g., uterine cancer), ependymomas, esophageal cancer, Ewing's family of tumors, eye cancers (e.g., intraocular melanoma and retinoblastoma), gallbladder cancer, stomach (gastric) cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors,GIST), germ cell tumors (e.g., extracranial, extragonadal, ovarian), gestational trophoblastic tumor, head and neck cancer, hepatocellular (liver) cancer (e.g., hepatic carcinoma and heptoma), hypopharyngeal cancer, islet cell carcinoma (endocrine pancreas), laryngeal cancer, laryngeal cancer, leukemia, lip and oral cancer, oral cancer, liver cancer, lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma), lymphoid neoplasms (e.g., lymphoma), medulloblastoma, ovarian cancer, mesothelioma, metastatic squamous neck cancer, oral cancer, multiple endocrine neoplasia syndrome, myelodysplastic syndrome, myelodysplastic / myeloproliferative disorders, nasal and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, neuroendocrine cancer, oropharyngeal cancer , ovarian cancer (e.g., epithelial ovarian cancer, ovarian germ cell tumor, ovarian low-grade malignant potential tumor), pancreatic cancer, parathyroid cancer, penile cancer, peritoneal cancer, pharyngeal cancer, pheochromocytoma, pinealoblastoma and supratentorial primitive neuroectodermal tumor, pituitary tumor, pleuropulmonary blastoma, lymphoma, primary central nervous system lymphoma (microglioma), pulmonary lymphangioleiomyomatosis, rectal cancer, kidney cancer, renal pelvis and ureter cancer (transitional cell carcinoma), rhabdomyosarcoma, salivary gland cancer, skin cancer (e.g., non-melanoma (e.g., squamous cell carcinoma), melanoma, and Merkel cell carcinoma) cell carcinoma), small intestinal cancer, squamous cell carcinoma, testicular cancer, laryngeal cancer, thymoma and thymic cancer, thyroid cancer, tuberous sclerosis, urethral cancer, vaginal cancer, vulvar cancer, Wilms' tumor and post-transplant lymphoproliferative disorder (PTLD), abnormal blood vessel proliferation associated with phakomatoses, edema (e.g. associated with brain tumors) or Meigs' syndrome.

[0368] The present disclosure relates to the treatment of autoimmune disorders and / or the use of autoimmune-related antigens. The autoimmune disease or antigen to be treated can be an antigen associated with any autoimmune disorder known in the art or, for example, diabetes, transplant rejection, GVHC, arthritis (rheumatoid arthritis such as acute arthritis, chronic rheumatoid arthritis, gout or gouty arthritis, acute gouty arthritis, acute immune arthritis, chronic inflammatory arthritis, degenerative arthritis, type II collagen-induced arthritis, infectious arthritis, Lyme arthritis, proliferative arthritis, psoriatic arthritis, Still's disease, vertebral osteoarthritis and juvenile rheumatoid arthritis, osteoarthritis, chronic progressive arthritis (arthritis chronica progrediente), deforming arthritis, primary chronic polyarthritis (polyarthritis chronica primaria), reactive arthritis and ankylosing spondylitis), inflammatory hyperproliferative skin diseases, psoriasis such as plaque psoriasis, guttate psoriasis, pustular psoriasis and nail psoriasis, atopy including atopic diseases such as hay fever and Job's syndrome, dermatitis including contact dermatitis, chronic contact dermatitis, exfoliative dermatitis, allergic dermatitis, allergic contact dermatitis, dermatosis herpetiformis dermatitis, nummular dermatitis, seborrheic dermatitis, nonspecific dermatitis, primary irritant contact dermatitis and atopic dermatitis, x-linked hyper-IgM syndrome, allergic intraocular inflammatory diseases, urticaria such as chronic allergic urticaria and chronic idiopathic urticaria, including chronic autoimmune urticaria, myositis, polymyositis / dermatomyositis, juvenile dermatomyositis, toxic epidermal necrolysis, scleroderma (including systemic sclerosis), sclerosis, such as systemic sclerosis, multiple sclerosis (MS), sclerosis (MS) such as spinal-ocular MS, primary progressive MS (PPMS) and relapsing-remitting MS (RRMS), progressive systemic sclerosis, atherosclerosis, arteriosclerosis, disseminated sclerosis, ataxia sclerosis (ataxicsclerosis), neuromyelitis optica (NMO), inflammatory bowel disease (IBD) (e.g. Crohn's disease, autoimmune-mediated gastrointestinal diseases, colitis such as ulcerative colitis, colitis ulcera, microscopic colitis, collagenous colitis, polyposis colitis, necrotizing enterocolitis and transmural colitis, and autoimmune inflammatory bowel disease), enteritis, pyoderma gangrenosum, erythema nodosum, primary sclerosing cholangitis, respiratory distress syndrome,Includes adult or acute respiratory distress syndrome (ARDS), meningitis, inflammation of all or part of the uvea, iris, choroid, autoimmune blood disorders, rheumatoid spondylitis, rheumatoid synovitis, hereditary angioedema, cranial nerve damage such as meningitis, herpes gestationis, pemphigoid gestationis pruritus, scrotal pruritus, autoimmune premature ovarian failure, sudden hearing loss due to autoimmune disease, IgE-mediated diseases such as allergies and allergic and atopic rhinitis, encephalitis such as Rasmussen's encephalitis (Rasmussen's encephalitis) and limbic and / or brainstem encephalitis, uveitis such as anterior uveitis, acute anterior uveitis, granulomatous uveitis, non-granulomatous uveitis, phacoantigenic uveitis, posterior uveitis or autoimmune uveitis, glomerulonephritis (GN) with and without nephrotic syndrome such as chronic or acute glomerulonephritis, such as primary GN, immune-mediated GN, membranous GN (membranous nephropathy), idiopathic membranous GN or idiopathic membranous nephropathy, membranous or membranous proliferative GN (MPGN) (including type I and type II) and rapidly progressive GN, proliferative nephritis, autoimmune polyglandular endocrine disorders, balanitis including balanitis circumscripta plasmacellularis, balanitis, erythema annulare centrifugum, erythema persica dyschromicumperstans), erythema multiforme, granuloma annulare, lichen nitidus, lichen sclerosus et atrophicus, lichen simplex chronicus, lichen spinulosus, lichen planus, lamellar ichthyosis, epidermolytic hyperkeratosis, premalignant keratosis, pyoderma gangrenosum, allergic conditions and responses, allergic reactions, eczema including allergic or atopic eczema, dry eczema, hygroma and vesicular palmoplantar eczema, asthma such as asthmatic bronchitis, bronchial asthma and autoimmune asthma, conditions involving T cell infiltration and chronic inflammatory responses, immune responses to foreign antigens such as fetal ABO blood type during pregnancy, chronic pulmonary inflammatory diseases, autoimmune myocarditis, leukocyte adhesion deficiency, lupus,These include lupus nephritis, lupus encephalitis, pediatric lupus, non-renal lupus, extrarenal lupus, discoid lupus and discoid lupus erythematosus, alopecia, systemic lupus erythematosus (SLE) such as cutaneous SLE or subacute cutaneous SLE, neonatal lupus syndrome (NLE) and disseminated lupus erythematosus infection, juvenile (type I) diabetes, including pediatric insulin-dependent diabetes mellitus (IDDM) and adult diabetes (type II diabetes), and autoimmune diabetes. Also contemplated are immune responses associated with acute and delayed hypersensitivity reactions mediated by cytokines and T-lymphocytes, sarcoidosis, granulomatous diseases including lymphomatoid granulomatosis, Wegener's granulomatosis, agranulocytosis, vasculitis including vasculitis, large vessel vasculitis (including polymyalgia rheumatica and giant T cell (Takayasu's) arteritis), medium vessel vasculitis (including Kawasaki's disease and polyarteritis nodosa / periarteritis nodosa), microscopic polyarteritis, immune vasculitis, CNS vasculitis, cutaneous vasculitis, hypersensitivity vasculitis, necrotizing vasculitis such as systemic necrotizing vasculitis and ANCA-associated vasculitis such as Churg-Strauss vasculitis or syndrome (CSS) and ANCA-associated small vessel vasculitis, temporal arteritis, aplastic anemia, autoimmune aplastic anemia, Coombs positive anemia (Coombs positive anemia, Diamond Blackfan anemia, hemolytic anemia or immune hemolytic anemia including autoimmune hemolytic anemia (AIHA), Addison's disease, autoimmune neutropenia, pancytopenia, leukopenia, diseases involving leukocytopenia, CNS inflammatory disorders, Alzheimer's disease, Parkinson's disease, multiple organ injury syndromes such as those secondary to sepsis, trauma or hemorrhage, antigen-antibody complex mediated diseases, anti-glomerular basement membrane disease, antiphospholipid antibody syndrome, allergic neuritis, Behcet's disease / syndrome, Castleman syndrome, Goodpasture syndrome, Reynaud syndrome, Sjogren syndrome, Stevens-Johnson syndrome,Pemphigoid such as bullous pemphigoid and cutaneous pemphigoid, pemphigus (including pemphigus vulgaris, pemphigus foliaceus, pemphigus mucosa pemphigoid and pemphigus erythematosus), autoimmune polyendocrinopathy, Reiter's disease or syndrome, thermal injury, preeclampsia, immune complex disorders such as immune complex nephritis, antibody-mediated nephritis, polyneuropathy, chronic neuropathies such as IgM polyneuropathy or IgM-mediated neuropathy, autoimmune or immune-mediated thrombocytopenia such as idiopathic thrombocytopenic purpura, purpura, ITP) including chronic or acute ITP, scleritis such as idiopathic cerato-scleritis, episcleritis, autoimmune diseases of the testicles and ovaries including autoimmune orchitis and oophoritis, primary hypothyroidism, hypoparathyroidism, autoimmune endocrine diseases including thyroiditis such as autoimmune thyroiditis, Hashimoto's disease, chronic thyroiditis (Hashimoto's thyroiditis) or subacute thyroiditis, autoimmune thyroid disease, idiopathic hypothyroidism, Graves' disease disease), polyglandular syndromes such as autoimmune polyglandular syndromes (or polyglandular endocrine syndromes), paraneoplastic syndromes, including neurological paraneoplastic syndromes such as Lambert-Eaton myasthenic syndrome or Eaton-Lambert syndrome, stiff-man or stiff-person syndrome, encephalomyelitis such as allergic encephalomyelitis or encephalomyelitis allergic and experimental allergic encephalomyelitis (EAE), experimental autoimmune encephalomyelitis, myasthenia gravis such as thymoma-associated myasthenia gravis, cerebellar degeneration, neuromyotonia, opsoclonus or opsoclonusmyoclonus syndrome (OMS), and sensory neuropathy, multifocal motor neuropathy, Sheehan syndrome, autoimmune hepatitis, chronic hepatitis, lupus hepatitis, giant cell hepatitis, chronic active hepatitis or autoimmune chronic active hepatitis, lymphoid interstitial pneumonia (lymphoid interstitial pneumonia), pneumonitis (LIP), bronchiolitis obstructive (non-transplant) vs NSIP, Guillain-Barre syndrome, Berger's disease (IgA nephropathy), idiopathic IgA nephropathy, linear IgA dermatosis, acute febrile neutrophilic dermatosis, subcorneal pustular dermatosis, transient acantholytic dermatosis, cirrhosis such as primary biliary cirrhosis and pulmonary cirrhosis, autoimmune enteropathy syndrome,Celiac or Coeliac disease, celiac sprue (gluten enteropathy), refractory sprue, idiopathic sprue, cryoglobulinemia, amyotrophic lateral sclerosis (ALS; Lou Gehrig's disease), coronary artery disease, autoimmune ear disease such as autoimmune inner ear disease (AIED), autoimmune hearing loss, polychondritis such as refractory or relapsing or relapsing polychondritis, pulmonary alveolar proteinosis, Cogan's syndrome / nonsyphilitic interstitial keratitis, Bell's palsy palsy), Sweet disease / syndrome, autoimmune rosacea, pain associated with herpes zoster, amyloidosis, noncancerous lymphocytosis, primary lymphocytosis including monoclonal B-cell lymphocytosis (e.g., benign monoclonal gammopathy and monoclonal gammopathy of undetermined significance, MGUS), peripheral neuropathies, paraneoplastic syndromes, channelopathies such as epilepsy, migraine, cardiac arrhythmias, muscle disorders, deafness, blindness, periodic paralysis, and CNS channelopathies, autism, inflammatory myopathies, focal or segmental glomerulosclerosis, or focal segmental glomerulosclerosis glomerulosclerosis (FSGS), endocrine eye diseases, uveoretinitis, chorioretinitis, autoimmune liver diseases, fibromyalgia, multiple endocrine failure, Schmidt's syndrome, adrenalitis, gastric atrophy, Alzheimer's disease, demyelinating diseases such as autoimmune demyelinating diseases and chronic inflammatory demyelinating polyneuropathy, Dressler syndrome, alopecia greata, alopecia totalis, CREST syndrome (calcification, Raynaud's phenomenon, esophageal dysmotility, sclerodactyl and telangiectasia), male and female autoimmune infertility, for example due to antisperm antibodies, mixed connective tissue disease, Chagas disease, rheumatic fever, recurrent miscarriage, farmer's lung, erythema multiforme, postcardiotomy syndrome, Cushing's syndrome, bird-fancier's lung), allergic granulomatous vasculitis, benign lymphocytic vasculitis, Alport syndrome, alveolitis such as allergic alveolitis and fibrosing alveolitis, interstitial lung disease, transfusion reaction,Leprosy, malaria, parasitic diseases such as leishmaniasis, kypanosomiasis, schistosomiasis, ascariasis, aspergillosis, Sampter's syndrome, Caplan's syndrome, dengue fever, endocarditis, endomyocardial fibrosis, diffuse pulmonary interstitial fibrosis, interstitial pulmonary fibrosis, pulmonary fibrosis, idiopathic pulmonary fibrosis, cystic fibrosis, endophthalmitis, erythema elevatum et diutinum, erythroblastosis fetalis, eosinophilic fasciitis, Shulman's syndrome, Felty's syndrome, fliasis, cyclitis such as chronic cyclitis, metachronous cyclitis, iridocyclitis (acute or chronic), or Fuch's cyclitis, Henoch-Schonlein purpura, human immunodeficiency virus (HIV) HIV infection, SCID, acquired immune deficiency syndrome (AIDS), echovirus infection, sepsis, endotoxemia, pancreatitis, thyroxicosis, parvovirus infection, rubella virus infection, post-vaccination syndrome, congenital rubella infection, Epstein-Barr virus infection, mumps, Evan syndrome, autoimmune gonadal failure, Sydenham's chorea, poststreptococcal nephritis, thromboangitis obliterans ubiterans, thyrotoxicosis, tabes dorsalis, chorioiditis, giant cell polymyalgia, chronic allergic pneumonitis, keratoconjunctivitis sicca, epidemic keratoconjunctivitis, idiopathic nephritic syndrome, minimal change nephropathy, benign familial and ischemia-reperfusion injury, transplant reperfusion, autoimmune retinal, joint inflammation, bronchitis, chronic obstructive airway / lung disease, silicosis, aphthous stomatitis, aphthous stomatitis, arteriosclerosis, asperniogenese, autoimmune hemolysis, Boeck's disease, cryoglobulinemia, Dupuytren's contracture, endophthalmia phacoanaphylactica, allergic enteritis, erythema nodosum leprosum, idiopathic facial palsy, chronic fatigue syndrome, febris rheumatica, Hamman-Rich disease, sensorineural hearing loss, haemoglobinuria paroxysmatica, hypogonadism,Ileitis regionalis, leukopenia, infectious mononucleosis, transverse myelitis, primary idiopathic myxedema, nephropathy, sympathetic ophthalmia symphatica), granulomatous orchitis, pancreatitis, acute polyradiculitis, pyoderma gangrenosum, Quervain's thyroiditis, acquired splenic atrophy, nonmalignant thymoma, vitiligo, toxic shock syndrome, food poisoning, disorders involving T-cell infiltration, leukocyte adhesion defects, immune responses associated with acute and delayed hypersensitivity reactions mediated by cytokines and T-lymphocytes, diseases involving leukocytic infiltration, multiple organ injury syndrome, diseases mediated by antigen-antibody complexes, anti-glomerular basement membrane disease, allergic neuritis, autoimmune polyendocrinopathy, oophoritis, primary myxedema, autoimmune atrophic gastritis, sympathetic ophthalmia, rheumatic diseases, mixed connective tissue disease, nephrotic syndrome, insulitis, polyendocrine failure, autoimmune polyglandular syndrome type I, adult-onset idiopathic hypoparathyroidism hypoparathyroidism (AOIH), cardiomyopathies such as dilated cardiomyopathy, epidermolysis bullosa acquisita (EBA), hemochromatosis, myocarditis, nephrotic syndrome, primary sclerosing cholangitis, suppurative or nonpurulent sinusitis, acute or chronic sinusitis, ethmoid, frontal, maxillary, or sphenoid sinusitis, eosinophil-related disorders such as eosinophilia, pulmonary infiltrate eosinophilia, eosinophilia-myalgia syndrome, Loffler's syndrome, chronic eosinophilic pneumonia, tropical pulmonary eosinophilia, bronchopneumonia with eosinophils, aspergillosis, or granuloma, allergies, seronegative spondyloarthritis, polyendocrine autoimmune disease, sclerosing cholangitis, sclera, episclera, chronic mucocutaneous candidiasis, Bruton's syndrome syndrome, transient hypogammaglobulinemia of infancy, Wiskott-Aldrich syndrome, ataxia-telangiectasia syndrome, vasculoectasia, autoimmune diseases associated with collagen diseases, rheumatism, neuropathy, lymphadenitis, decreased blood pressure response, vascular dysfunction, tissue damage, cardiovascular ischemia, hyperalgesia, renal ischemia, cerebral ischemia and diseases with angiogenesis, allergic hypersensitivity disorders, glomerulonephritis, reperfusion injury, ischemic reperfusion disorder, reperfusion injury of the myocardium or other tissues, lymphomatous tracheobronchitis, inflammatory skin diseases, skin diseases with acute inflammatory components, multiple organ failure, bullous diseases, renal cortical necrosis,Acute purulent meningitis or other inflammatory conditions of the central nervous system, inflammatory conditions of the eye and orbit, granulocyte transfusion-related syndrome, cytokine-induced toxicity, narcolepsy, acute severe inflammation, chronic refractory inflammation, pyelitis, intra-arterial hyperplasia, peptic ulcer, valvulitis, graft-versus-host disease, contact hypersensitivity, asthmatic airway hyperresponsiveness, and endometriosis.

[0369] Cells can be formulated in a manner suitable for delivery to a recipient without adverse effects. They may be formulated into a cell suspension, or may not be formulated into a cell suspension. In certain cases, they are formulated in a single dose form. They may be formulated for systemic administration or local administration. In some cases, cells are formulated for storage before use, and the cell preparation may include one or more cryopreservatives, such as DMSO (e.g., in 5% DMSO). Cell preparations may include albumin, including human albumin, and specific preparations include 2.5% human albumin. Cells may be formulated specifically for intravenous administration; for example, they are formulated for intravenous administration for less than 1 hour. Cells may be in a formulated cell suspension that is stable at room temperature for 1, 2, 3 or 4 hours or longer from the time of thawing.

[0370] The therapeutic compositions and treatments disclosed herein may include administering a combination of therapeutic agents (e.g., a first therapeutic composition or pharmaceutical composition or treatment and a second therapeutic composition or pharmaceutical composition or treatment). The treatment may be administered in any suitable manner known in the art. For example, the therapeutic composition or pharmaceutical composition or treatment may be administered sequentially (at different times) or simultaneously (at the same time). The therapeutic composition or pharmaceutical composition or treatment may be administered as a separate composition. The therapeutic composition or pharmaceutical composition or treatment may be in the same composition.

[0371] The present disclosure also describes compositions and methods, which include therapeutic compositions. Different therapeutic compositions or pharmaceutical compositions or treatments can be administered as one composition or as more than one composition, such as 2 compositions, 3 compositions, or 4 compositions. Various combinations of agents can be used.

[0372] The therapeutic composition or pharmaceutical composition and treatment disclosed herein can be carried out before, simultaneously with and / or after another treatment or medicament, with an interval of several minutes to several weeks. In the case where the medicament is applied to cells, tissues or organisms respectively, it will generally be ensured that the important period between the time of each delivery does not expire (expire), so that the therapeutic agent or medicament will still be able to play a favorable combined effect on cells, tissues or organisms. For example, in such a case, it is expected that cells, tissues or organisms can be contacted with two, three, four or more medicaments or treatments substantially at the same time (that is, in less than about one minute). In other aspects, one or more therapeutic agents or treatments can be administered or provided within 1 minute, 5 minutes, 10 minutes, 20 minutes, 30 minutes, 45 minutes, 60 minutes, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 22 hours, 23 hours, 24 hours, 25 hours, 26 hours, 27 hours, 28 hours, 29 hours, 30 hours, 31 hours, 32 hours, 33 hours, 34 hours, 35 hours, 36 hours, 37 hours, 38 hours, 39 hours, 40 hours, 41 hours, 42 hours, 43 hours, 44 hours, 45 hours, 46 hours, 47 hours, 48 ​​hours, 49 hours, 50 hours, 51 hours, 52 hours, 53 hours, 54 hours, 55 hours, 56 hours, 57 hours, 58 hours, 59 hours, 29 hours, 30 hours, 31 hours, 32 hours, 33 hours, 34 hours, 35 hours, 36 hours, 37 hours, 38 hours, 39 hours, 40 hours, 41 hours, 42 hours, 43 hours, 44 hours, 45 hours, 46 hours, 47 hours, 48 ​​hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks or 8 weeks or longer, and any range derivable therein.

[0373] Treatment may include multiple "unit doses". A unit dose is defined as a therapeutic composition containing a predetermined amount. The amount to be administered as well as the specific route and formulation are within the skill of those skilled in the clinical art to determine. A unit dose need not be administered as a single injection, but may comprise a continuous infusion over a set period of time. A unit dose may comprise a single administrable dose.

[0374] A therapeutically effective or sufficient amount of a therapeutic composition or treatment administered to a human may be about 10 2 Up to about 10 10 The range of cells, whether by one or more administrations. For example, the treatment used may be administered daily, for example, about 10 2 cells to about 10 9 cells / kg, about 10 2 cells to about 10 8 cells / kg, about 102 cells to about 10 7 cells / kg, about 10 2 cells to about 10 6 cells / kg, about 10 2 cells to about 10 5 cells / kg, about 10 2 cells to about 10 4 cells / kg, or about 10 2 cells to about 10 3 The treatment described herein may be carried out at about 10 2 cells, about 10 3 cells, about 10 4 cells, about 10 5 cells, about 10 6 cells, about 10 7 cells, about 10 8 cells, about 10 9 cells or about 10 10 The dosage of each cell is administered to the subject. The dosage can be administered as a single dose or as multiple doses (e.g., 2 or 3 doses), such as infusions. The progress of such treatment is easily monitored by conventional techniques.

[0375] When an "immunologically effective amount," "anti-tumor effective amount," "tumor suppressive effective amount," or "therapeutic amount" is indicated, the physician can determine the precise amount of the composition of the present disclosure to be administered taking into account individual differences in the patient's (subject's) age, weight, tumor size, degree and condition of infection or metastasis.

[0376] XI.Medicine Box

[0377] The present disclosure also describes kits containing the compositions of the present disclosure or the compositions for practicing the methods of the present disclosure. The kits can be used to evaluate the expression of a transgene, the incorporation of a transgene into a genome, or for differentiating stem cells or progenitor cells containing a transgene into mature cells. The kit may comprise, contain at least or contain at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 100, 500, 1,000 or more probes, primers or primer sets, synthetic molecules or inhibitors, or any value or range and combination derivable therefrom.

[0378] The kit may include components that may be packaged individually or placed in a container, such as a tube, bottle, vial, syringe or other suitable container means. The kit may include cell culture components, compositions and culture vessels described herein.

[0379] XII. Examples

[0380] The following examples are included to illustrate some preferred embodiments of the present invention. It will be appreciated by those skilled in the art that the techniques disclosed in the following examples represent techniques that the inventors have found to work well in the practice of the present invention, and therefore can be considered to constitute preferred modes for its practice. However, based on this disclosure, it will be appreciated by those skilled in the art that many changes may be made to some of the disclosed specific embodiments without departing from the spirit and scope of the present invention and still obtaining the same or similar results.

[0381] Example 1 - Regulated gene expression during differentiation of mature T cells

[0382] H1 hESCs were transduced with a lentiviral vector co-expressing CAR19LH and the reporter eGFP. Cells were sorted for high eGFP expression and clones were evaluated for average vector copy number. H1 CAR19LH-eGFP clone 2 (VCN:4.6) was differentiated in ATO along with wild-type H1 controls and analyzed at weeks 2 to 5. H1 CAR19LH-eGFP clone 2 exhibited aberrant differentiation with a low frequency of CD3+TCRab+ cells compared to wild-type ( Figure 2A In addition, clone 2 also showed developmental arrest of SP4 and SP8 cells, as well as a predominant CD4-CD8- “double negative” cell population ( Figure 2B ).

[0383] like Figure 3 As shown, cells show stage-specific expression of a transgene inserted downstream of GZMA. CRISPR / Cas9-mediated homology-directed repair was performed in the H1 hESC line to insert the reporter mCitrine in the 3'UTR of the endogenous GZMA gene. The heterozygous H1GZMA-mCitrine reporter was cloned and differentiated in ATO along with wild-type H1 controls. The level of reporter expression was evaluated at different stages of T cell development during weeks 1, 3, and 5. Analysis of H1 GZMA-mCitrine Het clone 9 is shown. Mcitrine was expressed in 23% of SP8 and 2% of SP4, but not in the precursor stages ISP4 or DP.

[0384] Expression of CAR19LH by controlling the endogenous GZMA promoter allows CAR T cells to develop normally. Using a similar knock-in protocol, the CAR19LH exogenous gene was inserted into the 3'UTR of the endogenous GZMA gene in the T-iPSC line "n11:". This approach will delay the expression of CAR19LH until T cells reach the SP8 and SP4 stages of maturity, as demonstrated by previous reporter experiments. N11 GZMA-CAR19LH homozygous clones undergo differentiation in ATO. Figure 3 a shows a high frequency of CD3+TCRab+ cells and the normal developmental pattern of DP cells (week 1), SP8 cells (week 3), and SP4 cells (week 4). Figure 3 b shows staining of CAR using anti-FMC63 scFv antibody. At week 4, 67.8% of SP8 and 6.37% of SP4 expressed CAR, and at week 9, 76.9% of SP8 and 7.15% of SP4 expressed CAR. This is shown in Figure 4. Example 2. Engineering circumvention of biological barriers to allogeneic iPSC-derived chimeric antigen receptor therapy

[0385] Chimeric antigen receptor (CAR) T cell therapy has produced remarkable results in otherwise refractory hematological malignancies. Currently, due to the need to generate each personalized treatment from scratch and the inherent variability in T cell biology between patients, the process of manufacturing autologous CAR T cells is challenging, resulting in unpredictable and inconsistent clinical responses. Therefore, there is an increasing interest in the in vitro generation of CAR T cells from human pluripotent stem cells (hPSCs) of infinitely renewed allogeneic sources.

[0386] However, there are several biological barriers to generating CART cells from hPSCs. First, expressing the CAR transgene early in T cell differentiation diverts development to the intrinsic lymphoid pathway rather than the conventional T lineage. Second, removing the endogenous T cell receptor (T cellreceptor, TCR) to prevent alloreactivity leads to developmental arrest because mature T cells can no longer undergo positive selection. This program attempts to overcome these challenges in generating non-allogeneic reactive stem cell-derived CART cells by deploying innovative stage-specific expression strategies and new in vitro methods (artificial thymic organoid (ATO) system) to induce mature, conventional T cell differentiation.

[0387] ATO is a pioneering in vitro method for effectively generating mature single positive (SP) CD8+ and CD4+ T cells from a variety of stem cell sources, including hPSC. Improved on previously established systems, ATO can robustly support positive selection and maturation to the SP stage. Our preliminary studies have shown that inherent fate can be overcome by achieving delayed, stage-specific expression of CAR transgenes that are limited to mature T cells. In this program, we will be based on a delayed CAR expression model and evaluate how the destruction of endogenous TCR affects the development of CAR T cells in ATO. Then, we will evaluate two different methods of delivering exogenous positive selection signals to rescue development in the absence of endogenous TCR. These methods include expressing tonic signaling CAR or transiently expressing exogenous TCR at a physiologically appropriate DP T cell stage. The method developed in this program will enable the generation of non-alloreactive T cell therapy derived from hPSCs, increasing the chances of more patients in need receiving treatment.

[0388] As shown in Figure 5, early expression of CAR redirects T cell differentiation to the intrinsic lymphoid pathway in the ATO system. The inventors identified granzyme A (GZMA) as a potential candidate locus for anti-CD19 CAR knock-in ( Fig. 6A To B). CAR19 stage-specific expression through the GZMA locus allows the production of mature, conventional CART cells (Figure 7A to D). Limiting CAR19 expression to only mature SP T cells prevents early diversion to the intrinsic lymphoid pathway and allows conventional development (Figure 7B). By week 5, most ATO-derived T cells were CD8ɑβ+ and CAR19+ ( Figure 7C ).like Fig.7D As shown, mature SP8 CAR T cells robustly released cytotoxic cytokines and upregulated activation markers in response to the antigen CD19.

[0389] Here, the inventors report the generation of non-allogeneic TCR-free CAR T cells from T-iPSC. A stage-specific expression system has been established, which provides robust and reproducible time control of transgene expression during T cell differentiation. On the basis of this platform, the inventors have produced mature, conventional CART cells from T-iPSC, which show antigen-specific killing, activation and proliferation.

[0390] Example 3 - Regulated gene expression during differentiation of mature T cells

[0391] The goal of the present invention is to achieve the regulated gene expression of the inserted (transgenic / exogenous) T cell receptor (TCR), which is limited to a specific stage of T cell differentiation. Normal T cell development in the thymus requires the expression of TCR so that mature T cells are produced by positive selection. The method of removing TCR expression in T cell precursors [in order to prevent allogeneic response (allo-response) (graft versus host disease)] also prevents the positive selection of mature T cells. The ability to limit TCR expression to a specific stage of T cell differentiation will allow "positive selection" and induce the maturation of T cell precursors, while avoiding the negative results of ongoing transgenic TCR expression in mature T cells. The most easily applied application of the present invention is to stably modify pluripotent stem cells (PSC), but the method can potentially be used to target any cell type produced in vitro or in vivo in early T cell development, such as PSC, hematopoietic stem cells or progenitor cells (HSPC) or thymocyte precursors. The most direct application of this technology is to allow the in vitro production of therapeutic T cells from allogeneic PSCs that have been removed from endogenous TCR expression. The specific genes identified as the endogenous loci for TCR insertion were RAG1 and RAG2.

[0392] Adoptive T cell therapy, such as FDA-approved CART therapy, currently relies on the use of autologous T cells harvested from the patient's blood. This logistically difficult and expensive process has led to the exploration of using allogeneic T cells, which provides a potential off-the-shelf alternative to current methods using T cells from autologous (patient-specific) sources.

[0393] Allogeneic T cells can also be derived from the blood of healthy donors or generated in vitro from HSPCs or iPSCs. Clinical use of any allogeneic T cell product requires genetic modification to eliminate 1) endogenous TCR expression to avoid graft versus host disease (GvHD), and 2) expression of major histocompatibility complex antigens (class I and class II) to prevent donor cells from being rejected by the unmatched recipient immune system. Methods for two types of functional modifications have been developed in the field of allogeneic cell therapy. For example, elimination of endogenous TCR expression can be achieved by disrupting the TRAC locus or deleting RAG1+ / -RAG2 gene expression. Disruption of MHC class I expression can be achieved by deleting specific class I genes or beta 2 microglobulin (b2m), which is a component of all MHC class I molecules. Deletion of class II can be achieved by deleting CIITA.

[0394] However, the same TCR-MHC interaction that causes alloreactivity / rejection problems in an allogeneic environment is also crucial to normal T cell development. The interaction of TCR on the surface of so-called double positive / DP T cell precursors (named because they co-express both CD8 and CD4) and MHC molecules (provided by the thymic microenvironment, and also on the developing T cells themselves) is crucial for the normal "positive selection" process that occurs in the endogenous thymus. Positive selection allows DPs with optimal TCR-MHC affinity to survive and differentiate into so-called single positive / SP T lineage cells (named because they express one of CD4 or CD8, not both). Positive selection can be reproduced in vitro in the "artificial thymic organoid" (ATO) technology previously developed by the Crooks group to induce stem cell differentiation into SP4 and SP8 cells.

[0395] One solution to the problem of removing endogenous TCR expression mechanisms before positive selection is to constitutively express exogenous (transgenic) TCRs throughout T cell differentiation (including during β selection and positive selection). However, continued expression of exogenous TCRs in mature T cells produced during differentiation and infused into patients can create new problems - the possibility of off-target effects of exogenous TCRs through antigen expression in healthy tissues or allogeneic responses / GvHD. The present invention provides methods for providing TCR expression that is limited to the precursor stage of β and positive selection, i.e., lost in mature T cells. The present application is most relevant in the field of T cell therapy based on PSC, where gene deletions and insertions are typically applied to self-renewing PSCs, but it can also be used for any manipulation of TCR expression during or before the positive selection stage.

[0396] The inventors have determined that the RAG1 and RAG2 genes have tightly regulated stage-specific expression that is high during beta selection and positive selection of T cell differentiation, but decreases in mature T cells. The approach is to insert a transgenic TCR into the regulatory region and / or downstream of the endogenous RAG1 or RAG2 gene. In this way, any transgenic TCR will be available for direct positive selection in any T cell differentiation system, but will be downregulated in the mature T cells subsequently generated, thereby avoiding potential GvHD.

[0397] The homology-directed repair mediated by CRISPR / Cas9 is used to insert the biologically active transgenic (i.e., reporter, chimeric antigen receptor) into the framework of the 3' untranslated region of the endogenous gene. In the process, the stop codon is replaced by a serine-arginine linker and a self-cleaving peptide sequence (P2A). This method allows the expression of the transgenic inserted by regulating the endogenous promoter, and the translation of the endogenous gene remains unchanged. In addition, the puromycin resistance gene cassette can be inserted to help enrich the pluripotent stem cells (Fig. 1A) edited. After CRE-TAT treatment is carried out in the iPSC stage, the puromycin resistance cassette can be removed, leaving a single loxp3 sequence (Fig. 1B).

[0398] Figure 8A to B shows a representative flow cytometry analysis of T cell differentiation dynamics in seven weeks for RAG1 / 2KO human pluripotent stem cells (hPSC) and their WT (unedited) parental lines (H1, HLA-A*0201+; ESI017, HLA-A*0201-). Figure 8A shows surface staining of TCRab and CD3 in seven weeks for T lineage committed progenitor cells (CD45+CD5+CD7+) from RAG1 / 2DKO hPSC and WT parental lines. Figure 8B shows the progress of T cell development in seven weeks from the same T lineage committed progenitor cell group shown by surface staining of CD8a and CD4. Progenitor cells differentiate in the following order: double negative (DN, CD4-CD8-), immature single positive 4 (ISP4, CD4+CD8-), double positive (DP, CD4+CD8+), single positive (SP8, CD4-CD8+; SP4, CD4+CD8-).

[0399] Figure 9 shows the effect of lack of endogenous TCR expression on positive selection during T cell differentiation from hPSC. RAG1 / 2KO hPSC and WT (unedited) parental lines (H1, HLA-A*0201+; ESI017, HLA-A*0201-) were stably transduced with a lentiviral vector expressing an HLA-A*0201-restricted NYESO TCR (detectable using VB13.1 antibody) ( Figure 3 ). Figure 9A shows a representative flow cytometric analysis of VB13.1 and CD3 surface expression from total hematopoietic cells (CD45+) over seven weeks of T cell differentiation. Figure 9B shows positive selection of SP8 T cells from NYESO TCR-transduced RAG1 / 2DKO and parental WT hPSC lines. Total hematopoietic cells (CD45+VB13.1+CD3+) expressing VB13.1 and CD3 are shown over seven weeks of differentiation. When RAG1 / 2 is knocked out, the rescue of SP8 development is unique to the HLA-A*0201+ line H1.

[0400] Fig.10It was shown that expression levels of RAG1 and RAG2 genes were restricted to multiple points during the in vitro ATO differentiation process, including positive selection in the DP population.

[0401] Example 4 - Regulated gene expression during differentiation of mature T cells

[0402] Chimeric antigen receptor (CAR) T-cell therapy has produced remarkable results in treating otherwise refractory hematological malignancies 1-2 Currently, the process of manufacturing autologous CAR T cells is challenging due to the need to generate each personalized therapy de novo and the inherent variability in T cell biology between patients, resulting in unpredictable and inconsistent clinical responses. 3-5 As a result, there has been increasing interest in the in vitro generation of CAR T cells from indefinitely renewing allogeneic sources of human pluripotent stem cells (hPSCs). One advantage of this approach is that hPSCs are highly amenable to genetic editing, providing multiple avenues to manipulate the function of the final T cell product.

[0403] However, there are two key biological barriers to generating CART cells from hPSCs.

[0404] 1.) Expression of the CAR transgene early in T cell differentiation redirects development to the innate lymphoid pathway 6-10 This result was similarly reported by the inventors' team using the artificial thymic organoid (ATO) system to differentiate PSCs into T cells, as the constitutive expression of the second-generation CD19 CAR led to the generation of innate lymphoid cell type 2 (ILC2) cells instead of conventional single positive CD8+ (SP8) and CD4 (SP4) T cells ( Figure 2A to B) 11 . To overcome this early fate switch, this approach utilizes CRISPR / Cas9 editing to achieve delayed, stage-specific expression of the same CD19 CAR. The CAR transgene is inserted into the 3' untranslated region (UTR) of the gene granzyme A (GZMA), placing its expression under the control of the endogenous GZMA promoter and its regulatory elements. GZMA is only expressed late in the differentiation of single positive CD8 (also known as "SP8") (CD3+TCR+CD8+CD4-) and single positive CD4+ (also known as "SP4") (CD3+TCR+CD8-CD4+) T cells and is absent in the early T stage. Stage-specific expression was experimentally validated using the engineered GZMA-mCitrine reporter ATO ( Figure 3 As a result, differentiation of GZMA-CAR19LH T-iPSCs using the ATO system demonstrated efficient development of mature, conventional SP8 and SP4 T cells with robust CAR expression ( Figure 4B ) without any sign of a fateful shift towards an established lineage ( Figure 4A ). The inventors have identified other endogenous genes with similar expression patterns that can be used as alternatives to GZMA: these genes are listed in Table 2A.

[0405] 2.) To address the alloreactivity of T cells generated from allogeneic hPSC sources, the endogenous T cell receptor (TCR) must be knocked out to prevent the risk of TCR / MHC mispairing during adoptive transfer and subsequent graft-versus-host disease. However, removal of TCR expression (e.g., by disruption of the TRAC locus) results in a complete block in T cell development, as mature T cells no longer undergo positive selection ( Fig.11A to D). The inventors' solution to restore positive selection relied on a similar 3'UTR knockout to transiently express an exogenous TCR (e.g., 1G4 TCR recognizing the NYESO antigen) during the positive selection phase (i.e., in double positive, aka DP, aka CD4+CD8+) cells. Based on their own RNA-Seq studies, they identified the RAG1 gene as a candidate endogenous gene with the correct expression pattern; RAG1 is expressed during double negative (DN) to double positive (DP) T cells, but is absent in SP8 and SP4 T cells after positive selection has been achieved. Using the same stage-specific approach, a reporter line was generated, and it was determined that RAG1-regulated reporter expression followed the RNA-Seq pattern during differentiation. ( Fig. 12A To C). In addition, even after activation, the reporter is not detectable in mature T cells ( Fig. 12C We then used the same strategy to drive 1G4 TCR expression from the RAG1 locus and showed that positive selection could be restored in cells generated from the TRAC-disrupted T-iPSC line. Thus, expression of the transgenic TCR occurs only during the critical period of positive selection (DP stage) and is absent after SP8 / SP4 maturation ( Fig.13A to C), ultimately leading to the generation of TCRɑβ-CD3-mature, naive SP8 T cells (Figure 6D). The inventors have identified other endogenous genes with similar expression patterns that can be used as alternatives to RAG1: these genes are listed in Table 2B.

[0406] We then combined two editing strategies in a TRAC-disrupted PSC line, 1) expressing CD19CAR via GZMA (GZMA-CAR19LH) to avoid the block and diversion to the intrinsic lineage that occurs in the case of constitutive expression, and 2) transiently expressing exogenous 1G4(NYESO)TCR via RAG1 (RAG1-NYESO TCR). The data show that we developed a platform to generate non-alloreactive TCRɑβ-CD3-naive CAR T cells ( Fig.14A to B). The resulting T cells exhibited robust antigen-specific activation and cytokine production ( Fig. 14C ), and the approach can be extended to different CAR designs ( Fig.14D ).

[0407]

[0408]

[0409] Table 2A to B: Expression profiles of select genes during T cell differentiation using the ATO system. (2A) Select genes show low to absent expression from pluripotent stem cells to DP T cell stages, and high expression in mature SP8 and SP4 T cells. (2B) Select genes show low to absent expression from pluripotent stem cells to CD34+ cell stages, and increasing expression from DN to DP T cell stages. After positive selection, expression levels in SP8 and SP4 T cells return to low or absent levels.

[0410] Expression profiles were generated from published ATOs from various cell sources (i.e., PSC-ATO, cord blood CD34+ATO, thymus) 1 All values ​​in the table are expressed in RPKM.

[0411] ***

[0412] ————

[0413] According to the present disclosure, all methods disclosed and claimed herein can be carried out and implemented without excessive experimentation. Although the compositions and methods of the present invention have been described according to some preferred embodiments, it is obvious to those skilled in the art that the steps or sequence of steps of the methods described herein and the methods described herein can be changed without departing from the concept, spirit and scope of the present invention. More specifically, it will be obvious that certain medicaments related in both chemical and physiological aspects can be used to replace the medicaments described herein, while achieving the same or similar results. All such similar substitutions and changes that are obvious to those skilled in the art are considered to be within the spirit, scope and concept of the present invention as defined by the appended claims.

[0414] References cited in this disclosure are specifically incorporated herein by reference to the extent they provide exemplary procedural or other details supplementary to those set forth herein.

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[0419] 5.CongL,Ran F,Cox D,LinS,Barretto R,Habib N,Hsu P,Wu X,Jiang W,Marraffini L,Zhang F.Multiplex Genome Engineering Using CRISPR / CasSystems.Science.2013Feb15;339(6121):819-23.PMCID:PMC3795411

[0420] 6.ThemeliM,Kloss C,Ciriello G,Fedorov V,Perna F,Gonen M,SadelainM.Generation of tumor-targeted human T lymphocytes from induced pluripotentstem cells for cancer therapy.Nat.Biotechnol.2013Oct;31(10):928-33.PMCID:PMC5722218

[0421] 7.Feucht J,Sun J,Eyquem J,Ho Y,Zhao Z,Leibold J,Dobrin A,Cabriolu A,Hamieh M,Sadelain M.Calibration of CAR activation potential directsalternative T cell fates and therapeutic potency.Nat.Med.2019 Jan;25(1):82-88.PMCID:PMC6532069

[0422] 8.Maluski M,Ghosh A,Herbst J,Scholl V,Baumann R,Heuhn J,Geffers R,Meyer J,Maul H,Eiz-Vesper B,Krueger A,Schambach A,van den Brink M,SuerM.Chimeric antigen receptor-induced BCL11B suppression propagates NK-likecell development.J Clin Invest.2019Dec2;129(12):5108-5122.PMCID:PMC6877334

[0423] 9.Albert S,Koristka S,Gerbaulet A,Cartellieri M,Arndt C,Feldman A,Berndt N,Loureiro L,Bonin M,Ehninger G,Eugster A,Bonifacio E,Bornhauser M,Bachmann M,Ehninger A.TonicSignaling and Its Effects on Lymphopoiesis of CAR-Armed Hematopoietic Stem and Progenitor Cells.J.Immunol.2019Mar15;202(6):1735-1746.PMID:30728213.

[0424] 10.Ueda T,Shiina S,Iriguchi S,Terakura S,Kawai Y,Kabai R,Sakamoto S,Watanabe A,Ohara K,Wang B,Xu H,Minagawa A,Hotta A,Woltjen K,Uemura Y,KodamaY,Seno H,Nakatsura T,Tamada K,Kaneko S.Optimization of the proliferation andpersistency of CAR T cells derived from human induced pluripotent stem cells.

[0425] 11.LiS,Wang CS,Montel-Hagen A,Casero D,Lopez S,Chen H-C,Zhou O,BoteroC,Tsai SC,Wong C,Crooks GM,Seet CS.J.Immunol.2022.208(1_Supplement):107.11.

[0426] 12.Montel-Hagen A,Seet S,Li S,Chick B,Zhu Y,Chang P,Tsai S,Sun V,Lopez S,Chen H,He C,Chin C,Casero D,Crooks G.Organoid-Induced Differentiationof Conventional Tcells from Human Pluripotent Stem Cells.Cell StemCell.2019Mar7;24(3):376-389.PMCID:PMC6687310

Claims

1. A nucleic acid comprising a promoter region and a transgene located downstream of the transcription start site of the promoter region; wherein the promoter region comprises a promoter region from a gene in Table 1.

2. The nucleic acid of claim 1, wherein the promoter region comprises a promoter region from a granzyme gene.

3. The nucleic acid of claim 2, wherein the granzyme promoter region comprises a promoter region from a granzyme A, B, H, K or M gene.

4. The nucleic acid of claim 3, wherein the promoter region comprises a promoter from the granzyme A gene.

5. The nucleic acid of any one of claims 1 to 4, wherein the promoter region comprises an endogenous promoter region.

6. The nucleic acid of any one of claims 1 to 5, wherein the transgene comprises a chimeric antigen receptor (CAR) or a T cell receptor (TCR).

7. The nucleic acid of any one of claims 1 to 6, wherein the transgene comprises FOXP3.

8. The nucleic acid of any one of claims 1 to 7, wherein the transgene comprises BCL11b.

9. The nucleic acid of any one of claims 1 to 8, wherein the nucleic acid comprises genomic DNA.

10. The nucleic acid of any one of claims 1 to 9, wherein the promoter region comprises a RAG1 promoter region or a RAG 2 promoter region.

11. The nucleic acid of claim 10, wherein the genomic DNA lacks a gene encoding one or both of a functional RAG1 protein and / or a RAG2 protein.

12. The nucleic acid of claim 10 or 11, wherein the transgene is inserted into the RAG1 coding region or the RAG2 coding region, and / or wherein the transgene at least partially replaces the RAG1 coding region or the RAG2 coding region.

13. The nucleic acid of any one of claims 1 to 12, wherein the nucleic acid comprises the coding region of a gene of Table 1 located downstream of the transcription start site of the promoter region.

14. The nucleic acid of claim 13, wherein transcription of both the transgene and the gene of Table 1 is regulated by a promoter of the gene of Table 1.

15. The nucleic acid of claim 13 or 14, wherein the transgene and the gene of Table 1 are expressed on the same mRNA.

16. The nucleic acid of any one of claims 13 to 15, wherein the nucleic acid comprises a self-cleaving peptide site or an IRES.

17. The nucleic acid of any one of claims 13 to 16, wherein the transgene is located 3' proximal to the gene of Table 1.

18. The nucleic acid of any one of claims 13 to 16, wherein the transgene is located 5' proximal to the gene of Table 1.

19. The nucleic acid of any one of claims 1 to 18, wherein the nucleic acid further comprises a 3'UTR region downstream of the coding region of the Table 1 gene and / or the transgene.

20. The nucleic acid of claim 19, wherein the 3'UTR comprises an endogenous 3'UTR.

21. The nucleic acid of any one of claims 1 to 20, wherein the promoter region, 3'UTR, transgene, and / or the coding region of the Table 1 gene are located in genomic DNA at an endogenous locus of the Table 1 gene.

22. The nucleic acid of any one of claims 9 to 21, wherein the genomic DNA lacks genes encoding one or more of a functional endogenous TCR-α (TCR-a) protein, a functional endogenous TCR-β (TCR-b) protein, a functional endogenous TCR-γ (TCR-g) protein, or a functional endogenous TCR-δ (TCR-d) protein.

23. The nucleic acid of any one of claims 9 to 22, wherein the genomic DNA lacks a gene encoding one or more of a functional CD3γ protein, a functional CD3δ protein, a functional CD3ε protein, or a functional CD3ζ protein.

24. A cell comprising at least one nucleic acid according to any one of claims 1 to 22.

25. The cell of claim 24, wherein the cell comprises a first nucleic acid and a second nucleic acid, wherein the first nucleic acid comprises a first promoter region and a first transgene located downstream of the transcription start site of the promoter region; wherein the promoter region comprises a promoter region from gene RAG1 or RAG2, and the second nucleic acid comprises a second promoter region and a second transgene located downstream of the transcription start site of the promoter region; wherein the promoter region comprises a promoter region selected from the group consisting of: GZMA, GZMB, GZMH, GZMK, GZMM, TNFSF14, XCL2, PTGDS, SAMD3, NCALD, or XCL1.

26. The cell of claim 25, wherein the second promoter region comprises a promoter region from a granzyme gene.

27. The cell of claim 26, wherein the granzyme promoter region comprises a promoter region from a granzyme A, B, H, K, or M gene.

28. The cell of claim 27, wherein the second promoter region comprises a promoter from the granzyme A gene.

29. The cell of any one of claims 25 to 28, wherein the first promoter region and / or the second promoter region comprises an endogenous promoter region.

30. The cell of any one of claims 25 to 29, wherein the first transgene or the second transgene comprises a CAR or a TCR.

31. The cell of claim 30, wherein the first transgene comprises a TCR and the second transgene comprises a CAR.

32. The cell of any one of claims 25 to 31, wherein the first transgene and / or the second transgene comprises FOXP3.

33. The cell of any one of claims 25 to 32, wherein the first transgene and / or the second transgene comprises BCL11b.

34. The cell of any one of claims 25 to 33, wherein the first promoter region comprises the RAG1 promoter region.

35. The cell of any one of claims 25 to 34, wherein the cell lacks a gene encoding one or both of a functional RAG1 protein and / or a RAG2 protein.

36. The cell of any one of claims 25 to 35, wherein the first transgene is inserted into the RAG1 coding region or the RAG2 coding region, and / or wherein the transgene at least partially replaces the RAG1 coding region or the RAG2 coding region.

37. The cell of any one of claims 25 to 36, wherein the first promoter region comprises a RAG1 promoter region or a RAG2 promoter region, and the first nucleic acid comprises a RAG1 gene or a RAG2 gene located downstream of the transcription start site of the RAG1 promoter region or the RAG2 promoter region, respectively.

38. The cell of any one of claims 25 to 37, wherein the second promoter region comprises a GZMA, GZMB, GZMH, GZMK, GZMM, TNFSF14, XCL2, PTGDS, SAMD3, NCALD or XCL1 promoter region, and the second nucleic acid comprises a GZMA, GZMB, GZMH, GZMK, GZMM, TNFSF14, XCL2, PTGDS, SAMD3, NCALD or XCL1 gene located downstream of the transcription start site of the GZMA, GZMB, GZMH, GZMK, GZMM, TNFSF14, XCL2, PTGDS, SAMD3, NCALD or XCL1 promoter region, respectively.

39. The cell of claim 37, wherein the first transgene is expressed on the same mRNA as the RAG1 gene or the RAG2 gene.

40. The cell of claim 38, wherein the second transgene is expressed on the same mRNA as the GZMA, GZMB, GZMH, GZMK, GZMM, TNFSF14, XCL2, PTGDS, SAMD3, NCALD, or XCL1 gene.

41. The cell of any one of claims 25 to 40, wherein the first nucleic acid or the second nucleic acid comprises a self-cleaving peptide site or an IRES.

42. The cell of any one of claims 25 to 41, wherein the first transgene or the second transgene is located 3' proximal to the gene of Table 1.

43. The cell of any one of claims 25 to 41, wherein the first transgene or the second transgene is located 5' proximal to the gene of Table 1.

44. The cell of any one of claims 25 to 41, wherein the first nucleic acid or the second nucleic acid further comprises a 3'UTR region downstream of the coding region of the gene and / or the first transgene or the second transgene.

45. The cell of claim 44, wherein the 3'UTR comprises an endogenous 3'UTR.

46. ​​The cell of any one of claims 24 to 45, wherein the cell lacks a gene encoding one or more of a functional endogenous TCR-alpha (TCR-a) protein, a functional endogenous TCR-beta (TCR-b) protein, a functional endogenous TCR-gamma (TCR-g) protein, or a functional endogenous TCR-delta (TCR-d) protein.

47. The cell of any one of claims 24 to 46, wherein the genomic DNA lacks a gene encoding one or more of a functional CD3γ protein, a functional CD3δ protein, a functional CD3ε protein, or a functional CD3ζ protein.

48. The cell of any one of claims 24 to 47, wherein the cell comprises a stem cell, a hematopoietic stem or progenitor cell (HSPC), an embryonic stem cell, an induced pluripotent stem cell (iPSC), a human embryonic mesodermal progenitor cell, or a pluripotent stem cell (PSC).

49. The cell of claim 48, wherein the cell comprises a pluripotent stem cell.

50. The cell of claim 48, wherein the cell comprises an embryonic stem cell or an induced pluripotent stem cell (iPSC).

51. The cell of any one of claims 24 to 50, wherein the cell is isolated or derived from umbilical cord blood, peripheral blood, bone marrow, peripheral blood, umbilical cord blood, placenta, adipose tissue, or umbilical cord tissue.

52. The cell of any one of claims 24 to 51, wherein the cell comprises a T cell.

53. The cell of any one of claims 24 to 51, wherein the cell comprises a mature cell.

54. The cell of claim 53, wherein the mature cell comprises a mature T cell, a mature regulatory T cell (Treg), an iNKT cell, an innate lymphocyte, or a NK cell.

55. The cell of claim 53, wherein the cell comprises a CD4+CD8- or CD8ab+CD4- T cell.

56. The cell of any one of claims 52 to 55, wherein the cell comprises an in vitro differentiated T cell, a mature Treg, an iNKT cell, an innate lymphocyte, or a NK cell.

57. The cell of any one of claims 24 to 56, wherein the cell is further defined as allogeneic.

58. A method for preparing a cell, comprising culturing the cell of any one of claims 24 to 57 under conditions suitable for differentiation of the cell into a mature cell in vitro.

59. The method of claim 58, wherein the mature cells comprise mature T cells, mature regulatory T cells (Tregs), iNKT cells, innate lymphocytes, or NK cells.

60. The method of claim 59, wherein the cells comprise CD4+CD8-, CD8ab+CD4- T cells, CD8aa+ T cells, TCRgd T cells, or innate immune cells.

61. The method of any one of claims 58 to 60, wherein the method comprises culturing the cells in three-dimensional (3D) cell aggregates, wherein the cell aggregates further comprise a selected population of stromal cells expressing Notch ligands, and wherein the 3D cell aggregates are cultured in a serum-free medium comprising insulin, biotin, transferrin, and albumin for a period of time sufficient to allow the stem or progenitor cells to differentiate into T cells in vitro.

62. The method of claim 61, wherein the stromal cells comprise MS5 stromal cells.

63. The method of claim 61 or 62, wherein the Notch ligand is an exogenous Notch ligand.

64. The method of any one of claims 61 to 63, wherein the method further comprises centrifuging the stem or progenitor cells and the stromal cells to form 3D cell aggregates.

65. The method of any one of claims 61 to 64, wherein the culture medium further comprises one or more of the following: externally added FLT3 ligand (FLT3L), interleukin 7 (IL-7), stem cell factor (SCF), thrombopoietin (TPO), thrombopoietin (TPO), IL-2, IL-4, IL-6, IL-15, IL-21, TNF-α, TGF-β, interferon-γ, interferon-λ, TSLP, thymopentin, pleiotrophic factor, pleiotrophin, midkine, or a combination thereof.

66. The method of any one of claims 61 to 65, wherein the stromal cells have an exogenous nucleotide sequence encoding a complete, partial or modified Notch ligand, and wherein the Notch ligand is DLL4, DLL1, JAG1, JAG2 or a combination thereof.

67. A method for treating a subject comprising administering the cell of any one of claims 24 to 57.

68. The method of claim 67, wherein the cells are allogeneic.

69. The method of claim 67 or 68, wherein the subject has cancer.

70. The method of claim 69, wherein the method is used to treat cancer in the subject.

71. The method of claim 69 or 70, wherein the cancer is selected from the group consisting of lung cancer, prostate cancer, ovarian cancer, testicular cancer, brain cancer, skin cancer, melanoma, colon cancer, rectal cancer, stomach cancer, esophageal cancer, tracheal cancer, head and neck cancer, pancreatic cancer, liver cancer, breast cancer, ovarian cancer, lymphoma including lymphoma and multiple myeloma, leukemia, osteosarcoma or soft tissue sarcoma, cervical cancer, and vulvar cancer.

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