Engineered cells and methods of use

Abstract

The present disclosure provides methods and compositions that result in the insertion of a transgene into stem or progenitor cells without deleterious effects on T cell differentiation by in vitro T cell differentiation methods. To achieve this, the inventors have found that expression of a transgene under the control of a promoter region such as granzyme A allows for a coordinated expression pattern that results in both: (1) high expression of the transgene in mature T cells, and (2) coordinated levels of transgene expression throughout the in vitro differentiation method that allow for the generation of a mature T cell population.

Description

[Technical field]

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

[0002] Sequence Listing This application contains a sequence listing prepared in accordance with ST.26 format, which is incorporated herein by reference in its entirety. The sequence listing, prepared on March 1, 2023, is named UCLAP0162WO and is 61,982 bytes in size.

[0003] 1. Field of the Invention The present invention relates generally to the field of cell culture and development. [Background technology]

[0004] 2. Description of Related Art The use of stem or progenitor cells as a source of T cells for adoptive cell therapy offers a potential alternative to currently used approaches that rely on the manipulation of mature peripheral blood T cells. The generation of T cells from PSCs and HSPCs offers advantages over the use of primary mature T cells harvested from blood due to the ability of stem cells to self-renew indefinitely, either in vivo (HSPCs) or in vitro (PSCs). The insertion of bioactive genes into the genome of stem or progenitor cells may allow in vitro or in vivo differentiation into T cells with modified functional properties. However, some specific bioactive genes may be detrimental to T cell development when expressed at developmental stages prior to mature T cells. Thus, there is a need in the art to engineer stem or progenitor cells that can carry transgenes and be successfully differentiated into mature T cells. Summary of the Invention

[0005] Quick Overview The present disclosure provides methods and compositions for constitutive expression of a transgene during in vitro or in vivo T cell differentiation that results in insertion of the transgene into stem or progenitor cells without deleterious effects on T cell differentiation. To achieve this, the inventors have found that expression of a transgene under the control of a promoter region such as granzyme A allows for a coordinated expression pattern that results in both: (1) high expression of the transgene in mature T cells, and (2) coordinated levels of transgene expression throughout the in vitro differentiation process that allow for the generation of a mature T cell population.

[0006] In the present disclosure, a nucleic acid is described that includes a promoter region and a transgene downstream of a transcription start site of the promoter region, the promoter region comprising a promoter region of a gene in Table 1. Also described is a cell that includes a nucleic acid of the present disclosure. The cell of the present disclosure may include a plurality of nucleic acids of the present disclosure. The cell may include: a first nucleic acid that includes a first promoter region and a first transgene downstream of a transcription start site of the promoter region, the promoter region comprising a promoter region of a gene in Table 1; and a second nucleic acid that includes a second promoter region and a second transgene downstream of a transcription start site of the promoter region, the promoter region comprising a promoter region selected from the genes in Table 1. The cell may comprise: a first promoter region and a first transgene downstream of the transcription start site of the promoter region, the promoter region comprises the promoter region of gene: RAG1 or RAG2; and a second nucleic acid, comprising a second promoter region and a second transgene downstream of the transcription start site of the promoter region, the promoter region comprises the promoter region selected from gene: GZMA, GZMB, GZMH, GZMK, GZMM, TNFSF14, XCL2, PTGDS, SAMD3, NCALD, or XCL1. Also described is a method for producing a cell, comprising culturing the cell of the present disclosure under conditions suitable for in vitro differentiation of the cell into a mature cell. Also described is a method for treating a subject, comprising administering the cell of the present disclosure.

[0007] (Table 1) Promoter region TIFF2025507864000001.tif100128

[0008] One or more genes and / or their promoter regions in Table 1 may be excluded in the disclosed method, nucleic acid, cell, or composition. The promoter region, first promoter region, or second promoter region may be the promoter region of a granzyme gene. The promoter region, first promoter region, or second promoter region may be that of a granzyme A, B, H, K, or M gene. The promoter region, first promoter region, or second promoter region may be that of one or more of the granzyme A, B, H, K, or M genes that have been excluded. The promoter, first promoter region, or second promoter region may be the promoter region of a granzyme A gene. The promoter region, first promoter region, or second promoter region may be the promoter region of a granzyme B gene. The promoter region, first promoter region, or second promoter region may be the promoter region of a granzyme H gene. The promoter region, the first promoter region, or the second promoter region may be the promoter region of the granzyme K gene. The promoter region, the first promoter region, or the second promoter region may be the promoter region of the granzyme M gene. The promoter region, the first promoter region, or the second promoter region may include the RAG1 or RAG2 promoter region. Either the RAG1 or RAG2 promoter region may be excluded in the embodiments of the present disclosure. The genomic DNA may be defective with respect to a gene encoding one or both of functional RAG1 and / or RAG2 proteins. The genomic DNA may encode one or both of functional RAG1 and / or RAG2 proteins. The transgene, the first transgene, or the second transgene may be inserted within the coding region of RAG1 or RAG2, and / or the transgene, the first transgene, or the second transgene may at least partially replace the coding region of RAG1 or RAG2.The promoter region, the first promoter region, or the second promoter region may comprise an endogenous promoter region. An endogenous promoter region refers to the situation where the promoter region is present in its endogenous genomic environment, so that the upstream sequence of the promoter (i.e., the 5' region) is substantially the same as that of wild-type cells. Substantially the same may refer to a region being at least 80, 85, 90, 95, 96, 97, 98, or 99% identical to the upstream region of the wild-type. An endogenous promoter region may also refer to the situation where the promoter is in the same genomic location as the wild-type promoter. An endogenous promoter may refer to a promoter in a cell that is unmodified or is 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% identical to the wild-type promoter, or is at least 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% identical.

[0009] The transgene, the first transgene, or the second transgene may comprise a chimeric antigen receptor (CAR) or a T cell receptor (TCR). The transgene, the first transgene, or the second transgene may comprise FOXP3. The transgene, the first transgene, or the second transgene may comprise BCL11b. The transgene, the first transgene, or the second transgene may comprise a T cell receptor (TCR). The TCR may be a tumor antigen-specific TCR, a virus-specific TCR, a foreign body-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 virus antigen-specific TCR (i.e., a TCR that recognizes a virus antigen). The CAR may be, for example, a tumor antigen-specific CAR, a virus-specific CAR, a foreign body-specific CAR, or a bacteria-specific CAR. The CAR can be a tumor antigen-specific CAR (i.e., a CAR that recognizes a tumor antigen. The transgene, the first transgene, or the second transgene can be a transcription factor.

[0010] The transgene may be a cytokine receptor or a cytokine. The transgene, the first transgene, or the second transgene may be chimeric antigen receptor (CAR) or T cell receptor (TCR). The transgene, the first transgene, or the second transgene may be FOXP3-free. The transgene, the first transgene, or the second transgene may be BCL11b-free. The transgene, the first transgene, or the second transgene may be T cell receptor (TCR-free). The TCR may be tumor antigen-specific TCR, virus-specific TCR, foreign body-specific TCR, cancer cell-specific TCR, bacteria-specific TCR, or cancer testis antigen-specific TCR-free. The TCR may be tumor antigen-specific TCR (i.e., TCR that recognizes tumor antigen). The TCR may be viral antigen-specific TCR (i.e., TCR that recognizes viral antigen). The CAR may be, for example, one that excludes a tumor antigen-specific CAR, a virus-specific CAR, a foreign body-specific CAR, or a bacteria-specific CAR. The CAR may be one that excludes a tumor antigen-specific CAR (i.e., a CAR that recognizes a tumor antigen). The transgene, the first transgene, or the second transgene may be one that excludes a transcription factor. The transgene may be one that excludes a cytokine receptor or a cytokine.

[0011] The size of the transgene, first transgene, or second transgene may be 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, 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, 875, 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, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4500, 50 The nucleic acid sequence may be, may be at least, or may be at most, 00, 5500, 6000, or 7000 amino acids or nucleic acids in length (or any range derivable therein), and / or may encode a protein that is, at least, or at most, 00, 5500, 6000, or 7000 amino acids or nucleic acids in length (or any range derivable therein).

[0012] 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, the coding region of the gene downstream of the transcription start site of the promoter region, the first promoter region, or the second promoter region, or Table 1. The transcription of the transgene and the gene of Table 1 may both be regulated by the promoter of the gene of Table 1. The transgene, the first transgene, or the second transgene and the gene of Table 1 are expressed in 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 be one that excludes the self-cleaving peptide or the IRES. The transgene, the first transgene, or the second transgene may be 3' proximal to the gene of Table 1. The transgene, the first transgene, or the second transgene may be 5' proximal to the gene of Table 1. If the first region is linked to a nucleic acid at the 3' end of the second region, the first region may be 3'-proximal to the second region. There may be additional intervening amino acid residues between the first and second regions. Also, intervening amino acid residues between the first and second regions may be excluded. Thus, the regions need not be directly adjacent, unless expressly stated as not having intervening amino acid residues, which is also envisaged. The term "5'-proximal" is similarly defined as the first region being 5'-proximal to the second region, if the first region is linked to a nucleic acid at the 5' end of the second region. Similarly, there may be additional intervening amino acid residues between the first and second regions, unless otherwise stated. The nucleic acid, first nucleic acid, or second nucleic acid may include, or may further include, a 3'UTR region downstream of the coding region of a gene in Table 1 and / or the transgene, first transgene, or second transgene. The 3'UTR may include an endogenous 3'UTR. The promoter region, first promoter region, second promoter region, 3'UTR, transgene, first transgene, second transgene, and / or coding region of a gene of Table 1 are at the endogenous locus of the gene of Table 1 within the genomic DNA.

[0013] The nucleic acid, first nucleic acid, second nucleic acid, genomic DNA, and / or the cell may be defective with respect to 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. The nucleic acid, first nucleic acid, second nucleic acid, genomic DNA, and / or the cell may be defective with respect to a gene encoding one or more of a functional CD3 gamma protein, a functional CD3 delta protein, a functional CD3 epsilon protein, or a functional CD3 zeta protein.

[0014] The nucleic acid, first nucleic acid, second nucleic acid, genomic DNA, and / or the cell may be competent for 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. The nucleic acid, first nucleic acid, second nucleic acid, genomic DNA, and / or the cell may be competent for a gene encoding one or more of a functional CD3 gamma protein, a functional CD3 delta protein, a functional CD3 epsilon protein, or a functional CD3 zeta protein.

[0015] 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 isolated or derived from umbilical 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. The mature cell may comprise a mature T cell, a mature regulatory T cell (Treg), an iNKT cell, an innate lymphoid cell, or an NK cell. The cell may comprise a CD4 single positive (SP) cell or a CD8 SP cell. The cell may comprise a CD4+ CD8- or a CD8ab+ CD4- T cell. The cell may comprise an in vitro differentiated cell. The in vitro differentiated cell can be a T cell, a mature Treg cell, an iNKT cell, a natural lymphocyte, or a NK cell. The cell can be in vivo and / or administered to a subject. The cell can be in vitro. The cell can be differentiated in vitro. The cell can be differentiated in vivo (i.e., in a human subject).

[0016] The cells of the present disclosure may be those excluding PSCs, iPSCs, HSPCs, embryonic stem cells, iPSCs, human embryonic mesodermal progenitor cells, or cells isolated or derived from umbilical cord blood, peripheral blood, bone marrow, peripheral blood, umbilical cord blood, placenta, adipose tissue, or umbilical cord tissue. The mature cells may be those excluding one or more of mature T cells, mature regulatory T cells (Treg), iNKT cells, innate lymphoid cells, or NK cells. The cells may include CD4 single positive (SP) cells or CD8 SP cells. The cells may include CD4+ CD8- or CD8ab+ CD4- T cells. The cells may be those excluding in vitro differentiated cells and / or mature Treg, iNKT cells, innate lymphoid cells, or NK cells.

[0017] The method may also or further comprise culturing cells in a three-dimensional (3D) cell aggregate, the cell aggregate further comprises a selected population of stromal cells expressing Notch ligand, and the 3D cell aggregate is cultured in a serum-free medium containing insulin, biotin, transferrin, and albumin for a period of time sufficient for the in vitro differentiation of the stem cell or progenitor cell into a mature cell, such as a mature T cell. The stromal cell may comprise MS5 stromal cell. The stromal cell may be a human stromal cell. The Notch ligand may comprise an exogenous Notch ligand. The stromal cell may comprise a transgene expressing a Notch ligand. The method may or further comprise centrifuging the stem cell or progenitor cell and the stromal cell to form a 3D cell aggregate. The cell culture medium may contain or further contain one or more of exogenously 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-alpha, TGF-beta, interferon-gamma, interferon-lambda, TSLP, thymopentin, pleotrophin, pleiotrophin, midkine, or combinations thereof.The stromal cell may have an exogenous nucleotide sequence encoding an intact, partial, or modified Notch ligand, and the Notch ligand is DLL4, DLL1, JAG1, JAG2, or combinations thereof. The cell culture medium may exclude one or more of exogenously added FLT3 ligand (FLT3L), interleukin 7 (IL-7), stem cell factor (SCF), thrombopoietin (TPO), IL-2, IL-4, IL-6, IL-15, IL-21, TNF-alpha, TGF-beta, interferon-gamma, interferon-lambda, TSLP, thymopentin, pleotrophin, midkine, or combinations thereof.The stromal cells may exclude one or more of the exogenous nucleotide sequences encoding an intact, partial, or modified Notch ligand, wherein the Notch ligand is DLL4, DLL1, JAG1, JAG2, or a combination thereof.

[0018] The cells may be allogeneic. The cells may be further defined as autologous. The subject may be a subject with cancer. The method may be for treating cancer. The method may be for treating autoimmune disease. The cancer or autoimmune disease may be as listed herein. The cancer may 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, lymphoid cancer including lymphoma and multiple myeloma, leukemia, sarcoma of bone or soft tissue, cervical cancer, and vulvar cancer. The cancer may be excluded from one or more 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, cancers of the lymphoid system including lymphoma and multiple myeloma, leukemia, sarcoma of bone or soft tissue, cervical cancer, and vulvar cancer.

[0019] One or more of the cells, particularly the stromal cells, may express an exogenous Notch ligand. The culture medium may include an exogenously added Notch ligand. The exogenously added Notch ligand may be bound to a solid support or immobilized. Non-limiting examples of Notch ligands include intact (full-length), partial (truncated form), or modified (comprising one or more mutations, e.g. conservative mutations) DLL4, DLL1, JAG1, JAG2, or combinations thereof. For example, the stromal cells have an exogenous nucleotide sequence encoding a Notch ligand that may be introduced into the cells by transfection or transduction. The culture composition may not include a Notch ligand, or may exclude a Notch ligand, or may not include an exogenously added Notch ligand. The Notch ligand may be a human Notch ligand. The Notch ligand may be a human DLL1 or DLL4 Notch ligand.

[0020] The stromal cells can be mouse stromal cell lines, human stromal cell lines, selected populations of primary stromal cells, selected populations of stromal cells differentiated in vitro from pluripotent stem cells, or combinations thereof. The stromal cells can be MS5, OP9, S17, HS-5, or HS-27a cells. Stromal cell lines such as MS5, OP9, S17, HS-5, or HS-27a cells may be excluded in the methods and compositions of the present disclosure. The stromal cells can be differentiated from human cells. The stromal cells can be differentiated from human pluripotent stem cells. The stromal cells can be differentiated from human or non-human HSPC or PSC cells.

[0021] The medium may be prepared using any of media used as a basal medium for culturing animal cells, such as AIM V, X-VIVO-15, NeuroBasal, EGM2, TeSR, BME, BGJb, CMRL 1066, Glasgow MEM, Improved MEM Zinc Option, IMDM, Medium 199, Eagle MEM, αMEM, DMEM, Ham, RPMI-1640, and Fischer's medium, and any combination thereof, but the medium may not be particularly limited thereto as long as it can be used to culture animal cells. In particular, the medium may be xeno-free or chemically defined.

[0022] The medium may be serum-containing or serum-free, or xeno-free. From the viewpoint of preventing contamination of components derived from different animals, the serum is preferably derived from the same animal as that of the stem cells. Serum-free medium refers to a medium that does not contain untreated or unpurified serum, and therefore may include a medium that contains purified blood-derived components or animal tissue-derived components (e.g., growth factors).

[0023] The medium may or may not contain any substitute for serum. Serum substitutes may include substances that suitably contain albumin (e.g., lipid-rich albumin, bovine albumin, albumin replacement fluids such as recombinant or humanized albumin, plant starch, dextran, and protein hydrolysates), transferrin (or other iron transporters), fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thiolglycerol, or equivalents. Serum substitutes may be prepared, for example, by the methods disclosed in International Patent Application Publication No. 98 / 30679, which is incorporated herein in its entirety. Alternatively, any commercially available product may be used for greater convenience. Commercially available products include Knockout Serum Replacement (KSR), Chemically-Defined Lipid Concentrated (Gibco), and Glutamax (Gibco).

[0024] The medium may be a serum-free medium suitable for neural cell development.For example, the medium may comprise B-27® supplement, Xenofree B-27® 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, incorporated herein in its entirety), GS21™ supplement (available on the World Wide Web at amsbio.com / B-27.aspx), or a combination thereof, at a concentration effective for generating mature cells from 3D cell aggregates.

[0025] The medium may contain one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty or more of the following B-27 supplement containing components: 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); L-carnitine HCl; linoleic acid; linolenic acid; progesterone; putrescine diHCl; sodium selenite; and T3 (triodo-I-thyronine).

[0026] The medium may include exogenously added ascorbic acid. The medium may also contain exogenously added fatty acids or lipids, amino acids (e.g., non-essential amino acids), simple sugars, vitamins, growth factors, cytokines, antioxidants, 2-mercaptoethanol, pyruvic acid, buffers, inorganic ions and inorganic salts. The vitamins may include biotin, DL-alpha tocopherol acetate, DL-alpha-tocopherol, vitamin A, choline chloride, calcium pantothenate, pantothenic acid, folic acid, nicotinamide, pyridoxine, riboflavin, thiamine, inositol, vitamin B12, or combinations thereof or salts thereof. The amino acids may include arginine, cysteine, isoleucine, leucine, lysine, methionine, glutamine, phenylalanine, threonine, tryptophan, histidine, tyrosine or valine, or combinations thereof. The inorganic ions may include sodium, potassium, calcium, magnesium, nitrogen or phosphorus, or combinations thereof or salts thereof. The medium can also contain molybdenum, vanadium, iron, zinc, selenium, copper or manganese, or combinations thereof.

[0027] The medium was supplemented with the following: B-27 supplement, biotin, DL-alpha-tocopherol 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 diHCl, sodium selenite, T3 (triodo-I-thyronine), ascorbic acid, fatty acids, lipids, amino acids, simple sugars, vitamins, growth factors, cytokines, antioxidants, 2-mercaptoethanolamine ... ethanolamine HCl, ethanolamine HCl, ethanolamine HCl, ethanolamine HCl, ethanolamine HCl, ethanolamine HCl, ethanolamine HCl, ethanolamine HCl, ethanolamine HCl, ethanolamine HCl, ethanolamine HCl, ethanolamine HCl, ethanolamine HCl, ethanolamine HCl, ethanolamine HCl, ethanolamine HCl, ethanolamine HCl, ethanolamine HCl, ethanolamine HCl, ethanolamine HCl, ethanolamine HCl, ethanolamine HCl, ethanolamine HCl, ethanolamine HCl, ethanolamine HCl, ethanolamine HCl, ethanolamine HCl, ethanolamine HCl, ethanolamine HCl, ethanolamine HCl, ethanolamine HCl, ethanolamine HCl, ethanolamine HCl, ethanolamine HCl, ethanolamine In some embodiments, the nutrient source may be selected from the group consisting of ethanol, pyruvic acid, buffers, inorganic ions, inorganic salts, choline chloride, calcium pantothenate, pantothenic acid, folic acid, nicotinamide, 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.

[0028] The one or more media components may be at least 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 and at most 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, 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, at least 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 / ml and at most 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, 1 00, 150, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900 or 1000 ng / ml, 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 / ml, at least 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 / ml.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 μg / ml, at most 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 μg / ml, 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 μg / ml, at least 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 mg / ml, at most 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, 30 It may be added at a concentration of 0, 350, 400, 450, 500, 600, 700, 800, 900 or 1000 mg / ml, 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 mg / ml, or any range derivable therein.

[0029] The medium used contains at least one exogenously 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 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, or at most 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 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, 40 0, 450, 500, 600, 700, 800, 900 or 1000ng / L, at least 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 1000ng / ml, at most 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 / ml, 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 / ml, at least 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 μg / ml, at most 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 μg / ml, 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 μg / ml, at least 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, 60 0, 700, 800, 900 or 1000 mg / ml, at most 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 mg / ml, or about 0.1, 0.In some embodiments, the compound may be supplemented at a concentration of 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 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-alpha, TGF-beta, interferon-gamma, interferon-lambda, TSLP, thymopentin, pleotrophin and midkine.In particular, the culture medium can contain at least one of FLT3L and IL-7.More particularly, the culture medium can contain both FLT3L and IL-7. In some cases, one or more of FLT3 ligand (FLT3L), interleukin 7 (IL-7), stem cell factor (SCF), thrombopoietin (TPO), IL-2, IL-4, IL-6, IL-15, IL-21, TNF-alpha, TGF-beta, interferon-gamma, interferon-lambda, TSLP, thymopentin, pleotrophin, and midkine may be excluded.

[0030] Other culture conditions may be appropriately specified. For example, the culture temperature may be about 20 to 40°C, for example, at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40°C, at most 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40°C, or about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40°C (or any range that can be derived therefrom), but is not particularly limited thereto. The CO2 concentration can be about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10% (or any range derivable therein), such as about 2%-10%, such as about 2-5%, or any range derivable therein. The oxygen partial pressure can be at least 1, 5, 8, 10, 20%, or about 1, 5, 8, 10, 20%, or any range derivable therein.

[0031] The stromal cells and the 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.

[0032] Any cell population, e.g., stromal cells, mature cells, stem cells, progenitor cells, or PSCs induced to differentiate into mature cells, may be at least 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, 1 × 10 3 , 2×10 3 , 3×10 3 , 4×10 3 , 5×10 3 , 6×10 3 , 7×10 3 , 8×10 3 , 9×10 3 , 1×10 4 , 2×10 4 , 3×104 , 4×10 3 , 5×10 4 , 6×10 4 , 7×10 4 , 8×10 4 , 9×10 4 , 1×10 5 , 2×10 5 , 3×10 5 , 4×10 5 , 5×10 5 , 6×10 5 , 7×10 5 , 8×10 5 , 9×10 5 , 1×10 6 Or 2 x 10 6 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, 1×10 3 , 2×10 3 , 3×10 3 , 4×10 3 , 5×10 3 , 6×10 3 , 7×10 3 , 8×10 3 , 9×10 3 , 1×10 4 , 2×10 4 , 3×10 4 , 4×10 3 , 5×10 4 , 6×10 4 , 7×10 4 , 8×10 4 , 9×10 4 , 1×10 5 , 2×10 5 , 3×10 5 , 4×10 5 , 5×10 5 , 6×10 5 , 7×10 5 , 8×10 5 , 9×10 5 , 1×10 6 Or 2 x 10 6or 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, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 1×10 3 , 2×10 3 , 3×10 3 , 4×10 3 , 5×10 3 , 6×10 3 , 7×10 3 , 8×10 3 , 9×10 3 , 1×10 4 , 2×10 4 , 3×10 4 , 4×10 3 , 5×10 4 , 6×10 4 , 7×10 4 , 8×10 4 , 9×10 4 , 1×10 5 , 2×10 5 , 3×10 5 , 4×10 5 , 5×10 5 , 6×10 5 , 7×10 5 , 8×10 5 , 9×10 5 , 1×10 6 Or 2 x 10 6 The cell may comprise a single cell.

[0033] Culturing can be for any length of time, for example, 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, 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 days, or 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 weeks, or any range derivable therein. Culturing may or may not involve passaging of the cells.

[0034] The subject may include a clinical trial animal, such as a mouse, rat, rabbit, dog, cat, horse, or pig. The subject may be a human.

[0035] Any gene modification composition or gene modification method, such as gene editing, homologous or non-homologous recombination, RNA-mediated gene delivery, or any conventional nucleic acid delivery method, can be used to introduce exogenous nucleic acid into cells.Non-limiting examples of gene modification methods can include, for example, CRISPR / CAS9, zinc finger nuclease or TALEN technology gene editing methods.

[0036] The compositions and methods described herein may be modified such that the methods are for preparing cells having a particular phenotype. + 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 +T cells with CD34+CD5+CD7+, CD34+CD5+CD7-, natural killer T cells, regulatory T cells, antigen-specific T cells, intraepithelial lymphocyte T cells, or CD45+, CD11b+, CD11b-, CD15+, CD15-, CD24+, CD24-, CD114+, CD114-, CD182+, CD182-, CD4+, CD4-, CD14+, CD14-, CD11a+, CD11a-, CD91+, CD91-, CD16+, CD16-, C The present invention is for preparing cells that are D3+, CD3-, CD25+, CD25-, Foxp3+, 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-, or a combination thereof. As an example, intraepithelial lymphocytes (IEL) can be prepared by expressing allogeneic antigens in stromal cells.

[0037] The cells may be positive or negative for 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; and / or Alternatively, or in combination, the cells may be negative for 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.

[0038] The methods, cells, and compositions are of the phenotype: 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 + T cells with CD34+CD5+CD7+, CD34+CD5+CD7-, natural killer T cells, regulatory T cells, antigen-specific T cells, intraepithelial lymphocyte T cells, or CD45+, CD11b+, CD11b-, CD15+, CD15-, CD24+, CD24-, CD114+, CD114-, CD182+, CD182-, CD4+, CD4-, CD14+, CD14-, CD11a+, CD11a-, CD91+, CD91-, CD16+, C Cells that are D16-, CD3+, CD3-, CD25+, CD25-, Foxp3+, 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- may be excluded.

[0039] The cell may be a typical mature cell, such as a typical mature T cell having a phenotype of CD4+CD8-(SP4) or CD4-CD8ab.The cell may also be an NK cell, an iNKT cell, a Treg, a TCRgd T cell, or an innate immune cell, and may express CD8aa.

[0040] Genetic modification may also include the introduction of selectable or screenable markers to aid in in vitro or in vivo selection or screening or imaging. In particular, in vivo imaging agents or suicide genes may be exogenously expressed or added to the starting cells or progeny cells. The method may involve image-guided adoptive cell therapy.

[0041] The terms "subject" and "patient" may be used interchangeably and may refer to a human subject. A subject may be defined as a mammalian subject. A subject may also be a mouse, rat, pig, horse, non-human primate, cat, dog, cow, etc.

[0042] Throughout this application, the term "about" is used according to its plain and ordinary meaning in the field of molecular and cell biology to indicate that a value includes the standard deviation of error for the device or method being employed to measure the value.

[0043] The use of the words "a" or "an" when used in conjunction with the term "comprising" can mean "one," but is also compatible with the meanings of "one or more," "at least one," and "one or more."

[0044] As used herein, the terms "or" and "and / or" are used to describe whether multiple elements are in combination or mutually exclusive. For example, "x, y and / or z" can refer to "x" only, "y" only, "z" only, "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 in certain embodiments or aspects, x, y or z may be specifically excluded.

[0045] The words "comprising" (and any form of "comprising", e.g., "comprise" and "comprises"), "having" (and any form of "having", e.g., "have" and "has"), "including" (and any form of "including", e.g., "includes" and "include"), "characterized by" (and any form of "including", e.g., "characterized as") or "containing" (and any form of "containing", e.g., "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0046] The compositions and methods for their use may "comprise", "consist essentially of", or "consist" of any components or steps disclosed throughout this specification. The phrase "consisting of" excludes any unspecified elements, steps, or ingredients. The phrase "consisting essentially of" limits the scope of the described subject matter to those materials or steps specified and that do not materially affect its basic and novel characteristics. It is contemplated that embodiments and aspects described in the context of the term "comprising" can also be practiced in the context of the term "consisting of" or "consisting essentially of".

[0047] Any method in connection with a therapeutic, diagnostic or physiological purpose or effect may also be expressed in "use" claim language, e.g., the "use of" any compound, composition or agent discussed herein to achieve or produce a stated therapeutic, diagnostic or physiological purpose or effect.

[0048] The use of said one or more sequences or compositions may be used for any method described herein. Other embodiments and aspects are discussed throughout this application. Any embodiment discussed with respect to one aspect of this disclosure applies to other aspects of this disclosure as well, and vice versa. Furthermore, any composition of the invention may be used in any method of the invention, and any method of the invention may be used to make or utilize any composition of the invention. Aspects of an embodiment shown in the examples are also embodiments that may be implemented in a different example or in conjunction with an embodiment discussed elsewhere in this application, such as the summary, detailed description of the embodiments, claims, and figure legends.

[0049] Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and specific examples, while indicating specific embodiments of the present invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the present invention will become apparent to those skilled in the art from this detailed description. [Brief description of the drawings]

[0050] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. The disclosure may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0051] [Figure 1]Figure 1A-B: An example of a general transgene knock-in approach to achieve stage-specific expression followed by expression of an endogenous gene. (A) A bioactive transgene (i.e., reporter, chimeric antigen receptor) is inserted in frame into the 3' untranslated region of the endogenous gene using CRISPR / Cas9-mediated homologous recombination repair. In this process, the stop codon is replaced with a serine-arginine linker and a self-cleaving peptide sequence (P2A). This approach allows expression regulation by the endogenous promoter of the inserted transgene, while the translation of the endogenous gene remains unaltered. Also, a puromycin resistance gene cassette is inserted to aid in enrichment of edited pluripotent stem cells. (B) After CRE-TAT treatment at the iPSC stage, the puromycin resistance cassette can be removed, leaving only the loxp3 sequence. [Figure 2A] Figure 2A-B: Lentiviral vector-mediated constitutive high expression of CAR19 blocks normal differentiation from pluripotent stem cells. H1 hESCs were transduced with lentiviral vectors co-expressing CAR19LH and the reporter eGFP. Cells were selected for high eGFP expression and cloned. Clone 2 was differentiated into artificial thymic organoids (ATO) and analyzed at 2, 3, 4 and 5 weeks. Aberrant differentiation is shown with low frequencies of (A) CD3+TCRab+ cells and SP4 and SP8, and (B) a population of mostly CD4-CD8- "DN" cells. [Figure 2B] See legend to Figure 2A. [Diagram 3]Figure 3: Demonstration of stage-specific expression of foreign genes inserted downstream of GZMA. CRISPR / Cas9 editing was performed in H1 hESC cell lines to generate GZMA reporter cell lines. Knock-in (KI) of mCitrine was performed downstream of the endogenous GZMA gene in undifferentiated H1 hESCs and cells were cloned: clones were then differentiated to ATO and analyzed at 1, 3, and 5 weeks to assess reporter expression in specific T cell populations. Analysis of heterozygous clones is shown. mCitrine is expressed in 23% of SP8 and 2% of SP4, but not at precursor stage ISP4 or DP. [Figure 4A] Figure 4A-B: Demonstration that delayed onset of CAR19 expression allows normal T cell differentiation. Expression of the exogenous gene CAR19LH in mature T cells was achieved by insertion downstream of endogenous GZMA. Homozygous clones were generated by knock-in of CAR19LH into the GZMA locus in undifferentiated cells of the T-iPSC line "n11". Edited T-iPSC clones were then differentiated in the ATO system. The figure shows (3a) normal differentiation of DP (week 1) and SP8 and SP4 cells (weeks 3 and 4). (3b) At week 4, 67% of SP8 and 6% of SP4 cells expressed CAR. [Figure 4B] See legend to Figure 4A. [Figure 5A] Figure 5A-D. (A) Scheme of the protocol for generating mature T cells from hPSCs. All lentiviral transduction or genetic modifications using CRISPR / Cas9 were performed at the pluripotent stage. (B) Schematic representation of lentiviral vector expressing second generation anti-CD19 CAR (CAR19) under the ubiquitin constitutive promoter. (C) Representative flow cytometry plots of WT and CAR19 lenti-transduced hPSC ATOs. Transduced ATOs showed a dramatic reduction in DP T cells and enrichment of DNs. (D) Further analysis of DNs in CAR19 lenti-transduced ATOs showed upregulation of type 2 innate lymphoid cell (ILC2) markers. All FACS plots are gated for CD45+CD56-. [Figure 5B] See legend to Figure 5A. [Figure 5C] See legend to Figure 5A. [Figure 5D] See legend to Figure 5A. [Figure 6A] Figure 6A-B. (A) Tabular summary of selected candidate genes and their expression levels (RPKM) at each T cell differentiation stage. This analysis identified candidate genes with zero to low expression from PSC to late DP stages, but high expression in both SP8 and SP4. (B) Kinetics of T cell differentiation of GZMA-mCitrine reporter PSCs. Reporter expression was driven using the endogenous GZMA promoter. Reporter mCitrine expression was only present in mature SPs, but not in early T cell stages. [Figure 6B] See legend to Figure 6A. [Figure 7A] Figure 7A-D. (A) Schematic of the donor template design used to achieve specific CRISPR / Cas9-mediated HDR knock-in of CAR19 into the 3' untranslated region (UTR) of the GZMA locus. (B) Representative flow cytometry plots of T cell differentiation of GZMA-CAR19LH T-iPSCs. (C) Flow cytometry plots of ATO-derived T cells at week 5. (D) Representative flow cytometry plots of in vitro co-culture assay with GZMA-CAR19LH T-iPSC-derived T cells. [Figure 7B] See legend to Figure 7A. [Figure 7C] See legend to Figure 7A. [Figure 7D] See legend to Figure 7A. [Figure 8] Figure 8A-B: RAG1 / RAG2 double knockout (DKO) arrests T cell development at the double positive stage of T cell development. [Figure 9] FIG. 9A-B: Constitutive expression of an exogenous TCR rescues RAG1 / 2 DKO hPSC T cell differentiation. [Figure 10]Figure 10: RNA sequencing data. [Figure 11A] FIG. 11A-B. (A) FACS analysis of GZMA-CAR19LH T-iPSCs after TRAC disruption compared to WT T-iPSCs and TRAC disruption only control ATO analyzed at 2, 4, and 5 weeks. TRAC disruption prevents surface expression of TCRαβ and CD3 expression throughout differentiation from T-iPSCs. (B) FACS analysis of T cell development of TRAC-disrupted GZMA-CAR19LH T-iPSC ATO analyzed at 2, 4, and 5 weeks compared to wild-type and TRAC disruption only controls. As a result of the absence of TCRαβ and CD3 expression, T cells were unable to undergo positive selection and development was arrested at the DP stage. (C) Intracellular staining for GZMA showed the absence of GZMA expression in both TRAC-disrupted DP T cells at week 5. Mature SP8 T cells generated from wild-type (WT) controls were used to generate an isotype positive control. (D) FACS analysis of CD19 CAR expression at week 5. DP T cells do not express GZMA and therefore CAR expression was absent. [Figure 11B] See legend to Figure 11A. [Figure 11C] See legend to Figure 11A. [Figure 11D] See legend to Figure 11A. [Figure 12A]Figure 12A-C. (A) FACS analysis of RAG1-mCitrine T-iPSC ATOs at each T cell developmental stage. The mCitrine fluorescent reporter protein was knocked-in within the 3'UTR of RAG1. Reporter expression was analyzed at 1, 2, and 7 weeks to capture DN, ISP4, DP, and SP T cell stages. mCitrine expression was absent (data not shown) before T cell commitment (i.e., T-iPSCs, hEMPs) but increased from the DN to DP stages. Once positive selection was made, reporter expression was completely absent in naive mature SP8 and SP4 T cells. (B) Histogram and quantification of mCitrine reporter expression in RAG1-mCitrine T-iPSC ATOs. Reporter expression was present only from the DN to DP stages, but was absent before T cell commitment (i.e., T-iSPCs and hEMPs) and after positive selection (i.e., SP8 and SP4 T cells). (C) Analysis of RAG1-mCitrine SP8 and SP4 T cells during ex vivo culture and artificial stimulation. RAG1-mCitrine T-iPSC ATOs were dissociated at week 7. SP8 and SP4 T cells were cultured with IL-2 and IL-7, and a subset was maximally stimulated with PMA / PTI to measure any aberrant RAG1 expression that may occur upon activation. There was no detectable mCitrine expression in either SP8 or SP4 T cells in either condition. [Figure 12B] See legend to Figure 12A. [Figure 12C] See legend to Figure 12A. [Figure 13A]Figure 13A-D. FACS analysis of TCRαβ and CD3 of TRAC-disrupted RAG1-NYESO TCR T-iPSC ATOs analyzed at 2, 4, and 6 weeks compared to wild-type and TRAC-disrupted only controls. Exogenous NYESO(1G4) TCR was knocked-in within the 3'UTR of RAG1. As a result, exogenous TCR expression was accurately reflected by the expression profile of RAG1. Over the course of 6 weeks, NYESO TCR was downregulated as developing T cells underwent positive selection, resulting in the generation of SP8 and SP4 T cells that express neither TCRαβ nor CD3 (see also Figure 13B). (B) FACS analysis of developing T cells derived from TRAC-disrupted RAG1-NYESO TCR T-iPSC ATOs analyzed at 2, 4, and 6 weeks. At 6 weeks, approximately 40% of T cells underwent positive selection and subsequently downregulated TCRαβ and CD3. Mature SP8 and SP4 T cells were present only in the TCRαβ-CD3- population, while the remaining TCRαβ+CD3+ cells arrested progression at the DP stage. (C) Analysis of TCRαβ expression in TRAC-disrupted RAG1-NYESO TCR T-iPSC ATO analyzed at week 6. During development, TCRαβ expression disappeared as cells underwent positive selection. (D) Characterization of TCR-SP8 T cells generated from TRAC-disrupted RAG1-NYESO TCR T-iPSC ATO compared to wild-type controls at week 6. The resulting TCRαβ-CD3- SP8 T cells robustly expressed CD45RA+ and CD62L+ and were CD8α+ / CD8β+. [Figure 13B] See legend to Figure 13A. [Figure 13C] See legend to Figure 13A. [Figure 13D] See legend to Figure 13A. [Figure 14A]Figure 14A-E. (A) FACS analysis of TRAC-disrupted RAG1-NYESO TCR, GZMA-CAR19LH T-iPSC ATO analyzed at 2 and 6 weeks. The three genetic modifications accomplished the following: (1) TRAC disruption prevented endogenous TCR expression, (2) RAG1-NYESO TCR provided transient TCR expression for positive selection and then disappeared, and (3) the resulting mature SP8 T cells then expressed the CAR via the GZMA promoter. The combination of these strategies ultimately led to robust generation of TCRαβ-CD3- SP8 CAR T cells. (B) Characterization of TCRαβ-CD3- SP CAR T cells generated from ATO at 6 weeks. Mature T cells robustly expressed typical naive mature T cell markers and CD19 CAR. (C) Functional characterization of TCRαβ-CD3- SP8 CAR T cells. T cells were dissociated from 6 week ATO and co-cultured with either antigen positive (CD19+ K562) or negative (K562 WT) aAPCs to assess antigen-specific responses. In response to antigen, CAR T cells upregulated activation markers CD25 and 4-1BB (CD137) (D) and T cells also expressed multiple cytokines and the degranulation marker CD107a. All plots are gated on CD45+CD7+CD8+. (E) Characterization of TCRαβ-CD3- SP8 CAR T cells generated using the same triple gene knock-in strategy but expressing different CAR constructs. The first T-iPSC line expresses a CD19 CAR containing a long hinge spacer (LH) and CD28 costimulatory domain (CD28CS) by GZMA. The second T-iPSC line, expresses a CD19 CAR without a long spacer (SH = short hinge) and containing a 4-1BB costimulatory domain (41BB) by GZMA. Both cell lines were able to generate TCRαβ-CD3- SP8 CAR T cells expressing similar levels of typical naive mature T cell markers. [Figure 14B] See legend to Figure 14A. [Figure 14C] See legend to Figure 14A. [Figure 14D] See legend to Figure 14A. [Figure 14E] See legend to Figure 14A. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0052] Description of Example Embodiments I. Introduction This disclosure provides a solution to the problem of expressing transgenes, such as FOXP3 and CAR, that are detrimental to normal T cell differentiation by restricting the expression of foreign genes to mature T cells. The inventors have identified granzyme A (GZMA) as an endogenous gene with stage-specific expression that is tightly regulated during T cell differentiation in vitro and in normal thymus. The inventors have inserted a reporter gene downstream of GZMA in pluripotent stem cells (PSCs) and demonstrated that expression during differentiation is highly restricted to mature CD8+CD3+TCRab+CD4- (SP8) and mature CD4+ CD3+TCRab+CD8- (SP4) T cells (Figure 3). The inventors have also inserted a CAR19 construct into the GZMA locus of PSCs and demonstrated normal differentiation of mature T cells expressing high levels of the CAR construct (Figure 4). Thus, this approach could be applied to in vitro differentiation procedures using any type of pluripotent or multipotent cell or lymphoid cell that is a precursor of mature T cells as described herein and known in the art.

[0053] II. Definition The term "transgene" refers to a gene that is transferred into a cell in vitro (i.e., by gene transfer / transduction / transfection techniques). A transgene can be a gene that is not naturally expressed in a cell, such as a CAR or an engineered TCR.

[0054] The term "chimeric antigen receptor" or "CAR" refers to an engineered receptor in which a specific portion of an immune effector cell is grafted. Such receptors are used to graft the specific portion of a monoclonal antibody onto T cells; the transfer of the coding sequence is facilitated by retroviral or lentiviral vectors. The receptor is called chimeric because it is composed of portions from different sources. The most common form of such molecules is a fusion of a single chain variable fragment (scFv) from a monoclonal antibody fused to the CD3-zeta transmembrane domain and endodomain; CD28 or 41BB intracellular domain, or a combination thereof. Such molecules result in the transmission of a signal in response to the recognition of its target by the scFv. One example of such a construct is 14g2a-zeta, which is a fusion of a scFv from hybridoma 14g2a, which recognizes disialoganglioside GD2. When T cells express this molecule (usually by transduction with an oncoretroviral vector), they recognize and kill target cells expressing GD2 (e.g., neuroblastoma cells). To target malignant B cells, researchers have redirected the specificity of T cells with a chimeric immune receptor specific for the B cell lineage molecule CD19. The variable parts of the heavy and light chains of immunoglobulins are fused by a flexible linker to form scFvs. The scFv is preceded by a signal peptide (which is cleaved off) that directs the nascent protein to the endoplasmic reticulum and subsequent surface expression. The flexible spacer allows the scFv to be oriented in different directions to allow antigen binding. The transmembrane domain is a typical hydrophobic alpha helix, usually derived from the original molecule of the signaling endodomain that protrudes into the cell and transmits the desired signal.

[0055] The term "antigen" refers to any substance that causes the immune system to produce antibodies or any substance to which T cells respond. Antigens are peptides that are 5-50 amino acids in length, or at least 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 in length, but not more than 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, The peptide may be 85, 90, 95, 100, 125, 150, 175, 200, 250 or 300, 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.

[0056] The term "allogeneic to the recipient" is intended to refer to cells that are not isolated from the recipient. The cells may be cells that are not isolated from the patient. The cells may be cells that are not isolated from a genetically matched individual (e.g., a relative with a compatible genotype).

[0057] The terms "xeno-free (XF)" or "animal component free (ACF)" or "animal free", when used in reference to a medium, extracellular matrix, or culture condition, refer to a medium, extracellular matrix, or culture condition that is essentially free of components derived from a xenogeneic animal. In the culture of human cells, any protein of a non-human animal, e.g., mouse, can be a xenogeneic component. A xeno-free matrix may be essentially free of any components derived from a non-human animal, and thus excludes mouse feeder cells or Matrigel™. Matrigel™ is a solubilized basement membrane preparation extracted from Engelbreth-Holm-Swarm (EHS) mouse sarcoma, a tumor rich in extracellular matrix proteins, including laminin (major component), type IV collagen, heparin sulfate proteoglycan, and entactin / nidogen.

[0058] The term "defined," when used in reference to a medium, extracellular matrix, or culture conditions, refers to a medium, extracellular matrix, or culture conditions in which the nature and amount of nearly all components are known.

[0059] "Chemically defined media" refers to media in which the chemical nature and amounts of nearly all ingredients are known. Such media are also called synthetic media. An example of a chemically defined medium is TeSR™.

[0060] As used herein, a cell is "substantially free" of a particular reagent or element, such as serum, signaling inhibitors, animal components or feeder cells, exogenous genetic elements or vector elements, if the cell has less than 10% of the element, and "essentially free" of a particular reagent or element if the cell has less than 1% of the element(s). Even more desirable, however, are cell populations in which exogenous genetic elements or vector elements make up less than 0.5% or less than 0.1% of the total cell population.

[0061] A culture, matrix or medium is "essentially free" of a particular reagent or component, such as serum, signal transduction inhibitors, animal components, or feeder cells, if the culture, matrix, or medium, respectively, has levels of such reagent below detectable levels using routine detection methods known to those of skill in the art, or if such agents are not exogenously added to the culture, matrix, or medium. Serum-free medium may be essentially free of serum.

[0062] "Peripheral blood cells" refers to the cellular components of blood found in the circulating blood pool, such as red blood cells, white blood cells and platelets.

[0063] "Hematopoietic stem and progenitor cells" or "hematopoietic progenitor cells" refer to cells that are committed to the hematopoietic lineage but can undergo further differentiation into the hematopoietic lineage, and include hematopoietic stem cells, multipotential hematopoietic stem cells (hemocyte blasts), myeloid progenitor cells, megakaryocytic progenitor cells, erythroid progenitor cells, and lymphoid progenitor cells. "Hematopoietic stem cells (HSCs)" are multipotent stem cells that give rise to all blood cell types, including myeloid (monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes / platelets, dendritic cells) and lymphoid cell lineages (T cells, B cells, NK cells).

[0064] A "vector" or "construct" (sometimes referred to as a "vehicle" for gene delivery or transfer) refers to a macromolecule, complex of molecules, or viral particle that contains a polynucleotide that is delivered to a host cell either in vitro or in vivo. The polynucleotide may be a linear or circular molecule.

[0065] A "plasmid" is a common type of vector, which is an extrachromosomal DNA molecule that is separate from the chromosomal DNA and can replicate independently of the chromosomal DNA. In some cases, it is circular and double-stranded.

[0066] By "expression construct" or "expression cassette" is intended a nucleic acid molecule capable of directing transcription. An expression construct contains 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.

[0067] The term "corresponding to" is used herein to mean that a polynucleotide sequence is homologous (i.e., identical, though 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. By way of illustration, the nucleotide sequence "TATAC" corresponds to the reference sequence "TATAC" and is complementary to the reference sequence "GTATA."

[0068] A "gene," "polynucleotide," "coding region," "sequence," "segment," "fragment," or "transgene" "encoding" a particular protein is a nucleic acid molecule that can be transcribed in vitro or in vivo and, optionally, translated into a gene product, e.g., a polypeptide, when placed under the control of appropriate regulatory sequences. The coding region can be in the form of either cDNA, genomic DNA, or RNA. If in the form of DNA, the nucleic acid molecule can be single-stranded (i.e., the sense strand) or double-stranded. The boundaries of a coding region are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus. A gene can include, but is not limited to, cDNA of prokaryotic or eukaryotic mRNA, genomic DNA sequences of prokaryotic or eukaryotic DNA, and synthetic DNA sequences. A transcription termination sequence is usually located 3' to the gene sequence.

[0069] The term "cell" is used herein in its broadest sense in the art to refer to a living organism that is a structural unit of tissue in 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. As used herein, a cell may be a naturally occurring cell or an artificially modified cell (e.g., a fused cell, a genetically modified cell, etc.).

[0070] As used herein, the term "stem cell" refers to the cell that has the ability of self-renewal and pluripotency or multipotency. Typically, stem cell can regenerate damaged tissue. The stem cell in this specification can be, but is not limited to, embryonic stem (ES) cell, induced pluripotent stem cell, or tissue stem cell (also called tissue-specific stem cell or somatic stem cell).

[0071] Embryonic stem (ES) cells are pluripotent stem cells derived from early embryos. ES cells were first established in 1981, and have been used to create knockout mice since 1989. Human ES cells were established in 1998, and are now available for use in regenerative medicine.

[0072] Unlike ES cells, tissue stem cells have limited differentiation potential. Tissue stem cells are present in specific locations within tissues and have undifferentiated intracellular structures. Thus, the pluripotency of tissue stem cells is typically low. Tissue stem cells have a high nucleus / cytoplasm ratio and few intracellular organelles. Most tissue stem cells have low pluripotency, long cell cycles and proliferation capacity over the life of an individual. Based on the site from which the cells are derived, tissue stem cells are divided into categories such as skin system, digestive system, bone marrow system, and nervous system. Tissue stem cells of the skin system include epidermal stem cells and hair follicle stem cells. Tissue stem cells of the digestive system include pancreatic (common) stem cells and liver stem cells. Tissue stem cells of the bone marrow system include hematopoietic stem cells and mesenchymal stem cells. Tissue stem cells of the nervous system include neural stem cells and retinal stem cells.

[0073] "Induced pluripotent stem cells", commonly abbreviated as iPS cells or iPSCs, refer to a type of pluripotent stem cell that is artificially prepared from non-pluripotent cells, typically adult somatic cells or terminally differentiated cells, such as fibroblasts, hematopoietic cells, muscle cells, nerve cells, epidermal cells, etc., by introducing specific factors called reprogramming factors.

[0074] "Pluripotency" refers to the potential of a stem cell to differentiate into any cell that constitutes one or more tissues or organs, or in particular any of the three germ layers: endoderm (stomach lining, digestive tract, lungs), mesoderm (muscle, bone, blood, urogenital) or ectoderm (epidermal tissue and nervous system). As used herein, "pluripotent stem cell" refers to a cell that can differentiate into a cell derived from any of the three germ layers, such as a direct descendant of a totipotent cell or an induced pluripotent cell.

[0075] As used herein, "embryonic mesodermal progenitor cells" or "EMP cells" refer to pluripotent stem cells (PSCs), which may include hematopoietic stem and progenitor cells, embryonic stem cells (ESCs), and induced pluripotent stem cells (iPSCs) that are committed to the mesoderm. Commitment to the mesoderm is initiated by epithelial-mesenchymal transition (EMT) and by reciprocal changes in the expression of the cell surface proteins EpCAM / CD326 and NCAM / CD56. EMP cells may be further defined as EMP CD326-CD56+. EMP CD326 - CD56 + The cells may be generated by culturing PSCs in the presence of components such as, but not limited to, one or more of activin A, BMP4, VEGF, FGF2, and combinations thereof.

[0076] By "operably linked" to a nucleic acid molecule is intended that two or more nucleic acid molecules (e.g., a nucleic acid molecule to be transcribed, a promoter and an enhancer element) are linked in such a manner as to allow transcription of the nucleic acid molecule. By "operably linked" to a peptide and / or polypeptide molecule is intended that two or more peptide and / or polypeptide molecules are linked in such a manner as to result in a single polypeptide chain, i.e., a fusion polypeptide, having at least one property of each peptide and / or polypeptide component of the fusion. The fusion polypeptide is in particular chimeric, i.e., composed of heterologous molecules.

[0077] III. Transgene The transgene, the first transgene or the second transgene may comprise an antigen targeting molecule, such as a CAR or a TCR.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 resulting from 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, MAGE-5, MAGE-6, RAGE, NY-ESO, pl85erbB2, pl80erbB-3, c-met, nm- 23H1, PSA, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, beta-catenin, CDK4, Mum-1, p 15, p 16, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, beta-HCG, BCA225, BTAA, CA 125, CA 15-3\CA 27.29\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, SDCCAG1 6, TA- 90\Mac-2 binding protein\cyclophilm C binding protein, TAAL6, TAG72, TLP, TPS, GPC3, MUC16, MUC18, LMP1, EBMA-1, BARF-1, CS1, CD319, HER1, B7H6, L1 CAM, IL6 and MET.

[0078] Examples of tumor antigens include glioma-associated antigens, carcinoembryonic antigen (CEA), EGFRvlll, IL-IIRa, IL-13Ra, EGFR, FAP, B7H3, Kit, CA LX, CS-1, MUC1, BCMA, bcr-abl, HER2, b-human chorionic gonadotropin, alpha fetoprotein (AFP), ALK, CD19, cyclin Bl, 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-CA IX, EPCAM, and EVT6- AML, TGS5, human telomerase reverse transcriptase, polysialic acid, PLAC1, RUI, RU2(AS), intestinal carboxylesterase, Lewis Y, sLe, LY6K, mut hsp70-2, M-CSF, MYCN, RhoC, TRP-2, CYPIBI, BORIS, prostase, prostate specific antigen (PSA), PAX3, PAP, NY-ESO-1, LAGE-la, LMP2, NCAM, p53, p53 mutant, Ras mutant, gpl00, prostein, OR51 E2, PANX3, PSMA, PSCA, Her2 / neu, hTERT, HMWMAA, HAVCR1, VEGFR2, PDGFR-beta, survivin and telomerase, legumain, HPV E6, E7, sperm protein 17, SSEA-4, tyrosinase, TARP, WT1, prostate cancer tumor antigen-1 (PCTA-1), ML-IAP, MAGE, and MAGE-A1.MAD-CT-1, MAD-CT-2, MelanA / MART 1, XAGE1, ELF2M, ERG (TMPRSS2 ETS fusion gene), NA17, neutrophil elastase, sarcoma translocation breakpoint, 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.

[0079] Further examples of tumor cell antigens to which CAR and / or TCR can be directed 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, CD44v 7 / 8, CD70, CD123, CD138, CD171, CD133, CEA, c-Met, CSPG4, EGFR, EGFR family, such as ErbB2 (HER2), EGF RvIII, EGP2, EGP40, ERBB3, ERBB4, ErbB3 / 4, EPCAM, EphA2, EpCAM, folate receptor-a, FAP, FBP, fetal AchR, FRα , GD2, G250 / CAIX, GD3, glypican-3 (GPC3), GUCY2C, HER1, HER2, ICAM-1, IL-13Rα2, IL-11Rα, Kras, Kras G12D, L1CAM, lambda, Lewis-Y, 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 Examples of the CAR include 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). The CAR can be a first, second, third, or later generation CAR. The CAR can be bispecific for any two non-identical antigens, or specific for more than two non-identical antigens.

[0080] Non-limiting examples of bacterial antigens that may be targeted by the CARs and / or TCRs of the present disclosure include at least the following: Aeromonas aminopeptidase; Arthrobacter globiformis mosaic protein; Arthrobacter globiformis DMGO protein; A. globiformis dimethylglycine oxidase; Bacillus inteins; Bacillus thermolysin; Bacillus anthracis lethal factor; Bacillus anthracis protective antigen PA63; B. anthracis protective antigen; B. circulans inteins; B. polymyxin B peptide; B. anthracis edema factor; B. anthracis edema factor (S447N mutant form); B. anthracis protective antigen PA20; B. anthracis protective antigen PA 63 (active form); Cry1Ab toxin of B. thuringiensis; OspA of Borrelia spp.; NapA of Borrelia burgdorferi; BmpA of Borrelia spp.; P41 of Borrelia spp.; p45 of Borrelia spp.; OspC of B. burgdorferi; OspA of B. burgdorferi; P41 of B. burgdorferi; P41 of B. burgdorferi; B31 of B. burgdorferi; P100 of B. burgdorferi; DbpA of B. burgdorferi; BmpA of B. burgdorferi; DbpB of B. burgdorferi; B. garinii (B. garinii);P58 from B. garinii;OspC from B. spielmanii;B. burgdorferi grade 2;B. abortus;B. pertussis toxin;B. pertussis;FHA from B. pertussis;165 LPS from B. pertussis;Adenylate cyclase antigen from B. pertussis;Pertussis toxin subunit, A promoter from B. pertussis;Pertactin from B. pertussis;Fimbriae 2 / 3 from B. pertussis;Adenylate cyclase toxoid from B. pertussis;Bordetella pertussis whole cells (Higashihama I strain);Bordetella pertussis, lipopolysaccharide;C. jejuni;C. albicansalbicans enolase;Candida albicans;C. pneumoniae outer membrane protein VD3;C. pneumoniae;Chlamydia trachomatis MOMP;Chlamydia trachomatis antigen;Chlamydia trachomatis HSP70 protein;C. trachomatis outer membrane protein;C. trachomatis HSP70;C. trachomatis;C. trachomatis MOMP;C. trachomatis LGV II;PGP-3D of C. trachomatis;active MOMP of C. trachomatis;MOMP protein of Chlamydia trachomatis;active MOMP of C. trachomatis;active HSP70 of C. trachomatis;W4 MOMP of C. trachomatis;W5 MOMP of C. trachomatis;MOMP, E78 of C. trachomatis;Clostridium difficile toxoid A;Clostridium difficile binary toxin A subunit;Clostridium difficile glutamate dehydrogenase;Clostridium difficile toxoid B;BoNT-A light chain of C. botulinum;BoNT-B light chain of C. botulinum;BoNT-E light chain of C. botulinum;C. difficile difficile toxin B, TcdB;BoNT-F light chain of C. botulinum;C. difficile toxin B;chitinase of Cl. paraputrificum;C3 toxoid of C. botulinum;C. difficile toxin A;C. difficile toxin A (ribotype 027);C. difficile toxin A (ribotype 078);C. difficile toxin B (ribotype 027);C. difficile toxin B (ribotype 078);diphtheria toxoid of C. diphtheriae;C. diphtheriae toxin (mutant CRM197);C. diphtheriae toxin;E. 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;H. pylori;H. pylori outer membrane protein;H. pylori Cag antigen;H. pylori flagellin A antigen;H. pylori urease small subunit antigen;H. pylori Vac (toxin) antigen;Helicobacter pylori protein;Helicobacter pylori urease large subunit protein;L. pneumophila (L. pneumophila;LipL32 lipoprotein of L. interrogans;L. biflexa;Internalin of L. monocytogenes;Flagellin of L. monocytogenes;Active listeriolysin;Mycobacterium spp.;Heat shock protein 65 of Mycobacterium spp.;Heat shock protein 70 of Mycobacterium spp.;PstS1 of Mycobacterium spp.;Secreted proteins of Mycobacterium spp.;DESAT6 of Mycobacterium spp.;Heat shock protein DnaK of Mycobacterium spp.;TB Ag85A of Mycobacterium spp.;Early secreted target 6 protein of Mycobacterium Tuberculosis;ESAT-6 of Mycobacterium tuberculosis;M. tuberculosis tuberculosis FbpA;M. tuberculosis;M. tuberculosis PPD protein;M. tuberculosis old tuberculin;M. bovis HSP65;M. tuberculosis heat shock protein 70;M. tuberculosis heat shock protein 65;M. tuberculosis MTB heat shock protein;M. tuberculosis MTB lipoprotein;M. tuberculosis MTB fragment and esat6 fragment;P. aeruginosa (P.aeruginosa exotoxin A;Urealyticum (serotype 3);Salmonella typhi OMP;Salmonella Typhiurium;Salmonella paratyphi A antigen;Salmonella paratyphi B antigen;Salmonella typhimurium antigen;Salmonella spp. 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;S. enterica (S. enterica flagellin;S. minnesota LPS;Staphylococcus aureus delta hemolysin;Staphylococcus aureus protease protein;Staphylococcus spp. protein A;S. aureus subsp. penicillin-binding protein 2;S. aureus staphylokinase;S. aureus protein A;S. aureus;S. aureus enterotoxin type B toxoid;S. aureus HLA;S. aureus Hlg B;S. aureus leukocidin-D;S. aureus leukocidin-E;S. aureus LukF-PV;S. aureus LukS-PV;S. aureus phenol-soluble modulin alpha 3;S. aureus toxic shock syndrome toxin-1;S. pneumoniae cell wall polysaccharide antigen of S. pneumoniae; cell wall polysaccharide antigen of S. pneumoniae; B(1-4)-galactosidase of S. pneumoniae; streptavidin of Streptomyces avidinii; tetanus toxoid antigen of C. tetani; TeNT light chain of C. tetani; C. tetani toxin C-fragment; treponemal membrane protein A of T. pallidum; active p41 of T. pallidum; active p47 of T. pallidum; T.P17 of T. pallidum;Treponemal membrane protein A;Active p15 of T. pallidum;T. pallidum antigen;Tp0453 antigen of T. pallidum;tpp17 of T. pallidum;Y. enterocolitica (subtype O:3);Y. enterocolitica (serotype O:8);Y. enterocolitica (serotype O:9);MEP4 of Arthroderma benhamiae;MEP5 of Arthroderma benhamiae;MEP3 of Arthroderma benhamiae;VPS10 of Arthroderma gypseum;AMPP of Arthroderma gypseum;DAPB of Arthroderma gypseum;Arthroderma otae otae; SEY1 from Arthroderma otae; VPS10 from Arthroderma otae; ferredoxin from Spirulina maxima; desA from Arthrospira platensis; ATP6 from Ascaris suum; ND5 from Ascaris suum; ND4L from Ascaris suum; V-type proton ATPase from Ascaris suum; PET8 from Ashbya gossypii; TIM21 from Ashbya gossypii; and ATG22 from Ashbya gossypii.

[0081] Non-limiting examples of viral antigens that may 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 grade 2; cytomegalovirus (CMV); CMV AD169; CMV grade 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 precursor membrane;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;EBV BFRF3;EBV BMRF1;EBV EBNA1;EBV early antigen;Enterovirus 71 (EV71);EV71 VP1;EV71 VP0;Enterovirus A Cox 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 type);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 Pre S1 / 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 antigen;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-1 IIIB gp120 protein;HIV-1 IIIB gp160 protein;HIV-1 451 gp120 protein;HIV-1 451 gp160 protein;HIV-1 IIIB p24 protein;HIV-1 gp120 and gp41 chimeric antigens;HIV-1 Gp160 protein;HIV-1 Gag polyprotein Pr55;HIV-1 p17 protein;HIV-1 p24 protein;Human papillomavirus (HPV);HPV 16;HPV16 E6 protein;HPV16 E7 protein;HPV18 E6 protein;HPV18 E7 protein;HPV 18 L1 protein;HPV 2, HPV 3;HPV 18;HPV 11;HPV 16 L1 protein;HPV 33 L1 protein;HPV 6 L1 protein;HPV 16 L1 protein;Herpes simplex virus (HSV);HSV type 1;HSV-2 gG peptide;HSV8 ORF8&ORF65 protein;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 grade III antigen;Human T-lymphotropic virus (HTLV);HTLV types 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 p24 protein type 1;HTLV glycoprotein 46 mosaic protein;HTLV mosaic protein;Human T-cell lymphotropic virus type I gp21+gp46 chimeric antigen;Human T-cell lymphotropic virus type II gp21+gp46 chimeric antigen;Human T-cell lymphotropic virus type I&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 grade 2 antigen;IAV H3N2 matrix protein 1;IAV H4N4 hemagglutinin protein;IAV H4N6 hemagglutinin protein;Recombinant IAV H4N8 hemagglutinin protein;Influenza type B (IBV);Influenza type B viral 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 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 co-capsid protein;Parvovirus B19 VP1 / 2;Parvovirus B19 VP1;Rabies virus (RV);Rabies virus glycoprotein;RV M2;RV (MRV strain) nucleoprotein;RV nucleoprotein;Respiratory syncytial virus (RSV);Respiratory syncytial virus (long strain) antigen;Y. 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 proteins;Rubella virus (RuV);RuV grade IV;RuV envelope protein 1;RuV grade 2;RuV K1S;RuV K2S;RuV nucleocapsid;RuV capsid;RuV E1 protein;RuV E2 protein;RuV E1 mosaic;RuV capsid C;Rotavirus grade 3;Rotavirus;Tick-borne encephalitis virus (TBEV);TBEV gE, TBEV NE proteins;TBEV gE, TBEV NE, 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;Volavirus 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;Huayangshan 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.

[0082] IV. Cells of the Disclosure The cells of the present disclosure include cells such as pluripotent stem cells or hematopoietic stem or progenitor cells, and mature cells such as mature T cells.The pluripotent stem cells that contain the transgene, the first transgene, or the second transgene 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 that promotes the differentiation of the cells into mature cells can use stromal cells that are co-cultured with the stem cells or progenitor cells.The cells of the present disclosure are further described below.

[0083] A. Stromal cells Stromal cells are cells of the connective tissue of any organ, such as bone marrow, thymus, uterine mucosa (endometrium), prostate, and ovaries. They are the cells that support the function of the parenchymal cells of that organ. Fibroblasts (also known as mesenchymal stromal cells / MSCs) and pericytes are the most common types of stromal cells.

[0084] Interactions between stromal cells and tumor cells are known to play a major role in cancer growth and progression. In addition, bone marrow stromal cells have been reported to participate in human hematopoiesis and inflammatory processes by regulating local cytokine networks (e.g., M-CSF, LIF).

[0085] Stromal cells of the bone marrow, thymus, and other hematopoietic organs regulate hematopoietic and immune cell development through cell-cell ligand-receptor interactions and through the release of soluble factors, including cytokines and chemokines. Stromal cells of these tissues form niches that regulate stem cell maintenance, lineage specification and commitment, and differentiation into effector cell types.

[0086] The stroma is composed of non-malignant host cells and provides an extracellular matrix in which tissue-specific cell types, and in some cases tumors, can grow.

[0087] B. Hematopoietic Stem and Progenitor Cells Due to the significant medical potential of hematopoietic stem and progenitor cells, considerable research has been conducted to try to improve methods for the differentiation of hematopoietic progenitor cells from embryonic stem cells. In human adults, hematopoietic stem cells, which reside primarily in the bone marrow, give rise to a heterogeneous population of hematopoietic (CD34+) progenitor cells that differentiate into all cells of the blood system. In adult humans, hematopoietic progenitor cells proliferate and differentiate daily, resulting in the generation of hundreds of billions of mature blood cells. Hematopoietic progenitor cells are also present in umbilical cord blood. In vitro, human embryonic stem cells can be differentiated into hematopoietic progenitor cells. Hematopoietic progenitor cells may also be expanded or enriched from a sample of peripheral blood as described below. Hematopoietic cells can be of human origin, murine origin, or any other mammalian species.

[0088] Isolation of hematopoietic progenitor cells can be performed by any method of choice, 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" using antibodies bound to a solid matrix, such as a plate, or any other convenient technique.

[0089] 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), those based on differences in cell surfaces (lectin and antibody affinity), and those based on differences in vital staining properties (mitochondrial binding dye rhol23 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 may have various sophistications, such as multiple color channels, small and obtuse angle light scattering detection channels, impedance channels, etc.

[0090] The antibodies utilized in the above methods or methods used to assess the purity of cell types (e.g., flow cytometry) can be conjugated to identifiable agents, such as, 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 β-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 additional fluorescent dyes that can be conjugated to antibodies, see Haugland, Molecular Probes: Handbook of Fluorescent Probes and Research Chemicals (1992-1994). Metal compounds that can be conjugated to antibodies include, but are not limited to, ferritin, gold colloid, and especially colloidal superparamagnetic beads.Haptens that can be conjugated to antibodies include, but are not limited to, biotin, digoxigenin, oxazalone, and nitrophenol.Radioactive compounds that can be conjugated or incorporated into antibodies are known in the art and include, but are not limited to, technetium 99m (99TC), 125I, and any radioactive nuclide, such as, but are not limited to, amino acids, including 14C, 3H, and 35S.

[0091] Other techniques for positive selection that allow for precise separation may also be used, such as affinity columns, etc. The method should allow for removal of the non-target cell population until less than about 20%, preferably less than about 5%, remains.

[0092] Cells can be selected based on light scattering properties and their expression of various cell surface antigens. Purified stem cells have low side scattering and low to moderate forward scattering profiles by FACS analysis. Cytospin preparations show enriched stem cells with a size between mature lymphoid cells and mature granulocytes.

[0093] In addition, CD34 + The seeded population of cells can be enriched prior to culturing, for example, using the methods described in U.S. Patent No. 4,714,680 to Sutherland et al. (1992). For example, the cells are subjected to negative selection to remove cells expressing lineage-specific markers. The cell population is enriched for non-CD34 + Hematopoietic cells and / or specific hematopoietic cell subsets can be subjected to negative selection for depletion.Negative selection can be performed based on the cell surface expression of various molecules, such as 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 cell lineage specific marker.Negative selection can be performed based on the cell surface expression of various molecules, such as the cocktail of antibodies (e.g., CD2, CD3, CD11b, CD14, CD15, CD16, CD19, CD56, CD123, and CD235a) that can be used for the separation of other cell types, for example, by MACS or column separation.

[0094] As used herein, lineage negative (LIN-) refers to cells that lack at least one marker associated with lineage-committed cells, such as markers associated with T cells (e.g., CD2, 3, 4, and 8), markers associated with B cells (e.g., CD10, 19, and 20), markers associated with myeloid cells (e.g., CD14, 15, 16, and 33), markers associated with natural killer ("NK") cells (e.g., CD2, 16, and 56), markers associated with RBCs (e.g., glycophorin A), markers associated with megakaryocytes (CD41), markers associated with 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 may be performed, for example, by positive selection of c-kit+ or Thy-1+. Further enrichment may be obtained by methods known in the art, using the mitochondria-binding dye rhodamine 123 and selecting for rhodamine+ cells. Highly enriched compositions may include CD34 + , preferably CD34 + LIN - , most preferably CD34 + Thy-1 + LIN - A highly enriched population of stem cells and methods for obtaining the same are well known to those skilled in the art, see, for example, the methods described in PCT / US94 / 09760, PCT / US94 / 08574, and PCT / US94 / 10501.

[0095] Various techniques can be used to separate cells by first removing lineage-specialized cells. Monoclonal antibodies are particularly useful for identifying markers associated with specific cell lineages and / or differentiation stages. The antibodies can be bound to a solid support to allow crude separation. The separation technique used should maximize the retention of viability of the collected fraction. Various techniques of varying effectiveness can be used to obtain a "relatively crude" separation. Such a separation is when the unwanted cells that remain with the cell population to be retained are up to 10%, usually no more than about 5%, and preferably no more than about 1% of the total cells present. The specific technique used depends on the efficiency of the separation, the associated cytotoxicity, the ease and speed of execution, and the need for sophisticated equipment and / or technical skill.

[0096] Selection of hematopoietic progenitor cells need not be performed solely using cell-specific markers: a combination of negative and positive selection may be used to obtain enriched cell populations.

[0097] C. Source of Blood Cells Hematopoietic stem cells (HSCs) normally reside in the bone marrow but can be pushed out into the blood in a process called mobilization that is used clinically to harvest large numbers of HSCs from peripheral blood. One example of the mobilizing agent of first choice is granulocyte colony-stimulating factor (G-CSF).

[0098] Circulating CD34 in peripheral blood + Hematopoietic stem or progenitor cells can be collected by apheresis either in an unperturbed state or after mobilization following exogenous administration of hematopoietic growth factors such as G-CSF. The number of stem or progenitor cells collected after mobilization is greater than that obtained in an unperturbed state after apheresis. The source of the cell population can be a subject in which cells have not been mobilized by exogenously applied factors, since hematopoietic stem or progenitor cells do not need to be enriched in vivo.

[0099] The cell population for use in the methods described herein can be mammalian cells, such as human cells, non-human primate cells, rodent cells (e.g., mouse or rat), bovine cells, ovine cells, porcine cells, equine cells, sheep cells, canine cells, and feline cells, or mixtures thereof. Non-human primate cells include rhesus monkey cells. The cells can be obtained from an animal, such as a human patient, or can be from a cell line. When the cells are obtained from an animal, they can be used as is, e.g., as unsegregated cells (i.e., a mixed population); they can be initially established in culture, e.g., by transformation; or they can have been subjected to a preliminary purification method. For example, the cell population can be engineered by positive or negative selection based on the expression of cell surface markers; stimulated in vitro or in vivo with one or more antigens; treated in vitro or in vivo with one or more biological modifiers; or a combination of any or all of these.

[0100] The cell populations include peripheral blood mononuclear cells (PBMCs), whole blood or fractions thereof including mixed populations, spleen cells, bone marrow cells, tumor infiltrating lymphocytes, cells obtained by leukapheresis, biopsy tissue, lymph nodes, such as tumor draining lymph nodes. Suitable donors include immunized donors, non-immunized (naive) donors, treated or untreated donors. A "treated" donor is one that has been exposed to one or more biological modifiers. An "naive" donor has not been exposed to one or more biological modifiers.

[0101] For example, peripheral blood mononuclear cells (PBMCs) can be obtained according to methods known in the art, as previously described, examples of which are reviewed in Kim et al. (1992); Biswas et al. (1990); Biswas et al. (1991).

[0102] Methods for obtaining hematopoietic progenitor cells from cell populations are also well known in the art. Hematopoietic progenitor cells may be expanded using various cytokines, such as hSCF, hFLT3 and / or IL-3 (Akkina et al., 1996), and CD34 + Cells may be enriched using MACS or FACS, or CD34 cells may be isolated using negative selection techniques as described above. + The cells may be concentrated.

[0103] It is also possible to obtain a cell sample from a subject and then enrich for the desired cell type. For example, PBMCs and / or CD34 + Hematopoietic cells can be isolated from blood as described herein. Cells can also be isolated from other cells using a variety of techniques, such as isolation and / or activation by antibody binding to epitopes on the cell surface of the desired cell type. Another method that can be used includes negative selection using antibodies against cell surface markers to selectively enrich for specific cell types without activating the cells by receptor ligation.

[0104] Bone marrow cells can be obtained from the iliac crest, femur, tibia, spine, rib, or other medullary cavity. Bone marrow can be removed from a patient and isolated by various separation and washing procedures. An exemplary procedure for the isolation of bone marrow cells includes the following steps: (a) centrifugation of bone marrow suspension into three fractions and collection of intermediate fraction or buffy coat; (b) centrifuging the buffy coat fraction of step (a) once more into a separation fluid, typically Ficoll (a trademark of Pharmacia Fine Chemicals AB), and collecting the intermediate fraction containing bone marrow cells; and (c) washing the fraction collected in step (b) for the recovery of bone marrow cells that can be retransfused.

[0105] D. Pluripotent stem cells Hematopoietic stem 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 and progenitor cells, embryonic stem cells or induced pluripotent stem cells) that are directly seeded into cell differentiation system, such as artificial thymus organoid (ATO), that differentiates cells into mature cells.The cells used in the methods and compositions described herein can be derived cells or progeny of PSC, such as but not limited to mesodermal progenitor cells, endothelial progenitor cells or hematopoietic progenitor cells.

[0106] The pluripotent stem cell can be an embryonic stem (ES) cell derived from the inner cell mass of a blastocyst.The pluripotent stem cell can be an induced pluripotent stem cell induced by reprogramming of somatic cells.The pluripotent stem cell can be an embryonic stem cell induced by somatic cell nuclear transfer.

[0107] Embryonic stem (ES) cells are pluripotent cells derived from the inner cell mass of a blastocyst. ES cells can be isolated by removing the trophectoderm, the outer layer of the developing embryo, and then culturing the inner cell mass on a feeder layer of non-growing cells. Under appropriate conditions, a colony of proliferating undifferentiated ES cells will result. This colony can be removed, dissociated into individual cells, and then replated on a new feeder layer. The replated cells will continue to grow, and new colonies of undifferentiated ES cells can result. This new colony can then be removed, dissociated, replated again, and cultured and expanded. This process of "subculture" or "passage" of undifferentiated ES cells can be repeated several times to create cell lines containing undifferentiated ES cells (U.S. Patent Nos. 5,843,780; 6,200,806; 7,029,913). A "primary cell culture" is a culture of cells obtained directly from a tissue, such as the inner cell mass of a blastocyst. A "subculture" is any culture derived from a primary cell culture.

[0108] Methods for obtaining mouse ES cells are well known. In one method, preimplantation blastocysts from mouse line 129 are treated with mouse antisera to remove the trophectoderm, and the inner cell mass is cultured on a feeder layer of chemically inactivated mouse embryonic fibroblasts in medium containing fetal bovine serum. Colonies of undifferentiated ES cells that develop are subcultured on a feeder layer of mouse embryonic fibroblasts in the presence of fetal bovine serum to generate a population of ES cells. In some methods, mouse ES cells can be grown in culture in the absence of a feeder layer by adding the cytokine leukemia inhibitory factor (LIF) to serum-containing culture medium (Smith, 2000). In other methods, mouse ES cells can be grown in culture in the presence of bone morphogenetic protein and LIF in serum-free medium (Ying et al., 2003).

[0109] 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 example method, day 5 human blastocysts are exposed to rabbit anti-human spleen cell antiserum, followed by a 1:5 dilution of guinea pig complement to lyse the trophectoderm cells. After removal of the lysed trophectoderm cells from the intact inner cell mass, the inner cell mass is cultured on a feeder layer of gamma-irradiation inactivated mouse embryonic fibroblasts in the presence of fetal bovine serum. After 9-15 days, clumps of cells derived from the inner cell mass can be chemically (i.e., exposed to trypsin) or mechanically dissociated and replated in fresh medium containing fetal bovine serum and a feeder layer of mouse embryonic fibroblasts. After further expansion, colonies with undifferentiated morphology are selected by micropipette, mechanically dissociated into clumps, and replated (see U.S. Patent No. 6,833,269). ES-like morphology is characterized as compact colonies with a distinctly high nuclear-cytoplasmic ratio and prominent nucleoli. The resulting ES cells can be routinely passaged by brief trypsinization or by selection of individual colonies with a micropipette. In some methods, human ES cells can be grown in culture without serum by culturing the ES cells on a feeder layer of fibroblasts in the presence of basic fibroblast growth factor (Amit et al., 2000). In other methods, human ES cells can be grown in culture without a feeder cell layer by culturing the cells on a protein matrix, such as Matrigel™ or laminin, in the presence of a "conditioned" medium containing basic fibroblast growth factor (Xu et al., 2001). The medium is preconditioned by co-culturing with fibroblasts.

[0110] Methods for the isolation of rhesus monkey and common marmoset ES cells are also known (Thomson, and Marshall, 1998; Thomson et al., 1995; Thomson and Odorico, 2000).

[0111] Another source of ES cells is established ES cell lines. A variety of mouse cell lines and human ES cell lines are known, and the conditions for their culture growth and propagation have been defined. For example, mouse CGR8 cell line is established from the inner cell mass of mouse 129 lineage embryos, and the culture of CGR8 cells can be cultured and propagated without feeder layer in the presence of LIF. As a further example, human ES cell lines H1, H7, H9, H13 and H14 were established by Thompson et al. Also, subclones H9.1 and H9.2 of H9 cell line have been developed.

[0112] The source of ES cell can be blastocyst, the cell derived by culturing the inner cell mass of blastocyst, or the cell obtained from the culture of established cell line.Therefore, as used herein, the term "ES cell" can refer to the cell of the inner cell mass of blastocyst, the ES cell obtained from the culture of inner cell mass, and the ES cell obtained from the culture of ES cell line.

[0113] Induced pluripotent stem (iPS) cells are cells that have characteristics of ES cells but are obtained by reprogramming differentiated somatic cells. Induced pluripotent stem cells have been obtained by various methods. In one method, adult human dermal fibroblasts are transfected with the transcription factors Oct4, Sox2, c-Myc, and Klf4 using retroviral transduction (Takahashi et al., 2007). The transfected cells are plated on SNL feeder cells (a fibroblast cell line of mouse cells that produces LIF) in medium supplemented with basic fibroblast growth factor (bFGF). After approximately 25 days, colonies resembling human ES cell colonies appear in culture. ES cell-like colonies are picked and expanded on feeder cells in the presence of bFGF.

[0114] Based on the cell characteristics, the cells of the ES cell-like colonies are induced pluripotent stem cells. The induced pluripotent stem cells are morphologically similar to human ES cells and express various human ES cell markers. In addition, when cultured and grown under conditions known to result in the differentiation of human ES cells, the induced pluripotent stem cells differentiate accordingly. For example, the induced pluripotent stem cells can differentiate into cells with neural cell structures and neural cell markers.

[0115] 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). 12-20 days after infection, colonies with human ES cell morphology become visible. These colonies are picked and expanded. The induced pluripotent stem cells that compose these colonies are morphologically similar to human ES cells, express various human ES cell markers, and form teratomas with neural tissue, cartilage, and intestinal epithelium after injection into mice.

[0116] Also known is a method for preparing induced pluripotent stem cells from mice (Takahashi and Yamanaka, 2006). Induction of iPS cells typically requires expression or exposure to at least one member of the Sox family and at least one member of the Oct family. Sox and Oct are considered to be central to the transcriptional regulatory hierarchy that specifies the identity of ES cells. For example, Sox can be Sox-1, Sox-2, Sox-3, Sox-15, or Sox-18; Oct can be Oct-4. Additional factors such as Nanog, Lin28, Klf4, or c-Myc can enhance reprogramming efficiency; a specific reprogramming factor set can be a set that includes Sox-2, Oct-4, Nanog, and optionally Lin-28; or a set that includes Sox-2, Oct4, Klf, and optionally c-Myc.

[0117] iPS cells, like ES cells, have characteristic antigens that can be identified or confirmed by immunohistochemistry or flow cytometry using antibodies against SSEA-1, SSEA-3 and SSEA-4 (Developmental Studies Hybridoma Bank, National Institute of Child Health and Human Development, Bethesda Md.) and antibodies against TRA-1-60 and TRA-1-81 (Andrews et al., 1987). The pluripotency of embryonic stem cells is approximately 0.5-10 × 10 6 This can be confirmed by injecting 1000 cells into the hind leg muscle of 8-12 week old male SCID mice, which develop teratomas that display at least one cell type of each of the three germ layers.

[0118] E. Embryonic mesoderm progenitor cells Embryonic mesoderm precursor (EMP) cells are cells derived from PSCs. EMP cells can be prepared by culturing PSCs, such as ESCs, 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 acquires cell surface expression of CD56 at day 3.5 of culture, and CD326 - CD56 + EMPs undergo the process of EMT. Significant upregulation of key transcriptional regulators of EMT, Snail-1, Snail-2 and Twist, is consistent with downregulation of E-cadherin and CD326, as well as tight junction-related genes such as claudins, syndecans and occludin. Significant reorganization of cytoskeletal proteins can be documented based on upregulation of vimentin. There is also upregulation of fibronectin expression, consistent with the role that this extracellular matrix protein plays in promoting cell migration. Furthermore, CD326 - CD56 +EMPs lose pluripotency as evidenced by the downregulation of cell surface markers CD9 and SSEA-4, which are often used to identify undifferentiated cells, and three key transcription factors, Nanog, Sox-2, and Oct-4 / Pou5f1, that are associated with ESC pluripotency. - CD56 + Expression of known mesodermal markers KDR, PDGFR-α and CD34 by EMP continues to increase up to 2 weeks after mesoderm induction. - CD56 + EMPs are a primitive cell population generated from PSCs by the process of epithelial-mesenchymal transition. - CD56 + Cells emerge when the full mesodermal potential is still present, prior to the more lineage restricted mesodermal population.

[0119] After mesoderm induction, CD326 - CD56 + Cells are isolated by fluorescence activated cell sorting (FACS) on day 3.5 and can optionally be further differentiated into mesodermal cell lineages under vascular endothelial stem cell, cardiac stem cell or mesenchymal stem cell conditions.

[0120] V. Transgenes A. Antigen-Targeting Transgenes The transgene, the first transgene or the second transgene may comprise an antigen targeting molecule, such as a CAR or a TCR.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 resulting from 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, MAGE-5, MAGE-6, RAGE, NY-ESO, pl85erbB2, pl80erbB-3, c-met, nm- 23H1, PSA, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, beta-catenin, CDK4, Mum-1, p 15, p 16, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, beta-HCG, BCA225, BTAA, CA 125, CA 15-3\CA 27.29\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, SDCCAG1 6, TA- 90\Mac-2 binding protein\cyclophilic C binding protein, TAAL6, TAG72, TLP, TPS, GPC3, MUC16, MUC18, LMP1, EBMA-1, BARF-1, CS1, CD319, HER1, B7H6, L1 CAM, IL6 and MET.

[0121] Examples of tumor antigens include glioma-associated antigens, carcinoembryonic antigen (CEA), EGFRvlll, IL-IIRa, IL-13Ra, EGFR, FAP, B7H3, Kit, CA LX, CS-1, MUC1, BCMA, bcr-abl, HER2, b-human chorionic gonadotropin, alpha fetoprotein (AFP), ALK, CD19, cyclin Bl, 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-CA IX, EPCAM, and EVT6- AML, TGS5, human telomerase reverse transcriptase, prisialic acid, PLAC1, RUI, RU2(AS), intestinal carboxylesterase, Lewis Y, sLe, LY6K, mut hsp70-2, M-CSF, MYCN, RhoC, TRP-2, CYPIBI, BORIS, prostase, prostate specific antigen (PSA), PAX3, PAP, NY-ESO-1, LAGE-la, LMP2, NCAM, p53, p53 mutant, Ras mutant, gpl00, prostein, OR51 E2, PANX3, PSMA, PSCA, Her2 / neu, hTERT, HMWMAA, HAVCR1, VEGFR2, PDGFR-beta, survivin and telomerase, legumain, HPV E6, E7, sperm protein 17, SSEA-4, tyrosinase, TARP, WT1, prostate cancer tumor antigen-1 (PCTA-1), ML-IAP, MAGE, and MAGE-A1.MAD-CT-1, MAD-CT-2, MelanA / MART 1, XAGE1, ELF2M, ERG (TMPRSS2 ETS fusion gene), NA17, neutrophil elastase, sarcoma translocation breakpoint, 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.

[0122] Further examples of tumor cell antigens to which the CAR and / or TCR can be directed include, for example, at least 5T4, 8H9, αvβ6 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, e.g., ErbB2 (HER2), EGFRvIII, EGP2, EGP40, ERBB3, ERBB4, ErbB3 / 4, EPCAM, EphA2, EpCAM, folate receptor-a, FAP, FBP, fetal AchR, FR□, GD2, G250 / CAIX, GD3, glypican-3 (GPC3), GUCY2C, HER1, HER2, ICAM-1, IL-13R□2, IL-11Rα, Kras, Kras G12D, L1CAM, lambda, Lewis-Y, 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 Examples of the CAR include 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). The CAR can be a first, second, third, or later generation CAR. The CAR can be bispecific for any two non-identical antigens, or specific for more than two non-identical antigens.

[0123] Non-limiting examples of bacterial antigens that may be targeted by the CARs and / or TCRs of the present disclosure include at least the following: Aeromonas aminopeptidase; Arthrobacter globiformis mosaic protein; Arthrobacter globiformis DMGO protein; A. globiformis dimethylglycine oxidase; Bacillus inteins; Bacillus thermolysin; Bacillus anthracis lethal factor; B. anthracis protective antigen PA63; B. anthracis protective antigen; B. circulans inteins; B. polymyxin B peptide; B. anthracis edema factor; B. anthracis edema factor (S447N mutant form); B. anthracis protective antigen PA20; B. anthracis protective antigen PA 63 (active form); Cry1Ab toxin of B. thuringiensis; OspA of Borrelia spp.; NapA of Borrelia burgdorferi; BmpA of Borrelia spp.; P41 of Borrelia spp.; p45 of Borrelia spp.; OspC of B. burgdorferi; OspA of B. burgdorferi; P41 of B. burgdorferi; P41 of B. burgdorferi; B31 in B. burgdorferi;P100 in B. burgdorferi;DbpA in B. burgdorferi;BmpA in B. burgdorferi;DbpB in B. burgdorferi;B. garinii;P58 in B. garinii;OspC in B. spielmanii;B. burgdorferi grade 2;B. abortus;B. pertussis toxin;B. pertussis;B. pertussis FHA;B. pertussis 165 LPS; B. pertussis adenylate cyclase antigen; B. pertussis pertussis pertussis toxin subunit, A promoter; B. pertussis pertactin; B. pertussis fimbriae 2 / 3; B. pertussis adenylate cyclase toxoid; Bordetella pertussis whole cells (Higashihama I strain); Bordetella pertussis lipopolysaccharide; C. jejuni; C. albicans enolase; Candida albicans; C. pneumoniae outer membrane protein VD3; C. pneumoniae; Chlamydia trachomatis MOMP; Chlamydia trachomatis antigen; Chlamydia trachomatis HSP70 protein; C. trachomatis outer membrane protein; C. trachomatis HSP70; C. trachomatis; C. trachomatis MOMP; C. trachomatis LGV II; C. trachomatis PGP-3D; C.Active MOMP of C. trachomatis;MOMP protein of Chlamydia trachomatis;Active MOMP of C. trachomatis;Active HSP70 of C. trachomatis;W4 MOMP of C. trachomatis;W5 MOMP of C. trachomatis;MOMP, E78 of C. trachomatis;C. difficile toxoid A;C. difficile binary toxin A subunit;C. difficile glutamate dehydrogenase;C. difficile toxoid B;BoNT-A light chain of C. botulinum;BoNT-B light chain of C. botulinum;BoNT-E light chain of C. botulinum;C. difficile toxin B, TcdB;BoNT-F light chain of C. botulinum;C. difficile toxin B;Cl. paraputrificum chitinase of;C3 toxoid of C. botulinum;C. difficile toxin A;C. difficile toxin A (ribotype 027);C. difficile toxin A (ribotype 078);C. difficile toxin B (ribotype 027);C. difficile toxin B (ribotype 078);diphtheria toxoid of C. diphtheriae;C. diphtheriae toxin (mutant CRM197);C. diphtheriae toxin;chaperone protein fimC of E. coli;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;H. pylori;H. pylori outer membrane protein;H. pylori Cag antigen;H. pylori flagellin A antigen;H. pylori urease small subunit antigen;H. pylori Vac (toxin) antigen;Helicobacter pylori protein;Helicobacter pylori urease large subunit protein;L. pneumophila;L. interrogans LipL32 lipoprotein;L. biflexa;L. monocytogenes internalin;L.monocytogenes flagellin;active listeriolysin;Mycobacterium spp.;Mycobacterium heat shock protein 65;Mycobacterium heat shock protein 70;Mycobacterium spp. PstS1;Mycobacterium spp. secreted proteins;Mycobacterium spp. DESAT6;Mycobacterium spp. heat shock protein DnaK;Mycobacterium spp. TB Ag85A;Mycobacterium tuberculosis early secretory target 6 protein;Mycobacterium tuberculosis ESAT-6;M. tuberculosis FbpA;M. tuberculosis;M. tuberculosis PPD protein;M. tuberculosis old tuberculin;M. bovis HSP65;M. tuberculosis heat shock protein 70;M. tuberculosis heat shock protein 65;M. tuberculosis MTB heat shock protein;M. tuberculosis MTB lipoprotein;M. tuberculosis MTB fragment and esat6 fragment of P. aeruginosa exotoxin A; urealyticum (serotype 3); OMP of Salmonella typhi; Salmonella typhiurium; Salmonella paratyphi A antigen; Salmonella paratyphi B antigen; Salmonella typhimurium antigen; beta-lactamase of Salmonella spp.; pagC antigen of Salmonella typhi; flag of Salmonella typhi; uridine phosphorylase of Salmonella typhimurium; flagellin protein of Salmonella typhimurium; lipid A of Salmonella minnesota (R595) monophosphoryl;LPS of Salmonella typhimurium;flagellin of S. enterica;LPS of S. minnesota;delta hemolysin of S. aureus;protease protein of S. aureus;protein A of Staphylococcus spp.;penicillin-binding protein 2 of S. aureus;staphylokinase of S. aureus;protein A of S. aureus;S. aureus;enterotoxin type B toxoid of S. aureus;HLA of S. aureus;Hlg B of S. aureus;leukocidin-D of S. aureus;leukocidin-E of S. aureus;LukF-PV of S. aureus;LukS-PV of S. aureus;phenol-soluble modulin alpha 3 of S. aureus;toxic shock syndrome toxin-1 of S. pneumoniae;cell wall polysaccharide antigen of S.cell wall polysaccharide antigen of S. pneumoniae;B(1-4)-galactosidase of S. pneumoniae;streptavidin of Streptomyces avidinii;tetanus toxoid antigen of C. tetani;TeNT light chain of C. tetani;C. tetani toxin C-fragment;Treponemal membrane protein A of T. pallidum;active p41 of T. pallidum;active p47 of T. pallidum;P17 of T. pallidum;Treponemal membrane protein A;active p15 of T. pallidum;Tp0453 antigen of T. pallidum;tpp17 of T. pallidum;Y. enterocolitica (subtype O:3);Y. enterocolitica (serotype O:8);Y. enterocolitica (serotype O:9);MEP4 of Arthroderma benhamiae;A These include MEP5 from Arthroderma benhamiae;MEP3 from Arthroderma benhamiae;VPS10 from Arthroderma gypseum;AMPP from Arthroderma gypseum;DAPB from Arthroderma gypseum;CHO2 from Arthroderma otae;SEY1 from Arthroderma otae;VPS10 from Arthroderma otae;ferredoxin from Spirulina maxima;desA from Arthrospira platensis;ATP6 from Ascaris suum;ND5 from Ascaris suum;ND4L from Ascaris suum;V-type proton ATPase from Ascaris suum;PET8 from Ashbya gossypii;TIM21 from Ashbya gossypii;and ATG22 from Ashbya gossypii.

[0124] Non-limiting examples of viral antigens that may 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 grade 2; cytomegalovirus (CMV); CMV AD169; CMV grade 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 precursor membrane;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;EBV BFRF3;EBV BMRF1;EBV EBNA1;EBV early antigen;Enterovirus 71 (EV71);EV71 VP1;EV71 VP0;Enterovirus A Cox 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-Naxamata);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 Pre S1 / 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-1 IIIB gp120 protein;HIV-1 IIIB gp160 protein;HIV-1 451 gp120 protein;HIV-1 451 gp160 protein;HIV-1 IIIB p24 protein;HIV-1 gp120 and gp41 chimeric antigens;HIV-1 Gp160 protein;HIV-1 Gag polyprotein Pr55;HIV-1 p17 protein;HIV-1 p24 protein;Human papillomavirus (HPV);HPV 16;HPV16 E6 protein;HPV16 E7 protein;HPV18 E6 protein;HPV18 E7 protein;HPV 18 L1 protein;HPV 2, HPV 3;HPV 18;HPV 11;HPV 16 L1 protein;HPV 33 L1 protein;HPV 6 L1 protein;HPV 16 L1 protein;Herpes simplex virus (HSV);HSV 1;HSV-2 gG peptide;HSV8 ORF8&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 grade III antigen;Human T-lymphotropic virus (HTLV);HTLV types 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 p24 protein type 1;HTLV glycoprotein 46 mosaic protein;HTLV mosaic protein;Human T-cell lymphotropic virus type I gp21+gp46 chimeric antigen;Human T-cell lymphotropic virus type II gp21+gp46 chimeric antigen;Human T-cell lymphotropic virus type I & 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 grade 2 antigen;IAV H3N2 matrix protein 1;IAV H4N4 hemagglutinin protein;IAV H4N6 hemagglutinin protein;Recombinant IAV H4N8 hemagglutinin protein;Influenza type B (IBV);Influenza type B viral 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 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 co-capsid protein;Parvovirus B19 VP1 / 2;Parvovirus B19 VP1;Rabies virus (RV);Rabies virus glycoprotein;RV M2;RV (MRV strain) nucleoprotein;RV nucleoprotein;Respiratory syncytial virus (RSV);Respiratory syncytial virus (long strain) antigen;Y. 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 grade IV;RuV envelope protein 1;RuV grade 2;RuV K1S;RuV K2S;RuV nucleocapsid;RuV capsid;RuV E1 protein;RuV E2 protein;RuV E1 mosaic;RuV capsid C;Rotavirus grade 3;Rotavirus;Tick-borne encephalitis virus (TBEV);TBEV gE, TBEV NE protein;TBEV gE, TBEV NE, 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;Volavirus 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;Huayangshan 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 antiviral protein;Echovirus type 9; MuV; African swine fever virus (ASFV); African swine fever virus p30 protein; and African swine fever virus p54 protein.

[0125] The transgene, first transgene or second transgene disclosed herein may be included in the nucleotide sequence encoding the polypeptide. As used herein, "protein" or "polypeptide" refers to a molecule that comprises at least 5 amino acid residues. As used herein, the term "wild type" refers to the endogenous form of a molecule that naturally occurs in an organism. Although wild type forms of proteins or polypeptides are used to generate an immune response, modified forms of proteins or polypeptides are also used. The above terms may be used interchangeably. "Modified protein" or "modified polypeptide" or "variant" refers to a protein or polypeptide whose chemical structure, particularly amino acid sequence, is modified compared to the wild type protein or polypeptide. A modified / variant protein or polypeptide may have at least one modified activity or function (recognizing that the protein or polypeptide may have multiple activities or functions). It is specifically envisaged that a modified / variant protein or polypeptide may be modified with respect to one activity or function but still retain wild type activity or function in other respects, such as immunogenicity.

[0126] The size of the transgene (wild type or modified) may be, but is not limited to, 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, 109, 109, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118 7, 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, The amino acid sequence may comprise 850, 875, 900, 925, 950, 975, 1000, 1100, 1200, 1300, 1400, 1500, 1750, 2000, 2250, 2500 or more amino acid residues, and any range derivable therein, or derivatives of the corresponding amino acid sequences described herein or referenced herein.

[0127] B. T Cell Receptors (TCRs) and Methods for Making Engineered TCRs T cell receptors or TCRs are molecules found on the surface of T lymphocytes (T cells) that are responsible for recognizing fragments of antigens as peptides bound to major histocompatibility complex (MHC) molecules. The TCR is composed of two different protein chains (i.e., it is a heterodimer). In 95% of human T cells, the TCR consists of alpha (α; also referred to herein as "a") and beta (β-also referred to herein as "b") chains, while in 5% of T cells the TCR consists of gamma and delta (γ / δ) chains. This ratio varies during ontogeny and in disease states as well as in different species.

[0128] When the TCR engages antigenic peptides and MHC (peptide / MHC), the T lymphocyte is activated by signal transduction, a series of biochemical events mediated by associated enzymes, co-receptors, specialized adaptor molecules and activated or released transcription factors. The TCR is a disulfide-linked membrane-anchored heterodimeric protein consisting of highly variable alpha (α) and beta (β) chains that are usually expressed as part of a complex with the invariant CD3 chain molecule. T cells expressing this receptor are referred to as α:β (or αβ or ab) T cells, while a minority of T cells express an alternative receptor formed by variable gamma (γ-also referred to herein as "g") and delta (δ-also referred to herein as "d") chains and are referred to as γδ (or gd) T cells.

[0129] Each chain is composed of two extracellular domains: a variable (V) region and a constant (C) region, both of which are immunoglobulin superfamily (IgSF) domains arranged in an antiparallel β-sheet. The constant region is proximal to the cell membrane, followed by a transmembrane region and a short cytoplasmic tail, while the variable region binds the peptide / MHC complex.

[0130] The variable domains of both the α-chain and β-chain of the TCR each have three hypervariable or complementarity determining regions (CDRs), while the variable region of the β-chain has an additional region of hypervariability (HV4) that does not normally contact the antigen and is therefore not considered a CDR.

[0131] The residues are located in two regions of the TCR, the α-chain and β-chain interface and within the β-chain framework region that is believed to be in close proximity to the CD3 signaling complex. CDR3 is the main CDR responsible for recognition of processed antigens, but CDR1 of the alpha chain has also been shown to interact with the N-terminal portion of antigenic peptides, whereas CDR1 of the β-chain interacts with the C-terminal portion of the peptide. CDR2 is believed to recognize MHC. CDR4 of the β-chain is not believed 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 linking sequence in which cysteine ​​residues form disulfide bonds that form the link between the two chains.

[0132] That the TCR is a member of the IgSF protein means that it can be compared to antibodies and BCRs. In terms of similarity, the TCR is like half an antibody with a heavy and light chain, except that the heavy chain lacks the crystallizable portion (Fc) (note: ontogenetically, TCR alpha undergoes VJ recombination, so it is like a light chain; TCR beta undergoes VDJ recombination, so it is like a heavy chain). So, ontologically, the TCR is like one of the antibody-binding fragments of an antibody. The two subunits of the TCR are twisted together. Antibodies use their Fc region to bind to Fc receptors on natural white blood cells, but the TCR is already docked on the cell membrane. However, due to the short cytoplasmic tail, it cannot mediate signaling itself, so the TCR still requires CD3 and Zeta for signaling to occur properly, just as antibodies require binding to FcR to initiate signaling. Thus, MHC-TCR-CD3 interactions in T cells are functionally similar to Ag-Ig-FcR interactions in myeloid leukocytes and Ag-Ig-CD79 interactions in B cells.

[0133] The foreign TCR comprises a protein that can be expressed from TCR-alpha and TCR-beta genes. The foreign TCR can comprise a protein that is expressed from TCR-gamma and TCR-delta genes. The foreign TCR can comprise a protein that is expressed from TCR-alpha and TCR-beta genes, and the antigen-recognizing receptor comprises a protein that is expressed from TCR-gamma and TCR-delta genes. The foreign TCR can comprise a protein that is expressed from TCR-gamma and TCR-delta genes, and the antigen-recognizing receptor comprises a protein that is expressed from TCR-alpha and TCR-beta genes.

[0134] Methods for generating antigen-specific TCRs are known in the art. The method may include, for example, (1) synthesizing a known or predicted HLA-restricted peptide epitope derived from a protein of interest (e.g., tumor antigen, neoantigen, etc. in sequencing data); (2) presenting this by antigen-presenting cells (for expansion) or tetramers (for direct selection) to a pool of T cells (e.g., tumor-infiltrating lymphocytes in the case of tumor antigen-specific T cells) from which the TCR sequences are extracted; (3) selecting or screening the antigen-specific T cells (e.g., FACS sorting of antigen-specific T cells based on tetramer binding); (4) cloning (by RT-PCR) and sequencing the TCR genes (i.e., the alpha and beta or gamma and delta chains of the TCR); cloning and sequencing may be performed at either the population level or single cell level; and (5) confirming and analyzing the specificity of the TCR by, for example, transducing peripheral blood T cells with such sequences and testing the functionality of the TCR clone by assessing its reactivity against target cells expressing cognate peptide-MHC complexes. Responsiveness is usually measured on the basis of cytokine production (eg, interferon gamma).

[0135] C. Chimeric Antigen Receptors (CARs) and Methods for Making CARs The term "chimeric antigen receptor" or "CAR" refers to an engineered receptor that is grafted with arbitrary specific parts onto immune effector cells. Such receptors are used to graft the specific parts of monoclonal antibodies onto immune cells; the transfer of the coding sequence is facilitated by retroviral or lentiviral vectors. The receptor is called chimeric because it is composed of parts from different sources.

[0136] The most common form of such molecules is a fusion of a single chain variable fragment (scFv) derived from a monoclonal antibody fused with the CD3-zeta transmembrane domain and endodomain, the CD28 or 41BB intracellular domain, or a combination thereof. Such molecules result in the transmission of a signal in response to the recognition of its target by the scFv. One example of such a construct is 14g2a-zeta, which is a fusion of the scFv derived from the hybridoma 14g2a (which recognizes the disialoganglioside GD2). When immune cells express this molecule (for example, by transduction with an oncoretroviral vector), the cells recognize and kill target cells expressing GD2 (e.g., neuroblastoma cells).

[0137] The variable parts of the immunoglobulin heavy and light chains are fused by a flexible linker to form the scFv. The scFv is preceded by a signal peptide (which is cleaved off) that directs the nascent protein to the endoplasmic reticulum and subsequent surface expression. The flexible spacer allows the scFv to be oriented in various ways to allow antigen binding. The transmembrane domain is a typical hydrophobic alpha helix, usually derived from the original molecule, with the signaling endodomain projecting into the cell to transmit the desired signal.

[0138] A CAR may comprise at least one extracellular domain and at least one intracellular domain. The extracellular domain may comprise a target-specific binding element, or a moiety referred to as an antigen-binding moiety or a ligand-binding moiety, that specifically binds to any particular antigen of interest.

[0139] The intracellular or cytoplasmic domain may comprise one or more costimulatory signaling regions and a zeta chain portion. The costimulatory signaling region refers to the portion that constitutes the intracellular domain of the costimulatory molecule of the CAR. The costimulatory molecule may be a cell surface molecule other than an antigen receptor or its ligand that is required for an efficient response of immune cells to an antigen.

[0140] 1. Signal peptide The polypeptide of the present disclosure may include a signal peptide. "Signal peptide" refers to a peptide sequence that directs the transport and localization of the protein within the cell, for example to a specific cell organelle (e.g., endoplasmic reticulum) and / or to the cell surface. The signal peptide may direct the protein being produced into the endoplasmic reticulum. This is essential when the receptor is glycosylated and anchored to the cell membrane. Generally, the signal peptide that is naturally attached to the most amino-terminal component is used (e.g., in scFv with a light chain-linker-heavy chain configuration, the natural signal of the light chain is used).

[0141] The signal peptide may be cleaved after passage through the endoplasmic reticulum (ER), i.e., it is a cleavable signal peptide. A restriction site may be present at the carboxy terminus of the signal peptide to facilitate cleavage.

[0142] 2. Antigen-binding domain The polypeptide of the present disclosure may comprise one or more antigen-binding domains. An "antigen-binding domain" refers to a region of a polypeptide that can bind to an antigen under appropriate conditions. The antigen-binding domain may be one or more antibody-based (e.g., CD20 antibody) single-chain variable fragments (scFvs). The antigen-binding domain may comprise a heavy chain variable (VH) region and a light chain variable (VL) region, where the VH and VL regions are present 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.

[0143] The variable region of the antigen-binding domain of the polypeptide of the present disclosure may be modified by mutating amino acid residues in the CDR 1, CDR 2 and / or CDR 3 regions of the VH and / or VL to improve one or more binding properties (e.g., affinity) of the antibody. The term "CDR" refers to a complementarity determining region based on a portion of the variable chain of immunoglobulins (antibodies) and T cell receptors produced by B cells and T cells, respectively, where the molecule binds to its specific antigen. Since most sequence variations associated with immunoglobulins and T cell receptors are found within the CDRs, this region is sometimes referred to as a hypervariable region. Mutations may be introduced by site-directed mutagenesis or PCR-mediated mutagenesis, and the effect on antibody binding or other functional properties of interest may be evaluated in suitable in vitro or in vivo assays. Preferably, conservative modifications are introduced, typically more than one, but 2, 3, 4 or 5 residues in the CDR regions are altered. Mutations may be amino acid substitutions, additions, or deletions.

[0144] Framework modifications can be made, for example, by "backmutating" one or more framework residues to the corresponding germline sequence to reduce immunogenicity to the antibody.

[0145] It is also envisaged that an antigen-binding domain may be made multispecific or multivalent by multimerising the antigen-binding domain with pairs of VH and VL domains that bind either the same antigen (multivalent) or different antigens (multispecific).

[0146] The binding affinity of an antigen-binding region, e.g., a variable region (heavy and / or light chain variable region) or a CDR, is at least 10M, 10M, 10M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 M, or 10 -13 The KD of the antigen-binding region, e.g., the variable region (heavy 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 M (or any range derivable therein).

[0147] The binding affinity, K or K can be determined by methods known in the art, such as by surface plasmon resonance (SRP)-based biosensors, Kinetic Exclusion Assay (KinExA), optical scanners for microarray detection based on polarization-modulated grazing incidence differential reflectance (OI-RD) or ELISA.

[0148] A polypeptide comprising a humanized binding region has 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% affinity for a host cell compared to a polypeptide comprising a non-humanized binding region, e.g., a binding region derived from a mouse. , 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 104, 106, 106, 108, 109, 110, 115 or 120% or 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%.

[0149] The framework regions of the human framework, e.g., FR1, FR2, FR3, and / or FR4, individually or in combination, have at least 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, 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, 161, 162, 163, 164, 1 65, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 6, 197, 198, 199 or 200, or 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, 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, 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, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 1 5, 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, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199 or 200, 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 ...10, 111, 112, 113, 114, 115, 1 3, 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, 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, 7, 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,The nucleic acid sequence may have 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199 or 200 amino acid substitutions, consecutive amino acid additions or consecutive amino acid deletions (or any range derivable therein).

[0150] The framework regions of the mouse framework, e.g., FR1, FR2, FR3, and / or FR4, individually or in combination, have at least 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, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 109, 109, 108, 109, 109, 102, 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, 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, 161, 162, 163, 164, 1 65, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 6, 197, 198, 199 or 200, or 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, 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, 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, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 1 5, 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, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199 or 200, 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 ...10, 111, 112, 113, 114, 115, 1 3, 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, 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, 7, 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,The nucleic acid sequence may have 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199 or 200 amino acid substitutions, consecutive amino acid additions or consecutive amino acid deletions (or any range derivable therein).

[0151] The substitutions may be made at 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, 109, 109, 108, 110, 111, 112, 113, 114, 115, 116, 117, 118, 120, 1 It may be at position 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.

[0152] 3. Extracellular peptide spacer Spacer domains may be incorporated between the extracellular domain and the transmembrane domain of the CAR and / or between the cytoplasmic domain and the transmembrane domain of the CAR. As used herein, the term "spacer domain" generally refers to any oligopeptide or polypeptide that serves to link the transmembrane domain to either the extracellular domain or the cytoplasmic domain in a polypeptide chain. The extracellular spacer may link the antigen-binding domain to the transmembrane domain. The peptide spacer may be flexible enough to allow the antigen-binding domain to be oriented in different directions to facilitate antigen binding.

[0153] The spacer may comprise a hinge region from IgG. The spacer may comprise, or may further comprise, a portion of an immunoglobulin CH2CH3 region and CD3. The CH2CH3 region may have an L235E / N297Q or L235D / N297Q modification, or 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.

[0154] As used herein, the term "hinge" refers to a flexible polypeptide junction region (also referred to herein as "hinge region" or "spacer") that provides structural flexibility and spacing between flanking polypeptide regions, and may be composed of natural or synthetic polypeptides. "Hinge" or "spacer" from immunoglobulins (e.g., IgG1) is generally defined as the portion of the chain from Glu216 to Pro230 of human IgG1, for example (Burton (1985) Molec. Immunol., 22:161-206). Hinge regions of other IgG isotypes may be matched to the IgG1 sequence by placing the first and last cysteine ​​residues that form the inter-heavy chain disulfide (SS) bond in the same position. Hinge regions may be naturally occurring or non-naturally occurring, for example, but not limited to, modified hinge regions as described in U.S. Pat. No. 5,677,425. The hinge region may include a complete hinge region derived from an antibody of a different class or subclass than that of the CH1 domain. The term "hinge" may also include regions derived from CD8 and other receptors that provide similar functions in providing flexibility and spacing with the flanking regions. Other alternatives include the CH2CH3 region of immunoglobulins and portions of CD3.

[0155] The extracellular spacer is at least 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, 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, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 6, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 260, 270, 280, 290, 300, 325, 350 or 400, at most 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, 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 pieces, 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, 11 0, 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, 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,The extracellular spacer may have a length of 350 or 400 amino acids (or any range derivable therein). The extracellular spacer may consist of or include a hinge region derived from an immunoglobulin (e.g., IgG). The amino acid sequences of immunoglobulin hinge regions 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.

[0156] The length of the extracellular spacer can have an effect on the signaling activity of the CAR and / or the expansion and proliferation properties of the CAR-T cells in response to antigen-stimulated CAR signaling. For example, a short spacer of less than 50, 45, 40, 30, 35, 30, 25, 20, 15, 14, 13, 12, 11, or 10 amino acids can be used. 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, 23 Longer spacers, such as 0, 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 expansion in vivo or in vitro.

[0157] As a non-limiting example, the immunoglobulin hinge region can comprise one of the following amino acid sequences: (Table) Exemplary hinge regions TIFF2025507864000002.tif98160

[0158] The extracellular spacer may comprise the amino acid sequence of the hinge region of human IgG1, IgG2, IgG3 or IgG4. The extracellular spacer may also comprise one or more amino acid substitutions and / or insertions and / or deletions compared to the wild-type (naturally occurring) hinge region. For example, His229 of the hinge of human IgG1 is located at the position 1 in the hinge region of human IgG1. It can be substituted with Tyr to include TIFF2025507864000003.tif4128.

[0159] The extracellular spacer can comprise an amino acid sequence derived from human CD8; for example, the hinge region has the amino acid sequence: It may include TIFF2025507864000004.tif4151 or a variant thereof.

[0160] The extracellular spacer may include, or may further include, a CH2 region. An exemplary CH2 region is TIFF2025507864000005.tif19159. The extracellular spacer may include, or further include, a CH3 region. An exemplary CH3 region is The file is TIFF2025507864000006.tif19159.

[0161] When the extracellular spacer comprises multiple portions, the number of amino acids between the various portions may be anywhere from 0 to 50. For example, between the hinge and CH2 or CH3 region, or between the CH2 region and the CH3 region, if both are present, there may be at least 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, and at most 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. There may be 5, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45 or 50 amino acids, 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 range derivable therein). The extracellular spacer may consist essentially of the hinge, CH2 and / or CH3 regions, meaning that only the hinge, CH2 and / or CH3 regions are identifiable regions present and not excluding any other domains or regions, and that there may be additional amino acids present that are not part of the identifiable regions.

[0162] 4. Transmembrane Domain Regarding the transmembrane domain, the CAR can be designed to include a transmembrane domain fused to the extracellular domain of the CAR. The transmembrane domain can be a hydrophobic alpha helix that spans the membrane. Different transmembrane domains can result in different receptor stabilities.

[0163] A transmembrane domain that is naturally associated with one of the domains of CAR may be used. In some cases, the transmembrane domain may be selected or modified by amino acid substitution to avoid such domain binding to the transmembrane domain of the same or a different surface membrane protein to minimize interaction with other members of the receptor complex.

[0164] The transmembrane domain may be derived from either natural or synthetic sources. When the source is natural, the domain may be derived from any membrane-bound or transmembrane protein. Illustrative but non-limiting examples of transmembrane regions specifically used in the CAR constructs envisaged herein may be derived from (i.e., at least include the transmembrane region(s) thereof) the alpha, beta or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD 16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154. Alternatively, the transmembrane domain may be synthetic, in which case it may comprise mainly hydrophobic residues, such as leucine and valine. Synthetic transmembrane domains may have triplets of phenylalanine, tryptophan and valine at each end. Optionally, a short oligopeptide or polypeptide linker, e.g., 2 to about 10 amino acids in length, may form the link between the transmembrane domain and the cytoplasmic signaling domain of the CAR. A glycine-serine doublet may provide a particularly suitable linker.

[0165] The transmembrane domain is sandwiched between the extracellular spacer and the cytoplasmic region. The transmembrane domain may be sandwiched between the extracellular spacer and one or more costimulatory regions. A linker may be between the transmembrane domain and the one or more costimulatory regions.

[0166] Any transmembrane domain that provides for insertion of the polypeptide into the cell membrane of a eukaryotic (e.g., mammalian) cell may be suitable for use. The transmembrane domain may be derived from CD28, CD8, CD4, CD3-zeta, CD134, or CD7.

[0167] Exemplary transmembrane domains useful in any of the compositions and methods of the disclosure include those in the table below. (Table) Exemplary transmembrane domain sequences TIFF2025507864000007.tif75160

[0168] 5. Cytoplasmic Domain The cytoplasmic domain or intracellular signaling domain of the CAR is responsible for activating at least one of the normal effector functions of the immune cell in which the CAR is placed. After recognition of antigen and / or ligand, the receptor clusters and signals are transmitted to the cell via the cytoplasmic domain. The cytoplasmic domain may include an intracellular signaling domain. The intracellular signaling domain may include a main signaling domain and one or more costimulatory domains. The costimulatory domain described herein may be part of the cytoplasmic domain.

[0169] The term "effector function" refers to a specialized function of a cell. For example, an effector function of a T cell can be cytotoxic activity or helper activity, such as secretion of a cytokine. Thus, the term "intracellular signaling domain" refers to a portion of a protein that transmits an effector function signal to direct a cell to perform a specialized function. Usually, the entire intracellular signaling domain can be used, but in many cases it is not necessary to use the entire chain. To the extent that a truncated portion of the intracellular signaling domain is used, such a truncated portion can be used in place of the intact chain as long as the effector function signal is transmitted. Thus, the term intracellular signaling domain is intended to include any truncated portion of the intracellular signaling domain sufficient to transmit the effector function signal.

[0170] The cytoplasmic and / or costimulatory regions suitable for use in the CARs of the present disclosure include any desired signaling domain that provides a distinct and detectable signal in response to activation by antigen binding to the antigen-binding domain (e.g., increased production of one or more cytokines by the cell; altered transcription of a target gene; altered activity of a protein; altered cell behavior, such as cell death; cell proliferation; cell differentiation; cell survival; modulation of cell signaling responses; etc.). The cytoplasmic region may include at least one (e.g., 1, 2, 3, 4, 5, 6, etc.) ITAM motif as described herein. The cytoplasmic region may include a signaling chain of the DAP10 / CD28 type. The cytoplasmic region may include a signaling chain of the CD3-zeta, DAP10, CD28, 2B4, DNAM-1, 4-1BB, OX40, CD30, CD40, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, and NKG2C type.

[0171] Cytoplasmic regions suitable for use in the polypeptides of the present disclosure include immunoreceptor tyrosine-based activation motif (ITAM)-containing intracellular signaling polypeptides. The ITAM motif is YX1X2(L / I), where X1 and X2 are independently any amino acid. In some cases, the cytoplasmic region contains 1, 2, 3, 4, or 5 ITAM motifs. In some cases, the ITAM motif is repeated twice in the endodomain, where the first and second ITAM motifs are separated from each other by 6-8 amino acids, e.g., (YX1X2(L / I))(X3)n(YX1X2(L / I)), where n is an integer between 6 and 8, and each of the 6-8 X3 can be any amino acid.

[0172] A suitable cytoplasmic region can be an ITAM motif-containing portion derived from a polypeptide containing an ITAM motif. For example, a suitable cytoplasmic region can be an ITAM motif-containing domain derived from any ITAM motif-containing protein. Thus, a suitable endodomain does not need to contain the entire sequence of the entire protein from which it is derived. Examples of suitable ITAM motif-containing polypeptides include, but are not limited to: DAP12, DAP10, FCER1G (Fc epsilon receptor I gamma chain); CD3D (CD3 delta); CD3E (CD3 epsilon); CD3G (CD3 gamma); CD3-zeta; and CD79A (antigen receptor complex-associated protein alpha chain).

[0173] Exemplary cytoplasmic regions are known in the art. The cytoplasmic regions shown below also provide examples of regions that can be incorporated into the CARs of the present disclosure.

[0174] In some cases, the cytoplasmic region is derived from DAP12 (also known as TYROBP; TYRO protein tyrosine kinase binding protein; KARAP; PLOSL; DNA AX activating protein 12; KAR associated protein; TYRO protein tyrosine kinase-binding protein; killer activating receptor associated protein; killer activating receptor associated protein; etc.). A suitable cytoplasmic region may include an ITAM motif-containing portion of the amino acid sequence of full-length DAP12.

[0175] The cytoplasmic region may be derived from FCER1G (also known as FCRG; Fc epsilon receptor I gamma chain; Fc receptor gamma chain; fc-epsilon Rl-gamma; fcR gamma; fceRI gamma; high affinity immunoglobulin epsilon receptor subunit gamma; immunoglobulin E receptor, high affinity, gamma chain; etc.). A suitable cytoplasmic region may comprise an ITAM motif-containing portion of the full-length FCER1G amino acid sequence.

[0176] The cytoplasmic region may be derived from the T cell surface glycoprotein CD3 delta chain (also known as CD3D; CD3-DELTA; T3D; CD3 antigen, delta subunit; CD3 delta; CD3d antigen, delta polypeptide (TiT3 complex); OKT3, delta chain; T cell receptor T3 delta chain; T cell surface glycoprotein CD3 delta chain; etc.). A suitable cytoplasmic region may comprise an ITAM motif-containing portion of the full-length CD3 delta amino acid sequence.

[0177] The cytoplasmic region may be derived from the T cell surface glycoprotein CD3 epsilon chain (also known as CD3e, T cell surface antigen T3 / Leu-4 epsilon chain, T cell surface glycoprotein CD3 epsilon chain, AI504783, CD3, CD3 epsilon, T3e, etc.). A suitable cytoplasmic region may comprise an ITAM motif-containing portion of the full-length CD3 epsilon amino acid sequence.

[0178] The cytoplasmic region may be derived from the T cell surface glycoprotein CD3 gamma chain (also known as CD3G, T cell receptor T3 gamma chain, CD3-GAMMA, T3G, gamma polypeptide (TiT3 complex), etc.). A suitable cytoplasmic region may comprise an ITAM motif-containing portion of the full-length CD3 gamma amino acid sequence.

[0179] The cytoplasmic region may be derived from the T cell surface glycoprotein CD3 zeta chain (also known as CD3Z, T cell receptor T3 zeta chain, CD247, CD3-ZETA, CD3H, CD3Q, T3Z, TCRZ, etc.). A suitable cytoplasmic region may comprise an ITAM motif-containing portion of the full-length CD3 zeta amino acid sequence.

[0180] The cytoplasmic region may be derived from CD79A (also known as B-cell antigen receptor complex-associated protein alpha chain; CD79a antigen (immunoglobulin-associated alpha); MB-1 membrane glycoprotein; ig-alpha; membrane-associated immunoglobulin-associated protein; surface IgM-associated protein; etc.). A suitable cytoplasmic region may comprise an ITAM motif-containing portion of the full-length CD79A amino acid sequence.

[0181] A suitable cytoplasmic region may include a signaling chain of the CD28 type. Further cytoplasmic regions suitable for use in the CARs of the present disclosure include the ZAP70 polypeptide.

[0182] Specific exemplary cytoplasmic regions are known in the art and are further set forth in the table below. (Table) Cytoplasmic region TIFF2025507864000008.tif237166TIFF2025507864000009.tif222166

[0183] 6. Co-stimulation area The term "costimulatory ligand", as the term is used herein, includes a molecule on an antigen-presenting cell (e.g., APC, dendritic cell, B cell, etc.) that specifically binds to a cognate co-stimulatory molecule or cognate co-stimulatory domain on an immune effector cell, thereby resulting in a signal, such as, but not limited to, proliferation, activation, differentiation, etc., in addition to the primary signal that mediates immune effector cell response. A costimulatory ligand also includes, among others, an antibody that specifically binds to a costimulatory molecule present on an immune effector cell. A "costimulatory molecule" refers to a cognate binding partner on an immune effector cell that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response by the immune effector, such as, but not limited to, proliferation and / or activation. A "costimulatory signal", as the term is used herein, refers to a signal that, in combination with a primary signal, results in immune cell activation, proliferation, and / or up-regulation or down-regulation of key molecules.

[0184] By the term "stimulation", we mean the primary response induced by stimulatory molecules binding to their cognate ligands, thereby mediating signal transduction events, including but not limited to signal transduction. Stimulation can mediate the modification of the expression of a particular molecule. "Stimulatory molecule", as this term is used herein, means a molecule on immune effector cells that specifically binds to the cognate stimulatory ligand present on antigen-presenting cells. "Stimulatory ligand", as this term is used herein, means a ligand that, when present on antigen-presenting cells (e.g., APCs, dendritic cells, B cells, etc.), specifically binds to the cognate binding partner (herein referred to as "stimulatory molecule") on immune effector cells, thereby mediating the primary response by immune effector cells, including but not limited to activation, initiation of immune response, proliferation, etc.

[0185] Non-limiting examples of suitable costimulatory regions, e.g., those that can be included 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.

[0186] A costimulatory region can have a length of at least 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200 or 300, at most 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200 or 300, 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.

[0187] The costimulatory region may be derived from DAP10 (also known as HCST, DAP10, KAP10, PIK3AP, hematopoietic cell signaling factor; etc.). The costimulatory 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; CD137; CDwl37; ILA; etc.). The costimulatory region may be derived from the intracellular portion of the transmembrane protein CD28 (also known as Tp44). The costimulatory region may be derived from the intracellular portion of the transmembrane protein ICOS (also known as inducible T cell costimulator AILIM, CD278 and CVID1). The costimulatory 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 costimulatory 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 costimulatory 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 costimulatory 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 costimulatory 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 costimulatory region may be derived from the intracellular portion of the transmembrane protein HVEM (tumor necrosis factor receptor superfamily member 14, TNFRSF14, also known as RP3-395M20.6, ATAR, CD270, HVEA, LIGHTR, and TR2).The costimulatory region may be derived from 2B4 (also known as CD244, NAIL, NKR2B4, Nmrk, SLAMF4, CD244 molecule, etc.). The costimulatory region may be derived from DNAM-1 (also known as CD226, DNAM1, PTA1, TLiSA1, CD226 molecule, etc.). The costimulatory region may be derived from CD40 (also known as Bp50, CDW40, TNFRSF5, p50, CD40 (protein), CD40 molecule, etc.). The costimulatory region may be derived from LFA-1 (also known as lymphocyte function associated antigen 1, integrin alpha L, ITGAL, CD11A, LFA1A, integrin subunit alpha L, etc.). The costimulatory region may be derived from CD2 (also known as lymphocyte function antigen-2, LFA-2, SRBC, T11, CD2 molecule, etc.). The costimulatory region may be derived from CD7 (also known as GP40, LEU-9, TP41, Tp40, CD7 molecule, etc.). The costimulatory region may be derived from LIGHT (also known as TNFSF14, CD258, HVEML, LIGHT, LTg, TR2, TNLG1D, tumor necrosis factor superfamily member 14, etc.). The costimulatory region may be derived from NKG2C (also known as KLRC2, CD159c, NKG2-C, killer cell lectin-like receptor C2, etc.).

[0188] Specific exemplary costimulatory domains are illustrated by the following amino acid sequences: (Table) Costimulatory domains TIFF2025507864000010.tif141159

[0189] 7. Detection peptide The CAR described herein may further comprise a detection peptide or detection molecule.Suitable detection peptides include hemagglutinin (HA; for example, YPYDVPDYA; SEQ ID NO:56); FLAG (for example, DYKDDDDK; SEQ ID NO:57); c-myc (for example, EQKLISEEDL; SEQ ID NO:58) and the like.Other suitable detection peptides are known in the art.

[0190] 8. Peptide Linker The polypeptides of the present disclosure may include a peptide linker (sometimes referred to as a linker). A peptide linker may be used to separate any of the peptide domains / regions described herein. By way of example, a linker may be present 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, in the flanking portion of a costimulatory region or the N- or C-region of a costimulatory region, and / or between a transmembrane domain and an endodomain. The peptide linker may have any of a variety of amino acid sequences. The domains and regions may be linked by peptide linkers, which are generally of a flexible nature, but do not exclude other chemical bonds. The linker may be a peptide of about 6 to about 40 amino acids in length, or about 6 to about 25 amino acids in length. Such linkers may be made and coupled to proteins by using synthetic linker-encoding oligonucleotides.

[0191] Peptide linkers with some degree of flexibility can be used. It should be noted that although the peptide linker can have virtually any amino acid sequence, suitable peptide linkers generally have sequences that result in flexible peptides. The use of small amino acids, such as glycine and alanine, is useful in creating flexible peptides. The creation of such sequences is routine for those skilled in the art.

[0192] 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 to 15 amino acids, from 3 to 12 amino acids, such as from 4 to 10 amino acids, from 5 to 9 amino acids, from 6 to 8 amino acids, or from 7 to 8 amino acids, and can be 1, 2, 3, 4, 5, 6 or 7 amino acids.

[0193] Suitable linkers may be readily selected and may 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, such as 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 may be 1, 2, 3, 4, 5, 6 or 7 amino acids.

[0194] Exemplary flexible linkers include glycine polymers (G), glycine-serine polymers (e.g., TIFF2025507864000011.tif4136, where n is an integer of at least 1. n can be at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, 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 therefore can serve as neutral tethering moieties between components. Glycine polymers can be used; glycine still reaches significantly more φ-ψ space than alanine and is much less constrained than residues with long side chains. Exemplary spacers include, but are not limited to, The linker may comprise an amino acid sequence such as TIFF2025507864000012.tif19159. The linker may comprise a repeat structure, such as a consecutive repeat structure of one or more of SEQ ID NOs: 61-66, e.g., the linker comprises an amino acid sequence corresponding to one of SEQ ID NOs: 61-66 repeated at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 times, at most 2, 3, 4, 5, 6, 7, 8, 9 or 10 times, or exactly 2, 3, 4, 5, 6, 7, 8, 9 or 10 times, or any range derivable therein.

[0195] The linker may comprise (EAAAK)n (SEQ ID NO:67), where n is an integer of at least 1. n may be at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (or any range derivable therein).

[0196] B. Treatment Control The transgene, the first transgene or the second transgene may be co-expressed with a therapeutic control. The therapeutic control may regulate cell proliferation, facilitate cell selection (e.g., selection of cells expressing the chimeric antigen receptor of the present invention), or a combination thereof. Regulating cell proliferation may include upregulating cell proliferation to promote cell proliferation spread. Regulating cell proliferation may include downregulating cell proliferation to reduce or abrogate cell proliferation spread. The agent acting as a therapeutic control may promote enrichment of cells expressing the chimeric antigen receptor, which provides a therapeutic benefit. The agent acting as a therapeutic control may biochemically interact with an additional composition such that the function of the therapeutic control is modulated. For example, EGFRt (therapeutic control) may biochemically interact with cetuximab such that the function of EGFRt in selection, tracking, cell removal, or a combination thereof is modulated.

[0197] Exemplary therapeutic controls include truncated epidermal growth factor receptor (EGFRt), chimeric cytokine receptor (CCR) and / or dihydroxyfolate receptor (DHFR) (e.g., mutant DHFR). The polynucleotides encoding the CAR and therapeutic control can be linked via an IRES sequence or via a polynucleotide sequence encoding a cleavable linker. The CAR of the present invention is constructed so that it can be expressed intracellularly and thus further proliferate in response to the presence of at least one molecule, e.g., an antigen, that interacts with at least one antigen-specific targeting region. The therapeutic control can include a cell surface protein that does not have an intracellular signaling domain. It is envisioned that any cell surface protein that does not have intracellular signaling or has been modified (e.g., by truncation) to eliminate intracellular signaling can be used. Further examples of therapeutic controls include truncated LNGFR, truncated CD19, etc., where the truncated protein does not have an intracellular signaling domain.

[0198] "Co-expressed" as used herein refers to the simultaneous expression of two or more genes. The genes can be, for example, nucleic acids encoding a single protein or a chimeric protein as a single polypeptide chain. For example, the transgene of the present disclosure can be co-expressed with a therapeutic control (e.g., a truncated epidermal growth factor (EGFRt)), where the CAR is encoded by a first polynucleotide strand and the therapeutic control is encoded by a second polynucleotide strand. The first and second polynucleotide strands can be linked by a nucleic acid sequence encoding a cleavable linker. The polynucleotide encoding the CAR and the polynucleotide encoding the therapeutic control system can be linked by an IRES sequence. Alternatively, the CAR and the therapeutic control are encoded by two different polynucleotides, for example, encoded by two different vectors, rather than linked via a linker. Additionally, the CAR of the present disclosure can be co-expressed with a therapeutic control and a CCR, a therapeutic control and a DHFR (e.g., a mutant DHFR), or a therapeutic control and a CCR and a DHFR (e.g., a mutant DHFR). CAR, therapeutic control and CCR can be co-expressed and encoded by a first, second and third polynucleotide sequence, respectively, where the first, second and third polynucleotide sequences are linked via an IRES sequence or a sequence encoding a cleavable linker (e.g., T2A). Alternatively, these sequences are not linked by a linker but are encoded, for example, by separate vectors. CAR, therapeutic control and DHFR (e.g., mutant DHFR) can be co-expressed and encoded by a first, second and fourth polynucleotide sequence, respectively, where the first, second and fourth polynucleotide sequences are linked via an IRES sequence or a sequence encoding a cleavable linker. Alternatively, these sequences are not linked by a linker but are encoded, for example, by separate vectors.CAR, therapeutic control, CCR and DHFR (e.g. mutant DHFR) can be co-expressed and are encoded by the first, second, third and fourth polynucleotide sequences, respectively, where the first, second, third and fourth polynucleotide sequences are linked via an IRES sequence or via a sequence encoding a cleavable linker. Alternatively, these sequences are not linked by a linker but are encoded, for example, by separate vectors. When the aforementioned sequences are encoded by separate vectors, these vectors can be transfected simultaneously or sequentially.

[0199] Further aspects of therapeutic control, CAR molecules and methods useful in the compositions of the present disclosure may be found in U.S. Pat. No. 9,447,194, which is incorporated herein by reference for all purposes.

[0200] VI. Gene transfer methods A. Gene Editing The engineered nuclease can be used to introduce transgenes into cells, such as progenitor cells, stem cells, HSPCs, ES cells, iPSCs and human embryonic mesoderm progenitor cells. When cells are genetically modified, for example, to add or reduce one or more characteristics, the genetic modification can be performed by any suitable method. For example, any genetic modification composition or genetic modification method, such as gene editing, homologous or non-homologous recombination, RNA-mediated gene delivery or any conventional nucleic acid delivery method, can be used to introduce exogenous nucleic acid into cells or edit genomic DNA. Genetic introduction techniques can include targeted integration into endogenous loci of the genome of cells, for example, into the loci of genes in Table 1. Non-limiting examples of genetic modification methods can include gene editing methods, such as CRISPR / CAS9, zinc finger nuclease or TALEN technology.

[0201] Genome editing or genome editing with engineered nucleases (GEEN) is a type of genetic engineering in which DNA is inserted, replaced, or removed from a genome using engineered nucleases or "molecular scissors." The nucleases create specific double-strand breaks (DSBs) at desired locations in the genome, and the cell's endogenous machinery is used to repair the induced breaks by the natural processes of homologous recombination (HR) and non-homologous end joining (NHEJ).

[0202] Non-limiting engineered nucleases include zinc finger nucleases (ZFN), transcription activator-like effector nucleases (TALEN), CRISPR / Cas9 system, and engineered meganucleases, re-engineered homing endonucleases.Any engineered nuclease known in the art can be used in the method and composition.

[0203] It is common in genetic analysis to disrupt gene or protein functions in a sequence-specific manner and monitor their effects on organisms to understand them. However, in some organisms, it is difficult or impossible to perform site-specific mutagenesis, and therefore more indirect methods must be used, such as silencing of the gene of interest by small RNA interference (siRNA). However, gene disruption by siRNA can be variable and incomplete. Genome editing using nucleases such as ZFNs differs from siRNA in that engineered nucleases can modify DNA binding specificity, thus in principle allowing cleavage at any desired location in the genome, and can introduce endogenous sequence modifications to genes that cannot be specifically targeted by conventional RNAi. Furthermore, the specificity of ZFNs and TALENs is improved, since two ZFNs are required for a portion to recognize the target and then be directed to adjacent sequences.

[0204] Meganucleases commonly found in microbial species have the unique property of having very long recognition sequences (>14bp), which makes them highly specific in the natural state. This can be exploited to perform site-specific DSBs in genome editing; however, the challenge is that not enough meganucleases are known, or may not have been known so far, to cover all possible target sequences. To solve this challenge, mutagenesis and high-throughput screening methods have been used to create meganuclease variants that recognize unique sequences. Others have been able to fuse different meganucleases to create hybrid enzymes that recognize new sequences. Others have attempted to design sequence-specific meganucleases by modifying the DNA-interacting amino acids of meganucleases, in a method named rationally designed meganucleases (U.S. Patent No. 8,021,867, incorporated herein by reference).

[0205] Meganucleases have the advantage of causing less toxicity to cells compared to methods such as ZFN, presumably due to more stringent DNA sequence recognition; however, constructing sequence-specific enzymes for all possible sequences is costly and time-consuming, as some of the possible combinations for which methods such as ZFN and TALEN are used are not beneficial. Thus, there are both advantages and disadvantages.

[0206] In contrast to meganucleases, the idea behind ZFNs and TALENs is more based on non-specific DNA cleavage enzymes that could then be linked to specific DNA sequence recognition peptides, such as zinc fingers and transcription activator-like effectors (TALEs). One method was to find an endonuclease in which the DNA recognition and cleavage sites are far from each other, a situation that is not common for restriction enzymes. Once this enzyme was found, its cleavage portion, which would be very non-specific since it would not have the recognition ability, could be separated. This portion could then be linked to a sequence that recognizes a peptide that could result in very high specificity. One example of a restriction enzyme with such properties is FokI. Furthermore, FokI has the advantage that it requires dimerization to have nuclease activity, which means that specificity is dramatically increased since each nuclease partner will recognize a unique DNA sequence. To improve this effect, FokI nuclease has been engineered to be able to function only as a heterodimer and have high catalytic activity. Heterodimeric functioning nucleases could avoid the possibility of unwanted homodimeric activity and thus increase specificity for DSBs.

[0207] Although the nuclease moieties of both ZFNs and TALENs have similar properties, the difference between these engineered nucleases lies in their DNA recognition peptides. ZFNs rely on Cys2-His2 zinc fingers, while TALENs rely on TALEs. Both of these DNA recognition peptide domains have the characteristic of being naturally found in combinations within the protein. Cys2-His2 zinc fingers typically exist in a repeat structure separated by 3 bp and are found in various combinations in various nucleic acid interacting proteins, such as transcription factors. On the other hand, TALEs are found in a repeat structure with a 1:1 recognition ratio between amino acids and recognized nucleotide pairs. Since both zinc fingers and TALEs exist in a repeat pattern, different combinations can be tried to create various sequence specificities. Zinc fingers are more established in these terms and approaches such as modular assembly (where zinc fingers correlating with triplet sequences are joined in a line to cover the required sequence), OPEN (low stringency selection of peptide domains versus triplet nucleotides in bacterial systems followed by high stringency selection of peptide combinations versus the final target), and bacterial one-hybrid screening of zinc finger libraries has been used to generate site-specific nucleases, among other methods.

[0208] B. Transgene Delivery Any cell used herein that is induced to differentiate into a mature cell, particularly a vector for genetic modification of a starting cell, such as a stem cell or progenitor cell, can be constructed to contain a nucleic acid sequence of a transgene. Details of the components and delivery methods of such vectors are disclosed below.

[0209] A cell can be engineered to contain one or more genetic modifications either before or after differentiation by genetically engineering the cell (US2002 / 0168766). A cell can be said to be "genetically engineered," "genetically modified," or "transgenic" if an exogenous nucleic acid or polynucleotide has been transferred into the cell by any suitable means of artificial manipulation, or if the cell is the descendant of an originally engineered cell from which the polynucleotide has been inherited. For example, a cell can be engineered to increase the replicative potential either before or after progression to a committed developmental lineage cell or a terminally differentiated cell by genetically engineering the cell to express telomerase reverse transcriptase (US2003 / 0022367).

[0210] C. Vector Those skilled in the art are well equipped 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).

[0211] Vectors may also contain other components or functional moieties that further regulate gene delivery and / or gene expression or that confer beneficial properties on the cells of interest, including, for example, components that affect binding or targeting to cells (e.g., components that mediate cell-type- or tissue-specific binding), components that affect uptake of vector nucleic acid by cells, components that affect localization of polynucleotides within cells after uptake (e.g., factors that mediate nuclear localization), and components that affect expression of polynucleotides.

[0212] Such components could also include markers, such as detectable and / or selectable markers, that can be used to detect or select cells that have incorporated and expressed the nucleic acid delivered by the vector. Such components can be provided as natural structural parts of the vector (e.g., using certain viral vectors with components or functional parts that mediate binding and uptake), or the vector can be modified to provide such functional parts. A wide variety of such vectors are known in the art and are generally available. When a vector is maintained in a host cell, it can either be stably replicated by the cell as an autonomous structural part during mitosis, integrated into the genome of the host cell, or maintained in the nucleus or cytoplasm of the host cell.

[0213] D. Regulatory Elements A eukaryotic expression cassette contained in a vector contains, inter alia, a eukaryotic transcription promoter operably linked (5' to 3') to the protein coding sequence, splicing signals with intervening sequences, and transcription termination / polyadenylation sequences.

[0214] 1. Promoter / Enhancer A "promoter" is a control sequence, a region of a nucleic acid sequence that controls the initiation and rate of transcription. A promoter or enhancer may include the promoter or enhancer of a gene in Table 1. It may contain genetic elements to which regulatory proteins and molecules, such as RNA polymerase and other transcription factors, may bind to initiate specific transcription of a nucleic acid sequence. The phrases "operably located," "operably linked," "under control," and "under transcriptional control" mean that the promoter is in the correct functional location and / or orientation relative to a nucleic acid sequence to control transcription initiation and / or expression of the sequence.

[0215] The promoter region of the present disclosure may be an endogenous promoter region. An endogenous promoter region refers to the situation where the promoter region is present in its endogenous genomic environment, so that the sequence upstream of the promoter (i.e., the 5' region) is substantially the same as that of wild-type cells. Substantially the same may refer to a region being at least 80, 85, 90, 95, 96, 97, 98 or 99% identical to the upstream region of the wild-type. An endogenous promoter region may also refer to the situation where the promoter is present in the same genomic location as the wild-type promoter. An endogenous promoter may also refer to a promoter in a cell that is unmodified or is 70, 75, 80, 85, 90, 95, 96, 97, 98 or 99% identical to the wild-type promoter, or is at least 70, 75, 80, 85, 90, 95, 96, 97, 98 or 99% identical.

[0216] Promoters generally contain sequences that function to position the start site for RNA synthesis. The best known example of this is the TATA box, but in some promoters lacking a TATA box, such as the mammalian terminal deoxynucleotidyl transferase gene promoter and the SV40 late gene promoter, separate elements that overlap the start site itself help to localize start. Additional promoter elements are those that regulate the frequency of transcription initiation. Typically, these are located in the region 30-110 bp upstream of the start site, although some promoters have been shown to contain functional elements downstream of the start site as well. To bring a coding sequence "under the control" of a promoter, the 5' end of the transcription start site of the transcriptional reading frame is positioned "downstream" (i.e., 3') of the selected promoter. The "upstream" promoter drives transcription of the DNA and promotes expression of the encoded RNA.

[0217] Spacing between promoter elements is often flexible, so that promoter function is maintained when elements are inverted or moved relative to one another. In the tk promoter, spacing between promoter elements can be increased up to 50 bp apart, after which activity begins to decline. Depending on the promoter, individual elements appear to function either cooperatively or independently to activate transcription. Promoters may or may not be used in conjunction with "enhancers," which refer to cis-acting regulatory sequences involved in the transcriptional activation of a nucleic acid sequence.

[0218] 2. Protease cleavage sites / self-cleaving peptides and internal ribosome binding sites Suitable protease cleavage sites and self-cleaving peptides are known to those of skill in the art (see, for example, Ryan et al., 1997; Scymczak et al., 2004). Examples of protease cleavage sites are the cleavage sites for potyvirus NIa protease (e.g., tobacco etch virus protease), potyvirus HC protease, potyvirus P1 (P35) protease, byovirus Nla protease, byovirus RNA-2 encoded protease, aphthovirus L protease, enterovirus 2A protease, rhinovirus 2A protease, picorna 3C protease, comovirus 24K protease, nepovirus 24K protease, RTSV (Rice Tungro Spherical Virus) 3C-like protease, PY\IF (Parsnip Yellow Mottle Virus) 3C-like protease, thrombin, factor Xa and enterokinase. Due to high cleavage stringency, the TEV (Tobacco Etch Virus) protease cleavage site can be used.

[0219] Exemplary self-cleaving peptides (also referred to as "cis-acting hydrolytic element," CHYSEL; see deFelipe (2002)) are derived from potyvirus and cardiovirus 2A peptides. Specific self-cleaving peptides may be selected from 2A peptides derived from FMDV (foot and mouth disease virus), Equine rhinitis A virus, Thosea asigna virus, and Porcine teschovirus.

[0220] Specific initiation signals for efficient translation of coding sequences in polycistronic messengers may also be used. Such signals include the ATG initiation codon or adjacent sequences. It may be necessary to provide exogenous translational control signals, such as the ATG initiation codon. Those skilled in the art can easily determine this and provide the necessary signals. It is well known that the initiation codon must be "in frame" with the reading frame of the desired coding sequence to ensure translation of the entire insert. Exogenous translational control signals and initiation codons may be natural or synthetic. The efficiency of expression may be improved by including appropriate transcriptional enhancer elements.

[0221] The use of internal ribosome entry site (IRES) elements can be used to create multigene or polycistronic messengers. IRES elements can bypass the ribosome scanning model of 5' methylated cap-dependent translation and initiate translation at internal sites (Pelletier and Sonenberg, 1988). IRES elements from two members of the picornavirus family (polio and encephalomyocarditis) have been reported (Pelletier and Sonenberg, 1988), as well as IRESs from mammalian messengers (Macejak and Sarnow, 1991). IRES elements can be linked to heterologous open reading frames. Multiple open reading frames, each separated by an IRES, can be transcribed together to create polycistronic messengers. Thanks to the IRES element, each open reading frame is ribosome accessible for efficient translation. Multiple genes can be efficiently expressed using a single promoter / enhancer and a single messenger can be transcribed (see US Pat. Nos. 5,925,565 and 5,935,819, each of which is incorporated herein by reference).

[0222] 3. Multiple cloning site A vector may contain a multiple cloning site (MCS), which is a nucleic acid region that contains multiple restriction enzyme sites, all of which can be used with standard recombinant techniques to digest the vector (see, e.g., Carbonelli et al., 1999; Levenson et al., 1998; and Cocea, 1997, incorporated herein by reference). "Restriction enzyme digestion" refers to the catalytic cleavage of a nucleic acid molecule by an enzyme that functions only at a specific location within the nucleic acid molecule. Many such restriction enzymes are commercially available. The use of such enzymes is well understood by those of skill in the art. Often, vectors are linearized or fragmented using a restriction enzyme that cuts within the MCS, allowing exogenous sequences to be ligated into the vector. "Ligation" refers to the process of forming phosphodiester bonds between two nucleic acid fragments that may or may not be contiguous with each other. Techniques involving restriction enzymes and ligation reactions are well known to those of skill in the recombinant arts.

[0223] 4. Splicing Sites Most transcribed eukaryotic RNA molecules undergo RNA splicing to remove introns from the primary transcript. Vectors containing eukaryotic genomic sequences may require donor and / or acceptor splice sites to ensure proper processing of the transcript for protein expression (see, e.g., Chandler et al., 1997, incorporated herein by reference).

[0224] 5. Termination Signal A vector or construct may contain at least one termination signal. A "termination signal" or "terminator" is composed of a DNA sequence that is involved in the specific termination of an RNA transcript by an RNA polymerase. Thus, a termination signal that ends the production of an RNA transcript is envisioned. A terminator may be necessary to obtain a desired messenger level in vivo.

[0225] In eukaryotic systems, the terminator region may also contain specific DNA sequences that allow for site-specific cleavage of the new transcript to expose a polyadenylation site. This signals a specialized endogenous polymerase to add a stretch of about 200 A residues (polyA) to the 3' end of the transcript. RNA molecules modified with this polyA tail are more stable and appear to be translated more efficiently. Thus, the terminator may contain signals for cleavage of the RNA, and the terminator signal promotes polyadenylation of the messenger. The terminator and / or polyadenylation site elements may function to increase messenger levels and minimize read-through from the cassette to other sequences.

[0226] Contemplated terminators include any known transcription terminator described herein or known to one of skill in the art, including, but not limited to, termination sequences of genes, such as the bovine growth hormone terminator, or termination sequences of viral systems, such as the SV40 terminator, etc. Termination signals may also be absent from transcribable or translatable sequences, for example due to truncated sequences.

[0227] 6. Polyadenylation Signals In expression, especially in eukaryotic expression, polyadenylation signals are typically included to ensure proper polyadenylation of the transcript.The nature of the polyadenylation signal is not believed to be crucial for successful implementation, and any such sequence can be used.Examples include the SV40 polyadenylation signal or the bovine growth hormone polyadenylation signal, which are convenient and known to function well in various target cells.Polyadenylation can increase the stability of the transcript or facilitate transport to the cytoplasm.

[0228] E. Vector Delivery The present disclosure of gene modification or introduction of transgene nucleic acid into cell can use any suitable method for nucleic acid delivery for cell transformation, as described herein or known by those skilled in the art.Methods for introducing and expressing genes into cells are known in the art.In the context of expression vectors, the vector can be easily introduced into host cells, such as mammalian cells, bacterial cells, yeast or insect cells, by any method in the art.For example, expression vectors can be transferred into host cells by physical, chemical or biological means.

[0229] Such methods include, but are not limited to, for example, 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), such as by microinjection (Harland and Weintraub, 1985; U.S. Pat. No. 5,789,215, incorporated herein by reference); electroporation (U.S. Pat. No. 5,384,253, incorporated herein by reference; Tur-Kaspa et al., 1989), by cytogenetic algebra (Clin. Sci. 1999, 14:131-135, 2001), by cytogenetic algebra (Clin. Sci. 2002, 14:131-135, 2002), by cytogenetic algebra (Clin. Sci. 2002, 14:131-135, 2003 ... 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 the use of DEAE-dextran followed by polyethylene glycol (Gopal, 1985); by direct ultrasound loading (Fechheimer et al., 1987); 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 particle bombardment (PCT Application Nos. WO 94 / 09699 and 95 / 06128; U.S. Patent 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. Patent Nos. 5,302,523 and 5,464,765, each of which is incorporated herein by reference); by Agrobacterium-mediated transformation (U.S. Patent 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. Patent 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., 1985), as well as direct delivery of DNA by any combination of such methods. By application of techniques such as these, organelles, cells, tissues or organisms may be stably or transiently transformed.

[0230] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, etc. Methods for producing cells containing vectors and / or exogenous nucleic acids are well known in the art (see, for example, 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.

[0231] Biological methods for introducing polynucleotide of interest into host cell can include the use of DNA vector and RNA vector.Virus vector, especially retrovirus vector, has become the most widely used method for inserting genes into mammalian cells, such as human cells.Other virus vectors can be derived from lentivirus, poxvirus, herpes simplex virus type I, adenovirus and adeno-associated virus, etc. (see, for example, U.S. Patent No. 5,350,674 and U.S. Patent No. 5,585,362, etc.).

[0232] Chemical means for introducing polynucleotides into host cells include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems, such as oil-in-water emulsions, micelles, mixed micelles, and liposomes. An illustrative colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).

[0233] 1. Liposome-mediated transfection When a non-viral delivery system is used, one illustrative delivery vehicle is lipid and / or liposome. The introduction of nucleic acid into host cells (in vitro, ex vivo or in vivo) envisages the use of lipid formulations. The nucleic acid can be associated with lipid. The nucleic acid associated with lipid can be encapsulated in the aqueous interior of liposome, interspersed within the lipid bilayer of liposome, bound to liposome via a linking molecule associated with both liposome and oligonucleotide, encapsulated within liposome, complexed with liposome, dispersed in a solution containing lipid, mixed with lipid, combined with lipid, contained in lipid as a suspension, contained in or complexed with micelles, or associated with lipid. The composition with lipid, lipid / DNA or lipid / expression vector is not limited to any particular solution state structure. For example, it can be present as a micelle in a bilayer structure or in a "collapsed" structure. It may also simply be scattered throughout the solution, forming clumps that may not be uniform in size or shape. Lipids are fatty substances, and may be naturally occurring or synthetic lipids. For example, lipids include the fatty droplets that naturally occur in the cytoplasm, as well as the class of compounds that contain long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, aminoalcohols, and aldehydes.

[0234] Nucleic acids may be encapsulated in lipid complexes, such as liposomes. Liposomes are vesicular structures characterized by a phospholipid bilayer membrane and an aqueous internal medium. Multilamellar liposomes have multiple lipid layers separated by aqueous media. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-reorganization before the formation of a closed structure, encapsulating water and dissolved solutes 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 may vary depending on the nature of the liposomes and cells used, for example, about 5 to about 20 μg vector DNA per 1 to 10 million cells may be envisaged.

[0235] 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), and the feasibility of liposome-mediated delivery and expression of foreign DNA in cultured chick embryo, HeLa cells, and hepatoma cells has also been demonstrated (Wong et al., 1980).

[0236] Liposomes may be complexed with hemagglutinating virus (HVJ), which has been shown to facilitate fusion with the cell membrane and promote cell entry of liposome-encapsulated DNA (Kaneda et al., 1989). Liposomes may be complexed with or used in conjunction with nuclear non-histone chromosomal proteins (HMG-1) (Kato et al., 1991). Liposomes may be complexed with or used in conjunction with both HVJ and HMG-1. The delivery vehicle may include a ligand and a liposome.

[0237] Lipids suitable for use may be obtained from commercial sources. For example, dimyristyl phosphatidylcholine ("DMPC") may be obtained from Sigma, St. Louis, Mo., dicetyl phosphate ("DCP") may be obtained from K&K Laboratories (Plainview, NY); cholesterol ("Choi") may be obtained from Calbiochem-Behring; dimyristyl phosphatidylglycerol ("DMPG") and other lipids may be obtained from Avanti Polar Lipids, Inc. (Birmingham, Ala.). Stock solutions of lipids in chloroform or chloroform / methanol may be stored at about -20°C. Chloroform may be used as the sole solvent since it evaporates more readily than methanol. "Liposome" is a generic term that encompasses a variety of single and multilamellar lipid vehicles formed by the formation of closed, closed lipid bilayers or aggregates. Liposomes may be characterized as having a vesicular structure with a phospholipid bilayer membrane and an aqueous interior medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-reorganization before forming closed structures, trapping water and dissolved solutes between the lipid bilayers (Ghosh et al. (1991) Glycobiology 5:505-510). However, compositions with solution-state structures different from the normal vesicle structure are also encompassed. For example, lipids may be in the form of micellar structures or simply exist as heterogeneous aggregates of lipid molecules. Lipofectamine-nucleic acid complexes are also contemplated.

[0238] 2. Electroporation Nucleic acids can be introduced into cells by electroporation. Electroporation involves exposing a suspension of cells and DNA to a high-voltage discharge. Recipient cells can be rendered more susceptible to transformation by mechanical damage. The amount of vector used can also vary depending on the nature of the cells used; for example, about 5 to about 20 μg vector DNA per 1 to 10 million cells can be envisaged.

[0239] Transfection of eukaryotic cells using electroporation has been fairly successful: in this manner, mouse pre-B lymphocytes have been transfected with the human kappa-immunoglobulin gene (Potter et al., 1984) and rat hepatocytes with the chloramphenicol acetyltransferase gene (Tur-Kaspa et al., 1986).

[0240] 3. Calcium phosphate Nucleic acids can be introduced into cells using calcium phosphate precipitation. Human KB cells have been transfected with adenovirus type 5 DNA using this technique (Graham and Van Der Eb, 1973). Mouse L(A9), mouse C127, CHO, CV-1, BHK, NIH3T3 and HeLa cells have also been transfected with the neomycin marker gene in this manner (Chen and Okayama, 1987), and rat hepatocytes have been transfected with various marker genes (Rippe et al., 1990).

[0241] 4. DEAE-Dextran Nucleic acids can be delivered into cells using DEAE-dextran followed by polyethylene glycol. In this manner, reporter plasmids have been introduced into mouse myeloma and erythroleukemia cells (Gopal, 1985).

[0242] VII. Selectable or Screenable Markers Regardless of the method used to introduce transgene nucleic acid into host cells or to expose cells to the inhibitors of the present disclosure, various assays can be performed to confirm the presence of recombinant DNA sequences in host cells. Such assays include, for example, "molecular biological" assays well known to those skilled in the art, such as Southern and Northern blotting, RT-PCR and PCR; "biochemical" assays, such as detecting the presence or absence of specific peptides by immunological means (ELISA and Western blot) or by the assays described herein to identify factors within the scope of the present disclosure.

[0243] Cells harboring exogenous nucleic acid can be identified in vitro or in vivo by including a marker in the expression vector or exogenous nucleic acid. Such a marker would confer an identifiable change to the cell, allowing easy identification of cells harboring the expression vector. In general, a selection marker can confer a property that allows selection. A positive selection marker can be one whose presence allows selection, while a negative selection marker is one whose presence prevents selection. One example of a positive selection marker is a drug resistance marker.

[0244] Cloning and identification of transformants are usually aided by including a drug selection marker; for example, genes that confer resistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin, and histidinol are useful selection markers. In addition to markers that confer a phenotype that allows the differentiation of transformants based on the application of conditions, other types of markers are also envisioned, such as colorimetrically based screenable markers, such as GFP. Alternatively, screenable enzymes such as herpes simplex virus thymidine kinase (tk) or chloramphenicol acetyltransferase (CAT) can be used as negative selection markers. Those skilled in the art will also know how to use immunological markers, possibly with FACS analysis. The marker used is not believed to be critical, so long as it can be expressed simultaneously with the nucleic acid encoding the gene product. Further examples of selection and screenable markers are well known to those skilled in the art.

[0245] Selectable markers may include reporter genes of the type used in experimental microbiology, molecular biology and genetic engineering to indicate the success of transfection or other procedures intended to introduce foreign DNA into cells.Selectable markers are often antibiotic resistance genes; cells subjected to a procedure for introducing foreign DNA are grown in culture in a medium containing antibiotics, and cells capable of growing in culture have successfully incorporated and expressed the introduced genetic material.Examples of selectable markers include: the Abicr gene or the Neo gene from Tn5, which confers antibiotic resistance to geneticin.

[0246] Screenable markers may include reporter genes that allow researchers to distinguish between wanted and unwanted cells. Reporter genes may be used to indicate specific cell lineages. For example, reporter genes may be placed within an expression element so that they are co-expressed under the control of ventricular- or atrial-selective regulatory elements that are normally associated with the coding region of a ventricular- or atrial-selective gene. The reporter allows cells of a specific lineage to be isolated without being subjected to drugs or other selection pressures or risking cell viability.

[0247] Examples of such reporters include genes encoding cell surface proteins (e.g., CD4, HA epitope), fluorescent proteins, antigenic determinants, and enzymes (e.g., β-galactosidase). Cells containing the vector can be isolated, for example by FACS, using fluorescently tagged antibodies against cell surface proteins or substrates that can be converted to fluorescent products by the vector-encoded enzyme.

[0248] The reporter gene can be a fluorescent protein. A wide range of fluorescent protein gene variants have been developed that feature fluorescence emission spectral profiles spanning almost the entire visible light spectrum. Mutagenesis efforts in the original Aequorea victoria jellyfish green fluorescent protein have yielded new fluorescent probes with colors ranging from blue to yellow, and some reporter molecules are the most widely used in biological studies in vivo. Long-wavelength fluorescent proteins that exhibit emission in the orange and red spectral regions have been developed from marine anemones, Discosoma striata, and reef-building corals belonging to the class Anthozoa. Still other species are being explored to create similar proteins with cyan, green, yellow, orange, and crimson fluorescence emissions. Developmental research efforts are currently underway to improve the brightness and stability of fluorescent proteins and therefore their overall utility.

[0249] VIII. Cell culture conditions Cell culture conditions can be provided for the cultivation of the 3D cell aggregates described herein and for the production of mature cells containing the transgene. The starting cells of the selected population are at least 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 Pieces, or about 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 The starting cell population may contain at least 10, 10 1 , 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 cells / ml, or approximately 10 1 , 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 The seeding density may be in cells / ml, or any range derivable therein.

[0250] A. Culture medium The cells may be cultured in a particular medium at any stage in the process to produce mature cells expressing the transgene. The cells may be formulated in a manner suitable for delivery to a recipient without adverse effects.

[0251] The medium may be prepared using any of media used as a basal medium for culturing animal cells, such as AIM V, X-VIVO-15, NeuroBasal, EGM2, TeSR, BME, BGJb, CMRL 1066, Glasgow MEM, Improved MEM Zinc Option, IMDM, Medium 199, Eagle MEM, αMEM, DMEM, Ham, RPMI-1640, and Fischer's medium, and any combination thereof, but the medium may not be particularly limited thereto as long as it can be used to culture animal cells. In particular, the medium may be xeno-free or chemically defined.

[0252] The medium may be serum-containing or serum-free, or xeno-free. From the viewpoint of preventing contamination of components derived from different animals, the serum is preferably derived from the same animal as that of the stem cells. Serum-free medium refers to a medium that does not contain untreated or unpurified serum, and therefore may include a medium that contains purified blood-derived components or animal tissue-derived components (e.g., growth factors).

[0253] The medium may or may not contain any substitute for serum. Serum substitutes may include substances that suitably contain albumin (e.g., lipid-rich albumin, bovine albumin, albumin replacement solutions such as recombinant or humanized albumin, plant starch, dextran, and protein hydrolysates), transferrin (or other iron transporters), fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thiol diisocyanol, or equivalents. Serum substitutes may be prepared, for example, by the methods disclosed in International Patent Application Publication No. 98 / 30679, which is incorporated herein in its entirety. Alternatively, any commercially available product may be used for greater convenience. Commercially available products include knockout serum replacement (KSR), Chemically-defined lipid concentrated (Gibco), and Glutamax (Gibco).

[0254] The medium may be a serum-free medium suitable for cell development.For example, the medium may comprise B-27® supplement, Xenofree B-27® 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, incorporated herein in its entirety), GS21TM supplement (available on the World Wide Web at amsbio.com / B-27.aspx), or a combination thereof, in a concentration effective for generating T cells from 3D cell aggregates.

[0255] The medium may contain one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty 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); L-carnitine HCl; linoleic acid; linolenic acid; progesterone; putrescine diHCl; sodium selenite; and / or T3 (triodo-I-thyronine).

[0256] The medium may further comprise vitamins. The medium may comprise one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve or thirteen of the following (and any range derivable therein): biotin, DL alpha tocopherol acetate, DL alpha-tocopherol, vitamin A, choline chloride, calcium pantothenate, pantothenic acid, folic acid, nicotinamide, pyridoxine, riboflavin, thiamine, inositol, vitamin B12, or the medium may comprise combinations thereof or salts thereof. The medium may comprise or consist essentially of biotin, DL alpha tocopherol acetate, DL alpha-tocopherol, vitamin A, choline chloride, calcium pantothenate, pantothenic acid, folic acid, nicotinamide, pyridoxine, riboflavin, thiamine, inositol and vitamin B12. The vitamins may comprise or consist essentially of biotin, DL-alpha tocopherol acetate, DL-alpha-tocopherol, vitamin A, or combinations or salts thereof. The medium may further comprise a protein. The protein may comprise albumin or bovine serum albumin, human albumin fraction, catalase, insulin, transferrin, superoxide dismutase, or combinations thereof. The medium may further comprise one or more of the following: corticosterone, D-galactose, ethanolamine, glutathione, L-carnitine, linoleic acid, linolenic acid, progesterone, putrescine, sodium selenite, or triiodo-I-thyronine, or combinations thereof. The medium may comprise one or more of the following: B-27® supplement, Xenofree B-27® supplement, GS21™ supplement, or combinations thereof. The medium may comprise or further comprise amino acids, simple sugars, inorganic ions. The amino acids may include arginine, cysteine, isoleucine, leucine, lysine, methionine, glutamine, phenylalanine, threonine, tryptophan, histidine, tyrosine, or valine, or combinations thereof.The inorganic ions may include sodium, potassium, calcium, magnesium, nitrogen, or phosphorus, or combinations or salts thereof. The medium may further include one or more of the following: molybdenum, vanadium, iron, zinc, selenium, copper, or manganese, or combinations thereof. The medium may comprise or consist essentially of one or more vitamins discussed herein and / or one or more proteins discussed herein, and / or one or more of the following: corticosterone, D-galactose, ethanolamine, glutathione, L-carnitine, linoleic acid, linolenic acid, progesterone, putrescine, sodium selenite or triiodo-I-thyronine, B-27® supplement, XenoFree B-27® supplement, GS21™ supplement, 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 salts thereof, and / or molybdenum, vanadium, iron, zinc, selenium, copper, or manganese.

[0257] The medium can include exogenously added ascorbic acid, and may contain one or more exogenously added fatty acids or lipids, amino acids (e.g., non-essential amino acids), vitamin(s), growth factors, cytokines, antioxidants, 2-mercaptoethanol, pyruvic acid, buffers, and / or inorganic salts.

[0258] One or more of the medium components may be at least 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, at most 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, or at most 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, 250ng / L, at least 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, 250ng / ml, at most 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 / ml, or at least 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 / ml , 150, 180, 200, 250 μg / ml, at most 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 μg / ml, 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 μg / ml, at least 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 μg / ml.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 mg / ml, at most 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 mg / ml, 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 mg / ml, or any range derivable therein.

[0259] The medium used contains 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 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, and at most 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, 250ng / L, 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, 250ng / L, at least 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, 250ng / ml, at most 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, 250ng / ml , 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 / ml, or at least 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 / ml. 80, 85, 90, 95, 100, 150, 180, 200, 250 μg / ml, at most 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 μg / ml, 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 μg / ml, at least 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 μg / ml.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 mg / ml, at most 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 mg / ml, 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 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-alpha, TGF-beta, interferon-gamma, interferon-lambda, TSLP, thymopentin, pleotrophin, and / or midkine. In particular, the culture medium may include at least one of FLT3L and IL-7. More particularly, the culture medium may include both FLT3L and IL-7.

[0260] Other culture conditions can be appropriately specified. For example, the culture temperature can be about 20-40°C, for example, at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40°C, at most 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40°C, or about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40°C (or any range derivable therein), but the temperature can be higher or lower than these values. The CO2 concentration can be about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10% (or any range derivable therein), such as about 2%-10%, such as about 2-5%, or any range derivable therein. The oxygen partial pressure can be at least 1, 5, 8, 10, 20% or about 1, 5, 8, 10, 20%, or any range derivable therein.

[0261] B. Culture vessel Culture vessels used to culture the cells and / or 3D cell aggregates or their progeny may include, but are not limited to: flasks, tissue culture flasks, dishes, Petri dishes, tissue culture dishes, multi-dishes, microplates, microwell plates, multi-plates, multiwell plates, microslides, chamber slides, tubes, trays, CellSTACK® chambers, culture bags, and roller bottles, so long as stem cells can be cultured therein. Stem cells may be cultured in a volume of at least 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 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 derivable therein, depending on the culture requirements. The culture vessel may be a bioreactor, which may refer to any device or system that supports a biologically active environment. The bioreactor may have a volume of at least 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 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 derivable therein.

[0262] The culture vessel may be cell-adhesive or non-adhesive, and may be selected according to purpose. The cell-adhesive culture vessel may be coated with any cell-adhesive substrate, such as an extracellular matrix (ECM), to improve the adhesion of the vessel surface to cells. The cell-adhesive substrate may be any material intended to attach stem cells or feeder cells (if used). The cell-adhesive substrate may include collagen, gelatin, poly-L-lysine, poly-D-lysine, laminin, and fibronectin, as well as mixtures thereof, such as Matrigel™, and dissolved cell membrane preparations.

[0263] C. Matrix Components Various defined matrix components can be used in the culture method or composition. For example, as described in Ludwig et al. (2006a; 2006b), which are incorporated by reference in their entirety, a combination of recombinant type IV collagen, fibronectin, laminin and vitronectin can be used to coat the culture surface as a means of providing a solid support for the culture growth of pluripotent cells.

[0264] A matrix composition may be immobilized on a surface to provide support for cells. The matrix composition may comprise one or more extracellular matrix (ECM) proteins and an aqueous solvent. The term "extracellular matrix" is recognized in the art. Its components include one or more of the following proteins: fibronectin, laminin, vitronectin, tenascin, entactin, thrombospondin, elastin, gelatin, collagen, fibrillin, merosin, anchorin, chondronectin, link protein, bone sialoprotein, osteocalcin, osteopontin, epinectin, hyaluronectin, undulin, epiligrin, and kalinin. Other extracellular matrix proteins are described in Kleinman et al., (1993), which is incorporated herein by reference. The term "extracellular matrix" is intended to encompass currently unknown extracellular matrices that may be discovered in the future, as their characteristics as extracellular matrices are readily determinable by those skilled in the art.

[0265] 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.

[0266] The extracellular matrix (ECM) proteins may be of natural origin and may be purified from human or animal tissue. Alternatively, the ECM proteins may be genetically engineered recombinant proteins or synthetic in nature. The ECM proteins may be intact proteins or in the form of native or engineered peptide fragments. Examples of ECM proteins that may be useful in matrices for cell culture include laminin, type I collagen, type IV collagen, fibronectin and vitronectin. The matrix composition may include synthetic peptide fragments of fibronectin or recombinant fibronectin.

[0267] The matrix composition may comprise a mixture of at least fibronectin and vitronectin.The matrix composition may comprise laminin.

[0268] The matrix composition preferably comprises a single type of extracellular matrix protein. The matrix composition may comprise fibronectin, particularly for use in the culture of hematopoietic stem or progenitor cells. For example, a suitable matrix composition may be prepared by diluting human fibronectin, such as human fibronectin (Cat#354008) sold by Becton, Dickinson & Co. (BD) of Franklin Lakes, NJ, to a protein concentration of 5 μg / mL to about 200 μg / mL in Dulbecco's phosphate buffered saline (DPBS). In one particular example, the matrix composition comprises a fibronectin fragment, such as RetroNectin®. RetroNectin® is a protein of about 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.

[0269] The matrix composition may include laminin. For example, a suitable matrix composition may be prepared by diluting laminin (Sigma-Aldrich (St. Louis, Mo.); Cat#L6274 and L2020) in Dulbecco's Phosphate Buffered Saline (DPBS) to a protein concentration of 5 μg / ml to about 200 μg / ml.

[0270] The matrix composition may be xeno-free in that the matrix or its component proteins are exclusively of human origin. This may be desirable in certain applied studies. For example, a xeno-free matrix for culturing human cells may use matrix components of human origin, in which case any non-human animal components may be excluded. Matrigel™ may be excluded from the culture composition as a substrate. Matrigel™ is a mixture of gelatinous proteins 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 frequently used by cell biologists as a substrate for cell culture, but may introduce unwanted foreign antigens or contaminants.

[0271] IX. Cell Culture Compositions and Methods 3D culture compositions, such as artificial thymic organoids (ATO), are optimized, highly efficient, and reproducible off-the-shelf solutions for the in vitro generation of mature cells from pluripotent starting cell sources. The 3D culture compositions can use serum-free conditions, avoid the use of human thymic tissue or proprietary scaffolds, and foster the positive selection and robust generation of fully functional, mature human cells, such as mature human T cells and NK cells, from their stem cells. As a potential commercial platform for in vitro cell generation, the 3D culture compositions offer efficiency, reproducibility, scalability, and reduced costs and labor compared to competing technologies. Non-limiting commercial applications may include in vitro experimental modeling of human cell development, and in vitro production of engineered antigen-targeting and / or transgene-expressing cells useful for cell therapy, such as immunotherapy, to treat cancer and autoimmune pathologies from various stem cell sources.

[0272] Also provided is an optimized three-dimensional (3D) culture system for the in vitro generation of functional mature cells. The resulting 3D cellular constructs can be referred to as artificial thymic organoids (ATOs).

[0273] The system may involve aggregation of PSCs with stromal cells expressing Notch ligands into 3D structures in the presence of optimized medium containing FLT3 ligand (FLT3L), interleukin 7 (IL-7), B27 and ascorbic acid, and conditions may also be present that allow for culture at an air-liquid interface.

[0274] Also provided are methods of 3D culture compositions (e.g., generation of ATOs) as developed, comprising the human DLL4-transduced MS-5 murine stromal cell line (hereinafter MS5-hDLL4) and stem or progenitor cells and / or CD34 cells isolated from human umbilical cord blood, bone marrow, whole blood samples, or peripheral blood mononuclear cells (PBMCs). + Accompanied by aggregation with PSC. Up to 1 × 10 6 stem or progenitor cells and / or CD34 + PSCs are mixed with MS5-hDLL4 cells at an optimized ratio (typically 1:10 HSPC to stromal cells).

[0275] For example, aggregation can be obtained by centrifugation of the mixed cell suspension ("compression aggregation") followed by aspiration of the cell-free supernatant. The cell pellet can then be aspirated as a slurry in 5-10 μl of differentiation medium and transferred as a droplet onto a 0.4 um nylon transwell culture insert, which is floated within a well of differentiation medium, thereby allowing the bottom of the insert to be in contact with the medium and the top with air.

[0276] For example, the differentiation medium consists of RPMI-1640, 5 ng / ml human FLT3L, 5 ng / ml human IL-7, 4% serum-free B27 supplement and 30 μM L-ascorbic acid. The medium can be completely exchanged from around the culture insert every 3-4 days. Variations in the protocol allow the use of alternative components with different effects on efficacy, specifically: the basal medium RPMI can be replaced by several commercially available alternatives (e.g., IMDM). The stromal cell line used is MS-5, a previously described mouse bone marrow cell line (Itoh et al, 1989), but MS-5 can be replaced by similar mouse stromal cell lines (e.g., OP9, S17), human stromal cell lines (e.g., HS-5, HS-27a), primary human stromal cells or stromal cells derived from human pluripotent stem cells.

[0277] The stromal cell line is transduced by lentivirus encoding human DLL4 cDNA; however, the method of gene delivery and the Notch ligand gene may be altered. Alternative Notch ligand genes include DLL1, JAG1, JAG2, etc. Notch ligands also include those described in U.S. Patent Nos. 7,795,404 and 8,377,886, which are incorporated herein by reference. Notch ligands further include Delta 1, 3 and 4 and Jagged 1, 2. Notch ligands also include functional fragments of Notch ligands. The type and source of PSCs may include bone marrow, whole blood, umbilical cord blood, peripheral blood, peripheral blood mononuclear cells or thymus, or PSCs or hematopoietic stem or progenitor cells may be differentiated in vitro from embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs). PSCs or hematopoietic stem or progenitor cells derived from primary tissue or ESCs or iPSCs may be human or non-human animal (eg, mouse) in origin.

[0278] X. Treatment of Disease The methods may be used on individuals who have tested positive for such disorders or who are believed to be at risk for developing such or related conditions. The compositions and methods described herein may be used to treat the disorders listed herein and / or disorders known in the art with inflammatory or autoimmune components.

[0279] The present disclosure relates to the use of cancer treatment and / or cancer antigen.The cancer or antigen to be treated can be any cancer known in the art, or the antigen associated with, for example, epithelial cancer, (e.g. breast, digestive tract, lung), prostate cancer, bladder cancer, lung (e.g. small cell lung) cancer, colon cancer, ovarian cancer, brain cancer, stomach cancer, renal cell carcinoma, pancreatic cancer, liver cancer, esophageal cancer, head and neck cancer, or colorectal cancer. The cancers or antigens to be treated include the following cancers: adrenocortical carcinoma, idiopathic myeloid metaplasia, 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 tumors (e.g., glioma, brain stem glioma, cerebellar or cerebral astrocytoma (e.g., pilocytic astrocytoma, diffuse astrocytoma, anaplastic (malignant) astrocytoma). ), malignant glioma, ependymoma, oligodenglioma, meningioma, meningeal sarcoma, craniopharyngioma, hemangioblastoma, medulloblastoma, supratentorial primitive neuroectodermal tumor, optic nerve hypothalamic glioma and glioblastoma), breast cancer, bronchial adenoma / carcinoid, carcinoid tumors (e.g., gastrointestinal carcinoid tumors), cancer of unknown primary, central nervous system lymphoma, cervical cancer, colon cancer, colorectal cancer, chronic myeloproliferative disorder, endometrial cancer (e.g., uterine , ependymoma, esophageal cancer, Ewing family tumors, eye cancer (e.g., intraocular melanoma and retinoblastoma), gallbladder cancer, gastric (stomach) cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors (GIST), germ cell tumors (e.g., extracranial, extragonadal, ovarian), gestational trophoblastic neoplasia, head and neck cancer, hepatocellular (liver) cancer (e.g., hepatoma and hepatoma), hypopharyngeal cancer, islet cell carcinoma (pancreatic endocrine), laryngeal cancer, leukemia, lip and oral cavity cancer, oral cancer, liver cancer, lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous cell carcinoma of the lung), lymphoid neoplasms (e.g., lymphoma), medulloblastoma, ovarian cancer, mesothelioma, metastatic squamous cell carcinoma of the head and neck, cancer of the mouth, multiple endocrine neoplasia syndromes, myelodysplastic syndromes, myelodysplastic / myeloproliferative disorders, cancer of the nasal cavity and paranasal sinuses, nasopharyngeal carcinoma, neuroblastoma, neuroendocrine carcinoma, oropharyngeal cancer, ovarian cancer (e.g., ovarian epithelial carcinoma,Ovarian germ cell tumors, ovarian low malignant potential tumors), pancreatic cancer, parathyroid cancer, penile cancer, cancer of the peritoneum, pharyngeal cancer, pheochromocytoma, pineoblastoma and supratentorial primitive neuroectodermal tumors, pituitary tumors, pleuropulmonary blastoma, lymphoma, primary central nervous system lymphoma (microglioma), pulmonary lymphangioleiomyomatosis, rectal cancer, kidney cancer, cancer of the renal pelvis and ureter (transitional cell carcinoma), rhabdomyosarcoma, salivary gland cancer, skin cancer (e.g., non-melanoma (e.g., For example, squamous cell carcinoma, melanoma and Merkel cell carcinoma, small intestine cancer, squamous cell carcinoma, testicular cancer, throat cancer, thymoma and thymic carcinoma, thyroid cancer, tuberous sclerosis, urethral cancer, vaginal cancer, vulvar cancer, Wilms' tumor, and one of the following: post-transplant lymphoproliferative disorder (PTLD), abnormal blood vessel proliferation associated with nevus syndrome, edema (e.g., associated with brain tumors), or Meigs' syndrome.

[0280] The present disclosure relates to the treatment of autoimmune conditions and / or the use of autoimmune associated antigens. The autoimmune disease or antigen to be treated may be any autoimmune condition known in the art or may be, for example, diabetes, transplant rejection, GVHC, arthritis (rheumatoid arthritis, e.g. 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, spondyloarthritis and juvenile onset rheumatoid arthritis, osteoarthritis, chronic progressive arthritis, osteoarthritis, chronic primary polyarthritis, reactive arthritis and ankylosing spondylitis), inflammatory hyperproliferative skin diseases, psoriasis, e.g. psoriasis vulgaris, guttate psoriasis, pustular psoriasis and psoriasis of the nail, atopy, e.g. atopic diseases such as hay fever and Job's syndrome, dermatitis, e.g. contact dermatitis, chronic contact dermatitis, exfoliative dermatitis, allergies, allergic dermatitis, allergic contact dermatitis, dermatitis herpetiformis, nummular dermatitis, seborrheic dermatitis, nonspecific dermatitis, temporary irritant contact dermatitis and atopic dermatitis, x-linked hyper IgM syndrome, allergic intraocular inflammatory disease, urticaria, e.g. chronic allergic urticaria and chronic idiopathic urticaria, e.g. chronic autoimmune urticaria, myositis, polymyositis / dermatomyositis, juvenile dermatomyositis, toxic epidermal necrolysis, scleroderma (e.g. systemic scleroderma), sclerosis such as systemic sclerosis, multiple sclerosis (MS), such as optic spinal MS, primary progressive MS (PPMS) and relapsing-remitting MS (RRMS), progressive systemic sclerosis, atherosclerosis Atherosclerosis, disseminated sclerosis, ataxic sclerosis, neuromyelitis optica (NMO), inflammatory bowel disease (IBD) (e.g., Crohn's disease, autoimmune-mediated gastrointestinal diseases, colitis, e.g. ulcerative colitis) colitis), ulcerative colitisulcerosa), microscopic colitis, collagenous colitis, polypoid colitis, necrotizing enterocolitis and transmural colitis, and autoimmune inflammatory bowel disease), intestinal inflammation, pyoderma gangrenosum, erythema nodosum, primary sclerosing cholangitis, respiratory distress syndromes, such as adult or acute respiratory distress syndrome (ARDS), meningitis, inflammation of all or part of the uvea, iritis, choroiditis, autoimmune hematopoietic disorders, rheumatoid spondylitis, rheumatoid synovitis, hereditary angioedema, cerebral neuropathy, such as in the case of meningitis. trauma, herpes gestationis, pemphigoid of gestationis, scrotal pruritus, autoimmune premature ovarian failure, sudden deafness due to autoimmune pathologies, IgE-mediated diseases such as anaphylaxis and allergic and atopic rhinitis, encephalitis such as 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 uveitic uveitis, uveitis or autoimmune uveitis, glomerulonephritis (GN) with and without nephrotic syndrome, e.g. chronic or acute glomerulonephritis, e.g. primary GN, immune-mediated GN, membranous GN (membranous nephropathy), idiopathic membranous GN or idiopathic membranous nephropathy, membranous (membrano- or membranous) proliferative GN (MPGN), e.g. types I and II and rapidly progressive GN, proliferative nephritis, autoimmune polyendocrine dysfunction, turtle balanitis, e.g. localized plasma cell balanitis, balanoposthitis, erythema annulare centrifugally, erythema dyschromicus perstans, erythema multiforme, granuloma annulare, lichen sclerosus atrophicus, lichen simplex chronicus, lichen spinous, lichen planus, ichthyosis phyllodes, epidermolytic hyperkeratosis, precancerous keratosis, pyoderma gangrenosum, allergic conditions and responses, allergic reactions, eczema, e.g. allergic or atopic eczema, asteatotic eczema, dyshidrotic eczema and bullous palmoplantar eczema, asthma, e.g. asthma bronchiale,asthma and autoimmune asthma, pathologies involving infiltration of T cells and chronic inflammatory responses, immune responses during pregnancy to foreign antigens, e.g. fetal ABO blood groups, chronic pulmonary inflammatory diseases, autoimmune myocarditis, leukocyte adhesion deficiency, lupus, e.g. lupus nephritis, lupus encephalitis, childhood lupus, non-renal lupus, extrarenal lupus, discoid lupus and discoid lupus erythematosus, lupus alopecia, systemic lupus erythematosus (SLE), e.g. cutaneous SLE or subacute cutaneous SLE, neonatal lupus syndrome (NLE) and systemic lupus erythematosus disseminatus, juvenile onset (type I) diabetes, e.g. childhood insulin-dependent diabetes mellitus (IDDM) and adult onset diabetes mellitus (type II diabetes), and autoimmune diabetes. Also, immune responses associated with acute and delayed hypersensitivity mediated by cytokines and T-lymphocytes, sarcoidosis, granulomatosis, e.g. lymphomatoid granulomatosis, Wegener's granulomatosis, agranulocytosis, vasculitis, e.g. vasculitis, large vasculitis (e.g. polymyalgia rheumatica and giant cell (Takayasu) arteritis), medium vasculitis (e.g. Kawasaki disease and polyarteritis nodosa / periarteritis nodosa), microscopic polyarteritis, immunovasculitis, CNS vasculitis, cutaneous vasculitis, hypersensitivity vasculitis, necrotizing vasculitis, e.g. systemic necrotizing vasculitis, and ANCA-associated vasculitis, e.g. Churg-Strauss vasculitis or syndrome (CSS) and ANCA-associated small vasculitis, temporal arteritis, aplastic anemia, autoimmune aplastic anemia, Coombs positive Anemia, Diamond-Blackfan anemia, haemolytic anemia or immune-mediated haemolytic anemia, e.g. autoimmune haemolytic anemia (AIHA), Addison's disease, autoimmune neutropenia, pancytopenia, leukopenia, diseases involving leukocyte extravasation, CNS inflammatory disorders, Alzheimer's disease, Parkinson's disease, multi-organ dysfunction syndromes, e.g. sepsis, secondary to injury or hemorrhage, antigen-antibody complex mediated diseases, antiglomerular basement membrane disease, antiphospholipid syndrome, allergic neuritis, Behcet's disease / syndrome, Castleman syndrome, Goodpasture's syndrome, Raynaud's syndrome, Sjogren's syndrome, Stevens-Johnson syndrome, pemphigoid, e.g. bullous pemphigoid and cutaneous pemphigoid, mucous membrane pemphigoid (e.g. pemphigus vulgaris, pemphigus foliaceus, mucous membrane pemphigoid) mucous membrane pemphigoid and pemphigus erythematosus), autoimmune polyendocrinopathy, Reiter's disease or syndrome, burns, pre-eclampsia, immune complex disorders such as immune complex nephritis, antibody mediated nephritis, polyneuropathy, chronic neuropathy such as IgM polyneuropathy or IgM mediated neuropathy, autoimmune or immune mediated thrombocytopenia such as idiopathic thrombocytopenic purpura (ITP), such as chronic or acute ITP, scleritis such as idiopathic cerato-scleritis, episcleritis, autoimmune diseases of the testes and ovaries such as autoimmune orchitis and oophoritis, primary hypothyroidism, hypoparathyroidism, autoimmune endocrine disorders such as thyroiditis, such as autoimmune thyroiditis, Hashimoto's disease,Chronic thyroiditis (Hashimoto's thyroiditis) or subacute thyroiditis, autoimmune thyroid disease, idiopathic hypothyroidism, Graves' disease, polyendocrine syndromes, e.g. autoimmune polyendocrine syndrome (or polyendocrine endocrinopathy syndrome), paraneoplastic syndromes, e.g. neurological paraneoplastic syndromes, e.g. Lambert-Eaton myasthenic syndrome or Eaton-Lambert syndrome, stiff or stiff-person syndrome, encephalomyelitis, e.g. allergic encephalomyelitis or allergic encephalomyelitis and experimental allergic encephalomyelitis (EAE), experimental autoimmune encephalomyelitis, severe Myasthenia, e.g. thymoma-associated myasthenia gravis, cerebellar degeneration, neuromyotonia, opsoclonus or opsoclonus-myoclonus syndrome (OMS) and sensory neuropathy, multifocal motor neuropathy, Sheehan syndrome, autoimmune hepatitis, chronic hepatitis, lupoid hepatitis, giant cell hepatitis, chronic active hepatitis or autoimmune chronic active hepatitis, lymphocytic interstitial pneumonia (LIP), bronchiolitis obliterans (non-transplant) vs. NSIP, Guillain-Barré syndrome, Buerger's disease (IgA nephropathy), idiopathic IgA nephropathy, linear IgA dermatosis, acute febrile neutrophilic dermatosis, keratoconjunctivitis ... Sublaminar pustulosis, Transient acantholytic dermatosis, Cirrhosis, e.g. primary biliary cirrhosis and pneumonocirrhosis, Autoimmune enteropathy syndromes, 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 diseases, e.g. Autoimmune inner ear disease (AIED), Autoimmune hearing loss, Polychondritis, e.g. Refractory or relapsing or relapsing polychondritis, Alveolar proteinuria Albinism, Cogan's syndrome / nonsyphilitic interstitial keratitis, Bell's palsy, Sweet's disease / syndrome, rosacea autoimmune zoster-associated pain, amyloidosis, non-cancerous lymphocytosis, primary lymphocytosis, including monoclonal B-cell lymphocytosis (e.g., monoclonal gammopathy and monoclonal gammopathy of undetermined significance, MGUS), peripheral neuropathies, paraneoplastic syndromes, channelopathies, e.g., epilepsy, migraine, cardiac arrhythmias, muscle disorders, hearing loss, blindness, periodic paralysis and CNS channelopathies, autism, inflammatory myopathies,Focal or segmental glomerulosclerosis (FSGS), endocrine ophthalmopathy, uveoretinitis, chorioretinitis, autoimmune hepatological disorders, fibromyalgia, polyendocrine deficiency, Schmidt's syndrome, adrenalitis, gastric atrophy, presenile dementia, demyelinating diseases such as autoimmune demyelinating diseases and chronic inflammatory demyelinating polyneuropathy, Dressler's syndrome, alopecia areata greata), alopecia totalis, CREST syndrome (calcinosis, Raynaud's phenomenon, esophageal dysmotility, digital sclerosis) and telangiectasia), male and female autoimmune infertility due to e.g. antisperm antibodies, mixed connective tissue disease, Chagas' disease, rheumatic fever, recurrent miscarriages, farmer's lung disease, erythema multiforme, post-cardiotomy syndrome, Cushing's syndrome, bird breeder's disease, allergic granulomatous vasculitis, benign lymphocytic vasculitis, Alport syndrome, alveolitis, e.g. allergic alveolitis and fibrosing alveolitis, interstitial lung disease, transfusion reactions, leprosy, malaria, parasitic diseases, e.g. leishmaniasis, kypanosomiasis, schistosomiasis, ascariasis, aspergillosis, Samter's syndrome, Caplan's syndrome, dengue fever, endocarditis, endomyocardial fibrosis, diffuse interstitial pulmonary fibrosis, interstitial pulmonary fibrosis, lung Fibrosis, idiopathic pulmonary fibrosis, cystic fibrosis, endophthalmitis, erythema elevatum, erythroblastosis fetalis, eosinophilic fasciitis, Charmant's syndrome, Felty's syndrome, filariasis, cyclitis, e.g. chronic cyclitis, heterochronic cyclitis, iridocyclitis (acute or chronic) or Fuchs' cyclitis, Henoch-Schönlein purpura, human immunodeficiency virus (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, parotitis, Evan's syndrome, autoimmune gonadal dysfunction, Sydenham's chorea, poststreptococcal nephritis, thromboangitis ubiterans), thyrotoxicosis, tabes dorsalis, choroiditis, giant cell polymyalgia, chronic hypersensitivity pneumonitis, keratoconjunctivitis sicca, epidemic keratoconjunctivitis, idiopathic nephritic syndrome, minimal change nephropathy, benign familial ischemia-reperfusion injury, transplanted organ reperfusion, retinal autoimmunity, arthritis, bronchitis, chronic obstructive airway / lung disease, silicosis,Aphtha, aphthous stomatitis, arteriosclerotic disorders, asperniogenese, autoimmune hemolysis, Beck's disease, cryoglobulinemia, Dupuytren's contracture, phacosensitivity endophthalmitis, allergic enteritis, erythema nodosum leprosum, idiopathic facial nerve palsy, chronic fatigue syndrome, rheumatic fever, Hamman-Rich disease, sensorineural deafness, paroxysmal hemoglobinuria, hypogonadism, focal ileitis, leukopenia, infectious mononucleosis, transverse myelitis, primary idiopathic myxedema, nephrosis, sympathetic ophthalmia, granulomatous orchitis, pancreatitis, acute polyradiculitis, gangrenous Pyoderma, Kervan thyroiditis, acquired splenic atrophy, nonmalignant thymoma, vitiligo, toxic shock syndrome, food poisoning, conditions involving T-cell infiltration, leukocyte adhesion deficiency, immune responses associated with acute and delayed hypersensitivity mediated by cytokines and T-lymphocytes, diseases involving leukocyte extravasation, multiorgan dysfunction syndrome, antigen-antibody complex-mediated diseases, anti-glomerular basement membrane disease, allergic neuritis, autoimmune polyendocrine disease, oophoritis, primary myxedema, autoimmune atrophic gastritis, sympathetic ophthalmia, rheumatic diseases, mixed connective tissue disease, nephrotic syndrome, islet inflammation, polyendocrine deficiency, autoimmune polyendocrine syndrome type I, adult-onset idiopathic hypoparathyroidism (AOIH), cardiomyopathies such as dilated cardiomyopathy, epidermolysis bullosa acquisita (EBA), hemochromatosis, myocarditis, nephrotic syndrome, primary sclerosing cholangitis, suppurative or non-suppurative sinusitis, acute or chronic sinusitis, ethmoid, frontal, maxillary or sphenoid sinusitis, eosinophil-related disorders such as eosinophilia, eosinophilic pulmonary infiltrates, eosinophilic myalgia syndrome, Löffler's syndrome, chronic eosinophilic pneumonia, tropical pulmonary eosinophilia, bronchopneumonia aspergillosis, aspergilloma or eosinophil-containing granulomas, anaphylaxis, seronegative spondyloarthritis, polyendocrine autoimmune diseases, sclerosing cholangitis, sclera, episcleral plate, chronic mucocutaneous candidiasis, Bratton syndrome, transient infantile hypogammaglobulinemia, Wiskott-Aldrich syndrome, ataxia-telangiectasia syndrome, vascular ectasia, autoimmune disorders associated with collagen diseases, rheumatism, diseases of the nervous system, lymphadenitis, hypotensive reactions, vascular insufficiency, tissue damage, cardiovascular ischemia, hyperalgesia, renal ischemia, cerebral ischemia, and diseases involving angiogenesis, allergic hypersensitivity disorders, glomerular nephropathy, reperfusion injury,Also contemplated are ischemic reperfusion injury, myocardial or other tissue reperfusion injury, lymphomatous tracheobronchitis, inflammatory dermatoses, dermatoses with acute inflammatory components, multiple organ failure, bullous disease, renal cortical necrosis, acute purulent meningitis or other inflammatory disorders of the central nervous system, ocular and orbital inflammatory disorders, granulocyte transfusion-associated syndrome, cytokine-induced toxicity, narcolepsy, acute severe inflammation, chronic refractory inflammation, pyelitis, intimal hyperplasia, peptic ulcer, valvulitis, graft-versus-host disease, contact hypersensitivity, asthmatic airway hypersensitivity, and endometriosis.

[0281] The cells can be formulated in a manner suitable for delivery to a recipient without adverse effects. The cells may or may not be formulated as a cell suspension. In a specific case, the cells are formulated in a single dose form. The cells can be formulated for systemic or local administration. In some cases, the cells are formulated for storage prior to use, and the cell formulation can include one or more cryopreservatives, such as DMSO (e.g., in 5% DMSO). The cell formulation can include albumin, such as human albumin, and one specific example formulation includes 2.5% human albumin. The cells can be specifically formulated for intravenous administration; for example, the cells are formulated for intravenous administration in less than 1 hour. The cells can 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.

[0282] The therapeutic compositions and treatments disclosed herein may include combinations of therapeutic agents, such as administering a first therapeutic or pharmaceutical composition or treatment with a second therapeutic or pharmaceutical composition or treatment. Multiple therapeutic agents may be administered in any suitable manner known in the art. For example, the therapeutic or pharmaceutical compositions or treatments may be administered sequentially (at different times) or in parallel (at the same time). The therapeutic or pharmaceutical compositions or treatments may be administered in separate compositions. The therapeutic or pharmaceutical compositions or treatments may be present in the same composition.

[0283] Also described in this disclosure are compositions and methods that include therapeutic compositions.The different therapeutic or pharmaceutical compositions or treatments can be administered in one composition or more than one composition, for example, two compositions, three compositions or four compositions.Various combinations of drugs can be used.

[0284] The therapeutic or pharmaceutical compositions and treatments disclosed herein can be prior to, concomitant with and / or after another treatment or drug, with intervals ranging from minutes to weeks.When drugs are applied separately to cells, tissues or organisms, it can be generally ensured that no significant period of time passes between each delivery time, so that the therapeutic or pharmaceutical drugs can still exert advantageous combined effects on the cells, tissues or organisms.For example, in such cases, it is envisioned that the cells, tissues or organisms can be contacted with two, three, four or more drugs or treatments at substantially the same time (i.e., within less than about one minute). The one or more therapeutic agents or treatments may be administered 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, 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, 60 minutes, 61 hours, 62 hours, 63 hours, 64 hours, 65 hours, 66 hours, 67 hours, 68 hours, 69 hours, 70 hours, 71 hours, 72 hours, 73 hours, 74 hours, 75 hours, 76 hours, 77 hours, 78 hours, 79 hours, 80 hours, 81 hours, 82 hours, 83 hours, 84 hours, 85 hours, 86 hours, 87 hours, 88 hours, 89 hours, 90 hours, 91 hours, 92 hours, 93 hours, 94 hours, 95 hours, 96 hours 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 any longer period, and any range derivable therein.

[0285] The treatment may include various "unit doses". A unit dose is defined as containing a predetermined amount of the therapeutic composition. The amount to be administered and the specific route and formulation are within the skill of those skilled in the clinical arts to determine. A unit dose need not be administered as a single injection, but may include continuous infusions over a set period of time. A unit dose may include a single administrable dose.

[0286] A therapeutically effective or sufficient amount of a therapeutic composition or treatment administered to a human, whether in a single dose or multiple doses, is about 10 2 up to about 10 10 The therapeutic agent used may range from, for example, about 10 cells / kg patient weight. 2 ~ approx. 10 cells 9 Cells / kg, approximately 10 2 ~ approx. 10 cells 8 Cells / kg, approximately 10 2 ~ approx. 10 cells 7 Cells / kg, approximately 10 2 ~ approx. 10 cells 6 Cells / kg, approximately 10 2 ~ approx. 10 cells 5 Cells / kg, approximately 10 2 ~ approx. 10 cells 4 cells / kg or approximately 10 2 ~ approx. 10 cells 3 The therapeutic agents described herein may be administered to a subject at a dose of about 10 cells / kg daily. 2 cells, approximately 10 3 cells, approximately 10 4 cells, approximately 10 5 cells, approximately 10 6 cells, approximately 10 7 cells, approximately 10 8 cells, approximately 10 9 cells or approximately 10 10 The cells may be administered in a dose of 100 μg / ml or 100 μg / ml of the therapeutic agent. The dose may be administered as a single dose or multiple doses (e.g., 2 or 3 doses), e.g., as an infusion. The progress of this therapy is easily monitored by conventional techniques.

[0287] When an "immunologically effective amount," "antitumor effective amount," "tumor inhibitory effective amount," or "therapeutic amount" is indicated, the exact amount of a composition of the present disclosure to be administered can be determined by a physician, taking into account individual differences in age, weight, tumor size, extent of infection or metastasis, and physical condition of the patient (subject).

[0288] XI. Kits The present disclosure also describes a kit comprising the composition of the present disclosure or a composition for carrying out the method of the present disclosure. The kit can be used to evaluate the expression of a transgene, the integration of a transgene into the genome, or the differentiation of stem or progenitor cells containing a transgene into mature cells. The kit can include 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 of the above-mentioned or derived therefrom. 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, 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, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148 , 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 therein, or 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 therein.

[0289] The kits can include components that can be individually packaged or placed within a container, such as a tube, bottle, vial, syringe, or other suitable container means. The kits can include cell culture components, compositions, and culture vessels described herein. EXAMPLES

[0290] XII. Working Examples The following examples are included to demonstrate preferred embodiments of the invention. Those skilled in the art will recognize that the procedures disclosed in the following examples represent procedures that the inventors have found to work well in the practice of the invention and therefore may be considered to constitute preferred embodiments for its practice. However, those skilled in the art should recognize in light of this disclosure that many changes can be made in the specific embodiments disclosed without departing from the spirit and scope of the invention and still obtain like or similar results.

[0291] Example 1 - Regulation of gene expression during differentiation of mature T cells 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 assessed for average vector copy number. H1 CAR19LH-eGFP clone 2 (VCN:4.6) was differentiated into ATO in parallel with wild-type H1 controls and analyzed at weeks 2-5. Compared to wild-type, H1 CAR19LH-eGFP clone 2 showed atypical differentiation with a low frequency of CD3+TCRab+ cells (Figure 2A). Furthermore, clone 2 also showed an arrest in the developmental progression of SP4 and SP8 cells or a population of mostly CD4-CD8- "double negative" cells (Figure 2B).

[0292] As shown in Figure 3, the cells showed stage-specific expression of the transgene inserted downstream of GZMA. CRISPR / Cas9-mediated homologous recombination repair was performed in H1 hESC cell line to insert the reporter mCitrine within the 3'UTR of the endogenous GZMA gene. Heterozygous H1 GZMA-mCitrine reporter was cloned in parallel with wild-type H1 control and differentiated into ATO. Reporter expression levels were assessed at different T cell development stages at 1, 3 and 5 weeks. Analysis of H1 GZMA-mCitrine Het clone 9 is shown. mCitrine is expressed in 23% of SP8 and 2% of SP4, but not in precursor stage ISP4 or DP.

[0293] Expression of CAR19LH under the control of the endogenous GZMA promoter allows for normal development of CAR T cells. Using a similar knock-in protocol, the CAR19LH exogenous gene was inserted into the 3'UTR of the endogenous GZMA gene of the T-iPSC line "n11:". With this approach, expression of CAR19LH would be delayed until T cells reached the mature stages of SP8 and SP4, as evidenced by previous reporter experiments. n11 GZMA-CAR19LH homozygous clones were differentiated into ATO. Figure 3a shows high frequency of CD3+TCRab+ cells and normal developmental patterns in DP cells (1 week), SP8 cells (3 weeks), and SP4 cells (4 weeks). Figure 3b shows staining of CAR with 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, as shown in Figure 4.

[0294] Example 2. Engineering circumventing biological barriers to allogeneic iPSC-derived chimeric antigen receptor therapy Chimeric antigen receptor (CAR) T-cell therapy has produced remarkable results in otherwise treatment-refractory hematopoietic malignancies. Currently, the process of manufacturing autologous CAR T cells is challenging due to the need for de novo generation of each individual treatment and the inherent variability of T-cell biology between patients, making clinical responses unpredictable and inconsistent. As a result, there is growing interest in generating CAR T cells in vitro from an infinitely renewable allogeneic source of human pluripotent stem cells (hPSCs).

[0295] However, there are several biological barriers to generating CAR T cells from hPSCs. First, expression of CAR transgenes early in T cell differentiation deviates development toward the innate lymphoid pathway rather than the typical T cell lineage. Second, removal of endogenous T cell receptors (TCRs) to suppress alloreactivity results in a developmental block, as maturing T cells cannot undergo positive selection. This proposal seeks to resolve such challenges to generating non-allo-reactive stem cell-derived CAR T cells by deploying innovative stage-specific expression strategies and novel in vitro methods to induce the differentiation of typical mature T cells, the artificial thymic organoid (ATO) system.

[0296] ATO is a first-in-class in vitro method for efficient generation of mature single positive (SP) CD8+ and CD4+ T cells from multiple stem cell sources, e.g., hPSCs. Improving on previously established systems, ATO can robustly support positive selection and maturation to the SP stage. Our preliminary studies have already shown that deviation from natural fate can be overcome by achieving stage-specific delayed expression of the CAR transgene restricted to mature T cells. In this proposal, we build a model of delayed expression of the CAR to evaluate how disruption of endogenous TCR affects CAR T cell development in ATO. We then evaluate two different ways to deliver an exogenous positive selection signal to rescue development in the absence of endogenous TCR. These methods include expression of a persistently signaling CAR or transient expression of an exogenous TCR at the physiologically relevant DP T cell stage. The approach developed in this proposal will enable the generation of hPSC-derived non-allo-reactive T cell therapeutics, increasing access to treatment for more patients in need.

[0297] As shown in Figure 5, early expression of CAR in the ATO system deviates T cell differentiation towards the innate lymphoid pathway. We identified granzyme A (GZMA) as a potential candidate locus for anti-CD19 CAR knock-in (Figure 6A-B). Stage-specific expression of CAR19 at the GZMA locus allows the generation of typical mature CAR T cells (Figure 7A-D). Restricting CAR19 expression only to mature SP T cells prevented early deviation towards the innate lymphoid pathway and allowed typical development (Figure 7B). By week 5, the majority of ATO-derived T cells were CD8αβ+ and CAR19+ (Figure 7C). As shown in Figure 7D, mature SP8 CAR T cells robustly released cytotoxic cytokines and upregulated activation markers in response to antigen CD19.

[0298] Here, we report the generation of CAR T cells lacking non-allogeneic TCRs from T-iPSCs. We established a stage-specific expression system that provides robust and reproducible temporal control of transgene expression during T cell differentiation. Building on this platform, we generated typical mature CAR T cells from T-iPSCs that exhibited antigen-specific killing, activation, and proliferation.

[0299] Example 3 - Regulation of gene expression during differentiation of mature T cells The aim of the present invention is to achieve the regulation of gene expression of an inserted (transgenic / foreign) T cell receptor (TCR) restricted to a specific T cell differentiation stage. Expression of the TCR is required to allow normal T cells to develop in the thymus and produce mature T cells by positive selection. Also, methods to eliminate TCR expression in T cell precursors [to prevent alloresponses (graft-versus-host disease)] also prevent positive selection of mature T cells. The ability to restrict TCR expression to a specific T cell differentiation stage allows for the "positive selection" and induction of maturation of T cell precursors while avoiding the negative consequences of ongoing transgenic TCR expression in mature T cells. Although the most readily applicable application of the present invention would be to stably modify pluripotent stem cells (PSCs), this approach could potentially be used to target any cell type produced in vitro or in vivo during early T cell development, e.g. PSCs, hematopoietic stem or progenitor cells (HSPCs), or thymocyte precursors. A first-priority application of this technology will be to enable the in vitro generation of therapeutic T cells from allogeneic PSCs in which endogenous TCR expression has been ablated. The specific genes identified as endogenous loci for TCR insertion are RAG1 and RAG2.

[0300] Adoptive T cell therapies, such as the FDA-approved CART therapy, currently rely on the use of autologous T cells harvested from the patient's blood. This logistically challenging and costly process has led to the exploration of the use of allogeneic T cell sources, which offer the potential to be off-the-shelf alternatives to currently used approaches that use autologous (patient-specific) sources of T cells.

[0301] Allogeneic T cells may also be sourced from healthy donor blood 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 rejection of donor cells by the immune system of the mismatched recipient. In the field of allogeneic cell therapy, methods for both types of functional modification have been developed. For example, elimination of endogenous TCR expression can be performed by disruption of the TRAC locus or deletion of RAG1 + / - RAG2 gene expression. Disruption of MHC class I expression can be performed by deletion of either specific class I genes or beta 2 microglobulin (b2m), a component of the entire MHC class I molecule. Class II deletion can be performed by deletion of CIITA.

[0302] However, the same TCR-MHC interactions that cause the problem of alloreactivity / rejection in an allogeneic context are also crucial for the normal T cell development process. The interaction between TCR and MHC molecules (provided by the thymic microenvironment and also by the developing T cell itself) on the surface of so-called double positive / DP T cell precursors (so named because they co-express both CD8 and CD4) 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 cell lineage cells (so named because they express either CD4 or CD8, but not both). Positive selection can be reproduced in vitro in the "artificial thymic organoid" (ATO) technology, previously developed by Crooks' research group, to induce differentiation of stem cells into SP4 and SP8 cells.

[0303] One solution to the problem of endogenous TCR expression machinery being removed before positive selection is to constitutively express exogenous (transgenic) TCR throughout T cell differentiation, including during beta selection and positive selection. However, persistent expression of exogenous TCR in mature T cells that occur during differentiation and are transferred to patients may create new problems, such as possible off-target effects of exogenous TCR due to either alloresponse / GvHD or antigen expression in healthy tissues. The present invention provides a method for resulting in TCR expression that is restricted to the precursor stage of beta selection and positive selection, i.e., lost in mature T cells. This application is of great importance in the field of PSC-based T cell therapy, where gene deletion and insertion are typically applied in self-renewing PSCs, but can also be used for any TCR expression manipulation during or before the positive selection stage.

[0304] The inventors have determined that RAG1 and RAG2 genes have tightly regulated stage-specific expression that is high during the beta-selection and positive selection stages of T cell differentiation, but is reduced in mature T cells. The approach is to insert a transgenic TCR into / downstream of the endogenous RAG1 or RAG2 gene regulatory region. In this way, any transgenic TCR can be used to direct positive selection in any T cell differentiation system, but is downregulated in the mature T cells that are subsequently produced, thus avoiding potential GvHD.

[0305] A biologically active transgene (i.e., reporter, chimeric antigen receptor) is inserted in-frame into the 3' untranslated region of the endogenous gene using CRISPR / Cas9-mediated homologous recombination repair. In this process, the stop codon is replaced with a serine-arginine linker and a self-cleaving peptide sequence (P2A). This approach allows expression regulation by the endogenous promoter of the inserted transgene, while the translation of the endogenous gene remains unaltered. Also, a puromycin resistance gene cassette can be inserted to aid in enrichment of edited pluripotent stem cells (Figure 1A). After CRE-TAT treatment at the iPSC stage, the puromycin resistance cassette can be removed, leaving only the loxp3 sequence (Figure 1B).

[0306] Figure 8A-B shows the effect of RAG1 / 2 KO human pluripotent stem cells (hPSCs) and their WT (unedited) parental cell lines (H1, HLA-A * 0201+;ESI017, HLA-A *Representative flow cytometry analysis of T cell differentiation kinetics of RAG1 / 2 DKO hPSCs (Figure 8A) and WT parental cell line derived T cell lineage-restricted precursors (CD45+CD5+CD7+) over a 7 week period. Figure 8B shows the progression of T cell development over a 7 week period from the same population of T cell lineage-restricted precursors as shown by CD8a and CD4 surface staining. Precursors 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-).

[0307] Figure 9 shows the effect of deleting endogenous TCR expression on positive selection during T cell differentiation from hPSCs. RAG1 / 2 KO hPSCs and the WT (unedited) parental cell line (H1, HLA-A * 0201+;ESI017, HLA-A * 0201-) is HLA-A * The hPSCs were stably transduced with a lentiviral vector expressing the 0201-restricted NYESO TCR (detectable using the VB13.1 antibody) (Figure 3). Figure 9A shows a representative flow cytometry analysis of VB13.1 and CD3 surface expression on all hematopoietic cells (CD45+) at 7 weeks of T cell differentiation. Figure 9B shows positive selection of SP8 T cells in NYESO TCR-transduced RAG1 / 2 DKO and parental WT hPSC lines. All hematopoietic cells expressing VB13.1 and CD3 (CD45+VB13.1+CD3+) are shown at 7 weeks of differentiation. Rescue of SP8 development when RAG1 / 2 is knocked out is due to HLA-A * 0201+ cell line, exclusive to H1.

[0308] FIG. 10 shows that expression levels of RAG1 and RAG2 genes are restricted at various in vitro ATO differentiation process time points, including positive selection in the DP population.

[0309] Example 4 - Regulation of gene expression during differentiation of mature T cells Chimeric antigen receptor (CAR) T-cell therapy has produced remarkable results in otherwise refractory hematopoietic malignancies 1~2 Currently, the process of manufacturing autologous CAR T cells is challenging due to the need for de novo generation of each individual treatment and the inherent variability in T cell biology between patients, making clinical responses unpredictable and inconsistent. 3~5 As a result, there has been growing interest in generating CAR T cells in vitro from an infinitely renewable, allogeneic source of human pluripotent stem cells (hPSCs). One advantage of this approach is that hPSCs are highly amenable to gene editing, providing multiple avenues to manipulate the function of the final T cell product.

[0310] However, there are two crucial biological barriers to generating CAR T cells from hPSCs.

[0311] (1) Expression of a CAR transgene early in T cell differentiation leads to a deviation of development toward the innate lymphoid pathway 6~10 This result was also reported by our research group using the artificial thymic organoid (ATO) system for differentiating PSCs into T cells, as constitutive expression of a second-generation CD19 CAR led to the production of type 2 innate lymphoid cell (ILC2) cells rather than typical single-positive CD8+ (SP8) and CD4 (SP4) T cells (Figure 2A-B). 11To address this early fate deviation, our approach uses CRISPR / Cas9 editing to achieve stage-specific delayed expression of the same CD19 CAR. The CAR transgene is inserted within the 3' untranslated region (UTR) of the Granzyme A (GZMA) gene, placing its expression under the control of the endogenous GZMA promoter and its regulatory elements. GZMA is expressed only late in differentiation in single positive CD8 (aka "SP8") (CD3+TCR+CD8+CD4-) and single positive CD4+ (aka "SP4") (CD3+TCR+CD8-CD4+) T cells and is absent in early T stages. Stage-specific expression was validated using an engineered GZMA-mCitrine reporter ATO experiment (Figure 3). As a result, differentiation of GZMA-CAR19LH T-iPSCs with the ATO system showed efficient generation of conventional mature SP8 and SP4 T cells, with robust CAR expression (Figure 4B) and no evidence of any fate deviation to innate cell lineages (Figure 4A). We identified other endogenous genes with similar expression patterns that could be used as alternatives to GZMA: these genes are shown in Table 2A.

[0312] (2) To address alloreactivity of T cells produced from allogeneic hPSC sources, endogenous T cell receptors (TCRs) must be knocked out to prevent TCR / MHC mismatches during adoptive transfer and the resulting risk of 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 maturing T cells are unable to undergo positive selection (Figure 11A-D). Our solution to restore positive selection relies on a similar 3'UTR knockout that transiently expresses an exogenous TCR (e.g., 1G4 TCR recognizing the NYESO antigen) in positively selected (i.e., double positive, a.k.a. DP, a.k.a. CD4+CD8+) cells. Based on our own RNA-Seq studies, we identified the RAG1 gene as a candidate endogenous gene with the correct expression pattern; RAG1 is expressed in double negative (DN) to double positive (DP) T cells, but is absent in SP8 and SP4 T cells after undergoing positive selection. We used the same stage-specific approach to generate reporter cell lines and confirmed that RAG1-regulated reporter expression follows the RNA-Seq pattern during differentiation (Figure 12A-C). Moreover, the reporter remained undetectable in mature T cells even after activation (Figure 12C). We then used the same strategy to drive 1G4 TCR expression from the RAG1 locus and showed that we could restore positive selection in cells generated from TRAC-disrupted T-iPSC lines. Thus, expression of the transgenic TCR only occurred during the crucial positive selection period (DP stage) and was absent after SP8 / SP4 maturation (Figure 13A-C), ultimately leading to the generation of TCRαβ-CD3- naive mature SP8 T cells (Figure 6D). We identified other endogenous genes with similar expression patterns that could be used as alternatives to RAG1: these genes are shown in Table 2B.

[0313] We then combined two editing strategies in TRAC-disrupted PSC lines: (1) CD19 CAR expression with GZMA (GZMA-CAR19LH) to avoid the block and deviation to innate cell lineages that accompanies constitutive expression, and (2) transient expression of exogenous 1G4(NYESO) TCR with RAG1 (RAG1-NYESO TCR). The data show that we have developed a platform for generating non-allo-reactive TCRαβ-CD3-, naive CAR T cells (Figure 14A-B). The resulting T cells showed robust antigen-specific activation and cytokine production (Figure 14C), and this approach may be generalizable to various CAR designs (Figure 14D).

[0314] (Table 2A) TIFF2025507864000013.tif243133

[0315] (Table 2B) TIFF2025507864000014.tif24545

[0316] Table 2A-B: Expression profile of selected genes during T cell differentiation using the ATO system. (2A) Selected genes show low to absent expression in pluripotent stem cells at the DP T cell stage and high expression in mature SP8 and SP4 T cells. (2B) Selected genes show low to absent expression in pluripotent stem cells at the CD34+ cell stage and progressively higher expression from DN to DP T cell stages. Upon positive selection, expression levels return to low to absent levels in SP8 and SP4 T cells.

[0317] Expression profiles have been published for various cell sources (i.e., PSC-ATO, cord blood CD34+ ATO, thymus) 1 and unpublished bulk RNA sequencing data. All values ​​in the table are given in units of RPKM.

[0318] All of the methods disclosed and claimed herein can be made and executed in light of the present disclosure without undue experimentation. Although the compositions and methods of the present invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the art that variations may be applied to the methods described herein and in the steps or sequence of steps of the methods described herein without departing from the spirit, spirit and scope of the invention. More specifically, it will be apparent that certain agents that are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutions and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and spirit of the invention as defined by the appended claims.

[0319] The references cited herein, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference. TIFF2025507864000015.tif55160TIFF2025507864000016.tif215160

Claims

1. A nucleic acid comprising a promoter region and a transgene downstream of a transcription start site of the promoter region, the promoter region comprising a promoter region of a gene in Table 1.

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

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

4. The nucleic acid of claim 3, wherein the promoter region comprises the promoter of 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, comprising genomic DNA.

10. The nucleic acid of any one of claims 1 to 9, wherein the promoter region comprises a RAG1 or RAG2 promoter region.

11. The nucleic acid of claim 10, wherein the genomic DNA is defective with respect to genes encoding one or both of the functional RAG1 and / or RAG2 proteins.

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

13. The nucleic acid according to any one of claims 1 to 12, comprising a coding region of a gene in Table 1 downstream of the transcription initiation site of said promoter region.

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

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

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

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

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

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

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

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

22. 22. The nucleic acid of any one of claims 9 to 21, wherein the genomic DNA is defective with respect to genes 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.

23. 23. The nucleic acid of any one of claims 9 to 22, wherein the genomic DNA is defective with respect to genes encoding one or more of a functional CD3 gamma protein, a functional CD3 delta protein, a functional CD3 epsilon protein, or a functional CD3 zeta protein.

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

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

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

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

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

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

30. The cell of any one of claims 25 to 29, wherein the first or 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 and / or second transgene comprises FOXP3.

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

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

35. The cell of any one of claims 25 to 34, which is defective with respect to genes encoding one or both of functional RAG1 and / or RAG2 proteins.

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

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 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 promoter region of GZMA, GZMB, GZMH, GZMK, GZMM, TNFSF14, XCL2, PTGDS, SAMD3, NCALD, or XCL1, and the second nucleic acid comprises a gene of GZMA, GZMB, GZMH, GZMK, GZMM, TNFSF14, XCL2, PTGDS, SAMD3, NCALD, or XCL1 downstream of a transcription start site of the promoter region of GZMA, GZMB, GZMH, GZMK, GZMM, TNFSF14, XCL2, PTGDS, SAMD3, NCALD, or XCL1, respectively.

39. The cell of claim 37, wherein the first introduced gene and the RAG1 or RAG2 gene are expressed in the same mRNA.

40. 39. The cell of claim 38, wherein the second introduced gene and the GZMA, GZMB, GZMH, GZMK, GZMM, TNFSF14, XCL2, PTGDS, SAMD3, NCALD, or XCL1 gene are expressed in the same mRNA.

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

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

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

44. 42. The cell of any one of claims 25 to 41, wherein the first or second nucleic acid further comprises a coding region of the gene and / or a 3'UTR region downstream of the first or 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, which is defective with respect to 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 is defective with respect to genes encoding one or more of a functional CD3 gamma protein, a functional CD3 delta protein, a functional CD3 epsilon protein, or a functional CD3 zeta protein.

48. 48. The cell of any one of claims 24 to 47, comprising 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, comprising a pluripotent stem cell.

50. 49. The cell of claim 48, comprising an embryonic stem cell or an induced pluripotent stem cell (iPSC).

51. 51. The cell of any one of claims 24 to 50, 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, comprising a T cell.

53. The cell of any one of claims 24 to 51, comprising 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 lymphoid cell, or an NK cell.

55. The cell of claim 53, comprising CD4+ CD8- or CD8ab+ CD4- T cells.

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

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

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

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. Culturing the cells in three-dimensional (3D) cell aggregates; the cell aggregate further comprises a selected population of stromal cells that express a Notch ligand; The 3D cell aggregates are cultured in serum-free medium containing insulin, biotin, transferrin and albumin for a period of time sufficient for in vitro differentiation of the stem or progenitor cells into T cells. The method of any one of claims 58 to 60.

62. The method of claim 61 , wherein said 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, further comprising centrifugation of said stem or progenitor cells and said stromal cells to form a 3D cell aggregate.

65. 65. The method of any one of claims 61 to 64, wherein the medium further comprises one or more of exogenously 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-alpha, TGF-beta, interferon-gamma, interferon-lambda, TSLP, thymopentin, pleotrophin, 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 an intact, partial or modified Notch ligand, wherein the Notch ligand is DLL4, DLL1, JAG1, JAG2, or a combination thereof.

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

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

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

70. 70. The method of claim 69, for treating cancer in a subject.

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

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