A novel method for producing large-scale CAR-T immune cells by lentiviral vector transduction

Abstract

The present disclosure provides novel and efficient methods and lentiviral vectors for producing populations of immune cells engineered to express nucleic acid sequences encoding chimeric antigen receptors (CARs), engineered T cell receptors (TCRs), and / or polypeptides that enhance immune cell function, or functional derivatives thereof, in less than 72 hours; engineered cells produced by the methods, compositions comprising the cells, and methods for treating diseases or conditions using the cells.

Description

[Technical Field]

[0001] This application claims priority from U.S. Provisional Application No. 63 / 414,829, filed October 10, 2022, the contents of which are incorporated herein by reference in their entirety for all purposes.

[0002] The present disclosure generally relates to methods for efficiently producing immune effector cells that express nucleic acid sequences encoding chimeric antigen receptors (CARs), and / or artificial T cell receptors (TCRs), and / or polypeptides that enhance immune cell function, or functional derivatives thereof. [Background technology]

[0003] Adoptive immunotherapy, in which ex vivo generated autoantigen-specific T cells are infused back into patients, has shown promise as a promising strategy for the treatment of cancer, infectious diseases, and autoimmune diseases. T cells used in adoptive immunotherapy are primary cells engineered to express chimeric antigen receptors (CARs) or recombinant T cell receptors (TCRs) and expanded ex vivo to redirect primary immune cells against diseased cells, such as cancer cells. CARs are synthetic antibody-like molecules consisting of a targeting moiety fused to one or more signaling domains, engineered to convey antigen specificity to T cells. CARs have been successfully used to redirect T cells against antigens expressed on the surface of tumor cells in a variety of malignancies, including lymphomas and solid tumors.

[0004] Currently, the production of genetically modified T cells is a complex process. There is a need for methods and processes to improve the production of CAR or TCR-expressing cell therapy products, enhance product quality, and maximize the therapeutic efficacy of CAR-T cell immunotherapy. The present invention provides methods and compositions that address these needs. Summary of the Invention

[0005] One aspect of the present disclosure provides a method for producing a population of engineered immune cells, the method comprising: (a) extracting a population of lymphocytes, a population of immune cells, or CD4 + and CD8 + (b) enriching the population of cells for a lymphocyte population, a population of immune cells, or CD4 + and CD8 + (c) combining the population of cells with one or more buffers; (d) determining whether the population of lymphocytes, the population of immune cells, or the population of CD4 + and CD8 + transfecting a population of cells with an effective amount of a modifying agent, thereby producing a population of modified lymphocytes, a population of modified immune cells, or a modified CD4 + and CD8 + In some embodiments, steps 1(a)-(c) are performed within 24 hours. In some embodiments, a population of immune cells or CD4 + and CD8 + Prior to enriching the population of cells, the blood is separated by apheresis into a plasma component, a mononuclear cell-containing layer, a platelet layer, and red blood cells to produce an apheresis product selected from red blood cell apheresis, thrombus apheresis, platelet apheresis, leukocyte apheresis, stem cell apheresis, plasma apheresis, and platelet apheresis. In some embodiments, the population of immune cells, or CD4 + cells and CD8 + The population of cells is enriched by apheresis, elutriation or gradient centrifugation.

[0006] Another aspect of the present disclosure provides a method for producing a population of engineered immune cells, the method comprising: (a) generating a population of lymphocytes, a population of immune cells, or a population of CD4 + cells and CD8 + (b) enriching the population of cells from the donor's white blood cells; (c) enriching the population of lymphocytes, the population of immune cells, or CD4 + cells and CD8 + (c) combining the population of cells with one or more buffers; (d) determining whether the population of lymphocytes, the population of immune cells, or the population of CD4+ and CD8 + transfecting a population of cells with an effective amount of a modifying agent, thereby producing a population of lymphocytes, a population of modified immune cells, or a modified CD4 + and CD8 + Generating a population of cells In some embodiments, steps 1(a)-(c) are performed within 24 hours.

[0007] Another aspect of the present disclosure provides a method of producing a population of engineered eukaryotic cells, the method comprising: (a) obtaining a population of eukaryotic donor cells from a subject; (b) combining the population of eukaryotic donor cells with one or more buffers; and (c) transfecting the population of eukaryotic donor cells with an effective amount of a modifying agent, thereby producing a population of modified eukaryotic donor cells. In some embodiments, steps 1(a)-(c) are performed on the same day.

[0008] In some embodiments of the methods described herein, prior to the transfection step (c), a population of immune cells, such as CD4 + cells and CD8 + The population of cells, or the population of eukaryotic donor cells, is stimulated and / or activated with one or more stimulatory agents.

[0009] In some embodiments of the methods described herein, the modifying agent is selected from the group consisting of a small molecule drug, a biologic drug, a therapeutic drug, a protein, a peptide, a protein therapeutic drug, a peptide therapeutic drug, a chimeric antigen receptor, a xenogeneic T cell receptor, a viral vector, a vector, a retroviral vector, a lentiviral vector, an adenoviral vector, and an adeno-associated viral vector.

[0010] In some embodiments of the methods described herein, the modifying agent is: (a) selected from a retroviral vector, a lentiviral vector, an adenoviral vector, or an adeno-associated viral vector; (b) a lentiviral vector; or (c) a retroviral vector.

[0011] In some embodiments of the methods described herein, a population of immune cells, CD4 + cells and CD8 + The population of cells, or a population of eukaryotic donor cells, is transfected with an effective amount of a lentiviral or retroviral vector.

[0012] In some embodiments of the methods described herein, the lentiviral or retroviral vector comprises a nucleic acid sequence encoding a chimeric antigen receptor (CAR), an engineered T cell receptor (TCR), and / or a polypeptide that enhances immune cell function, or a functional derivative thereof, or a nucleic acid sequence that produces a therapeutic protein.

[0013] In some embodiments of the methods described herein, the population of immune cells or the population of eukaryotic donor cells includes, but is not limited to, mononuclear cells, lymphocyte-rich cells, B lymphocytes, T lymphocytes, CD4 + T lymphocytes, CD8 + T lymphocytes, dendritic cells, monocytes, natural killer (NK) cells, natural killer T (NKT) cells, T regulatory cells, CD4 + T helper cells, CD8 + Cytotoxic T lymphocytes (CTLs), CD62L + cells, CD27 + cells, CCR7 + cells, CD45 RO - cells, CD45RA + Cells, neutrophils, basophils, eosinophils, megakaryocytes, stem cells, hematopoietic stem cells (HSCs), hematopoietic progenitor cells (HPCs), CD34 + cells, CD34 + The cells are selected from the group consisting of peripheral blood stem cells, lymphocyte-activated killer cells (LAK), tumor-infiltrating lymphocytes (TIL), mesenchymal stem cells, mast cells, monocytes, macrophages, neutrophils, basophils, eosinophils, dendritic cells, megakaryocytes, and combinations thereof.

[0014] In some embodiments of the methods described herein, a population of immune cells, CD4 + cells and CD8+ The concentration of the population of cells, or the population of eukaryotic donor cells, is (a) at least about 0.7 × 10 7 , at least about 0.8 × 10 7 , at least about 0.9 × 10 7 , at least about 1 x 10 7 , at least about 2 × 10 7 , at least about 4 × 10 7 , at least about 6 × 10 7 , at least about 8 × 10 7 , at least about 1 x 10 8 , or at least about 5 × 10 8 pieces / mL, (b) approx. 0.5×10 6 pieces / mL ~ approx. 4×10 6 pieces / mL, (c) approx. 0.5×10 6 pieces / mL ~ approx. 1×10 8 cells / mL, or (d) approximately 4.0 × 10 6 pieces / mL ~ approx. 1×10 8 pieces / mL.

[0015] In some embodiments of the methods described herein, the transfection is selected from the group consisting of: (a) viral transfection, transduction, non-viral transfection, and hybrids of viral and non-viral transfection; (b) electroporation, laser beam, gene injection, sonoporation, magentafection, metal-coated nanoparticles, magnetically coupled adeno-associated virus, micro / nanoparticle-mediated transfection, lipofection, lipid-based transfection, anionic liposomes, cationic liposome-mediated transfection. (c) electroporation of viral particles; (d) electroporation and viral transfection (transduction); (e) viral transfection and lipid-based transfection; or (f) viral transfection and liposome-based transfection.

[0016] In some embodiments of the methods described herein, (a) a population of modified immune cells, a modified CD4 + cells and CD8 + (b) the population of cells, or the population of modified eukaryotic donor cells, is not activated with one or more stimulatory agents after or before transfection; and (c) the population of modified immune cells, the modified CD4 + cells and CD8 + The population of cells, or the population of modified eukaryotic donor cells, is not expanded in vitro after transfection.

[0017] In some embodiments, the methods described herein provide a population of modified immune cells, such as modified CD4 + cells and CD8 +The population of cells, or the population of modified eukaryotic donor cells, is stimulated and activated with one or more stimulatory agents to produce a population of activated modified immune cells, such as activated modified CD4 + cells and CD8 + and producing a population of cells, or a population of activated modified eukaryotic cells.

[0018] In some embodiments, the methods described herein involve generating a population of activated lymphocytes, a population of activated modified immune cells, a population of activated modified monocytes, a population of activated modified CD4 + and CD8 + The population of cells, or the population of activated modified eukaryotic donor cells, is expanded for a predetermined period of time to produce a population of modified lymphocytes, a population of modified immune cells, a modified CD4 + and CD8 + The method further includes producing a population of cells, or a population of modified eukaryotic donor cells. In some embodiments, the expanding step is carried out (a) under shaking or rotating conditions; (b) in a closed system; (c) using serum-free culture medium; and / or (d) in the presence of one or more stimulatory agents.

[0019] In some embodiments, the population of activated modified immune cells, activated modified CD4 + cells and CD8 + The population of cells, or the population of activated modified eukaryotic donor cells, is expanded at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, or at least about 25-fold.

[0020] In some embodiments, the methods described herein provide for cryopreservation or administration of a population of modified lymphocytes, a population of modified immune cells, a modified CD4 + and CD8 + In some embodiments, the harvesting further comprises harvesting the population of cells, or the population of modified eukaryotic donor cells. ... lymphocytes, modified immune cells, modified CD4 + and CD8 +In some embodiments, the harvesting includes selecting and enriching the cells, or modified donor eukaryotic cells, for cryopreservation or administration to a subject in need thereof, such as artificial lymphocytes, artificial immune cells, artificial CD4 + and CD8 + The method further includes formulating the cells, or artificial donor eukaryotic cells.

[0021] In some embodiments, the modified activated cell populations described herein (lymphocytes, immune cells, monocytes, CD4 + cells and CD8 + The predetermined time for growing the cells (or eukaryotic donor cells) is as follows: (a) less than about 24 hours; less than about 30 hours; less than about 48 hours; less than about 72 hours; less than about 96 hours; or less than about 120 hours; (b) less than about 0.5 hours, less than about 1 hour, less than about 2 hours, less than about 3 hours, less than about 4 hours, less than about 5 hours, less than about 6 hours, less than about 7 hours, less than about 8 hours, less than about 9 hours, less than about 10 hours, less than about 11 hours, or less than about 12 hours. or (c) about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, or about 23 hours.

[0022] In some embodiments of the methods described herein, the population of lymphocytes, the population of immune cells, CD4 + cells and CD8 + Enrich and / or obtain a population of cells, or a population of eukaryotic donor cells, and then generate engineered immune cells, engineered CD4 + cells and CD8 +The time to harvest the cells, or engineered eukaryotic donor cells, is as follows: (a) about 72 hours or less, (b) about 18 hours to about 72 hours, about 18 hours to about 36 hours, about 18 hours to about 24 hours, about 24 hours to about 72 hours, about 24 hours to about 36 hours, or about 36 hours to about 72 hours; (c) less than about 2 hours, less than about 3 hours, less than about 4 hours, less than about 5 hours, less than about 6 hours, less than about 7 hours, less than about 8 hours, less than about 9 hours, less than about 10 hours, or less than about 11 hours. (d) about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, or about 23 hours; (d) about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, or more days; or (e) about 1 day, about 3 days, about 4 days, about 5 days, or about 6 days.

[0023] In some embodiments of the methods described herein, the electroporation, activation, and / or expansion steps are performed in a closed system, a semi-closed system, and / or a functionally closed system. In some embodiments, the closed system is selected from the group consisting of a closed bag system, an automated closed cell sample processing system, and a bioreactor. In some embodiments, (a) the one or more stimulatory agents are selected from the group consisting of an agonistic antibody, a cytokine, a recombinant costimulatory molecule, an anti-CD3 antibody or fragment thereof, an anti-CD28 antibody or fragment thereof, a small drug inhibitor, and / or a combination thereof; (b) the one or more stimulatory agents are selected from the group consisting of interleukin-2 (IL-2), interleukin-3 (IL-3), interleukin-6 (IL-6), interleukin-7 (IL-7), interleukin-7 receptor (IL-7R), or a combination thereof. , interleukin-11 (IL-11), interleukin-12 (IL-12), interleukin-15 (IL-15), interleukin-15 receptor (IL-15R), interleukin-18 (IL-18), interleukin-18 receptor (IL-18R), interleukin-21 (IL-21), granulocyte-macrophage colony-stimulating factor, alpha, beta, or gamma interferon, erythropoietin, and combinations thereof. In some embodiments, the one or more stimulating agents are bound to beads or nanostructures.

[0024] In some embodiments of the methods described herein, (a) the one or more stimulatory agents are an anti-CD3 antibody and an anti-CD28 antibody or fragments thereof; (b) the one or more stimulatory agents are an anti-CD3 antibody and an anti-CD28 antibody or fragments thereof and one or more cytokines; (b) the nanostructure is a nanomatrix; (c) the cytokine is selected from IL-2, IL-7, IL-6, IL-15, IL-15Ra, or IL-21; (d) the cytokine is selected from IL-15 and IL-7; IL-7 and IL-21; IL-7 and IL-2; IL-15 and IL-2; IL-7, IL-15 and IL-21; IL-15 and IL-15Ra; or IL-7, IL-15 and IL-15Ra; and / or (e) the one or more stimulatory agents are a nanomatrix and one or more cytokines. In some embodiments, the nanomatrix (a) comprises a matrix of flexible polymer chains and an anti-CD3 antibody and an anti-CD28 antibody or fragment thereof; or (c) is between 1 and 500 nm in size.

[0025] In some embodiments of the methods described herein, the effective amount of retroviral or lentiviral vector comprises a multiplicity of infection (MOI) of about 0.01, about 0.02, about 0.03, about 0.04, about 0.05, about 0.06, about 0.07, about 0.08, about 0.09, about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1.0, about 1.25, about 1.5, about 2.0, about 3.0, about 4.0, or about 5.0.

[0026] In some embodiments, the effective amount of retroviral or lentiviral vector comprises: (a) about 2 ul of lentiviral vector at an MOI of about 0.08; (b) about 5 ul of lentiviral vector at an MOI of about 0.2; or (c) about 10 ul of lentiviral vector at an MOI of about 0.4.

[0027] In some embodiments of the methods described herein, the lentiviral vector is based on a virus selected from the group consisting of a retrovirus, an alpharetrovirus, a betaretrovirus, a gammaretrovirus, a deltaretrovirus, and an epsilonretrovirus. In some embodiments, the lentiviral vector is based on a virus selected from the group consisting of human immunodeficiency virus (HIV), equine infectious anemia virus (EIAV), vizma-ezivirus (VMV), caprine arthritis-encephalitis virus (CAEV), feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), VISNA virus, and simian immunodeficiency virus (SIV).

[0028] In some embodiments, the lentiviral vector is pseudotyped with an envelope glycoprotein (Env) from a virus selected from the group consisting of murine leukemia virus (MLV), vesicular stomatitis virus (VSV) Indiana strain, VSV New Jersey strain, Cocalvirus, Chandipura virus, Pirie virus, spring of carp virus (SVCV), sigma virus, infectious hematopoietic necrosis virus (IHNV), Mokola virus, rabies virus, CVS virus, Isfahan virus, Alagoas virus, Calchaqui virus, Jurona virus, La Joya virus, Maraba virus, feline endogenous retrovirus (RD114) envelope protein, Perinet virus, Yugbugdanova virus, prototype foamy virus (PFV), and gibbon ape leukemia virus (GaLV).

[0029] In some embodiments, the lentiviral vector is pseudotyped with an envelope glycoprotein (Env) selected from the group consisting of vesicular stomatitis virus (VSV) Indiana strain, VSV New Jersey strain, and Cocal virus.

[0030] In some embodiments, the lentiviral vector comprises a heterologous viral envelope protein (Env) selected from the group consisting of Indiana strain VSV-G, New Jersey strain VSV-G, Kokar virus envelope protein, Isfahan virus envelope protein, Chandipura virus envelope protein, Pilibiri virus envelope protein, murine leukemia virus (MLV) envelope glycoprotein, SVCV virus envelope protein, and mutants thereof. In some embodiments, the lentiviral vector comprises a nucleotide sequence encoding a VSV-G envelope protein or a VSV G protein mutant. In some embodiments of the methods described herein, the lentiviral vector is a lentiviral particle.

[0031] In some embodiments of the methods described herein, the CAR comprises an antigen-binding domain, a transmembrane domain, a costimulatory domain, and an intracellular domain, and the antigen-binding domain is selected from the group consisting of: (a) a full-length antibody or an antigen-binding fragment thereof, (b) a Fab, (c) a single-chain variable fragment (scFv), and (d) a single-domain antibody.

[0032] In some embodiments, the antigen binding domain is selected from the group consisting of CD4, CD5, CD19, CD20, CD22, CD79b, CD79a, CD33, CD30, CD70, BCMA, GPC2, CD123, CD133, EGFR, EGFRvIII, mesothelin, HER2, PSMA, PSCA.FAP, CEA, GD2, IL-13Ra2, glypican-3, CIAX, LI-CAM, CA 125, CTAG1B, TnMUC1, mucin 1, folate receptor alpha (FRa), GFRα-4, NYESO, WT1, (AFP) / HLA-A2, AXL, B7-H3, CA-IX, CD3, CD7, CD8, CD38, CD44v6, CD80, CD86, CD117, CD147, CD276, CEA, claudin 18.2, c-Met, DLL3, DR5, EpCAM, EphA2, FAP, folate-binding protein (FBP), glycolipid F77, glypican-3 (GPC3), glypican-2, HLA-A2, ICAMI, IL3Ra, LAGE-I, Lewis Y, LMPI (EBV), MAGE-AI, MAGE-A3, MAGE-A4, Melan A, MG7 (glycated CEA), MMP, MUCI, Nectin4 / FAP, NKG2D-ligand, MIC-A, MIC-B, ULBPs I to 6, NY-ESO-1, P16, PD-L1, ROR1, ROR2, TIM-3, TM4SF1, VEGFR2, and combinations thereof.

[0033] In some embodiments, the CAR transmembrane domain is selected from the group consisting of an artificial hydrophobic sequence, a transmembrane domain of a type I transmembrane protein, the α, β, or ζ chain of the T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, OX40 (CD134), 4-1BB (CD137), ICOS (CD278), or CD154, and a transmembrane domain derived from a killer immunoglobulin-like receptor (KIR).

[0034] In some embodiments, the costimulatory domain is the intracellular domain of a protein selected from the group consisting of TNFR superfamily proteins, CD27, CD28, 4-1BB (CD137), OX40 (CD134), PD-1, CD7, LIGHT, CD83L, DAP10, DAP12, CD27, CD2, CD5, ICAM-1, LFA-1, Lck, TNFR-I, TNFR-II, Fas, CD30, CD40, ICOS (CD278), NKG2C, B7-H3 (CD276), and killer immunoglobulin-like receptors (KIR).

[0035] In some embodiments, the intracellular signaling domain comprises an intracellular domain selected from the group consisting of the cytoplasmic signaling domain of human CD3 zeta chain (CD3ζ), FcγRIII, the cytoplasmic tail of FcsRI, an Fc receptor, a cytoplasmic receptor with an immunoreceptor tyrosine-based activation motif (ITAM), TCRζ, FcRγ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, and CD66d. In some embodiments of the methods described herein, the CAR further comprises a hinge region.

[0036] Another aspect of the present disclosure provides a method for delivering a nucleic acid encoding a chimeric antigen receptor (CAR), an engineered T cell receptor, or a therapeutic protein to a cell, the method comprising introducing into the cell a transfer plasmid comprising: (a) a polynucleotide sequence encoding at least one heterologous viral envelope protein engineered by the methods described herein; (b) a polynucleotide sequence encoding at least one retroviral rev protein; (c) a polynucleotide sequence encoding at least one retroviral gag protein and a retroviral pol protein; and / or (d) a polynucleotide sequence encoding a chimeric antigen receptor, an engineered T cell receptor (TCR), or a therapeutic protein. In some embodiments, at least a portion of one or more regions of the retroviral genome essential for replication are mutated.

[0037] Another aspect of the present disclosure provides lentiviral vector particles produced by the methods described herein.

[0038] Another aspect of the present disclosure provides a method for introducing a variant into a cell, the method comprising electroporating the cell with an effective amount of lentiviral vector particles described herein or produced by the methods described herein, thereby producing a variant cell. In some embodiments, the cell is contacted with the effective amount of lentiviral vector before electroporation. In some embodiments, the cell is contacted with the effective amount of lentiviral vector up to about 4 hours after electroporation. In some embodiments, the cells are contacted with an effective dose of the lentiviral vector for: (a) at least about 5-30 minutes, at least about 25-50 minutes; at least about 5-60 minutes, at least about 5-12 minutes, at least about 60-120 minutes, at least about 120-240 minutes after electroporation; or (b) at least about 1 minute, at least about 2 minutes, at least about 5 minutes, at least about 10 minutes, at least about 20 minutes, at least about 30 minutes, at least about 45 minutes, at least about 60 minutes, at least about 75 minutes, at least about 90 minutes, at least about 100 minutes, at least about 110 minutes, at least about 120 minutes, at least about 150 minutes, at least about 160 minutes, at least about 170 minutes, at least about 180 minutes, at least about 190 minutes, at least about 200 minutes, at least about 220 minutes, or at least about 240 minutes after electroporation.

[0039] In some embodiments, the cells are immune cells, eukaryotic donor cells, monocytes, enriched lymphocytes, B lymphocytes, T lymphocytes, CD4 + T lymphocytes, CD8 + T lymphocytes, dendritic cells, monocytes, natural killer (NK) cells, natural killer T (NKT) cells, T regulatory cells, CD4 + T helper cells, CD8 + Cytotoxic T lymphocytes (CTL), CD62L + cells, CD 27 +cells, CCR 7 + cells, CD45RO - cells, CD45 RA + cells, neutrophils, basophils, eosinophils, megakaryocytes, stem cells, hematopoietic stem cells (HSCs), hematopoietic progenitor cells (HPCs), CD34 + cells, CD34 + The cells are selected from the group consisting of peripheral blood stem cells, lymphocyte-activated killer cells (LAKs), tumor-infiltrating lymphocytes (TIL), circulating tumor-specific T cells, mesenchymal stem cells, mast cells, monocytes, macrophages, neutrophils, basophils, eosinophils, dendritic cells, megakaryocytes, and combinations thereof.

[0040] In some embodiments, the cells are selected from the group consisting of (a) T cells, B cells, natural killer (NK) cells, CD8 + T cells, CD4 + (b) lymphocytes selected from the group consisting of T cells, cytotoxic T lymphocytes, regulatory T cells, and any combination thereof; (b) myeloid cells selected from the group consisting of monocytes, macrophages, neutrophils, basophils, eosinophils, dendritic cells, megakaryocytes, and any combination thereof; (c) stem cells, hematopoietic stem cells, hematopoietic progenitor cells, CD34 + cells, or CD34 + Peripheral blood stem cells;

[0041] In some embodiments, an effective amount of lentiviral vector particles comprises about 0.5ul, about 1ul, about 1.5ul, about 2ul, about 2.5ul, about 3ul, about 3.5ul, about 4ul, about 5ul, about 6ul, about 7ul, about 8ul, about 9ul, about 10ul, about 15ul, or about 20ul of lentiviral vector. In some embodiments, an effective amount of lentiviral vector particles comprises a multiplicity of infection of about 0.01, about 0.02, about 0.03, about 0.04, about 0.05, about 0.06, about 0.07, about 0.08, about 0.09, about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1.0, about 1.25, about 1.5, about 2.0, about 3.0, about 4.0, or about 5.0.

[0042] In some embodiments, an effective amount of lentiviral vector particles comprises: (a) about 2 ul of lentiviral vector particles at an MOI of about 0.08; (b) about 5 ul of lentiviral vector particles at an MOI of about 0.2; or (c) about 10 ul of lentiviral vector particles at an MOI of about 0.4.

[0043] One aspect of the present disclosure is directed to modified cells, modified immune cells, modified CD4 + and CD8 + A cell, or modified eukaryotic donor cell, is provided.

[0044] One aspect of the present disclosure is a population of modified cells, a population of modified immune cells, a modified CD4 + cells and CD8 + A population of cells, or a population of modified eukaryotic donor cells, is provided.

[0045] One aspect of the present disclosure is directed to modified cells, modified immune cells, modified CD4 + and CD8 + A cell, or modified eukaryotic donor cell, is provided.

[0046] One aspect of the present disclosure is a method for producing a population of modified cells, a population of modified immune cells, a modified CD4 + cells and CD8 + A population of cells, or a population of modified eukaryotic donor cells, is provided.

[0047] In some embodiments, the modified CD4 + cells and CD8 +The cells, or modified eukaryotic donor cells described herein, are engineered or generated by the methods described herein for use in producing a protein of interest. In some embodiments, the protein of interest is selected from the group consisting of an industrial protein or a therapeutic protein. In some embodiments, the protein of interest is selected from the group consisting of an enzyme, a regulatory protein, a receptor, a peptide, a peptide hormone, a cytokine, a membrane protein or transport protein, a vaccine antigen, an antigen-binding protein, an immunostimulatory protein, an allergen, a full-length antibody or an antibody fragment or derivative; a single-chain antibody (scFv), a Fab fragment, an Fv fragment, a single-domain antibody (VH or VL fragment), a domain antibody, a camelid single-domain antibody (VHH), a nanobody, and combinations thereof.

[0048] One aspect of the present disclosure provides a composition comprising: (a) a modified cell, modified immune cell, modified CD4 + and CD8 + (b) a population of modified cells, a population of modified immune cells, a modified CD4 + and CD8 + (c) a population of cells, or a population of modified eukaryotic donor cells engineered by the methods described herein; or (c) a lentiviral vector described herein. In some embodiments, the composition further comprises a pharmaceutically acceptable excipient.

[0049] One aspect of the present disclosure provides a method of treating a disease or condition in a subject, the method comprising administering to a subject in need thereof: (a) a modified cell, modified immune cell, modified CD4 + and CD8 + (b) a population of modified cells, a population of modified immune cells, a modified CD4 + and CD8+ or (c) a composition described herein, thereby treating the disease or condition in the subject.

[0050] In some embodiments, the disease or condition is selected from the group consisting of a viral infection, a bacterial infection, a parasitic infection, cancer, a malignancy, a non-cancerous condition, an autoimmune disease, a fibrotic disease, Alzheimer's disease, a protein deficiency state, and a factor VIII deficiency.

[0051] In some embodiments, the cancer is selected from the group consisting of breast cancer, triple-negative breast cancer, prostate cancer, ovarian cancer, glioma, glioblastoma, renal cell carcinoma, kidney cancer, mesothelioma, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, lung cancer, lung adenocarcinoma, gallbladder cancer, colon cancer, cervical squamous cell carcinoma, non-small cell lung cancer, small cell lung cancer, Merkel cell carcinoma, hepatocellular carcinoma, esophageal cancer, brain tumor, melanoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, urothelial cancer, gastric cancer, blood cancer lymphoma, leukemia, multiple myeloma, diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, acute myeloid leukemia, B-cell acute lymphoblastic leukemia (ALL), pre-B ALL, and any combination thereof. In some embodiments, the modified immune cells, modified CD4 + and CD8 + The cells, or modified eukaryotic donor cells, are: (a) autologous to the subject; (b) allogeneic to the subject; or (c) xenogeneic to the subject. In some embodiments, the modified cells, modified immune cells, modified CD4 + and CD8 + The cells, or modified eukaryotic donor cells, are allogeneic to the subject. In some embodiments, the subject is a human.

[0052] One aspect of the present disclosure provides a method for producing a therapeutic protein, comprising: (a) producing a population of artificial immune cells or a population of artificial eukaryotic cells comprising the therapeutic protein using a method described herein; (b) harvesting the therapeutic protein; and (c) isolating and purifying the therapeutic protein.

[0053] In some embodiments, the therapeutic protein is selected from the group consisting of an enzyme, a regulatory protein, a receptor, a peptide, a peptide hormone, a cytokine, a membrane protein or a transport protein, a vaccine antigen, an antigen-binding protein, an immunostimulatory protein, an allergen, a full-length antibody or an antibody fragment or derivative; a single chain antibody (scFv), a Fab fragment, an Fv fragment, a single domain antibody (VH or VL fragment), a domain antibody, a camelid single domain antibody (VHH), a nanobody, and combinations thereof.

[0054] One aspect of the present disclosure is a method for producing a modified immune cell population, or a modified CD4 + cells and CD8 + A kit comprising (a) a population of cells, or a population engineered by the methods described herein; or (b) a lentiviral vector described herein.

[0055] Both the foregoing summary and the following drawings and detailed description are exemplary and explanatory. They are intended to provide further details of the present disclosure and are not to be construed as limiting. Other objects, advantages, and novel features will be readily apparent to those skilled in the art from the following detailed description of the present disclosure. [Brief explanation of the drawings]

[0056] [Figure 1]Figure 1A is a schematic diagram showing a rapid T cell engineering platform (1A) using integrated lentiviral vector (LVV) transfection on day 0. This platform 1A includes donor leukocyte collection and processing, CD4+ and CD4+ cell selection, lentiviral vector transduction in a closed system on day 0, subsequent cell activation, and ex vivo culture and expansion for at least about 1 hour and up to about 72 hours prior to collection (e.g., culture and expansion from day 0 to day 3). The collected engineered T cells are either cryopreserved or administered to subjects in need.

[0057] [Figure 2] Figure 1B is a schematic diagram showing the rapid T cell engineering platform using integrated lentiviral vector (LVV) transfection at day 0 of Figure 1. The process begins with the donor's whole blood rather than donor leukocyte harvest.

[0058] [Figure 3] Figure 1C is a schematic diagram showing a rapid immune cell engineering platform using integrated lentiviral vector (LVV) transfection on day 0. This platform 1C involves obtaining and processing donor white blood cells, selecting immune cells (white blood cells and / or other immune cells), transfecting them with lentiviral vectors in a closed system on day 0, followed by cell activation, and culturing and expanding them in vitro for at least about 1 hour to about 72 hours before harvest (e.g., culturing and expanding from day 0 to day 3). The harvested engineered immune cells are either cryopreserved or administered to a subject in need.

[0059] [Figure 4] Figure 3. Schematic showing the rapid immune cell engineering platform (1D) using integrated lentiviral vector (LVV) transfection at day 0. In this case, the process starts with donor whole blood rather than donor leukocyte harvest.

[0060] [Figure 5]Schematic diagram showing a rapid culture-free T cell engineering platform (2A) using integrated lentiviral vector (LVV) transfection on day 0. Platform 2A includes donor leukocyte collection and processing, CD4+ and CD8+ selection, and lentiviral vector transfection in a closed system on day 0, followed by cell harvest without post-transfection culture or expansion. The harvested engineered T cells are cryopreserved or administered to subjects in need.

[0061] [Figure 6] Figure 5 is a schematic diagram illustrating a rapid culture-free T cell engineering platform (2B) using integrated lentiviral vector (LVV) transfection at day 0 in Figure 5. This process is initiated from donor whole blood rather than donor leukocyte harvest.

[0062] [Figure 7] This is a schematic diagram showing a rapid, culture-free immune cell engineering platform (3A) using integrated lentiviral vector (LVV) transfection on day 0. This platform 3A includes donor leukocyte collection and processing, immune cell (leukocyte and / or other immune cell) selection, lentiviral vector transduction in a closed system on day 0, and subsequent cell harvest. The harvested engineered cells are then cryopreserved or administered to subjects in need.

[0063] [Figure 8] Figure 5 shows a schematic diagram illustrating a rapid, non-ex vivo cultured immune cell engineering platform (3B) using integrated lentiviral vector (LVV) transfection at day 0. This process starts with donor whole blood rather than donor leukocyte harvest.

[0064] [Figure 9]Figure 4A shows a schematic diagram of a rapid eukaryotic cell processing platform using integrative lentiviral vector (LVV) transfection on day 0. This platform 4A involves obtaining and processing donor eukaryotic cells (e.g., mammalian cells, human cells), selecting a specific cell type (epithelial cells, mesenchymal cells, fibroblasts, neural cells, or stem cells), and transfecting with a lentiviral vector in a closed system on day 0, followed by cell activation and ex vivo culture and expansion for at least about 1 hour up to about 72 hours before harvest (e.g., culture and expansion on day 0-3). Harvested engineered eukaryotic cells can be cryopreserved or used immediately.

[0065] [Figure 10] Figure 4B is a schematic diagram showing a rapid, ex vivo culture-free eukaryotic cell engineering platform using integrated lentiviral vector (LVV) transfection on day 0. This platform 4B involves obtaining donor eukaryotic cells, processing them, selecting specific cell types (epithelial cells, mesenchymal cells, fibroblasts, neural cells, or stem cells), transfecting them with lentiviral vectors in a closed system on day 0, and subsequently harvesting the cells. The harvested eukaryotic cells can be cryopreserved or used immediately.

[0066] [Figure 11] 1 is a bar graph summarizing the results of a CD19CAR-T cell-Nalm6 coculture assay, demonstrating that CD19 CAR-T cells produced by the novel manufacturing process disclosed herein (Electric CAR-T cells) using lentiviral transfection exhibited high cytotoxicity against target tumor cells, as demonstrated by Nalm6 killing in vitro. Representative mean values ​​from 2-3 independent experiments (n=2-3) are shown. Mean ± SEM ***p<0.005. BEST MODE FOR CARRYING OUT THE INVENTION

[0067] I. Overview A. Regular manufacturing process Adoptive transfer therapy using T cells, particularly those transfected with chimeric antigen receptors (CARs), has shown promise in several hematological cancer clinical trials. Despite this success, the production of genetically modified T cells remains a complex, costly, and lengthy process. A typical CAR-T cell manufacturing process begins with enrichment of T cells from a fresh or cryopreserved white blood cell sample. Enrichment typically involves positive or negative selection. The enriched T cells are then activated using anti-CD3 / anti-CD28 antibody-coated beads (e.g., Dynabeads®), anti-CD3 / anti-CD28 antibody-coated polymers, nanoparticles, nanocolloids, and / or coactivators selected from the group consisting of ICOS, CD27, HVEM, LIGHT, CD40, 4-1BB, OX40, DR3, GITR, CD30, TIM1, CD2, or CD226-stimulating agents. Once activated, T cells are transfected with a nucleic acid molecule encoding a CAR molecule or an exogenous TCR either immediately after the activation process or up to 18 hours later. Typically, T cells are transduced with a lentiviral vector containing a nucleic acid molecule encoding a CAR molecule or an exogenous TCR. Optionally, the cells are electroporated with in vitro transcribed RNA. The transfected cells are then cultured (i.e., expanded) in vitro for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more days. After the desired number of days of culture, the cells are harvested. Cell harvesting involves mechanically resuspending the engineered cells (e.g., T cells) by swirling or pipetting and removing the mock / activation reagents using an appropriate buffer. The cells are also washed to remove unwanted reagents and are either reconstituted in cryopreservation medium or immediately administered to the subject. The cells are then cryopreserved until needed for administration.

[0068] The invention of the present disclosure is directed to a significant improvement over this routine manufacturing process, eliminating or shortening one or more manufacturing steps. It has surprisingly been discovered that this shortened, more efficient immune effector cell manufacturing process results in highly desirable and effective compositions. This shortened process relies on lentiviral transduction rather than traditional lentiviral transduction. B. Improved One-Day Manufacturing Process Using Lentiviral Transfection

[0069] Provided herein are novel and efficient methods for producing immune effector cells (e.g., T cells or NK cells) engineered to express a CAR or TCR, methods for treating a disease (e.g., cancer) in a subject using the engineered cells, and lentiviral vectors for use in the methods described herein. In some embodiments, the manufacturing process disclosed herein can produce immune effector cells engineered to express a CAR or TCR in less than about 24 hours (e.g., less than about 20 hours, less than about 15 hours, less than about 10 hours, less than about 5 hours, or less than about 3 hours, or any other time frame of less than about 24 hours described herein). In some embodiments, the manufacturing process disclosed herein can produce immune effector cells engineered to express a CAR or TCR in less than about 72 hours (e.g., less than about 24 hours, less than about 30 hours, less than about 40 hours, less than about 48 hours, less than about 60 hours, or less than about 72 hours, or any other time frame of less than about 24 hours described herein).

[0070] One aspect of the present disclosure provides a method for producing a population of engineered immune cells, the method comprising: (1) extracting a population of lymphocytes, a population of immune cells, or CD4 cells from donor whole blood or donor white blood cells (e.g., frozen or raw); + and CD8 + (2) enriching the population of cells, such as a population of lymphocytes, a population of immune cells, or a population of CD4 + and CD8 +(3) combining the population of cells with one or more buffers; and (4) determining whether the population of lymphocytes, the population of immune cells, or the population of CD4 + and CD8 + transfecting the population of cells with an effective amount of a modifying agent, thereby producing a population of modified lymphocytes, a population of modified immune cells, or a modified CD4 + and CD8 + Generating a population of cells In some embodiments, steps 1(a)-(c) are performed within about 24 hours.

[0071] Another aspect of the present disclosure provides a method for producing a population of engineered eukaryotic cells, the method comprising: (1) obtaining a population of eukaryotic donor cells; (2) combining the population of eukaryotic donor cells with one or more buffers; and (3) transfecting the population of eukaryotic donor cells with an effective amount of a modifying agent, thereby producing a population of modified eukaryotic donor cells.

[0072] In some embodiments, the population of modified lymphocytes, the population of modified immune cells, the modified CD4 + and CD8 + The population of cells, or the population of modified eukaryotic donor cells, is not activated or expanded with one or more stimulatory agents after transfection.

[0073] In some embodiments, the methods described herein include stimulating and / or activating with one or more stimulatory agents and generating a population of modified lymphocytes, a population of modified immune cells, a modified CD4 + and CD8 + The population of cells, or the population of modified eukaryotic donor cells, is expanded to produce a population of activated modified immune cells, such as activated modified CD4 + and CD8 + In one embodiment, the method described herein further comprises the step of generating a population of lymphocytes, a population of immune cells, CD4 + cells and CD8 +The method further comprises stimulating and / or activating the population of cells, or the population of eukaryotic donor cells, with one or more stimulatory agents.

[0074] The immunotherapy (e.g., adoptive cell transfer) methods for treating diseases or disorders described herein use a well-established and powerful system (apheresis and its products) to acutely, reliably, and efficiently produce clinical-grade CAR- or TCR-modified cells for immediate administration to subjects in situ. Immunotherapy using electrocompetent CAR-T cells produced by the methods disclosed herein can shorten the entire adoptive cell transfer process to approximately one day (e.g., approximately 24 hours), or approximately three days or less (e.g., approximately 72 hours or less). Thus, production time is reduced from approximately 12-15 days to approximately one day, approximately two days or less, or approximately three days or less. The manufacturing process disclosed herein is efficient because fewer cells are required to produce CAR-T cells. For example, while conventional manufacturing processes require up to approximately 300 million cells, the manufacturing process described herein works efficiently with approximately 3 million cells. This is because the cells produced by this method are significantly fresher and more intact, and therefore more potent (e.g., requiring less ex vivo / in vitro handling).

[0075] The manufacturing methods disclosed herein improve the production of CAR- or TCR-expressing cell therapy products, enhance the quality of the CAR-T cell product, and maximize the therapeutic efficacy of the CAR-T cell product in the following ways:

[0076] First, the manufacturing process disclosed herein shortens the turnaround manufacturing time to about 1 day (e.g., 24 hours) or less (or about 2 days or less, or about 3 days or less) compared to standard manufacturing processes (e.g., about 12 days). This short turnaround time allows for timely infusion of CAR-T cells (e.g., CD19, mesothelin, PSMA, TnMUC, BCMA, or GPC2 CAR-T cells) into patients. Furthermore, this manufacturing process preserves putative stem memory T (Tstem) cells, a cell subset associated with improved anti-tumor efficacy. The majority of unstimulated CAR-T cells generated by the manufacturing process disclosed herein maintained a less differentiated phenotype (e.g., greater than 50% of transfected CAR-T cells were naive CAR-T cells (CD45RO) compared to less than 10% of the CAR-T cell population in stimulated CAR-T cells). - CCR7 + )). A high population of naive electric CAR-T cells is a desirable improvement because these CAR-T cells preserve a non-activated (i.e., less differentiated) phenotype, which is known to be advantageous for the persistence and efficacy of CAR-T cells in cancer patients.

[0077] In some embodiments, CART cells produced by the methods disclosed herein can be administered to a subject with minimal in vitro expansion, e.g., less than about 1 day, less than about 12 hours, less than about 8 hours, less than about 6 hours, less than about 4 hours, less than about 3 hours, less than about 2 hours, less than about 1 hour, or no in vitro expansion. Optionally, in other aspects, production can be in less than about 5 days, less than about 4 days, less than about 3 days, or less than about 2 days. Thus, the methods described herein provide a rapid manufacturing process for producing improved CAR-expressing cell products for use in treating disease in a subject. C. Summary of Experimental Results

[0078] The shortened manufacturing process disclosed herein is made possible by a novel strategy of transducing immune cells with a lentiviral vector containing a nucleic acid encoding a CAR-TCR and / or a polypeptide that enhances immune cell function, or a functional derivative thereof.

[0079] First, in one embodiment, the manufacturing process relies on a hybrid transfection method that combines biological transfection (virus-based transfection or viral transduction) and physical transfection (electroporation). Specifically, lentiviral particles are electroporated into immune cells. Second, the lentiviral vector used to transfect cells is replication-incompetent. Electroporation of lentiviral particles into cells speeds up viral transfection / transduction, followed by standard cell culture processes or ultrafast processes without cell culture. Electroporating lentiviral vectors into cells also significantly reduces the amount of lentiviral vector used for optimal transfection using the process disclosed herein.

[0080] For example, the electroporation process can be performed with lentiviral nucleofection (Lentiviral Nucleofection®). Ultrafast electric CAR-T cells were produced using the lentiviral nucleofection workflow shown in Figures 7 and 8. The generation of electric CAR-T cells using this manufacturing process did not require cytokines or ex vivo culture steps. Furthermore, nucleofection of the CAR lentiviral vector resulted in efficient integration of the CAR transgene into the T cell genome. Further analysis showed that the vector copy number per cell of electric CAR-T cells was substantially similar to that of conventional CAR-T cells (e.g., approximately 1-1.5 copies / cell). Furthermore, mRNA encoding the CAR transgene (qRTPCR of the WPRE sequence) was expressed within approximately 1 hour after nucleofection. In general, electroporation of the lentiviral vector significantly improved the efficiency of CAR-T cell production using the process disclosed herein.

[0081] Third, this disclosure demonstrates for the first time that applying electricity to cells for up to 4 hours (e.g., up to 2 hours) before adding a lentiviral vector to the cells enhances CAR expression by approximately 10-15% compared to conventional electroporation processes (e.g., adding the expression vector to the cells before electroporation). Thus, lentiviral vectors are used as particle-shaped cargo carriers, and physical transfection, such as electroporation, is used to deliver the lentiviral particles into cells.

[0082] Tables 2 and 3 summarize the raw data from flow cytometry analysis of electroporated CD19 CAR-T cells produced by the disclosed methods, demonstrating that significant quantities of CAR-T cells were produced by electroporating lentiviral vectors into both unstimulated and stimulated primary human T cells at a very low multiplicity of infection (MOI). The transfection rates disclosed herein are significantly higher than those achieved by conventional methods (viral transduction alone), which yielded CAR expression in T cells of approximately 1 to 3%.

[0083] Figure 11 shows that stimulated or unstimulated lentiviral vector-transduced CD19 CAR-T cells (CART19 cells) efficiently killed target cells. Furthermore, lentiviral vector Nucleofection® significantly reduced the number of CAR T cells killed within the same period compared to lentiviral transduction. + Increased the proportion of T cells (e.g., CD19 CAR-T cells) by at least 10-fold (39.8% of CAR + T cells vs. 3.5% CAR + T cells).

[0084] Electric CAR-T cells generated by the process disclosed herein differ from conventional CAR-T cells at least in part because they were not activated ex vivo, whereas conventional CAR-T cells were generated using a 7-12 day process. Additionally, compared with Electric CAR-T cells stimulated with, for example, CD3 / CD28 Dynabeads®, the majority (more than 50%) of unstimulated Electric CAR-T cells maintained a non-activated or less differentiated phenotype (Tables 4 and 5). The high proportion of less differentiated CAR-T cells (e.g., naive T cell populations) is a highly desirable and unexpected improvement. Furthermore, Electric CAR-T cells exhibited a CD19 T cell phenotype within 48 hours of co-culture. +They were shown to be as effective as conventional CAR-T cells in killing Nalm6 cells (Table 6). Electrical CAR-T cells were effective in killing target cells even at effector:target ratios as low as 0.62:1 or less.

[0085] The novel manufacturing method described herein provides the most efficient CAR-T cell immunotherapy known to date for several reasons. The electric CAR-T cell manufacturing method shortens the entire CAR-T manufacturing process to one day, or at most three days, if expansion (e.g., culture) is desired. Thus, the manufacturing time for electric CAR-T cells is reduced from 12–15 days to approximately one day, or at most three days. Additionally, fewer cells are required. For example, while conventional manufacturing processes require up to approximately 300 million cells, the manufacturing process disclosed herein worked efficiently with approximately 3 million cells, because the cells are generally very fresh.

[0086] Finally, this manufacturing method is cost-effective because a batch of cells produced by conventional methods costs $1 million per batch, which is enough to treat approximately eight patients. However, the methods disclosed herein produce a batch of CAR-T cells sufficient to infuse approximately 20 patients. Thus, the methods disclosed herein double or triple the number of patients treated for the same cost, significantly reducing the CAR-T cell cost per patient.

[0087] Thus, the manufacturing processes described herein provide for the production of clinical-grade CAR- or TCR-modified cells effective for immediate administration in about 1 day or less (or in other embodiments about 3 days or less, or other time periods described herein), which is an improvement over manufacturing processes known in the art. II. Methods for generating modified T cells

[0088] The present disclosure provides a rapid and efficient manufacturing process for engineered cells (e.g., immune effector, electric CAR-T cells) containing a CAR, an exogenous TCR, and / or immune-enhancing factors that improve the compatibility of the engineered immune cells; compositions containing the engineered cells; and methods of using the engineered cells to treat diseases such as cancer in a subject. The rapid and efficient manufacturing method for engineered immune cells disclosed herein provides engineered CAR-T cells (i.e., electric) in less than 24 hours after transfection. The rapid turnaround is made possible by the combination of at least three factors: (1) the use of fresh apheresis product, (2) electroporation of a lentiviral vector at a very low MOI into purified apheresis product (e.g., purified T cells or purified immune cells), and / or (3) a freshly engineered lentiviral vector. CAR-T cells engineered by the methods disclosed herein are referred to as electric CAR-T cells. CARs engineered by the processes disclosed herein are referred to as electric CARs because electricity (e.g., electroporation) is used to drive a CAR-encoding vector (e.g., a lentiviral particle containing a nucleic acid sequence encoding a CAR) into cells. In particular, rather than passively introducing a lentiviral vector into cells, the lentiviral vector is actively introduced into cells by electroporation. A. New Electric Car Manufacturing Platform One aspect of the present disclosure provides a method for producing a population of engineered immune cells, the method comprising: (1) extracting a population of lymphocytes, a population of immune cells, or CD4 cells from blood obtained from a subject; + and CD8 + (2) enriching the population of cells; (3) enriching the population of lymphocytes, immune cells, or CD4 + and CD8 + (3) combining the population of cells with one or more buffers; and (4) determining whether the population of lymphocytes, the population of immune cells, or the population of CD4 + and CD8 +transfecting a population of cells with an effective amount of a modifying agent; thereby producing a population of modified lymphocytes, a population of modified immune cells, or a modified CD4 + and CD8 + Generate a population of cells.

[0089] In some embodiments, prior to the enrichment step (e.g., a population of lymphocytes, a population of immune cells, or CD4 + and CD8 + Prior to enriching or selecting a population of cells, the blood is separated by apheresis into a plasma component, a mononuclear cell-containing layer, a platelet layer, and red blood cells to produce an apheresis product selected from red blood cell apheresis, platelet apheresis, leukocyte apheresis, stem cell apheresis, plasma apheresis, and platelet apheresis. In some embodiments, the apheresis product (e.g., a population of lymphocytes, a population of immune cells, or CD4 + and CD8 + The resulting population of cells is enriched by apheresis, elution, or gradient centrifugation.

[0090] In some embodiments, the apheresis sample is used at the point-of-care site in the methods for producing CAR-T cells (i.e., Electric CARs) disclosed herein. Fresh apheresis samples are preferred because fewer immune cells are needed / required for optimal transfection using the methods disclosed herein. For example, conventional manufacturing processes require up to approximately 300 million cells, while the manufacturing process disclosed herein works efficiently with approximately 3 million cells. This difference is due to the freshness of the apheresis product.

[0091] In some embodiments, an apheresis sample (e.g., a leukapheresis sample) is collected from a subject and shipped to a cell manufacturing facility as a fresh product (e.g., an unfrozen product). The desired cells (e.g., immune cells, CD4 + T cells and / or CD8 + T cells) can be obtained using, for example, CliniMACS Enriched cells (e.g., immune cells, CD4 + T cells and / or CD8+ T cells) are seeded for CART production using the methods described herein.

[0092] In some embodiments, an apheresis sample (e.g., a leukapheresis sample) is collected from a subject and shipped to a cell manufacturing facility as a frozen sample (e.g., a cryopreserved sample). The frozen apheresis sample is then thawed and desired cells (e.g., immune cells, CD4 + T cells and / or CD8 + T cells) are selected. Enriched cells (e.g., immune cells, CD4 + T cells and / or CD8 + The enriched cells (e.g., CD4 T cells) are then seeded for CART production using the methods described herein. In some embodiments, at the end of the manufacturing process, the CART T cells are harvested, cryopreserved, and later thawed and administered to a subject. In some embodiments, enriched cells (e.g., CD4 T cells) are used to generate CART T cells. + T cells and / or CD8 + T cells) undergo one or more freeze-thaw cycles before being seeded for CAR-T manufacturing.

[0093] In some embodiments, an apheresis sample (e.g., a leukapheresis sample) is collected from a subject. + T cells and / or CD8 + T cells) can be obtained using, for example, CliniMACS Enriched cells (e.g., immune cells, CD4 + T cells and / or CD8 +The enriched cells (e.g., immune cells, CD4 T cells) are then shipped as frozen samples (e.g., cryopreserved samples) to a cell manufacturing facility. + T cells and / or CD8 + T cells) are then thawed and seeded for CART production using the methods described herein.

[0094] In some embodiments, after the cells (e.g., T cells) are seeded, one or more cytokines and one or more modifying agents (e.g., a vector encoding a CAR) are added to the cells. In such embodiments, the one or more cytokines may be selected from the group consisting of IL-2, IL-7, IL-15, hetIL-15 (IL15 / SIL-15Ra), IL-21, or IL-6 (e.g., IL-6 / SIL-6R). After incubation for at least about 5-72 hours, the cells are harvested, washed, and formulated for storage (e.g., cryopreservation) or administration.

[0095] One aspect of the present disclosure provides a method for producing a population of engineered immune cells, the method comprising: extracting a lymphocyte population, an immune cell population, or a CD4 + cells and CD8 + Enriching the cell population; lymphocyte population, immune cell population, or CD4 + cells and CD8 + combining the cell population with one or more buffers; determining whether the lymphocyte population, immune cell population, or CD4 + cells and CD8 + transfecting the cell population with an effective amount of a modifying agent; and detecting the transfected lymphocyte population, immune cell population, or CD4 + cells and CD8 + transfecting the cell population with an effective amount of a modifying agent; + and CD8 + Culturing and expanding the population of cells; and culturing the engineered lymphocytes, immune cells, or CD4 + and CD8 +harvesting the cells; thereby obtaining a population of modified lymphocytes, a population of modified immune cells, or a modified CD4 + and CD8 + In some embodiments, a population of immune cells, or CD4 + and CD8 + The population of cells is not stimulated and / or activated prior to transfection. In some embodiments, the transfected cells are cultured and expanded in the presence of one or more stimulatory agents described herein.

[0096] Another aspect of the present disclosure provides a method for producing a population of engineered immune cells, the method comprising: (1) generating a population of lymphocytes, a population of immune cells, or a population of CD4 + and CD8 + (2) enriching the population of cells from donor leukoagglutinin; (3) enriching the population of lymphocytes, immune cells, or CD4 + and CD8 + (3) combining the population of cells with one or more buffers; and (4) determining a population of lymphocytes, a population of immune cells, or a population of CD4 + and CD8 + transfecting a population of cells with an effective amount of a modifying agent, thereby producing a population of modified lymphocytes, a population of modified immune cells, or a modified CD4 + and CD8 + Generate a population of cells.

[0097] Another aspect of the present disclosure provides a method for producing a population of engineered immune cells, the method comprising: (1) generating a population of lymphocytes, a population of immune cells, or a population of CD4 + cells and CD8 + (2) enriching the population of cells from the donor's white blood cells; (3) enriching the population of lymphocytes, the population of immune cells, or CD4 + cells and CD8 + (3) combining the population of cells with one or more buffers; and (4) determining whether the population of lymphocytes, the population of immune cells, or the population of CD4 + cells and CD8 +(4) transfecting the population of cells with an effective amount of a modifying agent; and (5) detecting the transfected lymphocyte population, immune cell population, or CD4 + cells and CD8 + (5) culturing and expanding the cell population; and (6) culturing and expanding the engineered lymphocyte population, immune cell population, or CD4 + cells and CD8 + and harvesting the cells; thereby obtaining a modified lymphocyte population, a modified immune cell population, or a modified CD4 + cells and CD8 + In some embodiments, the transfected cells are cultured and expanded in the presence of one or more stimulatory agents described herein. In some embodiments, a population of lymphocytes, a population of immune cells, or a population of CD4 + and CD8 + The population of cells is not stimulated and / or activated prior to transfection.

[0098] In some embodiments, the apheresis product (e.g., a population of lymphocytes, a population of immune cells, or a population of eukaryotic donor cells) comprises mononuclear cells, lymphocyte-rich cells, B lymphocytes, T lymphocytes, CD4 + T lymphocytes, CD8 + T lymphocytes, dendritic cells, monocytes, natural killer (NK) cells, natural killer T (NKT) cells, T regulatory cells, CD4 + T helper cells, CD8 + Cytotoxic T lymphocytes (CTL), CD62L + cells, CD27 + cells, CCR7 + cells, CD45RO - cells, CD45RA + Cells, neutrophils, basophils, eosinophils, megakaryocytes, stem cells, hematopoietic stem cells (HSCs), hematopoietic progenitor cells (HPCs), CD34 + cells, CD34 +The cells are selected from peripheral blood stem cells, lymphocyte-activated killer cells (LAK), tumor-infiltrating lymphocytes (TIL), mesenchymal stem cells, mast cells, monocytes, macrophages, neutrophils, basophils, eosinophils, dendritic cells, megakaryocytes, and combinations thereof.

[0099] Another aspect of the present disclosure provides a method for producing a population of engineered eukaryotic cells, the method comprising: (1) obtaining a population of eukaryotic donor cells (e.g., from a subject or a cell line); (2) combining the population of eukaryotic donor cells with one or more buffers; and (3) transfecting the population of eukaryotic donor cells with an effective amount of a modifying agent, thereby producing a population of modified eukaryotic donor cells. In some embodiments, the transfected cells are cultured and grown in the presence of one or more stimulatory agents.

[0100] Another aspect of the present disclosure provides a method for producing a population of engineered eukaryotic cells, the method comprising: (1) obtaining a population of eukaryotic donor cells from a subject; (2) combining the population of eukaryotic donor cells with one or more buffers; (3) transfecting the population of eukaryotic donor cells with an effective amount of a modifying agent; (4) culturing and expanding the population of transfected eukaryotic donor cells; and (5) harvesting the modified eukaryotic cells, thereby producing a population of modified eukaryotic donor cells. In some embodiments, the transfected cells are cultured and expanded in the presence of one or more stimulatory agents. In some embodiments, the population of eukaryotic donor cells is not activated and / or stimulated prior to transfection.

[0101] In some embodiments, the methods disclosed herein provide for the production of a population of modified lymphocytes, a population of modified immune cells, or a modified CD4+ cell expressing a modifying agent such as a CAR in less than, within, or within about 24 hours. + cells and CD8 +In some embodiments, the methods disclosed herein can produce a population of modified lymphocytes, a population of modified immune cells, or a modified CD4+ cell expressing a modifying agent such as a CAR within about 24 hours. + and CD8 + In some embodiments, the methods disclosed herein can produce a population of modified lymphocytes, a population of modified immune cells, or a modified CD4+ cell expressing a modifying agent such as a CAR within about 48 hours. + cells and CD8 + In some embodiments, the methods disclosed herein can produce a population of modified lymphocytes, a population of modified immune cells, or a modified CD4+ cell expressing a modifying agent such as a CAR within about 72 hours. + cells and CD8 + A population of cells can be produced. B. Source of immune cells

[0102] The method for producing a population of engineered immune cells disclosed herein includes obtaining immune cells from a subject for in vitro manipulation. Sources of target cells for in vitro manipulation can include, for example, autologous or heterologous donor blood, umbilical cord blood, or bone marrow. For example, the source of immune cells can be obtained from the subject to be treated with the engineered immune cells of the present invention, such as the subject's blood, umbilical cord blood, or bone marrow. Non-limiting examples of subjects include humans, dogs, cats, mice, rats, and transgenic species thereof. Preferably, the subject is human.

[0103] Target cells can be obtained from many sources, including blood, peripheral blood mononuclear cells, bone marrow, lymph node tissue, spleen tissue, umbilical cord, lymph, and lymphoid organs. Immune cells are cells of the immune system, such as cells of innate or adaptive immunity (myeloid or lymphoid cells, including lymphocytes, typically T cells and / or NK cells). In some embodiments, the cells are human cells. With respect to the subject being treated, the cells can be allogeneic and / or autologous. The cells are typically primary cells, such as those isolated directly from the subject and / or isolated and cryopreserved from the subject. 1.Immune cells

[0104] In certain embodiments, the target cell is an immune cell, a T cell (e.g., a CD8 + T cells, CD8 + Naive T cells, central memory T cells, or effector memory T cells, CD4 + The cell is a T cell, a natural killer T cell (NKT cell), a regulatory T cell (Treg), a stem cell memory T cell, a lymphoid progenitor cell, a hematopoietic stem cell, a natural killer cell (NK cell), or a dendritic cell. In some embodiments, the cell is a monocyte or a granulocyte (e.g., a myelocyte, a macrophage, a neutrophil, a dendritic cell, a mast cell, an eosinophil, and / or a basophil).

[0105] In some embodiments, the cells are selected from the total T cell population, CD4 T cells, CD4+ ... + cells, CD8 + cells, and subpopulations thereof, including one or more subsets of T cells or other cell types, such as T cells and / or CD4 + and / or CD8 +Among the subtypes and subpopulations of T cells are naive T (TN) cells, effector T cells (TEFF), memory T cells and their subtypes, such as stem cell memory T (TSCM), central memory T (TCM), effector memory T (TEM), or terminally differentiated effector memory T cells, tumor-infiltrating lymphocytes (TIL), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosal-associated invariant T (MAIT) cells, naturally occurring adaptive regulatory T (Treg) cells, helper T cells (e.g., TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells, follicular helper T cells, α / β T cells, and δ / γ T cells). In certain embodiments, any number of T cell lines available in the art may be used. 2. Stem cells

[0106] Other exemplary cells that can be engineered using the manufacturing process of the present disclosure include stem cells, such as pluripotent stem cells and multipotent stem cells, including induced pluripotent stem cells (iPSCs). In one embodiment, the target cells are induced pluripotent stem (iPS) cells or iPS cell-derived cells, e.g., iPS cells generated from a subject. In some embodiments, the iPS cells are engineered to alter (e.g., induce mutations in) or induce expression of one or more target genes. In some embodiments, the iPS cells are engineered to transform or induce expression of T cells, CD8 + T cells (e.g., CD8 + naive T cells, central memory T cells, or effector memory T cells), CD4 + They can be differentiated into T cells, stem cell memory T cells, lymphoid progenitor cells, or hematopoietic stem cells. 3. Cell Isolation

[0107] In some embodiments, the manufacturing process of the present disclosure involves isolating, preparing, processing, and optionally culturing and / or transfecting target cells (e.g., immune cells; enriched apheresis product) from a subject. In some embodiments, the preparation of processed cells includes one or more culturing and / or preparation steps. Cells for the described processed manipulations can be isolated from a sample, such as a biological sample, e.g., obtained from or derived from a subject. In some embodiments, the subject from whom the cells are isolated has a disease or condition and is in need of, or will be administered, cell therapy. The subject in some embodiments is a human in need of a specific therapeutic intervention, such as adoptive cell therapy, from which cells are isolated, processed, and / or manipulated. Thus, in some embodiments, the cells are primary cells (e.g., primary human cells). Samples include tissues, body fluids, and other samples taken directly from a subject, as well as samples obtained after one or more processing steps, such as separation, centrifugation, genetic engineering (e.g., transduction with a viral vector), washing, and / or incubation. Biological samples may be samples obtained directly from a biological source or may be processed samples. Biological samples include, but are not limited to, body fluids (e.g., blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine, and sweat), tissue and organ samples, and processed samples derived therefrom.

[0108] In certain embodiments, the sample from which immune cells are derived or isolated is blood, a blood-derived sample, or an apheresis or leukapheresis product. Exemplary samples include whole blood, peripheral blood mononuclear cells (PBMCs), white blood cells, bone marrow, thymus, tissue biopsy, tumor, leukemia, lymphoma, lymph node, gut-associated lymphoid tissue, mucosa-associated lymphoid tissue, spleen, other lymphoid tissue, liver, lung, stomach, intestine, colon, kidney, pancreas, breast, bone, prostate, cervix, testis, ovary, tonsil, other organ, and / or cells derived therefrom. In the case of cell therapy (e.g., adoptive cell therapy), the sample may be of autologous or allogeneic origin.

[0109] In some embodiments, cell isolation involves one or more preparative and / or non-affinity-based cell separation steps. In some embodiments, cells are washed, centrifuged, and / or incubated in the presence of one or more reagents (e.g., to remove unwanted components), enriched for desired components, and lysed or removed cells sensitive to a particular reagent. In some embodiments, cells are separated based on one or more properties, such as density, adhesiveness, size, sensitivity and / or resistance to a particular component.

[0110] In some embodiments, cells are obtained from a subject's circulating blood by apheresis. The sample, in some embodiments, contains lymphocytes, including T cells, monocytes, granulocytes, B cells, and other nucleated white blood cells, red blood cells, and / or platelets; in some embodiments, cells other than red blood cells and platelets. In some embodiments, blood cells collected from a subject are washed to remove the plasma fraction and / or to place the cells in an appropriate buffer or medium for subsequent processing steps. In some embodiments, the cells are washed with phosphate-buffered saline (PBS). In some embodiments, the washing step is accomplished by tangential flow filtration (TFF) according to the manufacturer's instructions. In some embodiments, the cells are resuspended in various biocompatible buffers after washing. In some embodiments, components of the blood cell sample are removed, and the cells are resuspended directly in culture medium. In some embodiments, the method involves density-based cell separation, such as the preparation of white blood cells from peripheral blood by lysing red blood cells and centrifuging through a Percoll or Ficoll gradient.

[0111] In one embodiment, immune cells are obtained from an individual's circulating blood by apheresis or leukocytosis. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, red blood cells, and platelets. Cells collected by apheresis are washed to remove the plasma fraction, and for subsequent processing steps, the cells are placed in an appropriate buffer or medium, such as phosphate-buffered saline (PBS) or a wash solution, which may lack calcium, magnesium, or many, if not all, divalent cations. As will be readily apparent to those skilled in the art, washing steps can be accomplished by methods known to those skilled in the art, such as using a semi-automated "flow-through" centrifuge (e.g., a Cobe 2991 cell processor, a Baxter CytoMate, or a Haemonetics Cell Saver 5) according to the manufacturer's instructions. After washing, the cells are then lysed, for example, in a Ca 2+ buffer. 2+ Does not contain Mg 2+ The cells can be resuspended in a variety of biocompatible buffers, such as PBS without PlasmaLyte A, or another saline solution with or without buffer. In some embodiments, undesirable components of the apheresis sample can be removed and the cells can be resuspended directly in culture medium.

[0112] In some embodiments of the manufacturing processes described herein, cells are obtained from a subject's circulating blood by apheresis or leukapheresis using an extracorporeal apheresis system, and in some embodiments, cell isolation and transfection are performed on the same day. 4. Extracorporeal Apheresis

[0113] In some embodiments of the manufacturing processes described herein, cells are collected using a standard apheresis device, such as a Cobe® Spectra, Spectra Optia®, Fenwal™ Amicus®, or equivalent. In some embodiments, cells are obtained from a subject's circulating blood by erythrocyte apheresis, platelet apheresis, leukocyte apheresis, stem cell collection, plasma apheresis, or platelet apheresis. The leukapheresis process typically yields approximately 200-400 mL of apheresis product from the patient (i.e., subject). The apheresis product is subjected to a manufacturing process on-site (e.g., at the point of care).

[0114] In some embodiments, the concentrated apheresis product is a "leukocyte" product. As used herein, the term "leukapheresis" refers to the separation and collection of white blood cells (WBCs) from the bulk mononuclear cells present in blood, i.e., plasma and red blood cells.

[0115] In some embodiments, the enriched apheresis product comprises about 5% to about 25% of the total peripheral blood mononuclear cell component. In some embodiments, the enriched apheresis product is a population of lymphocytes or lymphoid cells. In this embodiment, lymphoid cells include T cells, B cells, natural killer (NK) cells, CD8 + T cells, CD4 + The antigen-binding protein is selected from the group consisting of T cells, cytotoxic T lymphocytes, regulatory T cells, and any combination thereof.

[0116] In some embodiments, the enriched apheresis product is a population of myeloid cells or myeloid cells, which may be selected from monocytes, macrophages, neutrophils, basophils, eosinophils, dendritic cells, megakaryocytes, and any combination thereof.

[0117] In some embodiments, the concentrated apheresis product has a predetermined volume and / or a predetermined hematocrit regardless of the number of mononuclear cell collection cycles performed by the apheresis system and / or the number of precursor products used to produce the concentrated apheresis product.

[0118] In some embodiments, the predetermined volume is about 120 mL to about 400 mL. In some embodiments, the predetermined volume is about 120 mL, about 150 mL, about 175 mL, about 180 mL, about 200 mL, about 225 mL, about 250 mL, about 275 mL, about 300 mL, about 325 mL, about 350 mL, about 375 mL, or about 400 mL or less. In some embodiments, apheresis is configured for a specific target yield of collected and treated mononuclear cells. The specific target yield of collected and treated mononuclear cells can be assessed by the apheresis system and / or by inputting the subject's mononuclear cell reserve count. Based on the target mononuclear cell yield and the number of mononuclear cells collected during each mononuclear cell collection cycle, the apheresis system controller can determine the number of mononuclear cell collection cycles to perform. As an example, in some embodiments, the target mononuclear cell yield is about 5 x 10 9 For mononuclear cells, the apheresis system can measure approximately 1 x 10 per mononuclear cell collection cycle. 9 The mononuclear cells are collected and the controller determines that it is appropriate to perform five mononuclear cell collection cycles.

[0119] In some embodiments, the target mononuclear cell yield is at least about 0.7 x 10 7 pieces, at least about 0.8 x 10 7 pieces, at least about 0.9 x 10 7 At least about 1 x 10 pieces 7 At least about 2 x 10 pieces 7 pieces, at least about 4 x 10 7 pieces, at least about 6 x 10 7 pieces, at least about 8 x 10 7 At least about 1 x 10 pieces 8 pieces, or at least about 5 x 10 8In some embodiments, the target mononuclear yield is about 0.5 x 10 6 cells / mL ~ approx. 4×10 6 In some embodiments, the target mononuclear cell yield is about 0.5 x 10 cells / mL. 6 cells / mL ~ approx. 1×10 8 In some embodiments, the target mononuclear cell yield is about 4.0 x 10 cells / mL. 6 cells / mL ~ approx. 1×10 8 cells / mL.

[0120] In certain embodiments, the predetermined hematocrit is about 0% to about 10%. In other embodiments, the predetermined hematocrit is about 2%. In some embodiments, the predetermined volume is about 200 mL and the predetermined hematocrit is about 2%. The predetermined volume and / or the predetermined hematocrit may vary without departing from the scope of the present disclosure. 5. Cell enrichment

[0121] In some embodiments, the manufacturing processes disclosed herein involve selecting specific cells to improve enrichment of desired immune effector cells suitable for CAR expression. Systems or devices used for cell enrichment and purification include, for example, BAXTER ISOLEX 300I™ and Miltenyi CLINIMACS™, which enrich peripheral blood progenitor cells (PBPCs) based on specific cell surface ligands (e.g., CD34 or CD133).

[0122] In certain embodiments, the selection comprises a positive selection, e.g., selection against desired immune effector cells. In some embodiments, the selection comprises a negative selection, e.g., selection against unwanted cells, e.g., removal of unwanted cells. In some embodiments, the positive or negative selection methods described herein are performed under flow conditions using a flow-through device or cell processing system to further enrich the cell preparation for desired immune effector cells. Column technology (e.g., CliniMACS® System, CliniMACS® Plus, or CliniMACS Negative T cell selection via removal of unwanted cells with CD19, CD14, and CD26 Miltenyi beads in combination with CliniMACS® System (CliniMACS® Plus, or CliniMACS Prodigy®) can be used. Positive T cell selection using CD4 and CD8 Miltenyi beads in combination with column technology (CliniMACS® System, CliniMACS® Plus, or CliniMACS Prodigy®) can also be used. Alternatively, column-free technology using releasable CD3 beads (GE Healthcare) can be used. Additionally, bead-free technology such as ThermoGenesis X-series instruments can be utilized. Other exemplary cell separation and debeading methods are known to those skilled in the art, for example, as set forth in WO 2017 / 117112.

[0123] In some embodiments, the enriched apheresis product is enriched for one or more target cell types selected from the group consisting of B lymphocytes, T lymphocytes, CD4 and CD8 T lymphocytes, dendritic cells, monocytes, natural killer (NK) cells, NKT cells, T regulatory cells, CD4 T helper cells, CD8 cytotoxic T lymphocytes (CTL) NKT cells, neutrophils, basophils, eosinophils, megakaryocytes, hematopoietic stem cells (HSCs), hematopoietic progenitor cells (HPCs), lymphocyte-activated killer cells (LAKs), tumor-infiltrating lymphocytes (TILs), mesenchymal stem cells, mast cells, subsets of these cells, and combinations thereof.

[0124] In some embodiments, the enriched apheresis product is enriched for one or more target lymphoid or myeloid cell populations. In some embodiments, the enriched apheresis product is enriched for T cells, B cells, natural killer (NK) cells, CD8 + T cells, CD4 + The cells are enriched for lymphocytes selected from the group consisting of T cells, cytotoxic T lymphocytes, regulatory T cells, and any combination thereof.

[0125] In some embodiments, the concentrated apheresis product is enriched for myeloid cells selected from monocytes, macrophages, neutrophils, basophils, eosinophils, dendritic cells, megakaryocytes, and any combination thereof.

[0126] In some embodiments, one or more markers, one or more "cell surface determinants" or "cell surface markers" are used to enrich the target cell population. In some embodiments, the one or more markers or cell surface determinants are selected from the following: CD19 and / or CD20 for B cells; CD3, CD56 for T cells. - , CD4, and / or CD8; CD25 and / or CD69 for activated T cells and / or regulatory T cells; CD1c, CD83, CD141, CD209, MHC II, and / or CD11c for dendritic cells; CD3, CD16, and / or CD56 for NK cells; CD34, CD90, and / or CD135 for hematopoietic stem or progenitor cells; CD11b, CD68, CD163, and / or CD33 for macrophages; CD14, CD16, and / or CD64 for monocytes; and CD15, CD16, and / or CD49d for neutrophils. - , 2D7 antigen, CD117-, CD123, CD203c, and / or FcεRIa in basophils, and CD11b, CD193, EMR1, and / or Siglec-8 in eosinophils.

[0127] Techniques for concentrating the enriched apheresis product are known to those skilled in the art and include, but are not limited to, magnetic separation, filtration, immunoaffinity separation, gravity separation, density gradient separation, elution separation, and any combination thereof. The cell separation module can employ any of these methods or other methods known in the art to further enrich and / or obtain a target population of nucleated blood cells from the patient. For example, binding to one or more selection or affinity agents, such as antibodies bound to degradable buoyant beads, magnetic beads, or microbubbles, can be used to enrich for specific target cell types or classes after cell separation.

[0128] In some embodiments, magnetic beads coated with antibodies against one or more specific cell surface antigens are used to enrich target cell populations from enriched apheresis products. Cells expressing the target antigens are then attached to the magnetic beads. Upon exposure to a strong magnetic field, the cells attached to the beads (expressing the cell surface marker) are retained on the column or sample tube, while other cells (not expressing the cell surface marker) pass through or remain in suspension. Using this method, cells can be positively or negatively selected for specific cell surface markers, or a combination of positive and negative selection. In some embodiments, cells remain bound to the microbead-bound antibodies during transfection. In some embodiments, cells are released from the microbead-bound antibodies prior to transfection.

[0129] In some embodiments, target cells are enriched using one or more methods known in the art, including, but not limited to, antigen capture. In some embodiments, antigen capture is selected from the group consisting of filters, beads, magnetic beads, fluorescence-activated cell sorting, microfluidics, solid support affinity, acoustics, bioluminescence, antibody tagging, and enzyme substrates. In some embodiments, target cells are enriched using a suitable solid support selected from the group consisting of ferromagnetic particles and density-modified particles. In some embodiments, solid supports comprised of affinity molecules, such as antibody domains, that bind to selected cell surface markers are available from, for example, Miltenyi Biotec and Dynal. Methods that can be used to release captured cells include competition with excess ligand, enzymatic digestion, changes in pH, changes in ionic strength, removal of the magnetic field, and / or physical agitation.

[0130] In some embodiments, the separation method involves separating different cell types based on the intracellular expression or presence of one or more specific molecules, such as surface markers, surface proteins, intracellular markers, or nucleic acids. In some embodiments, any known method for such marker-based separation can be used. In some embodiments, the separation is affinity- or immunoaffinity-based. For example, the separation can involve separating cells and cell populations based on the cellular expression or expression level of one or more markers. Typically, cell surface markers are incubated with an antibody or binding partner that specifically binds to the marker. This incubation step is followed by a washing step and a purification step to separate cells that are bound to the antibody or binding partner from those that are not. Such purification steps can be based on positive selection, which retains cells that bind to the reagent for further use, and / or negative selection, which retains cells that do not bind to the antibody or binding partner. In some instances, both fractions are retained for further use.

[0131] In certain embodiments, negative selection is particularly useful in the absence of antibodies that specifically identify cell types in a heterogeneous population, such that separation is best performed based on markers expressed by cells other than the desired population. Separation need not 100% enrich for or remove a particular cell population or cells expressing a particular marker. For example, positive selection, enrichment for a particular type of cell (e.g., cells expressing a marker) may increase the number or proportion of such cells, but need not completely remove cells that do not express the marker. Similarly, negative selection, removal, or depletion of a particular type of cell (e.g., cells expressing a marker) may decrease the number or proportion of such cells, but need not completely remove all such cells. In certain exemplary embodiments, separation steps can be performed multiple times, with positively or negatively selected fractions from one step subjected to a subsequent separation step, such as a positive or negative selection. In certain exemplary embodiments, cells expressing multiple markers can be simultaneously removed in a single separation step, such as by incubating cells with multiple antibodies or binding partners, each specific for a marker that is subject to negative selection. Similarly, multiple cell types can be positively selected simultaneously by incubating cells with multiple antibodies or binding partners expressed on different cell types.

[0132] Enrichment of a T cell population by negative selection can be achieved using a combination of antibodies directed against surface markers specific to the negatively selected cells. Exemplary methods include negative magnetic immunoadherence or flow cytometric cell sorting and / or selection using a cocktail of monoclonal antibodies directed against cell surface markers present on the negatively selected cells. For example, negative selection can enrich for CD4 + To enrich for cells, monoclonal antibody cocktails typically include antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8.

[0133] For isolation of desired cell populations by positive or negative selection, the concentration of cells and surfaces (e.g., particles such as beads) can be varied. In certain embodiments, it may be desirable to significantly reduce the volume in which the beads and cells are mixed together (i.e., increase the concentration of cells) to ensure maximum contact between the beads and cells. For example, in one embodiment, a concentration of 2 billion cells / mL is used. In one embodiment, a concentration of 1 billion cells / mL is used. In a further embodiment, a concentration of greater than about 100 million cells / mL is used. In a further embodiment, a cell concentration of about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, or about 50 million cells / mL is used. In yet another embodiment, a cell concentration of about 75, about 80, about 85, about 90, about 95, or about 100 million cells / mL is used. In further embodiments, concentrations of about 125 million cells / mL or about 150 million cells / mL may be used. The use of higher concentrations may result in increased cell yield, cell activation, and cell proliferation.

[0134] In some embodiments, different cell types are enriched using a cell sorter such as the CliniMACS Prodigy®. For example, T cells (e.g., CD4 + T cells and / or CD8 + T cells) are identified by CliniMACS Selected T cells (e.g., CD4 T cells) are selected from the apheresis product using a cell sorter such as the Prodigy® device. + T cells and / or CD8 + T cells) are then washed and transfected to produce artificial T cells as described herein.

[0135] In some embodiments, one or more of the T cell populations are positive for one or more particular markers, such as surface markers. + ) or high level (marker 高) or one or more markers are negative (marker - ) or at a relatively low level (marker 低 For example, in certain embodiments, the cells are enriched for or depleted for cells expressing one or more surface markers (e.g., CD28 + , CD62L + , CCR7 + , CD27 + , CD127 + , CD4 + , CD8 + , CD45RA + , and / or CD45RO + Specific subpopulations of T cells, such as cells expressing positive or high levels of markers (e.g., CD8 T cells), are isolated by positive or negative selection techniques. In some cases, such markers are absent or expressed at relatively low levels on certain populations of T cells (e.g., non-memory cells) but present or expressed at relatively high levels on other specific populations of T cells (e.g., memory cells). In certain embodiments, cells (e.g., CD8 + cells or T cells, e.g., CD3 + The apheresis product (e.g., cells) are enriched (i.e., positively selected) for cells that are positive for or express high surface levels of CD45RO, CCR7, CD28, CD27, CD44, CD127, and / or CD62L, and / or depleted (e.g., negatively selected) for cells that are positive for or express high surface levels of CD45RA. In some embodiments, the cells are enriched for or depleted from cells that are positive for or express high surface levels of CD122, CD95, CD25, CD27, and / or IL7-Ra (CD127). In some embodiments of the methods disclosed herein, the enrichment step of the apheresis product is performed by selecting cells that are positive for or express high surface levels of CD25 + In some embodiments of the methods disclosed herein, the enrichment step of the apheresis product comprises depletion of CD25 cells. +Does not involve cell depletion. See, e.g., WO2016 / 109410. CD25 + Depletion can increase the efficiency of lentiviral transduction and ultimately improve the therapeutic efficacy of CAR T therapy. However, this step may not be critical for the production of the electric CAR T cells described herein.

[0136] In certain exemplary embodiments, CD8 + T cells are enriched for CD45RO positive (or CD45RA negative) and CD62L positive cells. For example, CD3 + , CD28 + T cells can be positively selected using CD3 / CD28-conjugated magnetic beads (e.g., Dynabeads® M-450 CD3 / CD28 T Cell Expander).

[0137] In some embodiments, T cells are isolated from peripheral blood mononuclear cells (PBMC) samples by negative selection for markers expressed on non-T cells, such as B cells, monocytes, or other leukocytes, such as CD14. + Helper T cells and CD8 + To isolate cytotoxic T cells, CD4 + or CD 8 + A selection process is used. + and CD8 + The population can be further sorted into subpopulations by positive or negative selection for markers that are expressed, or expressed to a relatively high degree, on one or more naive, memory, and / or effector T cell subpopulations. In some embodiments, CD8 +The cells are further enriched or depleted for naive, central memory, effector memory, and / or central memory stem cells, such as by positive or negative selection based on surface antigens associated with each subpopulation. In some embodiments, enrichment for central memory T (TCM) cells is performed to enhance efficacy, such as improving long-term survival, proliferation, and / or engraftment following administration, and is, in certain aspects, particularly robust for such subpopulations.

[0138] In some embodiments, the TCM is enriched for CD8 + T cells and CD4 + Combining T cells further enhances efficacy. In some embodiments, memory T cells are CD8 + CD62L on peripheral blood lymphocytes + Subsets and CD62L - PBMCs were purified using anti-CD8 and anti-CD62L antibodies to detect CD62L-CD8 + fraction and / or CD62L + CD8 + The fraction can be enriched or depleted. In some embodiments, CD4 + T cell populations and / or CD8 + The T population is enriched for central memory (TCM) cells. In some embodiments, the enrichment for central memory T (TCM) cells is based on positive or high surface expression of CD45RO, CD62L, CCR7, CD28, CD8, and / or CD127. In certain embodiments, the enrichment may be based on negative selection for cells expressing or overexpressing CD45RA and / or granzyme B. In certain embodiments, the enriched TCM cells are based on negative selection for CD8 +Isolation of the population is achieved by depletion of cells expressing CD4, CD14, and CD45RA, and positive selection or enrichment for cells expressing CD62L. In one embodiment, enrichment of central memory T (TCM) cells begins with a negative fraction of cells selected on the basis of CD4 expression, followed by negative selection on the basis of CD14 and CD45RA expression, and positive selection on the basis of CD62L. Such selections are performed simultaneously in some embodiments and sequentially in other embodiments. In one embodiment, enrichment of central memory T (TCM) cells begins with depletion of cells expressing CD4, CD14, and CD45RA, followed by negative selection on the basis of CD62L expression. + The same CD4 expression-based selection process used in preparing the cell population or subpopulation may be used to select CD4 + It may also be used in preparing cell populations or subpopulations, where both the positive and negative fractions from CD4-based separation are retained and used in subsequent steps of the method.

[0139] CD4 + T helper cells are selected into naive, central memory, and effector cells by identifying cell populations bearing cell surface antigens. + Lymphocytes can be obtained by standard methods. In some embodiments, naive CD4 + T lymphocytes are CD45RO-, CD45RA + , CD62L + , CD4 + In some embodiments, central memory CD4 T cells. + The cells are CD62L + and CD45 RO + In some embodiments, the effector CD4 + The cells are CD62L- and CD45RO. In one example, negative selection results in CD4 +To enrich for cells, monoclonal antibody cocktails typically include antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CDS. In some embodiments, the antibodies or binding partners are attached to a solid support or matrix, such as magnetic or paramagnetic beads, to allow for separation of cells for positive and / or negative selection.

[0140] In some embodiments, cells are incubated and / or cultured prior to or in conjunction with genetic engineering. Incubation steps can include culturing, culturing, stimulating, activating, and / or expanding. In some embodiments, the composition or cells are incubated under stimulatory conditions or in the presence of a stimulatory agent. Such conditions include those designed to induce proliferation, growth, activation, and / or survival of cells in a population, mimic antigen exposure, and / or prime cells for genetic engineering, such as the introduction of a recombinant antigen receptor. Conditions can include one or more of a specific medium, temperature, oxygen content, carbon dioxide content, time, agents such as nutrients, amino acids, antibiotics, ions, and / or stimulatory factors such as cytokines, chemokines, antigens, binding partners, fusion proteins, recombinant soluble receptors, and other agents designed to activate cells. In some embodiments, the stimulatory conditions or agents include one or more agents. A ligand can activate the intracellular signaling domain of the TCR complex. In some embodiments, the agent turns on or initiates the TCR / CD3 intracellular signaling cascade in T cells. Such agents may include antibodies specific for TCR components (e.g., anti-CD3, anti-CD28), costimulatory receptors, and / or one or more cytokines. The agents may be bound to a solid support, such as beads. Optionally, the expansion method may further include adding an anti-CD3 antibody and / or an anti-CD28 antibody to the culture medium (e.g., at a concentration of at least about 0.5 ng / ml). In some embodiments, the stimulatory agent includes IL-2 and / or IL-15, e.g., the IL-2 concentration is at least about 10 units / ml. In some embodiments, the stimulatory agent includes IL-7 and / or IL-15. In some embodiments, the stimulatory agent includes IL-2, IL-7, and / or IL-15. In some embodiments, the stimulatory agent includes IL-2, IL-15, and / or IL-15Ra.In some embodiments, the stimulatory agent comprises IL-2 and / or heterodimeric IL-15 (ie, a polypeptide consisting of IL-15 and the IL-15 receptor alpha chain).

[0141] In another embodiment, T cells are isolated from peripheral blood by lysing red blood cells and depleting monocytes, for example, by centrifugation through a PERCOLL™ gradient. Alternatively, T cells can be isolated from umbilical cord blood. In either case, specific subpopulations of T cells can be further isolated by positive or negative selection techniques. Cord blood mononuclear cells isolated in this manner can be depleted from cells expressing specific antigens, including, but not limited to, CD34, CD8, CD14, CD19, and CD56. Removal of these cells can be achieved using isolated antibodies, antibody-containing biological samples such as ascites fluid, antibodies bound to physical supports, or antibodies bound to cells.

[0142] C. Activation before transfection In one embodiment of the methods disclosed herein, prior to the transfection step, a concentrated apheresis product (e.g., a population of immune cells, such as CD4 + and CD8 + It is not necessary to stimulate and / or activate the population of cells (or population of eukaryotic donor cells) with one or more stimulatory agents.

[0143] However, in some embodiments, the method further comprises the step of transfecting a concentrated apheresis product (e.g., a population of immune cells, such as CD4 + cells and CD8 +The method may further include stimulating and / or activating the primary human immune cell (e.g., a population of cells, or a population of eukaryotic donor cells) with one or more stimulatory agents. The stimulatory agents described herein (e.g., CD3, CD28, cytokines, and / or growth factors) can promote efficient transduction and / or electroporation of primary human immune cells (e.g., T cells), and supplementing the culture medium with cytokines selected from IL-7, IL-15, IL-15Ra, IL-7, and IL-15 and / or heterodimeric IL-15 (i.e., a polypeptide consisting of IL-15 and the IL-15 receptor alpha chain) can dramatically enhance the proliferation of transfected cells. Furthermore, pre- and / or post-transfection stimulation and / or activation can preserve undifferentiated T cells during CART production, which can improve the longevity of the produced T cells and thereby improve the therapeutic efficacy of CART therapy.

[0144] In some embodiments, the one or more stimulatory agents are selected from the group consisting of an agonistic antibody, a cytokine, a recombinant costimulatory molecule, an anti-CD3 antibody or fragment thereof, an anti-CD28 antibody or fragment thereof, a small drug inhibitor, and / or a combination thereof. In some embodiments, the one or more stimulatory agents are an anti-CD3 antibody and an anti-CD28 antibody or fragment thereof. In some embodiments, the one or more stimulatory agents are an anti-CD3 antibody and an anti-CD28 antibody or fragment thereof, and one or more cytokines. 1.CD3 / TCR complex

[0145] In some embodiments, the enriched apheresis product (e.g., a population of immune cells, CD4 + cells and CD8 +The population of cells, or a population of eukaryotic donor cells, is stimulated and / or activated with an agent that stimulates the CD3 / TCR complex and / or an agent that stimulates a cell surface costimulatory molecule and / or a growth factor receptor. In some embodiments, the agent that stimulates the CD3 / TCR complex is an agent that stimulates CD3. In some embodiments, the agent that stimulates the CD3 / TCR complex is selected from an antibody (e.g., a single domain antibody, a heavy chain variable domain antibody, a peptibody, a Fab fragment, or an scFv), a small molecule, or a ligand (e.g., a naturally occurring ligand, a recombinant ligand, or a chimeric ligand).

[0146] In some embodiments, the agent that stimulates the CD3 / TCR complex does not comprise beads. In some embodiments, the agent that stimulates a costimulatory molecule and / or a growth factor receptor does not comprise beads. In some embodiments, the agent that stimulates the CD3 / TCR complex comprises an anti-CD3 antibody. In some embodiments, the agent that stimulates the CD3 / TCR complex comprises an anti-CD3 antibody covalently bound to a colloidal polymer nanomatrix. In some embodiments, the agent that stimulates CD3 comprises one or more CD3 or TCR antigen binding domains (e.g., anti-CD3 antibodies or anti-TCR antibodies or antibody fragments) comprising one or more CDRs, heavy chains, and / or light chains, as known to those of skill in the art.

[0147] In some embodiments, the agent that stimulates the CD3 / TCR complex and the agent that stimulates a costimulatory molecule and / or a growth factor receptor comprise a T Cell Trans Act™. In some embodiments, the agent that stimulates the CD3 / TCR complex and the agent that stimulates a costimulatory molecule and / or a growth factor receptor are comprised in a multispecific binding molecule. In some embodiments, the multispecific binding molecule comprises a CD3 antigen-binding domain and a CD28 or CD2 antigen-binding domain. In some embodiments, the multispecific binding molecule comprises one or more heavy chains and / or light chains. In some embodiments, the multispecific binding molecule comprises a bispecific antibody. In some embodiments, one or more of the multiple bispecific antibodies are conjugated together to form a multimer. 2. Co-stimulatory molecules In some embodiments, the agent that stimulates a costimulatory molecule and / or growth factor receptor is an agent that stimulates CD28, ICOS, CD27, HVEM, LIGHT, CD40, 4-1BB, OX40, DR3, GITR, CD30, TIM1, CD2, CD226, or any combination thereof. In some embodiments, the agent that stimulates a costimulatory molecule and / or growth factor receptor is an agent that stimulates CD28. In some embodiments, the agent that stimulates a costimulatory molecule and / or growth factor receptor is selected from an antibody (e.g., a single domain antibody (e.g., a heavy chain variable domain antibody), a peptibody, a Fab fragment, or an scFv), a small molecule, or a ligand (e.g., a naturally occurring ligand, a recombinant ligand, or a chimeric ligand).

[0148] In some embodiments, the agent that stimulates a costimulatory molecule and / or growth factor receptor comprises an anti-CD28 antibody. In some embodiments, the agent that stimulates a costimulatory molecule and / or growth factor receptor comprises an anti-CD28 antibody covalently attached to a colloidal polymer nanomatrix. In some embodiments, the agent that stimulates a costimulatory molecule and / or growth factor receptor is an agent that stimulates CD28, ICOS, CD27, CD25, 4-1BB, IL6RA, IL6RB, or CD2. In some embodiments, the agent that stimulates a costimulatory molecule and / or growth factor receptor comprises one or more of the CD28, ICOS, CD27, CD25, 4-1BB, IL6RB, and / or CD2 antigen binding domains. For example, the agent is an anti-CD28, anti-ICOS, anti-CD27, anti-CD25, anti-4-IBB, anti-IL6RA, anti-IL6RB, anti-CD2 antibody, or an antibody fragment comprising one or more CDRs, heavy chains, and / or light chains thereof, as known to those skilled in the art.

[0149] In some embodiments, prior to the transfection step, enriched apheresis products (e.g., lymphocyte populations, immune cell populations, CD4 + cells and CD8 + In some embodiments, the enriched apheresis product (e.g., a population of lymphocytes, a population of immune cells, a population of eukaryotic donor cells) is stimulated and / or activated in vitro with an agent that stimulates the CD3 / TCR complex (e.g., an anti-CD3 antibody) and / or an agent that stimulates cell surface costimulatory molecules and / or growth factor receptors (e.g., an anti-CD28 antibody). In some embodiments, the enriched apheresis product (e.g., a population of lymphocytes, a population of immune cells, a CD4 + cells and CD8 + The population of cells, or population of eukaryotic donor cells) can be stimulated and / or activated for about 1 hour, about 1.5 hours, about 2 hours, about 2.5 hours, about 3 hours, about 3.5 hours, about 4 hours, about 4.5 hours, or about 5 hours or less.

[0150] In some embodiments, the enriched apheresis product (e.g., a population of lymphocytes, a population of immune cells, CD4+ cells and CD8 + The population of cells, or population of eukaryotic donor cells, is stimulated and / or activated in vitro with an agent that stimulates the CD3 / TCR complex (e.g., an anti-CD28 antibody) for about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 24 hours, about 25 hours, about 26 hours, about 27 hours, or about 28 hours.

[0151] In some embodiments, the agent that stimulates the CD3 / TCR complex and the agent that stimulates a costimulatory molecule and / or a growth factor receptor are comprised in a multispecific binding molecule. The multispecific binding molecule may be comprised of the agent that stimulates the CD3 / TCR complex and the agent that stimulates a costimulatory molecule and / or a growth factor receptor. For example, the multispecific binding molecule may be comprised of a CD3 antigen-binding domain and one or more of CD28, ICOS, CD27, CD25, 4-IBB, IL6RA, IL6RB, and / or CD2 antigen-binding domains. In some embodiments, the multispecific binding molecule comprises a CD3 antigen-binding domain and a CD28 or CD2 antigen-binding domain. 3. Cytokines

[0152] In some embodiments, the one or more stimulating agents are selected from the group consisting of interleukin-2 (IL-2), interleukin-3 (IL-3), interleukin-6 (IL-6), interleukin-7 (IL-7), interleukin-7 receptor (IL-7R), interleukin-11 (IL-11), interleukin-12 (IL-12), interleukin-15 (IL-15), interleukin-15 receptor (IL-15R), heterodimeric IL-15 (i.e., a polypeptide comprising IL-15 and an IL-15 receptor alpha chain), interleukin-18 (IL-18), interleukin-18 receptor (IL-18R), interleukin-21 (IL-21), granulocyte-macrophage colony-stimulating factor, alpha, beta, or gamma interferon, erythropoietin, and any combination thereof.

[0153] Cytokines can be selected from IL-2, IL-7, IL-6, IL-15, IL-15Ra, heterodimeric IL-15 (i.e., a polypeptide containing IL-15 and the IL-15 receptor α chain; hetIL-15), or IL-21. IL-2 is the cytokine most frequently used to generate lymphocytes for adoptive immunotherapy. IL-2 promotes T cell survival and proliferation and enhances their tumoricidal activity. IL-2 significantly increased the accumulation of CAR-T cells and their cytotoxicity, but IL-2-exposed CAR-T cells exhibited inferior antitumor immunity in vivo after adoptive transfer. IL-2-exposed CAR-T cells also exhibited lower expression of CD62L, CCR7, CD27, and CD28, exhibiting a relatively mature phenotype and poor in vivo persistence. Adoptive transfer of less differentiated T cells correlates with superior tumor regression, supporting the finding that IL-2-exposed CAR-T cells are less effective than other groups (Gattinoni et al., Nat Med, 2011, 17: 1290-7; and Markley et al., Blood, 2010, 115:3508-19).

[0154] IL-15, like IL-2, stimulated CAR-T cell proliferation and tumor lytic function, resulting in superior antitumor immunity in animal models. Additionally, IL-15 induced a less differentiated phenotype (high expression of CD27 and CD28). Therefore, IL-15 can support CAR-T cell persistence in vivo. IL-7 also promoted CAR-T cell proliferation in vitro.

[0155] IL-7 induced high expression of CD62L and resulted in the highest percentage of CAR-Tscm cells in an antigen-free environment. Exposing T cells or CAR-T cells to IL-7 ex vivo without antigen challenge enhanced the antitumor efficacy of CAR-T cells. However, IL-7-exposed CAR-T cells did not exhibit improved in vivo antitumor efficacy compared with IL-2. IL-7 was also less effective than IL-15, due to reduced proliferation of CAR-T cells under antigen challenge. The combination of IL-7 and IL-15 promotes Tscm production, beneficial for generating younger CAR-T cells. Therefore, combining IL-7 and IL-15 can promote CAR-T cell proliferation and induce the most therapeutically effective T cell phenotype.

[0156] IL-21 can induce the proliferation of less differentiated CAR-T cells, which have a phenotype that highly expresses CD62L, CCR7, CD27, and CD28. Therefore, CAR-T cells exposed to IL-21 exhibited the best persistence in animal models and in vivo after IL-21 injection, and also demonstrated superior efficacy in promoting tumor eradication compared with other cytokines, except for IL-15. See, e.g., WO 2016 / 109410.

[0157] Thus, in some embodiments, the cytokine may also be selected from IL-15 and IL-7; IL-7 and IL-21; IL-7 and IL-2; IL-15 and IL-2; IL-7, IL-15 and IL-21; IL-15 and IL-15Ra; or IL-7, IL-15 and IL-15Ra. In some embodiments, the cytokine is IL-2. In some embodiments, the cytokine is IL-15 (e.g., hetIL-15 (IL15 / sIL-15Ra)). In some embodiments, the cytokine is IL-6 (e.g., IL-6 / sIL-6Ra). In some embodiments, the cytokine is IL-7. In some embodiments, the cytokine is IL-7 and IL-15.

[0158] In some embodiments, the enriched apheresis product (e.g., a population of lymphocytes, a population of immune cells, CD4 + cells and CD8 + In some embodiments, the enriched apheresis product (e.g., a population of immune cells, or a population of eukaryotic donor cells) is stimulated and / or activated with about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 210, about 220, about 230, about 240, about 250, about 260, about 270, about 280, about 290, or about 300 U / ml of IL-2 (or any amount between these values). + cells and CD8 + In some embodiments, the enriched apheresis product (e.g., a population of immune cells, or a population of eukaryotic donor cells) is stimulated / activated with about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 ng / ml (or any amount between these values) of IL-7. + cells and CD8 +The population of cells, or population of eukaryotic donor cells, is stimulated and / or activated with about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 ng / ml (or any amount between these values) of IL-15. Cytokine stimulation maintains or increases the undifferentiated phenotype of the T cells during CAR-T (electric CAR-T cell) manufacturing disclosed herein, generating CAR-T cells (electric CAR-T cells) that persist longer in the subject after administration. Supplementing the culture medium with a cytokine selected from IL-7, IL-15, IL-15Ra, IL-7 and IL-15, and / or heterodimeric IL-15 (i.e., a polypeptide comprising IL-15 and the IL-15 receptor alpha chain) dramatically enhances proliferation of transduced cells by at least 200-fold over a 14-day period. See, e.g., WO 2016 / 109410. nanostructure In some embodiments, one or more stimulating agents are conjugated to beads or nanostructures. In some embodiments, the nanostructure is a nanomatrix. The nanomatrix can include a matrix of flexible polymer chains and an anti-CD3 antibody and an anti-CD28 antibody, or fragments thereof. The nanomatrix can be about 1 to about 500 nm in size (or any size between these two values). In some embodiments, the one or more stimulating agents are a nanomatrix and one or more cytokines described herein.

[0159] In some embodiments, the nanomatrix can comprise a polymeric, biodegradable, or biocompatible inert material. The inert material may be non-toxic to cells. In some embodiments, the nanomatrix can be composed of hydrophilic polymer chains, which achieve maximum mobility in aqueous solution through chain hydration. In some embodiments, the mobile nanomatrix can be collagen, purified proteins, purified peptides, polysaccharides, glycosaminoglycans, or extracellular matrix compositions. Polysaccharides include, for example, cellulose ethers, starch, gum arabic, agarose, dextran, chitosan, hyaluronic acid, pectin, xanthan gum, guar gum, alginic acid, and the like. Other polymers include polyesters, polyethers, polyacrylates, polyacrylamides, polyamines, polyethyleneimines, polyquaternium polymers, polyphosphazenes, polyvinyl alcohols, polyvinyl acetates, polyvinylpyrrolidones, block copolymers, or polyurethanes. In some embodiments, the mobile nanomatrix is ​​a polymer of dextran.

[0160] Another aspect of the present disclosure provides a method for producing a population of engineered eukaryotic cells, the method comprising: (1) obtaining a population of eukaryotic donor cells from a subject; (2) combining the population of eukaryotic donor cells with one or more buffers; (3) stimulating the population of eukaryotic donor cells with one or more stimuli; (4) transfecting the stimulated population of eukaryotic donor cells with an effective amount of a modifying agent; (5) culturing and expanding the population of transfected eukaryotic donor cells; and (6) harvesting the modified eukaryotic cells, thereby producing a population of modified eukaryotic donor cells. In some embodiments, the transfected cells are cultured and expanded in the presence of one or more stimuli.

[0161] Another aspect of the present disclosure provides a method for producing a population of engineered immune cells, the method comprising: (1) generating a population of lymphocytes, a population of immune cells, or a population of CD4 +and CD8 + (2) enriching the population of cells from the donor leukocytes; (3) enriching the population of lymphocytes, the population of immune cells, or CD4 + and CD8 + (3) combining the population of cells with one or more buffers; (4) stimulating the population of eukaryotic donor cells with one or more stimulatory agents; and (5) generating a population of lymphocytes, a population of immune cells, or CD4 + and CD8 + (5) transfecting the population of cells with an effective amount of a modifying agent; and (6) determining whether the population of transfected lymphocytes, immune cells, or CD4 + and CD8 + (6) culturing and expanding the population of cells; and (7) culturing and expanding the engineered lymphocytes, immune cells, or CD4 + and CD8 + harvesting the cells; thereby obtaining a population of modified lymphocytes, a population of modified immune cells, or a modified CD4 + and CD8 + Generating a population of cells. In some embodiments, the transfected cells are cultured and expanded in the presence of one or more stimulatory agents.

[0162] One aspect of the present disclosure provides a method for producing a population of engineered immune cells, the method comprising: (1) extracting a population of lymphocytes, a population of immune cells, or CD4 cells from blood obtained from a subject; + cells and CD8 + (2) enriching the population of cells; (3) enriching the population of lymphocytes, immune cells, or CD4 + cells and CD8 + (3) combining the population of cells with one or more buffers; and (4) determining whether the population of lymphocytes, the population of immune cells, or the population of CD4 + cells and CD8 + (4) stimulating the population of cells with one or more stimulatory agents; and (5) stimulating the population of lymphocytes, the population of immune cells, or the population of CD4 + and CD8 + (5) transfecting the population of cells with an effective amount of a modifying agent; and (6) determining whether the population of transfected lymphocytes, immune cells, or CD4+ and CD8 + (6) culturing and expanding the population of cells; and (7) culturing and expanding the engineered lymphocytes, immune cells, or CD4 + and CD8 + and harvesting the cells, thereby obtaining a population of modified lymphocytes, a population of modified immune cells, or a population of modified CD4 + and CD8 + Producing a population of cells. In some embodiments, the transfected cells are cultured and expanded in the presence of one or more stimulatory agents described herein.

[0163] In some embodiments, the enriched apheresis product (e.g., lymphocyte populations, immune cell populations, CD4 + cells and CD8 + A population of cells, or a population of eukaryotic donor cells, is stimulated and / or activated with anti-CD3 and anti-CD28 antibodies for about 12 hours in the presence of cytokines described herein, followed by transfection with a modifying agent (e.g., a vector encoding a CAR, an artificial TCR, a polypeptide that enhances immune cell function, or a functional derivative thereof, transduction with a lentiviral vector, and / or electroporation). Then, about 24 hours after the start of stimulation, the cells are washed and formulated for storage or administration. In other embodiments, about 12 hours, about 22 hours, about 30 hours, about 40 hours, about 45 hours, about 50 hours, about 60 hours, or about 72 hours after the start of stimulation, the cells are washed and formulated for storage or administration. D. Methods for Introducing Viral Vectors into Cells

[0164] Methods for introducing a modifying agent (e.g., an expression vector, a viral vector, a polynucleotide, or a nucleic acid) into a cell include physical, biological, chemical, and combinations thereof. Expression vectors, including the viral vectors or expression vectors of the present disclosure, can be introduced into host cells by any means known to those skilled in the art. The expression vector may optionally include a viral sequence for transfection. Alternatively, the expression vector may be introduced by fusion, electroporation, biolistics (e.g., a gene gun), transfection, lipofection (e.g., cationic liposomes), polymer encapsulation, etc. Host cells (e.g., immune cells) may be grown in culture before introduction of the expression vector and then treated appropriately for vector introduction and integration. The host cells (e.g., immune cells) can then be grown and screened for markers present in the vector. Methods for producing cells containing vectors and / or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York (2001).

[0165] In some embodiments, the enriched apheresis product and / or enriched target cell population can be modified using any method known in the art, such as activation, proliferation, induction of apoptosis, genetic manipulation, induction of antigen specificity, etc. In some embodiments, the enriched apheresis product and / or enriched target cell population can be modified by adding cytokines, crosslinking specific receptors, adding antigens, introducing nucleic acid molecules (DNA, RNA, and / or variants thereof), adding protein agents, drugs, or small molecules, or any combination thereof. In some embodiments, introduction of a modifying agent (e.g., an expression vector, a viral vector, an exogenous nucleic acid molecule, a polynucleotide, or a nucleic acid) includes viral transfection, non-viral transfection, electroporation, lipofection, cationic liposome-mediated transfection using lipofection, polymer encapsulation, peptide-mediated transfection, or a biological particle delivery system such as a "gene gun" (see, e.g., Nishikawa et al., Hum Gene Ther., 12(8):861-70 (2001)). 1. Biological methods

[0166] Biological methods for introducing a modifying agent of interest into a host cell (e.g., an immune cell) include the use of expression vectors (e.g., viral vectors, exogenous nucleic acid molecules, polynucleotides or nucleic acids (DNA and RNA)). Viral vectors, particularly retroviral vectors (viral transfection), have become the most widely used method for inserting genes into mammalian (e.g., human) cells. Viral vectors can be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses and adeno-associated viruses, etc. See, e.g., U.S. Pat. Nos. 5,350,674 and 5,585,362.

[0167] In some embodiments, nucleic acids encoding target CARs, target engineered TCRs, target KIRs, target antigen-binding polypeptides, target cell surface receptor ligands, target tumor antigens, target switch receptors, target dominant-negative receptors, and / or target polypeptides that enhance immune function (e.g., T cell priming or T cell infiltration) can be introduced into cells using expression vectors (viral transfection). Provided herein are expression vectors (e.g., lentiviral vectors or retroviral vectors) that contain nucleic acids encoding target CARs, target engineered TCRs, target KIRs, target antigen-binding polypeptides, target cell surface receptor ligands, target tumor antigens, target switch receptors, target dominant-negative receptors, and / or target polypeptides that enhance immune function (e.g., T cell priming or T cell infiltration). Suitable expression vectors include lentiviral vectors, gamma retroviral vectors, foamy virus vectors, adeno-associated virus (AAV) vectors, adenoviral vectors, artificial hybrid viruses, naked DNA, including, but not limited to, transposon-mediated vectors such as Sleeping Beauty and Piggyback, and integrases such as Phi31. Other suitable expression vectors include herpes simplex virus (HSV) and retroviral expression vectors.

[0168] Adenovirus expression vectors are based on adenovirus, and have low integration ability into genomic DNA, but high transfection efficiency into host cells. Adenovirus expression vectors contain sufficient adenovirus sequences to: (a) support the packaging of expression vectors, and (b) ultimately express in host cells a target CAR, a target artificial TCR, a target KIR, a target antigen-binding polypeptide, a target cell surface receptor ligand, a target tumor antigen, a target switch receptor, a target dominant-negative receptor, and / or a target polypeptide that enhances immune function (e.g., T cell priming or T cell infiltration). In some embodiments, the adenovirus genome is a linear double-stranded DNA with a foreign DNA sequence of 36 kb. For example, a nucleic acid encoding a subject CAR, a subject engineered TCR, a subject KIR, a subject antigen-binding polypeptide, a subject cell surface receptor ligand, a subject tumor antigen, a subject switch receptor, a subject dominant negative receptor, and / or a subject polypeptide that enhances immune function (e.g., T cell priming or T cell infiltration) can be inserted in place of a larger piece of adenoviral DNA to generate an expression vector of the invention.

[0169] Another type of expression vector is based on the adeno-associated virus, which utilizes the adenovirus attachment system. These AAV expression vectors integrate frequently into the host genome. Because they can infect non-dividing cells, they are useful, for example, for gene transfer into mammalian cells in tissue culture or in vivo. AAV vectors have a broad host range of infection. Further details regarding the construction and use of AAV vectors are described in U.S. Patent Nos. 5,139,941 and 4,797,368.

[0170] Retroviral expression vectors integrate into the host genome, deliver large amounts of foreign genetic material, infect a wide range of species and cell types, and can be packaged in specialized cell lines. Retroviral vectors introduce nucleic acids (e.g., a target CAR, a target engineered TCR, a target KIR, a target antigen-binding polypeptide, a target cell surface receptor ligand, a target tumor antigen, a target switch receptor, a target dominant-negative receptor, and / or a target polypeptide that enhances immune function (e.g., T cell priming or T cell infiltration)) into specific locations in the viral genome and produce replication-deficient viruses. While retroviral vectors can infect a wide variety of cells, host cell division is required for the integration and stable expression of the target CAR, a target engineered TCR, a target KIR, a target antigen-binding polypeptide, a target cell surface receptor ligand, a target tumor antigen, a target switch receptor, a target dominant-negative receptor, and / or a target polypeptide that enhances immune function (e.g., T cell priming or T cell infiltration).

[0171] Lentiviral vectors are derived from lentiviruses, which are complex retroviruses that contain the common retroviral genes gag, pol, and env, as well as other genes with regulatory and structural functions. See, for example, U.S. Patent Nos. 6,013,516 and 5,994,136. Examples of lentiviruses include human immunodeficiency virus (HTV-1, HTV-2) and simian immunodeficiency virus (SIV). Lentiviral vectors have been engineered by multiple attenuation of HIV pathogenic genes, such as deletion of the env, vif, vpr, vpu, and nef genes, resulting in biologically safe vectors. Lentiviral vectors can infect non-dividing cells and can be used for both in vivo and ex vivo gene transfer and expression of nucleic acids encoding target CARs, target engineered TCRs, target KIRs, target antigen-binding polypeptides, target cell surface receptor ligands, target tumor antigens, target switch receptors, target dominant-negative receptors, and / or target polypeptides that enhance immune function (e.g., T cell priming or T cell infiltration). See, for example, U.S. Patent No. 5,994,136.

[0172] In some embodiments, nucleic acids encoding a subject CAR, a subject engineered TCR, a subject KIR, a subject antigen-binding polypeptide, a subject cell surface receptor ligand, a subject tumor antigen, a subject switch receptor, a subject dominant-negative receptor, and / or a subject polypeptide that enhances immune function (e.g., T cell priming or T cell infiltration) are introduced into immune cells by viral transduction. In some embodiments, the viral transduction comprises contacting immune cells with a viral vector comprising one or more nucleic acids. In some embodiments, the viral vector is selected from the group consisting of a retroviral vector, a Sendai virus vector, an adenoviral vector, an adeno-associated viral vector, and a lentiviral vector. Various markers that can be used are known in the art and can include hprt, neomycin resistance, thymidine kinase, hygromycin resistance, etc.

[0173] The modified concentrated apheresis products (e.g., immune cells) of the invention (e.g., a subject CAR, a subject engineered TCR, a subject KIR, a subject antigen-binding polypeptide, a subject cell surface receptor ligand, a subject tumor antigen, a subject switch receptor, a subject dominant-negative receptor, and / or an immune function (e.g., T cell priming or T cell infiltration)) can be produced by stably transfecting host cells (e.g., immune cells) with an expression vector comprising a nucleic acid of the present disclosure.

[0174] Transfected cells (i.e., immune cells) expressing nucleic acids encoding a CAR, KIR, TCR, KIR, antigen-binding polypeptide, cell surface receptor ligand, tumor antigen, targeted switch receptor, targeted dominant-negative receptor, and / or a polypeptide of interest that enhances immune function (e.g., T cell priming or T cell infiltration) of the present disclosure can be expanded in vitro. In some embodiments, transfected cells (i.e., immune cells) expressing nucleic acids encoding a CAR, KIR, TCR, KIR, antigen-binding polypeptide, cell surface receptor ligand, tumor antigen, targeted switch receptor, targeted dominant-negative receptor, and / or a polypeptide of interest that enhances immune function (e.g., T cell priming or T cell infiltration) of the present disclosure are not expanded in vitro.

[0175] Additional methods for generating modified cells of the present disclosure include, but are not limited to, chemical transformation methods (e.g., using calcium phosphate, dendrimers, liposomes, and / or cationic polymers), non-chemical transformation methods (e.g., electroporation, phototransformation, gene electrotransfer and / or hydrodynamic delivery), and / or particle-based methods (e.g., imparefection, gene gun and / or magnetofection). 2.Physical method

[0176] Physical methods for introducing polynucleotides (RNA or DNA) or expression vectors into host cells include lipofection, particle bombardment, microinjection, electroporation, etc. Expression vectors or polynucleotides can be introduced into target cells using commercially available methods, including electroporation, such as 4D-Nucleofector™ Technology (Lonza Bioscience, Walkersville, MD), Amaxa Nucleofector-II (Amaxa Biosystems, Cologne, Germany), ECM 830 (BTX) (Harvard Instruments, Boston, MA), Gene Pulser II (BioRad, Denver, CO), or Multiporator (Eppendorf, Hamburg, Germany). a. Electroporation

[0177] In some embodiments, the enriched apheresis product and / or enriched target cell population are transfected. In some embodiments, the enriched apheresis product and / or enriched target cell population are electroporated. In some embodiments, the cell transfection device comprises a flow electroporation chamber. For example, chambers or systems are described in U.S. Patent Nos. 5,720,921, 6,074,605, 7,141,425, 7,521,224, and 8,673,623, as well as U.S. Patent Application Publication Nos. 2007 / 0128708A1, 2008 / 0182251A1, 2013 / 0196441, and 2017 / 0233716A1, and Kim et al., Biosens Bioelectron 2008 23(9):1353-60.

[0178] Electroporation involves applying an electric field to cells, opening pores (electropores) in the cell membrane, allowing (usually charged) macromolecules or drugs to enter the cell. When the electric field is removed, the pores reseal, allowing the introduced molecules to enter the cell. Critical parameters for successful electroporation include the maximum applied voltage and the duration of the current pulse. Voltage and capacitance settings should also be optimized for each cell type, and the resistance of the electroporation buffer is important in selecting the initial setup of the device. Optimal stable and transient transformation occur with similar device settings, so transient expression can be used to optimize conditions when adapting to new cell types.

[0179] Thus, electroporation-mediated administration of nucleic acids, including expression constructs, into cells offers a means for delivering RNA of interest to target cells. Electroporation-mediated administration can utilize any of the many available devices and electroporation systems known to those skilled in the art. Exemplary formulations and methodologies for electroporating nucleic acid constructs into mammalian cells are taught in US 2004 / 0014645, US 2005 / 0052630, US 2005 / 0070841, US 2004 / 0059285, US 2004 / 0092907, and US 2007 / 0128708. The various parameters, including the electric field strength required for electroporation of any known cell type, are generally known in the relevant research literature, as well as numerous patents and applications in the field. See, for example, U.S. Patent Nos. 6,678,556, 7,171,264, and 7,173,116.

[0180] In some embodiments, the cell transfection device is a commercially available device for therapeutic applications of electroporation selected from, but not limited to, the MedPulser™ DNA Electroporation Therapy System (Inovio / Genetronics, San Diego, Calif.), and described in patents such as U.S. Pat. Nos. 6,567,694; 6,516,223; 5,993,434; 6,181,964; 6,241,701; and 6,233,401.

[0181] In some embodiments, cells are not activated before transfection process.In some embodiments, before transfection, cells can be generally activated and proliferated, for example, by using the method described in U.S. Patent No. 6,352,694;6,534,055;6,905,680;6,692,964;5,858,358;6,887,466;6,905,681;7,144,575;7,067,318;7,172,869;7,232,566;7,175,843;5,883,223;6,905,874;6,797,514;6,867,041;and US 2006 / 0121005.

[0182] In some embodiments, the majority of unstimulated CAR-T cells generated by lentiviral electroporation (e.g., nucleofection) can maintain a less differentiated phenotype compared to the stimulated CAR-T cell population. For example, at least about 30% or more, at least about 40% or more, at least about 45% or more, at least about 50% or more, at least about 55% or more, at least about 60% or more, at least about 65% or more, or at least about 70% or more of the electric CAR-T cells can be naive CAR-T cells. In some embodiments, electroporation of the CAR lentiviral vector can result in efficient integration of the CAR transgene into the T cell genome. In some embodiments, the vector copy number per cell of electric CAR-T cells can be substantially similar to that of conventional (transduced) CAR-T cells. In some embodiments, the electroporated CAR transgene can be expressed within about 0.5 hours, about 0.75 hours, about 1 hour, about 1.5 hours, about 2.0 hours, about 2.5 hours, about 3.0 hours, about 3.5 hours, or at least about 5.0 hours after nucleofection.

[0183] In some embodiments, the enriched apheresis product and / or enriched target cell population is transfected using, for example, 4D-Nucleofector™ Technology (Lonza Bioscience, Walkersville, MD), Amaxa NUCLEOFECTOR™-II (Amaxa Biosystems, Cologne, Germany), ECM 830 (BTX; Harvard Instruments, Boston, Massachusetts), Gene Pulser II or Gene Pulser MXCELL™ (BioRad, Denver, Colorado), Multiporator (Eppendort, Hamburg, Germany), or FLOW ELECTROPORATION® technology (MaxCyte). In some embodiments, the transfection device is an ECM830 Electro Square Wave Porator (Harvard Apparatus BTX), and cells are electroporated in a 2 mm cuvette (Harvard Apparatus BTX, Holliston, MA).

[0184] Those skilled in the art will understand that the type of pulse, pulse duration, voltage, and frequency of application will depend on the type of device and cell type; and that optimization of transfection efficiency can be adjusted based on the pulse type, pulse duration, voltage, frequency of application, and concentration of nucleic acid or particle (e.g., DNA, RNA, expression vector, lentiviral vector, or lentiviral particle) being electroporated.

[0185] There are two methods of electroporation: batch electroporation and flow-through electroporation. i. Batch electroporation

[0186] Electroporation involves the transfer of a relatively small volume (approximately 1 ml, often approximately 1 × 10 cells) of cells and a suspension of macromolecules to be transfected into a cuvette containing two electrodes connected to a pulse generator and positioned to pass an electric current through the suspension. 6 This is most often performed in a batch format (on a single cell), where one or more electric field pulses are applied to the cells, and the treated cells are typically transferred to culture medium and allowed to recover.

[0187] In one embodiment, a batch processing mode can be used in the methods and systems described herein. In this embodiment, cells are electroporated by dispensing a suspension of the washed enriched apheresis product or enriched target cell population into an electroporation chamber when a predetermined concentration of target cells is reached in the cell separation module (e.g., as detected by a detector), and then adding a cell-modifying or cell-customizing agent, such as a drug (e.g., a lentiviral vector, lentiviral particles, expression vector, DNA, RNA, or protein) to generate CART cells (as detected by a detector in the cell separation module), and applying one or more electric pulses from a pulse generator to the cell suspension. The electroporated cells can be reintroduced into the patient after each batch of cells is electroporated, or can be continuously introduced if continuous electroporation is used. ii. Flow-through electroporation

[0188] In some embodiments, a flow-through or continuous-flow electroporation system can be used. In this embodiment, washed cells (e.g., concentrated apheresis product or concentrated target cells) and modifying agents (e.g., lentiviral particles, expression vectors, lentiviral vectors, DNA, or RNA) are passed through an electroporation unit where a voltage is constantly applied. In some embodiments, a flow-through or continuous-flow electroporation system can process up to 20 ml of cell suspension per minute, with transfection efficiencies as high as 75%. In some embodiments, a flow-through or continuous-flow electroporation system can process 1 to 10 cells per second in a single transfection procedure, with efficiency rates ranging from about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% or higher. 10 cells per second 4 ~10 7 cells per second 5 ~10 8 cells, or 10 per second 6 ~10 9 cells, or 1 cell to 10 10 Processing of a range of cell batches is achieved.

[0189] In some embodiments, the transfection devices disclosed herein incorporate fluid channels with a constant depth and / or variable width across their length, narrower in some sections and wider in others. The electric field at any point is determined by the width of the channel, with narrower sections experiencing a stronger electric field than wider sections. The width and current are selected so that the electric field exceeds the transmembrane potential, enabling electroporation only at the narrow points. By alternating between narrow and wide sections along the length of the channel, an effect approximating that of a pulsed electric field can be achieved without the need for a pulsed electric field generator. The flow rate through the channel and the respective lengths of the wide and narrow sections can be adjusted to control the duration of exposure of cells to a current strong enough to electroporate. Continuous flow electroporation systems are described, for example, in Wei & Li, Methods Mol. Biol. 1121: 99-110 (2014), Geng et al., J. Controlled Release 144: 91-100 (2010), U.S. Patent Nos. 10,253,316; 6,617,154; 6,673,669; 7,029,916; 7,771,984; 9,546,350; or 10,253,316.

[0190] In some embodiments, the flow-through electroporation system within the transfection device is fabricated from polydimethylsiloxane (PDMS) on a glass substrate and uses a fluidic system incorporating alternating wide (10,000-5,000 μm, e.g., approximately 7,500 μm) and narrow (500-700 μm, e.g., approximately 500 μm) channels. The inlet of the device can be connected to a conduit or tubing through which cells are transported from a buffer exchange device to the transfection device. Wire electrodes inserted into the inlet and outlet of the flow-through electroporation device can be connected to a constant-voltage power supply. Cells suspended in electroporation buffer and containing a lentiviral vector and / or a nucleic acid encoding a CAR or TRC are pumped through the fluidic channels.

[0191] In some embodiments, the apheresis product is transfected with about 0.5 μl, about 1 μl, about 1.5 μl, about 2 μl, about 2.5 μl, about 3 μl, about 3.5 μl, about 4 μl, about 5 μl, about 6 μl, about 7 μl, about 8 μl, about 9 μl, about 10 μl, about 15 μl, or about 20 μl of lentiviral vector. In some embodiments, an effective amount of lentiviral vector is an MOI of about 0.01 to about 5.0, including a multiplicity of infection (MOI) of about 0.01, about 0.02, about 0.03, about 0.04, about 0.05, about 0.06, about 0.07, about 0.08, about 0.09, about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1.0, about 1.25, about 1.5, about 2.0, about 3.0, about 4.0, or about 5.0. In some embodiments, the apheresis product is transfected with about 2 ul of lentiviral vector at an MOI of about 0.08; 5 ul of lentiviral vector at an MOI of about 0.2; or 10 ul of lentiviral vector at an MOI of about 0.4.

[0192] In some embodiments, the electroporation module comprises a flow electroporation chamber.For example, the chamber or system described in U.S. Patent Nos. 5,720,921; 6,074,605; and 7,141,425.The chamber or system described in U.S. Patent Nos. 5,720,921; 6,074,605; and 7,141,425.

[0193] Another aspect of the present invention provides a novel method of electroporation.

[0194] Typically, in electroporation, cells are contacted with an effective amount of a lentiviral vector before applying electricity to a cuvette containing the cells to be transfected. However, the present inventors have found that electroporation is toxic to expression vectors and reduces transfection efficiency. Therefore, to increase electroporation efficiency, the present inventors electroporated cells in the absence of an expression vector and observed improved transfection efficiency. Therefore, in some embodiments of the present disclosure, cells can be contacted with an effective amount of a lentiviral vector before electroporation. In alternative embodiments, cells can be contacted with an effective amount of a lentiviral vector for up to about 4 hours after electroporation (e.g., application of electricity). For example, cells can be contacted with an effective amount of a lentiviral vector for at least about 5-30 minutes, at least about 25-50 minutes, at least about 5-60 minutes, at least about 5-12 minutes, at least about 60-120 minutes, or at least about 120-240 minutes after electroporation. Alternatively, the cells can be contacted with an effective amount of lentiviral vector for at least about 1 minute, at least about 2 minutes, at least about 5 minutes, at least about 10 minutes, at least about 20 minutes, at least about 30 minutes, at least about 45 minutes, at least about 60 minutes, at least about 75 minutes, at least about 90 minutes at least about 100 minutes, at least about 110 minutes, at least about 120 minutes, at least about 150 minutes, at least about 160 minutes, at least about 170 minutes, at least about 180 minutes, at least about 190 minutes, at least about 200 minutes, at least about 220 minutes, or at least about 240 minutes after electroporation.

[0195] Adding the lentiviral vector to cells up to 4 hours after electroporation (e.g., 1 minute to 2 hours, or 1 minute to 4 hours) can reduce the amount of lentiviral particles killed by electroporation, which can be toxic to the lentiviral particles. Therefore, adding the lentiviral vector to cells up to 4 hours after electroporation can enhance CAR transfection. For example, adding the lentiviral vector to cells up to 4 hours after electroporation can enhance CAR expression by approximately 10-15% compared to conventional electroporation processes (e.g., adding the lentivirus to cells before electroporation).

[0196] Thus, in some embodiments of the manufacturing methods disclosed herein, transfecting the cells comprises electroporating the cells with the lentiviral vector and / or particles. In some embodiments, electroporation comprises adding the lentiviral vector to the cells before, simultaneously with, or after applying electricity to the cells. In some embodiments, electricity is applied to the cells after adding the lentiviral vector. In some embodiments of the manufacturing processes disclosed herein, electricity is applied to the cells before adding the lentiviral vector. For example, electricity is applied to the cells for at least about 5-30 minutes, at least about 25-50 minutes, at least about 5-60 minutes, at least about 5-12 minutes, at least about 60-120 minutes, or at least about 120-240 minutes before adding the lentiviral vector. Alternatively, the cells are electrified for at least about 1 minute, at least about 2 minutes, at least about 5 minutes, at least about 10 minutes, at least about 20 minutes, at least about 30 minutes, at least about 45 minutes, at least about 60 minutes, at least about 75 minutes, at least about 90 minutes, at least about 100 minutes, at least about 110 minutes, at least about 120 minutes, at least about 150 minutes, at least about 160 minutes, at least about 170 minutes, at least about 180 minutes, at least about 190 minutes, at least about 200 minutes, at least about 220 minutes, or at least about 240 minutes prior to addition of the lentiviral vector. b. Cell squeeze microfluidics

[0197] In some embodiments, cell squeeze microfluidics is used to introduce an effective amount of lentiviral vectors into concentrated apheresis products. In some embodiments, lentiviral vectors are introduced into mononuclear cells by forcing the cells under pressure through a small diameter constriction. Rapid extension, rapid compression, or high shear rate pulses lead to the uptake of molecules from the surrounding cell medium into the cell cytoplasm. This so-called cell squeeze microfluidic technology is applicable to a wide range of cell types and is suitable for the introduction of substances into mononuclear cells. Cell squeeze microfluidic technology is described, for example, in WO 2013 / 059343 and US 2014 / 287509. 3.Chemical methods

[0198] Chemical means for introducing expression vectors into host cells include polymer complexes, nanocapsules, microspheres, beads, and colloidal dispersion systems such as lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. Chemical means for introducing polynucleotides into host cells include polymer complexes, nanocapsules, microspheres, beads, and colloidal dispersion systems such as lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., artificial membrane vesicle).

[0199] Regardless of the method used to introduce exogenous nucleic acid into host cells or otherwise expose cells to the inhibitors of the present invention, various tests can be performed to confirm the presence of the nucleic acid in the host cells. Such tests include, for example, molecular biological tests well known to those skilled in the art, such as Southern blotting and Northern blotting, RT-PCR and PCR; biochemical tests such as detecting the presence or absence of specific peptides (e.g., immunological means (ELISA and Western blot)), or tests described herein to identify agents within the scope of the present invention.

[0200] Furthermore, nucleic acids can be introduced by any means, such as transducing proliferating host cells (e.g., immune cells; concentrated apheresis products), transfecting proliferating host cells (e.g., immune cells; concentrated apheresis products), or electroporating proliferating host cells (e.g., immune cells; concentrated apheresis products). Some nucleic acids may be introduced into host cells (e.g., immune cells; concentrated apheresis products) by one method, and other nucleic acids may be introduced into host cells (e.g., immune cells; concentrated apheresis products) by another method. In some embodiments, expression systems such as lentiviral or retroviral particles can be introduced using viral transfection and chemical or physical transfection. For example, lentiviral or retroviral particles can be transfected into cells using electroporation. 4. Lentiviral Vector Transfection

[0201] In some embodiments, the methods described herein include transfecting the stimulated and / or unstimulated apheresis or blood product, or the concentrated apheresis or blood product, with one or more modifying agents. In some embodiments, the one or more modifying agents are selected from the group consisting of small molecule drugs, biologics, therapeutic agents, proteins, peptides, protein therapeutics, peptide therapeutics, nucleic acids, DNA, RNA, mRNA, chimeric antigen receptors, xenogeneic T cell receptors, expression vectors, viral vectors, vectors, retroviral vectors, lentiviral vectors, adenoviral vectors, and adeno-associated viral vectors. The modifying agent may include a virus that is not permissive for human or eukaryotic cells (e.g., a virus that cannot naturally infect or invade human or eukaryotic cells). In some embodiments, the modifying agent may also be selected from a retroviral vector or a lentiviral vector. In some embodiments, the modifying agent may be a retroviral vector, a lentiviral vector, an adenoviral vector, or an adeno-associated viral vector. In one embodiment, the modifying agent is a lentiviral vector or a retroviral vector. In some embodiments, the lentiviral vector is a lentiviral particle.

[0202] In some embodiments, the transfection is viral transfection (e.g., viral transduction), or the transfection is electroporation of a lentiviral vector comprising a nucleic acid sequence encoding a chimeric antigen receptor (CAR), an artificial T cell receptor (TCR), and / or a polypeptide that enhances immune cell function, or a functional derivative thereof.

[0203] In some embodiments, the transfection is selected from the group consisting of viral transfection (e.g., viral transduction), non-viral transfection, and / or a hybrid of viral and non-viral transfection. In some embodiments, the transfection is selected from the group consisting of electroporation, laser beam, gene injection, spinoculation, sonoporation, magentofection, metal-coated nanoparticles, magnetically coupled adeno-associated virus, micro / nanoparticle-mediated transfection, lipofection, lipid-based transfection, anionic liposome- or cationic liposome-mediated transfection, cationic polymers, polymer encapsulation, peptide-mediated transfection, calcium phosphate, dendrimers, flowfection, photoporation, solvation, transient cell membrane disruption, deformation, squeezing, stretching, pinching, weakening, elongation, thinning, biological particle delivery systems, and combinations thereof.

[0204] In some embodiments of the aforementioned methods, the cells are transduced by spinoculation. For example, transducing the non-spherical cell product with a viral vector includes subjecting the non-spherical cell product and the viral vector to centrifugal force to promote cell renewal of viral particles, thereby increasing transduction efficiency.

[0205] In some embodiments, the apheresis product (e.g., a population of immune cells, CD4 + cells and CD8 + In some embodiments, the apheresis product (e.g., a population of immune cells, CD4 + and CD8 +In some embodiments, the apheresis product (e.g., a population of immune cells, CD4 + cells and CD8 + In some embodiments, the apheresis product (e.g., a population of immune cells, CD4 + cells and CD8 + A population of cells, or a population of eukaryotic donor cells, is transfected by viral transfection and liposome-based transfection.

[0206] In some embodiments, the population of apheresis products (e.g., a population of immune cells, CD4 + cells and CD8 + Transfection of a population of cells, or a population of eukaryotic donor cells, with one or more modifying agents (e.g., nucleic acid sequences encoding chimeric antigen receptors (CARs), engineered T cell receptors (TCRs) and / or polypeptides that enhance immune cell function, or functional derivatives thereof) is performed simultaneously with stimulating and / or activating the apheresis product population with one or more stimulatory agents (e.g., cytokines, recombinant costimulatory molecules, anti-CD3 antibodies or fragments thereof, anti-CD28 antibodies or fragments thereof, small molecule drug inhibitors, and / or combinations thereof).

[0207] In some embodiments, the population of apheresis products (e.g., a population of immune cells, CD4 + cells and CD8 +Transfection of the population of cells, or population of eukaryotic donor cells, with one or more modifying agents (e.g., nucleic acid sequences encoding chimeric antigen receptors (CARs), engineered T cell receptors (TCRs), and / or polypeptides that enhance immune cell function, or functional derivatives thereof) occurs within 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, 4.5 hours, 5 hours, 5.5 hours, 6 hours, 6.5 hours, 7 hours, 7.5 hours, 8 hours, 8.5 hours, 9 hours, 9.5 hours, or 10 hours after initiation of stimulation and / or activation of the apheresis product with one or more stimulating agents as described above.

[0208] In some embodiments, the population of apheresis products (e.g., a population of immune cells, CD4 + cells and CD8 + Transfection of a population of cells, or a population of eukaryotic donor cells, with one or more modifying agents (e.g., a nucleic acid sequence encoding a chimeric antigen receptor (CAR), an engineered T cell receptor (TCR), and / or a polypeptide that enhances immune cell function, or a functional derivative thereof) occurs no later than 5 hours after initiation of stimulation and / or activation of the apheresis product with one or more stimulating agents described above.

[0209] In some embodiments, the population of apheresis products (e.g., a population of immune cells, CD4 + cells and CD8 + Transfection of the population of cells, or population of eukaryotic donor cells, with one or more modifying agents (e.g., nucleic acid sequences encoding chimeric antigen receptors (CARs), engineered T cell receptors (TCRs), and / or polypeptides that enhance immune cell function, or functional derivatives thereof) occurs no later than four hours after initiation of stimulation and / or activation of the apheresis product with one or more stimulating agents described above.

[0210] In some embodiments, the population of apheresis products (e.g., a population of immune cells, CD4 + cells and CD8 +The activation of the apheresis product (a population of cells, or a population of eukaryotic donor cells) with one or more modifying agents (e.g., a nucleic acid sequence encoding a chimeric antigen receptor (CAR), an engineered T cell receptor (TCR), and / or a polypeptide that enhances immune cell function, or a functional derivative thereof) occurs no later than three hours after initiation of stimulation and / or activation of the apheresis product with one or more stimulating agents described above.

[0211] In some embodiments, the population of apheresis products (e.g., a population of immune cells, CD4 + cells and CD8 + Transfection of the population of cells, or population of eukaryotic donor cells, with one or more modifying agents (e.g., nucleic acid sequences encoding chimeric antigen receptors (CARs), engineered T cell receptors (TCRs), and / or polypeptides that enhance immune cell function, or functional derivatives thereof) occurs no later than two hours after initiation of stimulation and / or activation of the apheresis product with one or more stimulating agents described above.

[0212] In some embodiments, the population of apheresis products (e.g., a population of immune cells, CD4 + cells and CD8 + Transfection of the population of cells, or population of eukaryotic donor cells, with one or more modifying agents (e.g., nucleic acid sequences encoding chimeric antigen receptors (CARs), engineered T cell receptors (TCRs), and / or polypeptides that enhance immune cell function, or functional derivatives thereof) occurs no later than one hour after initiation of stimulation and / or activation of the apheresis product with one or more stimulating agents described above.

[0213] In some embodiments, the population of apheresis products (e.g., a population of immune cells, CD4 + cells and CD8 +Transfection of the apheresis product (population of cells, or population of eukaryotic donor cells) with one or more modifying agents (e.g., nucleic acid sequences encoding chimeric antigen receptors (CARs), artificial T cell receptors (TCRs), and / or polypeptides that enhance immune cell function, or functional derivatives thereof) occurs without stimulation and / or activation of the apheresis product with one or more stimulating agents described above.

[0214] In some embodiments, the transfected apheresis product (e.g., a population of immune cells or a population of eukaryotic donor cells) comprises mononuclear cells, lymphocyte-rich cells, B lymphocytes, T lymphocytes, CD4 + T lymphocytes, CD8 + T lymphocytes, dendritic cells, monocytes, natural killer (NK) cells, natural killer T (NKT) cells, T regulatory cells, CD4 + T helper cells, CD8 + Cytotoxic T lymphocytes (CTL), CD62L + cells, CD 27 + cells, CCR7 + cells, CD45RO - cell CD45RA + cells, neutrophils, basophils, eosinophils, megakaryocytes, stem cells, hematopoietic stem cells (HSCs), hematopoietic progenitor cells (HPCs), CD34 + cells, CD34 + The cells are selected from the group consisting of peripheral blood stem cells, lymphocyte-activated killer cells (LAKs), tumor-infiltrating lymphocytes (TILs), mesenchymal stem cells, mast cells, monocytes, macrophages, neutrophils, basophils, eosinophils, dendritic cells, megakaryocytes, and combinations thereof.

[0215] In some embodiments, the concentration of the transfected apheresis product (e.g., a population of immune cells, CD4 + cells and CD8 + The concentration of the donor cells (population of cells, or population of eukaryotic donor cells) is at least about 0.7 x 10 7 , at least about 0.8 × 10 7 , at least about 0.9 × 10 7 , at least about 1 x 107 , at least about 2 × 10 7 , at least about 4 × 10 7 , at least about 6 × 10 7 , at least about 8 × 10 7 , at least about 1 x 10 8 , or at least about 5 × 10 8 In some embodiments, the apheresis product (e.g., a population of immune cells, such as CD4 + cells and CD8 + The concentration of the donor cells (population of cells, or population of eukaryotic donor cells) is approximately 0.5 × 10 6 cells / mL ~ approx. 4×10 6 The apheresis product (e.g., a population of immune cells, CD4 + cells and CD8 + The concentration of the donor cells (population of cells, or population of eukaryotic donor cells) was also approximately 0.5 × 10 6 cells / mL ~ approx. 1×10 8 In some embodiments, the concentration of the apheresis product may also be about 4.0 x 10 cells / mL. 6 cells / mL ~ approx. 1×10 8 It can be cells / mL.

[0216] In some embodiments of the methods disclosed herein, the method further comprises adding an adjuvant or transduction-enhancing reagent to the cell culture medium to enhance transfection (e.g., transduction) efficiency. In some embodiments, the adjuvant or transduction-enhancing reagent comprises a cationic polymer. In some embodiments, the adjuvant or transduction-enhancing reagent is selected from LentiBOOST™ (Sirion Biotech), Vectofusin-1, F108 (Poloxamer 338 or Pluronic® F-38), protamine sulfate, hexadimethrine bromide (Polybrene), PEA, Pluronic F68, Pluronic F127, Synperonic, or LentiTrans™. In some embodiments, the transduction-enhancing reagent is LentiBOOST™ (Sirion Biotech). In some embodiments, the transduction-enhancing reagent is F108 (Poloxamer 338 or Pluronic® F-38).

[0217] The manufacturing methods (e.g., electric CAR-T cells) disclosed herein are made possible by a novel strategy of transducing immune cells with lentiviral vectors (containing nucleic acids encoding CAR-TCRs and / or polypeptides that enhance immune cell function, or functional derivatives thereof).

[0218] CAR-T cell manufacturing methods rely on a hybrid transfection method that combines biological transfection (virus-based transduction), as described in Example 1 below, with physical transfection, such as electroporation. Specifically, lentiviral particles are electroporated into immune cells or T cells. Electroporation of lentiviral particles into cells speeds up viral transfection / transduction and enables one-day production of CAR-T cells (e.g., electro-CAR-T cells) without post-transfection culture and / or expansion. As shown in Figures 5-8 and 10, electroporated CAR-T cells can be harvested within a few hours. As further discussed in Examples 3 and 4 and shown in Figure 11 and Tables 4-6, such CAR-T cells (e.g., electro-CAR-T cells) efficiently killed target cells.

[0219] Thus, in some embodiments of the manufacturing processes disclosed herein, the apheresis product (e.g., a population of immune cells, CD4 + cells and CD8 + A population of cells, or a population of eukaryotic donor cells, is transfected with an effective amount of a lentiviral or retroviral vector, which may comprise a nucleic acid sequence encoding a chimeric antigen receptor (CAR), an artificial T cell receptor (TCR), and / or a polypeptide that enhances immune cell function, or a functional derivative thereof.

[0220] In some embodiments, manufacturing processes contemplated herein comprise transfecting the concentrated apheresis product with an effective amount of a lentiviral or retroviral vector at a multiplicity of infection (MOI) of about 0.01, about 0.02, about 0.03, about 0.04, about 0.05, about 0.06, about 0.07, about 0.08, about 0.09, about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1.0, about 1.25, about 1.5, about 2.0, about 3.0, about 4.0, or about 5. In some embodiments, manufacturing processes contemplated herein comprise transfecting the concentrated apheresis product with an effective amount of a lentiviral or retroviral vector at an MOI of about 10 or 20. In a preferred embodiment, the manufacturing process contemplated herein involves transfecting the concentrated apheresis product with an effective amount of a lentiviral or retroviral vector at an MOI of about 0.08, 0.2, or 0.4.

[0221] In some embodiments, the manufacturing process contemplated herein comprises transfecting the concentrated apheresis product with about 0.5, about 1, about 1.5, about 2, about 2.5, about 3, about 3.5, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 15, or about 20 μl of lentiviral vector at a multiplicity of infection (MOI) of 0.01 to about 20.0.

[0222] In some embodiments, the manufacturing process contemplated herein comprises transfecting the concentrated apheresis product with 2 μl of lentiviral or retroviral vector at an MOI of about 0.08, with 5 μl of lentiviral or retroviral vector at an MOI of about 0.2, or with 10 μl of lentiviral or retroviral vector at an MOI of about 0.4.

[0223] The lentiviral vector may be based on a virus selected from the group consisting of retrovirus, alpharetrovirus, betaretrovirus, gammaretrovirus, deltaretrovirus, and epsilonretrovirus. For example, the lentiviral vector may be based on human immunodeficiency virus (HIV), equine infectious anemia virus (EIAV), Visna-Medivirus (VMV), caprine arthritis-encephalitis virus (CAEV), feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), Visna virus, and simian immunodeficiency virus (SIV). In some embodiments, the lentiviral vector may be pseudotyped with an envelope glycoprotein (Env) from a virus selected from the group consisting of murine leukemia virus (MLV), vesicular stomatitis virus (VSV) Indiana strain, VSV New Jersey strain, Cocal virus, Chandipura virus, Piry virus, spring of carp virus (SVCV), Sigma virus, infectious hematopoietic necrosis virus (IHNV), Mokola virus, rabies virus, CVS virus, Isfahan virus, Alagoas virus, Calchaqui virus, Jurona virus, La Joya virus, Maraba virus, feline endogenous retrovirus (RD114) envelope protein, Perinet virus, Yug Bugdanovac virus, prototype foamy virus (PFV), and gibbon ape leukemia virus (GaLV). In some embodiments, the lentiviral vector may be pseudotyped with an envelope glycoprotein (Env) selected from the group consisting of vesicular stomatitis virus (VSV) Indiana strain, VSV New Jersey strain, and Cocal virus.

[0224] In some embodiments, the manufacturing process contemplated herein comprises transfecting the concentrated apheresis product with 2 μl of lentiviral or retroviral vector at an MOI of about 0.08, 5 μl of lentiviral or retroviral vector at an MOI of about 0.2, or 10 μl of lentiviral or retroviral vector at an MOI of about 0.4.

[0225] In some embodiments, the lentiviral vector comprises a heterologous viral envelope protein (Env) selected from the group consisting of Indiana strain VSV-G, New Jersey strain VSV-G, Cocal virus envelope protein, Isfahan virus envelope protein, Chandipura virus envelope protein, Pilibiri virus envelope protein, murine leukemia virus (MLV) envelope glycoprotein, SVCV virus envelope protein, and mutants thereof.

[0226] In some embodiments, the lentiviral vector comprises a nucleotide sequence encoding a VSV-G envelope protein or a VSV G protein variant.

[0227] In some embodiments of the manufacturing processes disclosed herein, the apheresis product (e.g., a population of immune cells, CD4 + cells and CD8 + A population of cells, or a population of eukaryotic donor cells, is transfected with an effective amount of a lentiviral or retroviral vector containing the VSV G envelope protein. E. Ex vivo culture after transfection: activation and stimulation

[0228] In another embodiment, the methods disclosed herein involve the production of a population of transfected cells (e.g., a population of modified lymphocytes, a population of modified immune cells, a modified CD4 + and CD8 +a population of cells, or a population of modified eukaryotic donor cells) with one or more stimulatory agents and activated to produce a population of activated cells (e.g., a population of activated modified immune cells, activated modified CD4 + and CD8 + The method further comprises producing a population of cells, or a population of activated modified eukaryotic cells.

[0229] In yet another embodiment, the methods disclosed herein include administering to a patient a population of activated modified immune cells, a population of activated modified monocytes, an activated modified CD4 + and CD8 + The population of cells, or the population of activated modified eukaryotic donor cells, is cultured and / or expanded for a predetermined period of time to produce a population of engineered cells or an engineered CD4 + and CD8 + The method further includes producing a population of cells.

[0230] In some embodiments, the expansion step is performed under shaking or rotating conditions. In some embodiments, the expansion step is performed in a closed system. In some embodiments, the expansion step is performed using a serum-free culture medium and / or in the presence of one or more stimuli described herein. In some embodiments, the expansion step is performed in the presence of one or more stimuli described herein.

[0231] In some embodiments, a population of activated apheresis products (e.g., a population of activated modified immune cells, activated modified CD4 + and CD8 + The population of cells, and / or the population of activated modified eukaryotic donor cells) is expanded by at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, or at least about 25-fold compared to the population of cells before or immediately after transfection.

[0232] In some embodiments, the population of cells is expanded by about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, or about 60% or less compared to the population of cells before or immediately after transfection, e.g., as assessed by viable cell count. In some embodiments, the population of cells is expanded by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, or about 40% or less compared to the population of cells before or immediately after transfection, e.g., as assessed by viable cell count.

[0233] In some embodiments, the population of cells does not proliferate for more than about 1 hour, about 1.5 hours, about 2 hours, about 2.5 hours, about 3 hours, about 3.5 hours, about 4 hours, about 4.5 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 16 hours, about 20 hours, about 24 hours, about 36 hours, about 48 hours, about 55 hours, about 60 hours, about 65 hours, about 70 hours, about 72 hours, about 80 hours, about 90 hours, or about 96 hours, as assessed by viable cell count.

[0234] In some embodiments, during the culturing and expansion steps, the population of transfected cells is contacted in vitro with an agent that stimulates the CD3 / TCR complex (e.g., an anti-CD3 antibody) and / or an agent that stimulates a costimulatory molecule (e.g., an anti-CD28 antibody), and / or a cell surface growth factor receptor. In some embodiments, the population of transfected cells is stimulated throughout the entire expansion period. In some embodiments, the population of transfected cells is stimulated for at least about 20 hours, at least about 21 hours, at least about 22 hours, at least about 23 hours, at least about 24 hours, at least about 25 hours, at least about 26 hours, at least about 27 hours, or at least about 28 hours. In some embodiments, the population of transfected cells is cultured and expanded in medium that is about 0%, about 0.5%, about 1%, about 1.5%, about 2%, about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, about 5%, about 5.5%, about 6%, about 6.5%, about 7%, about 7.5%, or about 8% or more serum-free. In some embodiments, the cytokine processes provided herein are carried out in cell culture medium that includes an LSD1 inhibitor, a MALT1 inhibitor, or a combination thereof.

[0235] In some embodiments, the population of cells (i.e., the apheresis product) produced by the methods disclosed herein exhibits a higher proportion of naive immune cells among the CAR-expressing cells. For example, the proportion of naive immune cells among the CAR-expressing cells can be at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, or at least about 60% higher compared to cells produced by conventional methods of producing CAR T cells.

[0236] In some embodiments of the methods disclosed herein, the transfected population (e.g., a population of modified lymphocytes, a population of modified immune cells, a modified CD4 + and CD8 + In some embodiments, the population of cells (e.g., a population of modified immune cells, modified CD4 + cells and CD8 + In some embodiments, the population of cells (e.g., a population of modified immune cells, modified CD4 donor cells) is not activated with one or more stimulatory agents after transfection. + cells and CD8 + The population of cells, or the population of modified eukaryotic donor cells, is also not expanded in vitro after transfection. In that embodiment, the transfected population of cells is harvested within 24 hours of transfection. In this embodiment, an enriched population of cells (e.g., a population of immune cells, CD4 + cells and CD8 + The population of cells, or population of eukaryotic donor cells, may be stimulated and / or activated with one or more stimulatory agents prior to transfection. F. Collection

[0237] In another embodiment, the methods disclosed herein involve cryopreserving or administering a modified apheresis product population (e.g., a modified lymphocyte population, a modified immune cell population, a modified CD4 + and CD8 + The method further comprises the step of harvesting the population of cells, or the population of modified eukaryotic donor cells).

[0238] In some embodiments, the harvesting is performed using engineered lymphocytes, engineered immune cells, engineered CD4 + and CD8+ In some embodiments, the harvesting includes selecting and enriching the donor eukaryotic cells, or engineered donor eukaryotic cells, for cryopreservation or administration to a subject in need thereof, such as artificial lymphocytes, artificial immune cells, artificial CD4 + and CD8 + The method further includes formulating the cells, or artificial donor eukaryotic cells.

[0239] In some embodiments, a transfected apheresis product (e.g., a population of modified lymphocytes, a population of modified immune cells, a modified CD4 + cells and CD8 + If the transfected apheresis product (e.g., a population of modified lymphocytes, a population of modified immune cells, a modified CD4 + cells and CD8 + The modified donor cells (population of cells, or population of modified eukaryotic donor cells) may be cultured for a predetermined period of time before harvesting, such as a population of modified lymphocytes, a population of modified immune cells, a modified CD4 + and CD8 + The population of cells, or population of modified eukaryotic donor cells, may be an engineered population of desired cells (e.g., an engineered population of lymphocytes, an engineered population of immune cells, an engineered CD4 + and CD8 + The population of cells, or population of engineered eukaryotic donor cells, can be cultured for a predetermined period of time before being harvested.

[0240] In some embodiments, the predetermined growth time can be about 24 hours or less, about 30 hours or less, about 48 hours or less, about 72 hours or less, about 96 hours or less, or about 120 hours or less. In some embodiments, the predetermined growth time can be less than about 0.5 hours, less than about 1 hour, less than about 2 hours, less than about 3 hours, less than about 4 hours, less than about 5 hours, less than about 6 hours, less than about 7 hours, less than about 8 hours, less than about 9 hours, less than about 10 hours, less than about 11 hours, less than about 12 hours, less than about 13 hours, less than about 14 hours, less than about 15 hours, less than about 16 hours, less than about 17 hours, less than about 18 hours, less than about 19 hours, less than about 20 hours, less than about 21 hours, less than about 22 hours, or less than about 23 hours.

[0241] In some embodiments, the predetermined growth time can be about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, or more days.

[0242] In some embodiments of the methods of producing a population of engineered immune cells disclosed herein, the apheresis product (e.g., a population of immune cells, CD4 + and CD8 + Enrich and / or obtain a population of cells, or a population of eukaryotic donor cells, and then use engineered cells (e.g., engineered lymphocytes, engineered immune cells, engineered CD4 + cells and CD8 + The time to harvesting the cells, or engineered eukaryotic donor cells) can be about 12 hours or less, about 18 hours or less, about 20 hours or less, about 22 hours or less, about 24 hours or less, about 26 hours or less, about 28 hours or less, about 30 hours or less, about 32 hours or less, about 36 hours or less, about 40 hours or less, about 45 hours or less, about 48 hours or less, about 50 hours or less, about 55 hours or less, about 60 hours or less, about 65 hours or less, about 70 hours or less, or about 72 hours or less.

[0243] In some embodiments, the apheresis product (e.g., a population of immune cells, CD4 + and CD8 +Enrich and / or obtain a population of cells, or a population of eukaryotic donor cells, and then use engineered cells (e.g., engineered lymphocytes, engineered immune cells, engineered CD4 + cells and CD8 + The time to harvest the cells (or engineered eukaryotic donor cells) can be from about 18 hours to about 72 hours, from about 18 hours to about 36 hours, from about 18 hours to about 24 hours, from about 24 hours to about 72 hours, from about 24 hours to about 36 hours, or from about 36 hours to about 72 hours.

[0244] In some embodiments, the apheresis product (e.g., a population of immune cells, CD4 + and CD8 + Enrich and / or obtain a population of cells, or a population of eukaryotic donor cells, and then use engineered cells (e.g., engineered lymphocytes, engineered immune cells, engineered CD4 + cells and CD8 + The time to harvest of the cells, or engineered eukaryotic donor cells) can be less than about 2 hours, less than about 3 hours, less than about 4 hours, less than about 5 hours, less than about 6 hours, less than about 7 hours, less than about 8 hours, less than about 9 hours, less than about 10 hours, less than about 11 hours, less than about 12 hours, less than about 13 hours, less than about 14 hours, less than about 15 hours, and can be less than about 16 hours, less than about 17 hours, less than about 18 hours, less than about 19 hours, less than about 20 hours, less than about 21 hours, less than about 22 hours, less than about 23 hours, less than about 24 hours, less than about 30 hours, less than about 35 hours, less than about 40 hours, less than about 45 hours, less than about 50 hours, less than about 55 hours, less than about 60 hours, less than about 65 hours, less than about 70 hours, or less than about 72 hours.

[0245] In some embodiments, the apheresis product (e.g., a population of immune cells, CD4 + and CD8 + Enrich and / or obtain a population of cells, or a population of eukaryotic donor cells, and then use engineered cells (e.g., engineered lymphocytes, engineered immune cells, engineered CD4 + cells and CD8 +The time to harvest of the apheresis product (e.g., a population of immune cells, CD4+, or engineered eukaryotic donor cells) can be, for example, about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, or more. In some embodiments, the apheresis product (e.g., a population of immune cells, CD4+, or engineered eukaryotic donor cells) can be harvested in about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, or more. + cells and CD8 + Enrich and / or obtain a population of cells, or a population of eukaryotic donor cells, and then use engineered cells (e.g., engineered lymphocytes, engineered immune cells, engineered CD4 + cells and CD8 + The time to harvest the cells, or engineered eukaryotic donor cells) can be about 1 day, about 3 days, about 4 days, about 5 days, or about 6 days.

[0246] In some embodiments, the electroporation, activation, and / or expansion steps are performed in a closed system, a semi-closed system, and / or a functionally closed system. The manufacturing processes disclosed herein can be performed in a closed system where manual manipulation is limited and therefore the potential for contamination is minimized. Thus, a closed system can minimize the risk of contamination (e.g., environmental contamination). In some embodiments, T cell isolation, activation, transfer, incubation, and washing are all performed in a closed system. In some embodiments of the methods disclosed herein, the methods are performed in separate devices. In some embodiments, T cell isolation, activation and transfer, incubation, and washing are performed in separate devices. In some embodiments, the closed system is selected from the group consisting of a closed bag system, an automated closed cell sample processing system, and a bioreactor (e.g., Xuri™ Cell Expansion System W25 - Girgin Ltd (or any GE Healthcare WAVE Bioreactor™ technology), etc.).

[0247] In certain embodiments, the closed system is a closed-bag culture system using any suitable cell culture bag (e.g., Mitenyi Biotec MACS® GMP Cell Differentiation Bags, Origen Biomedical PermaLife™ Cell Culture bags, or Origen PermaLife™ PL240 bags). In some embodiments, the cell culture bag used in the closed-bag culture system is coated with recombinant human fibronectin protein during the transduction process. In certain embodiments, the cell culture bag used in the closed-bag culture system is coated with a recombinant human fibronectin protein fragment during the transduction process. The recombinant human fibronectin fragment may contain three functional domains: a central cell-binding domain, a heparin-binding domain II, and a CS1 sequence. Recombinant human fibronectin protein or a fragment thereof may be used to enhance the gene efficiency of retroviral transduction of immune cells by assisting in colocalization of the viral vector with the target cell. In certain embodiments, the recombinant human fibronectin fragment is RetroNectin® (Takara Bio, Japan). In certain embodiments, cell culture bags can be coated with recombinant human fibronectin fragments at a concentration of about 1 to 60 μg / mL, preferably 1 to 40 μg / mL, or about 1 to 20 μg / mL, 20 to 40 μg / mL, or 40 to 60 μg / mL.

[0248] In some embodiments of the methods disclosed herein, the concentrated apheresis product (e.g., T cells) is stimulated and / or activated and transfected in a cell culture flask containing a gas-permeable membrane at the bottom that supports a large volume of medium without substantially compromising gas exchange. In some embodiments, cell growth is achieved by convection, providing substantially uninterrupted access to nutrients. III. Lentiviral Vectors

[0249] One aspect of the present disclosure provides a lentiviral vector as described herein.

[0250] Another aspect of the present disclosure provides a lentiviral vector comprising a polynucleotide sequence encoding at least one heterologous viral envelope protein derived from a virus; a polynucleotide sequence encoding at least one viral rev protein; a polynucleotide sequence encoding at least one viral gag protein and at least one viral pol protein; and / or a polynucleotide sequence encoding a chimeric antigen receptor or an artificial T-cell receptor (TCR).

[0251] The lentiviral vector may be based on a virus selected from the group consisting of retrovirus, alpharetrovirus, betaretrovirus, gammaretrovirus, deltaretrovirus, and epsilonretrovirus. For example, the lentiviral vector may be based on human immunodeficiency virus (HIV), equine infectious anemia virus (EIAV), Visna-Medivirus (VMV), caprine arthritis-encephalitis virus (CAEV), feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), Visna virus, and simian immunodeficiency virus (SIV). In some embodiments, the lentiviral vector may be pseudotyped with an envelope glycoprotein (Env) from a virus selected from the group consisting of murine leukemia virus (MLV), vesicular stomatitis virus (VSV) Indiana strain, VSV New Jersey strain, Cocal virus, Chandipura virus, Piry virus, spring of carp virus (SVCV), Sigma virus, infectious hematopoietic necrosis virus (IHNV), Mokola virus, rabies virus CVS virus, Isfahan virus, Alagoas virus, Calchaqui virus, Jurona virus, La Joya virus, Maraba virus, feline endogenous retrovirus (RD114) envelope protein, Perinet virus, Yug Bugdanovac virus, prototype foamy virus (PFV), and gibbon ape leukemia virus (GaLV). In some embodiments, the lentiviral vector may be pseudotyped with an envelope glycoprotein (Env) selected from the group consisting of vesicular stomatitis virus (VSV) Indiana strain, VSV New Jersey strain, and Cocal virus.

[0252] In some embodiments of the lentiviral vectors described herein, the viral envelope protein (Env) comprises a VSV-G glycoprotein selected from the group consisting of Indiana strain VSV-G, New Jersey strain VSV-G, Kokar virus envelope protein, Isfahan virus envelope protein, Chandipura virus envelope protein, Pili virus envelope protein, murine leukemia virus (MLV) envelope glycoprotein, SVCV virus envelope protein, and mutants thereof. The lentiviral vector may also comprise a nucleotide sequence encoding a heterologous VSV-G envelope protein.

[0253] The heterologous VSV G envelope protein may be codon-optimized for expression in humans, or may be a VSV G protein variant.

[0254] In some embodiments, the lentiviral vector comprises a nucleotide sequence encoding a VSV-G envelope protein or a VSV G protein variant.

[0255] In some embodiments of the lentiviral vectors described herein, the heterologous envelope protein may be under the control of a transcriptional regulatory element, which may be a promoter selected from a eukaryotic promoter or an constitutive promoter.

[0256] The lentiviral vectors described herein may further comprise a transcriptional regulatory element, which may be upstream of the heterologous envelope glycoprotein (i.e., in the 5' direction of the nucleotide sequence encoding the heterologous envelope glycoprotein). For example, the transcriptional regulatory element may control the expression (i.e., transcription and, accordingly, but optionally, translation) of the nucleic acid encoding the heterologous envelope glycoprotein. In some embodiments, the transcriptional regulatory element is a constitutively active or essential promoter. In exemplary embodiments, the constitutively active transcriptional regulatory element or essential promoter can be a cytomegalovirus (CMV) promoter, e.g., a CMV major immediate early promoter (CMV IE1), a murine stem cell virus promoter, an elongation factor-1 alpha promoter (EF-1α), a viral simian virus 40 (SV40) (e.g., early or late), a Moloney murine leukemia virus (MoMLV), a ubiquitin C promoter, a phosphoglycerokinase (PGK) promoter, a Rous sarcoma virus (RSV), or a herpes simplex virus (HSV) (thymidine kinase) promoter.

[0257] In other embodiments, the activity of the transcriptional regulatory element may be inducible, or the promoter may be an inducible promoter. In some embodiments, the transcriptional regulatory element may be a eukaryotic promoter, such as a phosphoglycerate kinase promoter. Other transcriptional regulatory elements, including prokaryotic and eukaryotic, constitutive and inducible promoters, and replication origins, are described, for example, in MOLECULAR CLONING: A LABORATORY MANUAL (Joseph F. Sambrook and David W. Russell, eds.; 3rd ed.; Vols. 1, 2, and 3; Cold Spring Harbor Laboratory Press; 2001) and MOLECULAR CLONING: A LABORATORY MANUAL (Michael R. Green and Joseph F. Sambrook, eds.; 4th ed.; Vols. 1, 2, and 3; Cold Spring Harbor Laboratory Press; 2012).

[0258] In some embodiments, the lentiviral vectors described herein can be structured and positioned such that expression of proteins, enzymes, and viral elements (i.e., cis- and trans-acting genes) necessary for producing retroviral particles is under the control of transcriptional regulatory elements. In preferred embodiments, the lentiviral vector can further comprise a transcriptional regulatory element, which is upstream of the proteins, enzymes, and viral elements (i.e., in the 5' direction) necessary for producing retroviral particles. Optionally, the transcriptional regulatory element controls the expression (i.e., transcription or translation) of nucleic acids (i.e., cis- and trans-acting genes) encoding the proteins, enzymes, and viral elements necessary for producing retroviral particles. In some embodiments, the transcriptional regulatory element can be constitutively active or a constitutive promoter.

[0259] In some embodiments, nucleic acids encoding the lentiviral vectors and heterologous envelope proteins described herein can be amplified or produced prior to introduction into producer cells and, accordingly, prior to production of viral particles. In some embodiments, nucleic acids encoding lentiviral vectors and other proteins, enzymes, and elements required for retroviral particle production can be amplified or produced prior to introduction into producer cells and, accordingly, prior to production of retroviral proteins.

[0260] In some embodiments, lentiviral vectors and nucleic acids encoding heterologous envelope proteins can be structured and arranged such that transcriptional control elements drive transcription, and thus translation, of the heterologous envelope protein in producer cells to facilitate production of lentiviral particles. In some embodiments, lentiviral vectors and nucleic acids encoding proteins, enzymes, and viral elements (i.e., cis- and trans-acting genes including rev and gag / pol) necessary for production of retroviral particles can be structured and arranged such that transcriptional control elements can drive transcription, and thus translation, of proteins, enzymes, and viral elements (i.e., cis- and trans-acting genes including rev and gag / pol). Transcriptional control elements can be structured and arranged to drive transcription, and thus translation, of proteins, enzymes, and viral elements (i.e., cis- and trans-acting genes including rev and gag / pol) in producer cells such that the producer cells produce retroviral particles. In some embodiments of the manufacturing processes disclosed herein, the apheresis product (e.g., a population of immune cells, CD4 + cells and CD8 + A population of cells, or a population of eukaryotic donor cells, is transfected with an effective amount of a lentiviral or retroviral vector comprising a chimeric antigen receptor (CAR) or engineered TCR as described. In some embodiments, the lentiviral vector is a lentiviral vector particle. A. Lentivirus

[0261] Vectors derived from retroviruses, such as lentiviruses, are suitable tools for achieving long-term gene transfer because they can stably integrate transgenes and propagate in daughter cells. Compared to vectors derived from oncoretroviruses, such as murine leukemia viruses, lentiviral vectors have the advantage of being able to transduce non-proliferating cells, such as hepatocytes. They also have the advantage of being less immunogenic. Retroviral vectors may also be, for example, gammaretroviral vectors. Gammaretroviral vectors may contain, for example, a promoter, a packaging signal (ψ), a primer binding site (PBS), one or more (e.g., two) long terminal repeats (LTRs), and a transgene of interest, such as a gene encoding a CAR. Gammaretroviral vectors may lack viral structural genes, such as gag, pol, and env. Exemplary gammaretroviral vectors include Murine Leukemia Virus (MLV), Spleen-Focus Forming Virus (SFFV), Myeloproliferative Sarcoma Virus (MPSV), and vectors derived therefrom. Other gammaretroviral vectors are described, for example, in Tobias Maetzig et al., "Gammaretroviral Vectors: Biology, Technology and Application," Viruses. 2011 Jun; 3(6):677-713. Retroviruses provide a convenient platform for gene transfer systems. A gene of choice can be inserted into a vector and packaged into retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to cells of a subject either in vivo or ex vivo. Many retroviral systems are known in the art. In some embodiments, adenoviral vectors are used. Numerous adenoviral vectors are known in the art. In some embodiments, lentiviral vectors are used.

[0262] In some embodiments, the lentiviral vector is based on a virus selected from the group consisting of retroviral vectors, alpharetroviral vectors, betaretroviral vectors, gammaretroviral vectors, deltaretroviral vectors, and epsilonretroviral vectors. In some embodiments, the lentiviral vector is based on human immunodeficiency virus (HIV), equine infectious anemia virus (EIAV), Visna-Medivirus (VMV), caprine arthritis-encephalitis virus (CAEV), feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), VISNA virus, and simian immunodeficiency virus (SIV). In one embodiment, the viral vector is derived from EIAV. EIAV has the simplest genome structure of all lentiviruses.

[0263] The RNA encapsidation determinant of feline immunodeficiency virus (FIV) has been shown to be discontinuous, consisting of a region (R-U5) at the 5' end of the genomic mRNA and another region mapped to the proximal 311 nt of gag.

[0264] In some embodiments of the manufacturing methods disclosed herein, the lentiviral vector comprises a heterologous viral envelope protein (Env) selected from the group consisting of Indiana strain VSV-G, New Jersey strain VSV-G, Coca-Cola vecicloviral envelope protein, Isfahan virus envelope protein, Chandipura virus envelope protein, Pilibiriviral envelope protein, murine leukemia virus (MLV) envelope glycoprotein, SVCV virus envelope protein, and mutants thereof. B. Pseudotyping Lentiviral Vectors

[0265] Viral envelope proteins (env) determine the range of host cells that can ultimately be infected and transformed by the recombinant retroviruses produced from the cell line. In the case of lentiviruses such as FflV-1, FflV-2, SIV, FIV, and EIV, env proteins include gp41 and gp120. Preferably, the viral env proteins expressed by the packaging cells of the present disclosure are encoded on a vector separate from the viral gag and pol genes.

[0266] Examples of the envelope gene from retrovirus that can be used in the present disclosure include, but are not limited to, MLV envelope, 10A1 envelope, BAEV, FeLV-B, RD114, SSAV, Ebola, Sendai, FPV (fowl plague virus) and influenza virus envelope.In some embodiments, the envelope gene from retrovirus is selected from the gene encoding the envelope protein from RNA virus selected from Picornaviridae, Calciviridae, Astroviridae, Togaviridae, Flaviviridae, Coronaviridae, Paramyxoviridae, Rhabdoviridae, Filoviridae, Orthomyxoviridae, Bunyaviridae, Arenaviridae, Reoviridae, Birnaviridae, Retroviridae). In some embodiments, the env gene from a retrovirus is selected from genes encoding envelope proteins from a DNA virus selected from the families Hepadnaviridae, Circoviridae, Parvoviridae, Papoviridae, Adenoviridae, Herpesviridae, Poxyviridae, and Iridoviridae.

[0267] In some embodiments, the env gene from a retrovirus is selected from alfalfa mosaic virus (AMV), human papillomavirus (HPV), white spot syndrome virus (WDSV), Semliki Forest virus (SFV), rabies, avian leukosis virus (ALV), bovine immunodeficiency virus (BIV), bovine leukemia virus (BLV), Epstein-Barr virus (EBV), squirrel monkey retrovirus (SMRV), equine infectious anemia virus (EIAV), feline leukemia virus (FeLV), caprine arthritis encephalitis virus (CAEV), Sin Nombre virus (SNV), human T-cell lymphotropic virus (HTLV), simian T-cell leukemia viruses (STLVs), Venezuelan equine encephalitis virus (VEEV), Mason-Pfizer monkey virus (M-MMV), avian carcinoma virus MH2, avian encephalomyelitis virus (AEV), V-crk sarcoma virus CT10, and respiratory syncytial virus (RSV).

[0268] In some embodiments, envelope proteins for pseudotyping the lentiviruses of the present disclosure include, but are not limited to, any of the following viruses: H1N1, H1N2, H3N2, H5N Influenza A viruses such as avian influenza 1 (avian influenza), influenza B viruses, influenza C viruses, hepatitis A viruses, hepatitis B viruses, hepatitis C viruses, hepatitis D viruses, hepatitis E viruses, rotaviruses, viruses of the Norwalk virus group, enteric adenoviruses, parvoviruses, dengue virus, monkeypox, Mononegavirales, lyssaviruses such as rabies virus, Lagos bat virus, Mokola virus, Dubenhage virus, European bat virus 1 and 2, Australian bat virus, ephemeloviruses such as vesicular virus, vesicular stomatitis virus (VSV), herpesviruses such as herpes simplex virus 1 and 2, varicella zoster, cytomegalovirus, Epstein-Barr virus (EBV), human herpesvirus (HHV), human herpesvirus 6 and 8, human immunodeficiency virus (HIV), and human immunodeficiency virus (HIV). All viruses (HIV), papillomaviruses, murine gammaherpesviruses, arenaviruses such as Argentine hemorrhagic fever virus, Bolivian hemorrhagic fever virus, Sabia-associated hemorrhagic fever virus, Venezuelan hemorrhagic fever virus, Lassa fever virus, Machupo virus, and lymphocytic chorionitis virus (LCMV), Bunyaviridae such as Crimean-Congo hemorrhagic fever virus, Hantaviruses, viruses causing hemorrhagic fever with renal syndrome, Rift Valley fever virus, Filoviridae (filoviruses) such as Ebola hemorrhagic fever and Marburg hemorrhagic fever, Flaviviridae (Kaisanur Forest disease virus, Omsk hemorrhagic fever virus), tick-borne encephalitis virus, Paramyxoviridae (hendra virus, Nipah virus), alphaviruses such as major and minor variola virus (smallpox), Venezuelan equine encephalitis virus, eastern equine encephalitis virus, and western equine encephalitis virus, SARS-associated coronavirus (SARS-CoV), West Nile virus, and all encephalitis-causing viruses.

[0269] In some embodiments, lentiviral vectors can be pseudotyped with any molecule. In some embodiments, the lentiviral vectors of the present disclosure are pseudotyped with an envelope glycoprotein (Env) selected from the group consisting of murine leukemia virus (MLV), chimeric envelope glycoprotein mutants derived from MLV, vesicular stomatitis virus G glycoprotein (VSV-G), prototype foamy virus (PFV) modified envelope, and chimeric envelope glycoprotein mutants derived from gibbon ape leukemia virus (GaLV).

[0270] In some embodiments, the lentiviral vector is pseudotyped with an envelope glycoprotein (Env) viral vector selected from the group consisting of murine leukemia virus (MLV), vesicular stomatitis virus (VSV) Indiana strain, VSV New Jersey strain, Cocarveciclovirus, Chandipura virus, Pirie virus, carp virus (SVCV), sigma virus, infectious hematopoietic necrosis virus (IHNV), Mokola virus, rabies virus CVS virus, Isfahan virus, Alagoas virus, Calchaqui virus, Jurona virus, La Joya virus, Maraba virus, Perine virus, Yugubgdanova virus, prototype foamy virus (PFV), and gibbon ape leukemia virus (GaLV).

[0271] In some embodiments, the Env protein may be a modified Env protein, such as a mutant or engineered Env protein. The modifications may be made or selected to introduce targeting capabilities, reduce toxicity, or for another purpose. The Env protein may be a modified Env protein, such as a mutant or engineered Env protein. The modifications may be made or selected to introduce targeting capabilities, reduce toxicity, or for another purpose. 1.VSV-G

[0272] The envelope glycoprotein (G) of vesicular stomatitis virus (VSV), a rhabdovirus, is an envelope protein that has been shown to pseudotype certain enveloped viruses and viral vector virions. The ability of VSV-G to pseudotype MoMLV-based retroviral vectors in the absence of retroviral envelope proteins is known in the art. Any retroviral vector can be pseudotyped with VSV-G. These pseudotyped VSV-G vectors can be used to transduce a wide range of mammalian cells. Non-infectious retroviral particles can be made infectious by the addition of VSV-G. VSV-G pseudotyped vectors have been shown to infect not only mammalian cells but also cell lines derived from fish, reptiles, and insects. The VSV-G protein can be used to pseudotype certain retroviruses because its cytoplasmic tail can interact with the retroviral core.

[0273] Providing a nonretroviral pseudotyped envelope, such as the VSV-G protein, offers the advantage of allowing vector particles to be concentrated to high titers without losing infectivity. In contrast, the VSV glycoprotein is composed of a single unit. Pseudotyping with the VSV-G protein offers potential advantages for both efficient target cell infection / transduction and the manufacturing process, as the VSV glycoprotein is composed of a single unit and can withstand the shear forces during ultracentrifugation. In contrast, retroviral envelope proteins are composed of two noncovalently linked subunits and appear unable to withstand the shear forces during ultracentrifugation, as the intersubunit interactions may be disrupted by centrifugation. WO 2000 / 52188 describes the generation of pseudotyped retroviral vectors with the vesicular stomatitis virus G protein (VSV-G) as a membrane-associated viral envelope protein from stable production cell lines and provides the gene sequence for the VSV-G protein.

[0274] Pseudotyping offers several advantages. For example, in lentiviral vectors, the env gene product of HIV-1-based vectors limits infection to cells that express a protein called CD4. However, if the env gene of these vectors is replaced with env sequences from other RNA viruses, they may have a broader infectious spectrum. 2. Spherical vesiculovirus envelope glycoproteins

[0275] These particles and Cocal vesiculovirus envelope glycoprotein (Cocal-G) have low toxicity to the cells that produce them (i.e., "producer cells") and high transduction efficiency of cells infected by them (i.e., "target cells"). Cocal vesiculovirus envelope glycoprotein has higher particle titers than compositions consisting of other viral particles. Therefore, lentiviral vectors containing Cocal vesiculovirus envelope glycoprotein can be produced at higher concentrations. Furthermore, compositions containing Cocal vesiculovirus envelope glycoprotein have higher titers of mature and immature particles, higher titers of infectious particles, and higher titers of genetic information (e.g., CAR) carried within particles compared to envelope glycoproteins derived from non-Cocal vesiculoviruses. In some embodiments, the lentiviral vector comprises a nucleotide sequence encoding the Cocal-G envelope protein. In some embodiments, the Cocal-G envelope protein is a Cocal-G protein variant. 3. Ross River virus

[0276] Ross River virus (RRV) is a mosquito-borne alphavirus that is endemic in tropical and temperate regions of Australia. Antibody prevalence in the general population of temperate coastal areas tends to be low (6-15%), but reaches 27-37% in the plains of the Murray Valley River system. Between 1979 and 1980, RRV caused an epidemic in the Pacific Islands. The disease is not transmissible between humans and is not fatal. Initial symptoms are joint pain, and fatigue and lethargy are present in approximately half of patients (Fields Virology).

[0277] The Ross River virus envelope has been used to pseudotype non-primate lentiviral vectors (FIV) and primarily transduced the liver after systemic administration. The transduction efficiency of lentiviral vectors pseudotyped with the Ross River virus envelope was reported to be 20-fold higher than that obtained with vectors pseudotyped with VSV-G. Furthermore, lentiviral vectors pseudotyped with the Ross River virus envelope were less cytotoxic as measured by serum levels of liver enzymes, suggesting hepatotoxicity. 4. Baculovirus GP64 The baculovirus GP64 protein has been shown to be an attractive alternative to VSVG as a viral vector for large-scale production of high-titer viruses required for clinical and commercial applications. Compared to VSVG, GP64 vectors have a similar broad tropism and similar native titers. Because expression of GP64 does not kill cells, 293T-based cell lines that constitutively express GP64 can be generated. In some embodiments, a lentiviral vector contains a nucleotide sequence encoding the baculovirus GP64 protein. In some embodiments, the baculovirus GP64 protein is a variant baculovirus GP64 protein. 5. Other envelope glycoproteins

[0278] The lentiviral vectors of the present disclosure can be pseudotyped with at least a portion of the rabies G protein or a mutant, variant, homolog, or fragment thereof. Teachings regarding the rabies G protein and its mutants can be found in WO 1999 / 61639; EP 0445625. Other envelopes that provide reasonable titers when pseudotyped with EIAV include Mokola, rabies, Ebola, and LCMV (lymphocytic choriomeningitis virus). C. Lentiviral Vectors

[0279] In some embodiments of the manufacturing processes, CAR-T cell therapy methods, methods for engineered transduction of mononuclear cells, or lentiviral vectors of the present disclosure disclosed herein, the lentiviral vector is an infectious lentiviral vector or a lentiviral vector.

[0280] One aspect of the present disclosure provides a lentiviral vector comprising a polynucleotide sequence encoding at least one heterologous viral envelope protein derived from a virus; a polynucleotide sequence encoding at least one viral rev protein; a polynucleotide sequence encoding at least one viral gag protein and at least one viral pol protein; and a polynucleotide sequence encoding a chimeric antigen receptor or an artificial T cell receptor (TCR). In some embodiments, at least a portion of one or more regions of the viral genome essential for replication is mutated. In some embodiments, the at least a portion of one or more regions of the viral genome essential for replication is selected from the group consisting of the rev gene, the gag gene, the pol gene, the integrase gene, the 5'LTR, the 3'LTR, and combinations thereof. In some embodiments, the mutation is selected from the group consisting of a deletion, an insertion, or a substitution. 1. Non-replicating Vectors

[0281] In exemplary retroviral vectors of the present disclosure, at least a portion of one or more protein coding regions essential for replication may be removed from the virus. For example, gag / pol and env may be deleted or non-functional, rendering the viral vector replication-deficient. Portions of the viral genome may also be replaced with a library encoding candidate nucleic acid binding sequences operably linked to regulatory control regions and reporter genes in the vector genome to generate vectors comprised of candidate nucleic acid binding sequences capable of transducing target non-dividing cells and / or integrating into the host genome.

[0282] In the genome of replication-deficient lentiviral vectors, the gag / pol and / or env sequences may be mutated, deleted, and / or non-functional. In a typical lentiviral vector, at least a portion of the coding region for one or more proteins essential for viral replication may be removed from the vector, rendering the viral vector replication-deficient. Alternatively, portions of the viral genome may be replaced with nucleotides of interest to generate vectors containing the nucleotides of interest that are capable of transducing non-dividing target cells and / or integrating into the target cell genome.

[0283] In some embodiments, the lentiviral or retroviral vector of the present disclosure is a non-integrating vector and / or a non-replicating vector. See, for example, WO 2006 / 010834 and WO 2007 / 071994. In one aspect of the present disclosure, the lentiviral or retroviral vector of the present disclosure may be incapable of autonomous replication and specific integration in transduced cells. In some embodiments, the lentiviral or retroviral vector of the present disclosure comprises a recombinant genome comprising a lentiviral capsid sequence, an RNA nuclear export element, a transgene, and optionally a promoter and / or a sequence favoring nuclear import of RNA, between the LTR 5' and 3' lentiviral sequences. In some embodiments, the lentiviral vector comprises a mutation in at least a portion of one or more regions of the viral genome essential for replication. In such embodiments, the one or more regions are selected from the group consisting of the rev gene, the gag gene, the pol gene, the integrase gene, the 5' LTR, the 3' LTR, and combinations thereof. The mutation is selected from the group consisting of a deletion, an insertion, or a substitution.

[0284] In some embodiments, the lentiviral or retroviral vector comprises and / or further comprises a mutated integrase that prevents integration of the retroviral or lentiviral genome into the genome of the host cell, hi some embodiments, the lentiviral or retroviral vector comprises a modified pol sequence that generates a non-functional integrase.

[0285] In a further embodiment, the lentiviral vector has the ability to deliver sequences that do not contain or lack viral RNA.In a further embodiment, the heterologous binding domain (heterologous to gag) located on the delivered RNA and the cognate binding domain on Gag or Gag Pol can be used to ensure the packaging of the delivered RNA.Both of these vectors are described in WO 2007 / 072056.In some embodiments, the recombinant retrovirus is replication-incompetent, which means that the retrovirus cannot replicate after leaving the packaging cell. 2. Self-inactivating vectors

[0286] In some embodiments, the lentiviral vector is a non-replicating, self-inactivating minimal lentiviral vector derived from human immunodeficiency virus (HIV) or equine infectious anemia virus (EIAV), which may be pseudotyped with an env selected from the group consisting of VSV-G, Ebola, Flu-HA, Sendai virus envelope F or HN, baculovirus GP64, rabies G, cocaine vein cyclovirus envelope protein, or an alternative viral envelope protein.

[0287] Those skilled in the art will understand how to modify the methods disclosed herein for use with different retroviruses. For example, in some embodiments, the polynucleotide regions encoding HIV RRE and HIV Rev can be replaced with polynucleotide regions encoding an N-terminal RGG box RNA-binding motif and ICP27. In some embodiments, the polynucleotide region encoding HIV Rev can be replaced with one or more polynucleotide regions encoding adenovirus E1B 55kDa and E4 Orf6. In some embodiments, the recombinant retrovirus can be adenovirus, adeno-associated virus, herpesvirus, cytomegalovirus, poxvirus, avipoxvirus, influenza virus, vesicular stomatitis virus (VSV), or Sindbis virus.

[0288] In some embodiments, the retroviral or lentiviral vector disclosed herein is a self-inactivating vector. As used herein, the term "self-inactivating vector" refers to a vector in which the 3'LTR enhancer promoter region (U3 region) has been modified (e.g., by deletion or substitution). A self-inactivating vector can block viral transcription after the first round of viral replication. As a result, a self-inactivating vector can infect and then integrate into a host genome (e.g., a mammalian genome) only once, and cannot be further passaged. Therefore, a self-inactivating vector can significantly reduce the risk of producing a replication-competent virus.

[0289] In some embodiments, the viral particles can self-inactivate. Self-inactivating viral particles can prevent viral transcription after the first round of viral replication. As a result, the self-inactivating particles can infect cells, and the genetic information contained therein can be integrated into the host genome (e.g., a mammalian genome). This integration and transfer occurs only once and cannot be further inherited. Therefore, self-inactivating particles can significantly reduce the risk of producing replication-competent viruses. The most commonly used lentiviral vector system is the so-called third-generation self-inactivating system. A third-generation lentiviral vector system can contain four plasmids. The "transfer plasmid" encodes the polynucleotide sequence delivered to target cells by the lentiviral vector system. The transfer plasmid typically contains one or more transgene sequences of interest flanked by long terminal repeat (LTR) sequences, which facilitate integration of the transfer plasmid sequence into the host genome. For safety reasons, transfer plasmids are generally engineered to render the resulting vector replication-incompetent. For example, transfer plasmids lack genetic elements necessary for the production of infectious particles within host cells. Furthermore, transfer plasmids can also be engineered to delete the 3' LTF, rendering the virus "self-inactivating" (SIN).

[0290] Third-generation systems also typically include two "packaging plasmids" and an "envelope plasmid." The "envelope plasmid" typically encodes an Env gene operably linked to a promoter. In at least one embodiment of a third-generation system, the Env gene is VSV-G, Ebola env, Flu-HA, Sendai virus envelope F, HN, baculovirus GP64, rabies G, or cocaine vesiculovirus envelope protein, or a derivative thereof (e.g., a mutant described herein), and the promoter is a CMV promoter. As an additional safety feature, third-generation systems use two packaging plasmids: one encoding Gag and Pol, and the other encoding Rev, which represents an improvement over the single packaging plasmid used in so-called second-generation systems. While safer, third-generation systems can be more cumbersome to use and produce lower viral titers due to the addition of additional plasmids. Exemplary packaging plasmids include, but are not limited to, pMD2.G, pRSV-rev, pMDLG-pRRE, and pRRL-GOI.

[0291] In some embodiments, the lentiviral vector is a third generation self-inactivating (SIN) vector, does not contain any viral proteins, and is replication-incompetent, such that no infectious particles are produced by cells transduced and / or transfected with the vector. 3. Regulatory factors In some embodiments, the retroviral or lentiviral vectors described herein comprise a transcriptional regulatory element. In some embodiments, the transcriptional regulatory element is a promoter selected from eukaryotic promoters or constitutive promoters. Physiological promoters (e.g., the EF-1α promoter) are less likely to induce integration-mediated genotoxicity and may negate the ability of retroviral vectors to transform stem cells. Other physiological promoters suitable for use in retroviral or lentiviral vectors are known to those skilled in the art and can be incorporated into exemplary embodiments of nucleic acid vectors. In some embodiments, the promoter is the elongation factor-1α promoter (EF-1α promoter). Use of the EF-1α promoter can increase the expression efficiency of downstream transgenes (e.g., nucleic acid sequences encoding TCR and / or CAR).

[0292] In some embodiments, the lentiviral or retroviral vector further comprises non-essential cis-acting sequences that can improve titer and gene expression.One non-limiting example of a non-essential cis-acting sequence is the central polypurine tract and central termination sequence (cPPT / CTS), which is important for efficient reverse transcription and nuclear import.Other non-essential cis-acting sequences are known to those skilled in the art and can be incorporated into lentiviral or retroviral vector particles.

[0293] In some embodiments, the lentiviral or retroviral vectors disclosed herein further comprise a post-transcriptional regulatory element. The post-transcriptional regulatory element can improve RNA translation, improve transgene expression, and stabilize RNA transcripts. An example of a post-transcriptional regulatory element is the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE). Thus, in some embodiments, the nucleic acid vector further comprises a WPRE sequence. A variety of post-transcriptional regulatory elements are known to those skilled in the art and can be incorporated into lentiviral or retroviral vectors.

[0294] The lentiviral or retroviral vectors disclosed herein can further comprise additional elements, such as a Rev response element (RRE) for RNA transport, a packaging sequence, and 5' and 3' long terminal repeats (LTRs). The term "long terminal repeat" or "LTR" refers to the base-pair domains located at the ends of retroviral DNA that comprise the U3, R, and U5 regions. LTRs generally provide functions necessary for retroviral gene expression (e.g., promotion, initiation, and polyadenylation of gene transcripts) and viral replication. In one embodiment, the lentiviral or retroviral vector lacks a 3' U3-deleted LTR, a non-functional LTR, and / or a functional 3' or 5' LTR. Thus, the lentiviral or retroviral vectors disclosed herein can comprise any combination of the elements described herein to increase the efficiency of functional expression of the transgene. For example, the lentiviral or retroviral vector can comprise a WPRE sequence, a cPPT sequence, an RRE sequence, a 5' LTR, and a 3' U3-deleted LTR in addition to a nucleic acid encoding a TCR or CAR. D. Lentiviral Production

[0295] One aspect of the present disclosure provides a method for producing a lentiviral or retroviral vector particle described herein. Another aspect of the present disclosure provides a method for producing a lentiviral or retroviral vector particle, comprising introducing a lentiviral or retroviral vector particle disclosed herein into a host cell.

[0296] The production of lentiviral vectors relies on the use of "packaging cell lines." Generally, packaging cell lines are cell lines capable of producing infectious lentiviral particles when transfected with a transfer plasmid, a packaging plasmid, and an envelope plasmid. Various methods, such as transfection and electroporation, can be used to introduce plasmids into cells. In some cases, packaging cell lines are adapted for high-efficiency packaging of lentiviral vector systems into lentiviral particles. In one embodiment, the present disclosure provides packaging cells that produce recombinant retroviruses, e.g., lentiviruses, pseudotyped with the VSV-G glycoprotein or variants thereof disclosed herein. Large-scale viral particle production is often required to achieve reasonable viral titers. Viral particles are produced by introducing a transfection vector into a packaging cell line containing viral structural and / or accessory genes, such as gag, pol, env, tat, rev, vif, vpr, vpu, vpx, or nef genes, or other retroviral genes. 1. Production of retroviral therapeutic vectors

[0297] In some embodiments, the retroviral vectors of the present disclosure can be produced by transient transfection of HEK293T cells with four plasmids: (1) a recombinant retroviral vector genome plasmid encoding the required transgene(s) and a binding site capable of interacting with an RNA-binding protein; (2) a synthetic retroviral gag / pol expression plasmid; (3) an envelope (env) expression plasmid (e.g., VSV-G or a mutant thereof); and (4) an RNA-binding protein expression plasmid.

[0298] In some embodiments, the retroviral vector of the present disclosure is HIV. In such embodiments, the retroviral vector can be produced by transient transfection of HEK293T cells with five plasmids: (1) a recombinant HIV vector genome plasmid encoding the necessary transgene(s), a binding site capable of interacting with an RNA-binding protein, and an RRE sequence; (2) a synthetic gag / pol expression plasmid; (3) an envelope (env) expression plasmid (e.g., VSV-G, Cocal vesiculovirus, or a mutant thereof); (4) an RNA-binding protein expression plasmid; and (5) an REV expression plasmid.

[0299] In some embodiments, the retroviral vectors of the present invention can be produced by using packaging cells that stably express (1) gag / pol; (2) env (e.g., VSV-G or a mutant thereof); and (3) an RNA-binding protein, and for HIV vectors, a plasmid encoding a recombinant retroviral vector genome encoding Rev and a binding site capable of interacting with a required transgene (e.g., CAR) and the RNA-binding protein, and for HIV vectors, a plasmid containing an RRE sequence, is introduced into such cells by transient transfection.

[0300] In some embodiments, retroviral vectors of the present disclosure can be produced in producer cells that stably express a recombinant EIAV vector genome encoding (1) gag / pol, (2) env (e.g., VSV-G or a mutant thereof), (3) an RNA-binding protein, and (4) a required transgene (e.g., a CAR) and a binding site capable of interacting with the RNA-binding protein.

[0301] In some embodiments, the lentiviral vector is an HIV lentiviral vector. In this embodiment, the HIV vector can be produced in a producer cell stably expressing (1) gag / pol; (2) env (e.g., VSV-G or a mutant thereof); (3) an RNA-binding protein; (4) a recombinant HIV vector genome encoding a required transgene (e.g., CAR), a binding site capable of interacting with the RNA-binding protein, and an RRE sequence, and (5) REV. a. Codon optimization

[0302] In some embodiments, any of the polynucleotides used in the present disclosure to generate lentiviral vectors can be codon-optimized. Different cells vary in their use of specific codons. This codon bias corresponds to a bias in the relative abundance of certain tRNAs in a cell type. By changing the codons in the sequence to match the relative abundance of the corresponding tRNA, expression can be increased. Similarly, expression can be reduced by intentionally selecting codons whose corresponding tRNAs are known to be rare in a particular cell type. In this way, even more sophisticated translational control is possible.

[0303] Many viruses, including HIV and other lentiviruses, use a large number of rare codons, and by changing these to correspond to commonly used mammalian codons, increased expression of the gene of interest or packaging components in mammalian producer cells can be achieved. Codon usage tables are known in the art for mammalian cells and a variety of other organisms.

[0304] Codon optimization of viral vector components has many other benefits. The sequence modification eliminates RNA instability sequences from the nucleotide sequences encoding viral particle packaging components required for viral particle assembly in producer / packaging cells. At the same time, the amino acid coding sequences for the packaging components are retained, and the viral components encoded by the sequences remain the same, or at least similar enough that the functionality of the packaging components is not impaired. In lentiviral vectors, codon optimization also overcomes the Rev / RRE export requirement, making the optimized sequences Rev-independent. Codon optimization also reduces homologous recombination between different constructs within the vector system (e.g., between overlapping regions of the gag-pol and env open reading frames). Therefore, the overall effect of codon optimization is a significant increase in viral titer and improved safety.

[0305] In one embodiment, only the codons for the instability sequence are codon-optimized. In a preferred embodiment, the entire sequence is codon-optimized. In that embodiment, the sequence encompasses the gag-pol frameshift site (see below). The gag-pol gene contains two overlapping reading frames encoding the gag-pol proteins. Expression of both proteins is dependent on a frameshift during translation. This frameshift occurs as a result of ribosome "slippage" during translation. This slippage is thought to occur, at least in part, by the ribosome stripping off secondary structures in the RNA. Such secondary structures exist downstream of the frameshift site in the gag-pol gene. For example, derivations from optimal codon usage may be made to accommodate convenient restriction sites, or conservative amino acid changes may be introduced into the Gag-Pol protein.

[0306] In one embodiment, codon optimization is based on a lightly expressed mammalian gene. Due to the degenerate nature of the genetic code, it will be understood that numerous gag-pol sequences can be achieved by a skilled practitioner. Additionally, many retroviral mutants have been described that can be used as starting points for generating codon-optimized gag-pol sequences. Lentiviral genomes are highly variable. For example, HIV-1 has many quasispecies that are still functional, as is the case with EIAV. These mutants can be used to enhance specific parts of the transduction process. Examples of HIV-1 mutants can be found in the HIV Databases, operated by Los Alamos National Security, LLC, at hiv-web.lanl.gov. Details of EIAV clones can be found in the National Center for Biotechnology Information (NCBI) database at ncbi.nlm.nih.gov.

[0307] The strategy of codon-optimized gag-pol sequences can be used with any retrovirus. It will be applicable to all lentiviruses, including EIAV, FIV, BIV, CAEV, VMR, SIV, HIV-1, and HIV-2. Furthermore, this method can be used to increase expression of genes from HTLV-1, HTLV-2, HFV, HSRV, human endogenous retroviruses (HERVs), MLV, and other retroviruses.

[0308] Codon optimization can render gag-pol expression Rev-independent. However, to allow the use of anti-Rev and RRE elements in lentiviral vectors, the viral vector production system must be made completely Rev / RRE-independent. Therefore, the genome must also be modified. This is achieved by optimizing the genomic components of the vector. Advantageously, these modifications also lead to the production of a safer system, free of all additional proteins, both in the producer and in the transduced cells. B virus particle production Production of infectious viral particles and viral stock solutions can be carried out using conventional techniques, such as those described herein. Recombinant viruses (e.g., retroviral or lentiviral vectors or particles described herein) with titers of millions of transducing units per milliliter (TU / mL) can be produced by known techniques. After ultracentrifugation, approximately 10 8 TU / mL, 10 9 TU / mL, 10 10 TU / mL, 10 11 Concentrated stocks can be obtained at titers of 10 TU / mL, 10 TU / mL, or 10 TU / mL, or anywhere in between. Recombinant viruses (e.g., retroviral or lentiviral vectors or particles described herein) can be delivered according to viral titer (TU / mL), which can be measured, for example, using a commercially available p24 titer test, an ELISA for the p24 viral coat protein.

[0309] In some embodiments, the concentrated apheresis product has a viral transduction unit (TU) concentration of about 1 x 10 per concentrated apheresis product (cell). 8 ~Approx. 1×10 10 TU / 10 8 cells, approximately 5 x 10 8 ~Approx. 5×10 9 TU / 10 8 cells, approximately 1 x 10 9 ~Approx. 5×10 9 TU / 10 8 cells, approximately 1 x 10 9 ~Approx. 4×10 9 TU / 10 8 cells, approximately 1 x 10 9 ~Approx. 3×10 9 TU / 10 8 cells, approximately 1 x 10 9 ~Approx. 2×10 9 TU / 10 8 The cells are transfected with a lentiviral or retroviral vector, or TU in between.

[0310] In one embodiment, the manufacturing process contemplated herein comprises converting a concentrated apheresis product into a lentiviral or retroviral vector product having a lentiviral or retroviral vector concentration of about 1×10 8 TU / 10 8 cells, approximately 5 x 10 8 TU / 10 8 cells, approximately 6 x 10 8 TU / 10 8 cells, approximately 7 x 10 8 TU / 10 8 cells, approximately 8 x 10 8 TU / 10 8 cells, approximately 9 x 10 8 TU / 10 8 cells, approximately 1 x 10 9 TU / 10 8 cells, approximately 2 x 10 9 TU / 10 8 cells, approximately 3 x 10 9 TU / 10 8 cells, approximately 4 x 10 9 TU / 10 8 cells, approximately 5 x 10 9 TU / 10 8 cells, approximately 6 x 10 9 TU / 10 8 cells, approximately 7 x 10 9 TU / 10 8 cells, approximately 8 x 10 9 TU / 10 8 cells, approximately 9 x 10 9 TU / 10 8 cells, or approximately 1 x 10 10 TU / 10 8 In certain embodiments, the concentrated apheresis product is about 1 x 10 cells transfected with a lentiviral or retroviral vector, or a TU. 7 ~Approx. 2×10 9 TU / 10 8 The cells are transfected with a lentiviral or retroviral vector at a concentration of 1000 ng / ml.

[0311] The production of virus particles and virus stock solutions can be carried out using conventional techniques. Methods for preparing virus stock solutions are known in the art, for example, Soneoka et al. (1995) Nucl. Acids Res. 23:628-633, and Landau et al. (1992) J. Virol. 66:5110-5113. Recombinant viruses with titers of millions of transducing units per milliliter (TU / mL) can be produced by known techniques. After ultracentrifugation, approximately 10 8 TU / mL, 10 9 TU / mL, 10 10 TU / mL, 10 11 TU / mL, 10 12 Concentrated stocks can be obtained at titers of 100 TU / mL or in between. c. p24 titer measurement

[0312] Virus can be delivered according to viral titer (TU / mL), which can be measured, for example, using a commercially available p24 titration assay. The p24 titration assay is an ELISA for the p24 viral coat protein. Assuming there are approximately 2,000 molecules of p24 per lentiviral physical particle (PP), the pg / mL of p24 can be calculated using the following formula: (2 x 10 3 ) x ( 24 x 10 per PP 3 Da), 48 × 10 6 / Avogadro = (48 × 10 6 )I(6×10 23 ) = 8 x 10 per PP 17 g of p24, 1 x 10 of p24 16 Approximately 1 PP per g, 1 × 10 per pg of p24 4 PP. In some embodiments, a properly packaged, VSV-G pseudotyped lentiviral vector has an infectivity index ranging from about 1 TU per 1000 physical particles (PP) to about 1 TU per 100 PP (or less). Thus, the p24 range is about 10 to about 100 TU / pg. It is from this conversion that TU / mL is obtained.

[0313] Lentiviral titers can also be determined by analyzing transduced human osteosarcoma (HOS) cells. Briefly, transduced HOS cells are cultured in DMEM supplemented with 10% fetal bovine serum (FBS) for 7 days, after which genomic DNA is extracted using DNeasy (Qiagen, Venlo, Netherlands, Cat# 69506) and evaluated by quantitative PCR (qPCR). The primer / probe set in the qPCR protocol measures the vector copy number (VCN) of transduced cells by determining the number of lentiviral psi-gag region copies per number of endogenous human RNase P copies. Proviral integrity was assessed by sequencing individual proviral inserts. In some embodiments, viral titers are determined using an HOS cell line test. d. host cell

[0314] As used herein, a "host cell" is a cell that is transfected with a nucleic acid vector to replicate and produce more of the nucleic acid vector itself (i.e., produce more plasmids). In some embodiments, supernatants containing lentiviral vectors (LV) encoding the CAR or TCR disclosed herein are produced in HEK 293T cells. To produce the lentiviral vectors of the present disclosure, 293 cells are transiently transfected with four plasmids: a plasmid encoding HIV gag-pol, a plasmid encoding VSV-G envelope protein, a plasmid encoding HIV rev protein, and a lentiviral transfer vector encoding a CAR.

[0315] Bacterial, yeast, and animal cells can be used to amplify or produce nucleic acids and vectors encoding heterologous envelope proteins or proteins, enzymes, and viral elements (i.e., cis- and trans-acting genes, including rev and gag / pol) necessary for the production of retroviral particles. For amplification in bacterial cells, suitable promoters include, but are not limited to, lacI, lacZ, T3, T7, gpt, λP, and trc. For amplification or expression in eukaryotic cells, suitable promoters include, but are not limited to, the light or heavy chain immunoglobulin gene promoter and enhancer elements; the cytomegalovirus immediate early promoter; the herpes simplex virus thymidine kinase promoter; the early and late SV40 promoters; promoters present in long terminal repeats from retroviruses; the mouse metallothionein-I promoter; and various tissue-specific promoters known in the art. Suitable reversible promoters, including reversibly inducible promoters, are known in the art. Such reversible promoters can be isolated and derived from many organisms, e.g., eukaryotes and prokaryotes. It is well known in the art to modify a reversible promoter from a first organism for use in a second organism, e.g., a first prokaryote and a second eukaryote, a first eukaryote and a second prokaryote, etc.Such reversible promoters and systems based on such reversible promoters, which further comprise a regulatory protein, include alcohol-regulated promoters (e.g., but not limited to, alcohol dehydrogenase I (alcA) gene promoter, promoters responsive to alcohol transactivator protein (A1cR)), tetracycline-regulated promoters (e.g., promoter systems including TetActivators, TetON, TetOFF, etc.), steroid-regulated promoters (e.g., rat glucocorticoid receptor promoter system, human estrogen receptor promoter system, retinoid promoter system, thyroid promoter system, ecdysone promoter system, mifepristone promoter system, etc.), metal-regulated promoters (e.g., metallothionein promoter system), pathogenesis-related regulated promoters (e.g., salicylic acid-regulated promoters, ethylene-regulated promoters, benzothiadiazole-regulated promoters, etc.), temperature-regulated promoters (e.g., heat shock-inducible promoters (e.g., HSP-70, HSP-90, soybean heat shock promoter, etc.)), light-regulated promoters, synthetic inducible promoters, etc.

[0316] In some embodiments, the host cell and the producer cell can be derived from the same cell line. In some embodiments, the host cell and the producer cell are HEK293-T cells. Thus, in some embodiments, the promoter can be generally expressed in all cells, selectively expressed in producer cells, or specifically expressed in producer cells. In some embodiments, the promoter is a CD8 cell-specific promoter, a CD4 cell-specific promoter, a neutrophil-specific promoter, or an NK cell-specific promoter. For example, the CD4 gene promoter can be used; see, e.g., Salmon et al., Proc. Natl. Acad. Sci. USA (1993) 90:7739; and Marodon et al. (2003) Blood 101:3416. As another example, the CD8 gene promoter can be used. NK cell-specific expression can be achieved by using the NcrI (p46) promoter; see, e.g., Eckelhart et al., Blood (2011) 117:1565. For expression in yeast host cells for amplification, suitable promoters are constitutive promoters such as the ADH1 promoter, PGK1 promoter, ENO promoter, PYK1 promoter, etc., or controllable promoters such as the GAL1 promoter, GAL10 promoter, ADH2 promoter, PHOS promoter, CUP1 promoter, GALT promoter, MET25 promoter, MET3 promoter, CYC1 promoter, HIS3 promoter, ADH1 promoter, PGK promoter, GAPDH promoter, ADC1 promoter, TRP1 promoter, URA3 promoter, LEU2 promoter, ENO promoter, TP1 promoter, and AOX1 (e.g., for use in Pichia). Selection of an appropriate vector and promoter is within the level of ordinary skill in the art.Suitable promoters for use in prokaryotic host cells include the bacteriophage T7 RNA polymerase promoter; the trp promoter; the lac operon promoter; hybrid promoters, such as the lac / tac hybrid promoter, the tac / trc hybrid promoter, the trp / lac promoter, the T7 / lac promoter, the trc promoter, the tac promoter, and the like; the araBAD promoter; in vivo regulated promoters, such as the ssaG promoter or related promoters (US 2004 / 0131637), the pagC promoter, the nirB promoter, and the like (see, e.g., Dunstan et al., Infect. Immun. (1999) 67:5133-5141; McKelvie et al., Vaccine (2004) 22:3243-3255); sigma 70 promoters, such as the consensus sigma 70 promoter (see, e.g., GenBank Accession No. 10 ... See Nos. AX798980, AX798961, and AX798183); stationary phase promoters, such as the dps promoter and spv promoter; promoters from pathogenicity island SPI-2; the actA promoter; the rpsM promoter; the tet promoter; the SP6 promoter, etc. Strong promoters suitable for use in prokaryotes such as E. coli include, but are not limited to, Trc, Tac, T5, T7, PLambda, etc.

[0317] One aspect of the present disclosure provides a method of producing lentiviral vector particles, comprising introducing a lentiviral vector described herein into a host cell.

[0318] Another aspect of the present disclosure provides a method for delivering a nucleic acid encoding a chimeric antigen receptor (CAR), an engineered T cell receptor, or a therapeutic protein to a cell, the method comprising introducing into a cell a transfer plasmid comprising: a polynucleotide sequence encoding at least one heterologous viral envelope protein engineered by a method described herein; a polynucleotide sequence encoding at least one retroviral rev protein; a polynucleotide sequence encoding at least one retroviral gag protein and a retroviral pol protein; and / or a polynucleotide sequence encoding a chimeric antigen receptor, an engineered T cell receptor (TCR), or a therapeutic protein. In some embodiments, at least a portion of one or more regions of the retroviral genome essential for replication are mutated as described herein.

[0319] Another aspect of the present disclosure provides lentiviral vector particles produced by the methods described herein.

[0320] Another aspect of the present disclosure provides a method for introducing a variant into a cell, the method comprising electroporating the cell with an effective amount of a lentiviral vector particle described herein, thereby generating a variant cell. In some embodiments, the cell is contacted with an effective dose of the lentiviral vector prior to electroporation (e.g., application of electricity). Alternatively, the cell can be contacted with the effective dose of the lentiviral vector up to about 4 hours after electroporation (e.g., application of electricity). For example, the cell can be contacted with the effective amount of the lentiviral vector for at least about 5-30 minutes, at least about 25-50 minutes, at least about 5-60 minutes, at least about 5-12 minutes, at least about 60-120 minutes, or at least about 120-240 minutes after electroporation. Alternatively, the cells can be contacted with an effective amount of the lentiviral vector for at least about 1 minute, at least about 2 minutes, at least about 5 minutes, at least about 10 minutes, at least about 20 minutes, at least about 30 minutes, at least about 45 minutes, at least about 60 minutes, at least about 75 minutes, at least about 90 minutes, or for at least about 100 minutes, at least about 110 minutes, at least about 120 minutes, at least about 150 minutes, at least about 160 minutes, at least about 170 minutes, at least about 180 minutes, at least about 190 minutes, at least about 200 minutes, at least about 220 minutes, or at least about 240 minutes after electroporation.

[0321] Adding the lentiviral vector to cells up to 4 hours after electroporation (e.g., 1 minute to 2 hours, or 1 minute to 4 hours) can reduce the amount of lentiviral particles killed by electroporation, which can be toxic to the lentiviral particles. Therefore, adding the lentiviral vector to cells up to 4 hours after electroporation can enhance CAR transfection. For example, adding the lentiviral vector to cells up to 4 hours after electroporation can enhance CAR expression by approximately 10-15% compared to conventional electroporation processes (e.g., adding the lentivirus to cells before electroporation).

[0322] In some embodiments, the cells are immune cells, eukaryotic donor cells, monocytes, enriched lymphocytes, B lymphocytes, T lymphocytes, CD4 + T lymphocytes, CD8 + T lymphocytes, dendritic cells, monocytes, natural killer (NK) cells, natural killer T (NKT) cells, T regulatory cells, CD4 + T helper cells, CD8 + Cytotoxic T lymphocytes (CTLs), selected from the group consisting of monocytes, natural killer (NK) cells, natural killer T (NKT) cells, T regulatory cells, and CD4 + T helper cells, CD8 + Cytotoxic T lymphocytes (CTL), CD62L + cells, CD 27 + cells, CCR7 + cells, CD45RO - cells, CD45RA + cells, neutrophils, basophils, eosinophils, megakaryocytes, stem cells, hematopoietic stem cells (HSCs), hematopoietic progenitor cells (HPCs), CD34 + cells, CD34 + The cells are selected from peripheral blood stem cells, lymphocyte-activated killer cells (LAKs), tumor-infiltrating lymphocytes (TILs), circulating tumor-specific T cells, mesenchymal stem cells, mast cells, monocytes, macrophages, neutrophils, basophils, eosinophils, dendritic cells, megakaryocytes, and combinations thereof.

[0323] In some embodiments, the cells are T cells, B cells, natural killer (NK) cells, CD8 + T cells, CD4 + The cells may be lymphoid cells selected from the group consisting of T cells, cytotoxic T lymphocytes, regulatory T cells, and any combination thereof. In some embodiments, the cells may be myeloid cells selected from the group consisting of monocytes, macrophages, neutrophils, basophils, eosinophils, dendritic cells, megakaryocytes, and any combination thereof. In some embodiments, the cells may be stem cells, hematopoietic stem cells, hematopoietic progenitor cells, CD34 + cells, or CD34 + They may be peripheral blood stem cells.

[0324] In some embodiments of the method of introducing a variant into a cell, the method comprises electroporating the cell with an effective dose of lentiviral vector particles, wherein the effective dose comprises about 0.5, about 1, about 1.5, about 2, about 2.5, about 3, about 3.5, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 15, or about 20 μl of lentiviral vector.

[0325] In some embodiments, the effective dose of lentiviral vector particles is about 0.01, about 0.02, about 0.03, about 0.04, about 0.05, about 0.06, about 0.07, about 0.08, about 0.09, about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1.0, about 1.25, about 1.5, about 2.0, about 3.0, about 4.0, or about 5.0. In some embodiments, the effective dose of lentiviral vector particles comprises about 2 μl of lentiviral vector particles at an MOI of about 0.08. In some embodiments, the effective dose of lentiviral vector particles comprises about 5 μl of lentiviral vector particles at an MOI of about 0.2. In some embodiments, the effective dose of lentiviral vector particles consists of about 10 μl of lentiviral vector particles at an MOI of about 0.4. e. Methods for producing therapeutic proteins One aspect of the present disclosure provides a method of producing a therapeutic protein, the method comprising: producing a population of artificial immune cells or a population of artificial eukaryotic cells comprising the therapeutic protein using the methods described herein; harvesting the therapeutic protein; and isolating and purifying the therapeutic protein. In some embodiments, the therapeutic protein is selected from the group consisting of an enzyme, a regulatory protein, a receptor, a peptide, a peptide hormone, a cytokine, a membrane protein or a transport protein, a vaccine antigen, an antigen-binding protein, an immunostimulatory protein, an allergen, a full-length antibody or an antibody fragment or derivative; a single-chain antibody (scFv), a Fab fragment, an Fv fragment, a single-domain antibody (VH or VL fragment), a domain antibody, a camelid single-domain antibody (VHH), a nanobody, and combinations thereof. IV. Chimeric Receptors

[0326] One aspect of the present disclosure provides a method for producing a population of engineered immune cells, the method comprising extracting a population of lymphocytes, a population of immune cells, or a population of CD4 + and CD8 + Enriching a population of cells; a population of lymphocytes, a population of immune cells, or CD4 + and CD8 + combining the population of cells with one or more buffers; and determining the population of lymphocytes, the population of immune cells, or the population of CD4 + and CD8 + combining the population of cells with one or more buffers; + and CD8 + transfecting the population of cells with an effective amount of a modifying agent; thereby producing a population of modified lymphocytes, a population of modified immune cells, or a modified CD4 + and CD8 + Generate a population of cells.

[0327] Another aspect of the present disclosure provides a method for producing a population of engineered immune cells, the method comprising: producing a population of lymphocytes, a population of immune cells, or a population of CD4 +and CD8 + Enriching a population of cells from the donor's white blood cells; a population of lymphocytes, a population of immune cells, or CD4 + and CD8 + combining the population of cells with one or more buffers; + and CD8 + transfecting the population of cells with an effective amount of a modifying agent, thereby producing a population of modified lymphocytes, a population of modified immune cells, or a modified CD4 + and CD8 + Generate a population of cells.

[0328] Furthermore, another aspect of the present disclosure provides a method for producing a population of engineered eukaryotic cells, the method comprising: obtaining a population of eukaryotic donor cells from a subject; combining the population of eukaryotic donor cells with one or more buffers; and transfecting the population of eukaryotic donor cells with an effective amount of a modifying agent, thereby producing a population of modified eukaryotic donor cells. In some embodiments, the transfected cells are cultured and expanded in the presence of one or more stimulatory agents.

[0329] In some embodiments, the cells are transfected with one or more modifying agents selected from the group consisting of small molecule drugs, biologic drugs, therapeutic drugs, proteins, peptides, protein therapeutic drugs, peptide therapeutic drugs, nucleic acids, DNA, RNA, mRNA, chimeric antigen receptors, heterologous T cell receptors, retroviral vectors, lentiviral vectors, adenoviral vectors, and adeno-associated viral vectors.

[0330] In some embodiments, the lentiviral or retroviral vector comprises a nucleotide sequence encoding a chimeric antigen receptor (CAR); an engineered T cell receptor; and / or a nucleic acid sequence encoding a polypeptide that enhances immune cell function, or a functional derivative thereof.

[0331] In some embodiments, the lentiviral or retroviral vector comprises a nucleic acid encoding a chimeric antigen receptor (CAR), an artificial T cell receptor (TCR), a killer cell immunoglobulin-like receptor (KIR), an antigen-binding polypeptide, a cell surface receptor ligand, or a tumor antigen. In some embodiments, the nucleic acid encodes a chimeric antigen receptor (CAR). In some embodiments, the nucleic acid encodes an antigen-binding polypeptide. In some embodiments, the nucleic acid encodes a killer cell immunoglobulin-like receptor (KIR). In further embodiments, the exogenous nucleic acid encodes a cell surface receptor ligand or a tumor antigen.

[0332] In some embodiments, retroviral or lentiviral vectors can be used to introduce TCRs or CARs into immune cells or their precursors (e.g., T cells). In some embodiments, retroviral or lentiviral vector particles can contain additional elements that aid in the functional expression of the TCR or CAR encoded therein. In some embodiments, the expression vector comprising a nucleic acid encoding a TCR or CAR further comprises a mammalian promoter. A. Chimeric Antigen Receptor

[0333] The present invention provides engineered immune effector cells (e.g., T cells or NK cells) that contain one or more CARs that direct immune effector cells to cancer. In some embodiments, the CAR comprises an antigen-binding domain, a transmembrane domain, a costimulatory domain, and an intracellular domain. The CAR may comprise any of the antigen-binding domains, any of the hinges, any of the transmembrane domains, any of the costimulatory domains, and any of the intracellular signaling domains described herein.

[0334] The antigen-binding domain can be operably linked to another domain of the CAR, such as a transmembrane domain or an intracellular domain described herein, for expression in any of the immune cells described herein. In one embodiment, a first nucleic acid sequence encoding the antigen-binding domain is operably linked to a second nucleic acid encoding the transmembrane domain, which is further operably linked to a third nucleic acid sequence encoding the intracellular domain.

[0335] The antigen-binding domain described herein can be combined with any of the transmembrane domains described herein, the intracellular domains or cytoplasmic domains described herein, or any of the other domains described herein that can be included in the CAR of the present invention. The subject CAR of the present invention can also include a spacer domain described herein. In some embodiments, each of the antigen-binding domain, transmembrane domain, and intracellular domain is separated by a linker. 1. Antigen-binding domain The antigen-binding domain of a CAR is the extracellular region of the CAR that binds to a specific target antigen, including proteins, carbohydrates, and glycolipids. In some embodiments, a CAR comprises an affinity for a target antigen (e.g., a tumor-associated antigen) on a target cell (e.g., a cancer cell). The target antigen can include any type of protein associated with the target cell or an epitope thereof. For example, a CAR may comprise an affinity for a target antigen on a target cell that indicates a specific state of the target cell.

[0336] As described herein, the CAR of the present disclosure, which has affinity for a specific target antigen on a target cell, can include a target-specific binding domain. In some embodiments, the target-specific binding domain is a mouse target-specific binding domain, e.g., the target-specific binding domain is derived from a mouse. In some embodiments, the target-specific binding domain is a human target-specific binding domain, e.g., the target-specific binding domain is derived from a human.

[0337] The antigen-binding domain can comprise any domain that binds to an antigen, including, but not limited to, monoclonal antibodies, polyclonal antibodies, synthetic antibodies, human antibodies, humanized antibodies, non-human antibodies, and fragments thereof. Thus, in certain embodiments, the antigen-binding domain portion comprises a mammalian antibody or a fragment thereof. In certain embodiments, the antigen-binding domain comprises a full-length antibody. In some embodiments, the antigen-binding domain comprises an antigen-binding fragment (Fab), such as a Fab, Fab', F(ab')2, single-chain specific Fab2, bispecific Fab2, trispecific Fab2, single-chain variable fragment (scFv), dAb, tandem scFv, VhH, V-NAR, camelid body, diabody, minibody, triabody, or tetrabody. In some embodiments, the antigen-binding domain is selected from the group consisting of: (a) a full-length antibody or an antigen-binding fragment thereof, (b) a Fab, (c) a single-chain variable fragment (scFv), and (d) a single-domain antibody.

[0338] In some embodiments, the CARs of the present disclosure may have affinity for one or more target antigens on one or more target cells. In some embodiments, the CARs may have affinity for one or more target antigens on a single target cell. In such embodiments, the CARs are bispecific or multispecific CARs. In some embodiments, the CARs comprise one or more target-specific binding domains that confer affinity for one or more target antigens. In some embodiments, the CARs comprise one or more target-specific binding domains that confer affinity for the same target antigen. For example, a CAR composed of one or more target-specific binding domains with affinity for the same target antigen may bind to different epitopes of the target antigen. When a CAR has multiple target-specific binding domains, the binding domains may be arranged in tandem or separated by a linker peptide. For example, in a CAR composed of two target-specific binding domains, the binding domains are covalently linked to each other on a single polypeptide chain via a polypeptide linker, an Fc hinge region, or a membrane hinge region.

[0339] In some cases, the antigen-binding domain may be derived from the same species in which the CAR will ultimately be used, for example, for human use, the antigen-binding domain of the CAR may be composed of a human antibody, or fragment thereof, as described elsewhere herein.

[0340] Thus, a CAR encoded by the lentiviral or retroviral vector of the present disclosure may target one of the following cancer-associated antigens (tumor antigens): CD19; CD20; CD22 (Siglec-2); CD37; CD123; CD22; CD30; CD171; CS-1 (CD2 subset 1, also known as CRACC, SLAMF7, CD319, and 19A24); C-type lectin-like molecule-1 (CLL-1 or CLECL1); CD33; CD133; epidermal growth factor receptor (EGFR); epidermal growth factor receptor variant III (EGFRvIII); human epidermal growth factor receptor (HER1); ganglioside G2 (GD2); ganglioside GD3 (aNeu5Ac(2-8)aNeu5Ac(2-3)bDGalp(l-4)bDGlcp(ll)Cer); TNF receptor family member B-cell maturation (BCMA); Tn antigen ((Tn Ag) or (GalNAca-Ser / Thr)); prostate-specific membrane antigen (PSMA); receptor tyrosine kinase-like orphan receptor 1 (ROR1); Fms-like tyrosine kinase 3 (FLT3); tumor-associated glycoprotein 72 (TAG72); CD38; CD44v6; calcynoembrionic antigen (CEA); epithelial cell adhesion molecule (EPCAM); B7H3 (CD276); KIT (CD117); interleukin-13 receptor subunit α2 (IL-13Ra2 or CD213A2); mesothelin; interleukin-11 receptor α (IL-1 lRa); prostate stem cell antigen (PSCA); protease serine 21 (Testisin or PRSS21); vascular endothelial growth factor receptor 2 (VEGFR2); Lewis (Y) antigen; CD24; platelet-derived growth factor receptor beta (PDGFR-β); stage-specific embryonic antigen-4 (SSEA-4); folate receptor alpha; receptor tyrosine-protein kinase ERBB2 (Her2 / neu); cell surface-associated mucin 1 (MUC 1); GalNAca1-O-Ser / Thr(Tn)MUC1 (TnMUC1); neural cell adhesion molecule (NCAM); prostase; prostatic acid phosphatase (PAP); elongation factor 2 mutant (ELF2M); ephrin B2; fibroblast activation protein alpha (FAP); insulin-like growth factor 1 receptor (IGF-I receptor), carbonic anhydrase IX (CAIX);Proteasome (prosome, macropein) subunit, beta type 9 (LMP2); glycoprotein 100 (gp100); oncogene fusion protein consisting of breakpoint cluster region (BCR) and Abelson murine leukemia viral oncogene homolog 1 (Abl) (bcr-abl); tyrosinase; ephrin type A receptor 2 (EphA2); fucosyl GM1; sialyl Lewis adhesion molecule (sLe); ganglioside GM3 (aNeu5Ac(2-3)bDGalp(l-4)bDGl cp(ll)Cer); transglutaminase 5 (TGS5); high-molecular-weight melanoma-associated antigen (HMWMAA); o-acetyl-GD2 ganglioside (OAcGD2); folate receptor β; tumor endothelial marker 1 (TEM1 / CD248); tumor endothelial marker 7-related (TEM7R); claudin 6 (CLDN6); thyroid-stimulating hormone receptor (TSHR); G protein-coupled receptor class C group 5, member D (GPRC5D); chromosome X open reading frame 61 (CXORF 61); CD97; CD179a; anaplastic lymphoma kinase (ALK); polysialic acid; placenta-specific 1 (PLAC1); hexasaccharide moiety of globoH glycoceramide (GloboH); mammary differentiation antigen (NY-BR-1); uroplakin 2 (UPK2); tyrosine protein kinase Met (c-Met); hepatitis A virus cellular receptor 1 (HAVCR1); adrenergic receptor beta 3 (ADRB3); pannexin 3 (PANX3); G protein-coupled receptor 20 (GPR20); lymphocyte antigen 6 complex Merger, K locus 9 (LY6K); olfactory receptor 51E2 (OR51E2); TCRγ alternative reading frame protein (TARP); Wilms tumor protein (WT1); cancer / testis antigen 1 (NY-ESO-1); cancer / testis antigen 2 (LAGE-la); melanoma-associated antigen 1 (MAGE-A1); ETS translocation mutant gene 6 located on chromosome 12p (ETV6-AML); sperm protein 17 (SPA17); X antigen family, member 1A (XAGEl); angiopoietin-binding cell surface receptor 2 (Tie 2); melanoma cancer testis antigen-1 (MAD-CT-1); melanoma cancer testis antigen-2 (MAD-CT-2); Fos-related antigen 1; tumor protein p53 (p53); p53 mutant; prostein; survival; telomerase;Prostate cancer tumor antigen 1 (PCTA-1 or galectin-8), melanoma antigen 1 recognized by T cells (MelanA or MARTI); rat sarcoma (Ras) mutant; human telomerase reverse transcriptase (hTERT); sarcoma translocation margin; melanoma inhibitor of apoptosis (ML-IAP); ERG (transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene); N-acetylglucosaminyltransferase V (NA17); paired-box protein Pax-3 (PAX3); androgen receptor; cyclin B l; v-myc avian myelocytomatosis viral oncogene neuroblastoma-derived homolog (MYCN); Ras homolog family member C (RhoC); tyrosinase-related protein 2 (TRP-2); cytochrome P450 1B 1 (CYP1B 1); CCCTC-binding factor (zinc finger protein)-like (sibling of BORIS or regulator of imprinted sites), squamous cell carcinoma antigen 3 recognized by T cells (SART3); paired box protein Pax-5 (PAX5); proacrosin-binding protein sp32 (OY-TES l); lymphocyte-specific protein tyrosine kinase (LCK); A-kinase anchor protein 4 (AKAP-4); synovial sarcoma, X-breakpoint 2 (SSX2); receptor for advanced glycation end products (RAGE-1); renal ubiquitous 1 (RU1); renal ubiquitous 2 (RU2); legumain; human papillomavirus E6 (HPV E6); human papillomavirus E7 (HPV E7); intestinal carboxylesterase; heat shock protein 70-2 mutant (mutant hsp70-2); CD79a; CD79b; CD72; leukocyte-associated immunoglobulin-like receptor 1 (LAIR1); Fc fragment of IgA receptor (FCAR or CD89); leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2); CD300 molecule-like family member f (CD300LF); C-type lectin domain family 12 member A (CLEC12A); bone marrow stromal cell antigen 2 (BST2); EGF-like module-containing mucin-like hormone receptor-like 2 (EMR2); lymphocyte antigen 75 (LY75); glypican-2 (GPC2); glypican-3 (GPC3); NKG2D; KRAS; GDNF family receptor alpha 4 (GFRa4); IL13Ra2;Fc receptor-like 5 (FCRL5); and immunoglobulin lambda-like polypeptide 1 (IGLL1).

[0341] In some embodiments, the CAR targets CD19, CD20, CD22, BCMA, CD37, mesothelin, PSMA, PSCA, Tn-MUC1, EGFR, EGFRvIII, c-Met, HER1, HER2, CD33, CD133, GD2, GPC2, GPC3, NKG2D, KRAS, or WT1. In some embodiments, the antigen binding domain specifically binds to a target antigen selected from the group consisting of CD4, CD19, CD20, CD22, BCMA, CD123, CD133, EGFR, EGFRvIII, mesothelin, Her2, PSMA, CEA, GD2, IL-13Ra2, glypican-3, GPC2, TnMuc1, CIAX, LI-CAM, CA125, CTAG1B, mucin 1, and folate receptor alpha. 2. Transmembrane domain

[0342] The CAR encoded by the lentiviral vector or retroviral vector of the present disclosure can be designed to include a transmembrane domain that connects the antigen binding domain of the CAR to the intracellular domain.The transmembrane domain of the subject CAR is the region that can span the cell membrane of a cell (e.g., immune cell or its precursor).The transmembrane domain is for inserting into a cell membrane, for example, a eukaryotic cell membrane.In some embodiments, the transmembrane domain is interposed between the antigen binding domain and the intracellular domain of the CAR.

[0343] In one embodiment, the transmembrane domain naturally binds to one or more domains of the CAR. In some embodiments, the transmembrane domain can be selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins, in order to minimize interaction with other members of the receptor complex. In some embodiments, transmembrane domain can be derived from either natural or synthetic sources.When the source is natural, the domain can be derived from any membrane-binding protein or transmembrane protein, for example, type I transmembrane protein.When derived from synthetic sources, the transmembrane domain can be an artificial sequence, for example, an artificial hydrophobic sequence, that facilitates the insertion of CAR into cell membrane. In some embodiments, transmembrane domains of particular use in the present invention include, but are not limited to, transmembrane domains derived from (the α, β, or ζ chain of the T cell receptor, CD28, CD2, CD3ε, CD45, CD4, CD5, CD7, cd8, cd9, cd16, cd22, cd33, cd37, cd64, cd80, cd86, cd134(ox-40), cd137(4-1bb), cd154(cd40l), cd278(icos), cd357(gitr), Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, killer immunoglobulin-like receptors (KIR)).

[0344] In some embodiments, the transmembrane domain comprises at least the transmembrane region of a protein selected from the group consisting of the alpha, beta, or zeta chain of the T-cell receptor, CD28, CD2, CD3ε, CD45, CD4, CD5, CD7, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134 (OX-40), CD137 (4-1BB), CD154 (CD40L), CD278 (ICOS), CD357 (GITR), Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, and killer immunoglobulin-like receptors (KIR).

[0345] In some embodiments, the transmembrane domain may be synthetic. In some embodiments, the synthetic transmembrane domain comprises primarily hydrophobic residues such as leucine and valine. In certain exemplary embodiments, a triplet of phenylalanine, tryptophan, and valine will be found at each end of the synthetic transmembrane domain.

[0346] The transmembrane domains described herein can be combined with any of the antigen binding domains described herein, any of the costimulatory signaling domains described herein, any of the intracellular signaling domains described herein, or any of the other domains described herein that can be included in a subject CAR.

[0347] In one embodiment, the transmembrane domain comprises a CD8α transmembrane domain. In some embodiments, the transmembrane domain consists of a CD8α transmembrane domain consisting of the amino acid sequence set forth in SEQ ID NO: 33. In some embodiments, the transmembrane domain consists of the nucleotide sequence set forth in SEQ ID NO: 34.

[0348] In some embodiments, the transmembrane domain comprises a CD28 transmembrane domain. In some embodiments, the CAR comprises a CD28 transmembrane domain comprising the amino acid sequence set forth in SEQ ID NO: 37. In some embodiments, the CD28 transmembrane domain comprises the nucleotide sequence set forth in SEQ ID NO: 38.

[0349] Acceptable mutations of the transmembrane domain and / or hinge domain while maintaining its intended function will be known to those of skill in the art. In some embodiments, the transmembrane domain consists of an amino acid sequence having at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:33 and / or 37. In some embodiments, the transmembrane domain has at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity to any of the nucleotide sequences set forth in SEQ ID NOs:34 and / or 38. The transmembrane domain may be combined with any hinge domain and / or may be composed of one or more transmembrane domains described herein.

[0350] In some embodiments, a CAR comprises: a transmembrane domain of the alpha, beta, or zeta chain of the T cell receptor, any transmembrane domain selected from the group consisting of CD28, CD2, CD3ε, CD45, CD4, CD5, CD7, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134 (OX-40), CD137 (4-1BB), CD154 (CD40L), CD278 (ICOS), CD357 (GITR), Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, a killer immunoglobulin-like receptor (KIR); any costimulatory signaling domain, an intracellular or cytoplasmic domain described herein, or other domains described herein that may be included in a CAR, and optionally a hinge domain.

[0351] In some embodiments, the CAR further comprises a spacer domain between the extracellular domain and the transmembrane domain of the CAR, or between the intracellular domain and the transmembrane domain of the CAR. In some embodiments, the spacer domain can be a short oligo- or polypeptide linker, e.g., between about 2 and about 10 amino acids in length. For example, a glycine-serine doublet provides a particularly suitable linker between the transmembrane domain and the intracellular signaling domain of a subject CAR. Thus, the CAR of the present disclosure can comprise any of the transmembrane domains, hinge domains, or spacer domains described herein. 3. Hinge domain

[0352] In some embodiments, the CAR encoded by the lentiviral or retroviral vector of the present disclosure further comprises a hinge region. The hinge region of a CAR is a hydrophilic region located between the antigen-binding domain and the transmembrane domain. In some embodiments, the hinge region promotes proper protein folding of the CAR. In some embodiments, the hinge domain is an optional component of the CAR. In some embodiments, the hinge domain is comprised of a domain selected from an Fc fragment of an antibody, an antibody hinge region, an antibody CH2 region, an antibody CH3 region, an artificial hinge sequence, or a combination thereof. In some embodiments, the hinge domain is selected from, but is not limited to, a CD8a hinge, an artificial hinge comprised of a polypeptide that may be as small as three glycines (Gly). In some embodiments, the hinge region is a receptor-derived hinge region polypeptide. In some embodiments, the hinge region is a CD8-derived hinge region. In some embodiments, the hinge region is comprised of an amino acid sequence derived from human CD8 or a variant thereof. In some embodiments, the subject CAR comprises a CD8α hinge domain and a CD8α transmembrane domain. In some embodiments, the CD8α hinge domain consists of the amino acid sequence set forth in SEQ ID NO: 35. In some embodiments, the CD8α hinge domain consists of the nucleotide sequence set forth in SEQ ID NO: 36.

[0353] In some embodiments, the hinge domain comprises an amino acid sequence having at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity to any of the amino acid sequences set forth in SEQ ID NO: 35.

[0354] In some embodiments, the hinge domain is encoded by a nucleic acid sequence consisting of a nucleotide sequence having at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any of the nucleotide sequences set forth in SEQ ID NO:36.

[0355] In some embodiments, the hinge region connects the antigen-binding domain to the transmembrane domain connected to the intracellular domain. In exemplary embodiments, the hinge region can support the antigen-binding domain so that it recognizes and binds to a target antigen on a target cell. In some embodiments, the hinge region is a flexible domain, thus allowing the antigen-binding domain to have a structure that optimally recognizes the specific structure and density of the target antigen on a cell, such as a tumor cell. The flexibility of the hinge region allows the hinge region to adopt many different conformations.

[0356] In some embodiments, the hinge region has a length selected from about 4 to about 50, about 4 to about 10, about 10 to about 15, about 15 to about 20, about 20 to about 25, about 25 to about 30, about 30 to about 40, or about 40 to about 50 amino acids. A suitable hinge region can be readily selected and can be any of a number of suitable lengths, such as about 1, about 2, about 3, about 4, about 5, about 6, or about 7 amino acids, including about 1 amino acid (e.g., glycine (Gly)) to about 20 amino acids, about 2 amino acids to about 15 amino acids, about 3 amino acids to about 12 amino acids, about 4 amino acids to about 10 amino acids, about 5 amino acids to about 9 amino acids, about 6 amino acids to about 8 amino acids, or about 7 amino acids to about 8 amino acids.

[0357] In some embodiments, the amino acid is glycine (Gly). Glycine and glycine-serine polymers can be used; both Gly and Ser are relatively unstructured and can therefore function as neutral tethers between components. Glycine polymers can be used; glycine has access to significantly more φ-Ψ space than alanine and is much less restrictive than residues with long side chains. In some embodiments, the hinge region comprises a glycine polymer (G)n, a glycine-serine polymer. In some embodiments, the hinge region is comprised of a glycine-serine polymer selected from the group consisting of (GS)n, (GSGGS)n, and (GGGS)n, where n is an integer of at least 1. In some embodiments, the hinge region consists of an amino acid sequence including, but not limited to, GGSG (SEQ ID NO: 24), GGSGG (SEQ ID NO: 25), GSGSG (SEQ ID NO: 26), GSGGG (SEQ ID NO: 27), GGGSG (SEQ ID NO: 28), GSSSG (SEQ ID NO: 29). In some embodiments, the hinge region comprises a glycine-alanine polymer, an alanine-serine polymer, or other flexible linker known in the art.

[0358] In some embodiments, the hinge region is an immunoglobulin heavy chain hinge region. Immunoglobulin hinge region amino acid sequences are known in the art. In some embodiments, the immunoglobulin hinge region is selected from the group consisting of DKTHT (SEQ ID NO: 39); CPPC (SEQ ID NO: 40); CPEPKSCDTPPPCPR (SEQ ID NO: 41) (see, e.g., Glaser et al., J. Biol. Chem. (2005) 280:41494-41503); elktplgdttht (SEQ ID NO: 42); kscdkthtcp (SEQ ID NO: 43); kccvdcp (SEQ ID NO: 44); kygppcp (SEQ ID NO: 45); epkscdkthtcppcp (SEQ ID NO: 46) (human IgG1 hinge); ERKCCVECPPCP (SEQ ID NO: 47) (human IgG2 hinge); ELKTPLGDTTHTCPRCP (SEQ ID NO: 48) (human IgG3 hinge); SPNMVPHAHHAQ (SEQ ID NO: 49) NO:49) (human IgG4 hinge); and the like.

[0359] In some embodiments, the hinge region is an immunoglobulin heavy chain hinge region. In some embodiments, the hinge is selected from the CH1 and CH3 domains of IgG (such as human IgG4). In some embodiments, the hinge domain comprises the amino acid sequence of a human IgG1, IgG2, IgG3, or IgG4 hinge domain. In some embodiments, the hinge region may comprise one or more amino acid substitutions and / or insertions and / or deletions compared to a wild-type (naturally occurring) hinge region. In some embodiments, the histidine at position 229 (His229) of the human IgG1 hinge is substituted with tyrosine (Tyr). In some embodiments, the hinge domain comprises the amino acid sequence EPKSCDKTYTCPPCP (SEQ ID NO: 46). 4. Intracellular domain

[0360] The CAR encoded by the lentiviral or retroviral vector of the present disclosure also contains an intracellular domain. The intracellular domain, or otherwise the cytoplasmic domain, of the CAR is responsible for activating the cell in which the CAR is expressed. Thus, the term "intracellular domain" refers to any portion of the intracellular domain sufficient to transmit an activation signal. In one embodiment, the intracellular domain includes a domain responsible for effector function. The term "effector function" refers to a specialized function of a cell. For example, the effector function of a T cell may be cytolytic activity or helper activity, including cytokine secretion. In one embodiment, the intracellular domain of the CAR includes a domain responsible for signal activation and / or transduction. The intracellular domain can transmit signal activation through protein-protein interactions, biochemical changes, or other responses to change the cell's metabolism, shape, gene expression, or other cellular responses to the activation of the chimeric intracellular signaling molecule.

[0361] Examples of intracellular domains for use in the present invention include, but are not limited to, the cytoplasmic portion of the T cell receptor (TCR), and any costimulatory molecule, or any molecule that acts in concert with the TCR to initiate signaling in the T cell following engagement with the antigen receptor, as well as any derivatives or variants of these elements, and any synthetic sequences having the same functional capabilities.

[0362] In certain embodiments, the intracellular domain comprises an intracellular signaling domain. Examples of intracellular domains include TCR, CD3ζ, CD3γ, CD3δ, CD3 epsilon, CD86, common FcRγ, FcR beta (Fc epsilon rib), CD79a, CD79b, FcγR11a, DAP10, DAP12, T cell receptor (TCR), CD2, CD8, CD27, CD28, 4-1BB (CD137), OX9, OX40, CD30, CD40, PD-1, ICOS, KIR family proteins, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, and NKG2C. , B7-H3, CD83, CD5, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD127, CD160, CD19, CD4, CD8α, ligands that specifically bind to CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1Id, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD lib, ITGAX, CD11c, ITGBl, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, Syk family tyrosine kinase (Syk), ZAP70, etc.), src family tyrosine kinases (such as Lck, Fyn, Lyn), other costimulatory molecules described herein, derivatives, variants, or fragments thereof, any synthetic sequence of a costimulatory molecule having the same functional capability, and any combination thereof.

[0363] In some embodiments, the intracellular signaling domain is selected from the group consisting of the cytoplasmic signaling domain of human CD2, the CD3 zeta chain (CD3ζ), FcγRIII, the cytoplasmic tail of FcsRI, the cytoplasmic tail of an Fc receptor, a cytoplasmic receptor with an immunoreceptor tyrosine-based activation motif (ITAM), TCRζ, FcRγ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, and CD66d, or a variant thereof. In some embodiments, the intracellular signaling domain consists of the CD3ζ intracellular signaling domain.

[0364] Additional examples of intracellular domains include the intracellular signaling domains of several types of other immune signaling receptors, including, but not limited to, first-, second-, and third-generation T cell signaling proteins, including CD3, costimulatory members of the B7 family, and tumor necrosis factor receptor (TNFR) superfamily receptors. Additionally, intracellular signaling domains can include signaling domains used by NK cells and NKT cells, such as the signaling domains of NKp30 (B7-H6), DAP12, NKG2D, NKp44, NKp46, DAP10, and CD3z.

[0365] Intracellular signaling domains suitable for use in the CAR of the present invention include any desired signaling domain that transmits a signal in response to CAR activation (i.e., activation by an antigen and a dimerization agent). In some embodiments, clear and detectable signals include, for example, an increase in the production of one or more cytokines by the cell; a change in the transcription of a target gene; a change in the activity of a protein; a change in cell behavior (e.g., cell death); cell proliferation; cell differentiation; cell survival; and / or modulation of cell signaling responses. In some embodiments, the intracellular signaling domain comprises a DAP10 / CD28-type signaling chain. In some embodiments, the intracellular signaling domain is not covalently bound to the membrane-bound CAR, but instead diffuses within the cytoplasm.

[0366] Intracellular signaling domains suitable for use in the CARs of the invention include immunoreceptor tyrosine-based activation motif (ITAM)-containing intracellular signaling polypeptides. In some embodiments, the intracellular signaling domain contains at least one, at least two, at least three, at least four, at least five, or at least six ITAM motifs, as described below. In certain embodiments, the ITAM motif is repeated twice in the intracellular signaling domain, wherein the first and second instances of the ITAM motif are separated from each other by 6 to 8 amino acids. In one embodiment, the intracellular signaling domain of a subject CAR contains three ITAM motifs. In some embodiments, the intracellular signaling domain comprises a signaling domain of a human immunoglobulin receptor, including, but not limited to, an immunoreceptor tyrosine-based activation motif (ITAM), such as FcγRI, FcγRIIA, FcγRIIC, FcγRIIIA, or FcRL5.

[0367] A suitable intracellular signaling domain can be an ITAM motif-containing portion derived from a polypeptide containing an ITAM motif. For example, a suitable intracellular signaling domain can be an ITAM motif-containing domain derived from any ITAM motif-containing protein. Thus, a suitable intracellular signaling domain need not 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, FCER1G (Fcε receptor Iγ chain), CD3D (CD3δ), CD3E (CD3ε), CD3G (CD3γ), CD3Z (CD3ζ), and CD79A (antigen receptor complex-associated protein α chain).

[0368] In one embodiment, the intracellular signaling domain is derived from DAP12 (also known as TYROBP; TYRO protein tyrosine kinase-binding protein; KARAP; PLOSL; DNAX-activating protein 12; KAR-associated protein; TYRO protein tyrosine kinase-binding protein; killer activating receptor-associated protein; killer activating receptor-associated protein; etc.). In one embodiment, the intracellular signaling domain is derived from FCER1G (also known as FCRG; Fcε receptor Iγ chain; Fc receptor γ chain; fcεRIγ; fcRγ; fceR1γ; high-affinity immunoglobulin epsilon receptor subunit γ; immunoglobulin E receptor, high affinity, γ chain; etc.). In one embodiment, the intracellular signaling domain is 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.). In one embodiment, the intracellular signaling domain is 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 chain, T3e, etc.). In one embodiment, the intracellular signaling domain is 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.). In one embodiment, the intracellular signaling domain is derived from the T cell surface glycoprotein CD3ζ chain (also known as CD3Z, T cell receptor T3ζ chain, CD247, CD3-ζ, CD3H, CD3Q, T3Z, TCRZ, etc.). In one embodiment, the intracellular signaling domain is derived from CD79A (also known as B cell antigen receptor complex-associated protein α chain; CD79a antigen (immunoglobulin-associated α); MB-1 membrane glycoprotein; Ig-α; membrane-bound immunoglobulin-associated protein; surface IgM-associated protein; etc.).In one embodiment, the intracellular signaling domain suitable for use in a CAR of the present disclosure comprises a DAP10 / CD28-type signaling chain. In an embodiment, the intracellular signaling domain suitable for use in a subject CAR of the present disclosure comprises a ZAP70 polypeptide. In an embodiment, the intracellular signaling domain comprises the cytoplasmic signaling domain of TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3 epsilon, CD5, CD22, CD79a, CD79b, or CD66d. In one embodiment, the intracellular signaling domain of a CAR comprises the cytoplasmic signaling domain of human CD3ζ.

[0369] Typically, the entire intracellular signaling domain can be employed, although 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, so long as it transmits an effector function signal. The intracellular signaling domain includes a truncated portion of the intracellular signaling domain sufficient to transmit an effector function signal.

[0370] The intracellular signaling domains described herein can be combined with any of the costimulatory signaling domains described herein, any of the antigen-binding domains described herein, any of the transmembrane domains described herein, or any of the other domains described herein that can be included in a CAR. In some embodiments, the intracellular domain of a CAR comprises a dual signaling domain. The dual signaling domain can comprise a fragment or domain from any of the molecules described herein. In some embodiments, the intracellular domain consists of a 4-1BB costimulatory domain and a CD3ζ signaling domain; a CD28 costimulatory domain and a CD3ζ signaling domain; or a CD2 costimulatory domain and a CD3ζ signaling domain. In some embodiments, the intracellular domain of a CAR comprises at least one signaling domain from any portion of a costimulatory molecule, such as CD3, CD27, CD28, ICOS, 4-1BB, PD-1, T cell receptor (TCR), any derivative or variant thereof, any synthetic sequence thereof having the same functional capability, and any combination thereof.

[0371] Furthermore, mutant intracellular signaling domains suitable for use in the target CAR are known in the art.YMFM motif is found in ICOS, and is an SH2 binding motif that recruits both p85 and p50α subunits of PI3K, thereby enhancing AKT signal transduction.In one embodiment, CD28 intracellular domain mutants can be generated to contain YMFM motif.

[0372] In one embodiment, the intracellular domain of a subject CAR comprises a CD3ζ intracellular signaling domain consisting of the amino acid sequence set forth in SEQ ID NO: 50 or SEQ ID NO: 51, which can be encoded by a nucleic acid sequence consisting of the nucleotide sequence set forth in SEQ ID NO: 52 or SEQ ID NO: 53, respectively.

[0373] Acceptable mutations of the intracellular domain while maintaining specific activity will be known to those of skill in the art. In some embodiments, the intracellular domain comprises an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of the amino acid sequences set forth in SEQ ID NO:50 or 51. In some embodiments, the intracellular domain has sequence identity to any of the nucleotide sequences set forth in SEQ ID NO:52 or 53, having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. 5. Costimulatory Area

[0374] In some embodiments, the intracellular domain comprises a costimulatory signaling domain and an intracellular signaling domain. In one embodiment, the intracellular domain comprises a costimulatory signaling domain. In one embodiment, the intracellular domain of the CAR comprises a costimulatory signaling domain selected from the group consisting of a portion of the signaling domains from the TNFR superfamily of proteins, CD27, CD28, 4-1BB (CD137), OX40 (CD134), PD-1, CD7, LIGHT, CD83L, DAP10, DAP12, CD27, CD2, CD5, ICAM-1, LFA-1, Lck, TNFR-I, TNFR-II, Fas, CD30, CD40, ICOS (CD278), NKG2C, B7-H3 (CD276), and killer immunoglobulin-like receptors (KIRs, derivatives or variants thereof, synthetic sequences thereof having the same functional capabilities, and any combination thereof).

[0375] In some embodiments, the costimulatory domain comprises an intracellular domain from a protein of the TNFR superfamily, CD28, 4-1BB (CD137), OX40 (CD134), PD-1, CD7, LIGHT, CD83L, DAP10, DAP12, CD27, CD2, CD5, ICAM-1, LFA-1, Lck, TNFR-I, TNFR-II, Fas, CD30, CD40, ICOS (CD278), NKG2C, B7-H3 (CD276), and killer immunoglobulin-like receptor (KIR), or a variant thereof. In some embodiments, the costimulatory domain comprises one or more costimulatory domains of a protein selected from the group consisting of CD28, 4-1BB (CD137), OX40 (CD134), CD27, CD2, or a combination thereof. In some embodiments, the costimulatory signaling domain comprises a 4-1BB costimulatory domain. In some embodiments, the costimulatory signaling domain comprises a CD2 costimulatory domain. In some embodiments, the costimulatory signaling domain comprises a CD28 costimulatory domain.

[0376] In some embodiments, the costimulatory domain comprises an amino acid sequence set forth in SEQ ID NO: 54, 57, 59, 61, 64, 66, 68, or 70 that has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of the amino acid sequences set forth in SEQ ID NO: 54, 57, 59, 61, 64, 66, 68, or 70. In some embodiments, the intracellular domain has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of the nucleotide sequences set forth in SEQ ID NOs:55, 56, 58, 60, 62, 63, 65, 67, 69, or 71.

[0377] In one embodiment, the intracellular domain of the subject CAR comprises an ICOS costimulatory domain and a CD3ζ intracellular signaling domain. In one embodiment, the intracellular domain of the subject CAR comprises a CD28 costimulatory domain and a CD3ζ intracellular signaling domain. In one embodiment, the intracellular domain of the subject CAR comprises a CD28 YMFM mutant costimulatory domain and a CD3ζ intracellular signaling domain. In one embodiment, the intracellular domain of the subject CAR comprises a CD27 costimulatory domain and a CD3ζ intracellular signaling domain. In one embodiment, the intracellular domain of the subject CAR comprises an OX40 costimulatory domain and a CD3ζ intracellular signaling domain. In one exemplary embodiment, the intracellular domain of the subject CAR comprises a 4-1BB costimulatory domain and a CD3ζ intracellular signaling domain. In one exemplary embodiment, the intracellular domain of the subject CAR comprises a CD2 costimulatory domain and a CD3ζ intracellular signaling domain. B. Other Antigen-Binding Polypeptides

[0378] In some embodiments, the engineered T cells express an antigen-binding polypeptide, a cell surface receptor ligand, or a polypeptide that binds to a tumor antigen. In some embodiments, the antigen-binding domain consists of an antibody that recognizes a cell surface protein or receptor expressed on a tumor cell. In some embodiments, the antigen-binding domain consists of an antibody that recognizes a tumor antigen. In some embodiments, the antigen-binding domain consists of a full-length antibody or an antigen-binding fragment thereof, Fab, F(ab)2, monospecific Fab2, bispecific Fab2, trispecific Fab2, single-chain variable fragment (scFv), diabody, triabody, minibody, V-NAR, or VhH. C. Cell surface receptor ligands

[0379] In some embodiments, the lentiviral or retroviral vector of the present disclosure further comprises a nucleic acid encoding a cell surface receptor ligand. In some embodiments, the ligand binds to a cell surface receptor expressed on tumor cells. Optionally, the ligand consists of a wild-type protein or a mutant thereof that binds to the cell surface receptor. Optionally, the ligand consists of a full-length protein or a functional fragment thereof that binds to the cell surface receptor. In certain embodiments, the functional fragment is about 90%, 80%, 70%, 60%, 50%, or 40% of the length of the full-length version of the protein, but retains binding to the cell surface receptor. Optionally, the ligand is a de novo designed protein that binds to the cell surface receptor. Exemplary ligands include, but are not limited to, epidermal growth factor (EGF), platelet-derived growth factor (PDGF), or Wnt3A. D. Tumor antigens

[0380] In some embodiments, the lentiviral or retroviral vector of the present disclosure further comprises a nucleic acid encoding a polypeptide that binds to a tumor antigen. In some embodiments, the tumor antigen is associated with a hematological malignancy. Exemplary tumor antigens include, but are not limited to, CD19, CD20, CD22, CD33 / IL3Ra, ROR1, mesothelin, c-Met, PSMA, PSCA, folate receptor alpha, folate receptor beta, EGFRvIII, GPC2, Tn-MUC1, GDNF family receptor alpha-4 (GFRa4), fibroblast activation protein (FAP), and IL13Ra2. In some embodiments, the tumor antigen consists of CD19, CD20, CD22, BCMA, CD37, mesothelin, PSMA, PSCA, Tn-MUC1, EGFR, EGFRvIII, c-Met, HER1, HER2, CD33, CD133, GD2, GPC2, GPC3, NKG2D, KRAS, or WT1. In some embodiments, the polypeptide is a ligand for a tumor antigen, e.g., a full-length protein that binds to the tumor antigen, a functional fragment thereof, or a de novo artificial ligand that binds to the tumor antigen. In some embodiments, the polypeptide is an antibody that binds to the tumor antigen. E. Artificial T Cell Receptors

[0381] In some embodiments, the antigen-binding domain of a CAR described herein can be grafted onto one or more constant domains of a T cell receptor ("TCR") chain (e.g., a TCR alpha chain or a TCR beta chain) to create a chimeric TCR. The chimeric TCR can transmit a signal through the TCR complex upon antigen binding. For example, an scFv as disclosed herein can be grafted onto at least a portion of the constant domain, extracellular constant domain, or transmembrane domain of a TCR chain. As another example, an antibody fragment, such as a VL domain as described herein, can be grafted onto the constant domain of a TCR alpha chain. Such chimeric TCRs are produced, for example, by methods known in the art (e.g., Aggen et al., Gene Ther. 2012 Apr;19(4):365-74). F. Switch receptors and dominant-negative receptors

[0382] In one aspect, the lentiviral or retroviral vector of the present disclosure further comprises a nucleic acid encoding a dominant-negative receptor, a switch receptor, or a combination thereof. In some embodiments, the lentiviral or retroviral vector described herein comprises a chimeric antigen receptor (CAR) and / or a dominant-negative receptor. In some embodiments, the lentiviral or retroviral vector comprises a CAR and / or a switch receptor. In some embodiments, the lentiviral or retroviral vector described herein comprises an artificial TCR and a switch receptor. In some embodiments, the lentiviral or retroviral vector described herein comprises an artificial TCR and a dominant-negative receptor. In some embodiments, the lentiviral or retroviral vector described herein comprises a KIR and a switch receptor. In some embodiments, the lentiviral or retroviral vector described herein further comprises a KIR and a dominant-negative receptor. 1. Switch receptor

[0383] The present disclosure provides a rapid and efficient manufacturing process for engineering modified immune cells containing CARs or exogenous TCRs and / or switch receptors. In some embodiments, the CARs, TCRs, and / or switch receptors are encoded by one or more nucleic acids. In some embodiments, the lentiviral or retroviral vectors disclosed herein comprise one or more nucleic acid sequences encoding the CARs, TCRs, and / or switch receptors. In some embodiments, the nucleic acid sequence encoding the CAR is operably linked to the nucleic acid sequence encoding the switch receptor. In some embodiments, the switch receptor can increase the efficiency of the CAR or CAR-expressing cells.

[0384] Tumor cells generate an immunosuppressive microenvironment that protects them from immune recognition and elimination. This immunosuppressive microenvironment can limit the effectiveness of immunosuppressive therapies, such as CAR-T or TCR-T cell therapy. For example, the secreted cytokine transforming growth factor β (TGFβ) directly inhibits the function of cytotoxic T cells and further suppresses the immune response by inducing the formation of regulatory T cells. TGFβ-mediated T cell immunosuppression in prostate cancer has previously been demonstrated. To mitigate the immunosuppressive effects of TGFβ, immune cells can be engineered to express an artificial TGFβR, which combines the extracellular ligand-binding domain of the TGFβR with the intracellular signaling domain of, for example, the interleukin-12 receptor (IL12R; TGFβR-IL12R). Engineered immune cells containing the switch receptor can thus bind to negative signaling molecules in the engineered immune cell microenvironment and convert the negative signaling signals exerted by inhibitory molecules into positive signals that stimulate the engineered immune cells. The switch receptors of the present disclosure can be designed to reduce the effect of negative signaling molecules or to convert negative signals into positive signals by including intracellular domains associated with positive signals.

[0385] As used herein, the term "switch receptor" refers to a molecule designed to reduce the effect of a negative signaling molecule on the engineered immune cells of the present invention. A switch receptor consists of a first domain derived from a first polypeptide associated with a negative signal (a signali...

Claims

1. 1. A method for producing a population of artificial immune cells, comprising: (a) A population of lymphocytes, a population of immune cells, or CD4 + cells and CD8 + enriching the population of cells; (b) lymphocyte populations, immune cell populations, or CD4 + and CD8 + combining the population of cells with one or more buffers; and (c) lymphocyte populations, immune cell populations, or CD4 + cells and CD8 + transfecting the population of cells with an effective amount of a modifying agent; thereby producing a population of modified lymphocytes, a population of modified immune cells, or a modified CD4 + cells and CD8 + A method for generating a population of cells, The steps (a) to (c) are carried out within 24 hours.

2. Immune cell population or CD4 + cells and CD8 + 10. The method of claim 1, wherein prior to enriching the cell population, the blood is separated by apheresis into a plasma component, a mononuclear cell-containing layer, a platelet layer, and red blood cells to produce an apheresis product selected from red blood cell apheresis, platelet apheresis, leukocyte apheresis, stem cell apheresis, plasma apheresis, and platelet apheresis.

3. A population of immune cells, or CD4 + Cells and CD8 + 3. The method of claim 1 or 2, wherein the population of cells is enriched by apheresis, elutriation, or gradient centrifugation.

4. 1. A method for producing a population of artificial immune cells, comprising: (a) A population of lymphocytes, a population of immune cells, or CD4 + Cells and CD8 + enriching the population of cells; (b) lymphocyte populations, immune cell populations, or CD4 + and CD8 + combining the population of cells with one or more buffers; and (c) lymphocyte populations, immune cell populations, or CD4 + cells and CD8 + transfecting the population of cells with an effective amount of a modifying agent; thereby providing a population of lymphocytes, a population of modified immune cells, or a modified CD4 + cells and CD8 + A method for generating a population of cells, Here, steps 1(a) to (c) are carried out within 24 hours.

5. 1. A method for producing a population of engineered eukaryotic cells, comprising: (a) obtaining a population of eukaryotic donor cells from a subject; (b) combining the population of eukaryotic donor cells with one or more buffers; and (c) transfecting the population of eukaryotic donor cells with an effective amount of a modifying agent, thereby producing a population of modified eukaryotic donor cells, wherein steps 1(a) through (c) are performed on the same day.

6. Prior to the transfection step (c), a population of immune cells, CD4 + cells and CD8 + The method of any one of claims 1 to 5, wherein the population of cells, or the population of eukaryotic donor cells, is stimulated and / or activated with one or more stimulating agents.

7. 7. The method of any one of claims 1 to 6, wherein the modifying agent is selected from the group consisting of a small molecule drug, a biological drug, a therapeutic drug, a protein, a peptide, a protein therapeutic drug, a peptide therapeutic drug, a chimeric antigen receptor, a xenogeneic T cell receptor, a viral vector, a vector, a retroviral vector, a lentiviral vector, an adenoviral vector, and an adeno-associated viral vector.

8. The modifying agent is (a) selected from a retroviral vector, a lentiviral vector, an adenoviral vector, or an adeno-associated viral vector; (b) a lentiviral vector; or (c) Retroviral vectors The method according to any one of claims 1 to 7, wherein

9. CD4, a population of immune cells + cells and CD8 + The method of any one of claims 1 to 8, wherein the population of cells, or the population of eukaryotic donor cells, is transfected with an effective amount of a lentiviral or retroviral vector.

10. 10. The method of claim 9, wherein the lentiviral or retroviral vector comprises a nucleic acid sequence encoding a chimeric antigen receptor (CAR), an artificial T cell receptor (TCR), and / or a polypeptide that enhances immune cell function, or a functional derivative thereof, or a nucleic acid sequence that produces a therapeutic protein.

11. The population of immune cells or eukaryotic donor cells may include monocytes, lymphocyte-rich cells, B lymphocytes, T lymphocytes, CD4 + T lymphocytes, CD8 + T lymphocytes, dendritic cells, monocytes, natural killer (NK) cells, natural killer T (NKT) cells, T regulatory cells, CD4 + T helper cells, CD8 + Cytotoxic T lymphocytes (CTL), CD62L + cells, CD27 + cells, CCR7 + cells, CD45 RO- cells, CD45RA + Cells, neutrophils, basophils, eosinophils, megakaryocytes, stem cells, hematopoietic stem cells (HSCs), hematopoietic progenitor cells (HPCs), CD34 + cells, CD34 + The method of claims 1-10, wherein the cells are selected from peripheral blood stem cells, lymphocyte-activated killer cells (LAK), tumor-infiltrating lymphocytes (TIL), mesenchymal stem cells, mast cells, monocytes, macrophages, neutrophils, basophils, eosinophils, dendritic cells, megakaryocytes, and combinations thereof.

12. CD4, a population of immune cells + cells and CD8 + The concentration of the population of cells, or the population of eukaryotic donor cells, (a) at least about 0.7 × 10 7 , at least about 0.8 × 10 7 , at least about 0.9 × 10 7 At least about 1 x 10 pieces 7 , at least about 2 × 10 7 , at least about 4 × 10 7 , at least about 6 × 10 7 , at least about 8 × 10 7 , at least about 1 x 10 8 , or at least about 5 × 10 8 pieces / mL; (b) About 0.5×10 6 / mL~about 4×10 6 pcs / mL; (c) Approximately 0.5 ×10 6 pieces / mL ~ approx. 1 × 10 8 cells / mL; or (d) Approximately 4.0×10 6 cells / mL to approximately 1 x 10 8 pieces / mL, The method according to any one of claims 1 to 11, wherein

13. Transfection is (a) selected from the group consisting of viral transfection, transduction, non-viral transfection, and hybrids of viral and non-viral transfection; (b) selected from the group consisting of electroporation, laser beam, gene injection, sonoporation, magentofection, metal-coated nanoparticles, magnetically coupled adeno-associated virus, micro / nanoparticle-mediated transfection, lipofection, lipid-based transfection, anionic liposomes, cationic liposome-mediated transfection, cationic polymers, polymer encapsulation, peptide-mediated transfection, calcium phosphate, dendrimers, flowfection, photoporation, solvation, transient cell membrane disruption, deformation, squeezing, stretching, pinching, weakening, elongation, thinning, biological particle delivery systems, and combinations thereof; (c) electroporation of viral particles; (d) electroporation and viral transfection (transduction); (e) viral transfection and lipid-based transfection; or (f) viral transfection and liposome-based transfection; The method according to any one of claims 1 to 12, wherein

14. (a) A population of engineered immune cells, engineered CD4 + cells and CD8 + The population of cells, or the population of modified eukaryotic donor cells, is not activated with one or more stimulatory agents after or before transfection; and (b) A population of engineered immune cells, engineered CD4 + cells and CD8 + the population of cells, or the population of modified eukaryotic donor cells, is not expanded in vitro after transfection; The method according to any one of claims 1 to 13.

15. A population of engineered immune cells, engineered CD4 + cells and CD8 + The population of cells, or the population of modified eukaryotic donor cells, is stimulated and activated with one or more stimulatory agents to produce a population of activated modified immune cells, such as activated modified CD4 + cells and CD8 + 14. The method of any one of claims 1 to 13, further comprising producing a population of cells or a population of activated modified eukaryotic cells.

16. Activated lymphocyte populations, activated modified immune cell populations, activated modified monocyte populations, activated modified CD4 + and CD8 + The population of cells, or the population of activated modified eukaryotic donor cells, is expanded for a predetermined period of time to produce a population of modified lymphocytes, a population of modified immune cells, a modified CD4 + and CD8 + 16. The method of claim 15, further comprising producing a population of cells or a population of modified eukaryotic donor cells.

17. 17. The method of claim 16, wherein the growing step comprises: (a) Under vibration or rotation conditions; (b) In a closed system; (c) using serum-free culture medium; and / or (d) in the presence of one or more stimulants; The method of claim 16, wherein

18. A population of activated modified immune cells, activated modified CD4 + and CD8 + 18. The method of claim 16 or 17, wherein the population of cells, or the population of activated modified eukaryotic donor cells, is expanded at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, or at least about 25-fold.

19. For cryopreservation or administration, modified lymphocyte populations, modified immune cell populations, modified CD4 + and CD8 + 19. The method of any one of claims 1 to 18, further comprising harvesting the population of cells or the population of modified eukaryotic donor cells.

20. The harvesting of engineered lymphocytes, engineered immune cells, engineered CD4 + and CD8 + 20. The method of claim 19, comprising selecting and enriching for cells or engineered donor eukaryotic cells.

21. The collection may be performed to freeze or administer to a subject in need thereof engineered lymphocytes, engineered immune cells, engineered CD4 + and CD8 + 21. The method of claim 19 or 20, further comprising formulating the cells or engineered donor eukaryotic cells.

22. The specified time is (a) less than about 24 hours; less than about 30 hours; less than about 48 hours; less than about 72 hours; less than about 96 hours; or less than about 120 hours; (b) less than about 0.5 hours, less than about 1 hour, less than about 2 hours, less than about 3 hours, less than about 4 hours, less than about 5 hours, less than about 6 hours, less than about 7 hours, less than about 8 hours, less than about 9 hours, less than about 10 hours, less than about 11 hours, less than about 12 hours, less than about 13 hours, less than about 14 hours, less than about 15 hours, less than about 16 hours, less than about 17 hours, less than about 18 hours, less than about 19 hours, less than about 20 hours, less than about 21 hours, less than about 22 hours, or less than about 23 hours; or (c) about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, or more days; The method according to any one of claims 16 to 21, wherein

23. Lymphocyte population, immune cell population, CD4 + and CD8 + A population of cells, or a population of eukaryotic donor cells, is enriched and / or obtained, followed by the production of engineered immune cells, engineered CD4 + and CD8 + The time to harvest the cells or engineered eukaryotic donor cells is (a) Approximately 72 hours or less; (b) from about 18 hours to about 72 hours, from about 18 hours to about 36 hours, from about 18 hours to about 24 hours, from about 24 hours to about 72 hours, from about 24 hours to about 36 hours, or from about 36 hours to about 72 hours; (c) less than about 2 hours, less than about 3 hours, less than about 4 hours, less than about 5 hours, less than about 6 hours, less than about 7 hours, less than about 8 hours, less than about 9 hours, less than about 10 hours, less than about 11 hours, less than about 12 hours, less than about 13 hours, less than about 14 hours, less than about 15 hours, less than about 16 hours, less than about 17 hours, less than about 18 hours, less than about 19 hours, less than about 20 hours, less than about 21 hours, less than about 22 hours, or less than about 23 hours; (d) about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, or more days; or (e) about 1 day, about 3 days, about 4 days, about 5 days, or about 6 days; The method according to any one of claims 1 to 22, wherein

24. The method according to any one of claims 1 to 23, wherein the electroporation step, the activation step and / or the propagation step are carried out in a closed system, a semi-closed system and / or a functionally closed system.

25. 25. The method of claim 24, wherein the closed system is selected from the group consisting of a closed bag system, an automated closed cell sample processing system, and a bioreactor.

26. (a) the one or more stimulatory agents are selected from the group consisting of agonistic antibodies, cytokines, recombinant costimulatory molecules, anti-CD3 antibodies or fragments thereof, anti-CD28 antibodies or fragments thereof, small drug inhibitors, and / or combinations thereof; (b) the one or more stimulatory agents are cytokines selected from the group consisting of interleukin-2 (IL-2), interleukin-3 (IL-3), interleukin-6 (IL-6), interleukin-7 (IL-7), interleukin-7 receptor (IL-7R), interleukin-11 (IL-11), interleukin-12 (IL-12), interleukin-15 (IL-15), interleukin-15 receptor (IL-15R), interleukin-18 (IL-18), interleukin-18 receptor (IL-18R), interleukin-21 (IL-21), granulocyte-macrophage colony-stimulating factor, alpha, beta, or gamma interferon, erythropoietin, and combinations thereof; The method according to any one of claims 6 to 25, wherein

27. 27. The method of claim 26, wherein the one or more stimulatory agents are bound to beads or nanostructures.

28. 28. The method of claim 26 or 27, wherein (a) the one or more stimulatory agents are an anti-CD3 antibody and an anti-CD28 antibody or fragment thereof; (b) the one or more stimulatory agents are anti-CD3 and anti-CD28 antibodies or fragments thereof, and one or more cytokines; (b) the nanostructure is a nanomatrix; (c) the cytokine is selected from IL-2, IL-7, IL-6, IL-15, IL-15Ra, or IL-21; (d) the cytokines are selected from IL-15 and IL-7; IL-7 and IL-21; IL-7 and IL-2; IL-15 and IL-2; IL-7, IL-15 and IL-21; IL-15 and IL-15Ra; or IL-7, IL-15 and IL-15Ra; and / or (e) the one or more stimulating agents are a nanomatrix and one or more cytokines; 28. The method of claim 26 or 27.

29. The nanomatrix is (a) a matrix of flexible polymer chains and an anti-CD3 antibody and an anti-CD28 antibody or fragment thereof; or (c) size is between 1 and 500 nm; 29. The method of claim 28.

30. 30. The method of any one of claims 9-29, wherein the effective amount of retroviral or lentiviral vector comprises a multiplicity of infection (MOI) of about 0.01, about 0.02, about 0.03, about 0.04, about 0.05, about 0.06, about 0.07, about 0.08, about 0.09, about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.6, about 0.07, about 0.08, about 0.09, about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1.0, about 1.25, about 1.5, about 2.0, about 3.0, about 4.0, or about 5.

0.

31. An effective dose of a retroviral or lentiviral vector is (a) Approximately 2 ul of lentiviral vector at an MOI of approximately 0.08; (b) about 5 ul of lentiviral vector at an MOI of about 0.2; or (c) approximately 5 ul of lentiviral vector at an MOI of approximately 0.08; The method according to any one of claims 9 to 30, comprising:

32. 32. The method of any one of claims 9 to 31, wherein the lentiviral vector is based on a virus selected from the group consisting of a retrovirus, an alpharetrovirus, a betaretrovirus, a gammaretrovirus, a deltaretrovirus, and an epsilonretrovirus.

33. 33. The method of any one of claims 9 to 32, wherein the lentiviral vector is based on human immunodeficiency virus (HIV), equine infectious anemia virus (EIAV), vizma-ezivirus (VMV), caprine arthritis-encephalitis virus (CAEV), feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), VISNA virus, and simian immunodeficiency virus (SIV).

34. 34. The method of any one of claims 9 to 33, wherein the lentiviral vector is pseudotyped with an envelope glycoprotein (Env) from a virus selected from the group consisting of murine leukemia virus (MLV), vesicular stomatitis virus (VSV) Indiana strain, VSV New Jersey strain, Cocal virus, Chandipura virus, Pirie virus, spring of carp virus (SVCV), Sigma virus, infectious hematopoietic necrosis virus (IHNV), Mokola virus, rabies virus, CVS virus, Isfahan virus, Alagoas virus, Calchaqui virus, Jurona virus, La Joya virus, Maraba virus, feline endogenous retrovirus (RD114) envelope protein, Perine virus, Yugbagdanova virus, prototype foamy virus (PFV), and gibbon ape leukemia virus (GaLV).

35. 32. The method of any one of claims 9 to 31, wherein the lentiviral vector is pseudotyped with an envelope glycoprotein (Env) selected from the group consisting of vesicular stomatitis virus (VSV) Indiana strain, VSV New Jersey strain, and Cocal virus.

36. 36. The method of any one of claims 9 to 35, wherein the lentiviral vector comprises a heterologous viral envelope protein (Env) selected from the group consisting of Indiana strain VSV-G, New Jersey strain VSV-G, Kokar virus envelope protein, Isfahan virus envelope protein, Chandipura virus envelope protein, Pilibiri virus envelope protein, murine leukemia virus (MLV) envelope glycoprotein, SVCV virus envelope protein, and mutants thereof.

37. The method of any one of claims 9 to 36, wherein the lentiviral vector comprises a nucleotide sequence encoding a VSV-G envelope protein or a VSV G protein variant.

38. The method of any one of claims 9 to 37, wherein the lentiviral vector is a lentiviral particle.

39. 39. The method of any one of claims 10-38, wherein the CAR comprises an antigen-binding domain, a transmembrane domain, a costimulatory domain, and an intracellular domain, and the antigen-binding domain is selected from the group consisting of: (a) a full-length antibody or an antigen-binding fragment thereof, (b) a Fab, (c) a single-chain variable fragment (scFv), and (d) a single-domain antibody.

40. Antigen-binding domains include CD4, CD5, CD19, CD20, CD22, CD79b, CD79a, CD33, CD30, CD70, BCMA, GPC2, CD123, CD133, EGFR, EGFRvIII, mesothelin, HER2, PSMA, PSCA, FAP, CEA, GD2, IL-13Ra2, glypican-3, CIAX, LI-CAM, CA125, CTAG1B, TnMUC1, mucin 1, folate receptor alpha (FRa), GFRα-4, NYESO, WT1, (AFP) / HLA-A2, A XL, B7-H3, CA-IX, CD3, CD7, CD8, CD38, CD44v6, CD80, CD86, CD117, CD147, CD276, CEA, claudin 18.2, c-Met, DLL3, DR5, EpCAM, EphA2, FAP, folate-binding protein (FBP), glycolipid F77, glypican-3 (GPC3), glypican-2, HLA-A2, ICAMI, IL3Ra, LAGE-I, Lewis Y, LMPI (EBV), MAGE-Al, MAGE-A3, MAGE-A4, Melan 40. The method of claim 39, wherein the antibody specifically binds to a target antigen selected from the group consisting of: A, MG7 (glycated CEA), MMP, MUCI, Nectin4 / FAP, NKG2D-ligand, MIC-A, MIC-B, ULBPs I to 6, NY-ESO-1, P16, PD-L1, ROR1, ROR2, TIM-3, TM4SF1, VEGFR2, and combinations thereof.

41. The CAR transmembrane domain is selected from the group consisting of an artificial hydrophobic sequence, a transmembrane domain of a type I transmembrane protein, the α, β, or ζ chain of the T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, OX40 (CD134), 4-1BB (CD137), ICOS (CD278), or CD154, and a transmembrane domain derived from a killer immunoglobulin-like receptor (KIR).

41. The method of claim 39 or 40.

42. The method of any one of claims 39 to 41, wherein the costimulatory domain is an intracellular domain of a protein selected from the group consisting of TNFR superfamily proteins, CD27, CD28, 4-1BB (CD137), OX40 (CD134), PD-1, CD7, LIGHT, CD83L, DAP10, DAP12, CD27, CD2, CD5, ICAM-1, LFA-1, Lck, TNFR-I, TNFR-II, Fas, CD30, CD40, ICOS (CD278), NKG2C, B7-H3 (CD276), and killer immunoglobulin-like receptors (KIR).

43. The method of any one of claims 39 to 42, wherein the intracellular signaling domain is selected from the group consisting of the cytoplasmic signaling domain of human CD3 zeta chain (CD3ζ), FcγRIII, FcsRI, the cytoplasmic tail of an Fc receptor, a cytoplasmic receptor with an immunoreceptor tyrosine-based activation motif (ITAM), TCRζ, FcRγ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, and CD66d.

44. The method of any one of claims 39 to 43, wherein the CAR further comprises a hinge region.

45. 1. A method for delivering a nucleic acid encoding a chimeric antigen receptor (CAR), an engineered T cell receptor, or a therapeutic protein to a cell, the method comprising introducing into the cell a transfer plasmid comprising: (a) a polynucleotide sequence encoding at least one heterologous viral envelope protein engineered by the method of any one of claims 32 to 37; (b) a polynucleotide sequence encoding at least one retroviral rev protein; (c) a polynucleotide sequence encoding at least one retroviral gag protein and a retroviral pol protein; and / or (d) a polynucleotide sequence encoding a chimeric antigen receptor, an artificial T cell receptor (TCR), or a therapeutic protein; Here, at least a portion of one or more regions of the retroviral genome essential for replication is mutated.

46. 46. ​​A lentiviral vector particle produced by the method of claim 45.

47. A method for introducing a variant into a cell, comprising electroporating the cell with an effective amount of the lentiviral vector particle of claim 46, thereby producing a variant cell.

48. 48. The method of claim 47, wherein the cells are contacted with an effective amount of a lentiviral vector prior to electroporation.

49. 48. The method of claim 47, wherein the cells are contacted with the effective amount of the lentiviral vector for up to about 4 hours after electroporation.

50. infecting the cells with an effective amount of a lentiviral vector; (a) at least about 5-30 minutes, at least about 25-50 minutes; at least about 5-60 minutes, at least about 5-12 minutes, at least about 60-120 minutes, at least about 120-240 minutes after electroporation; (b) at least about 1 minute, at least about 2 minutes, at least about 5 minutes, at least about 10 minutes, at least about 20 minutes, at least about 30 minutes, at least about 45 minutes, at least about 60 minutes, at least about 75 minutes, at least about 90 minutes, at least about 100 minutes, at least about 110 minutes, at least about 120 minutes, at least about 150 minutes, at least about 160 minutes, at least about 170 minutes, at least about 180 minutes, at least about 190 minutes, at least about 200 minutes, at least about 220 minutes, or at least about 240 minutes after electroporation; 50. The method of claim 49, wherein the contacting is performed for a period of time.

51. The cells include immune cells, eukaryotic donor cells, mononuclear cells, concentrated lymphocytes, B lymphocytes, T lymphocytes, CD4 + T lymphocytes, CD8 + T lymphocytes, dendritic cells, monocytes, natural killer (NK) cells, natural killer T (NKT) cells, T regulatory cells, CD4 + T helper cells, CD8 + Cytotoxic T lymphocytes (CTL), CD62L + cells, CD 27 + cells, CCR 7 + cells, CD45RO - cells, CD45 RA + cells, neutrophils, basophils, eosinophils, megakaryocytes, stem cells, hematopoietic stem cells (HSCs), hematopoietic progenitor cells (HPCs), CD34 + cells, CD34 + The method of any one of claims 47 to 50, wherein the cells are selected from the group consisting of peripheral blood stem cells, lymphocyte-activated killer cells (LAKs), tumor-infiltrating lymphocytes (TIL), circulating tumor-specific T cells, mesenchymal stem cells, mast cells, monocytes, macrophages, neutrophils, basophils, eosinophils, dendritic cells, megakaryocytes, and combinations thereof.

52. The cells (a) T cells, B cells, natural killer (NK) cells, CD8 + T cells, CD4 + lymphocytes selected from the group consisting of T cells, cytotoxic T lymphocytes, regulatory T cells, and any combination thereof; (b) myeloid cells selected from the group consisting of monocytes, macrophages, neutrophils, basophils, eosinophils, dendritic cells, megakaryocytes, and any combination thereof; (c) Stem cells, hematopoietic stem cells, hematopoietic progenitor cells, CD34 + cells, or CD34 + peripheral blood stem cells; The method according to any one of claims 47 to 51, wherein

53. 53. The method of any one of claims 47-52, wherein the effective amount of lentiviral vector particles comprises about 0.5ul, about 1ul, about 1.5ul, about 2ul, about 2.5ul, about 3ul, about 3.5ul, about 4ul, about 5ul, about 6ul, about 7ul, about 8ul, about 9ul, about 10ul, about 15ul, or about 20ul of lentiviral vector.

54. 54. The method of any one of claims 47-53, wherein the effective amount of lentiviral vector particles comprises a multiplicity of infection (MOI) of about 0.01, about 0.02, about 0.03, about 0.04, about 0.07, about 0.08, about 0.07, about 0.08, about 0.09, about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1.0, about 1.25, about 1.5, about 2.0, about 3.0, about 4.0, or about 5.

0.

55. an effective amount of lentiviral vector particles, (a) Approximately 2 ul of lentiviral vector particles at an MOI of approximately 0.08; (b) about 5 ul of lentiviral vector particles at an MOI of about 0.2; or (c) approximately 10 ul of lentiviral vector particles at an MOI of approximately 0.4; 55. The method of any one of claims 47 to 54, comprising:

56. Modified cells, modified immune cells, modified CD4 cells engineered by the method of any one of claims 1 to 38. + and CD8 + cells, or modified eukaryotic donor cells.

57. A modified population of cells, a modified population of immune cells, a modified CD4 + cells and CD8 + A population of cells, or a population of modified eukaryotic donor cells.

58. A modified cell, a modified immune cell, or a modified CD4 comprising the lentiviral vector of claim 46. + and CD8 + cells, or modified eukaryotic donor cells.

59. A modified population of cells, a modified population of immune cells, a modified CD4 + cells and CD8 + A population of cells, or a population of modified eukaryotic donor cells.

60. The modified cells, modified immune cells, modified CD4 cells according to any one of claims 56 to 59 for use in the production of a protein of interest. + and CD8 + Manipulation of cells, or modified eukaryotic donor cells.

61. The modified cell, modified immune cell, or modified CD4 cell of claim 60, wherein the protein of interest is selected from the group consisting of an industrial protein or a therapeutic protein. + and CD8 + cells, or modified eukaryotic donor cells.

62. 61. The modified cell, modified immune cell, or modified CD4+ antibody of claim 60, wherein the protein of interest is selected from the group consisting of an enzyme, a regulatory protein, a receptor, a peptide, a peptide hormone, a cytokine, a membrane protein or transport protein, a vaccine antigen, an antigen-binding protein, an immunostimulatory protein, an allergen, a full-length antibody or an antibody fragment or derivative, a single-chain antibody (scFv), a Fab fragment, an Fy fragment, a single-domain antibody (VH or VL fragment), a domain antibody, a camelid single-domain antibody (VHH), a nanobody, and combinations thereof. + and CD8 + Cells, or modified eukaryotic donor cell manipulations.

63. (a) the modified cells, modified immune cells, or modified CD4 cells according to claim 56 or 58 + and CD8 + cells, or modified eukaryotic donor cells; (b) a modified cell population, modified immune cell population, or modified CD4 + and CD8 + a cell population, or a modified eukaryotic donor cell population; or (c) a lentiviral vector according to any one of claims 5 to 44; A composition comprising:

64. 64. The composition of claim 63, further comprising a pharmaceutically acceptable excipient.

65. 1. A method of treating a disease or condition in a subject, comprising: A therapeutically effective amount of (a) the modified cells, modified immune cells, or modified CD4 cells according to claim 56 or 58 + and CD8 + cells, or modified eukaryotic donor cells; (b) the modified cell population, modified immune cell population, or modified CD4 + and CD8 + a cell population, or a modified eukaryotic donor cell population; or (c) a composition according to claim 60 or 63; to a subject in need thereof, thereby treating a disease or condition in a subject.

66. 66. The method of claim 65, wherein the disease or condition is selected from the group consisting of a viral infection, a bacterial infection, a parasitic infection, cancer, a malignancy, a non-cancerous condition, an autoimmune disease, a fibrotic disease, Alzheimer's disease, a protein deficiency state, and a factor VIII deficiency.

67. 68. The method of any one of claims 65 to 67, wherein the cancer is selected from breast cancer, triple-negative breast cancer, prostate cancer, ovarian cancer, glioma, glioblastoma, renal cell carcinoma, kidney cancer, mesothelioma, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, lung cancer, lung adenocarcinoma, gallbladder cancer, colon cancer, cervical squamous cell carcinoma, non-small cell lung cancer, small cell lung cancer, Merkel cell carcinoma, hepatocellular carcinoma, esophageal cancer, brain tumor, melanoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, urothelial cancer, gastric cancer, blood cancer lymphoma, leukemia, multiple myeloma, diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, acute myeloid leukemia, B-cell acute lymphoblastic leukemia (ALL), pre-B ALL, and combinations thereof.

68. Modified immune cells, modified CD4 + and CD8 + the cell, or modified eukaryotic donor cell, (a) is autologous to the subject; (b) allogeneic to the subject; or (c) Allogeneic to the subject The method according to any one of claims 65 to 67, wherein

69. Modified cells, modified immune cells, modified CD4 + and CD8 + 69. The method of any one of claims 66 to 68, wherein the cells or modified eukaryotic donor cells are allogeneic to the subject.

70. 70. The method of any one of claims 66 to 69, wherein the subject is a human.

71. 1. A method for producing a therapeutic protein, comprising: (a) producing a population of engineered immune cells or a population of engineered eukaryotic cells comprising a therapeutic protein using the method of any one of claims 1 to 38; (b) harvesting the therapeutic protein; and (c) isolating and purifying the therapeutic protein; A method comprising:

72. 72. The method of claim 71, wherein the therapeutic protein is selected from the group consisting of an enzyme, a regulatory protein, a receptor, a peptide, a peptide hormone, a cytokine, a membrane or transport protein, a vaccine antigen, an antigen-binding protein, an immunostimulatory protein, an allergen, a full-length antibody or an antibody fragment or derivative, a single chain antibody (scFv), a Fab fragment, an Fv fragment, a single domain antibody (VH or VL fragment), a domain antibody, a camelid single domain antibody (VHH), a nanobody, and combinations thereof.

73. Modified immune cell populations, or modified CD4 + cells and CD8 + A population of cells, or a population manipulated by the method of any one of claims 1 to 38; or (b) the lentiviral vector of claim 46; Kit including:

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